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1.  Experimental Bovine Genital Ureaplasmosis II. Granular Vulvitis, Endometritis and Salpingitis Following Uterine Inoculation 
Twenty-three virgin Holstein heifers received uterine inoculations with ureaplasma and were necropsied one to thirteen days later. Three heifers inoculated intracervically were necropsied on days 3, 5 and 11.
Granular vulvitis was produced on average by 3.6 days in fourteen of sixteen uterine inoculated heifers monitored for four or more days. Two cervically inoculated heifers monitored for over four days also developed granular vulvitis by the fourth day.
At necropsy, ureaplasma was recovered from 94% of uterine horn cultures for the first four days postinoculation and 50% during days 5 to 7. Thereafter all uterine cultures were negative. The percentage of positive ureaplasma recoveries from uterine tube flushings was lower than for uterine horns but remained positive for a longer period. By day 7, three of four uterine tube flushings were still positive. No bacterial pathogens were isolated from the uterine horns or uterine tube flushings.
On histopathology 50% of uterine inoculated heifers had endometritis up to six days postinoculation and a slightly higher percentage (58%) had salpingitis. Endometritis was not found in any heifers after day 6. Residual salpingitis was present in one heifer on day 7. Endometritis was present in cervically inoculated heifers necropsied on days 3 and 5 but not on day 11. Salpingitis was not found in any of the three cervically inoculated animals.
The study concluded that some strains of ureaplasma are pathogenic for the upper reproductive tract of the cow and should be considered significant when isolated from cases of granular vulvitis, endometritis or salpingitis.
PMCID: PMC1320071  PMID: 7427773
2.  Isolation of Ureaplasma from bovine granular vulvitis. 
Cultures for mycoplasmatales, viruses and bacteria were made from bovine vulvar swabs to determine whether ureaplasma was associated with a clinical granular vulvitis observed in 16 Ontario dairy herds. Ureaplasma was isolated from 23.5% of 34 clinically normal cows, 74% of 27 cows with mild to moderate vulvar hyperemia but no discharge and 100% of 20 cows with acute vulvar hyperemia accompanied by purulent discharge. There were statistically significant differences in rates of isolation among clinical groups. Mycoplasma bovigenitalium was isolated from 7.7% and 20% of cows with moderate or acute vulvitis respectively but not from normal cows. Haemophilus somnus was isolated from 25% of cows with acute vulvitis. There were no significant differences in isolations of Escherichia coli, Corynebacterium pyogenes and alpha-hemolytic streptococcus between normal and clinically affected animals. Cultures of 135 repeat samples from 33 cows revealed that ureaplasma persisted in some animals for at least three months. No viruses were isolated from any of the animals in this study.
PMCID: PMC1277608  PMID: 352491
3.  Bovine Granular Vulvitis Associated with Ureaplasma Infection 
A granular vulvitis syndrome associated with ureaplasma infection was first recognized in Ontario dairy herds in 1972.
The acute form of the disease was characterized by a purulent vulvar discharge, an inflamed hyperemic vulvar mucosa and varying degrees of granularity. In the chronic form, there was an absence of a purulent discharge and a gradual decline in the severity of the hyperemia and granularity. Epithelial inclusion cysts were observed in the vulvar epithelium of approximately 10% of affected cows.
A seasonal variation in the incidence of the disease was observed. Herd morbidities during the summer months reached a low of 37% and increased to 75% during the winter months with constant housing.
When widespread in herds, the acute form of the disease had a significant effect on fertility. In four herds examined, first service conceptions dropped on average by 27%.
The chronic form of the disease had a less detrimental effect on fertility with first service conceptions being reduced on average by 13%.
Intrauterine infusions of a tetracycline 24 hours postbreeding were found to be of value in improving conception rates in acutely affected herds.
PMCID: PMC1789526  PMID: 427710
4.  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.
PMCID: PMC1481172  PMID: 17423929
5.  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).
PMCID: PMC1320213  PMID: 7340908
6.  Vulvitis plasmacellularis: two new cases. 
Genitourinary Medicine  1995;71(5):311-313.
Vulvitis chronica plasmacellularis or Zoon's vulvitis is a rare benign circumscribed inflammation of the vulvar mucosa. It is found in women ranging in age from 26 to 70 years. Shiny, macular erythematous lesions, which are irregular in shape and sharply marginated are usually observed. The histologic findings show chronic subepithelial dense inflammation composed largely of plasma cells. We here report two cases of vulvitis plasmacellularis with typical clinical manifestations, courses and histopathologic findings.
PMCID: PMC1195548  PMID: 7490049
7.  Characterization of a Murine Model of Ureaplasma urealyticum Pneumonia  
Infection and Immunity  2002;70(10):5721-5729.
Ureaplasma urealyticum respiratory tract colonization in preterm infants has been associated with a high incidence of pneumonia and the development of bronchopulmonary dysplasia. However, study of this human pathogen has been hampered by the absence of animal models. We have developed the first juvenile mouse model of Ureaplasma pneumonia and characterized the histopathology during the month following inoculation. C3H/HeN mice were inoculated intratracheally with a mouse-adapted clinical Ureaplasma isolate (biovar 2) or sham inoculated with 10B broth. Culture of lung homogenates and PCR of DNA from bronchoalveolar lavage fluid (BAL) confirmed the presence of Ureaplasma in 100% of inoculated animals at 1 day, 60% at 2 days, 50% at 3 days, and 25% at 7 and 14 days. Ureaplasma was undetectable 28 days postinoculation. There were marked changes in BAL and interstitial-cell composition with increased number of polymorphonuclear leukocytes 1 to 2 days and 14 days postinoculation and macrophages at 2 and 14 days postinoculation. The Ureaplasma infection caused a persistent focal loss of airway ciliated epithelium and a mild increase in interstitial cellularity. There were no differences in BAL protein concentration during the first 28 days, suggesting that pulmonary vascular endothelial barrier integrity remained intact. Comparison of BAL cytokine and chemokine concentrations revealed low levels of tumor necrosis factor alpha (TNF-α) at 3 days and monocyte chemoattractant protein 1 at 7 days in Ureaplasma-infected mice but a trend toward increased TNF-α at 14 days and increased granulocyte-macrophage colony-stimulating factor and interleukin-10 at 28 days. These data suggest that Ureaplasma alone may cause limited inflammation and minimal tissue injury in the early phase of infection but may promote a mild chronic inflammatory response in the later phase of infection (days 14 to 28), similar to the process that occurs in human newborns.
PMCID: PMC128302  PMID: 12228302
8.  Prolonged eradication of urogenital mycoplasmas after administration of tetracycline to men in the Antarctic. 
Meatal swabs were obtained at intervals over 1 year from 23 men in the Antarctic. A 5-day course of tetracycline was given to twelve of them. In retrospect it was found that the antibiotic had been received by two men who were harbouring ureaplasmas, one of whom also had M. hominis. After treatment, these organisms were not found in any of the swabs taken over the next year, except in a swab from one of the men following sexual contact after this time. One of the twelve men developed N.S.U. just before arriving in the Antarctic. He responded clinically to a shorter course of tetracycline and ureplasmas were not recovered from a meatal swab immediately thereafter. However, without further sexual contact, ureaplasmas and disease recurred about a month later. This time, after a 5-day course of tetracycline, disease was not seen, and ureaplasmas were not isolated, over the next year. In contrast, ureaplasmas were isolated consistently over a year from two men who were not given the antibiotic. The evidence strongly suggests that, under natural conditions, the most likely cause of mycoplasmas, particularly ureaplasmas, recurring in the genital tract after apparently adequate tetracycline therapy, is re-infection as a result of sexual re-exposure.
PMCID: PMC1045296  PMID: 1036463
9.  Ureaplasma serovars & their antimicrobial susceptibility in patients of infertility & genital tract infections 
Background & objectives:
Ureaplasmas have been implicated in a variety of clinical conditions. However, only certain serovars of ureaplasmas are disease associated. Only a few classes of antimicrobial agents are available for the treatment of mycoplasmal infections in humans. Increase of resistance of genital mycoplasmas to antimicrobials has been reported worldwide. The aim of the present study was to determine the occurrence of Ureaplasma serovars in patients with infertility and genital tract infections with polymerase chain reaction (PCR)–based serotyping. The antimicrobial susceptibilities of Ureaplasma spp. and Mycoplasma hominis were also assessed to determine the most suitable treatment strategy.
Sexually active adults (n=147) with symptoms of genital tract infections and 115 infertile women were enrolled. Endocervical swabs from women and urethral swabs from men were subjected to culture and multiplex PCR for detection of genital mycoplasmas. Serotyping of Ureaplasma was done by PCR and antimicrobial susceptibility to doxycycline, azithromycin, josamycin and ofloxacin was done by microbroth dilution method.
Ureaplasma was detected in 25.8 per cent patients with genital tract infections and 20.8 per cent in infertile women. Serovar 3/14 was the most frequent isolate followed by serovar 1 and serovar 6. The majority of Ureaplasma isolates were susceptible to doxycycline (91%) and josamycin (86%) followed by ofloxacin (77%) and azithromycin (71%). All the isolates of M. hominis were uniformly susceptible to doxycycline, josamycin and ofloxacin.
Interpretation & conclusions:
The predominance of Ureaplasma serovar 3/14 suggests their possible pathogenic role in genital tract infections and infertility. For empirical treatment, doxycycline could be the drug of choice for genital mycoplasmas.
PMCID: PMC3612329  PMID: 23391795
Antimicrobial susceptibility; PCR; ureaplasma serovars
10.  Patients with usual vulvar intraepithelial neoplasia-related vulvar cancer have an increased risk of cervical abnormalities 
British Journal of Cancer  2009;101(1):27-31.
Vulvar squamous cell carcinoma (SCC) originates the following two pathways, related to differentiated (d) vulvar intraepithelial neoplasia (VIN) or to human papillomavirus (HPV)-related usual (u) VIN. Multicentric HPV infections (cervix, vagina and vulva) are common. We hypothesise that patients with a uVIN-related vulvar SCC more often have cervical high-grade squamous intraepithelial lesions (HSILs) compared with women with dVIN-related vulvar SCC.
All vulvar SCCs (201) were classified to be dVIN- (n=164) or uVIN related (n=37). Data with regard to the smear history and cervical histology were retrieved from PALGA, the nationwide Netherlands database of histo- and cytopathology. For HSIL cervical smears of which histology was taken, HPV DNA analysis on both the vulvar and cervical specimens was performed.
At least one smear was available in 145 (72%) of the 201 patients. Patients with a uVIN-related vulvar SCC more often had an HSIL compared with patients with a dVIN-related SCC (35 vs 2%, P<0.001). A total of 10 of the 13 HSILs were histologically assessed and identical HPV types were found in the vulva and cervix.
These data emphasise the necessity to differentiate between dVIN- and uVIN-related vulvar tumours and to examine the entire lower female ano-genital tract once an uVIN-related lesion is found.
PMCID: PMC2713690  PMID: 19513077
vulvar squamous cell carcinoma; usual vulvar intraepithelial neoplasia; cervical high-grade squamous intraepithelial lesion; human papillomavirus
11.  Colonization of the lower urogenital tract with Ureaplasma parvum can cause asymptomatic infection of the upper reproductive system in women: a preliminary study 
Archives of Gynecology and Obstetrics  2013;289(5):1129-1134.
Genital ureaplasmas are considered opportunistic pathogens of human genitourinary tract involved in adverse pregnancy sequelae and infertility. While association of Ureaplasma urealyticum with urogenital tract infections is well established, the role of Ureaplasma parvum in these infections is still insufficient. In the study, we compared how often cervicovaginal colonization with U. parvum is associated with the presence of these microorganisms in the upper genitourinary tract of fertile and infertile women.
We used PCR assay to determine the prevalence of U. parvum and U. urealyticum in pairs of specimens, i.e., vaginal swabs and Douglas’ pouch fluid samples from consecutive 40 women with no symptoms of genital tract infection.
In total, 19 (47.5 %) of the 40 samples were positive for ureaplasmas. U. parvum was simultaneously detected in pairs of samples in five (55.5 %) of the nine (47.4 %) women positive in PCR assay. As many as 5 (18.5 %) of the 27 infertile women and 1 (7.7 %) of the 13 fertile women showed infection of the upper genital tract with U. parvum.
The results of the study demonstrated that colonization of the lower genital tract with U. parvum can produce asymptomatic infection of the upper reproductive system in women. These findings also imply that U. parvum may be present in the upper genital tract at the time of conception and might be involved in adverse pregnancy outcomes.
PMCID: PMC3984420  PMID: 24318169
Ureaplasma parvum; Ureaplasma urealyticum; Upper urogenital tract; Asymptomatic infection
12.  The Role of the Multiple Banded Antigen of Ureaplasma parvum in Intra-Amniotic Infection: Major Virulence Factor or Decoy? 
PLoS ONE  2012;7(1):e29856.
The multiple banded antigen (MBA) is a predicted virulence factor of Ureaplasma species. Antigenic variation of the MBA is a potential mechanism by which ureaplasmas avoid immune recognition and cause chronic infections of the upper genital tract of pregnant women. We tested whether the MBA is involved in the pathogenesis of intra-amniotic infection and chorioamnionitis by injecting virulent or avirulent-derived ureaplasma clones (expressing single MBA variants) into the amniotic fluid of pregnant sheep. At 55 days of gestation pregnant ewes (n = 20) received intra-amniotic injections of virulent-derived or avirulent-derived U. parvum serovar 6 strains (2×104 CFU), or 10B medium (n = 5). Amniotic fluid was collected every two weeks post-infection and fetal tissues were collected at the time of surgical delivery of the fetus (140 days of gestation). Whilst chronic colonisation was established in the amniotic fluid of animals infected with avirulent-derived and virulent-derived ureaplasmas, the severity of chorioamnionitis and fetal inflammation was not different between these groups (p>0.05). MBA size variants (32–170 kDa) were generated in vivo in amniotic fluid samples from both the avirulent and virulent groups, whereas in vitro antibody selection experiments led to the emergence of MBA-negative escape variants in both strains. Anti-ureaplasma IgG antibodies were detected in the maternal serum of animals from the avirulent (40%) and virulent (55%) groups, and these antibodies correlated with increased IL-1β, IL-6 and IL-8 expression in chorioamnion tissue (p<0.05). We demonstrate that ureaplasmas are capable of MBA phase variation in vitro; however, ureaplasmas undergo MBA size variation in vivo, to potentially prevent eradication by the immune response. Size variation of the MBA did not correlate with the severity of chorioamnionitis. Nonetheless, the correlation between a maternal humoral response and the expression of chorioamnion cytokines is a novel finding. This host response may be important in the pathogenesis of inflammation-mediated adverse pregnancy outcomes.
PMCID: PMC3257234  PMID: 22253806
13.  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.
PMCID: PMC1255382  PMID: 3453276
14.  Comparison of the new Mycofast Revolution assay with a molecular assay for the detection of genital mycoplasmas from clinical specimens 
BMC Infectious Diseases  2013;13:453.
Genital mycoplasmas are opportunistic bacteria that are associated with undesirable gynaecologic and reproductive events. Mycoplasmas are fastidious bacteria with increasing resistance to routine antimicrobials and often fail to grow on conventional culture methods. The commercial Mycofast Revolution assay permits the phenotypic detection and identification of genital mycoplasmas. Antimicrobial susceptibility testing against five antimicrobial agents with MICs corresponding to the CLSI guidelines can also be performed. This study aimed to compare the new commercially available Mycofast Revolution assay with a multiplex PCR assay.
Self-collected swabs were obtained from pregnant women attending the antenatal clinic of a tertiary academic hospital in Pretoria, South Africa from October 2012 to November 2012. These swabs were used to seed UMMt and modified Amies transport media. The seeded UMMt transported medium was used to inoculate the Mycofast Revolution assay for the identification, enumeration and antimicrobial susceptibility testing of genital mycoplasmas. Following DNA extraction from the modified Amies transport medium, specimens were subjected to a multiplex PCR assay for the detection of genital mycoplasmas.
The Mycofast Revolution kit had a sensitivity and specificity of 77.3% (95% CI: 62.15% to 88.51%) and 80% (95% CI: 28.81% to 96.70%), respectively, against the PCR assay. The positive and negative predictive values were 97.1% (95% CI: 85.03% to 99.52%) and 28.6% (95% CI: 8.57% to 58.08%). Genital mycoplasmas were detected in 71.4% (35/49) of samples with the Mycofast Revolution assay with 49% (24/49) being Ureaplasma spp. and 22.4% (11/49) mixed strains. The multiplex PCR assay had a positivity rate of 89.8% (44/49) for genital mycoplasmas; mixed strains were present in 51% (25/49) of samples, Ureaplasma spp. in 16.3% (8/49) and M. hominis in 22.4% (11/49) of samples.
There was a fair agreement (κ = 0.319) between the Mycofast Revolution assay and the mPCR assay. With the high prevalence rates of genital mycoplasmas, fast and efficient diagnostic methods are imperative to treat infections and minimise complications. The Mycofast Revolution assay is simple to use, has a short turn-around time and interpretation of results are straightforward. This assay circumvents common problems experienced with conventional culture and molecular methods in diagnostic laboratories where skilled personnel are limited and can be used as an alternative diagnostic assay.
PMCID: PMC3849776  PMID: 24079603
Mycoplasma hominis; Ureaplasma spp; Mycofast; Antimicrobial susceptibilities; Multiplex PCR assay
15.  Characterization and Comparative Genital Tract Pathogenicity of Bovine Mycoplasmas 1 
Infection and Immunity  1970;2(1):101-104.
Sixteen bovine genital mycoplasmal isolates obtained from semen and prepuce of bulls and from aborted fetuses were compared physiologically and serologically with the Donetta strain (tentatively Mycoplasma agalactiae var. bovis), a known pathogen. All isolates were distinct from the Donetta organism. Four appeared to be saprophytes, and the remainder were placed in one group which could not be further separated by the biochemical or serological methods used. Two of the organisms in the latter group have been subsequently identified as M. bovigenitalium. Uterine infusion of broth cultures of four isolates into virgin heifers failed to produce clinical evidence of disease, and significant lesions were not present at necropsy. The mycoplasmas were recovered from cervicovaginal mucus of only three heifers, and never for more than 3 days postinfusion. Since the organisms were not recovered from any organs at necropsy, it appears that the mycoplasmas were incapable of surviving in the clinically normal virgin female reproductive tract.
PMCID: PMC415971  PMID: 16557786
16.  Ureaplasma infection of cell cultures. 
Infection and Immunity  1986;52(2):437-444.
Studies were performed to characterize the effects of ureaplasmas in HeLa, 3T6, and CV-1 cell cultures. The ureaplasmas studied were human Ureaplasma urealyticum T960 (serotype VIII), bovine U. diversum T95, simian strain T167-2, ovine strain 1202, canine strain D1M-C, and feline strains 382 and FT2-B. FT2-B was the only ureaplasma to grow in the cell free culture medium, Dulbecco modified Eagle-Earle medium containing 10% fetal bovine serum. The growth pattern of the ureaplasmas varied in the different cell cultures, but each strain grew in at least two of the cell cultures, suggesting a requirement for a product of the cell culture and for low concentrations of urea. When growth occurred, organisms grew to concentrations that approached, but did not equal, those observed in 10B broth. Most, but not all, ureaplasmas grew quickly, reaching peak titers 2 days after infection. Canine strain D1M-C did not grow in 3T6, but showed rapid growth in HeLa and CV-1 cells, killing both cultures, In some systems, e.g., U. urealyticum T960 and simian strain T167-2, the infection persisted, and ureaplasmas could be recovered from cell cultures four passages after infection, when studies were terminated. The cell culture ureaplasmas grew on T agar, but not on mycoplasma agar medium.
PMCID: PMC261018  PMID: 3699891
17.  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)
PMCID: PMC1255543  PMID: 2469532
18.  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.
PMCID: PMC1263674  PMID: 8004534
19.  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)
PMCID: PMC1255303  PMID: 3607652
20.  Circinate vulvitis in Reiter's syndrome. 
Two cases of Reiter's syndrome in women are described. The diagnosis was based on the presence of increased vaginal and cervical discharge containing excess leucocytes, arthritis, conjunctivitis, and HLA B27 tissue-typing antigen. In addition circinate lesions developed on the vulva similar to those seen on the glans penis. No previous description of these lesions has been traced and the name 'circinate vulvitis' is suggested for these lesions.
PMCID: PMC1045412  PMID: 922460
21.  Ulcerative vulvitis in Reiter's syndrome. A case report. 
In a case of acute Reiter's syndrome with severe vulvitis the diagnosis was based on the presence of a vaginal discharge and dysuria, arthritis, conjunctivitis, buccal ulceration, keratodermia blenorrhagica, and HLA B27 tissue-typing antigen. The vulval lesions were similar in appearance to those of circinate vulvitis. The acute histological change were confined to shallow ulceration with an inflammatory infiltration of the subjacent dermis. Coincidential lichen sclerosus et atrophicus was present, which could have been masked by the acute lesions.
PMCID: PMC1046112  PMID: 7171984
22.  Infection of Calves with Bovine Norovirus GIII.1 Strain Jena Virus: an Experimental Model To Study the Pathogenesis of Norovirus Infection ▿ 
Journal of Virology  2011;85(22):12013-12021.
The experimental infection of newborn calves with bovine norovirus was used as a homologous large animal model to study the pathogenesis of norovirus infection and to determine target cells for viral replication. Six newborn calves were inoculated orally with Jena virus (JV), a bovine norovirus GIII.1 strain, and six calves served as mock-inoculated controls. Following infection, calves were euthanized before the onset of diarrhea (12 h postinoculation [hpi]), shortly after the onset of diarrhea (18 to 21 hpi), and postconvalescence (4 days pi [dpi]). Calves inoculated with JV developed severe watery diarrhea at 14 to 16 hpi, and this symptom lasted for 53.5 to 67.0 h. Intestinal lesions were characterized by severe villus atrophy together with loss and attenuation of villus epithelium. Viral capsid antigen (JV antigen) was detected by immunohistochemistry in the cytoplasm of epithelial cells on villi. In addition, granular material positive for JV antigen was detected in the lamina propria of villi. Lesions first appeared at 12 hpi and were most extensive at 18 to 19 hpi, extending from midjejunum to ileum. The intestinal mucosa had completely recovered at 4 dpi. There was no indication of systemic infection as described for norovirus infection in mice. JV was found in intestinal contents by reverse transcription-PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) as early as 12 hpi. Fecal shedding of the virus started at 13 hpi and stopped at 23 hpi or at necropsy (4 dpi), respectively. Throughout the trial, none of the control calves tested positive for JV by ELISA or RT-PCR.
PMCID: PMC3209315  PMID: 21880760
Twelve normal monkeys inoculated on the mucous membranes of the nose or nose and mouth with a strain of Bacillus influenzæ; originally isolated in pure culture from the pleural exudate of a case of empyema following influenzal pneumonia in man and subsequently raised in virulence by animal passage, developed an acute self-limited respiratory disease of from 3 to 5 days duration, characterized by sudden onset with profound prostration, the development of rhinitis and tracheobronchitis, with sneezing, cough, and the outpouring of a scanty mucoid, or mucopurulent exudate, a variable febrile reaction, and either a leucopenia or no significant change in the leucocyte count. This disease was complicated in five instances by purulent sinusitis of one or both antra, in three by bronchopneumonia. Bacillus influenzæ was recovered at autopsy from the lesions of the disease either in pure culture or in association with organisms that are normal inhabitants of the upper respiratory tract of monkeys. Of ten normal monkeys injected intratracheally with the same strain of Bacillus influenzæ, seven developed bronchopneumonia, two developed tracheobronchitis without pneumonia, and one resisted infection. The general symptoms and duration of the disease were similar to those of the preceding group. There were a severe cough and accelerated respirations. Bacillus influenzæ was recovered in pure culture from the lungs, bronchi, or trachea in the animals killed during the active stage of the disease. It disappeared promptly from the respiratory tract with recovery. The significance of the first series of experiments in which monkeys were inoculated in the upper respiratory tract is twofold. First, they establish the fact that Bacillus influenzæ can initiate in monkeys an acute infection of the normal mucous membranes of the upper respiratory tract; that is, it can act as a primary incitant of respiratory infection without the assistance of a preceding or concomitant contributing cause. In this respect it differs radically from the pneumococcus and Streptococcus hæmolyticus, since experiments previously reported2, 4 have shown that neither of these organisms possesses the property of initiating an infection of the normal mucous membranes of the upper respiratory tract of monkeys, even though the strains used were incalculably more virulent for monkeys than the strain of Bacillus influenzæ used in the foregoing experiments. Secondly, the experiments show that Bacillus influenzæ infection of the mucous membranes of the upper respiratory tract may spread by continuity to the paranasal sinuses, setting up an acute sinusitis, that it spreads readily to the lower respiratory tract, producing a tracheobronchitis and permitting the ready invasion of secondary bacteria, and that it may penetrate as far as the terminal bronchioles, alveolar ducts, atria, and alveoli, there setting up a bronchiolitis and true bronchopneumonia. In these respects it likewise differs radically from the pneumococcus and Streptococcus hæmolyticus which do not possess these pathogenic properties as previous experiments have shown.2, 4 The bearing of these facts on the possible etiologic relation of Bacillus influenzæ to influenza is important, since they show that Bacillus influenzæ possesses certain definite primary pathogenic properties which distinguish it and therefore separate it from the group of recognized secondary organisms in influenzal complications, of which the pneumococcus and the streptococcus are the most frequent. The possible etiologic relation of Bacillus influenzæ to influenza is further supported by the character of the respiratory disease that occurred in the monkeys. The sudden onset with profound prostration, the absence of leucocytosis or often a leucopenia, the congestion of the mucous membranes of the respiratory tract, the development on the 2nd or 3rd day of an irritative cough due to an inflammatory tracheitis or tracheobronchitis, the brief self-limited course of the infection, and the irregular febrile reactions are all characteristic of influenza. Many of these symptoms were in striking contrast with the symptoms and course of pneumococcus or streptococcus infections in monkeys in which there were no prostration at onset, invariable leucocytosis, and infrequent cough developing only late in the disease. While all the above features of the disease produced in monkeys are characteristic of influenza in man, none are pathognomonic and, in fact, it is doubtful whether uncomplicated influenza possesses any pathognomonic features by which it may be diagnosed certainly in the absence of an epidemic. Even during epidemic times many respiratory infections arise which, though presumably influenza, it is impossible to diagnose as such with certainty. Nor does pathology help in this respect, since there would appear to be no established distinctive lesions of uncomplicated influenza in man, nor for that matter of the complications of influenza, apart from the complications which have been ascribed by Pfeiffer,5 MacCallum,6 Wolbach,7 and others to infection with Bacillus influenzæ because of the association of Bacillus influenzæ in pure culture with these complications. For these reasons, although the disease produced in monkeys appears to be essentially identical with influenza in man with respect to its clinical course and complications, it is impossible to determine certainly whether it is actually so. The experiments are advanced, therefore, as evidence in favor of the etiologic relation of Bacillus influenzæ to influenza, though they do not permit of a definite conclusion in this respect. Their bearing upon the relation of Bacillus influenzæ to certain of the complications of influenza would appear to be reasonably conclusive. The recovery of Bacillus influenzæ in pure culture at autopsy from the antra, from the trachea and bronchi, and from the lungs in some of the animals developing sinusitis, bronchiolitis, and a characteristic type of bronchopneumonia confirms by animal experiment the etiologic relation of Bacillus influenzæ to these complications of influenza, which hitherto has rested solely upon the frequent association of the influenza bacillus with these lesions in man. The production of tracheobronchitis and the same type of bronchopneumonia by the intratracheal injection of Bacillus influenzæ in the second series of experiments serves as additional confirmation of this, but has no direct bearing on the etiologic relation of Bacillus influenzæ to uncomplicated influenzæ.
PMCID: PMC2128159  PMID: 19868470
24.  Influence of vulvar hygiene on cytology of vaginal smears after sham artificial insemination in sows. 
The description of vaginal cytology in the sow in relation to artificial insemination (AI) has never been reported. Poor vulvar hygiene is frequently imputed as a cause for the development of endometritis after AI and could thus enhance the inflammatory response of the genital tract. The goal of this study was to use cytology as an objective tool to evaluate the vulvar hygiene during sham AI. Sixty-eight sows were matched according to their parity and week of mating and divided into 2 groups: treatment sows (CVS) had their vulva cleaned with a 1:2000 Hibitane solution and control sows (SVS) had theirs soiled with feces. Both groups of sows were inseminated twice with saline following this vulvar treatment, once at detection of estrus and a 2nd time 24 hours later. Vestibular smears were taken before each AI, and vaginal smears were taken after each AI from the material present on the insemination spirette. Cytological smears were described by the predominant type of cells, namely epithelial, neutrophil, or no predominance. Results showed no significant differences between the 2 groups and no evolution in the predominance of neutrophils from the 1st to the 2nd AI (P > 0.05). The pooled results from the 2 groups showed an increase in cellularity from the 1st to the 2nd AI (P > 0.05). Neither the cellularity nor predominant cell type in vestibular or vaginal smears from estrus sows are predictors of vulvar hygiene during sham AI (P > 0.05).
PMCID: PMC1263765  PMID: 8521352
25.  Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis and Mycoplasma genitalium infections and semen quality of infertile men 
Genital ureaplasmas (Ureaplasma urealyticum and Ureaplasma parvum) and mycoplasmas (Mycoplasma genitalium and Mycoplasma hominis) are potentially pathogenic species playing an etiologic role in both genital infections and male infertility. Reports are, however, controversial regarding the effects of these microorganisms infections in the sperm seminological variables. This study aimed at determining the frequency of genital ureplasmas and mycoplasmas in semen specimens collected from infertile men, and at comparing the seminological variables of semen from infected and non-infected men with these microorganisms.
A total of 120 semen samples collected from infertile men were investigated. Semen specimens were examined by in-house PCR-microtiter plate hybridization assay for the presence of genital ureaplasmas and mycoplasmas DNA. Semen analysis was assessed according to the guidelines of the World Health Organization. Standard parametric techniques (t-tests) and nonparametric techniques (Wilcoxon tests) were used for statistical analysis.
The frequency of genital ureaplasmas and mycoplasmas detected in semen samples of infertile men was respectively 19.2% (23/120) and 15.8% (19/120). The frequency of Ureaplasma urealyticum (15%) was higher than that of Mycoplasma hominis (10.8%), Ureaplasma parvum (4.2%) and Mycoplasma genitalium (5%). Mixed species of mycoplasmas and ureaplasmas were detected in 6.7% of semen samples.
Comparison of the parameters of the standard semen analysis between the male partners of the infertile couples with and without genital ureaplasmas and mycoplasmas infection showed that the presence of Mycoplasma hominis DNA in semen samples is associated with low sperm concentration (p = 0.007) and abnormal sperm morphology (p = 0.03) and a negative correlation between sperm concentration and the detection of Mycoplasma genitalium in semen samples of infertile men (p = 0.05). The mean values of seminal volume, pH, vitality, motility and leukocyte count were not significantly related either to the detection of genital mycoplasmas DNA or to the detection of ureaplasmas DNA in semen specimens.
Our results demonstrate that genital mycoplasmas and ureaplasmas seem to be widespread among the male partners of infertile couples in Tunisia. Genital mycoplasmas infections of the male genital tract could negatively influence semen quality. Our results also indicate that PCR-microtiter plate hybridization assay method provides a rapid and effective technique to detect human genital mycoplasmas and ureaplasmas which is useful for etiological and epidemiological studies of these pathogens.
PMCID: PMC2194714  PMID: 17988404

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