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1.  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
2.  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
3.  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
4.  Experimental bovine genital ureaplasmosis. I. Granular vulvitis following vulvar inoculation. 
Granular vulvitis was reproduced in ten virgin heifers following vulvar inoculation with strains of ureaplasma previously isolated from natural cases. The disease appeared one to three days postinoculation and was characterized by vulvar swabs but not from the upper mucopurulent discharge. At necropsy 13 to 41 days later, ureaplasmas were recovered consistently from vulvar swabs but not from the upper reproductive tract. It was concluded that some strains of ureaplasma are pathogenic and should be viewed as a cause of bovine granular vulvitis.
PMCID: PMC1320070  PMID: 7427772
5.  Bovine Genital Mycoplasmosis 
The Canadian Veterinary Journal  1981;22(11):339-343.
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.
PMCID: PMC1789999  PMID: 7337908
6.  The indirect hemagglutination test for the detection of antibodies in cattle naturally infected mycoplasmas. 
Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titer greater than or equal to 1:80 were detected in 148 animals, 76 of these having antibody titers greater than or equal to 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera of ten of the 26 animals. However, the IHA test detected antibody titers of greater than or equal to 1:160 in 13 animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12 species of bovine mycoplasmatales. Homologous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA antibody titers of less than or equal to 1:20. Also eight type-specific bovine antisera were reacted with M agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homoogous reactions showed IHA antibody titers greater than or equal to 1:320, whereas heterologous reactions showed IHA titers of less than or equal to 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in sera from cattle infected with M. agalactiae subsp. bovis or M. bovigenitalium. Thes test is sensitive, reproducible and specific and the technique is relatively simple and rapid. The antigens were stable for at least seven months.
PMCID: PMC1277514  PMID: 1000374
7.  Mycoplasma species recovered from the reproductive tracts of western Canadian cows. 
Samples of cervico-vaginal mucus from 633 animals from 110 herds were cultured and yielded the following mycoplasmas: T-strain--88: Mycoplasma bovigenitalium--79, Mycoplasma spp. (Leach Group 7)--7, Acholeplasma laidlawii--4, Mycoplasma bovirhinis--2 and one not typable. Uterine exudates and endometrial scrapings from 80 infertile cows in two herds were examined. Four animals were positive, M. bovigenitalium was isolated three times, A. laidlawii and Mycoplasma arginini once each. Sixty-five normal uterine contents from pregnant cows were examined, one yielded M. bovigenigalium and the same organism was recovered from the fetal kidney. T-strain mycoplasma, M. bovigenitalium and other Mycoplasma spp. appear to be a part of the normal flora of the cervico-vaginal region of clinically normal one and two year old bred heifers in Alberta and Saskatchewan. Although M. arginini was not recovered from the cervico-vaginal region, a single recovery was made from the uterus of an infertile cow.
PMCID: PMC1277433  PMID: 1125831
8.  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
9.  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
10.  Clinical presentation, auscultation recordings, ultrasonographic findings and treatment response of 12 adult cattle with chronic suppurative pneumonia: case study 
Irish Veterinary Journal  2013;66(1):5.
Auscultation is considered the critical component of the veterinary clinical examination for the diagnosis of bovine respiratory disease but the accuracy with which adventitious sounds reflect underlying lung pathology remains largely unproven. Modern portable ultrasound machines provide the veterinary practitioner with an inexpensive, non-invasive tool with which to examine the pleural surfaces and superficial lung parenchyma. Simultaneous recording of sounds overlying normal lung and defined pathology allows critical assessment of auscultated sounds in the same animal removing confounding factors such as respiratory rate and thickness of the chest wall (body condition). Twelve cows, referred to the University of Edinburgh Veterinary School, were diagnosed with chronic suppurative pneumonia and enrolled into this prospective study to record and monitor lung sounds, ultrasonographic findings, and response to a standardised antibiotic treatment regimen.
Most cows (8/12) had a normal rectal temperature on presentation but all cows had received antibiotic therapy at some time in the previous two weeks and six animals were receiving antibiotic treatment upon admission. All cattle were tachypnoeic (>40 breaths per minute) with frequent and productive coughing, halitosis, and a purulent nasal discharge most noticeable when the head was lowered. Ultrasonographic examination of the chest readily identified pathological changes consistent with severe lung pathology subsequently confirmed as chronic suppurative pneumonia in four cows at necropsy; eight cows recovered well after antibiotic treatment and were discharged two to six weeks after admission. It proved difficult to differentiate increased audibility of normal lung sounds due to tachypnoea from wheezes; coarse crackles were not commonly heard. In general, sounds were reduced in volume over consolidated lung relative to normal lung tissue situated dorsally. Rumen contraction sounds were commonly transmitted over areas of lung pathology.
Trueperella (formerly Arcanobacterium) pyogenes was isolated from three of four lung tissue samples at necrospy. Treatment with procaine penicillin for 42 consecutive days resulted in marked improvement with return to normal appetite and improvement in body condition in 8 of 12 cows (67%) where lesions did not extend more than 10-15 cm above the level of the olecranon on both sides of the chest.
PMCID: PMC3671146  PMID: 23547784
Ultrasonography; Respiratory disease; Cattle; Auscultation; Diagnosis; Trestment
11.  Bovine mastitis in Ontario due to Mycoplasma agalactiae subsp. bovis. 
Bovine mastitis caused by Mycoplasma agalactiae subsp. bovis was first diagnosed in 16 of 55 cows in an Ontario herd in Feburary 1972. A total of 182 of 598 (30.4%) cows from 33 of 64 (51.5%) farms in widely separated areas of the province were culturally positive. Herd incidence varied from 15 to 40% with one closed herd having an incidence of 61%. Four herds were investigated culturally and serologically by the growth inhibition test for 15 months. In the acute phase the organism was present in the milk in extremely high numbers and could still be isolated from a few cows after eight to 12 months. The sera from 89.5% of the animals with clinical mycoplasma mastitis produced a zone of surface "film" and/or colony inhibition and some cows remained positive for six to 12 months. The disease was experimentally reproduced with a pure culture of the organism isolated from the milk of a cow from one of the herds.
PMCID: PMC1277538  PMID: 1000385
12.  Association of herd BRSV and BHV-1 seroprevalence with respiratory disease and reproductive performance in adult dairy cattle 
The aim of this study was to detect the associations between bovine herpesvirus 1 (BHV-1) status of a herd and respiratory disease (BRD) occurrence and reproductive performance in pregnant heifers and cows. The association between management-related factors and higher BRD occurrence was also estimated.
Serum samples, collected from cows and youngstock from 103 dairy cattle herds, were analyzed for antibodies against BHV-1, bovine respiratory syncytial virus (BRSV), bovine viral diarrhoea virus (BVDV), and Mycoplasma bovis. A questionnaire was used to collect data concerning herd management factors and reproductive performance, as well as the occurrence of clinical signs of respiratory disease in the last two years, as evaluated by the veterinarian or farm manager. Multiple correspondence analysis (MCA) and logistic regression analysis were performed to identify and quantify the risk factors.
A low to moderate prevalence (1-49%) of BRSV antibodies among youngstock was associated with a high occurrence of respiratory disease (OR = 6.2, p = 0.010) in cows and in-calf heifers. Employees of the farm may participate in the spread of such disease. Larger herd size, loose-housing of cows, housing youngstock separately from cows until pregnancy, and purchasing new animals were factors possibly related to a high occurrence of respiratory disease symptoms in pregnant heifers and cows. The highest risk of abortions (> 1.3%) and increased insemination index (number of inseminations per pregnancy) (> 1.9) occurred in herds with a moderate prevalence of BHV-1 antibodies (1-49%) in cows.
BHV-1 was not associated with acute respiratory disease in adult dairy cattle, however was significantly related to reproductive performance. BRSV possesses the main role in respiratory disease complex in adult dairy cattle.
PMCID: PMC3298528  PMID: 22289165
Bovine respiratory disease; reproduction; dairy cattle; bovine herpesvirus 1; bovine respiratory syncytial virus
13.  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
14.  Assessment of Early Postpartum Reproductive Performance in Two High Producing Estonian Dairy Herds 
Acta Veterinaria Scandinavica  2003;44(3):131-143.
Early postpartum (6 weeks) ovarian activity, hormonal profiles, uterine involution, uterine infections, serum electrolytes, glucose, milk acetoacetate and blood urea nitrogen (BUN) levels were studied in 2 Estonian high producing dairy herd with annual milk production of 7688 (Farm A) and 9425 (Farm B). From each farm 10 cows, with normal calving performance were used. Blood samples for the hormonal (PGF2α-metabolite, progesterone) analyses were withdrawn. On day 25 PP blood serum samples were taken for the evaluation of metabolic/electrolyte status. On the same day estimation of milk acetoacetate values was done. The ultrasound (US) was started on day 7 PP and was performed every 3rd day until the end of experiment. Uterine content, follicular activity and sizes of the largest follicle and corpus luteum were monitored and measured. Vaginal discharge and uterine tone were recorded during the rectal palpation. Each animal in the study was sampled for bacteriological examination using endometrial biopsies once a week. Two types of PGF2α-metabolite patterns were detected: elevated levels during 14 days PP, then decline to the basal level and then a second small elevation at the time of final elimination of the bacteria from the uterus; or elevated levels during first 7 days PP, then decline to the basal level and a second small elevation before the final elimination of bacteria. Endometritis was diagnosed in 5 cows in farm A and in 3 cows in farm B respectively. In farm A, 5 cows out of 10 ovulated during experimental period and in 1 cow cystic ovaries were found. In farm B, 3 cows out of 10 ovulated. In 3 cows cystic ovaries were found. Altogether 40% of cows had their first ovulation during the experimental period. Three cows in farm A and 5 cows in farm B were totally bacteria negative during the experimental period. The most frequent bacteria found were A. pyogenes, Streptococcus spp., E. coli., F. necrophorum and Bacteroides spp. The highest incidence of bacteriological species was found during the first 3 weeks in both farms. All animals were free from bacteria after 5th week PP in farm A and after 4th week in farm B respectively. Serum electrolytes and glucose levels were found to be within the reference limits for the cows in both farms. No significant difference was found between farms (p > 0.05). Low phosphorus levels were found in both farms. Significant difference (p < 0.05) was found in BUN levels between farms. In both farms milk acetoacetate values were staying within the reference range given for the used test (<100 μmol/l). The uterine involution and bacterial elimination in the investigated cows could consider as normal but more profound metabolic studies could be needed to find reasons for later resumption of ovarian activity. Some recommendations to changing feeding regimes and strategies should also be given.
PMCID: PMC1831562  PMID: 15074626
Postpartum cow; milk production; ovarian activity; PGF2α; progesterone; uterine bacteriology; blood electrolytes; glucose; blood urea nitrogen
15.  Experimental abortion and the systemic immune response to "Haemophilus somnus" in cattle. 
Infection and Immunity  1986;54(2):555-560.
"Haemophilus somnus" has been identified in the etiology of bovine abortion on the basis of the isolation of the organism from aborted fetal and placental tissues. To investigate the role of hematogenous dissemination of "H. somnus" in the pathogenesis of abortion and to monitor the humoral immune response to infection, 19 pregnant cows (gestation ages, 1.4 to 7 months) were challenged intravenously (11 cows) or intrabronchially (8 cows). Five cows challenged intravenously aborted, and one cow challenged intrabronchially resorbed her fetus. "H. somnus" was isolated in large numbers from aborted tissues, and placental lesions were similar to those reported in a field case of "H. somnus" abortion. Antibody titers in serum were measured by the microagglutination test (MAT) and by enzyme-linked immunosorbent assay (ELISA). A response to challenge was measured by MAT; it was also measured by ELISA within the immunoglobulin G1 (IgG1), IgG2, and IgM isotypes. On comparison of pre- and postchallenge antibody titers, the greatest and most persistent response was detected within the IgG2 isotype. Prechallenge antibody titers (measured by MAT and by IgG2 ELISA) were lower in animals that aborted than in normal calving animals, indicating that IgG2 antibody may have a role in limiting hematogenous dissemination of "H. somnus."
PMCID: PMC260197  PMID: 3770951
16.  Longitudinal Analysis of Prototheca zopfii-Specific Immune Responses: Correlation with Disease Progression and Carriage in Dairy Cows 
Journal of Clinical Microbiology  2003;41(3):1181-1186.
In order to characterize the humoral and cellular immune responses to bovine mammary protothecosis, serum and whey samples obtained from 72 dairy cows assigned to four different clinical stages of infection were examined for specific antibodies by indirect enzyme-linked immunosorbent assay techniques. Milk samples were analyzed for the total numbers of excreted algal cells and somatic cells. After characterization of the course of immune induction in bovine protothecal mastitis, a long-term sentinel study was performed in an affected herd in order to investigate disease progression. A total of 61 dairy cows with protothecal mastitis were examined for shedding of algae cells and for local immune responses three times in 6-month intervals. During acute and chronic stages of protothecosis, significantly elevated specific antibody activities in sera were detected. A strong correlation of whey immunoglobulin A (IgA) and whey IgG1 antibody activity with the total counts of somatic cells in milk was observed, whereas only a weak correlation of whey IgA and whey IgG1 concentrations to the number of algal cells excreted with the milk was seen. Our results from the sentinel long-term study of infected cows revealed that 70.5% of the persistently infected animals were continuously shedding the pathogen. About 4.9% of the animals showed an intermittent shedding, whereas 18% of the cows were tested culturally negative throughout the study. It can be assumed that Prototheca zopfii mastitis in dairy cows is maintained on the herd level by subclinically infected alga-shedding cows.
PMCID: PMC150299  PMID: 12624049
17.  Transgenic Cows That Produce Recombinant Human Lactoferrin in Milk Are Not Protected from Experimental Escherichia coli Intramammary Infection▿  
Infection and Immunity  2006;74(11):6206-6212.
This is the first study describing an experimental mastitis model using transgenic cows expressing recombinant human lactoferrin (rhLf) in their milk. The aim of the study was to investigate the concentrations in milk and protective effects of bovine and recombinant human lactoferrin in experimental Escherichia coli mastitis. Experimental intramammary infection was induced in one udder quarter of seven first-lactating rhLf-transgenic cows and six normal cows, using an E. coli strain isolated from cows with clinical mastitis and known to be susceptible to Lf in vitro. Clinical signs were recorded during the experimental period, concentrations of human and bovine Lf and indicators of inflammation and bacterial counts were determined for milk, and concentrations of acute-phase proteins and tumor necrosis factor alpha were determined for sera and milk. Serum cortisol and blood hematological and biochemical parameters were also determined. Expression levels of rhLf in the milk of transgenic cows remained constant throughout the experiment (mean, 2.9 mg/ml). The high Lf concentrations in the milk of transgenic cows did not protect them from intramammary infection. All cows became infected and developed clinical mastitis. The rhLf-transgenic cows showed milder systemic signs and lower serum cortisol and haptoglobin concentrations than did controls. This may be explained by lipopolysaccharide-neutralizing and immunomodulatory effects of the high Lf concentrations in their milk. However, Lf does not seem to be a very efficient protein for genetic engineering to enhance the mastitis resistance of dairy cows.
PMCID: PMC1695535  PMID: 16954396
18.  Diagnosis of vulvar lesions by non-invasive optical analysis: a pilot study 
Rare Tumors  2009;1(1):e8.
A procedure that could allow an early in vivo and non-invasive detection of vulvar lesions would be extremely useful. We tested an innovative optical method (Optiprobe), which uses a harmless, visible light source for the in vivo, on-line detection of minimal alterations in the structure of vulvar epithelium. A group of 3 female volunteers without gynecological symptoms were first screened to evaluate optical properties of normal vulvar tissue. Next, a group of 16 patients undergoing gynecological examination for vulvar lesions was evaluated by the Optiprobe at suspected sites before these sites were biopsied for histological analysis. Adjacent, non-involved sites were also measured to provide internal controls. Histological analysis of the biopsies identified one case that did not show obvious alterations, 4 cases of high-grade vulvar intraepithelial neoplasia (VIN), 5 cases of vulvitis, and 6 cases of lichen sclerosis (LS).
The optical properties of the VIN cases were significantly different from those of controls, due to a decrease in the absorption spectra and an increase in the scattering spectra. In contrast, a significant increase in the absorption spectra and a decrease in the scattering spectra were observed in the cases of vulvitis. In the LS cases, the absorption spectra were as in controls, whereas the scattering spectra were significantly decreased. We conclude that the Optiprobe provides a useful tool for a rapid and non-invasive detection of vulvar alterations. The method should contribute to reduce the number of biopsies and to facilitate the long-term follow-up of vulvar lesions.
PMCID: PMC2994441  PMID: 21139902
vulvar intraepithelial neoplasia; non invasive diagnosis; optical analysis; absorption spectra; scattering spectra.
19.  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
20.  Treatment of nongonococcal urethritis with rifampicin as a means of defining the role of Ureaplasma urealyticum 
The results of a double-blind therapeutic trial on 217 men with nongonococcal urethritis (NGU) show that minocycline was more effective than rifampicin. Before treatment Chlamydia trachomatis was isolated from 43% of men, Ureaplasma urealyticum from 59%, and Mycoplasma hominis from 22%. Chlamydiae and ureaplasmas were isolated less frequently from men with a recent history of NGU. Minocycline was given to 94 patients, and after treatment chlamydiae were isolated from only one of 40 initially chlamydia-positive patients and ureaplasmas from only five of 57 initially ureaplasma-positive patients. Although most patients responded clinically, failure and partial recovery rather than complete recovery were observed more often among those who were infected with ureaplasmas. Rifampicin was given to 123 patients, after which chlamydiae were isolated from only one of 53 initially chlamydia-positive men whereas ureaplasmas, insensitive to the antibiotic in vitro, were isolated from 55 of 68 men who had initially positive results. Patients infected with ureaplasmas failed to respond to rifampicin treatment significantly more often than those who were not infected. This was also observed when only patients who had never had NGU or who had not had a recent episode were considered. Furthermore, 24 (44%) of the 55 men whose ureaplasmas persisted failed to recover whereas only one (7·7%) of 13 men whose ureaplasmas disappeared did not respond to treatment. These results suggested that ureaplasmas were a cause of urethritis in some of the men (an estimated 10% at least). In addition, Reiter's disease developed in two men treated with rifampicin from whom only ureaplasmas had been isolated initially. M. hominis did not seem to have an important pathogenic role in NGU and there was evidence that ureaplasmas were an unlikely cause of urethritis in some men since the organisms persisted despite complete clinical recovery.
PMCID: PMC1045579  PMID: 371746
21.  Detection of ureaplasmas by the polymerase chain reaction in the amniotic fluid of patients with cervical insufficiency 
Journal of perinatal medicine  2010;38(3):261-268.
The purpose of this study was to determine the clinical significance of detecting microbial footprints of ureaplasmas in amniotic fluid (AF) using specific primers for the polymerase chain reaction (PCR) in patients presenting with cervical insufficiency.
Amniocentesis was performed in 58 patients with acute cervical insufficiency (cervical dilatation, ≥1.5 cm) and intact membranes, and without regular contractions (gestational age, 16–29 weeks). AF was cultured for aerobic and anaerobic bacteria as well as genital mycoplasmas. Ureaplasmas (Ureaplasma urealyticum and Ureaplasma parvum) were detected by PCR using specific primers. Patients were divided into three groups according to the results of AF culture and PCR for ureaplasmas: those with a negative AF culture and a negative PCR (n=44), those with a negative AF culture and a positive PCR (n = 10), and those with a positive AF culture regardless of PCR result (n=4).
1) Ureaplasmas were detected by PCR in 19.0% (11/58) of patients, by culture in 5.2% (3/58), and by culture and/or PCR in 22.4% (13/58); 2) Among the 11 patients with a positive PCR for ureaplasmas, the AF culture was negative in 91% (10/11); 3) Patients with a negative AF culture and a positive PCR for ureaplasmas had a significantly higher median AF matrix metalloproteinase-8 (MMP-8) concentration and white blood cell (WBC) count than those with a negative AF culture and a negative PCR (P<0.001 and P<0.05, respectively); 4) Patients with a positive PCR for ureaplasmas but a negative AF culture had a higher rate of spontaneous preterm birth within two weeks of amniocentesis than those with a negative AF culture and a negative PCR (P<0.05 after adjusting for gestational age at amniocentesis); 5) Of the patients who delivered within two weeks of amniocentesis, those with a positive PCR for ureaplasmas and a negative AF culture had higher rates of histologic amnionitis and funisitis than those with a negative AF culture and a negative PCR (P<0.05 after adjusting for gestational age at amniocentesis, for each); (6) However, no significant differences in the intensity of the intra-amniotic inflammatory response and perinatal outcome were found between patients with a positive AF culture and those with a negative AF culture and a positive PCR.
1) Cultivation techniques for ureaplasmas did not detect most cases of intra-amniotic infection caused by these microorganisms (91% of cases with cervical insufficiency and microbial footprints for ureaplasmas in the amniotic cavity had a negative AF culture); 2) Patients with a negative AF culture and a positive PCR assay were at risk for intra-amniotic and fetal inflammation as well as spontaneous preterm birth.
PMCID: PMC3085903  PMID: 20192887
Amniotic fluid (AF); cervical insufficiency; chorioamnionitis; intra-amniotic infection; intra-amniotic inflammation; polymerase chain reaction (PCR); pregnancy; preterm birth; spontaneous abortion; ureaplasmas
22.  Mycoplasmas and ureaplasmas in patients with hypogammaglobulinaemia and their role in arthritis: microbiological observations over twenty years. 
Annals of the Rheumatic Diseases  1994;53(3):183-187.
OBJECTIVES--To study the occurrence of mycoplasmas and ureaplasmas in patients with hypogammaglobulinaemia and the relationship of these micro-organisms to septic arthritis. METHODS--Over a period of about 20 years, 53 men and 38 women with hypogammaglobulinaemia, most of whom were less than 50 years old, were examined clinically and microbiologically. Mycoplasmas and ureaplasmas were sought in the throat, urogenital tract and joints by standard cultural methods, although not consistently in the three sites of all patients. RESULTS--Arginine-hydrolysing mycoplasmas and ureaplasmas occurred with similar frequency in the sputum/throat of the hypogammaglobulinaemic patients, but no more often than might be expected in immunocompetent patients. Ureaplasmas, however, dominated in the urogenital tracts of both men and women, being found in 75% of vaginal specimens. Arginine-hydrolysing mycoplasmas occurred two to six times more frequently and ureaplasmas two to three times more frequently in urine specimens from hypogammaglobulinaemic patients than they did in such specimens from sex- and age-matched non-venereal disease, hospital patients or healthy subjects; these differences were statistically significant (p < 0.05). Enhanced mucosal colonisation probably increases the chance of spread to distant sites, such as the joints. Of the 91 patients, 21 (23%) had septic arthritis involving one or more joints. Mycoplasmas and/or ureaplasmas, but not bacteria, were isolated from the joints of eight (38%) of these patients. However, dissemination to joints apparently had not occurred in some despite the opportunity by virtue of mycoplasmal or ureaplasmal colonisation at a mucosal site. Sometimes antibiotic therapy failed clinically, and microbiologically and recommendations for management are outlined. CONCLUSIONS--Hypogammaglobulinaemic patients appear to be more susceptible to colonisation of mucous membranes, especially of the urogenital tract, with mycoplasmas and ureaplasmas than are immunocompetent individuals. These micro-organisms are responsible for about two fifths of the septic arthritides occurring in these patients.
PMCID: PMC1005283  PMID: 8154936
23.  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
24.  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
25.  Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvum strains 
BMC Microbiology  2012;12:88.
Ureaplasma urealyticum (UUR) and Ureaplasma parvum (UPA) are sexually transmitted bacteria among humans implicated in a variety of disease states including but not limited to: nongonococcal urethritis, infertility, adverse pregnancy outcomes, chorioamnionitis, and bronchopulmonary dysplasia in neonates. There are 10 distinct serotypes of UUR and 4 of UPA. Efforts to determine whether difference in pathogenic potential exists at the ureaplasma serovar level have been hampered by limitations of antibody-based typing methods, multiple cross-reactions and poor discriminating capacity in clinical samples containing two or more serovars.
We determined the genome sequences of the American Type Culture Collection (ATCC) type strains of all UUR and UPA serovars as well as four clinical isolates of UUR for which we were not able to determine serovar designation. UPA serovars had 0.75−0.78 Mbp genomes and UUR serovars were 0.84−0.95 Mbp. The original classification of ureaplasma isolates into distinct serovars was largely based on differences in the major ureaplasma surface antigen called the multiple banded antigen (MBA) and reactions of human and animal sera to the organisms. Whole genome analysis of the 14 serovars and the 4 clinical isolates showed the mba gene was part of a large superfamily, which is a phase variable gene system, and that some serovars have identical sets of mba genes. Most of the differences among serovars are hypothetical genes, and in general the two species and 14 serovars are extremely similar at the genome level.
Comparative genome analysis suggests UUR is more capable of acquiring genes horizontally, which may contribute to its greater virulence for some conditions. The overwhelming evidence of extensive horizontal gene transfer among these organisms from our previous studies combined with our comparative analysis indicates that ureaplasmas exist as quasi-species rather than as stable serovars in their native environment. Therefore, differential pathogenicity and clinical outcome of a ureaplasmal infection is most likely not on the serovar level, but rather may be due to the presence or absence of potential pathogenicity factors in an individual ureaplasma clinical isolate and/or patient to patient differences in terms of autoimmunity and microbiome.
PMCID: PMC3511179  PMID: 22646228

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