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ummary For many years, epizootic bovine abortion in California has been recognized as the cause of extensive calf losses, particularly among heifers in their first pregnancy. Fetal pathology revealed subcutaneous edema, ascites, generalized hemorrhage, and hepatopathy. Earlier workers excluded all known bacterial causes of abortion. Five viral isolates were recovered from various tissues of aborted fetuses originating from herds in widely separated areas. Cultures of bovine embryonic kidney cells and embryonated chicken eggs were employed as the means for isolation. Four isolates were studied comparatively. The behavior in chicken embryos, mice, and guinea pigs identified these isolates as members of the Psittacosis-Lymphogranuloma-Venereum group. Smears showed typical elementary bodies after staining by the Macchiavello, Castaneda, Giemsa and the Stamp technique. Electronmicroscopic observations and serological investigations confirmed these findings. A disease similar to epizootic bovine abortion was reproduced experimentally by inoculating cows in various stages of pregnancy by the intrafetal, intravenous, intramuscular, and subcutaneous exposure. After aerosal exposure of a pregnant cow the EBA-agent was recovered from the fetus at slaughter 18 days later. Oral administration of the agent and contact with infected animals were without effect. The inoculated cows aborted or gave birth to weak calves 11 to 126 days after inoculation. The virus was reisolated from uterine contents, and from the internal organs of the fetuses. Intrauterine infection of the fetuses was thus demonstrated. Cows were bred and conceived after experimentally produced abortion. The immunity of two of these cows was challenged. Virus was not recovered either from uterine contents or fetal tissues subsequently, although these animals, as well as others that recovered from experimental abortion, responded with fever to the challenge exposure. Comparative studies in one cow with Miyagawanella bovis were carried out. They revealed certain differences in the response of the cow to the agent. Intrauterine infection of the calf could be demonstrated 84 days after inoculation. It is recommended that the agent isolated from epizootic bovine abortion be considered a separate entity until exact antigenic analysis reveals its relation to other agents of this group associated with cattle infections. Zusammenfassung Atiologische Studien am epizootischen Abort der Rinder Seit vielen Jahren auftretendes Verkalben bei Rindern mit betrachtlichen Verlusten an Kalbern, besonders bei Erstlingsfarsen, wurde in Kalifornien als epizootischer Abort der Rinder bezeichnet. Fotale pathologische Veranderungen waren subkutanes Odem, Aszites, generalisierte petechiale Blutungen und Hepatopathie. Andere Bearbeiter hatten fruher die bekannten bakteriellen Ursachen von Aborten bei Rindern ausgeschlossen. Funf Virusstamme wurden von Organen abortierter Kalber, die aus geographisch weit getrennten Herden stammten, mittels embryonaler Rindernierenzellen und Huhnerembryonen isoliert. Vier Stamme wurden vergleichend untersucht. Das Verhalten im embryonierten Huhnerei, in Mausen und in Meerschweinchen sowie elektronenmikroskopische und serologische Befunde charakterisierten diese Stamme als zur Psittakose-Lymphogranuloma-Venereum-Gruppe gehorend. Eine dem epizootischen Rinderabort ahnliche Krankheit wurde experimentell erzeugt durch intrafotale, intravenose, intramuskulare und subkutane Inokulation von Kuhen in verschiedenen Trachtigkeitsstadien. Aerosolinfektion einer Kuh fuhrte zur Infektion ihres Fotus, wahrend orale Verabreichung des Virus und Kontakt mit infizierten Tieren ohne klinische Folgen blieben. Die inokulierten Tiere verkalbten oder hatten lebensschwache Kalber 11 bis 126 Tage nach der Injektion. Intrauterine Infektion wurde durch Virusisolierung vom Uterusinhalt und von den inneren Organen der Kalber nachgewiesen. Kuhe, die nach experimenteller Infektion verkalbten, wurden wieder tragend. Die Immunitat zweier dieser Tiere wurde gepruft. Virus wurde weder vom Uterusinhalt noch von fotalen Geweben isoliert, obwohl diese Tiere wie auch andere, die sich vom experimentellen Abort erholt hatten und nicht tragend waren, mit Fieber auf die Testinfektion reagierten. Vergleichende Studien an einer tragenden Kuh mit Miyagawanella bovis zeigten eine mildere Erkrankung dieser Kuh nach der Inokulation, doch konnte 84 Tage spater intrauterine Infektion ihres Kalbes nachgewiesen werden. Resume Etudes sur l'etiologie d'un avortement epizootique des bovides Un avortement observe depuis plusieurs annees chez des bovins et provoquant une perte considerable en veaux, surtout chez les primipares est appele en Californie “avortement epizootique bovin”. A l'autopsie du foetus on trouve un oedeme sous-cutane, une ascite, des hemorragies petechiales generalisees et une hepatopathie. D'autres auteurs ont exclu dans de precedents travaux les causes bacteriennes connues de l'avortement chez les bovins. Cinq souches de virus furent isolees des organes des veaux nes avant terme provenant de troupeaux geographiquement eloignes les uns des autres, au moyen de cellules renales bovines embryonales et sur des embryons de poulet. Quatre souches furent l'objet d'une etude comparee. Leur comportement dans l'oeuf embryonne de poulet, chez la souris et le cobaye ainsi que les resultats obtenus au microscope electronique et l'etude serologique caracteriserent ces souches comme appartenant au groupe psittacose-lymphogranuloma-venereum. Une maladie analogue a “l'avortement epizootique bovin” put etre reproduite experimentalement par inoculation intrafoetale, intraveineuse, intramusculaire et sous-cutanee chez des vaches a differents stades de gestation. Une infection par aerosol chez une vache provoqua l'infection du foetus tandis que l'administration orale du virus et le contact avec des animaux infectes resta sans consequences cliniques. Les animaux inocules avorterent ou donnerent naissance a des veaux debiles 11–126 jours apres l'injection. Des infections intrauterines furent mises en evidence par l'isolement du virus dans le contenu uterin et dans les visceres. Des vaches ayant avorte apres l'infection experimentale devinrent a nouveau portantes. L'immunite de deux de ces animaux fut verifiee. On ne put isoler le virus ni dans le contenu uterin ni dans les tissus du foetus, bien que ces animaux comme ceux qui avaient surmonte un avortement experimental et ne portaient pas reagirent par une poussee de fievre a l'infection test. Des etudes comparees effectuees sur une vache portante avec Miyagawanella bovis montrerent une maladie plus benigne chez cette vache apres l'inoculation, mais on put mettre en evidence 84 jours plus tard une infection intrauterine chez le veau qu'elle portait. Resumen Estudios etiologicos sobre el aborto epizootico del ganado vacuno En California se denomina aborto epizootico del ganado vacuno a un tipo de aborto en vacas que desde hace muchos anos viene ocasionado grandes perdidas entre las terneras, sobre todo en novillas. Las alteraciones patologicas fetales eran edema subcutaneo, ascitis, hemorragicas petequiales generalizadas y hepatopatia. Otros autores ya habian excluido con anterioridad las conocidas causas bacterianas de abortos en vacas. Se aislaron 5 cepas de virus de organos de terneras abortadas, procedentes de vacadas geograficamente bastante distantes entre si, mediante cultivos en celulas embrionarias de rinones vacunos y embriones de pollo. 4 cepas se estudiaron comparativamente. El comportamiento en el huevo embrionado de gallina, en ratones y en cobayas, asi como los hallazgos electonicomicroscopicos y serologicos caracterizaban a estas cepas como pertenecientes al grupo de la psitacosis-linfogranuloma venereo. Una enfermedad semejante al aborto epizootico vacuno se produjo experimentalmente por medio de inoculacion intrafetal, intravenosa, intramuscular y subcutanea de vacas en diversos estadios de gestacion. La infeccion aerosolica de una vaca condujo a la infeccion de su feto, mientras que la administracion oral del virus y el contacto con animales infectados quedaron sin consecuencias clinicas. Los animales inoculados abortaron o tenian terneras con escasa vitalidad a los 11–126 dias despues de la inyeccion. La infeccion intrauterina se comprobo mediante aislamiento del virus del contenido uterino y de ios organos internos de las terneras. Las vacas que abortaron tras la infeccion experimental, volvieron a concebir. Se comprobo la inmunidad en dos de estos animales. Del contenido uterino y de los tejidos fetales no se aislo virus alguno, aunque estos animales, al igual que otros que se habian recuperado del aborto experimental y no estaban gestantes, reaccionaban con fiebre frente a la infeccion patron. Estudios comparativos en una vaca gestante con Miyagawanella bovis presentaban una enfermedad benigna de esta vaca tras la inoculacion, pero al cabo de 84 dias se pudo identificar la infeccion intrauterina de su ternera.

Zusammenfassung Intestinale Psittakose-Lymphogranuloma-Infektion bei Schafen, durch Isolierung von Erregern in Dottersackkulturen sich entwickelnder Huhnerembryonen diagnostiziert, wurde mit den komplementbindenden, spezifischen Antikorpern der betreffenden Tiere in Zusammenhang gebracht. Dabei hatten 20 von 29 Schafen, die Psittakoseerreger im Kot ausschieden, einen Antikorpertiter von weniger als 1:8. Der Titer der restlichen 9 Schafe schwankte zwischen 1:8 und 1:32. Andererseits vermochten in 4 Schafen nach enzootischem Abort hohe Antikorpertiter die intestinale Infektion weder zu unterdrucken noch die enterale Ausscheidung der Psittakose-Lymphogranuloma-Erreger zu verhindern. Die Psittakose-Lymphogranuloma-Erreger wurden bei 7 eingehend untersuchten, klinisch normalen Schafen aus Duodenum und den Darmabschnitten abwarts isoliert. Eines dieser Schafe beherbergte diese Erreger im Leberlymphknoten und dem Lymphknoten des Ileum, ein weiteres nur im letztgenannten Lymphknoten. Ein drittes Tier hatte neben der intestinalen Infektion auch infizierte Nieren. Die Schafe mit parenteraler Infektion hatten Antikorperstimulation. Die intestinale Psittakose-Lymphogranuloma-Infektion wurde in allen 4 untersuchten Schafherden festgestellt. Schafe im Alter von 2 Monaten bis zu 6 Jahren wurden als Ausscheider aetiologisch diagnostiziert. Einzelne Schafe schieden diese Erreger mit moglichen Unterbrechungen uber Zeitspannen von mehreren Jahren enteral aus. Summary Serological and aetiological studies on sheep with intestinal infection with psittacosis-lymphogranuloma agents Intestinal psittacosis-lymphogranuloma infection in sheep was demonstrated by isolation of the agents from faecal samples by means of developing chick embryos and was correlated with the complement-fixing, specific antibodies of these animals at the time of sampling. The antibody level was below 1:8 in 20 of 29 animals which were shedding psittacosis-lymphogranuloma agents in the faeces. The titre of the remaining 9 sheep ranged from 1:8 to 1:32. A high antibody titre, on the other hand, did not inhibit intestinal infection and faecal shedding of these agents in 4 ewes following natural cases of enzootic abortion. Among 7 clinically normal sheep which were studied aetiologically in detail, the psittacosis-lymphogranuloma agents were isolated from composite samples of digestive contents and the mucosa of the duodenum and all lower parts of the intestine. One of these sheep harboured the agent also in the hepatic and ileal lymph nodes, a second one only in the ileal lymph node, and a third sheep had infected kidneys. The sheep with parenteral infection had evidence of antibody stimulation. The intestinal psittacosis-lymphogranuloma infection was identified in all 4 herds of sheep examined. Sheep aged 2 months to 6 years were identified aetiologically as intestinal shedders. Selected sheep were found to shed those agents in the faeces during a period of 4 years with possible negative intervals. Resume Etudes serologiques et etiologiques d'une infection intestinale du Mouton par un virus du groupe psittacose-lymphogranulomatose L'infection intestinale du mouton par un virus du groupe psittacoselymphogranulomatose fut mise en parallele avec la presence d'anticorps specifiques deviant le complement chez les memes animaux. Le diagnostic de l'infection avait ete etabli par l'isolement des germes dans des cultures sur sac vitellin d'embryons de poulet en developpement. On constata a ce sujet que 20 sur 29 moutons qui eliminaient le virus dans les feces avaient un titre inferieur a 1:8. Le titre des 9 autres moutons variait entre 1:8 et 1:32. D'autre part des titres d'anticorps eleves trouves chez 4 moutons apres un avortement enzootique ne suffisaient pas a empecher l'infection intestinale ni l'elimination enterale des germes du groupe psittacose-lymphogranulomatose. Les germes du groupe psittacose-lymphogranulomatose ont ete isoles a partir du duodenum et d'autres portions du tractus intestinal de 7 moutons examines tres completement et cliniquement sains. Un de ces moutons hebergeait le virus dans les ganglions lymphatiques du foie et de l'ileum, un autre seulement dans les ganglions de l'ileum. Un troisieme animal presentait outre l'infection intestinale une infection renale. Chez les moutons atteints d'une infection parenterale on constatait une stimulation d'anticorps. L'infection intestinale par le virus du groupe psittacose-lymphogranulomatose fut constatee dans les 4 troupeaux de moutons examines. On diagnostiqua une elimination specifique de virus chez des moutons âges de 2 mois a 6 ans. Quelques moutons eliminerent ces germes par voie enterale, avec des interruptions possibles, pendant une periode s'etendant sur plusieurs annees. Resumen Estudios serologicos y etiologicos sobre la infeccion de psitacosis-linfogranuloma intestinal en las ovejas La infeccion de psitacosis-linfogranuloma intestinal en ovejas, diagnosticada mediante aislamiento de germenes en cultivos de sacos vitelinos de embriones de pollo en desarrollo, se relaciono con los anticuerpos especificos fijadores del complemento de los animales correspondientes. Con ello, 20 de 29 ovejas, las cuales eliminaron agentes de la psitacosis con el estiercol, tenia un titulo de anticuerpos menor que 1:8. El titulo de las 9 ovejas restantes oscilaba entre 1:8 y 1:32. Por otro lado, en 4 ovejas tras aborto enzootico titulos elevados de anticuerpos no lograron contener la infeccion intestinal ni impedir la difusion enterica del agente etiologico de la psitacosis-linfogranuloma. Los agentes de la psitacosis-linfogranuloma se aislaron en 7 ovejas normales, exploradas con detenimiento, a partir del duodeno y los tramos intestinales descendientes. Una de estas ovejas albergaba estos agentes etiologicos en los ganglios hepaticos y en los iliacos, mientras que otra solo en los ganglios citados en ultimo lugar. Un tercer animal tambien tenia al lado de la infeccion intestinal infectados los rinones. Las ovejas con infeccion parenteral tenian estimulacion de anticuerpos. La infeccion intestinal de psitacosis-linfogranuloma se comprobo en todos los 4 rebanos examinados. Ovejas de edades comprendidas entre 2 meses y 6 anos se diagnosticaron etiologicamente como difusores. Ciertas ovejas eliminaron estos agentes por via enteral con posibles interrupciones a lo largo de varios anos.

Summary The ovine chlamydial abortion strain B-577 was adapted and maintained for 101 serial passages in cell cultures. Rates of chlamydial infection of cells ranged between 8.2 and 100%, and the infectivity in developing chicken embryos of the tissue culture-adapted strain varied from 103.5 to 107.833 CELD50/0.5 ml. at different passage levels. Unexpected reductions of chlamydial infectivity occurred at the 55th, 56th and 93rd passages. The tissue culture-adapted chlamydial strain B-577 remained consistently pathogenic for chicken embryos, and caused placental and fetal infection after inoculation of pregnant ewes with material of the 54th passage in L cells. Furthermore, this strain caused an accidental laboratory infection of man after the 94th passage in tissue culture. These facts indicate that this chlamydial strain did not become attenuated during serial passage in culture cells. Zusammenfassung Pathogenitat eines Chlamydienstammes vom Schafabort nach Adaption an Gewebekulturen Der Chlamydienstamm B 577 vom Schafabort wurde an L-Zellen adaptiert und in 101 Passagen darin weitergezuchtet. Wahrend dieser Passagen schwankte die Rate infizierter Zellen zwischen 8,2 und 100%. Der Infektionstiter in Huhnerembryonen lag zwischen 10−3,5 und 10−7,833 LD50. In der 55., 56. und 93. Gewebekulturpassage waren die Titer am niedrigsten. Die Pathogenitat des Stammes fur die Huhnerembryonen blieb unverandert. Nach der 54. L-Zellen-Passage infizierte dieser Stamm Plazenta und Fotus in tragenden Schafen. Auserdem verursachte der Stamm B 577 wahrend der 94. Passage eine Augeninfektion beim Menschen. Diese Befunde weisen darauf hin, das der Chlamydienstamm B 577 durch Passagen in L-Zellen nicht attenuiert wurde. Resume Pathogenite d'une souche de Chlamydia, isolee lors d'une avortement chez le mouton, apres une adaptation aux cultures de tissue On a adapte aux cellules-L la souche Chlamydia B 577 d'un avortement de mouton et on l'a reproduite lors de 101 passages. Le taux de cellules infectees alla de 8,2 a 100% durant ces passages. Le titre infectieux se situa chez l'embryon de poulet entre 10−3,5 et 10−7,833 LD50. Les titres furent les plus bas lors du 55e, 56e et 93e passage en culture de tissu. La pathogenite de la souche pour l'embryon de poulet resta inchangee. Cette souche infecta le placenta et le foetus de brebis gravides apres le 54e passage sur cellules-L. La souche B 577 provoqua en outre une infection oculaire chez l'homme pendant le 94e passage. Ces resultats montrent que la souche Chlamydia B 577 n'a pas ete attenuee par des passages sur cellules-L. Resumen Patogeneidad de una estirpe de clamidios del aborto ovino tras adaptacion a cultivos histicos Se adapto la estirpe de clamidios B 577 del aborto ovino a celulas L y se siguio cultivando en ellas durante 101 pases. A lo largo de estos pases oscilaba la tasa de celulas infectadas entre 8,2 y 100%. El titulo de infeccion en los embriones de pollo se hallaba entre 10−3,5 y 10−7,833 DL50. Los titulos eran minimos en los pases 55°, 56° y 93° por cultivo histico. La patogeneidad de la estirpe permanecio inalterada para los embriones de pollo. Tras el pase 54° por celulas infectaba esta estirpe la placenta y feto en ovejas gestantes. Ademas ocasiono la estirpe B 577 durante el pase 94° una infeccion ocular en una persona. Estos hallazgos indican que la estirpe de clamidios B 577 no fue atenuada mediante los pases por celulas L.

Summary The dynamics of the bacteremic phase of vibrio infection of non-pregnant and pregnant ewes was investigated using vibrio organisms which differed distinctly in catalase activity, hydrogen sulfide production and cellular morphology. The following facts emerged: 1 The rate of clearance of vibrio organisms from the blood is the same in pregnant and non-pregnant ewes. The rate is determined by the morphologic characteristics of the vibrio cells. Coccoid Vibrio fetus are cleared relatively slowly from the blood while typical comma-shaped cells are eliminated at a significantly faster rate. The catalase activity of an organism and its ability to produce hydrogen sulfide do not influence the rates of clearance. 2 The coccoid Vibrio fetus strain, which remained for a long period in the blood stream, was lethal to ewes 5½ to 12 hours after inoculation. The animals developed a shock-like syndrome and died. Generalized thrombosis was observed in these ewes. 3 A duration of detectable bacteremia of as little as 15 minutes was sufficient to establish uterine infection in pregnant ewes. Catalase activity seemed to be a determining factor in the establishment of uterine infection. 4 Gall bladder infection with Vibrio fetus was observed after bacteremia. Vibrio fetus was also present in the intestinal tract in cases where gall bladder infection had been established. Zusammenfassung Uber Ereignisse im Fruhstadium der Vibrio fetus-Infektion bei Mutterschafen Die Dynamik der bakteriamischen Phase der Vibrioinfektion tragender und nichttragender Schafe wurde untersucht. Verwendet wurden Vibrionen, die sich eindeutig voneinander in ihrer Katalase-Aktivitat, ihrer Schwefelwasserstoffproduktion und ihrer Zellmorphologie unterschieden. Folgende Befunde wurden erhoben: 1 Die Geschwindigkeit, mit der Vibrionen aus dem Blut eliminiert werden, ist bei tragenden und nichttragenden Schafen die gleiche und hangt anscheinend von morphologischen Eigenschaften der Vibrionen ab. Coccoide Vibrio fetus-Organismen werden langsam aus dem Blut eliminiert, wahrend typisch kommaformige Vibrionen signifikant schneller aus dem Blut beseitigt werden. Die Katalase-Aktivitat der Erreger und Befahigung zur Schwefelwasserstoffbildung beeinflussen die Ausscheidungsrate nicht. 2 Der hier verwendete coccoide Vibrio fetus-Stamm verursacht 5½ bis 12 Stunden nach intravenoser Inokulation ein todlich verlaufendes, schockartiges Syndrom. Bei diesen Schafen wird eine generalisierte Thrombose fest-gestellt. 3 Eine fur nur 15 Minuten nachweisbare Bakteriamie fuhrt bei tragenden Schafen zur uterinen Infektion. Hierfur erscheint die Katalaseaktivitat bestimmend zu sein. 4 Nach der bakteriamischen Phase ist eine Infektion der Gallenblase mit Vibrio fetus feststellbar. In diesen Fallen last sich Vibrio fetus auch im Intestinaltrakt nachweisen. Resume Phenomenes accompagnant le stade precoce de l'infection par «vibrio fetus» chez les moutons femelles L'examen a porte sur la dynamique de la phase bacteriemique de l'infection par vibrions chez les moutons gravides et ceux qui ne le sont pas. Les vibrions utilises se distinguaient nettement les uns des autres par leur activite catalasique, leur production d'hydrogene sulfure et leur morphologie cellulaire. Les constats suivants ont ete enregistres: 1 La rapidite avec laquelle les vibrions sont elimines du sang est la meme chez les moutons gravides et ceux qui ne le sont pas et semble dependre des proprietes morphologiques des vibrions. Les vibrions de forme coccoide sont elimines lentement du sang tandis que ceux qui sont de forme nettement en virgule disparaissent beaucoup plus rapidement du sang. L'activite catalasique des microorganismes et leur faculte de produire de l'hydrogene sulfure n'influence en rien leur taux d'elimination. 2 La souche de vibrions coccoides utilises ici provoque 5½ a 12 heures apres l'inoculation intraveineuse, un syndrome de choc avec issue mortelle. Chez ces moutons on constate une trombose generalisee. 3 Une bacteriemie decelable pendant 15 minutes seulement entraine chez les moutons gravides une infection uterine. A cet egard, l'activite catalasique semble etre determinante. 4 Apres la phase bacteriemique, on peut constater une infection de la vesicule biliaire par «vibrio fetus». Dans ces cas-la, le microorganisme peut egalement etre identifie dans le tractus intestinal. Resumen Sobre sucesos en el estadio precoz de la infeccion por Vibrio fetus en ovejas madres Se estudio la dinamica de la fase bacteriemica de la vibrioinfeccion en ovejas prenadas y vacias. Utilizaronse vibriones que se diferenciaban con evidencia entre si en su actividad catalasica, su produccion de acido sulfhidrico y su morfologia celular. Se evidenciaron los hallazgos siguientes: 1 La velocidad, con la que se eliminan los vibriones de la sangre, es la misma en ovejas gravidas que vacias, dependiendo al parecer de las propiedades morfologicas de los vibriones. Los organismos Vibrio fetus cocoides se eliminan con lentitud de la sangre, mientras que los vibriones en forma tipica de coma se eliminan del torrente sanguineo con una velocidad significativa mucho mayor. La actividad catalasica de los agentes etiologicos y la capacidad de formacion de acido sulfhidrico no influencian la cuota de eliminacion. 2 La cepa cocoide de Vibrio fetus aqui empleada ocasiona entre las 5½ y 12 horas a contar de la inoculacion intravenosa un sindrome en forma de choque, de curso mortal. En estas ovejas se comprobo la existencia de una trombosis generalizada. 3 La bacteriemia solo identificable durante 15 minutos conduce en ovejas prenadas a la infeccion uterina. A este respecto parece ser decisiva la actividad catalasica. 4 Tras la fase bacteriemica se puede comprobar una infeccion de la vesicula biliar con Vibrio fetus. En estos casos tambien se puede identificar Vibrio fetus en el tracto intestinal.

Summary Seven conventional newborn calves were inoculated orally with the coronavirus strain LY-138, originally recovered from naturally-infected diarrheic calves. All calves developed diarrhea during the first 24 h after inoculation. The presence and excretion of virus was monitored by electron microscopy. Grossly, the changes were subtle and not striking. The intestinal tract was distended and contained a yellow clear fluid. The small intestinal wall was thin and transparent. Histologic changes were characterized by atrophy and fusion of the villi, decreased height and vacuolation of the intestinal epithelial cells, by a reduced number of goblet cells, crypt hypertrophy and dilatation, and by edema and increased cellularity of the lamina propria mucosae. Ultrastructurally the viral agent infected the intestinal absorptive epithelial cells, undifferentiated epithelial cells and goblet cells of the villi, and occasionally fibroblasts and endothelial cells of the lamina propria mucosae. Morphologic evidence indicated that virus multiplication occurred only in the cytoplasm of infected cells. The rough and smooth endoplasmic reticulum became dilated, and electron-dense, granulo-fibrillar material filled their cysternae to form smooth membrane-bounded viral factories in the supranuclear cytoplasm of infected intestinal epithelial cells. Ultrastructural cytologic changes in this viral infection consisted of dilation of the endoplasmic reticulum network, cellular ribosome depletion, extensive endoplasmic vacuolation and vesiculation of Golgi elements of intestinal epithelial cells, swelling and degeneration of mitochondria, loss of normal nuclear polarity with pyknosis and karyolysis of the nuclei, loss of cytoplasmic matrix, and a broad spectrum of pathologic changes involving the microvilli and terminal web. Zusammenfassung Lichtmikroskopische und Ultrastrukturelle pathologische Veranderungen bei intestinaler Coronavirusinfektion neugeborener Kalber Sieben konventionelle neugeborene Kalber wurden oral mit dem Coronavirus-Stamm LY-138, der ursprunglich von naturlich infizierten Kalbern mit Diarrhoe stammt, infiziert. Alle Kalber entwickelten Durchfall wahrend der ersten 24 Stunden nach Inokulation. Vorhandensein und die Ausscheidung des Virus wurden elektronenoptisch verfolgt. Sichtbare Veranderungen waren gering und wenig auffallend. Der Darmtrakt war erweitert und enthielt eine gelbe, klare Flussigkeit. Die Dunndarmwand war dunn und durchscheinend. Histologische Veranderungen waren charakterisiert durch Atrophie und Verschmelzung der Zotten, verminderte Hohe und Vakuolisierung der intestinalen Epithelzellen, durch eine reduzierte Anzahl von Gobletzellen, Kryptenhypertrophie und -erweiterung, sowie Odematisierung und erhohte Zellularitat der lamina propria mucusae. Ultrastrukturell infizierte das Virus die intestinalen absorptiven Epithelzellen, nichtdifferenzierte Epithelzellen und Gobletzellen der Zotten, und gelegentlich Fibroblasten sowie Endothelzellen der Lamina propria mucosae. Die morphologischen Veranderungen weisen darauf hin, das die Virusvermehrung nur im Zytoplasma infizierter Zellen stattfand. Das rauhe und glatte endoplasmatische Retikulum war erweitert, und elektronendichtes, granulo-fibrillares Material fullte die Zisternen aus, die feine Membran-gebundene Virusvermehrungsfabriken in dem supranuklearen Zytoplasmabereich infizierter intestinaler Epithelzellen bildeten. Ultrastrukturelle zytologische Veranderungen bestanden bei dieser Virusinfektion aus Erweiterung des endoplasmatischen Retikulumnetzwerks, zellularem Ribosomenverlust, extensiver endoplasmatischer Vakuolisierung der Golgielemente der intestinalen Epithelzellen, Schwellung und Degeneration von Mitochondrien, Verlust der normalen nuklearen Polaritat mit Pyknose und Karyolyse der Zellkerne, Verlust der Zytoplasmamatrix, und aus einem breiten Spektrum pathologischer Veranderungen an den Mikrozotten und der terminalen Netzstruktur. Resume Lesions pathologiques ultrastructurelles et au microscope optique lors d'infection intestinale a Coronavirus chez des veaux nouveau-nes Sept veaux nouveau-nes conventionnels ont ete infectes oralement par la souche LY-138 de Coronavirus, isolee precedement chez des veaux naturellement infectes presentant une diarrhee. Tous les veaux developperent une diarrhee durant les premieres 24 heures suivant l'inovulation. La presence et la secretion du virus ont ete suivies au microscope electronique. Les lesions apparentes furent faibles et peu apparentes. Le tractus digestif fut elargi et contenait un liquide jaune et clair. La paroi de l'intestin grele etait mince et transparente. Les lesions histologiques furent caracterisees par une atrophie et une fusion des villi, une hauteur diminuee et une vacuolisation des cellules epitheliales de l'intestin, par un nombre reduit des cellules caliciformes, une hypertrophie et un elargissement des cryptes ainsi qu'un oedeme et un nombre de cellules augmente de Lamina propria mucosae. Le virus a infecte ultrastructurellement les cellules epitheliales absorbantes de l'intestin, des cellules epitheliales non differenciees et des cellules caliciformes des villi, de temps en temps des fibroblastes et des cellules endotheliales de Lamina propria mucosae. Les lesions morphologiques ont montre que la multiplication du virus n'avait lieu que dans le cytoplasme des cellules infectees. Le Reticulum endoplasmique grossier et fin fut elargi et un materiel granuleux et fibrillaire remplissait les citernes qui formaient de fines regions de replication virale liees a la membrane dans le voisinage cytoplasmique supranucleaire des cellules epitheliales infectees de l'intestin. Les lesions cytologiques ultrastructurelles lors de cette infection virale consisterent en un elargissement du reseau endoplasmique, une perte des ribosomes cellulaires, une vacuolisation endoplasmique extensive des elements de Golgi dans les cellules epitheliales de l'intestin, une enflure et une degenerescence des mitochondries, une perte de la polarite nucleaire normale avec une picnose et une carylolyse des noyaux cellulaires, une perte de la matrice cytoplasmique et un large eventail de lesions pathologiques des microvilli et des tissus terminaux. Resumen Modificaciones patologicas fotomicroscopicas y ultraestructurales en la infeccion intestinal de terneros recien nacidos por virus Corona Se infectaron por via oral siete terneros convencionales con la estirpe de virus Corona LY-138, la cual es originaria de terneros, infectados naturalmente, con diarrea. Todos los terneros manifestaban diarrea durante las 24 horas primeras despues de la inoculacion. Se seguia electronicoopticamente la presencia y eliminacion de virus. Las modificaciones visibles eran escasas y se destacaban poco. El tracto intestinal se hallaba dilatado, conteniendo un liquido amarillo claro. La pared del intestino delgado era tenue y translucida. Las modificaciones histopatologicas se caracterizaban por atrofia y soldadura de las villosidades, altura reducida y vacuolizacion de las celulas epiteliales intestinales, por una cantidad reducida de celulas caliciformes, hipertrofia y ensanchamiento de las criptas, y por edematizacion y celularidad aumentada de la lamina propia mucosa. El virus infectaba ultraestructuralmente las celulas epiteliales intestinales absortivas, celulas epiteliales no diferenciadas y celulas caliciformes de las villosidades, y, en ocasiones fibroblastos y celulas endoteliales de la lamina propia mucosa. Las modificaciones morfologicas senalan que la multiplicacion de virus solo tuvo lugar en el citoplasma de las celulas infectadas. El reticulo endoplasmatico basto y fino se hallaba dilatado y material granulosofibrilar, opaco a los electrones, ocupaba las cisternas, las cuales formaban lugares finos de replicacion antiviral, ligados a las membranas en el ambito citoplasmatico supranuclear de celulas epiteliales intestinales infectadas. Las modificaciones citologicas ultraestructurales consistian en esta infeccion a virus en dilatacion del reticulo endoplasmatico, perdida celular de ribosomas, vacuolizacion endoplasmatica extensiva de los elementos de Golgi de las celulas epiteliales intestinales, tumefaccion y degeneracion de mitocondrias, perdida de la polaridad nuclear normal con picnosis y cariolisis de los nucleos celulares, perdida de la matriz citoplasmatica, y en un espectro amplio de modificaciones patologicas en las microvillosidades y tejidos terminales.

The serum antibody responses of cattle with respiratory coronavirus infections during the pathogenesis of shipping fever pneumonia were analyzed with different bovine coronavirus antigens, including those from a wild-type respiratory bovine coronavirus (RBCV) strain (97TXSF-Lu 15-2) directly isolated from lung tissue from a fatally infected bovine, a wild-type enteropathogenic bovine coronavirus (EBCV) strain (Ly 138-3), and the highly cell culture-adapted, enteric prototype strain (EBCV L9-81). Infectivity-neutralizing (IN) and hemagglutinin-inhibiting (HAI) activities were tested. Sequential serum samples, collected during the onset of the respiratory coronavirus infection and at weekly intervals for 5 weeks thereafter, had significantly higher IN and HAI titers for antigens of RBCV strain 97TXSF-Lu15-2 than for the wild-type and the highly cell culture-adapted EBCV strains, with P values ranging from

Respiratory bovine coronaviruses (RBCV) emerged as an infectious agent most frequently isolated from respiratory tract samples of cattle with acute respiratory tract diseases. Infectivity-neutralizing (IN) and hemagglutinin-inhibiting (HAI) antibodies induced by RBCV infections were monitored in sequential serum samples collected from cattle during a naturally evolving and experimentally monitored epizootic of shipping fever pneumonia (SFP). Cattle nasally shedding RBCV at the beginning of the epizootic started with low levels of serum IN and HAI antibodies. An increase in serum IN antibody after day 7 led to reduction of virus shedding in nasal secretions by the majority of the cattle between days 7 and 14. A substantial rise in the serum HAI antibody was observed during the initial phase among the sick but not the clinically normal cattle which were infected with RBCV. The RBCV isolation-positive cattle that developed fatal SFP had minimal serum IN and HAI antibodies during the course of disease development. Cattle that remained negative in RBCV isolation tests entered this epizootic with high levels of serum IN and HAI antibodies, which dramatically increased during the next two weeks. Protection against SFP was apparently associated with significantly higher levels of serum IN antibodies at the beginning of the epizootic. The RBCV-neutralizing activity is associated with serum immunoglobulin G (IgG), particularly the IgG2 subclass, while RBCV-specific HAI antibody is related to both serum IgG and IgM fractions.

Summary. Antibody responses against respiratory bovine coronavirus (RBCV) infections were monitored in cattle from the onset of a naturally occurring severe shipping fever (SF) epizootic to complete recovery of affected cattle or fatal outcomes. The infection with RBCV was detected in nasal secretions of 86 cattle, and 81 of them developed acute respiratory tract disease, including fatal pneumonia. Cattle nasally shedding RBCV at the beginning of the epizootic experienced characteristic primary immune responses with specific antibodies for hemagglutinin-esterase (HE) and spike (S) glycoproteins. Virus shedding in nasal secretions of the majority of the cattle ceased between days 7 and 14 with the appearance of HE- and S-specific antibodies. Nasal samples and lung tissues from 9 of the 10 fatal cases had high titers of RBCV, but these cattle had only IgM responses to RBCV infections. Cattle remaining negative in RBCV isolation tests entered this epizootic with antibodies against HE and S. Protection against respiratory tract disease was apparently associated with high level of opsonic and virus-neutralizing IgG2. The HE and S glycoproteins were recognized earliest by the bovine immune system while the N protein induced antibody responses during the later stage of initial infection and the early stage of reinfection. The membrane (M) glycoprotein was the least immunogenic of the major viral structural proteins.

Objective—To identify cytocidal viruses and Pasteurella spp that could be isolated from cattle involved in 2 natural outbreaks of shipping fever. Design—105 and 120 castrated male 4- to 8-monthold feedlot cattle involved in 1997 and 1998 outbreaks, respectively. Animals—Nasal swab specimens and blood samples were collected, and cattle were vaccinated on arrival at an order-buyer barn from 4 local auction houses. Four days later, they were transported to a feedlot, and additional nasal swab specimens and blood samples were collected. Nasal swab specimens were submitted for virus isolation and bacterial culture; blood samples were submitted for measurement of respiratory bovine coronavirus (RBCV) hemagglutinin inhibition titers. Results—93 of 105 cattle and 106 of 120 cattle developed signs of respiratory tract disease during 1997 and 1998, respectively, and RBCV was isolated from 81 and 89 sick cattle, respectively, while at the orderbuyer's barn or the day after arrival at the feedlot. During the 1997 outbreak, bovine herpesvirus 1 was isolated from 2 cattle at the order-buyer's barn and from 5 cattle 7 and 14 days after arrival at the feedlot, and parainfluenza virus 3 was isolated from 4 cattle 14 days after arrival at the feedlot. During the 1998 outbreak, bovine herpesvirus 1 was isolated from 2 cattle at the order-buyer's barn and on arrival at the feedlot and from 5 cattle 7 and 14 days after arrival at the feedlot, and parainfluenza virus 3 was isolated from 1 animal the day of, and from 18 cattle 7 and 14 days after, arrival at the feedlot. Pasteurella spp was cultured from 4 and 6 cattle at the order-buyer's barn and from 92 and 72 cattle on arrival at the feedlot during the 1997 and 1998 outbreaks, respectively. Conclusion and Clinical Relevance—Results suggest that RBCV may play a causative role in outbreaks of shipping fever in cattle. More than 80% of the sick cattle shed RBCV at the beginning of 2 outbreaks when the Pasteurella spp infection rate was low. (J Am Vet Med Assoc 2000;216:1599–1604)

The 3′-ends of the genomes (9538 bp) of two wild-type respiratory bovine coronavirus (RBCV) isolates LSU and OK were obtained by cDNA sequencing. In addition, the 3′-end of the genome (9545) of the wild-type enteric bovine coronavirus (EBCV) strain LY-138 was assembled from available sequences and by cDNA sequencing of unknown genomic regions. Comparative analyses of RBCV and EBCV nucleotide and deduced amino acid sequences revealed that RBCV-specific nucleotide and amino acid differences were disproportionally concentrated within the S gene and the genomic region between the S and E genes. Comparisons among virulent and avirulent BCV strains revealed that virulence-specific nucleotide and amino acid changes were located within the S and E genes, and the 32 kDa open reading frame.

DNA sequences coding for 81% of the ompA gene from 24 chlamydial strains, representing all chlamydial species, were determined from DNA amplified by polymerase chain reactions. Chlamydial strains of serovars and strains with similar chromosomal restriction fragment length polymorphism had identical ompA DNA sequences. The ompA sequences were segregated into 23 different ompA alleles and aligned with each other, and phylogenetic relationships among them were inferred by neighbor-joining and maximum parsimony analyses. The neighbor-joining method produced a single phylogram which was rooted at the branch between two major clusters. One cluster included all Chlamydia trachomatis ompA alleles (trachoma group). The second cluster was composed of three major groups of ompA alleles: psittacosis group (alleles MN, 6BC, A22/M, B577, LW508, FEPN, and GPIC), pneumonia group (Chlamydia pneumoniae AR388 with the allele KOALA), and polyarthritis group (ruminant and porcine chlamydial alleles LW613, 66P130, L71, and 1710S with propensity for polyarthritis). These groups were distinguished through specific DNA sequence signatures. Maximum parsimony analysis yielded two equally most parsimonious phylograms with topologies similar to the ompA tree of neighbor joining. Two phylograms constructed from chlamydial genomic DNA distances had topologies identical to that of the ompA phylogram with respect to branching of the chlamydial species. Human serovars of C. trachomatis with essentially identical genomes represented a single taxonomic unit, while they were divergent in the ompA tree. Consistent with the ompA phylogeny, the porcine isolate S45, previously considered to be Chlamydia psittaci, was identified as C. trachomatis through biochemical characteristics. These data demonstrate that chlamydial ompA allelic relationships, except for human serovars of C. trachomatis, are cognate with chromosomal phylogenies.

Hemagglutinating and acetylesterase functions as well as the 124 kDa glycoprotein were present in the highly cell-culture adapted, avirulent bovine coronavirus strain BCV-L9, in the Norden vaccine strain derived from it, and in 5 wild-type, virulent strains that multiplied in HRT-18 cells but were restricted in several types of cultured bovine cells. The BCV-L9 and the wild-type strain BCV-LY-138 agglutinated chicken and mouse erythrocytes. The acetylesterase facilitated break-down of the BCV-erythrocyte complex with chicken but only to a minimal extent with mouse erythrocytes in the receptor-destroying enzyme test. Purified preparations of the vaccine and the wild-type strains agglutinated chicken erythrocytes at low titers and mouse erythrocytes at 128 to 256 times higher titers whereas receptor destroying enzyme activity was detectable only with chicken erythrocytes. When wild-type strains were propagated in HRT cells at low passage levels, they produced 5 x 10(5) to 4.5 x 10(6) plaque forming units per 50 microliters which agglutinated erythrocytes from mice but not from chickens. Diisopropylfluoro-phosphate moderately increased the hemagglutination titers, but completely inhibited the receptor destroying enzyme of purified virus of all strains. It had virtually no influence on the plaque-forming infectivity of the different BCV strains. The acetylesterase of strain BCV-L9 reacting in the receptor-destroying enzyme test was stable for 3 h at 37 and 42 degrees C. It was inactivated within 30 min at 56 degrees C while the hemagglutinin function of this strain was stable for 3 h at 37, 42, and 56 degrees C, but it was inactivated at 65 degrees C within 1 h.

The entire nucleotide sequences of the hemagglutinin/esterase (HE) genes specified by the highly virulent strain LY138 and the avirulent strain L9 of bovine coronavirus (BCV) were determined. These sequences were compared with recently published sequences of the HE genes of the Quebec and Mebus strains. A large open reading frame of 1272 nt encoding a protein of 424 amino acid residues was predicted. The putative esterase active site was conserved in the virulent and avirulent BCV strains, indicating that this domain is probably not a determinant for BCV virulence. Four amino acid substitutions occurred between the HE proteins of BCV-L9 and BCV-LY138 (leu to Pro at 5, Leu to Val at 103, Ser to Pro at 367, and Thr to Asn at 379). Monoclonal antibodies specific for the HE glycoprotein inhibited the hemagglutination and acetylesterase activities of BCV-L9, but showed no inhibitory effect on the acetylesterase activity of BCV-LY138. These results suggest that at least one epitope is located proximal to one of the three strain-specific amino acids. Four S-specific monoclonal antibodies inhibited hemagglutination but not acetylesterase activity of BCV-L9, implying that the S glycoprotein can promote hemagglutination of chicken erythrocytes in addition to the HE glycoprotein.

The entire nucleotide sequences of the spike glycoprotein (S) genes of the highly virulent bovine coronavirus (BCV) strain BCV-LY138, the avirulent BCV-L9 and related Norden Vaccine (BCV-Vaccine) strains were determined using the polymerase chain reaction (PCR) to amplify cDNAs obtained by reverse transcription of viral RNA, and to produce single strand cDNAs for DNA sequencing. The S gene sequences of these viral strains were compared with those of recently published strains BCV-Mebus, BCV-Quebec, and BCV-F15. An open reading frame of 4092 nucleotides, encoding a protein of 1363 amino acid residues, was found in all six strains. Frameshifts and insertions or deletions were not observed except for the BCV-Fl 5. The S gene sequences were more than 98% conserved overall inspite of different origins of the six viruses. There were 45 to 56 nt differences between the virulent and avirulent groups while there were 6 to 14 nt differences among four avirulent strains. Comparison of the deduced amino acid sequences indicated that the S proteins had typical properties of membrane glycoproteins. Nineteen Winked glycosylation sites were predicted in five strains, and 18 of them were conserved in the avirulent strain BCV-L9. The sequence KRRSRR at the predicted proteolytic cleavage site was identified in five strains while the sequence KRRSVR was found in BCV-Fl 5. Substitutions of few amino acids in the putative fusogenic domains and two prolines at 507 and 567 in the antigenic domains may cause altered immunogenic and other functional properties of the S proteins specified by the virulent and avirulent BCV strains. Nine amino acid substitutions between the virulent and avirulent groups may correlate with BCV virulence.

Monoclonal antibodies (MAbs) against two major structural proteins of the cell-adapted Mebus strain of bovine coronavirus (BCV-L9) were produced and characterized. Seven MAbs reacted with the peplomeric glycoprotein, gp100/S, while three MAbs reacted with the nucleoprotein p53/N in Western blot analysis of BCV polypeptides. MAbs to gp100/S reacted with discontinuous epitopes of gp100/S in Westerns under mild but not under standard denaturing conditions. In contrast, MAbs to p53/N reacted in both types of Westerns, and those epitopes were thus continuous. MAbs to p53/N failed to neutralize BCV infectivity, while 4 MAbs to gp100/S neutralized BCV effectively. Cross reactivity of MAbs to gp100/S specified by five virulent wild-type strains and two high passage, cell-culture-adapted strains in mildly denaturing Westerns and neutralization assays indicated that two epitopes were conserved in all seven strains, while two epitopes of the avirulent strains were not detected in the wild-type strains. Non-neutralizing MAbs of gp100/S reacted with all seven strains in Westerns with the exception of one MAb that was specific for the highly cell-adapted strain BCV-L9.

Expression of bovine coronavirus (BCV) antigen in the plasmalemma of epithelioid human rectal tumor (HRT-18) and fibroblastic bovine fetal spleen (BFS) cell lines was traced by immunofluorescence and immunoelectron microscopy facilitated by colloidal gold. Cytoplasmic fluorescence was first observed at 12 hr postinfection (h.p.i) in infected HRT-18 cultures. This fluorescence coincided with the appearance of cell surface antigen reacting with colloidal gold-labeled antibodies to BCV antigens. At 24 h.p.i the amount of viral antigens at the surface of HRT-18 had increased, although cytoplasmic fluorescence remained constant. Infected BFS cells but not HRT-18 cells formed polykaryons when incubated in the presence of trypsin. One viral antigen in the plasma membrane of BFS cells was thus identified as the S glycoprotein with a fusion domain. In contrast to HRT-18 cells, the overall amount of BCV antigens at the surface of BFS cells remained constant after the onset of fusion. Analysis of the labeling characteristics established that the goldmarked-sites represented de novo expression of BCV antigen in the plasma membrane of infected cells.

Summary The early events in the infection of human rectal tumor cells by bovine coronavirus were investigated by colloidal gold-mediated immunoelectron microscopy and by analysis of the effect of lysosomotropic weak bases on virus yield. Electron microscopic studies revealed sites of fusion between the virus envelope and the plasmalemma but fusion events along intracellular membranes were not observed despite extensive searches. Virion-antibody-colloidal gold complexes were, in fact, endocytosed by synchronously infected cells. These complexes were apparently non-infectious, and they accumulated in vacuoles that resembled secondary lysosomes. Exposure of cells to ammonium chloride or to methylamine during the first hour of infection had little inhibitory effect on the production of infectious virus. Chloroquine treatments were inhibitory but this effect depended on relatively late events in the infectious process. The chloroquine inhibitory step blocked infection of virus adsorbed to cells that were exposed to buffers in the pH range of 4.4 to 8.4. These findings indicate that BCV penetrates its host cell by direct fusion with the plasmalemma and does not require an acidic intracellular compartment for infectious entry.

Approximately 60% of the outer membrane of chlamydial elementary bodies (EBs) consists of the major outer membrane protein (MOMP) that has structural and metabolic functions. The antigenic properties of MOMPs from mammalian strains of serovars 1 and 2 and an avian strain of Chlamydia psittaci were analyzed. Polyclonal-monospecific antisera (PMAs), one monoclonal antibody (MAb), and polyclonal antisera (PAs) were produced against reduced polyacrylamide gel electrophoresis-separated MOMPs and against infectious EBs. Three PMAs and the MAb, which were induced by reduced polyacrylamide gel electrophoresis-separated MOMPs, reacted strongly in Western blot (immunoblot) assays with MOMPs of serovar 1 and 2 strains as well as with that of the avian strain 6BC, and two of these PMAs reacted weakly (dilution, 1:20) with the MOMP of strain LGV-2. The third PMA and the MAb against the MOMP of the serovar 2 strain did not react with the MOMP of LGV-2. Four PAs were produced against infectious EBs of the serovar 1 strain. One of these PAs reacted with the homologous MOMP and that of the avian strain 6BC but did not recognize MOMPs of other chlamydial strains. Three of the PAs reacted with MOMPs of homologous strains only and failed to recognize MOMPs of avian, serovar 2, and LGV-2 strains. Five PAs induced against infectious EBs of the serovar strain 2 reacted only with the MOMPs of the homologous strains and failed to recognize MOMPs of other strains of chlamydiae. Consequently, MOMPs of C. psittaci strains possess genus-, species-, and serovar-specific epitopes whereby the immune response to serovar-specific epitopes of MOMP predominate when infectious EBs are used for immunization.

Summary The ecology of cytopathic expression of bovine coronavirus (BCV) in HRT-18 cells was analyzed within virus-induced plaques by scanning electron microscopy. Virus replication was cytocidal for many HRT-18 cells, a function enhanced in the presence of trypsin. A monolayer of cells remained that imparted a characteristic turbidity to the plaque. These structurally normal, lysisresistant cells did not stain with fluorescent antibodies specific for BCV antigens, failed to adsorb virus particles or mouse erythrocytes in contrast to the susceptible cells. The survival of cells in the plaque interior reflects a non-productively infected population with evidence of viral persistence.

The complete genome sequences are reported here of two field isolates of bovine coronavirus (BCoV), which were isolated from respiratory and intestinal samples of the same animal experiencing fatal pneumonia during a bovine shipping fever epizootic. Both genomes contained 31028 nucleotides and included 13 open reading frames (ORFs) flanked by 5′- and 3′-untranslated regions (UTRs). ORF1a and ORF1b encode replicative polyproteins pp1a and pp1ab, respectively, that contain all of the putative functional domains documented previously for the closest relative, mouse hepatitis virus. The genomes of the BCoV isolates differed in 107 positions, scattered throughout the genome except the 5′-UTR. Differences in 25 positions were non-synonymous and were located in all proteins except pp1b. Six replicase mutations were identified within or immediately downstream of the predicted largest pp1a-derived protein, p195/p210. Single amino acid changes within p195/p210 as well as within the S glycoprotein might contribute to the different phenotypes of the BCoV isolates.

Over the last few years numerous respiratory bovine coronaviruses (RBCV) were isolated from respiratory infections of cattle including two shipping fever epizootics (Storz et al., 1996; Storz et al., 2000). The RBCV strain 98TXSF-110-LU (LU) and the enteric strain 98TXSF-110-ENT (ENT) were isolated from lung and intestinal samples, respectively, from an animal experiencing fatal respiratory disease during a 1998 shipping fever epizootic. We obtained and compared the entire genomic sequences of the LU and ENT isolates to delineate nucleotide (nt) and amino acid (aa) differences between them. In addition, we analyzed the leader-mRNA junction sequences of the S and 12.7 kDa mRNAs specified by different BCV strains.

Numerous respiratory bovine coronaviruses (RBCV) were isolated recently from nasal swab samples and lung tissues of feedlot cattle with acute respiratory tract disease. These newly emerging RBCV isolates exhibited distinct phenotypic features that differentiated them from enteropathogenic bovine coronaviruses (EBCV). The RBCV strains had a receptor-destroying enzyme function mediated by acetylesterase (AE) activity of the haemagglutinin-esterase (HE) glycoprotein. The HE genes of wild-type EBCV strain LY138 and RBCV strains OK-0514 (OK) and LSU-94LSS-051 (LSU) were cloned, sequenced and transiently expressed in COS-7 cells. The enzymic properties of HE proteins in COS-7 cellular extracts and in purified virus preparations were assayed at room temperature, 37 degrees C and 39 degrees C by two different assays. One assay used p-nitrophenyl acetate (PNPA) as substrate and detected serine-esterase activity; the second assay monitored AE function with bovine submaxillary mucin (BSM) as substrate. The PNPA tests confirmed that HE proteins of EBCV and RBCV were functionally expressed in transfected COS-7 cells. Time-dependent determination of the AE activity of purified RBCV OK and LSU particles showed lower AE activity at 39 degrees C than at 37 degrees C, whereas the purified EBCV LY particles retained full AE activity at both 37 degrees C and 39 degrees C. Transiently expressed RBCV HE exhibited a marked reduction of AE activity after 40 min of assay time at 37 degrees C. In contrast, the AE activity of the transiently expressed EBCV HE remained stable beyond 40 min. The deduced amino-acid sequences of the HE proteins specified by the RBCV strains OK and LSU contained specific amino-acid changes in comparison with the EBCV LY strain, which may be responsible for the observed enzymic differences. These results are consistent with the hypothesis that RBCV strains have evolved to selectivelyreplicate in respiratory tissues and that HE may play a role in this tissue tropism.

A monoclonal antibody-based capture enzyme-linked immunosorbent assay (ELISA) was developed to detect respiratory bovine coronavirus (RBCV) antigens in nasal swabs collected from cattle showing signs of respiratory tract disease following shipping. These samples had been previously tested for RBCV by inoculation of G clone cultures of human rectal tumor cells (HRT-18G) and for bovine herpes virus 1, parainfluenza virus 3, bovine adenovirus, bovine respiratory syncytial virus, and bovine viral diarrhea virus on other specifically permissive cell cultures. RBCV has not previously been recognized as an important etiological factor in the bovine respiratory disease complex of feedlot cattle. Thirty of 100 samples tested positive for RBCV antigen by capture ELISA in contrast to 38 of 100 samples that yielded RBCV isolates in G clone cells. Samples yielding other bovine respiratory viruses in the absence of RBCV were negative in the capture ELISA, which was based on the use of a single monoclonal antibody that recognizes one RBCV epitope on the S glycoprotein with the broadest reactivity with different strains of RBCV tested. Some RBCV strains may not be detected by this ELISA, which may account for the higher percentage of RBCV-infected cattle detected by RBCV isolation. However, the ELISA was simple to perform, sensitive, and specific and was more rapid than virus isolation. This assay will be useful for processing large numbers of field samples in future epidemiologic and diagnostic studies of RBCV infections of cattle.

The coronavirus strain HECV‐4408 was isolated from diarrhea fluid of a 6‐year‐old child with acute diarrhea and propagated in human rectal tumor (HRT‐18) cells. Electron microscopy revealed coronavirus particles in the diarrhea fluid sample and the infected HRT‐18 cell cultures. This virus possessed hemagglutinating and acetylesterase activities and caused cytopathic effects in HRT‐18 cells but not in MDBK, GBK and FE cells. One of four S‐specific monoclonal antibodies reacted in Western blots with HECV‐4408, BCV‐L9 and BCV‐LY138 but not with HCV‐OC43, and two reacted with BCV‐L9 but not with HECV‐4408, BCV‐LY138 and HCV‐OC43. One S‐specific and two N‐specific monoclonal antibodies reacted with all of these strains. cDNA encompassing the 3′ 8.5 kb of the viral RNA genome was isolated by reverse transcription followed by polymerase chain reaction amplification had size and restriction endonuclease patterns similar to those of BCV‐L9 and BCV‐LY138. In contrast, the M gene of HCV‐OC43 differed in restriction patterns from HECV‐4408 and BCV. A genomic deletion located between the S and M within the nonstructural genes of HCV‐OC43 was not detected in HECV‐4408. DNA sequence analyses of the S and HE genes revealed more than 99% nucleotide and deduced amino acid homologies between HECV‐4408 and the virulent wild‐type BCV. Forty‐nine nucleotide and 22 amino acid differences were found between the HE genes of HECV‐4408 and HCV‐OC43, while only 16 nucleotide and 3 amino acid differences occurred between the HE genes of HECV‐4408 and BCVLY138. We thus conclude that the strain HECV‐4408 is a hemagglutinating enteric coronavirus that is biologically, antigenically and genomically more closely related to the virulent BCVLY138 than to HCV‐OC43. © 1994 Wiley‐Liss, Inc.

Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis.

The complete nucleotide sequences of the hemagglutinin/esterase (HE) genes of human coronavirus (HCV) strain OC43 and bovine respiratory coronavirus (BRCV) strain G95 were determined from single-stranded cDNA fragments generated by reverse transcription of virus-specific mRNAs and amplified by polymerase chain reaction. An open reading frame of 1272 nucleotides was identified as the putative HE gene by homology to the bovine coronavirus HE gene. This open reading frame encodes a protein of 424 amino acids with an estimated molecular weight of 47.7 kDa. Ten potential N-linked glycosylation sites were predicted in the HE protein of HCV-OC43 while nine of them were present in BRCV-G95. Fourteen cysteine residues were conserved in the HE proteins of both viruses. Two hydrophobic sequences at the N-terminus and the C-terminus may serve as signal peptide and transmembrane anchoring domain, respectively. The predicted HE protein of HCV-OC43 was 95% identical to the HEs of BRCV-G95 and other bovine coronaviruses, and 60% identical to the HEs of mouse hepatitis viruses. Phylogenetic analysis suggests that the HE genes of coronaviruses and influenza C virus have a common ancestral origin, and that bovine coronaviruses and HCV-OC43 are closely related.

A relatively simple and sensitive method is described which enables the effect of monoclonal antibodies (MAbs) on the receptor-destroying enzyme (RDE) and the haemagglutination (HA) activity of bovine coronavirus (BCV) to be analysed in one assay. A lysate of HRT-18 cells infected with the L9 strain of BCV was found to have a higher RDE:HA ratio than purified virus. At 4 degrees C the lysate induced an HA pattern which completely disappeared upon raising of the temperature to 37 degrees C. This L9-infected cell lysate was used to determine the HA inhibition (HAI) titres of MAbs directed against the surface glycoproteins S and HE of BCV. Thereafter, the test plates were incubated at 37 degrees C to enable the ability of the MAbs to prevent elution of virus from BCV-erythrocyte complexes to be assessed. No inhibition of RDE was detectable with MAbs against glycoprotein S, which had HAI titres ranging from 1:16 to 1:128. On the other hand, MAbs directed against glycoprotein HE had similar HAI titres, but they inhibited elution of 8 HA units of BCV at titres of up to 1:65,000.

The morphology and morphogenesis of virus strain LY-138 recovered from neonatal diarrheic calves were investigated by electron microscopy using negativestaining techniques and ultrathin sectioning. Purified viral particles were spherical in shape and measured 90 nm in average diameter in negatively stained preparations. Pleomorphic forms were also present. The virions had envelopes with petal-shaped projections characteristic of coronaviruses. In ultrathin sections, cores in viral factories were round with a diameter of 50–60 nm. Most of these cores were electron dense but some had an electron-lucent center. In cytoplasmic vacuoles, Golgi vesicles, and on the apical plasmalemma of intestinal epithelial cells, the virions were round or ellipsoidal in shape, measuring 70–80 nm in diameter, and had fine thread-like projections on their surfaces. Uptake of virus occurred through fusion of viral envelopes with the plasmalemma of the microvillous border or by entry into intercellular spaces and interaction with the lateral cell membranes of adjacent intestinal epithelial cells. As a result of this interaction, the lateral cell membranes became altered and ill-defined. During the early stage of infection, the rough andasmooth elements of the endoplasmic reticulum became distended with electron-dense granulofibrillar material. This material accumulated subsequently as well-defined, smooth membrane-bound areas mainly in the apical cytoplasm of infected cells. These structures were considered to be viral factories. The morphogenesis of virus occurred mainly through condensation of the electron-dense, granulo-fibrillar material into viral cores in cytoplasmic viral factories or within the distended cisternes of the rough endoplasmic reticulum. Viral envelopment occurred on membranes of cytoplasmic vacuoles, Golgi vesicles, or in association with membranes of viral factories. Release of virus from infected cells occurred by lysis and fragmentation of the apical plasmalemma and flow of the cytoplasm with its contents into the gut lumen. Release also occurred by digestion and lysis of extruded infected cells or by fusion of virus-containing cytoplasmic vacuoles with the apical plasmalemma and liberation of their contents.

Chlamydial isolates of bovine origin were serotyped by a plaque reduction method. Of the two major serotypes observed, type 1 included isolates from bovine abortion and enteric infections, whereas type 2 isolates were associated with polyarthritis or encephalomyelitis. These two serotypes were identical to those with a similar disease distribution previously observed in isolates of ovine origin. The two groups did not cross-react and they were serologically unrelated to chlamydiae of avian origin. Thus, it appears that many chlamydial isolates causing intestinal infections or abortion in sheep or cattle are closely related antigenically, as are those producing polyarthritis, encephalomyelitis, and conjunctivitis, and that the two groups are distinct.

Summary Bovine Herpesvirus Type 4: Studies on the Biology and Spread in Cattle Herds and in Insemination Bulls A serological survey with approx. 3,000 bovine sera revealed that 18.4% of the animals tested from West Germany and 8.7% from the USA harboured antibodies against bovine herpesvirus-4. A large number of BHV-4 reactors was found among healthy bulls in different German bull studs. In one stud with almost 70% seropositive animals an incidence rate of ca. 5% was observed. Virus could not be isolated from the semen of some of these animals. No correlation between the number of BHV-4 positive animals and respiratory or genital distress, Infectious Bovine Rhinotracheitis or Malignant Catarrhal Fever was detectable. The virus has a comparatively long replication cycle in tissue culture and a wide host cell range. Eight isolates were indistinguishable by serological means and no cross-reactions with BHV-1, —2, —3 and —6 were detectable, when tested by indirect fluorescence assays. Using this test, virus specific antigens could be stained in the nucleus of infected cells from 10 hours p. i. onwards and four hours later also in the cytoplasm. Zusammenfassung Eine serologische Untersuchung mit etwa 3000 Rinderseren zeigte, das in der Bundesrepublik 18,4%, in den USA 8,7% aller getesteten Tiere Antikorper gegen das bovine Herpesvirus-4 besasen. Auffallig viele Reagenten befanden sich dabei unter den gesunden Bullen verschiedener deutscher Besamungsstationen. In einer Station mit fast 70% Reagenten traten in einem Jahr knapp 5% Neuinfektionen auf. Versuche, aus dem Sperma dieser Tiere Virus zu isolieren, blieben erfolglos. Zusammenhange zwischen der Anzahl BHV-4-positiver Tiere und dem Auftreten von Erkrankungen des Respirations- oder Genitaltraktes, infektioser boviner Rhinotracheitis und bosartigem Katarrhalfieber liesen sich nicht ermitteln. In vitro zeichnet sich dieses Virus durch eine relativ langsame Replikation und ein breites Wirtszellspektrum aus. Wahrend 8 untersuchte Isolate serologisch einheitlich reagierten, liesen sich Kreuzreaktionen mit BHV-1, —2, —3 und —6 im indirekten Fluoreszenzantikorpertest nicht feststellen. Mit Hilfe dieses Testes waren virusspezifische Antigene in infizierten Zellen ab 10 Stunden p.i. im Kern, 4 Stunden spater auch im Cytoplasma nachweisbar.

Summary Polykaryon formation in bovine fetal spleen (BFS) cells infected with bovine coronavirus L9 occurred only in media supplemented with trypsin. A single 1 to 2 h trypsin treatment 10 h and later after infection induced formation of polykaryons. Trypsin treatment at pH 7.5 and 8.0 induced polykaryons while treatments at lower or higher pH levels did not. Cell fusion activity was partially suppressed by the presence of antibody.

The growth characteristics of Chlamydia psittaci serotypes 1 and 2 (ovine and bovine origin) were studied in mouse L cells. Formation of inclusions and yield of infectious progeny for serotype 1 were maximal when host cells were treated with cycloheximide and the pH in the cell culture medium was 7.2-7.4. The number of cells that contained inclusions and the infectivity yield for serotype 2 were maximal when the pH was 6.6-7.0. Treatments with diethylaminoethyl dextran and cycloheximide increased inclusion formation in serotype 2 but decreased the yield per infected cell. For both serotypes, centrifugation of the chlamydiae onto monolayers infected 50-1,200 times the number of cells infected with use of stationary adsorption. An enumeration method for L-cell infectious units was developed that gave higher titers than the chicken embryo 50% lethal dose after yolk sac inoculation.

Indirect enzyme-linked immunosorbent assays were applied to differentiate Chlamydia (C.) psittaci -specific IgG1 and IgG2 levels in 143 individual serum samples from cattle with naturally occurring chlamydial infections and in 190 sequential serum samples from 26 experimentally infected pregnant cows, calves, and a bull. The mean IgG1:IgG2 ratio of naturally infected cattle was 1:4 indicating a significant (p C. psittaci and Coxiella (C.) burnetii simultaneously. The predominance of C. psittaci -specific IgG2, in contrast to the predominance of C. burnetii -specific IgG1 detected in these same individual serum samples under identical conditions, indicates that the ability to preferentially produce either IgG1 or IgG2 was not limited in these individual cattle. A transient yet significant IgG1 response was also developed in cows following chlamydia-induced abortions (immunotype 1) or in cattle infected with the polyarthritis-serositis-encephalomyelitis agents (immunotype 2). IgG1 levels decreased faster than IgG2 levels. These findings have diagnostic implications and identify the need for determining the immunoglobulin classes and subclasses of the humoral immune responses of animals and man to chlamydial infections.

Summary Biological properties and restriction enzyme patterns of the slowly replicating herpesviruses isolated from cattle affected with different diseases in North America and Europe were analysed. These virus isolates induced identical plaques that developed within 7 to 9 days in bovine foetal spleen cells and within 5 days in actively growing Georgia bovine kidney cells. These virus isolates were found to be antigenically related when tested in the indirect immunofluorescence test, and antigenic relationships with bovine herpesvirus 1 (BHV-1), BHV-2, BHV-3 or BHV-6 were not detected. The genomes of these strains were shown to have virtually identical cleavage sites when treated with restriction enzymes EcoRI, BamHI, SstII, SphI and HindIII. The resulting restriction enzyme patterns differed strikingly from those of BHV-1, BHV-2, BHV-3 and BHV-6. Because the herpesviruses tested become enveloped on the nuclear as well as on endoplasmic membranes, a process through which they induce cytoplasmic vesicles filled with enveloped viral particles, and because of the unique cytoplasmic inclusions that are induced, we classify them tentatively as bovine cytomegaloviruses.

Antigens of the immunotype 1 strain B-577 of Chlamydia psittaci, which were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes were used to probe sequential serum samples of cattle with experimentally induced or naturally occurring chlamydial infections. Applying IgGl- and IgG2-specific markers in an enzyme immunoassay procedure, a predominance of IgG2 reactions with different proteins was determined. The interaction of IgGl with antigens such as the genus-specific lipopolysaccharide and the major outer membrane protein was usually limited to periods immediately following overt clinical disease. Some other antigens like the 60,000 and 62,000 D proteins, for example, were recognized by both subclasses over the entire period of investigation. This indicates that it may be possible to determine the phase of infection through analysis of the IgGl and IgG2 responses with the Western blot technique. The different IgGl and IgG2 responses of cattle infected with different strains of Chlamydia psittaci as well as the diverse reactions of cattle from different herds with naturally occurring chlamydial infections further indicate that it may be feasible to distinquish the strains causing these chlamydial infections using different antigens in the Western blot technique. The results obtained by this method may have implications for the production of a subunit vaccine as well as for serodiagnostic purposes.

Initiation of autonomous parvovirus replication depends on the S phase of host cells actively traversing the cell cycle. The parvoviruses Lu III and H-1 inhibit synchronized cells from entering mitosis, implying that parvoviruses rapidly shut down cell cycle traverse during G2 phase. Bovine parvovirus (BPV) did not inhibit the entry into mitosis of hydroxyurea synchronized bovine fetal spleen cells. Mitotic indexes of infected cultures were as much as 60-fold higher than those of mock-infected controls. Mitosis in control and infected cultures peaked at 10 hr after infection corresponding to the end of the BPV eclipse period. Cytopathic changes, including morphological alteration of mitotic chromosomes, were detected in mitotic cells from infected cultures by light and electron microscopy. Arrest of BPV-infected cells in mitosis may explain these results. Not all infected cells were killed in mitosis, since some developed intranuclear inclusions and became pycnotic as nucleated, interphase cells. Inclusion formation was coincident with viral morphogenesis in interphase nuclei at 16 to 18 hr postinfection, late in the viral replication cycle. The cell cycle stage at which parvovirus-infected cells are arrested and cytopathic events ensue may be determined by the cellular progression rate from S phase through G2 and M.

One-step growth curves of a bovine enterovirus and the virus of bovine diarrhea (BVD) in singly and doubly infected bovine embryonic kidney (BEK) and bovine embryonic spleen (BES) cells were determined. In BEK cells, bovine enterovirus infectivity increased exponentially in both tissue culture fluid and cells 4 to 10 hours after inoculation and then levelled off. In BES cells, the exponential increase lasted 14 to 17 hours. In the growth curve of the BVD virus in BEK cells, the extracellular and cell-associated viruses increased exponentially until hour 12, increasing gradually thereafter. In BES cells, BVD virus infectivity increased exponentially 24 hours after infection and then levelled off. The peak infectivity in BES cells was reached 24 hours before that in BEK cells. Growth of bovine enterovirus was studied in cells of BEK and BES monolayers preinfected with the BVD virus for 20 hours. The infectivity titer in preinfected BEK cells was approximately 2 logs less than in preinfected BES cells. A decreased yield in infectivity was observed compared to the bovine enterovirus one-step growth curve.

In assessing the host cell range of bovine parvoviruses, these viruses were found to replicate optimally in actively dividing bovine fetal lung and spleen cells. Other primary bovine fetal cells supported growth to a lesser extent, but bovine line cells and line cells of other animal species tested did not. Minimal infectivity remained after passage of bovine parvovirus in cells from chicken embryos and guinea pig fetuses. During bovine parvovirus replication in bovine fetal lung and spleen cells, production kinetics of infectious virus and hemagglutinins were determined. An eclipse period of 16 h occurred, and viral release from cells was not detected until 30 h after inoculation of bovine fetal lung cells and 36 h after inoculation of bovine fetal spleen cells. Cell-associated virus titers were always higher than extracellular virus titers. Hemagglutinins were detected in parallel to infectious virus.

Tritiated uridine was selectively incorporated into the cytoplasm but not the nucleus of BEK cells infected with bovine enterovirus. This was accompanied by complete shutdown of cellular RNA synthesis. Bovine embryonic spleen cells infected with the BVD virus incorporated3H-uridine into the nucleus and cytoplasm, while uninfected cells showed only nuclear and nucleolar incorporation. Bovine embryonic spleen cells that were preinfected with BVD virus for 20 hours and then superinfected with bovine enterovirus showed complete absence of nuclear and nucleolar labelling in the early stages of superinfection with the bovine enterovirus, but nuclear labelling returned in reduced amounts 8 to 10 hours after superinfection and persisted throughout the remainder of the double infection ending in cell death.

Abstmct. Ultrastructural changes induced by chlamydia1 infection of niucosal cells of the ileum of ncwborn calves after oral inoculation were investigated. Depending on the stage of chlamydia1 development, the organelles of all infected cells became damaged. The damage was degenerative and included vesiculation of microvilli and swelling of the terminal web of absorptive epithelial cells. The mitochondria were swollen and had fragmented cristae. The endoplasmic reticulum was dilated and vesiculated, and infected cells gradually lost their ribosomes. The lateral junctional complex between infected cells became occasionally dislocated and fragmented. The basal lamina was thrown into folds, became discontinuous and separated from the basal border of the epithelial cells. The nuclei of infected cells were affected last, lost their chromatin pattern, and ultimately became pyknotic. Enteric disease represents a serious problem for the newborn of many different animal species and man. The newborn encounters numerous infectious agents during the postnatal period when the intestinal microbial flora is being established. Intestinal infections with viruses and other obligate intracellular parasites such as chlamydiae are also known to occur during this period. Chlamydia1 agents have been isolated from feces of clinically normal young calves [ 151 and from specimens of newborn calves with diarrhea and polyarthritis [ 121. After oral inoculation with specific chlamydia1 strains, young calves developed diarrhea and died rapidly because of water and electrolyte losses, but only minor gross and histopathologic lesions were detected in the intestine [5]. Since chlamydiae replicate by going through a welldefined developmental cycle, infected cells can be identified ultrastructurally. The purpose of this investigation was to analyze ultrastructural changes induced by chlamydiae in the ileal mucosa of calves that developed diarrhea after oral inoculation.

The uptake and release of chlamydiae in infections of highly specialized intestinal, absorptive epithelial cells of the ileum of newborn calves were studied. Infection occurred only through the brush border of the enterocytes. The chlamydial elementary bodies were adsorbed onto the microvilli, a process that induced pinocytosis of the elementary bodies and formation of endosomes containing one dense-centered chlamydial form. The endosomes moved towards the supranuclear region of the enterocytes, and the cell membrane entrapping the chlamydial agent gradually dissolved so that inclusions with reticulate and dense-centered forms appeared to be free in the cytoplasm. After replication, chlamydiae were shed into the intestinal lumen in three different ways. Some infected enterocytes ruptured and liberated their contents into the lumen. In other instances, entire infected cells were extruded into the lumen, where their membranes degenerated to release chlamydial forms. In the third mode of release, chlamydiae were liberated in cytoplasmic fragments surrounded by cell membranes.

After oral inoculation of calves with chlamydial agents, infection of cells in the abomasum and the lower portions of the gastrointestinal tract occurred. The range of different cells infected in the ileum was analysed by electron microscopic examination. In decreasing order of frequency, the following types of cells were found to contain chlamydiae at different stages of the developmental cycle: absorptive epithelial cells at the tip of the villi, absorptive epithelial cells of the intervillous zone, macrophages of the intestinal lamina propria, endothelial cells of the central lacteals, enterochromaffin cells and goblet cells of the villi, fibroblasts of the lamina propria, rarely undifferentiated cells of the crypts of Lieberkuhn. Chlamydial forms were seldom seen in neutrophilic leukocytes which were increased in number in the neigh bourhood of chlamydia-infected cells. Plasma-cells were numerous in the intestinal lamina propria. These cells often contained degenerating chlamydial forms in cytoplasmic phagosegrosomes.

Five identical viral isolates were recovered from bovine fetal spleen cells maintained in culture for 30 to 67 days. During this time the cells were sub-cultured 3 to 8 times. Cells cultured from other organs of the 3 donor fetuses did not evidence lytic viral infections. The viral isolates were identified serologically, morphologically, culturally and by characterization of the virus DNA as the herpesvirus of infectious bovine rhinotracheitis. The available data indicate latent infection of the donor fetuses as the source of these viral isolates.

A hemadsorbing enteric virus (HADEN), isolated from the gastrointestinal tract of cattle and previously classified as a bovine enterovirus, was found to induce Feulgen-positive intranuclear inclusions and to be inhibited in its replication by FUDR, BUDR and actinomycin D, indicating DNA as genetic material. Because of this fact and the other known properties of HADEN virus it should be considered a bovine parvovirus.

Eight chlamydial isolates of ovine origin were tested in a plaque reduction system using homologous and heterologous rooster antisera. The eight isolates could be separated into two separate immunotypes. Type 1 included isolates associated with ovine abortion and one agent recovered from the feces of an apparently normal sheep. Type 2 isolates were associated with polyarthritis and conjunctivitis. These two serotypes were not cross-reactive with several chlamydiae of avian origin. Further application of the plaque reduction test may provide a useful means of typing chlamydiae.

Cornonaviruses cause severe and economically important intestinal infections in newborn calves which become manifest as enteritis with diarrhea and additional clinical consequences. Studies concerning the pathogenesis of enteropathogenic bovine coronavirus (EBC-virus) enteritis have led to a better understanding of morphological alterations of the intestinal tract as well as of the individual cells, helping to explain the functional disorders observed with this disease (for references see Storz and Rott, 1980). Relatively little is known, however, about structural details of the virus and about the biochemical events of EBC-virus replication, principally a result of the difficulties encountered in growing the virus in tissue culture. After we succeeded in adapting EBC-virus strain L-9 isolated by Mebus et al. (1973) to grow in bovine fetal thyroid or brain cells in reasonable amounts it became possible to study some of the many unanswered questions. As a beginning, efforts were made to determine optimal growth conditions in order to provide an in vitro source for viral antigen. In addition, biochemical analysis of the virus was undertaken.