Replication of bovine viral diarrhoea virus in the bovine reproductive tract and excretion of virus in semen during acute and chronic infections.

Five mature bulls were studied during an acute transient infection with bovine viral diarrhoea virus (BVDV). The bulls had been infected experimentally by the intranasal instillation of blood and serum from a cow which was a persistent carrier of the virus. Infection was confirmed by the demonstration of a low titred viraemia in four of the five animals and by the seroconversion of all five. Semen samples were collected from each bull on four occasions between seven and 14 days after infection. The virus was isolated from the semen of three of the five bulls and from nine of 12 batches of semen from them. In contrast to other studies of the infection of semen, BVDV was isolated with similar efficiency from raw, unprocessed semen and from diluted, extended semen. The titres of virus in the semen ranged from 5 to 75 TCID50/ml. The infection did not appear to affect the quality of the semen. Shedding of virus continued after the end of the period of viraemia and appeared to be a consequence of the replication of the virus in the reproductive tract and its subsequent excretion in the seminal fluid. Virological studies of the reproductive tracts of these bulls suggested that the most productive sites of virus replication were the seminal vesicles and the prostate gland. Concurrent studies in a persistently infected bull supported these findings.     

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.     

Insemination of Susceptible Heifers with Semen from a Non-Viraemic Bull with Persistent Bovine Virus Diarrhoea Virus Infection Localized in the Testes

Bulls shedding bovine viral diarrhoea virus (BVDV) in semen and simultaneously having a high concentration of circulating antibodies may cause reproductive problems and spread the viral infection within cattle populations. To investigate this in detail, three heifers were inseminated with BVDV‐infected semen from a non‐viraemic, seropositive Holstein–Friesian bull, named `Cumulus'. One control heifer was inseminated with semen from a healthy bull that was free of BVDV. All four heifers remained clinically healthy throughout the experiment. The conception succeeded in the control animal and in two of the three heifers inseminated with semen containing BVDV. The heifer with the failed conception was the only one that became systemically infected with BVDV. This animal was deemed non‐pregnant by ultrasonic examination on day 34 after insemination and showed no signs of subsequent oestrus during the entire experimental period. At slaughter, 42 days after insemination, there were no histopathological changes in the ovaries and virus was not detected in ovarian tissue. The fact that seronegative dams served with semen from persistently infected bulls have occasionally produced persistently infected calves together with the present findings and the fact that non‐viraemic, seropositive bulls can constantly shed BVDV, suggest that the use of semen from such bulls in BVDV‐free herds could have far‐reaching consequences, especially if it led to the birth of persistently infected (P1) calves.     

Molecular detection of BHV-1 in artificially inoculated semen and in the semen of a latently infected bull treated with dexamethasone

Two polymerase chain reaction (PCR) assays specific for glycoprotein B (gB) and glycoprotein E (gE) gene detection, respectively, were adopted for the detection of bovine herpesvirus-1 (BHV-1) in naturally infected bulls. The methods were tested on bovine semen artificially inoculated with BHV-1 and were compared with an optimised virus isolation method. Raw and extended semen samples were diluted in minimal essential medium (MEM) and spiked with equal dose of BHV-1. The extended semen was found to be more toxic for the cells than the raw semen, while the viral DNA could be detected by the PCR method in all tested dilutions of raw and extended semen samples. The sensitivity of both methods was compared also for BHV-1 detection in semen, nasal swabs and leucocytes of a seropositive bull in a different time period after virus reactivation with dexamethasone treatment. The sensitivity of virus detection by the PCR method was equivalent to that of virus isolation in cell culture. However, PCR was shown to be faster and easier to perform and may be a good alternative to virus isolation especially when bovine semen has to be screened for BHV-1 prior to artificial insemination.     

牛精液中基因I型牛病毒性腹泻病毒荧光探针定量RT-PCR检测方法的建立

为建立牛精液中基因I型牛病毒性腹泻病毒(BVDV-1)的快速检测方法,本研究采用Sephycral S-400凝胶对牛精液过滤处理后提取病毒核酸,根据BVDV-1 5'UTR保守区基因序列,设计特异性引物和荧光探针,通过反应条件的优化,建立了牛精液中BVDV-1荧光定量RT-PCR检测方法.该方法可以检测到牛精液中含量为0.0125 TCID50的病毒,灵敏度比病毒分离方法高200倍～2000倍,比常规RT-PCR方法高10倍.对从同一个牛场6个月内采集的120份新鲜牛精液和40份冷冻牛精液用该荧光RT-PCR方法检测,没有检测到BVDV-1阳性样品.对其中的10头牛定期检测精液中病毒的同时,并同步检测了全血中的病毒和血清抗体,结果血清抗体阳性牛精液中没有检测到病毒,本研究结果表明,不能以血清抗体阴阳性做为精液是否带毒的依据.     

Some observations on the semen of bulls persistently infected with bovine virus diarrhoea virus

As a result of screening procedures employed for animals entering the AI service, two bulls were identified as being persistently infected with bovine virus diarrhoea virus by isolation of the virus from blood. Semen was collected on two occasions from these bulls; its quality as measured by density and motility was poor. Gross abnormalities of the sperm head, termed 'collapsed' heads, were seen in 28 to 45 per cent of sperm from one bull and in 1 per cent of sperm from the other. The collapsed heads were small and the whole head or its anterior part had the appearance of a dried pea. Electron microscopy showed the defect to consist of convoluted nuclear material with membrane-bound vacuoles and invaginations containing membranous debris and lamellar structures. In the 'high incidence' bull there was a corresponding increase in enlarged sperm heads. The 'low incidence' bull had sperm with heads of similar mean size to sperm from control bulls but with an increased variance. The semen was diluted in a lactose diluent, frozen and stored in liquid nitrogen. The distribution of viral antigen was determined and virus was isolated from several fractions of the semen, both before and after processing and cryopreservation. In one animal raw semen failed to yield virus but virus was recovered after processing, suggesting that raw semen may not be suitable for the efficient detection of the virus.     

Elimination of toxicity and enhanced detection of lumpy skin disease virus on cell culture from experimentally infected bovine semen samples

Lumpy skin disease virus (LSDV), a poxvirus of the genus Capripoxvirus, is shed in the semen of infected bulls. The screening of semen for infectious virus requires a sensitive diagnostic method. The isolation of the virus on cell cultures and/or the polymerase chain reaction (PCR) are sensitive diagnostic tests which may be used to screen semen for LSD viral DNA prior to artificial insemination. Although cell culture detects infectious virus and is a sensitive method, there are major difficulties in using this method due to the toxic effect of semen on the cells. The aim of this study was to find a method that decreases the toxic effect of semen and enhances the isolation of LSDV on cell culture. Semen samples from LSDV sero-negative bulls were collected and infected with a field isolate of LSDV, strain V248/93, with a titre of 6.5 log TCID50. The semen samples were treated with one of four different methods: centrifugation, serial dilution, filtration and chemical treatment with kaolin. The samples subjected to centrifugation, serial dilution and filtration were supplemented with gentamycin. Semen toxicity on cell cultures was eliminated when supernatants of semen samples centrifuged at 2000 rpm for 1, 3 and 5 min and serially diluted were used to inoculate confluent monolayer bovine dermis cells. The toxicity recorded when the pellet fractions of semen samples centrifuged for 5 min at 2000 rpm was comparable to results obtained from serially diluted samples supplemented with gentamycin. Filtration and kaolin treatment of semen samples did not remove the toxic effect.     

Detection of bovine immunodeficiency virus DNA in the blood and semen of experimentally infected bulls

Five 18- to 24-month-old bulls were inoculated with either a cell suspension containing bovine immunodeficiency virus (BIV-FL112; 3 bulls) or a BIV-free cell suspension (2 bulls). Blood and semen specimens were collected once a week for 14 weeks, and seroconversion was confirmed by indirect immunofluorescent antibody (IFA) testing. The presence of BIV in blood and semen was determined by virus isolation and/or polymerase chain reaction (PCR) assays. Antibodies to BIV were detected in the 3 experimentally infected bulls as early as day post inoculation (DPI) 17, and levels peaked at DPI 37-58. BIV was isolated from the peripheral blood mononuclear cells (MNCs) of the infected bulls at DPI 9 (2 bulls) and DPI 23 (1 bull), and could be isolated from one animal up to DPI 65. PCR analysis of MNC DNA, using BIV pol gene primers, detected virus in all three of the experimentally infected bulls from DPI 9 until the termination of the experiment at DPI 98. Efforts to isolate a significant number of non-spermatozoal cells (NSC) by gradient separation from the semen of the experimentally infected bulls were unsuccessful. Two methods for the extraction of total NSC DNA from up to 2 ml of non-extended semen were employed; however, no BIV pol fragment was amplified from these DNA preparations. Additionally, 30 bulls from artificial insemination (AI) centers were evaluated for BIV infection by PCR. No amplification products were obtained from MNC DNA from the AI submissions using primer sets for both the BIV pol and env genes.     

An international inter-laboratory ring trial to evaluate a real-time PCR assay for the detection of bovine herpesvirus 1 in extended bovine semen

Six laboratories participated in a ring trial to evaluate the reliability of a real-time PCR assay for the detection of bovine herpesvirus 1 (BoHV-1) from extended bovine semen. Sets of coded samples were prepared and distributed to each of the laboratories. The sample panel contained semen from naturally and artificially infected bulls, serial dilutions of positive semen with negative semen, semen from uninfected seronegative bulls, negative semen spiked with virus, as well as serial dilutions of reference virus. The samples were tested using a previously validated real-time PCR assay for the detection of BoHV-1 in each participating laboratory. The PCR tests were conducted with four different real-time PCR amplification platforms, including RotorGene 3000, Stratagene MX 3000/4000, ABI 7900, and Roche LightCycler 2.0. Virus isolation using one set of samples was performed in one laboratory. The results of the laboratories were compared with one another, and with those of virus isolation. It was found that the sensitivity and specificity of the real-time PCR test was greater than those of virus isolation (82.7% versus 53.6% and 93.6% versus 84.6%, respectively). A high level of agreement on PCR testing results between the laboratories was achieved (kappa value 0.59-0.95). The results of this study indicate that the real-time PCR assay is suitable for the detection of BoHV-1 in extended semen, and would be a good substitute for the slow and laborious virus isolation, for the screening testing at artificial insemination centres and for international trade.     

Persistent bovine pestivirus infection localized in the testes of an immuno-competent,non-viraemic bull

A post-pubertal bull on an artificial insemination station was found to be persistently shedding bovine viral diarrhoea virus (BVDV) in semen over a period of eleven months, while demonstrating no viraemia. Circulating antibodies to BVDV were consistently high, suggesting that the immune system was challenged repeatedly. Post-mortem findings confirmed that the virus was sequestered in the testes of the bull. It is hypothesized that the BVDV in this immuno-competent bull was protected from the bull's immune response by the blood-testes barrier. The barrier becomes functional only at puberty when tight junctions form between adjacent Sertoli cells, suggesting that this bull became persistently infected with BVDV during puberty.     

Transmission of bovine virus diarrhoea virus (BVDV) by artificial insemination (AI) with semen from a persistently-infected bull

Twelve heifers that did not have antibodies to bovine virus diarrhoea virus (BVDV) were inseminated with semen from a bull that was persistently infected with the virus and contained 10(4.0)-10(6.5) TCID50 0.1 ml-1. All 12 became infected, as indicated by seroconversion within 2 weeks of insemination. Four control heifers were inseminated with virus-free semen. The virus was not transmitted to these animals in spite of close contact with the heifers inseminated with the infected semen. All the heifers became pregnant and gave birth to clinically normal calves at term. However, one calf was born persistently infected with BVDV. After the birth of this persistently-infected calf the control heifers and their calves seroconverted. The study demonstrates that BVDV may be transmitted in cattle by artificial insemination (AI). Therefore entry of persistently-infected animals into AI centres should be prevented.     

Bovine viral diarrhea virus in embryo and semen production systems.

Although BVDV-free offspring have been produced from persistently infected bulls and heifers via advanced reproductive techniques, embryos and semen can potentially transmit the virus. Due to this potential for transmission, appropriate testing is necessary to ensure freedom of semen and embryos from BVDV. In the future, less constraining quality control measures may ensure freedom of embryos and semen from BVDV. These quality control measures require additional research to be validated.     

Insemination of susceptible and preimmunized cattle with bovine viral diarrhoea virus infected semen

Six susceptible and six preimmunized heifers were inseminated with semen from a bull persistently infected with bovine viral diarrhoea virus (BVDV). They had poor rates of conception, but ultimately, all but one conceived. Eleven, apparently normal, calves were born, none of which showed evidence of BVDV infection.     

Pregnancy rate of heifers bred by an immunotolerant bull persistently infected with bovine viral diarrhoea virus

Twenty four maiden heifers were bred by natural route by a specific immunotolerant bull, that was persistently infected with Bovine Viral Diarrhoea virus (BVD virus). The quality of the bull's semen was normal. Twelve heifers became pregnant in the first oestrus cycle and the remaining twelve in the second oestrus cycle. This leads to the conclusion that such persistently infected bulls may have good fertilisation results. Nevertheless, it is felt that bulls persistently infected with the BVD virus must be excluded from artificial insemination centres because of the risk of introducing BVD virus in a herd by the semen.     

Prevalence of bovine viral diarrhea virus infection in bulls in artificial insemination centers in Poland

This study was directed at the evaluation of the prevalence of bovine viral diarrhea virus (BVDV) infection in bulls in artificial insemination centers. Both serological and virological examinations were performed. Blood samples were tested in micro-seroneutralization test for BVDV antibodies. Virus isolation was performed in cell culture and BVDV antigen was detected in an indirect immunofluorescence assay with monoclonal antibodies. One hundred and seventy-five serum samples and 219 whole blood samples for virus isolation were tested. Neutralizing antibodies were found in 86% of the bulls. Persistent infection (PI) was detected in 0.9% of the analyzed blood samples.     

Evidence of excretion of Schmallenberg virus in bull semen

Schmallenberg virus (SBV) is a novel orthobunyavirus, discovered in Germany in late 2011. It mainly infects cattle, sheep and goats and could lead to congenital infection, causing abortion and fetal abnormalities. SBV is transmitted by biting midges from the Culicoides genus and there is no evidence that natural infection occurs directly between ruminants. Here, we could detect SBV RNA in infected bull semen using qRT-PCR (three bulls out of seven tested positive; 29 positive semen batches out of 136). We also found that highly positive semen batches from SBV infected bulls can provoke an acute infection in IFNAR^-/- mice, suggesting the potential presence of infectious virus in the semen of SBV infected bulls.     

Sites of Persistence of Lumpy Skin Disease Virus in the Genital Tract of Experimentally Infected Bulls

The objectives of this work were to determine the site of persistence of lumpy skin disease virus (LSDV) in bulls shedding the virus in semen for a period longer than 28 days, to determine if the virus is present in all fractions of semen and to study lesions that developed in the genital tract. Six serologically negative postpubertal bulls were experimentally infected with a virulent field isolate of LSDV. The polymerase chain reaction (PCR) was performed on sheath washes, vesicular fluid, supernatant and cell‐rich fractions of semen from day 10 to day 26 postinfection (p.i.). Bulls that were positive by PCR on the whole semen sample collected on day 28 p.i. were slaughtered and tissue samples from their genital tracts submitted for histopathological evaluation, immunoperoxidase staining, virus isolation and PCR. Two of the bulls developed severe lumpy skin disease (LSD) and were found to be shedding viral DNA in their semen on day 28 p.i. Viral DNA was identified in all semen fractions from all bulls, but mostly from the cell‐rich fraction and from the severely affected bulls. The PCR assay was positive on postmortem samples of testes and epididymides from the two severely affected bulls. Virus could be recovered from the testes of these two bulls and from the epididymis of one of them. Immunoperoxidase staining was positive for LSDV staining in sections of testes and epididymides exhibiting necrosis. This study suggests that the testis and epididymis are sites of persistence of LSDV in bulls shedding virus in semen for prolonged periods and revealed that viral DNA is present in all fractions of the ejaculate.