Detection of Leptospira spp. from pure cultures and from experimentally contaminated bovine semen by polymerase chain reaction

Considerando a importância do sêmen na transmissão da leptospira bovina, foi realizado o presente estudo que teve como objetivo aplicar a reação em cadeia pela polimerase (PCR) para a detecção de leptospiras em sêmen bovino experimentalmente contaminado. A reação de PCR foi capaz de amplificar um fragmento de DNA específico de 330 pares de bases a partir de cultivos puros de 26 sorovares de Leptospira spp. A contaminação experimental de sêmen com Leptospira interrogans serovar hardjo revelou que a técnica de PCR conseguiu detectar 10 bactérias/ml, concentração sensivelmente mais baixa que as 1.000 bactérias/ml detectadas através do cultivo microbiológico. Os resultados observados revelam o grande potencial da reação de PCR para a detecção de Leptospira spp. em sêmen bovino, notadamente em centrais de inseminação artificial.

Interference of vaccinal antibodies on serological diagnosis of leptospirosis in vaccinated buffalo using two types of commercial vaccines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Resposta humoral, recuperação bacteriana e lesões histológicas em camundongos geneticamente selecionados para bons e maus produtores de anticorpos e Balb/c, frente à infecção por Leptospira interrogans sorovar icterohaemorrhagiae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pesquisa de suínos portadores renais de leptospiras pelo isolamento microbiano e reação em cadeia pela polimerase em amostras de rins de animais sorologicamente positivos e negativos para leptospirose

Com o intuito de se estudar a presença de suínos portadores renais de leptospiras foram colhidas 131 amostras sanguíneas e os respectivos rins de animais durante o abate em abatedouro da região de Botucatu-SP. Pela prova de Soroaglutinação Microscópica obteve-se 48 amostras sorológicas positivas para um ou mais sorovar de Leptospira spp., com uma taxa de ocorrência de anticorpos anti-leptospira de 36,64%, e maior importância para o sorovar icterohaemorrhagiae. Para a pesquisa do agente nos rins, das 88 amostras renais submetidas a cultura em meio de EMJH e analisadas pela prova de PCR, foi isolado e detectado o agente em uma única amostra renal, pertencente a um animal soropositivo. Embora não tenha sido possível a comparação estatística, em termos de sensibilidade e especificidade das duas provas de detecção do agente a partir de amostras renais, a PCR mostrou-se mais rápida e prática na pesquisa de portadores renais. Pelo isolamento obtido, ressalta-se a importância desses animais como possíveis transmissores da doença para trabalhadores de abatedouro e inspetores de carne.

Identificação da Leptospira interrogans sorovar pomona em camundongos geneticamente selecionados, para a alta e baixa produção de anticorpos, através da técnica de imunoperoxidase em tecido renal e isolamento bacteriano em meio de Fletcher

O presente trabalho teve por objetivo identificar a presença da Leptospira interrogans sorovar pomona em camundongos geneticamente selecionados para a alta e baixa resposta a anticorpos. Todos os animais foram submetidos ao isolamento bacteriano, imunohistoquímica (imunoperoxidase) em cortes de tecido renal e coloração através da hematoxilina-eosina. A técnica de imunoperoxidase apresentou-se pouco mais sensível em relação ao cultivo, entretanto, ambas foram bons parâmetros de identificação do agente. Presença de lesões renais mais intensas ocorreram em períodos em que houve maior número de bactérias isoladas em meio de cultivo. Camundongos da linhagem HIV-A conseguiram eliminar as leptospiras com maior eficiência e rapidez em relação as linhagem LIV-A, entretanto o estudo demonstrou que ambas linhagens da seleção IV-A foram eficientes em controlar o processo infeccioso.

Evaluation of isopathic treatment of Salmonella enteritidis in poultry

Background. Salmonellosis is a common problem worldwide in commercially reared poultry. It is associated with human Salmonellosis. No fully satisfactory method of control is available.Method: Nosodes to an antibiotic-resistant strain of Salmonella enterica serovar Enteritidis in D30 (30X) potency were prepared. One day old chicks (N = 180) were divided into four groups: two control and two different preparations of the nosode. Treatments were administered in drinking water for 10 days. The birds were challenged by a broth culture of the same Salmonella, by mouth, on day 17. Cloacal swabs were taken twice weekly for Salmonella enterica serovar Enteritidis.Results: Birds receiving active treatment were less likely to grow the strain of Salmonella from cloacal swabs compared to control.Conclusion: Isopathy is low cost and non-toxic. It may have a role to play in the widespread problem of Salmonella in poultry. Further research should be conducted.

Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide 0 side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Detection of leptospires in bovine semen by polymerase chain reaction

Objective: In view of the considerable importance of venereal transmission of bovine leptospirosis, the objective of the present study was to compare the polymerase chain reaction (PCR), culture/isolation and serology to detect leptospire infection in bovine semen. Design: Blood for serologic examination and semen for bacterial culture and PCR were collected from 20 bulls at artificial insemination centres in Brazil. Each animal was sampled twice for serology. Result: Forty-five percent (9/20) of the serum samples collected showed agglutinin titers to serovar hardjo in the first sample and 25% (5/20) had agglutinin titers to serovar hardjo in the second sample. Eighty percent (16/20) of semen samples were positive by PCR. Leptospires could not be isolated from any of the semen samples examined. Conclusion: Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.

Cryptococcosis outbreak in psittacine birds in Brazil

An outbreak of cryptococcosis occurred in a breeding aviary in São Paulo, Brazil. Seven psittacine birds (of species Charmosyna papou, Lorius lory, Trichoglossus goldiei, Psittacula krameri and Psittacus erithacus) died of disseminated cryptococcosis. Incoordination, progressive paralysis and difficulty in flying were seen in five birds, whereas superficial lesions coincident with respiratory alterations were seen in two birds. Encapsulated yeasts suggestive of Cryptococcus sp. were seen in faecal smears stained with India ink in two cases. Histological examination of the birds showed cryptococcal cells in various tissues, including the beak, choana, sinus, lungs, air sacs, heart, liver, spleen, kidneys, intestines and central nervous system. High titres of cryptococcal antigen were observed in the serum of an affected bird. In this case, titres increased during treatment and the bird eventually died. Yeasts were isolated from the nasal mass, faeces and liver of one bird. Cryptococcus neoformans var. gattii serovar B was identified based on biochemical, physiological and serological tests. These strains were resistant (minimum inhibitory concentration 64 μ g/ml) to fluconazole. This is the first report of C. neoformans var. gattii occurring in psittacine birds in Brazil. © 2004 ISHAM.

Cytokine and antibody production during murine leptospirosis

The aim of the present study was to investigate the kinetics of humoral and cellular responses during leptospirosis. We observed that the presence of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) was associated with antibody production and bacterial recovery, and the compromising of both TNF-α and IL-6 in the immunopathogenesis of leptospirosis during an experimental infection of BALB/c mice inoculated with Leptospira interrogans serovar Canicola was verified. Results showed higher levels of TNF-α and IL-6 in the initial phase of infection, in which the greatest bacterial clearance was observed. However, when the bacterial recovery was compared with the kinetics of the production of antibodies, the results revealed a kinetics proportionally inverted to antibody production. This fact may be related to some inhibitory factor which could be responsible for the selective suppression of the cellular immune response. We concluded that during leptospirosis there was a greater mobilization of the cellular immune response activity, mainly in the initial phase of the infectious process, for posterior involvement of the humoral response, and that both TNF-α and IL-6 could be associated with the immunopathogenesis of the disease.

Avaliação de fatores de risco de cães sororreagentes à leptospira spp. e sua distribuição espacial, em área territorial urbana

We aimed to evaluate the risk factor of serum reactive dogs to antileptospire agglutinin and their spatial distribution in an urban area. We collected 1,000 blood samples from dogs at 20 immunization centers, homogeneously distributed in the urban area (32 km²) of Botucatu, Sao Paulo, Brazil. Diagnosis was made by microscopic serum agglutination using 24 serovars of Leptospira spp. Statistical analysis was performed by Goodman's test with 5% of significance. Spatial clusters were tested by spatial scan statistic using SaTScan. We found that 17.9% dogs reacted to Leptospira spp. (p<0.0001). Dogs with street access (22.14%) were more reactive (p<0.05) than those without (14.83%). The scan test indicated only one significant cluster influenced by age. Incorporating each one of the co-variables gender, breed, and street access to age, we found that street access was the most important. Street access and age were the most important risk factors in the large number of reactive animals in the cluster, with the castellonis serovar being the most reactive in urban dogs. The identification of a cluster with more reactive dogs than expected allows local preventive measures to be taken.

Experimental infection of commercial layers using a Salmonella enterica sorovar Gallinarum strain: Blood serum components and histopathological changes

The purpose of the study was to evaluate the blood serum components and histopathological findings of commercial layers experimentally infected with Salmonella Gallinarum (SG), the microorganism responsible for the fowl typhoid. 180 commercial layers were distributed into three groups (G): G1 and G2 received 0.2mL of inoculum containing 3.3x10 8 and 3.3x10 5 CFU of resistant SG to the nalidix acid (Nal r)/mL, respectively, directly into their crops; G3 did not receive the inoculum (control group). The birds were inoculated when they were 5 days old and the euthanasia was performed 24 hours before and after infection and 3, 5, 7 and 10 days after the administration of the inoculum. In each day of collection, blood samples were obtained for biochemical tests of the blood serum besides macroscopic and histopathological examination of the birds. Data were submitted to analysis of variance by the SAS statistical program and the means were compared by Tukeýs test (P<0,05). In the serum biochemical profile it was observed that the infection interfered in the values of total protein, albumin, calcium, phosphorus, cholesterol, triglycerides, GGT and ALT in the infected groups. The macroscopic examination showed hepatomegaly, alteration of the hepatic color and hemorrhagic spots in the kidneys of animals from G1. The histopathology showed degeneration of hepatocytes in G1 and G2 although other lesions like multifocal hepatic necrosis and inflammatory infiltrate on the liver and kidneys were restricted to G1. The alterations were more evident on G1 which received a higher concentration of bacteria/mL when compared to G2. The results showed that the correlation between biochemical alterations and macroscopic and histopathological lesions can assist the comprehension of the pathophysiology of fowl typhoid, supplying important information for the diagnosis and prognosis of this disease.

Evaluation of three sampling methods for the microbiological analysis of broiler carcasses after immersion chilling

Countries have different official programs and implement different sampling methods for the detection of Salmonella on poultry carcasses. In Brazil, a 25-g sample of skin and muscle excision (SME) from the wings, neck, and pericloacal parts is used; in the European Union (EU), a 25-g sample of neck skin (NSE) is used; and, in the United States, the whole carcass is rinsed with 400 ml of diluent (WCR). In the present study, these methods were evaluated to compare Salmonella occurrence and counts of hygiene indicator microorganisms (Escherichia coli, Enterobacteriaceae, and total viable count of aerobic mesophilic bacteria) using different carcasses from the same flock and also using different analytical units taken from the same carcass. Eighty flocks, with four broiler carcasses from each, were included in this study; three broilers were sampled according to protocols from Brazil, the EU, and the United States, and the last one by all three methods. SME, NSE, and WCR provided equivalent results (P > 0.05) for Salmonella detection on broiler carcasses when using different carcasses from the same flock and when using the same carcass. The predominant serovar was Salmonella Enteritidis. For the enumeration of hygiene indicator microorganisms, WRC provided higher counts than SME or NSE (P < 0.05), when using both the same or different carcasses. Therefore, it is possible to directly compare Salmonella results in poultry carcasses when using the methods recommended by the legislative bodies of Brazil, the United States, and the EU. However, WCR provides the best results for hygiene indicator microorganisms. Copyright © International Association for Food Protection.

Isolamento e reação em cadeia pela polimerase (PCR) para Leptospira spp. em amostras renais e hepáticas de ovinos sorologicamente positivos e negativos para leptospirose, abatidos em frigorífico, procedentes do estado de São Paulo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Associação entre concepção e enfermidades infecciosas da reprodução em matrizes nelore

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)