Enzyme-linked immunosorbent assay (ELISA) for detection of IgY Anti-Borrelia anserina antibodies in Gallus gallus domesticus / Ensaio de imunoadsorção enzimático (ELISA) indireto para detecção de anticorpos IgY Anti-Borreliaanserina em Gallus gallus domesticus

This study aimed to promote the standardization of an indirect, enzyme-linked immunosorbent assay (ELISA) for the serological detection of B. anserina in Gallus gallusdomesticus. An aliquoted sera from vaccinated chicken with B. anserina antigen (GI), experimental infected chickens with B. anserina (GII) and rustic poultry rearing of G. gallus (GIII) were tested with in-house ELISA developed to detect serum antibodies against B. anserina in G. gallus domesticus. On average, the experimentally infected chickens became positive at 9 DPI a mean ± standard deviation (SD) ODI value of 163.11 ± 70.65. The highest observed Optical Density Index (ODI) was 372.54 ± 132.39, at 26 DPI, and the highest overall ODI value was 626.51. The vaccinated chickens became positive between 8 and 10 DPV, with an ODI of 245.59 at 10 DPV, with an overall maximum ODI of 543.13. A total of 108 blood samples were collected from poultry raised on rustic farms. Of the total samples collected, 58.33% (63/108) were considered positive for B. anserina. The maximum ODI found among these rustic chickens was 283.24. This stardardization provided a sensitivity and specificity of 100%.

Este estudo teve como objetivo promover a padronização de um ensaio imunoenzimático indireto (ELISA) para a detecção sorológica de Borrelia anserina em Gallus gallus domesticus. Um frango vacinado com antígeno de B. anserina (GI), frangos infectados experimentalmente com B. anserina (GII) e frangos criados de forma rústica (GIII) foram testados com ELISA indireto in house desenvolvido para a detecção sorológica contra B. anserina em G. gallus domesticus. Em média, os frangos infectados experimentalmente tornaram-se positivos aos 9º dia pós-inoculação (DPI), um valor do índice de densidade óptica (ODI) médio ± desvio padrão (SD) de 163,11 ± 70,65. O maior ODI observado foi 372,54 ± 132,39, em 26ºDPI, e o maior valor geral de ODI foi 626,51. Os frangos vacinados tornaram-se positivos entre 8º e 10° DPV, com um ODI de 245,59 a 10 DPV, com um ODI máximo geral de 543,13. Um total de 108 amostras de sangue foram coletadas de aves criadas em fazendas rústicas. Do total de amostras coletadas, 58,33% (63/108) foram consideradas positivas para B. anserina. O ODI máximo encontrado entre essas galinhas rústicas foi 283,24. Essa padronização proporcionou sensibilidade e especificidade de 100%.

Detection of Rickettsia spp. in ring-tailed coatis (Nasua nasua) and ticks of the Iguaçu National Park, Brazilian Atlantic Rainforest.

Wild animals are of considerable importance in the ecology of infectious agents, as they can function as hosts and even as possible vectors. In this study, DNA from Rickettsia spp. was detected on ticks and fragments of skin collected from wild coatis with synanthropic habits in the Iguaçu National Park (INP) in the state of Paraná in southern Brazil. Testing was carried out on a total of 566 ticks, comprising Amblyomma spp. larvae, nymphs of Haemaphysalis juxtakochi, Amblyomma brasiliense, Amblyomma coelebs, and adults of Amblyomma ovale. The samples were tested by polymerase chain reaction (PCR) by amplifying htrA, gltA, ompA, and ompB gene fragments to detect Rickettsia spp. A fragment of each positive sample was sequenced in both directions, submitted to Genbank for a homology search, and also used for phylogenetic analyses. Samples of A. coelebs (1.90%, 8/420), A. ovale (13%, 6/45), and ring-tailed coati skin (1%, 1/75) amplified Rickettsia spp. DNA. Through sequencing, Rickettsia bellii was observed in A. ovale, Rickettsia amblyommatis in A. coelebs, while Rickettsia rhipicephali was detected in the skin samples. Wild ring-tailed coatis with synanthropic habits in the INP and their ticks are infected by Rickettsia spp., and associations with new hosts have been described.

Hemoparasites in ticks of wild birds of Serra dos Órgãos National Park, state of Rio de Janeiro, Brazil.

The aim of this study is to detect the presence of tick-borne agents of genera Rickettsia, Borrelia, Babesia, Ehrlichia and Anaplasma in ticks collected from native wild birds in the state of Rio de Janeiro. Birds were captured and observed carefully to find the ectoparasites. DNA detection of hemoparasites was performed by means of the polymerase chain reaction (PCR). The sequences obtained were analyzed and their homologies were compared to the available isolates in the GenBank platform database. A total of 33 birds were captured from 20 different species, of which 14 were parasitized by Amblyomma longirostre (n = 22). There was absence of DNA from agents of the genera Babesia, Anaplasma and Ehrlichia in the evaluated samples. The phylogenetic analysis indicated that one sample had 100% identity with Rickettsia bellii (KJ534309), the other two samples showed 100% identity with Rickettsia sp. Aranha strain and strain AL (EU274654 and AY360216). The positive sample for R. bellii was also demonstrated to be positive for Borrelia sp., which presented a similarity of 91% with Borrelia turcica (KF422815). This is the first description of Borrelia sp. in ticks of the genus Amblyomma in South America.

Hemoparasites in ticks of wild birds of Serra dos Órgãos National Park, state of Rio de Janeiro, Brazil / Hemoparasitos em carrapatos de aves silvestres do Parque Nacional da Serra dos Órgãos, estado do Rio de Janeiro, Brasil

Abstract The aim of this study is to detect the presence of tick-borne agents of genera Rickettsia, Borrelia, Babesia, Ehrlichia and Anaplasma in ticks collected from native wild birds in the state of Rio de Janeiro. Birds were captured and observed carefully to find the ectoparasites. DNA detection of hemoparasites was performed by means of the polymerase chain reaction (PCR). The sequences obtained were analyzed and their homologies were compared to the available isolates in the GenBank platform database. A total of 33 birds were captured from 20 different species, of which 14 were parasitized by Amblyomma longirostre (n = 22). There was absence of DNA from agents of the genera Babesia, Anaplasma and Ehrlichia in the evaluated samples. The phylogenetic analysis indicated that one sample had 100% identity with Rickettsia bellii (KJ534309), the other two samples showed 100% identity with Rickettsia sp. Aranha strain and strain AL (EU274654 and AY360216). The positive sample for R. bellii was also demonstrated to be positive for Borrelia sp., which presented a similarity of 91% with Borrelia turcica (KF422815). This is the first description of Borrelia sp. in ticks of the genus Amblyomma in South America.

Resumo Este trabalho teve como objetivo detectar evidências moleculares da presença de agentes dos gêneros Rickettsia, Borrelia, Babesia, Anaplasma e Ehrlichia transmitidos por carrapatos coletados de aves silvestres no estado do Rio de Janeiro. Aves foram capturadas e observadas cuidadosamente a procura de ectoparasitos. A detecção de DNA de hemoparasitos foi realizada por meio da reação em cadeia da polimerase (PCR). As sequências obtidas foram analisadas e sua homologia comparada aos isolados disponíveis na base de dados da plataforma GenBank. Foram capturadas 33 aves, de 20 espécies diferentes das quais 14 estavam parasitadas por Amblyomma longirostre (n = 22). Houve ausência de DNA de agentes dos gêneros Babesia, Anaplasma e Ehrlichia nas amostras avaliadas. A análise filogenética indicou que uma amostra apresentou 100% de identidade com Rickettsia bellii (KJ534309), as outras duas amostras apresentaram 100% de identidade com Rickettsia sp. cepa Aranha e Cepa AL (EU274654 e AY360216.). A amostra positiva para R. bellii também apresentou positividade para Borrelia sp. que apresentou similaridade de 91% com Borrelia turcica (KF422815). Esta é a primeira descrição de Borrelia sp. em carrapatos do gênero Amblyomma na América do Sul.

Morphological, molecular and phylogenetic characterization of Borrelia theileri in Rhipicephalus microplus.

This study aimed to perform a morphological, molecular and phylogenetic characterization of Borrelia theileri obtained from infected Rhipicephalus microplus in Brazil. Fifty engorged R. microplus females from cattle in the municipality of Seropédica, Rio de Janeiro, were analyzed for spirochetes by hemolymph smear. Macerated eggs and positive ticks, as well as blood from the bovine infested by these ticks, were analyzed the glpQ, flaB and hpt genes by PCR. The PCR products were purified and sequenced for analysis and construction of a phylogenetic tree. Only 2% (1/50) of the ticks generated a positive result by both smear and PCR. The spiral forms (n = 50) had (media ± SD) a mean length of 19.17 ± 4.12 µm, diameter of 0.2935 ± 0.0469 and number of turns 8.44 ± 2.59. Sequence alignments of the three evaluated genes exhibited 98% similarity to B. theileri isolates, occurring in a clade highly related to B. theileri strain KAT. Egg maceration samples were positive for the three evaluated genes, whereas bovine blood was negative by PCR. This is the most detailed characterization of B. theileri in the Americas to-date, presenting morphological, molecular and phylogenetic data, including the transovarial transmission of the spirochete in the host tick.

Ocorrência de Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae) em Rhinella icterica (Spix, 1824) (Anura: Bufonidae) em Maricá, Rio de Janeiro, Brasil / Occurrence of Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae) in Rhinella icterica (Spix, 1824) (Anura: Bufonidae) in Maricá, Rio de Janeiro, Brazil / Ocurrencia de Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae) en Rhinella icterica (Spix, 1824) (Anura: Bufonidae) en Maricá, Río de Janeiro, Brasil

Anfíbios anuros são comumente parasitados por carrapatos da espécie Amblyomma rotundatum, os quais possuem alta predileção por animais ectotérmicos. Fêmeas e ninfas ingurgitadas de A. rotundatum foram coletadas em condições naturais a partir de um espécime de "sapo-cururu" da espécie Rhinella icterica. A identificação dos carrapatos foi feita por meio de características morfológicas. Este é o primeiro relato do parasitismo deste carrapato em sapos desta espécie no Município de Maricá, Região Metropolitana do Estado do Rio de Janeiro, Brasil.(AU)

Anuran amphibians are commonly parasitized by ticks of the species Amblyomma rotundatum, which have high predilection for ectothermic animals. Engorged females and nymphs of A. rotundatum were collected under natural conditions from a "toad-cururu" specimen of the species Rhinella icterica. The identification of ticks was made through morphological characteristics. This is the first report of the parasitism of this tick in toads of this species in the Municipality of Maricá, Metropolitan Region of the State of Rio de Janeiro, Brazil.(AU)

Los anfibios anuros son comúnmente parasitados por garrapatas de la especie Amblyomma rotundatum, los cuales poseen alta predilección por animales ectotermos. Las hembras y ninfas ingurgitadas de A. rotundatum fueron recolectadas en condiciones naturales a partir de un espécimen de "sapo cururú" de la especie Rhinella icterica. La identificación de las garrapatas se ha hecho a través de características morfológicas. Este es el primer relato del parasitismo de esta garrapata en sapos de esta especie en el Municipio de Maricá, Región Metropolitana del Estado de Río de Janeiro, Brasil.(AU)

Morphological, molecular and phylogenetic characterization of Borrelia theileri in Rhipicephalus microplus / Caracterização morfológica, molecular e filogenética de Borrelia theileri em Rhipicephalus microplus

Abstract This study aimed to perform a morphological, molecular and phylogenetic characterization of Borrelia theileri obtained from infected Rhipicephalus microplus in Brazil. Fifty engorged R. microplus females from cattle in the municipality of Seropédica, Rio de Janeiro, were analyzed for spirochetes by hemolymph smear. Macerated eggs and positive ticks, as well as blood from the bovine infested by these ticks, were analyzed the glpQ, flaB and hpt genes by PCR. The PCR products were purified and sequenced for analysis and construction of a phylogenetic tree. Only 2% (1/50) of the ticks generated a positive result by both smear and PCR. The spiral forms (n = 50) had (media ± SD) a mean length of 19.17 ± 4.12 µm, diameter of 0.2935 ± 0.0469 and number of turns 8.44 ± 2.59. Sequence alignments of the three evaluated genes exhibited 98% similarity to B. theileri isolates, occurring in a clade highly related to B. theileri strain KAT. Egg maceration samples were positive for the three evaluated genes, whereas bovine blood was negative by PCR. This is the most detailed characterization of B. theileri in the Americas to-date, presenting morphological, molecular and phylogenetic data, including the transovarial transmission of the spirochete in the host tick.

Resumo O estudo teve como objetivo realizar a caracterização morfológica, molecular e filogenética de Borrelia theileri obtida de Rhipicephalus microplus naturalmente infectado em bovino no estado do Rio de Janeiro, Brasil. Um total de 50 fêmeas de R. microplus ingurgitadas foram analisadas para espiroquetas por meio de esfregaço de hemolinfa. Ovos macerados e carrapatos, assim como sangue de bovinos infectados por esses carrapatos, foram analisados os genes glpQ, flaB e hpt por PCR. Os produtos de PCR foram purificados e sequenciados para análise e construção de uma árvore filogenética. Apenas 2% (1/50) dos carrapatos geraram um resultado positivo tanto pelo esfregaço como pela PCR. As formas espirais (n = 50) apresentaram (média ± DP) comprimento médio de 19,17 ± 4,12, diâmetro de 0,2935 ± 0,0469 e número de voltas de 8,44 ± 2,59. Os alinhamentos das sequências dos três genes avaliados exibiram 98% de similaridade aos isolados de B. theileri, ocorrendo em um clado altamente relacionado à linhagem de B. theileri KAT. As amostras de maceração de ovos foram positivas para os três genes avaliados, enquanto o sangue bovino foi negativo pela PCR. Esta é a mais completa caracterização de B. theileri nas Américas, apresentando dados morfológicos, moleculares e filogenéticos, incluindo a transmissão transovarial da espiroqueta no carrapato hospedeiro.

Epidemiological investigation of Borrelia burgdorferi in horses in the municipality of Sinop-MT, Brazil.

Borrelia burgdorferi sensu stricto is the main etiological agent of Lyme disease (LD) in the USA. In Brazil, it is believed that a similar spirochete is the causal agent of the Baggio-Yoshinari syndrome (BYS), a zoonosis also transmitted by ticks, whose clinical manifestations are similar to those of LD. Despite the epidemiological importance, there are no studies reporting the presence and the prevalence of B. burgdorferi among horses in Mato Grosso State. The aim of this study was to detect and measure the frequency of IgG antibodies anti-B. burgdorferi American strain G39/40 in horses in the municipality of Sinop, MT-Brazil, using the indirect enzyme-linked immunosorbent assay (ELISA) for serological diagnosis. Blood samples from 367 horses were collected in 81 farms. An epidemiological questionnaire was applied during the visits to obtain information related to the animals and the farms. From the 367 horses, 214 were positive for B. burgdorferi sensu stricto according to the results of the ELISA test, representing an apparent prevalence of 54.04% [CI = 0.4548051-0.6237234]. Concomitantly, 89 blood samples were taken for molecular analysis by nested polymerase chain reaction (PCR). According to the PCR test results, none of the samples were reactive, although 53 of these samples were reactive according to ELISA. Seventy five farms (92.59%) had at least one reactive horse for B. burgdorferi. Our results support the hypothesis of the presence of anti-Borrelia spp. antibodies in horses in Mato Grosso, reaching a high animal prevalence. Besides that, leisure/sport purposes proved to be a risk factor, with an odds ratio of 3.16. These findings clearly indicate the need of borreliosis control in Sinop and make a significant contribution to the knowledge of the disease in Mato Grosso.

Soroprevalência de anticorpos homólogos anti-Borrelia burgdorferi em equinos de uso militar no Brasil / Seroprevalence of homologous antiBorrelia burgdorferi antibodies in horses of military use in Brazil

A maioria das atividades de treinamento militar transcorre em ambiente de risco para aquisição de doenças de transmissão vetorial, incluindo importantes zoonoses e doenças emergentes/reemergentes. O objetivo deste estudo foi relatar a ocorrência de anticorpos homólogos da classe IgG anti-Borrelia burgdorferi em equinos de uso militar no Brasil, assim como analisar os fatores de risco associados à soropositividade nos animais. Foram estudados dois plantéis de equinos, sendo um no município de Resende, estado do Rio de Janeiro, tendo sido encontrada prevalência de 29,9%, e outro no município de São Borja, estado do Rio Grande do Sul, com prevalência de 44,7%, totalizando 474 animais e prevalência de 39,24%. Na análise dos fatores de risco associados, observou-se que a infestação por carrapatos constituiu-se no fator de risco para a soropositividade. A evidência de circulação de Borrelia spp. com soroprevalência nos equinos reforça seu papel como sentinela, sendo fator de alerta para potencial ocorrência de casos humanos. Os resultados obtidos reforçam a importância da vigilância epidemiológica dos carrapatos vetores e sua associação tanto com agentes infecciosos de risco para a saúde do efetivo militar humano, como nos equinos de uso militar nas regiões avaliadas. Isto também realça a necessidade da adoção de medidas de proteção coletivas e individuais visando mitigar o risco de doenças transmitidas por carrapatos em atividades militares.