€LIN|COPATHQLOGICAL CHANGES IN CATTLE, SHEEP. ANS RABBITS CAUSED BY A NORTH DAKOTA BOVINE ViRAL ESOLANT Thesis for The Degree of D“. D. MICHIGAN STATE UNIVERSITY Hansjakob Rothenbacher 1962 THESHS This is to certify that the thesis entitled CLINICOPATHOLOGICAL CHANGES IN CATTLE, SHEEP, AND RABBITS CAUSED BY A NORTH DAKOTA BOVINE VIRAL ISOLANT. presented by Hansjakob Rothenbacher has been accepted towards fulfillment of the requirements for Ph.D. degree in Veterinary Pathology C’QMW Major! professor Date June 18, 1962 0-169 LIBRARY Michigan State University ABSTRACT CLINICOPATHOLOGICAL CHANGES IN CATTLE, SHEEP, AND RABBITS CAUSED BY A NORTH DAKOTA BOVINE VIRAL ISOLANT by Hansjakob Rothenbacher Mucosal disease, a sporadic but highly fatal condition in cattle resembling rinderpest has been recognized for the past ten years. In spite of extensive research efforts its cause and mode of transmission have remained unknown. In 1958 the isolation of a virus from fecal material of affected cattle was announced and it was considered the causative agent. The characterization of this new bovine viral isolant-- the North Dakota mucosal disease virus--was attempted in this dissertation. Studies included clinical, hematological, immunological, serological, gross and histopathological obser- vations on experimentally inoculated cattle, sheep, and rabbits. All experimental animals proved susceptible to the bovine embryo kidney-cell-culture propagated virus. The experimental infection in cattle was characterized by an acute monophasic febrile reaction combined with a severe leukopenia and heteropenia. Other clinical signs of the experimental infeCtion included partial anorexia, nervousness, tachycardia, increased respiration, ocular discharge, depression and Hansjakob Rothenbacher constipation. The experimental infection in cattle lasted from six to ten days and resulted in the production of virus neutralizing antibodies measurable in tissue culture systems and in immunity to challenge 14 days after the initial inoculation. The major gross and histOpathological lesions produced by the North Dakota virus in experimental animals were con- fined to the adrenal glands, the gastroin estinal tract, the lymphatic tissues, the kidneys, the liver, and the bone marrow. The epitheliotropism of the virus was evidenced by inflammatory and necrotizing changes of the epithelial tissues of the gastrointestinal tract and of the parenchy— m matous organs. Inflammatory, necrotizing and regaessiv } changes were noted in the lymphatic system. Simila» re .. U A PBS“ S b 0Q sive changes were also seen in the bone marrow. 7— Contact transmission did not occur. experimental cattle inoculated with fresh blood and organ emulsions of infected animals failed to contract the infection. Three or more inoculations of calves with organ emulsions resulted in immunity to challenge with the tissue—culture—grown virus. Numerous virus isolation attempts failed to reveal the presence of the virus in fresh blood, nasal and ocular swabs, mucosal scrapings and fecal specimens of infected cttcle. and lungs H (I) (T) *1 \o |r_.l P] Q :x m ‘4 H) The virus was recovered from the sp of two calves six and nine days, respectively, after virus inoculation. Hansjakob Rothenbacher The experimental North Dakota virus infection in cattle did not resemble any of the classical descriptions of Iowa mucosal disease, malignant catarrhal fever, Ume disease, parainfluenza-3 respiratory infection, virus diarrhea, or infectious bovine rhinotracheitis. h The experimental North Dakota virus infection in sheep and rabbits was similar but milder in degree and of shorter dura ,ion . CLINICOPATHOLOGICAL CHANGES IN CATTLE, SHEEP, AND RABBITS CAUSED BY A NORTH DAKOTA BOVINE VIRAL ISOLANT by HansJakob Rothenbacher Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Veterinary Pathology 1962 ACKNOWLEDGMENTS The author wishes to express his sincere gratitude for the kind guidance and assistance offered by Dr. R. D. Earner during the course of this work. The benevolent cooperation of Dr. I. A. Schipper in supplying the North Dakota virus isolant is herewith appreciated. The helpful collaboration of Dr. C. H. Cunningham and Mrs. C. E. Church of the Virology Section, Department of Microbiology and Public Health made these studies possible. Sincere thanks are due to Dr. C. C. Morrill for his aid in the preparation and review of this manuscript. The help of Mrs. Nancy Anderson, Mrs. Marge Landtiser, Mrs. Joyce Trier, Mrs. Anne Mahr, Mrs. Betty Myers, and Mrs. Carolyn McLean in the processing of the tissue sections and in the hematological studies is gratefully acknowledged. TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . INTRODUCTION . . . . . CHAPTER I. REVIEW OF LITERATURE General Remarks . . Viral Isolancs from Syndromes of the Virus Diarrhea ‘HWCO‘dl Disease Complex with Probable Viral Etiolo ogy. Syndrome of the Virus Diarrhea-Muss , Disease Complex with Questionable o Undetermined Etiology - . Summary. . . . II. MATERIALS AND MET “O S . . . . . . Materials Used for Ar~mml Inoculation. Experimental Animals . Hematological Studies . . Virus Inoculations and Transmission Clinical Observations . . . Gross Pathological Observations and Necropsy Procedure HistOpathological Oc . . . Serological Studies. . . . . . . Bacteriological Examinations. . . Other Post-Mortem Observations G O 0 Stud 0) (ll < Q) d“ ° F“ O :1 (/1 General Clinical Ob servatior.s in Virus~ Inoculated Experirnental Ania als . . Cattle . . . . . . . . . . Sheep. . . . . . . . . . . Rabbits . . . . .. . . c . Hematological Findings. . . . . . Transmission Attempts . . . . . . Routes of Transmission and Infectious Agent . . . . . . . . . . Contact Transm‘ssion Studies . . . Koch‘s Postulates. . . . . . . 9 CD (IN (D 4;. ‘\ A.“ R) h) -4 F1 TD (D U) LA) LU m R) «art—J OK) (1‘) (A) LA) LA.) W LL) CO CD CD ‘1 U1 b. +— D D U) C) (a :- .IT.‘ 41‘ em #4:; 9“. —+._ C7 O‘\ Q \ ‘V‘ \* \—' CA ‘1 Chapter Gross Pathological Findings Cattle. . . . . Sheep . Rabbits HistOpathological Findings. Cattle. . . . . . Sheep . Rabbits Other Post— Mortem Findings Serological and Immunological Finding IV. DISCUSSION Transmission Studies. Gross and Histopathologic a1 Findings Lymphatic Tissues . Bone Marrow. . . Respiratory Tract. Adrenal Glands. . Summary of Discussions V. SUMMARY AND CONCLUSIONS. BIBLIOGRAPHY APPENDICES Ki) \0 ‘0 ‘21) .) KO \0 \JW trout») ID H 0 k0 \O \f {\O N P‘“ H C) TABLE 10. ll. 12. LIST OF TABLES North Dakota virus infection of a 4-months- old calf. Average hematological data from healthy 1- to 2-months-old calves before and after North Dakota virus inoculation Average hematological data from healthy 3-month- old calves before and after North Dakota virus inoculation. Hematological and clinical changes in a typical North Dakota virus experimental infection of a 3-months-old calf. Hematological changes 72 hours after North Dakota virus inoculation of 3-month—old calves with clinical signs of pneumonia and coccidiosis. . . . Average hematological data from healthy 4- to 8—months—old calves before and after North Dakota virus inoculation Hematological and clinical changes in a typical North Dakota virus experimental infection of a 4-months-old calf. Hematological data from four yearling cattle be— fore and after North Dakota virus inoculation North Dakota virus infection of a yearling heifer . . . . . . . . . . . . Hematological and clinical changes in a typical North Dakota virus experimental infection of a l-year—old bull Hematological and clinical changes in a typical North Dakota virus experimental infection of a 2-year-old cow. Hematological and clinical changes in a l-day— old calf after North Dakota virus inoculation PAGE 42 45 46 47 48 49 SO 51 53 54 55 TABLE 13. 14. Average hematological data from healthy adult sheep before and after North Dakota virus inoculation. Average hematological data from healthy adult rabbits before and after North Dakota virus inoculation. vi PAGE 56 57 LTQ’T‘I C) .111 131 r—i C.) h (1) FIGURE PAGE 1. Swollen jeguno-ileal lvmph node plus edema and hyperplasia of Peyer‘s patches as seen from serOSal surface. . . . . . . . . . . 111 2. Swollen and edematous mesenteric lymph node with corti::al and subcapsular ecchym oses . . lll 3. Linear demarkation hemorrhage along the lateral edge of a jejun nal Peyer‘s patch as seen from the serosal surface . . . . . . 111 4. SubcapSu 1ar and cortical ecchymotic hemor— rhages in a poscerior jejunal lymph node . . 111 5. Cortical ecchymotic hemorrhages in a congested and edematous ileocecal lymph node . . . . 111 6. Cortical andparenchymal hemorrhages in edematous and congested mesenteric lymph nodes lll 7, Hemorrhagic marbling in a Peyer‘s patch of the Jejunum . . . . . . . . . . . . 111 8. Ecchymotic hemorrhages in a hyperplastic jejunal Peyer s patch. . . . . . . . . 111 9. Severe catarrhal to hemorrhagic enteritis of the posterior jejunum, and ileum (above). Less affected cecum and colon below . . . . 113 10. Hemorrhagic-necrotic seam around the ileocecal ostium . . . . . . . . . 113 11. Patch* hemorrhagic congestion of the ileum; petechial hemorrLag s in the mucosa of the ileocecal ostium and surrounding cecum . . . 113 12. Ecc hymotic hemorrhages and cys stic degeneratioi in the mucosa of the cecocolic junction. . . 113 13. Petechial hemorrhages in the cortex of a k:idney0 O O I I O 0 O O O O I O O 113 14. Subcapsular suffusion in the adrenal gland. . 113 17. 18. 19. 20. 24. 25. 26. 28. Rumen: focal hyperkeratotic and prolifera- tive changes in the mucosa. H. & E. x 200. . Jejunum; catarrhal enteritis with necrosis and denudation of surface epithelium. HO & E. X 200 O O O 0 O O 0 O O O O Posterior jejunum: catarrhal enteritis with inflammatory hyperemia of capillaries in den‘dded xJ'j_]..lj.c Ho & E. X 200 o o o o o o Lymph node: hemorrhage in reaction center showing lymphocytic depletion. H. & E. x 200. f l3nm phoid cell n d reaction center. ED X 200 O I O O 0 O I I O O Q Spleen; necrotic focus in react ' n center with heterophil ic cell infiltration. H. & E. x 200 . . . . . . . . . . . Lymph node: Ter i r7 lymph nodule: marginal sinus congested w t. lymphoid and reticulo- histiocvtic cell is rents. Capsule shows thinning and distention. H. & E. x 200. . . 5'“ p— r(I) H; p) Lymph node: conglomerated lymphoid and reticulohistiocytic cells. H. & E. x 200 . . Liver; focal areas of reticuloendothelial and lymphocytic infiltr rations in the vicinity of pyknotic parenc hyma 1 cells. H. & E. x 200. Liver: focal area of parenchymal necrosis with reactive ret icu loendothelial cell proliferation. Sinusoidal spaces show distention. H. & E. x 200 . . . . . . . Kidney: shruxflcen glomerular tuft and dis- tended Bowman 5 capsule. Focal interstitial lymph ocytic infiltration. H. & E. x 200 . . Kidney: thickened Bowman‘s capsule and focal lymphocytic infiltration. H. & E. x 200 . . Adrenal: small necrotic focus in the central zona fasciculata. H. & E. x 200 . . . . . Adrenal: pattern of vacuolar cell degener- ation in the peripheral zona fasciculata. H. & E. x 400 . . . . . . . . . . . V111 PAGE 115 115 116 116 117 118 118 119 120 120 121 30. PAGE Adrenal: foci of coagulation necrosis in the zona fasciculata with reactive lymphocytic and heterophilic infiltrations. H. & E. x 200 . 122 ands of darkly staining cells in iculata with hemogeneous pyknotic & E. X 400. o o o o o o o t 1.22 Adrenal; str the zona fasc nuclei. H. INTRCDU CTTCN The name Chivers (1953) for an apparently new disease syndrome recog- ota (Scm pper and EJ. 5 (D 1.4 \ 0 L51 H H 3 Z O P—j (T .3” U m FR nized in Iowa s Noyce, 1959c) possibly since 1945, and in other states for several years prior to tr e an ove n1entioned report in 1953 Pritchard (1955) stated that the difference bet ween the easily transmissible virls diarrhea ar d mucosal disease was quantitative rather than quali at i e, though he errone— ously thought the described so-cal led ”lndiana” virus diarrhea (Pritchard e§_al., 1956) to be different from the ed by Clafson et 1. (1946) in —.——. (T. virus diarrhea earlier regmo New York. it was later proved by Gillespie and Baker (1959 that the existing strains of Indiana and New York virus diarrhea were identical. With the increasing recognition of clinically and ('1‘ pathologically similar ransmissible disease syndromes in this country by Wheat t a . (1954) in California; Hoag t al. (1956) and Rooney (1957) in Virginia; Huck (1957), Dow et a1. (1956) and Jarrett (19 8) in En and; Hedstrom and \J1 (W }_J \ Isaksson (1951) in Sweden; Feinoers (1959) in Holland; and \ Johnston (1959) in Australia--to name ju a few-—the term (0 "mucosal disease complexH has been u sed by many author s to 2 include the "Iowa" type mucosal disease and the easily trans- missible virus diarrheas resembling the description by Olaf- son _t_a1. (1946). Due to the similarity of both mucosal disease and virus diarrhea to the potentially dangerous foreign disease, rinderpest, extensive investigational work has been carried out in several states to find exact means of differentiation. To date, four doctoral dissertations have been written des- cribing the clinical, gross, and microscopic pathology of the Iowa-type mucosal disease (Ramsey, 1956; Whiteman, 1960; Trapp, 1960; and Bajwa, 1961). In spite of vigorous research efforts the cause of this mucosal disease is still an enigma and all attempts to experimentally reproduce the disease seen in the field, or isolate its etiological agent have failed to yield conclusive results. Serological studies have shown ( lafson and Rickard, 1947; Ramsey, 1956) that mucosa disease and virus diarrhea have no antigenic and/or immuno- logical relationship to rinderpest. Schipper and Noyce (1959A) and Noyce and Schipper(l959) announced the isolation of a mucosal disease agent in bovine embryo kidney cell culture. This was the first report of a cytopathogenic virus isolated repeatedly from field cases of mucosal disease, and subsequent investigations by Schipper and Noyce (19593) and Earner e_ a1. (1959) indicated a possi- ble etiological relationship of this virus to the mucosal disease syndrome. 3 It was the purpose of this investigation to character~ ize this North Dakota mucosal disease virus by its clinical, gross, and microscopic pathology produced in calves, sheep, and rabbits. The virus was generously made available by the original investigators to Dr. R. D. Barner under whose guidance this work was conducted. CHAPTER I REVIEW OF THE LITERATURE General Remarks uring the past few years many "new" viral agents have been isolated from the bovine species. In order to correlate research and outline standard methods of classi- fication f animal viruses, a Committee on Virus Research was formed by the United States Livestock Sanitary Associa- tion (1960). A proposed standard method for serological identification of viruses was drawn up by this committee in order to facilitate comparison of results obtained by dif— ferent groups of research workers. A coordinator for each of the following animal species was chosen: bovine, porcine, feline, canine, and poultry. As a part of the future program, it was recommended that investigators should be willing to supply virus and antiserum to a repository for use as reference in future research. It was also recommended that the National Animal Disease Laboratory at Ames,lowa, act as the repository and In as a central agency for these viruses and antisera. studying the causative agents of any disease the first step, after the isolation of the agent, should be to determine whether the new isolant is identical to, or different from, ..1 .4» .AU P—a H, J‘ P—. agents already obtained by other groups of investigators. Our present methods to identify a virus still vary widely and comparisons between laboratories are therefore difficult. Too frequently, when a virus is isolated, it is studied from various approaches without too much consider- ation to its possible relationship to agents isolated else- where from the same species of animal. In the rapidly expanding field of virus infections in the bovine, an effective virus classification method is urgently needed to overcome the already existing confusion between the various etiological agents isolated to date and to avoid similar confusion in the future. Viral Isolants from Syndromes of the Virus Diarrhea- Mucosal Disease Complex with Probable Viral Etiology Since the original reports by Schipper and Noyce (1959A), and Noyce and Shipper (1959), research workers from Indiana (Claflin §t_g1., 1959) and Iowa (Ramsey gt _1., 1959) announced the isolation of two nonculturable and noncytopathogenic infectious agents from mucosal disease field cases. These agents were named the ”Merrell” (Indiana) and "Sanders” (Iowa) mucosal disease agents. Both agents are under investigation and their comparison in cross protection studies proved them to be similar or identical (Claflin_et 1., 1959; Ramsey gt _g1., 1959) and possibly also identical to the noncytopathogenic virus diarrhea agent (Rothenbacher §£._l-: 1961) described by Lee and Gillespie (1957). All three agents are noncytopathogenic and produce a characteristic diphasic temperature elevation combined with similar hematological and clinical changes in experimentally inoculated cattle. The experimental disease resembles the description of virus diarrhea by the American (ciarsoh_et_a;., 1946; Pritchard _t_gl., 1956; Carlson_et_al., 1957; and EunnellSI§£_§1., 1960), British (Huck, 1957; Dow _§t_§1., 1956; and Jarrett,1958) and Australian (Johnston, 1959; Edwards and Sier, 1960) workers. Kniazeff (1960) reported the neutralization of Sanders post—inoculation sera by the Oregon C-24-V virus diarrhea virus (Gillespie et_11., 1960) indicating a close relationship between the two agents. More recently, Cunningham and Church (1960) announced the isolation of a bovine cytOpathogenic enterovirus in Michigan from a field case of mucosal disease clinically diagnosed by Beck (1959) and reported by Rothenbacher and U) Earner (1960) and Barner e 1. (1960). ubsequent studies by Earner et a1. (1960) showed that this ”Dibble” virus was nonpathogenic to calves and that it had no clinical, anti- genic, or immunological relationship to the North Dakota mucu— sal disease virus. Its possible etiological role in a bovine dysentery syndrome reported by MQOre et a1. 1962) is in doubt. Antisera against the Dibble virus did not show any close relationship with the Oregon Cr24~V virus (York, 1361) or with the Michigan LC—4 strain ECBO virus (Cunningham and Church, 1960). With the discovery by Gillespie gt a1. (1960) that the cytopathogenic Oregon C-24—V virus was antigenically and immunologically identical to the noncytopathogenic strains described by Lee and Gillespie (1957) a valuable tool was obtained for future serological studies. For many years this comparative research on bovine virus diarrhea had been hin- dered by the fact that all the isolated agents were noncyto— pathogenic in tissue culture systems. Kniazeff and Pritchard (1960) used this new strain (C—24-V) to investigate the anti- genic relationships in the ”virus diarrhea—mucosal disease complex." Dow _t _1. (1956) and Kniazeff and Pritchard (1958) first used this term of wider definition which has been adopted by many workers. Numerous recent reports from all over the world describe mucosal—type disease syndromes which are easily transmissible and, in their epizootiology and path- ologY, resemble virus diarrhea rather than the classical A 101? 4.5-) NA) description of mucosal disease by Ramsey and Ch vers ,. Virus diarrhea antisera from Florida, Indiana, New York, and Nebraska all showed high and approximately equal neutral- izing capacities against Oregon C-E4—V strain virus. Similar results were obtained with antisera to mucosal disease syn- dromes from Indiana, Iowa, North Dakota, and England. on the other hand, Kniazeff and Pritchard (1960) did not discover any degree of neutralization from antisera against the fol- lowing diseases: bluetongue, hog cholera, infectious bovine rhinotracheitis, infectious ulcerative stomatitis, mycotic stomatitis, malignant catarrhal fever, sporadic bovine en- cephalitis, and winter dysentery. In their discussion of the 8 immunological evidence from these tests, the authors concluded that the virus diarrhea and mucosal disease agents considered are members of an antigenically related group. This sero- logical relationship, in conjunction with the highly similar pathogenicities exhibited, led the authors to consider all the agents (as tested by their antisera) as the etiological agents of a single disease. They also felt that differences in epizootiological and pathological characteristics did not invalidate their conclusion. They speculated that the appar- entimmunological relationship between these agents did not imply that they must be antigenically related. They cone ceded, however, that studies by York (1960) have already shown that a certain percentage of antisera collected in field outbreaks of virus diarrhea show no neutralization titer against the C-24-V prototype virus. Kniazeff and Pritchard (1960) also conceded that their experimental design did not permit exact evaluation of antigenic interrelation- ships between the viral agents in question. Such evaluations could only be accomplished by quantitative crosstitration tests, tissue—culture studies and animal inoculations. Results of such studies presently in progress have not been reported at this time. The reported antigenic relationship between hog cholera and mucosal disease by Darbyshire (1960) which seems in con~ tradiction to the negative relationship reported by Kniazeff and Pritchard (1960) was explained by the latter authors to be due to antibodies not participating in the virus-serum neutralization phenomenon. Beckenhauer__t._l. (1961) seem to confirm Darbyshire's (1960) report to the extent that they consider the hog cholera virus as another serological type of the virus diarrhea group of agents. This group of agents, in their opinion-~in contrast to Kniazeff's and Pritchard's (1960) findings of a close relationship between all members of the group--shows quite a wide divergence of serological types. This is evidenced by their finding that ' only one of three bovine enteroviruses, the C-24-V virus diarrhea prototype, was able maprotect swine against hog cholera challenge. The immunity produced in swine by the virus diarrhea agent (C-E4-V) was shown to be due to anti- body formation and not to some blocking mechanism. The vac- cination of pigs with the Oregon C-24-V virus did not cause any clinical symptoms and no contact infection of the vac- cinal virus from pig to pig could be observed. Beckenhauer __§_g1. (1961) emphasized that if titered against the hog cholera virus, various virus diarrhea agents did not seer closely related. Serum neutralization tests conducted by York (1961) between antisera to the Michigan (Dibble) and the North Dakota mucosal disease viruses revealed no close relation- ship of either virus to the Oregon C—24—V prototype virus. Also, convalescent sera from a dysentery syndrome in young cattle and dairy cows reported by Moore at g1.(1962) did not 10 show a significant neutralization titer to the C-24-V virus. York's (1961) finding pertaining to the North Dakota virus was not in agreement with Kniazeff‘s and Pritchard's (1960) data but was a confirmation of clinical and pathological findings as presented in this thesis. The controversial views of various authors point out the confusion in the field of bovine enteric virus infections. It is obvious that our present means of classifying and com- paring viruses need standardization and refinement. The relative accuracy of present serological tests and their questionable comparative value is self-evident. It is hoped that the work presented in this thesis will fill a research gap in the investigation of the virus diarrhea—mucosal dis— ease complex. It should further point out the need for clinical and pathological characterization of an individual virus and its pathogenicity in the experimental animal as a necessary supplementation of gggyitgg virological studies. York e_t_ _a_1_. (1960) evaluated a modified—live, and a rabbit-adapted vaccine produced from the prototype Oregon C-24—V virus. Of 112 vaccinated feedlot cattle, nine later developed signs of virus diarrheaafter field exposure while 67 of 265 control animals became ill. Their extensive sero— logical studies of vaccinated cattle and of cattle from field outbreaks confirmed the findings made by Robson_et_gl. (1960) that antibodies determined by neutralization tests are indicators of immunity to virus diarrhea. This is also in ll agreement with the studies on infectious bovine rhinotra- cheitis by Schwarz_et a1. (1957), and on canine distemper by Gillespie gt g1. (1958), both of whom presented evidence that neutralizing antibodies when present denote resistance to the specific virus. It was the opinion of Hagan (1958) that Olson and hoer- lei n(1956 ) probably saw virus diarrhea in their observations on mucosal disease epizootics in Nebraska, involving mainly beef calves. Hagan (1958)a1so believed that Hoag et a1. (1956) and Rooney (1957) procably described virus diarrhea in Virginia. though no viius comparisons were made. Also, the Swedish epizootic enteritis described by Hedstrom and Isaksson (1951) and the British reports by Dow §t_g;, (1936) and Huck (1957), according to Hagan (1958), resembled virus diarrhea even though some of their reported features did not exactly match the description by Olafson 33 gl. (1946), who first saw the clinical signs of epizootic diarrhea primarily in adult dairy cows. Olafson and Rickard (1947) named the disease "virus diarrhea. -agan (1958) enumerated the original descriptions of the sepa ate dis ease entities WJTCh were later grouped to- gether in the virus diarrhea-mucosal disease complex as follows: virus di_arrhea (New York), virus diarrhea (Indiana), mucosal disease (Iowa), infectiozs bovine "hinotrucntitii, mycotic stomatitis, ulcerative stomatitis (Indiana),e epi o otic enteritis (Sweden), malignant catarrhal fever. and rinderpest. 13 1961; Robson _t__1., 1961) who assumed a subclinical form of the disease. York and Rosner (1961) furnished the serological confirmation for this assumption. It is now generally con- cluded that a large percentage of all cattle show an antibody titer against the prototype C-QM—V virus as reported by Runnells.et_a1. (1960). Underdahl §£_al, (1957) emphasized that many cases of what they considered to be mucosal disease obviously went undiagnosed. For their transmission studies they reported difficulty in finding animals without some degree of immunity. They succeeded in isolating a viral agent from tissues col— lected in Iowa and Nebraska outbreaks. The cytopathogenic effect of the virus on bovine embryo kidney cells was the same from both tissue pools. The virus could be passaged in tissue culture but not eggmadapted. Preliminary neutralis- ation tests indicated a cross neutralization of the two iso— lants. They also found antibody titers commonly in herds with no history of mucosal disease or virus diarrhea. The ‘— authors concluded that the evidence presented in their studies 0 w 3 _ ‘V .1113 O :3 (3.1. did not prove this virus to be the causative agent U) disease. It is hoped that further studies in progre s will soon show the possible relationship between this virus and the already mentioned Dibble virus (Barner_et.a1., 1960), the North Dakota mucosal disease virus (Schipper and Noyce, 1959A; and Noyce and Schipper, 1959) and the cytopathogenic prototype virus diarrhea strain C-EM-V (Gillespie t al., 1960). 14 Van Bekkum (1959) in Holland reported a cytopathogenic agent isolated from a cow suffering from a mucosal disease syndrome. This agent was isolated on bovine fetal skin epi— thelium. Virus passaged on skin epithelium proved pathogenic also for embryo kidney cells, while primary culturing attempts with the original material on kidney cells were negative. This fact led Van Bekkum (1959) to speculate that skin cells might be more suitable for primary isolation attempts. Ex— perimentally inoculated calves showed fever combined with a leukopenia between the second and fourth days after inocul— ation. The described lesions in the oral cavity and the occasional diarrhea compared well to the existing descriptions of experimental virus diarrhea by Baker et_ 1. (1954) and Carlson et__l. (1957). Contact transmission was also seen in experimental animals and the discrepancy between the condition in the field and the experimental disease was pointed out. Van Bekkum (1959) also acknowledged the similarity of his experimental disease with the virus diarrhea reported in North America. From this description one cannot deny the possible relationship to the C—24~V virus described by Gi lespie_e§ a (1960), which future serological studies may well confirm. Bbgel and Nussgay (1960) reported the isolation of a cytopathogenic virus from calf feces which differed sero- logically from the "ZCBO" LC—R4 virus, and caused blood- flaked feces in colostrum—deprived calves after experimental infection. 15 In his characterization of an ECBO virus isolation from healthy cattle, Soliman (1958) observed no signs of illness after inoculation of a three-month—old calf. However, there was a significant rise in antibody titer after inoculation and the virus could be cultured from the calf‘s feces for 13 days. Intracerebral inoculation of suckling mice and hamsters produced paralytic symptoms similar to those induced by Coxsackie A or Lansing~type polio viruses. Soliman (1958) did not inoculate colostrum-deprived calves. This latter procedure, if used, might shed more light on the obscure nature of the numerous isolations of enteric cyto— pathogenic bovine orphan (ECBO) viruses. Another instance ofan incompletely characterized virus is the isolation of a cytopathogenic agent from bovine feces by Moll and Finlayson (1958). Their isolant was grown in bovine kidney cell cultures and produced high fever and res~ piratory symptoms in calves. Other clinicopathological data are not given, and the etiological role of the virus in calves or cattle has not been established. Referring back to Hedstrom and Isaksson‘s (1951) de- scription of a virusmdiarrhea-like epizootic in Sweden, Fakes and Dinter (1960A) reported the isolation of a cytopathogenic agent from respiratory tract exudate of animals with a mucosal type disease. This disease outbreak at Umeg, Sweden, was in— vestigated by Bakos, Isaksson and Nystedt (1958), and showed a mortality of up to 70% in individual herds. Outstanding 15 In his characterization of an ECBO virus isolation from healthy cattle, Soliman (1958) observed no signs of illness after inoculation of a three—month-old calf. However, there was a significant rise in antibody titer after inoculation and the virus could be cultured from the calf's feces for 13 days. Intracerebral inoculation of suckling mice and hamsters produced paralytic symptoms similar to those induced by Coxsackie A or Lansing—type polio viruses. (1) J Soliman (1958) did not inoculate colostrum-deprived calv This latter procedure, if used, might shed more light on the obscure nature of the numerous isolations of enteric cyto— pathogenic bovine orphan (ECBO) viruses. Another instance ofan incompletely characterized virus is the isolation of a cytopathogenic agent from bovine feces *4. by Moll and Finlayson (1958). Their sol nt was grown in {13 bovine kidney cell cultures and produced high fever and res- piratory symptoms in calves. Other clinicopathological data are not given, and the etiological role of the virus in calves or cattle has not been established. Referring back to Hedstrom and Isaksson‘s (195l) de— scription of a virusxdiarrhea-like epizootic in Sweden, Fakes and Dinter (1960A) reported the isolation of a cytopathogenic agent from respiratory traCt exudate of animals with a mucosal type disease. This disease outbreak at Umeg, Sweden, was in— vestigated by Bakos, Isaksson and Nystedt (1958), and showed a mortality of up to 70% in individual herds. Outstanding 16 clinical signs were conjunctivitis, rhinitis, fever, sali- vation,diarrhea, coughing, and buccal erosions. In comparing this disease outbreak with the one described by Hedstrom and Isaksson, they pointed out a primarily respiratory involve- ment in the outbreak at Umeg 1958, while Hedstrom and Isaksson in 1951 saw predominantly gastrointestinal signs with respira- tory involvement in only some cases. The virus isolated by Eakos and Dinter (1960A) from this so—called "Ume" disease was cultivated in bovine embryo kidney cells and, when in- Jected into experimental .“1ves, produced a mild form of the k (D ed Q) n.) disease seen in the field. Serological studies also rev 4:) an antibody titer in convalescent sera. The results 01 r O U) o) l O neutralization tests indicated a close relationship between the Umeg virus and the parainfluenza—B virus strain HA-l. Further work on Ume disease was reported by Nystedt (1960). A cytopathogenic strain of the virus diarrhea virus (C-QM-V) isolated in the U.S.A. was used to test cattle sera from Umea. Sera taken at the later stage of Ume disease, as well as convalescent cattle sera, contained antibodies against the virus diarrhea agent in addition to antibodies against the parainfluenza—3 virus. Sera from the early stages 0. disease were generally negative. The discovery of neutralizing antibodies against the C—EQ-V prototype virus in Ume disease convalescent sera tends to confirm Hagan‘s (1958) earlier supposition that epizootic diarrhea in Sweden (Hedstrom and Isaksson, 1951) is identical 17 with the American virus diarrhea described by Olafson e£__l. (1946). However, it does not seem clear at this time whether two viruses, parainfluenza and virus diarrhea,are involved in Ume disease, or whether the C~24~V virus is closely related to the parainfluenza virus; there might be only an immunological relationship without antigenic similarity or identity. Such immunological relationships have been found to exist between rinderpest and canine distemper (Goret et al., 1960), hog cholera and mucosal disease (Darbyshire, 1960; Beckenhauer et al., 1961), and between human measles and canine distemper *“ (Ca'lstrdm, 1960). *3 Further studies by Bakos and Dinter (19603) revealed antibodies against the parainfluenza—3 virus in 39 out of 41 suspect herds with primarily respiratory symptoms. A posi- tive titer was found in 70% of random cow sera from different parts of Sweden and in 50% of all calf sera. The calves had lower titers than did adult cattl . Experimentally inocu- lated calves developed fever and rhinotracheitis very similar to the disease seen in the field. The antibody response in the calves was prompt and reached high titers U) K'F‘ «:1 This finding of respiratory signs in calves ('0 to the report by N011 and Finlayson (1958) before th para- influenza relationship was discovered. Runnells_et_al. (1960) stated that ”there is considerable evidence to indicate that infectious rhinotracheitis in many instances, is probably nothing more than virus diarrhea.” While details to this 18 mentioned evidence are not given, the authors proceed to state that there is also reason to believe that outbreaks of calf di pr ith er ia may be preceded by a mild infection of virus {1) *3 ('D diarrhea. Again, the reasons for this belief not given. Runnells et a1. (1960) go so far as to say that not only in- 1 Q factious bovine rhinotracheitis, but mucosal disease (Hamsez 7 U, and tnivers, 1953), mycotic stomatitis (Pritchard and Nassenaar, 195;),a nd malisncnt cats rr.h a1 fever are "similar . u _ H — . . . ., . ,1 m" if not identica to virus diarrhea. while the similarit: st matitis (Pritchard and Nassenaar 1959 ‘ 1 t ) \ v \o d :93“. (D rhea y be well established (Olson and Hoerlein, l9 author of this thesis takes excep ion to malignant catarrhal fever as a disease similar identical with, virus diarrhea. A clear differe tie 1 diagntsis between the virus diap- rhea- mucosal disease complex and malignant cat arrrlal fever has been given by Eerkman (1958) and Eerkman 3; e1. (1960). Ac C) 4 .~ ' H‘ ‘ 1 1‘“ '7‘: ‘ .1 4 ~ 3" .7“ ' Rn ‘- ‘fiv ' v -‘ ‘\ 1 .7“ a 7 A] ‘1 ording to Smith and Jones (199,), malignant caoairhal lever -J ‘5 1 7‘ F 4 - - p 1 4 . ‘fi .- I“ w a --' ‘ 7" ~‘ Vy" ’\ .1“ -$ ‘ -? ‘ V ,' ca. b oiiferentlated irom virus diarrhea cy its lax morbid ty (D and the characteristic ocular, nasal and brain lesions. That our present definition of some of the viral dis- eases formerly counted to the mucos a1 disease calm ex is still somewhat arbitrary was shown by Abinar .ti and Plumer (1961), wb o recovered the infectious bovine rhinotracheitis (IBH) virus from field cases of keratoconjunctivitis. Two— 19 thirds of 125 yearling cattle showed signs of keratoconjunc- tivitis but without respiratory involvement or corneal Opacity. Koch's postulates were fulfilled with the isolated virus and neutralizing antibodies dis covered after inoculation. Since the 1BR virus may cause infectious pustular vulv07aginitis 'T I T ‘A.; .0 M 7_“ ‘7 N f ‘1‘ 1‘1 (lFV), conjuctivitis and TUuniClo, the authors uQECldflcfl, ' ‘ ‘ f‘. v" 7 - ‘ ' i ‘ .. — z to ge her with faker et ii. (1500), that the original cesgrip— tive name, mlixotracheitis, may be too restrictive. \oinosti and Plumer (1901) stated that the diverse pathogenicity of th (D lER—IPV virus resettled the hu:na1 ade novir lll. Dmochowski (1961) recently also isolate d a cytopatho— f cattle with a mucosal type cyn— 0 1c age nt from the eyes 3 m drome, nd comparative studnss to classify this virus aze in , 7 ' . ’1 ~'\ 9 r‘ R' . z . . ’ .-. 4- <9 ' , C‘ 'r f‘ fi‘ 7 the m3XOVlIdS par ainfluenzua~3 in bQVine lnlepulOflo. In omléu 1 1 ‘ fl .9 ,1 '_ V q- 1., ‘1 .0 U‘ a F: ‘ ,1 '1 , n .m “ .~~, 4- . . 1 ' :1 _ - . outoreans of respiratory in1”CLLth Ol Ca1Ves and cattle they recovered parainfluenza virus on 15 trials. Serologiccl .7 f) ' 1 ~ A ‘1 Avr‘, '\ r - . I" ‘L l i,“ ‘2“, ’3 evid uence of iniection was OcMOnStPatdd in some caLtio, 1”; authors concluded that at this tine it could hat be said 1; +_ (J a L; O I 7hether the calves became infected iLon expori LLrC t< to other cattle. Serological comparisons to the lER-lEV virus or to the 0-24-V virus were not made. Rosen and Abinanti (1950) showed that calves could be infected with each of four types of human reovir sea. They also proved serologically that infected animals could transmit their 20 infection to uninoculated calves by contact. Reoviruses anti- genically identical with the human parainfluenza-3 were isolated from a group of calves which became irmi cted under natural conditions. Again, serological comparisons to the virus diarrhea virus or to the IER- IPV virus were rot ma deg The great importance of a serological diff3~~ni al diagnosis in the virus diarrhea—mucosal disease mp ex becoxes evident from these reports. 't se:ms quite possible ’ ' " ‘ it' 1 r- w ‘ H P; .1 71 ’Jfin ' w’) —, V)”. ‘ “‘ »‘. in the m2n7.ulture to eXalude the tufiary leapiiatoix INIup“ called virus diarnr a yesterdov may turn out to be an ih~ faction caused by more than one Iirus. . ‘ .‘ Pa“ A '- 5-: IF -~ r-s rs « I’ § 4- , i" {#:1,- A z . -‘ _. .‘ :r‘_"\‘fi ,, The bwedish PupOPLH (:uKO; and Dinter, l; Cm aul lac b, vx f“,,-—* ’- ’ “V _ “__1 9 a . ‘3. .V,‘ A. rakos, e a ., 1735) Wigfib be tue IJPLB in a series a ~1e 1‘ . 1 -1 . fl "\ x f“ .- \ -\ \ A j SEWOlO&10al Stdklbc have : o z the. “ire trah or: not all by Hedstrom and Isaksson (1951), Pritchard et a1. \3y i), Hoag et al. (1956), Rooney (19%7), Carlson et ale (1} T), .p ”(N .l_ . \ _ .3 ‘3‘,“ Bamos et a . (1930), and Eako and ointer (l,toE). For further clarii cation. clinical, gross and ii,to- _l pathological studies of the individual pure virus infection in x0 erimcntal animals-~as was abt mgttd with the N rth Dakota mucosal disease virus in this thesis—~would be h giiy 21 desirable. While Carlson et_al. (1957) gave a classical pathological description of what they called "Indiana" virus diarrhea, it would be interesting to test serologically whether they worked with a pure virus since they used de- fibrinated whole blood for their experimental infections. <“rdcome of the Virus Diarrhea-Mucosal Disease .i'gir‘ nith Que s.tio nable or Undetermined Viral Etidlvgv The last section discussed primarily viral agents-— cytopathogenic, noncytopathogenic, or nonculturable-—from virus diarrhea syndromes which are able to reproduce a pre- dictable sequence of clinical and patnol oggical charge and an immunological response demonstrable by cross -immunization D or serum-neutralization tests. The syndrome discussed in this section has not yet been reproduced experimentally, according to Ramsey (1956), and its cause and transmissi- bility are to a large extent as yet unknown. In spite of vigorous research efforts, irwc u1ing four Ph. D. theses (Ramsey, lQEt; Whiteman, 1960; Trapp, ljfit; Bajwa 1961) describing the gross and microscopic pathology J‘s 1; . of mucosal disease as reported in Iowa (Tamsey and Ctivcrs. r A ., ‘ “1.; .....,-,..._g ,Vr. ‘, " ' . - 1,,,,-.,.a 1953), its cause and pitntgeresis aie to date not an) n. O l ( n ‘_ I I O r‘f b y—a J 3 The search for a viral agent immunologicall‘ ‘ ~ '~ ' P-c ‘- -. ‘1 \,VV 1 vyn ~ L‘ r' . T C‘ n:"7 1“ I“ f‘ virus diarrhea, as suggested by Dmibfl and sones (la;(), has r Ct 071 not led to any conclusive eV1dence. None of the viral agen' discussed in the previous chapter has reproduced the clissi" mlndrome as described by Tamsey and Chivers (1:53)- L.— (\ m L...’ | 22 Nielsen §£._l- (1955) described a similar or identical disease in Canada. Transmission attempts were reportedly un- successful. Swope and Luedke (1956) had similarly negative results in their transmission experiments of a mucosal dis- ease in Pennsylvania. Voss (1959) gave the first description of the Iowa or classical type mucosal disease from Germany. Ten animals died out of a herd of 13 six- to ten-months~old calves. A clear differential diagnosis from malignant catarrhal fever was given by the author. Stober (1959) made further clinical observations of mucosal disease in Germany and reported .‘fi Tr) transmission attempts with 100 ml.o1 citrated blood irom acute cases as inconclusive or negative. He also reported negative results in differential diagnostic examinations for paratuberculosis and salmonellosis. His finding of hen - sized skin encrustations with epidermal s1 oug hing and alo- pecia in two animals that recovered corresponded with Rothen~ b acher's and Barne 's (1960) repart of a clirical n-co 11 disease case from Mic‘ni ga1i. Stdber (1959) coniirnci 9 mor- tality rate of 90% from several disease outbreaks. An interesting statement was his remark that at the date of the report (1959) the other members of the mucosal disease com- plex had not be en diagnm ed in Germany. Schulz (1959) gave a pathological-anatomical description of mucosal disease in Germany which closely correlated to Ramsey's (1956) findings. Both Schultz (195'9 ) and Stober 23 (1959) pointed out the greatest similarity to (or identity with) rinderpest lesions as presented by Albrecht (1929), and Hutyra et'al. (1954). Stober (1959) also mentioned the possibility that some of the cases fo merly diagnosed as atypica a1 malignant catarrhal fever (MCF) may actually have been mucosal disease: Goret and Pilet (1958) reporting on "mu cosal diseases" from France called attention to the fact that what they termed the "tr ue mucosal disease" could also in certain as- pects resemble foot and mouth disease. Seibold (1956) in Alabama described what Rooney (1957) called the "Ramsey-type” mucosal disease. The lesions re- ported matched those reported from Iowa. Barner et_§1. (1959, 1960) reported 23 Iowa- t ype mucosal disease cases from Michigan in a two-year period. Attempts to transmit the diseasevwnxeunsuccessful. While these classical reports described the Iowa—type mucosal disease with its clinical and post mor em changes they also agreed with Ramsey's (1956) differential diagnostic exclusion of virus diarrhea on the basis of high morbidity, low mortality, and easy transmissibility. It is interesting, however, that in the progress reports of the regional NC- 34 research project on ”mucosal diseases in cattle" virus diar- rhea was not reported from those collaborating states which reported muc sal disease (Ramsey t al., 1959, 1960; Barner et al., 1958, 1959, 1960; Schipper and Noyce, 1959C; Jones, 24 1959). The only state of the North Central region which for the last six years has consistently reported virus diarrhea and mucosal disease is Indiana (Claflin_et.al., 1957; Prit- chard, 1955). Pritchard (1955) and Pritchard s£.a1- (1955) were the first to state that the differences between the two diseases are quantitative rather than qualitative. This view was later supported by the research workers in Great Britain (Huck, 1957; Dow §£_al., 1956; Jarrett, 1958), who described mild syndromes resembling the descriptions of virus diarrhea (Olafson gt 1., 1946; Baker et al., 1954) and more severe syndromes resembling mucosal disease. According to Pritchard (1955) it was Ramsey and Chivers (1953) who first presented evidence that mucosal disease might be transmissible. They obtained a temperature rise in seven calves two to eight days after inoculation. Pritchard (1955) reproduced mucosal disease by the intravenous inoculation of defibrinated whole blood from ucosal disease field cases taken during the leuxopenic early phase of the illness. A typical temperature and leukocyte curve presented in Pritchard‘s (1955) paper for the experi- mental mucosal disease is identical with the clinical course reported for experimental virus diarrhea (Carlson t o d s4 1957; Baker t 1., 1954) as well as for the Merrell *— fl Sanders experimental mucosal disease (Claflin et al., 195‘; Ramsey gt al., 1959). A diphasic temperature response was 25 described by Pritchard (1955) with a first peak and leukopenia around the third day and a second higher fever peak for a short period (24 hours) around the seventh day. The symptoms included depression, lacrimation, nasal discharge and mouth lesions. Cross protection tests with two strains of vi rus— diarrhea virus were reported as inconclusive by Pritchard (1955). Later tests by Rothenbacher and Whiteman (1961) indicated a cross immunization between the Senders, Merrell, and virus diarrhea (Indiana) agents. Reports from North akot a (sw.1pper t al., 1955; Schipper, 1957), and South Dakota (Harshfield, 1957) confirmed Pritchard's (1955) ‘ earlier transmission studies. Sc hipper et a . (1955) described mu cosal disease in cattle and found a morbidity of 1 to 89% and a mortality of 100% in sick animals. They reported subacute and chronic cases with hyperkeratosis correlating with later reports from Michigan (Rothenbacher and Barner, 1960) and Germany (Stdber, 1959). The fact that hyperkeratosis was noted in herds two to three weeks a Wt mucosal disease outbreaks, was interpreted by Schipper t al (1955) to suggest ih1t numerous mild muco osal dis se cases go Ufanthf d with per— haps only a transient temperature elevation. In their study on the incidence and mortality of muco- A sal disease in Iowa, Ramsey et a1. 1958) gave a mortality rate of 1 to 50%. They admitted that insufficient herd observations were primarily me Mons ble for insufficient 26 information on morbidity. Ramsey §t_al. (1958) also conceded that a necropsy is usually necessary to make a specific diag- nosis of mucosal disease. In some instances they failed to diagnose the condition in the antemortem state. Discussing the virus diarrheas of cattle and similar diseases in Australia, Johnston (1959) stated that mucosal disease was first seen in 1956 with high mortality, and that there was some evidence of a high incidence of inapparent infections. Transmission attempts with defibrinated blood succeeded in some animals and not in others. Epizootic diar- rhea (Edwards and Sier, 1960), an apparently new disease in Australia, spread throughout that country, affecting cattle of all ages. Whittem (1959) pointed out that the lesions of mucosal disease--as first observed in Sidney 1956—-1ooked like rinderpest. 0n the other hand, the epizootiology of epizootic diarrhea closely resembled rinderpest. Whittem (1959) wondered whether the three viruses (rinderpest, epizo- otic diarrhea, mucosal disease) had arisen from a common ancestor and developed different degrees of pathogenicity. In their studies on bovine mucosal disease, Schipper and Noyce (1959C), reported the reisolation of their earlier reported mucosal-disease virus (Schipper and Noyce, 1959A) from six animals showing typical signs of mucosal disease during the first nine months of 1959. They emphasized that in all instances the tissues prepared for virus culturing were obtained from diseased animals immediately prior to, 27 or upon the first appearance of, the acute symptoms. In no case were they able to isolate the virus when the animal had displayed the signs of the disease for several days or after death of the animal. Summary In summarizing the review of the literature pertaining to this thesis it can be said that virus diarrhea as first described in New York seems to be a better explored syndrome than the Iowa mucosal disease. Its viral etiology has been confirmed and Koch's postulates fulfilled. There remains, however, a certain discrepancy between the severity of the syndrome seen in the field and the reportedly mild experi- mental disease. The possibility of more than one etiological factor in the pathogenesis of virus diarrhea has to be con- sidered. There is indication that in severalcases a combi- nation of two viruses was involved in the syndrome. The pathogenesis of Iowa-type mucosal disease has re- mained a challenge to many investigators. While several viruses have been isolated from field cases, these viruses are either nonpathogenic in pure infection, or resemble the experimental virus diarrhea, causing only mild and transient symptoms. The supposition by several research workers that Iowa mucosal disease represents nothing but a severe form of virus diarrhea is being carefully investigated. The possi- bility of additional and as yet unknown factors in its patho- genesis must be considered. CHAPTER II MATERIALS AND METHODS Materials Used for Animal Inoculation The North Dakota mucosal disease virus isolated and reported by Schipper and Noyce (1959A)and Noyce and Schipper (1959) was grown and maintained in tissue culture by Cunning- ham and Church (1960). Bovine embryo kidney cells in primary culture according to the procedure given by the original investigators (Noyce and Schipper, 1959) were required for viral multiplication. Virus was harvested 24 hours after inoculation of a six—day-old tissue culture when ap- proximately 75% of the cells showed cytopathogenic effects (CPE). The virus was then stored at -60 C until used. The titer of the virus was generally 105 tissue culture infectious doses (TCID). Tissue culture fluid from a preliminary experiment aimed at adapting the virus to rabbit kidney cell culture (Rothenbacher, 1960) was harvested, stored, and used in like manner. Sterile lactalbumin hydrol sate plus 2% horse serum, the growth medium for the virus, was used to inoculate con- trol animals to test for possible antigenicity or patho~ genicity. 29 Organ emulsions of spleen, liver, lungs, kidneys, adrenals, amdlymph nodesof infected calves euthanatized during the acute, subacute, or recovery phase of the experi- mental disease were similarly used to inoculate calves. A mixture of approximately equal amounts of the ground tissues was suspended lle in sterile physiological saline solution; antibiotics were added to the emulsion to a concentration of 1000 units of penicillin and lOOO micrograms of streptomycin per ml. Organ emulsion was used fresh, or stored like the virus until further use. Fresh blood, without addition of preservative, from mg i of the experimental disease was S13 :s *4. ’i co H U) m :5 O 2 i4. :3 U: m a :3 fl (D 01 0Q & used for transmission studies. The blood was taken from donor calves two to nine days alter their inoculation with the virus. Experimental Animals Thirty-seven healthy dairy cattle with no history of virus diarrhea or mucosal disease were used for experimental inoculations. The majority of these were calves from threw to six months old. The youngest animal inoculated was a one- day-old colostrum-deprived call and the oldest animal was a two-year—old Herford cow that had recovered from clinical signs of mucosal disease. Thirty-one of the 37 animals originated from the Michigan State University dairy farm; the rest were purchased from outside sources. 30 Ten sheep (five six-year-old ewes, four 18-month-old lambs, and one 4-year-old ram) served as experimental animals for studies with the North Dakota virus. The six- year-old ewes originated from the Michigan State University sheep herd. Four lambs were purchased from outside sources, and one five-year-old ram was donated by the United States Department of Agriculture. Ten rabbits ranging in age between one and three years were used for similar studies with the North Dakota virus. A11 rabbits were raised in the Department of Veterinary Pathology animal colony. Case histories and materials of 11 calves from negative malignant catarrhal fever transmission studies (Barner t 1., 1958) served as controls for comparative .—_—-—- studies. Hematological Studies It was found that in the present literature only sparse data on the hematological valuescfthe young calf exist (Dukes, 1955; Coffin, 1953; Krolling and Grau, 1960; Schalm, 1961). Since data were available in this study, the preinoculation values of #1 healthy experimental cattle were divided into four age groups, averaged and considered as normal values for the respective age groups. The average hematological data of five healthy sheep were used as normal values. 31 Eight rabbits were similarly used for normal prein- oculation blood data. Blood examinations including total erythrocyte (RBC) and leucocyte (WBC) counts, hemoglobin and hematocrit deter- minations and a NBC-differential count were made once to twice daily. The hemoglobin determination was done by the acid-hematin (Dukes, 1955) and the cyanmethemoglobin (Schalm, 1961) spectrophotometric methods. Hematocrit values were determined by means of a microhematocrit centrifuge. Wright's stain was used on the blood smears for the differential counts. Blood sugar and nonprotein nitrogen were determined in some animals on Folin Wu protein-free filtrates according to the methods given by Bray (1957). Virus Inoculations and Transmission Studies On the first five calves, various routes of inoculation including intravenous, subcutaneous, intracutaneous, intra- muscular, intranasal, intramucosal, and intranodal (pre— scapular lymphnode) were used. After the infectivity of the virus and the susceptibility of the eXperimental animals were well established, the main routes of inoculation used were intravenous and subcutaneous. Five m1. of the freshly thawed virus constituted the basic dose ofijuxnfhxnfor calves and sheep. A few drops of the virus which remained in the inoculation needle and syr— inge were given intraocularly and/or intranasally in some animals. 32 Inoculation with virus. Twenty-six calves from two to eight months old, four yearling cattle, one two—year-old cow, and one day-old calf were inoculated with the North Dakota mucosal disease virus. Four calves received repeated injec- tions of the virus: one of the four (#25) received three virus injections at weekly intervals. Another calf (#04) received two virus injections 69 days apart; the third calf (#35) received two virus injections 36 days apart; and the fourth calf (#02) received four virus inoculations with one each on days O, 23, 51, and 121 of the experiment. Epine- phrin was usually given at repeated injections to counteract anaphylactic reactions. Inoculation with rabbit kidney cell culture harvest. Rabbit kidney cell culture harvest (Rothenbacher, 1960) was similarly inoculated into three Calves. Inoculations with fresh whole blood. Six calves were inoculated with fresh, whole blood (without addition of preservative) from animals showing acute signs of the experi- mental North Dakota mucosal disease (Chp.II, p.28). Twenty m1. of the fresh blood were given intravenously as a standard dose. The blood was drawn and reinjected within ten seconds. One of the six calves received blood from a sheep showing acute signs of the experimental infection. One calf received a second blood inoculation five days later. To test for immunity all calves were challenged with the pure virus ten to 60 days later. 33 Inoculationsfiwith organ emulsions. Three calves were given organ emulsions from seven animals euthanatized from two to eight days after inoculation, during the acute or subacute phase of the experimental North Dakota mucosal dis- ease. Each inoculated animal received an organ emulsion mixture (Chp. II,p.28)from one or several donor animals. The inoculum for sheep and calves consisted of 20 m1. of the emulsion given subcutaneously or intramuscularly. One of the four animals (#32) received three consecutive injections of the organ emulsion mixture at weekly intervals. Another (#27) received consecutive injections on days O, 14, 32, and 39. One ml. epinephrin was usually administered subcutaneously at the time of repeated injections. To check for the onset of immunity, two out of these three calves were challenged with the pure virus from seven to 60 days after inoculation. Inoculations with sterile tissue culture medium. Three calves and one sheep received the sterile tissue culture medium (Chp.II, p.28) in the identical dosage and by the same routes of inoculation as for the virus. Contact transmissi-n studies. Two calves were kept in intimate contact with an experimentally inoculated animal during the acute phase and recovery period of the experi- mental disease. Each calf was placed into the same stall with an experimental calf that had just been given the virus. The calves drank and ate from the same troughs and were kept 34 in contact for ten days. Both calves were challenged with the pure virus from five to eight weeks after this contact period to check for a possible subclinical infection result- ing in immunity. Inoculation of sheep. Seven sheep ranging in age from one and one-half to six years were inoculated with the same virus dose subcutaneously. One lB-month-old lamb was inocu— lated with sterile tissue culture medium. Inoculation of rabuits. Eight rabbits from one to three ' £3 \z 1d each received 0.2 m1. of the virus intravenously, O 1” S Q) 3 subcutaneously, intramuscularly, and intraocularly. Clinical Observations All experimental animals were allowed to adjust to the isolation quarters for one to several weeks before in- oculation. No change in diet, environment, or management occurred during the experimental period. Preinoculation 1) observations included those on body temperature, pulse, res— l 7) r-< ' ) .4 ) piration, food and water consumption. and the consist;*- . .I ’1 0 a 7n -1 ’\ .- 7.“ 4 ‘f‘. )8]. Vs; l’ulll; .w , CT‘ the feces. Blood sam les for hematological ob Q r taken daily for several days prior to inoculation. During an experiment, strict isolation of the experi- mental animals was maintained. The temperature of inoculated animals was taken from one to three times daily. Other clinical observations before and after inoculation included complete daily hemograms (Chp.II, p.30) and periodic but less frequent urinalysis and fecal examination. Gross Pathological Observations and Necropsy_Procedures At various time intervals ranging from one to 134 days after inoculation, animals were euthanatized by exsanguina- tion, usually following electrical stunning. Gross lesions were observed as soon as possible after death. The necropsy procedure was modified routinely as to the sequence of body tracts and regions examined; alternatively, gastrointestinal tract, respiratory tract, or urogenital tract were examined first, in order to allow an immediate and fresh post-morten view of each tract. In the case of the gastrointestinal tract, different parts of it were examined first on various necropsies, in order to obtain a clear picture of the patho~ logical changes occurring. Eighteen calves were necropsied during the acute clinical phase of the experimental North Dakota mucosal dis- ease infection from one to six days after inoculation. One I and another 14 days after L calf was necropsied eight d H“ ('0 [JV inoculation. Three calves that were allowed to recover frcm “>“ acute experimental diselse were necropsied 54, 64, and if -3 day alter inoculation. All three of these calves had been 0) L. hyperimmunized with additional inoculations of virus, rabbis cell culture harvest, or organ emulsion. One calf was necropsied six days after inoculation with sterile lactalbumin hydrolysate, the tissue culture growth medium. 36 Five calves and one two-year—old cow were commercially slaughtered at the M. S. U. meats laboratory 22, 33, MO, 45, 118, and 218 days, respectively, after virus inoculation. All animals had completely recovered from the experimental disease and none showed signs of illness. Gross observations were made and tissue specimens obtained at the time of slaughter. Six calves, after complete recovery from the North Dakota experimental disease (21, 22, 28, 32, 33, and 67 days after inoculation) were released to Dr. C. K. Whitehair* for nitrate-nitrite toxicity studies and were necropsied in the course of this work by Dr. R. Naghshineh.* Of seven sheep inoculated with the North Dakota virus, two six-year-old ewes were necropsied six days, another two six-year—old ewes five days, and two l8-month-old lambs four days after inoculation. Three sheep (one six-year-old ewe, and two l8-month-old lambs) were necropsied as healthy control animals. One of the control lambs was necropsied four days after inoculation with sterile tissue culture growth medium. Two one~year~old rabbits were necropsied two days, and another three one—year-old rabbits three days after the North Dakota virus inoculation. Three three-year-old rabbits were necropsied three days after inoculation. One two-year-ol" erinary Pathology, Michigan State *Department of Vet ing, Michigan. University, East Lars 37 and one one-year-old rabbit were necropsied as normal con— trol animals. Histopathological Observations Representative tissue sections of the adrenal, thy— roid, parathyroid and pituitary glands, brain, spinal cord, liver, kidneys, spleen, oral and nasal mucosae, lung, heart, stomach(s), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, urethra, vagina, uterus, ovaries, testes, penis, musculature, skin, bone marrow, pancreas, salivary glands, thymus, aorta and pulmonary arteries, and lymph nodes from various body regions were fixed in buffered 10% formalin solution (A.F.I.P., 1960). In View of the distribution of the gross lesions, special emphasis was given to the sections of the posterior small intestine, adrenal glands, spleen, bone marrow, kidneys, liver, tonsils, and mesenteric, mandibular, parotid, supra— pharyngeal, bronchial, mediastinal, “rescapular and pre- femoral lymph nodes. Sections of the sternum were deca cified after fixation in buffered 10% formalin. Decalcification was accomplishad by the formic acid-sodium citrate method (A.F.I.P., 19:0). The histological technique used for all tissue scc~ tions followed the outline given by the Manual 2: HistOLocic and Special Stainin:r Tschnics (A.F.I.P., l9ct) unless other- wise noted. After fixation the tissues were dehydrated in a 38 series of graded alcohol solutions, cleared in xylene and embedded in paraffin. The paraffin sections were cut at six to seven microns thickness and stained with hematoxylin and eosin (A.F.I.P., 1960). Special staining procedures used on selected tissue sections included the periodic acid-Schiff reaction (PAS), Mallory's iron reaction, Lendrum's, Heidenhain's anilin blue, crystal violet, Schorr's, Weil's, Verhoef's, Von Kossa's, Scharlach red (Sudan IV), and oil—red-O stains. The latter two stains were used on frozen sections of adrenal glands. Serological Studies Pre- and post-inoculation sera were saved from each animal and stored at -60 C for serum neutralization studies and virus comparison work. Bacteriological and Virological Examinations Fresh tissue specimens were submitted for examination to the microbiological diagnostic laboratory at Michigan State University. For virus isolation attempts fresh tissue specimens and fecal scrapings of the intestines were submitted to the virology section, Department of Microbiology and Public Health, within ten minutes after death of the experimental animal. Other Post-Mortem Observations Fecal samples were routinely checked for parasite eggs, Strongyloides, nematode larval stages, coccidia and other protozoa. 3’9 Urine specimens were also taken in some cases during necropsy of the experimental animal and submitted for exam- ination. CHAPTER III RESULTS 0 neral Clinical Observations in Virus— Inoculated Experimental Animals Cattle. Thirty calves (five weeks to eight months old), two yearling bulls, two yearling heifers, one tw yea r- old cow and one day—old calf were inoculated with the North Dakota virus by various routes of inoculation (see Chapter II, p. 31). A predictable and rather constant sequence of sv mptoms was produced in all cattle including the day~ old calf. Pyrexia started after 16 to A8 hours and reached a peak after 24 to 72 hours. The average preinoculation tem- perature of the experimental cattle was 102.28 F. The average peak temperature reached after 24 to 72 hours was 105.2A F. The peak temperature was 107.0 F. in one calf and 11 out of 30 calves had peak tewnperat res of 106 F or higher. Concomitant with the rising temperature were nerv us- ness and restlessness evidenced 16 to A8 hours aft er inocul- ation, slight to pro Mi se serous ocular discharge, tachycardia and an increased rate of respiration. These clinical signs appe ared to be more pronounced in the younge er calves. Usually there was an erythema of the oral mucous membranes during Al the period of high fever. Increased salivation and champing were also observed in some calves during this period. Starting generally at the time of the highest temperature (24 to 48 hours after inoculation), partial to complete anorexia, lasting from one to three days, was observed in the experimental cattle. Colic was noted in some calves, evidenced by restless- ness and by kicking of the abdomen with the hind feet. In several calves a moderate degree of bloating of the rumen was also seen beginning during this period of high fever, and lasting from one to four days. . Dehydration and constipation during the time of high temperature was evidenced by a decrease in the number of defecations to one-half or less and by the passing of small amounts of dry feces in formed spheres two to three cm. in diameter. The water consumption decreased during the time of the highest temperature elevation and increased one to three days after the peak in temperature was reached. Pulse, respiration, water and feed consumption, and amount and con- sistency of the feces, as well as frequency of defecation, returned to normal approximately two to five days after the highest temperature elevation. The whole course of the experimental infection lasted from five to nine days (see Tables 1, A, 7, 9, 10, and 11). The initial period of nervousness and restlessness, which occurred between one and two days after inoculation, while 1+2 25.. e... :8 233 323.3; m>m maommm l zo_mmm¢muo l mmuzmao>zmz I a :ooon “‘ I” \\\.I..| I. :83. \\\\ , ..ooom \ I \\ ’ .vooow \ I \s x :82. \\ "I-““"'#v°°°° \ omillai\\ .oooa kqfib agonmikzoiufi < to 22593;: mat; 3:335 1.2324 3.2K .meF on; 143 the fever was rising, generally gave way to a period of depression lasting from one to two days, usually until the high fever began to subside. Diarrhea was never seen in observations extending up to 60 days after virus inoculation. Respiratory symptoms were not observed except for a transitory increas in respiration during the period of restlessness and high tem- perature. The ocular signs described were very mild in the older calves and rarely lasted 1 nger than 48 hours. Sheep. Four six-year—old ewes, one four—year—old ram, and three one-andxnmrhalf-year-old lambs were inoculated with the North Dakota virus by various routes of inoculation (see Chp.ll, p.34). The clinical symptoms produced in sheep ‘1 were milder in degree than in the cattle and less obvio s to i the observer. Temperature rises of one to two F. were ob— served from one to four days after inoculation. The average preinoculation temperature of the experimental sheep was 102.80 F. The average peak temperature reached from one to four days after inoculation was 104.15 F. The highest tem— perature in one sheep was 104.8 and two other sheep had peak temperatures of 104 F. No temperature elevation was oetected in one sheep. Depression and partial anorexia were observed from two to four days after inoculation. The visible muc us membranes appeared slightly congested during the febrile period. Simul— taneous to the period of depression and anorexia, dehydration 4M and constipation were evidenced by a decrease in the defeca- tion rate and by dehydrated and smaller amounts of feces with a smaller than normal pellet size. In general, the onset of the temperature rise occurred later in sheep and the course of the experimental infection was shorter than in cattle, lasting only from three to five d £13 ys. Rabbits. Eight rabbits (three two-and-one-half year— olds, twc one-and-one—half-year-olds, and three one—year—olds) received the North Dakota virus by various routes of inocu— lation including intravenOIs, intramuscular, subcutaneous, and intraocular. Similarly to the sheep, the rabbits displayed a very mild degree of clinical disturbance. Pyrexia occurred from one to three days after inoculation and temperature rises from 0.8 to 2.5 F. were noted. The average preinjection temperature in rabbits was 102.5 F. and the average peak temperature measured three days after inoculation was 104.9 F. No temperature elevation was noted in one rabbit. Partial anorexia was seen during the febrile period from one to four days after inoculation. The feces passed during this period were less in amount, smaller in pellet size and showed a marked dehydration as compared to those of control animals. Hematological Findings Due to the paucity of specific hematological data for the young bovine (see Chp. II ), the preinoculation data of 45 34 healthy experimental cattle were divided into four age groups and the hematological values averaged for each group. These values are compared with the hematological levels observed during the period of lowest depression of the total leukocyte count and the highest temperature elevation, both of which occurred from two to four days after inoculation. TABLE 2 AVERAGE HEMATOLOGICAL DATA FROM HEALTHY 1- TO E-MONTHS-OLD CAL’ES BEFORE AND AFTER NORTH DAKOTA VIRUS INOCULATION 6 Calves Before 4 Calves 78 Hrs. Inoculation After Inoculation Average Per cu. Per cu. mm. % mm. % Hemoglobin éGm./100 ml.) 9.99 7.52 Hematocrit volumes %) 29.0 7 5 Total leukocytes 8200 3667 Heterophils 2186.66 2 .66 617.65 16.75 Stab cells 273.3 3.33 138 2 3.7) Lymphocytes 5589.66 6 .16 2996.09 81.25 Monocytes 369.00 4.50 55.31 1.50 Eosinophils 54.66 0.66 18.43 0.50 Basophils 0.00 0.00 0.00 0.80 TABLE 3 46 AVERAGE HEMATOLOGICAL DATA FROM HEALTHY 3-MONTHS-OLD CALVES BEFORE AND AFTER NORTH DAKOTA VIRUS INOCULATION 15 Calves Before 9 Calves 51 Hours Inoculation After Inoculation Average Per cu. Per cu mm. % mm. % Hemoglobin éGm./100 ml.) 11.6 10.86 Hematocrit volumes %) 31.06 32.0 Total leukocytes 7323.00 3111 1 Heterophils 1854,91 25.33 60 .44 19.33 Stab cells 375.66 5.13 152.06 4.98 Lymphocytes 5008.93 68.40 2309.3i 74.22 Monocytes 263.62 3.60 159 00 5.11 Eosinophils 185.49 2.53 37.35 1.22 Basophils 9.76 0.13 3.45 0.11 47 0 Q .4 I4- . K.“ a... 4 “Air m .5. 3:1 up —I o .1» r L ;. MU mmaflgaocflmom H m mumpmooxoa u z ”mop;ooaAIAH H A muaaom n.3h r n mmaflcaoaopog H m Mmaamc Good; opflrs H 0mg m.pfi .:@\maamm woman was no mCOHHHHE H 0mm m.HE ©0fl\cHQonOEm£ .83 H .p: Mcoflpcfldoocfl go new H 0 am; ”mcoflpme>mpppd E as E a: E :s . em -- 036 seam -- mama ooh.m m.m m.eu e.mca o+ 8V 3 E 3.6 3; 33 .. - _ -- me new com: sew ewsa 666 b e.@ w.m c w,a s+ 2: 8V 5, 3:. A: $8 I . -- -- com ooam ms mmsa com.» ..s o.w o.oca 6+ 2: A8 2; a: is $3 - .. -- -- mom comm baa coca co» s m a m.c m. we m+ as as E 3...; E a: - ., - - III III m.M®H m.m@Hx n.3w m.ct®a on:.w m.b H.0 0.2;H 3+ 8V A8 A8 . 3m- E 8: -- -- -- easy emu 66mm 660.6 t.m 6.0 m.moH m+ 8V 8V 5 :3 E, G: I \ - . III III OOH m.:mmm m.aur maea 0,3au m.® C.s ©.moH m+ A8 as E a: E 3.: ; . -- -- me memm mo mm- oma.m s.@ m.m m.aoa H+ s E is es 3 as . -- HHH -- eamo -- mesa ooa Ha H.s H.0H m.moa 8 gas has Ame lame Ase lame . - -- -- mam 3mm memo was came com 0H 0.» 0.0 o.mcH H- A8 E E £8. E 2.3 . -- -- we mmmm mmu msam 00: m s.» 0.0 m.moa m- m m 2 a m m cm; 0mm .Qz .ane mama Ammmmpcmscmmv ecmWOOHm so .sa .zp\maamo mgdo QQQImmBZOEIm d m0 ZOHBDmmZH Q dBOMdQ Wbmoz QQQHQNH < fiH mmozoocoE H E “mopAQoBQE%H H q mmaamo omen H m mmaasmopmpoc n m mmaamo oooap opmrz n wmz M.EE .:p\mHHoo oooan cop mo mcoHaHaE u 0mm M.HE oow\cflnoamoEos .Ew H .9: “we ofipma>oinp< --- -- Ame game Ame -Aev o o mwa Amos msa who oomm H.m m.o m age e+ --- --- lee lees lee legs . o o sow game we «owe oowo m o o.o o.eo 2+ -- --- gov Aoew gee Fame 0 o mmm come 6 mmoa come m.» o.oH m.eoa ++ --- -- Ase gees Ame Ammv I o o mqa manm as wmoa oosm ©.m m.oa m.moH w+ -- has Ase flees Ame fleas \ 0 mm mam smwm as wpm ommm m.s H.0H O.woa m+ -- -- AHV Amev Ase heme o O s: :Hmm owe Hmma 00s: m.m m.® :.mofi H+ --- Ame Age Ammw Nae Aemw O :Na em mqou aw womw oosm :.m s.oH ©.moH o m m 2 g m m om: 0mm .Qm .QEoB soapmHSooQH Ampmd mama Amomwpcoopomv 62m nooam Ho .55 .5w\mmaoo C RQ «HOK<- TE moz quHme ¢ ZH mmwztchnux 1 to 20:. Duns): mbkta ShOk‘N Ikkcz um Sank 2:5 u. : o. m o s m m e n u _ _ m . _ . . .r . _ _ d . .95 + P A' no- .N m. A ‘- AVO- n. n . t. .. no. u o a n N .. 00. H .95... o u \\I \ a g. a $000» \ ; \\\ I er \s i. x .. o. .58.. k 1. \ r. 1 i s r. 1. a \\ h. .x. ' .1 Z :88. \ ’ O I. \‘ t O 0* O \ a 00 O I \ +.. 00 W . .. m. +88. \ 0000 4. 10 O \ 000 0 ++ O 1.0 a on; It \\os ooonfi. onswoooo floooo Jo: oooIl\ ooo ++ .7... ++ + wooooooooo? n_ .OOON. 000000 +++ ***** ** I'-'II'L # + L. c. 288. .32. Ill .91 um) ._o> :SoSEogoBE .. .3: .mm .on 53 .m H5H00m9 n w HmHH :ro Hmom H m ”mommwccoe H E ”mop co.w. HH u H HwHHmo Qmpm H m M,HHHQOH0Hor H m mmHHoo mooHH opH£3 H mmz .flo HlHo o> HHLQOHmEmL n .pom M.HE Q©fl\cho HmoEmL .aw H .nm “msoHHmH>mLoH< 228 2H8 Ammo -AHV 2028 , mm mm ewes mew 0mm ooma Hm _.HH r.mcH 6+ --- Amy stv Hmv Hews \ , \ o mHH game How wqu omom Hw H.HH o :.H 0+ --- Ass Ammo ”so Ammo - - . o :wm :mpm mam s:«H com: 26 m.WH m.uoH 2+ --- H68 Hoes HqHV Hflmv o mHm mwmm Hmm quH 0mm: mm 3.0H 3.60H w\H-m+ Hwy - HmsH Hal HMHV \ as o mmmw as man ocmm mm m.mH 3.00H m+ --- 228 Home --- Hall 0 Hm 000m 0 moH QQHm mm o.mH o.HpH m\H-m+ HHV Hmv Homo Hmv HOV NH mm oomH mm mmH oosH mm m.m 0 so m+ --- AHV Homo “we AHHV . 0 mm mmom HH can ommm am m.HH 3.:JH m\H H+ --- Ame A068 Ame Ammo - o mmH mmm: Hmm mmmH omww mm H.mH q.moH H+ HHV Hmv Hfimv --- Away _ 0s on owom o ouoH coop mm 6.mH :.moH o m 2 Nu m m om; .pom .Qm .gsm coHpmHSQOcH Hmpwfi mama Amommpcoohmmv 0cm voon mo .EE.5p\mHHmo HHDH QHOI m< m% H < HO ZOHBOMHZH H H0L H m MmHHco UooHH oszz H omz HR 0E Ho> HH900pw;oL H .Ho. H.HE OQfl\QHQonoE0; .nc 1;.m ”mLOHHwH>mHHQ¢ --- Ass Ame 2208 Amy Hews _ 0 Com 03H owmq on ommH QOOH mm m.mH 3.HQH 6+ --- Hsv Hes HHHV HsH HmHV o mom new www: mow Hmm 6000 mm m.mH m.HoH m+ --- ANHV 2:8 HHHH Has Ammo - 0 mo» 0mm smHm mam HHHH comm Hm o.eH u.m H m+ --- Hmv Hmv quv HHH HHmv 0 :0m 0mm @mmm mmm mmHH saga mm m.MH o TrH H+ --- AOHV 2H8 Ammo Ame Ammo 0 03m :m mme mQH mam Qo;m mm o.mH m.:QH m+ --- Home 2H8 Amps Has ”[8 - 0 com mm @Hom mm 00H 03mm 30 H.HH :.mOH m+ --- HHHV Amy Homv Hmv Hcmv O 03w omH Doom omH OQwH ooow 3m H.:H J.moH H+ --- Haas 2N8 23ml 208 AHHV . - O wmmH 30m momm me wme 00m OH mm O.mH ®.Ho o --- Homo HHV HNHH HHV Homv o ozmH we :mmw vs mbwm CONN mm O.mH m.HOH H: m m E H m m omz .pom .Qm .QEmE :oHpmHSoocH Ammmmpcoohomv cam moon L0HH< mmma Ho .EE .Sp\mHHmo H <6 Od<© zoo GHQ: m Q If I\— Q (u C! (U Cu ‘0 CU Hm Hi!" OJ IZTVC) r—I' CU I—I-TJ” r-ICI) H r-I' H r-{VHVCUV H\/H\_/r_j\_/ VCUV L(\ I0. 0 A0,) A "ACID Ni: A L_f-\\/\ (“Id A L’\. A \C A O \C'C9<7VifITWCflr~IOJCQ:IJLf\~1:rx£9uw»:>0Icu ’11— (YH C-\ {NW Ln CU ufix (\J m r.__{ kj\ [.4 (i) («\4' C) KY“. (3‘ (U NV(Y‘3VC\JVHVHV x/(V‘\Vojvfjuv O C) LU. C) Q LO 0 Lfl ) CU C» [\._ O\ J (yj V“ CC q 3 a) H (I) “\C) it) :P\- H VC) {v} H (”‘1 --—4 H N LO. :1- CU H k ) H J\ ,__I m (WW KY) 01 I YW ) IV" ‘3 (U (V ’3 ‘£3 CT\ ct) r4 ,4 (:3 r4 :3’ rvw H H O r—I r—I G\. O\ O\ D r—I H H r—I H r4 CU 0 CH 0 L3“ 0 \O CO (13 m QJ (a qx 4' In 4; .3 ow C) C) O ( ) O O i) O O .—-I H H H r-1 H r—I I-H r—“{ O +1 + +'I +0 WBC : white J Volume % L : lymphocytes; Fit "3 H hematoI uab n1»— jv’t HI c J 1/100 ml. 1’ L i hemoglob : Gm. M: ells; '3 u ( U) U1 TABLE 13 AVERAGE HEMATOLOGICAL DATA FROM HEALTY ADULT SHEEP BEFORE AND AFTER NORTH DAKOTA VIRUS INOCULATION 6 Sheep Before A Sheep 96 Hours Inoculation After Inoculation Average Per cu. Per cu. mm. % mm. % Hemoglobin (Gm./ioo ml.) 12.55 1: A2 Erythrocytes (millions) 11.52 10.45 Total Leukocytes 58Al.66 3375.00 Heterophils 1915.8 32.8 995.62 29.50 Stab cells 70.1 1.2 92.81 2.75 Lymphocytes 3551.3 60.8 2160.0 64.0 Monooytes 93.4 1.6 67.5 2.0 Eosinophils 198.6 3.A 52.05 1.75 Easophils ll.6 0.2 0.0 0.0 b \ 57 TABLE 1A AVERAGE HEMATOLOGICAL DATA FROM HEALTHY ADULT HABEITS BEFORE AND AFTER NORTH DAKOTA VIRUS INOCULATION 12 Rabbits Before 7 Rabbit; 58 firs Inoculation After lhooalation Average Per cu. Per cu. mm. % mm % Hemoglobin Ecm./100 m1.) 13.05 1.64 Hematocrit volume %) 38.08 30 1L Total Leukocytes 9087.50 5593.00 Heterophils 3331.47 36.66 17A1.66 31.1A Stab cells 7.27 0.08 7.83 0.14 Lymphocytes A982.67 5A.83 2931.85 02.42 Monocytes 454.37 5.0 758.j7 13.57 Eosinophils 90.87 1.0 55.93 1.0 Basophils 219.0 2.Al 95.90 1.71 (1 57 Group of one-to two-months—old calves. The outstanding hematological change seen in these calves was the pronounced leuk0penia between the second and fourth day after inoculation. The average depression from the preinoculation total leukocyte count was 3837.5 in all four calves. The lowest individual lPI/O”"G count was 1200 n one calf reached three days after inoculation (Table 2). 1 Deoressi on of the absolute out not the relative .nvl t (3 ‘A; _ ,' o . _‘ ". I; C .. of lywnnopvt,o was eVldent by an increase irom Lu.l to Cl 3 in he relative difierential perc nta ge for lymphocytes. ‘he absolute and re1.:ative counts or eosinophils dropped markedly during the period of high fever and leuKOpenia. An .) absolute and relative drop of the average count of monocytes was also encountered. Anemia was evidenced during the iebril and leoiLopeni: period Group of three—months-old calves. Pronounced leukopenia was the outsta Md ng hematological change also in this age group leuKo yt count (a\er2ge 7323) was A322. The 10~ : *ru.v u,al leukocyte count was 1950 in one calf reached after 3w hours. There was a severe abl“ O'u e and relative depreo ion of the total count of heterophils (Tables 3 and A). The average counts of step cells revealed also a; 59 leukocytes was not seen at this time (50 hours after virus L inoculation). An absolute depression of the total lymphocyte counts was evident. This depression of lymphocytes, like in the one- to two-month-old calves was absolute; the differential percentage showed a relative increase in lymphocytes from 68.4 to 74.2. While the absolute count of monocytes showed a dr0p after inoculation, this cell category similarly increased slightly in its relative differential percentage- A constant and severe reduction of both the absolute and relative counts of eosinophils was noted in all inocu- lated calves of this age group. This eosinopenia was more pronounced than in the calves of the one- to two—months age group. The post-inoculation drop in the absolute and rela— tive numbers of bas0phils appeared to follow the rate of reduction reported for the heterophils and eosinophils. Fifty hours after inoculation the reduction of the average preinoculation total leukocyte count was 57.A%; however, the average reduction for the absolute hetercphil count was 76.6%, for the stab cells 59%, and for the absolute eosinophil count 79.9%. The corresponding reduction of the average absolute lymphocyte count was 53.8% and for the abso- lute monocyte count 38.9%. Comparison of pre- and post—inoculation values of hemo— globin revealed a slight anemia. 0n the other hand, the 60 hematocrit value increased slightly from an average of 31.0% to 32.1% volumes. Tables 1, 7, 9, 10, and 11 illustrate that the inception and degree of hematological changes closely follow the tem- perature curve of the clinical course of the experimental infection. The depression of the total leukocyte counts in the two calves with pneumonia (Table 5) as well as the depression of their absolute heterophil, eosinOphil, and monocyte counts 1) iv m CI L.) 3 followed the pattern seen in the inoculated healthy c R) The relative lymphocytosis was also noted. Calf No. was euthanatized three days after virus inoculation and calf No. 1 made a slower but uneventful recovery, the clinical course of both the experimental virus infection and the pneumonia taking about ten days. Group of four- to eight-months-old calves. Like in the previous age groups, a pronounced leukopenia was the out- standing change in the four— to eight—months-old calves. The average depression of the preinoculation total leukocyte count (average 8732) was by 3790. The lowest individual WED count in one calf was 3550 (Tables 6 and 7). A severe absolute and relative depression of the total count of heterophils was evident. The pre- and post~incoulation comparison of the stab cells also showed an absolute and rela~ tive depression. An increase of immature leukocytes was not seen at this time (60 hours after inoculation). {7 61 There was an absolute depression of the total lympho— cyte count as in the two previous age groups of calves, but the differential percentage showed a relative increase. Similarly to the lymphocytes, the relative percentage of monocytes also showed an increase. As in the previous age (3 alve ([1 groups of , the depression of the absolute and relative counts of eosinophils was a constant finding also in this age roup. (W2 (D ( 3 {D k-) (A (D (I) O ["3 cf {3' 7‘1 U) (:3 1'7; (1) (V H C; (J 'U ' r ' , .2 F. 3 +- relative increase in ch C" . ‘n a '5 a « ~1~ r\ , . 1 , o ~r~_ ---. \_ A. V» -\ olxty hours alter inocu‘ation tee reduction 0. tLe average pre-inoculation 00* leukocyte eo.wt was A; #3; however, the reduction of the avera e total heterophils was 64.1%, that of the average total strh cells 49.1%, and that Group of yearling cattle. Comparing the pre- and post— inoculation hematological averages of this age group, similar changes were encountered as in the previous grouos o: calves. An average depression of 3626 Tom the pre~inoculaiiow total leukocyte count was noted. A slight anemia was also evident in this see srcun of C) Q Q t cattle. Also in this group the most outstanding hematological change was the absolute and relative depression of the heterophil counts (Tables 8 to 10). While there was a depression of the absolute count of lymphocytes, the relative percentage value of this cell category showed a substantial increase as in the other gro of calves. Monocytes decreased in absolute and relative numbers. A marked drop was noted in absolute and relative numbers of eosinophils. "7'7 Two-year-old cov. As can no seen from latle 11, tv .‘r‘\ [O ups experimental infection of a two—year—old cow with North Dakota virus produced similar clinical and hematological changes as seen in the experimental cattle repor ed previously- Tbs depression of the absolute and relative counts of heterophils was most outstanding during the febrile periid. A corresponding increase of the relative lymphocyte coun was noted. The (D erratic behavior of the relative and absolut eosinophil in this case may be explained by a chronic pediculosis o the animal. One-day—old calf. Even in a one-day—old colostrum changes were similar to those in older cat. Sheen. A pronounced leukopenia was evidenced in t;w {0 experimental sheep. The lowest individual WEc count wa and the average depression from the preinoculation total leukoc'te count was by 2A66 or 42.2%. As in the cattle, J / count f ll” -\r rrvw-r ;/’r\ -m & ’x/ 6 k0 but somewhat less pronounced, there was an absolute and re~ lative depression of the total heterophil count. The depres- sion of heterOphils was 48.1%. Corresponding to similar findings in the experimental cattle, the relative lymphocyte and monocyte counts showed increases after inoculation. The depression of the absolute and relative counts of eosinOphils was a constant finding also in the sheep. Post—inoculation hemoglobin values showed a slight increase and post—inoculation REC counts a slight decrease as compared to the pre—inoculation normals. A slight increase in the number of stab cells was also evident (Table 13). fiabbits. As can be seen from Table 14, the rabbit post— inoculation hemogram showed essentially similar changes after North Dakota virus inoculation as did the hemograms of cattle and sheep. The absolute and relative depression of the heterOphils and basophils was the most outstanding change in the post~ L_J O :3 0 Hi i 3 inoculation hemogram of rabbits. The depressi eosinophils and lymphocytes was absolute and not relative. A pronounced absolute and relative monocytosis was an addi~ tional finding in the rabbits. The depression of the average total leukocyte count from the pre-inoculation average was by 3494 cells or 38.48%. The corresponding depression far the average heterophil count was by 1590 cells or A7.73 and the depression oftflmaaverage lymphocyte count from the 6. pre-inoculation average by 2051 cells or Al.0%. A mild anemia was also seen. Transmission Attempts Routes of transmission and infectious a a to establish the susceptibility of the available native calves to the North Dakota virus, the following routes of inoculation were used simultaneously: intraveno‘s, subcu— taneous, intracutaneous, intramuscular, intranasal, intra~ mucosal, rectal, intratesticular, and intranodal (prescapular lymph node). After the susceptibility of the experimental {-2 ~ ‘A ‘ fl 1‘ I I‘ ,I ,‘ __ Oi. 11AM.» 43.4%“ calves had been proved in two calves, the route U) tion were restricted to either the intravenous or subcutaneous in the following calves. The infectivity of the bovine tissue-culture virus was confirmed by the inoculation oi Eh calves from two to eight months old, four yearlings and one two-year~old cow. All of these experimental cattle showed the typical sequence of clinicopathological changes outlined earlier. Attempts to transmit the experimental disease by us”‘ rd P 0733 materials other than the bovine tissue~culture~grown virus lailed. Fresh blood (Chapter II) without preservative, taken from experimental calves during the febrile andlruko— penic period of the experimen,al infection, and reinjected within ten seconds, did not reproduce the experimental infection in six calves. The blood was taken from the 65 experimental calves from one to nine days after inoculation. One of the six calves received the blood of a sheep donor. The challenge of the blood-inoculated calves two to four weeks later with the pure virus produced the typical clinico- pathological signs of the experimental North Dakota virus infection in each case. Other attempts were aimed at transmitting the experi~ mental disease by the inoculation of organ emulsions prepared from spleen, kidneys, lungs, liver, lymph nodes, and adrenals of experimental cattle sacrificed during the acute, subacute, or recovery period of the experimental infection. Three calves from two to four months of age were inoculated with organ emulsion and showed no clinical signs 01 inlet— tion. One of these calves (No. 17) was challenged seven days after organ-emulsion inoculation with the pure virus and developed the typical sequence of clinicopathological changes as described. The second calf (No. 27) received k) {'1 repeated injections of organ emulsions on days O, 14, ~c, L and 39 and showed no signs of infection. Sixty days fter the last injection the calf was challenged with the pure virus and proved to be resistant. The third cali (No. 3:) was given three injections of organ emulsion at weekly intervals and showed no signs of infection. Challenge with the pure virus two weeks after the last inoculation with organ emulsion proved this calf to be immune also. Rabbit kidney-cell culture harvest from an experi- ment to adapt the North Dakota virus to rabbit 66 kidney—cell culture (Rothenbacher, 1960) was ino ulated into three three~months-old calves. A three—months—old calf (No. 16) was given 10.0 ml. of this inoculum intravenously. While no clinical signs of infection developed, this calf proved resistant to challenge with the original (bovine- embryo-kidney-cell culture) virus three weeks later. The second three- -months -old calf (No. 25) received the same rabbit—cell-culture inoculum and developed mild clinical signs consisting of slight leukopenia and a temperature elevation of 1.5 F. Challenge of this calf with the bovine~ kidney—cell virus seven days later did not produce any }_J clinical chan nges . A second chal enge with the virus aiter one more week was also negative. The third three-months-old calf (No. 26) was given c-c m1. of the rabbit-cell culture intravenously. No clinico- pathological changes were observed within four days after inoculation. Challenge of this calf after two weeks with the bovine~ce11~culture virus did not produce any of the characteristic changes of the North Dakota experimental infection. The inoculation of one calf and one sheep with sterile tissue-culture medium did not produce ary cliricai, gross and histopathological changes in the exp~11 entai Contact transmission studies. A three—months—old and a five-months-old calf were placed in a stall together with an experimental calf which had just been inoculated with the North Dakota virus. The caltes ate and drank from the same trough and were kept in contact for ten days during the whole course of the experimental infection, Whi-e the inoculated calf in each case developed the typical sig ns of the experi- mental infection, none of the contact calves showed any LU clinicopathological changes. oth contact calves proved su sc “pt mile to the North Dakota virus challenge when inocu- lated five and eight weexs_, mp cti W.el;, after their contact period. No accidental contact transmissions occurred during the two year period of exrerinen tal work with the North Dakota virus. During this period an accidental transmission of virus diarrhea was reported by Rothenbacher t l. (1961) working -———— under similar experimental conditions with the nonculturable virus diarrhea agents from Iowa and Indiana. Koch‘s postulates. From the results of experimental studies reported, it is obvious hat the typical acute experi- mental North Dakota virus inflection could not be reproduced by any material other than the virus grown in bovine-embryo kidney—cell culture. The inoculation of sterile lactalbu:ni n hydrolysate plus 2% horse serum, the growth medium for the virus, did not produce any clinicopathological changes in two calves and one shee . Re iso olation of the virus (Earner t al., 1959) proved very difficult? Numerous specimens of internal organs, heparinized blood, nasal and ocular swabs, mucosal scrapings, and fecal specimens taken from experimental calves from two to 15 days after North Dakota virus inoculation O\ (1) failed to reveal the presence of the virus. The virus was recovered in only two instances from the lung, spleen, and kidney of experimental calves six and nine days after inocu- lation (Earner etval_, 1959)° The recovered virus was neutralized by North Dakota virus antiserum, and, upon re- inoculation into susceptible calves, produced the typical (D i H: '3 changes of the descrited exp gross Pathologica Eindings Experimental animals were euthanatized at various time intervals ranging from one to 134 days after virus inoculation (Shapter II). The most pronounced gross lesions were en- countered in animals necropsied during the acute phase of ection from two to six davs after inocu- , the experimental inf lation. Catt Sixteen calves from one to six months of age, 1 one yearling steer and one yearlin; heifer were necropsied from one to l4 days after North Dakota Virus inoculation and showed the following gro s lesions: U) External bodv surface;—-The con‘unctivae showed variable a J degrees of congestion, Occasionally, mucopurulent exudate and/or encrustations were present in the medial canthi of the eyes. Serous discharge was evidenced by matting of hair below the eyes of some animals necropsied from one to two days after virus inoculation“ A moderate degree of edematous swelling was usually seen in the submaxillary, prefemoral, and prescapular lymph nodes. .‘. is Oh» s9 Digestive tract.--The oral mucosae showed moderate to severe and patchy congestion. The parotid and the supra— pharynge eal lymph nodes were enlarged and congested. The tonsils similarly appeared swollen and congested. No could be discovered in the pharynx, rumen, reticulum, omasum. A moderate to severe catarrhal gastritis was present in the abomasum. The duodenum and th; miterior half of the jejunum showed catarrhal enteritis with patchy areas of marked congestion. A severe catarrhal to catarrn h: oichgic enteri- tis was noted in the posterior half of th, jejunum and extendirg oscasio.all to the ileum. Areas of marked ccnge 3 ion Wer more nu merous in this region and, in some animals, gave a "tiger stripe" appearance. An abundance of catarrhal c—x;d te ranging from clear to blood-tinged was usually ercoartrred 1n the jejuhua. The Peyer's -atc hes of the pos teriorje:unum and ileum appeared hyperplastic. Lesions in the Peyer's pitchr: varied from scattered petechiae and ecchymoses to patchy con— gestion and hemorrhagic marbling in the lympha 1c tir‘ua lager Hemorrhagic se ms of 4 to E cm. width were 0L“civ\d in tie submucosa and subs erosa around Peyer's patches of the pc~t3-:o* Ueianum The mucosa covering the Peyer's patches hnwgd mild degrees of fibrinous exudation and/pr fibrinonecroti “nangrs Similar changes in the form of a blackish-bro ;n nor~ rhagic to fib rinonecrotic seam o: 5 to lC mm. width wcie observed on the ile ocecal Scattered ecchymoses were lymphatic tissue of the os ostium protruding ofte n found in the tium (Fig. 10 and into the sut o: :muc 11). 09.3fo1. a1 70 The large and continuous Peyer's patches of the ileum were occasionally covered with cream-colored fibrinonecrotic epithelial layers that wiped off easily from the underlyin lymphatic tissue. The latter showed varying degrees of hem- rrhage and/or congestion (Fig. 7 and 11). L). L_J ‘ 3 (“r .3“ (D Only a mild catarrhal inflammation was note 0 cf 3“ (D H [.1 (D O O (D F) 9) t...’ O U) 1 t '1 A J cecum. From 30 to 45 cm. posterior t an area of small cystic degeneration interspersed with ecchyrotic hemorrhages was r G }_J n) w |.._1 K‘ M O 3 :5 CL y.) :5 (1“ .J (r) 'l g k) 0 I) )1) O l’*‘, eg the colon over an rea from 10 to 25 cm. in lergth. Only q ‘4‘ ' 1 . A—. 1 . ~ A' V ~ I ' A J "l‘ -. ‘ 1’... mild catarrhal Changes were encountered in the c0lon. in colon and rectum. Lon itudinal areas of merged congestion were seen on the rugae of the rectum. A band~shaped area from 2 to 5 cm. in length adfaceat and anterior to ;he and the cecum showed various degrees of edematous swelling and congestion of medulla and cortex. Scattered petechial and ecchymotic hemorrhages were seen in subcapsular and cortical areas of the lymph nodes of the posterior jegu a and ileum. Lymph nodes of the duodenum, coloA, and rectur were less severely affected (Fig. l, 2, 4, 5, and 6)- Respiratory tract.--There was a slight hyperemia of tn nasal mucosae with a corresponding mild edema and con- (1) gestion of the suprapharyngeal lymph nodes. The lung lymph 71 0 '3 nodes similarly showed a moderate degree 0: edema and conges— tion. No lesions could be found in the larynx, trachea, bronchi, or lungs 9 "I Heart.—~cubendocardiai suffusions were seen in two calves. No other visible lesions could be noted. Liver.--The liver showed a generalized congestion and a moderate swelling as evidenced by rounded ventral eo ge s. The gall bladder was distended up to twi3e its normal size. The b ile appeared dark and thickened. Spleem.-—The spleen was enlarged. A few petechial hemor- rhages were occasionally seen subcapsularly. The cut surface A. bulged and showed congeztion. Th mus.--Numerous petech ia l and ec chymotic hemorrha age were scattered subcapsularly and on the cut surface of the thymus gland. To a lesser degree these lesions were also present in glands from control animals. Urogenital trgct.-—A iew pe techia l hemorrhages were occasionally seen subcapsxlarly and in the cortex of the kid- neys (Fig. 13). Cortex and medul_a of the kidneys appeared '0 £1 cf 5. O I,_._J O anemic. No gross gical changes could be seen in the ureters, bladder, urethra, testes,or ovaries. Endocr ne lar s.--The adrenal glands appeared enlarged and showed variable subcapsular hemorrhage in the form of suf- fusions or scattered ecchymoses (Fig. 14). On cut section, areas of hemorrhage and/pr dar k brown necrotic foci of l to 5 mm. in diameter were discovered in the zona fasciculata. No gross lesions were seen in the thyroid gland and in the hypophysis. Central nervous system.~-No gross lesions could be found. Sheep. Six adult sheep from one and one~half to six years of age were necropsied from four to six days after North Dakota virus inoculation. The gross pathological findings seen in sheep compared closely to those described in cattle. The degree of the lesions 'was somewhat milder. No gross lesions could be found in the eyes, in the upper digestive tract, and in the respiratory tract. A catarrhal abomasits was present in all sheep and, unlike the syndrome in cattle, there was severe catarrhal duodenitis. The jejunum showed a moderate catarrhal inflam- mation with areas of pronounced congestion. The Peyer's patches of the posterior jejunum appeared hyperplastic and some were covered with a fibrinonecrotic pseudomembrane. In contrast to the mild involvement of the large intestine in cattle, the spiral colon of the sheep was empty of feed and showed a severe catarrhal colitis with abundance of clear and foamy viscid mucus. The mesenteric lymph nodes appeared slightly swollen and congested. Ecchymotic cortical hemor- rhages were noted occasionally. The liver was congested and the gall bladder distended. A moderate swelling and congestion were seen in the spleen. The kidneys appeared anemic. No other gross lesions were present in the urogenital tract. The central nervous system 73 did not reveal any gross lesions. On cut section, the adrenals revealed similar lesions as were des ribc d in cattle except that no subcapsular or cortical hemorrhages were present. nabbits. Seven adult rabbits ranging in age from one to three years were necropsied two to three days after North Dakota virus inoculation. The stomachs of the rabbits were empty and various amounts of hair were billed up in the furdus. r“; O k) 5: ‘) ‘3 a) x) (‘1‘ (D *3 *4. U) (n U) tic gro lesions could be definitely associatrd . v x" q t— 1 _.~ (:7 Li J .1. (x) ‘ ‘. x r—r, with the expe ri mental NOIWt Dakota virus in .Digestive tract.— diild patchy hyperemia of the lamina propria could be occasionally found in the stratified sq anous mucous membranes of the digestive tract glands of these membranes were found in a state Oi hroer~ secretion or secretory exhaustion in animals euthanatized during the acute or subacute ph se of the ex crincntcl in— C.) [at n J J) (.1 3 ('f‘ )3 (D (,7 CT fl k‘\ ——? ,2? 3 '4 f .1 ‘ .I X n fection. Occasional hydropic ch‘ and stratum cor neum of the esophag is, rumen, reticulum, and omasum were found. Rarely encountered focal hypcckeratotic changes of the rumen and esophagus were also present (Fig. 15). Abomasum The abomasum showed an acute catarrhal gastritis with a variable degree of infl rma*07v edema and 74 congestion in the lamina propria and in the submucosa. Copious amounts of seromucous exudate were found in the fundic and pyloric regions. Small intestine,- A similar acute catarrhal enteritis was seen in the duodenum, Numerous cystic submucosal glands (Brunner's glands) were observed filled with seromucous p. E:1”C€ O- (V) exudate and desquamated epithelial cells. The d catarrhal enteritis increased posteriorly in the jejunum and was fourxc to be catarrho—hemorrhacic to fibrinous in areas of the posterior jejunum. lnf‘la mmatory edema and n m; rem.i1 were marked, especially in the lamina propria of the villi and in the submucosa. Necrosis and desquamation were noted in the epithelial covering of the mucosal villi. Cellular “3 in iltrations were observed in the prOpria, including the villi, and consisted primarily of lymphocytes, macrophages, plasma cells, histiocytes, eosinophils, and a few ne utrop‘nil s The goblet cells appeared to be increased in size and nu.ber, especially in the posterior part of the jejunum and in the ileum, Catarrhal to hemorrhagic exudate of the posterior jejunum also contained monouclear cells, heterophils, and a few eosinophils. The hemorrhagic exudation was found to be primarily diffuse in distribution and probably derived (3 U o C, api lie "5 V‘J from diapedesis through the extremely congested of the denuded intestinal villi. Occasionally, petechial and ecchymotic hemorrha es could be seen in the lamina UT ‘7 propria and submucosa of the jejunum and ileum(Fig. 16 & 17). The posterior Jejunum and ileum, in many instances, also presented the histopathological picture of a fibrinous enteritis. This was particularly frequent in the mucosal areas covering the larger Peyer's patches. Fibrinous exudate on the mucosal surface was mixed with muons, necrotic inflax~ matory cells as described, and desquamated epithelial QCLiZ. s a n _‘\ \ v v .— .2 ‘y \ _" . A M‘ v .‘1 “K . r\ 7 ‘\ "x ‘ ‘fi ,. .i' A ”a A _ - . A vilii shened inilammatory edema, hyperehia, and cellula: H i, J U (D Q (D p) ’"i 1...] F...» (D h) *3 D. (D W F) ( .. infiltration similar to that deSC‘ patches showed inflammatory edema and hyperemia and were found in a state of lymphocytic hyperplasia during the acute phase of the experimental infection. Lymphocytic depletion and/or exhaustion was seen in animals euthanatized during the later stages of the acute phase. The severe and patchy congestion corresponded well to the described gross findings. Scattered petechial and ecchymotic hemorrhages were often present in the lymphatic tissue of the ileal Peyer‘s patches. Occasionally necrotic foci were observed in reaction centers forming micro-abscesses with lymphorrhexi and heterophilic infiltration. Extensive submucosal edema and congestion, combined with a fibrinonecrotic to hemorrhagic iiflammation of the mucosa,vwn%3consistently observed in sections of the iieo— cecal ostiun. Large intestine.- The cecal mucosa showed similar patgh O) congestion with a relatively mild catarrhal typhliti ‘ 76 mild catarrhal enteritis continued into the colon. The area over an approximately 25-cm.-long stretch of colon mucosa near the ceco-colic junction (as described in gross findings) con— tained numerous cystic mucosal crypts filled with mucus and cells, primarily heterophils and lymphocytes. Fibrinonecrotic anges of the mucosa of thi area were similar to Hae ones described in the ileum and posterior jejunum- The underlying Peyer's patches also showed occasional foci of lymphorrhex-‘ heterophilic infiltration of the reaction centers. The .ucosa of the remaining posterior portion of the colon dis~ played only mild catarrhal changes with increased mucous se— cretion. Areas of congestion were seen especially on the crests of the rugae of the rectum. Severe cor nge stion was also noted in the rectal mucosa immediately anterio to the anorectal junction. Lymph nodes of the diges ive tract— Similarly to the lymphatic tissue of the Peyer's patches, the lymph nodes of the digestive tract showed the histopathological picture of a reactive stimulation with va ria131e degr:.es o: irflrn- matory hyperemia, edema, and petechH l and ecchymet1c hemorm rhages. In the cortical and medullary sinuses of trr modes, reticulohistiocytic proliferation and lymphocytic hyper- plasia frequently created the classic picture of the "sinus~ catarrh",(Pallaske, 1960) with all sinus oidal spac es crammed by proliferated reticulohistiocytic (mononuclear) cells, 77 mature lymphocytes, heterophils, plasma cells, and eosinophils. The nuclei of these cells, congesting the marginal sinuses, were often found flattened in the tangential plane of the capsule. The capsule frequently showed various degrees of thinning and infiltration with mononuclear cell elements. Petechial hemorrhages were most commonly encountered in the reaction centers of secondary lymph nodules (Krellirg and Grau, 1960) and in the vicinity of small cortical arteriole: showing mild hyalinization of their wall structure(Fflg 18), The medulla of the lymph nodes commonly showed an inflammatory edema with an increase of reticulohistiocytic, plasmocytic and heterophilic elements. Increased hemosider— osis was occasionally seen in macrophages in the medullary sinuses, cortical reaction centers of secondary lymph nodules, and in cortical sinuses. Circular to fusiform bodies with onion-skin structure of 20 to 50 microns in diameter were also occasionally found in reaction centers. These bodies stained blue on hematoxylin—eosin stains and gave a positive reaction for calcium with the Von Kossa stain. They were also found in control animals and were considered a fortuitous finding (Trapp, 1960). A state of lymphocytic depletion was often seen in the primary and secondary lymph nodules of the cortex as evidenced by a severe narrowing or complete disap— "caps" of maturing lymphocytes. In pearance of the polar some lymph nodes the reactive reticulohistiocytic prolifera- tion led to a uniformalization of the cortical tissue with a resulting disappearance of the primary and secondary folli- cular structures. Balled-up proliferating histiocytic cells and an increased amount of nuclear debris was often seen in such lymph nodes. This was a common finding in animal 1 euthanatized during the later stages of the acute phas Cf. (D h experimental infection (Fig. 21 and 22). Reaction centers in many lymph nodes showed an :1. + :3 :3 *4 (x H ‘D Q.) '3 “3 F5 U 9:.) 3 (D :3 U) D .3 Q; Q) O. (D f) "5 \D Ll) U) (D O *4.) H \D T“ f] f FJ U) ’3 f) C) }__J l”“' rj) r" "S L k r’w Q) m ((4 C.) *3 n , J ( L >4 *d 7;) < (D *3 (D (D 'i k I O 5; d (D *3 (0 Li; H1. 1'5 ’U H .i k") 9. D *5 }_J 5 *0 :3 .1 ( \ L) -. -s. ‘ ‘1 “ ‘ . f- A. (“an tn these necrotic leci. iLe O < 3 H. c) C—f (D LL 9‘ were usually 353 pronounced of these described l sions were seen in the I) .4 at 0 Th ( .3 of the ileocecal region, the ileum, the posterio jejunum, and the head region (Fig. 19). mild hyperemic and hypersecretory cnanges similar to t described for the upper digestive tract. igs - Even as the gross pathological examinati the lungs did not yield any pertinent findinss, the his ’ ‘I g u 1 n 1‘. P‘ , ’- ’ .. ‘ , , — 9’" .t ,- -. '- 4—1‘,7 v\ \ pathological findings were primarily coniined to -_e t H) O "5 Ct J” (D H d {1; U- L) :3 O Q (D U) 0 H.) of I T “D . T \D u) L) Q) :3 L1. c t .3 (D d) u: 0‘: (' ( i r L) ‘t ’ 3 L3 ( 3 ‘ 4_ . r7 ‘,..‘ a m.._ / rs of tne cortex. heteropniiii Essr' S D “V‘. L {I} v! -J O O f.‘ "<1 0 7") Slight evi ence of peribronchial and interstitial monov*. D .filtration and congestion was Otcuéionallv noted. H) ( I) Q t {D \ . r A . , _. r . Q ~.. , J ‘ < .. t '» ‘ 7‘3 .; n 0') ’1 -\1 O -J U} (111 ‘k/L '1’) (f (D Cardiova ar sv, m.—-Besides a few subendocardial ecchymoses and suffusions no other histopathological changes were seen. Such endocardial hemorrhages are frequently present in electrically stunned animals. Liver.--A mild edematous swelling of the liver paren- chyma was evidenced by intra— and extracellular edema and edematous distention of the Disse-space (Krolling and Grau, U) 1960). The sinusoids and intralobular capillaries appeared distended and were sometimes filled with a serous fluid. Microfoci of reticulohistiocytic and mononuclear cells cculd be seen intralobularly in the parenchyma or perilobularly, ct adjacen to the portal triads. The Kupffer cells appeared in a state of active proliferation. Occasionally the liver parenchyma showed microfoci of necrosis with pyknosis, karyorrhexis, and karyolysis. lnfiltrations of mononuc ear cells tended to assemble around the necrotic foci and sinus- oidal and capillary congestion was common in the vicinity of the foci (Fig. 25 and 24). Lymphatic system.-—Similarly to the lymphatic tissue of the digestive tract, the spleen showed lymphocytic hyper- plasia with extensive reticulohistiocytic proliferation. In the later stage of the acute phase of the experimental infection, lymphocytic depletion and exhaustion, edema of the reaction centers and depression of the reticulocytic mitotic activity could be noted. Microfoci of lymphorrhexis 80 with neutrophilic infiltration were a fairly common occur- rence in the spleen. Petechial and ecchymotic hemorrhages were also seen in splenic follicles as described in the reaction centers of mesenteric lymph nodes. An increased hemosiderosis of the free macrOphages and sessile reticu— r—‘f- "‘3 locytes could also be seen during the acute phase of t. infection (Fig. 20), As in the spleen, numerous petechial and ecchymo is hemorrhages were noted in the thyn al medullaiy light sub- stance or at its periphery. A depletion of the medulla was evident and focal IP‘°POthllC infiltration was ire- U ' if“ . ”r" Y ”3 i" j r‘ r‘, 1 fr Ir ‘1 r5 .3 Oi lili Eu .6! c..'. 1-31., .L...‘ \D quent tly seen. ihe Hassrll lodi appeared to be fewer in number and at an immature stage. The other body lymph nodes showed similar or identical histopathological changes as alrea d described for the C«:‘ gastrointestinal and respiratory lymphatic tissue. Bone marrow.-—Limited histopathological studies of the bone marrow of experimental cattle appeared to correlate -. v.1:1 )3 . 4'L‘~‘.’.“ a Wfr.J..J...€-:.4 in J.,a‘..'_C with the hematological findings described (I) Mn g>ter . A depression of the myelomenous activity was ([1 \ evident and there was a pauCi ty Oi mature and immature h~ ophils and eosinophils as cozpared to the control animals. ) )1 Kidneys.—-As in the cpse of the liver, the Kidney responded with mild focal or diffuse nononuc lear prolifera- tion in the cortex. Large numbers of the glomerular tufts 81 were shrunken and appeared nonfunctional. Bowman's capsules were often found to be thickened. There was capillary hyper— emia in the vicinity of the affected glomeruli. Petechial hemorrhages in the cortex were rarely observed (Fig, 25 and 26), Ballooned Bowman‘s capsules were occasionally seen. A focal interstitial nephritis was evidenced by proliferated interstitial cells obstructing tubules and capillaries, expeoially in the area of the corticomedullary junction, and resulting in severe focal congestion in this region and dis- tention of renal tubules anterior to the obstruction sites. Obstructed tubules frequently contained hvaline or granular casts. A small number of hyaline casts resulting from foeal interstitial nephritis was observed to a lesser degree in tissues from control animals. andocrine glands.-- Adrenal ~land.- The most ronounced histo atholooical O changes in the experimental North Dakota virus infection were encountered in this gland. In accord with the gross lesions, effusions could be seen in the loose pericapsular connective C0 tissue. What appeared on gross inspection as peteehial or ecchymotic capsular hemorrhages turned out to be numerous distinct foei of necrosis with severe paripheral capillary congestion and extravasations in the cortex close to the capsule. These distinct cortical foci of necrosis were present in l2 out of 14 calves euthanatized during the acute phase of 'f 82 the experimental infection. They consisted of spherical foci in the zona fasciculata showing coagulation necrosis, karyolysis, and occasional karyorrhexis in the center and primarily vacuolar, cytolytic, and karyolytic changes at the periphery. Pyknotic nuclear changes in dark red~ to purple- staining cells were also occasionally seen at the periphery of a necrotic focus. The necrotic foci were surrounded by severe congestion, infiltration of mononuclear and hetero- \ nilic cells and serous exudation into interstitial spaces 'U in the vicinity of capillaries. In some cases the picture of coagulation necrosis predominated while in other: karyor~ rhexis and more extensive heterophilic infiltration could be seen. These necrotic foci varied from 100 to more than 1,000 rnicrons in diameter. They were always located in the zona fasciculata, sometimes infringing upon the pe‘ipheral zona glomerulosa and the more central zona reticularis (Fig. 29). The parenchymal cells of the zona fasciculata and its neighboring zones also showed marked alterative parenchymal changes consisting of vacuolation and reticulation of cyto~ lasm and numerous eccentric nuclei. A foam“ reticu p J V ~ . ' o h v ' 7 .- o . ‘7»5 A -~ fi","_’“~‘«” ,§‘,‘ fi 17 c*toplasmic structure and partial cytolysis hurt liaiuentiy ~ . ‘ ~ A P! -\ ‘0 ~" f‘ 0 ~ ‘ A u . 3““—\ r'.<‘; seen. This oiange was most pronounced in the per gflpfal zona fasciculata. The zona glomerulosa rarely showed the vacuolar changes (Fig. 28). Small colloid-like drOplets were occasionally seen in the zona fasciculata. These were probably similar to or 83 identical with the hyaline—like droplets described by White- man (1960) in Iowa mucosal disease field cases. Whiteman (1960) ascribed these droplets to dehydration and hemoconcen— tration, clinical signs also present to a moderate degree in the North Dakota virus experimental infection. Between the cell cords of the zona fasciculata showing vacuolar and/br cytolytic-reticular changes, strands or microfoci of elongated, histiocyte—like dark red- or purple~ staining cells with pyknotic nuclei could frequently be observed, primarily in radial arrangement. This cell type was also occasionally observed at the periphery of the necrotic foci as described previously. Similarly staining cells could sometimes be seen in adrenals of control animals. Identical cells were documented by Whiteman (lQQO) in the adrenal cortices of Iowa mucosal disease field cases and of n erotic (D described as scattered cell necrosis consistinT cells with increased cytoplasmic staining, cell shrinkage, and pyknosis (Fig. 30). Increased mitotic activity, as described by Whiteman (1960)in the peripheral zona fasciculata-intcrmedia of Iowa mucosal disease field cases, could not be observed in the North Dakota virus experimental infection. A constant finding in the adrenal cortices oi Dakota virus—inoculated cattle was the marked inner progres- sive transformation (Matthias, 195M; Krolling and Grau, 1960) consisting of a hypertrophic widening and “xtension (A) 84 of the zona fasciculata at the expense of the zona glomeru- losa and zona reticularis. In all the adrenals studied, a narrowing and compression of the zona glomerulosa was a rather constant finding. As a result, the adrenal capsule .1 appeared stretched and thin as compared to control anima U) *— As a consequence of this progressive tr ns uAth” the zona intermedia could no longer be distinguished. Though 1m1ite~ man (1960) did not use the term, he accuraiel: described a ‘ "\I’”"’ C ‘4 . n. a u r— 04 P: a_ ,_ f b V. -‘ T “I... progness.ve tr an S=formation in ij 01 the examined losa . " A? . . A 9 .. , -. , .—. 1,. ,j. .7 “ 'v “ .— mucocal disease field cases. In other cases he seemed .o or.- '. 1“ * "r. r< 'v . ‘ . 1"}- De P9113»: islve Gnarlges . Griem (1955) saw similar progressive changes in ho UN cholera—infected pigs with acute gastrointestinal involve- ment. Special stains applied to the described sdreral charges (N V\' 757‘.“ (1. a (JU) *‘3 r)-’ U }__1 F (‘1‘ L~ £2; ( ) Cf J *5 \J (.4 F" I re J I Q l r ther variable firdir () }--J c) ts of scattered D ,C "5 1 arms p) O and Sudan IV, yielded only minim positive-staining globules in the cells of the glomerulo a, fasciculata and in the medulla. The uniformly are n oii~r¢c~ O-stained adrenal sections appeared somewhat darker in the .rciculata and medulla. In no instance canld a 7cm- parable lipidosis be obtained as documented by White in (1960) for Iowa mucosal disease. Whiteman (19cc) reportec that the lipids of his oil—red—O-stained adrenal sections corresponded in location and amount with the fatty alterative changes described from the hem atoxylin-eosin-stainwd paraffir CD 5 sections. The fatty changes seen by Whiteman (1960) are, in location and description, similar to or identical with the vacuolar and reticular degenerative changes described here. However, no lipidosis was seen in the experimental North Dakota virus infection. An increased accumulation of lipid droplets in the (D C’) U! U) P (’1‘ r... ,L; .1) (‘7‘ F4 (J (1" a 3 j) bovine adrenal as a result of chronic str reported by Bell and Weber (1959). This mi (1‘ ""3ij y~ r‘ .A— C‘ u e stress of short duration as caused by the experimental North Dakota virus infection. On oil~red-O—stained sections occasionally some increased reddish—brown staining amorphous matter was noted at the periph— ery of the described necrotic foci. Some necrotic foci showed a slightly positive reaction for amyloid when stained with crystal violet. PAS-positive particles were Often seen in the centers of the iecrotic foci. Distinct foci of coagu- lation necrosis in the zona fasciculata, as frequently seen in the experimental North Dakota virus infection, took on similar tingibility as did the connective tissue of the adrenal capsule when stained with Heidenhain's aniline llue stain. Globular and rod—shaped, bright red-staining bodies of l to 2 microns in diameter were described by Whiteman (l9ti) in the zona glomerulosa cells of Iowa mucosal disease field cases when stained with Crossman‘s modification of Mallory's connective tissue stain. Similar or identical bodies were 86 seen in the same cells in North Dakota virus—infected cattle as well as in control animals when stained with Heidenhain's aniline blue stain. Thyroid gland.- Upon histopathological examination, no definite changes could be found in the thyroids of experi— mental animals. In several instances extensive vacuolation was seen at the periphery of the colloid in the smaller (I) acini. The vacuoles varied in size from lO to EC micron This change was present to a mild degree in the thyroids of control animals. Hypophysis.- The histopathological changes of the *ongestion was noted in (\ hypophysis were indistinct. Severe the adenohypophysis and in the pars intermedia. Occasionally microfoci of pyknosis and coagulation necrosis were present in the pars intermedia. Patchy areas of eosinophilic and basophilic degranulation were encountered in the anterior (1) part of the adenohypophysi . Quantitative and morphological studies, such as those attempted by Whiteman (196C), were found to be unreliable and were not pursued. Other body systems and tracts ~-No significant nisto~ pathological changes could be found in the central nervous system, the spinal cord, the musculo-skeletal system, the .‘ l ’- ""( male or female reproductive tracts, or th, ski C1?) ‘4 J Histopathological findings in North Dakota virus— inoculated sheep. The histopathological changes seen in the tissues of experimental sheep resembled those described in cattle. They tended to be somewhat milder in the respiratory tract, the liver, and the kidneys. Five out of five inocu- lated sheep showed pronounced changes in their adrenal glands similar to those in experimental cattle. excessive nemosiderosis of the spleen was present in all experimental (D sheep, but not in the controls. The changes in the ga tro~ intestinal tract, the spleen, and the adrenals tended to be more severe than those reported in calves. inoculated rabbits. Corresponding to the mild hematological changes in the experimental rabbits, the histopathological lesions were also slight and difficult to evaluate. The lesions in the lymphatic system and in the adrenal glands appeared to be similar to those described for the cattle and sheep. Changes found in the liver, spleen, and kidneys were variable. In general, the lesions were considered to be too ) mild in degree and too variable to make the rabbit a useful experimental animal. Other Post-Nortem Findings UFifiespecimens of cattle and sheep taken at the time of necropsy and submitted for urinalysis did not show ny changes from the normal. Periodical fecal examinations were similarly negative. No significant changes were found in limited blood—sugar and nonprotein~nitrogen determinations. Aseptically removed organ specimens from euthanatized experi- mental animals were submitted for bacteriological examination (OSaPUEP 31). No pathogenic organisms were isolated. Serological and Immunological Findings The antibody response to North Dakota virus inoculation was studied in several calves. Pre— and posteinoculation sera were submitted to the virology section, Department of F.) Nicrobio ogy and Public Health. No neutralizing antibodies were present in the pre- inoculation sera of experimental calves as indicated by tissue—culture studies. Eight days after inoculation, less than 101 neutralizing antibodies were present in the serum. On days 15 and 23 after inoculation the neutralization index was 102. Following reinoculation on day 23, sera taken on day 33 through day 65 had indices equal to or greater 4 than 10 in one animal. Four calves proved resistant to reinoculation from 14 to 130 days after the experimental North Dakota virus in— fection. One calf, inoculated with the rabbit tissue culture harvest (Rothenbacher, 1960), proved resistant to challenge with the bovine-embryo kidney-cell virus given 7 and 14 days after the original inoculation. Another calf similarly 89 inoculated resisted challenge with the North Dakota virus (bovine tissue-culture origin) 14 days later. Two calves having received three or more injections of organ emulsions (three injections at weekly intervals in one instance and four injections on days 0, 14, 32, and 39 in the other) proved resistant to challenge with the North Dakota virus. A third calf was fully susceptible to virus challenge seven days after inoculation with organ emulsions. CHAPTER IV DISCUSSION Transmission Studies Limited transmission attempts by contact and by using inoculation materials other than the tissue culture grown virus were unsuccessful. This indicated that sufficient quantities of the infectious agent were not present in the body discharges and in the blood and organ emulsions taken from experimental cattle during the febrile and leukOpenic phase of the infection. The fact that two calves receiving three or more inoculations with organ emulsions proved resistant to challenge with the tissue culture virus indi- cated that some form of a viral antigen may have been present in these organ emulsions from experimentally infected cattle. The difficulties in reisolating the virus from blood, organs, and body discharges of experimentally infected animals have been reported (Barner et al., 1959). In two instances, however, the virus was recovered from kidneys, spleen, and lungs of calves euthanatized six and nine days, respectively, after virus inoculation. The identity of the virus was proved by neutralization tests with North Dakota antiserum,; and by reinoculation into several calves. 91 Comparison of the degree of gross and microscOpic pathological findings in cattle, sheep, and rabbits may indi- cate that, of the three animal species the bovine might act as the most probable host of the virus in a possible but as yet unknown natural transmission of epizootic importance. The pathogenicity exhibited and the lesions produced by the virus in cattle strongly suggest that it does not fit into the bovine enterovirus (ECBO) classification like the one described by Soliman (1958). Gross and HistOpathological Findings Acute catarrhal inflammation was evident in the gastro- intestinal tract starting from the abomasum. Rarely seen hyperkeratotic foci of the ruminal, reticular, and esophageal mucosae were interpreted as being due to causes other than the North Dakota virus inoculation. The possibility of a hypovitaminosis A was considered as a cause (Pallaske, 1960). Hydropic changes encountered in the stratum lucidum and corneum of the esophagus,rumen, reticulum, and omasum may be interpreted as a normal finding according to Krolling and Grau (1960). Catarrhal inflammatory changes often increased to fibrinous and hemorrhagic in the posterior half of the small intestine. The hemorrhagic to fibrinous exudation was thought to be due to diapedesis through extremely con— gested capillaries of denuded intestinal villi, since petechial and ecchymotic hemorrhages were rarely observed in the lamina propria and submucosa of the jejunum and ileum. 92 Lymphatic tissues. An initial state of lymphocytic hyperplasia during the acute phase giving way to lymphocytic depletion and/or exhaustion was the common histopathological picture seen in the Peyer's patches as well as in all lymphatic tissues of the body. Necrotic foci that were often present in secondary reaction centers of Peyer's patches and lymph nodes were considered a sequence to lymphoid exhaustion. Lymphor- rhexis and an abundance of nuclear fragments with pronounced heterophilic infilitration were found in the necrotic foci. Pallaske's (1960) description of the so-called "sinus-catarrh" consisting of densely crammed peripheral sinuses with various stages of lymphoid cells mixed with neutrophils, plasma cells, eosinophils, and reticulohistiocytic cell elements was often observed in lymph nodes during the subacute and/or recovery phase of the experimental infection. Petechial hemorrhages frequently seen in secondary reaction centers of, especially, the mesenteric lymph nodes and corresponding to those reported in Peyer's patches were thought to be due to a possible attack of the virus on the peripheral capillaries. In lymph nodes from experimental cattle euthanatized during the subacute and recovery phases of the North Dakota virus infection, the disappearance of primary and secondary lymph nodules which took place and the uniformalization of the cortical architecture resembled the tertiary stage of 93 Krolling and Grau‘s (1960) description of lymph nodules. The cortical tissue consisted mainly of the reticulohistiocytic, large lymphoid and plasmocytic elements and a paucity of mature lymphocytes was noted in such nodes. The similarity to Raffel‘s (1961) description of the antibody—producing lymph node cortex was evident. The significance of conglomer- ated reticulohistiocytic cells into globular dark-staining structures of 20 to 100 microns in diameter was considered uncertain. Bone marrow. In limited histopathological bone marrow studies it appeared that the hematological findings of hetero- penia and eosinopenia were due, not merely to a depression of circulating cells, but to a generalized depression of the myelogenous activity resulting in a paucity of mature and immature heterophils and eosinophils. This fact correlated to the absence of immature cell forms noted in the hemogram of virus inoculated cattle and pointed to the bone marrow as a major target of the experimental North Dakota virus. Respiratory tract. In the respiratory tract very mild catarrhal and hyperemic changes were considered secondary to the febrile reaction of the experimental infection. Focal areas of congestion and the already described lesions in the lymphatic tissues were the only findings in the lungs. Based on the questionable significance of these lesions, it is believed that the North Dakota virus is not primarily a 914 respiratory virus such as the parainfluenze—3 (Abinanti 33 _al., 1960) and the infectious bovine rhinotracheitis viruses (Hagan and Bruner, 1961). Adrenal glands. As in the liver and kidney, the epitheliotropism of the North Dakota virus was most evident in the parenchyma of the adrenal cortex. The most pronounced lesions were encountered in the zona fasciculata. The similarity of the vacuolar parenchymal change with Whiteman's (1960) description of adrenal lesions in Iowa mucosal disease was pointed out. However, a lipidosis could not be found in these affected cells. The increased accumulation of lipid droplets in the bovine adrenal as a result of chronic stress reported by Bell and Weber (1959) could be confirmed by White— man (1960) for Iowa mucosal disease. The reason why this change was not found in the adrenals of North Dakota virus- inoculated cattle may be due to the fact that this experimental infection constitutes a more acute stress. Distinct and ex- tensive necrotic foci of the zona fasciculata were not common in the adrenals of cattle affected with Iowa mucosal disease as reported by Whiteman (1960). A cell form of elongated histiocyte-like shape and with a uniformly dark red or purple staining cytoplasm and uniformly dark blue homogenous and pyknotic-appearing nucleus was seen in the zona fasciculata. The darker cells occurred individ— ually, in small aggregations or in radial strands between the lighter cells of the fasiculata. They were identical with the cell form documented and described by Whiteman (1960) as "scattered necrotic cells with increased cytoplasmic staining, cell shrinkage and nuclear pyknosis.” This author tends to believe that these cell forms may not constitute necrosis but resting, (Matthias, 1954) regenerative, or reve*sible physiological exhaustion stages of parenchymal fasciculata cells. Further degeneration stages of these darkly—staining cells were not commonly seen in the adrenals of North Dakota virus inoculated cattle nor were they reported in adrenals of Iowa mucosal disease field cases (Whiteman, 1960). This cell form was not commonly involved in the necrotic foci of the vesicular lightly-staining fasciculata cells reported here. The progressive inner transformation of the adrenal cortex was found to compare well to the descriptions by Matthias (1954) and Griem (1955) of the adrenal changes in pigs caused by the acute infectious stress of hog cholera. Summary of Discussion In summarizing this discussion of the clinical, gross, and histopathological changes it may be stated that the experimental North Dakota virus infection resembled that of a pantropic virus, affecting most notably the mucous mem- branes of the intestinal tract, the parenchymatous organs, the lymphatic tissues, and the bone morrow. The depression of circulating eosinophils, heterophils, and lymphocytes with 96 depletion and exhaustion of lymphatic tissues is compatible with the theory of adrenocortical hyperactivity during acute infections. The possibility of a direct effect of the virus upon the adrenocortical cells may be assumed since the described focal adrenocortical lesions have not been reported in other acute infections such as virus diarrhea, malignant catarrhal fever, infectious bovine rhinotracheitis, calf pneumonia, bovine salmonellosis, pasteurellosis, and enter— otoxemia. A primary attack by the vi°us or a secondary hormonal effect upon the hypophysis, thyroid, the lymphatic system and the bone marrow may also be assumed. From the difficulties in transmitting the experimental disease and in reisolating the virus, and from the absence of neutralizing antibodies in a large number of native cattle, the true origin of the virus as a bovine enterovirus may be doubted or an obscure mode of transmission assume CHAPTER V SUMMARY AND CONCLUSIONS The characterization of a disease caused by a new re bovine viral isolant~-the North Dakota mucosal disease ‘1 virus—-was attempted. Studies included clinical, hemato- logical, immunological, serological, and pathological i" — v I L: observations on experimentally inoculated cattle, sheep, and rabbits. The experimental infection in cattle was character- ized by an acute monophasic febrile reaction combined with a severe leukopenia and heteropenia. Clinical signs of the experimental infection in cattle included partial anorexia, nervousness, tachycardia, increased respiration, ocular discharge, depression, and constipation. The experimental infection lasted from six to ten days and resulted in im— munity measurable by serum—neutralizing antibodies and resistance to challenge 14 days after the initial inocula- tion. The major gross and histopathological lesions produced by the North Dakota mucosal disease virus were confined to the adrenal glands, the gastrointestinal tract, the lymphatic tissues, the kidneys, the liver, and the bone marrow. They consisted of inflammatory and necrotizing changes of the 98 epithelial tissues of the gastrointestinal tract and the parenchymatous organs. Inflammatory, regressive, and necrotizing changes were noted in the lymphatic system. Regressive changes were also present in the bone marrow. Contact transmission did not occur. Experimental cattle inoculated with fresh blood and organ emulsions of .VT infected animals failed to contract the infection. Three or more inoculations of calves with organ emulsions resulted in immunity to challenge with the tissue-culture—grown virus. . .' 1.! 0",.” Virus reisolation attempts failed to reveal the presence of the virus in fresh blood, nasal and ocular swabs, mucosal scrapings, and fecal specimens of infected cattle. The virus was recovered from the spleen, kidneys, and lungs of two calves six and nine days, respectively, after virus inoculation. The experimental North Dakota virus infection in cattle did not resemble in all respects the classical descrip— tions of Iowa mucosal disease, malignant catarrhal fever, Ume disease, parainfluenza-3 respiratory infection, virus diarrhea, or infectious bovine rhinotracheitis. The experimental disease in sheep and rabbits was similar but milder in degree and of shorter duration. BIBLIOGRAPHY BIBLIOGRAPHY Abinanti, F. R., Byrne, R. J., Watson, R. L , Poelma, L. J., Lucas, F. R., and Huebner, R. J. 1960. Observations on infections of cattle with myxovirus parainfluenza-3. Am. J. Hyg., 71:52 58. 3‘ Abinanti, F. R., and Plumer, G. J. 1961. Infectious bovine rhinotracheitis virus in cattle with conjunctivitis Am. J. Vet. Res., 22:13—17. A.,! s ‘ 1'3". Albrecht‘s. 1929. Handbuch der pathogenen Likroo organismen, Vol. 9, 1929- Edited by Kolle, W., Kraus, R., and Uhlenhut P. Gustai Fischer, Jena. Cited by Schultz, 1959 Pathologisch-anatomische Befunde beider sogen ten ”mucosal disease“ (Schleimbautkrankheit) des Rindes. Deutsche Tierarztl, Wsch. 66:586—588. \ Armed Forces Institute of Pathology. 1960. Manual of histo— logic and special staining technics. Second ed., McGraw-Hill, Inc., New York. Bajwa, c. s. 1961. Unpublished data. Thesis (in preparation), Michigan State University, East Lansing, Michigan. Baker, J. A., McEntee, K., and Gillespie, J. H. 1960. Effects of IBR-IPV (infectious pustular vulvovaginitis) on newborn calves. Cornell Vet., 50:156-170. Baker, J. A., York, C. J., Gillespie, J. H., and Mitchell, G. B. 1954. Virus diarrhea in cattle. Am. J. Vet. Res., 15: EBB-Ejl. Bakos, K., and Dinter, Z. 1960A. Identification of a bovine mucosal disease virus isolated in Sweden as Myxovirus parainfluencae-3. Nature, 185:549~ 1960B. Antikorperreaktion des Rin nde Iniektion mit dem Virus der Parainfluenza -3. Zbl. Bact. I (ori .), lBOzl-ll. Bakos, K., Isaksson, A., and Nystedt, H. 1958. Cited by Bakos and Dinter, 1960A. Identification of a bovine mucosal disease virus isolated in Sweden as myxovirus parainfluencae-3. Nature, 185; 549-550. Barner, R. D., 1955-60 chief investigator, Dept. Vet. Pathology; project NCe34: Mucosal-respiratory diseases of cattle. Personal communication. lOl Barner, R. D., Cunningham, C. H., Morrill, C. C., and Rothen- bacher, H. J. 1958. Annual progress report for 1957- 58; project NC—34: Mucosal diseases of cattle. M S.U. . 1959. Annual progress report for 1958—59; project NC—34: Mucosal diseases of cattle. M.S.U. Barner, R. D., Whiteman, C. E., Cunningham, C. H., Rothen— bacher, H. J., and Church, C. C. 1960. Annual prog— ress report for 1959-60; project NC-34: Mucosal diseases of cattle. M.S.U. Beck, C. C. 1959. Dept. of Surgery and Medicine, M.S U. Personal communication. Beckenhauer, W. H., Brown, A. L., Lidolph, A. A., and Norden, C. J. 1961. Immunization of swine against hog cholera with a bovine enterovirus. Vet. Med., 56: 108-112. Bell, J. T., Jr., and Weber, A. F. 1959. A comparati study of lipid accumulation in the adrenal glar d mature nonpregnant dairy heifer, nonpregnant la dairy cows, and pregnant lactating dairy cows. Jour. Vet. Res. 20:53-60. 0 1 ve r; I cta ti Am Berkman, R. N. 1958. Bovine malignant catarrhal fever in Michigan; I. Occurrence, II. Pathology, III. Differen— tial diagnosis, IV. Comparison with similar syndromes in other countries. Thesis, M.S.U. Berkman, R. N., Barner, R. D., Morrill, C. C., and Langham, R. F. 1960. Bovine malignant catarrhal fever in Michigan. II. Pathology. Am. J. Vet. Res., 2121015— 1027. Blood, D. C., and Henderson, J. A. 1960. Veterinary Medicine. The Williams and Wilkins Co., Baltimore. Bogel, K., and Mussgay, M. 1960. Isolation and properties of an enterovirus from cattle. Zbl. Vet. Med , 7:534- 552. Bray, W. E. 1957. Clinical Laboratory Methods, 5th ed., The C. V. Mosby Co., St. Louis. Carlson, R. G., Pritchard, W. R., and Doyle, L. P. 1957. The pathology of virus diarrhea of cattle in Indiana. Am. J. Vet. Res., 18:560—568. Carlstrbm, G. 1960. Comparative studies on measles and distemper viruses. Acta Paediatr. 8:25— 32. 102 Claflin, R. M., Gillette, K. G., Gustafson, D. P., Moses, H. E., Tietz, W. J., and Tyler, D. E. 1957. Annual progress report, Project NC-34: Mucosal diseases of cattle. Purdue University, Lafayette, Indiana. Claflin, R. M., Gillette, K. G., Gustafson, D. P., Moses, H. E., Taylor, D. 0. N., and Tyler, D. E. 1959. Annual progress report for 1958-59; project NC—34: Mucosal diseases of cattle. Purdue University, Lafayette, Indiana. Coffin, D. L. 1953. Manual of veterinary clinical patho- logy. Third, ed., Comstock Pub1., Ithaca, New York. Cunningham, C. H., and Church, 0. C. 1960. Virology section, ept. of Microbiology and Public Health, M.S.U. ersonal communicat13n. Darbyshire, J. H. 1960. A serological relationship between swine fever and mucosal disease of cattle. Vet. Rec , 72:331-334. DeLay, P. D. 1959. l"m Island, N. Y. Minutes of t technical committee metting, Univ. of Wis., Madi Wisconsin, 1959. Dmochowski, L. L961. Chief, Virology and Electron Micro— sc0py Section, M.D. Anderson Hospital and Tumor Insti— tute, Univ. of Texas Medical Center, Houston, Texas. Personal communication. Dow, C., Jarrett, W. F. H., and McIntyre, W.I.M. 1956. A disease of cattle in Britain resembling the virus iarrhoea-mucosal disease complex. Vet. Rec., 68: 620-627. Dukes, H. H. 1955. The physiology of domestic animals. Seventh ed., Comstock Pub1., Ithaca, New York. Edwards, M. J., and Sier, A. M. 1960. Bovine epizootic diarrhea in Western Australia. Austral. Vet. J., 36:402-404. Gillespie, J. H., Baker, J. A., Burgher, J., Robson, D. 8., and Gilman, B. 1958. The immune response of dogs to distemper virus. Cornell Vet., 38:103-108. Gillespie, J. H., and Baker, J. A; 1959. Studies on virus diarrhea- Cornell Vet., 49:439-443. Gillespie, J. H., Baker, J. A., and McEntee, K. 1960. A cytopathogenic strain of virus diarrhea virus. Cornell Vet., 50:73-79. 103 Goret, P., Brion, R., Fontaine, M., Pilet, 0., Girard, M., and Moraillon, R. 1960. Echec des essays de preven- tion et de traitement de la maladie de Carre par 1e serum contre la peste bovine. Bull. Acad. Vet., France, 33:343—347. Goret, P., and Pilet, C. H. 1958. Maladies des muqueuses. Affections a ultravirus oe s bovides nouvellement decrits. Rec. Med. Vet., 13 4: 53-80. Griem. 195 . Cited by Pallaske (1960). Patr olo gis sche Histologie. Second ed., Gustav Fischer, Jena. Hagan, W. A. 1958. The mucosal disease complex in cattle. Mededelingen der Veeartsenijschool van de Rijksuniver- siteit te Gent. 4:51-67. Hagan, W. A., and Bruner, D. W. 1961. The infectious dis- eases of domestic animals. Fourth ed., Comstock Pub1., Ithaca, New York. Harshfield, G. S. 1957. Annual progress report for 1956-57; project NC-34: Mucosal diseascs of cattle. South Dakota Agr. Ex t. Sta tion, Brookin_s, South Dakota. C Hedstrom, H., and Isaksson A. 1951. Epizootic enteritis in cattle in Sweden. Cornell Vet., 41:251-253. Hoag, W. G., Rooney, J. R., and Williams, W. J. 1956. A mucosal type disease of cattle in Virginia. J. Am. Vet. Med. Assn., 129:105-110. Huck, R. A. 1957 Mucosal disease complex. J. Com p. Path. and Therap. 67:267—276. Hutyra, F. V., Marek, J., Manninger, R., and Mocsy, J. 1954. Spezielle Pathologie und Therapie der Haustiere. 10th ed., Fischer, Jena. Jarrett, W. F. H. 1958. British mucosal disease. Vet. Rec., 70:48-50. Johnston, K. G. 1959. The virus diarrhoeas of cattle and similar diseases. Austral. Vet. J., 35:323-324. Jones, L. D. 1959. Annual progress report for 1958—59; project WG-34; Mucosal diseases of cattle. South Dakota Agr. Expt. Station, Brookings, South Dakota. Kniazeff, A. J. 1960. Communication to the annual NC- technical committee meeting, Brookings, South Dakota. 104 Kniazeff, A. J., and Pritchard, W. R. 1958. Annual progress report for 1957-58; project NC—34: Mucosal diseases of cattle. Florida Agr. Expt. Stations. 1960. Antigenic relationships in the bovine viral diarrhea—mucosal disease complex. Proc. U. S. Livestock San. Assn. 64th meeting: 344-350. Krdlling, 0., and Grau, H. 1960. Lehrbuch der Histologie und vergleichenden mikroskopischen Anatomie der Haustiere. Tenth ed. Paul Parey, Berlin and Hamburg. Lee, K. M., and Gillespie,J. H. 1957. Propagation of virus '51 dirarrhea virus of cattle in tissue culture. Am. J. ' Vet. Res., 18 952-953. Matthias, D. 1954. Das Histologische Verhalten der 5'" ‘ Nebennieren bei der Schweinepest. rch. exptl. Vet. 1 Med., 8. 226- 262. r!” Moll, T., and Finlayson, A. v. 1958. Isolation of cyto- pathogenic viral agent from feces of cattle. Science, 126:401-402. Moore, G. R., Rothenbacher, H. J., Bennett, M. V., and Barner, R. D. 1962. Bovine sa1monellosis. In press. J. Am. Vet. Med. Assn. Nielson, S. W., Horney, F. D., Hulland, R. J., and Roe, C. K. 1955. Mucosal disease of cattle in Ontario. Can. J. Comp. Med., 19:318-324. Noyce, F. N., and Schipper, I. A. 1959. Isolation of mucosal disease virus by tissue cultures in mixture 199, Morgan, Morton, and Parker. Proc. Soc. Exp. Biol. and Med., 100:84-86. Nystedt, H. 1960. Ume—disease, a mucosal infection of cattle in Vasterbotten, caused by para influenza- 3 vir1s Medlemsbl. Sverig. Vet. Fdrb , 122129-132, 135. Olafson, P., MacCallum, A. D. and Fox, F. H. 1946 An apparently new transmissible disease 0: cattle. Cornell Vet. 36: 205- 213. Olafson, P., and Rickard, C. G. 1947. Further obser va on the virus diarrhea (new transmissible disease) cattle. Cornell Vet , 37: 104- 106. tio of Olson G., and Hoerlein, A. B. 1956. Observations on mucosal disease of cattle. J. Am. Vet. Med. Assn., 129:466-470. Pallaske, G. 1960. Pathologiscne Histologie. Second ed., Gustav Fischer, Jena. Pritchard, W. R. 1955. The mucosal disease of cattle—- epizootiology, symptomatology and experimental studies. Proc. A.V.M.A., 92:37-42. Pritchard, W. R., Bunnell, D., Taylor, D. B., Moses, H. E., and Doyle, L. P. 1956. A transmissible disease affecting the mucosae of cattle. J. Am. Vet. Med. Assn., 128:1-5. Pritchard, W. R., Carlson, R. G., Moses, H. E., and Taylor, D. E. B. 1955. Virus diarrhea and mucosal disease. Proc. U. S. Livestock San. Assn.59th meeting: 173-188. Pritchard, W. R., and Wassenaar, P. W. 1959. Studies on the syndrome called mycotic stomati is of cattle. J. Am. Vet. Med. Assn., 135:274~277. Raffel, S. 1961. Immunity. Second ed. Appleton-Century- Crofts, Inc., New York. Ramsey, F. K. 1956. Pathology of a mucosal disease of cattle. Dissertation, Iowa State College, Ames, Iowa. Ramsey, F. L., and Chivers, W. H. 1953. Mucosal disease of cattle. North Am. Vet., 34:629-633. Ramsey, F. K., Chivers, W. H., Trapp, A. L., and Whiteman, C. E. 1958. Incidence and mortality of mucosal disease in Iowa. Iowa State College Vet., 20:101-103. Ramsey, F. K., Trapp, A. L., Richter, W. R., Whiteman, C. E. 1959. Annual progress report for 1958—59; project NC-34: Mucosal diseases of cattle. Iowa State University, Ames, Iowa. Ramsey, F. K., Trapp, A. L., Tyler, D. E. Davison, D., Van Der iaaten, M. J., Switzer, W. P., and Richter, W. R. 1960. Annual progress report for 1959-60; project NC-34: Mucosal diseases of cattle. Iowa State University. Reinders, J. S. 1959. irus diarrhea in cattle. Tijdschr. Diergeneesk., 84:81-89. Robson, D. 8., Gillespie, J. H., and Baker, J. A. 1960. The neutralization test as an indicator of immunity to virus diarrhea. Cornell Vet., 50:503-509. Rooney, J. R. 1957. Pathology of a bovine mucosal-type disease. Am. J. Vet. Res., 67:283‘291. Rosen, L., and Abinanti, F. R. 1960. Natural and experimental 0e Am. J. Hyg., 71:250-257. 106 Rothenbacher, H. J., and Barner, R. D. 1960. Case report D 1598, Dept. of Vet, Pathology, M. S. U. Rothenbacher, H. J. 1960. Attempt to adapt the North Dakota mucosal disease virus to rabbit kidney cell culture. Unpublished, report by Barner et a1. 1960. Annual Progress report for1959-1960; project NC- 34: Mucosal diseases of cattle. M.S.U Rothenbacher, H. J., Whiteman, C. E., Cunningham, C. H., and Church, C. C. 1961. Annual progress report for 1960— 61; project NC-34: Mucosal diseases of cattle, M.S.U. Runnells, R. A., Monlux, W S., and Monlux, A. W. 1960. F88 Principles of Veterinary pathology. Iowa State Univ. Press, Ames, lowa. Schalm, O. W. 1961. Veterinary hematology. Lea and 1 Febiger, Philadelphia. » Schipper, I. A. 1957. Annual progress report for 1956-57; project NC-34: Mucosal diseases of cattle. North Dakota Exp. Station, Fargo, North Dakota. Schipper, I. A., Eveleth, D. F., Schumard, R. F., and Richards, S. H. 1955. Mucosal disease of cattle. Vet. Med., 50:431-435, 450. Schipper, I. A., and Noyce, F. N. 1959A. Mucosal disease agent--isolation, transmission, and tissue culture studies. A.V.M.A. 196th annual meeting, Kansas City, Missouri. . 1959B. Intra—herd trans- mission of mucosal disease. Vet. Med., 54:442-445. . 1959C. Annual progress report for 1958-59; project NC-34: Mucosal diseases of cattle. North Dakota Agr. Expt. Station, Fargo, North Dakota. Schultz, L. C. 1959. Pathologisch-anatomische Befunde bei der soganannten "mucosal disease” (Schleimhautkrankheit) des Rindes. Deutsche Tiera.rztl. Wschr. , 66: 586- 588. Schwarz, A. F. J., York, C. J., Zirbel, L. W., and Estela, L. A. 1957. Modification of infectious bovine rhinotracheitis (1BR) virus in tissue culture and development of a vaccine. Proc. Soc. Exp. Biol. and Med., 96:453-458. Seibold, H. R. 1956. The pathology of mucosal disease in Alabama. J. Am. Vet. Med. Assn., 128-:21-26. 107 Smith, H. A., and Jones, T. C. 1957. Veterinary pathology. Lea and Febiger, Philadelphia. Soliman, A. M. 1958. The host range of an enteric cyto- pathogenic orphan "ECBO" virus isolated from healthy dairy-cattle. Thesis, M.S.U. Stdber, M. 1959. Die klinische Seite der sogenannten "mucosal disease" (Schleimhautkrankheit) des Rindes. Deutsche Tierarztl. Wschr., 66:582-586. Swope, R. E., and Luedke, A. J. 1956. A mucosal disease in cattle in Pennsylvania. J. Am. Vet. Med. Assn., Fa 129:111-115. Trapp, A. L. 1960. Pathology of the blood-vascular and lymphatic systems of cattle affected with mucosal , disease. Dissertation, Iowa State University, Ames, 1 Iowa. v Underdahl, N. R., Grace, 0. D., and Hoerlein, A. B. 1957. Cultivation in tissue culture of cytopathogenic agent from bovine mucosal disease. Proc. Soc. Exp. Biol. and Med., 94:795-797. ‘U. S. Livestock San. Assn. 64th Ann. Meeting. 1960. Report of the Committee on Virus Research: 351-353. Van Bekkum, J. G. 1959. A cytopathogenic agent isolated from a cow suffering of a syndrome similar to mucosal disease. Proc. 16th Int. Vet. Congr., Madrid. 2:477- 478. Voss, H. J. 1959. Beobachtungen uber die "Schleimhauterkran- kung“ (mucosal disease) der Rinder in Deutschland. Deutsche Tierarztl. Wschr., 66:149-151. Wheat, J. D., McKercher, D. J., and York, C. J. 1954. Virus diarrhea in California. California Veterinarian 7:26—29. Whiteman, C. E. 1960. Histopathology of the adrenal cortex and adeno-hypcphysis in cattle with mucosal disease. Dissertation, Iowa State University, Ames, Iowa. Whittem, J. H., cited by Johnston, K. G. 1951. The virus diarrheas of cattle and similar diseases. Austral. Vet. J., 35:323-324. York, C. J. 1960. Cited by Kniazeff, A. J., and Pritchard, W. R. 1960. Antigenic relationships in the bovine viral d1arrhea--mucosal disease complex. Proc. U. S. Livestock San. Assn. 64th meeting: 344-350. 108,"J York, C. J., and Rosner, S. F. 1961. Virus diarrhea of cattle--Serologica1 identification of disease and an incidence survey. To be published. Cited by York, Rosner, and MacLean, 1960. Evaluation of vaccines for virus diarrhea of cattle. Proc. U.S. Livestock San. Assn. 64th meetingz339-343. York, 0. J., Rosner, s. F., and MacLean, F. J. 1960. Evaluation of vaccines for virus diarrhea of cattle. Proc. U. S. Livestock San. Assn. 64th meeting: 339- 343. York, C. J. 1961. Research Div., Pitman-Moore Co., Indianapolis, Indiana. Personal communication. APPENDICES tlj * J. 09 Fig. Fig. Fig. Fig. l.--cwollen jejuno-ileal lymph node plus edema and hyper- plasia of Peyer's patches as seen from serosal surface. 2.--Swollen and edematous mesenteric lymph node with cortical and subcapsular ecchymoses. 3.--Linear demarkation hemorrhage along the lateral edge of a jejunal Peyer‘s patch as seen from the serosal surface. 4.-—Subcapsular and cortical ecchymotic hemorrhages in a posterior jejunal lymph node. 5.--Cortical ecchymotic hemorrhages in a congested and edematous ileocecal lymph node. 6--Cortica1 and parenchymal hemorrhages in edematous and congested mesenteric lymph nodes. 7.-—Hemorrhagic marbling in a Peyer's patch of the jejunum. 8.--Ecchymotic hemorrhages in a hyperplastic jejunal Peyer's patch. r—J r4 k’v‘ Fig. 9.--Severe catarrhal to hemorrhagic enteritis of the posterior jejunum, and ileum (above). Less affected cecum and colon beIOW. .--Hemorrhagonecrotic seam around the ileocecal ostium. Fig. 11 --Patchy hemorrhagic congestion of hemorrhages in the surrounding cecum. the ileum; petechial mucosa of the ileocecal ostium and Fig. l2.--Ecchymotic hemorrhages and cystic degeneration in the mucosa of the cecocolic junction. Fig. 13 --Petechia1 hemorrhages in the cortex of a kidney. Fig. l4.--Sucapsular suffusion in the adrenal 1;..J 11. :3 Q m Fig. Fig. 115 l5.--Rumen: changes focal hyperkeratotic and proliferative in the mucosa H. & E. x . , \.- ' ,‘ (“"“~6"I .. ,‘Vry , ,_ 5E_. 16.—-Jejunum: catarrhal enteritis with necrosis and denudation of surface epithelium. H. & E. x 200. Fig. l7.--Posterior jejunum; catarrhal enteritis with inflammatory hyperemia of capillaries in denuded villi. H. & E. x 200. Fig. 18.-—Lymph node: Hemorrhage in reaction center showing lymphocytic depletion. H. & E. x 200. Fig. l9.-—Lymph node: necrosis of lymphoid cell elements in an exhausted reaction center. H. & E. x 200. . , .- ‘Inv ’, . Fig. 20.--Spleen: necrotic focus in reaction center with heterophilic cell infiltration. H. & E. x 200. 118 Fig. 21.--Lymph node: Tertiary lymph nodule: marginal sinus congested with lymphoid and reticulo- histiocytic cell elements. Capsule shows thinning and distention. H. a s. x zoo. Fig. 22.-—Lymph node: conglomerated lymphoid and reticu1onistiocytic cells. H. & E. x 200. 119 Fig. 23.--Liver: focal areas of reticuloendothelial and lymphocytic infiltrations in the vicinity of Pyknotic parenchymal cells. H. & E. x 200. Fig. 24.--Liver: focal area of parenchymal necrosis with reactive reticuloendothelial cell proliferation. Sinusoidal spaces snow distention. H. & E. x 200. 120 Fig. 25.--Kidney: shrunken glomerular tuft and distended Bowman‘s capsule. Focal interstitial lymphocytic infiltration. H. & E. x 200. Fig. 26.--Kidney: thickened Bowman's capsule and focal lymphocytic infiltration. H. & E. x 200. 121 Fig. 27.——Adrena1; small necrotic focus in the central zona fasciculata. H. & E. x 200. Fig. 28.--Adrenal: pattern of vacuolar cell degeneration in the peripheral zona fasciculata. H. & E. x 400. 122 Fig. 13.--Adrenal: foci of coagulation necrosis in the zona fasciculata with reactive lymphocyti an? c heterophilic infiltrations. H.‘& E. x 200. Fig. 30.--Adrenal: strands of darkly staining cells in the zona fasciculata with homogeneous pyknotic nuclei. H. & E. x 400.