Antibacterial activity of Crude Momordica charantia, Cassia alata, and Allium sativum methanolic extracts on Leptospira interrogans serovar Manilae

Background and Objective@#Leptospirosis is a disease caused by pathogenic Leptospira prevalent in tropical countries like the Philippines. Some studies have shown that the role of currently used antibiotics for leptospirosis is unclear since trials have found no significant benefit to patient outcomes compared to placebo. This signals the need for alternative therapies, such as herbal medicines, which may provide effective therapeutic regimens in treating this infection. In this study, we characterized the antibacterial potential of three Philippine herbal medicines against Leptospira interrogans. @*Methods@#Crude methanolic extracts of Momordica charantia, Cassia alata, and Allium sativum were subjected to an optimized broth microdilution assay against L. interrogans, utilizing the resazurin-resorufin reaction as a cell proliferation and viability indicator. @*Results@#The respective minimum inhibitory concentrations of the plants were found to be as follows: 1.25 mg/mL (M. charantia), 2.5 mg/mL (C. alata), and >5 mg/mL (A. sativum). @*Conclusions@#Among the three herbal medicines, M. charantia and C. alata proved to have antibacterial activity against L. interrogans. Given the promising potential of two of these plant extracts, exploring the use of other solvents to extract natural compounds from these plants, and discovering possible synergistic effects between these plants and conventional antibiotics may be worthwhile.

Evidencia molecular de Leptospira interrogans sensu stricto en Cavia porcellus (cuyes) destinados para el consumo humano en el municipio de Pasto, Nariño / Molecular evidence of Leptospira interrogans sensu stricto in Cavia porcellus (guinea pig) grown for human consumption in Pasto, Nariño

Introducción: La leptospirosis es una zoonosis emergente, endémica en Colombia, que afecta tanto animales domésticos como silvestres. Es considerada de riesgo laboral, ya que la transmisión al ser humano está asociada a la exposición con animales o ambientes infectados. En el departamento de Nariño, la producción de cuyes para el consumo humano se realiza en sistemas de crianza tradicionales que podrían favorecer la infección por Leptospira interrogans en esta especie. Objetivo: Detectar molecularmente la infección natural por especies patógenas del género Leptospira en cuyes que son destinados para el consumo humano en el municipio de Pasto. Materiales y métodos: Se tomaron 270 muestras de tejido renal en cuyes sacrificados en dos mataderos. Las muestras fueron analizadas mediante Reacción en Cadena de la Polimerasa (PCR) convencional y coloración diferencial de Warthin Starry (W-S). Resultados: En la evaluación de las 270 muestras, 4 (1,5%) fueron positivas para PCR y una de las muestras demostró la presencia de Leptospira bajo tinción W-S. Conclusiones: Mediante el uso de técnicas moleculares se evidenció L. interrogans en el tejido renal de Cavia porcellus. La circulación del patógeno en esta población representa un riesgo de infección para humanos y animales domésticos en contacto con estos sistemas productivos.

Introduction: Leptospirosis is an emerging zoonosis that is endemic in Colombia and affects both domestic and wild animals. It is considered an occupational risk since human transmission is associated with exposure to infected animals or environments. In the department of Nariño, the production of guinea pigs for human consumption applies traditional rearing systems that could cause animals to get infected with Leptosipira interrogans. Objective: To molecularly identify natural infection by pathogenic species of the genus Leptospira in guinea pigs used for human consumption in the municipality of Pasto (Colombia). Materials and methods: 270 kidney tissue samples were taken from guinea pigs slaughtered in two facilities. Samples were analyzed through conventional polymerase chain reaction (PCR) and Warthin Starry (W-S) differential staining. Results: While 4 (1.5%) out of the 270 samples were categorized as positive using PCR, only 1 sample showed the presence of Leptospira through W-S staining. Conclusions: Molecular techniques were useful to identify L. interrogans in kidney tissue of Cavia porcellus. Dissemination of this pathogen within this population represents an infection risk for humans and domestic animals that are in close proximity to these productive systems.

Caracterização funcional de proteínas hipotéticas de Leptospira interrogans como adesinas e potenciais antígenos vacinais e para diagnóstico / Functional characterization of hypothetical proteins in Leptospira interrogans as adhesins and potential vaccine and diagnostic antigens

Leptospira spp. constitui um grupo de bactérias espiroquetas gram-negativas englobando espécies saprofíticas, intermediárias e patogênicas, sendo as últimas agentes causadores da leptospirose, doença zoonótica de alcance mundial e endêmica em regiões tropicais em desenvolvimento. O crescente número de espécies identificadas de leptospiras destaca ainda mais sua diversidade genética e mecanismos de virulência únicos, muitos deles com função ainda desconhecida. Esforços para o desenvolvimento de novas vacinas com proteção cruzada e efeito duradouro revelaram possíveis candidatos vacinais que necessitam ser adequadamente validados, sendo assim, há ainda uma urgente necessidade de uma vacina universal contra a leptospirose capaz de controlar e reduzir os surtos cada vez mais frequentes da doença. Adesinas são importantes fatores de virulência em diversos patógenos, constituindo antígenos promissores para o desenvolvimento de vacinas contra a leptospirose, assim como para o desenvolvimento de métodos diagnósticos mais rápidos e precisos. Previamente, foram identificadas três proteínas hipotéticas conservadas em L. interrogans pela técnica de phage display, denominadas arbitrariamente como LepA069, LepA962 e LepA388. A expressão do gene codificador da proteína LepA069 apresentou aumento de aproximadamente 70 % em animais infectados por leptospiras virulentas, representando a primeira evidência funcional desta proteína ainda desconhecida. Porções recombinantes da lipoproteína hipotética LepA962 (LepA962_Nt e LepA962_Phg) foram obtidos, sendo demonstrada a forte interação da proteína LepA962_Phg, contendo a sequência identificada por phage display, com laminina, fibronectina plasmática, colágeno I e fibrinogênio de maneira dose-dependente. Adicionalmente, LepA962_Phg apresentou ligação às células VERO e à sua matriz extracelular secretada, e o soro obtido a partir desta proteína recombinante foi capaz de se ligar à superfície de leptospiras virulentas, indicando que LepA962_Phg pode representar um importante domínio de interação entre as leptospiras e seu hospedeiro. Finalmente, a proteína LepA388 pertencente a uma extensa família de proteínas modificadoras de virulência com função desconhecida (DUF_61), presente apenas nas leptospiras patogênicas mais virulentas, apresentou aumento na expressão de seu gene codificador em animais infectados por leptospiras virulentas de acordo com dados na literatura. Além disso, porções recombinantes da região Nterminal desta proteína apresentaram ligação a laminina, colágenos I e IV, vitronectina e fibronectinas plasmática e celular, principalmente considerando a sequência identificada por phage display. Estes dados reforçam as predições de modelos tridimensionais da proteína LepA388 e de outros membros da família DUF_61, as quais identificam domínios semelhantes a toxinas (como abrina e CARDS) responsáveis pela ligação e internalização celulares nos hospedeiros. Dados recentes sugerem um possível papel citotóxico desempenhado pelas proteínas desta família em leptospiras, as quais podem também ser consideradas potenciais candidatas vacinais e para diagnóstico da leptospirose, devido à sua distribuição restrita em espécies e cepas patogênicas de importância para saúde humana.

Leptospira spp. constitutes a group of gram-negative spirochete bacteria comprising saprophytic, intermediate and pathogenic species, the last being causative agents of leptospirosis, a zoonotic disease of worldwide extent and endemic in developing tropical regions. The growing number of identified leptospiral species further highlights their genetic diversity and unique virulence mechanisms, many of them with unknown function. Efforts to develop new vaccines with cross-protection and long-lasting effect have revealed possible vaccine candidates that need to be properly validated. Therefore, there is still an urgent need for a universal vaccine against leptospirosis capable of controlling and reducing the increasing outbreaks of the disease. Adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines against leptospirosis, as well as for the development of faster and more accurate diagnostic methods. Previously, three conserved hypothetical proteins in L. interrogans were identified by phage display technique, arbitrarily named as LepA069, LepA962 and LepA388. Expression of the LepA069 encoding gene showed an increase of approximately 70 % in animals infected by virulent leptospires, representing the first functional evidence of this still unknown protein. Recombinant portions of the hypothetical lipoprotein LepA962 (LepA962_Nt and LepA962_Phg) were obtained, demonstrating the strong interaction of the LepA962_Phg protein, containing the sequence identified by phage display, with laminin, plasma fibronectin, collagen I and fibrinogen in a dose-dependent manner. Furthermore, LepA962_Phg showed binding to VERO cells and its secreted extracellular matrix, and the serum obtained from this recombinant protein was able to bind to the surface of virulent leptospires, indicating that LepA962_Phg may represent an important domain of interaction between leptospires and its host. Finally, LepA388 protein belonging to an extensive family of virulence modifying proteins with unknown function (DUF_61), present only in the most virulent pathogenic leptospires, showed an increase in the expression of its encoding gene in animals infected by virulent leptospires according to data in literature. Moreover, recombinant portions of the N-terminal region of this protein showed binding to laminin, collagens I and IV, vitronectin and plasma and cell fibronectins, especially considering the sequence identified by phage display. These data support the predictions of three-dimensional models of the LepA388 protein and other members of the DUF_61 family, which identify toxin-like domains (such as abrin and CARDS) responsible for cellular binding and internalization in hosts. Recent data suggest a possible cytotoxic role played by proteins of this family in leptospires, which can also be considered potential vaccine candidates and antigens for diagnosis, due to their restricted distribution in pathogenic species and strains of importance to human health

Hematúria macroscópica em equinos associada à infecção por Leptospira interrogans / Macroscopic haematuria in horses associated with Leptospira interrogans infection

Hematúria é uma grave manifestação clínica de doença do sistema urinário, ocorrendo sob as formas micro ou macroscópica. Neste artigo relatam-se dois casos de hematúria macroscópica associada à infecção por Leptospira interrogans sorogrupo Canicola. O exame clínico inicial revelou hematúria macroscópica, taquicardia, taquipneia, febre, elevação do tempo de perfusão capilar, hipomotilidade intestinal, além de icterícia da mucosa oral. Leucocitose, proteinúria, glicosúria, piúria e azotemia foram achados comuns aos dois casos. Teste de Soroaglutinação Microscópica foi realizado para titulação de anticorpos contra Leptospira interrogans. Tratamento incluiu medidas terapêuticas de suporte (fluidoterapia), controle da hematúria e antibioticoterapia. Sete dias após manifestação dos sinais clínicos iniciais, ambos animais receberam alta hospitalar após remissão dos sinais clínicos.

Haematuria is a serious clinical manifestation of urinary system disease, occurring in micro or macroscopic forms. In this article two cases of macroscopic haematuria associated with Leptospira interrogans serogroup Canicolainfection are related. The initial clinical examination revealed macroscopic haematuria, tachycardia, tachypnea, fever, increased capillary perfusion time, intestinal hypomotility, in addition to jaundice of the oral mucosa. Leukocytosis, proteinuria, glycosuria, pyuria and azotemia were common findings in both cases. Microscopic serum agglutination test was performed for titration of antibodies against Leptospira interrogans. Treatment included supportive therapeutic measures (fluid therapy), hematuria control and antibiotic therapy. Seven days after the manifestation of the initial clinical signs, both animals were discharged from the hospital without complications.

High level of serovar copenhageni detection in dog sera from Patrocínio, MG, Brazil / Alto nível de detecção do sorovar copenhageni em soros de cães de Patrocínio, MG, Brasil

This study aimed to evaluate the occurrence of canine leptospirosis and the possible risk factors associated with the disease in the municipality of Patrocínio, MG, Brazil. A cross-sectional observational study was carried out from July through August 2017. The municipality was divided into four regions (north, south, east and west) and a predefined number of neighborhoods (25) were randomly sampled in each region. Samples of blood serum were collected from 241 domiciled male and female dogs of different breeds and ages. To investigate the risk factors for canine leptospirosis, the owners of the animals were asked to fill out an epidemiological questionnaire. The following factors were evaluated: breed, sex, age, presence of rodents, type of diet, access to the street, vaccination, presence of flooded areas, and educational level of the owners. Blood serum samples were evaluated by the Microscopic Agglutination Test (MAT), using a collection of 24 live antigens. Of the 241 dogs evaluated, 32 (13.2%) were reactive. The most frequent serovars were: Copenhageni (37.5%) and Canicola (21.8%), followed by Icterohaemorrhagiae and Grippotyphosa (12.5%), Pomona, Tarassovi and Butembo (9.3%) and Hardjo (6.2%). The presence of canine leptospirosis was associated with purebred dogs (OR=0.3059 [95% CI: 0.1430 ­ 0.6547]) and vaccination (OR=2.581 [95% CI: 1.198 ­ 5.563]). It was concluded that some dogs in the municipality of Patrocínio, MG have anti-Leptospiraspp. antibodies and that the serovars most frequently identified were Copenhageni (37.5%) and Canicola (21.8%). Pure breeds and vaccination were factors associated with the prevalence of infection.

O objetivo deste estudo foi avaliar a ocorrência da leptospirose canina e os possíveis fatores de riscos associados à doença no município de Patrocínio ­ MG. Foi realizado um estudo observacional transversal, durante os meses de Julho à Agosto de 2017. O município foi divido em quatro regiões (norte, sul, leste e oeste) e um número predefinido de bairros (25) foi amostrado aleatoriamente em cada região. Foram colhidas 241 amostras de soro sanguíneo de cães domiciliados de ambos os sexos e de diferentes raças e idades. Para investigação dos fatores de risco da leptospirose canina foi aplicado um questionário epidemiológico aos tutores dos animais, foram avaliados os fatores: raça, sexo, idade, presença de roedores, tipo de dieta, acesso à rua, vacinação, presença de áreas alagadas, terrenos baldios e grau de escolaridade dos tutores. As amostras de soro sanguíneo foram avaliadas pelo exame de Soroaglutinação Microscópica (SAM), com uma coleção de vinte e quatro antígenos vivos. Dos 241 cães avaliados, 32 (13,2%) apresentaram-se reagentes. Os sorovares de maior frequência foram: Copenhageni (37,5%) e Canicola (21,8%), seguido por Icterohaemorrhagiae e Grippotyphosa (12,5%), Pomona, Tarassovi e Butembo (9,3%) e Hardjo (6,2%). A presença de leptospirose canina foi associada em cães com raça definida (OR = 0,3059 [IC 95%: 0,1430 ­ 0,6547]) e vacinação (OR = 2,581 [IC 95%: 1.198 ­ 5.563]). Concluiu-se que existem cães que apresentam anticorpos anti-Leptospira spp., no município de Patrocínio-MG e que os sorovares Copenhageni (37,5%) e Canicola (21,8%) foram os de maior ocorrência. Apresentar raça definida e a vacinação foram fatores associados à prevalência da infecção.

Leptospirosis in slaughtered cows in the Triangulo Mineiro, Minas Gerais: prevalence, serological profile and renal lesions / Leptospirose em vacas abatidas no Triângulo Mineiro, Minas Gerais: prevalência, perfil sorológico e lesões renais

Leptospirosis is a zoonosis that affects several species of domestic and wild animals and is an important cause of economic losses in cattle in Brazil. In this study, we determined the prevalence of bovine leptospirosis in the Triângulo Mineiro region, Minas Gerais, Brazil, identified the most frequent serovars of Leptospira interrogans, and examine the renal pathological changes associated with the disease. Samples of blood serum and kidney fragments from 100 bovine females were collected in cattle abattoir. In the serological investigation 48% of the cows were positive. The serovars for which there were more reactive animals were Wolffi (24%), Hardjo (21%) and Hebdomadis (18%). Among the positive samples, 14/48 showedantibody titers greater than 1:100, and 70.83% of the seropositive animals responded to more than one Leptospira interrogans serovar. Only one farm did not have seropositive cows and in nine farms studied, six (66.66%) presented seropositive animals to the Hebdomadis serovar. At the histopathological examination, the most frequent microscopic lesions in positive animals were hyalinization (81.25%), congestion (81.25%) and hydropic degeneration (70.83%). However, these histopathological alterations were also found in kidneys of animals negative to serology, such as hyalinization (80.77%), congestion (48.07%) and hydropic degeneration (55.77%) and these findings are unrelated to positivity. Histopathological examination of the kidneys is not indicated to replace the serological diagnosis of leptospirosis, and may be used only as a complementary examination. Despite the low frequency of seropositive animals in the Triângulo Mineiro region, the disease is present in a large number of farms. Noteworthy is the high frequency of serovar Hebdomadis and it can be considered an emerging serovar in the region. The evaluation of the frequency of this serovar in other regions becomes important, and once verified should result in the recommendation of the inclusion of this serovar in the leptospirosis control.

A leptospirose é uma zoonose que afeta várias espécies de animais domésticos e silvestres e importante causa de prejuízos à bovinocultura nacional. Objetivou-se determinar a prevalência da leptospirose e identificar os sorovares de Leptospira interrogans mais freqüentes na região do Triângulo Mineiro, além de avaliar microscopicamente as lesões renais e correlacionar estas lesões com sorovares específicos. Foram coletadas amostras de soro sanguíneo e fragmentos de rim de 100 fêmeas bovinas em abatedouro e, destas 100 amostras, 48% foram positivas. Os sorovares para os quais houve mais animais reagentes foram Wolffi (24%), hardjo (21%) e Hebdomadis (18%). Dentre as amostras positivas, 14/48 apresentaram títulos de anticorpos aglutinantes superiores a 1:100 e notou-se que 70,83% dos animais soropositivos reagiram a mais de um sorovar de Leptospira interrogans. Somente uma propriedade não possuía vacas sororreagentes para leptospirose e das nove propriedades estudadas, seis (66,66%) apresentaram animais soropositivos ao sorovar Hebdomadis. Ao exame histopatológico, as alterações microscópicas mais encontradas em animais positivos foram hialinização (81,25%), congestão (81,25%) e degeneração hidrópica (70,83%). Porém, essas alterações histopatológicas também foram encontradas em rins de animais negativos à sorologia, como hialinização (80,77%), congestão (48,07%) e degeneração hidrópica (55,77%) e estes achados não apresentaram correlação com positividade. O exame histopatológico dos rins não é indicado para substituir o diagnóstico sorológico da leptospirose, podendo ser utilizado somente como um exame complementar. Apesar da frequência de animais sororreagentes ser baixa na região do Triângulo Mineiro, a doença está presente em um grande número depropriedades. Chama a atenção a alta frequência do sorovar Hebdomadis, podendo ser considerado um sorovar emergente na região. A avaliação da frequência deste sorovar em outras regiões se torna importante, pois uma vez verificada, deve resultar na recomendação da inclusão deste sorovar no controle da leptospirose.

DNA nanovaccines prepared using LemA antigen protect Golden Syrian hamsters against Leptospira lethal infection

BACKGROUND Nanoparticles (NPs) are viable candidates as carriers of exogenous materials into cells via transfection and can be used in the DNA vaccination strategy against leptospirosis. OBJECTIVES We evaluated the efficiency of halloysite clay nanotubes (HNTs) and amine-functionalised multi-walled carbon nanotubes (NH2-MWCNTs) in facilitating recombinant LemA antigen (rLemA) expression and protecting Golden Syrian hamsters (Mesocricetus auratus) against Leptospira interrogans lethal infection. METHODS An indirect immunofluorescent technique was used to investigate the potency of HNTs and NH2-MWCNTs in enhancing the transfection and expression efficiency of the DNA vaccine in Chinese hamster ovary (CHO) cells. Hamsters were immunised with two doses of vaccines HNT-pTARGET/lemA, NH2-MWCNTs-pTARGET/lemA, pTARGET/lemA, and empty pTARGET (control), and the efficacy was determined in terms of humoral immune response and protection against a lethal challenge. FINDINGS rLemA DNA vaccines carried by NPs were able to transfect CHO cells effectively, inducing IgG immune response in hamsters (p < 0.05), and did not exhibit cytotoxic effects. Furthermore, 83.3% of the hamsters immunised with NH2-MWCNTs-pTARGET/lemA were protected against the lethal challenge (p < 0.01), and 66.7% of hamsters immunised with HNT-pTARGET/lemA survived (p < 0.05). MAIN CONCLUSIONS NH2-MWCNTs and HNTs can act as antigen carriers for mammalian cells and are suitable for DNA nanovaccine delivery.

Diferenciación de serovares de leptospiras patógenas mediante PCR del gen ligB y secuenciación / Differentiation of pathogenic leptospires spp by PCR of ligB gene and sequencing

La leptospirosis es la zoonosis de mayor distribución mundial. El objetivo del preReacción en cadena sente trabajo fue desarrollar una técnica molecular que diferencie leptospiras patógenas. Se amplificó mediante PCR y se secuenció una región de la adhesina ligB, presente solo en las especies patógenas. Se utilizaron los iniciadores ligBFpet y ligBRpet. Dichos iniciadores lograron amplificar el ADN blanco de 6 cepas patógenas referenciales de Leptospira interrogans (serovar Pomona cepa Pomona, serovar Canicola cepa Hond Utrecht IV, serovar Copenhageni cepa M 20, serovar Wolffi cepa 3705, serovar Pyrogenes cepa Salinem y serovar Hardjo cepa Hardjoprajitmo) y el de Leptospira borgpetersenii serovar Castellonis cepa Castellon 3; también el de 4 cepas patógenas aisladas de bovino, porcino, rata y comadreja. En las 2 cepas referenciales no patógenas utilizadas, Leptospira biflexa serovar Patoc cepa Patoc i y L. biflexa serovar Andamana cepa Andamana, no hubo amplificación. La secuenciación de los productos amplificados expuso una suficiente variación entre los serovares estudiados, que permite diferenciarlos.

Leptospirosis is a zoonosis having worldwide distribution. The objective of this work was to develop a molecular technique to differentiate pathogenic Leptospira spp. A region of adhesin ligB, present only in the pathogenic species was amplified by PCR and sequenced. ligBRpet and ligBFpet primers were used, which amplified the target DNA from pathogenic L. interrogans reference strains serovars Pomona strain Pomona, Canicola strain Hond Utrecht IV, Copenhageni strain M 20, Wolffi strain 3705, Pyrogenes strain Salinem, Hardjo strain Hardjoprajitmo, L. borgpetersenii serovar Castellonis strain Castellon 3 and 4 pathogenic strains isolated from bovines, pigs, rats and opossums. L. biflexa serovars Patoc strain Patoc i and Andamana strain Andamana were not amplified. Sequencing of the amplified products exhibited sufficient variation among serovars, which differentiates them.

Whole-genome sequencing of Leptospira interrogans from southern Brazil: genetic features of a highly virulent strain

BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.

Survey and Phylogenetic Analysis of Rodents and Important Rodent-Borne Zoonotic Pathogens in Gedu, Bhutan

Rodents are well-known reservoirs and vectors of many emerging and re-emerging infectious diseases, but little is known about their role in zoonotic disease transmission in Bhutan. In this study, a cross-sectional investigation of zoonotic disease pathogens in rodents was performed in Chukha district, Bhutan, where a high incidence of scrub typhus and cases of acute undifferentiated febrile illness had been reported in people during the preceding 4–6 months. Twelve rodents were trapped alive using wire-mesh traps. Following euthanasia, liver and kidney tissues were removed and tested using PCR for Orientia tsutsugamushi and other bacterial and rickettsial pathogens causing bartonellosis, borreliosis, human monocytic ehrlichiosis, human granulocytic anaplasmosis, leptospirosis, and rickettsiosis. A phylogenetic analysis was performed on all rodent species captured and pathogens detected. Four out of the 12 rodents (33.3%) tested positive by PCR for zoonotic pathogens. Anaplasma phagocytophilum, Bartonella grahamii, and B. queenslandensis were identified for the first time in Bhutan. Leptospira interrogans was also detected for the first time from rodents in Bhutan. The findings demonstrate the presence of these zoonotic pathogens in rodents in Bhutan, which may pose a risk of disease transmission to humans.

Primer informe de Leptospira interrogans en el roedor sigmodontino Scapteromys aquaticus / First report on Leptospira interrogans in the sigmodontine rodent Scapteromys aquaticus / Primeiro relato da presença de Leptospira interrogans em roedores sigmodontíneos Scapteromys aquaticus

RESUMEN La leptospirosis es una enfermedad zoonótica de distribución mundial que puede transmitirse por contacto directo o indirecto con orina o tejidos de animales infectados. En Argentina, la leptospirosis es endémica en la provincia de Santa Fe y presenta brotes epidémicos durante las inundaciones. Sin embargo, se sabe muy poco sobre el papel que cumplen los roedores silvestres en la diseminación de la enfermedad en el país. El objetivo de este estudio fue identificar las especies hospederas de leptospiras patógenas entre los roedores presentes en un asentamiento ribereño de la provincia de Santa Fe. Se realizó un muestreo de roedores durante octubre de 2015. Los riñones de los animales capturados se analizaron por real-time PCR para el gen LipL32 de leptospiras patógenas. En los animales que resultaron positivos, se realizó test de microaglutinación (MAT) y tipificación molecular por amplificación del gen 16S rRNA y dos esquemas de MLST. Se capturaron 37 roedores de las especies Akodon azarae, Cavia aperea, Oligoryzomys flavescens, Rattus rattus y Scapteromys aquaticus. En el análisis por real-time PCR resultó positivo un macho de Scapteromys aquaticus. El suero de este individuo y del resto de los S. aquaticus capturados (n = 18) se analizaron por test de microaglutinación (MAT), y fueron no reactivos para los 10 serovares probados. La amplificación del gen 16S rRNA identificó la especie infectante como Leptospira interrogans, mientras que no se obtuvo amplificación para los dos esquemas de MLST. El hallazgo de este estudio aporta nueva información acerca de presencia de leptospiras patógenas en roedores silvestres, que es relevante para la zona por tratarse de una especie ampliamente distribuida en ambientes pantanosos e inundables de América del Sur.(AU)

ABSTRACT Leptospirosis is a globally distributed zoonosis that can be transmitted through direct or indirect contact with the urine or tissues of infected animals. In Argentina, leptospirosis is endemic in the province of Santa Fe and epidemic outbreaks occur during floods. However, very little is known about the role that wild rodents play in the spread of the disease in Argentina. The objective of this study was to identify the host species of pathogenic Leptospira among rodents in a riverine settlement in the province of Santa Fe. We conducted a trapping session in October 2015. Kidneys of the captured animals were analyzed by real-time PCR for the LipL32 gene of pathogenic Leptospira. Animals that were positive were subjected to microscopic agglutination test (MAT) and molecular typing by amplification of the 16S rRNA gene and two multilocus sequence typing (MLST) schemes. A total of 37 rodents of the species Akodon azarae, Cavia aperea, Oligoryzomys flavescens, Rattus rattus, and Scapteromys aquaticus were captured. Real-time PCR found one male Scapteromys aquaticus that was positive. The serum of this individual and of the rest of the S. aquaticus captured (n = 18) were analyzed by MAT and were non-reactive for the 10 serovars tested. Amplification of the 16S rRNA gene identified the infective species as Leptospira interrogans, while amplification could not be obtained for the two MLST schemes. The findings of this study contribute new information concerning the presence of pathogenic Leptospira in wild rodents, which is relevant in this region because the species is widely distributed in swampy and flood-prone environments of South America.(AU)

RESUMO A leptospirose é uma doença zoonótica de distribuição mundial transmitida pelo contato direto ou indireto com a urina ou os tecidos de animais infectados. Na Argentina, a leptospirose é endêmica na Província de Santa Fé com surtos epidêmicos ocorrendo com as enchentes. Sabe-se pouco sobre o papel dos roedores silvestres na propagação da doença no país. O objetivo deste estudo foi identificar as espécies hospedeiras de leptospiras patogênicas em roedores encontrados em um núcleo de povoamento ribeirinho na Província de Santa Fé. A amostragem dos roedores foi feita no mês de outubro de 2015. Os tecidos dos rins dos animais capturados foram analisados com a técnica de reação em cadeia da polimerase em tempo real (PCR-RT) quanto à presença do gene LipL32 de leptospiras patógenas. Para os animais com resultados positivos, foi realizado o teste de microaglutinação (MAT) e tipagem molecular baseada na amplificação do gene 16S rRNA e dois esquemas de tipagem por sequenciamento de locos múltiplos (MLST). Ao todo, foram capturados 37 roedores das espécies Akodon azarae, Cavia aperea, Oligoryzomys flavescens, Rattus e Scapteromys aquaticus. O ensaio de PCR-RT foi positivo em um roedor macho da espécie Scapteromys aquaticus. Os soros deste animal e dos outros S. aquaticus capturados (n = 18) foram analisados com o MAT e os resultados foram não reagentes para os 10 sorovares testados. A amplificação do gene 16S rRNA permitiu identificar a espécie infetante como sendo Leptospira interrogans e não houve amplificação nos dois esquemas de MLST. O achado deste estudo fornece um novo dado quanto à presença de leptospiras patogênicas em roedores silvestres, importante para esta área por se tratar de uma espécie de ampla distribuição em terras pantanosas e inundáveis da América do Sul.(AU)

A cross sectional study of leptospirosis and fetal death in Yucatan, Mexico / Estudio transversal de leptospirosis y muerte fetal en Yucatán, México

Introduction: Leptospirosis is a zoonotic disease affecting mainly to low income human population. Acute leptospiral infection during pregnancy has been associated with spontaneous abortion and fetal death during the first trimester and the abortion may occur as consequence of systemic failure. Objective: To estimate the frequency of Leptospira interrogans infection in women with spontaneous abortion in the state of Yucatan, Mexico. Methods: A cross sectional study on women with spontaneous abortion was conducted. Serum samples were tested for Leptospirosis by the microaglutination test, to estimate the frequency of the infecting serovar. The indirect ELISA IgM was used to detect recent infection by L. interrogans. DNA was extracted from paraffin-embedded tissue of placenta for PCR detection of L. interrogans. Results: Overall frequency of infection with L. interrogans in the 81 women with abortion was 13.6%. Five of the 12 serovars evaluated were found and included. Two of the 11 women with abortion and positive to microaglutination test were also positive to the ELISA IgM test. None samples were positive for PCR Leptospira diagnosis. Conclusion: two women could be associated with spontaneous abortion due to leptospirosis, because they showed antibodies against L. interrogans in the microaglutination test and ELISA IgM assays. Differences between regions were found with respect to the prevalences of lesptospirosis.

Introducción: Leptospirosis es una enfermedad zoonótica que afecta principalmente la población humana de bajos recursos. Infección aguda por leptospirosis durante el embarazo se ha asociado con aborto espontáneo y muerte fetal durante el primer trimestre del embarazo. Objetivo: Estimar la frecuencia de infección por Leptospira interrogans en mujeres con aborto espontáneo en el estado de Yucatán, México. Métodos: Se efectuó un estudio trasversal en 81 mujeres con aborto espontáneo. La prueba de referencia para Leptospirosis, prueba de microaglutinación, se utilizó para estimar la frecuencia de la serovar infectante. El ELISA IgM indirecto se utilizó para detectar infección reciente por L. interrogans. Se extrajo ADN a partir de tejido embebido en parafina de placenta para la detección de L. interrogans por PCR. Resultados: La frecuencia global de la infección con L. interrogans en 81 mujeres con aborto, fue del 13.6%. Se encontró cinco de 12 serovares de Leptospira. Dos de las 11 mujeres con aborto espontáneo y positivo a microaglutinación también fueron positivas a la prueba ELISA IgM. Ninguna muestra fue positiva hacia el diagnóstico PCR de Leptospira. Conclusion: Dos mujeres podrían estar asociados con el aborto espontáneo debido a la leptospirosis, porque mostraron anticuerpos contra L. interrogans en la microaglutinación y ensayos de ELISA IgM. Diferencias entre regiones fueron encontradas con respecto a las prevalencias de leptospirosis.

Clinical Evaluation of Rapid Diagnostic Test Kit Using the Polysaccharide as a Genus-Specific Diagnostic Antigen for Leptospirosis in Korea, Bulgaria, and Argentina

Leptospirosis, a zoonotic disease that is caused by many serovars which are more than 200 in the world, is an emerging worldwide disease. Accurate and rapid diagnostic tests for leptospirosis are a critical step to diagnose the disease. There are some commercial kits available for diagnosis of leptospirosis, but the obscurity of a species- or genus-specific antigen of pathogenic Leptospira interrogans causes the reduced sensitivity and specificity. In this study, the polysaccharide derived from lipopolysaccharide (LPS) of nonpathogenic Leptospira biflexa serovar patoc was prepared, and the antigenicity was confirmed by immunoblot and enzyme linked immunosorbent assay (ELISA). The performance of the rapid diagnostic test (RDT) kit using the polysaccharide as a diagnostic antigen was evaluated in Korea, Bulgaria and Argentina. The sensitivity was 93.9%, 100%, and 81.0% and the specificity was 97.9%, 100%, and 95.4% in Korea (which is a rare region occurring with 2 serovars mostly), Bulgaria (epidemic region with 3 serovars chiefly) and Argentina (endemic region with 19 serovars mainly) respectively. These results indicate that this RDT is applicable for global diagnosis of leptospirosis. This rapid and effective diagnosis will be helpful for diagnosis and manage of leptospirosis to use and the polysaccharide of Leptospira may be called as genus specific antigen for diagnosis.

Clinical Evaluation of Rapid Diagnostic Test Kit Using the Polysaccharide as a Genus-Specific Diagnostic Antigen for Leptospirosis in Korea, Bulgaria, and Argentina

Leptospirosis, a zoonotic disease that is caused by many serovars which are more than 200 in the world, is an emerging worldwide disease. Accurate and rapid diagnostic tests for leptospirosis are a critical step to diagnose the disease. There are some commercial kits available for diagnosis of leptospirosis, but the obscurity of a species- or genus-specific antigen of pathogenic Leptospira interrogans causes the reduced sensitivity and specificity. In this study, the polysaccharide derived from lipopolysaccharide (LPS) of nonpathogenic Leptospira biflexa serovar patoc was prepared, and the antigenicity was confirmed by immunoblot and enzyme linked immunosorbent assay (ELISA). The performance of the rapid diagnostic test (RDT) kit using the polysaccharide as a diagnostic antigen was evaluated in Korea, Bulgaria and Argentina. The sensitivity was 93.9%, 100%, and 81.0% and the specificity was 97.9%, 100%, and 95.4% in Korea (which is a rare region occurring with 2 serovars mostly), Bulgaria (epidemic region with 3 serovars chiefly) and Argentina (endemic region with 19 serovars mainly) respectively. These results indicate that this RDT is applicable for global diagnosis of leptospirosis. This rapid and effective diagnosis will be helpful for diagnosis and manage of leptospirosis to use and the polysaccharide of Leptospira may be called as genus specific antigen for diagnosis.

Heterogenic colonization patterns by Leptospira interrogans in Rattus norvegicus from urban slums

Abstract We evaluated the renal colonization by Leptospira interrogans in Rattus norvegicus (rats), as it is the major natural reservoir of urban leptospirosis. We caught 72 R. norvegicus, out of which 32 were found to be positive for L. interrogans by immunofluorescence assay. From these rats, we selected 17 and divided them into six groups based on the mass-age/sex. We performed the immunohistochemistry test against L. interrogans in the kidney sections of the rats and systematically counted the colonized tubules (CTs) in 20 fields. The proportion of positive fields varied from 5% to 95%. The number of CTs in 20 fields varied from 0.5 to 85.5. These differences were not related to age or sex of the animals. The characterization of leptospiral colonization patterns in the natural reservoirs is important to better understand the host-pathogen interactions in leptospirosis.

Occurrence of antibodies anti -Toxoplasma gondii, Neospora caninum and Leptospira interrogans in a captive deer herd in Southern Brazil / Ocorrência de anticorpos anti -Toxoplasma gondii, Neospora caninum e Leptospira interrogans em um rebanho de cervídeos de cativeiro do sul do Brasil

Abstract A large number of Brazilian zoos keep many endangered species of deer, however, very few disease surveillance studies have been conducted among captive cervids. Blood samples from 32 Brazilian deer (Blastocerus dichotomus, Mazama nana and Mazama americana) kept in captivity at Bela Vista Biological Sanctuary (Foz do Iguaçu, Brazil) were investigated for 10 ruminant pathogens, with the aims of monitoring deer health status and evaluating any potential zoonotic risk. Deer serum samples were tested for Brucella abortus, Leptospira (23 serovars), Toxoplasma gondii, Neospora caninum, bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, foot-and-mouth disease virus, western equine encephalitis virus, eastern equine encephalitis virus and Venezuelan equine encephalitis virus. Antibodies against T. gondii (15.6%), N. caninum (6.2%) and L. interrogans serogroup Serjoe (3.1%) were detected. The serological results for all other infectious agents were negative. The deer were considered to be clinically healthy and asymptomatic regarding any disease. Compared with studies on free-ranging deer, the prevalences of the same agents tested among the captive deer kept at the Sanctuary were lower, thus indicating good sanitary conditions and high-quality management practices at the zoo.

Resumo Um grande número de zoológicos brasileiros abriga espécies de cervídeos ameaçados de extinção, entretanto, estudos de vigilância de doenças em cervídeos de cativeiro são escassos. Amostras de sangue de 32 cervídeos brasileiros (Blastocerus dichotomus, Mazama nana e Mazama americana), mantidos em cativeiro no Refúgio Biológico Bela Vista (Foz do Iguaçu, Brasil), foram investigados para 10 patógenos de ruminantes, visando monitorar o estado de saúde dos cervídeos e avaliar a presença de agentes zoonóticos. As amostras de soro foram testadas para Brucella abortus, Leptospira (23 sorovares), Toxoplasma gondii, Neospora caninum, diarreia viral bovina, rinotraqueíte infecciosa bovina, febre aftosa, encefalomielite equina do oeste, encefalomielite equina do leste e encefalomielite equina venezuelana. Foram detectados anticorpos para T. gondii (15,6%), N. caninum (6,2%) e para L. interrogans sorogrupo Serjoe (3,1%). As sorologias apresentaram resultado negativo para as demais doenças. Os cervídeos foram considerados clinicamente sadios e assintomáticos para doenças. Comparados aos estudos de populações de vida livre, as soroprevalências para os mesmos agentes testados foram menores para os cervídeos mantidos no Refúgio, indicando as boas condições sanitárias e a qualidade das práticas de manejo no zoológico.

Presence of antibodies against Leptospira spp. in dogs of Uberlandia, MG, Brazil / Presença de anticorpos contra Leptospira spp. em cães no município de Uberlândia, MG, Brasil

The aim of this study was to investigate the occurrence of antibodies against Leptospira spp. and risk factors related to the epidemiology of leptospirosis in dogs living in the urban area of the municipality of Uberlandia, Minas Gerais, Brazil. Blood serum samples were examined from 268 dogs from the municipality's North, South, East, West and Central sanitary districts. The samples were collected during an animal vaccination campaign against rabies in August 2008. The occurrence of antibodies against specific leptospiral serovars was determined by the microscopic agglutination test (MAT). Multivariable logistic regression was used to evaluate the association between sex, age, previous vaccination, habitation, presence of rats, and history of human and/or animal leptospirosis on the occurrence of infection. The occurrence of canine leptospirosis was 28.36% (76/268), the most prevalent serovars were: Autumnalis (34.21%), Tarassovi (23.58%), Canicola (17.11%) and Grippotyphosa (14.47%). No risk factors were found to be statistically significant (p> 0,05 for all examined variables). The fact that close to 30% of dogs showed evidence of past infection with Leptospira points to the need for the adoption of preventive measures, as the disease may represent a public health risk.

O objetivo deste estudo foi investigar a ocorrência de anticorpos contra Leptospira spp. e os fatores de risco relacionados à epidemiologia da leptospirose em cães que vivem na área urbana do município de Uberlândia, Minas Gerais, Brasil. Amostras de soro foram examinados a partir de 268 cães dos distritos sanitários Norte, Sul, Leste, Oeste e Central. As amostras foram colhidas durante a campanha de vacinação contra a raiva animal em agosto de 2008. A ocorrência de anticorpos contra sorovares específicos foi determinada pelo teste de Soroaglutinação Microscópica (SAM). Foi utilizada a regressão logística multivariada para avaliar a associação entre sexo, idade, a vacinação, habitação, presença de ratos e história de humanos e/ou leptospirose animal na ocorrência da infecção. A ocorrência da leptospirose canina foi de 28,36% (76/268), os sorovares mais prevalentes foram: Autumnalis (34,21%), Tarassovi (23,58%), Canicola (17,11%) e Grippotyphosa (14,47%). Quanto aos fatores de risco analisados, não foi encontrada diferença estatística significativa (p> 0,05 para todas as variáveis analisadas). O fato de 30% dos cães terem apresentado evidência de infecção causada por Leptospira aponta para a necessidade da adoção de medidas preventivas, pois a doença pode representar um risco para a saúde pública.

Molecular characterisation and disease severity of leptospirosis in Sri Lanka

Leptospirosis is a re-emerging zoonotic disease all over the world, important in tropical and subtropical areas. A majority of leptospirosis infected patients present as subclinical or mild disease while 5-10% may develop severe infection requiring hospitalisation and critical care. It is possible that several factors, such as the infecting serovar, level of leptospiraemia, host genetic factors and host immune response, may be important in predisposition towards severe disease. Different Leptospira strains circulate in different geographical regions contributing to variable disease severity. Therefore, it is important to investigate the circulating strains at geographical locations during each outbreak for epidemiological studies and to support the clinical management of the patients. In this study immunochromatography, microscopic agglutination test and polymerase chain reaction were used to diagnose leptospirosis. Further restriction fragment length polymorphism and DNA sequencing methods were used to identify the circulating strains in two selected geographical regions of Sri Lanka. Leptospira interrogans, Leptospira borgpetersenii and Leptospira kirschneri strains were identified to be circulating in western and southern provinces. L. interrogans was the predominant species circulating in western and southern provinces in 2013 and its presence was mainly associated with renal failure.

Serosurvey of Leptospira interrogans, Brucella abortus and Chlamydophila abortus infection in free-ranging giant anteaters (Myrmecophaga tridactyla) from Brazil / Inquérito sorológico de Leptospira interrogans, Brucella abortus e Chlamydophila abortus em tamanduás-bandeira (Myrmecophaga tridactyla) de vida livre no Brasil

A serological survey for antibodies against Leptospira interrogans, Brucella abortus, and Chlamydophila abortus was conducted in 21 clinically healthy, free-ranging giant ant- eaters (Myrmecophaga tridactyla) from Parque Nacional das Emas (Goiás State, Brazil; n=6), Parque Nacional da Serra da Canastra (Minas Gerais State, Brazil; n=9), and RPPN SESC Pantanal (Mato Grosso State, Brazil; n=6) between July 2001 and September 2006. Sera were screened for antibodies against 22 serovars of Leptospira interrogans with a microscopic agglutination test. Twelve tested positive for L. interrogansserovars sentot (n=5 in PN Emas, n=2 in PN Serra da Canastra), butembo (n=2 in PN Serra da Canastra), autumnalis, bataviae, and shermani/icterohaemorrhagiae(n=1 each in SESC Pantanal)One adult female tested positive for B. abortus with the buffered plate antigen test. All sera were negative for C. abortususing the complement fixation text. This is the first report of pathogens that may interfere with the reproduction and population dynamics of free-ranging giant anteaters.

Inquéritos sorológicos para detecção de anticorpos contra Leptospira interrogans, Brucella abortus, e Chlamydophila abortus foram realizados em 21 tamanduás-bandeira (Myrmecophaga tridactyla) de vida livre do Parque Nacional das Emas (Goiás, Brasil, n=6), o Parque Nacional da Serra da Canastra (Minas Gerais, Brasil, n=9) e RPPN SESC Pantanal (Mato Grosso, Brasil, n=6) entre julho de 2001 e setembro de 2006. Os sor os foram testados para anticorpos contra 22 sorotipos de Leptospira interrogans com um teste de aglutinação microscópica. Doze animais foram considerados positivos para L. interrogans sorovares sentot (n=5 em PN Emas, n=2 em PN Serra da Canastra), butembo (n=2 em PN Serra da Canastra), autumnalis, bataviae e shermani/icterohaemorrhagiae(n=1 para cada sorovar em SESC Pantanal). Uma fêmea adulta testou positivo para B. abortuscom o teste do antígeno tamponado. Todos os soros se mostraram negativos para C. abortusatravés do teste de fixação do complemento. Este é o primeiro relato de patógenos que podem interferir na dinâmica reprodutiva de populações de tamanduás em estado selvagem.