Detection of bovine herpesvirus type 4 antibodies and bovine lymphotropic herpesvirus in New Zealand dairy cows

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.     

Malignant catarrhal fever: polymerase chain reaction survey for ovine herpesvirus 2 and other persistent herpesvirus and retrovirus infections of dairy cattle and bison.

Using a polymerase chain reaction (PCR) test for sequences of ovine herpesvirus 2 (OHV2), this virus was shown to be significantly associated with sheep-associated malignant catarrhal fever (SA-MCF) in terminal cases of disease in 34 cattle and 53 bison. Ovine herpesvirus 2 was not detected in cattle (38) and bison (10) that succumbed to other diseases. Other persistent herpesviruses, retroviruses, and pestivirus, some of which have been previously isolated from cases of SA-MCF, were not associated with the disease. These included bovine herpesvirus 4 (BHV4), bovine lymphotrophic herpesvirus (BLHV), bovine syncytial virus (BSV, also known as bovine spumavirus), bovine immunodeficiency virus (BIV), and bovine viral diarrhea virus (BVDV). A PCR survey for OHV2 in DNA from individual cow's peripheral blood lymphocytes in 4 dairies showed that the 1 dairy that was in close contact to sheep had a prevalence of OHV2 of 21.3%, whereas the 3 other dairies had no OHV2. Prevalence of the other herpesviruses and retroviruses in the dairy cows was variable, ranging from 2% to 51% for BHV4, 52% to 78.7% for BLHV, and 10% to 34% for BSV. Bovine lymphotrophic herpesvirus and BSV were also found in a few (1-4 of 21 tested) cases of terminal SA-MCF, but BIV and BVDV were not found in either the dairy cows sampled, or in the cases of SA-MCE No significant correlation was found between the presence of any 2 viruses (OHV2, BHV4, BLHV, BSV) in the dairy cows or terminal cases of SA-MCE     

Bovine lymphotropic herpesvirus and non-responsive post-partum metritis in dairy herds in the UK

Bovine lymphotropic herpesvirus (BLHV) was detected for the first time in the UK in December 2005 in a dairy herd suffering from chronic, non-responsive post-partum metritis (NPPM). A small-scale investigation was undertaken in order to determine whether this was an isolated case. Samples of vaginal exudates or vaginal swabs were collected from cows in 13 UK dairy herds with a history of post-partum metritis that had not responded to standard treatment regimes for this condition. Cows in 9/13 herds and 1/13 herds were positive for BLHV and bovine herpesvirus-4, respectively, by pan-herpesvirus polymerase chain reaction. No consistent pattern of infectious agents or nutritional/metabolic factors commonly associated with post-partum metritis was observed at the times of sampling. The detection of BLHV in association with NPPM indicates that further work is warranted to determine the impact this virus has on cattle health.     

Impact of metritis on the generation of reactive oxygen species by circulating phagocytes and plasma lipopolysaccharide concentration in peripartum dairy cows

This study examined the relationship between postpartum metritis, reactive oxygen species (ROS) generation, and plasma lipopolysaccharide (LPS) concentration in peripartum dairy cows. Blood was collected twice weekly from 2 weeks prepartum through 6 weeks postpartum. Whole blood chemiluminescence (WBCL) was measured using the luminol‐enhanced zymosan‐stimulated chemiluminescence assay. Cows were examined for uterine health disorders and classified into two groups, healthy (n = 11) and metritis (n = 5). Metritis had a significant effect on WBCL, with cows with metritis having a higher WBCL. Plasma LPS concentrations in cows with metritis were significantly higher than in healthy cows. To examine the effect of LPS on WBCL, blood was sampled in healthy peripartum cows (1 to 2 weeks prepartum, n = 8; 0 to 3 weeks postpartum, n = 11; and 4 to 8 weeks postpartum, n = 8) and incubated with LPS. At 1 endotoxin units/mL of LPS, similar to the plasma LPS concentration in cows with metritis, the WBCL increased in cows at 0 to 3 weeks postpartum. Results indicate that the increase in ROS generation and plasma LPS concentration are associated with metritis, and LPS may be responsible for enhanced ROS generation in early postpartum dairy cows.     

A rapid and sensitive PCR-based diagnostic assay to detect bovine herpesvirus 1 in routine diagnostic submissions

We describe a rapid, sensitive and specific polymerase chain reaction (PCR) assay for the detection of BHV1 DNA in a range of routine diagnostic submissions without the need for prior virus isolation. The assay, which is based on the selected amplification of a portion of the viral tk gene, detected both BHV1.1 and BHV1.2 subtypes in a panel of 15 characterised field isolates, and its sensitivity was estimated to be <0.125 TCID(50). BHV2, alcephaline herpesvirus, BHV4, equine herpesvirus 1 (EHV1), EHV4 and pseudorabies virus were not detected confirming the specificity of the assay. One hundred and five diagnostic submissions, including tissues, nasal secretions and nasal swabs were taken from cattle with respiratory disease and tested using the routine methods of virus isolation (VI) and the fluorescent antibody test (FAT), and the results were compared with those obtained by PCR. The PCR assay detected BHV1 DNA in all samples that were positive by VI. BHV1 DNA was also detectable by PCR in raw and extended semen samples at a sensitivity of 1 TCID(50) per 50microl. The assay also detected BHV5, permitting differentiation between it and BHV1 by virtue of the size of the amplified PCR product. The PCR assay is more sensitive and independent of sample quality than either virus isolation or FAT, and it is faster than virus isolation. The sample preparation method is simple with few steps involved. There are no extra post-amplification blotting/hybridisation steps and the assay is not based on a nested PCR strategy that might otherwise exacerbate the problem of oversensitivity/contamination in the routine use of such a test in a diagnostic laboratory. This assay would permit discrimination between those animals naturally infected with wild type BHV1 and those vaccinated with tk-BHV1 strains.     

BHV4 (bovine herpes virus 4) related disorders in Belgian cattle: A study of two problem herds

Two cattle farms, with a ten year history of BHV4 related postpartum metritis accompanied by fertility problems, were monitored during the winter season 1985–1986. BHV4 was isolated from the lochia from 55% of the animals on farm A and 66% of those on farm B. Respectively 59% and 30% of the animals presented postpartum metritis. In some animals virus multiplication was followed by severe leucopenia lasting several weeks. Indirect immunofluorescence (IIF) BHV4 seropositive as well as IIF seronegative animals were affected. The latter responded with a rapid or late IIF antibody reaction. No BHV4 seroneutralizing antibodies could be detected.The authors also suggest a possible role of BHV4 in the respiratory problems observed during the study.     

Eradication of infectious bovine rhinotracheitis in Switzerland: review and prospects

Ten years after the first outbreak of infectious bovine rhinotracheitis (IBR) in Swiss dairy cows, the national cattle herd is almost free from infection with IBR virus (bovine herpesvirus 1, BHV 1). The national programme for the eradication of IBR was divided into four phases: (1) Prevention of transmission of the infection by restrictions on trade of bovines and assessment of the prevalence of cattle with antibodies to BHV 1. (2) Slaughtering animals with antibodies to BHV 1 in order to eradicate BHV 1 from breeding herds. (3) Detection and eradication of further BHV 1 reservoirs (e.g. fattening cattle). (4) Monitoring programme and legal actions in order to maintain the favourable situation. Approximately 50,000 animals were slaughtered in the course of the eradication of IBR. The total costs amounted to approximately SFr. 110,000,000 over 10 years. The costs for maintaining the situation are estimated at approximately SFr. 5,000,000 per annum.     

A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen

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%     

Risk factors for delayed conception in Korean dairy herds

Delayed conception is defined as an interval of greater than 90 days postpartum before a cow becomes pregnant again. In this study, the risk factors for delayed conception in Korean dairy herds were determined by evaluating several reproductive factors in individual cows. The following data was recorded from 1,012 pregnancies in eight dairy herds (designated A-H) from July 2001 to June 2006: herd, cow parity, repeated animal (cows included 2, 3, or more times), calving season, calving condition (abnormal partus), postpartum disorders (retained placenta, metabolic disorders, metritis and ovarian cysts) and conception. Logistic regression was used to evaluate the effects of these factors on delayed conception. A stepwise procedure was used to obtain the appropriate model (α = 0.05), which revealed the herd, metritis and ovarian cysts to be significant risk factors for delayed conception. The odds ratio showed that the likelihood of delayed conception increased by 3.3 and 2.0 fold for each incidence of metritis and ovarian cysts, respectively. Delayed conception was significantly more likely in 2 herds, in herd A by 2.0 fold and in herd B by 2.4 fold, compared with herd H. These results suggest that the prevention of postpartum metritis and ovarian cysts, as well as improved herd management, will be needed to maintain a short interval between calving and conception in Korean dairy herds.     

Genomic characterization of Arcanobacterium pyogenes isolates recovered from the uterus of dairy cows with normal puerperium or clinical metritis

Arcanobacterium pyogenes is considered to be the most relevant bacterium involved in the establishment of puerperal uterine infection in cattle due to its persistence in utero, resistance to treatment and synergic action with Gram negative anaerobes. Once the infection is established, A. pyogenes is responsible for the persistence of the infection. The objective of this study was to characterize A. pyogenes field isolates recovered from the uterus of cows with either normal puerperium or clinical metritis, in an attempt to identify factors that might be associated with the establishment and persistence of the disease. This characterization was based on BOX-PCR typing and on screening of eight virulence factor genes (plo, nanP, nanH, cbpA, fimA, fimC, fimE, fimG) by conventional PCR. Finally, a relationship between clonal types, virulence factors and presence of disease was investigated. A. pyogenes clonal types identified from isolates recovered from the uterus of postpartum dairy cows differed among herds. Although some clonal types were strictly associated with the development of clinical metritis, others were identified from isolates recovered from normal puerperium and clinical metritis cows. Moreover, the presence of the eight virulence factor genes was not related with the ability to induce clinical metritis, suggesting that the type of A. pyogenes may not be a determinant factor in the development of the disease. We suggest that host intrinsic factors, the synergism between A. pyogenes and other bacteria and the differential gene expression of virulence factor genes may play a more relevant role in the establishment of puerperal uterine infections.     

Simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4 induce subclinical mastitis

In this study, we examined whether an experimental bovine herpesvirus 4 (BHV4) infection can induce bovine mastitis, or can enhance bovine mastitis induced by Streptococcus uberis (S. uberis). Four lactating cows were inoculated intramammarily and intranasally with BHV4, and four lactating control cows were mock-inoculated. After 14 days, two of four cows from each group were inoculated intramammarily with S. uberis. No clinical signs were recorded in cows inoculated only with BHV4, and their milk samples showed no abnormal morphology, despite the fact that BHV4 replicated in inoculated quarters. Somatic cell count increased significantly in milk from three of six BHV4-inoculated quarters, compared to the non-inoculated quarters of the same cows (within-cow) and the quarters of mock-inoculated cows (control group) on days 8, 9 and 11 post-inoculation (pi). BHV4 was isolated from nasal swabs between days 2 and 9 pi. Clinical mastitis was observed in all four cows intramammarily inoculated with S. uberis. A preceding BHV4 infection did not exacerbate the clinical mastitis induced by S. uberis. S. uberis infections appeared to trigger BHV4 replication. From one quarter of each of two cows inoculated with BHV4 and S. uberis, BHV4 was isolated, and not from quarters inoculated with BHV4 only. In conclusion, BHV4 did not induce bovine clinical mastitis after simultaneous intranasal and intramammary inoculation. However, the BHV4 infection did induce subclinical mastitis in 50% of the cows and the quarters.     

The possible role of bovine herpesvirus type‐4 infection in cow infertility

Experimental and field studies have proven that the bovine herpesvirus type‐4 (BHV‐4) infection leads to various reproductive system problems. In this study, the role of BHV‐4 infection in repeat breeding was investigated serologically. Eighty‐four samples were obtained from repeat‐breeding diagnosed cows in two organized dairy herds; an equal number of healthy cows were sampled from the same farms. The rest of the samples (105) were obtained from reproductively normal cows that were breeding in 18 small enterprises as a control group. The seropositivity proportion in repeat‐breeding diagnosed cows was found to be significantly higher (69% (58/84)) than other cows (44% (37/84)) on the same farms. The lowest antibody positivity value for BHV‐4 was detected as 24.7% (26/105) in the samples from family‐type small farms. The odds ratio (OR) value was calculated as 2.834 in repeat‐breeding diagnosed and healthy cows on the same farms, while 6.778 was determined in cows with and without reproductive problems on organized farms compared to small farms. As a result, the BHV‐4 infection can be considered one of the reasons for repeat breeding besides other reproductive disorders.     

奶牛产后子宫感染与子宫免疫

非特异性子宫感染会降低奶牛的生产性能和奶牛场的潜在效益。发生子宫感染时常并发感染化脓性放线杆菌或混合感染其他细菌。当从产后21 d的奶牛子宫液中分离到化脓性放线杆菌时,说明奶牛发生了严重的子宫内膜炎,并且将导致产后第一个发情期不孕。然而引起子宫感染的确切原因尚不清楚,但可以肯定与多种因素有关。发生难产、胎衣不下、怀有双胎或死胎以及患有各种代谢紊乱疾病的奶牛比其他奶牛更容易发生子宫感染。分娩前后异常的免疫功能更容易导致发生严重的子宫感染。子宫感染一般不会引起奶牛死亡,但是发生子宫感染的奶牛其生产性能会受到很大影响。另外,子宫发生感染还会降低产奶量,并且对子宫感染的一些治疗措施还会对牛奶产生污染。因为感染的非特异性,所以子宫感染很难预防;注意加强卫生设施以及围产期的卫生,特别是在助产过程中的卫生,才可能是最好的预防方法。异常免疫功能导致奶牛发生子宫感染的现象,说明通过对免疫功能的调节来预防和治疗围产期奶牛子宫感染是有潜力的。     

Postpartum uterine disease and dairy herd reproductive performance: a review

This paper reviews the causes, impact, treatment, and prevention of retained placenta (RP), metritis, and endometritis in dairy cows. The occurrence of each of these diseases largely depends on immune function in the transition period. Retained placenta affects 5-10% of calvings and greatly increases the risk of metritis and endometritis. More field studies are needed to validate criteria for treatment of metritis, but cows with at least two of RP, fever, dullness, and fetid uterine discharge appear to merit treatment with systemic antibiotics. Clinical endometritis affects 15-20% of cows at 4-6 weeks postpartum; an additional 30-35% have subclinical endometritis between 4 and 9 weeks postpartum. Under specific conditions, treatment of cows with endometritis improved pregnancy rate. Systematic use of prostaglandin F(2alpha) at 5 and 7 weeks postpartum may improve pregnancy rate. The economic benefit of efforts to identify and treat endometritis is herd-specific.     

Bovine herpes mammillitis in two New York dairy herds

Bovine herpes mammillitis was diagnosed in 2 New York dairy herds. Lesions ranged from vesiculation and ulceration of large (up to 10 cm wide) areas of udder and teat skin to single small (2-3 cm wide) plaques of edema. Some lesions resembled "umbilicated pocks" characteristic of cowpox virus infections. Recently freshened heifers were the most severely affected; older cows and heifers with less turgid udders had milder lesions. In 2 cows, incurable mastitis developed. In other cows, the lesions healed by centripetal growth of epidermis into the lesions. Diagnosis was made by isolation of bovid herpesvirus 2 from lesions in both herds and by serum-neutralization testing. Virus isolated from a cow in 1 herd was injected into 9 members of the same herd and may have been responsible for the absence of lesions on these animals; all other members of the herd were affected.     

Risk factors for metritis in Michigan dairy cattle using herd- and cow-based modelling approaches

A prospective study, using the Michigan portion of the National Animal Health Monitoring System, was conducted to determine disease and management factors that affect the incidence of metritis in Michigan dairy herds. A stratified random sample representing 1% of all Michigan dairy herds was observed. Data related to herd, feeding, and reproduction management, disease and environmental risk factors, and cow-based disease and reproductive data were collected monthly for a 12 month period. Cows with complete management records that were observed for at least 3 months postpartum were included in the study (85 herds, 5278 cows). A multivariate linear regression model was developed for lactational incidence of metritis by herd, and a multivariate random-effects logistic model was developed for the occurrence of metritis by cows. Significant factors associated with increased occurrence of metritis included larger herds, problem calvings, overconditioning, underconditioning, and more active reproductive management. Factors associated with reductions in metritis involved good nutritional management, and working relationships with veterinarians for disease management. Implications for the significance of these factors are discussed, with emphasis on differences in interpretation and use of results from herd and cow-based analyses.     

Comparison of swabbing and biopsy for studying the flora of the bovine uterus

The objectives of this study were to evaluate the efficacy of uterine biopsy as a sampling procedure for bacteriological examination, and to assess the importance of obligate anaerobes in the bovine uterus. The aerobic and anaerobic uterine flora of cows with postpartum metritis, cows in postpartum period without metritis and repeat-breeder cows was examined by using swab and biopsy sampling techniques. Obligate anaerobes were isolated in all the 11 cows with postpartum metritis and in three of the five normal cows. No obligate anaerobes were isolated from the six repeatbreeder cows. There was a significant difference (p < 0.01) in the number of bacterial isolates obtained from samples collected by biopsy and by swabbing. A total of 72 isolates was obtained with the biopsies compared to 48 by swabbing. Obligate anaerobes make up an important part of the postpartum uterine bacterial flora, and it seems that in some instances uterine biopsy would be more satisfactory than swabbing for bacteriological examination of the uterus.     

Long-term alteration of follicular steroid concentrations in relation to subclinical endometritis in postpartum dairy cows

The focus of this study was to investigate the effect of subclinical endometritis (scEndo) on ovarian follicular steroid concentrations in early postpartum pasture-fed dairy cows. Mixed-age lactating dairy cows (n = 169) were examined to ascertain uterine health status on d 21 postpartum (±3 d). From this herd, a cohort of scEndo and uninfected cows (n = 47) were selected using uterine cytology to determine scEndo. To ensure cows with scEndo were selected for the study, a conservative threshold [>18% polymorphonuclear (PMN) cells among uterine nucleated cells] was chosen as a selection threshold. Ovarian follicular dynamics were assessed by ultrasonography on d 21, 42, and 63 postpartum. On the latter 2 d, all follicles >4 mm in diameter were ablated, and 4 d later, the largest (F1) and second largest (F2) follicles were measured and their follicular fluid aspirated. Hematological variables and plasma metabolites were measured also on these days to further characterize scEndo cows. On d 21, the prevalence of scEndo was approximately 9% in this herd; by d 42 infections had self-resolved in the majority (81%) of those cows classified as having scEndo on d 21. The scEndo cows had a delayed return to cyclicity; however, no effect was evident on ovarian follicle size or growth rate. Weeks after scEndo had self-resolved and cyclicity was restored, decreased (P = 0.07) testosterone and increased (P = 0.07) cortisol concentrations were evident in F1 follicles of scEndo compared with uninfected cows. Progesterone concentrations of F1 increased (P < 0.05) in 11- to 16-mm diameter follicles of scEndo cows, whereas estradiol, androstendione, and dehydroepiandrosterone concentrations were decreased (P < 0.05) in F1 8- to 10-mm diameter follicles of scEndo cows. These 3 steroids also differed (P < 0.05) between F1 follicle size categories of scEndo but not uninfected cows. On d 21, mean plasma albumin concentration was decreased (P = 0.02) in scEndo cows. In summary, early postpartum scEndo had surprisingly long-term influences on the steroid concentrations of ovarian follicles long after infections had self-resolved. This is likely to affect oocyte quality and may partially explain the reduced conception rates and longer interval between calving and conception that are often associated with scEndo, although more detailed investigations are required to substantiate this theory.     

Comparison of performance of dairy herds that were or were not vaccinated with a bovine herpes virus 1 marker vaccine in 1998

This study analysed the effects of the use of bovine herpesvirus 1 (BHV1) marker vaccine on the performance of dairy cattle. In Spring of 1999, vaccination of 12 herds with the BHV1 marker vaccine resulted in severe animal health problems and mortality. The vaccines used on these farms were all from a batch that appeared to be contaminated with bovine virus diarrhoea virus type 2. This led to a general call to farmers and veterinary practitioners to report side-effects of this vaccine. As a result, more than 7000 farmers reported symptoms. The information was obtained by means of a questionnaire; there was no control group. To determine the effects of the use of the marker vaccine, it was necessary to perform a study based on objectively acquired information. The information collected by the Royal Dutch Cattle Syndicate and the office of Identification and Registration was complied into herd indices on production, udder health, reproduction, and culling. Two groups of dairy farms that had used the BHV1 marker vaccine (attenuated and inactivated vaccine) were compared with farms that were certified BHV1-free. The analyses were performed based on intra-herd comparisons, meaning that per herd each index calculated over a certain period of time after the use of the marker vaccine was compared to a similar period of time prior to the use of the marker vaccine. A total of 144 comparisons were made. Seven comparisons were statistically significant. In two comparisons, the results were in favour of the BHV1-free farms and in five comparisons, the result were in favour of the vaccinated farms. Thus use of the BHV1 marker vaccine could not be proven to affect herd performance. The sensitivity of the tests was very high, so with a high level of probability even very small differences in indices between groups would have been detected.