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.     

Rapid detection of bovine herpesvirus 1 in the semen of infected bulls by a nested polymerase chain reaction assay.

A nested polymerase chain reaction (PCR) assay was developed for the detection of bovine herpesvirus 1 (BHV-1) in bovine semen and compared with the virus isolation method. When extended semen, commonly used in the bovine artificial insemination industry, was inoculated with BHV-1, the PCR assay detected BHV-1 DNA in semen inoculated at 0.25-2.5 TCID50 per 0.5 mL. In contrast, the lower limit of detection for virus isolation was 250 TCID50 of BHV-1 inoculated in 0.5 mL of extended semen. These methods were also used to detect BHV-1 in the semen of four bulls which were experimentally infected with BHV-1. All infected bulls demonstrated balanitis at 3 d post-inoculation (DPI) and severe balanoposthitis at 4 DPI. BHV-1 was detected in raw semen by virus isolation and PCR at 2 DPI, before balanitis was evident. For virus isolation, the last day that BHV-1 was detected during primary infection was 7 DPI for two bulls and 9 and 11 DPI for the other two bulls. In contrast, PCR detected BHV-1 in the bulls' semen until 14 or 18 DPI. For individual animals, PCR detected BHV-1 during primary infection for at least 1-10 d longer than virus isolation. Reactivation of BHV-1 from latency without the presence of visible lesions was promoted twice by two series of 5 d dexamethasone injections. For the first series of dexamethasone treatments, a positive virus isolation result was obtained on the 5th d of treatment for only one bull. In contrast, two bulls demonstrated evidence of viral reactivation on this day by PCR. All bulls shed BHV-1 in semen on d 4 after dexamethasone treatment, as evidenced by positive virus isolation and PCR results. One bull was still PCR positive 13 d later. For the second series of dexamethasone treatments, a small amount of virus was isolated from semen collected on d 3 or 4 after treatment for two bulls but not from the other two bulls. In contrast, semen samples from all bulls were PCR positive for either or both of these 2 d. In total, from 80 semen samples, 45 were PCR positive and 26 were virus isolation positive. Thus, the PCR assay detected BHV-1 shedding in bulls earlier, more often, and for a longer duration, than did the virus isolation method.     

Specificity of the indirect enzyme-linked immunosorbent assay for detection of pseudorabies virus antibodies in pigs exposed to bovine herpesvirus-1

Six 5-week-old pigs were inoculated intranasally (IN) with 10(7.6) TCID50 of bovine herpesvirus-1 (BHV-1). Three of the pigs also were inoculated IV with a similar dose of BHV-1. Clinical responses were not observed in these 6 pigs before oronasal challenge exposure with 10(7.8) TCID50 of virulent pseudorabies virus (PRV) at postinoculation day 42. Two pigs inoculated IN with BHV-1 and challenge exposed with PRV remained healthy, whereas the remaining 4 pigs developed severe clinical signs of pseudorabies and were moribund at postinoculation day 50 (8 days after challenge exposure). Anti-BHV-1 antibodies were demonstrable by ELISA in all 6 pigs and by serum neutralization (SN) in 5 pigs before challenge exposure with PRV. Anti-PRV antibody was not detected by ELISA or SN before challenge exposure to PRV. After challenge exposure to PRV, pigs with humoral antibody to BHV-1 responded anamnestically, and anti-PRV antibody activity was demonstrable by ELISA and SN in the 2 surviving pigs.     

Molecular diagnosis of alcelaphine herpesvirus (malignant catarrhal fever) infections by nested amplification of viral DNA in bovine blood buffy coat specimens

A fragment of alcelaphine herpesvirus-1 (AHV-1; malignant catarrhal fever) DNA was subcloned into pUC 18 and sequenced. The subclone hybridized strongly to AHV-1 DNA, weakly to alcelaphine herpesvirus-2 (AHV-2) DNA, and not at all to DNA from bovine herpesvirus-1 (BHV-1; infectious bovine rhinotracheitis [IBR] virus), bovine herpesvirus-2 (BHV-2; bovine herpes mamillitis [BHM] virus), and bovine herpesvirus-4 (BHV-4; isolate DN599). A 2-stage (nested) polymerase chain reaction (PCR) diagnostic test was devised based on a portion of the subcloned AHV-1 DNA sequence. First and second stage amplified AHV-1 DNA targets were 487 and 172 base pairs (bp) in length, respectively. Unique Pvu II and Stu I restriction endonuclease cleavage sites confirmed the identity of amplified AHV-1 DNA. Five AHV-1 and 2 AHV-2 isolates were identically and specifically PCR positive. BHV-1, BHV-2, and BHV-4 viruses were negative by the same procedure. As little as 0.01 TCID50 AHV-1 was detected using the nested amplification procedure. Simple methods of buffy coat isolation from bovine blood were employed to prepare specimens for PCR. An AHV-1-infected calf was PCR positive from 3 to 77 days postinoculation (PI), with rising seroconversion first noted 14 days PI. The AHV-1 DNA sequence was 62% homologous to a portion of the Epstein-Barr virus genome. The nested PCR procedure may improve the viral diagnosis of clinical and subclinical alcelaphine herpesvirus infections.     

Presence of bovine herpesvirus 1 gB-seropositive but gE-seronegative Dutch cattle with no apparent virus exposure

Two hundred and thirty-seven of 2052 cattle which had not been vaccinated against bovine herpesvirus 1 (BHV-1) were seropositive in a glycoprotein B (gB)-blocking ELISA, but seronegative in a glycoprotein E (gE)-blocking ELISA. In order to detect whether they were latently infected with BHV-1, 10 of them were treated with corticosteroids in an attempt to reactivate putatively latent virus. After successive treatments with dexamethasone and prednisolone, no virus excretion was detected and they showed no increase in antibody titres. In contrast, one gE-seropositive animal re-excreted BHV-1 and had a four-fold increase in antibody titre after the corticosteroid treatments. After slaughter, no BHV-1 DNA could be detected with a sensitive PCR in samples of the trigeminal, cervical and sacral ganglia and spinal cords of the gE-seronegative cattle.     

Comparison of dot blot hybridization, polymerase chain reaction, and virus isolation for detection of bovine herpesvirus-1 (BHV-1) in artificially infected bovine semen.

Bovine semen samples spiked with bovine herpesvirus 1 (BHV-1) were used to compare dot blot hybridization, polymerase chain reaction (PCR), and virus isolation for detection of BHV-1 in bovine semen. The PCR amplification used primers targeting the BHV-1 thymidine kinase gene and a nucleic acid releasing cocktail (GeneReleaser); the PCR product was used as the DNA probe in dot blot hybridization; virus isolation was done in primary bovine fetal testis (BFT) cell cultures. Semen diluted 1:20 in tissue culture medium had the least cytotoxicity and inhibition of viral cytopathic effects in BFT cells, allowing detection of 1 TCID50/100 microL of BHV-1 suspension by virus isolation. The presence of foreign DNA such as bovine sperm DNA or salmon sperm DNA increased the sensitivity of dot blot hybridization in detecting BHV-1, allowing detection of 20,000 TCID50/100 microL of neat semen. The inhibition of PCR amplification of BHV-1 DNA in bovine semen was eliminated by diluting the samples 1:20 in tissue culture medium. The best PCR amplification was obtained when semen was diluted 1:20 and when a reaction buffer of pH 9.0, with 1.0 mM MgCl2 was used. Under these conditions, the PCR followed by ethidium bromide staining of agarose gels could detect 1 TCID20/100 microL of sample, whereas PCR followed by Southern blot hybridization could detect 0.01 TCID50/100 microL of sample.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of respiratory bovine coronavirus, other cytocidal viruses, and Pasteurella spp from cattle involved in two natural outbreaks of shipping fever

OBJECTIVE: To identify cytocidal viruses and Pasteurella spp that could be isolated from cattle involved in 2 natural outbreaks of shipping fever. ANIMALS: 105 and 120 castrated male 4- to 8-month-old feedlot cattle involved in 1997 and 1998 outbreaks, respectively. PROCEDURES: 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 order-buyer'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. CONCLUSIONS 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.     

Virological and serological studies on the role of PI-3 virus, BRSV, BVDV and BHV-1 on respiratory infections of cattle. I. The detection of etiological agents by direct immunofluorescence technique

Nasal cells extracted from nasal swabs obtained from 95 cattle with signs of respiratory disease, out of eleven different herds, were tested for BHV-1, PI-3 virus, BRSV and BVDV using direct immunofluorescence technique. Viral antigen positive samples were detected in seven out of eleven herds examined. Of the 95 individual diseased cattle, 19 were found positive for at least one viral antigen. It was found that especially BHV-1 and PI-3 virus are important causative agents in cattle respiratory disease, both or in combination with other pathogenic agents. Multiple infection in virologically positive herds were observed in six (9.8%) of 61 animals tested. The findings reveal that single or multiple infections of selected viruses may be present in an important range in cattle and that direct immunofluorescence technique as a rapid method, based on the detection of viral antigen in nasal swab samples, is useful to establish the viral aetiology of acute bovine respiratory disease caused by these viruses, particularly in the diagnosis of mixed viral infections.     

Vaccination of calves against bovine herpesvirus-1: assessment of the protective value of eight vaccines

Eight separate, but related experiments, were carried out in which groups of six calves were vaccinated with one of eight commercial vaccines. In each experiment the vaccinated calves were subsequently exposed to three calves infected with virulent bovine herpesvirus-1 (BHV-1). In each experiment, all infected donor calves developed a typical severe infectious bovine rhinotracheitis (IBR) infection and excreted virus in their nasal secretions of up to 10(8.00) TCID50/0.1 ml. One live BHV-1 gE-negative vaccine (A) and three modified live vaccines (B, C, D), administered intranasally, all protected against clinical disease. The calves vaccinated with one vaccine (C) also did not excrete virus in the nasal secretions, whereas the calves protected by vaccines A, B and D excreted virus in their nasal secretions but at low titres (10(0.66)-10(1.24) TCID50/0.1 ml). A fourth modified live vaccine (E), given intramuscularly, failed to prevent mild clinical disease in the calves which also excreted virus in the nasal secretions at titre of 10(1.00) TCID50/0.1 ml. An analogous result was given by the calves vaccinated with either of the two inactivated vaccines (F and G) or with a BHV-1 subunit vaccine (H). All calves developed mild clinical signs and excreted virus at titres of 10(2.20)-10(3.12) TCID50/0.1 ml. Calves vaccinated with C vaccine were subsequently given dexamethasone, following which virus was recovered from their nasal secretions. The virus isolates did not cause disease when calves were infected and appeared to be closely related to the vaccine strain.     

Synergistic effects of concurrent challenge with bovine respiratory syncytial virus and 3-methylindole in calves.

OBJECTIVE: To evaluate the potential synergy between bovine respiratory syncytial virus (BRSV) and 3-methylindole (3MI) in inducing respiratory disease in cattle. ANIMALS: 20 mixed-breed beef calves. PROCEDURE: A 2 X 2 factorial design was used, with random assignment to the following 4 treatment groups: unchallenged control, BRSV challenge exposure (5 X 10(4) TCID50 by aerosolization and 5.5 X 10(5) TCID50 by intratracheal inoculation), 3MI challenge exposure (0.1 g/kg of body weight, PO), and combined BRSV-3MI challenge exposure. Clinical examinations were performed daily. Serum 3MI concentrations, WBC counts, PCV, total plasma protein, and fibrinogen concentrations were determined throughout the experiment. Surviving cattle were euthanatized 7 days after challenge exposure. Pulmonary lesions were evaluated at postmortem examination. RESULTS: Clinical respiratory disease was more acute and severe in cattle in the BRSV-3MI challenge-exposure group than in cattle in the other groups. All 5 cattle in this group and 3 of 5 cattle treated with 3MI alone died or were euthanatized prior to termination of the experiment. Mean lung displacement volume was greatest in the BRSV-3MI challenge-exposure group. Gross and histologic examination revealed that pulmonary lesions were also most severe for cattle in this group. CONCLUSIONS AND CLINICAL RELEVANCE: Feedlot cattle are commonly infected with BRSV, and 3MI is produced by microflora in the rumen of all cattle. Our results suggest that there is a synergy between BRSV and 3MI. Thus, controlling combined exposure may be important in preventing respiratory disease in feedlot cattle.     

Prevalence and specificity of antibodies to bovine respiratory syncytial virus in sera from feedlot and range cattle

The specificity of serum antibodies for the polypeptides of bovine respiratory syncytial virus (BRSV) was examined, using sera obtained from feedlot and range cattle. Test results in sera from feedlot cattle indicated a 60% rate of seroconversion and 95% seropositivity to BRSV, associated with lack of clinical signs indicative of respiratory tract disease. Exposure to other common respiratory tract viruses also was high (greater than or equal to 92% to bovine herpesvirus type 1, bovine viral diarrhea virus, and para-influenza virus type 3). Test results in sera from range cattle indicated BRSV seropositive rates of 28% in calves, 49% in yearling cattle, and 70% in mature cows; clinical signs of respiratory tract disease were not observed in these cattle. Antibodies to BRSV in sera from cattle in both environments reacted predominantly with polypeptides of molecular weight 80,000 through 85,000, 40,000, and 28,000. Reactivity to a glycoprotein of molecular weight between 43,000 and 44,000 and to several glycopolypeptides of smaller molecular weight increased in serum specimens obtained from feedlot cattle between time of entry into the feedlot and slaughter.     

Seroprevalence of bovine respiratory viruses in North-Western Turkey

Bovine respiratory disease complex is a very important health problem around the world. Present study describes serological distribution of bovine major respiratory viruses in non -vaccinated cattle population of Marmara region in north-western Turkey. Neutralising antibodies specific to bovine viral diarrhoea virus (BVDV), bovine herpesvirus 1 (BHV-1), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus 3 (PI-3), bovine adenovirus serotype 1 (BAV-1) and serotype 3 (BAV-3) were investigated. Among 584 serum samples collected from 39 establishments in 7 provinces, 41.4% were positive for BVDV, 17.1% for BHV-1, 73.0% for BRSV, 43.0% for PI-3, 89.5% for BAV-1 and 92.3% for BAV-3. There were significant differences observed between seroprevalence rates detected in neighbouring provinces. Serological prevalence of BVDV, BHV-1 and BRSV were extremely higher in large capacity dairy farms than of small capacity farms (p < 0.0001). This study demonstrates that herd capacity is a very important risk factor for respiratory viruses and, on the other hand bovine adenoviruses and BRSV are the common reason of respiratory diseases in the region.     

Immunological relationships between malignant catarrhal fever virus (alcelaphine herpesvirus 1) and bovine cytomegalovirus (bovine herpesvirus 3)

Strains of malignant catarrhal fever virus (alcelaphine herpesvirus 1 (AHV-1)) and bovine cytomegalovirus (bovine herpesvirus 3 (BHV-3)) were compared for serological relatedness by cross-titration in an indirect immunofluorescent (IIF) antibody assay. There was definite cross-reactivity between these 2 viruses, with heterologous sera staining intracellular and membrane antigens of infected cells. Heterologous antibody titres were approximately 50-fold lower than homologous titres and could be removed by absorption with either homologous or heterologous virus-infected cells, but not with uninfected cells. Regression analyses of IIF antibody titres to AHV-1 and BHV-3 virus in 3 groups of wild ungulate sera also indicated a serological relationship between these herpesviruses. In a cross-immunity trial, 2 of 3 cattle immunized with a BHV-3 virus and 2 of 3 cattle immunized with avirulent AHV-1 resisted challenge with virulent AHV-1-infected blood which killed 3 unimmunized controls. These results are discussed particularly with respect to the involvement of BHV-3 in malignant catarrhal fever.     

Epidemiology and genetic characterization of BVDV,BHV-1, BHV-4, BHV-5 and Brucella spp. infections in cattle in Turkey

The aim of the study was to determine the epidemiological data of bovine viral diarrhea virus (BVDV), bovine herpesvirus-1 (BHV-1), bovine herpesvirus-4 (BHV-4), bovine herpesvirus-5 (BHV-5) and Brucella–associated cattle that were previously reported to have abortion and infertility problems in Ankara, Corum, Kirikkale and Yozgat provinces, Turkey. Whole blood and sera samples were obtained from 656 cattle, and antibodies against Brucella spp. were detected in 45 (6.86%) and 41 (6.25%) animals by Rose Bengal plate and serum tube agglutination tests, respectively. The seropositivity rates against BVDV, BHV-1 and BHV-4 were 70.89%, 41.3% and 28.78%, respectively. RT-PCR and PCR were performed to detect RNA and DNA viruses in blood samples, respectively. The BVDV 5′-untranslated region and BHV-1 gB gene detected in this study were phylogenetically analyzed. The BVDV strains analyzed in this study were closely related to those previously reported from Turkey. The nucleotide sequence from the BHV-1 strain detected in this study is the first nucleotide sequence of BHV-1 circulating in this area of Turkey deposited in the GenBank. The presence of Brucella spp. and prevalence of BHV-1, BHV-4 and BVDV in cattle should be further investigated throughout these regions.     

Antibody titers against bovine coronavirus and shedding of the virus via the respiratory tract in feedlot cattle

OBJECTIVE: To describe patterns of seroconversion to bovine coronavirus (BCV) and shedding of BCV from the respiratory tract in feedlot cattle. ANIMALS: 1,074 calves in feedlots in Ohio, Texas, and Nebraska. PROCEDURE: Nasal swab specimens were obtained at time of arrival (day 0) and at various times during the initial 28 days after arrival at feedlots. Specimens were tested for BCV, using an antigen-capture ELISA. Serum samples were obtained at time of arrival and again 28 days after arrival; sera were analyzed for antibodies to BCV, using an antibody-detection ELISA. RESULTS: Samples from 12 groups of cattle entering 7 feedlots during a 3-year period revealed that 78 of 1,074 (7.3%) cattle were shedding BCV (range, 0 to 35.9% within specific groups). At time of arrival, 508 of 814 (62.4%) cattle had low (< 50) or undetectable BCV antibody titers. Seroconversion to BCV during the initial 28 days after arrival was detected in 473 of 814 (58%) cattle tested (range, 20.3 to 84.1 % within specific groups). In cattle shedding BCV from the nasal passages, 49 of 68 (72.1 %) seroconverted, and 472 of 746 (63.3%) cattle that were not shedding the virus seroconverted. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine coronavirus can be detected in populations of feedlot cattle in the form of viral shedding as well as seroconversion to the virus. Although only a few cattle were shedding the virus at the time of arrival at a feedlot, most of the cattle seroconverted to BCV by 28 days after arrival.     

Evaluation of concurrent shedding of bovine coronavirus via the respiratory tract and enteric route in feedlot cattle

OBJECTIVE: To assess the relationship between shedding of bovine coronavirus (BCV) via the respiratory tract and enteric routes and the association with weight gain in feedlot cattle. ANIMALS: 56 crossbred steers. PROCEDURES: Paired fecal samples and nasal swab specimens were obtained and were tested for BCV, using antigen-capture ELISA. Paired serum samples obtained were tested for antibodies to BCV, using antibody-detection ELISA. Information was collected on weight gain, clinical signs, and treatments for enteric and respiratory tract disease during the study period. RESULTS: Number of samples positive for bovine respiratory coronavirus (BRCV) or bovine enteric coro navirus (BECV) was 37/224 (17%) and 48/223 (22%), respectively. Some cattle (25/46, 45%) shed BECV and BRCV. There were 25/29 (86%) cattle positive for BECV that shed BRCV, but only 1/27 (4%) cattle negative to BECV shed BRCV. Twenty-seven of 48 (56%) paired nasal swab specimens and fecal samples positive for BECV were positive for BRCV. In contrast, only 10/175 (6%) paired nasal swab specimens and fecal samples negative for BECV were positive for BRCV. Only shedding of BECV was associated with significantly reduced weight gain. Seroconversion to BCV during the 21 days after arrival was detected in 95% of the cattle tested. CONCLUSIONS AND CLINICAL IMPLICATIONS: Feedlot cattle infected with BCV after transport shed BCV from the respiratory tract and in the feces. Fecal shedding of BCV was associated with significantly reduced weight gain. Developing appropriate control measures for BCV infections could help reduce the decreased weight gain observed among infected feedlot cattle.     

Prevalence of bovid herpesvirus-4 and its antibody in cattle in Minnesota

Serologic analyses and virus isolation studies were carried out to determine the role of bovid herpesvirus-4 (BHV-4) in infections in cattle, principally those of the reproductive tract. Serologic analyses were performed, using an indirect fluorescent antibody test on thoracic fluid specimens from aborted fetuses and on sera from 3 sources of adult cattle. Virus isolation was attempted from field cases of abortion, early embryo death, and postpartum vulvovaginitis/metritis, using uterine discharge and buffy coat preparations obtained from cows and tissues obtained from aborted fetuses. Of 420 fetal thoracic fluid specimens examined, 5 were positive for BHV-4 antibodies. Seventeen percent of adult cattle from 2 sources ie, clinically normal herds and abattoir cattle, were seropositive for BHV-4 antibodies. Cattle from a third source, 4 herds with high incidence of reproductive tract disorders, had a seroprevalence rate between 36 and 88%. Two isolates of BHV-4 were also obtained from this group. The overall incidence of BHV-4 antibodies in clinically normal cattle was higher than previously recognized, with relatively higher prevalence in herds having reproductive problems (chi 2 = 156.5, P less than 0.005). At least 10% of the BHV-4 antibody-positive sera did not have neutralizing antibody against bovine viral diarrhea virus and/or bovid herpesvirus-1, both important causes of bovine reproductive tract disorders.     

Malignant catarrhal fever. I. Response of American cattle to malignant catarrhal virus isolated in Kenya.

Fifty-three American cattle were inoculated with malignant catarrhal fever virus isolated from a wildebeest in Kenya. Three animals showed the mild form of the disease and recovered, and 47 showed the severe form of the disease. The other three did not react. Of the 47 cattle, 28 died, 16 were killed for the collection of specimens and three recovered. The incubation period for the 47 cattle ranged from 16 to 29 days and the course of the fatal disease for 28 cattle averaged three to 23 days. Virus titration of specimens from nine infected steers yielded a mean titer of 10(4)/TCID50 per gm for lymph nodes, 10(3) TCID50 per mL for buffy coats and 10(2.3) TCID50 per gm for spleens. Smaller amounts of virus were found in the liver, kidneys, adrenals and thyroids. Malignant catarrhal fever virus was also found in nasal secretions and saliva of viremic cattle. Viral infectivity was shown in bovine buffy coat cells stored at 4 degrees C for two days but was immediately destroyed upon freezing even when glycerine or dimethylsulfoxide was added. Viral particles were not found in infected animal tissues by electron microscopy. The disease was successfully transmitted in steers by intratracheal intubation and by aerosol inhalation but not by contact.     

Diagnosis of persistent bovine viral diarrhea virus infection by immunohistochemical staining of formalin-fixed skin biopsy specimens.

The objective of this study was to evaluate the efficacy of immunohistochemical (IHC) staining for diagnosis of persistent bovine viral diarrhea virus (BVDV) infection using formalin-fixed, paraffin-embedded skin biopsy specimens. Skin from 41 of 42 calves shown to be persistently infected (PI) with BVDV by repeated virus isolation more than 3 weeks apart were immunohistochemically positive for BVDV antigen. Positive IHC staining was most pronounced in the keratinocytes and in hair follicle epithelium, hair matrix cells of the hair bulb, and the dermal papilla. All of the skin sections from 10 calves experimentally infected postnatally with BVDV (10(5) median tissue culture infective doses [TCID50]) and biopsied on days 0, 5, 7, and 9 postinfection were negative for viral antigen. Ten calves from a second group experimentally infected with a higher dose of BVDV (10(8) TCID50) were biopsied when viremic between 10 and 14 days postinfection and 4 calves exhibited positive IHC staining for BVDV; however, staining in these skin biopsies was confined to small foci in the nonfollicular epidermis and follicular ostia. This staining was distinct from that observed in skin obtained from PI cattle. Skin biopsy represents an effective method for identifying animals PI with BVDV.     

Inactivation of bovine herpesvirus 1 in semen using a hyperimmune egg yolk semen extender

Hyperimmune egg yolk semen extender was used for the inactivation of bovine herpesvirus (BHV-1) in experimentally infected bovine semen. As much as 5 x 10(4) TCID50/ml of virus was inactivated in semen as assayed by tissue culture. Moreover the hyperimmune egg yolk semen extender did not produce any adverse effect on the quality of the semen after being frozen/thawed in comparison with normal egg yolk semen extender (P > 0.05). The hyperimmune egg yolk semen extender is considered an important tool for containing the spread of BHV-1 from infected semen.