In vitro exposure of bovine morulae to Ureaplasma diversum.

Ureaplasma diversum has been associated with infertility in the cow experimentally and in naturally occurring cases. However, the pathogenic mechanism is undetermined. The purpose of this study was to determine whether ureaplasmas are pathogenic for bovine morulae in vitro. Twenty-one morulae were recovered from three superovulated, mature, Holstein cows six or seven days postestrus. The embryos were divided into three groups (A,B,C) and incubated for 16 hours at 37 degrees C in humidified air with 10% CO2. Group A was incubated in embryo culture medium alone, Group B was incubated in culture medium with sterile ureaplasma broth added and Group C was incubated in culture medium containing 1.7 X 10(6) colony forming units Ureaplasma diversum strain 2312. After incubation, the morulae were examined using an electron microscope. Structures morphologically identical to U. diversum were present on the outer surface of the zonae pellucidae of all the morulae exposed to the organism and none were present on the unexposed control embryos. No other morphological differences were observed in either the ureaplasma-exposed embryos or the two groups of control embryos. Ureaplasma diversum was isolated from three of the five embryos incubated in culture medium with sterile ureaplasma broth added. These three embryos were recovered from one donor cow which cultured positive for U. diversum from the vulva and flush fluid. This finding suggests that the contaminating organisms entered the embryo culture wells either in the embryo collection medium or attached to the embryos.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Presence of Ureaplasma diversum in the Australian cattle population

In cattle, Ureaplasma diversum has been associated with decreased fertility, granular vulvovaginitis, endometritis, salpingitis and spontaneous abortion in cows and seminal vesiculitis, balanoposthitis and changes in bull sperm. The presence of U. diversum within the Australian cattle population has not been established. One of the aims of this study was to determine if U. diversum was present in Australian cattle, using culture and polymerase chain reaction (PCR), both of which are considered to be gold standards for bacterial identification. Of 100 samples collected from 66 male and 34 female cattle, 15 were positive for U. diversum. Therefore, Australia can no longer be considered free of U. diversum. Further studies should be conducted to ascertain the effects of U. diversum within Australian cattle herds and, if warranted, to investigate prevention, treatment and eradication protocols.     

Modulation of murine macrophages and T lymphocytes by lysozyme dimer

The effects of lysozyme dimer (2 and 20 microg/kg) administered i.p. once and four times to mice on the phagocytic and killing ability of peritoneal macrophages, interleukin-1 (IL-1) production by murine macrophages stimulated in vitro with lipopolisaccharide of E. coli and expression of thymocyte, splenocyte and mesenteric lymphonode cell CD3+, CD4+ and CD8+ markers were studied. It was found that lysozyme dimer administered once or four times at doses of 2 microg/kg and 20 microg/kg augments the phagocytic and killing activity of peritoneal macrophages. The strongest stimulating effect was noted after four injections of lysozyme dimer at a dose of 20 microg/kg. Moreover, lysozyme dimer is able to modulate the production of IL-1 by murine macrophages stimulated in vitro with LPS. Exposure to four doses of lysozyme dimer (20 microg/kg) enhances the synthesis and release of IL-1, but this drug administered once (2 microg/kg and 20 microg/kg) or four times (2 microg/kg) decreases IL-1 production by peritoneal macrophages. It was also found that administration of lysozyme dimer at a dose of 20 microg/kg, irrespective of the number of doses applied, increases the percentage of CD4+ thymocytes and splenocytes. Moreover, exposure to four doses of lysozyme dimer (2 and 20 microg/kg) increases the percentage of CD4+ and CD8+ mesenteric lymphonode cells.     

The effect of intrauterine inoculation with Ureaplasma diversum on bovine fertility.

To determine the influence of Ureaplasma diversum on bovine fertility 11 uninfected virgin heifers with normal ovarian cyclic activity were randomly allocated to test or control groups. At a synchronized estrus, five test heifers were given an intrauterine broth inoculum containing 1.09 x 10(8) to 1.4 x 10(9) colony forming units of U. diversum and six control animals were infused with sterile ureaplasma broth medium. All animals were artificially inseminated within one hour of infusion. Pregnancy was diagnosed in one of five test heifers and all of six controls by serum progesterone concentrations measured to 25 days postinsemination. The difference in pregnancy rates between the two groups was statistically significant (p = 0.0152). It was concluded that under the conditions of this experiment U. diversum is capable of causing infertility in cattle.     

The effects of two Ureaplasma diversum strains on early pregnancy in heifers.

Two field isolates of Ureaplasma diversum spp. were used to infect heifers at the time of insemination in a preliminary study to observe the effect of infection on early pregnancy. M84-14c-1 was a field isolate from a bull's prepuce typed by immunofluorescence to be similar to U. diversum strain T-44 (Group C). M84-477c-4 was a field isolate from bovine semen typed by immunofluorescence to be similar to U. diversum strain T-288 (Group A). All three heifers infected with M84-477c-4 had a mild granular vulvitis at some time during the trial. None was pregnant when slaughtered 27 days after infection. The result of infection with M84-14c-1, a preputial isolate, was not consistent. One heifer had no infection and a normal pregnancy, one heifer was infected with an abnormal pregnancy, and one heifer was open but ureaplasmas were not detected until day 17 of the trial.     

Detection of Ureaplasma diversum in the upper airways of Australian feedlot cattle

Bovine respiratory disease (BRD) exerts a major impact on the beef cattle industry nationally and worldwide, with a range of aetiological factors impacting its pathogenesis. Previous research has focussed on an increasing number of bacteria and viruses that have been shown to play a role in eliciting disease. Recently, additional agents have been emerging as potential contributors to BRD, including the opportunistic pathogen Ureaplasma diversum. To determine if U. diversum was present in Australian feedlot cattle and if that presence was linked to BRD, nasal swabs were collected from a cohort of 34 hospital pen animals and compared to 216 apparently healthy animals sampled contemporaneously at feedlot induction and again after 14 days on feed at an Australian feedlot. All samples were subjected to a de novo polymerase chain reaction (PCR) assay targeting U. diversum in combination with other BRD agents. U. diversum was detected at a low prevalence in cattle at induction (Day 0: 6.9%, Day 14: 9.7%), but in a significantly greater proportion of cattle sampled from the hospital pen (58.8%). When considering the presence of other BRD-associated agents, co-detection of U. diversum and Mycoplasma bovis was most common in hospital pen animals receiving treatment for BRD. These findings suggest that U. diversum may be an opportunistic pathogen involved in the aetiology of BRD in Australian feedlot cattle, in combination with other agents, with further studies are warranted to identify if a causal relationship exists.     

Intraspecific sequence variation in 16S rRNA gene of Ureaplasma diversum isolates

Ureaplasma diversum infection in bulls may result in seminal vesiculitis, balanoposthitis and alterations in spermatozoids. In cows, it can cause placentitis, fetal alveolitis, abortion and the birth of weak calves. U. diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. These microorganisms were submitted to Polymerase Chain Reaction for 16S gene sequence determination using Taq High Fidelity and the products were purified and bi-directionally sequenced. Using the sequence obtained, a fragment containing four hypervariable regions was selected and nucleotide polymorphisms were identified based on their position within the 16S rRNA gene. Forty-four single nucleotide polymorphisms (SNP) were detected. The genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains.     

Humoral and secretory antibodies to Ureaplasma diversum in heifers following subcutaneous vaccination and vaginal infection.

We measured antibody levels in serum and cervicovaginal mucus (CVM) of four heifers vaccinated with two inoculations of killed Ureaplasma diversum strain 2312 in incomplete Freund's adjuvant (IFA) two weeks apart, and six heifers given a placebo. Two weeks later, the vaccinates and four placebo heifers, were challenged by intravaginal inoculation with 6.4 x 10(8) colony-forming units of the homologous U. diversum strain. The remaining two placebo heifers served as unvaccinated, unchallenged controls. Antibody levels in serum and CVM of all heifers were determined by an enzyme-linked immunosorbent assay (ELISA). Vaccination stimulated specific IgG1 and IgG2 responses in serum and CVM but only a slight IgM and no IgA response. In both vaccinate and placebo heifers, subsequent intravaginal challenge resulted in a granular vulvitis (GV) with a predominant IgA response in the CVM. The GV gradually subsided during the 35 day observation period but ureaplasmas were consistently demonstrated by culture. We concluded that subcutaneous vaccination stimulated a specific, albeit nonprotective, IgG response in serum and CVM. In contrast, vaginal infection primarily induced a mucosal IgA response.     

Effects of Ureaplasma diversum on bovine oviductal explants: quantitative measurement using a calmodulin assay.

Calmodulin (CAM) acts as an intracellular regulator of calcium, an important mediator of many cell processes. We used the CAM assay and electron microscopy to investigate the effects of Ureaplasma diversum on bovine oviductal explants obtained aseptically from slaughtered cows. A stock suspension of U. diversum (treated specimens) and sterile broth (controls) was added to replicates of cultured explants and incubated at 38 degrees C in an atmosphere of 5.5% CO2 for 48 hours. Explants were examined for ciliary activity, extracellular CAM loss, and for histological and ultrastructural changes. Explants and their culture media were examined for changes in CAM concentration. All experiments were replicated three times. In addition, U. diversum, medium and broth were assayed for CAM content. The concentrations of CAM in explants and media changed significantly (p < 0.05) in samples which were inoculated with U. diversum when compared to controls. The controls and infected specimens did not differ histologically or ultrastructurally, but U. diversum was seen to be closely associated with infected explant tissue. In view of this close affinity it is assumed the loss of CAM from the oviductal cells was causally related, but this was not proven. The failure to show cell membrane injury on light and electron microscopic examination was probably related to the short duration of the experiment and may only point out the sensitivity of the CAM assay in detecting early cell membrane injury. Compromise in characteristics of the medium to support both, the viability of oviductal cells and U. diversum limited the experimental time to 48 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immune response of heifers to vaginal submucosal or subcutaneous vaccination and intravaginal challenge with Ureaplasma diversum.

Twenty beef heifers were randomly assigned to five equal groups and vaccinated: Group 1--in vaginal submucosa (VM) with Ureaplasma diversum ultrasonicated whole cells (WC) in complete Freund's adjuvant (CFA); Group 2--in VM with U. diversum cell membranes (CM) in CFA; Group 3--subcutaneously (SC) with CM in CFA; Group 4--in VM with CM alone; and Group 5--in VM with phosphate buffered saline (PBS) in CFA. A second vaccination with the same antigens in incomplete Freund's adjuvant was given after four weeks, and three weeks later, all heifers were challenged intravaginally with 3.6 x 10(7) colony-forming units (CFU) of U. diversum strain 2312. Immunoglobulins that reacted with U. diversum were measured in serum and cervicovaginal mucus (CVM) by an enzyme-linked-immunosorbent assay. In groups 1 and 2, vaccination by the VM route with WC or CM antigens, stimulated high levels of U. diversum-reactive IgG1 and IgG2 antibodies in serum as well as CVM, but a low IgA response only in CVM. In group 4, VM vaccination with CM (no adjuvant) elicited a minimal IgG1 and IgG2 response in serum and CVM. In group 3, SC vaccination with CM antigen stimulated high IgG1 and IgG2 reactivity in both serum and CVM, but no IgA reactivity. Very little IgM reactivity was detected in the four vaccinated groups. Intravaginal challenge resulted in characteristic granular vulvitis in all vaccinated and control heifers, with all animals remaining culture-positive for the 35 day observation period. The infection stimulated a marked increase in the specific IgA response in CVM of the three groups vaccinated with either, adjuvanted antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Isolation of Ureaplasma diversum and mycoplasmas from genital tracts of beef and dairy cattle in Saskatchewan

We report herein a survey in which cultures of bovine reproductive tracts for Ureaplasma diversum and mycoplasmas were carried out in order to better understand the role of these organisms in granular vulvitis (GV). Samples cultured were vulvar swabs from clinically normal cows or ones with GV, preputial swabs or raw semen from bulls, and abomasal contents of aborted fetuses.

Ureaplasma diversum was isolated from 104 (43.3%) of 240 dairy cows, 32 (27.1%) of 118 beef cows, 43 (47.2%) of 91 beef heifers, 23 (67.6%) of 34 beef bulls, and three (60%) of five dairy bulls. Mycoplasmas were isolated from 18 (7.5%) dairy cows, two (1.6%) beef cows, three (8.8%) beef bulls, and one dairy bull. No isolation was made from 97 aborted fetuses. For 65 dairy cows and 30 beef heifers with vulvar lesions, the isolation rates for ureaplasmas of 62.5% and 69.7%, respectively, were significantly higher (X²) than those for normal animals (37.5% and 30.3%). On immunofluorescent serotyping of 137 of the 205 isolates, there were 66 in serogroup C (strain T44), 18 in serogroup B (strain D48), eight in serogroup A (strain A417 or strain 2312), 14 cross-reacting, and 31 that were not identified. It was concluded that U. diversum is commonly present in the lower reproductive tract of beef/dairy cattle in Saskatchewan and is associated with granular vulvitis.

     

Polymerase chain reaction for detection of Ureaplasma diversum from urogenital swabs in cattle in Australia

Background Ureaplasma diversum has been associated with various reproductive problems in cattle, including granular vulvovaginitis, endometritis, salpingitis, early embryonic death, weak calves, decreased conception rates, balanoprosthitis, impaired spermatozoids and seminal vesiculitis in bulls. Methods This study briefly outlines the use of polymerase chain reaction (PCR) for the rapid detection of U. diversum directly from urogenital swabs collected from Australian beef cattle. Results The 16S ribosomal RNA gene sequences obtained from the PCR products of the clinical samples were closely related to U. diversum strain A417. Conclusion The present test enabled detection of the organism directly from clinical swabs collected from animals with or without lesions.     

Activation of macrophages

The role of macrophages is essential in the development of a normal immune response. Not only are these cells involved in the initiation of this response by presenting antigens to lymphocytes and by producing Interleukin I, but they also participate in the various phenomena of cellular co-operation and regulation. It is also evident that macrophages can act as cytotoxic effector cells, especially against micro-organisms and tumor cells. This last function is restricted to activated macrophages. The aim of this review is to summarize our present knowledge concerning this "macrophage activation".     

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.     

The Effect of Two Antibiotic Mixtures on Haemophilus somnus, Campylobacter fetus ssp. venerealis, Mycoplasma bovis, and Ureaplasma diversum in Frozen Bovine Semen

A two-step semen-extending protocol was compared to a one-step protocol in its efficiency in inhibiting growth of Haemophilus somnus, Campylobacter fetus ssp. venerealis, Mycoplasma bovis , and Ureaplasma diversum in experimentally infected semen. In both protocols, the effect of an antibiotic mixture of 500 μg gentamycin, 100 μg tylosin, 300 μg lincomycin, and 600 μg spectinomycin (GTLS) was compared to a mixture of 500 IU penicillin, 500 IU streptomycin, 150 μg lincomycin, and 300 μg spectinomycin (PSLS). The one-step extending method was as effective as the two-step extending method. Both antibiotic mixtures were equally effective in controlling C. fetus . For H. somnus and U. diversum , the PSLS mixture was more effective than the GTLS mixture. It was striking that both antibiotic mixtures had no effect in decreasing the numbers of M. bovis .     

Expression of procoagulant activity by equine lung macrophages: stimulation by blood lymphocytes

Increases in procoagulant activities (PCA) in equine lung macrophages were induced by non-adherent blood lymphocytes which were prestimulated with phytohaemagglutinin for 48 to 72 hours or by supernatants harvested from prestimulated blood lymphocyte cultures. However, prestimulated lymphocyte suspensions themselves expressed PCA which was most probably derived from contaminating monocytes. Because non-adherent cells from lymphocyte suspensions may have attached to adherent macrophages, cells within lymphocyte suspensions might have contributed to the PCAs expressed by lymphocyte-stimulated lung macrophages. Stimulation of lung macrophages for 24 hours by supernatants of phytohaemagglutinin-prestimulated blood lymphocytes induced a significantly greater PCA increase than stimulation by phytohaemagglutin alone. Thus, cytokines from lymphocyte cultures might have triggered or enhanced PCA induction. Direct stimulation of lung cell preparations with phytohaemagglutinin for 48 hours resulted in a progressive increase of PCA in only two of five specimens tested. The failure to induce PCA in three specimens could be due to the absence of sufficient numbers of T cells within the adherent lung cell preparations. In conclusion, PCA response of equine lung macrophages might be lymphocyte-stimulated in which case PCA might be a useful tool for monitoring the processes of cell-mediated immunity in horses.