230535E Q? g LI}. I: ! 3L.I E‘: .~ VNG a-v 0"‘1' ram LL‘ VSTRO 51%?) : f=.;::::;__e_ e d. :v... H. .. :‘s' L. E Li‘ég‘w. 12:? 535 or {+29 Dag?” cf A a O 1 image £1033 a ‘8 M g: fr { L315 LIBRARY Michigan Stan: University THE 31 VITRO AND M VIVO RESPONSE OF MYCOPLASMA GALLISEPTICUM T0 DEOXYCORTTCOSTERONE BY -George A. Padgett AN ABSTRACT Submitted to the College of Vetvv nary Hedicine of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of'Microbiology and Public Health 1961 ££Z/( /C+v {,{C’h/k-{t L 5. Approved by Abstract George A. Padgett The purpose of this study was to determine the ig_!it§g and 32' zizg response of’Mycoplasma_gallisepticum to deoxycorticosterone (DOC) and deoxycorticosterone trimethylacetate (DOCT). Attempts were made, using various dilutions of DOC to inhibit growth ig_zit£g of four separate cultures of?!, gallisepticum, an avian pleuropneumonia-like organism. Growth was inhibited by a minimum of O.h mg. of DOC; the growth characteristics of surviving colonies were modified.— In the i§_zizg experiments 1 cc. of cultures of !, gallisepticum, containing about 106 organisms, was injected via the trachea into chickens, causing chronic respiratory disease. The birds were treated either by injecting DOC into the abdominal airsaos or by injecting DOCT into the breast muscle. To evaluate the response 6: birds infected with g. gallisepticum and treated with DOC or DOCT these criteria were used: (1) number of isolable organisms; (2) weight gains of the birds; (3) degree of gross pathologys (h) feed conversion. ‘flhen birds infected and treated were compared with infected and non-treated birds, the number of isolable organisms decreased, weight gains increased, gross pathology decreased, and feed conversion improved to the treated birds. The most effective dosage level found was 20 mg. of DOCT. A statistical analysis using the t test was performed, and this dose was found to be significant at the 0.1% level. Treatment at O, 7, 1h, or 21 days post-inoculation with the organisms appears to control the disease. Toxicity of this hormone for the chicken, resulting from admin- istration via either the infraorbital or intramuscular routes, with one exception, was not observed at the dosage levels used in these experiments. THE 9; VITRO AND E VIVO RESPONSE OF MYOOPLASMA GAILISEPTICUM T0 DEDHCORTICOS TERON E By George A. Padgett A THESIS Submitted to the College of Veterinary Medicine of Michigan State university of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1961 )(fitflfl/ // -"/~5‘w'( Approved by /S'I‘§'Cs ;3&;AQ4 ACKNOWLEDGEMENT The author wishes to express his appreciation to Dr. D. E. Schoenhard for suggesting this problem and for his kindness and encouragement in this study. The advice given by Dr. J. J. Stockton and Dr. R. F. Langham is also very much appreciated. The author thanks the Ciba Pharmaceutical Company for its financial support of this study. Chapter WDUCTION . . . . MATERIAIS AND METHODS EDI-18.... DISCUSSION.. 3111414131.... BIBLIOGRAEI. mun ... TABLE OF CONTENTS Page 10 18 25 26 30 INTRODUCTION Chronic respiratory disease (CRD) is a respiratory infection of chickens. It is characterized by coughing, tracheal rales and a pasty nasal discharge. The appetite of affected birds drops off, with a re- sultant loss in weight; the birds become depressed and egg production decreases in laying flocks. Gross lesions of the disease consist primarily of a mucopurulent exudate in the nasal passages, trachea, bronchi, and airsacs. The airsacs may appear beaded and contain a caseous yellow exudate. The tracheal mucosa may be thickened and some degree of pneumonia may be observed. The infective organism is transmitted by carriers, through the egg, by contact, and by airborne dust or droplets. The incubation period varies from h to 2l days. Bergey's Manual classifies the etiological agent of 0RD as Mycoplasma gallinarum. Nelson (1935) described a "coryza of slow onset" caused by cocoo- bacilliform.bodies varying in size from 0.1 to 0.5 microns. This is probably the first report of the disease which Delaplane and Stuart (19h3) called chronic respiratory disease. Markham and'Hong (1952) identified the pleuropneumonia-like organisms (PPTO) as the etiologic agent of 0RD. Both pathogenic and nonpathogenic species of PPID have been isolated from the respiratory tracts of chickens. Edward and Kanarek (1960) suggest that the virulent pathogenic PPDO of avian -1- origin be given the species name gallisepticum. It is estimated that CRD causes an annual loss of 25 million dol— lars to poultry raisers of the United States. The seriousness of this loss to poultrymen is reflected in the work of researchers in attempt- ing to prevent, control, or cure the disease. As shown in Table l a wide range of antibiotics and chemotherapeutic agents have been used with varying results. More than hO agents and/or combinations thereof have been tested ig 33333, in 333 or in 3113, or by all of these meth- ods, in attempts to discover effective therapy for CRD. The results have been somewhat confusing and even at times con- tradictory. For example, Table 1 shows that at least 15 different investigators have worked with the antibiotic terramycin. ‘With the ig,zizg work two of these researchers found the drug to be effective as a cure for the disease or at least to show excellent results in decreasing losses due to CRD. Four investigators obtained somewhat promising results from in 3139 tests with the drug. Three, however, found it to be of little or no therapeutic value. Several investigators have suggested possible explanations for variations in experimental results using terramycin and other anti- biotics, among these being, strain differences in the parasite and host, variation in degree or seriousness of the infection, number and kinds of secondary invaders, variations in housing, light and nutri- tion, amount of stress to which the birds are subjected and differences in methodology employed in the research and interpretation of results. It is generally agreed by investigators in the field that these prob- lems must be worked out before real correlative work can be accomplished. Lester and Hechter (1958) have shown that deoxycorticosterone (DOC) inhibits Micrococcus aureus, Sarcina lutea, Bacillus megaterium, Bacillus subtilis, Corynebacterium pseudodiphtheriticum and Mycobac- terium ranae as well as a number of Gram-positive yeasts and molds. They found that Gram-negative bacteria are generally insensitive to this steroid. Jefferson and Sisco (1959) have reported the inhibition of Aspergillus niger with DOC. Lester gt a1. (1958) with Neurospora crassa and Maxwell gt El. (1960) with Saccharomyces fragilis have shown these organisms to be inhibited by steroids. This thesis will report the in 31332 and in 1313 reaponse of Mycoplasma gallisepticum, the agent of CRD, to the action of deoxycorticosterone and its trimethylacetate ester (DOCT). This study was divided into six parts: Experiment I: The effect of DOC on the growth of PPLO in 31332. Experiment II: The toxicity of DOC for the chicken.. Experiment III: A pilot study on the effect of DOC on the growth of PPLO in zizg. Experiment IV: The in zizg response ofiM. gall_- septicum to DOCT. Experiment V: The effect of dosage on the in zizg response of M. gallisepticum to DOCT. Experiment VI: The effect of time of treatment on the ig_zizg reaponse of'M. gallisep- ticum to DOCT. MATERIALS AND METHODS Experiment I: The PPLO in this study were cultured using PPLO broth or agar (Difco) enriched with one per cent PPLO serum fraction (Difco). DOC was added to 125 ml. Erlenmeyer flasks in 0.8, O.h, 0.2, 0.1, and 0.05 mg. amounts per 20 ml. of culture. It was prepared by either one of two methods. In the first, (solvent 1) ethanol-chloroform in a 1:1 ratio was added as a solvent. The solvent utilized in the second method, (solvent 2) was 0.85 per cent NaCl-ethanol solution in a h:l ratio. In both cases the solvent was evaporated prior to inoculation of the flasks with the test organisms. Four strains of PPLO were used in this experiment. CRD-l and -2 ferment arabinose, dextrose, galactose, levulose and xylose. CED-7 ferments arabinose, dextrose, dextrin, galactose, levulose, maltose, mannose, starch, trehalose and xylose. The fourth strain, isolated in the diagnostic laboratory at Michigan State University, is designated as CRD-ll; fermentation reactions for this strain are not available. CRD-l and -2 were used with solvent 1, CED-7 and -11 with solvent 2. The PPLO were subcultured every 72 hours using 0.2 ml. of inoculum in 20 m1. of the PPLO broth. Following 72 hours of growth the cultures i3 tgtg were transferred to five flasks containing the various amounts of DOC. At the same time PPLO agar and the next broth subculture (which was used as a control) were inoculated. After 72 hours 0.2 ml. of both the control and the DOC flasks were transferred to PPLO agar plates. The plates were incubated for 72 hours, then observed. -h- All cultures were incubated at 37° 0. Experiment II: Solvent 2 was utilized in the toxicity tests. The birds were inoculated with 0.25 ml. of the solvent containing the DOC levels of 0.8, 0.h, 0.2, 0.1, and 0.05 mg. Two tests were run eight days apart on chickens. Two birds were used with each of the DOC levels. The route of inoculation was the infraorbitol sinus of the right eye. Toxicity was measured by inflamation and/or swelling of the sinus. Experiment III: CED-ll, the PPLO strain used in this experiment, was serially passed three times in the allantoic fluid of ten-day-old chicken embryos, incubating five days in each passage, immediately prior to inoculation of the birds. The birds were inoculated intra- trachially using sterile blunt 2% inch lB-gauge needles. The DOC used in this trial was diluted with solvent 2 so that 0.5 ml. contained O.h mg. of DOC. The birds were treated by injecting the DOC into the ab- dominal airsac, alternating sides with each inoculation, using sterile 20-gauge needles. The design of this experiment is shown in Table 2. To determine whether infection had occurred, tracheal swabs on all 39 birds were taken using six-inch sterile cotton swabs twelve days and twenty-two days after inoculation. The swabs were then streaked on PPID agar plates containing one per cent PPLO serum frac- tion with thallium acetate at a concentration of 1:1000 added as an inhibitor. 'When infection was established, twenty-two days after inocula- tion, treatment with DOC was started and the effect of DOC was ascer- tained on the basis of whether PPLO could be isolated two days after the last day of treatment. Isolation attempts at the time of post mortem, were made by the swab method described above and by excising a section of the airsacs. Each portion of airsac was placed in a flask containing 20 ml. of PPLO broth with serum fraction and no in- hibitors; the flasks were incubated for 2h hours at 37° C; the contents 'were then filtered through a Seitz-type S-3 filter. Identification of the PPDO isolates was based upon their characteristic growth and their inability to grow in or on medium not enriched with serum fraction. Experiment IV: TheIM, gallisepticum used in this experiment were isolated in the diagnostic laboratory at Michigan State university from.a field case of CRD. These organisms were typical of PPIO asso- ciated with CRD, as shown by serological, cultural, and pathogenic properties. Prior to inoculation of the birds, the PPIO were serially passed eight times in the allantoic fluid of seven-day—old embryos. Using a blunt h-inch, 20-gauge needle, fouramonth old, apparently diseaserfree leghorn cockerels, found to be negative for'Newcastle: disease by'theIHLI. test, were inoculated intratracheally with 0.5 cc. of pooled PPLO-containing allantoic fluid from.25 twelve-day-old embryos. The birds were housed in isolation pens, with no contact between pens. They were fed a commercial antibiotic-free grower ration. Food and water consumption were measured in all pens begin- ning 22 days preinfection until termination of the experiment. All birds were weighed at the beginning of the experiment and immediately before they were sacrificed. The experimental design is shown in Table 3. Each group of birds (pens 2, 3, and h) to receive treatment with DOCT (Ciba) (25 mg./cc. as commercially prepared) was divided, one half receiving 0.5 cc. of DOCT diluted with 0.5 cc. of a commercial diluent (Ciba) at each treatment and the other half receiving 0.6 cc. of DOCT with 0.h cc. of the same diluent. All treated birds were given DOCT twice, 22 and h3 days after inoculation with the PPUO- containing allantoic fluid. The DOCT was injected into the right side of the breast, the left side serving as a control to determine degree of absorption of the DOCT. Twenty-one and bl days posttreatment, respectively, one-half the birds in each pen were sacrificed and examined for lesions of 0RD. At the same time an attempt was made to isolate PPLO from the abdom- inal, diaphragmatic and thoracic airsacs of the birds. Portions of the airsacs were excised, weighed, and placed in 1 cc. of PPLO broth (Difco) enriched with one per cent serum fraction (Difco) and con- taining thallium acetate in a 1:2000 concentration. The tissue was then ground and three serial tenfold dilutions made. One-hundredth cc. of each dilution was dropped on PPDO agar (Difco) enriched with four per cent serum fraction and containing a 1:2000 concentration of thallium acetate. The plates were incubated at 37° c for 72 hours, after which the number of organisms contained in each 0.01 cc. drop was counted at 1001 magnification. From this the number of organisms per mg. of tissue was calculated. Experiment V: The M. gallisepticum used in this experiment was supplied by Dr. Henry Van Roekel (University of Massachusetts) and designated by him as 19h. This culture has been and has continued to be maintained in young chickens. The inoculum used to produce the disease was harvested from five infected birds by placing the airsacs and tracheas in 115 ml. of PPLO broth (Difco) containing in serum fraction (Difco) and 1800 units of procaine penicillin per ml. One cc. of this material containing approximately 106 organisms was inoc- ulated intratracheally in 18 birds using a blunt h-inch 20-gauge needle. The birds utilized were five-to-six-week-old, apparently disease- free, leghorn cockerels. 'Prior to inoculation, serum plate aggluti- nation tests using a standard PPDO antigen on blood samples from eight of these birds selected at random were negative. The experimental design is shown in Table b. 0n the 2lst day post-inoculation each group of birds to receive treatment with DOCT (pens 3, h, 5, 6, 7) were given intramuscular in- jections of 1.6 ml. of diluent (Ciba) containing various quantities of poor (Ciba) in the right side of the breast. At 0, 7, lb, and 21 days post-treatment, respectively, birds from each pen were sacrificed and examined for the lesions of 0RD. At the some times, an attempt was made to isolate M, gallisepticum from the airsacs of the birds. Isolation, housing, and feeding of the birds werethe same as that described in Experiment IV. Experiment VI: The M, gallisepticum used in this experiment was the culture 19h supplied by Dr. Van Roekel following four passages in young chickens since the previous experiment. The preparation of the inoculum and method of inoculation were the same as in experiment V with the exception that each cc. of the 6 PPID. inoculum contained approximately 1.5 x 10 The birds used in this experiment were four-week-old, apparently disease-free leghorn cockerels. On the day of inoculation and at 7 and 1h days thereafter 20 mg. of DOCT in 0.8 ml. of diluent (Ciba) (a total volume of 1.6 cc.) was injected intramuscularly in the right side of the breast of treated birds. The birds in pen 3 were treated with 1.6 ml. of diluent as a control at the time of inoculation. At 7, 1h, 21, 28, and 35 days post-inoculation, respectively, birds were sacrificed and examined for the lesions of 0RD. At the same time an attempt was made to isolate M, gallisepticum from the airsacs of the birds. Isolation, housing, and feeding of the birds werethe same as that described under Experiment IV. RESULTS Experiment I: The normal growth of all four strains of PPLO used in this study appears. in two distinct colonial forms. One is a large colony, approximately 0.0h-0.05 mm. in diameter, with an entire margin, and a ”grainy” center. This form grows down into the agar. The other is a very small colony, approximately 0.01-0.02 mme in diameter, with an entire margin, a less "grainy" appearing center, and less penetra- tion of the agar than that characteristic of the larger colony form. . DOC in 0.8, O.h, and 0.2 mg. amounts per 20 ml. almost entirely inhibited the growth of the small colonial form of PPLO. The large colonies treated with the same amounts of DOC lost their "grainy" appearance, the margin became jagged, and a reduction in size was noted. Inhibition occurred with 0.1 and 0.05 mgs. of DOC per 20 m1. of medium but to a lesser extent than when treated with the larger amounts. Controls using either DOC solvent showed no appreciable dif- ference in growth when compared with the non-solvent controls. Experiment II: Toxicity was determined by inflammation and/or swelling of the sinus. The toxicity tests, with two exceptions, were negative. The controls using either solvent showed no toxicity. Experiment III: Tracheal rales were noted in the infected birds beginning on the lhth day following inoculation; none were observed in the control pen. A pasty nasal discharge was noted from some of the birds in the infected pen twenty—two days after inoculation. 0n post mortem examination of the birds in group 1, thirty days after -10- -11- inoculation, the gross lesions were typical for CRD. Thirty-three days after inoculation and after the birds had received a total of 2.h.mg. of’DOC the coughing and rales tended to decrease in the pen containing groups 2 and 3, although they did not completely stop throughout the course of this experiment. From group 2, which was posted 38 days after inoculation, four birds did not show the typical lesions of CRD. Their airsacs were clear and the birds appeared to be in good condition. In the six remaining birds of this group the typical cloudy thickened airsacs were present and a mucous exudate was found in the trachea; no pneumonia, however, was noted. Group 3 birds were posted h6 days after inoculation along with the nine con- trol birds. 'Uith the exception of two of the control birds, all of birds examined at this time appeared healthy and showed no lesions. The two control birds showed involvement of the airsacs, but no tracheal exudate and no pneumonia. Results of attempts to isolate PPLO from all the groups of birds both pro and post treatment are shown in Table 6. ‘Experiment IV: Approximately 12,000 organisms per.mg. of tissue were isolated from the infected birds immediately prior to treatment. (See Table 7.) From this table it can be seen that at the first post treahent sacrifice PPID was not isolated from eight of the 18 birds that had been infected and treated (pens 3 and h): from the other ten birds an average of less than 100 organisms per mg. of tissue was re- covered. At the same time, an average of 5,700 organisms per mg. of tissue was isolated from the infected, non-treated control birds (pen 5); all birds sacrificed from this pen were found to be infected. -12- In the final sacrifice, hl days post treatment, PPLO could not be recovered from the infected, treated birds, while an average of 100 PPLO per mg. of tissue was isolated from six of the nine infected, non- treated birds. At this time no PPLO were isolated from the remaining three birds. (See Table 7.) No organisms could be isolated from birds in either of the non- infected groups (pens l and 2) at any time during the course of the experiment. The number of'PPIO per mg. of tissue isolated before and after treatment, shown in Table 7, are presented in graphic form in Figure 1. Except for slight enlargement of the testes, no evidence of tox- icity due to DOCT was noted in any of the treated birds, infected or non-infected. ‘When the birds were sacrificed prior to treatment, the typical lesions of CRD, i. 3., tracheitis and aerosacculitis with an accumu- lation of exudate in the trachea and airsacs, were marked in all of the inoculated birds. Twenty-one days later, when one-half the re- maining birds were sacrificed, the airsacs and trachea of all except two of the infected, treated birds appeared normal; the airsacs of these two birds were slightly cloudy, but no exudates were present. The infected, non-treated birds showed the typical lesions of CRD at this thus. At the termination of the experiment, when all the remaining birds were sacrificed, all birds in the infected, treated groups appeared normal. 'Four of the nine infected, non-treated birds had slightly cloudy airsacs; the rest appeared normal. During the entire -13- experiment, no lesions of CRD were noted in the non-infected birds. As shown in Table 8, average weight gains of the infected, non- treated birds were considerably less than in any of the other four groups of birds. A considerably higher percentage of feed was required to pro- duce a pound of gain in the infected, non-treated birds than was needed in the infected, treated birds when compared with the "normal" controls.(Table 8). All of the DOCT did not appear to be absorbed at the time of the first post treatment sacrifice; the amount of DOCT remaining at the final sacrifice appeared considerably greater than that remaining at the first post treatment sacrifice. No observable difference in effect between the 0.5 cc. and 0.6 cc. dosages of'DOCT was detected throughout the course of the experiment. Experiment'V: The first symptoms of CRD in the inoculated birds occurred seven days post inoculation. Seven days later tracheal roles, coughing, depression, and listlessness were marked in these birds. Coughing did not stop in the treated pens throughout the course of this experiment; however, coughing was considerably alleviated in the treated pens as compared to the non-treated pens during the course of treatment. An analysis of variance (F test) was completed on the data given in Table 9. An.F ratio of 5.001 with 65 degrees of freedom allowed the conclusion that it is improbable that sampling variation alone could account for the observed differences between the treatment group means (Batson, 1956). ~1h- To compare each of the DOCT treated groups of birds with the un- treated, infected control birds (pen 2) the t test was applied. Com— paring the isolation results (Table 9) of pen 2 with pens 5, 6, and 7, with 22, 21, and 22 degrees of freedom respectively, it was found that the above three pens of birds differed significantly from the birds Du pen 2 at the 0.5%, 0.1% and 1.0% levels respectively. According to Randall (1958) if there is greater than a 2% chance that a value is a normal variation (in biological work) then that value is generally not considered significant. The levels of signi- ficance for pens 3 and h are 5% and 2.5% respectively; as they are above the 2% level, the values for these pens are not considered to be significant. Height gains and feed conversion were considerably better in the treated birds than in the non-treated birds (Table 10). The DOCT treated birds gained a minimum of 18% (2.5 mg. DOCT) and a maximum of 39% (20.mg. DOCT) better than the infected untreated controls when compared with the "normal" birds. Furthermore, less feed per pound of gain was required to produce this weight than was necessary for the infected and untreated controls (Table 10). The airsacs of all of the infected, non-treated birds, except one, were extremely thickened and cloudy. The gross pathology of the airsacs of the infected, treated birds was considerably less extensive and marked than the non-treated birds; however, none of the airsacs of the infected birds returned to a completely normal appear- ance during the 21-day treatment period. It should be noted that the “airsacs of ‘the noninfected, -15- non-treated controls remained clear. 'Hith the exception of the noninoculated control birds (pen 1), M, gallisepticum was isolated from every bird sacrificed in this experiment. At the final sacrifice, 21 days after treatment, a slight amount of DOCT remained unabsorbed in one bird in pen 3, one bird in pen h and two birds in pen 5. A greater amount was unabsorbed and was found in the four remaining birds sacrificed in each of pens 6 and 7. No evidence of toxicity was observed in this experiment. The testicular enlargement mentioned in discussing experiment IV was not observed with these birds. The PPID per mg. of tissue isolated post-inoculation, shown in Table 9, are presented in graphic form in figure 2. Experiment VI: Nine days following inoculation the first symp- toms were observed: a slight amount of coughing from the birds in all pens except pen 1, feed consumption slightly off, water consump- tion normal. Six days later the typical 0RD symptoms, tracheal rales, coughing,depression and relative listlessness, were noted in all pens except pen 1, where a slight amount of coughing had started. These marked signs of CRD persisted throughout the course of the experiment in the birds in pens 2 and 3. 'Hith the exception of the birds in pan 1 where marked symptoms were not seen at any time, the symptomatology (i.e. rales and coughing) tended to decrease eight to ten days after treatment although they had not stopped at the term- ination of the experiment. As can be seen from Table 11, treatment -16- appears to reduce the number of infecting organisms a minimum of ten- fold. ‘Height gains and feed conversion were considerably better in the treated birds than in either the non-treated birds or those treated with diluent (Table 12). Due to a shortage of pen space uninfected controls were not maintained for this experiment. The “normal" bird figures used in Table 12 have been taken from."Feeding Poultry" by Heuser (1955) and represent the normal gain and feed per pound of gain expected from four-week-old leghorns maintained for 35 days, the length of this experiment. The DOCT treated birds gained approximately the same as the Heuser "normals" and 0.69 pounds more than the birds not treated with DOCT. The feed conversion results were not as good when the DOCT treated birds were compared with the “normals" requiring an average of 19% more feed per pound of gain. However, the birds not treated with DOCT required a minimum of 96% more feed per pound of gain than the "normal" birds. 'iith the exception of one bird treated at the time of infection (pen 1) and sacrificed 11; days later, 31. gallisepticum was isolated from every bird sacrificed in this experiment. DOCT was found in the right side of the breast of all treated birds (excluding diluent treated controls) sacrificed in this experi- ment except for two birds treated at time of inoculation (pen 1) and sacrificed 35 days later. The other two birds in this pen sacrificed at the same time had very slight amounts of DOCT remaining. It appears that a minimum of 35 days is required for absorption of 20 mg. of DOCT diluted to 1.6 ml. -17- No evidence of toxicity was observed in this experiment. The testicular enlargement remarked in experiment IV was not noted with these birds. The PPLO per mg. of tissue isolated post-inoculation, shown in Table 11, are presented in graphic form in figure 3. Treatment with 1.6 ml. of diluent (Ciba) produced results essentially no different from those obtained with complete absence of treatment in the infected, non-treated control birds (see Tables 11 and 12, figure 3). DISCUSSION From.the results previously presented it can be said that DOC, a mineralocorticoid adrenal hormone, appears to behave as an anti- microbial agent in.zit£g, and in_zizg in the chicken. In the mammal, DOC activity is said to be limited to an influence on sodium, potas- sium.and water metabolism (Beckman 1958), any benefit accruing to an animal treated with this hormone being attributed to the "salt effect." In large doses DOC has been shown to be toxic to mammals. In experiment II infraorbital injections of DOC did not elicit toxic responses when 0.25 ml. containing O.h mg. or less of the in- oculum was used. Toxicity was noted when 0.8.mg. of DOC with either of the solvents was used and was characterized by both inflamation and swelling of the area around the infraorbital sinus of the birds. The author believes, however, that this reaction may not be entirely due to DOC toxicity, as 0.5 m1. of the solvent containing 0.8 mg. of DOC was used in this case. Thus, a bulging of the skin covering the sinus was produced mechanically. The rest of the birds were inoculated with only 0.25 ml. of the preparation and no swelling was produced. ‘Hhen a total of 6.0 mg. of DOC per bird was administered via the abdominal airsac over a period of 2h days no deleterious effects upon the birds were observed (experiment III). ‘Iith the exception of a slight testicular enlargement when a total of either 25 mg. or 30 mg. of DOCT had been injected into the breast of both infected and noninfected birds, there was no evidence of toxicity -18- -19- due to this hormone (experiment IV). No testicular enlargement or other evidence of toxicity was noted in experiments V and VI. This difference in glandular reaction may be due to the 2.5 and 3.months age difference between these birds and those in experiment IV at time of treatment. This apparent lack of toxicity for the chicken is supported by the work of both Boas (1958) and Conner (1959) who have reported a similar conclusion. DOC appears to inhibit the growth of PPID ig_!it£g. In experi- ment I not only was the density of the colonial population markedly reduced but the appearance of the surviving colonies was altered and did not resemble the typical colonial morphology. The fact that D00 is an effective antimicrobial agent in 13252 suggests that it also acts directly upon the microorganisms in 3112. Therefore, the bene- ficial therapeutic activity of DOC is probably not simply a result of the physiological response of the host. This conclusion seems to have merit in the light of the work of Maxwell 93 a_l. (1960) who have shown that l-androstene -3, l7 dione is lethal, not just inhibitory to growing cells of g. fragilis. All of the in zizg_experiments III, IV, V, VI indicate that DOCT effectively inhibits the organisms associated with CRD at least to the degree that the pathological responses of host (ire., sinusitis, tracheitis and aerosacculitis with exudate accumulation in the airsacs) are reversed. If the chief cause of the pathology in CRD is indeed the secondary invaders, especially Escherichia coli and gseudomonas aerugi- nosa as suggested by many workers in the field, then any effect of DOCT in alleviating this pathology would be very difficult to explain, as -20- both of these organisms are Gram-negative. As pointed out previously, Lester and Hechter (1958) have shown that Gram-negative organisms are generally insensitive to this hormone. Since these experiments have shown that DOCT is effective in the treatment of CRD, it leads one to suspect that the secondary invaders may be saprophytes and when the primary etiological agent, PPLO, are reduced in numbers the normal de- fense mechanisms of the host are sufficient to overcome the secondary invaders. Price 93 91., (1957) in their work with can practically ig- nored PPID when measuring the effect of terramycin on the disease (as was done in the present work as far as the secondary invaders are con- cerned). Their work was concentrated on measuring the response of the secondary invaders and they showed terramycin to be fairly effective in controlling CED. In the light of the present work and that of Price ‘23 31, one may well wonder then about the role of both PTID and the secondary invaders in CRD. Fron.figures 1, 2, and 3 it can be seen that DOCT reduces the nor- mal recovery time of birds affected with CRD. As the name implies CRD is a chronic disease with a course varying from a minimum of about 30 days to a maximum of about 180 days. Any significant shortening of the term of the disease would, of course, be highly desirable to the poultryman. ‘Ueight gains in the infected and DOCT treated groups of birds in experiments IV, V, and'VI were respectively 80, 83 (at the 20 mg. level of DOCT) and 102 (average of 3) per cent of normal, the latter increase of some 20 per cent probably being due to the earlier commencement of treatment (Tables 8, 10, 12). -21- -00nsidering all three experiments we find that treated birds gained an average of hl per cent more weight than non-treated birds when compared with 'normal' controls. Feed conversion (i.e., the amount of feed utilized to produce 1 pound of gain) in the infected and DOCT treated groups of birds in experiments IV, V, and VI were an average of 7% higher (more feed) than the normal controls, while the infected, non-DOCT-treated birds required an average of twice as much feed (103% more) as the nonmal controls to produce 1 pound of gain. The noninfected treated birds (experiment IV, Table 8) needed 9.8% less feed than the normal birds to produce a pound of gain. The fact that better feed conversion is obtained in those birds nonin- fected but treated suggests that DOCT may act as a growth stimulant in normal birds. However, considerably more experimentation would be necessary to establish this point. The use of number of isola- .able organisms as an indication of degree of infection and rate of recovery or even of just sickness and health.may be questionable. In experiments IV, V, and VI about 12,000, 3700, and 6000 organisms, respectively, per mg. of airsac (see Tables 7, 9, and 11) were iso- 1ated from the infected, non-treated control birds. However, little or no difference in degree of gross pathology was noted between the birds of the three different experiments. The pathology was as marked in the birds in experiment V as in those of experiment IV even though fourfold the number of organisms was isolated from the birds in experiment IV as from the later experiment. Differences in the results of these experiments may perhaps be -22- attributed to differences in age, susceptibility of the birds, or numbers and virulence of the organisms in the inoculums used. In addition, Adler and Shifrine (1960) have already pointed out many of the difficulties encountered when isolating g. gallisepticum. If one uses the number of organisms isolated as an indication of the effect of’DOCT in 0RD, then feed consumption, weight gain, and gross pathology should probably be correlated with it. In these ex- periments when the number of isolatable infecting organisms decreased with DOCT treatment then the gain increased, feed per pound of gain decreased and gross pathology was considerably less marked. 0n the basis of the results presented for these four factors it appears that DOCT is effective in the treatment of CRD. In experiment IV no organisms could be isolated hl days post- treatment from previously infected and treated birds (Table 7). 'HhetherIE, gallisepticum were totally absent or whether theyhmay have been present but in too few numbers to be isolated by the meth- ods used is unknown. In experiments V and VI organisms were isolated from.all infected and treated birds at the termination of the experi- ments; however, these experiments were terminated earlier than experiment IV. The question arises as to whether DOCT is lethal or merely'in- hibitory'to fl. gallisepticum, and as to whether the disease will recur when treatment is stopped. The answer to neither of these points can be found in the data presented. Further work is necessary to determine lethality and recurrence. Cholesterol has been shown to be a growth requirement for certain -23- PPID. Considering the recent work of Smith and Rothblat (1960), who have found that the major portion of this sterol was located in the cell membrane and that only steroids possessing a side chain similar to cholesterol are utilized, indicating that the adsorption of the steroid occurs through the 8 carbon side chain, one may speculate as to the mode of action of DOCT. The present work has shown that DOCT inhibits PPID both.ig_zizg and ig_!it£g, Cholesterol and DOCT differ structur- ally in their side chains and at the cholesterol 3-fi-hydroxy site. The specificity of the 349-hydroxy group for growth has been shown by Smith and Eynn (1958). Its nonrequirement for initial adsorption (Smith and Rothblat, 1960), and the cholesterol esterase activity of the cells (Smith, 1959) suggest an additional metabolic function particularly for parasitic strains (Smith, 1960) as well as a probable structural role (Smith and Rothblat, 1960). The utilization of cholesterol may be prevented by DOCT in much, but not necessarily, the same way that sulfas interfere with the uti- lization of para-aminobenzoic acid. Further evidence along this line is suggested by the fact that saprophytic PPID (i.e. those not re- quiring cholesterol) are not inhibited by DOCT (Padgett and Schoenhard, unpublished data). Negative evidence concerning this hypothesis is found in the work of Lester and Hechter (1958) who have shown that organisms notdependent on cholesterol are inhibited by DOC. The mechanism of action of DOCT is unknown. The above hypothesis ties together some information about PPLO, cholesterol and DOCT, but further work will be necessary to determine the interactions of them. -2h- It is hoped that the present thesis will stimulate further research on the subject. SUMMARY Avian pleuropneumonia-like organisms (PPID) were inhibited by 0.h.mg. of deoxycorticosterone in zit£g5 the growth characteristics of surviving colonies were modified. In three separate ig_zivg experiments avian PPLD were inhibited by deoxycorticosterone trimethylacetate; symptoms and pathology of chronic respiratory disease in chickens were reduced, with a resultant increase in weight in infected, treated chickens when compared with infected, non-treated control birds. Toxicity of this hormone for the chicken, with one exception, was not observed at the dosage levels used in these experiments. -25- BIBLIOGRAPHY Adler, H. E., and Shifrine, M., 1960, Nutrition, Metabolism, and Pathogenicity of Mycoplasmas.: Ann. Rev. of Micro. 1h, 1141-160. Batson, H. C., 1956, An Introduction to Statistics in the Medical Sciences.: Burgess Pub. Co., Minn., Minn., 25. Beckman, M., 1958, Drugs Their NatureW.: W. B. Saunders Co., Phil., Pa., 591-593. Bergey's Manual of Determinative Bacteriology... Seventh Edition, 1957, Williams and Wilkins Co., Baltimore, Md. Boas, M. P., 1958, The Effect of Deoxycorticosterona Acetate on Testis Size and Function in the Cookere1.: Endocrin., 63, 323-328. Boone, M. A., Richey, n. J., and Morgan, 0. L, 1957. Effects of Antibiotics on Egg Production When Chickens are Naturally Infected with Chronic Respiratory Disease.: Poult. Sci., 36, 13h0-13hh. Chalquest, R. H., and Fabricant, J., 1959, Survival of PPID Injected Into Eggs Previously Dipped in Antibiotic Solutions.z Avian Diseases, 3, 257-271. Conner, M. H., 1959, Effect of Various Hormone Preparations and Nutritional Stresses in Chicks.x Poult. Sci., 38, l3hO-l3h5. Cover, MI. 3., 1955, The Therapeutic Use of Antibiotics for Chronic Respiratory Disease in Three Laying Flocks.: Poult. Sci., 31;, Dardas, Richard B., 1959, A Comparison of the Morphology, Fermenta- tion, and Serology of Eleven Mycilasma and two Corynebacterium '32. Reversion Products.: A Thesis, Mich. State—L Univ. Delaplane, J. P., and Stuart, H. 0., l9h3, The Propagation of a Virus in Bnbryonated Chicken Eggs Causing a Chronic Respiratory Disease of Chickens.: Am. J. Vet. Res., )4, 325-332. Domermuth, C. H., 1958, In V_i__tro Resistance of Avian PPID to Anti- bacterial Agents. : Avian Diseases, 2, M24416. Edward, D. G., ff. and Fitzgerald, W. A., 1951, Cholesterol in the Growth of Organisms of the Pleuropneumonia Group.: J. Gen. H101‘0biOle, s, 576-586e -26.. -27- Edward, D. G., and Kanarek, A. D., 1960, Organisms of the Pleuro- pneumonia Group of Avian Origin: Their Classification into Species.: Ann. N. Y. Acad. Sci., 79, 696-702. Fahey, J. 3., and Crawley, J. F., l95h, Studies on Chronic Res- piratory Disease of Chickens III. Egg Transmission of a Pleuropneumonia-like Organisms.: Canad. Jour. Comp. Med., 18, 67-75. Fahey, J. E., and Crawley, J. F., 1955, Studies on Chronic Res- piratory Disease of Chickens VI. The Effects of Antibiotics on the Clinical and Serological Course of CRD.: Canad. Jour. Comp. Med., 19, 281-286. Gross, Y. B., and Johnson, E. P., 1953, Effect of Drugs on the Agents Causing Infectious Sinusitis of Turkeys and Chronic Respiratory Disease (Air-Sac Infection) of Chickens.: Poult. Sci., 32, 260-263. Hamdy, A. H., Ferguson, L. C., Sanger, V. L., and Bohl, E. H., 1957, Susceptibility of Pleuropneumonia-like Organisms to the Action of the Antibiotics Erythromycin, Chlortetracycline, Hygromycin, Magnamycin, Oxytetracycline and Streptomycin.: Poult. Sci., 36, 7h8-75h. Heuser, c. F., 1955, {ceding Poultry.: Second Edition, wiley and Sons, N. 2., 350. Jefferson,‘H3 C., and Sisco, G., 1959, The Response of As er illus niger to.Added Steroids.: Federation Proc., 18, 253. Kiser, J. S., Popken, F., Clemente, J., 1960, Antibiotic Control of an Experimental Mycoplasma Gallinarum (PPID) Infection in Chickens.: Ann. N. I. Acad. Sci., 79, 593-607. Iecce, J. G., and Sperling, F. G., 1955, Chronic Respiratory Disease III. The Effect of Treatment on the Pleuropneumonia-like Or- ganism Flora of Avian Tracheas.: Jour. Am.'Vetn Med. Assn., 127, Sh'Séo Lester, G., and Hechter, 0., 1958, Effect of Deoxycorticosterone on the Growth of'Microorganisms.: J. Bacteriol., 76, 365-367. Lester, G., Stone, D., and Hechter, 0., 1958, The Effects of Deoxy- corticosterone and Other Steroids on Neurospora crassa.: Arch. Biochem. Biophys., 75, 196-21h. ‘Markham, F. S., and‘Wong, S. C., 1952, Pleuropneumonia-like Organ- isms in the Etiology of Turkey Sinusitis and Chronic Respira- tor, 13136836 Of ChiCkenSo: P011113. 501., 31, 902-90’40 -28- Maxwell, E. S., McGuire, J. S., and Tomkins, G. M., 1960, Antibiotic Effect of Steroids on Saccharomyces fragilis and the Isolation of a Resistant Mutant.: Bacterial, 80, 1-6. Nelson, J. B., 1935, Cocco-bacilliform.Bodies Associated With an Infectious Fowl Coryza.: Science, 82, hB-hb. Olesiuk, o. M., Van Roekel, H., and Beninato, L. P., 1957, Influence of Chemotherapeutic Agents on Experimental Chronic Respiratory Disease in Chickens and Turkeys.: Poult. Sci., 37, 383-395. Olesiuk, O. M., and Van Roekel, H., 1959, The Effects of Antibiotics on Experimental Chronic Respiratory Disease in Chickens.: Avian Diseases, 3, h57-h70. Olson, N. 0., Shelton, D. C., and Heishman, J. 0., 1959, Control of Chronic Respiratory Disease I. The Effects of Chlortetracycline (arc) and Terephthalic Acid (TPA).: Avian Diseases, 3, hh3-h51. Padgett, G. A., and Schoenhard, D. E., 1959, Effect of Deoxycorti- costerone on the Agent of Chronic Respiratory Disease.: M.S.U. vets , 20, 27-310 Perek, M., and Perk, K., 1958, Studies of the Effect of Dihydro- streptomycin Propylene-Glycol Spray on Chronic Respiratory Disease in Chickens.: Avian Diseases, 2, 269-278. Price, K. E., 20111, 2., Hardie, W. B., and Gallian, M. J., 1957, Respiratory Tract Flora in CRD and Effect of Antibiotics in the Feed.: Poult. Sci., 36, 219-225. ‘ Price, K. E., 20111, 2., Hardie, W, B., and Owens, R., 1958, The Influence of Penicillin and Oxytetracyline in the Ration on Mortality, Lesions and Predominating Microflora in Experi- mental CRD.: Poult. Sci., 37, h72-h78. Randall, J. E., 1958, Elements of Biophysics.: Year Book Pub. Inc., Chi., 111., III. Smith, P. F., and Lynn, R. J., 1958, Lipid Requirements for the Growth of Pleuropneumonia-like Organisms.: Bacteriol., 76, 26h-269. Smith, P. F., 1959, Cholesterol Esterase Activity of Pleuropneumonia- like Organisms.: Bacteriol., 77, 682-689. Smith, P. F., and Rothblat, G. H., 1960, Incorporation of Cholesterol by Pleuropneumonia-like Organisms.: Bacteriol, 80, 8h2-850. Smith, Paul F., 1960, Nutritional Requirements of PPDO and their Rela- tion to Metabolic Function.: Ann. N. Y., Acad. Sci., 79, 508-520. -29- Van Roekel, H., and Olesiuk, o. M., 1953, The Etiology of Chronic Respiratory Disease.: Proc. Am. Vet. M. A., 289-303. Van Roekel, H., Gray, J. E., Shipkowitz, N. L., Clarke, M. K., Enchini, R. M., 1957, Etiology and Pathology of the Chronic Respiratory Disease Complex in Chickens.: Exp. Sta. Bull. h86, Univ. Mass. white-Stevens, R., and Zeibel, H. G., 195h, The Effect of Chlor- tetracycline (Aureomycin) on the Growth Efficiency of Broilers in the Presence of Chronic Respiratory Disease.: Poult. Sci., 33, 116h-ll7h. weng, S. C., and James, C. G., 1953, The Susceptibility of the Agent of Chronic Respiratory Disease of Chicken and Infectious Sinus- itis of Turkeys to Various Antibiotics.: Poult. Sci., 32, 589- 593. Yamamoto, R., and Adler, H. E., 1956, The Effect of Certain Anti- biotics and Chemical Agents on Pleuropneumonia-like Organisms of Avian Origin.: Am. Jour. Vet. Res., 17, 538-5h2. Hmdwm H modHUPOdwom one connedoesmvmodpo wmepeu some as 49¢Ho. Hm oeo. one pp dado so Ease: .2. seconds ass senses on 3.8 on .eflfiflpmf I sees a. has a. me. .. . a. Em zed MW: mas-mega Mammal 20+. was moses-33.. fiance: zed mu... mos—mares uncle“: mmodpdo mmweodpdc wade Hmodpdm Mammndpdo ;¢edm moanwdm HammedwoHe HomemaweHo . e . assessesopse . hope mammal 20¢ NH: moammomd ¢H.m.~ out. _ u m mo.m.u asp. » n _m ae.m. man. u a n: ee.w. _. zcaonmnOw _:e_oonam zcauow ca .smoonem w_amu eases-sauce _sn.nenau «can. zcaooe om «_wmu n. ..>.1. a were .amonnon >._esno_n m.c.m w. >.m. u >__o:no_n m.c.m :. zen ozonxom - nedem d. 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Hnwmodma. moon _zmwmwé omHm one noose : mp0, new sta Hummumm 00:¢H0Hu . on «an n A Qme oqes deuaeHndHom Hm possum H: possum azouamHs oopHHOHm azoeSmHa oomHHOHm H. ., zoanmeHma H.mm .mm nu In zeodummdma m. . mosHbmmodma H.0m .mw HOHR nw.m& Hammaoa ; 0 one :. Hseccaca H.0ms .mws moms eHm.0ma esmmdoa -: m. ,, Huwmoama .00 :1 :0m amm.mm zoodsmmdma . abdmwmmm om mean 0 one : Table 9. Average number of PPLO per mg. of airsac per group of birds at each time of sacrifice. Experiment V. Pen No. and Time Treatment No. of Average.No. of of Sacrifice Birds PPLO Per mg. Airsac 1 Pretreatment 2 O 2 3 h 5 6 7 Pretreatment 9 3,350 7 days Posttreatment 1 O h 0 2 .0 . h 3,300 3 2.5 mg. 1; 2,000 b 5 mg. h 550 5 10 mg. b 1,200 6 20 mg. )4 1100 7 110 mg. )4 1,600 114 days Posttreatment 1 0 h 0 2 0 h 2,500 3 2.5 mg. 1; 650 h 5 mg. 11 1,100 5 10 mg. 11 330 6 20 mg. 3 380 7 140 mg. h 350 21 days Posttreatment 1 O h 0 2 0 h 3,600 3 2.5 mg. h 320 h 5 mg. 11 lho s 10 mg. n 120 6 20 mg. 11 220 7 1.0 mg. 11 380 Host H0. 040300 £00.03. mambo mun ween oondmsuHom. 53503 d. . Home 20. edema—.80 .3de .3de N on $5 when new. a Hosea-ma 0b .. no.0 HmH 03:. H3.— flmHmse £00.03 0mg ”250 on... Moon 08. 0050 .UcmoandHon momeHbooon.. commence mmHs 00 0:0 mzosJ . 00pm omHo odes . : HmHHoov Ho 005000 Ho nausea immH oomeonms. Huuuomnam axousmHm.uob«u0Hu H. - - - . aoonwooema 0 0.00 H.00 In: 0.00 as- acacsomwoa .. A m. success 0 0.5 .3. E 050. H000 mowHHomHaa .. .- u. . 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