Polymerase chain reaction for the diagnosis of bovine genital campylobacteriosis

Bovine genital campylobacteriosis is a common venereal disease of cattle; the prevalence of this disease can be underestimated mostly because of the nature of the etiological agent, the microaerobic Campylobacter fetus subspecies venerealis. The purpose of the current study was to evaluate the utilization of polymerase chain reaction (PCR) in the diagnosis of genital campylobacteriosis in samples obtained from bull prepuce aspirate, cow cervical mucus, and abomasum contents of aborted fetuses, collected into enrichment medium. Five different DNA extraction protocols were tested: thermal extraction, lysis with proteinase K, lysis with guanidine isothiocyanate, lysis with DNAzol, and lysis with hexadecyltrimethylammonium bromide (CTAB). The specificity, sensitivity, and technical application of the PCR assay were also evaluated with clinical samples and compared to bacterial isolation by standard culture. DNA extraction by the CTAB protocol provided better results in PCR, and it was able to detect 63 colony-forming units per ml of C. fetus. Out of 277 clinical samples tested, 68 (24%) were positive for Campylobacter fetus using PCR, while only 8 (2.8%) of the samples were positive by bacterial isolation in solid medium, proving the superiority of the PCR technique when compared to the standard isolation method, and providing evidence for its usefulness as a better screening test in cattle for the diagnosis of bovine genital campylobacteriosis.

Immunohistochemical detection of Tritrichomonas foetus in experimentally infected mice

The need to intensify knowledge of the pathogenesis of bovine genital trichomoniasis (BGT) led to the use of alternative animal models such as the mouse. Nevertheless, it is necessary to elucidate the dynamics of the infection in this animal species, evaluating different stages of the colonization and evolution of the pathological alterations. The immunohistochemistry (IHC) offers advantages over the routine histopathological staining techniques for the detection of the protozoan in tissues, cellular detritus and inside the macrophages. The goal of the present study was to demonstrate the presence of Tritrichomonas foetus in the reproductive tract of infected mice using an IHC technique. Female BALB/c mice were infected with a suspension of T. foetus by intravaginal route, in the estrum phase, detected by exfoliative vaginal cytology. After 10 weeks, the animals were sacrificed; uterus and vagina were fixed and histologically processed. Some slides were stained with HE. The rest of the slides were processed for IHC. An immunoadsorbed polyclonal serum against T. foetus was used. The avidine-biotine technique (HistoMouse, Zymed™) was employed. The histopathological studies showed a dilation of the uterine glands, presence of macrophages in the lumen of the organ and inner part of the endometrial glands. No T. foetus was identified using this method. The IHQ allowed additionally the identification of the protozoan in the endometrium, endometrial glands, uterine lumen and inside neutrophils and macrophages. The cytological studies stained with IHC showed either isolated T. foetus adhered to epithelial cells or inside macrophages. This technique proves to be a useful tool for the study of the pathogenesis of bovine genital trichomoniasis (BGT) in an experimental model.

Production and characterization of monoclonal antibodies against Campylobacter fetus subsp. venerealis

Myeloma cells Sp2/0-Ag14 and spleen cells from BALB/c mouse immunized with sonicated Campylobacter fetus subsp. venerealis NCTC 10354 were fused with polyethylene glycol (PEG) for the selection of clones producing antibodies. Clones were obtained by limiting dilution and screened for the production of specific antibodies to C. fetus subsp. venerealis NCTC 10354 by indirect ELISA and western blot against a panel of bacteria: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352, and Arcobacter skirrowii LMG 6621 for the ELISA and C. fetus subsp. venerealis NCTC 10354 and C. sputorum biovar sputorum LMG 6647 for the western blotting. Fifteen clones producing monoclonal antibodies (MAbs) anti-C. fetus subsp. venerealis of the IgM (1) and IgG (14) classes were further screened for species-specificity. Four clones of the 15 obtained were producers of species-specific monoclonal antibodies (MAbs): two were specific for C. fetus subsp. venerealis and two were specific for C. fetus subsp. fetus. None of the clones were reactive against C. sputorum biovar sputorum LMG 6647. All clones recognized a protein with molecular mass of approximately 148 kDa from lysed C. fetus subsp. venerealis NCTC 10354.

Genital immunization of heifers with a glycoprotein Edeleted, recombinant bovine herpesvirus 1 strain confers protection upon challenge with a virulent isolate

Venereal infection of seronegative heifers and cows with bovine herpesvirus type 1.2 (BoHV-1.2) frequently results in vulvovaginitis and transient infertility. Parenteral immunization with inactivated or modified live BoHV-1 vaccines often fails in conferring protection upon genital challenge. We herein report an evaluation of the immune response and protection conferred by genital vaccination of heifers with a glycoprotein E-deleted recombinant virus (SV265gE-). A group of six seronegative heifers was vaccinated with SV265gE- (0,2mL containing 10(6.9)TCID50) in the vulva submucosa (group IV); four heifers were vaccinated intramuscularly (group IM, 1mL containing 10(7.6)TCID50) and four heifers remained as non-vaccinated controls. Heifers vaccinated IV developed mild, transient local edema and hyperemia and shed low amounts of virus for a few days after vaccination, yet a sentinel heifer maintained in close contact did not seroconvert. Attempts to reactivate the vaccine virus in two IV vaccinated heifers by intravenous administration of dexamethasone (0.5mg/kg) at day 70 pv failed since no virus shedding, recrudescence of genital signs or seroconversion were observed. At day 70 pv, all vaccinated and control heifers were challenged by genital inoculation of a highly virulent BoHV-1.2 isolate (SV56/90, 10(7.1)TCID50/animal). After challenge, virus shedding was detected in genital secretions of control animals for 8.2 days (8-9); in the IM group for 6.2 days (4-8 days) and during 5.2 days (5-6 days) in the IV group. Control non-vaccinated heifers developed moderate (2/4) or severe (2/4) vulvovaginitis lasting 9 to 13 days (x: 10.7 days). The disease was characterized by vulvar edema, vulvo-vestibular congestion, vesicles progressing to coalescence and erosions, fibrino-necrotic plaques and fibrinopurulent exudate. IM vaccinated heifers developed mild (1/3) or moderate (3/4) genital lesions, lasting 10 to 12 days (x: 10.7 days); and IV vaccinated heifers developed mild and transient vulvovaginitis (3/4) or mild to moderate genital lesions (1/4). In the IV group, the clinical signs lasted 4 to 8 days (x: 5.5 days). Clinical examination of the animals after challenge revealed that vaccination by both routes conferred some degree of protection, yet IV vaccination was clearly more effective in reducing the severity and duration of clinical disease. Furthermore, IV vaccination reduced the period of virus shedding in comparison with both groups. Taken together, these results demonstrate that SV265gE- is sufficiently attenuated upon IV vaccination in a low-titer dosis, is not readily reactivated after corticosteroid treatment and lastly, and more importantly, confers local protection upon challenge with a high titer of a virulent heterologous BoHV-1 isolate. Therefore, the use of this recombinant for genital immunization may be considered for prevention of BoHV-1-associated genital disease in the field.

A glycoprotein E gene-deleted bovine herpesvirus 1 as a candidate vaccine strain

A bovine herpesvirus 1 (BoHV-1) defective in glycoprotein E (gE) was constructed from a Brazilian genital BoHV-1 isolate, by replacing the full gE coding region with the green fluorescent protein (GFP) gene for selection. Upon co-transfection of MDBK cells with genomic viral DNA plus the GFP-bearing gE-deletion plasmid, three fluorescent recombinant clones were obtained out of approximately 5000 viral plaques. Deletion of the gE gene and the presence of the GFP marker in the genome of recombinant viruses were confirmed by PCR. Despite forming smaller plaques, the BoHV-1△gE recombinants replicated in MDBK cells with similar kinetics and to similar titers to that of the parental virus (SV56/90), demonstrating that the gE deletion had no deleterious effects on replication efficacy in vitro. Thirteen calves inoculated intramuscularly with BoHV-1△gE developed virus neutralizing antibodies at day 42 post-infection (titers from 2 to 16), demonstrating the ability of the recombinant to replicate and to induce a serological response in vivo. Furthermore, the serological response induced by recombinant BoHV-1△gE could be differentiated from that induced by wild-type BoHV-1 by the use of an anti-gE antibody ELISA kit. Taken together, these results indicated the potential application of recombinant BoHV-1 △gE in vaccine formulations to prevent the losses caused by BoHV-1 infections while allowing for differentiation of vaccinated from naturally infected animals.

Detecção de HPV na mucosa oral e genital pela técnica PCR em mulheres com diagnóstico histopatológico positivo para HPV genital

A infecção do papilomavírus humano (HPV) é uma das mais freqüentes doenças sexualmente transmissíveis em todo o mundo. A relação entre o HPV genital e oral permanece incerta, assim como o seu papel na carcinogênese oral. O objetivo deste estudo foi verificar a presença do DNA do HPV na mucosa oral e genital de mulheres com infecção genital por HPV, pela técnica de reação em cadeia de polimerase (PCR). FORMA DE ESTUDO: Coorte transversal. MATERIAL E MÉTODO: Trata-se de um estudo piloto, prospectivo, com 30 mulheres, idade de 14 a 51 anos, portadoras de infecção genital por HPV confirmada pelo exame de histopatológico. Todas as pacientes foram submetidas a exame e coleta por raspagem da cavidade oral e genital para pesquisa do DNA do HPV pela técnica PCR. RESULTADOS: Nenhuma das amostras da cavidade oral foi positiva para HPV, enquanto no genital, o HPV foi detectado em 17 (57%) das 30 pacientes, principalmente o HPV 6b e 16. CONCLUSÃO: Os resultados mostraram maior porcentagem do HPV genital em relação à cavidade oral, e sugerem que o HPV genital não parece ser fator predisponente para a infecção oral no mesmo paciente.

Antibodies against Bovine herpesvirus 1 in dairy herds in the state of Espirito Santo, Brasil

Bovine herpesvirus 1 (BoHV-1) causes major losses in worldwide livestock, affecting the respiratory and reproductive tracts of bovine. In the past decades, the number of cases in Brazil has been gradually increasing. Therefore, it is important to assess the distribution of infection in different regions of the country. In the state of Espírito Santo (ES) the BoHV 1 infection rate in dairy cattle herds is unknown. Thus, the aim of this study was to detect neutralizing antibodies against BoHV-1 in serum samples from 1,161 non-vaccinated cows from 59 dairy cattle herds in 23 municipalities of the Metropolitan, North, Northwest and South macro-regions. The identification of seropositive cows was evaluated by the virus neutralization test. The results showed that of all serum samples evaluated 775 (66.75%) had neutralizing antibodies against BoHV-1. Moreover, all herds were found positive; however, the percentage of positive cows varied among regions; 49.06%, 62.15%, 67.21% and 80.04% for the Metropolitan, South, North and Northwest macro-regions, respectively. In this study, the results clearly indicate the dissemination of the viral agent in dairy cattle in the ES state, requiring the monitoring and control of diseases related to BoHV-1 infection.

Infectivity of cysts of the ME-49 Toxoplasma gondii strain in bovine milk and homemade cheese

OBJECTIVE: Analyze the infectivity and storage resistance of cysts of the ME-49 strain of Toxoplasma gondii in artificially infected bovine milk and homemade fresh cheese. METHODS: Pasteurized bovine milk was infected with 10 cysts/ml of the ME-49 strain of T.gondii and inoculated in different groups of mice, immediately or after storage at 4ºC for 5, 10 and 20 days. Homemade fresh cheese was prepared with artificially infected milk, and also tested in groups of mice, using the same storage process. Infection was identified by the presence of cysts in the brain or serological testing in challenged mice after 5 weeks, confirmed by Western Blot and histology. RESULTS: The infectivity of cysts of the ME-49 strain of T.gondii was maintained in the milk even after storage for 20 days at refrigerator temperatures. Cysts were also able to survive the production process of homemade fresh cheese and storage for a period of 10 days in the same conditions. CONCLUSIONS: These data demonstrated that milk and dairy products could be an important source of T.gondii in human contamination, reinforcing the importance of milk pasteurization before any processing or ingestion.

Prevalencia de microorganismos asociados a secreción genital femenina, Argentina

OBJETIVO: Hay un aumento significativo de pacientes con Secreción Genital Femenina, en el Sector Público del gran Buenos Aires. Fue necesario actualizar la prevalencia de los microorganismos asociados a los efectos de revisar el apoyo necesario de laboratorio y ajustar las medidas de prevención y control. MÉTODOS: Se incorporan a este estudio, la totalidad de los casos atendidos (1997-1998): 84 adolescentes (15 a 19 años) y 784 adultas (20 a 60 años) sintomáticas. El protocolo incluye (secreción vaginal y endocervical) detección de Neisseria gonorrhoeae, Streptococcus agalactiae, Trichomonas vaginalis, Candida spp y vaginosis bacteriana. Aplicando métodos específicos directos y cultivo, Chlamydia trachomatis (detección de antígeno), Ureaplasma urealitycum y Mycoplasma hominis (cultivos) fueron estudiados en parte de la población total. RESULTADOS: El aumento de la demanda de consulta fue continuo desde 1997 y aumentó 2.10 veces del primero al último semestre de 1998. En las mujeres adultas se encontró: vaginosis bacteriana, 23,8%; Candida spp 17,8%; S. agalactiae 5,6%; T. vaginalis 2,4%. En 50,3% no se detecto ninguno. En adolescentes se detectó: vaginosis bacteriana, 17,8%; Candida spp 29,7%; S. agalactiae 3,6%, T. vaginalis 2,4%: En 46,4% de los casos el resultado fue negativo. En el grupo de mujeres adultas sintomáticas, no en la totalidad, se detectó: C. trachomatis (7/400) 1,76%, U. urealyticum (209/340) 61,4% y M. hominis (45/272) 16,5%. CONCLUSIONES: El aumento significativo de consultas se debe a problemas sociales en la población, no al aumento de ninguna patología en especial. Impacta como problema clínico concreto la prevalencia de vaginosis bacteriana y Candida spp. Llama la atención, la nula incidencia de N. gonorrhoeae y la baja circulación de T. Vaginalis y C. trachomatis, en este tipo de población. La prevalencia de U. urealyticum y M. hominis es alta, pero su real participación en la patología genital de adultas debe ser reconsiderada. Las prevalencias establecidas motivan la necesidad de adecuar los manuales de procedimientos apuntando a la aplicación de metodologías simples de alto valor predictivo.

Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE). As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80) than the latter (1:80 to 1:160). Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

Paracoccidioidomicose genital feminina. Descrição de um caso clínico

A paracoccidioidomicose é doença sistêmica que atinge mais o sexo masculino do que o feminino. Modelos experimentais mostram maior sensibilidade dos machos do que as fêmeas à dsseminação da doença. A literatura médica é rica em relatos de casos de Pbmicose do trato genital masculino. A raridade da Pbmicose sistêmica na mulher devido à ação hormonal tornou inusitada a descrição de um caso com envolvimneto genital. Paciente de 57 anos, branca, procedente de Conchas (SP), queixando-se de dor abdominal há 10 anos, difusa, seguida de eólica periumbelical periódica, com diarréia e obstipação alternadas. Piorou desse quadro e evoluiu para abdome agudo cirúrgico. O anátomo patológico revelou Pbmicose no epiplon e ovário direito. A biópsia do endométrio mostrou endometrite crônica granulomatosa. A radiologia e a planigrafia revelaram nódulo parahilar direito. Os achados clínicos e laboratoriais permitiram explicar a provável origem do envolvimento genital.

Transmission of Schistosoma mansoni under experimental conditions using the bovine - Biomphalaria glabrata - bovine model

Three calves experimentally infected with Schistosoma mansoni, and passing viable eggs in feces, as well as 5 normal calves (coming from a non-endemic area for schistosomiasis) kept as controls, were maintained in an enclosure (850 m² in area). In this enclosure, a tank with water received 500 laboratory reared Biomphalaria glabrata. All the control calves were infected for a period ranging from 79 to 202 days after the beginning of the experiment, and afterwards presented viable S. mansoni eggs in feces. The mean worm recovery was 555. The snail population increased throughout the experimental period, showing a high number of B. glabrata infected with S. mansoni (42% on average). According to the present study, bovine has been suggested as having potentially a role in the maintenance of the life cycle of S. mansoni

Paracoccidioidomycosis of the male genital tract. Report of eleven cases and a review of Brazilian literature

Eleven cases of involvement of the genital tract in paracoccidioidomycosis were collected in a retrospective study of the clinical records of 683 patients seen in Porto Alegre, Rio Grande do Sul, Brazil. These cases are herein summarily reported. Eighteen similar cases were gathered in review of the Brazilian literature. Obtained data are discussed.

Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA

The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF) obtained from fertile (FC) and non-fertile cysts (NFC). Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC) and NFC (PNFC), respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates) were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates.