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.

     

Campylobacter fetus in artificial insemination unit and slaughterhouse bulls in Ontario.

Preputial fluid samples were collected from 90 bulls in two Ontario artificial insemination units using a penial glove swab technique previously developed by one of us for use in donor bulls. No Campylobacter fetus organisms were identified from the prepuce or from samples of semen collected at the same time from these bulls. The distal genitalia of 200 bulls were collected at a slaughter house. One isolation of a Campylobacter fetus subspecies venerealis was obtained on a culture from the fornix area of the prepuce of one of these bulls.     

STUDIES ON VENEREAL TRANSMISSION OF CAMPYLOBACTER FETUS BY IMMUNISED BULLS

Temporary contamination of the penis and prepuce with Campylobacter fetus subsp. venerealis and Campylobacter fetus subsp. venerealis biotype intermedius may occur when immunised bulls mate with infected heifers. However, 2 experiments showed that this contamination was not a significant cause of genital infection in susceptible heifers that subsequently mated with bulls.     

In vitro antimicrobial sensitivity of Mycoplasmas isolated from the bovine genital tract.

The in vitro antimicrobial sensitivity of 14 mycoplasma and 13 ureaplasma strains isolated from the genital tracts of bulls was examined. It was found that at relatively low concentrations, tetracycline, declomycin and tylosin were lethal to both types of organisms. Lincospectin, berenil, streptomycin and erythromycin were lethal to mycoplasmas but were only inhibitory to the ureaplasma strains at the same concentrations. Polymyxin B and novobiocin were ineffective at the levels tested.     

Sites of Persistence of Lumpy Skin Disease Virus in the Genital Tract of Experimentally Infected Bulls

The objectives of this work were to determine the site of persistence of lumpy skin disease virus (LSDV) in bulls shedding the virus in semen for a period longer than 28 days, to determine if the virus is present in all fractions of semen and to study lesions that developed in the genital tract. Six serologically negative postpubertal bulls were experimentally infected with a virulent field isolate of LSDV. The polymerase chain reaction (PCR) was performed on sheath washes, vesicular fluid, supernatant and cell‐rich fractions of semen from day 10 to day 26 postinfection (p.i.). Bulls that were positive by PCR on the whole semen sample collected on day 28 p.i. were slaughtered and tissue samples from their genital tracts submitted for histopathological evaluation, immunoperoxidase staining, virus isolation and PCR. Two of the bulls developed severe lumpy skin disease (LSD) and were found to be shedding viral DNA in their semen on day 28 p.i. Viral DNA was identified in all semen fractions from all bulls, but mostly from the cell‐rich fraction and from the severely affected bulls. The PCR assay was positive on postmortem samples of testes and epididymides from the two severely affected bulls. Virus could be recovered from the testes of these two bulls and from the epididymis of one of them. Immunoperoxidase staining was positive for LSDV staining in sections of testes and epididymides exhibiting necrosis. This study suggests that the testis and epididymis are sites of persistence of LSDV in bulls shedding virus in semen for prolonged periods and revealed that viral DNA is present in all fractions of the ejaculate.     

Prevalence of mycoplasmas in the semen and vaginal swabs of Danish stallions and mares

The reproduction rate of horses is one of the lowest within domestic livestock despite advances the veterinary medicine. Infertility in horses may be due mainly to the lack of suitable selection criteria in the breeding of horses. However, acquired infertility due to genital, bacterial infections may occur. Mycoplasmas have been implicated in genital disorders and infertility of many species including humans and horses. However, their role as commensals or pathogens of the genital tract of horses is still not determined. Bacteriological examinations made on the fossa glandis, urethra, penis and semen of stallions, showed the presence of different Mycoplasma species. Therefore our study aimed to find the prevalence of Mycoplasma species and a possible association with fertility problems in Danish riding horses. Eighty semen samples from stallions and 19 vaginal swab samples from mares were tested by PCR for presence of mycoplasmal DNA. The vaginal swab samples were also cultured in the Mycoplasma specific medium. None of the samples were positive for presence of genital mycoplasmas during the screen. The lack of genital mycoplasmas observed in this study may be due to a very extensive use of artificial insemination of modern sport horses.     

High prevalence of mycoplasmas in the genital tract of asymptomatic stallions in Austria

Mycoplasma equigenitalium and M. subdolum have been implicated in genital disorders and infertility of horses. The reported cytopathic effects of M. equigenitalium observed in vitro underscore its potential pathogenic role in reproductive dysfunction in mares. This study was initiated to determine the prevalence of mycoplasmas in the genital tract of stallions in relationship to age, clinical signs, geographic location and semen quality. For this purpose the mycoplasma flora of the genital tract of 116 stallions of the Noric breed was determined by isolation and colony immunoblotting and by polymerase chain reaction (PCR) assays. Of 438 swabs from the genital tract, pre-ejaculatory fluid and semen samples, 352 (80%) samples were positive by PCR and 125 (29%) were positive by culture. Mycoplasmas were isolated predominantly from the fossa glandis and urethra and less frequently from the penis shaft and from semen. M. equigenitalium (89 isolates) and M. subdolum (70 isolates) were the predominant species identified. M. equirhinis and M. felis were detected in 27 and 8 samples, respectively. Comparison of these isolations with clinical signs, semen quality, and age of the stallions revealed no significant correlation. However, geographical location of the stallion significantly correlated with mycoplasma detection. These results suggest that mycoplasmas are present as commensals in the genital tract of stallions. Thus, clinically healthy stallions may present a permanent reservoir for infection of mares via venereal transmission.     

Sample collection factors affect the sensitivity of the diagnostic test for Tritrichomonas foetus in bulls

The current diagnostic test for Tritrichomonas foetus involves the culture of collected preputial or vaginal samples. In an earlier study, which evaluated sampling tools for use with bulls, it was observed that the sensitivity of the diagnostic test was higher for 2nd samples collected from the right side of the prepuce than it was for samples collected 1st from the left side. The study described in this paper was conducted to evaluate which of these factors was responsible for the effect on diagnostic sensitivity. Twenty-nine bulls infected with T. foetus were repeatedly sampled in a 2-factor cross-over design. Samples taken from the right side of the prepuce were 4 times as likely to be positive as samples taken from the left side (P = 0.03). Other factors did not have a significant effect on the outcome of the diagnostic test. Unexpected factors may affect the sensitivity of the diagnostic test for T. foetus.     

Mycoplasmosis: experimental inoculation of calves with a Danish strain of Mycoplasma bovigenitalium

During the fall of 1966 several strains of Mycoplasma were isolated from semen samples of bulls. A strain, “K”, isolated from a bull with vesiculitis was selected for a series of experiments and proved to be pathogenic for the mammary gland of cows and for the genital tract of bulls (Ernø 1967, Blom & Εrnø 1967). The “K” strain has now been examined serologically by the author, and it must be considered as belonging to the species M. bovigenitalium as determined by complement fixation and indirect haemagglutination tests. This communication reports the results of experimental inoculation of calves.     

Mycoplasmosis: Demonstration of Pathogenicity of a Danish Strain of Mycoplasma

During the fall of 1966 several strains of Mycoplasma were isolated from semen samples of bulls. A strain “K” isolated from a bull with simultaneous occurrence of leucocytes and mycoplasmas in the semen was selected for a series of experiments; the mammary gland was chosen as pilot organ.     

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.     

Bovine Genital Mycoplasmosis

Infection, lesions and clinical significance of Acheloplasmas, Mycoplasma bovis and Mycoplasma bovigenitalium in genital disease of cattle are described. A more detailed account is given of ureaplasma infections. Acute and chronic forms of granular vulvitis in both field and experimental disease are described as well as the role of the organism in abortion.

Recovery rates of ureaplasma and mycoplasma from semen and preputial washings in bulls are outlined and their significance in disease is discussed. There are problems in differentiating pathogenic from nonpathogenic isolates. Methods are being developed to treat semen for these organisms.

This paper provides a concise summary of clinical and microbiological aspects of bovine genital mycoplasmosis.

     

The course of mastitides in cows infected into the mammary gland with strains of Mycoplasma bovigenitalium and M. bovis]

Six cows were inoculated into the mammary gland with eight mycoplasma strains isolated from the genital tract of bulls and two type strains. The milk of cows infected with Mycoplasma bovigenitalium strains isolated from the genital tracts of bulls showed a change in the appearance and contained large quantities of mycoplasmas and specific antibodies. The mastitis was most intense in about 9 days and began to subside in 17 days infection. The type strain of M. bovigenitalium PG11 failed to produce mastitis. On the other hand, the type strain of M. bovis PG45 produced severe mastitis after a 14-day latency period, with the infection spreading to the uninoculated quarters, causing atrophy of the mammary gland, and persisting till slaughter. The sera of all cows that developed mastitis after experimental infection contained high titres of specific antibodies. The two infecting mycoplasma species were recovered from the inner organs and mammary glands of these cows after slaughter.     

Investigation of Chlamydiaceae in semen and cauda epididymidis and seroprevalence of Chlamydophila abortus in breeding bulls

Background

Reproductive disorders associated with chlamydial infection have been reported worldwide in cattle and there are indications of potential venereal transmission.

Methods

Semen samples from 21 dairy bulls and cauda epididymidis tissue samples from 43 beef bulls were analysed for chlamydial agent by real-time polymerase chain reaction (PCR) including an internal amplification control (mimic). Additionally, presence of antibodies against Chlamydophila (Cp.) abortus among the bulls was investigated with the commercial Pourquier^® ELISA Cp. abortus serum verification kit.

Results

No chlamydial agent was detected by PCR in either the semen samples or in the tissue samples. Additionally, no antibodies against Cp. abortus were detected.

Conclusions

The results suggest that Cp. abortus is very rare, or absent in Swedish bulls and thus the risk for venereal transmission of chlamydial infection through their semen is low. However, because Chlamydophila spp. infection rates seem to differ throughout the world, it is essential to clarify the relative importance of transmission of the infection through semen on cattle fertility.     

CONGENITAL AND DEVELOPMENTAL ANOMALIES OF THE GENITALIA OF SLAUGHTERED BULLS

The genital tracts of 968 slaughtered bulls (46% of which were young post-puberal animals) were examined for defects of a congenital or developmental nature. The overall occurrence of such lesions was 7%. These comprised persistent penile frenulum (0.5%), hypospadias (0.3%), detached urethral process (0.4%), testicular hypoplasia (0.2%), cryptorchidism (0.6%), mesonephric duct abnormalities (1.1%) and bulbourethral cysts, fusion and aplasia (3.6%). Segmental aplasia of the mesonephric duct, not previously recorded in the study area, was found in 4 Shorthorn bulls (0.4%); 2 affected animals were from one herd. In 3 cases of hypospadias (2 from one herd), the urethra communicated with the ventral surface of the penis at the junction of the body and glans through a slit-like orifice. The occurrence of defects observed was generally comparable to that found in bull populations elsewhere but elevated occurrence of several defects in particular herds emphasized the need for further study.     

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.     

Effects of reduced glutathione supplementation in semen freezing extender on frozen-thawed bull semen and in vitro fertilization

During cryopreservation, spermatozoa may suffer cold and cryo-induced injuries ―associated with alterations in cell defense systems― that are detrimental to their function and subsequent fertility. This study aimed to determine the efficacy of supplementing the semen freezing extender with the antioxidant reduced glutathione (GSH) in cattle. Semen was collected from four bulls and diluted in a freezing extender supplemented with or without GSH (0, 1, 5, and 10 mM) before the cooling step of the cryopreservation process. After thawing, the quality of the frozen-thawed semen was investigated for motility, viability, acrosomal and DNA integrity, and subsequent embryo development after in vitro fertilization of bovine oocytes. Additionally, semen from one of the bulls was used to analyze semen antioxidative potential, sperm penetration into oocytes, male pronucleus formation rate, and embryo DNA integrity. The sperm quality varied among bulls after GSH supplementation. One bull had decreased sperm total motility, and two bulls had decreased sperm DNA integrity. GSH supplementation had positive effects on embryo development for three bulls. Two of them showed both improved cleavage and blastocyst formation rates, while the other one only showed an improved cleavage rate. We observed positive effects on early male pronucleus formation and no negative effects on DNA integrity and cell number in blastocyst stage embryos. Although the effect varies depending on individual bulls and GSH concentration, GSH supplementation in semen may improve in vitro embryo production from frozen semen.     

Evaluation of a Campylobacter fetus subspecies venerealis real-time quantitative polymerase chain reaction for direct analysis of bovine preputial samples

The detection and subspeciation of Campylobacter fetus subsp. venerealis (CFV) from veterinary samples is important for both clinical and economic reasons. Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, a venereal disease that can lead to serious reproductive problems in cattle, and strict international regulations require animals and animal products to be CFV-free for trade. This study evaluated methods reported in the literature for CFV detection and reports the translation of an extensively tested CFV-specific polymerase chain reaction (PCR) primer set; including the VenSF/VenSR primers and a real-time, quantitative PCR (qPCR) platform using SYBR Green chemistry. Three methods of preputial sample preparation for direct qPCR were evaluated and a heat lysis DNA extraction method was shown to allow for CFV detection at the level of approximately one cell equivalent per reaction (or 1.0 × 10³ CFU/mL) from prepuce. The optimized sample preparation and qPCR protocols were then used to evaluate 3 western Canadian bull cohorts, which included 377 bulls, for CFV. The qPCR assay detected 11 positive bulls for the CFV-specific parA gene target. DNA sequence data confirmed the identity of the amplified product and revealed that positive samples were comprised of 2 sequence types; one identical to previously reported CFV parA gene sequences and one with a 9% sequence divergence. These results add valuable information towards our understanding of an important CFV subspeciation target and offer a significantly improved format for an internationally recognized PCR test.     

Isolation of Acholeplasma laidlawii and Mycoplasma equigenitalium from stallion semen

Forty semen samples from 40 stallions were examined for the presence of mycoplasmas. Mycoplasmas were isolated from 18 samples of semen, either immediately after collection or 3 months after storage at -20°C or in both cases. Ten of the isolates could be typed, nine as Mycoplasma equigenitalium and one as Acholeplasma laidlawii. Eight of the 18 isolates were lost during cultivation and storage; they were all glucose-positive and arginine-negative.     

Evidence of excretion of Schmallenberg virus in bull semen

Schmallenberg virus (SBV) is a novel orthobunyavirus, discovered in Germany in late 2011. It mainly infects cattle, sheep and goats and could lead to congenital infection, causing abortion and fetal abnormalities. SBV is transmitted by biting midges from the Culicoides genus and there is no evidence that natural infection occurs directly between ruminants. Here, we could detect SBV RNA in infected bull semen using qRT-PCR (three bulls out of seven tested positive; 29 positive semen batches out of 136). We also found that highly positive semen batches from SBV infected bulls can provoke an acute infection in IFNAR^-/- mice, suggesting the potential presence of infectious virus in the semen of SBV infected bulls.