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1.
AIM: To detect the presence of bovine herpesvirus (BoHV) type 4 in New Zealand dairy cows with clinical metritis.

METHODS: Serum samples taken from 92 dairy cows with clinical metritis, each from a different farm, were tested for the presence of antibodies against BoHV-4 using a commercially available, indirect ELISA. Peripheral blood mononuclear cells (PBMC) were collected from 10 BoHV-4 seropositive cows, and PBMC were examined by a pan-herpesvirus nested PCR to detect herpesvirus. PCR products were sequenced directly and a proportion of the PCR products were cloned and sequenced to identify the virus present.

RESULTS: Antibodies to BoHV-4 were detected in 23/92 (25%) serum samples. The pan-herpesvirus PCR was positive in 8/10 PBMC samples. Cloning and sequencing identified that all of the eight PCR-positive PBMC contained bovine lymphotropic herpesvirus (BLHV); no BoHV-4 DNA was detected.

CONCLUSIONS: This study reports the finding of the presence of apparent antibodies to BoHV-4, and BLHV DNA in New Zealand dairy cows affected by metritis.

CLINICAL RELEVANCE: Bovine herpesvirus type 4 and BLHV are reported to have the potential to cause reproduction failure in cows. This is the first report of apparent BoHV-4 antibodies, and BLHV in New Zealand. The importance and epidemiology of these viruses in cattle in New Zealand requires further investigation.  相似文献   

2.
AIM: To genotype bovine herpesvirus type 1 (BHV-1) isolates from cattle in New Zealand. METHODS: Twenty-eight BHV-1 isolates were collected from clinical samples from cattle over 28 years. They were characterised and compared using restriction endonuclease analysis (REA), and polymerase chain reaction (PCR) and DNA sequencing. RESULTS: Twenty-four isolates were classified as bovine herpesvirus subtype 1.2b (BHV-1.2b) by REA. The remaining four isolates were distinct from the others in REA profiles of one of the major enzymes (HindIII) by which the classification was made. However, these four isolates were closely related to others when the REA profiles of other restriction enzymes were studied, and therefore were regarded as divergent strains of BHV- 1.2b. All BHV-1 isolates were detectable by PCR, and sequence analysis of selected PCR products did not indicate any significant differences between isolates. CONCLUSION: BHV-1.2b appears to be the predominant strain of BHV-1 in cattle in New Zealand. There was no evidence that more virulent strains of BHV-1, e.g. subtype 1.1 and BHV type 5, are, or have been, present in New Zealand. Genetic variations exist among these BHV-1.2b isolates.  相似文献   

3.
Serological comparisons were made using related herpesviruses from cattle (bovid herpesvirus 1), red deer (herpesvirus of cervidae 1) and goats (bovid herpesvirus 6) by virus neutralization and enzyme-linked immunosorbent assays. The test samples comprised field sera from British cattle, red deer and goats and sera from experimentally infected or immunized animals. Both the cervine and caprine viruses appeared to be more closely related to bovid herpesvirus 1 than they were to each other. Cattle sera reacted most strongly with the bovine virus and deer sera with the cervine virus. Antibodies to the caprine virus were not detected in the samples from British goats.  相似文献   

4.
Bovine herpesvirus type 5 (BoHV-5), which is potentially neuropathogenic, was detected in clinical samples of bovine semen, both directly and after isolation in cell culture, using a nested PCR system for amplifying the US4 gene. Nucleotide sequences generated from the amplicons were analysed and deposited at GenBank (NCBI, Bethesda, MD, USA) under the accession numbers AF298174 and AF330157. Alignment of these sequences and previously deposited sequences of BoHV-1 and BoHV-5 showed 82% and 98% similarity, respectively. The bulls, which were maintained at an artificial insemination centre, had presented no clinical signs, indicating that bovine semen should be screened for BoHV-5 to prevent transmission of the virus.  相似文献   

5.
Fourteen hybridoma cell lines secreting monoclonal antibodies (Mab) to cervine herpesvirus-1 (CerHV-1) produced following the fusion of NSO myeloma cells with splenocytes of BALB/c mice previously immunized with gradient purified CerHV-1 were selected using an indirect enzyme-linked immunosorbent assay (ELISA) employing CerHV-1 antigen and tested by the ELISA against four other ruminant alphaherpesviruses from cattle (bovine herpesvirus type 1.1 and 1.2) goat (caprine herpesvirus-2) and reindeer (rangiferine herpesvirus-1). Comparison of all five ruminant alphaherpesviruses with these Mabs confirmed their close antigenic relationships, with two Mabs reacting against all viruses. Ten Mabs which were able to differentiate between the viruses reacted with a 64 kDa polypeptide in a western blot. Four Mabs including two specific only for CerHV-1 with neutralizing activity against the virus used for immunization were directed against a 74 kDa viral protein.  相似文献   

6.
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.  相似文献   

7.
Herding semi-domesticated reindeer has economic and social value for Sami people in the northern territories of Fennoscandia. However, with the intensification of reindeer husbandry, interspecies transmission of pathogens between reindeer and domestic animals may become a problem, especially for countries such as Sweden, Norway, and Finland where pestivirus and alphaherpesvirus have been eradicated in domestic ruminants. This study, which included 1158 Swedish reindeer, showed relatively high prevalence of antibodies against bovine viral diarrhoea virus (BVDV) (32%) and bovine herpesvirus-1 (BoHV-1) (53%). Adult animals were more often seropositive for BVDV and BoHV-1 (50% and 78%, respectively) than were calves (18 and 11%, respectively). While the seroprevalence of alphaherpesvirus was similar in different herding districts, pestivirus seropositivity was highest in the South and diminished towards the North of the Swedish reindeer herding area. High correlation of the seropositivity against both pathogens at both individual and herd levels may indicate possible mutual synergetic effects and may be explained by the immunosuppressive nature of the viruses. While alphaherpesvirus seroprevalence was probably related to putative cervid herpesvirus 2 (CvHV-2), the pestivirus infecting reindeer remains undefined. The virus neutralisation test of reindeer sera using different pestivirus strains, revealed higher titres against Border disease virus strains like 137/4 (BDV-1) and Reindeer-1 (BDV-2) than against BVDV-1. However, the virus was not identified by real time RT-PCR in any of the samples (n=276) from seronegative reindeers. The study showed that pestivirus and alphaherpesvirus infections are endemic in the Swedish reindeer population.  相似文献   

8.
Artificial insemination is widely used in the cattle industry and a major challenge is to ensure that semen is free of infectious agents. A healthy donor bull was tested for freedom from infectious agents. A bovine herpesvirus was isolated in testis cells and identified as bovine herpesvirus type 5 (BoHV-5) by polymerase chain reaction and by direct amplicon sequencing. The amplicon sequence shared 100% similarity with the published sequence of BoHV-5. This is the first report in Australia of BoHV-5 in semen. The implications of this finding are discussed.  相似文献   

9.
AIM: To investigate the prevalence of bovine polyomavirus (BPyV) DNA in commercial batches of bovine serum products, cell lines and cattle in New Zealand and to characterise the viral DNA detected. METHODS: Two nested polymerase chain reaction (PCR) assays were applied to detect BPyV in bovine sera. One was used to screen for the VP1 gene of BPyV DNA in: 140 batches of commercial bovine serum products, including 66 batches of fetal bovine serum (FBS), 34 batches of calf serum, and 40 batches of adult bovine serum (ABS)/plasma; 112 individual adult bovine sera; and 16 cell lines of various species origin. Fifty batches of serum samples were also tested, using the second nested PCR assay that screened for the Large T gene. Restriction fragment length polymorphism (RFLP) was conducted with 36 PCR products amplified from the VP1 gene of BPyV using EcoRI. Five selected VP1 PCR products were subjected to DNA sequencing and phylogenetic analysis. RESULTS: BPyV DNA was detected in 46 (70%) batches of FBS, 11 (32%) batches of calf sera and two (5%) batches of ABS/plasma, an overall prevalence of 42%. None of 112 adult bovine sera was BPyV-positive. RFLP analysis demonstrated a uniform digestion pattern in the majority (31/36) of amplicons tested, while the remaining PCR amplicons did not show enzyme cleavage. Sequence analysis of the PCR products (a 263 base pair (bp) fragment of the VP1 gene) obtained from five batches of FBS showed 96.2-98.9% homology to that of published sequences of BPyV. CONCLUSION: BPyV is a frequent contaminant of commercial bovine serum in New Zealand. The incidence of BPyV in adult bovine serum products is much lower than in FBS and calf serum. Genomic variations exist among different viruses. The clinical significance of the high prevalence of BPyV DNA in bovine serum products is yet to be determined.  相似文献   

10.
As the first step in the development of a cervine IFN-gamma assay for the diagnosis of tuberculosis in deer, cervine IFN-gamma, cDNA was amplified by polymerase chain reaction using primers based on the bovine IFN-y sequence. A high level of amino acid homology was found between the cervine and the ovine and bovine sequences (94% and 91% respectively). There was less identity with the porcine, human, mouse and rat sequences (78%, 62%, 37% and 39%, respectively). The amino terminus of the mature IFN-gamma protein, which is critical for interaction with its receptor and for triggering biological activity, is highly conserved between the cervine, bovine and ovine proteins. A monoclonal antibody-based sandwich enzyme immunoassay (EIA) specific for bovine IFN-gamma also detects ovine but not cervine IFN-gamma. This suggests that the antibodies recognise epitopes common to the bovine and ovine protein but not cervine IFN-gamma. Seven amino acid residues that were common to the bovine and ovine sequence differed in the cervine sequence, suggesting that the specificity of the monoclonal antibodies may be dependent on one or more of these residues. The possibility of the development of an EIA for cervine IFN-gamma as a commercial in vitro diagnostic assay for tuberculosis in deer is discussed.  相似文献   

11.
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.  相似文献   

12.
As the first step in the development of a cervine IFN-γ assay for the diagnosis of tuberculosis in deer, cervine IFN-γ cDNA was amplified by polymerase chain reaction using primers based on the bovine IFN-γ sequence. A high level of amino acid homology was found between the cervine and the ovine and bovine sequences (94% and 91% respectively). There was less identity with the porcine, human, mouse and rat sequences (78%, 62%, 37% and 39%, respectively). The amino terminus of the mature IFN-γ protein, which is critical for interaction with its receptor and for triggering biological activity, is highly conserved between the cervine, bovine and ovine proteins. A monoclonal antibody-based sandwich enzyme immunoassay (EIA) specific for bovine IFN-γ also detects ovine but not cervine IFN-γ. This suggests that the antibodies recognise epitopes common to the bovine and ovine protein but not cervine IFN-γ. Seven amino acid residues that were common to the bovine and ovine sequence differed in the cervine sequence, suggesting that the specificity of the monoclonal antibodies may be dependent on one or more of these residues. The possibility of the development of an EIA for cervine IFN-γ as a commercial in vitro diagnostic assay for tuberculosis in deer is discussed.  相似文献   

13.
AIMS: To review the number of microbiologically-confirmed cases of Johne's disease in farmed deer since 2000, and determine the prevalence of the bovine and ovine subtypes of Mycobacterium avium subsp paratuberculosis (M. paratuberculosis), using a highly specific polymerase chain reaction (PCR) test on samples from infected herds. METHODS: The number of cases of M. paratuberculosis in farmed deer identified by culture or IS900 PCR was documented. A highly specific PCR test was applied to subtype M. paratuberculosis from BACTEC 12B cultures selected on the basis of one culture per deer herd, to give a wide coverage of herds in New Zealand. RESULTS: From January 2001 to October 2005, M. paratuberculosis was isolated from 1,141 farmed deer, and has now been identified by microbiological testing in over 600 deer herds in New Zealand. The bovine subtype of M. paratuberculosis was shown by a highly specific PCR test to be present in 91/95 herds examined; the ovine subtype was found in the remaining four herds. CONCLUSIONS: Since 2000, there has been a substantial increase in both the number of microbiologically-confirmed cases of Johne's disease in farmed deer and the number of infected herds. Johne's disease is now widespread and common in deer herds throughout New Zealand. Whilst the bovine subtype of M. paratuberculosis predominates in deer herds in New Zealand in which Johne's disease has been confirmed, the occasional finding of the ovine subtype highlights the need to consider both sheep and cattle as potential sources of infection for farmed deer.  相似文献   

14.
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)  相似文献   

15.
Rabies and herpetic encephalitis are the main viral infections in bovines with neurological symptoms. Bovine rabies has a high prevalence in Central and South America, while bovine encephalitis associated with herpesvirus is especially important in South America. Viral isolation is the classical way to confirm herpesvirus infection, but molecular evidence of the presence of the virus in affected animals is gaining importance in the diagnosis of the disease in the laboratory. This study investigated the presence of herpesvirus type 1 and 5 (BoHV-1 and BoHV-5) in 182 encephalon of rabies-suspected cattle in Rio Grande do Sul state (RS), Brazil using multiplex real-time polymerase chain reaction (mRT-PCR). The rabies virus was investigated by direct fluorescent antibody assay and intracerebral suckling mouse inoculation. The genomes of BoHV-1 and BoHV-5 were detected in 17% of samples. BoHV-5 and BoHV-1 were detected in 100% and 19% of BoHV positive samples, respectively, indicating the circulation of the pathogens in cattle herds in RS. The high Ct values and the absence of isolation suggest viral latency. Coinfection of herpesvirus and the rabies virus was detected in 28% of samples, although no significant association between pathogens was observed. Rabies was detected in 57.7% of suspected samples, confirming the importance of the disease in the state. Concerning the method by which samples were conserved, no significant difference was observed between the number of positive results in frozen and refrigerated samples.  相似文献   

16.
The presence and numbers of bovine herpesvirus 4 (BoHV-4) infected CD11b+ leukocytes were investigated during experimental infections of New Zealand White rabbits by Fluorescence Activated Cell Sorter (FACS) analysis. Peripheral blood leukocytes (PBL) were collected every second day, and the cells were stained with phycoerythrin-labelled CD11b-specific mouse monoclonal antibody and fluorescein-conjugated bovine herpesvirus 4-specific mouse monoclonal antibody. The numbers of double-stained cells from PBLs of the control and inoculated groups were measured and compared in FACSTREK analyser. Double-stained cells were detected in the virus-inoculated group on postinoculation days (PID) 2-5 and 9-12. The results indicated that CD11b+ PBLs were permissive for BoHV-4 infection, and are probably the main reservoir of the virus during the latent period. The data did not indicate production of infectious viral particles, but virus-specific proteins were expressed on the surface of CD11b+ cells. The two waves of double-stained cells gave similar results to the PCR assays from serum samples, which showed the presence of viral DNA in the serum on the same days when virus-infected CD11b cells were also present. Productive BoHV-4 infection of mast cells or undifferentiated leukocytes in the bone marrow and the antiviral immune response might be responsible for this periodic appearance of the virus in CD11b+ PBLs and in the serum. The paper provides evidence that CD11b+ PBLs are the main target cell populations in the blood for BoHV-4.  相似文献   

17.
Two healthy buffaloes (Bubalus bubalis) in a herd which had not been vaccinated against infectious bovine rhinotracheitis (IBR), were selected for their seropositivity for anti-bovine herpesvirus type 1 (BoHV-1) glycoprotein E antibodies, and injected intramuscularly daily with dexamethasone for five consecutive days (day 1 to day 5) to reactivate any latent herpesvirus. Blood samples and nasal and vaginal swabs were collected daily from day 5 to day 15 from each buffalo for virological examination. All the vaginal swabs and blood samples were negative, but 13 of the 22 nasal swabs were positive; a cytopathic effect was observed in primary cultures of bovine fetal lung cells, and the viral isolates were identified as a herpesvirus by PCR. The viral strains were characterised by the sequence analysis of the genes coding for glycoproteins D and B, and the gene sequences were then used for phylogenetic analysis. The isolates from both buffaloes appeared identical at the level of the two genes, and were more closely related to bovine herpesvirus type 5 than to BoHV-1.  相似文献   

18.
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.  相似文献   

19.
Uteri from 31 infertile cattle were examined for the presence of bovine herpesvirus 4 (BoHV-4) by nested polymerase chain reaction (PCR). Samples were also tested for bacteria, including chlamydiae and Mycoplasma bovis. BoHV-4 was detected by PCR in 27/31 (87.1%) samples, but the presence and amount of viral DNA was not correlated with histological and bacteriological findings. Arcanobacterium pyogenes, Histophilus somni and Pasteurella multocida were isolated from five cows with endometritis. Chlamydiae were detected in four cases (12.9%), but only two of these had endometritis. The study does not support a role for BoHV-4 as primary agent in bovine endometritis.  相似文献   

20.
A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reaction. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.Abbreviations AI artificial insemination - BHV1 bovine herpesvirus type 1 - PCR polymerase chain reaction - TCID50 tissue culture infective dose, 50%  相似文献   

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