Frequency of and risk factors associated to Leptospira spp. Seropositivity in goats in the state of Sergipe, Northeastern Brazil / Frequência e fatores de riscos associados a caprinos soropositivos a Leptospiras spp. no Estado de Sergipe, Nordeste do Brasil

ABSTRACT: The objective of this study was to determine the frequency and factors associated with the occurrence of seropositivity for Leptospira spp. in goat herds in the state of Sergipe, Brazil, on the basis of the analysis of 675 samples collected from 41 properties in 2013-2014. Analysis of anti-Leptospira spp. antibodies revealed that 25.74% goats (194) were seropositive and that 90.24% (37) of the properties had at least one infected goat. The most prevalent serogroup was Icterohaemorrhagiae (85.57%); although, the study animals were reactive to other serogroups: Australis (5.15%), Pomona (3.61%), Sejroe (3.09%), and Pyrogenes (2.58%) had titers ≤400 in 96.91% of cases. The origin of the water source [still water source (OR=3.86)] was associated with seropositivity. Results reflected the importance of adopting appropriate management practices for herds in Sergipe.

RESUMO: O objetivo deste estudo foi determinar a frequência e os fatores associados à soropositividade para Leptospira spp. em caprinos do Estado de Sergipe, Nordeste do Brasil, utilizando 675 animais de 41 propriedades no período de 2013 e 2014. A frequência de positividade entre os animais foi de 25,74% (194), e em 90,24% (37) das propriedades houve pelo menos um animal soropositivo. O sorogrupo reagente predominante foi o Icterohaemorrhagiae (85,57%), com presença também de animais reagentes para Australis (5,15%), Pomona (3,61%), Sejroe (3,09%) e Pyrogenes (2,58%) com títulos ≤400 em 96,91% dos casos. A origem da água fornecida (água parada) (OR=3,86) foi o fator associado à presença de caprinos positivos. Ressalta-se a importância da adoção de práticas de manejo adequadas nos rebanhos caprinos sergipanos.

Discriminación genotípica de serovariedades del serogrupo Icterohaemorrhagiae pertenecientes a Leptospira interrogans (Spirochaetales: Leptospiraceae) y el análisis de distancias génicas mediante coordenadas principales

La leptospirosis es una zoonosis de amplia distribución mundial y de importancia en salud pública y veterinaria. El serogrupo Icterohaemorrhagiae toma importancia, ya que ha sido aislado con frecuencia en roedores y en casos clínicos de humanos, caninos, bovinos y porcinos; y se ha logrado aislar a partir de agua del rio Reconquista, provincia de Buenos Aires, Argentina. El objetivo de este estudio fue discriminar molecularmente entre las serovariedades Copenhageni e Icterohaemorrhagiae del serogrupo Icterohaemorrhagiae de importancia en Argentina, utilizando la técnica del análisis de repeticiones en tándem en múltiples locus (MLVA) y analizar las distancias génicas entre los genotipos determinados a partir de cepas aisladas de animales. Para ello, genotipificamos las 4 cepas referenciales del serogrupo Icterohaemorrhagiae de la especie Leptospira interrogans serovariedad Copenhageni cepa M20 y cepa Ictero I y de la serovariedad Copenhageni cepa RGA y cepa Fiocruz L1-130. Para este estudio se incluyeron 24 cepas aisladas de animales domésticos como silvestres. Logramos mediante esta técnica determinar un código numérico para cada serovariedad y así pudimos discriminar entre las serovariedades de este serogrupo. El análisis de coordenadas principales (PCoA) demostró la variabilidad génica existente entre las 4 cepas referenciales pertenecientes al serogrupo Icterohaemorrhagiae, el genotipo con mayor representación fue el serovar Copenhageni cepa Fiocruz L1-130 con un total de 13 cepas aisladas con ese perfil genético. Discriminar las serovariedades de este importante serogrupo nos permitirá en un futuro investigar si existen diferencias entre la sintomatología clínica y/o respuesta al tratamiento en los casos clínicos producidos por las distintas serovariedades del serogrupo Icterohaemorrhagiae discriminadas en este trabajo.

Leptospirosis is the world`s most widely distributed zoonosis and of great importance in animal health and in public health. The relevance of the serogroup Icterohaemorrhagiae relies on the frequent isolation from rodents and from clinical cases of humans, canines, bovines and swine; and this serogroup was also isolated from water samples of the Reconquista river, Buenos Aires province, Argentina. The objective of this study was to discriminate molecularly between serovars Copenhageni and Icterohaemorrhagiae being of importance in Argentina, using Multiple Locus Variable number tandem repeats analysis (MLVA) and analyzing the genetic distances among genotypes obtained from animal isolates in this study. We genotyped the four referential strains of the serogroup Icterohaemorrhagiae belonging to the species Leptospira interrogans serovar Copenhageni strain M20 and strain Ictero I and of the serovar Copenhageni strain RGA and strain Fiocruz L1-130. In this study we included 24 strains isolated from animals domestic and wildlife. Using this technique we could determine numeric codes for each serovar and in this way, discriminate between serovars of this serogroup. The principal coordinates analysis (PCoA) showed the genetic variability of the four referential strains from the serogroup Icterohaemorrhagiae, the genotype with better representation was serovar Copenhageni strain Fiocruz L1-130 with 13 isolated strains with this genetic profile. Discrimination of the serovars from this important serogroup allows to establish differences between clinical signs and/or response to treatment in the clinical cases produced by different serovars of the serogroup Icterohaemorrhagiae successfully distinguished in this study.

Caracterização das leptospiras isoladas dos pacientes atendidos no Hospital Couto Maia / Characterization of isolated leptospires of patients seen in the hospital Couto Maia

A leptospirose é uma zoonose de distribuição mundial, com 1,2 milhões de casos registrados a cada ano. De 1996 a 2013, o grupo de pesquisa de leptospirose do CPqGM, realiza uma vigilância ativa no Hospital Couto Maia em Salvador-Ba, onde foi recrutado 4612 casos suspeitos para leptospirose. Destes 4612 foi confirmado o diagnóstico de 1853 (40%) utilizando pelo menos um dos três métodos de diagnóstico (MAT, Hemocultura, qPCR). Dentre os casos confirmados, 1759 (95%) foram confirmados pelo MAT. A sensibilidade do MAT foi diferente entre as amostras aguda e convalescente, sendo 60% na fase aguda e 97% na fase convalescente. O sorogrupo Icterohaemorrhagiae foi o mais prevalente (90%) dos casos confirmados para MAT. Durante o período do estudo foram coletadas 1133 hemoculturas e destas 203 (18%) foram positivas, sendo possível isolar leptospiras de 93/203 (45%) das hemoculturas, as quais foram soro-agrupadas com soros heterológos de coelho. A concordância entre o sorogrupo encontrado no MAT e na soro-agrupagem foi de 80%. Os achados mostram que existe uma concordância significante entre o sorogrupo encontrado pelos dois métodos, o que indica que o painel de cepas utilizado no MAT apresenta uma ótima cobertura para os sorogrupos prevalentes na região. A predominância de um sorogrupo facilitou quanto a tomadas de decisões para prevenção e controle, assim como facilita para o desenvolvimento de novos testes de diagnóstico e vacinas mais direcionados.

Leptospirosis is a re-emerging zoonotic disease of global importance, with 1,2 million cases reported each year. Diagnosis of leptospirosis is often difficult given the nonspecific disease presentation. In order to compare the performance of the two gold standard diagnostic tests for leptospirosis, the group enrolled 4612 patients with suspected leptospirosis during active surveillance at the state infectious disease reference hospital, Hospital Couto Maia, in Salvador, Bahia between 1996 and 2013. Of these, was confirmed Leptospira infection in 1853 (40%) using at least one of three diagnostic methods (microagglutination (MAT), blood culture, and qPCR). Was confirmed 1759 (95%) cases using only the MAT assay, and identified the serogroup Icterohaemorrhagiae as the infective agent in 90% of MAT positive samples. It was determined the sensitivity of the MAT was 60% for acute phase samples and increased to 97% for convalescent samples. Within this study period, it was possible to collect 1133 blood cultures and was isolated leptospires from 93 of 203 (45%) of blood cultures, and determined the serotype using heterologous rabbit sera. The concordance between the infective serogroup identified using hemoculture and MAT techniques was 80%. This result indicates that the panel of 11 strains used in the MAT represents a majority of the infective serogroups causing disease in our study population. The predominance of a single serogroup in symptomatic cases informs the development of new diagnostic tests and novel vaccines to prevent leptospirosis in Brazil.

Application of multiple-locus variable-number tandem repeat analysis(MLVA) for molecular typing of Leptospira interrogans serogroup Icterohaemorrhagiae / 中华微生物学和免疫学杂志

Objective To establish the method of multiple loci VNTR(variable numbers tandem-repeats) analysis (MLVA) for genotyping Leptospira interrogans serogroup ieterohaemorrhagiae . Methods Seven VNTR loci were chosen for genotyping 117 strains of L. interrogans serogroup Icterohaemorrhagiae by PCR-electrophoresis-based VNTR analysis and the results were analyzed by software BioNumerics( Version 4.0). Results One hundred and seventeen isolates of L. interrogans serogroup Icterohaemorrhagiae detec-ted with 7 VNTR loci were classified into three clusters(A,B,C), twenty-eight types were found, type A 11.97% (14/117), type B 0.85% (1/1 17), type C 87.18% (102/117). Diversity Indexes for the loci varied between 0.0831 and 0.8005. Clinical strains isolated from the same geographic area and belonging to the same serogroup shared a common VNTR pattern. Conclusion MLVA could be used to classify and identify Leptospira interrogans preliminarily. With the improvement of technology, this rapid and easy method should greatly contribute to a better knowledge of the epidemiology of Leptospira.

Alterações na bioquímica hepática em cães com leptospirose aguda determinada por amostras do sorogrupo Icterohaemorrhagiae / Hepatic biochemistry alterations in dogs with naturally occurring acute leptospirosis determined by Icterohaemorrhagiae serogroup

Foram testadas 120 amostras séricas de cães com títulos (≥100) perante Leptospira spp. serovar Icterohaemorrhagiae para a determinação da atividade séricade alanina aminotransferase (ALT), aspartato aminotransferase(AST), fosfatase alcalina (FA), gamaglutamiltransferase (GGT)e dos níveis séricos de colesterol total, triglicerídeos, proteínatotal, albumina, globulinas e bilirrubinas total, direta e indireta. Um grupo de 34 animais sem qualquer sintomatologiaclínica e idade semelhante ao grupo testado foi utilizado como controle, tendo sido submetido às mesmas dosagens bioquímicas. Os animais com títulos ≥200 apresentaram dosagens de FA e bilirubina direta superiores ao grupo controle e também aos animais com títulos menores que 200 (P<0,05).Observou-se uma clara correlação entre dano hepático, sem necrose hepatocelular, com a sororeatividade específica para leptospirose, em animais acometidos de infecção por amostrasdo sorogrupo Icterohaemorrhagiae.

One hundred and twenty serum samples obtained from dogs with specific titres (≥100) against Leptospira spp.serovar Icterohaemorrhagiae were tested in order to determine the enzymatic activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamaglutamyltransferase (GGT), as well as of the total cholesterol, triglycerides, total protein, albumin, globulins and total, direct and indirect bilirubins serum concentrations. Thirtyfour animals free from clinical signs with the same age of the tested group were used as control group and submitted to the same biochemical tests. The animals with titres ≥200 showed increased ALP activity and greater direct bilirubin concentration when compared to the control group and also to the group ofdogs with titres <200 (P<0.05). A clear correlation was observed between hepatic damage, without hepatocellular necrosis, and specific seroreactivity for leptospirosis caused by serogroupIcterohaemorrhagiae.

Níveis séricos de uréia e creatinina em cães com leptospirose aguda determinada por amostras do sorogrupo Icterohaemorrhagiae / Blood urea nitrogen and creatinine levels in dogs with acute leptospirosis determined by Icterohaemorrhagiae serogroup

Foram testadas 120 amostras séricas de cães com títulos (³100) perante Icterohaemorrhagiae para a determinação dos níveis séricos de uréia e de creatinina. Os animais com títulos ³800 apresentaram dosagens de uréia bastante superiores aos valores de referência e os com títulos ³200 e £400 apresentaram valores de uréia significativamente menores (P<0,05) do que os anteriores, porém ainda acima dos valores de referência. As amostras com títulos menores que 200 apresentaram dosagens de uréia significativamente menores (P<0,05) do que os observados nos outros grupos e dentro dos valores de referência. No que se refere à dosagem de creatinina sérica, verificou-se que apenas os animais com títulos ³800 apresentaram dosagens acima do padrão normal. Observou-se uma clara correlação entre dano renal e a sororreatividade específica para leptospirose.

One hundred and twenty serum samples from dogs with specific titres (³100) against Leptospira sp. serovar Icterohaemorrhagiae for the dosage of blood urea nitrogen (BUN) and creatinine levels were tested. Animals presenting titres ³800 showed BUN levels higher than reference values while samples with titres ³200 and £400 presented BUN levels significantly lower (P<0.05) than the others, but still higher than the reference values. Samples presenting titres lower than 200 showed BUN dosages according to the reference values. In relation to creatinine dosage, only samples presenting titres ³800 showed dosages higher than the reference values. A correlation between renal damage and specific seroreactivity for leptospirosis was demonstrated.

Distribuição dos serovares de leptospira em caninos clinicamente suspeitos no Rio de Janeiro

A leptospirose é uma doença de distribuição mundial que acomete várias espécies de animais domésticos e de vidaselvagem. Diversos serovares podem infectar o cão, podendo ou não determinar manifestações clínicas. Assim, oobjetivo do presente estudo foi relatar resultados de provas de imunodiagnóstico em cães clinicamente suspeitos deleptospirose no município do Rio de Janeiro e adjacências. Foram analisadas pelo teste da Soroaglutinação Microscópicacom Antígenos Vivos 120 amostras de cães com clínica sugestiva de leptospirose aguda no período de março anovembro de 2004. Destas, 88 (73,3%) foram reativas. Baseando-se nos títulos alcançados, 25 animais (20,8%)apresentaram título mínimo de 800, 63 (52,5%) entre 200 e 400, e 32 animais (26,7%) inferiores a 200, sendo portantoconsideradas negativas. No que se refere à distribuição por serovares, verificou-se amplo predomínio para a amostraIcterohaemorrhagiae, com 60 amostras reativas (50%), seguido de Copenhageni, com 19 amostras reativas (15,8%) eCanicola, com nove amostras reativas (7,5%). Ao somar-se Icterohaemorrhagiae e Copenhageni, por serem ambasestirpes do mesmo sorogrupo, obtiveram-se 79 amostras sororreativas para este sorogrupo, o que corresponde a78,4% das amostras reativas e 57,5% dos animais estudados. Nenhuma amostra apresentou sororreatividade para osserovares Autumnalis, Bataviae, Grippotyphosa, Pomona ou Bratislava.

Characterization of Korean Leptospira interrogans Isolates by Pulsed-Field Gel Electrophoresis of Not I Digests of DNA / 감염과화학요법

BACKGROUND: Many strains of Leptospira interrogans have been isolated in Korea since 1984. Most isolates were identified as serovar lai by serological methods. The pulsed field gel electrophoresis (PFGE) patterns of Korean isolates have not been investigated currently. METHODS: 29 reference strains and 29 Korean isolates of Leptospira interrogans were characterized by PFGE. Chromosomes were digested by the Not I restriction enzyme and subsequently PFGE was performed in CHEF-DRII (Bio Rad Lab) with 3 pulse times (30 seconds 13 hours, 60 seconds 13 hours, 120 seconds 14 hours) at 150 V (4.5 V/cm). RESULTS: 12 serogroup reference strains and most 17 serovars reference strains in the serogroup Icterohaemoffhagie showed the unique Not I restriction patterns. Most isolates identified serologically as serovar lai showed the same PFGE patterns as the serovar lai reference strain. The strain HM3 and 18R identified serologically as new serovars yeonchon and hongchon respectively showed the same PFGE patterns as serovar lai. The strain AP31, CH88-19 and NR13 that were different from serovar lai by serological methods showed the PFGE patterns indistinguishable from serovar lai reference strain. The strain HY2 that was identified as serovar lai, and the strain 30R that was different from serovar lai serologically showed the PFGE patterns slightly different from serovar lai reference strain. CONCLUSION: The PFGE profile of most Korean isolates Leptospira interrogans serologically identified as serovar lai is identical to the reference strain serovar lai. PFGE analysis thus may be applied to identify serovar of isolates and to investigate the genetic diversity of related serovar.

Characterization of Korean Leptospira interrogans Isolates by Pulsed-Field Gel Electrophoresis of Not I Digests of DNA / 감염과화학요법

BACKGROUND: Many strains of Leptospira interrogans have been isolated in Korea since 1984. Most isolates were identified as serovar lai by serological methods. The pulsed field gel electrophoresis (PFGE) patterns of Korean isolates have not been investigated currently. METHODS: 29 reference strains and 29 Korean isolates of Leptospira interrogans were characterized by PFGE. Chromosomes were digested by the Not I restriction enzyme and subsequently PFGE was performed in CHEF-DRII (Bio Rad Lab) with 3 pulse times (30 seconds 13 hours, 60 seconds 13 hours, 120 seconds 14 hours) at 150 V (4.5 V/cm). RESULTS: 12 serogroup reference strains and most 17 serovars reference strains in the serogroup Icterohaemoffhagie showed the unique Not I restriction patterns. Most isolates identified serologically as serovar lai showed the same PFGE patterns as the serovar lai reference strain. The strain HM3 and 18R identified serologically as new serovars yeonchon and hongchon respectively showed the same PFGE patterns as serovar lai. The strain AP31, CH88-19 and NR13 that were different from serovar lai by serological methods showed the PFGE patterns indistinguishable from serovar lai reference strain. The strain HY2 that was identified as serovar lai, and the strain 30R that was different from serovar lai serologically showed the PFGE patterns slightly different from serovar lai reference strain. CONCLUSION: The PFGE profile of most Korean isolates Leptospira interrogans serologically identified as serovar lai is identical to the reference strain serovar lai. PFGE analysis thus may be applied to identify serovar of isolates and to investigate the genetic diversity of related serovar.