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)

Studies on inactivation of pathogenic microorganisms in culture media and in bovine semen by photosensitive agents.

The application of three photosensitive agents for disinfection of bovine semen was investigated. Bovine microbial pathogens suspended in tissue culture medium and/or PBS and also added to bovine semen were exposed to the photosensitive agents followed by irradiation. Hematoporphyrin, hematoporphyrin derivative and thiopyronine were effective against bovine herpes virus-1, bovine viral diarrhoea virus, Mycoplasma bovigenitalium, Mycoplasma canadense, and Ureaplasma diversum in culture media. In addition, thiopyronine was effective against Leptospira pomona. Similar treatments were not effective against Leptospira hardjo, Mycoplasma bovis, or Campylobacter fetus subsp. venerealis. When microorganisms were added to bovine semen, only bovine herpes virus-1 was controlled by the photosensitive agents used at concentrations which did not appear harmful to sperm cells.

Mycoplasma phocidae sp. nov., isolated from harbor seals (Phoca vitulina L.).

In 1979 and 1980, more than 400 harbor seals (Phoca vitulina) along the New England coast of the United States died of epizootic pneumonia that was attributed to an influenza virus. Six mycoplasma isolates that were recovered from the respiratory tracts of affected seals were investigated and were found to be serologically identical and distinct from previously described species. These isolates required serum for growth, did not possess a cell wall, and did not hydrolyze urea. Arginine was hydrolyzed, glucose was not fermented, film and spots were observed on horse serum agar, phosphatase was produced, tetrazolium was not reduced, and serum and casein were not digested. The guanine-plus-cytosine content of the DNA was 27.8 mol%. We propose the name Mycoplasma phocidae for these isolates. The type strain of M. phocidae is strain 105 (= ATCC 33657).

Common sulfoglycolipid receptor for mycoplasmas involved in animal and human infertility.

Sulfoglycolipids are ubiquitous components of the male germ cell membrane. Sulfogalactoglycerolipid (SGG) is restricted to mammalian cells and has recently been implicated in sperm/egg interactions. Mycoplasma infections have been implicated in infertility in a variety of species, including humans. Four such species-specific mycoplasmas, Ureaplasma urealyticum and Mycoplasma hominis (humans), Mycoplasma pulmonis (rodents), and Ureaplasma diversum (cattle) are not shown to specifically recognize SGG and the sphingolipid counterpart, sulfogalactosyl ceramide. This glycolipid receptor binding may relate to the reproductive pathogenesis of these organisms.

Isolation of Mycoplasma bovis from intact and microinjected preimplantation bovine embryos washed or treated with trypsin or antibiotics.

Incubation of day 7 bovine embryos with 10(4) or 10(6) CFU/ml of Mycoplasma bovis (M. bovis) or microinjection of M. bovis into the cells of day 7 embryos did not influence embryonic development. M. bovis was recovered from all embryos washed 10 times by a standard pipetting method or vortexed and pipeted 10 times. M. bovis was also recovered from zonae pellucidae removed and washed from microinjected embryos. Neither treatment with trypsin nor exposure of embryos to combinations of penicillin, streptomycin, lincomycin and spectinomycin, or gentamicin, tylosin, lincomycin, and spectinomycin, inactivated M. bovis.

Protein A gold identification of ureaplasmas on the bovine zona pellucida.

The object of this study was to develop a prefixation protein A gold labelling technique for Ureaplasma diversum and to apply this to bovine embryos. Sixteen hour cultures of Ureaplasma diversum strain 2312 were incubated with either specific antiserum or nonimmune serum, followed by exposure to protein A gold and negative staining. The ureaplasmas which were incubated with specific antiserum were labelled with gold particles while those ureaplasmas which were incubated with nonimmune serum were not labelled. Twenty-three unhatched, day 7 bovine embryos were then incubated in either embryo culture medium (ECM) alone, ECM with sterile ureaplasma broth added or ECM with 1.7 X 10(6) colony forming units of Ureaplasma diversum strain 2312 per embryo. After 16 hours, the embryos were washed twice and incubated with either specific antiserum or nonimmune serum. The embryos were then incubated with medium containing protein A gold and examined by electron microscopy. No ureaplasmas were identified on the zona pellucida of the control embryos. Ureaplasmas were identified on the outer surface of the zona pellucida of 13 of the 17 embryos which had been exposed to the organism. Of these, the embryos which were incubated with specific antiserum had labelled ureaplasmas while the embryos which were incubated with nonimmune serum had unlabelled ureaplasmas on the zona pellucida. It was concluded that the protein A gold method was a suitable technique for the identification of ureaplasmas in EM preparations. The presence of ureaplasmas on the outer surface of the bovine zona pellucida following in vitro exposure to the organism was confirmed.(ABSTRACT TRUNCATED AT 250 WORDS)

A new antibiotic combination for frozen bovine semen 1. Control of mycoplasmas, ureaplasmas, Campylobacter fetus subsp. venerealis and Haemophilus somnus.

Systematic evaluations of new combinations of antibiotics for the control of bovine mycoplasmas, ureaplasmas, Campylobacter fetus subsp. venerealis and Haemophilus somnus in a bovine frozen semen process were made. These organisms were standardized to 10(5) to 10(6) colony forming unit (CFU) and inoculated into each ml of raw semen. Antibiotics in a final volume of 0.02 ml were added to each ml of the raw semen and were contained at the same concentration in the nonglycerol portion of the extenders (whole milk, 20% egg yolk citrate, 20% egg yolk tris, Plus-X, and 28% egg yolk tris). The combination of gentamicin (500 ug/ml) tylosin (100 ug/ml) and Linco-Spectin (300/600 ug/ml) was more effective for the control of mycoplasmas and ureaplasmas and equally effective for the control of C. fetus subsp. venerealis and Haemophilus somnus than the standard combination of penicillin, dihydrostreptomycin and polymyxin B sulfate.

The recovery of ureaplasmas from bovine embryos following in vitro exposure and ten washes.

Twenty-six unhatched embryos and ova were exposed to Ureaplasma diversum strain 2312 in vitro for 16 h and subsequently washed ten times. Fifteen of the embryos and their wash fluids were cultured for ureaplasmas. Of the remaining 11 embryos, six were incubated with rabbit anti-Ureaplasma immunoglobulin (RAI) and five were incubated with serum from naive rabbits(NRS), after which all were incubated with protein A gold and prepared for electron microscopy. On ultrastructural examination, ureaplasmas were observed on the outer surface of the zona pellucida of all 11 embryos. The ureaplasmas on the six embryos incubated with RAI were labeled with gold particles, while those on the five embryos incubated with NRS were not labeled. Ureaplasmas were recovered from all 15 of the cultured embryos and all of the first and second wash fluids as well as intermittently from the third, fourth, sixth, seventh, eighth and ninth wash but not fom the fifth or tenth wash. It was concluded that viable ureaplasmas adhered to the zona pellucida during in vitro exposure of bovine embryos and were not removed by ten washes.

Mycoplasma infection of the hand acquired from a cat.

Mycoplasmas, the smallest known organism capable of a free existence, have been recognized as human pathogens for 25 years. However, a soft tissue cellulitis caused by a mycoplasma has never been reported in a human subject. This case report of a mycoplasma infection of the hand acquired from an infected cat describes the clinical presentation, operative findings, mycoplasmology, and treatment of this infection.

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.

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)

Experimental studies on the pathogenicity of Mycoplasma ovipneumoniae and Mycoplasma arginini for the respiratory tract of goats.

Mycoplasma ovipneumoniae and Mycoplasma arginini were the species of Mollicutes most commonly isolated from 175 goats with respiratory disease in Ontario. The pathogenicity of M. ovipneumoniae, strain B321B and M. arginini, strain D53e, was assessed in goats following endobronchial inoculation. One out of three two year old goats developed fever after inoculation with a pure culture of strain B321B, and it had extensive subacute fibrinous pleuritis when necropsied three weeks later. Neither of the remaining goats had lesions in the respiratory tract. Mycoplasma ovipneumoniae was recovered from one of the animals four days after inoculation, but not at necropsy from any of the goats, at which time a marked humoral immune response with growth inhibiting antibodies was detected. In a second experiment three four to five week old goats were inoculated with the same strain and three other goats were given placebo treatment. One experimental goat developed fever and coughing, and it had extensive subacute fibrinous pleuritis in the right side and pneumonia. Another goat had focal pneumonia in the left diaphragmatic lobe. Microscopically there was subacute hyperplastic suppurative bronchiolitis, atelectasis and nonsuppurative alveolitis. The infected animals did not clear the mycoplasma and not all of them produced antibodies. Mycoplasma arginini, strain D53e, did not induce lesions in any of four goat kids within 14 days after inoculation but did cause transient elevations in rectal temperature, circulating monocytes, circulating neutrophils and blood fibrinogen. Mycoplasma arginini was infective and immunogenic for all inoculated animals and showed a particular affinity for the tonsil. Thus, this study provides the first evidence that M. ovipneumoniae is pathogenic for goats causing pneumonia and pleuritis.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of antibiotics against bovine mycoplasmas and ureaplasmas.

A combination of lincomycin-spectinomycin-tylosin was tested against several strains of mycoplasmas and acholeplasmas as might be encountered in bovine semen and shown to be effective against them. This combination as well as minocin , rosaramicin, rosoxacin, tiamulin, gentamicin and declomycin were tested in vitro against 58 isolates of ureaplasma from the bovine urogenital tract. The lincomycin-spectinomycin-tylosin combination, minocin , rosaramicin, tiamulin and declomycin were quite active, while rosoxacin and gentamicin were much less active against the test strains.

Bovine abortion and neonatal death associated with Ureaplasma diversum.

Ureaplasma diversum was isolated from the lungs and/or stomach fluid and placentas of five aborted bovine fetuses and four newborn calves. All isolates were serotype D48. Placentitis was observed in all instances in which the placenta was examined. Gross lesions consisted of focal or diffuse reddening of the chorioallantois and amnion and thickening of the amnion. Microscopically there were fibrosis, edema and inflammation of the amnion. Microscopic lesions in the lung consisted of diffuse pneumonitis with thickening of the alveolar walls and in some cases peribronchiolar lymphoid accumulations. Macrophages and granulocytes were present in the alveoli. Inoculation of the vulva of a virgin heifer with one of the isolates from a fetal lung produced hyperemia and profuse purulent discharge with slight granularity.

The effects of Ureplasma diversum inoculated into the amniotic cavity in cows.

Ureaplasma diversum was inoculated into the amniotic cavity in four cows. Two calves were aborted and two were born alive. One of the latter died shortly after birth and the other was killed. The cows remained clinically normal except that three retained their placenta. On microscopic examination there was a severe placentitis and an alveolitis was present in the lungs of all calves. Ureaplasma was recovered from four placentas and three lungs. Cows remained infected for a maximum of 132 days following inoculation and the organism was recovered in urine and vulvar swabs for a maximum of 17 and 60 days respectively following expulsion of the calf. Ureaplasma diversum has been isolated from natural cases of abortion with similar lesions. This experiment strongly supports a causal relationship between abortion, birth of calves with pneumonia and U. diversum infection.

Isolation of Mycoplasma mycoides subspecies mycoides from Polyarthritis and Mastitis of Goats in Canada.

The clinical signs, pathomorphological changes, and microbiological findings in Canadian goats infected with Mycoplasma mycoides subspecies mycoides are discussed. The disease affected mainly young goats and was characterized by septicemia and polyarthritis. Mastitis followed by septicemia was seen in two mature goats. The diagnosis was made by culture and identification of the mycoplasma. Infected goats without clinical signs were identified by cultural and serological (complement fixation) techniques. Healthy carriers are presumably able to transmit the infection and may have brought the disease to Canada.

The pulmonary clearance of Pasteurella haemolytica in calves infected with bovine virus diarrhea or Mycoplasma bovis.

Based on current literature which commonly associates bovine virus diarrhea virus and Mycoplasma bovis with "pneumonic pasteurellosis," an investigation was conducted into the effect of these two pathogens on the capacity of bovine lung to clear inhaled Pasteurella haemolytica. There was no significant effect (p less than 0.05) of either bovine virus diarrhea virus or M. bovis on the mean clearance rate of P. haemolytica, nor did the time interval of three, five or seven days between the first inoculation and exposure to P. haemolytica and adversely affect the lung clearance rates. However, it was found that the left lungs and a higher bacterial retention (p less than 0.05) than the right lungs.

Mass mortality of harbor seals: pneumonia associated with influenza A virus.

More than 400 harbor seals, most of them immature, died along the New England coast between December 1979 and October 1980 of acute pneumonia associated with influenza virus, A/Seal/Mass/1/180 (H7N7). The virus has avian characteristics, replicates principally in mammals, and causes mild respiratory disease in experimentally infected seals. Concurrent infection with a previously undescribed mycoplasma or adverse environmental conditions may have triggered the epizootic. The similarities between this epizootic and other seal mortalities in the past suggest that these events may be linked by common biological and environmental factors.

The genital Mycoplasma and Ureaplasma flora of healthy and diseased dogs.

The genital mycoplasma and ureaplasma flora was compared in 136 dogs with varied reproductive histories. Mycoplasmas were recovered from 88% of vulvovaginal swabs, 85% preputial swabs and 72% semen samples. Isolation rates were slightly higher from dogs that were infertile or had evidence of genital disease but the differences from those that were fertile or clinically normal were statistically significant only in the male. Ureaplasmas were recovered from half the females sampled. Higher, but not statistically significant isolation rates (75%) were made from infertile females with purulent vulvar discharge versus those that were clinically normal and fertile (40%). In the male dog there was a significantly higher incidence of ureaplasmas in the prepuce of infertile animals (69%) than those that were fertile (0%) (p less than or equal to 0.05). Semen isolations although not significantly higher in infertile males, were all made from ejaculates, with subnormal motility, low sperm counts and/or a high percentage of midpiece and tail abnormalities (bent or tightly coiled).