The recovery of Mycoplasmas from the genital tracts of bulls in artificial breeding units in Ontar?o.

One hundred and thirty-two penial-preputial swabbings, 140 raw and 42 processed semen samples were cultured for mycoplasmas. Mycoplasma or acholeplasma were recovered from 87, 32 and one respectively, while ureaplasmas were recovered from 46, 34 and six respectively.     

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.     

Some observations on the semen of bulls persistently infected with bovine virus diarrhoea virus

As a result of screening procedures employed for animals entering the AI service, two bulls were identified as being persistently infected with bovine virus diarrhoea virus by isolation of the virus from blood. Semen was collected on two occasions from these bulls; its quality as measured by density and motility was poor. Gross abnormalities of the sperm head, termed 'collapsed' heads, were seen in 28 to 45 per cent of sperm from one bull and in 1 per cent of sperm from the other. The collapsed heads were small and the whole head or its anterior part had the appearance of a dried pea. Electron microscopy showed the defect to consist of convoluted nuclear material with membrane-bound vacuoles and invaginations containing membranous debris and lamellar structures. In the 'high incidence' bull there was a corresponding increase in enlarged sperm heads. The 'low incidence' bull had sperm with heads of similar mean size to sperm from control bulls but with an increased variance. The semen was diluted in a lactose diluent, frozen and stored in liquid nitrogen. The distribution of viral antigen was determined and virus was isolated from several fractions of the semen, both before and after processing and cryopreservation. In one animal raw semen failed to yield virus but virus was recovered after processing, suggesting that raw semen may not be suitable for the efficient detection of the virus.     

Mycoplasma mycoides subsp. mycoides SC identification by PCR in sperm of seminal vesiculitis-affected bulls.

In this study, by using the polymerase chain reaction (PCR) diagnosis for the detection and identification of Mycoplasma, we investigated mycoplasmas contaminating the semen of yearling bulls affected by seminal vesiculitis. The bulls presented neither subclinical nor clinical contagious bovine pleuropneumonia signs and the complement fixation test for specific antibodies was negative. Furthermore, we have investigated mycoplasmas isolated from semen of healthy breeding bulls of several breeds and origins, which routinely underwent breeding soundness examinations and presented no clinical signs of seminal vesiculitis. We were able to demonstrate mycoplasma infection in all tested samples by i) growth on mycoplasma-specific media and ii) a PCR-based method using a mycoplasma-specific MGSO/GPO1 primer set to amplify the 16S fragment rDNA. In addition, the identification of Mycoplasma species was made by PCR using the MSC1/MSC2 primer set that specifically amplifies M. mycoides subsp. mycoides SC or the MM450/MM451 primer set followed by AsnI digestion analysis in order to identify M. mycoides subsp. mycoides LC. The data presented herein clearly show that M. mycoides subsp. mycoides SC infection was associated with seminal vesiculitis while M. mycoides subsp. mycoides LC was only found in bull semen from healthy control animals. Our findings confirm that the M. mycoides subsp. mycoides SC is shed in the sperm making the ejaculate a valuable biological sample for the isolation of these bacteria from serologically negative animals. Although the pathogenic role of M. bovigenitalium in bull seminal vesiculitis has been established, our clinical findings, semen characteristics, microbiological and bacterial genomic analysis strongly suggest that M. mycoides subsp. mycoides SC may contribute to induce vesicular adenitis in the bull.     

Isolation of picornavirus from feces and semen from an infertile bull.

Virus was isolated from semen and fecal samples from a bull with orchitis, testicular degeneration, aspermatogenesis, and loss of libido. Both isolates were classified as picornavirus, bovine enterovirus serotype I, on the basis of physical, chemical, and serologic characteristics. Veterinary practitioners that may suspect viral infection as a cause of bovine infertility should submit both semen and fecal samples for virus isolation and identification.     

Rapid detection of bovine herpesvirus 1 in the semen of infected bulls by a nested polymerase chain reaction assay.

A nested polymerase chain reaction (PCR) assay was developed for the detection of bovine herpesvirus 1 (BHV-1) in bovine semen and compared with the virus isolation method. When extended semen, commonly used in the bovine artificial insemination industry, was inoculated with BHV-1, the PCR assay detected BHV-1 DNA in semen inoculated at 0.25-2.5 TCID50 per 0.5 mL. In contrast, the lower limit of detection for virus isolation was 250 TCID50 of BHV-1 inoculated in 0.5 mL of extended semen. These methods were also used to detect BHV-1 in the semen of four bulls which were experimentally infected with BHV-1. All infected bulls demonstrated balanitis at 3 d post-inoculation (DPI) and severe balanoposthitis at 4 DPI. BHV-1 was detected in raw semen by virus isolation and PCR at 2 DPI, before balanitis was evident. For virus isolation, the last day that BHV-1 was detected during primary infection was 7 DPI for two bulls and 9 and 11 DPI for the other two bulls. In contrast, PCR detected BHV-1 in the bulls' semen until 14 or 18 DPI. For individual animals, PCR detected BHV-1 during primary infection for at least 1-10 d longer than virus isolation. Reactivation of BHV-1 from latency without the presence of visible lesions was promoted twice by two series of 5 d dexamethasone injections. For the first series of dexamethasone treatments, a positive virus isolation result was obtained on the 5th d of treatment for only one bull. In contrast, two bulls demonstrated evidence of viral reactivation on this day by PCR. All bulls shed BHV-1 in semen on d 4 after dexamethasone treatment, as evidenced by positive virus isolation and PCR results. One bull was still PCR positive 13 d later. For the second series of dexamethasone treatments, a small amount of virus was isolated from semen collected on d 3 or 4 after treatment for two bulls but not from the other two bulls. In contrast, semen samples from all bulls were PCR positive for either or both of these 2 d. In total, from 80 semen samples, 45 were PCR positive and 26 were virus isolation positive. Thus, the PCR assay detected BHV-1 shedding in bulls earlier, more often, and for a longer duration, than did the virus isolation method.     

Study of the role of Chlamydia,Mycoplasma, Ureaplasma and other microaerophilic and aerobic bacteria in uterine infections of mares with reproductive disorders

In six healthy mares and 24 mares showing reproductive disorders swab samples were taken from the fossa clitoridis to isolate Taylorella equigenitalis, and from the uterus to isolate mycoplasmas, ureaplasmas and other aerobic bacteria. Swab samples were also taken from the uterus for Chlamydia antigen ELISA and Chlamydia PCR studies. The uterus of 27 mares was examined cytologically, and biopsy samples were taken from the endometrium for histological examinations and for immunohistochemical examinations aimed at the detection of chlamydiae. T. equigenitalis, mycoplasmas, ureaplasmas and chlamydiae could not be detected from any of the mares examined. Aerobic facultative pathogenic bacteria were isolated from mares with endometritis in four cases. In 18 out of 22 mares with endometritis (82%) no infective agents could be demonstrated. Further studies are needed to elucidate the relative importance of non-infectious causes of endometritis and of anaerobic bacteria often detectable in the uterus in the aetiology of the reproductive disorders observed.     

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.

     

Mycoplasma species and related organisms isolated from ruminants in Britain between 1990 and 2000

Between 1990 and 2000, more than 1600 mycoplasmas and the related acholeplasmas were identified from ruminant animals by the Mycoplasma Group at the Veterinary Laboratories Agency--Weybridge. Mycoplasma bovis was the most commonly identified pathogen, mostly from pneumonic calves but occasionally from cattle with mastitis and arthritis. Mycoplasma canis was first isolated in Britain in 1995 from pneumonic calves and the number of isolates increased to 18 per cent of the total mycoplasmas isolated from cattle in 1999. The ELISA for antibodies to M. bovis detected 1971 positive samples (22 per cent) among 8959 serum samples, mainly from pneumonic cattle. Other mycoplasmas identified included Mycoplasma dispar from the lungs of cattle with respiratory disease, and Mycoplasma bovigenitalium from the reproductive tract of cows with vulvovaginitis and infertility. Mycoplasma bovirhinis and Acholeplasma species were found commonly but are thought to be more opportunistic than pathogenic. In sheep and goats, the majority of Mycoplasma species isolated were identified as Mycoplasma ovipneumoniae from pneumonic sheep, Mycoplasma conjunctivae from sheep with keratoconjunctivitis, and the ubiquitous Mycoplasma arginini.     

Microbiological and serological studies on caprine pneumonias in Oman

Eight of 10 typical cases of contagious caprine pleuro-pneumonia in Oman yielded strain F38-like mycoplasmas from the lungs in high titre, but no other mycoplasmas: both negative animals had been treated with tylosin shortly before death. Among 21 other lungs examined three of six cases of acute pneumonia yielded Mycoplasma ovipneumoniae; one also yielded M capricolum. M ovipneumoniae was also isolated from all eight cases of chronic pneumonia sampled from an abattoir, and from the lungs of three animals which died without overt signs of pneumonia. A single isolate of M arginini and three of unidentified mycoplasmas were also obtained from goats with and without pneumonia. Various bacterial species were isolated, none of which predominated. Antibodies to M mycoides subspecies capri (M m capri) and strain F38 were detected in sera from eight different sources. Assuming titres of 1 in 40 or more as positive in the indirect haemagglutination test used, 29 per cent of 422 serum samples had antibodies to M m capri alone, 2.6 per cent to strain F38 alone and 3.6 per cent to both organisms. These results confirm the presence of F38-like mycoplasmas in Oman, and indicate also widespread infection with M m capri. The role of the latter in caprine pneumonias in Oman requires elucidation.     

Prevalence, causes and laboratory diagnosis of subclinical mastitis in the goat

Five goat herds were examined to determine the prevalence and causes of subclinical mastitis and to assess the value of some laboratory tests currently used on milk samples as aids in the diagnosis of caprine mastitis. In the 170 samples taken from the pairs of mammary glands of 85 goats, the prevalence of infection in the different herds ranged from 15 per cent to 79 per cent of halves. Just over one-third (36 per cent) of all halves were infected, the organisms isolated being coagulase-negative staphylococci (80 per cent), coagulase-positive staphylococci (16 per cent), alpha-haemolytic streptococci (2 per cent) and Pasteurella haemolytica (2 per cent). Neither anaerobic organisms nor mycoplasmas were found. Tests confirmed that the coagulase-positive staphylococci were pathogens but that the coagulase-negative staphylococci rarely caused detectable disease in the caprine udder. The large between-herd variation in the geometric mean cell counts of uninfected milk samples means that somatic cell counts, the Whiteside test and the California mastitis test, are unreliable as aids in the diagnosis of caprine mastitis.     

Effects of various antibiotics on the control of bacteria in boar semen

Nine antibiotics were tested for the control of 11 bacterial genera isolated from boar semen. Of the antibiotics tested, the aminoglycosides dibekacin, amikacin and gentamicin proved fairly effective. Minimum inhibitory concentrations of those antibiotics were low (less than 6.25 micrograms/ml) compared to those of antibiotics commonly used for semen diluents. Seventy-four semen samples were stored at 15 degrees C by means of dialysis in the presence of dibekacin (100 micrograms/ml). After seven days' storage, about 80 per cent of the samples showed no bacterial growth and the mean values for sperm motility an normal acrosomes were 75.4 per cent and 82.7 per cent respectively. Twenty sows which were inseminated with semen stored for up to 14 days produced 16 litters and the average litter size was 9.9.     

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.     

Isolation of mycoplasma species from the lower respiratory tract of healthy cattle and cattle with respiratory disease in Belgium

Between 1997 and 2000, a total of 150 healthy cattle and 238 animals with respiratory disease were examined for six Mycoplasma species. Attempts were made to detect Mycoplasma canis, Mycoplasma dispar and Ureaplasma diversum in calves with recurrent disease, and all three of these species were identified in calves with recurrent disease and in healthy lungs. In healthy calves, 84 per cent of bronchoalveolar lavage fluids were mycoplasma free; when cultures were positive, Mycoplasma bovirhinis was the only species isolated. Mycoplasmas were isolated from 78 per cent of animals suffering recurrent respiratory disease and from 65 per cent of acute respiratory cases. Mycoplasma bovis was isolated from bronchoalveolar lavages from 35 per cent of calves suffering recurrent respiratory disease, and from 50 per cent of acute cases, and from 20 per cent of pneumonic cases examined postmortem. M bovis was associated with other Mycoplasma species in 44 per cent of cases. M dispar was also isolated from 45.5 per cent of calves suffering recurrent respiratory disease, often in association with M bovis. M canis was identified for the first time in diseased Belgian cattle. Other mycoplasmas, including Mycoplasma arginini, Mycoplasma alkalescens and U diversum, were isolated less frequently. Associations between mycoplasmas and other pathogens were often observed. Among lungs infected with Pasteurella and/or Mannheimia species, more than 50 per cent were mixed infections with M bovis.     

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.     

ECOLOGY OF MYCOPLASMAS IN CLINICALLY HEALTHY CATS

Mycoplasmas were isolated from various sites and organs of a series of 319 clinically healthy cats. They include M. felis, M. gateae, M. Arginini, A. laidlawii, feline ureaplasmas, M. pulmonis, M. arthritidis, and M. gallisepticum. In addition, there were 10 strains of mycoplasmas which could not be identified with the specific antiserums by growth inhibition tests. Antibody against M. felis was demonstrated by haemagglutination-inhibition and complement-fixation tests in cats which were over 6 months of age. However, no such antibody against M. felis was detected in animals which were less than 6 months old. No antibody against A. laidlawii, M. gateae, M. arginini and feline ureaplasmas was demonstrated by the same serological methods. The significance of these mycoplasmas in cats is described.     

Libido, hormone concentrations in blood plasma and semen characteristics in Holstein bulls

Relationships among bull libido, serum hormone concentrations and semen characteristics were studied using 18 Holstein bulls that were 4 to 5 yr old. The hormones studied included testosterone, estradiol (E2), prolactin (PRL), LH and cortisol. Two ejaculates were collected three times per week from each bull during a 5-wk trial. During the last week of the trial, on a day semen was not collected, blood was collected from indwelling catheters every 15 min for 6 h to determine the hormonal profiles of each bull. On the following day, blood was sampled every 10 min before and after the time of semen collection. Libido factors were quantified, and semen volumes and sperm concentrations were recorded. The libido factors included reaction time to first service, latency time between the first and second semen collections, and duration of time the bull mounted the teaser prior to the first (TM1) and second (TM2) semen collection. Average reaction and latency times were correlated (r = .524; P = .026), as were TM1 and TM2 (r = .597; P = .015). Latency times were correlated with average TM2 (r = .669; P = .003). Average PRL concentrations were correlated with average latency times (r = .467; P = .05). Low libido bulls tended to have higher E2:testosterone ratios than did high libido bulls. Both PRL and cortisol concentrations increased at semen collection.     

Boar Semen Studies: II. Laboratory and Fertility Results of a Method for Deep Freezing

A successful method for low temperature preservation of bull semen was modified for use with boar semen and resulted in recovery of twenty to fifty per cent motile cells immediately after thawing. Recovered cells did not survive five hours incubation at 37° C. and no pregnancies resulted following insemination of twenty-four sows and gilts with frozen semen.