Genetic typing of bovine pestiviruses from England and Wales.

Using RNA purified directly from stored clinical specimens, a collection of 62 pestiviruses were typed by RT-PCR and sequencing within the 5'-untranslated region of the genome. All the specimens had been obtained in 1966/1967 from diary cattle in England and Wales. Eight further pestiviruses, grown in cell culture, were characterised in the same way. Seven of these viruses were representatives of a panel of British isolates, obtained from cattle ten years before. The eighth was the virus used in a British bovine viral diarrhoea (BVD) vaccine. Most of the viruses were genetically unique and were of BVDV type Ia. One recent isolate was BVDV type Ib, two others were intermediate between Ia and Ib. No BVDV type II or border disease virus (BDV) isolates were found. There was no overall association between geographical and phylogenetic clustering, suggesting long-distance virus dispersal, presumably via trading of infected cattle. The sequences of the recently obtained cattle viruses were very similar or, in one case, identical to the older isolates in the region studied. Their close similarity to some previously characterised pestiviruses from British sheep suggests that a common pool of BVDV Ia is shared by these two livestock species, although another pestivirus--BVDV--is confined to sheep. The British cattle viruses were mostly distinct from continental European isolates, but more similar to type Ia isolates from North American cattle.     

Diversity among bovine pestiviruses

Bovine viral diarrhoea virus (BVDV) isolates are characterized by an important genetic, antigenic and pathogenic diversity. The emergence of new hypervirulent BVDV strains in North America has provided clear evidence of pathogenic differences between BVDV strains. The origin of BVDV diversity is related to high mutation rate occurring in RNA viruses but the consequences of mutations obviously depend on the genes which are involved. Mutations in genes encoding for structural proteins of immunological importance may have practical implications.Knowledge of BVDV diversity is important for understanding the wide variety of pathogenesis of diseases caused by the virus, for monitoring the epidemiology of the different types and for the design of optimum laboratory tests and vaccines.This review focuses on the origin and consequences of BVDV diversity with regard to pathogenesis, biotypes, and antigenic and genetic variations.     

Genetic typing of pestiviruses from New Zealand

AIM: To investigate the genetic type of 20 pestiviruses collected from New Zealand over the period 1967-97. METHODS: The pestiviruses were genetically typed by the sequencing of polymerase chain reaction (PCR) products. The primers selected were from the 5'-untranslated region (5'-UTR) of the pestivirus genome and consistently amplified a 288 bp fragment from all samples tested. RESULTS: Sequencing and phylogenetic analysis of PCR products revealed that all samples obtained from cattle represented bovine viral diarrhoea (BVDV) type I. Two sheep isolates were characterised as border disease virus (BDV). A pestivirus isolated from foetal calf serum of USA origin was typed as BVDV type II. CONCLUSIONS: The findings show that the evolution of pestiviruses in New Zealand has been similar to Europe and North America, indicating the occurrence of a conservative phylogenetic branch of BVDV type I in cattle and the presence of BDV in the sheep population.     

Genetic heterogeneity of pestiviruses of ruminants in Switzerland

We have genetically analyzed ruminant pestiviruses. All >150 bovine viral diarrhea (BVD) viruses isolated from cattle in Switzerland belonged to genotype 1, with subgenogroups e, h, k and b found in decreasing frequency. To date, representatives of subgenogroup k have been detected in Switzerland only. Despite serological evidence of Border disease in sheep, only few Border disease viruses have been isolated, all of which belong to the novel group 3. Serological evidence suggested that pestivirus infections may occur also in wild ruminants in Switzerland but no isolates are available for analysis. In addition, we describe two pestiviruses, one a cell culture contaminant and the other isolated from a buffalo, that cluster with a recently proposed novel pestivirus species.     

Examination of bovine and ovine fetuses

Diagnosis of the causes of bovine and ovine abortion is difficult and frustrating and requires a systematic and thorough approach. Laboratory assistance is required in all cases. Herd and individual histories occasionally help, as does knowledge of the gestational age and autolytic condition of the fetus when aborted. Tissues from mummified fetuses should be cultured and examined by fluorescent antibody techniques for viruses. The placental tissue from mummified fetuses should be examined for fungi and lesions. Lung, liver, kidney, spleen, abomasal content, body cavity fluid or serum, and fetal placenta from all but completely mummified fetuses should be submitted to a diagnostic laboratory.     

Serological comparison of type strains of porcine, bovine, and ovine mycoplasmas with atypical colony morphology

The type strains of Mycoplasma hyopneumoniae, M. flocculare, M. dispar, and M. ovipneumoniae, all characterized by nipple-less colonies on solid media, were compared serologically. By indirect hemagglutination and by complement fixation tests they were found to constitute a related group. By crossed immunoelectrophoresis a sharing of common antigens was demonstrated, whereas no cross reactivity was noted by the metabolism inhibition test.The type strains of Mycoplasma hyorhinis and Mycoplasma bovirhinis were included in the study for comparison. Although some cross reaction was noted, they appeared just moderately related to the nipple-less group as well as to each other.     

Characterisation of bovine strains of Pasteurella multocida and comparison with isolates of avian, ovine and porcine origin

One hundred and fifty-three bovine Pasteurella multocida strains recovered primarily from cases of pneumonia and mastitis in England and Wales over an 11-year period were characterised by capsular PCR typing, comparison of outer membrane protein (OMP) profiles, and multilocus sequence analysis. All of the strains were of capsular type A with the exception of a single capsular type F isolate. Thirteen distinct OMP profiles (OMP-types) were identified based mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins. However, 85% of the isolates were represented by just five OMP-types and 39% of the strains were of a single OMP-type. Multilocus sequence analysis revealed a limited degree of genetic diversity among bovine P. multocida isolates; strains of the same OMP-type have identical genetic backgrounds and represent distinct clones. Analysis of OMP variation was more discriminating than multilocus sequence analysis because strains of different OMP-types had the same, or similar, genetic backgrounds. The association of a small number of clones with the majority of cases of bovine pneumonia suggests that these clones have an increased capacity to cause disease compared to less frequently recovered clones. Molecular mass heterogeneity of OmpA and OmpH, in strains of the same or similar genetic background, suggests that these proteins are subject to diversifying selection within the host and might play important roles in host-pathogen interactions. Comparison of the OMP profiles of bovine isolates with those of avian, ovine and porcine strains showed that a high proportion of the respiratory tract infections in each of these species are caused by different strains of P. multocida. However, the presence of small numbers of closely related strains in more than one host species suggests that transmission of bacteria between different host species is also a factor in the population biology of P. multocida.     

Attempted reactivation of latent bovine herpesvirus 1 infection in calves by infection with ruminant pestiviruses

Three calves, latently infected with bovine herpesvirus 1 (BHV 1), were each inoculated intranasally with 9 strains of ruminant pestivirus (BVDV). All three calves developed a biphasic pyrexia and a lymphopenia followed by a neutrophilia. They did not shed BHV 1 in their nasal secretions in the 14 days following BVDV inoculation, and their BHV 1 antibody levels remained static, as did those of 2 control calves not given BVDV. All five calves were subsequently shown to be latently infected with BHV 1 by the production of recrudescent infections following the administration of dexamethasone. BHV 1 was recovered from nasal secretions and there was a marked rise in BHV 1 antibody titres in the second week after dexamethasone administration.     

Genetic study of Andalusia's ovine and caprine breeds

SUMMARY: Two different breeds of Andalusian sheep, 'Grazalema Merino' and 'Lebrijan Churro', and two different breeds of Andalusian goats, 'Andalusian White' and 'Andalusian Black', chosen by previous studies (Rodero et al. 1992a) as priority breeds for conservation, were studied. The systems used corresponded to ethnozootechnic characteristics, as well as the different biochemical-polymorphism variables. Farms were differentiated within breeds, or between themselves, and different tests were used of genetic and genotypic frequencies: Wright's indices, medium heterozygosities, Whalund's variances, G test of probability of reason, etc. Also Cavalli-Sforza's genetic distance was obtained. In the Andalusian Black and Grazalema Merino breeds, the Whalund's variances obtained were a result of selection, that has divided the breeds into distinct populations differentiated spatially. Medium heterozygosities of each breed do not differ much within themselves, but when each system is considered alone, discrepancies between ethnic groups are relevant. Wright's F indices demonstrated in the Andalusian White and Grazalema Merino breeds, genetic heterozygosities between populations or studied herds can be deduced, but this is not possible in the Andalusian Black. The F(IS) values indicated, despite the small size of the populations, that inbreeding has been avoided, probably because of the entry of foreign sires. In none of the breeds is there a significant excess of heterozygosis. The genetic distances between flocks within breeds do not differ from those found between breeds. RéSUMé: On a travallé avec, differents troupeau des races de montons de l'Andalusie, Grazalema Merino et Lebrija Churro, et avec les races caprines Andalusian White et Andalusian Black, choisie entre les races Andaluciennes comme prioritaires pour la conservation, dans un etudie avant (Rodero et col. 1992a). Les sistémes utilicés dans cette travaille correspondent á charactérés etnozootechniques et á differents variables de polymorphism biochimique. Lorsque on fait differences entre troupeau, dedans de races, ou entre elles, on a utilicés differents preuves, á partir des fréquences géniques et génotipiques: l'index de Wright, hétérozygotie moyennes, variances de Whalund, preuve G de raison de probabilité, etc. Aussi le distance de Cavalli-Sforza. Comme conclusion, dans les races Andalusian Black et Grazalema Merino les variances de Whalund obtenues sont cosequences de l'action de la selection, donant different populations avec differentiation spaciale. Les hétérozygoties moyennes de chaque race sont parus, mais lorsque on considérent chaque systéme separé, les differences entre groupes ethniques sont importantes. Les indexes F de Wright demonstrent que, dans le races Andalusian White et Grazalema Merino on peuvent déduire d'heterozygoties génétique entre les populations ou troupeau analicées, dans le race Andalusian Black les differences valeurs de FIS indiquent que, malgré les petites dimensions des populations, on a evité la consanguinitée, due, probablement, á l'entrée d'étalons externes. Il n'y a pas, chez auqune race, d'un signifivative accroissement d'hétérizygosis. Les distances génétiques entre troupeau, dedans des races, en different pas des distances obtenues entre races. RESUMEN: Se ha trabajado con diferentes ganaderias de las razas ovinas andaluzas Merino de Grazalema y Churra Lebrijana, y con las caprinas Blanca Serrana y Negra Serrana, elegidas entre el resto de las razas de Andalucia como prioritarias pra la conservación, por estudio previo (Rodero y col., 1992a). Los sistemas utilizados en este trabajo corresponden tanto a caracteres etnozootécnicos como a diferentes variables de polimorfismos bioquimicos. Cuando se han diferenciado las ganaderias dentrde razas, o las ganaderias entre si, se han utilizado diferentes pruebas, a partir de las frecuencias genéticas y genotipicas: indices de Wright, heterocigosidades media, varianzas de Whalund, prueba G de razón de probabilidad, etc. También se obtuvieron las distancia genéticas de Cavalli-Sforza. Se concluye que en las razas Negra Serrana y Merino de Grazalema las varianzas de Whalund obtenidas son consecuencia de la acción de la selección que ha actuado dividiendo las razas en distintas poblaciones con diferenciación espacial. Las heterocigosidades medias de cada raza no difieren mucho entre si, pero cuando se considera cada sistema aisladamente, las discrepancias entre grupos étnicos son acusadas. Los indices F de Wright ponen de manifiesto que, mientras en las razas Blanca Serrana y Merino de Grazalema se pueden deducir heterocigosidades genéticas entre las problaciones o ganaderias estudiadas, no ocurre otro tanto en la raza Negra Serrana. Los valores de F(IS) parecen indicar que, a pesar del tama?o peque?o de las poblaciones, se ha evitado la consanguinidad, probablemente por la entrada de sementales externos. No se produce en ninguna de las razas un exceso significativo de heterocigosis. Las distancias genéticas entre ganaderias dentro de razas no difieren de lashalladas entre razas. ZUSAMMENFASSUNG: Es wurde mit verschiedenen andalusischen Zuchten der Schafrassen 'Grazalema Merino' and 'Lebrijan Churro' und der Ziegenrassen 'Andalusian White' und 'Andalusian Black' gearbeitet, die man von den andalusichen Rassen im Hinblick auf Erhaltung ausgew?hlt hat. Die benutzten Systeme in dieser Forschungsarbeit entsprechen Merkmalen, die sich sowohl auf ethnisch wie auch auf die verschiedenen Variablen des biochemischen Polimorfismius bezichen. Zur Unterschlidung von Zuchten innerhalb die Rassen oder von zuchten untereinander wurden verschiedene Tests benutzt, die von den genetischen und genotypischen Frequenzen ausgehen: Wright-Index, Durchschnittsheterozygosit?ten, Wahl und Varianz G-Test der Wahrscheinlichkeit, etc. Au?erdem wurden die genetischen Distanzen nach Cavalli-Sforza errechnet. Man Kommt zum Schlu?, da? in den Andalusian Black und Grazalema Merino die Wahl und Varianz das Ergebnis einer Selektionsaktivit?t ist, die die Rassen in Verschiedenen Populationen und unterschiedlichen R?umen aufgeteilt hat. Die durchschnittlichen Heterozygosit?ten jeder Rasse unterscheiden sich wenig voneinander, aber wenn man jedes System für sich betrachtet, stell man doch erhebliche Diskrepanzen zwischen den ethnischen Gruppen fest. Der Wright-Index offenbart, da? man in den Rassen Andalusian White und Grazalema Merino genetische Heterozygosit?ten ableiten kann zwischen den Populationen oder den untersuchten Zuchten; dies ist nicht der Fall inder Rasse Andalusian Black. Die F(IS) werte scheinen anzugeben, da trotz der kleinen Gr??e der Populationen die Blutsverwandschaft vermieden wurde, wahrscheinlich durch von au?erhalb kommenden B?cken. In keiner der Rassen existiert überm?ssige Heterozygotie Die genetischen Distanzen zwischer der Zuchten unterscheiden sich nicht van dener der Rassen.     

Cytopathic bovine viral diarrhea viruses (BVDV): emerging pestiviruses doomed to extinction

Bovine viral diarrhea virus (BVDV), a Flaviviridae pestivirus, is arguably one of the most widespread cattle pathogens worldwide. Each of its two genotypes has two biotypes, non-cytopathic (ncp) and cytopathic (cp). Only the ncp biotype of BVDV may establish persistent infection in the fetus when infecting a dam early in gestation, a time point which predates maturity of the adaptive immune system. Such fetuses may develop and be born healthy but remain infected for life. Due to this early initiation of fetal infection and to the expression of interferon antagonistic proteins, persistently infected (PI) animals remain immunotolerant to the infecting viral strain. Although only accounting for some 1% of all animals in regions where BVDV is endemic, PI animals ensure the viral persistence in the host population. These animals may, however, develop the fatal mucosal disease, which is characterized by widespread lesions in the gastrointestinal tract. Cp BVD virus, in addition to the persisting ncp biotype, can be isolated from such animals. The cp viruses are characterized by unrestrained genome replication, and their emergence from the persisting ncp ones is due to mutations that are unique in each virus analyzed. They include recombinations with host cell mRNA, gene translocations and duplications, and point mutations. Cytopathic BVD viruses fail to establish chains of infection and are unable to cause persistent infection. Hence, these viruses illustrate a case of “viral emergence to extinction” – irrelevant for BVDV evolution, but fatal for the PI host.     

牛、羊、人叠朊基因的克隆与分析

为了解我国哺乳动物叠朊基因Prnd的生物信息，本实验采用PCR方法克隆了秦川黄牛、甘肃土种绵羊和汉族人的Prnd，运用分子生物学软件对这些序列进行了分析比较。结果表明，获得的三种哺乳动物的Prnd均位于1个单一的外显子内，与GenBank登录的相应序列具有高度同源性。秦川黄牛与甘肃土种绵羊Prnd的同源性为96．8％，推导氨基酸序列同源性为93．3％。汉族人Prnd与秦川黄牛和甘肃土种绵羊Prod的同源性均为80％，推导的氨基酸序列同源性均为76．3％。氨基酸一级结构分析显示3种Prod编码的叠朊均由氨基端的信号肽、中间的成熟蛋白和羧基端的GPI锚结合区组成。二级结构预测表明，3种Prod编码的叠朊均由3个α-螺旋和2个β-片层组成。本研究获得的这3种主要哺乳动物的Prnd信息为一进步研究叠朊的结构与功能奠定了基础。     

A comparative study of bovine and ovine Haemophilus somnus isolates.

Bacterial isolates (including 17 Haemophilus somnus isolates and an H. somnus-like isolate) from asymptomatic or diseased cattle and sheep, were evaluated for markers associated with virulence and host predilection. The isolates were separated into 6 distinct biovariants, 3 for sheep and 3 for cattle, based on reactions in a battery of 21 test media. Three bovine isolates associated with disease caused hemolysis of bovine blood. The rest of the isolates did not hemolyze either bovine or ovine erythrocytes. Protein profiles of all H. somnus isolates were similar with the exception of the major outer membrane proteins (MOMPs). The MOMPs of isolates associated with disease in cattle had a relative molecular weight of approximately 41 kDa compared with 33 kDa for the MOMPs of isolates from asymptomatic cattle. The MOMPs from sheep isolates were either slightly higher or lower than the 41 kDa MOMPs of bovine isolates. Major antigens detected by Western blotting were similar in all isolates except the H. somnus-like isolate. An immunodominant 40 kDa antigen was conserved in all H. somnus isolates. Antibodies to this antigen have previously been found to be protective in cattle and may also be protective for sheep. Marked differences between cattle and sheep isolates were revealed by use of restriction enzyme analysis, which separated the isolates into 12 ribotypes and 15 unique DNA profiles. Thus, cattle and sheep isolates in this collection had distinctive differences in biochemical reactions, MOMP profiles, and DNA analyses. Such differences have potential value for epidemiological studies and may also be used to evaluate host specificity of H. somnus isolates.     

Differences in fluorescent antibody staining of bovine respiratory syncytial virus-infected cells by ovine and bovine sera

In indirect fluorescent antibody tests in which sera from cattle and sheep with respiratory disease problems were used to stain foetal bovine lung cells infected with a bovine respiratory syncytial virus strain, differences were noted in the pattern of fluorescence produced by some sheep sera and that produced by positive bovine sera. In serum neutralisation tests, also using a bovine respiratory syncytial virus strain, 4 of 7 sera giving this atypical pattern of fluorescence had very low neutralising antibody titres (highest 1/4), and 3 were negative. It is suggested that two related but antigenically distinguishable respiratory syncytial virus types are present in sheep, one of which is similar to bovine strains.     

Cross reactions between porcine, bovine and ovine interleukin-2 preparations

Optimum conditions for the production of porcine interleukin-2 were found to include a delay of 24 hours before the addition of mitogen. Porcine and bovine interleukin-2 responded optimally in homologous systems whereas bovine interleukin-2 gave a better response in the ovine system than homologous ovine interleukin-2. Interleukin-2 produced from a continuous gibbon cell line reacted well with porcine, ovine and bovine T cell blasts indicating that it could act as a universal growth factor for T cell clones produced from these species.     

Milk whey induction of agglutination in ovine and bovine mastitis Staphylococcus aureus

A total of 59 mastitis staphylococcic strains were tested for growth agglutination upon supplementation of growth media with ovine and bovine milk whey and mammary secretions from dry cows. Differences were observed when comparing bacterial species or origins (ovine vs. bovine) of bacteria and whey. All of the ovine and bovine S. aureus strains tested, but only 4 among 22 other ovine mastitis staphylococcic strains, showed growth agglutination in Todd Hewitt broth (THB) supplemented with greater than or equal to 30% (v/v) ovine milk whey. None of the strains agglutinated during growth in regular THB medium. Ovine whey had an agglutination induction capacity higher than bovine whey (P less than 0.005), concerning the number of responsive ovine and bovine S. aureus strains. There were no differences between whey samples from different ewes with regard to their capacity to induce agglutination. Ovine S. aureus strains were more responsive than bovine strains of this bacterial species, concerning the number of responsive strains (P less than 0.001) to bovine whey (greater than or equal to 30% in THB), the proportion of responsive strains at low (10%) ovine whey concentration (P less than 0.001), and the strength of reaction (precipitation timing and clump size). Secretions from dry cows systematically induced agglutination in all of the bovine and ovine S. aureus strains tested.     

Observations on the aetiology of bovine and ovine salmonellosis in New Zealand

Extract

Salmonella infections occurring in cattle and sheep have been identified in New Zealand since July, 1948, and March, 1949, respectively. Two hundred and twenty bovine outbreaks and 100 sheep outbreaks have been confirmed up to the present. In order to gain knowledge of the epidemiology of these diseases, the co-operation of field veterinary officers of the Animal Industry Division was obtained in order to gather histories of outbreaks, and so that detailed field and laboratory investigations could be made on a few selected outbreaks. It is proposed in this paper to record the histories of one bovine and one ovine outbreak which were selected for special study, and to deal also with the facts elicited in the course of general investigation.     

Molecular genetics of pestiviruses.

Recent developments in understanding the molecular genetics of pestiviruses are reviewed. The genomic RNA of several pestiviruses has been molecularly cloned and sequenced. The genetic organization of the viral protein coding region has been refined, and the complete complement of virus-encoded proteins has been elucidated. Viral gene expression involves both co-translational and post-translational precursor polyprotein processing. Biogenesis of the virion structural proteins requires the proteolytic activity of the first protein of the open reading frame (p20), as well as host cell enzymes. A second virus-encoded proteinase (p80) is required for processing viral nonstructural proteins. Molecular and biochemical data indicate pestiviruses share many features with both the flaviviruses and human hepatitis C viruses, while at the same time, also reveal their distinct nature.