RESUMO
Clinical manifestations of leptospirosis are diverse and very similar to other febrile diseases, hence early and accurate detection of subclinical infections is a key element in disease control. We evaluated immunomagnetic separation (IMS) capture technology coupled with a standard quantitative PCR (qPCR) system for the detection of pathogenic Leptospira in urine samples from 803 cows from dairy herds with a history of clinical cases of leptospirosis. The urine samples were first processed in a purification step, then subdivided into 2 subsamples, one that continued to DNA extraction and direct qPCR, and one that was pretreated by IMS before continuing to DNA extraction and qPCR. Overall, 133 of 803 (16.6%) samples were IMS-qPCR positive, whereas only 92 of 803 (11.5%) were positive when using direct qPCR. Statistically significant differences were observed between the mean estimated Leptospira load between the IMS-qPCR and the direct qPCR positive urine samples. The IMS-qPCR technology revealed a larger number of positive results and higher bacterial loads than direct qPCR. This difference is most likely the result of the high antigen-binding capacity and capture efficiency of the IMS system. The use of polyclonal antibodies produced by the inoculation of 3 synthetic peptides, which make up the extracellular regions of the LipL32 protein, provided a high detection capacity to the IMS-qPCR technique, resulting in performance superior to direct qPCR.
Assuntos
Criação de Animais Domésticos , Doenças dos Bovinos/diagnóstico , Leptospira/isolamento & purificação , Leptospirose/veterinária , Animais , Bovinos , Doenças dos Bovinos/urina , Chile , Indústria de Laticínios , Feminino , Separação Imunomagnética/veterinária , Leptospira/genética , Leptospira/imunologia , Leptospirose/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sensibilidade e Especificidade , Urinálise/veterináriaRESUMO
A domestic cat dwelling in a dairy cattle farm with haematuria was referred for a physical examination. The examination showed no abnormalities therefore complementary exams were performed. Leukocytosis with neutrophilia, monocytosis and hyperproteinaemia were detected. The urine analysis showed a bacterial infection without ultrasound findings. Serological titers to Leptospira interrogans serovar Pomona and Autumnalis were detected. Molecular analysis demonstrated the presence of Leptospira spp. in urine. The findings were consistent with subclinical leptospirosis. The cattle herd had evidence of Leptospira infection. The microbiological exams confirmed the presence of the Leptospira spp. in urine and serum. According to the evidence presented in this study, cats that dwell within a dairy farm could play a role in the Leptospira infection epidemiologically. The importance of feline leptospirosis must be evaluated with leptospirosis control in livestock.
Assuntos
Doenças do Gato/transmissão , Doenças dos Bovinos/transmissão , Leptospira/isolamento & purificação , Leptospirose/veterinária , Animais , Anticorpos Antibacterianos , Doenças do Gato/sangue , Doenças do Gato/urina , Gatos , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/urina , Fazendas , Leptospirose/sangue , Leptospirose/transmissão , Leptospirose/urina , Gado , SorogrupoRESUMO
Most epidemiologic studies on bovine leptospirosis are based on serological tests that use antibodies against several serotypes, including the serovar Hardjo, which is widespread and considered to be the most adapted to bovine hosts. However, using only serological studies is not sufficient to identify and distinguish species of leptospires. The aim of this study was report the first isolation in Brazil of two strains serovar Hardjo obtained in urine samples from naturally infected cows in a small Brazilian dairy herd and find the genetic species and consequently the type strain Hardjobovis by molecular characterization. Fifteen dairy cows with a history of reproductive failure, such as abortion and infertility, were selected. Urine samples obtained from each animal were immediately seeded in tubes containing Ellinghausen-McCullough-Johnson-Harris culture medium. The identification of the isolates was performed by Multilocus variable-number tandem-repeat analysis (MLVA) technique and phylogenetic analysis of partial sequence of gene sec Y. From the 15 urine samples evaluated, two Leptospira were found and identified as the Londrina 49 and Londrina 54 strains. The MLVA profiles and sequencing of gene sec Y characterized the isolates as L. borgpetersenii serovar Hardjo strain Hadjobovis because it has different genetic pattern of Leptospira interrogans serovar Hardjo strain Hardjoprajitno. Therefore, more studies are needed including isolation and molecular characterization from regional strains to obtain a better knowledge about epidemiology of serovar Hardjo in bovine which may assist in future strategies of prevention and control of bovine leptospirosis.
Assuntos
Anticorpos Antibacterianos/urina , Doenças dos Bovinos/microbiologia , Genes Bacterianos , Infertilidade Feminina/microbiologia , Leptospira/genética , Leptospirose/microbiologia , Leptospirose/veterinária , Animais , Técnicas de Tipagem Bacteriana , Brasil , Bovinos , Doenças dos Bovinos/patologia , Doenças dos Bovinos/urina , Feminino , Infertilidade Feminina/patologia , Infertilidade Feminina/urina , Leptospira/classificação , Leptospira/isolamento & purificação , Leptospirose/patologia , Leptospirose/urina , Tipagem de Sequências Multilocus , Filogenia , Análise de Sequência de DNA , SorogrupoRESUMO
The aim of this study was to identify Leptospira in urine samples of cattle by direct sequencing of the secY gene. The validity of this approach was assessed using ten Leptospira strains obtained from cattle in Brazil and 77 DNA samples previously extracted from cattle urine, that were positive by PCR for the genus-specific lipL32 gene of Leptospira. Direct sequencing identified 24 (31·1%) interpretable secY sequences and these were identical to those obtained from direct DNA sequencing of the urine samples from which they were recovered. Phylogenetic analyses identified four species: L. interrogans, L. borgpetersenii, L. noguchii, and L. santarosai with the most prevalent genotypes being associated with L. borgpetersenii. While direct sequencing cannot, as yet, replace culturing of leptospires, it is a valid additional tool for epidemiological studies. An unexpected finding from this study was the genetic diversity of Leptospira infecting Brazilian cattle.
Assuntos
Proteínas de Bactérias/genética , Doenças dos Bovinos/epidemiologia , Genótipo , Leptospira/genética , Leptospirose/veterinária , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Brasil/epidemiologia , Bovinos , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/urina , Leptospira/metabolismo , Leptospirose/epidemiologia , Leptospirose/microbiologia , Leptospirose/urina , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/veterináriaRESUMO
The aim of the present study was to consider the wide usage of urinary PCR as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock. A total of 512 adult animals (300 cattle, 138 horses, 59 goats and 15 pigs), from herds/flocks with reproductive problems in Rio de Janeiro, Brazil was studied by serology and urinary PCR. From the 512 serum samples tested, 223 (43.5 %) were seroreactive (cattle: 45.6 %, horses: 41.3 %, goats: 34%and pigs: 60 %). PCR detected leptospiral DNA in 32.4 % (cattle: 21.6 %, horses: 36.2 %, goats: 77.4 % and pigs: 33.3 %. To our knowledge there is no another study including such a large number of samples (512) from different species, providing a comprehensive analysis of the usage of PCR for detecting leptospiral carriers in livestock. Serological and molecular results were discrepant, regardless the titre, what was an expected outcome. Nevertheless, it is impossible to establish agreement between these tests, since the two methodologies are conducted on different samples (MAT - serum; PCR - urine). Additionally, the MAT is an indirect method and PCR is a direct one. In conclusion, we have demonstrated that urinary PCR should be considered and encouraged as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock.
Assuntos
Doenças dos Bovinos/urina , Doenças das Cabras/urina , Doenças dos Cavalos/urina , Leptospirose/veterinária , Gado/parasitologia , Reação em Cadeia da Polimerase/veterinária , Doenças dos Suínos/urina , Testes de Aglutinação/normas , Testes de Aglutinação/veterinária , Animais , Bovinos , Cabras , Cavalos , Leptospirose/diagnóstico , Leptospirose/urina , Limite de Detecção , Gado/urina , Reação em Cadeia da Polimerase/normas , Reprodutibilidade dos Testes , SuínosRESUMO
Foram estudados casos espontâneos de intoxicação crônica por samambaia (Pteridium aquilinum) em bovinos nas formas clinicopatológicas de carcinoma de células escamosas (CCE) no trato alimentar superior (TAS) e de hematúria enzoótica bovina (HEB), provenientes da Mesorregião Centro Ocidental Rio-Grandense e encaminhados ao Laboratório de Patologia Veterinária da Universidade Federal de Santa Maria. Para o estudo clínico foram avaliados os sinais clínicos de bovinos com CCEs no TAS e com HEB e realizados hemogramas na fase terminal da doença. Os principais sinais clínicos nos bovinos com CCEs no TAS foram emagrecimento progressivo, atonia ruminal, tosse, disfagia, timpanismo e regurgitação. Nos bovinos com HEB, hematúria foi o principal sinal, observado em todos os casos, seguido de emagrecimento progressivo. No exame hematológico, 33,33 por cento dos bovinos com CCEs no TAS e 66,67 por cento dos bovinos com HEB apresentaram anemia arregenerativa...(AU)
Spontaneous cases of chronic poisoning by Pteridium aquilinum in cattle were studied. The clinical forms of the disease were squamous cell carcinoma (SCCs) of the upper digestive tract (UDT) and bovine enzootic hematuria (BEH). The cases were from the midland Region of the Midwest of Rio Grande do Sul State, Brazil, and were submitted to the Laboratório de Patologia Veterinária of the Universidade Federal de Santa Maria. Clinical signs and blood work were evaluated at terminal phase of disease. Cattle with UDT SCCs had progressive weigth loss, ruminal atony, cough, dysphagia, bloating, and regurgitation. In cattle with BEH, hematuria was observed in all cases, followed by progressive weight loss. Non-regenerative anemia was detected in 33.33 percent of the cattle with UDT SCCs form and in 66.66 percent of the cattle with BEH form. Changes in white blood count occurred in some cases but drop in lymphocyte numbers was uncommon in both forms of disease. For the morphological study, urinary bladders from 46 cattle with UDT SCCs and 11 cattle with BEH were analyzed. Grossly, 16/46 bladders from the UDT SCCs form had gross lesions (red or pale vesical nodules, hemorrhages, and papilomas; red urine was detected at necropsy of only three cases). In BEH form, the bladder had nodules, large neoplastic masses, red urine, papilomas, and hemorrhages. Pyelonephritis and hydronephrosis were seen in a few cases. Microscopically, in the UDT SCCs form, 44/46 (95.65 percent) bladders had 22 different types of morphological changes, characterized by neoplastic lesions (5/22) and non-neoplastic lesions (17/22); the latter were subdivided in non-neoplastic epithelial changes (6/17), general changes of the lamina propria (6/17), and inflammatory changes (5/17). The bladder changes in BEH form were of 19 different types, characterized by neoplastic lesions (5/19) and non-neoplastic lesions (14/19), which were subdivided in non-neoplastic epitelial changes (9/14),...(AU)
Assuntos
Animais , Pteridium/intoxicação , Intoxicação por Plantas/sangue , Intoxicação por Plantas/urina , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/complicações , Carcinoma de Células Escamosas/patologia , Hematúria/patologia , Hematúria/veterinária , Testes Hematológicos/métodos , Doenças dos Bovinos/etiologia , Intoxicação por Plantas/veterinária , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/urina , Doenças dos Bovinos/sangue , BovinosRESUMO
Papillomavirus infection in bovines is associated with cutaneous papillomatosis on the hide, udders and other epithelial tissues, as well as in oral respiratory, alimentary and urinary tract mucosa. Bovine papillomavirus (BPV) is also considered the etiological agent of esophageal tumors and the malignant bladder tumors that characterize the clinical condition associated with chronic enzootic hematuria. After infective viral DNA was found in cattle blood and BPV1, 2 and 4 DNA in cattle reproductive and embryonic tissues, we looked for and found BPV DNA in blood, milk, urine, seminal fluid, and spermatozoa of BPV-infected animals. Peripheral blood lymphocyte cultures from BPV-infected animals had high rates of chromosome aberrations, including radial rearrangements that signal oncogenic potential and viral interaction with telomeric regions. The finding of BPV DNA in body fluids and tissues other than the epithelium demonstrates co-infection of other tissues or cell types by papillomavirus and shows the potential role of lymphocytes, seminal fluid and spermatozoa in BPV transmission. Our findings reinforce a peremptory need for prophylactic and therapeutic instruments to curtail this disease in bovine livestock.
Assuntos
Doenças dos Bovinos/virologia , DNA Viral/análise , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/veterinária , Animais , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/urina , Feminino , Masculino , Leite/virologia , Papillomaviridae/genética , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/virologia , Espermatozoides/virologiaRESUMO
The purpose of the present study was to establish a practical, fast, precise and low-cost procedure to estimate the degree of metabolic acidosis in cattle with acute rumen lactic acidosis for further treatment. The rumen acidosis was induced experimentally in 40 crossbreed rumen-cannulated 1.5-year-old steers. The induction caused the development of the most characteristic clinical signs of acute rumen lactic acidosis, severe rumen acidosis and a moderate metabolic acidosis, which was evidenced by low blood pH, and blood bicarbonate concentration and base excess (BE). A highly positive correlation (r=0.80) between urinary pH and BE concentration, and between urinary pH and blood pH (r=0.75) was observed. The BE concentration estimated by urinary pH was similar to that determined by venous blood gas analysis (P>0.99). Furthermore, the results presented by the predictive formula were very significant. In conclusion, urinary pH is a good tool to predict the quantity of buffers needed to treat metabolic acidosis in cattle with acute rumen lactic acidosis.(AU)
O presente estudo teve como objetivo desenvolver um procedimento de baixo custo, preciso, rápido e prático para estimar o grau de acidose metabólica, para tratar bovinos com quadros de acidose láctica ruminal. A acidose ruminal foi induzida experimentalmente em 40 novilhos mestiços de 1,5 anos de idade, implantados com cânula ruminal. Essa indução causou o surgimento de sinais clínicos muito típicos da enfermidade aguda, com o aparecimento de pronunciada acidose ruminal e acidose metabólica de grau moderado, caracterizado por baixo pH sangüíneo e diminutos teores de bicarbonato e excesso de base (BE) no sangue. Verificou-se uma alta correlação positiva (r = 0,80) entre o pH urinário e o BE e entre o pH urinário e o pH sangüíneo (r = 0,75). A concentração de BE estimado pelo pH urinário foi similar à obtida pela análise do hemogasômetro (P = 0,99). Além disso, os resultados apresentados pela fórmula de predição foram muito significativos. Dessa forma, conclui-se que a mensuração do pH urinário é uma boa alternativa para estimar a quantidade necessária de tampão para tratar o quadro de acidose metabólica em bovinos com acidose láctica ruminal aguda.(AU)
Assuntos
Animais , Bovinos , Acidose Láctica/veterinária , Doenças dos Bovinos/urinaRESUMO
A nested polymerase chain reaction (PCR) using primers from the LipL32 sequence of Leptospira spp. was used to detect shedding of pathogenic leptospires in urine from naturally infected cattle. Amplicons (497bp) were obtained from 21 pathogenic reference serovars belonging to four species (L. interrogans, L. borgpetersenii, L. santarosai, L. kirschneri). DNA was amplified from 26/30 urine samples taken from cattle with suspected leptospirosis and from leptospires cultivated from 10 of these samples. The limit of detection of DNA in the clinical samples was 200pg and the nested PCR detected all pathogenic reference serovars of Leptospira spp. tested. No PCR products were amplified using DNA from other common bacterial species from the bovine urogenital tract or urine, or from the non-pathogenic L. biflexa Andamana serovar. The nested PCR exhibited high specificity and sensitivity for detection of pathogenic serovars in urine from cattle.
Assuntos
Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/urina , Leptospira/isolamento & purificação , Leptospirose/veterinária , Animais , Bovinos , Feminino , Leptospirose/microbiologia , Leptospirose/urina , Urina/microbiologiaRESUMO
We evaluated the use of low-stringency single specific primer PCR (LSSP-PCR) for genetically typing Leptospira directly from urine samples of cattle with clinical suspicion of leptospirosis. Urine samples obtained from 40 cattle with clinical suspicion of leptospirosis were amplified by specific PCR using the following primers: Internal 1/Internal 2 and G1/G2. The internal primers were designed from the gene sequence of the outer membrane lipoprotein Lip32 from Leptospira kirschneri, strain RM52. The PCR products were amplified with these two pairs of primers, which had approximately 497 and 285bp, respectively, and were subsequently used as a template for LSSP-PCR analysis. The genetic signatures from the leptospires which were present in the urine samples allowed us to make a preliminary identification of the leptospires by comparing the LSSP-PCR profiles obtained directly from urine samples with those from reference leptospires. The LSSP-PCR profiles obtained with the Internal 1 primer or with the G1 primer allowed the grouping of the leptospires into serogroups. LSSP-PCR was found to be a useful and sensitive approach capable of identifying leptospires directly from biological samples without the need for prior bacterial isolation. In conclusion, the LSSP-PCR technique may still be helpful in discriminating serogroups of Leptospira from different animal reservoirs, since the early identification of carrier animals and information on the shedding state are crucial to prevent the spread of leptospiral infection to other animals and humans.