Association of Ureaplasma diversum with reproductive disease in cattle.

Aims: To examine the association between the detection of Ureaplasma diversum in vaginal swabs from dairy cows in north western Spain with the diagnosis of granular vulvovaginitis (GVV) and reproductive performance, and the association with subclinical endometritis (SE) in slaughterhouse material. The presence of this microorganism in cases of abortion was also investigated. Methods: From 106 dairy farms in the province of Lugo, 40 herds were randomly selected. Vaginal swabs were obtained from 10 randomly selected cows per farm, then pooled for analysis to detect the presence of U. diversum by quantitative real-time PCR (qPCR). In five of these herds samples from the 10 animals were individually tested for U. diversum, and the presence of GVV lesions and their reproductive efficiency (number of inseminations to achieve pregnancy over two subsequent pregnancies) were determined. Vaginal swabs from uteri of cattle obtained at a slaughterhouse (n = 100) were tested for U. diversum and the presence of SE, defined as >5% polymorphonuclear cells in cytobrush smears, was determined. Sixteen farms with abortion problems submitted samples for culture and PCR testing including for U. diversum. Results: Of the 40 herds, 39 (98%) tested positive for U. diversum. On the five farms, 25/50 (50%) cows tested positive for U. diversum, and more cows with GGV-lesions (16/25; 64%) tested positive than cows without lesions (9/25; 36%) (p = 0.047). There were more cows with poor reproductive efficacy that tested positive (8/11; 57%) than tested negative (3/17; 18%) for U. diversum (p = 0.029). Of the 100 uteri, five tested positive for U. diversum and there were more uteri with SE that tested positive (3/19; 16%) than uteri without SE (2/81; 2%) (p = 0.036). U. diversum was also diagnosed in 4/16 farms with abortion problems and liver appeared to be the best tissue for detecting U. diversum DNA in the fetuses analysed. Conclusions and Clinical Relevance: Infection with U. diversum was present in most of herds investigated and it was statistically associated with GVV, SE and poor reproductive performance. It was also detected in abortions and the liver may also be an additional tissue to be considered in the diagnosis of U. diversum abortion by PCR. The possible association with different diseases in the same area suggests that different presentations should be considered when studying the implications of U. diversum on the reproductive diseases of cattle.

Parapoxviral vulvovaginitis in Holstein cows.

A group of Holstein first-calved heifers developed small pustules and ulcers on the vulva and in the vagina during the first 1-4 wk postpartum. The lesions varied from small red pinpoint foci to pustules and ulcers, 3-5 mm diameter. Some ulcers coalesced to form large ulcerated areas up to 15 mm diameter. In some animals, these ulcers progressed to become deep ulceration of the vaginal and vulvar mucosa with >50% of the mucosa involved. Vaginal biopsies from 4 heifers and vaginal individual swabs from 8 heifers for a combined sampling of 9 heifers were taken for clinical assessment. Six of the 9 heifers had parapoxvirus based on histopathology and/or PCR. Histologic examination of the biopsies of the pustules identified ballooning degeneration of the epithelium with degenerate epithelium containing eosinophilic intracytoplasmic inclusions consistent with a parapoxvirus in 3 of 4 biopsies. Testing for bovine herpesvirus 1, 2, and 4, bovine viral diarrhea virus, bovine papular stomatitis virus, and orf virus remained negative.

Bovine herpesvirus-1 in three major milk sheds of Ethiopia: Serostatus and association with reproductive disorders in dairy cattle.

Bovine herpesvirus-1 (BHV-1) causes infectious bovine rhinotracheitis (IBR), and infectious pustular vulvovaginitis (IPV) in cows and infectious pustular balanopostitis (IPB) in bulls worldwide. Infection of seronegative cattle with BHV-1 leads to abortion, retention of fetal membranes, increased service per conception, metritis and oophoritis. As part of an ongoing study on infectious causes of reproductive disorders in Ethiopia, this investigation aims at assessing the role of BHV-1 in the disorders and the risk factors affecting its seroprevalence. A cross-sectional study was conducted on a total of 1379 randomly selected dairy cattle from 149 herds. These dairy cattle were sampled from milks sheds of central (n = 555), western (n = 195) and southern (n = 629) Ethiopia. Blocking enzyme-linked immunosorbent assay (B-ELISA) was applied to detect antibodies specific to BHV-1. Additionally, a semi-structured questionnaire was administered and farm records were assessed to capture potential risk factors associated with BHV-1 seropositivity. Univariable and multivariable random-effects logistic regression analyses were used to assess potential risk factors associated with BHV-1 serostatus. Model fitness and reliability were assessed using the Hosmer and Lemeshow method and the receiver operating curve (ROC) respectively. An overall herd level BHV-1 seroprevalence of 81.8% (95% confidence interval (CI): 74.7-87.7%) and individual animal level seroprevalence of 41.0% (95% CI: 38.4-43.7%) were found. In a random-effects multivariable logistic regression model, the seroprevalence of BHV-1 exposure was higher in dairy cattle from breeding (Odds ratio [OR] = 1.3; p = 0.036) than in commercial (OR = 0.9; p = 0.137) and small-holder farms. Geographically, the prevalence was higher in western (OR = 1.4; p < 0.001) and southern Ethiopia (OR = 1.2; p < 0.001) than in central regions. BHV-1 seropositive cows had higher (p < 0.05) odds of clinical reproductive disorders including abortion, retained fetal membranes, stillbirth, birth of weak calf and metritis compared to seronegative cows. Thus, it is suggested that BHV-1 should be considered as differential diagnosis among improved dairy cattle herds with reproductive disorders in Ethiopia.

A nested-PCR strategy for molecular diagnosis of mollicutes in uncultured biological samples from cows with vulvovaginitis.

Bacteria classified in Mycoplasma (M. bovis and M. bovigenitalium) and Ureaplasma (U. diversum) genera are associated with granular vulvovaginitis that affect heifers and cows at reproductive age. The traditional means for detection and speciation of mollicutes from clinical samples have been culture and serology. However, challenges experienced with these laboratory methods have hampered assessment of their impact in pathogenesis and epidemiology in cattle worldwide. The aim of this study was to develop a PCR strategy to detect and primarily discriminate between the main species of mollicutes associated with reproductive disorders of cattle in uncultured clinical samples. In order to amplify the 16S-23S rRNA internal transcribed spacer region of the genome, a consensual and species-specific nested-PCR assay was developed to identify and discriminate between main species of mollicutes. In addition, 31 vaginal swab samples from dairy and beef affected cows were investigated. This nested-PCR strategy was successfully employed in the diagnosis of single and mixed mollicute infections of diseased cows from cattle herds from Brazil. The developed system enabled the rapid and unambiguous identification of the main mollicute species known to be associated with this cattle reproductive disorder through differential amplification of partial fragments of the ITS region of mollicute genomes. The development of rapid and sensitive tools for mollicute detection and discrimination without the need for previous cultures or sequencing of PCR products is a high priority for accurate diagnosis in animal health. Therefore, the PCR strategy described herein may be helpful for diagnosis of this class of bacteria in genital swabs submitted to veterinary diagnostic laboratories, not demanding expertise in mycoplasma culture and identification.

An unusual presentation of pseudocowpox associated with an outbreak of pustular ulcerative vulvovaginitis in a Swedish dairy herd.

Species Pseudocowpox virus (PCPV; family Poxviridae) is known to cause pustular cutaneous disease in cattle. We describe an outbreak of pseudocowpox with an unusual clinical picture in a free-stall dairy herd of ~80 cows. Approximately 90% of the cows had vesicles, erosions, papules, and scabs on the vulva and vaginal mucosa. Histologic analysis of biopsy tissues indicated a primary, although not specified, viral infection. Transmission electron microscopy revealed parapoxvirus particles in both tissue and vesicular materials. Deep sequencing analysis of extracted DNA from swabbed vesicle areas gave a contig of nearly 120,000 nucleotides, matching the PCPV strain VR 634 with 100% identity. Analyses confirmed the absence of other potential causes of pustular vulvovaginitis such as bovine herpesvirus 1 and Ureaplasma diversum. A rolling cow brush was suspected to be the fomite.

A novel bovine papillomavirus type in the genus Dyokappapapillomavirus.

Papillomaviruses are a diverse group of viruses that are known to infect a wide range of animal species. Bovine papillomaviruses (BPVs) are divided into at least 21 genotypes (BPV1 to BPV21),  with most BPV isolates/strains described to date belonging to one of four genera, including Deltapapillomavirus, Xipapillomavirus, Epsilonpapillomavirus and Dyoxipapillomavirus. Here, we describe the identification and genetic characterization of a new BPV type in the genus Dyokappapapillomavirus. A farm in the state of New York, USA, reported chronic cases of vulvovaginitis in Holstein cows in 2016. Biopsies and/or swab samples collected from the vaginal mucosa were subjected to diagnostic investigation. Conventional diagnostic assays yielded negative results, and vaginal swab samples were subjected to viral metagenomic sequencing. Notably, BLAST searches revealed a papillomavirus genome with 7480 bp in length (67% nt sequence identity to BPV16). Additionally, phylogenetic analysis of the L1 gene of the papillomavirus identified here (tentatively named BPV22) revealed that it clusters with members of the genus Dyokappapapillomavirus. Interestingly, the recently identified BPV16, which was detected in fibropapilloma lesions in cattle also clusters within the Dyokappapapillomavirus group. Each virus, however, forms a separate branch in the phylogenetic tree. These results indicate that the putative BPV22 represents the second BPV within the genus Dyokappapapillomavirus.

Characterization and identification of microbial communities in bovine necrotic vulvovaginitis.

Bovine necrotic vulvovaginitis (BNVV) is a severe and potentially fatal disease of post-partum cows that emerged in Israel after large dairy herds were merged. While post-partum cows are commonly affected by mild vulvovaginitis (BVV), in BNVV these benign mucosal abrasions develop into progressive deep necrotic lesions leading to sepsis and death if untreated. The etiology of BNVV is still unknown and a single pathogenic agent has not been found. We hypothesized that BNVV is a polymicrobial disease where the normally benign vaginal microbiome is remodeled and affects the local immune response. To this end, we compared the histopathological changes and the microbial communities using 16S rDNA metagenetic technique in biopsies taken from vaginal lesions in post-partum cows affected by BVV and BNVV. The hallmark of BNVV was the formation of complex polymicrobial communities in the submucosal fascia and abrogation of neutrophil recruitment in these lesions. Additionally, there was a marked difference in the composition of bacterial communities in the BNVV lesions in comparison to the benign BVV lesions. This difference was characterized by the abundance of Bacteroidetes and lower total community membership in BNVV. Indicator taxa for BNVV were Parvimonas, Porphyromonas, unclassified Veillonellaceae, Mycoplasma and Bacteroidetes, whereas unclassified Clostridiales was an indicator for BVV. The results support a polymicrobial etiology for BNVV.

Presence of Ureaplasma diversum in the genital tracts of female dairy cattle in Mato Grosso State, Brazil.

Ureaplasma diversum infection in bovine females may result in various reproductive problems, including granular vulvovaginitis, abortion, weak calves, salpingitis, and spontaneous abortion. The presence of U. diversum in a dairy bovine population from midwestern Brazil has not been established. The aim of this study was to determine whether U. diversum was present in dairy cattle from midwestern Brazil using polymerase chain reaction (PCR). Vulvovaginal mucus was analyzed from 203 cows located in six municipalities in the north region of Mato Grosso State, Brazil. A total of 25% of dairy cows with vulvovaginitis were positive for U. diversum. The factors evaluated were included in a multivariable logistic regression model with the presence of at least one positive cow in the herd serving as the dependent variable. Three variables were significantly associated with a U. diversum-positive PCR and were included in the final multivariable model: number of parities, vulvar lesions, and reproductive problems. For each new parity, the chance of U. diversum infection decreased 0.03-fold, indicating that cows with the highest number of parities were more protected. The presence of vulvar lesions was increased 17.6-fold in females positive for U. diversum, suggesting that this bacterium could be related to the red granular lesions in the vulvar mucosa, whereas reproductive problems were increased 7.6-fold. However, further investigations should be conducted to ascertain the effects of U. diversum in association with other mycoplasma species in the herds studied.

Ureaplasma diversum as a cause of pustular vulvovaginitis in bovine females in Vale Guapore, Mato Grosso State, Brazil.

Ureaplasma diversum has been associated with various reproductive problems in cattle that include granular vulvovaginitis, weak calves, and abortion. This study was conducted in a beef herd situated in the Middle-West region of Brazil, and the objectives were to verify the presence of U. diversum and to elucidate its possible relationships with independent variables in this bovine herd population. A total of 134 vaginal mucous swabs were taken for polymerase chain reaction (PCR). Of these, 51 (38 %) were PCR positive for U. diversum. Of the 58 heifers with vulvovaginal lesions characterized by hyperemia, granulated lesions, and edema distributed throughout the vulvar mucosa, 37 (64 %) were U. diversum positive; of the 76 heifers without reproductive lesions, 14 (18 %) were U. diversum positive. All tested samples were negative for bovine herpesvirus 1 (BoHV-1). Multivariate logistic regression revealed that the following two variables were significantly associated with the presence of U. diversum: the presence of vulvar lesions (p = 0.001) and the presence of a progesterone (P4) device (p = 0.001). These findings indicate that U. diversum should be considered a pathogen that is associated with pustular vulvovaginitis in heifers from the Mato Grosso state and that additional studies of the risk factors associated with intravaginal P4 device transmission should be performed.

Seminal transmission of lumpy skin disease virus in heifers.

It is known that lumpy skin disease virus (LSDV) can be shed in bull semen following infection and also that artificial insemination (AI) poses a biosecurity risk. However, it is not known whether the use of LSDV infected semen in AI poses a biosecurity risk. The aim of this study was to investigate whether LSDV, transmitted through semen, can infect cows and their embryos. Two controlled trials were performed simultaneously. Eleven young beef heifers, naïve to LSDV, were synchronized using an OvSynch protocol and inseminated on Day 0 with fresh semen spiked with a field strain of LSDV on day 0. Six of the heifers were superovulated on Day 1 using pregnant mare serum gonadotropin, and embryos were flushed from these heifers on Day 6. Blood and serum samples were collected from Day 4 until Day 27 to determine the presence of LSDV by PCR and virus isolation, and the presence of antibodies against LSDV by SNT. The first clinical signs of LSD were noticed on Day 10, followed by severe generalized LSD in three heifers and mild LSD in two more heifers. Two heifers were humanely euthanized due to severe unresponsive stranguria. LSDV was detected by PCR, virus isolation or electron microscopy in blood, embryos and organs of experimentally infected animals; and eight heifers had seroconverted by Day 27. Two control animals were not affected. This is the first report of experimental seminal transmission of LSDV in cattle.

Ocorrência de Mollicutes e Ureaplasma spp. em surto de doença reprodutiva em rebanho bovino no Estado da Paraíba / Occurrence of Mollicutes and Ureaplasma spp. in outbreak of reproductive disease in cattle herds, State of Paraíba, Brazil

Em março de 2012 foi diagnosticado um surto de doença reprodutiva em rebanho bovino no Estado da Paraíba, Brasil. Foram examinadas 32 vacas e dois touros da raça Girolando. As vacas apresentaram sinais de doença reprodutiva como repetição de cio, vulvovaginite granular, infertilidade e abortos. As amostras de suabes vaginais e prepuciais foram colhidas e submetidas a isolamento bacteriano e PCR. As reações da PCR para Mollicutes e Ureaplasma spp. foram realizadas com os iniciadores MGSO-GPO3 e UGP'F-UGP'R, respectivamente. Na Nested PCR para Ureaplasma diversum, os iniciadores usados foram UD1, UD2, UD3 e UD4. Para isolamento bacteriano, as amostras foram diluídas de 10-1 até 10-5, semeadas em meio "UB", líquido e placa, sendo incubadas por até 21 dias a 37ºC em jarra de microaerofilia. A frequência de Mollicutes detectada na PCR foi de 65,6% e para Ureaplasma spp. foi de 50,0%, enquanto que para U. diversum foi de 15,6%. No isolamento a frequência de Mollicutes foi de 57,1% e para Ureaplasma spp. foi de 28,6%. No ágar "UB" foi visualizado o crescimento misto de Mycoplasma spp. e Ureaplasma spp. em seis amostras. Foi confirmado o envolvimento de micro-organismos da Classe Mollicutes em surto de doença reprodutiva em vacas no sertão paraibano.

In March of 2012 was investigated a reproductive disease outbreak in cattle herds from Paraíba State, Brazil. Were examined 32 cows and two bulls Giroland breed. The cows showed signs and symptoms of reproductive failure such as repeat breeding, granular vulvovaginitis, infertility and abortions. Vaginal and preputial mucous samples were collected for analysis by PCR and isolation. The PCR reactions for Mollicutes and Ureaplasma spp. were realized with primers MGSO and GPO3, and UGP'F and UGP'R respectively. The nested PCR assay for Ureaplasma diversum was realized with primers UD1, UD2, UD3 and UD4. For bacteriologic isolation, obtained samples were diluted up to 10-1 at 10-5, inoculated into liquid and solid "UB" medium, and incubated for up to 21 days, at 37ºC in microaerophilie jar. In the PCR reactions the frequency of Mollicutes detected in the analyzed vaginal mucous samples was 65.6, for Ureaplasma spp. was 50.0, while for U. diversum was 15.6. The frequency for isolation of Mollicutes was of 57.1 and for Ureaplasma spp. was of 28.6. In the UB agar was visualized growth of Mycoplasma spp. and Ureaplasma spp., associated in six of the samples. In the cows the presence of Mollicutes and Ureaplasma spp. was confirmed for the reproductive disease outbreak in the semiarid region of Paraiba.

The effects of bovine necrotic vulvo-vaginitis on reproductive and production performance of Israeli 1st calf heifers.

Bovine necrotic vulvovaginitis (BNVV) is a syndrome unique to Israel characterized by necrotic lesion in the caudal vagina mainly in first calf heifers after calving, associated with Porphyromonas levii. The objectives of this study were to analyze the impact of BNVV on reproductive performance, milk production and survival in the heard of first calf dairy heifers in affected farms, and to verify if the effects of BNVV are severity-dependent. For assessment of the severity level a scale of 4 degrees was formed, and cows were scored 4 to 6 d after calving. Data were obtained from two dairy farms during 2006-07, consisting of 603 lactations. The incidence and the severity of BNVV declined between 2006 and 2007, and severe BNVV tended to be more prevalent in the summer. The odds to conceive in the first artificial insemination of BNVV cow tended to be lower than healthy cows (OR = 0.676, P = 0.052). Cows with BNVV had longer empty period (145.8 d vs. 135.1 d of healthy cows, P = 0.031), but only severe BNVV had a negative effect on the odds of the cow to be empty at 150 d in milk (DIM) (OR = 2.05, P = 0.052). Severe BNVV also affected the mean survival time to conception (155.9 d vs. 142.3 d, P = 0.042). All BNVV severity degrees had a negative effect on milk production. The effect on milk production was not limited only to the beginning of the lactation, cows with BNVV produced 338.1 kg milk less than healthy cows (P = 0.016) in 305 d corrected lactation. The effect on milk production was not severity depended. No effect on survival time in the herd was demonstrated.

Identification of Mycoplasma bovigenitalium and Mycoplasma canadense from outbreaks of granulopapular vulvovaginitis in dairy cattle in Israel.

A syndrome in which white foci and granulopustular lesions appeared on the vaginal mucous membranes of Holstein cows in several dairy herds in Israel is described. During clinical and diagnostic investigations, Mycoplasma bovigenitalium was isolated from 11 of 20 clinical cases. Vaginal swabs taken from the same cows yielded three isolates of Mycoplasma canadense, which were all associated with the M bovigenitalium infection. Two isolates of small, round, non-enveloped viral particles were approximately 25 nm in diameter and characteristic of enteroviruses on negative-staining electron microscopy.

Granular Vulvovaginitis Syndrome in Nelore pubertal and post pubertal replacement heifers under tropical conditions: role of Mycoplasma spp., Ureaplasma diversum and BHV-1.

In order to determine the role of Mycoplasma spp, Ureaplasma diversum and BHV-1 as causal agents of Granular Vulvovaginitis Syndrome in Nelore heifers raised under tropical conditions and based on the hypothesis that stressful conditions during puberty or breeding season would be a determinant factor for the infection, 340 heifers not vaccinated against BHV-1 were divided in Post-pubertal, in the beginning of the first breeding season, and Pubertal heifers. The vaginal lesion score (VLS) Grade 1 to 4 was giving according to lesion area and severity. Vaginal mucus was used to isolate Mycoplasma spp., Ureaplasma diversum and BHV-1. The predominant VLS was 2. No sample was positive for BHV-1; 48% were positive for Mycoplasma spp., Ureaplasma diversum, or both, with predominance of Ureaplasma diversum. Serum neutralization for BHV-1 showed more positive animals in pubertal group (23%); 3 of the paired sera demonstrated seroconversion. These data indicated that post-pubertal and pubertal Nelore heifers raised under extensive conditions are more susceptible to Mycoplasma spp. and Ureaplasma diversum. The hypothesis that the stress of pubertal period could lead to an acute vaginal infection by HBV-1 was not proofed.

Isolation of caprine herpesvirus 1 from a major outbreak of infectious pustular vulvovaginitis in goats.

We describe an outbreak of infectious pustular vulvovaginitis caused by Caprine herpesvirus 1 (CpHV1) in a group of approximately 200, 8 month old virgin does that were imported to Victoria from New Zealand. CpHV1 was isolated in cell cultures from vaginal swabs from three of three affected does but not from two bucks that had been with the does. The identity of the virus as a herpesvirus was confirmed by negative stain electron microscopy. Restriction endonuclease DNA fingerprint analysis showed that the DNA fingerprints were similar, but not identical, to previously described CpHV1 isolates made in New Zealand, New South Wales, and in other parts of the world. Acute and convalescent phase sera from selected does supported the diagnosis of CpHV1 infection. It is most likely that the disease was initiated by reactivation of latent virus in at least one of four bucks that served the does, since each was positive for CpHV neutralising antibody when first tested. This is the first report of CpHV infectious pustular vulvovaginitis in goats in Victoria and to our knowledge appears to be one of the largest outbreaks recorded anywhere.

Effects of bovine necrotic vulvovaginitis on productivity in a dairy herd in Israel.

Bovine necrotic vulvovaginitis (BNVV) is characterized by the development of a necrotic vulvovaginal lesion, almost exclusively in post-parturient first-lactation cows, associated with Porphyromonas levii. The scope of this survey was to evaluate the impact of BNVV on herd productivity as a means to rationally evaluate the resources that should be allocated in dealing with the syndrome. During an outbreak of BNVV in a dairy herd, following the introduction of a large number of cows from another farm, the impact of the animals' origin (local or transferred) and BNVV (positive or negative) upon involuntary culling rate, milk yield and days between pregnancies were assessed. The results indicated that the number of days between pregnancies was significantly higher in first-lactation cows with BNVV but was not influenced by the other independent variables. None of the other variables included in this survey had any effect on the involuntary culling rate and milk yield.

Sample-based assessment of the microbial etiology of bovine necrotic vulvovaginitis.

A semiquantitative evaluation of potential bacterial pathogens was correlated to the severity of lesions during an outbreak of bovine necrotic vulvovaginitis (BNVV) on an Israeli dairy herd. Bacteriologic examination of 287 vaginal swabs from 104 post-calving heifers showed a highly significant correlation between Porphyromonas levii colony forming unit numbers and the clinical scores of the lesions, when assessed by an ordinal regression statistical model. No such correlation was found for the other bacteria included in the study. Nineteen samples taken for virological examinations resulted negative for bovine herpes viruses 1, 2, 4 and 5. Thus the results of this study substantiate the essential role of P. levii in the etiology of BNVV and indicate that BHV4 is not required as a predisposing factor to the syndrome.

Bovine necrotic vulvovaginitis associated with Porphyromonas levii.

An outbreak of bovine necrotic vulvovaginitis associated with Porphyromonas levii, an emerging animal and human pathogen, affected 32 cows on a dairy farm in the northeast of Israel. Five animals had to be culled. This report appears to be the first that associates P. levii with bovine necrotic vulvovagnitis.