Molecular detection and characterization of vector-borne agents in common opossums (Didelphis marsupialis) from northeastern Brazil.

Opossums are synanthropic marsupials able to interchange among wild, peri­urban and urban environments, playing an epidemiologically important role as hosts for emerging pathogens and ectoparasites of relevance in public health. The present study aimed to detect and molecularly characterize vector-borne agents in a population of common opossums (Didelphis marsupialis) from the Island of São Luís do Maranhão, northeastern Brazil. Of the 45 animals analyzed, one (2.22%) was positive in the nested PCR assay based on the 18S rRNA gene of piroplasmids. The obtained sequence was phylogenetically positioned in a clade containing sequences of Babesia sp. previously detected in Didelphis aurita, Didelphis albiventris and associated ticks from Brazil. Eight (17.77%) samples were positive in PCR for Ehrlichia spp. based on the dsb gene; four samples were sequenced and positioned into a new clade, sister to E. minasensis and Ehrlichia sp. clade detected in Superorder Xenarthra mammals. No samples tested positive in the screening PCR assays based on the 16S rRNA gene of Anaplasma spp. Two samples were positive in the qPCR for Bartonella spp. based on the nuoG gene. Seven animals (15.56%) were positive in the nPCR based on the 16S rRNA gene of hemoplasmas. Of these, three were positive in a PCR based on the 23S rRNA gene. The phylogenies based on both 16S rRNA and 23S rRNA genes corroborated to each other and positioned the sequences in the same clade of hemoplasmas previously detected in D. aurita and D. albiventris sampled in Brazil. Finally, three (6.66%) animals were positive in the PCR for Hepatozoon spp.; the obtained 18S rRNA sequence was positioned into the H. felis clade.The present study showed, for the first time, the circulation of piroplasmids, Hepatozoon spp., Ehrlichia spp., hemoplasmas and Bartonella spp. in D. marsupialis sampled in northeastern Brazil, with description of putative novel genotypes of Ehrlichia and Hepatozoon and copositivity by different vector-borne agents. The present work consolidates the "South American Marsupialia" piroplasmid clade, adding one more genotype of Babesia sp. to this clade.

Detection of Rickettsia tamurae-like and other spotted fever group rickettsiae in ticks (Acari: Ixodidae) associated with wild birds in the Western Amazon, Brazil.

Ticks are vectors for several pathogens, including bacteria belonging to the Rickettsia genus, such as Rickettsia rickettsii and Rickettsia parkeri, the causative agents of spotted fever. The aim of the present study was to investigate the tick species richness and rickettsial agents associated with wild birds captured in the Humaita Forest Reserve, Acre, in the Western Amazon region. Wild birds were captured with ornithological nets for visual inspection with the purpose of collecting ticks, which were identified through morphological analyses and molecular tests for several genes (12S rDNA, 16S rDNA, gltA, ompA, and sca4). A total of 607 wild birds were captured, 12% of which were parasitized by 268 ticks of the Amblyomma genus, with new host-parasite associations reported for Amblyomma calcaratum, Amblyomma geayi, Amblyomma longirostre, Amblyomma naponense, Amblyomma nodosum, and Amblyomma varium. Of the total ticks collected, 113 were tested for the presence of rickettsial DNA fragments, with 19 testing positive for R. parkeri in A. geayi, Rickettsia tamurae-like in Amblyomma sp., and Rickettsia amblyommatis in A. geayi, A. longirostre, and Amblyomma sp. We detected R. tamurae-like in Amblyomma larvae for the first time in the Western Brazilian Amazon biome, and registered spotted fever group rickettsiae, although the relevance of the detected species in a public health context should be further explored in South America, as well as new host-parasite interactions in this underexplored region.

Exposure to and infection by Leishmania infantum among domestic dogs in an area of the Cerrado biome, Maranhão, Brazil.

Visceral Leishmaniasis is a serious public health problem and dogs are considered to be the main source of infection in urban areas. In Brazil, this disease is present in all regions, but with high concentration of cases in the Northeast, and the state of Maranhão is considered to be an endemic region. The aim of this study was to conduct an epidemiological, spatial, molecular and serological survey on Leishmania infantum among domestic dogs in the municipality of Belágua, Maranhão. Blood samples were collected from dogs and questionnaires were applied to their owners to obtain epidemiological data and risk factors relating to this zoonosis in the region. The coordinates of the dogs' homes were obtained to produce a disease risk map. Serological diagnoses were made using the indirect immunofluorescence reaction (IFAT) and the dual-path platform chromatographic immunoassay test (DPP®) (Bio-Manguinhos/FIOCRUZ, Brazil). A molecular investigation was undertaken using the polymerase chain reaction (PCR). Georeferencing was performed using the global positioning system (GPS) and cases of canine visceral leishmaniasis in the municipality were spatially represented and analyzed using QGIS version 3.16.6 (QGIS Development Team, 2021). A total of 205 blood samples were collected, of which 122 (59.51%) were seroreactive for L. infantum through IFAT, while the DPP test showed 84 reactive samples (40.97%). IFAT and DPP detected 16 positive animals simultaneously. One sample that was seroreactive through IFAT was also positive through PCR. In the clinical evaluation, it was observed that among the seropositive dogs, 112 (91.80%) were symptomatic and 10 (8.20%) were asymptomatic. In the spatial analysis, the Kernel density estimator enabled determination of the place at greatest risk of occurrence of the disease. The areas with the highest concentrations of cases were in districts with large quantities of precarious housing and lack of basic sanitation. This was the first report on the occurrence of L. infantum among dogs in the municipality of Belágua. The results show that canine visceral leishmaniasis is well dispersed in this municipality, thus putting at risk the human population.

Litomosoides brasiliensis (Nematoda: Onchocercidae) infecting chiropterans in the Legal Amazon region, Brazil.

Chiropterans play an important role in the maintenance of the environmental balance, since they are pollinators, seed dispersers and predators. They contribute to transmission and spreading of microorganisms such as helminths, fungi, protozoa, bacteria and virus. The aim of the present study was to investigate natural filariid infection among bats in the Legal Amazon region, Brazil, by means of parasitological and molecular analyses. Blood samples were collected from 82 bats for blood smears and for DNA extraction via the polymerase chain reaction (PCR) assay. Microfilariae were observed in blood smears from Carollia perspicillata (2), Artibeus lituratus (1), Artibeus fimbriatus (2), Dermanura gnoma (2) and Glossophaga soricina (1). Five positive samples were detected through the PCR assay and four of these were also positive in blood smears. From genome sequencing and comparative analysis with sequences deposited in GenBank, one sample showed 99.31% similarity to the species Litomosoides brasiliensis. The present study expands the geographical distribution of L. brasiliensis, to include the state of Maranhão as an area of occurrence of this species and includes D. gnoma and A. fimbriatus as hosts in Brazil.

Chiropterans as a potential hosts of Leishmania spp. in endemic areas for leishmaniasis in northeastern Brazil.

The species of the genus Leishmania are protozoa that are widely distributed from Asia to the Americas, affecting humans and wild and domestic animals. Little is known about infection by Leishmania in bats in the state of Maranhão, Brazil. The objective of this study was to investigate the presence of Leishmania in bats in Maranhão. Blood samples were collected from bat species for parasitological diagnosis. Samples of spleen and liver were collected for molecular analysis. All the blood cultures were negative. In two blood smears, organisms similar to amastigotes of Leishmania sp. were detected. Of the 116 samples, two spleen samples were positive and showed similarity to Leishmania infantum. Therefore, further studies are needed to elucidate whether bats take part in the epidemiological chain of leishmaniasis.

Rickettsia amblyommatis-infected Amblyomma coelebs parasitizing a human traveler in Rio Grande do Sul, southern Brazil, after returning from the Amazon.

With increased national and international transportation, humans as well as domestic and wild animals are able to get from one end to the other within hours, accidently carrying transboundary vectors and pathogens. We report here, the introduction of Amblyomma coelebs in Rio Grande do Sul, the southernmost state of Brazil, via a man travelling to the Carajás National Forest in the state of Pará (2661 km away). Upon return to his residence in Porto Alegre, RS, he realized that he was parasitized by one tick. The tick was identified as a nymph of Amblyomma sp., placed in a Biological Oxygen Demand incubator, and after ecdysis was identified as a male of Amblyomma coelebs. The tick's DNA was screened for Rickettsia spp., where Rickettsia amblyommatis DNA was detected. The parasitized man was seropositive to spotted fever group rickettsiae, by immunofluorescence assay, in a single serum sample collected 45 days after the tick-bite. Amblyomma coelebs is found in some Brazilian states, but Parana is the southernmost state in Brazil where it occurs. This species has already been reported to carry some species of Rickettsia, including species of the spotted fever group. It may risk public health if established in Rio Grande do Sul, with the introduction of tick and pathogenic rickettsias.

Novel genotypes of Hepatozoon spp. in small mammals, Brazil.

BACKGROUND: Small mammals (rodents and marsupials) have been poorly explored for the occurrence of apicomplexan (genus Hepatozoon and genera of the order Piroplasmorida) and Anaplasmataceae agents in Brazil. Thus, this study investigated the occurrence of Hepatozoon spp., Piroplasmorida, and Anaplasmataceae agents in small mammals in seven forest fragments in Brazil. METHODS: During 2015-2018, small mammals were captured in six forest fragments in the State of São Paulo (Cerrado and Atlantic Forest biomes) and one fragment in the State of Mato Grosso do Sul (Pantanal biome). Mammal blood, liver, spleen, and lung samples were tested molecularly for the presence of DNA of Hepatozoon, Piroplasmorida, and Anaplasmataceae agents. RESULTS: A total of 524 mammals were captured, comprising seven species of marsupials, 14 rodents, two carnivores, and one Cingulata. Four novel haplotypes (1, 2, 3, 4) of Hepatozoon spp. were detected in small mammals from different biomes. In São Paulo state, haplotype 1 was detected in rodents from Cerrado and a transition area of Cerrado and Atlantic Forest biomes, whereas haplotype 2 was detected in rodents from the Atlantic Forest biome. On the other hand, haplotypes 3 and 4 were restricted to rodents and marsupials, respectively, from the Pantanal biome of Mato Grosso do Sul. No host species shared more than one haplotype. Despite these distinct geographical and host associations, our phylogenetic analyses indicated that the four Hepatozoon haplotypes belonged to the same clade that contained nearly all haplotypes previously reported on rodents and marsupials, in addition to several reptile-associated haplotypes from different parts of the world. No mammal samples yielded detectable DNA of Piroplasmorida agents. On the other hand, the Anaplasmataceae-targeted polymerase chain reaction (PCR) assay amplified a sequence 100% identical to the Wolbachia pipientis endosymbiont of the rodent filarid Litomosoides galizai. CONCLUSIONS: We report a variety of Hepatozoon haplotypes associated with small mammals in three Brazilian biomes: Cerrado, Atlantic Forest, and Pantanal. Through phylogenetic analyses, the Hepatozoon agents grouped in the rodent-marsupial-reptile large clade of Hepatozoon spp. from the world. The detection of a W. pipientis associated with the rodent filarid L. galizai indicates that the rodent was infected by filarial nematodes.

Seroprevalence and detection of Trypanosoma cruzi in dogs living in a non-endemic area for Chagas disease in the legal Amazon region, Brazil.

Trypanosoma cruzi, the etiological agent for Chagas disease, is widely distributed in the Americas. Its hosts are humans and wild and domestic mammals, and its vectors are triatomine insects. Studies have indicated that domestic dogs are sentinel animals in the epidemiology of Chagas disease in endemic regions, including states in the Legal Amazon region of Brazil. In São Luís, the capital of Maranhão, a non-endemic state, the existence of a domestic cycle involving domestic rats has been proven, along with a wild cycle maintained by didelphids. However, no studies on T. cruzi infection in domestic animals in this locality have been conducted. The aim of this study was to investigate occurrence of T. cruzi in dogs living in the Itaqui Bacanga district of São Luís, Maranhão, by means of serological and molecular tests. Blood samples were obtained from 330 dogs and structured epidemiological questionnaires were applied to their keepers. These samples were used in the indirect immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Fisher's exact test was used for statistical calculations with the aim of identifying risk factors. Out of the 330 animals, 105 (31.8%) were reactive in IFAT, 46 (13.0%) in ELISA and 20 (6.0%) in both serological tests. The results were not significant (p > 0.05) when submitted to statistical analysis for the studied variables. From PCR, 58 samples (17.5%) were found to be positive and, of these, one (0.3%) showed similarity to T. cruzi after sequencing. These data demonstrate that dogs were exposed to and infected by T. cruzi. Thus, they can be considered sentinel animals for Chagas disease in the locality studied, which signals that there is a need for epidemiological surveillance actions.

High level of infection by Toxoplasma gondii in pigs slaughtered in the city of São Luís, Maranhão.

A serological, molecular and histopathological study was carried out in order to investigate occurrences of Toxoplasma gondii in pigs slaughtered with and without inspection service. Serum samples were collected from 60 pigs to detect anti-T. gondii antibody by indirect fluorescent antibody (IFAT). Tongue, masseter and diaphragm fragments were also collected for parasite DNA detection by means of the polymerase chain reaction (PCR) and histopathological analysis. The serological results showed that 77% (44/60) of the pigs were positive. Regarding PCR, 66.67% (40/60) were positive for T. gondii. Among the tissues evaluated, the diaphragm was the one with the highest frequency of positivity (40%; 24/60), followed by the masseter (38.33%; 23/60) and tongue (33.3%; 20/60). Histopathological changes were only observed in the diaphragm, which presented inflammatory infiltrates of lymphohistiocytic and neutrophilic types. These results not only show the potential threat of T. gondii to human health, but also demonstrate the dynamic epidemiological situation of toxoplasmosis in pigs in the city of São Luís, providing support for food security regarding pigs and for T. gondii control programs in Brazil.

Endoparasites of capybaras (Hydrochoerus hydrochaeris) from anthropized and natural areas of Brazil.

This study aimed to evaluate the occurrence of endoparasites in fecal samples of capybaras from anthropized areas in seven cities in the state of São Paulo, southeastern Brazil, and natural areas in two locations in the Pantanal biome. For the parasitological evaluation of samples, feces were subjected to centrifugal sedimentation in water-ether and centrifugal flotation in sucrose solution. Out of the 113 samples examined, 97.3% (110/113) were positive for the presence of helminth eggs and/or protozoan oocysts, with 96.7% (89/92) and 100% (21/21) in capybaras from anthropized and natural areas, respectively. Helminth eggs belonging to the Class Cestoda (Monoecocestus spp.), Class Digenea, and Class Nematoda (Trichostrongyloidea, Strongyloides chapini, Protozoophaga obesa, and Capillaria hydrochoeri) were detected. Coccidia oocysts of Eimeria spp. and Cryptosporidium spp. were detected. The difference found was in the occurrence of S. chapini between anthropized and natural areas, as S. chapini was not detected in natural areas. Capybaras from anthropized areas showed a greater species richness of endoparasites, including species such as Fasciola hepatica and Cryptosporidium spp., that might have zoonotic potential.

High level of infection by Toxoplasma gondii in pigs slaughtered in the city of São Luís, Maranhão / Alto nível de infecção por Toxoplasma gondii em suínos abatidos na cidade de São Luís, Maranhão

Abstract A serological, molecular and histopathological study was carried out in order to investigate occurrences of Toxoplasma gondii in pigs slaughtered with and without inspection service. Serum samples were collected from 60 pigs to detect anti-T. gondii antibody by indirect fluorescent antibody (IFAT). Tongue, masseter and diaphragm fragments were also collected for parasite DNA detection by means of the polymerase chain reaction (PCR) and histopathological analysis. The serological results showed that 77% (44/60) of the pigs were positive. Regarding PCR, 66.67% (40/60) were positive for T. gondii. Among the tissues evaluated, the diaphragm was the one with the highest frequency of positivity (40%; 24/60), followed by the masseter (38.33%; 23/60) and tongue (33.3%; 20/60). Histopathological changes were only observed in the diaphragm, which presented inflammatory infiltrates of lymphohistiocytic and neutrophilic types. These results not only show the potential threat of T. gondii to human health, but also demonstrate the dynamic epidemiological situation of toxoplasmosis in pigs in the city of São Luís, providing support for food security regarding pigs and for T. gondii control programs in Brazil.

Resumo Realizou-se um estudo sorológico, molecular e histopatológico com o objetivo de verificar a ocorrência de Toxoplasma gondii em suínos abatidos com e sem serviço de inspeção. Foram coletados soros de 60 suínos para a pesquisa de anticorpos anti-T. gondii pela reação de imunofluorescência indireta (RIFI). Também foram coletados fragmentos de língua, masseter e diafragma para a detecção do DNA do parasito por meio da reação em cadeia da polimerase (PCR) e análise histopatológica. A análise sorológica demonstrou que 77% (44/60) dos suínos apresentaram anticorpos anti-T. gondii. Com relação ao PCR, 66,67% (40/60) foram positivos para T. gondii. Dentre os tecidos avaliados, o diafragma foi o que obteve maior frequência de positividade (40%; 24/60), seguidos de masseter (38,33%; 23/60) e língua (33,3%; 20/60). Alterações histopatológicas foram observadas apenas no diafragma, que apresentou infiltrado inflamatório do tipo linfohistiocitário e neutrofílico. Esses resultados não evidenciam apenas a ameaça potencial de T. gondii à saúde humana, mas também demonstram a dinâmica situação epidemiológica da toxoplasmose em suínos na região da cidade de São Luís, fornecendo suporte para a segurança alimentar de suínos e programas de controle de T. gondii no país.

Coxiella burnetii in slaughterhouses in Brazil: A public health concern.

Q fever is an important zoonosis, yet it is often neglected and can present large outbreaks, as observed in the Netherlands. In the past few years, cases of Q fever have been described in Brazil; however, the epidemiological situation of Q fever in ruminants, the main reservoir of the pathogen, is unknown in this country. Our study aimed to estimate the prevalence of C. burnetii in cattle sent to slaughterhouses using an immunofluorescence assay (IFA) and quantitative real-time PCR (qPCR). From 1515 cattle serum samples collected from nine slaughterhouses, 23.8% (360/1515) were serologically positive by IFA (cutoff titer>1:64), indicating past or recent exposure to C. burnetii infection. Among the 54 cities sampled during the study, 83.3% (45/54) had at least one seropositive animal. Subsequently, all seropositive samples were submitted to qPCR for C. burnetii DNA, and 12.2% (44/360) of the sera were qPCR positive, which indicates bacteremia and suggests active or recent infection. The results highlight the risk for abattoir workers that results from exposure to contaminated aerosols produced during slaughter procedures. Moreover, the heat maps that were construction from the positive samples demonstrate the widespread distribution of C. burnetii in the State of São Paulo, Brazil and denotes the need for surveillance and preventive measures to reduce the prevalence in cattle.

Occurrence of Bartonella genotypes in bats and associated Streblidae flies from Maranhão state, northeastern Brazil.

Bartonella is a genus of emerging zoonotic bacteria that are mainly associated with mammalian erythrocytes and endothelial cells. Bats are natural reservoirs for a variety of important pathogens that impact human and animal health. Recent reports have highlighted the role of bats and bat flies in the maintenance of Bartonella. Here, we showed that none of the 29 bat DNA blood samples obtained from five bat species in São Luís Island, state of Maranhão, northeastern Brazil, were positive for Bartonella in qPCR assays targeting nuoG. On the other hand, three out of 15 DNA samples (20%) from flies in the family Streblidae were positive for Bartonella. The BLASTn results showed that the gltA and rpoB sequences shared identities ranging from 97.2% to 100%, with Bartonella sequences amplified from bats or bat flies from Costa Rica and Brazil. These findings were supported by phylogenetic analyses based on Bayesian inferences. The present study showed that Bartonella genotypes are present in bat flies, thus shedding some light on the distribution of bat fly-related Bartonella genotypes in South America.

Retrospective and new records of ticks (Acari: Argasidae, Ixodidae) from the state of Maranhão, an Amazon-Cerrado transition area of Brazil.

The Brazilian state of Maranhão is located in a transition area of the Amazon and Cerrado biomes, where there is a rich fauna of vertebrates. This study aimed to update the list of the ticks occurring in Maranhão, through a compilation of literature records and examination of three tick collections, plus the addition of unpublished collections of ticks from road-killed animals during recent years. Our results indicate that the tick fauna of Maranhão includes 26 species: seven in the family Argasidae (Antricola guglielmonei, Argas miniatus, Ornithodoros cavernicolous, Ornithodoros hasei, Ornithodoros mimon, Ornithodoros rietcorreai and Ornithodoros rudis); and 19 in the family Ixodidae (Amblyomma auricularium-provisional, Amblyomma cajennense sensu stricto, Amblyomma calcaratum, Amblyomma dissimile, Amblyomma geayi, Amblyomma longirostre, Amblyomma naponense, Amblyomma nodosum, Amblyomma oblongoguttatum, Amblyomma ovale, Amblyomma parvum, Amblyomma rotundatum, Amblyomma sculptum, Amblyomma triste, Dermacentor nitens, Ixodes luciae, Haemaphysalis juxtakochi, Rhipicephalus microplus, and Rhipicephalus sanguineus sensu lato). Eleven of the above species are reported for the first time in Maranhão. We consider previous reports of Ornithodoros talaje and Amblyomma pseudoconcolor in Maranhão as misidentification with O. hasei and A. auricularium, respectively. Until 1958, only 11 tick species were reported in Maranhão, with no additional reports until 2009. During 2010-2019, 15 additional species were reported. We also highlight the importance of ticks for public and veterinary health in Maranhão.

Ticks biting humans in the Brazilian savannah: Attachment sites and exposure risk in relation to species, life stage and season.

Information about human tick bites in Brazil is mostly anecdotal. Published information is typically restricted to single tick infestation episodes and does not address human exposure occurring on a daily basis in natural, rural, or green urban areas. We present a comprehensive 2-yr study on human parasitism during a broad survey on ticks within a Brazilian savannah reserve. Overall, 439 tick bites were recorded from the following species: Amblyomma sculptum (n = 331 bites), Amblyomma parvum (n = 64), Amblyomma auricularium (n = 3), Rhipicephalus microplus (n = 2), Dermacentor nitens (n = 1) and 38 Amblyomma spp. ticks. Nymphs accounted for most tick bites (n = 292, 66.5 % of all bites) and these were overwhelmingly A. sculptum (92.8 % of nymphal bites). The main adult tick biting humans was A. parvum (n = 61, 50 % of all adult tick bites) followed closely by A. sculptum (n = 58, 47.5 % of all adult tick bites). Winter was the season with the highest percentage of tick bites (39.7 % of all bites), followed by spring (32.4 %); summer (18.9 %) and autumn (9 %). Amblyomma sculptum adult bites peaked in summer whereas nymphal bites occurred primarily in winter and spring. Amblyomma parvum adult bites peaked in spring and summer. The most common tick attachment sites included the waist, legs and belly, but A. parvum adults were recorded from the head of humans as well. A noteworthy observation was the lack of human parasitism by Amblyomma triste, the third most prevalent species in the environment. This tick species is a frequent human biter in both Uruguay and Argentina where it transmits a pathogen, Rickettsia parkeri, to humans.

Occurrence of Bartonella genotypes in bats and associated Streblidae flies from Maranhão state, northeastern Brazil / Ocorrência de genótipos de Bartonella em morcegos e moscas Streblidae no Estado do Maranhão, Nordeste do Brasil

Abstract Bartonella is a genus of emerging zoonotic bacteria that are mainly associated with mammalian erythrocytes and endothelial cells. Bats are natural reservoirs for a variety of important pathogens that impact human and animal health. Recent reports have highlighted the role of bats and bat flies in the maintenance of Bartonella. Here, we showed that none of the 29 bat DNA blood samples obtained from five bat species in São Luís Island, state of Maranhão, northeastern Brazil, were positive for Bartonella in qPCR assays targeting nuoG. On the other hand, three out of 15 DNA samples (20%) from flies in the family Streblidae were positive for Bartonella. The BLASTn results showed that the gltA and rpoB sequences shared identities ranging from 97.2% to 100%, with Bartonella sequences amplified from bats or bat flies from Costa Rica and Brazil. These findings were supported by phylogenetic analyses based on Bayesian inferences. The present study showed that Bartonella genotypes are present in bat flies, thus shedding some light on the distribution of bat fly-related Bartonella genotypes in South America.

Resumo Bartonella é um gênero de bactérias zoonóticas emergentes associadas principalmente a eritrócitos e células endoteliais de mamíferos. Morcegos são reservatórios naturais para uma variedade de patógenos importantes que afetam a saúde humana e animal. Além disso, estudos recentes destacaram o papel dos morcegos e de moscas associadas a morcegos na manutenção de Bartonella. No presente estudo, nenhuma das 29 amostras de DNA obtidas a partir do sangue de cinco espécies de morcegos amostrados na ilha de São Luís, estado do Maranhão, Nordeste do Brasil, foi positiva para Bartonella nos ensaios de qPCR direcionados ao gene nuoG. Por outro lado, três das 15 (20%) amostras de DNA de moscas da família Streblidae foram positivas para Bartonella. Os resultados do BLASTn mostraram que as sequências dos genes gltA e rpoB compartilharam identidade, variando de 97,2% a 100%, com as sequências de Bartonella amplificadas em morcegos ou moscas amostrados na Costa Rica ou Brasil. Tais resultados corroboraram as análises filogenéticas realizadas por Inferência Bayesiana. O presente estudo mostrou a ocorrência de Bartonella em moscas de morcegos, auxiliando a esclarecer a distribuição dos genótipos de Bartonella relacionadas a moscas Streblidae na América do Sul.

Diagnosis and epidemiology of Leishmania infantum in domestic cats in an endemic area of the Amazon region, Brazil.

Visceral leishmaniasis is a zoonotic disease caused by Leishmania infantum for which dogs are the main reservoir. In South America, presence of this disease is expanding along with increasing dispersion of its principal vector, the sand-fly Lutzomyia longipalpis. Feline leishmaniasis is an emerging disease in domestic cats, but epidemiological studies in endemic areas of the Amazon region of Brazil are scarce and the role of cats as reservoirs of L. infantum has been debated. The aim of this study was to investigate L. infantum infection in cats living in the Amazon biome region, using serological and molecular methods. A total of 105 cats were subjected to clinical examination and blood samples were taken for immunofluorescent-antibody (IFAT) serological evaluation, to determine anti-Leishmania antibody titers. Conventional PCR and Sanger's sequencing targeting L. infantum chitinase and Leishmania species ribosomal internal transcribed spacer (ITS-1) encoding genes were performed on conjunctival swabs from these cats. Seropositivity was detected in 32 animals (30.48%), thus confirming that contact between these cats and the parasite was occurring. PCR followed by amplicon sequencing showed that three samples (2.86%) were positive for a chitinase gene and six (5.71%) were positive for the ITS-1 gene. Parasite-positive diagnoses presented a statistically significant association with free access to the streets (p = 0.0111), cohabitation with dogs affected previously by VL (p = 0.0006) and absence of backyard cleaning and garbage collection (p = 0.00003). These results emphasize that cats should be included in epidemiological surveys of leishmaniasis, especially in endemic areas, if not as the reservoir host (unproven), at least as a "sentinel host" that is useful for revealing situations of endemic circulation of L. infantum. Moreover, in these areas, feline leishmaniasis needs to be considered in the differential diagnosis among domestic cats presenting alopecia, rarefied hair, lacerations and ulcerative dermatitis.

Amblyomma sculptum Salivary PGE2 Modulates the Dendritic Cell-Rickettsia rickettsii Interactions in vitro and in vivo.

Amblyomma sculptum is an important vector of Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever and the most lethal tick-borne pathogen affecting humans. To feed on the vertebrate host's blood, A. sculptum secretes a salivary mixture, which may interact with skin resident dendritic cells (DCs) and modulate their function. The present work was aimed at depicting the A. sculptum saliva-host DC network and the biochemical nature of the immunomodulatory component(s) involved in this interface. A. sculptum saliva inhibits the production of inflammatory cytokines by murine DCs stimulated with LPS. The fractionation of the low molecular weight salivary content by reversed-phase chromatography revealed active fractions eluting from 49 to 55% of the acetonitrile gradient. Previous studies suggested that this pattern of elution matches with that observed for prostaglandin E2 (PGE2) and the molecular identity of this lipid mediator was unambiguously confirmed by a new high-resolution mass spectrometry methodology. A productive infection of murine DCs by R. rickettsii was demonstrated for the first time leading to proinflammatory cytokine production that was inhibited by both A. sculptum saliva and PGE2, a result also achieved with human DCs. The adoptive transfer of murine DCs incubated with R. rickettsii followed by treatment with A. sculptum saliva or PGE2 did not change the cytokine profile associated to cellular recall responses while IgG2a-specific antibodies were decreased in the serum of these mice. Together, these findings emphasize the role of PGE2 as a universal immunomodulator of tick saliva. In addition, it contributes to new approaches to explore R. rickettsii-DC interactions both in vitro and in vivo.

Rickettsia spp. among wild mammals and their respective ectoparasites in Pantanal wetland, Brazil.

The genus Rickettsia comprises obligatory intracellular bacteria, well known to cause zoonotic diseases around the world. The present work aimed to investigate the occurrence of Rickettsia spp. in wild animals, domestic dogs and their respective ectoparasites in southern Pantanal region, central-western Brazil, by molecular and serological techniques. Between August 2013 and March 2015, serum, whole blood and/or spleen samples were collected from 31 coatis, 78 crab-eating foxes, seven ocelots, 42 dogs, 110 wild rodents, and 30 marsupials. Serum samples from canids, felids, rodents and marsupials were individually tested by indirect fluorescent antibody test (IFAT) in order to detect IgG antibodies to Rickettsia rickettsii, Rickettsia parkeri and Rickettsia amblyommatis. DNA samples from mammals and ectoparasites were submitted to a multiplex qPCR assay in order to detect and quantify spotted fever group (SFG) and typhus group (TG) rickettsiae and Orientia tsutsugamushi. Positive samples in qPCR assays were submitted to conventional PCR assays targeting gltA, ompA, ompB and htrA genes, followed by sequencing and phylogenetic analyses. The ticks collected (1582) from animals belonged to the species Amblyomma sculptum, Amblyomma parvum, Amblyomma ovale, Amblyomma tigrinum, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus sensu lato and Amblyomma auricularium. Overall, 27 (64.2%) dogs, 59 (75.6%) crab-eating foxes and six (85.7%) ocelots were seroreactive (titer≥64) to at least one Rickettsia species. For 17 (40.4%) dogs, 33 (42.3%) crab-eating foxes, and two (33.3%) ocelots, homologous reactions to R. amblyommatis or a closely related organism were suggested. One hundred and sixteen (23.5%) tick samples and one (1.2%) crab-eating fox blood sample showed positivity in qPCR assays for SFG Rickettsia spp. Among SFG Rickettsia-positive ticks samples, 93 (80.2%) belonged to A. parvum, 14 (12%) belonged to A. sculptum species, three (2.5%) belonged to A. auricularim, and six (5.2%) were Amblyomma larval pools. Thirty samples out of 117 qPCR positive samples for SFG Rickettsia spp. also showed positivity in cPCR assays based on gltA, htrA and/or ompB genes. The Blast analyses showed 100% identity with 'Candidatus Rickettsia andeanae' in all 30 sequences obtained from gltA, htrA and/or ompB genes. The concatenated phylogenetic analysis based on gltA and 17-kDa htrA genes grouped the Rickettsia sequences obtained from tick samples in the same clade of 'Candidatus Rickettsia andeanae'. The present study revealed that wild and domestic animals in southern Pantanal region, Brazil, are exposed to SFG rickettsiae agents. Future studies regarding the pathogenicity of these agents are necessary in order to prevent human cases of rickettsiosis in Brazilian southern Pantanal.

Exposure of Baixadeiro horses to Rickettsia spp. and to ticks infected by Rickettsia amblyommatis in the Baixada Maranhense micro-region, Maranhão, Brazil / Exposição dos cavalos Baixadeiro a Rickettsia spp. e carrapatos infectados pela Rickettsia amblyommatis na Baixada Maranhense, microrregião do Estado do Maranhão, Brasil

ABSTRACT: The aim of this study was to investigate exposure of Baixadeiro horses to Rickettsia spp. and to ticks infected by Rickettsia in the Baixada Maranhense (lowlands) micro-region, state of Maranhão. A total of 258 horses were tested for Rickettsia rickettsii, Rickettsia amblyommatis and Rickettsia bellii using the immunofluorescence assay (IFA). Overall, 58.91% (152/258) of the horses were seroreactive for at least one Rickettsia species, and 85.27% of the horses were infested with one or more species of tick, which were identified as Dermacentor nitens (93.63%), Amblyomma cajennense sensu stricto (4.55%) and Rhipicephalus (Boophilus) microplus (1.82%). These ticks were subjected to DNA extraction and were tested using the polymerase chain reaction (PCR), targeting two rickettsia genes: citrate synthase gene (gltA) and 190kDa outer membrane protein gene (ompA). Three specimens of A. cajennense s.s. were positive. BLAST analyses on the nucleotide sequences obtained from the PCR products showed that these were 99-100% identical to the corresponding sequences of R. amblyommatis. Thus, results indicate that R. amblyommatis and/or a strain very close to this is circulating in ticks in this micro-region.

RESUMO: O objetivo deste estudo foi verificar a exposição e a infecção de cavalos baixadeiro e carrapatos por Rickettsia spp. na micro-região da Baixada Maranhense do Estado Maranhão. Um total de 258 cavalos foi testado pelo ensaio de Imunofluorescência Indireta (IFI) para Rickettsia rickettsii, Rickettsia amblyommatis e Rickettsia bellii. Deste total, 58,91% (152/258) foram sororreativos para pelo menos uma espécie de Rickettsia sp., e 85,27% cavalos estavam infestados por uma ou mais espécies de carrapatos identificados como Dermacentor nitens (93,63%), Amblyomma cajennense sensu stricto (4,55%) e Rhipicephalus (Boophilus) microplus (1,82%). Estes carrapatos foram submetidos à extração de DNA e testados pela reação em cadeia pela polimerase (PCR) alvejando os genes citrato sintase (gltA) e o de proteína de membrana externa de 190kDa (ompA). Três espécimes de A. cajennense s.s. foram positivos para Rickettsia amblyommatis. As sequências dos nucleotídeos obtidas a partir dos produtos de PCR mostraram 99-100% de identidade com as sequências correspondentes de R. amblyommatis quando analisadas pelo BLAST. Desta forma, os resultados indicam a circulação de R. amblyommatis nos carrapatos e/ou uma cepa muito próxima circulando na microrregião.