RESUMO
Bacteria classified in Mycoplasma (M. bovis and M. bovigenitalium) and Ureaplasma (U. diversum) genera are associated with granular vulvovaginitis that affect heifers and cows at reproductive age. The traditional means for detection and speciation of mollicutes from clinical samples have been culture and serology. However, challenges experienced with these laboratory methods have hampered assessment of their impact in pathogenesis and epidemiology in cattle worldwide. The aim of this study was to develop a PCR strategy to detect and primarily discriminate between the main species of mollicutes associated with reproductive disorders of cattle in uncultured clinical samples. In order to amplify the 16S-23S rRNA internal transcribed spacer region of the genome, a consensual and species-specific nested-PCR assay was developed to identify and discriminate between main species of mollicutes. In addition, 31 vaginal swab samples from dairy and beef affected cows were investigated. This nested-PCR strategy was successfully employed in the diagnosis of single and mixed mollicute infections of diseased cows from cattle herds from Brazil. The developed system enabled the rapid and unambiguous identification of the main mollicute species known to be associated with this cattle reproductive disorder through differential amplification of partial fragments of the ITS region of mollicute genomes. The development of rapid and sensitive tools for mollicute detection and discrimination without the need for previous cultures or sequencing of PCR products is a high priority for accurate diagnosis in animal health. Therefore, the PCR strategy described herein may be helpful for diagnosis of this class of bacteria in genital swabs submitted to veterinary diagnostic laboratories, not demanding expertise in mycoplasma culture and identification.
Assuntos
Doenças dos Bovinos/diagnóstico , Infecções por Bactérias Gram-Negativas/veterinária , Tenericutes/isolamento & purificação , Vulvovaginite/veterinária , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Feminino , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Especificidade da Espécie , Tenericutes/genética , Vagina/microbiologia , Vulvovaginite/diagnóstico , Vulvovaginite/microbiologiaRESUMO
Ureaplasma diversum infection in bovine females may result in various reproductive problems, including granular vulvovaginitis, abortion, weak calves, salpingitis, and spontaneous abortion. The presence of U. diversum in a dairy bovine population from midwestern Brazil has not been established. The aim of this study was to determine whether U. diversum was present in dairy cattle from midwestern Brazil using polymerase chain reaction (PCR). Vulvovaginal mucus was analyzed from 203 cows located in six municipalities in the north region of Mato Grosso State, Brazil. A total of 25% of dairy cows with vulvovaginitis were positive for U. diversum. The factors evaluated were included in a multivariable logistic regression model with the presence of at least one positive cow in the herd serving as the dependent variable. Three variables were significantly associated with a U. diversum-positive PCR and were included in the final multivariable model: number of parities, vulvar lesions, and reproductive problems. For each new parity, the chance of U. diversum infection decreased 0.03-fold, indicating that cows with the highest number of parities were more protected. The presence of vulvar lesions was increased 17.6-fold in females positive for U. diversum, suggesting that this bacterium could be related to the red granular lesions in the vulvar mucosa, whereas reproductive problems were increased 7.6-fold. However, further investigations should be conducted to ascertain the effects of U. diversum in association with other mycoplasma species in the herds studied.
Assuntos
Doenças dos Bovinos/epidemiologia , Infecções por Ureaplasma/veterinária , Ureaplasma/isolamento & purificação , Vulvovaginite/veterinária , Aborto Animal/epidemiologia , Animais , Brasil/epidemiologia , Bovinos , Doenças dos Bovinos/microbiologia , Indústria de Laticínios , Feminino , Reação em Cadeia da Polimerase/veterinária , Gravidez , Ureaplasma/genética , Infecções por Ureaplasma/epidemiologia , Infecções por Ureaplasma/microbiologia , Vulvovaginite/epidemiologia , Vulvovaginite/microbiologiaRESUMO
Ureaplasma diversum has been associated with various reproductive problems in cattle that include granular vulvovaginitis, weak calves, and abortion. This study was conducted in a beef herd situated in the Middle-West region of Brazil, and the objectives were to verify the presence of U. diversum and to elucidate its possible relationships with independent variables in this bovine herd population. A total of 134 vaginal mucous swabs were taken for polymerase chain reaction (PCR). Of these, 51 (38 %) were PCR positive for U. diversum. Of the 58 heifers with vulvovaginal lesions characterized by hyperemia, granulated lesions, and edema distributed throughout the vulvar mucosa, 37 (64 %) were U. diversum positive; of the 76 heifers without reproductive lesions, 14 (18 %) were U. diversum positive. All tested samples were negative for bovine herpesvirus 1 (BoHV-1). Multivariate logistic regression revealed that the following two variables were significantly associated with the presence of U. diversum: the presence of vulvar lesions (p = 0.001) and the presence of a progesterone (P4) device (p = 0.001). These findings indicate that U. diversum should be considered a pathogen that is associated with pustular vulvovaginitis in heifers from the Mato Grosso state and that additional studies of the risk factors associated with intravaginal P4 device transmission should be performed.
Assuntos
Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/microbiologia , Infecções por Ureaplasma/veterinária , Ureaplasma/isolamento & purificação , Vulvovaginite/veterinária , Animais , Brasil/epidemiologia , Bovinos , Feminino , Modelos Lineares , Reação em Cadeia da Polimerase/veterinária , Gravidez , Ureaplasma/genética , Infecções por Ureaplasma/epidemiologia , Vulvovaginite/epidemiologiaRESUMO
Em março de 2012 foi diagnosticado um surto de doença reprodutiva em rebanho bovino no Estado da Paraíba, Brasil. Foram examinadas 32 vacas e dois touros da raça Girolando. As vacas apresentaram sinais de doença reprodutiva como repetição de cio, vulvovaginite granular, infertilidade e abortos. As amostras de suabes vaginais e prepuciais foram colhidas e submetidas a isolamento bacteriano e PCR. As reações da PCR para Mollicutes e Ureaplasma spp. foram realizadas com os iniciadores MGSO-GPO3 e UGP'F-UGP'R, respectivamente. Na Nested PCR para Ureaplasma diversum, os iniciadores usados foram UD1, UD2, UD3 e UD4. Para isolamento bacteriano, as amostras foram diluídas de 10-1 até 10-5, semeadas em meio "UB", líquido e placa, sendo incubadas por até 21 dias a 37ºC em jarra de microaerofilia. A frequência de Mollicutes detectada na PCR foi de 65,6% e para Ureaplasma spp. foi de 50,0%, enquanto que para U. diversum foi de 15,6%. No isolamento a frequência de Mollicutes foi de 57,1% e para Ureaplasma spp. foi de 28,6%. No ágar "UB" foi visualizado o crescimento misto de Mycoplasma spp. e Ureaplasma spp. em seis amostras. Foi confirmado o envolvimento de micro-organismos da Classe Mollicutes em surto de doença reprodutiva em vacas no sertão paraibano.
In March of 2012 was investigated a reproductive disease outbreak in cattle herds from Paraíba State, Brazil. Were examined 32 cows and two bulls Giroland breed. The cows showed signs and symptoms of reproductive failure such as repeat breeding, granular vulvovaginitis, infertility and abortions. Vaginal and preputial mucous samples were collected for analysis by PCR and isolation. The PCR reactions for Mollicutes and Ureaplasma spp. were realized with primers MGSO and GPO3, and UGP'F and UGP'R respectively. The nested PCR assay for Ureaplasma diversum was realized with primers UD1, UD2, UD3 and UD4. For bacteriologic isolation, obtained samples were diluted up to 10-1 at 10-5, inoculated into liquid and solid "UB" medium, and incubated for up to 21 days, at 37ºC in microaerophilie jar. In the PCR reactions the frequency of Mollicutes detected in the analyzed vaginal mucous samples was 65.6, for Ureaplasma spp. was 50.0, while for U. diversum was 15.6. The frequency for isolation of Mollicutes was of 57.1 and for Ureaplasma spp. was of 28.6. In the UB agar was visualized growth of Mycoplasma spp. and Ureaplasma spp., associated in six of the samples. In the cows the presence of Mollicutes and Ureaplasma spp. was confirmed for the reproductive disease outbreak in the semiarid region of Paraiba.
Assuntos
Animais , Feminino , Bovinos , Infecções por Ureaplasma/veterinária , Tenericutes/isolamento & purificação , Ureaplasma/isolamento & purificação , Aborto Animal , Doenças dos Genitais Femininos/veterinária , Infertilidade/veterinária , Vulvovaginite/veterináriaRESUMO
Em março de 2012 foi diagnosticado um surto de doença reprodutiva em rebanho bovino no Estado da Paraíba, Brasil. Foram examinadas 32 vacas e dois touros da raça Girolando. As vacas apresentaram sinais de doença reprodutiva como repetição de cio, vulvovaginite granular, infertilidade e abortos. As amostras de suabes vaginais e prepuciais foram colhidas e submetidas a isolamento bacteriano e PCR. As reações da PCR para Mollicutes e Ureaplasma spp. foram realizadas com os iniciadores MGSO-GPO3 e UGP'F-UGP'R, respectivamente. Na Nested PCR para Ureaplasma diversum, os iniciadores usados foram UD1, UD2, UD3 e UD4. Para isolamento bacteriano, as amostras foram diluídas de 10-1 até 10-5, semeadas em meio "UB", líquido e placa, sendo incubadas por até 21 dias a 37ºC em jarra de microaerofilia. A frequência de Mollicutes detectada na PCR foi de 65,6% e para Ureaplasma spp. foi de 50,0%, enquanto que para U. diversum foi de 15,6%. No isolamento a frequência de Mollicutes foi de 57,1% e para Ureaplasma spp. foi de 28,6%. No ágar "UB" foi visualizado o crescimento misto de Mycoplasma spp. e Ureaplasma spp. em seis amostras. Foi confirmado o envolvimento de micro-organismos da Classe Mollicutes em surto de doença reprodutiva em vacas no sertão paraibano.(AU)
In March of 2012 was investigated a reproductive disease outbreak in cattle herds from Paraíba State, Brazil. Were examined 32 cows and two bulls Giroland breed. The cows showed signs and symptoms of reproductive failure such as repeat breeding, granular vulvovaginitis, infertility and abortions. Vaginal and preputial mucous samples were collected for analysis by PCR and isolation. The PCR reactions for Mollicutes and Ureaplasma spp. were realized with primers MGSO and GPO3, and UGP'F and UGP'R respectively. The nested PCR assay for Ureaplasma diversum was realized with primers UD1, UD2, UD3 and UD4. For bacteriologic isolation, obtained samples were diluted up to 10-1 at 10-5, inoculated into liquid and solid "UB" medium, and incubated for up to 21 days, at 37ºC in microaerophilie jar. In the PCR reactions the frequency of Mollicutes detected in the analyzed vaginal mucous samples was 65.6, for Ureaplasma spp. was 50.0, while for U. diversum was 15.6. The frequency for isolation of Mollicutes was of 57.1 and for Ureaplasma spp. was of 28.6. In the UB agar was visualized growth of Mycoplasma spp. and Ureaplasma spp., associated in six of the samples. In the cows the presence of Mollicutes and Ureaplasma spp. was confirmed for the reproductive disease outbreak in the semiarid region of Paraiba.(AU)
Assuntos
Animais , Feminino , Bovinos , Tenericutes/isolamento & purificação , Ureaplasma/isolamento & purificação , Infecções por Ureaplasma/veterinária , Doenças dos Genitais Femininos/veterinária , Vulvovaginite/veterinária , Infertilidade/veterinária , Aborto AnimalRESUMO
In order to determine the role of Mycoplasma spp, Ureaplasma diversum and BHV-1 as causal agents of Granular Vulvovaginitis Syndrome in Nelore heifers raised under tropical conditions and based on the hypothesis that stressful conditions during puberty or breeding season would be a determinant factor for the infection, 340 heifers not vaccinated against BHV-1 were divided in Post-pubertal, in the beginning of the first breeding season, and Pubertal heifers. The vaginal lesion score (VLS) Grade 1 to 4 was giving according to lesion area and severity. Vaginal mucus was used to isolate Mycoplasma spp., Ureaplasma diversum and BHV-1. The predominant VLS was 2. No sample was positive for BHV-1; 48% were positive for Mycoplasma spp., Ureaplasma diversum, or both, with predominance of Ureaplasma diversum. Serum neutralization for BHV-1 showed more positive animals in pubertal group (23%); 3 of the paired sera demonstrated seroconversion. These data indicated that post-pubertal and pubertal Nelore heifers raised under extensive conditions are more susceptible to Mycoplasma spp. and Ureaplasma diversum. The hypothesis that the stress of pubertal period could lead to an acute vaginal infection by HBV-1 was not proofed.
Assuntos
Doenças dos Bovinos/etiologia , Herpesvirus Bovino 1 , Mycoplasma , Ureaplasma , Vagina/patologia , Vulvovaginite/veterinária , Animais , Brasil , Bovinos , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/patologia , Doenças dos Bovinos/virologia , Feminino , Testes de Neutralização/veterinária , Fatores de Risco , Estresse Fisiológico/fisiologia , Síndrome , Clima Tropical , Vagina/microbiologia , Vagina/virologia , Vulvovaginite/etiologia , Vulvovaginite/microbiologia , Vulvovaginite/patologia , Vulvovaginite/virologiaRESUMO
A detecçäo de anticorpos anti-Herpesvírus Bovino tipo 1 (BHV-1) foi realizada, através da técnica de soroneutralizaçäo, em 1235 amostras de soro de bovinos adultos, näo vacinados contra Rinotraqueíte Infecciosa Bovina. As amostras de soro analisadas foram colhidas em 81 rebanhos, com histórico de problemas reprodutivos, incluindo animais com aptidäo para carne e leite, provenientes de 30 municípios do Estado do Paraná. Na amostragem proveniente de rebanhos leiteiros, 41,9 por cento (409/977) das amostras de soro e 90,5 por cento (57/63) dos rebanhos foram considerados positivos. Em bovinos de corte, o índice de soropositividade foi de 50,8 por cento (131/258) e 100 por cento (18/18) para amostras de soro e rebanhos, respectivamente. As frequências de 43,7 por cento (540/1235) de animais e 92,6 por cento (75/81) de rebanhos soropositivos demonstram que as infecçöes por BHV-1 apresentam-se amplamente disseminadas nas regiöes estudadas.