Isolation of bovine adenovirus serotype 6 from a calf in the United Kingdom

Two viruses, designated 99-8130(C) and 99-8130(I), were isolated in calf testis cells from the colon and ileum, respectively, of a suckled beef calf which had developed dysentery and died. Electron microscopy indicated that the mean (sd) size of the viral particles, 83 (2.5) nm, and their morphology were consistent with their being members of the family Adenoviridae. They were confirmed as adenoviruses by PCR when products of the expected size (608 bp) were amplified from both isolates by using a primer pair specific for members of the genus Atadenovirus. A comparison of the sequence of a 567 bp segment of the 99-8130(C) amplicon with that of other prototype bovine adenovirus (BAdV) strains of atadenoviruses identified the isolate as BAdV serotype 6 (BAdV-6), which had 99.3 per cent and 100 per cent identities at the nucleotide and amino acid levels, respectively, with the prototype BAdV-6 strain 671130. A virus neutralisation test was developed and indicated a high prevalence of antibody to BAdV-6 in Northern Irish cattle. There was no evidence of adenoviral inclusions in tissues from the affected calf and no antigen was detected when the tissues were stained by an immunoperoxidase technique, using a homologous antiserum raised in rabbits. The two viruses were the third reported isolation of BAdV-6, and the first from a clinically ill bovine animal.     

Infectivity of bovine adenovirus type 5 recovered from a polyarthritic calf with weak calf syndrome.

Bovine adenovirus type 5, isolated from newborn calves with a polyarthritic disease known as "weak calf syndrome" caused a mild, self-limiting illness in susceptible calves. The induced illness was characterized by marked pyrexia and occasionally by mild diarrhea. It was concluded that the virus may contribute to morbidity and mortality associated with the weak calf syndrome by adding to the stress and debilitation caused by cold wet weather and by bovine viral diarrhea (BVD) virus, which has also been isolated from tissues of calves affected with weak calf syndrome.     

Immunofluorescent cell assay of neonatal calf diarrhea virus.

A reliable plaque assay procedure has not yet been described for the neonatal calf diarrhea virus. Therefore, a previously developed immunofluorescent cell counting procedure was adapted to assay this virus. Adsorption of the virus to bovine kidney cells plateaued at 60 minutes. The optimal staining time was between 20 and 24 hours postinfection. Infected cells begun releasing from the coverslips if the cultures were incubated longer than 24 hours. This procedure has proven successful with virus grown in cell culture as well as virus present in fecal samples.     

Effect of lymphocytes and broncho-alveolar washing cells on the replication of bovine herpesvirus type 1 in tracheal organ cultures

The daily addition of lymphocytes collected from a calf between 7 and 11 days after experimental infection with bovine herpesvirus type 1 (BHV-1) to bovine fetal tracheal organ cultures after infection with BHV-1 did not inhibit virus replication. The daily addition of normal lymphocytes, together with a low concentration of serum antibody against BHV-1, had a slight viral inhibitory effect which was believed to be due to antibody-dependent cell-mediated cytotoxicity. The addition of broncho-alveolar washing (BAW) cells, collected before infection or 30 days after infection of a calf with BHV-1, together with lymphocyte culture supernatant, to tracheal organ cultures immediately after infection with BHV-1 produced some inhibition of virus replication. Virus replication was markedly inhibited when BAW cells collected from the calf 18 days after infection were used in a similar manner.     

Sensitivity of seven different types of cell cultures to three serotypes of foot-and-mouth disease virus.

The ability of bovine tongue origin foot-and-mouth disease virus serotypes A, O and C to replicate in seven different types of cell cultures was studied. Primary and secondary calf thyroid cells were equivalent in susceptibility to bovine kidney cell cultures passaged up to five times. Calf thyroid cells lost their susceptibility after two passages. Cryopreserved bovine kidney cell cultures passaged three and four times were equivalent in susceptibility to sensitive calf thyroid and bovine kidney cells. Susceptibility to foot-and-mouth disease virus serotype C was most variable among the cells tested. Lamb testicle and porcine kidney cells were susceptible to foot-and-mouth disease virus while goat and calf testicle and calf lung cells were refractory.     

Hemagglutination by calf diarrhea coronavirus

The virus was grown in BEK-1 cells, a stable cell line from bovine embryo kidney, and tested for hemagglutination (HA) with erythrocytes of a variety of species at 4°C, room temperature and 37°C. HA was observed at all temperatures with chicken, mouse, rat, and hamster erythrocytes but not with erthyrocytes of human (O), cattle, horses, sheep, guinea pigs, geese, ducks, pigeons and 1-day-old chicks. Chickens showed an individual variation in agglutinability of their erythrocytes, requiring selection of birds to obtain erythrocytes for HA. HA reaction was inhibited by specific antiserum. Some factors involved in HA and HA inhibition (HI) were investigated and standard HA and HI tests were worked out.     

Bold fusion: transtracheal wash from a Jersey calf

A 10-week-old, female, Jersey calf was referred to the University of Minnesota Veterinary Medical Center for evaluation of difficult breathing and inappetence of 18-hours duration. Cytologic examination of a transtracheal wash specimen revealed pyogranulomatous inflammation, with increased numbers of multinucleated cells. Differential diagnoses included established bacterial infection, viral infection, fungal infection, and foreign body reaction. Immunocytochemical staining was done on a direct smear of the transtracheal wash using a cocktail of 4 mouse monoclonal antibodies against 3 human respiratory syncytial virus proteins. Strong intracytoplasmic staining within mononuclear cells was observed and a diagnosis of bovine respiratory syncytial virus infection was made. Immunocytochemistry may be used as a rapid, inexpensive, adjunctive diagnostic tool for the antemortem diagnosis of bovine respiratory syncytial virus on transtracheal wash specimens.     

A case of an embryo transfer calf infected with bovine leukemia virus from the recipient cow

A case was discovered where the embryo transfer (ET) calf had been infected with bovine leukemia virus (BLV) from the recipient cow. The embryo was transferred from the BLV-uninfected donor cow to the recipient cow. However, the BLV test had not been performed to the recipient cow before ET was performed. The ET calf was raised in a calf hatch from birth to 1-month old and was given the recipient cow's colostrum and milk artificially. The ET calf was raised with the two other calves from a 1-month old to a 6-month old. The BLV test was performed to the ET calf by agar gel precipitation (AGP) and passive haemagglutination (PHA) assay when the ET calf was 6 months old. Because the ET calf was positive, the BLV test was performed to the recipient cow, the two other calves raised with the ET calf and the two dams of the two other calves. Because the recipient cow only was positive at the time of the first test, we judged that the ET calf had been infected with BLV from the recipient cow. The importance of the BLV test being carried out on the recipient cow for the prevention of enzootic bovine leukemia in a case of ET was recognised.     

Prevalence of neutralizing antibody to the calf rotavirus in New York cattle.

In March 1973, a modified live virus vaccine was released for sale in the United States for the protection of neonatal calves from infection with calf rotavirus. At that time no published evidence existed that this agent was present in New York or the New England states. Serum neutralizing antibodies for the calf rotavirus (reovirus-like agent of neonatal calf diarrhea) were detected in serum from 108 of 110 dairy cattle in New York State representing 78 different herds. To exclude the possibility that the demonstrated serologic response may have been stimulated by vaccine virus, 36 samples were included that had been collected prior to the date of vaccine release. Neutralizing antibodies were demonstrated in 108 of the 110 sera ranging from titers of 4 to greater than 1024, thereby offering indirect evidence of the ubiquitous nature of calf rotavirus in New York.     

Diabetes mellitus in a 6-month-old Charolais heifer calf

This unthrifty heifer calf was thin, weak and had a dull haircoat. Urinalysis and blood work revealed glycosuria, ketonuria, hypoproteinemia, and hyperglycemia. Euthanasia and necropsy were performed, revealing multifocal intersitial lymphocytic infiltration, an absence of islet cells, and a positive stain for bovine viral diarrhea virus in the pancreas.     

Studies on the cytopathology of bovine adenoviruses in calf testis and calf kidney cell cultures

The sequential development of intranuclear inclusions in calf kidney and calf testis cells infected with nine bovine adenovirus (BAV) serotypes is described. Haematoxylin and Eosin (H & E), immunofluorescent and electron microscope (EM) studies indicated two distinct subgroups of viruses. Serotypes 1, 2, 3, 7 and 9 comprised subgroup-1, while types 4, 5, 6 and 8 comprised subgroup-2. Differences were noted in the early stages of infection. With subgroup-1 viruses, irregular patches of eosinophilic material were first to appear, followed by small refractile inclusions and basophilic inclusions. In EM studies, the eosinophilic material was thought to correspond to the irregular type II inclusions, and the refractile bodies to type I inclusions. Eventually a basophilic inclusion, consisting of aggregated virus-associated inclusions and virus particles, was formed in the centre of the nucleus.With subgroup-2, the refractile inclusions were more prominent, larger, and were the first to appear. These were thought to correspond to type I inclusions, which were larger and denser than with subgroup-1. Circular basophilic bodies developing later were similar in size and distribution to type II inclusions, which with subgroup-2 viruses were seen in prominent circular or lobulated aggregates.Several other types of inclusion including tubular structures and paracrystals, which have thus far not been reported in BAV infected cells, are described.     

Diagnosis of viral agents associated with neonatal calf diarrhea.

During this study, 134 samples have been examined for the detection of the viruses associated with neonatal calf diarrhea. The presence of Nebraska viruses (rotavirus and coronavirus) has been demonstrated by using the electron microscope and the fluorescent antibody techniques while the presence of other viruses has been detected by the observation of a cytopathic effect on monolayer cells of calf testis. The Nebraska viruses have been demonstrated in 107 (80%) out of 134 field case specimens. An association of rotaviruses and coronaviruses was found in 58 cases (54%) whilst the coronaviruses and the rotavirus were found singly in 34 cases (53%) and in 15 cases (14%) respectively. Four bovine virus diarrhea viruses, two infectious bovine rhinotracheitis viruses and two enteroviruses have also been isolated in the preceding 107 Nebraska positive specimens. For the detection of the Nebraska viruses, the fluorescent antibody techniques were more sensitive than the electron microscopy. However, those two techniques must be used simultaneously for a better detection of a greatest possible number of cases.     

Effect of viral dose on experimental pneumonia caused by aerosol exposure of calves to bovine herpesvirus 1 and Pasteurella haemolytica.

The effect of various aerosol doses of bovine herpesvirus 1, followed four days later by aerosol exposure to a constant level of Pasteurella haemolytica, was studied in 16 crossbred Hereford range calves. A Collision nebulizer was used to generate aerosols from virus suspensions with concentrations of 10(8.2) (high), 10(5.2) (moderate) or 10(2.2) (low) TCID50/mL. The bacterial suspension contained 10(7) colony forming units/mL. Control calves exposed only to P. haemolytica developed no pulmonary lesions. Calves in the low, moderate and high virus exposure groups developed lobular areas of atelectasis; in addition, one calf in the moderate and all four in the high virus exposure group developed fibrinous pneumonia. One of the latter calves died. The 50% effective dose for fibrinous pneumonia under these experimental conditions was 10(6.0) TCID50 bovine herpesvirus 1/mL of suspension in the nebulizer reservoir, and approximately 10(4.0) infectious units inhaled per calf.     

A microbiological study of pneumonic calf lungs.

BITSCH, V., N. F. FRIIS and H. V. KROGH: A microbiological study of pneumonic calf lungs. Acta vet. scand. 1976, 17, 32–42.–Fifty pneumonic calf lungs were subjected to microbiologic screening with regard to bacteria, mycoplasmas, and viruses.Of bacteria the species most commonly found were Pasteurella multocida (eight lungs), Pasteurella hemolytica (eight lungs), and Corynebacterium pyogenes (13 lungs). Of special interest was the demonstration of Neisseria spp. in five lungs. Mycoplasma dispar was found in 31 lungs, Mycoplasma bovirhinis in 16 lungs, and Urea-plasma in 26 lungs. Cytopathogenic agents were demonstrated in 14 lungs. Four isolates were found to be bovine respiratory syncytial virus, three were bovine viral diarrhea virus, and two were bovine parainfluenza 3 virus. The remaining five cytopathogenic agents were not identified.     

Studies with an unclassified virus isolated from diarrheic calves

A transmissible agent (Breda agent) was isolated from a calf with diarrhea and shown to be infectious by inoculation orally into gnotobiotic and conventionally reared calves. The “Breda” agent had the morphology of a virus and possessed a hemagglutinin. Antigenic studies showed the virus to be antigenically different from bovine coronavirus, parainfluenza 3 virus, bovine rotavirus, bovine parvovirus and bovine pestivirus (BVD). Attempts to culture the virus in cell or organ cultures or in embryonated eggs, were unsuccessful. The virus was either spherical or kidney shaped, with 7–9 nm peplomers on the surface. A few particles possessed coronavirus processes of 17–20 nm, but these were arranged irregularly and were thought to be tissue debris. Three out of eight experimental calves developed severe diarrhea and the lesions in the small and large intestines were similar to those reported for coronavirus. The virus replicated in the jejunal and ileal regions of the small intestine and in the spiral colon, as judged by immunofluorescence. The virus multiplied in all experimental calves and was excreted in the feces; excretion correlating with the onset of diarrhea or a change in the appearance of the feces. There was little or no malabsorption measured by the uptake of D-xylose and the fact that infection of both the crypt and villus epithelial cells was observed, suggests that the pathogenesis may be different from rotavirus and coronavirus. Fourteen of fortyseven calves in the outbreak were infected with the virus, virus was not identified in other farm outbreaks of the disease.     

Bovine viral diarrhea in a newborn calf

Bovine viral diarrhea virus was believed to be the cause of ill-thrift since birth, resulting in death of a Holstein calf. Bovine viral diarrhea virus was isolated from Peyer's patches and mesenteric lymph nodes, but serum neutralizing antibodies were not detected. The lymphoid depletion and myeloid suppression seen in this case may be a factor in the immune system dysfunction described for bovine viral diarrhea. Typical ulcerative lesions within the alimentary tract were not observed.     

Separation of a Soluble Antigen and Infectious Particles of Bovine Viral Diarrhea Viruses and their Relationship to Hog Cholera

A soluble antigen present in infectious tissue culture fluids was separated from the infective virus particle by ultracentrifugation of two serologically related strains of bovine viral diarrhea viruses, NADL-MD and Oregon C24V.

Neutralizing antibodies against the two viruses were absent in four hog cholera antisera, but present in significant titer in the commercially prepared antiserum. Precipitin tests utilizing the agar double diffusion technique formed a single line of identity between the concentrated soluble antigen of both viruses and NADL-MD and hog cholera antisera. No lines were observed using concentrated virus pellet and noninfected BEK cell antigens or control SPF calf and swine sera.

     

The influence of normal guinea-pig serum and tissue culture assay system on foot-and-mouth disease virus neutralisation

The inclusion of normal guinea-pig serum in neutralisation reactions involving foot-and-mouth disease virus (FMDV) increased the neutralisation titre and rate of neutralisation by guinea-pig antiserum derived from animals convalescent from FMDV. Such inclusion had little or no effect on neutralisation involving guinea-pig antiserum collected early in infection or early or convalescent bovine antisera. Higher neutralisation titres and more rapid neutralisation were found from assay in bovine thyroid cells than in IB-RS-2 cells. Not all the increase in neutralising activity was removed by heating at 56 degrees C, indicating that other factors in addition to complement were responsible. In some tests a virus fraction resisting neutralisation was found and, when rabbit anti-guinea-pig gamma-globulin was added, an increase in neutralisation resulted, suggesting that most of the persistent virus fraction was due to the presence of infective virus-antibody complexes.     

Negative contrast electron microscopic diagnosis of viruses of neonatal calf diarrhea.

Ninety-one cases of neonatal calf diarrhea were examined for viruses with negative contrast electron microscopy. Viruses were demonstrated in 41% of the cases. Reo-like viruses and corona-like viruses, and mixed virus populations were observed in 12%, 20% and 9%, respectively. Twenty-six of the cases were examined by negative contrast electron microscopy, and by virus isolation or by fluorescent antibody technique. There was an 81% agreement in obtained results. The disagreements resulted from the demonstration of a viral agent by negative contrast electron microscopy while the other techniques did not indicate a virus. The results suggest that negative contrast electron microscopy is a more sensitive diagnostic tool for demonstration of viruses associated with neonatal calf diarrhea than are viral isolation or the fluorescent antibody technique.