Detection of Bartonella sp. and a novel spotted fever group Rickettsia sp. in Neotropical fleas of wild rodents (Cricetidae) from Southern Brazil.

The Neotropical region shows a great diversity of fleas, comprising more than 50 genera. The importance of the study of fleas is linked to their potential role as disease vectors. The aim of this study is to investigate the presence of Rickettsia spp. and Bartonella spp. in Neotropical fleas collected from wild rodents in Southern Brazil. From 350 rodents captured, 30 were parasitized by fleas. A total of 61 fleas belonging to two genera and six different species were collected (Craneopsylla minerva minerva, Polygenis occidentalis occidentalis, Polygenis platensis, Polygenis pradoi, Polygenis rimatus, and Polygenis roberti roberti). In 13 % of fleas of three different species (C. minerva, P. platensis, and P. pradoi) Rickettsia sp. DNA was found. Phylogenetic analysis of concatenated sequences of gltA, htrA, and ompA genes showed that Rickettsia sp. found in rodent fleas (referred as strain Taim) grouped together with Spotted Fever Group Rickettsia. In reference to Bartonella spp., five genotypes were identified in seven fleas of two species (C. minerva and P. platensis) and in five rodent spleens. Also, 207 frozen samples of wild rodents were screened for these pathogens: while none was positive for Rickettsia spp.; five rodent spleens were PCR-positive for Bartonella spp.. Herein, we show the detection of potential novel variants of Bartonella sp. and Rickettsia sp. in fleas collected of wild rodents from Southern Brazil. Further studies are needed to fully characterize these microorganisms, as well as to improve the knowledge on the potential role of Neotropical flea species as diseases vectors.

Molecular survey of Rickettsia spp. in the Neotropical deer tick Haemaphysalis juxtakochi from Brazilian Pampa.

Spotted fever (SF) is a tick-borne disease associated with Rickettsia spp.. In the Pampa biome, Southern Brazil, cases of SF seem to be strongly linked with the practice of hunting wild animals. An investigation of rickettsiae in tick species found on wild animals could provide more information regarding the rickettsiosis enzootic cycle. The aim of this study is to describe the results of a molecular survey of Rickettsia spp. in the Neotropical deer tick, Haemaphysalis juxtakochi Cooley, 1946 (Acari: Ixodidae), from the Brazilian Pampa. Ticks were obtained from 14 road-killed gray brocket deer, Mazama gouazoubira (Artiodactyla: Cervidae), found in nine different municipalities of Rio Grande do Sul state, Southern Brazil. Ticks were processed individually to obtain genomic DNA, and then Rickettsia spp. was investigated using a set of PCR reactions that amplified the rickettsial fragments of the gltA, ompA, and htrA genes. Of the 24 tick samples tested, DNA of Rickettsia parkeri sensu stricto (s.s.) was found in 11 H. juxtakochi specimens collected in two different areas of the Brazilian Pampa. This is the first report of R. parkeri s.s. (the main agent associated with SF in the Uruguayan, Argentinian, and Brazilian Pampa) in H. juxtakochi ticks. These findings indicate that R. parkeri s.s. may be much more common and widely distributed in the Pampa biome than previously assumed. Moreover, H. juxtakochi ticks and gray brocket deer could participate in the potential spillover of R. parkeri s.s. from endemic to non-endemic areas in the South American Pampa.

Records of ticks on humans in Rio Grande do Sul state, Brazil.

More than seventy tick species have been reported in Brazil. Despite the emergence of tick-borne diseases in Neotropical region, there are still limited data available on tick species parasitizing humans in Brazil. Rio Grande do Sul is the southernmost state of Brazil, comprising the only part of Brazilian territory inside the Pampa biome, as well as the transition between subtropical and temperate zones. Here, we report on human parasitism by ticks in Rio Grande do Sul state between 2004 and 2017. Seventy cases of human parasitism by ticks were recorded, with a total of 81 tick specimens collected. These included 11 tick species belonging to three genera of Ixodidae (hard-ticks), Amblyomma, Haemaphysalis and Rhipicephalus; and one genus of Argasidae, Ornithodoros. The most prevalent tick species associated to cases of human parasitism were Amblyomma parkeri (24%), Rhipicephalus sanguineus sensu lato (22%), Amblyomma aureolatum (15%) and Amblyomma ovale (12%). A spatial analysis showed two major hot spots of human parasitism by ticks in Rio Grande do Sul state. The findings of this study highlight the need for permanent monitoring of human parasitism by ticks in order to provide a better understanding of tick and tick-borne disease eco-epidemiology, and the early identification of potential cases of tick-borne diseases, particularly in spotted fever endemic regions.

Borrelia burgdorferi sensu lato in Ixodes longiscutatus ticks from Brazilian Pampa.

Borrelia burgdorferi sensu lato (s.l.) complex includes the agents of Lyme disease/borreliosis in North America, Europe, and Asia, such Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia bavariensis, Borrelia spielmanii, Borrelia bissettiae, and Borrelia mayonii. In 2013 B. burgdorferi s.l. was reported for the first time in the Neotropical region, from Ixodes aragaoi ticks in Uruguayan Pampa. In addition, from 2011 to 2016, 17 suspected human cases of borreliosis-like syndrome were reported in Rio Grande do Sul (RS) state, Brazil, which contains only part of country in the Pampa biome. The goal of this work is to report the results of a state surveillance program conducted in order to investigate the presence of B. burgdorferi s.l. in its classic vector, Ixodes spp. ticks, from the Brazilian Pampa. For this, we searched for Ixodes spp. ticks in 307 rodents from 11 municipalities of RS state. We then tested the ticks for the presence of B. burgdorferi s.l. DNA using PCR analysis. Of 35 Ixodes spp. ticks tested, one larva and one nymph of Ixodes longiscutatus ticks tested positive for Borrelia sp. DNA. The phylogenetic analysis of the flaB fragment grouped our samples (referred as Borrelia sp. haplotype Pampa) into B. burgdorferi s.l. group in a particular branch with other South American haplotypes, and this group was close to Borrelia carolinensis, B. bissettiae, and Borrelia californiensis. This is the first evidence of B. burgdorferi s.l. circulation in ticks of the genus Ixodes in Brazil. These results highlight the need for the implementation of public health policies for the diagnosis and prevention of potential cases of human borreliosis in Brazil. Further studies are needed to fill the gaps in our knowledge of the distribution, pathogenicity, reservoirs, and vectors of these emerging South American B. burgdorferi s.l. haplotypes.

DETECTION OF BARTONELLA SP. IN DEER LOUSE FLIES (LIPOPTENA MAZAMAE) ON GRAY BROCKET DEER (MAZAMA GOUAZOUBIRA) IN THE NEOTROPICS.

Louse flies or deer keds, Lipoptena spp., are widespread in Neotropical cervids, but the vector-borne pathogens of louse flies had only been previously reported in the Northern hemisphere. This is the first report of Bartonella spp. in deer louse flies (Lipoptena mazamae) in the neotropics collected from gray brocket deer ( Mazama gouazoubira ) in Southern Brazil. DNA from Bartonella sp. was detected in all 429 L. mazamae collected from 11 road-killed gray brocket deer. The same sequences of DNA of Bartonella spp. were identified in samples. Gray brocket deer are abundant in Brazil, so Bartonella-infected Lipoptena spp. may be widely distributed in the neotropics.

Nasal swab real-time PCR is not suitable for in vivo diagnosis of bovine tuberculosis / A PCR em tempo real de swab nasal não é adequada para o diagnóstico in vivo de tuberculose bovina

Bovine tuberculosis (bTB) is a zoonosis causing economic losses and public health risks in many countries. The disease diagnosis in live animals is performed by intradermal tuberculin test, which is based on delayed hypersensitivity reactions. As tuberculosis has complex immune response, this test has limitations in sensitivity and specificity. This study sought to test an alternative approach for in vivo diagnosis of bovine tuberculosis, based on real-time polymerase chain reaction (PCR). DNA samples, extracted from nasal swabs of live cows, were used for SYBR® Green real-time PCR, which is able to differentiate between Mycobacterium tuberculosis and Mycobacterium avium complexes. Statistical analysis was performed to compare the results of tuberculin test, the in vivo gold standard bTB diagnosis method, with real-time PCR, thereby determining the specificity and sensitivity of molecular method. Cervical comparative test (CCT) was performed in 238 animals, of which 193 had suitable DNA from nasal swabs for molecular analysis, as indicated by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and were included in the study. In total, 25 (10.5%) of the animals were CCT reactive, of which none was positive in the molecular test. Of the 168 CCT negative animals, four were positive for M. tuberculosis complex at real time PCR from nasal swabs. The comparison of these results generated values of sensitivity and specificity of 0% and 97.6%, respectively; moreover, low coefficients of agreement and correlation (-0.029 and -0.049, respectively) between the results obtained with both tests were also observed. This study showed that real-time PCR from nasal swabs is not suitable for in vivo diagnosis of bovine tuberculosis; thus tuberculin skin test is still the best option for this purpose.(AU)

A tuberculose bovina (bTB) é uma zoonose que causa perdas econômicas e riscos à saúde pública em muitos países. O diagnóstico da doença em animais vivos é realizado pelo teste intradérmico da tuberculina, que é baseado em reações de hipersensibilidade tardia. Como a tuberculose tem resposta imunológica complexa, este teste tem limitações em termos de sensibilidade e especificidade. Este estudo procurou desenvolver uma abordagem alternativa para o diagnóstico in vivo da tuberculose bovina, com base na reação em cadeia da polimerase (PCR) em tempo real. As amostras de DNA, extraídas de suabes nasais de vacas vivas, foram usadas para PCR em tempo real com SYBR® Green, capaz de diferenciar os complexos Mycobacterium tuberculosis e Mycobacterium avium. A análise estatística foi realizada para comparar os resultados de teste de tuberculina, padrão ouro para o diagnóstico in vivo da bTB, com PCR em tempo real, determinando-se assim a especificidade e sensibilidade do método molecular. O teste cervical comparativo (TCC) foi realizado em 238 animais, dos quais 193 tiveram DNA dos suabes nasais adequados para análise molecular, como indicado pela amplificação do gene gliceraldeído-3-fosfato-desidrogenase (GAPDH), e foram incluídos no estudo. No total, 25 (10,5%) animais foram reativos no TCC, dos quais nenhum foi positivo no teste molecular. Dos 168 animais negativos no TCC, quatro foram positivos para o complexo M. tuberculosis na PCR em tempo real a partir dos suabes nasais. A comparação destes resultados gerou valores de sensibilidade e especificidade de 0% e 97,6%, respectivamente; além disso, baixos coeficientes de concordância e correlação (-0,029 e -0,049, respectivamente) entre os resultados obtidos com ambos os testes também foram observados. Este estudo mostrou que a PCR em tempo real a partir de suabes nasais não é adequada para o diagnóstico in vivo da tuberculose bovina; portanto, o teste da tuberculina ainda é a melhor opção para este fim.(AU)