Isolation of bovine herpesvirus-3 from African buffaloes (Syncerus caffer)

Eleven virus isolations were made from the blood of 45 free living healthy African buffaloes by long term cocultivation of their leucocytes with bovine thymus or spleen cells. The isolates were indistinguishable from each other or from herpesviruses isolated from a severely ill buffalo calf and from a dead buffalo. These viruses possessed the characteristics of the bovine herpesvirus-3 (BHV-3) group and were indistinguishable by serology and restriction endonuclease analysis from the BHV-3 type strains Movar 33/63 and DN599. There was a 93.6 per cent prevalence of indirect immunofluorescent antibody to BHV-3 in the sera of 94 buffaloes in the sample population. No clinical signs or viraemia were detected in five cattle inoculated with 10(8.7) log10 TCID50 of the isolate from the sick buffalo calf. Two of three cattle hyperimmunised with this virus resisted challenge with malignant catarrhal fever herpesvirus, which proved fatal for the other immunised animal and for three control cattle.     

Epizootic diarrhoea of adult cattle associated with a coronavirus-like agent

An epizootic of acute diarrhoea of adult cattle occured in Japan during the winter of 1976 to 1977. A majority of adult cattle clinically diagnosed as having the disease, showed a significant rises in antibody titres to bovine coronavirus, whereas only a smal; minority showed serological evidence of recent infection with calf rotavirus, bovine adenovirus type 7, parainfluenza virus type 3, or bovine viral diarrhoea-mucosal disease virus. A coronavirus-like agent was detected by electron-microscopy in faecal material from a cow with diarrhoea, and was subsequently isolated in primary bovine kidney cell cultures.     

Protection studies on winter dysentery caused by bovine coronavirus in cattle using antigens prepared from infected cell lysates.

Cells infected with bovine coronavirus (BCV) were solubilized with Triton X-100 to yield a cell lysate (CL) antigen having high hemagglutinating (HA) titers. The antigen gave high HA titers using rat erythrocytes, suggesting that it contained large amounts of hemagglutinin esterase (HE) antigen. The CL antigen, combined with an oil adjuvant, was tested for protective and antibody-inducing activities in cattle. Four groups (2 cattle/group) of cattle were inoculated with CL antigen having HA titers of 16 000, 4000, 1000, and 250. Another group served as untreated controls. Two intramuscular inoculations were given at an interval of 3 wk. The animals were challenged with virus 1 wk after the second inoculation. The groups immunized with the CL antigen having an HA titer of 4000 or 16 000 produced hemagglutination inhibition (HI) antibody titers of > 320 and serum neutralizing (SN) antibody titers of > 1280. These groups of animals showed no clinical abnormalities after challenge. In the groups immunized with CL antigen at an HA titer of 1000 or 250, HI antibody titers were 40 to 160 and SN titers were 80 to 640. The cattle with HI antibody titers of > or = 160 and the SN titers of > or = 640 showed no clinical signs, but the cattle with the HI antibody titer < 80 and the SN antibody titer < 160 developed watery diarrhea and fever after challenge. These results indicate that CL antigen with high HA titer induces antibody production in cattle that provides effective protection against winter dysentery.     

Cell Culture Studies of a Neonatal Calf Diarrhea Virus

The effects of a neonatal calf diarrhea virus on cell cultures were investigated. Bovine embryonic kidney cell cultures were the most satisfactory for production of virus. Cytoplasmic changes detected after inoculation with a high multiplicity of virus were: 1) cytoplasmic vacuoles; 2) some eosinophilic cytoplasmic inclusions; and 3) some degeneration of cells and detachment from the monolayer. Cultures stained with fluorescein-labeled antibody showed cytoplasmic fluorescence as early as four hr after infection with the maximum fluorescence at five days. No cross reactions were observed between the neonatal calf diarrhea virus and reovirus type 1 or type 3 by the fluorescent antibody technique. Plaques were small and were not produced consistently. The optimal adsorption time was one to two hr. The maximum titer was reached at 18 hr, with the cell-associated titer remaining higher than the cell-free titer until that time. An interferon was produced by cultures infected with either ultraviolet-inactivated or untreated virus.     

牛传染性鼻气管炎病毒的分离鉴定与灭活疫苗免疫效果研究

由2例疑似牛传染性鼻气管炎（IBR）病例的荷斯坦奶牛分离到一株病毒,命名为IBRV—C1株。该病毒可被IBR标准阳性血清完全中和;接种MDBK细胞可出现IBR病毒典型细胞病变效应;选取IBR病毒gB蛋白基因序列设计引物进行PCR检测和基因测序,结果可扩增出特异性目的片段;动物回归试验显示,3头牛均可见体温升高、鼻流粘液、呼吸困难等典型的IBR临床症状。在此基础上制备了三批牛传染性鼻气管炎灭活疫苗,并进行了疫苗安全性和效力试验,结果表明三批疫苗对靶动物安全,免疫效果较好,免疫牛中和抗体效价几何平均值可达1：41以上,攻毒保护率达5／5。     

Evaluation of techniques to demonstrate foot-and-mouth disease virus in bovine tongue epithelium: comparison of the sensitivity of cattle, mice, primary cell cultures, cryopreserved cell cultures and established cell lines

Tongue epithelia infected with each of the 7 serotypes of foot-and-mouth disease virus (FMDV) were used to evaluate in vivo and in vitro systems for the detection of FMDV. Cattle inoculated by the intradermal route in the tongue (IDL) and suckling mice inoculated intraperitoneally were compared for susceptibility to FMDV with freshly prepared bovine thyroid cell cultures; cultures from cryopreserved bovine thyroid, bone marrow, mammary gland, myocardium, tongue, ovary and kidney cells; cultures from cryopreserved embryonic ovine kidney, newborn ovine kidney, ovine testicle, bone marrow, and chloroid plexus cells; and the continuous porcine kidney cell lines MVPK-1 and S6. The mean titers determined for each serotype in each system were statistically compared. The FMDV titers obtained in freshly prepared bovine thyroid cell cultures and by cattle IDL inoculation were the highest and were statistically indistinguishable. The titers obtained by suckling mouse inoculation were significantly lower than the titers obtained in thyroid cultures for serotypes A, C, Asia 1, and SAT 3. The cattle IDL assay was significantly more sensitive than the mouse assay for serotype A. The cell cultures from the cryopreserved newborn ovine kidney and embryonic ovine kidney were significantly less susceptible to serotype Asia 1 when compared with the fresh bovine thyroid cultures, but not significantly different when compared with the cattle assay for all serotypes. Cryopreservation of bovine thyroid cells directly after trypsinization resulted in the loss of susceptibility to FMDV serotype SAT 2. The other cryopreserved cell culture systems exhibited no or minimal susceptibility to all 7 serotypes, or exhibited considerable inconsistency. The established cell lines MVPK-1 and S6 were not susceptible to serotype A, and were less sensitive to serotype C than other culture systems. Quality control of cell cultures used to evaluate field specimens for FMDV was critical. The cell cultures of cryopreserved ovine kidney cells provided the most practical diagnostic system.     

Experimental inoculation of cattle with bovine herpesvirus-4: evidence for a lymphoid-associated persistent infection

A strain of bovine herpesvirus-4 isolated from cows with mammary pustular dermatitis was used for experimental inoculation of cattle. This strain is serologically indistinguishable from the group prototype Movar 33/63 and from strain DN-599. Seronegative cattle were inoculated IV or by simultaneous intranasal, IV, intramammary (via teat channel), and intradermal inoculations. All inoculated cattle seroconverted. Clinical signs of disease or lesions were not evident, except for a dermal lesion corresponding with one intradermal inoculation site. Virus was recovered from the dermal lesion and was excreted in the milk for 17 days. Virus was recovered from esophagopharyngeal fluid at 9 and 13 days after inoculation. At different times of euthanasia (2 to 14 months after inoculation), virus was recovered from cocultures with bovine lung cells and/or explant cultures of lymph nodes, spleen, tonsils, and, in one case, kidney. In 2 animals, the virus was recovered repeatedly during 1 year from peripheral blood leukocytes by cocultivation with bovine lung cells. The number of infectious leukocytes, as determined by infectious center assay, ranged from less than 1 to 6 infectious cells/10(7) leukocytes.     

A Study of Bovine Virus Diarrhea Mucosal Disease Virus by Plaque Technique

Bovine virus diarrhea-mucosal disease (BVD-MD), NADL, strain formed 3-4 mm plaques on monolayers of bovine embryo kidney (BEK), lung and testicular (BET) cell cultures on post inoculation day four. Plaques were 1.5 mm on the post inoculation day five in lamb testicular cell cultures. Neutral red incorporated in first overlay had inhibitory effect on plaque formation in these cell-virus systems. The study of effects of environmental variables on plaquing efficiency indicated that virus adsorption rate was temperature dependent and approximately 80% virus was adsorbed onto BET monolayers in two hours. Rate of adsorption was slightly superior in BEK monolayers than the ones recorded in BET cell cultures. Virus diluent should contain calcium and magnesium ions for maximum plaquing efficiency. Cultures maintained under lamb serum should be washed for the development of maximum number of plaques. Virus particles could diffuse through agar overlay to initiate infection and form delayed plaques. Size of the plaques was proportional to the concentration of agar in overlay medium. Plaquing efficiency was also dependent upon pH of the overlay and optimum pH for maximum efficiency was 7.3 - 7.7.

NADL strain of BVD-MD virus was sensitive to trypsin but resistant to 5'-Bromodeoxyuridine. Thermostability studies showed that 0.5% virus survived when incubated at 37°C for 48 hours. The virus was sensitive to freezing and thawing.

Comparative titers of virus determined and expressed as PFU and TCID₅₀ were almost similar.

     

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.     

一株MDBK细胞的低血清悬浮驯化研究

研究选取一株背景清晰、纯净的MDBK贴壁细胞,通过直接驯化和逐级降低血清相结合的办法,获得了一株可以在低血清培养基中全悬浮培养的MDBK细胞。该细胞在含0.5%胎牛血清的培养基中悬浮培养48 h后的细胞密度稳定在5.0×10⁶/mL以上,培养72 h后细胞密度最高可达1.16×10⁷/mL,且细胞形态良好,活力在93%以上,具有良好的传代稳定性。应用筛选获得的悬浮培养MDBK细胞株分别接种伪狂犬病毒和传染性牛鼻气管炎病毒后,检测病毒敏感性。细胞在24 h均发生了皱缩,72 h大部分死亡。悬浮培养的MDBK细胞对传染性牛鼻气管炎病毒的滴度在24 h达到最大值107.6TCID50/mL,对伪狂犬病毒的滴度在48 h达到最大值107.6 TCID50/mL,说明虽然细胞的培养方式发生了改变,但并没有影响上述两种病毒在该细胞上的增殖特性。对MDBK细胞的全悬浮驯化研究可为相关病毒类疫苗规模化生产及工艺的升级改进提供细胞学基础资料。     

Comparisons between two isolates of stomatitis papulosa virus

The cytopathology and length of latency in single-step growth curves of two isolates of stomatitis papulosa virus are compared in this report. Isolate 721 was obtained from a calf with oral ulcers and isolate 8665 was obtained from a calf with respiratory disease and oral ulcers. In single-step growth curves, the latency period of isolate 721 was 8 h while that of isolate 8665 was 6 h. The cytopathic effect produced by isolate 721 in bovine lung cells was characterized by enlargement of the cell, cell-to-cell adherence and large intracytoplasmic accumulations of viral inclusion material. Isolate 8665 caused rapid cell degeneration and detachment, with small accumulation of viral inclusion material. Neither of the two strains grew in bovine alveolar macrophage cultures or in the respiratory epithelium of fetal bovine tracheal explants. Intragingival inoculation of these isolates in cattle resulted in oral lesions without clinical signs of respiratory of systemic involvement. Virus was recovered from the oral lesions and from nasal secretions for as long as 10 days. Inoculation of dexamethasone-treated cattle resulted in a similar clinical condition although virus was recovered for 20 days from oral lesions and nasal secretions. Seroconversions from negative to 1 : 2560 were detected in inoculated cattle by indirect immunofluorescence.     

Duration of protection of calves against rhinovirus challenge exposure by infectious bovine rhinotracheitis virus-induced interferon in nasal secretions

Six calves inoculated intranasally with a vaccinal strain of infectious bovine rhinotracheitis (IBR) virus and 6 control calves were given a placebo. All calves were subsequently challenge exposed (by aerosol) with rhinovirus--3 of the calves from each group at 2 days after they were inoculated with IBR virus or with placebo and the remaining calves at 6 days. Nasal excretion of viruses, interferon (IFN) concentrations in nasal secretions (NS), and neutralizing antibody in sera and NS were determined. All calves given the vaccinal IBR virus subsequently had IFN in their NS. Interferon was detected as early as 1 day, reached maximal titers at 2 to 4 days, and persisted in individual calves for 5 to 10 days after inoculation. Rhinovirus shedding was not detected from IBR virus-inoculated calves whose NS contained both rhinovirus antibody and IFN at the time of challenge exposure; such calves were protected at either 2 or 6 days after IBR virus inoculation. The outcome of rhinovirus challenge exposure of calves whose NS contained IFN, but not rhinovirus antibody, varied with the day of challenge exposure. Rhinovirus excretion was detected from 2 of these calves challenge exposed 2 days after IBR virus inoculation, but was not detected from a calf challenge exposed 6 days after inoculation. However, while IFN was present in NS from the former 2 calves, rhinovirus shedding was markedly reduced as compared with that from control calves without IFN or NS antibody at the time of challenge exposure. Consistent relationship was not observed between the rhinovirus neutralizing antibody titer of calves' sera and NS. The antibody titer of NS more closely correlated with protective immunity to rhinovirus infection than did the serum antibody titer.     

ISOLATION AND CHARACTERISATION OF BOVINE HERPESVIRUS MAMMILLITIS VIRUS AND ITS PATHOGENICITY FOR CATTLE

Ten viruses isolated from swabs and vesicular fluid collected from the teats of dairy cattle on 4 properties in Northern Victoria were identified as bovine herpes mammillitis (BHM) viruses by their physico-chemical and morphological properties and serological relationship to each other and a Scottish Strain of BHM virus. The viruses, isolated in bovine kidney and testicular cell cultures, produced cytopathic effects characterised by very large syncytia and eosinophilic intranuclear inculsion bodies. The intradermal inoculation of BHM virus into two cattle produced necrosis and ulceration of the skin of the teats about the area of inoculation and the development of serum neutralising antibody. After healing of the ulcers on day 37 after inoculation, the cattle were intravenously inoculated with corticosteroid for 6 days but BHM virus was not re-isolated from the teat skin or vaginal or nasal swabs.     

Properties of non-neurovirulent plaque-forming mutants of Newcaslte disease virus.

The mesogenic Hertfordshire (H) strain, a vaccine strain of Newcastle disease virus, represents a heterogeneous virus population differing in physical, chemical, and biological properties. Small (S) and large (L) plaque-forming mutants were isolted from strain H in chick embryo fibroblast cell cultures. Part of the S mutants lacked neurovirulence, with an intracerebral pathogenicity index for day-old chicks of 0 to 0.46 vs 1.0 to 1.4 for L mutants. S mutants grew only to a very low titer in the brain of day-old chicks and cleared from the brain by the sixth day, whereas L mutants killed the chicks after a 1000-fold titer rise. The S mutants caused a complete cell destruction in fibroblast culture by 12 hours, but they had 10--20 times lower virus yields than the L mutants. The infective titers of S and L mutants grown in allantoic cells were identical, but the infectivity to hemagglutinin ratio was 10 to 50 times lower for S than for L. The thermostability of infectivity and hemagglutinin varied with the different mutants. As for the immunogenicity of the mutants, the minimum dose of S mutants inducing full protection by subcutaneous inoculation was 10(6) plaque-forming units per chicken, thus being about 100 times less immunogenic than either the L mutants or the parent strain H.     

A CELL CULTURE VACCINE AGAINST BOVINE EPHEMERAL FEVER

SUMMARY A vaccine was prepared from cell culture fluids harvested from the twelfth passage of the 919 strain of bovine ephemeral fever (BEF) virus in Vero cell cultures. Cattle were vaccinated subcutaneously with various combinations of strain 919 virus and adjuvants. Neutralising antibodies were assayed at various times after vaccination and some cattle were challenged by intravenous inoculation with the virulent 417WBC strain of BEF virus. Strain 919 virus of the third and twelfth passage levels in Vero cells produced neither fever, clinical illness nor detectable viraemia in 5 calves inoculated intravenously. Nor could viraemia be detected in 5 heifers receiving vaccine subcutaneously. When the vaccine was administered mixed with aluminium hydroxide adjuvant, the production of neutralising antibodies increased with an increase in the volume of vaccine from 2.5 ml to 10 ml and the response to 2 injections was significantly better than the response to a single injection. The neutralising antibody response was decreased when vaccine was diluted in phosphate buffered saline. The neutralising antibody response following 2 subcutaneous vaccinations with strain 919 virus mixed with aluminium hydroxide adjuvant was higher than that following intravenous inoculation with virulent virus. The vaccine-induced antibodies persisted for at least 12 months, and revaccination at this time led to an increase in the titre of neutralising antibody. Antibodies induced by a single subcutaneous administration of strain 919 virus mixed with Freund's complete adjuvant persisted for at least 40 weeks; those induced by vaccine containing Freund's incomplete adjuvant had virtually disappeared within 16 weeks. All these calves responded to vaccination with aluminium hydroxide-containing vaccine with increases in levels of neutralising antibodies. Of 26 vaccinated calves challenged with virulent BEF virus, 24 remained clinically normal. Two developed brief periods of pyrexia on the seventh day after challenge, but no other clinical signs. One of these calves had a viraemia that was demonstrated only by intravenous inoculation of a susceptible calf. The remaining calf had no detectable viraemia. All of 7 unvaccinated calves developed severe clinical BEF within 5 days of challenge. No disease attributable to the 919 virus occurred in 24 vaccinated pregnant heifers or their newborn calves.     

A short incubation serum neutralization test for bovine viral diarrhea virus.

A three day serum neutralization (SN) test for the detection of antibodies to bovine viral diarrhea virus (BVDV), which is an improvement on the existing five day test, is described. The improved test results in a more rapid viral cytopathic effect and utilizes Madin Darby kidney (MDBK) cells, and horse serum as a medium supplement. A comparison of tests utilizing the NADL and the Singer strains of BVDV and the use of either secondary bovine kidney cells with calf serum (BKCS) or continuous MDBK cells with horse serum (MDHS) was performed. Analysis of the SN results of 685 serum samples from 445 Quebec and Ontario cattle showed that there was no difference, as expected, in the means of the SN antibody titers when the NADL strain was used in either the BKCS or MDHS system but SN antibody titers were elevated (p less than 0.01) when the Singer strain was used in the MDHS system. The SN test with the Singer strain also yielded significantly higher titers for sera from 200 Alberta cattle.     

Comparative study on responses of cattle and water buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) to experimental inoculation of <Emphasis Type="Italic">Brucella abortus</Emphasis> biovar 1 by the intraconjunctival route—a preliminary report

The preliminary study was conducted to assess the virulence of a strain of Brucella abortus (1969D) and to compare the susceptibility of water buffalo and cattle calves to infection by the intraconjunctival route. Seven of each cattle and water buffalo (Bubalus bubalis) calves aged 3–6 months were inoculated intraconjunctivally with counts ranging from 1.5 × 10⁷ to 1.7 × 10¹⁰ colony forming units of B. abortus. Animals were monitored over an 8-week period for clinical manifestations and serological and hematological evidence of infection. At slaughter, eight lymph nodes from each animal were sampled for bacteriological and histopathological assessments. Lymph nodes from three water buffalo (43%) and five cattle (71%) yielded B. abortus (P = 0.048). Parotid/prescapular lymph nodes were most sensitive in detecting B. abortus. Our data suggest that B. abortus strain 1969D may be used as challenge strain, and water buffalo appeared to have a lower susceptibility to B. abortus infection than cattle.     

An investigation into the basic virus-antibody neutralization reaction, with special regard to the reaction in the constant-virus/varying-serum neutralization test.

A study of the basic reaction in neutralization of virus (V) by virus-neutralizing antibody (VNA) was performed with infectious bovine rhinotracheitis virus and serum collected from naturally and experimentally infected cattle after the primary immunization phase. In constant-virus/varying-serum neutralization tests a direct proportionality between VNA titer and length of preincubation was observed and found to be in accordance with basic laws of neutralization. A deviation from this direct proportionality, which was partly attributed to the presence of a dissociable V-VNA complex, was seen with relatively short preincubation. Expressing a relationship between VNA titer, length of preincubation, and virus dose under conditions where a dissociable V-VNA complex can be ignored, a log. VNA/log. V equivalence factor of neutralization was introduced. A linear relationship was found between VNA titer, taken logarithmically, and preincubation temperature. A rise in temperature by 10°C gave an increase in VNA titer of approx. 1.2 in log₂. Formulae are presented for the neutralization rate factor corrected for a demonstrated invalidity of the percentage law, and for the relation between the neutralization rate factor and VNA titer. It is concluded that the results presented have elucidated the possibilities of improving the sensitivity of neutralization tests.     

Foot-and-mouth disease and the African buffalo (Syncerus caffer). II. Virus excretion and transmission during acute infection

Three groups of young buffalo in captivity were infected by exposing them to similar buffalo in the acute stages of infection induced by needle inoculation with SAT 1 or 2 viruses. Clear foot lesions developed in most of the buffalo from which the relevant virus types were re-isolated. During the first week following infection virus was found in blood, nasal secretions, saliva, preputial secretions and faeces. Air samples collected in the immediate vicinity of acutely infected buffalo were also found to contain virus. However, the regularity of virus detection as well as the quantity of virus in buffalo specimens was generally lower than for cattle infected with viruses of the same type. Conversely, virus was detected in the nasal secretions or saliva of 3 buffalo up to 4 weeks after infection, a situation which has not been encountered in cattle. Susceptible cattle and impala (Aepyceros melampus) were penned together with or in the immediate vicinity of infected buffalo and shared feeding and watering facilities with the buffalo. The pattern of transmission which emerged indicated that transfer of these viruses from buffalo to other species probably occurs only in the acute stages of infection and where there is direct physical contact between the species.     

The persistence of bovine enterovirus and pseudorabies virus in liquid cattle manure at different storage temperatures

The tenacity of viruses in liquid manure of cattle was examined in a total of five samples inoculated with ECBO-virus (strain LCR-4) representing viruses without envelope and Aujeszky virus (field isolate) representing enveloped viruses. The titers were examined at regular intervals over a period of 26 weeks. On the day of inoculation each sample had a titer of 10(5) ID50/ml. After 16 weeks complete inactivation was observed in the Aujeszky virus sample stored at 20 degrees C. The Aujeszky virus sample wich was kept at 4 degrees C at 26 weeks had a titer of 10(1,75) ID50/ml. In the samples inoculated with ECBO virus after 26 weeks of inoculation a titer of 10(3) ID50/ml was found in the manure stored at 20 degrees C. No influence on the virus titers in the liquid manure samples was observed either from pH or the number of bacteria (3,4 x 10(7)-1.16 x 10(8)/ml during the examination period.