r... .2 \u... .1." A". .319. . .3 , sum? .12; . fl. 4 VIMMMLJN. J‘. 5.»? ‘J. . THESIS 3004 54530997 LIBRARY Michigan State . . . lJnhmnsfiy Tfi:§$§%§éfiifiw COMPARISON OF PRODUCTION, MANAGEMENT, AND ANTIMICROBIAL SUSCEPTIBILITY OF BACTERIA FROM ORGANIC AND CONVENTIONAL DAIRY HERDS presented by Kenji Sato has been accepted towards fulfillment of the requirements for me Ph. D. degree in Large Animal Clinical Science (Epidemiology) /M KW MajorP essor’s lg ture a a 4L fag” Date/ MSU is an Affirmative Action/Equal Opportunity Institution COMPARISON OF PRODUCTION, MANAGEMENT, AND ANTIMICROBIAL SUSCEPTIBILITY OF BACTERIA FROM ORGANIC AND CONVENTIONAL DAIRY HERDS By Kenji Sato A DISSERTATION Submitted to Michigan State University in partial fiilfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Large Animal Clinical Sciences (Epidemiology) 2003 ABSTRACT COMPARISON OF PRODUCTION, MANAGEMENT, AND ANTIMICROBIAL SUSCEPTIBILITY OF BACTERIA FROM ORGANIC AND CONVENTIONAL By Kenji Sato The production and management, prevalence and antimicrobial susceptibility of E. coli, Salmonella spp. , Enterococcus spp. and Campylobacter spp. isolated fi'om bovine feces, Staphylococcus aureus isolated from bulk tank milk were compared between organic and conventional dairy herds. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms, where antimicrobials were routinely used for animals for all ages. A seven-page questionnaire was used to assess management and production during 2000-2001. The organic farms had significantly fewer cattle than did the conventional herds. The average daily milk production per cow in organic dairy herds was lower than that of conventional herds. The incidence of clinical mastitis and bulk tank somatic cell count on organic farms was not statistically different from that of on conventional farms. There was little evidence of other fimdamental differences between two farm types in other major management and production parameters. Fecal specimens from ten cows and ten calves on 120 farm visits yielded 1,120 E. coli isolates, 7 Salmonella spp, which were tested for resistance to 17 antimicrobials. A total of 332 Campylobacter spp. isolates were tested to four antimicrobials (ciprofloxacin, erythromycin, gentamicin, and tetracycline). A total of 2,049 Enterococcus spp were tested to 3 antimicrobials (Quinupristin / dalfopristin, gentamicin, and vancomycin). Of the 118 bulk tank milk samples in Wisconsin, 71 samples (60%) yielded at least one Staphylococcus aureus isolate, and a total of 331 S. aureus were collected and tested for resistance to 15 antimicrobials. The susceptibility of S. aureus were also compared with Danish study. Our study shows significantly lower prevalence rates of AR in E. coli for seven antimicrobials (ampicillin, streptomycin, kanamycin, gentamicin, chloramphenicol, tetracycline, and sulphamethoxazole) in organic dairy herds, as compared to conventional herds. Two Campylobacter isolates from conventional dairy farms were resistant to ciprofloxacin and none of the isolates were resistant to gentamicin or erythromycin. Tetracycline-resistance in Campylobacter was 41.5% (66/159) for organic and 47.4% (82/173) for conventional herds, which was not statistically significant. A significant lower rate of resistant Staphylococcus aureus was detected to only one antimicrobial on organic farms in our Wisconsin study (ciprofloxacin) and on conventional farms in the parallel study in Denmark (avilamycin). Staphylococcus aureus isolates from Wisconsin had higher probability of reduced susceptibility to 7 out of 14 comparable antimicrobials, whereas Danish isolates had higher probability of reduced susceptibility to only two drugs. Differences in antimicrobial susceptibility between organic and conventional farms were small relative to the differences observed between the two countries. Although the organic farms had converted to organic farming methods at least 3 years before our study, antimicrobial resistance clearly presented long after antimicrobial selective pressure had been withdrawn. ACKNOWLEDGEMENT I would like to acknowledge the people whose contributions made this work possible. Especially my major professor, Dr. Paul C. Bartlett require a very special thanks for giving me this opportunity and for his support, sense of direction, input and patience. I will always be in great debt. Also, many thanks to rest of my graduate committee, Dr. John B. Kaneene, Dr. Ronald J. Erskine, Dr. Mahdi A. Saeed and Dr. Frances Pouch Downes for their insightful comments and advice. I gratefully acknowledge Michigan Department of Community Health for providing laboratory space, supplies and technical advice. In particular, I would like to thank Dr. James T. Rudrik, Robert Jacobson, Hao The Trinh, Carrie Anglewicz for their technical input. I thank Andrea Cedras for her laboratory work. Special thanks for Linda Reese, who instructed me in Salmonella serotyping and passed away in year 2000. I am greatly appreciated that William Beaumont Hospital provided Enterococcus isolation and test service and Atlantic Veterinary College, University of Prince Edward Island provided test service of antibodies against Ostertagia ostertagi. A big thanks to Wisconsin organic and conventional dairy producers because without their cooperation, this work would never been possible. I hope that this work contribute to improve their life and keep the industry healthy. The study was made possible by grants from FDA/CDC (Grant #USl/ccuS 16219 03 2 CCUSl). I am also sincerely grateful for comments and advice from Dr. Fred Angulo. Finally, a very warm and special thanks to my wife, Hiroko and my son, Remo. She always supported and encouraged me toward achieving this goal. iv TABLE OF CONTENTS LIST OF TABLES ix LIST OF FIGURES xi LIST OF ABBREVIATIONS xii CHAPTER 1: INTRODUCTION, STUDY OBJECTIVES AND STUDY METHOD INTRODUCTION 1 STUDY OBJECTIVES 5 STUDY METHOD 5 REFERENCE 7 CHAPTER 2: LITERATURE REVIEW Antimicrobial resistant bacteria from animals 8 Herd size 10 Milk production 11 Culling rate 11 Mastitis rate 11 Somatic Cell Count 12 Antimicrobial use in dairy production 13 Antimicrobial resistance 15 Avilamycin 15 Bacitracin 15 Ceftiofur 16 Chloramphenicol l 6 Fluoroquinolone 1 7 E. coli 17 C ampylobacter 1 8 Antimicrobial resistance in Campylobacter 21 Staphylococcus 21 Effect of management factors on resistance 22 References 23 CHAPTER 3: Milk Production and Mastitis on Midwestern Organic Dairy Farms Abstract 34 Introduction 35 Materials and Methods 36 Results 36 Discussion 39 References 43 Appendix 1. 49 CHAPTER 4: Comparison of Production and Management between Organic and Conventional Dairy Herds in Wisconsin Abstract 50 Introduction 5 1 Materials and Methods 53 Statistical Analysis 55 Results 56 Discussion 59 vi Conclusion 64 References 65 CHAPTER 5: Comparison of Antimicrobial Susceptibility of E. coli Isolated from Organic and Matched Conventional Dairy Cattle in Wisconsin Abstract 73 Introduction 74 Materials and Methods 75 Results 78 Discussion 80 Conclusion 86 References 87 CHAPTER 6: Comparison of Prevalence and Antimicrobial Susceptibility of Campylobacter Spp. Isolated from Organic and Conventional Dairy Herds in Wisconsin, USA Abstract 95 Introduction 96 Materials and Methods 98 Data and fecal sample collection 98 Bacteria isolation 99 Antimicrobial susceptibility testing 99 Statistical Analysis 100 Results 101 Discussion 103 vii Antimicrobial Resistance in Campylobacter spp. 106 Conclusion 107 References 109 CHAPTER 7: Comparison of Antimicrobial Susceptibility of Staphylococcus aureus Isolated from Bulk Tank Milk in Organic and Conventional Dairy Herds in Midwestern USA and Denmark Abstract 118 Introduction 1 19 Materials and Methods 121 Results 124 Discussion 125 Conclusion 130 References 13 1 CONCLUSIONS 142 APPENDICES Questionnaire used for interviews 147 SAS Control Commands 155 viii LIST OF TABLES Table 3-1. Estimated production parameters based on a presumable calving interval of 13 months and the dry period of two months with available Wisconsin state averages. Table 4-1. Comparison between organic and conventional dairy farms. Table 4-2. Comparison of discrete variables between organic and conventional dairy farms. Table 5-1. Frequency distributions of antimicrobial resistance E. coli. Table 5-2. Logistic Regression Analysis of resistance to antimicrobials among E coli isolates from organic and conventional dairy farms. Table 5-3. Frequency distribution of multiple antimicrobial resistance among 1,120 E. coli isolates. Table 6-1. Number of C ampylobacter isolates (percent of culture positive samples) in each group of samples. Table 6-2. Odds ratios for C ampylobacter sp. isolation (Generalized Linear Model analysis). Table 6-3. Generalized Linear Model analysis of management factors on Campylobacter sp. prevalence. Table 6-4. Proportion (%) of isolates which were inhibited by antimicrobials at each concentration. Table 7-1. Number of Staphylococcus aureus isolates examined in this study. Table 7-2. Antimicrobial susceptibility to 15 drugs in Staphylococcus aureus which were isolated from bulk tank milk samples collected from Wisconsin organic and conventional dairy farms. Table 7-3. Distribution of antimicrobial susceptibility to 15 drugs in Staphylococcus aureus which were isolated from bulk tank milk samples collected from Danish organic and conventional dairy farms. ix PAGE 48 68 69 91 93 94 113 114 115 116 135 136 137 Table 7-4. Logistic regression analysis with correlated data on dichotomized susceptibility result in Staphylococcus aureus from Wisconsin. 138 Table 7-5. Logistic regression analysis with correlated data on dichotomized susceptibility result in Staphylococcus aureus from Denmark. 139 Table 7-6. Logistic regression analysis with correlated data on dichotomized susceptibility result in Staphylococcus aureus. 140 LIST OF FIGURES PAGE Figure 3-1. Frequency distribution on number of lactating cows in July 1999. 45 Figure 3-2. Frequency distribution of average milk production per cow per day. 46 Figure 3—3. Frequency distribution on bulk tank SCC in July 1999. 47 Figure 1. Frequency distribution of mastitis rate (lOO-cow-year) of organic farms (n=3 0) and conventional farms (n=30). 71 Figure 4-2. Frequency distribution of milk somatic cell count (SCC) of organic farms (n=30) and conventional farms (n=3 0). 72 Figure 6-1. Distribution of MICs to tetracycline of Campylobacter spp. from conventional and organic dairy farms. 117 Figure 7-1. Comparison of minimum inhibitory concentration to bacitracin in Staphylococcus aureus isolated from bulk tank milk in Wisconsin and Denmark. 141 xi ATCC BCS BTSCC CDC CM DANMAP EASS ELISA ESBL GEE IVfDCH NIRSA MIC NAHMS NARMS NCCLS OD SBA SCC LIST OF ABBREVIATIONS Antimicrobial Resistance American Type Culture Collection Body Condition Score Bulk Tank milk Somatic Cell Count Center for Disease Control and Prevention Clinical Mastitis Danish Integrated Antimicrobial Resistance Monitoring and Research Programme Environmental and Animal Sanitation Score Enzyme-Linked Immunosorbent Assay Extended-spectrum B-lactamase Generalized Estimating Equation Michigan Department of Community Health Methicillin Resistant Staphylococcus aureus Minimal Inhibitory Concentration National Animal Health Monitoring System National Antimicrobial Resistance Monitoring System National Committee for Clinical Laboratory Standards Optical Density 5% Sheep Blood Agar Somatic Cell Count xii CHAPTER ONE INTRODUCTION, STUDY OBJECTIVES AND STUDY METHOD INTRODUCTION The wide use of penicillin began in 1942 and by 1945 the first report were made of antibiotic resistance bacteria. Resistant Staphylococcus aureus was described in a London hospital, and by 1949, approximately 59% of the S. aureus isolated in this hospital were reportedly resistant to penicillin. Some bacteria naturally developed resistance to antibiotics, long before the development of commercial antibiotics. Bacteria can develop resistance to certain drugs through mutation or can acquire resistant genes from other bacteria (plasmid, transposon, and transformation). The genetic traits for resistance has been shown to spread from one species of bacteria to other species. Antimicrobial resistance was not recognized as a major threat to human medicine until 1970’s, when new antimicrobials developed during 1950~1960’s were used to combat these resistant bacteria. For example, nosocomial infections with penicillinase producing S. aureus were already observed in 1950’s, but were controlled by methicillin, and cephalosporins. One of the most important challenges was recognized in 1980’s when MRSA (Methicillin Resistant Staphylococcus aureus) and other multiple-drug resistant bacterial infections were recognized in hospitals all over the world. Several strains of S. aureus had even acquired resistance to heavy metals and disinfectants used in hospitals. Advances in medical technology and an increased population of immunocompromised people, (cancer treatments, transplantations, diabetes, old age, and HIV) has further exacerbated the problem of antimicrobial resistance in the hospital and nursing home environment. The effects of antimicrobials as grth promoters for food animals were recognized soon after the initiation of antibiotic mass production, when higher grth rate were observed if the residue of tetracycline production were fed to mouse, poultry and swine. Also, the prophylactic use of antimicrobials for food animals became a common practice and allowed large scale livestock production systems. Soon thereafter, large scale livestock productions were accused of using antibiotics as a substitute for good hygiene and husbandry, however, consumers undoubtedly benefited from this mass productions which resulted in a high quality product at a relatively inexpensive price. Reports came from Norway (Klare, 1995) and Denmark (Aarestrup, 1995) on the possible association between avoparcin, used as a growth promoter, and the occurrence of vancomycin resistant enterococci in livestock. Such reports reversed the European trend toward antibiotic use as feed additives in animal production. Currently, the European Union has banned feed grade antimicrobials as growth promoters. Although the DANMAP 2002 data showed that feed-grade antimicrobials was decreased, therapeutic antibiotic use for livestock did increase. Other research data from Europe, however, showed mixed results and it still remains to be seen what benefits to human society might be realized by curtailing the use of antimicrobials for food animal growth promotion. The assessment of human health risk and benefits associated with the uses of antimicrobials in livestock industry had been the main issue for many years. There is still disagreement regarding the impact on public health caused by use of antimicrobials for food animals. Some have suggested that antibiotics should be banned from animal agriculture and be reserved exclusively for human clinical treatments (Mudd, 1996). Others contend that the prescribing practices in human medicine [not animal agriculture] are the major contributing factor to the current antimicrobial resistance problem (Cook, 1997; Schwartz et al., 1997). Assessments are needed regarding the degree to which use of antibiotics in animal agriculture is contributing to the proliferation of antibiotic resistant bacteria, and the extent to which animal agriculture can fiinction with reduced dependence on antibiotics (Dowling, 1997; Wiedemann and Grimm, 1996). It needs to be determined if a reduction in antimicrobial use in animal agriculture could eventually lead to a reduction, or at least a deceleration, in developing antibiotic resistance. Also, it is important to estimate the effect of policy changes on animal welfare and productivity. Some people insist the possibility of our food coming from diseased animal would increase or human food supplies would be reduced due to impact of death and disease. Population-based field data on antimicrobial resistance should be used as the basis for all policy changes regarding antibiotic usage. Organic dairy production may provide us the opportunity to evaluate one of possible consequences of the reduction in antibiotics usage on dairy farms. Organic dairy operations, which have long histories without the use of antimicrobials, allow us to simultaneously observe the effect of this management change on the prevalence of antimicrobial resistant bacteria and disease incidence in the farm animals. Intervention studies with antibiotic withdrawal for only a year or two may result in premature conclusions, since such studies make the assumption that the selective pressure will rapidly act upon the micro flora of the farm. The resistance to tetracycline in Escherichia coli was known to have been remained 126 months afier disuse of antimicrobials (Langlois et al. 1988). Although antibiotic usage for food animals increased in the 19605 ~ 1990s, most attempts to institute the judicious use of antibiotics were aimed at the human medical community, and hospitals in particular. As consumers and physicians learned of the extensive use of antibiotics for animal agriculture, they began to pressure the agricultural community to abandon the use of some antibiotics and to limit the use of antibiotics. It was, and still is, unclear how much of the human medical problem with antimicrobial resistance is caused by antibiotics fed to our food animals. It was also unclear how difficult it would be for animal agriculture to function efficiently without antibiotics, or how quickly antibiotic sensitivity might return to bacterial populations after the selective pressure for antibiotic resistance was removed. It was also clear that one answer to these questions could not be given for all antibiotic resistance mechanisms found in all species of bacteria. Rather, the answers would have to specifically address each “bug — drug” combination for the various uses in livestock production. For example, it may be that the use of fluoroquinolone is not very essential to poultry production, resistance genes are readily transferred to human pathogens such as Campylobacter, but that the genes that enable this resistance rapidly disappear in the population when the use of fluoroquinolone is ceased. A very different set of answers may exist for the use of tetracycline in swine operations. This dissertation attempts to answer some of the above-stated questions regarding the role of agricultural antibiotics in the epidemiology of antimicrobial traits. STUDY OBJECTIVES The overall objective of this study is to determine if dairy farms with at least a three year history of zero or minimal use of antibiotics have lower rates of antimicrobial resistant bacteria than do region-matched conventional dairy farms which make frequent use of antibiotics. Production and management parameters were also collected to estimate production, disease and management factors which enables organic operation feasible. The study was intended to provide scientific base to assess a possible outcomes of reduced use of antimicrobials on dairy farms. STUDY METHOD Thirty organic dairy farms and thirty neighboring conventional dairy farms in Wisconsin was selected for the study. As such, each organic farm was matched with a conventional farm based on region of the country. All farms were administered a seven- page questionnaire to measure overall frequency of disease and other production and management factors. From each farm, fecal samples were obtained from 10 calves and 10 cows, and a milk sample will be obtained from bulk tank. Escherichia coli, Salmonella spp., Enterococcus spp., C ampylobacter spp. from fecal samples and Staphylococcus aureus from milk samples were selected for this study. Since only a few isolates of Salmonella and a few resistant Enterococcus were obtained, the result of Salmonella and Enterococcus were not included in this dissertation. Because of the need to detect changes in degrees of susceptibility rather than dichotomized resistance, micro broth dilution methods (Sensititre‘m) was used for E. coli, Salmonella, Staphylococcus and Enterococcus, and gradient disk diffusion minimal inhibitor concentration method (Etestx') was used for Campylobacter. By having a hill range dilution scheme, it became possible to detect minor differences in MICs for a number of different bacteria. Analysis of the data was multi-factorial including susceptibility profiles, distribution of MICs, MICsos, MIC9oS, and model MICs in addition to categories of susceptibility of specific bacteria. REFERENCE Aarestrup, F. M., 1995. Occurrence of glycopeptide resistance among Enterococcus faecium isolates from conventional and ecological poultry farms. Microb. Drug Resist, 12255-257. Cook, R. R. 1997. Antimicrobial resistance - use in veterinary and human medicine. J. Antimicrob. Chemother. 39:435-439. Dowling, P. 1997. Antimicrobial Resistance. Bov. Vet. Nov-Dec. p. 14-16. Klare, I., Heier, H., Claus, H., Reissbrodt, R. and Witte, W., 1995. vanA-mediated high- level glycopeptide resistance in Enterococcusfaecium from animal husbandry. FEMS Microbiol. Lett., 125:165-172. Langlois BE, Dawson KA, Leak 1, Aaron DK. Antimicrobial resistance of fecal coliforms from pigs in a herd not exposed to antimicrobial agents for 126 months. Vet Microbiol. 1988a; 1 8: 147-53. Mudd, A. J. 1996. Is it time to ban all antibiotics as animal growth-promoting agents? Lancet. 348:1454-1456. Schwartz, D. M. Bell, and J. M. Hughes. 1997 . Preventing the emergence of antimicrobial resistance. A call for action by clinicians, public health officials, and patients. JAMA 278: 944-945. Wiedemann, B. and Grimm, H. 1996. Susceptibility to antibiotics: species incidence and trends. In: Antibiotics in Laboratory Medicine, 4th Ed. (Lorian, V. ed.) pp. 900-1168. Williams & Wilkins. Baltimore, MD. CHAPTER TWO LITERATURE REVIEW Antimicrobial resistant bacteria from animals Food animal agriculture has been suspected of fostering antimicrobial resistant (AR) bacteria, which can then be transmitted to people through direct contact, food of animal origin, and environment contamination (McEwen, 2002). Some resistant bacteria isolated from human infections have reportedly been traced back to farm animals (Lyons, 1980; O'Brien, 1982; Spika, 1987; Molbak, 1999; Fey, 2000; Van Den Bogaard, 2001). There is increased interest in research that addresses food animal agriculture’s relative contribution to the antimicrobial resistant problem (Sorum et al., 2001; Terrence, 2001). Farm-level studies have shown an association between antimicrobial usage in food animals and rates of antimicrobial resistant in animals (Mathew, 2001; Van Den Bogaard, 2001). Generally, farms with higher antimicrobial usage have a higher proportion of resistant bacteria, as well as the presence of multi-drug resistance strains. However, the magnitude of the contribution of livestock production practices to the growing antimicrobial resistant problem is unclear. The studies based on diagnostic submissions from ill animals that have recently undergone antimicrobial therapy may have resulted in higher measure of antimicrobial resistance than what might be present in the normal population (Schroeder et al., 2002). Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP, 1996 - 2001) reported higher prevalence of resistant bacteria in diagnostic submission samples and lower resistance rates in samples from animals at slaughter. For example, a high proportion (80-86%) of E. coli isolates from Denmark were resistant to ampicillin in diagnostic submission, whereas only 0-8 % of E. coli from slaughter cattle were resistant to ampicillin when measured on presumably health cattle (Bager, 1997). Geographic or temporal variations in the prevalence of resistant bacteria are believed to occur. A study conducted by Singer et al. (1998) suggested AR might be clustered in time and space, though their data were derived from a clinical diagnostic database, and so AR distributions from one region may not be applicable to other region. Organic dairy production Organic dairy production is drawing increasing attention because of public concerns about food safety, animal welfare and the environmental impacts of intensive livestock systems (Weller, 1996; Sundrum, 2001). Though the U. S. organic food market is approximately $6 billion, which is less than 1% of total food consumption in the USA, the organic market has been growing at 20-30 percent per year (Greene, 2000; Greene, 2001). In contrast, the organic milk market in Denmark is approximately 14% of the total milk consumption (Mann, 1999). In Denmark, national certification of organic “okologisk” farms began in 1988 and organic farms now comprise 8.4% of all Danish dairy farms and produced about 22% of the fluid (drinking) milk in 2000 (Bennedsgaard, 2003). More than 25% of total sales of dairy products in Switzerland is labeled as organic (Busato, 2000). In the UK, a 30 to 40% per annum increase of organic products was observed (Weller, 2000). Organic agriculture is being recognized by governmental bodies as a tool to improve rural income diversity and stability (F A0, 2000). The definition of "organic" has varied from country to country and among U. S. certifying organizations, thereby causing consumer confiision. Farms in the US had been certified as organic dairies by independent certifying organizations based on their own standards. The USDA had established the federal standard, and a revised National Organic Standard was published in the Federal Register (Federal Register, 2000a; USDA, 2001) which was fully implemented in October 2002. Organic dairies must use organic feedstuffs which are grown without any chemically synthesized fertilizer, herbicides or insecticides. The use of antibiotics for therapy or prophylaxis and hormones for grth promotion or production enhancement are prohibited. Since many conventional veterinary prophylactic and therapeutic medicines are not permitted, organic dairy farmers have adopted a variety of management practices to prevent clinical disease. Herd size Ogini et a1. (1999) studied six organic dairies in Ontario and found that organic dairy producers had comparable tillable land base and herd size (48 cows per herd) as did conventional dairies. The study in Switzerland indicated the organic herd size was equal to the national average (Busato, 2000). Large organic dairy operations can be found in other parts of the U. S. (Organic Trade Association, 1999). Average number of cows per herd and milk production per cow were 67.0 cows and 25.5kg/day in Wisconsin, but the national average were bigger than Wisconsin average (93.4 cows and 27.0 kg/day; USDA, 2002). Thirty one per cent (31%) of conventional operations used free-stall housing and 52 % of the operations used tie stall or stanchion housing (USDA, 2002). No statistical data on organic operations were available. 10 Milk production Lower milk production per cow was reported in organic dairies when compared to the conventional dairies in the same region (Busato, 2000; Krutzinna, 1996; Reksen, 1999). Lower milk yield per cow is likely attributable to more pasture and less grain being used for organic dairy production (Krutzinna, 1996). Grazing farms usually produce less milk per cow than do non-grazing farms (Hanson, 1998). Culling rate It has been speculated that organic dairies had higher culling rates, primarily due to the development of udder infections and reproductive problems (AHI, 1998). However, Norwegian organic dairies had a 23% cull rate, whereas conventional dairies had a 35% cull rate, which resulted in more multiparous cows in organic dairies as compared with conventional dairy farms (Reksen, 1999). The U. S. average culling rate for herds of less than 100 was 24.9 (USDA, 2002). A culling rate of 20-30% is thought to optimize producer profit, however, the culling rates are difficult to compare because of different definitions of culling (Radke, 2000). Mastitis rate Mastitis is a major cause of economic loss in the dairy industry and the primary reason for which antibiotics are used in dairy operations (Kaneene, 1992). Field studies in Pennsylvania, Ohio, and California indicated that the average annual herd incidence of clinical mastitis was 45 to 50 cases per 100 cows (Hady, 1993). Mastitis incidence is 11 usually highest in July and August (Erskine, 1988). Milking hygiene and environmental sanitation are traditional ways to prevent the disease (Bartlett, 1992a). Weller and Bowling (2000) reported that the incidence of clinical mastitis in 10 organic dairies in the UK was not significantly different from the rate of clinical mastitis in conventional dairies. Busato et al. (2000) reported the prevalence of subclinical mastitis in organic dairies in Switzerland to be lower than the national average. Barlow (2001) reported bacteriological analysis of 109 clinical mastitis quarters on 6 organic farms in Vermont, however no mastitis rate was reported. The mastitis rates from different studies are difficult to compare because of the regional differences, poor standardization of case definition and high variance in diagnostic acumen among the studies (Bartlett, 2001). Berry (2002) reported that farmers converting to organic status in the UK were less likely to report cases of clinical mastitis, which implies that a possible information bias may exist in comparison between organic and conventional dairy farms. Somatic Cell Count The mean bulk tank somatic cell counts (SCC) in Wisconsin for 1995 to 1998 was 335,000 cells/ml for grade A herd and 480,000 cells/ml for grade B herd (Ruegg, 2000). Highest SCC were observed in the month of July (USDA, 1999; Erskine, 1988). Although Weller et al. (1998) reported a higher SCC in one organic herd in a six-year longitudinal study as compared to conventional herds, other studies reported lower SCC in organic herds than what was found in conventional herds (Busato, 2000; Bennedsgaard, 2003). Busato (2000) studied 152 certified organic farms in Switzerland and found the 12 geometric mean of 85,600 cells/ml, which was 15% lower than the Swiss average of 100,000 cells/ml. Antimicrobial use in dairy production Dairy cattle rarely receive antimicrobials in the feed over long periods of time, except for medicated milk replacer which is often fed to calves. Antimicrobial use as feed additive is probably a principal reason why the total amount of antimicrobial use in swine and poultry production is much greater than what is seen in the dairy industry. Consequently, a higher proportion of resistant bacteria are found in swine and poultry isolates as compared with isolates from dairy cattle (Salmon, 1995; Bager, 1997; Mathew, 1998; Schroeder, 2002). In the United States, five antibiotics (i.e., bacitracin methylene disalicylate, chlortetracycline, oxytetracycline, tylosin, and virginiamycin) are approved and commonly used for beef cattle or calves for prevention of liver abscesses in feedlot cattle (Nagaraja, 1998). Most antibiotic use in adult dairy cows is for treatment and prevention of clinical mastitis (Hady, 1993; Kaneene, 1992) and in dairy calves, for treatment and prophylaxis of diarrhea/pneumonia, and as a medicated milk replacer (McEwen, 2002; USDA, 2002). Calf milk replacers, which may contain antimicrobials such as chlortetracycline, oxytetracycline and neomycin, are widely used by dairy producers in the United States (Heinrichs, 1995; USDA, 2002a). Milk replacer with antimicrobials have been prohibited in Denmark since the early 1970's (Larsen, 1972). Routine intramammary treatment for all cows with long-acting antimicrobials after the end of lactation (dry-cow-treatment) is widely adopted in the US as a preventive method 13 (Jayarao 1999; Hardeng 2001). According to the NAHMS study (USDA, 1996), 88 % of dairy operations use udder infusion for all four quarters on almost all cows at the end of lactation. Antibiotics are often ineffective for some mastitis pathogens such as S. aureus and non-severe coliforrn. (Kirk, 1994). Dry cow therapy infusions are effective against contagious mastitis pathogen, but are ineffective against environmental coliforms (Berry, 1997). A retrospective study of 9,007 cases of subclinical mastitis in New York and northern Pennsylvania showed the overall spontaneous bacteriological cure rate was 65% and the cure rate with antimicrobial treatment was not much better at 75% (Wilson, 1999). Recent work in Denmark showed that organic (ekologisk) farmer had less frequently asked veterinarians to treat their mastitis cows, however treatment fi'equency and antibiotic selection is not substantially different among two type of Danish herds (Bennedsgaard, 2003). Organic dairy operations are also prohibited to use anthelmintics. According to a recent NAHM study, over 60% of dairy operations in the U. S. normally use deworrners for at least some lactating cattle (USDA, 2002). The optical density value of ELISA has been found to be a reasonable overall measure of parasite burden, and bulk tank milk is useful for testing whole-worm antigen (Guitian, 2000; Sanchez, 2002). The worm burden were significantly associated with seasons, highest in late summer and lowest in spring, on the study of serum antibody (Borgsteede, 2000). 14 Antimicrobial resistance Avilamycin Avilamycin a mixture of oligosaccharides of the orthosomycin group, and had been used for growth promoter primarily of broilers and some for pigs, but not for dairy cattle in Denmark (Aarestrup, 1998; Aarestrup, 2001). Avilamycin was used as growth promoter for broilers and pigs, but its use was voluntarily abandoned in Denmark by the end of 1999, to avoid the possible cross-resistance to eveminomycin, which was investigated for use in humans (EU, 1998; Aarestrup, 2000; Butaye, 2003). Bacitracin Bacitracin is a polypeptide product of Bacillus Iicheniformis and Bacillus subtilis, and is commonly used in the topical treatment of human and animal skin infection. It is also commonly used as growth promoter in poultry, swine, and feedlot beef production (Aarestrup, 1998; Prescott, 2000b; FDA 2003c). The European Union banned bacitracin as animal growth promoter in 1998 because analogs of bacitracin can sometimes be used for the treatment of vancomycin-resistant enterococci (EU, 1998; Chia, 1995; O’Donovan, 1994). Relatively low levels of S. aureus resistance to bacitracin in cattle were reported in Europe before the ban (Devriese, 1980; Aarestrup, 1998), and reduced susceptibility to bacitracin (83% with MIC232ug/ml by e-test) was also reported in S. aureus from human skin and soft-tissue infections in Norway (Afset, 2003). 15 Ceftiofur Ceftiofur, a third-generation cephalosporin, has been developed strictly for veterinary use (Homish, 2003). It is approved for treatment of pneumonia in dairy cattle with zero withdrawal time, as well as for acute bovine interdigital necrobacillosis (Online Green book, 2003). Cefiiofur has worldwide approvals for respiratory disease in swine, ruminants (cattle, sheep and goats) and horses and has also been approved for foot rot and metritis infections in cattle. Since the extralabel use of cefiiofirr for severe mastitis is not very efficacious (Erskine, 2002), ceftiofirr is not widely used for mastitis. However, the zero withdrawal time of milk and meat still make the drug attractive. Chloramphenicol Chloramphenicol has been prohibited for food producing animal in the US since January 1986 (Veterinary Newsletter, 1996) and has been rigorously enforced by the FDA. The resistance to chloramphenicol is rendered by the inactivation with chloramphenicol acetyl transferase (CAT) (Prescott, 2000a) and by enhanced efflux. White et al. (2000) investigated 48 E. coli recovered from calves with diarrhea and found the majority of resistant E. coli had enhancing efflux genes (flo and chA ), which may be disseminated via plasmids or a mobile trasposon(s). They suggested the extra-label use of florfenicol in calves was the major selection pressure of those resistant traits. Florfenicol is a structurally similar antimicrobial to chloramphenicol, and was approved for bovine respiratory treatment in 1996 (Online Green book, 2003). 16 Fluoroquinolone Fluoroquinolone is not commonly used in dairy industry. Sarafloxacin was approved in the United States for the poultry in 1995, but the approval was withdrawn in 2001 (Federal Register, 2001). The Center for Veterinary Medicine of FDA proposed to withdraw approval of enrofloxacin for poultry use because of the possible transfer of fluoroquinolone-resistant Campylobacter spp. from poultry to human (Federal Register, 2000b). Enrofloxacin belongs to the fluoroquinolones group of antimicrobials and has been approved for treatment of bovine respiratory disease since 1998 in the United States (FDA, 2003a). The extralabel use of any fluoroquinolones in dairy cattle has been prohibited by the FDA in the United States (FDA, 2003b). However, extralabel drug use is practiced if the product being used is approved in that species for a different disease or if the product is used at a different dose or with an altered withdrawal time (Bateman, 2000) E. coli Osterblad et al. (2000) isolated the Enterobacteriaceae from human fecal samples and compared the antimicrobial resistance in E. coli to other enterobacteria, such as C itrobacter, Klebsiella and Proteus. They found little antimicrobial resistance in other enterobacteria and speculated that E. coli were the main carrier of antimicrobial resistance in fecal flora. Oppegaard et al. (2001) also suggested that E. coli is a major carrier of resistance traits in the coliform flora of cattle. E. coli have an ability to horizontally transfer their resistant determinants to other genera (Kruse, 1994). E. coli have been used in this and other studies as indicator organisms for antimicrobial 17 resistance monitoring activities among Gram-negative bacteria (Bager, 2001; NARMS, 2000). Escherichia coli are conditional pathogens causing urinary tract infections, wound infections, septicemia, and also are important agents of environmental clinical mastitis in dairy cows. They are present in the normal intestinal tract flora of most animals (Scrum, 2001). Indicator isolates of E. coli from Danish cattle (n=85) had resistance to tetracycline 4.7%, streptomycin 1.2% (modified based on our breakpoints), sulfamethoxazole 3.5%, and ampicillin 0% in DANMAP 2001. Extended-spectrum B-lactamase (ESBL) producing strains of Salmonella have been noted in many countries since 19905 (Jing-Jou, 2003). ESBL is produced by transmissible plasmids, which could be acquired from other multidrug —resistant enterobacteriaceae, such as Klebsiella pneumoniae or Escherichia coli. Fey et al. (2000) reported that the ceftriaxone-resistant Salmonella isolate from the child was indistinguishable from one of the isolates from cattle, which was also resistant to ceftn'axone. Winokur et al. (2001) reported in their broad study of human isolates that high levels of co-resistance to aminoglycosides, tetracycline, trimethoprim- sulfamethoxazole, and ciprofloxacin were observed in ESBL producing strains of E. coli. Campylobacter Campylobacter spp. has been recognized as a cause of septic abortion, infectious infertility and diarrhea in cattle and sheep (Radostits, 2000). Abortions in cattle can be caused by C ampylobacter fetus subsp. veneralis or C. fetus subsp. fetus, however C. jejuni and C. coli also recognized as causal agents of abortions (Larson, 1992; Welsh, 1984). Campylobacter hyointestinalis was reported as a cause of ileitis in pigs (Gebhart, 18 1983), bovine diarrhea (Diker, 1990) and human gastroenteritis (Gorkiewicz, 2002). Some Campylobacter species, such as C. fetus, were known cause of mastitis (Akhtar, 1993; Logan, 1982). Campylobacter was not recognized as a cause of human enteritis until the mid- 1970s when selective isolation media were developed for human stool culture. At present, campylobacteriosis is the most commonly reported human bacterial gastroenteritis in the United States, and the majority of infections are with C. jejuni (Nachamkin, 1999). The incidence of laboratory-diagnosed campylobacteriosis was 15.7 per 100,000 person-years in FoodNet surveillance sites (CDC, 2001) and an estimated 2 to 2.4 million infections occur in the United States each year (Friedman, 2000). The majority of sporadic cases of C ampylobacter infections are foodbome and undercooked poultry is the most likely source of infections (Friedman, 2000; Pearson, 2000). Contaminated water and unpasteurized milk are common sources of outbreaks; nine percent of bulk tank milk was found culture positive for Campylobacterjejum‘ in a study of 131 dairy herds in South Dakota and Minnesota (Jayarao, 2001). The Campylobacter spp. isolations were decreased approximately 16% by storing feces at 4°C for 24 hours (Ladron de Guevara, 1989). Cary-Blair transport medium with icepacks enabled Campylobacter spp. to maintain sufiicient viability to be isolated at the laboratory (Luechtefeld, 1981; Wang, 1983, Wasfy, 1995). Nielsen (2002) found 9 out of 77 positive samples were only positive after growth in enrichment broth. The use of enrichment broth may increase the rate of Campylobacter spp. isolation (Bolton, 1982; Martin, 1983). Campylobacterjejum' subsp. doylei, C. fetus subsp. fetus, C. upsaliensis, and C. hyointestinalis are known to be inhibited by cephalothin (Nachamkin, 1999). 19 Other studies of Campylobacter spp. used an incubation temperature of 42°C to optimize the growth of thermophilic Campylobacter species, such as C. jejuni, C. coli or C. Iari, with the decreased ability to isolate non-thermophilic species (C. fetus, and C. jejuni subsp. doylei). Atabay et al. (1998) used three kinds of media, enrichment technique, membrane filtration technique, and at three different incubation temperatures. They found 62% overall prevalence in 136 cattle in three farms in the U. K. Giacoboni et a1 (1993) found C. fetus subsp. fetus in 17% of cattle and C. hyointestinalis in 19% of cattle, whereas dominant species was C. jejuni found in 29% of 94 cattle in Japan. Nielsen (2002) found that 23% of 332 animals, and 83% of 24 farms were positive for Campylobacterjejuni in Denmark. The prevalence was significantly higher in calves (42%) than young cattle (20%) or cows (9%). Wesley, et al. (2000) found Campylobacterjejum’ in fecal specimens fi'om 37.7% of 2,085 individual dairy cattle fecal samples, and 80.6% of 31 farms in the USA. Samples collected before May 1 contains more C. jejuni in cull cows than those taken later in the year. They also reported that C. jejuni were more frequently recovered from large herds (more than100 cattle) than from smaller dairy herds. Hoar, et al. (2001) also observed a higher prevalence in larger beef herds in California than from smaller herds, although the prevalence of Campylobacter spp. in these beef herds was much lower (5%). Heuer et al. (2001) reported a 100% broiler flock prevalence of thermophilic Campylobacter spp. in Danish organic farms compared with a 36.7% prevalence in conventional broiler flocks. They suggested that the high prevalence of Campylobacter spp. in organic flocks may have been due to the aggregate effects of age, breed, housing, feed, and rearing system, and not related to the use of antimicrobials. 20 Antimicrobial resistance in Campylobacter The agar dilution test was recently set by NCCLS as a reference standard susceptibility testing method for veterinary isolates of Campylobacter spp. (NCCLS, 2002). Ge, et al. (2002) recently reported that MIC values measured with the Etest were generally lower than the results obtained with the agar dilution method. Huang, et al. (1992) also compared the Etest method with the agar dilution method and reported slightly lower MIC values with the Etest than with the agar dilution. Resistance to ciprofloxacin in human isolates of Campylobacterjejuni is reportedly increasing (Allos, 2001; Gaudreau, 1998; Smith, 1999). Ten percent of 604 C. jejuni isolated in NARMS EB 2000 program were resistant on Etest to ciprofloxacin, 52% were resistant to tetracycline, 0.5% were resistant to erythromycin and none were resistant to gentamicin (USDA, 2000). Staphylococcus Numerous reports have been published on antimicrobial susceptibility in Staphylococcus from dairy cattle, which were usually collected from individual quarter milk samples from cows with clinical mastitis (Devriese, 1980; De Oliveira, 2000). Bulk tank cultures for S. aureus could also be used as inexpensive and convenient monitoring methods for udder health (J ayarao, 2003). Antimicrobial resistance in S. aureus has primarily been studied on isolates from clinical mastitis that had been submitted to various veterinary diagnostic laboratories (Makovec, 2003; Erskin 2002b; De Oliveira, 2000; Bager, 2002). Multiple samples from a single farm may be common, and clonal isolates might have been included in the data. Most surveys did not account for multiple 2] observations from the same cow or herd, so clonal isolates of resistant bacteria from the same cow or herd might have greatly influenced the results. To avoid these complicated issues, De Oliveira et al. (2000) used only one isolates from a single herd. Samples submitted for diagnosis of clinical mastitis cases tend to exhibit more AR, probably because cases that are refiactory to treatment are more likely to be cultured in an attempt to identify the causative agent and its AR traits (Aarestrup, 1998). Also, such studies have mainly attributed AR to antibiotics commonly used for mastitis therapy (Rossitto, 2001; Watts, 1995). Effect of management factors on resistance Langlois et a1. (1988) reported that pigs on pasture have lower AR E. coli prevalence than pigs in finishing or farrowing house. They speculated the exposure to antimicrobials is not the only factor that influences the prevalence of AR bacteria. Hinton et al. 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Comparison of Mle of Ceftiofirr and Other Antimicrobial Agents Against Bacterial Pathogens of Swine from the United States, Canada, and Denmark. Journal of Clinical Microbiology, 33(9): 243 5-2444. 30 Schroeder, C. M., Zhao, C., DbRoy, C ., Torcolini, J., Zhao, S., White, D. G., Wagner, D. D., McDermott, P. F ., Walker, R. D., 2002. Antimicrobial Resistance of Escherichia coli 0157 isolated from human, cattle, swine, and food. Applied and Environmental Microbiology 68(2): 576-581. Singer, R. 8., Case, J. T., Carpenter, T. E., Walker, R. L., Hirsh D. C., 1998. Assessment of Spatial and Temporal Clustering of Ampicillin- and Tetracycline-Resistant Strains of Pasteurella multocida and P. haemolytica Isolated from Cattle in California. J. Am. Vet. Med. Assoc. 212(7): 1001-5. Smith, K.E., J. M. Besser, G. W. Hedberg, F. T. Leano, J. B. Bender, J. H. Wicklund, B. P. Jhonson, K. A. Moore, M. T. Osterholm, and the Investigation team. 1999. Quinolone-resistant Campylobacterjejuni Infections in Minnesota, 1992-1998. New Engl. J. Med. 340:1525-1532. Spika, J. S., Waterman, S. H, Hoo, G. W., St Louis, M. E., Pacer, R. E., James, S. M., Bissett, M. L., Mayer, L. W., Chin, J. Y., Hall, B., et al., 1987. Chloramphenicol- resistant Salmonella newport Traced through Hamburger to Dairy Farms. New Engl. J. Med. 316:565-570. Radke, B. R., and J. W. Lloyd. 2000. Sixteen Dairy Culling and Replacement Myths. Compendium on Continuing Education for the Practicing Veterinarian. 22:2:Food Animal 836-41. Reksen, 0., A. Tverdal, and E. Ropstad. 1999. A Comparative Study of Reproductive Performance in Organic and Conventional Dairy Husbandry. J. Dairy Sci. 8222605- 2610. Ruegg, P. L., and T. J. Tabone. 2000. The Relationship between Antibiotic Residue Violations and Somatic Cell Counts in Wisconsin Dairy Herds. J. Dairy Sci. 83:2805-2809. Sanchez, J ., I. R. Dohoo, F. Markham, K. Leslie, G. Conboy. 2002. Evaluation of the Repeatability of a Crude Adult Indirect Ostertagia ostertagi ELISA and Methods of Expressing Test Results. Vet. Parasitolo. 109:75-90. Scrum, H., Sunde M., 2001. Resistance to Antibiotics in the Normal Flora of Animals. Veterinary Research 32: 227-241. Sundrum, A. 2001. Organic Livestock Farming, A Critical Review. Livest. Prod. Sci. 67:207-215. Torrence, M. E., 2001. Activities to Address Antimicrobial Resistance in the United States. Preventive Veterinay Medicine, 51: 37-49. 31 USDA. 1996. Part HI: Reference of 1996 Dairy Health and Health Management. USDA:APHIS:VS, CEAH, National Animal Health Monitoring System. Fort Collins, CO. USDA. 1999. USDA-APHIS, U. S. Milk Quality Monitoring Using Bulk Tank Somatic Cell Counts. Info Sheet. Fort Collins, CO. USDA. 2000. NARMS - EB 2000 Veterinary Isolates - Campylobacterjejuni 2000. [Online]http://www.arru.saa.ars.usda.gov/narms/narms_2000/veterinary_isolates/nar ms-campylobacterjejuni.pdf. Accessed on March 3, 2003. USDA. 2001. National Organic Standard, Agriculture Marketing Service. [ONLINE] httpz/lwww.ams.usda.gov/nop/NOP/standards/FullText.pdf, Accessed on May 26 2003. USDA. 2002a. Highlights of NAHMS Dairy 2002: Part 1, Centers for Epidemiology and Animal Health, Veterinary Service, USDA. http://www.aphis.usda.gov/vs/ceah/cahm/Dairy_Cattle/DairyOZ/Highlights-of- NAHMS-Dairy-2002pdf USDA. 2002b. Part H: Changes in the United States Dairy Industry, 1991-2002. USDA:APHIS:VS, CEAH, National Animal Health Monitoring System, Fort Collins, CO #N388.0603. Van Den Bogaard, A. E., London, N., Driessen, C., Stobberingh, E. E., 2001. Antibiotic Resistance of Faecal Escherichia coli in Poultry, Poultry Farmers and Poultry Slaughters. Journal of Antimicrobial Chemotherapy, 47: 763-771. Veterinary Newsletter. 1996. Why veterinary drugs are not always available. Center for Veterinary Medicine, Food and Drug Administration, Veterinarian Newsletter, 11, 1. Wang, W.-L. L., L. B. Reller, B. Smallwood, N. W. 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Campylobacterjejuni Abortion in a Heifer. J AVMA. 185:549-551. Wesley, I. V., S. J. Wells, K. M. Harmon, A. Green, L. Schroeder-Tucker, M. Glover, and I. Siddique. 2000. Fecal Shedding of Campylobacter and Arcobacter spp. in Dairy Cattle. Appl. Environ. Microbiol. 66:1994-2000. White, D. G., C. Hudson, J. J. Maurer, S. Ayers, S. Zhao, M. D. Lee, L. Bolton, T. Foley, and J. Sherwood. 2000. Characterization of Chloramphenicol and Florfenicol Resistance in Escherichia coli Associated with Bovine Diarrhea. J. Clin. Microbiol. 38:4593-4598. Wilson, D. J., R. N. Gonzalez, K. L. Case, L. L. Garrison, and Y. T. Grohn. 1999. Comparison of Seven Antibiotic Treatments with no Treatment for Bacteriological Efficacy Against Bovine Mastitis Pathogens. J. Dairy Sci. 82: 1664-1670. Winokur, P. L., R. Canton, J. —M. Casellas, and N. Legakis, 2001. Variations in the Prevalence of Strains Expressing an Extended-Spectrum b-Lactamase Phenotype and Characterization of Isolates from Europe, the Americas, and the Western Pacific Region, Clinical infectious diseases 2001;32:894-103. 33 CHAPTER THREE MILK PRODUCTION AND MASTITIS ON MIDWESTERN ORGANIC DAIRY FARMS ABSTRACT Organic dairy farms in and around Wisconsin (n=101) were contacted in July 1999 with a one page questionnaire. A total of 69 responses were sufficiently complete for analysis. Herd size averaged 61.3 co'ws. Mean milk production was estimated as 17.2 kg per cow per day, which is less than the Wisconsin state average of 20.73 kg per cow per day. The average bulk tank somatic cell count was 329,000 cells per ml, which was almost identical to the Wisconsin average of 335,000 cells per ml. The overall rate of mastitis was 42 cases per 100 cow years-at-risk, which is within the range of rates reported in the literature. The cull rate based on July 1999 was 25% per year, which is low-normal for the Midwestern dairy industry. Only 16% of responding organic farms usually used non-conventional remedies, and 50% of farms never used non-conventional remedies. This preliminary survey suggested that the responding organic herds had similar herd size, mastitis rate and SCC as did conventional herds in the same geographic area. In contrast, their milk production and culling rates were lower than those reported for most conventional herds. 34 INTRODUCTION Organic dairy production is drawing increasing attention because of public concerns about food safety, animal welfare and the environmental impacts of intensive livestock systems (Weller and Cooper, 1996). Though the U. S. organic food market is approximately $6 billion, which is less than 1% of total food consumption in the USA, the organic market has been growing at 20-30 percent per year (Greene, 2000). In contrast, the organic milk market in Denmark is approximately 14% of the total milk consumption (Mann, 1999). More than 25% of total sales of dairy products in Switzerland is labeled as organic (Busato et al., 2000). In the UK, a 30 to 40% per annum increase of organic products was observed (Weller and Bowling, 2000). Organic agriculture is being recognized by governmental bodies as a tool to improve rural income diversity and stability (Anonymous, 2000a). The definition of "organic" has varied from country to country and among U. S. certifying organizations, causing consumer confusion, farms were certified as organic dairies by independent certifying organizations based on their own standards. The USDA has attempted to establish federal standards since 1990, and a revised National Organic Standard was recently published in the Federal Register (Anonymous, 2000b). Organic dairies must use organic feedstuffs which are grown without any chemically synthesized fertilizer, herbicides or insecticides. The use of antibiotics for therapy or prophylaxis and hormones for grth promotion or production enhancement are prohibited. Since many conventional veterinary prophylactic and therapeutic medicines are not permitted, organic dairy farmers have adopted a variety of management practices to prevent clinical disease. It has been reported that organic dairies had higher culling rates, primarily due to 35 the development of udder infections and reproductive problems (Anonymous, 1998). However, Wellers et. a1 (2000) reported that the rate of clinical mastitis in 10 organic dairies in the UK was not significantly different from the rate of clinical mastitis in conventional dairies. Busato et al. (2000) reported the prevalence of subclinical mastitis in organic dairies in Switzerland to be lower than the national average. The purpose of this survey was to determine if herd size, milk production, mastitis incidence, SCC and culling rate of organic dairy farms differ from values reported for conventional dairy farms in the same region. MATERIALS AND METHODS In August 1999, a one-page questionnaire regarding milk production, animal movement and mastitis was mailed to 101 dairies (80 in Wisconsin, 13 in Minnesota, seven in Iowa and one in Illinois). Non-responders received a second questionnaire six weeks later. Wisconsin state averages were used as comparison data. Statistical analysis 1(a) was performed using Exce . A Z test was used to test the hypothesis that the organic farms did not differ from the Wisconsin state average for milk production and herd size. RESULT A total of 73 responses were received, four of which were excluded due to missing data. There were 3,581 cows contributing bulk tank milk on responding farms in July 1999. Based on an assumed 13 months calving interval and two months dry period (Anonymous, 1998), the total number of milking cows (cows which have had at least one calf) was estimated at 4,232 or 61.3 head per farm. Only three farms had more 36 than 100 milking cows, and two farms had less than 20 milking cows (Figure 3-1). In 1999, the average number of milking cows per farm was 62.3 and 82.1 for Wisconsin and the U. S. dairy herds, respectively (Anonymous, 2000c). Therefore, the organic farms we studied had about the same herd size as the average dairy farm in Wisconsin (p=0.87), however they were much smaller than US average (p=0.0011). Production parameters were summarized in table 3-1. The amount of bulk tank milk produced per day per cow in the 69 organic dairy farms was 17.16 Kg (75.6 metric ton / 4,232 cows) which was lower than the Wisconsin state average of 20.73 kg (p=0.001) (8). There was considerable variation reported for milk yield; herd average milk production per cow ranged from 8.5 Kg to 25.6 Kg (Figure 3-2). Herd bulk tank SCC averaged 329,000 cells/ml of milk. The lowest SCC was 104,000 cells/ml and the highest SCC was 935,000 cells/ml. Two farms reported SCC >900,000 cells/ml during single month, and five farms had approximately 500,000 cells/ml (Figure 3-3). The mean bulktank SCC in Wisconsin for 1995 to 1998 was 335,000 cells/ml for grade A herd and 480,000 cells/ml for grade B herd (Ruegg and Tabone, 2000); SCC values for responding organic herds approximated the mean reported values for all Wisconsin dairy herds. The producers were asked to recall the number of mastitis cases in the "last month" (July 1999) and also the number of mastitis cases in the "last year" (June 1998 - July 1999). Clinical mastitis was defined in this questionnaire as "a period of disease when a cow has noticeable clots or strings in its milk". When based on "last month", the mastitis rate was estimated at 42.4 cases per 100 cow-years at risk, but when based on "last year", the mastitis rate was only 17.7 cases per 100 cow-years at risk. 37 When the organic producers found mastitic quarters, 72% usually treated the quarter by keeping it milked out. Sixty two percent of responding farms usually massaged the mastitic quarter, but 22 producers (33%) occasionally massaged the quarter and three farms (5%) never massaged the quarter. Sixty three percent of producers usually or sometimes used vitamins, but 36% of producers never used vitamins for mastitis treatment. Whey colostrum products for mastitis treatment were usually used by 28% of producers, 29% sometimes used whey products and 43% of producers never used these products. Sixty-four percent of producers had never used any udder infusions for mastitis treatment, but 36% of producers occasionally used various kind of non-antibiotic udder infusions, such as aloe products. Only 16% usually used non-conventional remedies, 34% sometimes used non-conventional remedies and half (50%) of the organic farms never used non-conventional remedies for mastitis. Sixty nine responding organic producers reported the number of cows sold for slaughter in the month of July 1999 and between June 1998 and July 1999. The total number of milking-age cows sold for slaughter were 88 in July 1999 and 795 in the previous year. The cull rate was therefore 25.0 % (88/4232* 12) and 18.8 % (795/4232) for July 1999 and for the previous year, respectively. Four organic producers reported selling a total of 13 milking-age cows to other organic dairy farms during 12 months in 1998. Eight organic producers sold a total of 32 cows to conventional dairy farms in 1998. Five organic dairy producers treated a total of nine organic cows with antibiotics and sold them to conventional farms. Organic producers reported that 15 cows died in the month of July 1999, and 92 died in previous year. Fifty four producers reported no dead cattle in July 1999 and 19 38 reported no death in the previous year. The mortality rate was estimated at 4.3% (15*12/4232) based on "last month" and 2.2% (92/4232) based on the "previous year". DISCUSSION The data for this study was from the membership of one particular organic dairy cooperative in Wisconsin and surrounding states. Therefore, the results of this analysis may not be applicable to other organic dairy organizations. The response to our questionnaire was moderately high (72%). However, non-respondents may have differed from respondents in their management and production parameters (Bartlett, 1992). Although defined on the survey form, the definition of clinical mastitis is subjective and open to interpretation, therefore reporting bias may have occurred. Our study found that organic dairy farms were about the same size as other farms in the region. Ogini et al. (1999) studied six organic dairies in Ontario and found that organic dairy producers had comparable tillable land base and herd size (48 cows per herd) as did conventional dairies. The study in Switzerland indicated the organic herd size was equal to the national average (Busato et al., 2000). Herd size may be determined by the regional conditions rather than the type of dairy husbandry. Though the organic dairies in our study were much smaller than the U. S. average, this does not imply that larger organic dairy operations are impractical; large organic dairy operations can be found in other parts of the U. S. (Anonymous, 1999a). The mean milk production reported by the organic dairy farms was lower than the Wisconsin state average. Lower milk production per cow when compared to the conventional dairies in the same region was supported by other studies (Busato et al., 39 2000, Krutzinna et al., 1996, Reksen et al., 1999). Lower milk yield per cow is likely attributable to more pasture and less grain being used for organic dairy production (Kmtzinna et al., 1996). Grazing farms usually produce less milk per cow than do nongrazing farms (Hanson et al., 1998). The rate of clinical mastitis based on data from July 1999 was slightly higher than rates reported from other regions (Bartlett et al., 1992, Weller and Bowling, 2000); however, the results based on data from the previous year were lower than what has been reported for other regions. The mastitis rates from other populations are difficult to compare because of the regional differences, poor standardization of case definition and high variance in diagnostic acumen among the studies (Bartlett et al. 2001). The mastitis rate, based on July data, may be higher than the rate based on the "previous year" because the more recently occurring cases were more easily recalled (recall bias). Also, mastitis incidence is usually highest in July and August (Erskine et al., 1988). In and around Wisconsin, July 1999 was a record breaking hot months. Therefore, the mastitis rate may be overestimated when the July rate is extrapolated to a whole year. The bulk tank SCC average of the 69 organic dairies, based on July, was within the range of the Wisconsin state average. The SCC data for the organic herds may be overestimated because it was based on the month of a year (July) which has been reported to have the highest SCC (Anonymous, 1999b; Erskine et al., 1988). Although Weller and Davies (1998) reported a higher SCC in one organic herd in a six-year longitudinal study as compared to conventional herds, other studies reported lower SCC in organic herds than what was found in conventional herds (Busato et al., 2000). The 40 SCC data reported here suggested that responding organic dairies may have lower average bulk tank SCC than do most conventional dairies. Radke and Lloyd (2000) reported that culling rates are difficult to compare because of different definitions of culling. The culling rate in our study was defined as number of cows culled per year divided by the average milking herd size. The culling rate in the reports of the Dairy Herd Improvement Associations (DHIA) include the average herd size plus the number of animals culled in the denominator (Radke and Lloyd, 2000), so the rates become lower than they would be using our definition. The statewide DHIA culling rate of Holstein cattle in Wisconsin was reported as 37.6% (AgSource CRI, personal communication). Therefore, the estimated culling rate of the organic dairies was considerably lower than the Wisconsin average. The estimated culling rate in the organic dairies based on "last month" was almost identical to the national average of 25.0% (Anonymous, 1996), but the 18.8% estimate based on "last year" was lower than the national average. Recall bias in failing to remember animals culled 2 to 12 months ago may have caused some of the difference between our two rates estimates. Also important is the previously discussed issues of extrapolating data from a particularly hot month of July to an annual rate. Reksen et al. (1999) reported a significant difference between organic and conventional dairy farms in annual replacement. Norwegian organic dairies had a 23% cull rate, whereas conventional dairies had a 35% cull rate, which resulted in more multiparous cows in organic dairies as compared with conventional dairy farms. A culling rate of 20-3 0% optimizes producer profit (Radke and Lloyd, 2000), therefore the culling rate in the organic dairies (18.8- 25.0%) may be economically optimized. 41 This preliminary survey suggested that organic dairy farm in Midwestern have lower or at least similar bulk tank SCC and mastitis rates as compared conventional dairy farms in the same region. Lower milk yield per cow may reduce stress on the udder, causing a lower mastitis rate in organic dairies. Culling rates appeared to be lower, or at most similar, in the organic herds as compared with state and national averages. Non- Non-conventional remedies were not widely used by the organic farms, and there was little movement of sick cows from organic to conventional farms. (a) Microsoft Excel 2000, Microsofi Corp, Redmond, Wash 42 REFERENCES Anonymous. 2000a. Food safety and quality as affected by organic farming. 22nd FAO regional conference for Europe. Food and Agriculture Organization. Rome. Anonymous. 2000b. National Organic Program; Final Rule. Federal Register, Vol. 65, No. 246, December 21, 2000. [Intemet, WWW], ADDRESS: httpz/lwww. access. gpo. gov/su_docs/fedreg/a00 1 22 1c.html Anonymous. 2000c. USDA_NASS Agricultural Statistics 2000. [Intemet, WWW], ADDRESS: http://www.usda.gov/nass/pubs/agr00/00_ch8.pdf Anonymous. 2000d. Annual milk production. Wisconsin Agricultural Statistics Service. [Intemet, WWW], ADDRESS: http://www.nass.usda.gov/wi/dairy/annmkpd.pdf Anonymous. 1999a. The Organic Pages: 1999 North American Resource Directory. Organic Trade Association. Greenfield, MA. [Intemet, WWW], ADDRESS: http://www.ota.com/ Anonymous. 1999b. USDA-APHIS, U. S. Milk quality monitoring using bulk tank somatic cell counts. Info Sheet. Fort Collins, CO. Anonymous. 1998. Antibiotic Resistance. AHI Quarterly. 19:2. Animal Health Institute, Washington, D. C. Anonymous. 1996. USDA-APHIS, Part 1: Reference of 1996 Dairy Management Practices. : National Animal Health Monitoring System [Intemet, WWW], ADDRESS: http://www.aphis.usda. gov/vs/ceah/cahm/Dairy_Cattle/dr96des1 .pdf Bartlett, P. C., J. F. Agger, H. Houe, G. L. Lawson. 2001. Incidence of clinical mastitis in Danish dairy cattle and screening for non_reporting in a passively collected national surveillance system. Prev. Vet. Med. 48(2):73_8 Bartlett, P. C., G. Y. Miller, S. E. Lance, and L. E. Heider. 1992. Clinical mastitis and intramammary infections on Ohio dairy farms. Prev. Vet. Med. 12:59-71. Busato, A., P. Trachsel, M. Schallibaum, J. W. Bluma. 2000. Udder health and risk factors for subclinical mastitis in organic dairy farms in Switzerland. Prev. Vet. Med. 44:205-220. Erskine, R. J ., R. J. Eberhart, L. J. Hutchinson, S. B. Spencer, and M. A. Campbell. 1988. Incidence and types of clinical mastitis in dairy herds with high and low somatic cell counts. J. Am. Vet. Med. Assoc. 192:6: 761_5. Greene, C. 2000. U. S. Organic agriculture gaining ground. Economic Research Service/ USDA. Agricultural Outlook. 9-14, [Intemet, WWW], ADDRESS: 43 httpzl/www.ers.usda.gov/epubs/pdf/agout/apr2000/a0270d.pdf Hanson, G. D., L. C. Cunningham, M. J. Morehart, and R. L. Parsons. 1998. Profitability of moderate intensive grazing of dairy cows in the Northeast. J. Dairy Sci. 81 :821- 829. Krutzinna, C., E. Boehncke, H. J. Herrmann. 1996. Organic Milk Production in Germany. Biol. Agric. Hortic. 132351-358. Mann, E. 1999. Organic dairy products. Dairy Ind. Int. 62:2:11-12. Ogini, Y. O., D. P. Stonehouse, and E. A. Clark. 1999. Comparison of organic and conventional dairy farms in Ontario. American Journal of Alternative Agriculture. 14-3: 122-128. Radke, B. R., J. W. Lloyd. 2000. Sixteen dairy culling and replacement myths. Compendium on continuing education for the practicing veterinarian. 22:2:Food Animal S36-41. Reksen, O., A. Tverdal, and E. Ropstad. 1999. A comparative study of reproductive performance in organic and conventional dairy husbandry. J. Dairy Sci. 8212605- 2610. Ruegg, P. L., T. J. Tabone. 2000. The relationship between antibiotic residue violations and somatic cell counts in Wisconsin dairy herds. J. Dairy Sci. 83:2805-2809. Weller, R. F., P. J. Bowling. 2000. Health status of dairy herds in organic farming. Vet. Rec. 146:80-81. Weller, R. F., A. C00per. 1996. Health status of dairy herds converting from conventional to organic dairy farming. Vet. Rec. 139: 141-142. Weller, R. F., D. W. R. Davies. 1998. Somatic cell counts and incidence of clinical mastitis in organic milk production. Vet. Rec. 143:365-366. 44 Figure 3-1. Frequency distribution on number of lactating cows in July 1999 Number of farms (y axis) for each herd size (x axis). Number of famrs A-A—L—I—LN Qua-05000 1 10 Herd size 45 Figure 3-2. Frequency distribution of average milk production per cow per day Number of farms (y axis) for each production class (x axis). 14) Number of farms Milk production (Kg/cow/day) 46 Figure 3-3. Frequency distribution on bulk tank SCC in July 1999 Number of farms (y axis) for each SCC class (x axis). Number of farms 30 10 H I l l 1 0 E}?egifiiliiéli’ifg? . L , 551i25231715151353" 200,000 300,000 400,000 500,000 600,000 700,000 800.000 900.000 Somatic cell count 47 Table 3-1. Estimated production parameters based on a presumable calving interval of 13 months and the dry period of two months with available Wisconsin state averages. The Wisconsin state average was extracted from USDA_NASS Agricultural Statistics 2000 (Anonymous, 2000c) and SCC from Ruegg and Tabone (2000). Organic farms (n=69) Wisconsin state average Number of cattle per herd 61.3 62.3 Milk production per cow (Kg per day) 17.8 20.3 SCC (cells/ml) 329,000 328,000-335,000 (Based on (based on last month) last year) Mastitis (case/ 100 cow-year) 42.4 17.7 Not Available Culling rate (%) 25.0 18.8 Not Available Mortality rate (%) 4.3 2.2 Not Available 48 Appendix 1. Questionnaire Initial Survey - August 1999 How many cows are now contributing milk to your bulk tank? How many years has your dairy farm been organic? years If a "case” of mastitis is defined as a period of disease when a cow has noticeable clots or strings in its milk, about how many cases of mastitis have you had in: the past week? the past month? the past year? About how many milking-age cows: last month? last year? died were sold for slaughter were sold to an organic farm received antibiotics and were sold to a non-organic farm were NOT treated, but were sold to a non-organic dairy farm Other Please indicate how you usually treat your routine cases of clinical mastitis in which the cow's milk has clots or strings, but the cow is not sick. Circle one Keep the quarter milked out. usually sometimes never Massage the quarter. usually sometimes never Use vitamins (B, C, etc.) usually sometimes never Use lmpro (or similar whey colostrum usually sometimes never product) Use other udder infusions (describe type) usually sometimes never Other usually sometimes never 49 CHAPTER FOUR COMPARISON OF PRODUCTION AND MANAGEMENT BETWEEN ORGANIC AND CONVENTIONAL DAIRY HERDS IN WISCONSIN ABSTRACT An observational study was conducted in Wisconsin to compare production and management on organic and conventional dairy farms. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms on which antimicrobials were routinely used for animals of all ages. A seven-page questionnaire regarding milk production, milking practices, housing, incidence of the major dairy diseases and medical treatments was used to assess management and production during 2000-2001. Body condition scores of lactating cows and environmental and animal sanitation scores were also collected on each of two farm visits. The organic herds had significantly fewer cattle than did the conventional herds (p=0.017). The average daily milk production per cow in organic dairy herds (20.2 kg/day) was lower than that of conventional herds (23.7 kg/day). The incidence of clinical mastitis on organic farms (28 cases per 100-cow-years at risk) was not statistically different from that of on conventional farms (32 cases per lOO-cow-year at risk). No significant difference in bulk tank somatic cell count was observed between organic (262,000 cells per ml) and conventional farms (285,000 cells per ml) farms. The average annual cull rate was 18.0 cases per 100-cow-years for the conventional farms and 17.2 for the organic farms (p = 0.426). There was little evidence of other fundamental differences between two farm types in other major management and production parameters. 50 INTRODUCTION Organic dairy production is drawing increasing attention because of public concerns about food safety, animal welfare and the environmental impacts of intensive livestock systems (Weller, 1996; Sundrum, 2001). The organic food market is approximately $6 billion, which is less than 1% of total food consumption in the USA. However, the organic market has been growing at 20-30 percent per year (Green, 2000; 2001). In contrast, the organic (okologisk) milk market in Denmark is approximately 14% of the total milk consumption (Mann, 1999) and more than 25% of total sales of dairy products in Switzerland is labeled as organic (Busato, 2000). In the UK, a 30 to 40% per annum increase of organic products was observed (Weller, 2000). Organic agriculture is increasingly being recognized by governmental bodies as a tool to improve rural income diversity and stability (FAO, 2000). The standards for using antibiotics in organic dairies in the EU are less strict than those in the USA. The USDA Organic Standard prohibits the use of any animal drug in the absence of demonstrated clinical illness (USDA, 2001). The standard also stipulates that all appropriate medications and treatments must be applied to restore an animal to health when methods acceptable to organic production standards fail, however, this means that the animal will lose its organic status. Mastitis is a major cause of economic loss in the dairy industry and the primary reason for which antibiotics are used in dairy operations (Kaneene, 1992). Field studies in Pennsylvania, Ohio, and California indicated that the average annual herd incidence of CM was 45 to 50 cases per 100 cows (Hady, 1993). Milking hygiene and environmental sanitation are traditional ways to prevent the disease (Bartlett, 1992a). Antimicrobial use 51 for treating CM is a common practice on most US dairy farms. Routine intramammary treatment for all cows with long-acting antimicrobials after the end of lactation (dry-cow- treatment) is widely adopted in the US as a preventive method. (Jayarao 1999; Hardeng 2001). Though mild cases of CM may not always receive antibiotic treatment during the lactational period, dairy producers and veterinarians often treat severe cases with supportive therapies and intramammary antibiotic infusion or injection. Organic dairy herds may have higher culling rates, primarily due to the development of intramammary infections and reproductive problems. However, Weller and Bowling (2000) reported that the incidence of CM in 10 organic dairies in the UK was not significantly different from the rate of CM in conventional dairies. Busato et al. (2000) reported that the prevalence of subclinical mastitis in organic dairies in Switzerland was lower than the national average. Few studies have compared the incidence of CM on organic and conventional dairy farms in the US. Barlow (2001) reported bacteriological analysis of 109 CM quarters on 6 organic farms in Vermont, however, no incidence of CM was given or compared with conventional dairy farms. In Wisconsin, the organic dairy farmers sell their milk for almost twice as much as what conventional farmers receive. As such, organic milk production has created a niche market that has allowed many small dairy farms to stay in business during a time when profit margins are small, the dairy industry is consolidating and many small and moderately sized dairy herds are going out of business. In general, the organic restrictions on the use of insecticides and herbicides for producing animal feed have induced most Midwestern organic dairy farms to employ grazing to a much greater extent than do conventional farms. This generally creates less 52 intensive feeding and housing management systems compared with what is seen in the mainline dairy industry. It is generally assumed by the dairy industry that organic farms have lower milk production and higher culling and disease rates because farmers are not allowed to treat sick cows with antibiotics. However, valid comparisons are not available and are confounded by comparisons between small extensive organic dairies and large total-confinement dairy operations which employ a very different intensive nutritional and management strategy. The overall purpose of this study was to compare the major health, management and milk production parameters between organic and neighboring conventional Wisconsin farms. MATERIALS AND METHODS A geographical cluster of 30 organic dairy farms in Southwestern Wisconsin were selected from 110 members of an organic dairy association (Sato, 2002). All farms were certified as complying with the US national organic standards and had been selling organic milk through their organic association for at least 3 years before the start of our study. For each organic farmer selected, a neighborhood "conventional" dairy farmer was asked to volunteer their farm to serve as a control. Thirty of 37 organic farmers who were contacted, agreed to participate in the study. The conventional dairy farms were chosen from the nearest neighboring conventional farms, with only five cases of a farmer declining to participate. All herds were visited twice; once in March and once in September. Fecal specimens were collected for comparison of antimicrobial resistance, as described 53 elsewhere (Sato, K., submitted for publication). Numbers of cows sold or culled were recorded. Herd average milk production, Bulk Tank Somatic Cell Count (BTSCC) and bacteria counts of the previous month were obtained from the milk production receipts or other production records whenever available. Average daily milk production per cow was computed from the amount of bulk tank milk per day divided by number of lactating cows. A seven-page questionnaire regarding milk production, milking practices, housing, incidence of the major dairy diseases, medical treatments and other management factors was conducted at the first visit; 10 pairs of herds in the Spring of 2000, 10 pairs in the Fall of 2000 and 10 pairs in the Spring of 2001. On the second farm visit, previously collected management and production data were reviewed and verified. Clinical mastitis (CM) was defined in this study as a cow having a swollen or hard udder or noticeable clots or strings in its milk . The dairy producers were asked to retrieve from their records (or recall from memory) the number of CM cases in the three months before the interview. Recurrent episodes of disease were counted as one case if the episodes occurred within two weeks afier the initial case. Cows with multiple quarters affected with CM were counted as one case. The CM rate for each herd was calculated as the number of CM cases per lOO-cows-years at risk. Bulk tank milk samples from each of the two visits were collected and sent to the laboratory at Atlantic Veterinary College, University of Prince Edward Island for examination of antibodies against Ostertagia ostertagi by an indirect enzyme-linked immunosorbent assay (ELISA) (Guitian, 2000). The optical density (OD) values of ELISA test were adjusted with positive and negative controls, and the adjusted OD data were used for the rest of analysis. 54 At each visit, the body condition score (BCS) was measured on ten systematically selected lactating cows. Cows were scored from 1 (thin) to 5 (fat), with increments of 0.25, (Wildman, 1982). A Body Condition Score Guide with photographs (Church & Dwight Co. Inc., Princeton, NJ) was used for standardization. The mean of ten BC 85 was used as a representative value for each farm for each season. Environmental and animal sanitation was measured with subjective scores of cow cleanliness and the amount of moisture and manure in the bedding and exercise areas, as previously described (Bartlett, 1992a). Subjective sanitation score were graded as 1 (presumed upper 1/3 of all dairy farms in Wisconsin), 2 (middle 1/3) and 3 (lower 1/3), given current weather conditions. The scores were considered to be discrete variable rather than ordered categorical data, and mean value of all environmental and animal sanitation score (EASS) were computed for each farm in each season. Statistical Analysis The questionnaire was proofread twice for correct data entry and were evaluated for reasonableness. No data were excluded as being unreasonable or impossible responses. The milk production per cow, CM rate, BTSCC, BCS, EASS, and adjusted OD data from ELISA test were tested for normality with the Shapiro-Wilk test and no transformations were deemed necessary (W > 0.95 in each instance). Bacteria count and total cow per herd (herd size) were transformed with a log firnction and the transformations yielded W-values of 0.96 and 0.98, respectively. A mixed linear model with random effects was used to estimate the difference in continuous production variables between organic and conventional dairy farms. Farm type, season, and 55 interaction of farm type and season were included in the model as fixed effects. Pairs (each organic farm and its conventional neighbor farm) were included as a random effect to reflect the pairwise matching. For discrete variables, such as grazing intensity, milking methods and mastitis treatments, McNemar’s test was used to examine differences between the two farm types. For culling and mortality analysis, Poisson regression was used to estimate the difference between farm types. The adjusted OD values of ELISA test for each season were tested by paired t-test, and then analyzed with mixed liner model which included farm type, season, grazing intensities and milk production per cow as fixed effects and pairs as a random effect. All statistical analyses were performed using SAS statistical software (SAS Institute, Cary, NC). RESULTS The mean herd size was 72 cows (range 35 - 223) for conventional herds and 51 cows (range 37 - 132) for the organic herds. The organic herds were significantly smaller than the conventional herds (p=0.017). Eighteen organic herds and 22 conventional dairy herds consisted of 100 % Holstein breed, whereas the other 12 organic and 8 conventional herds consisted of Jersey, Brown Swiss, Guernsey or mixed breed. McNemar’s test showed there was no statistical difference in breed choice between the two types of farms. However, estimations of herd breed composition (percentage of Holstein breed) showed preference on organic dairy farms for non-Holstein and mixed breeds (p=0.10). Organic farms had lower milk production per cow (20.2 kg/day) compared to conventional farms (23.7 kg/day)(p=0.013). The CM rates were 27.7 (SD=20.7) and 56 32.1 (SD=21.8) cases per 100-cows-year at risk for organic and conventional herds (p=0.65), respectively. The frequency distribution of herd CM rates is shown at figure 4- 1. The overall mean BTSCC was 274,000 cells /ml (SD=89,700) and was approximately normally distributed (Shapiro-Wilk = 0.968). The arithmetic mean BTSCC for organic herds was 263,000 cells /ml (SD= 101,000) and 285,000 cells /ml (SD= 137,000) for conventional herds (p=0.333), as shown in figure 4-2. Half of the organic herds (15 farms) applied intensive grazing during summer, whereas only two conventional herds used intensive grazing (p=0.008). Free stalls were used on 9 conventional herds, in contrast to being used on only four organic herds (p=0.059). The total number of milking-age cows culled for any reason were 367 per year from the conventional herds and 247 per year from the organic herds. The average annual cull rate were thereby calculated as 18.0 cases per lOO-cows-years (SD=9.00) for the conventional farms and 17.2 cases per 100-cows-years (SD=9.76) for the organic farms (p > 0.426). The incidence of cow deaths on farms were 90 (4.2 cases per 100- cow-yeasr at risk) on conventional and 47 (3.1 cases per 100-cow years) on organic dairy herds (p=0.082). Only four cows in two organic farms were sold to conventional farms as milking cows in a year. The most common type of milking systems were stanchion or tie stall systems (51/60). Only one organic farm used a milking parlor compared to 8 conventional farms (p=0.008). There was no significant difference in 9 milking procedures which were assessed and compared between paired organic and conventional dairy farms (Table 4-2). Fourteen organic farms and 17 conventional farms used pre-milking teat disinfections 57 (pre-dipping) and most farms (27 organic and all conventional farms) use post-milk teat disinfections. Though 26 conventional dairy farms use udder infusions for dry cow treatment, only 18 producers reported regular use of antimicrobial udder infusion for CM treatment. Udder infirsion of cephapirin was frequently used by nine conventional producers and penicillin was frequently used by four conventional producers, whereas the remaining others conventional producers did not report which specific antibiotic products were used. One conventional producer did not know what treatments were given by his veterinarian and two producers use only injectable antimicrobials for treatment of CM. Seven other conventional dairy producers reported that they usually do not use antimicrobials for CM. Thirteen conventional producers used oxytocin and 25 conventional producers reported frequent stripping out (frequent emptying of the udder) as CM treatment. None of the organic producers used antimicrobials to treat CM. They reported using anti- inflammatory drugs and stripping out the quarters at frequent intervals as the most common CM treatments. On 19 organic farms, natural remedies (whey products, herb, mineral oil, vinegar), vitamin E (parenteral), C and selenium were occasionally used. Two organic producers allocated mastitis cows for sucking by calves as the mastitis therapy and one organic producer provided no special care for CM for several years. Table 4-2 shows the major differences among the two farming types in the treatment of CM. In the Spring, organic dairy farms had significantly lower (thinner) BCS than did the conventional herds (p=0.001), however no significant difference was observed in September (p=0.66). We did not find any difference in environmental and animal 58 sanitation score among the two farm types. Neither CM nor BTSCC had shown any association with BCS and EASS in the mixed liner analysis. A paired t-test of adjusted optical density data from ELISA test had shown significant difference between organic and conventional farms in both seasons (p=0.0059 for March and p=0.0067 for September). However, a mixed liner analysis indicated that the type of farm (organic or conventional) was not significant (p=0.8730) when controlling season, grazing intensity and milk production. The season (p=0.0081), grazing intensity (p=0.0217) and average milk production per cow (p=0.0053) were highly associated with adjusted OD value of the milk ELISA test. DISCUSSION Though using neighboring conventional herds as a comparison group helped control for regional differences, the selection of the control farms was not random and was certainly not representative of Wisconsin conventional herds or the national dairy industry. Due to their proximity, it is likely that organic and conventional farms shared many management characteristics. For example, milking procedures were very similar between matched pairs, so the effect of milking procedures on CM rate or BTSCC could not be estimated. According to the NAHMS study (USDA, 1996), 88 % of dairy operations use udder infusion for all four quarters on almost all cows, which agrees relatively closely with our observation that 26 conventional dairy farms (87%) used udder infirsions for dry cow treatment. However, the percent of operations which use injectable antimicrobials for milking cows was lower in our study (60%) compared to the 1996 NAHMS study (93.5%). It was difficult to identify the amount and type of antimicrobials 59 used on conventional farms. Two conventional dairy producers in our study claimed they had not used any antimicrobials for any purpose for several years. Animal numbers per herd (71.7 cows) and milk production per cow (23.7 kg/day) in our conventional herds were similar to the Wisconsin average (67.0 cows and 25.5kg/day), but were smaller than the national average (93.4 cows and 27.0 kg/day; USDA, 2002). We did see some evidence that organic farms tended to have smaller cows. They had a greater proportion of smaller non-Holstein breeds. They were less likely to use artificial insemination, which tends to result in a gene pool of smaller cows compared with what is commonly seen in today’s herds that have used A1 to produce large cows with high milk production per cow. Therefore, because smaller cows eat less feed, comparisons in milk production between organic and conventional herds must consider this lower cost of production. Also, other differences in nutrition and management most likely act to reduce the per-cow cost of feeding and maintaining smaller grazing cows, as compared with larger genetically selected cows being fed a high energy diet in total confinement. Dairy cow mortality (4.2 deaths per 100 cow-years) on the conventional dairy herds was compatible with the US. average (4.4 for less than 100 herd size), however the culling rate (18.0) was somewhat lower than the U. S. average (24.9 for less than 100 herd size; USDA, 2002). Another unique feature of this region of Wisconsin regarded the preponderance of the more traditional style of tie stall or stanchion housing. Twenty two percent (13/60) of operations used freestall housing, which was lower than the national average of 31 %, and 75 % (45/60) of the operations used tie stall or stanchion 6O housing, which was higher than the national average of 52% (USDA, 2002). This probably is reflective of the small herd size and harsh winter climate in Wisconsin. We found significantly lower BCS in organic dairy cows in early spring. Because organic dairy producers are required to feed “organic feed”, which must be grown without herbicides and pesticides. Purchase of feed is therefore difficult, and it may sometimes be difficult for organic producer to prepare sufficient concentrated rations for a long winter. Cows in year-round confinement may have a less variable feed supply as compared with herds which employ substantially more grazing. Internal parasites are one of the main causes for lower heifer growth and reduced milk production in older cows. Anthelmintic treatment is prohibited on organic dairy farm, so higher prevalence of gastrointestinal nematodes in organic cows could expectedly be higher than what is commonly found in conventional dairy herd. The frequency and type of deworming was not measured in our conventional farms. According to a recent NAHM study, over 60% of dairy operations in the U. S. normally use dewonners for at least some lactating cattle (USDA, 2002). Fecal egg counts (FEG) are commonly used for detecting gastrointestinal nematodes, however the test is time consuming and expensive, and the result is highly variable. The ELISA test uses a crude antigen of Ostertagia ostertagi that has been evaluated in number of previous studies (Kloostennan, 1996; Borgsteede, 2000). The optical density value of ELISA has been found to be a reasonable overall measure of parasite burden, and bulk tank milk is useful for testing whole-worm antigen (Guitian, 2000; Sanchez, 2002). Our paired t-test results indicated significantly higher parasite burden on organic dairy farms, however, no significant difference between the two farm 61 types when controlling for season (March and September), grazing intensity (no grazing, little grazing, grazing with access to housing, and grazing only) and herd average milk production per cow. The result may indicate that the organic farms may have a greater worm burden because of the increased use of grazing. Our data shows significant association with seasons, highest in late summer and lowest in spring. The observation agrees with the study result on serum antibody (Borgsteede, 2000). Herd average milk production per cow was also a significant predictor of the adjusted OD value in our study (i.e. higher producing herds were associated with lower worm burdens). Though mean CM incidence on organic farms was lower than that of conventional farms (28 and 32), the difference was not statistically significant. The measure of herd CM in this study may have been affected by reporting bias. Conventional farmers must keep meticulous records of antimicrobial treatments, especially mastitis treatments, in order to avoid the substantial penalty resulting from contaminating an entire milk truck with antimicrobial residues. Organic farms have no such impelling need to record or remember their non-antimicrobial treatments and, therefore, incomplete reporting may have existed for organic farms. Also, farmers generally record or remember episodes of clinical treatment rather than episodes of disease. Clinical signs not sufficiently advanced to warrant medical treatment may not considered to be case of that particular disease. As such, disease reports on both organic and conventional farms likely reflect episodes for which the farmer decided that clinical signs were sufficiently severe to warrant treatment. Diagnostic acumen may therefore differ between organic and conventional farms because antimicrobial treatment generally entails withholding milk from sale for several days, and is usually not given to mild cases. Although we have no 62 evidence of this speculation, Berry (2002) reported that farmers converting to organic status in the UK were less likely to report cases of CM. It is therefore certainly possible that underreporting of clinical mastitis may have been greater on the organic farms as compared to conventional farms. The arithmetic mean of BTSCC on 30 organic farm (263,000 cells per ml) was lower than that of conventional herds (285,000), however the difference (79% lower) was not statistically significant. Reporting of BTSCC from the milk receipts was uninfluenced by farmer reporting. Busato (2000) studied 152 certified organic farms in Switzerland and found the geometric mean of 85,600 cells/ml, which was 15% lower than the Swiss average of 100,000 cells/ml. The difference we found in BTSCC between organic and conventional farms (7.9%) was not statistically significant given the relatively small number of herds that we studied. Despite of the possible reporting bias speculated for CM, the results of both CM rate and BTSCC suggested that organic dairy farms managed to successfully control mastitis without the use of antibiotics. For some mastitis pathogens such as S. aureus and non-severe coliform, antibiotics are often ineffective. (Kirk, 1994). Dry cow therapy infusions are effective against contagious mastitis pathogen, but are ineffective against environmental coliforms (Berry, 1997). A retrospective study of 9,007 cases of subclinical mastitis cases in New York and northern Pennsylvania showed the overall spontaneous bacteriological cure rate was 65% and the cure rate with antimicrobial treatment was not much better at 75% (Wilson, 1999). The majority of CM may be cured by udder immune mechanisms without much benefit of antimicrobial treatment, as witnessed by the fact that only 18 conventional producers reported regular use of 63 antimicrobial udder infusion for CM treatment. Some organic producers claimed that they keep their cows at a less stressfiil milk production level and thus maintain high immune firnction. Effective mastitis control should rely on prevention rather than treatment (Erskine, 1993) and early identification, culling, and segregation is probably the best management approach for controlling mastitis (Kirk, 1994). Higher producing cows and herds tend to have greater problems with mastitis. Grohn et al. (1995) studied 8,070 cows in 25 herds and found high producing cows are more susceptible to mastitis. Schukken (1990) studied 125 herds and found a higher incidence of CM in herds with high milk production. The average daily milk production per cow in organic dairy herds was about 15% lower than that of conventional dairy herd in our study. This lower milk production in organic herds could account for at least some of the non-significant trend to reduction in mastitis and BTSCC which we observed for organic farms. Milk production without increasing CM may be attained by those principles in the organic herds. CONCLUSION The study showed that organic dairy farms were producing milk without significantly increasing reported CM rate, BTSCC or culling rate as compared with matched conventional farms. Notable differences between organic dairy farms and conventional farms were lower milk production per cow and smaller herd size. Organic herds were more likely to use intensive grazing, which may have accounted for their higher rate of gastrointestinal nematodes as compared with conventional herds. 64 REFERENCES Barlow, J. 2001. Organic dairies - Mastitis and milk quality challenges. National Mastitis Council Annual Meeting Proceedings. Washington, National Mastitis Council. 143- 151. Bartlett, P.C., Miller, G. Y., Lance, S. E., and Heider, L. E., 1992a. Environmental and managerial determinants of somatic cell counts and clinical mastitis incidence in Ohio dairy herds. Prev. Vet. Med. 14, 195-207. Bartlett, P.C., Miller, G. Y., Lance, S. E, and Heider, L. E., 1992b. 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Med. 44, 205-220. Erskine, R. J ., Kirk, J. H, Tyler, J. W., DeGraves, F. J ., 1993. Advances in the therapy for mastitis. Vet. Clin. N. Am-Food A. 9, 499-517. FAO, 2000. Food safety and quality as affected by organic farming. 22nd F AO regional conference for Europe. Food and Agriculture Organization. Rome. Greene, C. R., 2000. U. S. Organic agriculture gaining ground. Economic Research Service, USDA. Agricultural Outlook. 9-14, ONLINE: http://www.ers.usda.gov/epub5/pdf/agout/apr2000/ao270d.pdf Greene, C. R., 2001. U. S. Organic Farming Emerges in the 19905: Adoption of Certified Systems. Agriculture Information Bulletin. 770. Grohn, Y. T., Eicker, S. W., Hertl, J. A, 1995. The association between previous 305-day milk yield and disease in New York state dairy cows. J. Dairy Sci. 78, 1693-1702. 65 Guitian, F. J ., Dohoo, I. R., Markham, R. J. F., Conboy, G., Keefe, G. P., 2000. Relationship between bulk-tank antibodies to Ostertagia ostertagi and herd- management practices and measures of milk production in Nova Scotia dairy herds. Prev. Vet. Med. 47 279-89. Hady, P. J ., Lloyd, J. W., Kaneene, J. B., 1993. Antibacterial use in lactating dairy cattle. JAVMA. 203, 2, 210-220. Hardeng, F., Edge, V. L., 2001. Mastitis, ketosis, and milk fever in 31 organic and 93 conventional Norwegian dairy herds. J. Dairy Sci. 84, 2673-2679. Jayarao, B. M., Cassel, E. K., 1999. Mastitis prevention and milk hygiene practices adopted by dairy producer. Large Animal Practice. 20, 5, 6-14 Kaneene, J. B., Miller, R., 1992. Description and evaluation of the influence of veterinary presence on the use of antibiotics and sulfonamides in dairy herds. JAVMA. 201:68- 76. Kirk, J. H., DeGraves, F., Tyler, J., 1994. Recent progress in treatment and control of mastitis in cattle. JAVMA 204, 8, 1152-1158. Kloosterman, A, Ploeger, H. W., Pieke, E. J ., Lam, T. J. G. M., Verhoeff, J., 1996. The value of bulk milk ELISA Ostertagia antibody titres as indicators of milk production response to anthelmintic treatment in the dry period. Vet. Parasitol. 64:197-205. Mann, E., 1999. Organic dairy products. Dairy Ind. Int. 62:2:11-12. Sanchez, J ., Dohoo, I. R., Markham, F ., Leslie, K., Conboy, G., 2002. Evaluation of the repeatability of a crude adult indirect Ostertagia ostertagi ELISA and methods of expressing test results. Vet. Parasitolo. 109275-90. Sato, K., Bartlett, P., Ruegg, P., Robinson-Dunn, B., Downes, F. P., Erskine, R., 2002. Milk production and mastitis on Midwestern organic dairy farms. Dairy, Food and Environmental Sanitation. 22, 3, 178-183. Schukken, Y. H, Grommers, F. J., Van-de-Geer, D., Erb, H. N., Brand, A, 1990. Risk factors for clinical mastitis in herds with a low bulk milk somatic cell count. 1. Data and risk factors for all cases. J. Dairy Sci. 73, 12, 3463-3471. Sundrum, A. 2001. Organic livestock farming, A critical review. Livest. Prod. Sci. 67:207-215. USDA. 2001. National Organic Standard, Agriculture Marketing Service. [ONLINE] httpz/lwww.am5.u5da.gov/nop/NOP/standards/FullText.pdf, Accessed on May 26 2003. 66 USDA. 1996. Part HI: Reference of 1996 Dairy Health and Health Management. USDA:APHIS:VS, CEAH, National Animal Health Monitoring System. Fort Collins, CO. USDA. 2002. Part H: Changes in the United States Dairy Industry, 1991-2002. USDA:APHIS:VS, CEAH, National Animal Health Monitoring System, Fort Collins, CO #N388.0603. Weller, R. F ., Cooper, A., 1996. Health status of dairy herds converting from conventional to organic dairy farming. Vet. Rec. 1392141-142. Weller, R. F., Bowling, P. J., 2000. Health status of dairy herds in organic farming. Vet. Rec. 146, 80-81. Wildman, E. E., Jones, G. M., Wagner, P. E., Boman, R. L., Trout, H. F., Lesch, T. N., 1982. A dairy cow body condition scoring system and its relationship to selected production variables in high producing Holstein dairy cattle. J. Dairy Sci. 65, 495- 501. Wilson, D. J., Gonzalez, R. N., Case, K. L., Garrison, L. L., Grohn, Y. T., 1999. Comparison of seven antibiotic treatments with no treatment for bacteriological efficacy against bovine mastitis pathogens. J. Dairy Sci. 82:1664-1670. 67 Table 4-1. Comparison between organic and conventional dairy farms. Means of continuous variables regarding management and production variables. The data was analyzed by PROC MIXED procedure with a random factor of “pairs”. Continuous variables Organic Conventional Type 111 test (n=30) (n=30) p-value Number of lactating and dry cows 51.1 71.7 0.017 Average milk production per cow 20.2 23.7 0.025 (kg/day) Mastitis rate per 100 cow-years 27.7 32.0 0.654 BTSCC (cells/ml) 263,000 285,000 0.328 Bacteria count of bulk tank milk 4,200 4,800 0.433 (cells/ml) Body condition score in March 2.58 2.81 0.001 Environmental Sanitation score in 1.88 1.79 0.548 March ‘ Body condition score in September 2.81 2.84 0.662 Environmental Sanitation score in 1.83 2.02 0.157 September Average labor (person-minutes) per 2.66 2.68 0.925 cow per milking 68 Table 4-2. Comparison of discrete variables between organic and conventional dairy farms. . . McNemar‘s (5:233? Colrzzzggcna test p-value 100% Holstein breed 18 22 0.248 Purchase of any cows in previous 12 months 4 9 0.132 Dry cow treatment with antibiotic infusion 0 26 NA Grazing No grazing 1 0.096 Outside exercise area with little grazing 3 0.593 Grazing pasture with access to housing 11 13 0.001 Intensive grazing (grazing only) 15 2 0.008 Housing Free stalls 4 9 0.059 Loose housing 2 0 NA Tie stalls 24 21 0.257 Bedding No bedding 3 1 0.317 Straw or com shredder 20 13 0.090 Wood shavings or sawdust 3 10 0.008 Sand 3 5 0.480 Rubber mat 1 1 1.000 Milking Cows were milked in stanchion/tie stalls 29 22 0.008 Dry massage or wipe with no water used 6 6 1.000 Wash bucket usually used 10 6 0.206 Individual cow paper towel or cloths used 14 11 NA for washing Shared towel or cloths used for washing 3 1 0.317 Pre-milking teat dipping usually used 14 17 0.405 Post-milking teat dipping always used 27 30 NA Gloves used for washing udder 7 12 0.197 Individual towels (cloth or paper) used for 15 21 0.058 drying Teats are usually not dried before milking 4 1 0.180 69 Table 4-2 (cont’d) Mastitis prevention and treatment Dry cow treatment with antibiotic infusion Strip out the quarter at frequent intervals Anti-inflammatory or antipyretics drugs Administer oxytocin to assist milkout Udder infusions of antibiotics Systemic antibiotic injection 20 10 26 25 13 18 NA 0.132 0.317 0.002 NA NA NA: McNemar's test cannot be performed because some cells contain zero. 70 Figure 4-1. Frequency distribution of mastitis rate (100-cow-year) of organic farms (n=30) and conventional farms (n=30). Mean mastitis rate were not statistically different among two farm types (p=0.432) Number of farms a: I I I 7.5 22.5 37.5 52.5 67.5 82.5 Mastitis rate (100-cow-year) IflOrganic IConventional I 71 Figure 4-2. Frequency distribution of milk somatic cell count (SCC) of organic farms (n=30) and conventional farms (n=30). Arithmetic mean were not statistically different between two farm type (p=0.333). Number of farms N 00 h 01 G \l on (O -§ O 120 180 240 300 360 420 480 Somatic Cell Counts (1,000 cells/ml) IOOrganic IConventional I 72 CHAPTER FIVE COMPARISON OF ANTMCROBIAL SUSCEPTIBILITY OF E. Coli ISOLATED FROM ORGANIC AND MATCHED CONVENTIONAL DAIRY CATTLE IN WISCONSIN ABSTRACT A cross-sectional study was conducted in Wisconsin to compare antimicrobial resistance patterns in fecal Escherichia coli on 30 organic dairy farms, where antimicrobials are rarely used for calves and never used for cows, and 30 neighboring conventional dairy farms, where antimicrobials are routinely used for animals of all ages. Fecal specimens from ten cows and ten calves on each farm yielded 1,120 E coli isolates, which were tested for resistance to 17 antimicrobials using a micro-broth dilution test. The highest overall rates of resistance were to tetracycline (26%), streptomycin (15%), kanamycin (14%), sulfamethoxazole (14%), and ampicillin (13%). Prevalence of antimicrobial resistance in E. coli was lower in organic dairy herds for ampicillin, amoxicillin-clavulanic acid, streptomycin, kanamycin, chloramphenicol, tetracycline, sulphamethoxazole, and trimethoprim—sulphamethoxazole. Although the organic farms had converted to organic farming methods at least 3 years before our study (mean = 8.0 years), antimicrobial resistance clearly presented long after antimicrobial selective pressure had been withdrawn. 73 INTRODUCTION Some resistant bacteria isolated from human infections have reportedly been traced back to farm animals (Fey, 2000; Larsen, 1972; Molbak, 1999, O’Brien, 1982; Spika, 1987). F arm-level studies have shown an association between antimicrobial usage in food animals and rates of antimicrobial resistant in animals (Mathew, 2001; Van Den Bogaard, 2001). Generally, farms with higher antimicrobial usage have a higher proportion of resistant bacteria, as well as the presence of multi-drug resistance strains. However, the magnitude of the contribution of livestock production practices to the growing antimicrobial resistant problem is unclear. (Sorum, 2001; Torrence, 2001). Antimicrobial agents are used less frequently in dairy cattle than they are in other food-producing animals. Because milk from antibiotic-treated cows must be withheld fi'om sale, most antimicrobials are used relatively conservatively in the dairy industry. Most antibiotic use in adult cows is for treatment and prevention of clinical mastitis (Hady, 1993). In dairy calves, most antimicrobial usage is for treatment and prophylaxis of diarrhea/pneumonia, and as a medicated milk replacer (McEwen, 2002; NAHMS, 2002) Escherichia coli most commonly are conditional pathogens causing urinary tract infections, wound infections, septicemia, and hemorrhagic colitis in humans and also are important agents of environmental clinical mastitis in dairy cows. They are present in the normal intestinal tract flora of most animals (Scrum, 2001). As commensal bacteria, E. coli are of concern to human health because they are likely to be transferred to humans through food or water. Osterblad et al. (2000) isolated Enterobacteriaceae from human fecal samples and compared the antimicrobial resistance in E. coli to other enterobacteria 74 such as Citrobacter, Klebsiella and Proteus. They found lack of antimicrobial resistance in other enterobacteria and speculated that E. coli were a principal carrier of antimicrobial resistance in fecal flora. Oppegaard et al. (2001) also suggested that E. coli is a major reservoir of resistance traits in the coliform flora of cattle. Escherichia coli have an ability to horizontally transfer their resistant determinants to other genera (Kruse, 1994). Escherichia coli have been used in this and other studies as indicator organisms for antimicrobial resistance monitoring activities among Gram-negative bacteria (Bager, 2001; NARMS, 2000). Our primary research interest was to determine if antimicrobial resistant in E. coli isolates on dairy farms is associated with the type and the quantity of antimicrobials being used. A second objective was to provide background information on antimicrobial resistance of E. coli on dairy farms and to estimate the degree of AR persistence after farms converted from conventional to organic farms. MATERIALS AND METHODS Cattle fecal samples were collected from 30 organic dairy farms and 30 conventional farms in southwestern Wisconsin. The organic farms were members of an organic dairy association of 325 dairy farms. All organic farms were certified by a certification agency as not having used antimicrobials on cows for at least 3 years before the start of our study. For each organic farm selected, the geographically closest "conventional" dairy farm, for which the farm owner would agree to participate, was included in this study as a control. All herds were visited twice; once in March and once in September, during 2000-2001. 75 Management information was collected at the first visit using an orally administered questionnaire. Questions were administered and observations were made regarding milk production, milking practices, housing, incidence of the major dairy diseases, medical treatments and other management factors. At each visit, fecal samples were collected from five lactating cows and five calves under approximately 6 months of age. Animals were excluded if they had diarrhea or were under treatment for another illness. Adult cows were sampled by walking among the cows and waiting for one to defecate. The fecal sample was taken from the freshly voided fecal pile, taking care to not contact the ground beneath. If this could not be done with certainty, the next cow to defecate was sampled. Fecal samples were obtained directly fi'om calves when they defecated following anal stimulation. A clean sterile latex glove was used for each specimen to avoid cross-contamination. Approximately five grams of fecal sample were placed into a Cary-Blair Transport Media tube (Medical Chemical Corp., Torrance, CA) and another five grams were placed into a sterile plastic tube. The specimens were kept on ice and sent to the Michigan Department of Community Health laboratory by overnight courier service. The fecal samples were processed within 72 hours of sample collection by streaking directly on McConkey agar plates (REMEL, Lenexa, KS) and incubating at 35 °C for 24 hours. If no suspect E. coli colonies were evident, the raw fecal sample was inoculated in a McConkey broth tube and incubated at 35 °C for 24 hours and then streaked on McConkey agar. One typical E. coli colony was selected and identified following standard methods (Gray, 1995). 76 Escherichia coli isolates were tested for AR against 17 antimicrobial agents (Table 5-1) by semi-automatic micro broth dilution methods (Sensititre; Trek diagnostic Systems Inc., OH). The Mle were tested in accordance with the manufacture’s instruction. In summary, E. coli were inoculated on TSA with 5% sheep blood agar (REMEL, Lenexa, KS) and incubated at 35 °C for 24 hours. The sub-cultured colonies were examined for purity and emulsified in 4 ml de-mineralized distilled water, adjusting the turbidity to that of a 0.5 McFarland standard. A 10 micro-liter suspension was transferred into a Mueller Hinton broth tube (11 ml) and 50 pl of the broth suspension was transferred to Sensititre panels, which were incubated at 35 °C for 18 hours prior to determining the minimum inhibitory concentration (MIC). Quality control was performed by testing E. coli of American Type Culture Collection (ATCC) 29212, Staphylococcus aureus (ATCC29213) and Pasturella aeruginosa (ATCC27853). The MIC results were dichotomized based on the clinical breakpoints set by National Committee for Clinical Laboratory Standards (NCCLS). Isolates with intermediate susceptibility were categorized as being susceptible. Logistic regression was used to estimate the odds ratio for each antimicrobial, comparing the organic and conventional farms. Animal age (cow/calf) and season (September/March) were also included in these models as possible confounders. Management factors, such as the number of cattle, amount of milk produced, intensive grazing, animals purchased during the past year (yes/no), mastitis rate, calf mortality rate and the number of years that each farm had been organic were tested as predictors of AR prevalence in logistic regression analysis. 77 The dichotomized data were used to construct tables of the multiple drug resistance. The number of E. coli isolates which were resistant to 0, 1, 2, and 23 antimicrobials were compared among conventional and organic dairy farms. Each table was analyzed separately by extended Mantel-Haenszel mean score statistic (Stokes, 2000) All statistical analysis was performed using SAS statistical software (version 8.02; SAS Institute, Cary, NC). RESULTS Nine fecal samples were not available from five farms due to an insufficient number of calves on farms. A total of 1,120 E. coli were isolated from 1,191 fecal samples (94.1%) collected at the 120 farm visits. Escherichia coli were not isolated from 45 of 596 organic samples (7.6%) and 25 of 595 conventional samples (4.2%). Each organic dairy had converted to organic farming methods at least 3 years before our study (mean=8.0 years). Organic farmers indicated that no antimicrobials were used on their dairy cows, but four organic farmers reported antimicrobials were used for calves with serious diarrhea or pneumonia. In 26 of the 30 conventional dairy herds, cows routinely received antimicrobial infusions into the udder at the cessation of each lactation cycle (dry-cow treatment). Cephapirin or penicillin were the most frequently used products. Eighteen conventional dairy producers reported using udder infiision of antimicrobials for the treatment of clinical mastitis. For the severe clinical mastitis cases, eight conventional dairy producers used systemic antimicrobials. 78 The frequency distributions of E. coli MIC and the NCCLS breakpoints for 17 antimicrobial agents were shown in Table 5-1. There were no clinically resistant isolates to ceftriaxone, amikacin, nalidixic acid, or ciprofloxacin. Though not resistant at NCCLS break points, five isolates from calves on three conventional dairy farms had MIC of more than 4 ,ug/ml of ceftriaxone, Those five isolates also had MIC of more than 4 ,ug/ml of ceftiofur and were resistant to eight other antimicrobials; ampicillin, amoxicillin- Clavulanic acid, cephalothin, cephoxitin, streptomycin, chloramphenicol, tetracycline, and sulphamethoxazole. Two of the decreased susceptibility isolates to ceftriaxone were from one farm, but were collected six month apart from each other. Another notable finding was that 27 resistant isolates to chloramphenicol were isolated from calves on 18 farms. All but two of these isolates were from conventional dairy farms and all 27 isolates had multiple AR patterns (3-11 drugs, median=5). Among those 27 chloramphenicol resistant isolates, 26 isolates, 25 isolates, 22 isolates and 22 isolates were resistant to sulphamethoxazole, tetracycline, streptomycin and kanamycin, respectively. Table 5-2 summarizes the logistic regression analysis of the NCCLS dichotomized data. The type of farm (conventional/organic) and age (cow/calf) were included in all logistic regression models. The season and the interaction between farm type and age (cow/calf) were evaluated in the model, but were not included in the final model because they were not statistically significant. The farm size (number of cattle), amount of milk produced, intensive grazing, animals purchased during the past year (yes/no), mastitis rate, calf mortality and the number of years that each farm had been organic, were not significant predictors for E. coli antimicrobial resistance to each 79 antimicrobial. The results of the analyses show that conventional dairy farms as compared to organic farms had a significantly higher rates of resistant isolates of E. coli to each of seven antimicrobials: ampicillin, streptomycin, kanamycin, gentamicin, chloramphenicol, tetracycline, sulphamethoxazole, and trimethoprim-sulfamethoxazole. The analysis of multiple antimicrobial resistance (Table 5-3 ), using the extended Mantel- Haenszel mean score statistic, indicated that prevalence of multiple AR was not significantly different between E. coli isolates from conventional and organic dairy herds (QSMH=0.6125, p=0.4338). However, multiple AR in E. coli isolates from calves was significantly higher in conventional dairy herds than it was in organic dairy herds (QSMH=24.1 193, p<0.0001). DISCUSSION The fecal samples were collected from presumably healthy cows and calves after having excluded animals with obvious diarrhea or that, to our knowledge, were under treatment for some other disease. This selection policy may have resulted in lower measures of antimicrobial resistance in our study compared to studies based on diagnostic submissions from ill animals, many of which may have recently undergone antimicrobial therapy (Schroeder, 2002). The Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DAN MAP, 1996 - 2001) reported a higher prevalence of resistant bacteria in diagnostic submission samples as compared with specimens from animals at slaughter. For example, a high proportion (SO-86%) of E. coli isolates fi'om Denmark were resistant to ampicillin in diagnostic submission, however only 0-8 % of E. coli from presumably healthy slaughter cattle were resistant to ampicillin (Bager, 1997). 80 The higher prevalence of antimicrobial resistance in diagnostic bacterial isolates may be due to the selection pressure from antimicrobial treatments. Like DANMAP and NARMS, we avoided the difficult issue of clustering of isolates in cows within herds by taking only one representative E. coli isolate from each animal. A non-differential selection bias may have existed if the E. coli isolates that we were able to culture were, in some unknown way, not representative of all the E. coli that were present. Such a non-differential bias should not have effected our measures of effect. Our organic dairy farm isolates were from a geographic cluster of about 110 organic dairy farms belonging to a particular organic dairy association at farm selection. The conventional dairy farms were not selected randomly, but were the nearest, in sequence of proximity, to the selected organic dairy farms that agreed to participate. A study conducted by Singer et al. (1998) suggested AR might be clustered in time and space, though their data was derived from a clinical diagnostic database. The logistic regression analysis indicated that the season of sampling was not significant predictor of antimicrobial resistance prevalence on dairy farms, although the six-month interval may be too short to detect the change of antimicrobial resistance E. coli prevalence in time. Assessing the amount of antimicrobials used on the farms was difficult. Generally, in spite of our requests, farmers may not have recorded the details of antimicrobials used. Some farmers may not have known what treatments were rendered by veterinarians. Also, our assumption that organic dairy herds used no antimicrobials was dependent upon the farmers’ compliance to the rules of their organic association. The certified organic dairy farms maintained the compliance records. If there were 81 serious compliance issues, the dairy farm would have lose their organic status and would not be able to sell the milk at the premium organic price. Moreover, the majority of organic producers started organic dairy production based on an individual philosophy of environmental impacts and biological soundness. The farms’ compliance to the rules of their organic association is assumed to be well regarded. Regardless of issues of absolute compliance, the evidence is overwhelming that antimicrobial use was much greater on the conventional farms than it was on the organic farms. Adult dairy cattle almost never receive antimicrobials in the feed over long periods of time, but medicated milk replacer is often used for calves. Antimicrobial use as feed additive is probably a principal reason why the total amount of antimicrobials used in swine and poultry production is much greater than what is used in the dairy industry. Consequently, a higher proportion of resistant bacteria are found in swine and poultry isolates as compared with isolates from dairy cattle (Salmon, 1995; Bager, 1997; Mathew, 1998; Schroeder, 2002). Our five E. coli isolates with reduced susceptibility to ceftriaxone were resistant to all beta-lactams and cephalosporins, and were co-resistant to aminoglycosides, chloramphenicol, tetracycline and sulfonamide. Winokur et al. (2001) reported in their study of human isolates that high levels of co-resistance to aminoglycosides, tetracycline, trimethoprim-sulfamethoxazole, and ciprofloxacin were observed in extended-spectrum B-lactamase (ESBL) producing strains of Enterobacteriaceae. Our isolates shared the resistant pattern with the human ESBL strains. Farm could be a source of resistant plasmids in human ESBL isolates. It must be noted that all five isolates came from conventional dairy calves, to which substantial amount of antimicrobials are administered 82 as feed additives and injections. CCfilOfUl' was developed for veterinary use, and is approved for treatment of pneumonia in dairy cattle with zero withdrawal time, as well as for acute bovine interdigital necrobacillosis (FDA, Green book). Since the extralabel use of ceftiofur for severe mastitis is not very efficacious (Erskine, 2002), ceftiofiir is not widely used for mastitis. However, the zero withdrawal time of milk and meat make the drug attractive. Though we could not assess the amount of ceftiofitr used on the farms, we speculate the use of third-generation cephalosporin is the selective pressure for ESBL isolates on these farms. Chloramphenicol has been prohibited for food producing animal in the US since January 1986 (FDA Veterinarian Newsletter, 1996) and has been rigorously enforced by the USDA. The resistance to chloramphenicol is rendered by the inactivation with the chloramphenicol transacetylase (Prescott, 2000) and by enhanced efflux. White et al. (2000) investigated 48 E. coli recovered from calves with diarrhea and found the majority of resistant E. coli had enhancing efflux genes (flo and chA ), which may be disseminated via plasmids and/or a mobile trasposon(s). They suggested the extra-label use of florfenicol in calves was the major selection pressure of those resistant traits. Florfenicol is a structurally similar antimicrobial to chloramphenicol, and was approved for bovine respiratory treatment in 1996 (Online Green book, 2003). Other possible selection pressure on farm is the co-selection by other antimicrobials. Our observation of high multiple AR supports the co-selection by non-chloramphenicol antimicrobials. Further research is required to identify the selective pressure on farm that has caused chloramphenicol resistant trait to be reserved for so long on both organic and conventional dairy farms. 83 The overall prevalence of resistant bacteria in calf fecal samples was significantly higher than that of cows in both conventional and organic dairy herds. Calf milk replacers, which may contain antimicrobials such as chlortetracycline, oxytetracycline and neomycin, are widely used by dairy producers in the United States (USDA/APHIS, 2002; Heinrichs et al., 1995). The possible use of medicated milk replacer in conventional dairies may, in part, explain the high prevalence of resistant E. coli in calves. Though organic dairy producers may use antimicrobials in animals under one year of age for life threatening situations, medicated milk replacer is prohibited. The organic dairy producers usually treated their sick calves with oral electrolyte solutions, whey products and probiotic products. Only four of the 30 organic producers responded that they had used antimicrobials for sick calves in the past one year. Hinton et al. ( 1985) reported a high prevalence of resistant E. coli in calves which did not receive oral antimicrobials. They found a continual tum-over of E. coli strains in the fecal flora, the majority of which were isolated only a few times. The prevalence of antimicrobial resistant isolates became highest on approximately the 10th day of age, and gradually reduced in following 140 days to the level observed in the cows. Bennedsgaard et al. (Bennedsgaard, T. W., S. M. Thamsborg, F. M. Aarestrup, C. E. Enevoldsen, and M. Vaarst, submitted for publication) recently reported a higher prevalence of AR E. coli in calves as compared to cows. This was observed in both organic and conventional dairy herds in Denmark, where milk replacer with antimicrobials have been prohibited since the early 1970's (Larsen, 1972). Higher percentages of resistant bacteria in young animals, as compared with adults, were observed in pigs (Hinton, 1987; Langlois, 1988; Larsen, 1972). Larsen et al. (1972) speculated that bacteria in young animals have the 84 increased potential for resistance transfer, and Langlois et al. (1988) suggested the AR E. coli can more easily colonize in intestinal tract in young animals than adult. In any case, animal age could be an important factor when investigating AR in enteric E. coli in populations. Our analysis did not find any significant effect of most management factors on AR E. coli prevalence when controlling for farm type (organic or conventional) and animal age. Langlois et al. ( 1988) reported that pigs on pasture have lower AR E. coli prevalence than pigs in finishing or farrowing house. They speculated the exposure to antimicrobials is not the only factor that influences the prevalence of AR bacteria. When we do not control for farm type and animal age in logistic regression analysis, intensive grazing becomes a significant predictor for AR prevalence. However, this is probably because intensive grazing is highly correlated with farm type, i.e. intensive grazing was a confounding factor. Less than 2% of indicator E. coli from cattle were multi-resistant in Denmark where a similar set of antimicrobials was tested. In our study, 23.9% (268/1, 120) had multiple antimicrobial resistant isolates in cows and calves combined, and 7.6% (3 7/486) in cows. Differences in multiple-AR between the Danish study and our study may be due to a more restrictive policy on use of antimicrobial agents in Denmark. Indicator isolates from Danish cattle (n=85) had resistance to tetracycline 4.7%, streptomycin 1.2% (modified based on our breakpoints), sulfamethoxazole 3.5%, and ampicillin 0% in DANMAP 2001. Our AR results for E. coli in cow was tetracycline 7.3%, streptomycin 2.5%, sulfamethoxazole 3.6% and ampicillin 2.0%. AR proportions in our Wisconsin isolates were slightly higher than in Danish isolates (or=0.05). 85 CONCLUSION Our study shows significantly lower prevalence rates of AR for seven antimicrobials in organic dairy herds as compared to conventional herds. However, the odds ratios for having resistant E. coli were relatively small, suggesting that AR persists for many years in organic herds after exposure to antimicrobials is withdrawn. Differences in AR prevalence between cows and calves was large in the both organic and conventional herds; therefore animal age (cow/calf) should be taken into account when AR E. coli prevalence in animals is studied. 86 REFERENCES Bager, F. (ed.), 1997. 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Cary, NC. Torrence, M. E. 2001. Activities to address antimicrobial resistance in the United States. Prev Vet Med. 51:37-49. 89 Van Den Bogaard, A. E., N. Bruinsma, and E. E. Stobberingh. 2000. The effect of banning avoparcin on VRE carriage in The Netherlands. J Antimicrob Chemother. 46: 145-153. Van Den Bogaard, A. E., N. London, C. Driessen, and E. E. Stobberingh. 2001. Antibiotic resistance of faecal Escherichia coli in poultry, poultry farmers and poultry Slaughters. J Antimicrob Chemother. 47:763-771. Veterinary Newsletter. 1996. Why veterinary drugs are not always available. Center for Veterinary Medicine, Food and Drug Adminstration, Veterinarian Newsletter, 11, 1. White, D. G., C. Hudson, J. J. Maurer, S. Ayers, S. Zhao, M. D. Lee, L. Bolton, T. Foley, and J. Sherwood. 2000. Characterization of chloramphenicol and florfenicol resistance in Escherichia coli associated with bovine diarrhea. J Clin Microbiol. 38:4593-4598. Winokur, P. L., R. Canton, J. M. Casellas, and N. Legakis. 2001. Variations in the prevalence of strains expressing an extended-spectrum B-lactamase phenotype and characterization of isolates from Europe, the Americas, and the Western Pacific Region, Clin Infect Dis. 32:894-103. 90 Table 5-1. Frequency distributions of antimicrobial resistance E. coli. Conventional Organic Antimicrobials MIC Cow Calf Cow Calf Beta-lactam Ampicillin <= 109 74 105 102 4 158 1 12 147 121 8 10 11 12 7 16 2 1 2 3 32 0 2 0 1 >32 7 84 4 47 Amoxicillin Clavulanic Acid <=0.5 0 0 1 0 1 1 1 2 9 1 1 2 49 27 44 37 4 174 144 176 155 8 45 96 34 70 16 6 6 5 4 32 1 2 1 3 >32 0 7 0 1 Cephalosporins (1" generation) Cephalothin 2 9 2 7 11 4 6O 53 43 51 8 154 153 147 161 16 54 62 64 44 32 4 5 4 8 <32 5 9 5 6 (2MI generation) Cefoxitin <=4 247 236 216 252 8 36 36 49 23 16 2 5 5 4 32 1 3 0 2 >32 0 4 0 0 (3"I generation) Ceftiofur 0.5 283 276 266 277 1 O 1 0 3 2 1 1 0 1 4 1 1 3 0 8 0 4 1 0 16 0 1 0 0 >16 1 0 0 0 Ceflriaxone <=0.25 284 278 270 280 0.50 1 1 0 1 2.00 1 0 0 0 4.00 0 2 0 0 (resistance 2 32ug/ml) 8.00 0 3 0 0 Aminoglycosidee Streptomycin <=32 279 186 263 220 64 5 46 4 35 128 1 27 1 18 256 1 22 2 7 >256 0 3 0 1 91 Table 5-1 (cont’d). Conventional Organic Antimicrobials MIC Cow Calf Cow Calf Kanamycin <=16 281 179 269 230 32.00 0 1 0 0 64.00 4 104 1 51 Gentamicin <=0.25 25 16 21 21 0.50 198 185 192 192 1 .00 59 57 54 59 2.00 2 3 2 2 4. 00 0 1 1 0 8. 00 0 2 0 0 16. 00 2 5 0 0 >16 0 15 0 7 Apramycin <=2 12 19 15 10 4 183 184 171 179 8 81 72 76 87 16 7 5 5 5 32 2 1 2 0 >32 1 3 1 0 Arnikacin <=4 282 282 269 277 (resistance 2 64pg/ml) 8 4 2 1 4 Chloramphenicol Chloramph <=4 261 252 249 267 8 25 10 21 9 32 0 6 0 0 >32 0 16 0 5 Tetracycline Tetracycline <= 252 1 13 249 169 8 9 16 5 12 16 0 2 1 4 32 0 24 3 21 >32 25 129 12 75 SulfonamidelTrimethoprim Sulphamethoxazole <=128 277 201 259 230 256 0 2 0 0 512 0 15 4 7 >512 9 66 7 44 Trimethoprim-SuIfamethoxazole <=0. 12 264 196 250 227 0.25 19 40 12 26 0.5 2 22 6 21 1 0 8 0 1 2 0 1 O 0 >4 1 17 2 6 Fluoroquinolones Nalidixic acid <=4 264 266 253 269 (resistance 2 32pg/ml) 8.00 22 18 17 12 Ciprofloxacin <=0.015 283 278 269 280 0.030 2 6 1 1 (resistance 2 4pg/ml) 0.060 1 0 0 0 92 Table 5-2. Logistic Regression Analysis of resistance to antimicrobials among E coli isolates from organic and conventional dairy farms. Conventional Organic Antimicwbia's 855.2315!) 8558:1115 OddsRatio $938238: Beta-lactams Ampicillin 93/477 52/499 2.055 0.0002 Qggx'c'm" ' C'avu'amc 10/560 5/546 1.98 0.2138 Cephalosporins (1 st generation) Cephalothin 23/547 23/528 0.969 0.9178 (2nd generation) Cefoxitin 8/562 2/549 3.997 0.081 1 (3rd generation) Cefliofur 6/564 1/550 5.910 0.1006 Ceftriaxone 0/570 0/551 NA NA Aminoglycosides Streptomycin 1 05/465 68/483 1 .755 0.0018 Kanamycin 1 07/462 52/499 2.588 <0.0001 Gentamicin 22/548 7/544 3.241 0.0077 Apramycin 7/563 3/548 2.262 0.2386 Amikacin 0/570 0/551 NA NA Chloramphenicol Chloramphenicol 22/548 5/546 4.384 0.0031 Tetracycline Tetracycline 180/390 1 16/434 2.009 <0.0001 Sulfonamide/Trimethoprim Sulphamethoxazole 90/480 63/488 1 .531 0.0209 Trimetho rim - Sulfa m etii ox az 0| e 18/552 37498 2.272 0.0573 Fluoroquinolones Nalidixic Acid 0/570 0/551 NA NA Ciprofloxacin 0/570 0/551 NA NA NA: Not available because there was no resistant bacteria. 93 Table 5-3. Frequency distribution of multiple antimicrobial resistance among 1,120 E. coli isolates COW Calf Number of antimicrobials to Conventional Organic Conventional Organic which isolates were (n=284) (n=270) (n=285) (n=281) resistant 0 245 241 121 167 1 19 12 20 27 2 11 10 24 20 3 7 6 36 24 4 2 0 43 19 5 0 1 11 17 6 0 0 14 3 7 0 0 7 2 8 0 0 4 1 9 0 0 1 0 10 0 0 1 1 11 0 0 2 0 12 0 0 1 0 The frequency of multiple antimicrobial resistant in cow and calf isolates were analyzed separately with the extended Mantel-Haenszel mean score statistic. The frequency distribution of cow isolates was not different among conventional and organic (QSMH=0.6125, p=0.4338), however the distribution of calf isolates was significantly different (QSMH=24. l 193, p<0.0001). 94 CHAPTER SIX COMPARISON OF PREVALENCE AND ANTIMICROBIAL SUSCEPTIBILITY OF CAMPYLOBACTER SPP. ISOLATED FROM ORGANIC AND CONVENTIONAL DAIRY HERDS IN WISCONSIN, USA ABSTRACT The prevalence and antimicrobial susceptibility of Campylobacter spp. isolated from bovine feces was compared between organic and conventional dairy herds. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms, where antimicrobials were routinely used for animals for all ages. Fecal specimens from ten cows and ten calves on 120 farm visits yielded 332 Campylobacter isolates. The prevalence of Campylobacter spp. in organic and conventional farms were 26.7% and 29.1%, and the prevalence was not statistically different between the two types of farms. Campylobacter prevalence was significantly higher in March than in September, higher in calves than in cows, and higher in smaller farms than in large farms. The rates of retained placenta, pneumonia, mastitis and abortion rate were associated with the proportion of Campylobacter isolation from fecal samples. Gradient disk diffusion minimal inhibitor concentration method (Etest) was used for testing susceptibility to four antimicrobial agents: ciprofloxacin, gentamicin, erythromycin and tetracycline. Two isolates were resistant to ciprofloxacin and none of isolates were resistant to gentamicin or erythromycin. Resistance to tetracycline was 45% (148/332 isolates). Tetracycline resistance was found more frequently in calves than in cows (p=0.042), but no difference 95 was observed between organic and conventional farms. We saw no evidence that restriction of antimicrobial use on dairy farms was associated with prevalence of antimicrobial resistance in Campylobacter spp. to ciprofloxacin, gentamicin, erythromycin and tetracycline. INTRODUCTION Campylobacter spp. has been recognized as a cause of septic abortion, infectious infertility and diarrhea in cattle and sheep (Radostits, 2000). Abortions in cattle can be caused by Campylobacter fetus sub sp. veneralis or C. fetus subsp. fetus, however C. jejuni and C. coli also recognized as causal agents of abortions (Larson, 1992; Welsh, 1984). Campylobacter hyointestinalis was reported as a cause of ileitis in pigs (Gebhart, 1983), bovine diarrhea (Diker, 1990) and human gastroenteritis (Gorkiewicz, 2002). Campylobacterjejuni, and C. coli can be found in the rumens and small intestines of normal calves and adult cattle (Stanley, 1998), so that the bacteria are considered commensal in cattle. Campylobacter was not recognized as a cause of human enteritis until the mid- 19705 when selective isolation media were developed for human stool culture. At present, campylobacteriosis is the most commonly reported human bacterial gastroenteritis in the United States, and the majority of infections are with C. jejuni (Nachmkin, 1999). The incidence of laboratory-diagnosed campylobacteriosis was 15.7 per 100,000 person-years in FoodNet surveillance sites (CDC, 2001; Friedman, et. al, 2000) and an estimated 2 to 2.4 million infections occur in the United States each year (Friedman, 2000). Though antimicrobials are not essential for the treatment of most routine human cases of 96 campylobacteriosis, severe or prolonged cases are usually treated with fluoroquinolone or erythromycin. Resistance to ciprofloxacin in human isolates of Campylobacterjejuni is reportedly increasing (Allos, 2001; Gaudreau, 1998; Smith, 1999). The majority of sporadic cases of Campylobacter infections are foodbome and undercooked poultry is the most likely source of infections (Friedman, 2000; Pearson, 2000). Contaminated water and unpasteurized milk are common sources of outbreaks; nine percent of bulk tank milk was found culture positive for Campylobacterjejuni in a study of 131 dairy herds in South Dakota and Minnesota (Jayarao, 2001). Critical control points are largely unknown for reducing pre—harvest Campylobacter prevalence. Most animal-specific factors (age, gender, breed, etc.) are not amenable to intervention. Herd-level management factors (bedding, sanitation, feeding, stocking rate, etc.) can often be changed, albeit sometimes only with considerable investment in labor and physical facilities. The influence on Campylobacter prevalence of the management factors that constitute “organic dairy production” has not heretofore been investigated. Organic dairy milk production has been previously described (Sato, 2002). Organic farms in Wisconsin usually graze their cattle during the warm season, do not use hormones, herbicides, insecticides, or anthelmintics, and no antibiotics are permitted for one year before milk is marketed. This antibiotic restriction means that dairy calves may receive antibiotics, but antibiotic usage for calves is reportedly very low due to the overall management philosophy of these farmers. It is not known to what extent the management practices embodied in the organic approach may lead to a lower rate of antimicrobial resistance among Campylobacter isolates from cattle on these farms. 97 The objective of the study was to describe the prevalence and antimicrobial resistance patterns of Campylobacter spp. in healthy calves and cows in organic and conventional dairy farms in Wisconsin. MATERIALS AND METHODS Datflnd fecaliample collection Cattle fecal samples and management & production data were collected fi'om 30 organic dairy farms and 30 conventional farms in southwestern Wisconsin. The organic farms were from an association of about 325 organic dairy farms. All organic farms were certified by an approved certification agency as not using antimicrobials for cows for at least 3 years (mean = 8.0 years) before the start of our study. For each organic farm selected, the nearest "conventional" dairy farmer (in sequence of geographical proximity) was asked to serve as a control farm. All herds were visited twice; once in March and once in September. Management and production information was collected at the first visit using an orally administered questionnaire. Questions and investigator observations regarded milk production, milking practices, housing, grazing, incidence of the major diseases, medical treatments and other management factors. Also at each visit, environmental and animal sanitation was assessed with a subjective score of cow cleanliness and the amount of moisture and manure in the bedding and exercise areas, as previously described (4). At each of the two visits, fecal specimens were collected from five lactating cows and five calves (under approximately 6 months of age). Animals were excluded if they had obvious diarrhea or were under treatment for another illness. Adult cows were 98 sampled by walking among the cows and waiting for one to defecate. The fresh fecal sample was taken from the freshly voided fecal pile, taking care to not contact the ground beneath. Fecal samples were obtained from calves when they defecated following anal stimulation. A sterile latex glove was used for each specimen to avoid cross- contamination. Approximately five grams of fecal sample were collected into a Cary- Blair Transport Media tube (Medical Chemical Corp, Torrance, CA). The specimens were kept on ice and mailed. to the Michigan Department of Community Health (MDCH), by overnight courier service for processing within 32 hours from the time of sampling. mm The fecal samples from the Cary-Blair tube were streaked directly on Campy Blood Agar (REMEL, Lenexa, KS). The inoculated plates were incubated under microaerophilic atmosphere (Campy-Pak, BBL Microbiology Systems, Cockeysville, MD) at 37 °C for 48 hours. One typical colony was selected and identified to genus level by testing by Gram stain, microscopic cell morphology, catalase production, oxidase production, and hippurate hydrolysis in accordance with the standard methods at MDCH (24). Antimicrobial susceptibility testing Bacterial isolates were tested for resistance using gradient disc diffusion minimal inhibitory concentration to ciprofloxacin (CI: 0.002-32.0 ,ug ’ml), erythromycin (EM: 0.016-256 pgmrl), gentamicin (GM: 0.016-256 ,ug’ml) and tetracycline (TC: 0.016-256 fig/in!) by Etest (AB Biodisk, Piscataway, NJ). Sample bacteria were streaked from the frozen stock onto SBA (SBA) plates (REMEL, Lenexa, KS) and incubated for 48 hours at 37 °C under microaerophilic atmosphere. The colonies were restreaked to a new SBA 99 and incubated for another 24 hours to allow recovery after being frozen. The sub- cultured colonies were examined for purity and emulsified in 4 ml Mueller-Hinton broth, adjusting the turbidity to that of a 1.0 McFarland standard. The suspension was then inoculated evenly on 150 mm Mueller-Hinton agar plates supplemented with 5% defibrinated sheep blood (REMEL, Lenexa, KS) by swabbing evenly in accordance with the Etest manufacture’s instructions. Etest strips containing CI, GM, EM and TC were placed on the surface of agar plate in a radial pattern with the lowest concentration toward the center. The plates were incubated for 72 hours at 37 °C under the microaerophilic conditions and the MICs were read directly from the test strip point where the growth inhibition zone intersected with the test strip, in accordance with manufacture’s instruction. Quality control was performed daily using Campylobacter coli of American Type Culture Collection (ATCC) 33559. Since no break points for Campylobacter MIC were defined by National Committee for Clinical Laboratory Standards (NCCLS), our test results were dichotomized based on the breakpoints used by the National Antimicrobial Resistance Monitoring System (N ARMS): CI 24 pg/ml, GM 216pg/ml, EM 28pg/ml, and TC 216pg/ml. Sjgtisticgl Agglysis The prevalence of Campylobacter spp. in herds was analyzed using a generalized linear model with logit link function, based on the binomial distribution. The outcome variable was Campylobacter negative (0) or positive (1). Explanatory (independent) variables were farm type (organic/conventional), cow or calf, season, herd size (number of milking cows), purchase of animals during the past year (yes/no), grazing intensity 100 during summer (no grazing/ little grazing / intensive grazing), abortion rate (per 100- cows/year), metritis rate, retained placenta rate (retained over 12 hours after calving), calf population, calf mortality rate, and calf diarrhea rate. “Farm” was included as a random effect variable with an independent correlation matrix. A regression model (generalized linear model with logit link fimction) was used to estimate the effect of farm type, animal age and season on the prevalence of antimicrobial resistant bacteria. The data were also analyzed using a proportional odds model with a generalized estimating equation (GEE). The proportional odds model with GEE provides a method to analyze an ordinal-level repeated dependent variable and several categorical and continuous-level explanatory variables with fixed or random effects (Stokes, 2000). Farm type, season and animal age were included as fixed effects and the farm was included as a random effect. All statistical analysis was performed using SAS statistical software (version 8.02. SAS Institute, Cary, NC). RESULTS The organic dairies had converted to organic farming methods at least 3 years before the initiation of our study (mean=8.0 years). Organic farmers indicated that no antimicrobials were used for cows on their dairy farms, but four organic farmers reported using antimicrobials for calves if they had serious diarrhea or pneumonia. In 26 of the 30 conventional dairy herds, cows routinely received antimicrobial infusions into the udder at the cessation of each lactation cycle (“dry-cow treatment”). Cephapirin or penicillin was used most for this purpose. Eighteen conventional dairy producers reported using 101 udder infusion of antimicrobials for the treatment of clinical mastitis. For the severe clinical mastitis cases, eight conventional dairy producers used systemic antimicrobials. A total of 332 Campylobacter spp. isolates were obtained from 1,191 fecal specimens (27.9%). A total of 234 (70.5%) was identified as Campylobacterjejuni, however the remainder of isolates, which were hippurate-negative, were not identified to species level. C. jejuni and others distributed evenly in both organic and conventional dairy herd (Mantel-Haenszel p=0.25). No Campylobacter isolates were obtained from one conventional farm or from three organic farms, thus 6.7% of farms were culture negative. On the 56 Campylobacter-positive farms, 5% to 70% of the collected specimens were culture positive. The prevalence was significantly higher in calves (32.7%) than in cows (23.2%), and significantly higher in March (36.8%) than in September (18.9%; Table 6-1). There was no significant difference in Campylobacter prevalence between organic and conventional farms in the multivariate analysis (p=0.5253; Table 6-3). Rates of retained placenta, pneumonia incidence rate, and abortion were positively associated with Campylobacter prevalence, whereas herd size (number of lactating cows and dry cows) and mastitis rate were negatively associated with Campylobacter prevalence (p < 0.05). The calf mortality was nearly significantly associated with the prevalence (p=0.0511). Only two isolates of Campylobacter spp. from geographically distant conventional dairy herds were resistant to ciprofloxacin (>32 and 24 fig/ml). The other 330 isolates had MIC values between 0.012-0.25 ,ug’ml. None of the 332 isolates was resistant to gentamicin or erythromycin. The ranges of MIC were 0.047 - 2 pg/ml for 102 gentamicin and 0.047 - 4 pg/ml for erythromycin (Table 6-4). A total of 148 resistant isolates (44.6%) to tetracycline were obtained (Table 6-4). The analysis of the dichotomized tetracycline resistant data indicated a higher prevalence of resistant Campylobacter spp. in calf isolates as compared with cow isolates (p=0.0419), with the estimated odds ratio of 1.81 (1.0221 < OR < 3.2059). Farm type (organic or conventional) and season of specimen collection were not significant predictors of tetracycline resistance (p=0.4971 and 0.1729, respectively). The proportional odds model analysis using all antimicrobial dilution levels did not find significant difference of MIC distributions to tetracycline between two types of farm (Fig 6-1). For ciprofloxacin, gentamicin and erythromycin resistance, the proportional odds model found no significant effect on MIC distribution by farm type (organic or conventional), animal age or season of specimen collection. DISCUSSION The estimation of Campylobacter spp. prevalence may be affected by factors, such as location, season, use of transport medium, time before processing, use of enrichment media, and the use of various isolation methods (media, temperature, atmosphere, and time). The selection of farms in the current study was not random, but rather constituted a cluster of organic herds and neighboring conventional herds in a particular region of Wisconsin. The fecal samples were collected from presumably healthy cows and calves, after having excluded animals with obvious diarrhea or that were under treatment for some other disease. This selection strategy may have resulted in lower measures of Campylobacter spp. prevalence in our study as compared with other 103 studies, if cows with diarrhea are more likely to have been infected with Campylobacter spp. It has been reported that Campylobacter spp. isolation rate was decreased approximately 16% by storing feces at 4°C for 24 hours (Ladron de Guevara, 1989) and our samples took 24 to 36 hours to be transported to the laboratory. However, the Cary- Blair transport medium with icepacks should have enabled Campylobacter spp. to maintain sufficient viability (Luechtefeld, 1981; Wang, 1983; Wasfy, 1995). The enrichment techniques are beneficial for the detection of Campylobacter spp. when present in low concentration. Perhaps our measured prevalence estimate would have been higher had we used enrichment technique (Bolton, 1982; Martin, 1983). Nielsen (2002) found 9 out of 77 positive samples were only positive after growth in enrichment broth. We used Campy Blood Agar plate, which contains cephalothin, polymyxin B, vancomycin, trimethoprim and amphotericin B. The culture media is optimized for C. jejuni and C. coli, but not for other Campylobacter spp. in cattle. Campylobacterjejuni subsp. doylei, C. fetus subsp. fetus, C. upsaliensis, and C. hyointestinalis are known to be inhibited by cephalothin (Nachamkin, 1999). Though we used an incubation temperature of 37 °C, other studies of Campylobacter spp. used an incubation temperature of 42°C to optimize the growth of thermophilic Campylobacter species, such as C. jejuni, C. coli or C. lari, with the decreased ability to isolate non- thermophilic species (C. fetus, and C. jejuni subsp. doylei). The incubation temperature of 37 °C may have resulted in lower prevalence of Campylobacter in our study. Atabay et al. (1998) used three kinds of media, enrichment technique, membrane filtration technique, and three different incubation temperatures. They found 62% overall prevalence in 136 cattle in three farms in the U. K. The major species in their study were 104 C. hyointestinalis (32%), C. sputorum biovar paraureolyticus (21%), C. fetus subsp. fetus (11%) and C. jejuni subsp. jejuni (7%). Giacoboni et al. (1993) also found C. fetus subsp. fetus in 17% of cattle and C. hyointestinalis in 19% of cattle, whereas dominant species was C. jejuni found in 29% of 94 cattle in Japan. Our study design emphasized the isolation of C. jejuni and C. coli which are species of public health importance. Higher Campylobacter spp. prevalence was found on dairy farms in March compared to September, and higher in calves compared to cows. These observations generally agreed with previous population-based studies (Nielsen, 2002; Wesley, 2000). The housing and grazing styles in our study were very different between organic and conventional dairy herds. Free stalls were used on 9 conventional herds, in contrast to being used on only four organic herds. Half of the organic herds (15 farms) applied intensive grazing during summer, whereas only two conventional herds used intensive grazing. We saw no evidence that antimicrobial use on dairy farms had any effect on Campylobacter spp. prevalence, since farm type was not significantly associated with prevalence after controlling for housing and grazing in the regression analysis. It may be reasonable to find retained placenta rate and abortion rate were positively associated with Campylobacter spp. prevalence, since Campylobacter spp. was originally found as a cause of abortion, and even C. jejuni and C. coli were recognized as causal agents of abortions (Larson, 1992; Wesley, 2000). Positive association between pneumonia and Campylobacter spp. prevalence may be explained by a shared common cause such as poor environmental sanitation. Some Campylobacter species, such as C. fetus, were known cause of mastitis (Akhtar, 1993; Logan, 1982). However, the negative 105 association between mastitis rates and Campylobacter prevalence was observed with borderline significance (p=0.0486) in our study. Antimicrobigl Resistance in Camlobacter spp, Agar disk diffusion, broth dilution, agar dilution and the gradient disk diffusion (Etest) have commonly been used to determine Campylobacter susceptibilities in vitro. The agar dilution test was recently set by NCCLS as a reference standard susceptibility testing method for veterinary isolates of Campylobacter spp. (Nccls, 2002), however the agar dilution test requires more materials and considerable labor, and therefore is not ideal for most surveillance purposes. Ge, et al. (2002) recently reported that MIC values measured with the Etest were generally lower than the results obtained with the agar dilution method. The agreement (i1 dilution range) between MICs between two test methods depended on antimicrobials used: Ciprofloxacin (85%), Gentamicin (92.6%), Erythromycin (65.6%), and tetracycline (57.7%). The Etest MIC results of the quality control strain (C. jejuni ATCC33560) were consistently one to several dilutions lower than the corresponding agar dilution results. Huang, et al. (1992) also compared the Etest method with the agar dilution method and reported slightly lower MIC values with the Etest than with the agar dilution. The percent agreement (:51 dilution range) were 90.4% for ciprofloxacin, 83.0% for gentamicin, 94.1% for erythromycin, and 77.5% for tetracycline. In our study, any bias due to the testing procedure should not have affected our comparison between organic and conventional farms. Any such systematic error or bias would have been a non-differential misclassification bias that would have equally affected the organic and conventional farms (Rothman, 1998). However, direct 106 comparisons of MIC values obtained from different methods should be interpreted with caution. We isolated two ciprofloxacin resistant Campylobacter spp. fiom conventional dairy farms. Fluoroquinolone is not commonly used in dairy industry. Sarafloxacin was approved in the United States for the poultry in 1995, but the approval was withdrawn in 2001 (Federal Register, 2001). The Center for Veterinary Medicine of FDA proposed to withdraw approval of enrofloxacin for poultry use because of the possible transfer of fluoroquinolone-resistant Campylobacter spp. from poultry to human (Federal Register, 2000). Though the use of enrofloxacin in beef cattle is approved for treatment of bovine respiratory disease, the extralabel use of any fluoroquinolones in dairy cattle has been clearly prohibited by the FDA. The resistance to fluoroquinolone is rendered by (a) decreased permeability of bacterial cell wall; (b) increased efflux pump activity; and (c) mutation of the DNA gyrase. Thus, the decreased permeability and/or the increased efflux pump can also confer resistance to other antimicrobial agents, such as tetracycline (Walker, 2000). Since our ciprofloxacin resistant Campylobacter spp. were not resistant to other three antimicrobials, it is speculated that the resistant isolates were arose by point mutation. CONCLUSION The prevalence of Campylobacter spp. was not significantly different between organic and conventional dairy farms in Wisconsin. Campylobacter prevalence was significantly higher in March than in September, higher in calves than in cows, and higher on smaller farms than on larger farms. Rates of retained placenta, pneumonia, and 107 abortion were positively associated with the Campylobacter spp. prevalence. 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Osterholm, and the Investigation team. 1999. Quinolone-resistant Campylobacterjejuni infections in Minnesota, 1992-1998. New Engl. J. Med. 340:1525-1532. Walker, R. D. 2000. Fluoroquinolones, p. 315-338. In J. F. Prescott, J. D. Baggot, and R. D. Walker (ed.), Antimicrobial therapy in veterinary medicine, third edition, Iowa State University Press, Ames, IA. Wang, W.-L. L., L. B. Reller, B. Smallwood, N. W. Luechtefeld, and M. J. Blaser. 1983. Evaluation of transport media for Campylobacterjejuni in human fecal specimens. J. Clin. Microbiol. 18:803-807. 11] Wasfy, M., B. Oyofo, A. Elgindy, and A. Churilla. 1995. Comparison of preservation media for storage of stool samples. J Clin Microbiol, 33:2176-2178. Welsh, R. D. 1984. Campylobacterjejuni abortion in a heifer. JAVMA. 185:549-551. Wesley, I. V., S. J. Wells, K. M. Harmon, A. Green, L. Schroeder-Tucker, M. Glover, and I. Siddique. 2000. Fecal shedding of Campylobacter and Arcobacter spp. in dairy cattle. Appl. Environ. Microbiol. 66:1994-2000. 112 Table 6-1. Number of Campylobacter isolates (percent of culture positive samples) in each group of samples. Conventional dairy farms (n=30) Organic dairy farms (n=30) Calf Cow Calf Cow March 56 (37.8%) 54 (36.0%) 65 (43.3%) 45 (30.0%) September 42 (28.6%) 21 (14.0%) 30 (20.5%) 19 (12.7%) 113 Table 6-2. Odds ratios for Campylobacter sp. isolation (Generalized Linear Model analysis) Risk factors Campylobacter Odds Odds 95% confidence Type 3 positive / negative ratio interval GEE Chi- Sq (p-value) Animal age Calf 193/ 398 (48.5%) 0.485 1.635 1.180 > OR > 2.656 0.0031 Cow 139 / 461 (30.2%) 0.302 1 Season March 220/ 378 (58.2%) 0.582 2.524 1.748 > OR > 3.646 <0.0001 September 112/ 481 (23.3%) 0.233 1 Farm type Conventional 173 / 422 (41.0%) 0.410 1.138 0.742 > OR > 1.743 0.5541 Organic 159 / 437 (36.4%) 0.368 1 114 Table 6-3. Generalized Linear Model analysis of management factors on Campylobacter sp. Prevalence. Parameters Estimates p-values Season (March/September) 1.0225 <0.0001 Retained placenta incidence rate 0.0460 <0.0001 Herd size -0.0234 0.0031 Cow or calf (Calf/Cow) 0.5304 0.0032 Pneumonia incidence rate 0.0266 0.0187 Mastitis rate -0.0131 0.0486 Abortion rate 0.0531 0.0437 Calf mortality 0.5909 0.0511 Metritis rate 0.0133 0.1532 Open herd (cow purchased) 0.1691 0.4359 Milk production per cow 0.0001 0.5165 Organic or conventional 0.1166 0.5253 Grazing with housing -0.1697 0.5204 No grazing (tie stall, free stall) 0.1178 0.7051 SCC 0.0003 0.7693 Cow mortality -0.0062 0.8391 115 Table 6-4. Proportion (%) of isolates which were inhibited by antimicrobials at each concentration. The dotted lines indicate the NARMS breakpoints (CI 24 ,ug/ml, GM .216,ug/ml, EM 28pg/ml, and TC 216pg/ml). Antimicrobial concentration Ciprofloxacin Gentamicin Erythromycin Tetracycline (lg/ml) (n=332) (n=332) (n=332) (n=332) 0.012 0.3 3.6 0.016 1.8 2.7 0.023 6.9 7.8 0.032 28.6 19.6 0.047 29.2 0.6 0.3 11.4 0.064 22.9 0.9 0.9 4.5 0.094 6.0 7.5 1.8 1.2 0.125 2.4 17.2 7.2 1.5 0.19 0.9 18.1 24.7 0.6 0.25 0.3 28.0 23.2 0.6 0.38 13.3 14.5 0.9 0.5 6.9 12.7 0.3 0.75 3.9 6.6 1 0.9 3.3 1.5 1.5 1.8 2 1.2 1.8 3 _____________ 0.9 4 0.3 6 _____________ 0.3 8 12 _ _ _ _ 0.3 16 1.8 24 0.3 1.5 32 3.9 48 2.1 64 0.3 3.3 96 3.3 128 2.4 192 0.6 256 0.6 >256 25.0 100% 100% 100% 100% 116 Figure 6-1. Distribution of MICs to tetracycline of Campylobacter spp. from conventional and organic dairy farms. The proportional odds model analysis using all antimicrobial dilution levels did not find significant difference of MIC distributions to tetracycline between two types of farm. Comparison of tetracycline resistance 12 on Percent of Isolates (%) O) 2 ll. 0 I 0.01 0.02 0.02 0.03 0.05 0.06 0.09 0.13 0.19 0.25 0.38 0.5 6 12 16 24 Minimun Inhibitory Concentration (pg/ml) Ll Conventional 0 Organic I 117 CHAPTER SEVEN COMPARISON OF ANTIMICROBIAL SUSCEPTIBILITY OF Staphylococcus aureus ISOLATED FROM BULK TANK MILK IN ORGANIC AND CONVENTIONAL DAIRY HERDS IN MIDWESTERN USA AND DENMARK ABSTRACT An observational study was conducted to compare the antimicrobial susceptibility patterns of Staphylococcus aureus isolated from bulk tank milk in organic and conventional dairy farms in Wisconsin, U. S. A. and in Southern Jutland, Denmark. Bulk tank milk samples and data regarding management and production were collected from 30 organic dairy farms and 30 conventional farms in Wisconsin and 20 organic and 20 conventional dairy herd in Denmark. S. aureus isolates were tested for resistance against 15 antimicrobial agents by semi-automatic micro broth dilution methods in each country. Of the 118 bulk tank milk samples in Wisconsin, 71 samples (60%) yielded at least one S. aureus isolate, and a total of 331 isolates were collected. Of the 40 bulk tank milk samples from Denmark, 27 samples (55%) yielded at least one S. aureus isolate and a total of 152 isolates were collected. Significant difference between organic and conventional dairy was detected to only Ciprofloxacin in Wisconsin and only avilamycin in Denmark. Significant difference between two countries was detected to nine antimicrobials. Denmark had higher probability of having reduced susceptibility to ciprofloxacin and streptomycin (p=0.015 and 0.003). Wisconsin isolates had higher probability of having reduced susceptibility to other seven antimicrobial agents 118 (bacitracin, gentamicin, kanamycin, penicillin, sulphamethoxazole, tetracycline, and trimethoprim). We found small differences between organic and conventional farms types in each country and larger differences between the two national agricultural systems. INTRODUCTION Staphylococcus aureus is a common cause of contagious mastitis in dairy cattle. Such infections often persist for weeks, months or years, with infected animals becoming the main source and reservoir of infection for herd mates. Antibiotics are commonly used to control S. aureus infection, by treating cases of clinical mastitis and by udder infusion at the cessation of each lactation cycle (dry-cow treatment). This type of mastitis treatment and prevention is the predominant reason for antibiotic use in dairy industry (Kaneene, 1992). Food animal agriculture has been suspected of fostering antimicrobial resistant (AR) bacteria, which can then be transmitted to people through direct contact, food of animal origin, and environment contamination (McEwen, 2002). Generally, farms with higher antimicrobial usage have been found to have a higher proportion of resistant bacteria, and farm-level studies have usually shown an association between antimicrobial usage in food animals and rates of antimicrobial resistance in several bacterial species (Mathew, 2001; Van Den Bogaard, 2001). The use of antibiotics has been prohibited for organic dairy cows, and now this prohibition is specified by the National Organic Standard (USDA, 2001). Though sick cows have to be treated with all necessary medications including antimicrobials, these 119 treated cows have to be excluded fiom organic herds in the United States. In Denmark, national certification of organic “ekologisk” farms began in 1988 and organic farms now comprise 8.4% of all Danish dairy farms and produced about 22% of the fluid (drinking) milk in 2000 (Bennedsgaard, 2003). In contrast to the organic farms in the USA, antimicrobial drugs can be used for Danish okologisk cows, although the ekologisk standards require a three fold longer milk withdrawal time than cows on conventional farms. In both Denmark and the US, it is not known to what extent the restriction or ban on antimicrobial use in the organic or ekologisk approach may lead to a lower rate of antimicrobial resistance among bulk tank Staphylococcus isolates from these farms in two very different agricultural systems. Antimicrobial resistance in S. aureus has primarily been studied on isolates fi'om clinical mastitis that had been submitted to various veterinary diagnostic laboratories (Bager, 2002; De Oliveira, 2000; Erskine, 2002; Makovec, 2003). Such studies have mainly attributed AR to antibiotics commonly used for mastitis therapy (Rossitto, 2001; Watts, 1995). In most studies, antimicrobial susceptibility was determined by agar disk diffusion method. Results have usually been reported as being either positive or negative, so susceptibility changes below the clinical breakpoints have usually gone unreported. The objective of the current study was to determine and compare the prevalence and antimicrobial susceptibility patterns of Staphylococcus aureus isolated from bulk tank milk in organic and conventional dairy farms in Wisconsin and Denmark. 120 MATERIALS AND METHODS Bulk tank milk samples and data regarding management and production were collected from 30 organic dairy farms and 30 conventional farms in Wisconsin, USA. The organic farms were from an organic association with about 120 member dairy farms at the start of the study. All 30 organic farms had been organic for at least 3 years before the start of our study. For each organic farmer selected, a neighborhood "conventional" dairy farmer was asked to allow their farm to serve as a control. All herds were visited twice; once in March and once in September. Management and production data were collected at the first visit using in-person administered questionnaires. Questions and investigator observations included milk production, milking practices, housing, grazing, incidence of the major diseases, medical treatments, and other management factors (Sato, Bartlett, Erskine, and Kaneene, submitted for publication). Also at each visit, environmental and animal sanitation was assessed with a subjective score of cow cleanliness and the amount of moisture and manure in the bedding and exercise areas, as previously described (Bartlett, 1992). In Southern Jutland, Denmark, 20 organic herds which had converted to organic dairy management before 1995 were selected for the study. Twenty conventional dairy herds were randomly selected from a group of 120 eligible herds which had participated in a previous research project. Information on parturition, culling, veterinary diagnostics, and disease treatments were readily available from the Danish Cattle Database (Bartlett, 2001). These dairy herds were visited by investigators from April to May 2000 and bulk tank milk samples were collected and processed (Danish Veterinary Institute) in accordance with the same procedure used in the USA. 121 The bulk tank milk was agitated for about five minutes before collecting samples. At each farm visits, approximately 10 ml of bulk tank milk (BTM) were collected from the top of the bulk tank with a stainless steel dipper into a sterile tube. The sample tubes were kept in ice and sent to a laboratory by overnight courier service. The BTM samples were frozen within 24 hours and stored for variable time at —72 °C until processing. Sample tubes were brought to room temperature, placed into water bath at 35-37 °C for 10 minutes, and mixed thoroughly with a mechanical platform shaker. One milliliter of milk was diluted to ten (10") and hundred (102) dilutions with physiological saline. An inoculum of 0.3 ml from each dilution was spread with a bent-glass streaking rod on the surface of 140 mm Baird-Parker Agar plate (Bacto/Difco, Detroit, Michigan, USA) supplemented with egg yolk tellurite (Ollis, 1995). The plates were incubated at 32 °C for 48 hours. Typical black, shiny, convex colonies with white edge surrounded by a clear zone were counted as presumptive staphylococci and the number of colonies on each dilution plate were recorded. Presumptive colonies were collected, starting from the more diluted plates (102) and moving to the undiluted plate, until up to ten isolates had been obtained. Then each colony was transferred to trypticase soy agar (TSA) with 5% sheep blood (REMEL, Lenexa, KS) and incubated at 32 °C for 24 hours. The colonies were examined for purity and identified as Staphylococcus aureus by Gram stain, catalase production, and tube coagulase test (coagulase rabbit plasma with EDTA, Bacto/Difco, Detroit, Michigan, USA) in accordance with the standard methods (Flowers, 1993). The material and microbiological procedures were standardized among American and Danish portions of the study as much as possible by Danish investigators visiting the project site in the USA at the initiation of the study. 122 Staphylococcus aureus isolates were tested for AR against 15 antimicrobial agents by semi-automatic micro broth dilution methods (Sensititre; Trek diagnostic Systems Inc., Cleveland, OH). The minimum inhibitory concentrations (MIC) of S. aureus were tested for the same antimicrobials and the same range of concentrations in both countries in accordance with the manufacture’s instruction, except that avilamycin was only tested in Denmark and cephapirin was only tested in the U. S. In summary, S. aureus were inoculated on TSA with 5% sheep blood agar and incubated at 35 °C for 24 hours. The sub-cultured colonies were examined for purity and emulsified in 4 ml de-mineralized distilled water, adjusting the turbidity to that of a 0.5 McFarland standard. A 10 micro- liter suspension was transferred into a Mueller Hinton broth tube (11 ml) and 50 pl of the broth suspension was transferred to Sensititre panels, which were incubated at 35 °C for 18 hours prior to determining the MIC. Quality control was performed by testing Staphylococcus aureus (ATCC29213) and Pasteurella aeruginosa (ATCC27853). A initial descriptive statistical analysis ignored the clustering of clonal isolates within the same farm and summarized the MIC results by country and type of farm. The interpretive standards set by National Committee for Clinical Laboratory Standards (NCCLS, 2002) and the breakpoints used by DANMAP 2001 (Bager, 2002) were utilized. Because the majority of isolates had MIC values far below threshold values for clinical resistance, the minimum inhibitory concentration for 90% of all isolates tested (MIC90) to each antibiotic was used as a break point upon which to dichotomize the susceptibility for purposes of comparison between the conventional and organic/ekologisk farms. Isolates with MIC values lower than the NIle value were categorized as a “high susceptibility”, otherwise categorized as a “reduced susceptibility” (Table 7-4 and 7-5). Logistic 123 regression analysis was used to estimate the effect of farm type (organic/okologisk or conventional) on rates of reduced S. aureus susceptibility to the different antimicrobials. The data were considered unbalanced with repeated values for each farm. All statistical analysis was performed using SAS statistical software (version 8e. SAS Institute, Cary, NC). RESULTS Of the 118 bulk tank milk samples in Wisconsin, 71 samples (60%) yielded at least one S. aureus isolate, and a total of 331 S. aureus were collected. Twenty six organic farms (87%) yielded at least one isolate in comparison to 22 conventional farms (73%) which yielded at least one isolate. Of the 40 bulk tank milk samples from Denmark, 27 samples (55%) yielded at least one S. aureus isolate: 10 (50%) for okologisk farms and 17 (85%) for conventional farms, and a total of 152 S. aureus were collected (Table 7-1). The MIC data for the S. aureus isolates is shown in Table 7-2. Resistant isolates were only found to bacitracin, penicillin, streptomycin, sulphamethoxazole, and trimethoprim based on NCCLS or DANMAP interpretive standards in our samples, and resistance to streptomycin and trimethoprim were only found in Wisconsin samples. A notable finding was that a very large proportion of isolates (162/331, 49%) exhibited high resistance to sulphamethoxazole (512 pg/ml) in Wisconsin, whereas only one Danish isolates (0.7%) had the same resistance level. Only one significant difference between organic and conventional/ekologisk farms in each country was found by a logistic regression with the correlated data model. 124 Conventional dairy herds in Wisconsin had 3.3 times higher probability (95% confidence interval [1.21 — 9.14]) of having S. aureus isolates with reduced susceptibility to ciprofloxiacin compared with organic dairy herds in Wisconsin (Table 7-4). In Denmark, rakologisk dairy farms had 6.78 time higher probability (95% confidence interval [1.30 — 35.3 1]) of having isolates with reduced susceptibility to avilamycin compared with Danish conventional dairy farms (Table 7-5). The logistic regression model with farm type, country and interaction of farm type and country was used for comparing the sub-therapeutic susceptibility differences between Wisconsin and Denmark. No difference was found among the two countries for chloramphenicol, erythromycin, oxacillin, quinupristin/dalfopristin and vancomycin. Denmark had higher probability of having reduced susceptibility to Ciprofloxacin and streptomycin (p=0.015 and 0.003). Wisconsin isolates had higher probability of having reduced susceptibility to other seven antimicrobial agents (bacitracin, gentamicin, kanamycin, penicillin, sulphamethoxazole, tetracycline, and trimethoprim at p<0.05; Table 7-6). DISCUSSION Organic dairy farmers in Wisconsin were chosen by geographical clusters and were asked to participate. Eight of 38 organic farmers who were contacted refused to participate in the study. The conventional dairy farms were chosen from neighboring farms, with only five cases of farmer declining to participate. Antimicrobial usage on conventional farms was difficult to quantify. Thus, the dose, route of administration, and frequency of dosing was unknown. Although somewhat larger than the organic farms, the conventional farms were small by US. standards and their philosophy regarding 125 antibiotic usage may have been influenced by the many organic dairy farms in the region. The farms in Denmark were selected randomly from their prescribed location, and are therefore presumably representative of Danish ekologisk and conventional farms. Bulk tank cultures for S. aureus, and somatic cell count (SCC) are widely used as inexpensive and convenient monitoring methods for udder health (Jayarao, 2003). Isolates of S. aureus from bulk tank milk are generally presumed to have come from the cow’s teats or udders. However, it is also possible that they may have originated from contamination or even from the humans working in the milking parlor, although given the sanitation of the milking equipment after each milking, it is probable that the overwhelming number of S. aureus isolates originated in cows’ teats or udders. Numerous reports have been published on antimicrobial susceptibility in Staphylococcus from dairy cattle, which were usually collected from individual quarter milk samples from cows with clinical mastitis (De Oliveira, 2000; Devriese, 1980). These studies focused on antimicrobial susceptibility of S. aureus as mastitis pathogens and the focus of the studies has been on the therapeutic implications for mastitis therapy. Study outcomes have usually been reported as a percentage of resistant isolates. Most surveys did not account for multiple observations from the same cow or herd, so clonal isolates of resistant bacteria from the same cow or herd might have greatly influenced the results. To avoid these complicated issues, De Oliveira et al. (2000) used only one isolates from a single herd. Our study included up to 10 isolates from a bulk tank on each of two separate sampling, separated by approximately a half year. Given the mixing of milk in the bulk tank, some of the isolates for one particular herd may have come from the same cow. Our logistic regression analysis for correlated data (repeated data) should have 126 helped to control for the possible effect of clonal isolates having an undue effect on the results . The proportion of resistant S. aureus in our study were generally much lower than what was found in recent studies of isolates fiom clinical submission from Wisconsin Makovec, 2003) and Michigan (Erskine, 2002). Samples submitted for diagnosis of clinical mastitis cases tend to exhibit more AR, probably because cases that are refractory to treatment are more likely to be cultured in an attempt to identify the causative agent and its AR traits (Aarestrup, 1998). Multiple samples from a single farm may be common, and clonal isolates might have been included in the data. Organic/ekologisk farms and conventional farms differed statistically with respect to: (1) reduced susceptibility to Ciprofloxacin in conventional farms in Wisconsin and (2) reduced susceptibility to avilamycin in akologisk farms in Denmark. The reduced susceptibility to Ciprofloxacin in Wisconsin could be related to the use of enrofloxacin in beef cattle. Enrofloxacin belongs to the fluoroquinolones group of antimicrobials and is approved for treatment of bovine respiratory disease since 1998 in the United States (FDA, 2003a). The extralabel use of any fluoroquinolones in dairy cattle has been prohibited by the FDA in the United States (FDA, 2003b). However, extralabel drug use is practiced if the product being used is approved in that species but for a different disease, or if the product is used at a different dose or with an altered withdrawal time (Bateman, 2000). The reduced susceptibility to Ciprofloxacin on conventional dairies relative to organic dairies could be explained by the use of enrofloxacin on conventional farms for dairy or beef cattle, by exposure to purchased feeds on conventional farms, or exposure to other sources of resistant bacteria. 127 Even though all isolates were susceptible to avilamycin with the interpretive standard of DANMAP, the difference of MIC distributions between organic and conventional farms in Denmark is hardly explained. Avilamycin is a mixture of oligosaccharides and had been used for growth promoter primarily of broilers and some for pigs, but not for dairy cattle in Denmark (Aarestrup, 1998; Aarestrup, 2001). Avilamycin use as growth promoter for broilers and pigs was voluntarily stopped by the end of 1999 in Denmark, to avoid the possible cross-resistance to eveminomycin, which was investigated for use in humans (Aarestrup, 2000; Butaye, 2003; EU, 1998). Organic dairy regulation in Denmark permits sick cows to be treated with antimicrobials. These cows are permitted to stay in their herds while they are receiving treatment, although the milk must be withheld from sale for three times longer than it is for conventional cows. Recent work showed that akologisk herds had less frequently asked veterinarians to treat their mastitis cows, however treatment fi'equency and antibiotic selection is not substantially different among two type of herds (Bennedsgaard, 2003). Avilamycin and its structurally related substances were generally not used on either okologisk or conventional dairy farms. Thus, the statistical significance we observed in avilamycin could have been by chance alone. Though bacitracin is not commonly used for dairy cattle in either country, we found that S. aureus from dairy herds in Wisconsin were more likely to have reduced susceptibility to bacitracin than were herds in Denmark (Figure 7-1). Bacitracin is a polypeptide product of Bacillus licheniformis and Bacillus subtilis, and is commonly used in the topical treatment of human and animal skin infection. It is also commonly used as growth promoter in poultry, swine, and feedlot beef production (Aarestrup, 1998; FDA, 128 2003c; Prescott, 2000). The European Union banned bacitracin as animal grth promoter in 1998 because analogs of bacitracin can sometimes be used for the treatment of vancomycin resistant enterococci (Chia, 1995; EU, 1998; O’Donovan, 1994). Relatively low levels of S. aureus resistance to bacitracin in cattle were reported in Europe before the ban (Aarestrup, 1998; Devriese, 1980), however, reduced susceptibility to bacitracin (83% with MIC232ug/ml by e-test) was also reported in S. aureus from human skin and soft-tissue infections in Norway (Afset, 2003). In the United States, five antibiotics (i.e., bacitracin methylene disalicylate, chlortetracycline, oxytetracycline, tylosin, and virginiamycin) are approved and commonly used for beef cattle or calves for prevention of liver abscesses in feedlot cattle (Nagaraja, 1998). It is speculated that, although bacitracin is not used for dairy animals in the US, dairy cattle may become colonized with resistant strains by environmental and foodbome exposure to their surrounding agricultural environment. Besides bacitracin, our study found significantly higher probability of having reduced susceptibility to other seven antimicrobials in Wisconsin, in contrast to Danish isolates which showed reduced susceptibility to only two antimicrobials. Differences in reduced susceptibility patterns between Wisconsin and Denmark may be due to a more restrictive policy on use of antimicrobial agents in Denmark. In general, we found small differences between organic/ekologisk and conventional farms types in each country and larger differences between the two national agricultural systems. 129 CONCLUSION The difference between Wisconsin and Denmark was large and isolates from Wisconsin had higher probability of reduced susceptibility to 7 out of 14 comparable antimicrobials, whereas Danish isolates had higher probability of reduced susceptibility to only two drugs. 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Available at: http://europa.eu.int/comm/food/fs/afs/authorisation/authoO8_en.pdf, Accessed on 5 June, 2003. Food and Drug Administration (FDA). 2003a. Code of Federal Regulations (CF R) Title 21, Volume 6, 522.812. Enrofloxacin solution. Food and Drug Administration (FDA). 2003b. Code of Federal Regulations (CFR) Title 21, Volume 6, 530.41. Drugs prohibited for extralabel use in animals. Food and Drug Administration (FDA). 2003c. Code of Federal Regulations (CFR) Title 21, Volume 6, 558.76. Bacitracin methylene disalicylate. Flowers, R. 8., W. Andrews, C. W. Donnelly, and E. Koenig. 1993. Pathogens in milk and milk products, p. 103-212. In: R. T. Marshall, (ed.), Standard methods for the microbiological examination of dairy products, 16th ed. American Public Health Association, Washington, DC. Jayarao, B. M., and D. R. Wolfgang 2003. Bulk-tank milk analysis: A useful tood for improving milk quality and herd udder health. Vet. Clin. North Am. Food Anim. Pract. 19:7 5-92. Kaneene, J. B., and R. Miller 1992. Description and evaluation of the influence of veterinary presence on the use of antibiotics and sulfonamides in dairy herds. J. Am. Vet. Med. Assoc. 201:68-76. 132 Makovec, J. A, andP. L. Ruegg 2003. Antimicrobial resistance of bacteria isolated from dairy cow inilk samples submitted for bacterial culture: 8, 905 samples (1994- 2001). J. Am. Vet. Med Assoc. 222: 1582- 1589. Mathew, A. G., M. A. Beckmann, and A. M. Saxton. 2001. A comparison of antibiotic resistance in bacteria isolated from swine herds in which antibiotics were used or excluded. J Swine Health Prod, 9: 125-129. McEwen, S. A, and P. Fedorka-Cray 2002. Antimicrobial use and resistance in animals. Clin. Infect. Dis. 342893-8106. Nagaraja, T. G., and M. M. Chengappa 1998. Liver Abscesses in Feedlot Cattle: A Review J. Anim. Sci. 76:287-298. National Committee for Clinical Laboratory Standards (NCCLS). 2002. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Test for Bacteria Isolated from Animals; Approved Standard-Second Edition, M31-A2 [ISBN 1- 5623 8-461-9]. NCCLS, 940 West Valley Road, Suite 1400, Wyne, Pensylvania 19087-1898 USA. O’Donovan, C. A, P. Fan-Havard, F. T. Tecson-Tumang, S. M. Smith, and R. H. Eng. 1994. Enteric eradication of vancomycin-resistant Enterococcus faecium with oral bacitracin. Diagn. Microbiol. Infect. Dis. 18:105-109. Ollis, G. W., S. A. Rawluk, M. Schoonderwoerd, and C. Schipper 1995. Detection of Staphylococcus aureus in bulk tank milk using modified Baird-Parker culture media. Can. Vet. J. 36:619-623. Prescott, J. F. 2000. Peptide Antibiotics: Polymyxins, Glycopeptides, Streptogramins, and Bacitracin, p. 177-190. In: J. F. Prescott, J. D. Barrot, and R. D. Walker (ed.), Antimicrobial Therapy in Veterinary Medicine. Third Edition. Iowa State University Press, Ames, IA. Rossitto, P. V., L. Ruiz, Y. Kikuchi, K. Glenn, K. Luiz, J. L. Watts, and J. S. Cullor. 2001. Antibiotic Susceptibility Patterns for Environmental Streptococci Isolated from Bovine Mastitis in Central California Dairies. J. Dairy Sci. 85:132-138. U. S. Department of Agriculture. 2001. National Organic Standard, Agriculture Marketing Service. Available at: http://www.ams.usda.gov/nop/NOP/standards/FullText.pdf, Accessed 5 June 2003. Van Den Bogaard, A. E., N. London, C. Driessen, and E. E. Stobberingh. 2001. Antibiotic resistance of faecal Escherichia coli in poultry, poultry farmers and poultry Slaughters. J. Antimicrob. Chemother. 47:763-771. 133 Watts, J. L., S. A. Salmon, J. R. J. Yancey, S. C. Nicerson, L. J. Weaver, C. Holmberg, J. W. Pankey, and L. K. Fox. 1995. Antimicrobial Susceptibility of Microorganisms Isolated from the Mammary Glands of Dairy Heifers. J. Dairy Sci. 78: 1637-1648. 134 Table 7-1. Number of Staphylococcus aureus isolates examined in this study. Wisconsin, USA March September Positive Positive Number S. aureus Number S. aureus milk milk of farms isolates of farms isolates samples samples Conventional 29 15 64 30 18 88 farms Organic farms 29 18 73 30 20 106 Denmark April-May Positive Number S. aureus milk of farms isolates samples Conventional 20 17 77 20 10 75 135 Table 7-2. Antimicroibal susceptibility to 15 drugs in Staphylococcus aureus which were isolated from bulk tank milk samples collected from Wisconsin organic and conventional dairy farms. Antimicrobial Farm Percent of isolates at each indicated MIC (pg/ml) agents type 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256 5 Bacitracin 0°" 8.4 21.7 23.8 39.9 6.3 Org A1721591197397 15.2 Ce ha irin 0°" / p p Org Chloram- Con 07 171 76.3 59 phenrcol Org , 26 8 70.4 2 8 Ciprofloxacin Con 83'615'1 1'3 Org 94.4 5.6 . Con 40.8 52.0 6.6 0.7 E hrom cm M y Org 35.2 60.3 4.5 I Gentamicin 0°" 93" 66 95.0 5.0 . 86213.8 Kanam crn y ///,87.7112%1 / Oxacillin 99.3 0.7 / /// , 100 / / Penidmn 67.1105 1.3 2.0 7.2 5.9 3.3 1.3 1.3 7 .1123 5.0 1.1 0.6 0.6 6.7 1.7 Streptomycin 52.0 44.1 3.3 55.9 36.9 5.6 ., Sulpha- // 5.9 1.3 2.0 1.3 5.3 84.2 methoxazole //i 10.1 % Quinupristin/ 100 % / Dalfopristin 100 / Tetracydine 86.2 13.2 0.7 91.1 8.9 Trimethoprim Vancomycin 3.3 19.7 42.1171 5.3 12.5 2.2 33.0 32414.0 5.0 13.4 98.7 1.3 I 96.1 3.9 Con: Conventional farms, Org: Organic farms *Interpretive standards were based on NCCLS M3 l-A2, otherwise based on DANMAP 2001. Dilution range were indicated as un-shaded cells and break points were shown as a vertical border. 136 le7 'al Susce “phyla: cu au eus ed tank co ect Danis an an con arm age . /2/ / //’///' //:///’Z /‘7///" 547.42% / /// // / // / '/ / /// / //// Table 7-4. Logistic regression analysis with correlated data on dichotomized susceptibility result in Staphylococcus aureus from Wisconsin. Odds ratio (OR) of having reduced susceptibility in conventional dairy herds Logistic regression with repeated observation Break point 95% Wald confidence (118/ml) OR interval Bacitracin 64 0.89 [0.32 — 2.47] Cephapirin 2* 1.31 [0.55 — 3.14] Chloramphenicol 8 1.70 [0.77 - 3.75] Ciprofloxacin 0.5 3.33 [1.21 — 9.14] 1 Erythromycin 0.5 0.79 [0.40 — 1.56] Gentamicin 2* 1.33 [0.49 — 3.64] Kanamycin 8 1.14 [ 0.63 - 2.08] Penicillin 1 2.25 [0.41 — 12.37] Streptomycin 8 1.17 [0.66 — 2.09] Sulphamethoxazole 512 1.79 [0.76 — 4.23] Tetracycline 1* 1.63 [0.73 — 3.67] Trimethoprim 16 0.96 [0.42 - 2.16] Vancomycin 2* 3.05 [0.74 — 12.51] 1 : Isolates fi'om conventional dairy herd had significantly higher probability to have reduced susceptibility * : MIC90 were used to dichotomize the MIC value, except for those antimicrobials with only two or three MIC categories. 138 Table 7-5. Logistic regression analysis with correlated data on dichotomized susceptibility result in Staphylococcus aureus from Denmark. Odds ratio (OR) of having reduced susceptibility in conventional dairy herds. Logistic regression with repeated observation Break point OR 95% Wald confidence (pg/ml) interval Avilamycin 8 0.15 [0.028 — 0.769] 1 Bacitracin 64 0.49 [0.09 — 2.56] Chloramphenicol 8 3.74 [0.99 — 14.17] Ciprofloxacin 0.5 0.37 [0.09 — 1.49] Erythromycin 0.5 2.07 [0.55 -- 7.87] Gentamicin 2* NE NE Kanamycin 8 7.40 [0.93 — 58.92] Penicillin 1 0.23 [0.013 - 4.26] Streptomycin 8 1.39 [0.42 -— 4.57] Sulphamethoxazole 64T 1.72 [ 0.37 — 7.94 ] Tetracycline 1* 1.03 [0.07 — 15.72] Trimethoprim 41 1.27 [0.18 — 9.10] Vancomycin 2* 0.23 [0.02 — 2.40] NE : Not Estimable, because all okologisk isolates had the same response. 1 : Isolates from organic dairy herd had significantly higher probability to have reduced susceptibility. * : MIC90 were used to dichotomize the MIC value, except for those antimicrobials with only two or three MIC categories. I : MIC90 of Danish isolates only were used to dichotomize the data. 139 Table 7-6. Logistic regression analysis with correlated data on dichotomized susceptibility result in Staphylococcus aureus. Odds ratio (OR) of having reduced susceptibility in Wisconsin, compare to Denmark. Logistic regression with repeated observation Break point OR 95% Wald (pg/ml) confidence lnterval Bacitracin 64 6.76 [2.56 — 17.82] 1 Chloramphenicol 8 0.58 [0.27 — 1.26] 3 Ciprofloxacin 0.5 0.25 [0.11 - 0.59] 2 Erythromycin 0.5 0.94 [0.44 — 1.99] 3 Gentamicin 2* 9.20 [1.16 - 72.28] I Kanamycin 8 4.08 [1.39 — 12.00] I Oxacillin - NE NE 3 Penicillin 1 5.78 [107—31.11]l Streptomycin 8 0.48 [0.25 — 0.94] 2 Sulphamethoxazole - NE NE ' Quinupristin/Dalfopristin - NE NE 3 Tetracycline 1* 4.64 [1.12 — 19.25] ‘ Trimethoprim - NE NE1 Vancomycin 2* 0.85 [0.21 — 3.34] 3 NE: Not Estimable. However, the difference of susceptibility to sulphamethoxazole and trimethoprim among two countries were determined from the Table 7-1 and 7-2. 1: Isolates from the U. S. A. had significantly higher probability to have reduced susceptibility. 2: Isolates from Denmark had significantly higher probability to have reduced susceptibility. 3: No significant difference were observed. *: MIC90 were used to dichotomize the MIC value, except for those antimicrobials with only two or three MIC categories. 140 //// ./ // 972/ .w.,,....y,..,,%//a ////c/ 70 ,0... 50 7 Figure 7-1. Comparison of minimum inhibitory concentration to bacitracin in 60 Staphylococcus aureus isolated from bulk tank milk in Wisconsin and Denmark 0 0 4 3 88.8. 8 as O 0 0 2 41 128 32 MIC (pg/ml) 16 II Wisconsin I Denmarlq 141 CONCLUSIONS Organically produced milk sells for almost twice the price of conventionally produced milk, creating an interesting economic comparison between the two different management styles of milk production Our findings suggest the need to more completely assess the cost effectiveness of some interesting differences between the organic and conventional farms. Our study showed that organic dairy farms were producing milk without significantly increasing reported CM rate, BTSCC or culling rate as compared with matched conventional farms. For organic and conventional farms, respectively, mastitis rate (28 and 32 cases per 100-cow-years at risk), bulk tank milk somatic cell count (262,000 and 285,000 cells per ml) and culling rate (17.2 and 18.0 cases per 100-cow years) were lower in organic than in the conventional farms, although the differences were not statistically significant. The milking procedures were very similar between matched pairs, so the effect of milking procedures on CM rate or BTSCC could not be estimated. The organic herds were significantly smaller than the conventional herds (51.1 and 71.1 cows) and lower milk production per cow (20.2 and 23.7 kg/day). Organic herds were more likely to use intensive grazing, which may have accounted for their higher rate of gastrointestinal nematodes as compared with conventional herds. We subjectively observed that organic farms tended to have smaller cows and had a greater proportion of smaller non-Holstein breeds. Organic farms used less artificial insemination, which may have created a gene pool of smaller cows compared with what is commonly seen in today’s mainstream dairy industry where AI has been used to produce large cows with high milk production per cow. Because smaller cows eat less feed, comparisons in milk production between organic and conventional herds must consider this lower cost of 142 production. Also, other differences in nutrition and management most likely act to reduce the per-cow cost of feeding and maintaining the smaller grazing cows, as compared with larger cows being fed a high energy diet in total confinement. Lip—oh; We used E. coli as an indicator organism to represent the selective pressure on a commensal Gram negative bacteria Other antimicrobial resistance studies, e. g. DAN MAP and NARMS, have used E. coli as their Gram negative indicator bacterial species, thereby allowing us to compare our results. Our study shows significantly lower prevalence rates of AR for seven antimicrobials (ampicillin, streptomycin, kanamycin, gentamicin, chloramphenicol, tetracycline, and sulphamethoxazole) in organic dairy herds, as compared to conventional herds. However, the odds ratios for having resistant E. coli were relatively small (OR=1.5 — 4.3). Although the organic farms had converted to organic farming methods at least 3 years before our study (mean = 8.0 years), antimicrobial resistance clearly persisted long after antimicrobial selective pressure had been withdrawn. Antimicrobial resistance tended to be larger in calves than in cows in the both organic and conventional herds; therefore animal age (cow/calf) should be taken into account when AR in animals is studied. Salmonella spp. We had only 7 (seven) isolates (4 from organic & 3 from conventional) from 1,191 samples. Three isolates were from cows, and four isolates from calves. Only two isolates were from the same organic farm, and the rest of isolates were from different farms. The lower rate of Salmonella isolation may be the real prevalence on farm, or due to the low detecting power of our sampling method. One isolate from a 143 conventional farm showed high resistance to Amoxicillin+Clavu1anic acid (>32), Ampicillin (>32 pg/ml), Apramycin (>32 pg/ml), Cefoxitin (32 pg/ml), Ceftiofur (16 pg/ml), Ceftriaxon (8 pg/ml), Cephalotin (>32 pg/ml), Kanamycin (>64 pg/ml), Streptomycin (256 pg/ml), Sulphamethoxazole (>512 pg/ml), tetracycline (>32 pg/ml), but was susceptible to Ciprofloxacin, and nalidixic acid. Campylobacter spp. The prevalence of Campylobacter spp. was not significantly different between organic and conventional dairy farms in Wisconsin. Campylobacter prevalence was significantly higher in March than in September, higher in calves than in cows, and higher on smaller farms than on larger farms. Rates of retained placenta, pneumonia, and abortion were positively associated with the Campylobacter spp. prevalence. The proportion of tetracycline-resistant Campylobacter spp. was higher in isolates derived from calves. Two isolates from conventional dairy farms were resistant to Ciprofloxacin and none of the isolates were resistant to gentamicin or erythromycin. Resistance to tetracycline was 41.5% (66/159) for organic and 47 .4% (82/ 17 3) for conventional herds, which was not statistically significant. We saw no evidence that restricted antimicrobial use on dairy farm had any association with antimicrobial resistance to Ciprofloxacin, gentamicin, erythromycin and tetracycline in Campylobacter spp. Staphylococcus aureus A significant difference in antimicrobial resistance in Staphylococcus aureus was detected to only one antimicrobial in our Wisconsin study and in the parallel study in Denmark. Our study showed that Wisconsin organic dairies had more 144 susceptible isolates to Ciprofloxacin as compare to conventional farms. Danish conventional dairies had more susceptible isolates to avilamycin as compared to Danish organic (okologisk) dairy farms. The differences in AR were large between Wisconsin and Denmark. Isolates from Wisconsin had higher probability of reduced susceptibility to 7 out of 14 comparable antimicrobials (bacitracin, gentamicin, kanamycin, penicillin, sulphamethoxazole, tetracycline, and trimethoprim), whereas Danish isolates had higher probability of reduced susceptibility to only two drugs (Ciprofloxacin and streptomycin). Differences in antimicrobial susceptibility between organic/okologisk and conventional farms were small relative to the differences observed between the two countries. Enterococcus 331p. Isolation, identification and MIC measurement was done at William Beaumont Hospital. However, the result shows the rates of resistance were very low for Quinupristin / dalfopristin (9 for organic and 16 for conventional) and Gentamicin (2 for organic and 7 for conventional), and no isolates were resistant to Vancomycin, which make a further analysis diflicult. Though it was not included in the original plan, we tested a stratified random sample (type of farms, cow/calf, faecalis/faecium/others, and season, in total 600) on a custom Sensititre panel (CMVSACDC) which is similar to the Danish gram positive panel. This will facilitate comparisons with the Danish data and give us more information regarding Enterococci as an indicator species of gram positive bacteria. The results will be published later. 145 This study confirmed that milk can be produced without antibiotics, and that disease rates and measures of production are roughly comparable between herds that don’t use antibiotics and small conventional dairy farms that do use antibiotics. Generally, AR prevalence in dairy animals in Wisconsin was very low compared to previous reports from poultry and swine production. We found lower rate of AR in organic herds for some “bug-drug” combinations, but not for others. The impact on public health caused by use of antimicrobials for dairy industry was assessed as relatively very low. In general, differences in AR between organic and conventional farms were mild to moderate, if they existed at all. Our results indicated that the persistence of AR on organic farms was such that AR remained for many years after antibiotics were no longer being used. 146 APPENDICES QUESTIONNAIRE Producer's name: Date of interview: Farm name: Farm address: Tel: Fax: E-mail: Type of farm: __ Organic _ Not organic 1) Usual number of animals making milk 2) Usual number of dry cows 3) Breed of cattle: Holstein _ Jersey _ Guernsey __ 4) Number of times per day cows are milked: 2 3 Based on monthly bulk tank remrt of CO-OP 5) Pounds of bulk tank milk 6) Usual SCC in cells/ml 7) Bacterial count (SP0 or Raw) On the average, how many times per year does each cow have: 8) The hair clipped around her teats and udder? 9) Feet trimmed? Mastitis: On the average day, how many lactating cows would not be put in the bulk tank because of mastitis or treatment for other diseases? 10) In Summer 11) In Winter 147 Dry cow treatment: 12) Udder infusions are used for drying cow? Yes / No 13) What is your treatment? Milking: 14) Usual milking procedure (check all that apply) Mnter Summer Dry massage or wipe with no water used Wash bucket usually used Running water (hose) usually used Pre-milking teat dipping usually used Individual-cow paper towels or cloths used for washing (each towel used for only one cow) Shared towels or cloths used for washing Gloves used for washing? Individual towels (cloth or paper) used for drying Teats are usually not dried before milking Post-milking teat dipping almost always used 15) Are cows milked in a stanchion or in a parior? Stanchion/Tie stalls Milking Parlor 16) For each milking, it usually takes people about hours to milk the cows using milking units (claws) at a time. 17) Approximately what percent of the labor for milking and cow-care is hired (non-family) labor? % 18) How many years old is your milking machine? years old 148 Cattle numbers and movement: 19) About how many animals were purchased last year? How many cows: last month? last year? 20) died 21) culled 22) were sold to an organic farm 23) were sold to a non-organic farm or auction 24) Not counting heifers, what percent of your cows were sired by: Herd bull % Artificial Insemination % 25) Average days in milk? days 26) Usual herd calving interval? months Housing: 27) WINTER Lactating cow housing (Check all that apply) _ Strictly pasture _ Pasture with access to housing _ Tie stalls _ Loose housing (manure pack/straw pack) _ Free stalls _ other 28) SUMMER Lactating cow housing (check all that apply) _ Outside exercise area with little grazing _ Grazing pasture with access to housing _ Strictly grazing pasture _ Tie stalls _ Loose housing (manure pack/straw pack) _ Free stalls _ other 29) If pastured, how many months do cows have access to grazing pasture? 30) What type of bedding is usually used for the milking herd? n0 bedding _ % sand _ % straw _ % wood shavings or sawdust _ % other % Feed 149 31) Do you feed a ration balanced for protein,energy, fiber and minerals? 32) Do you calculate dry matter intake for each cow/milking group? Yes _ No Yes __ No 33) Do you feed a TMR? Yes __ No 34) Are any feeds fed separately? Yes __ No 35) Do you get forage analyis done? Yes __ No 36) Do you feed a separate balanced ration for dry cows? Yes __ No 37) For pre-calving (pre-fresh, close-up, steam-up)? Yes _ No Diseases and Treatments: 38) How many cases of the following diseases were seen in your herd in the past 3 months? Mastitis (flakes or strings in the milk or a swollen hard udder) Metritis (smelly vaginal discharge usually following calving) Displaced abomasum Retained placenta (retained over 12 hours after calving)\ __ Abortion (observed loss of the fetus) 39) What is your standard treatment for routine cases of mastitis in which the cow IS NOT systemically sick with a fever, depression or other signs outside of the udder? _ Strip out the quarter at frequent intervals _ Anti-inflamatory or antipyretics drugs (steriods, aspirin, banamine, etc.) _ IV fluids _ Administer oxytocin to assist milkout _ Udder infusions of antibiotics? other? _ Systemic Antibiotics _ other _ other 150 40) What is your standard treatment for routine cases of mastitis in which the cow IS systemically sick with a fever, depression or other signs outside of the udder? _ Strip out the quarter at frequent intervals _ Anti-inflamatory or antipyretics drugs (steriods, aspirin, banamine, etc.) _ IV fluids _ Administer oxytocin to assist milkout _ Systemic Antibiotics _ Udder infusions of antibiotics? other? _ other _ other 41) What is your standard treatment for metritis (smelly vaginal discharge after calving)? __ Infuse iodine _ Manually remove as much fluid as possible and remove any placenta which may be retained. __ Antibiotics _ Prostagrandin F2 (Lutalyse‘) _ Estradiol (ECP‘) _ Other 42) What do you do when cows with severe disease do not respond to treatment? number of cows do not respond to treatment last year number of cows I sold the cow for slaughter number of cows I sold the cow to a non-organic dairy herd 151 Calf Section: 43) About what percent of Iive-bom calves die before they are weaned? % 44) What percent of the calves get colostrum by the following methods? _ Only by nursing the dam _ Always bottle or tube fed even if seen nursing the dam _ Bottle or tube fed only if not seen nursing _ Other 45) How many cases of calf diarrhea do your farm have per year? number of calves number of cases 46) How do you treat these diarrhea calves? I use 47) How many cases of calf pneumonia do your farm have per year? number of calves number of cases 48) How do you treat these pneumonia calves? Iuse Pre-weaning calf housing: 49) What kind of calf housing do you use? Calf housing (winter) Calf housing (summer) individual hutches group hutches or pens indoor individual pens indoor group housing on pasture Other 152 Direct Observations of Facilities by the Investigator: Random Selection of 10 cows Cow Body Cond. Score 1. 10. Lameness Score Lactating Cow Environmental Sanitation Scores: Lactating cow bedding area: Amount of moisture and manure Clean Lactating cow exercise area: Clean Amount of moisture and manure Lactating cow cleanliness: Clean Amount of manure above the knees Pre-weaning calf scores: Pre-weaning: housing Amount of moisture Clean and manure Pre-weaning: manure on Clean calves 153 Top third Middle third Bottom third _ dirty dirty dirty dirty _ dirty Cow numbers or names of all cows present on the farm. This includes all dry and lactating animals which have had at least one calf in their lifetimes. 154 SAS Control Commands SAS Control Command for Table 4-1 Pair wise analysis for continuous variables options nocenter; DATA mastitis; INFILE 'c:\SAS\mastitis.csv' DLM = DSD MISSOVER FIRSTOBS=2 OBS=61; INPUT pairtype season cow drycow Holstein milk SCC bacteriaNB_S NB_W drycowlnfuse Drywipe bucket predip ind_towel sha_towel glove dry_towel nodry postdip stanchion people hours Iaborpercow unit hired purchase OPENHIRD ai dim house grazing bedding straw sand wood corn mastitis Stripout antiinf Oxytocin infusions AB BC_Mar Env_Mar BC_Sep Env__Sep; BTM=milk*0.4536; l* milk in Kg */ totalcow=cow+drycow; masrate=mastitis*400/totalcow; milkpercow=BTMlcow; /*milk per cow in Kg */ if holstein=1then breed=1; else breed=0; I‘ type=1 is organic and type=0 is conventional*/ proc mixed; class pair type season; model totalcow = type /solution; random pair“, proc mixed; class pair type season; model ai = type /so|ution; random pair; proc mixed; class pair type season; model milkpercow = type Isolution; random pair; proc mixed; class pair type season; model Iaborpercow = type Isolution; random pair; proc mixed; class pair type season; model masrate = type Isolution; random pair; proc mixed; class pair type season; model SCC = type Isolution; random pair; proc mixed; class pair type season; model bacteria = type Isolution; random pair; proc chart data=mastitis; hbar SCC lgroup=type freq; 155 proc chart; hbar masrate lgroup=type freq; proc univan'ate; var totalcow; bytype; run; quit; SAS Control Command for Table 4-2 Comparison of discrete variable between organic and conventional dairy farms Pair wise analysis (McNemar’s test) options nocenter; DATA paired; INFILE 'c:\SAS\paired.csv' DLM = DSD MISSOVER FIRSTOBS=2 OBS=31; INPUT pair type season cow drycow Holstein milk SCC bacteriaNB_S NB_W drycowlnfuse Drywipe bucket predip ind_towel sha_towel glove dly_towel nodry postdip stanchion people hours Iaborpercow unit hired purchase OPENHIRD ai dim house grazing bedding straw sand wood corn mastitis Stripout antiinf Oxytocin Infusions AB BC_Mar Env_Mar BC_Sep Env_Sep o_cow o_drycow o_HoIstein o_milk o_SCC o_bacleria o_NB_S o_NB_W o_drycowlnfuse o_Drywipe o_bucket o_predip o_ind__towel o_sha_towe| o_glove o_dry_towel o_nodry o_postdip o_stanchion o_people o_hours o_Iaborpercow o_unit o_hired o_purchase o_OPENHIRD o_ai o_dim o_house o_grazing o_bedding o_straw o_sand o_wood o_com o_mastitis o_Stripout o_antiinf o_Oxytocin o_lnfusions o_AB o_BC_Mar o_Env_Mar o_BC_Sep o_Env_Sep; l‘assign 100% holstein as 1, else hostein=0*/ if holstein=1 then breed=1; else breed=0; if o_HoIstein=1 then o_breed=1; else o_breed=0; I'assign housing to each category *I if house='0' then freestall='1'; else freestall='0'; if house='1' then loose='1'; else loose='0'; if house='2' then tiestall='1'; else tiestall='0'; if o_house='0' then o_freestall='1'; else o_freestall='0'; if o_house='1' then o_Ioose='1'; else o_Ioose='0'; if o_house='2' then o_tiestall='1 '; else o_tiestall='0'; /*assign bedding to each category*/ if bedding='0' then nobedding='1'; else nobedding='0'; if bedding='1' then strawg='1'; else strawg='0'; if bedding='2' then woodg='1'; else woodg='0'; if bedding='3' then sandg='1'; else sandg='0'; if bedding='4' then rubberg='1'; else rubberg='0'; l‘assign bedding to each categoly*l if 0_bedding='0' then 0_nobedding='1'; else 0_nobedding='0'; if 0_bedding='1' then o_strawg='1'; else o_strawg='0'; if 0_bedding='2' then o_woodg='1'; else o_woodg='0'; if 0_bedding='3' then o_sandg='1'; else o_sandg='0'; if 0_bedding='4' then o_rubberg='1'; else o_rubberg='0'; 156 proc freq data=paired; run; quit; tables breed*o_breed lagree; tables drycowlnfuse*o_drycowinfuse lagree; tables openhird*o_openhird lagree; tables freestall*o_freestall lagree; tables loose*o_loose lagree; tables tiestall*o_tiestall lagree; tables stanchion*o_stanchion lagree; tables drywipe*o_drywipe lagree; tables bucket*o_bucket lagree; tables predip*o__predip lagree; tables postdip*o_postdip lagree; tables ind_towel*ind_towel lagree; tables sha_towel‘o_sha_towel lagree; tables glove*o_glove lagree; tables dry_towel*o_dry_towel Iagree; tables nodry*o_nodry lagree; tables stripout*o_stripout lagree; tables antiinf‘o_antiinf lagree; tables oxytocin*o_oxytocin lagree; tables infusions*o_infusions lagree; tables AB*0_AB lagree; tables nobedding*o_n0bedding lagree; tables strawg*o_strawg lagree; tables woodg*o_w00dg lagree; tables sandg*o_sandg lagree; tables rubberg*o_rubberg lagree; SAS Control Command for Table 5-2 Logistic Regression Analysis of resistance to antimicrobials among E. coli isolates from organic and conventional dairy farms options nocenter; Title 'The Analysis of MIC data with proportional odds model 8 GEE (for Table 4)’; %MACRO ecoli (variablet=); proc genmod data=ecoli descending; class farm organic CowCalf collection; model 8variablet = organic CowCalf/ Iink=clogit dist=mult typ63; repeated subject=faml /type=ind; estimate 'ORzo-C' organic1 -1 lexp; estimate 'ORzo-C' CowCaIf 1 -1 lexp; %MEND ecoli; DATA ecoli; INF ILE 'c:\SASZ\ecoli03.csv' DLM = DSD MISSOVER FIRSTOBS=2; INPUT SamplelD Farm Organic CowCalf season collection Amikacin AmoxClav Ampicillin 157 Apramycin Cefoxitin Ceftiofur Ceflriaxone Cephalothin Chloramph Ciprofiox Gentamicin Kanamycin Nalidixic Streptomycin Sulphamethox Tetracycline TrimetSulf; %ecoli (variable1= Amikacin ) %ecoli (variable1= AmoxClav) %ecoli (variable1=Ampicillin) %ecoli (variable1=Apramycin) %ecoli (variable1=Cefoxitin) %ecoli (variable1=Ceftiofur) %ecoli (variable1=Ceftriaxone) %ecoli (variable1=Cephanthin) %ecoli (variable1=Ch|oramph) %ecoli (variable1=Ciproflox) %ecoli (variable1=Gentamicin) %ecoli (variable1=Kanamycin) %ecoli (variable1=Nalidixic) %ecoli (variable1=Streptomycin) %ecoli (variable1=Sulphamethox) %ecoli (variable1=Tetracycline) %ecoli (variable1=TrimetSulf) run; quit; SAS Control Command for Table 5-3 title 'Multiple antimicrobial resistance Analysis with the extended Mantel-Haenszel mean score statistic'; 1* Table 5 was analyzed according to SAS manual Categorical Data Analysis p73- */ data multidrugcalf; input cowcalf 8 organic 5 response count @@; datalines; calf conv 6 30 calf conv 5 11 calf conv 4 43 calf conv 3 36 calf conv 2 24 calf conv 1 20 calf conv 0 121ca|forg 6 7 calforg 517 calf org 419 calforg 3 24 calforg 2 20 calforg127 calforg 0167; proc freq data=multidrugcalf order=data; weight count; tables cowcalf'organic*response lcmh nocol nopct; run; quit; data multidrugcow; input cowcalf 8 organic 5 response count @@; datalines; cow conv 3 9 cow conv2 11 cow conv1 19 cow conv 0245 coworg 37coworgz10coworg112coworg 0241; proc freq data=multidrugcow order=data; weight count; tables cowcalf‘organic*response Icmh nocol nopct; run; quit; 158 8A8 Control Command for Table 6-2 options nocenter; Title 'The Analysis of Campylobacter MIC data'; DATA campy; INFILE 'c:\SAS3\isolatesoz.csv' DLM = DSD MISSOVER FIRSTOBS=2; INPUT SampleID Organic CowCalf farm campy Season; proc freq; table organic*campy/ chisq relrisk; table cowcalf'campy/ chisq relrisk; table season*campy/ chisq relrisk; proc genmod data=campy descending; class farm organic CowCalf season campy; model campy = organic cowcalf season /LINK=LOG|T DlST=BINOMIAL type3; repeated subject=farrn / type=ind; estimate 'OR:Org-Convl' organic 1 -1 lexp; estimate 'Ochow-calf‘ CowCalf 1 -1 lexp; estimate 'ORzmar-sep' season 1 -1 /exp; run; quit; 8A8 Control Command for Table 6-3 options nocenter; Title 'The Analysis of Campylobacter MIC data'; DATA campy; INFILE 'c:\SAS3\manageO1.csv' DLM = DSD MISSOVER FIRSTOBS=2; INPUT SamplelD Organic CowCaIf farm campy Season cow milk SCC openherd die_year grazing mastitis metritis placenta abortion calf_die Calf diarrha pneumo; Proc sort; by organic cowcalf; l‘Overall estimates; Backward selection was performed"'/ Proc genmod data=campy descending; class farm organic cowcalf campy season openherd grazing; model campy = Organic CowCalf Season cow milk SCC openherd die _year grazing mastitis metritis placenta abortion calf_die Calf diarrha /LINK=LOGIT DlST=BINOMIAL type3; repeated subject=farrn ltype=ind; I‘Only Calf data; Backward selection was performed*/ Proc genmod data=campy descending; where cowcalho; class farm organic cowcalf campy season openherd grazing; model campy = season calf_die calf diarrha /LINK=LOGIT DlST=BINOMIAL type3; repeated subject=farrn / type=ind; 159 /*Only Cow data; Backward selection was perfonned*/ Proc genmod data=campy descending; where cowcalf=1; class farm organic cowcalf campy season openherd grazing; model campy = season cow openherd grazing metritis placenta abortion/LINK=LOGIT DlST=BINOMIAL type3; repeated subject=farrn /type=ar; estimate 'ORzgrazing' grazing 1 0 -1 lexp; run; quit; SAS Control Command for Table 7-4 options nocenter; Title 'The Analysis of Staphylococcus MIC data'; DATA staph; INFILE 'c:\SAS4\SAUSDEN.csv' DLM = ',' FIRSTOBS=2; INPUT Number ID country Farm Organic Season Avilamycin Bacitr Cepha Chlora Ciprof Eryth Gentam Kanamy Oxacil Penici Strept Sulfam Synerc Tetra Trim Vancom; lf Bacitr=4 then Bacitr=8; If country=1 and organic=1 then group=1; /"Organic in \Msconsing*/ if country=1 and organic=0 then group=2; PConventionaI in Wrsoconsin*/ if country=0 and organic=1 then group=3; /*Organic in Denmark*/ if country=0 and organic=0 then group=4; I‘Conventional in Denmark*/ l‘dichotomized by MICQO, high sensitibity=Ml090 */ If Avilamycin >=8 then AVI = 1; If Avilamycin < 8 then AVI = 0; If Bacitr >=64 then BAC = 1; If Bacitr < 64 then BAC = 0; If Cepha >=2 then CEF = 1; If Cepha < 2 then CEF = 0; If Chlora >= 8 then CHL = 1; If Chlora < 8 then CHL = 0; If Ciprof >=0.5 then CIP = 1; If Ciprof < 0.5 then CIP = 0; If Eryth >=0.5 then ERY = 1; If Eryth < 0.5 then ERY = 0; If Gentam >=2 then GEN = 1; If Gentam < 2 then GEN = 0; If Kanamy >=8 then KAN = 1; If Kanamy < 8 then KAN = 0; If Oxacil >=1 then OXA = 1; If Oxacil < 1 then OXA = 0; If Penici >=1 then PEN = 1; If Penici < 1 then PEN = 0; If Strept >=8 then STR = 1; If Strept < 8 then STR = 0; If Sulfam >=512 then SUL = 1; If Sulfam < 512 then SUL = 0; If Tetra >=1 then TET = 1; If Tetra < 1 then TET = 0; If Trim >=16 then TRl = 1; If Trim < 16 then TRl = 0; If Vancom >=2 then VAN = 1; If Vancom < 2 then VAN = 0; proc genmod data=staph descending; where country=1; class farm organic; model BAC = organic/ link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; 160 where country=1; class farm organic; model CEF = organic/ link=logit dist=binomial type3; repeated subject=fann ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=1; class farm organic; model CHL = organic! link=logit dist=binomial typeS; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=1; class farm organic; model CIP = organic/ link=logit dist=binomial type3; repeated subject=farrn / type=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=1; class farm organic; model ERY = organic! link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=1; class farm organic; model GEN = organic/ link=logit dist=binomial type3; repeated subject=farrn / type=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=1; class farm organic; model KAN = organic/ link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=1; class farm organic; model PEN = organic/ link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=1; class farm organic; model STR = organic/ link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; 161 proc genmod data=staph descending; where country=1; class farm organic; model SUL = organic/ link=logit dist=binomial type3; repeated subject=farrn / type=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=1; class farm organic; model TET = organic/ link=logit dist=binomial type3; repeated subject=fann ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=1; class farm organic; model TRI = organic/ link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=1; class farm organic; model VAN = organic! link=logit dist=binomial type3; repeated subject=fann ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; quit; SAS Control Command for Table 7-5 options nocenter; Title 'The Analysis of Staphylococcus MIC data'; DATA staph; INFILE 'c:\SAS4\SAUSDEN.osv' DLM = FIRSTOBS=2; INPUT Number ID country Farm OrganicSeason Avilamycin Bacitr Cepha Chlora Ciprof Eryth GentamKanamy Oxacil Penici Strept Sulfam Synerc Tetra Trim Vancom; If Bacitr=4 then BacitFB; If country=1 and organic=1 then group=1; /*Organic in Vlfisconsingfl if country=1 and organin then group=2; I‘Conventional in Wlsoconsin"! if country=0 and organic=1 then group=3; /*Organic in Denmark*/ if country=0 and organic=01hen group=4; I‘Conventional in Denma11<*/ Pdichotomized by MIC90, high sensitibity=MIC90 */ If Avilamycin >=8 then AVI = 1; If Avilamycin < 8 then AVI = 0; If Bacitr >=64 then BAC = 1; If Bacitr < 64 then BAC = 0; If Cepha >=2 then CEF = 1; If Cepha < 2 then CEF = 0; If Chlora >= 8 then CHL = 1; If Chlora < 8 then CHL = 0; If Ciprof >=0.5 then CIP = 1; If Ciprof < 0.5 then CIP = 0; If Eryth >=0.5 then ERY = 1; If Eryth < 0.5 then ERY = 0; 162 If Gentam >=2 then GEN = 1; If Gentam < 2 then GEN = 0; If Kanamy >=81hen KAN = 1; If Kanamy < 8 then KAN = 0; If Oxacil >=1 then OXA = 1; If Oxacil < 1 then OXA = 0; If Penici >=1 then PEN = 1; If Penici < 1 then PEN = 0; If Strept >=8 then STR = 1; If Strept < 8 then STR = 0; If Sulfam >=64 then SUL = 1; If Sulfam < 64 then SUL = 0; If Tetra >=1 then TET = 1; If Tetra < 1 then TET = 0; If Trim >=4 then TRI = 1; If Trim < 4 then TRI = 0; If Vancom >=2 then VAN = 1; If Vancom < 2 then VAN = 0; proc genmod data=staph descending; where country=0; class farm organic; model AVI = organic / link=logit dist=binomial type3; repeated subject=farrn / type=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=0; class farm organic; model BAC = organic! link=logit dist=binomial type3; repeated subject=farrn / type=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=0; class farm organic; model CEF = organic1 link=logit dist=bin0mial type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=0; class farm organic; model CHL = organic! link=logit dist=binomia| typeS; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=0; class farm organic; model CIP = organic! link=logit dist=binomia| type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=0; class farm organic; model ERY = organic/ link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; 163 run; proc genmod data=staph descending; where country=0; class farm organic; model GEN = organic! link=logit dist=binomial type3; repeated subject=farrn I type=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=0; class farm organic; model KAN = organic! link=logit dist=binomial type3; repeated subject=fann ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=0; class farm organic; model PEN = organic! link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=0; class farm organic; model STR = organic1 link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional’ organic 1 -1/exp; run; proc genmod data=staph descending; where country=0; class farm organic; model SUL = organic/ link=logit dist=binomial typ63; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=0; class farm organic; model TET = organic! link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=0; class farm organic; model TRI = organic! link=logit dist=binomial type3; repeated subject=fann ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; run; proc genmod data=staph descending; where country=0; class farm organic; model VAN = organic! link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'organic versus conventional' organic 1 -1/exp; 164 run; SAS Control Command for Table 7-6 options nocenter; Title 'The Analysis of Staphylococcus MIC data'; DATA staph; INFILE 'c:\SAS4\SAUSDEN.csv' DLM = FIRSTOBS=2; INPUT Number ID country Farm OrganicSeason Avilamycin Bacitr Cepha Chlora Ciprof Eryth GentamKanamy Oxacil Penici Strept Sulfam Synerc Tetra Trim Vancom; If Bacitr=4 then BacitF8; If country=1 and organic=1 then group=1; l‘Organic in VWsconsing"! if country=1 and organin then group=2; /*Conventional in Wrsoconsin‘! if country=0 and organic=1 then group=3; l‘Organic in Denmark*/ if country=0 and organic=0 then group=4; I‘Conventional in Denmark"! l'dichotomized by M1090, high sensitibity=MlC90 */ lf Avilamycin >=8 then AVI = 1; If Avilamycin < 8 then AVI = 0; If Bacitr >=64 then BAC = 1; If Bacitr < 64 then BAC = 0; If Cepha >=2 then CEF = 1; If Cepha < 2 then CEF = 0; If Chlora >= 8 then CHL = 1; If Chlora < 8 then CHL = 0; If Ciprof >=0.5 then CIP = 1; If Ciprof < 0.5 then CIP = 0; If Eryth >=0.5 then ERY = 1; If Eryth < 0.5 then ERY = 0; If Gentam >=2 then GEN = 1; If Gentam < 2 then GEN = 0; If Kanamy >=8 then KAN = 1; If Kanamy < 8 then KAN = 0; If Oxacil >=1 then OXA = 1; If Oxacil < 1 then OXA = 0; If Penici >=1 then PEN = 1; If Penici < 1 then PEN = 0; If Strept >=8 then STR = 1; If Strept < 8 then STR = 0; If Sulfam >=512 then SUL = 1; If Sulfam < 512 then SUL = 0; If Tetra >=1 then TET = 1; If Tetra < 1 then TET = 0; If Trim >=16 then TRI = 1; If Trim < 16 then TRI = 0; If Vancom >=2 then VAN = 1; If Vancom < 2 then VAN = 0; /" proc freq data=staph; where country=1; table organic*Avilamycin lchisq nocol nopct; table organic*Bacitr lchisq nocol nopct; table organic*Cepha lchisq nocol nopct; table organic*Chlora lchisq nocol nopct; table organic*Ciprof /chisq nocol nopct; table organic*Eryth /chisq nocol nopct; table organic*Gentam lchisq nocol nopct; table organic'Kanamy lchisq nocol nopct; table organic*OxaciI lchisq nocol nopct; table organic*Penici /chisq nocol nopct; table organic'Strept lchisq nocol nopct; table organic*Squam lchisq nocol nopct; table organicfi'etra lchisq nocol nopct; 165 table organic'T rim lchisq nocol nopct; table organic'Vancom lchisq nocol nopct; run; quit; */ proc genmod data=staph; class farm organic country; model BAC = organic country organic*country / link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'US versus Denmark' country 1 -1/exp; run; proc genmod data=staph; class farm organic country; model CHL = organic country organic*country / link=logit dist=binomial type3; repeated subject=farrn / type=ind; estimate 'US versus Denmark' country 1 -1/exp; run; proc genmod data=staph; class farm organic country; model CIP = organic country organic*country / link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'US versus Denmark' country 1 -1lexp; run; proc genmod data=staph; class farm organic country; model ERY = organic country organic*country / link=logit dist=bin0mial type3; repeated subject=farrn ltype=ind; estimate 'US versus Denmark' country 1 -1lexp; run; proc genmod data=staph; class farm organic country; model GEN = country / link=logit dist=binomial typeS; repeated subject=farrn ltype=ind; estimate 'US versus Denmark' country 1 -1/exp; run; proc genmod data=staph; class farm organic country; model KAN = organic country organic*country / link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'US versus Denmark' country 1 -1/exp; run; proc genmod data=staph; class farm organic country; model PEN = organic country organic*country / link=logit dist=binomial type3; repeated subject=fann ltype=ind; estimate 'US versus Denmark' country 1 -1lexp; run; proc genmod data=staph; class farm organic country; model STR = organic country organic*country / link=logit dist=binomial typea; repeated subject=farrn ltype=ind; estimate 'US versus Denmark' country 1 -1/exp; run; proc genmod data=staph; 166 class farm organic country; model SUL = country / link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'US versus Denmark' country 1 -1/exp; run; proc genmod data=staph; class farm organic country; model TET = organic country organic*country / link=logit dist=binomial typeS; repeated subject=farrn ltype=ind; estimate 'US versus Denmark' country 1 -1/exp; run; It proc genmod data=staph; class farm organic country; model TRI = organic country organic*country / link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'US versus Denmark' country 1 -1/exp; run;*/ proc genmod data=staph; class farm organic country; model VAN = organic country organic*country / link=logit dist=binomial type3; repeated subject=farrn ltype=ind; estimate 'US versus Denmark' country 1 -1/exp; run; quit; 167