Ureaplasma diversum and Ureaplasma sp. in nasal cavity of pigs: Distribution among herds and individual frequency based on pooled samples.

The role of Ureaplasma diversum in the porcine respiratory disease complex (PRDC), its circulation among herds and prevalence in live pigs is unknown; thus, the objectives of this study were: to determine the presence of U. diversum in indoor intensive pig herds and to determine the individual frequency of pigs with U. diversum from pooled samples. A cross sectional study was carried out in 16 indoor intensive herds from Córdoba and La Pampa provinces, collecting eight nasal swabs specimens that were further processed by two pools of four specimens each by a PCR targeting a fragment of 16S ribosomal RNA gene. Four PCR products were sequenced and aligned against data bases. U. diversum - Ureaplasma sp. were detected in 56.3% of the analyzed herds, with 16.5% (95% CI 8.2-30.8) of positive pigs. It was concluded that U. diversum - Ureaplasma sp. are present in the nasal cavity of live pigs, being widely distributed among herds.

Multilocus sequence typing characterizes diversity of Ureaplasma diversum strains, and intra-species variability induces different immune response profiles.

BACKGROUND: Ureaplasma diversum is a pathogen found in the genital tract of cattle and associated with genital disorders such as infertility, placentitis, abortion, birth of weak calves, low sperm motility, seminal vesiculitis and epididymitis. There are few studies evaluating the genetic diversity of U. diversum strains and their influence on the immune response in cattle. Therefore, to better understand genetic relationships of the pathogenicity of U. diversum, a multilocus sequence typing (MLST) scheme was performed to characterize the ATCC 49782 strain and another 40 isolates recovered from different Brazilian states. RESULTS: Primers were designed for housekeeping genes ftsH, polC, rpL22, rpoB, valS and ureA and for virulence genes, phospholipase D (pld), triacylglycerol lipase (tgl), hemolysin (hlyA), MIB-MIP system (mib,mip), MBA (mba), VsA (VsA) and ribose transporter (tABC). PCRs were performed and the targeted gene products were purified and sequenced. Sequence types (STs), and clonal complexes (CCs) were assigned and the phylogenetic relationship was also evaluated. Thus, a total of 19 STs and 4 CCs were studied. Following the molecular analysis, six isolates of U. diversum were selected, inoculated into bovine monocyte/macrophage culture and evaluated for gene expression of the cytokines TNF-α, IL-1, IL-6, IL-10 and IL-17. Differences were detected in the induction of cytokines, especially between isolates 198 and BA78, promoted inflammatory and anti-inflammatory profiles, respectively, and they also differed in virulence factors. CONCLUSION: It was observed that intra-species variability between isolates of U. diversum can induce variations of virulent determinants and, consequently, modulate the expression of the triggered immune response.

A novel quantitative polymerase chain reaction to monitor urinary tract mycoplasma infection in a dog.

The aim of the study was to develop a quantitative real-time PCR assay for diagnosis and monitoring of mycoplasma urinary tract infections (UTI) in a dog. An English Cocker Spaniel dog with the history of urinary tract infection was physically examined and laboratory findings identified chronic renal insufficiency and urinary tract infection. Attempts to culture organisms from pyuric urine failed, and empirical antibiotic therapy did not resolve the pyuria. A mycoplasma species most closely resembling Ureaplasma canigenitalium was identified in urine samples by conventional PCR and sequencing. A quantitative PCR method was developed to monitor and finally verify successful treatment. This novel approach to monitoring mycoplasma urinary tract infections is conceptually simple, and provides rapid results. It may have wider application in monitoring treatment efficacy for infections with other Mycoplasma spp. as well as additional organisms that are difficult to culture. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we highlight two different findings, detection of Ureaplasma canigenitalium in a dog with chronic urinary tract infection and development of a quantitative real-time PCR test to track treatment results in an infected dog. This report is the first report of detection of U. canigenitalium in one dog in Australia. This novel qPCR method for monitoring mycoplasma urinary tract infections is conceptually simple and provides results fast. It will have wider applications in monitoring treatment efficacy for infections with mycoplasmas and mycoplasma-like organisms that are difficult to culture, and provides a sensitive guide to treatment progress.

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.

Can Ureaplasma diversum be transmitted from donor to recipient through the embryo? Two case reports outlining U. diversum losses in bovine embryo pregnancies.

Two bovine embryo recovery results are outlined from different herds. Both cases involve significant late gestational loss from embryos relating back to a single donor. Ureaplasma diversum was confirmed in 3 of 4 cases submitted for postmortem examination. Natural infection originating from the donor and transmitted to the recipient has not previously been documented.

Peut-on transmettre Ureaplasma diversumdu donneur au récipiendaire par l'embryon? Deux rapports de cas présentant des pertes associées à U. diversumlors de gestations d'embryons bovins. Deux résultats de récupération d'embryons bovins provenant de différents troupeaux sont présentés. Les deux cas portent sur la perte gestationnelle considérablement tardive d'embryons provenant d'un seul donneur. Ureaplasma diversum a été confirmé dans 3/4 des cas soumis à l'examen post mortem. Une infection naturelle provenant du donneur transmise au récipiendaire n'a pas été documentée antérieurement.(Traduit par Isabelle Vallières).

[Identification of species of Mycoplasma and Ureaplasma diversum from Argentinian dairy herds]. / Identificación de especies de Mycoplasma y de Ureaplasma diversum en rodeos lecheros de Argentina.

Several species of Mycoplasma and Ureaplasma diversum can cause diseases in dairy cattle, which can be associated or not with clinical manifestations. In our country, the presence of Mycoplasma bovis, Mycoplasma californicum and Mycoplasma canadense has been detected, being the only mycoplasma species identified so far. The objective of this study was to identify other species of the Mycoplasmataceae family. Thirty-five Mycoplasma spp.-like isolates obtained from different samples from cattle, with or without clinical symptoms, from eight herds located in the provinces of Santa Fe, Cordoba, Buenos Aires and San Luis were utilized in the present study. Through the use of species-specific polymerase chain reactions (PCR) Mycoplasma bovigenitalium, Mycoplasma alkalescens, Mycoplasma bovirhinis and U. diversum were identified and through amplification and further sequencing of the 16-23S rRNA intergenic spacer regions, Mycoplasma arginine and M. californicum were identified. The identification of these species represents an important advance in knowledge in order to include these pathogens in the differential diagnosis of certain clinical and pathological entities of cattle from Argentina.

Assessment of cow and farm level risk factors associated with Ureaplasma diversum in pasture-based dairy systems - A field study.

Potential risk factors for Ureaplasma diversum in the vaginal mucus of 1,238 dairy cows were included in a multivariate logistic regression model, based on the cow level (i.e., granular vulvovaginitis [+GVV], yearly milk production [4500 kg or more], pregnancy, predominance of Bos taurus [+Bos Taurus], score of corporal condition [at least 2.5], concomitant positivity for Escherichia coli [+E.coli]), and farm level i.e., milking room hygiene (-Milking room), dunghill location, and replacement female). Ureaplasma diversum was present in 41.1% of the samples. Independent risk factors for U. diversum were +GVV (odds ratio [OR], 1.31); +Mycoplasma spp (OR, 5.67); yearly milk production (4500 kg or more) (OR, 1.99); +Bos taurus (OR, 1.68); +E. coli (OR, 4.96); -milking room (OR, 2.31); and replacement females (OR, 1.89). Ureaplasma diversum vaginal colonization was strongly associated with Mycoplasma spp., E. coli, and number of pregnant cows.

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 parvum undergoes selection in utero resulting in genetically diverse isolates colonizing the chorioamnion of fetal sheep.

Ureaplasmas are the microorganisms most frequently isolated from the amniotic fluid of pregnant women and can cause chronic intrauterine infections. These tiny bacteria are thought to undergo rapid evolution and exhibit a hypermutatable phenotype; however, little is known about how ureaplasmas respond to selective pressures in utero. Using an ovine model of chronic intraamniotic infection, we investigated if exposure of ureaplasmas to subinhibitory concentrations of erythromycin could induce phenotypic or genetic indicators of macrolide resistance. At 55 days gestation, 12 pregnant ewes received an intraamniotic injection of a nonclonal, clinical Ureaplasma parvum strain followed by (i) erythromycin treatment (intramuscularly, 30 mg/kg/day, n = 6) or (ii) saline (intramuscularly, n = 6) at 100 days gestation. Fetuses were then delivered surgically at 125 days gestation. Despite injecting the same inoculum into all the ewes, significant differences between amniotic fluid and chorioamnion ureaplasmas were detected following chronic intraamniotic infection. Numerous polymorphisms were observed in domain V of the 23S rRNA gene of ureaplasmas isolated from the chorioamnion (but not the amniotic fluid), resulting in a mosaiclike sequence. Chorioamnion isolates also harbored the macrolide resistance genes erm(B) and msr(D) and were associated with variable roxithromycin minimum inhibitory concentrations. Remarkably, this variability occurred independently of exposure of ureaplasmas to erythromycin, suggesting that low-level erythromycin exposure does not induce ureaplasmal macrolide resistance in utero. Rather, the significant differences observed between amniotic fluid and chorioamnion ureaplasmas suggest that different anatomical sites may select for ureaplasma subtypes within nonclonal, clinical strains. This may have implications for the treatment of intrauterine ureaplasma infections.

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.

Maternal administration of erythromycin fails to eradicate intrauterine ureaplasma infection in an ovine model.

Erythromycin is the standard antibiotic used for treatment of infection with Ureaplasma spp. during pregnancy; however, maternally administered erythromycin may be ineffective at eliminating intra-amniotic ureaplasma infections. We examined whether erythromycin would eradicate intra-amniotic ureaplasma infections in pregnant sheep. At Gestational Day (GD) 50 (term, GD 150), pregnant ewes received intra-amniotic injections of erythromycin-sensitive Ureaplasma parvum serovar 3 (n = 16) or 10B medium (n = 16). At GD 100, amniocentesis was performed; five fetal losses (ureaplasma group, n = 4; 10B group, n = 1) had occurred by this time. Remaining ewes were allocated into treatment subgroups: medium only (n = 7), medium and erythromycin (n = 8), ureaplasma only (Up; n = 6), or ureaplasma and erythromycin (Up/E; n = 6). Erythromycin was administered intramuscularly (500 mg) every 8 h for 4 days (GDs 100-104). Amniotic fluid samples were collected at GD 105. At GD 125, preterm fetuses were surgically delivered, and specimens were collected for culture and histology. Erythromycin was quantified in amniotic fluid by liquid chromatography-mass spectrometry. Ureaplasmas were isolated from the amniotic fluid, chorioamnion, and fetal lung of animals from the Up and Up/E groups, however, the numbers of U. parvum recovered were not different between these groups. Inflammation in the chorioamnion, cord, and fetal lung was increased in ureaplasma-exposed animals compared to controls but was not different between the Up and Up/E groups. Erythromycin was detected in amniotic fluid samples, although concentrations were low (<10-76 ng/ml). This study demonstrates that maternally administered erythromycin does not eradicate chronic, intra-amniotic ureaplasma infections or improve fetal outcomes in an ovine model, potentially because of the poor placental passage of erythromycin.

Chronic Intra-Uterine Ureaplasma parvum Infection Induces Injury of the Enteric Nervous System in Ovine Fetuses.

Background: Chorioamnionitis, inflammation of the fetal membranes during pregnancy, is often caused by intra-amniotic (IA) infection with single or multiple microbes. Chorioamnionitis can be either acute or chronic and is associated with adverse postnatal outcomes of the intestine, including necrotizing enterocolitis (NEC). Neonates with NEC have structural and functional damage to the intestinal mucosa and the enteric nervous system (ENS), with loss of enteric neurons and glial cells. Yet, the impact of acute, chronic, or repetitive antenatal inflammatory stimuli on the development of the intestinal mucosa and ENS has not been studied. The aim of this study was therefore to investigate the effect of acute, chronic, and repetitive microbial exposure on the intestinal mucosa, submucosa and ENS in premature lambs. Materials and Methods: A sheep model of pregnancy was used in which the ileal mucosa, submucosa, and ENS were assessed following IA exposure to lipopolysaccharide (LPS) for 2 or 7 days (acute), Ureaplasma parvum (UP) for 42 days (chronic), or repetitive microbial exposure (42 days UP with 2 or 7 days LPS). Results: IA LPS exposure for 7 days or IA UP exposure for 42 days caused intestinal injury and inflammation in the mucosal and submucosal layers of the gut. Repetitive microbial exposure did not further aggravate injury of the terminal ileum. Chronic IA UP exposure caused significant structural ENS alterations characterized by loss of PGP9.5 and S100ß immunoreactivity, whereas these changes were not found after re-exposure of chronic UP-exposed fetuses to LPS for 2 or 7 days. Conclusion: The in utero loss of PGP9.5 and S100ß immunoreactivity following chronic UP exposure corresponds with intestinal changes in neonates with NEC and may therefore form a novel mechanistic explanation for the association of chorioamnionitis and NEC.

Protection of the Ovine Fetal Gut against Ureaplasma-Induced Chorioamnionitis: A Potential Role for Plant Sterols.

Chorioamnionitis, clinically most frequently associated with Ureaplasma, is linked to intestinal inflammation and subsequent gut injury. No treatment is available to prevent chorioamnionitis-driven adverse intestinal outcomes. Evidence is increasing that plant sterols possess immune-modulatory properties. Therefore, we investigated the potential therapeutic effects of plant sterols in lambs intra-amniotically (IA) exposed to Ureaplasma. Fetal lambs were IA exposed to Ureaplasma parvum (U. parvum, UP) for six days from 127 d-133 d of gestational age (GA). The plant sterols ß-sitosterol and campesterol, dissolved with ß-cyclodextrin (carrier), were given IA every two days from 122 d-131 d GA. Fetal circulatory cytokine levels, gut inflammation, intestinal injury, enterocyte maturation, and mucosal phospholipid and bile acid profiles were measured at 133 d GA (term 150 d). IA plant sterol administration blocked a fetal inflammatory response syndrome. Plant sterols reduced intestinal accumulation of proinflammatory phospholipids and tended to prevent mucosal myeloperoxidase-positive (MPO) cell influx, indicating an inhibition of gut inflammation. IA administration of plant sterols and carrier diminished intestinal mucosal damage, stimulated maturation of the immature epithelium, and partially prevented U. parvum-driven reduction of mucosal bile acids. In conclusion, we show that ß-sitosterol and campesterol administration protected the fetus against adverse gut outcomes following UP-driven chorioamnionitis by preventing intestinal and systemic inflammation.

Etiology of respiratory disease in non-vaccinated, non-medicated calves in rearing herds.

The aim of this study was to examine the occurrence of bacterial, mycoplasmal and viral pathogens in the lower respiratory tract of calves in all-in all-out calf-rearing units. According to clinical status, non-medicated calves with and without respiratory disease signs were selected of the 40 herds investigated to analyse the micro-organisms present in healthy and diseased calves. Tracheobronchial lavage (TBL) and paired serum samples were analysed for bacteria, mycoplasmas, respiratory syncytial virus (RSV), parainfluenza virus 3 (PIV3), bovine corona virus (BCV) and bovine adenovirus (BAV). Pasteurella multocida was the most common bacterial pathogen. It was isolated from 34% of the TBL samples in 28 herds and was associated with clinical respiratory disease (p < 0.05) when other pathogenic bacteria or mycoplasma were present in the sample. Mannheimia spp. and Histophilus somni were rarely found. Mycoplasma bovis was not detected at all. Ureaplasma diversum was associated with clinical respiratory disease (p < 0.05). TBL samples from healthy or suspect calves were more often negative in bacterial culture than samples from diseased calves (p < 0.05). No viral infections were detected in six herds, while 16-21 herds had RSV, BCV, BAV or PIV3. In the herds that had calves seroconverted to BCV, respiratory shedding of BCV was more frequently observed than faecal shedding. This study showed that the microbial combinations behind BRD were diverse between herds. M. bovis, an emerging pathogen in many countries, was not detected.

Genotyping of Ureaplasma diversum isolates using pulsed-field electrophoresis.

Ureaplasma diversum has been associated with reproductive disorders in cattle and in the present study genotypic variations among U. diversum isolates obtained from the vaginal mucus of healthy cattle and sick animals were analyzed by enzymatic digestion and pulsed-field gel electrophoresis (PFGE). The influence of time and broth volume was important in obtaining sufficient cell sediment and DNA for PFGE. The method presented a high discriminatory power and satisfactory reproducibility for the analysis of detected variations among U. diversum isolates and strains. Different band profiles and wide genotypic heterogeneity were detected but no association between DNA polymorphism and sick or healthy animals could be established.

Molecular characterization of ureaplasmas isolated from reproductive tract of goats and sheep from Brazil.

Ovine/caprine ureaplasmas have not yet been assigned a species designation, but they have been classified into nine serotypes. Herein ureaplasmas were searched for in 120 samples of vulvo vaginal mucous from sheep and 98 samples from goats at 17 farms. In addition, semen samples were collected from 11 sheep and 23 goats. The recovered ureaplasma were from sheep and goats from animals without any reproductive disorder symptoms, but not all animals presented positive cultures. In sheep, 17 (68%) cultures of vulvovaginal mucous were positive for ureaplasma and 11 (27%) samples of semen presented positive cultures in animals with clinical signs of orchitis, balanoposthitis or low sperm motility. In goats four ureaplasma isolates were obtained from vulvovaginal mucus, but the semen samples were all negative. The isolates were submitted to Pulsed-field gel electrophoresis methodology and their 16S rRNA genes were sequenced. Fifty percent of ureaplasma recovered from sheep allowed for PFGE typing. Eleven isolates showed eight profiles genetically close to the bovine ureaplasmas. The 16S rRNA gene sequencing showed differences or similarities of isolates from sheep and goats, and the reference strains of bovine and human ureaplasma. Four clinical isolates from sheep were grouped separately. The studied ureaplasma isolates showed to be a diverse group of mollicutes.

Detection of Ureaplasma diversum in cattle using a newly developed PCR-based detection assay.

Ureaplasma diversum has been associated with different clinical manifestations including bovine vulvitis, endometritis, salpingitis, spontaneous abortion and infertility. Because the isolation of this ureaplasma from clinical samples is difficult, there is a need for improved detection methods. We developed a PCR assay based on amplification of a region of the gene encoding 16S rRNA. The specificity of the amplification was verified by sequence analysis. Female bovine vaginal swabs (n=168) were collected and the presence of U. diversum evaluated by both culture methods and by the PCR assay. Culture was positive for 60 samples (35.7%), and PCR-specific amplification was obtained for 89 samples (52.9%). These results indicated a high prevalence of U. diversum in the selected animals and the higher sensitivity of this PCR assay as compared to culture.

Ureaplasma diversum and reproductive disorder in Brazilian cows and heifers; first report.

The species Ureaplasma diversum is associated with bovine reproductive illnesses, in particular granular lesions of the vulva and vagina or granular vulvovaginitis (GVV). In Brazil, this pathology is unknown and, until this point in time, the presence of U. diversum in the Brazilian herds has been ignored. With the intention of detecting the microorganism, vulvovaginal mucuses of 152 animals located on seven farms in the São Paulo State, Brazil were analyzed. Those animals had evidence of reproductive disorders at the time of the sample collection. The technique used for microorganism detection was bacterial isolation. Statistical analysis assessed: the exposure of studied farms to U. diversum, relative risks for different symptoms, susceptibility of the animals according to age and breed. The frequency of that microorganism in tested animals was 38.8% and this frequency suggests that U. diversum can be related to GVV in Brazilian herds and possibly with other reproductive illnesses. As a result, the U. diversum differential diagnosis could be very important.

Prevalence and reproductive effects of Ureaplasma diversum in beef replacement heifers and the relationship to blood urea nitrogen level.

A systematic sample of replacement heifers from 5 herds underwent prebreeding vaginal swab cultures for Ureaplasma diversum. Heifers from three of the herds were subsequently sampled at pregnancy examination. Sampled heifers were given a vaginal lesion score (VLS), reproductive tract score (RTS) and body condition score (BCS), and peripheral blood was collected for serum blood urea nitrogen (BUN) estimation. Culture results revealed an overall prevalence of Ureaplasma diversum of 51% (87/171) at prebreeding and 65% (64/98) at pregnancy examination. Within herd prevalence ranged from 36% to 64% at prebreeding and 54% to 76% at pregnancy examination. Prevalence tended to differ between herds (P=0.08). At the prebreeding examination, heifers with a BCS of 5.5 or less were more likely to be culture positive than heifers with a BCS greater than 5.5 (p<0.05). No relationship was noted between BUN, VLS, RTS, or pregnancy status and prebreeding culture status. There was little variability among the heifers for any of these variables, with vaginal lesion scores generally being mild, RTS scores being high and BCS scores being moderate. At pregnancy examination, heifers that were culture negative tended to be more likely to be pregnant (odds 3.7, p=0.10) than culture positive heifers.