Molecular and serological detection of Anaplasma spp. in small ruminants in an area of Cerrado Biome in northeastern Brazil.

Anaplasmosis, caused by bacteria of the genus Anaplasma, is an important tick-borne disease that causes economic losses to livestock farms in many countries. Even though Anaplasma spp. have been detected in goats and sheep worldwide, few studies investigate the occurrence and genetic identity of these agents in small ruminants from Brazil. Thus, this work aimed to detect and determine the genetic identity of Anaplasma spp. in small ruminants from the Baixo Parnaíba region, state of Maranhão, northeastern Brazil. For this purpose, blood samples were collected from 161 animals (91 goats; 70 sheep) from 4 municipalities in the Baixo Parnaíba region. Sheep and goat serum samples were subjected to recombinant membrane surface protein (MSP5)-based iELISA. Whole blood samples were subject to DNA extraction and molecular diagnosis using PCR assays for Anaplasma spp. targeting msp1ß, msp1α, 16S rRNA and msp4 genes. Positive samples were sequenced and then subjected to Anaplasma marginale msp1α genetic diversity analysis and phylogenetic inferences based on the 16S rRNA and msp4 genes. The serological survey detected the presence of anti-A. marginale IgG antibodies in 18 animals (11.1%): 2.9% (2/70) sheep and 17.4% (16/91) goats. Anaplasma marginale DNA was detected in 2 goats (1.2%) using qPCR based on the msp1ß gene. Two distinct A. marginale msp1α strains, namely α ß and α ß ΓγΓγΓγΓγ were found in the infected goats, each one found in a different animal, both belonging to the H genotype. Phylogenetic analysis based on the 16S rRNA gene showed the sequences positioned in three different clades and grouped with sequences from 'Candidatus Anaplasma boleense', A. platys and A. marginale. Phylogenetic inferences based on the msp4 gene positioned the sequence variants in the A. marginale clade. The present work represents the first molecular detection of sequence variants phylogenetic associated to 'Candidatus Anaplasma boleense' and A. platys and α ß and α ß ΓγΓγΓγΓγ in goats from Brazil.

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.

Diagnosis and phylogenetic analysis of bovine leukemia virus in dairy cattle in northeastern Brazil.

Enzootic bovine leukosis (EBL) is a chronic viral disease of wide distribution in cattle herds and may take several years for the first manifestation of clinical signs. Most animals do not present clinical signs. However, the economic losses are underestimated due to this disease. Thus, this work aimed to detect and characterize BLV in dairy cattle in the Maranhão state, northeastern Brazil. Blood samples were collected from 176 animals from 8 municipalities in the southeastern state of Maranhão. Bovine blood samples were subjected to DNA extraction and molecular diagnosis using nested PCR assays for BLV, targeting gp51 gene. Positive samples were then sequenced and then subjected to phylogenetic inferences. BLV DNA was detected in 16 cattle (16/176, 9.09%) in 4 municipalities. Phylogenetic analyzes showed that the sequence obtained clustered in a clade containing BLV sequences classified as genotype 6, with a high degree of support. Our data shows BLV occurrence in the Northeast of Brazil and the identification of genotype 6 in this region. These findings contribute to the molecular epidemiology of this agent in Brazil.

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.

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.

First report of detection of Toxoplasma gondii DNA in oysters (Crassostrea sp.) in the state of Maranhão.

The aim of this study was to report on detection of Toxoplasma gondii DNA in oysters (Crassostrea sp.) in the state of Maranhão. To conduct this study, 200 farmed oysters were acquired in the municipality of Raposa and 100 in Paço do Lumiar; and a further 100 oysters were taken from the natural stock in the municipality of Primeira Cruz. This total of 400 specimens sampled was divided into 80 pools composed of five animals each. The gills and visceral mass of each oyster were removed for DNA extraction (per pool of oysters), using a commercial kit. The nested PCR technique (with the primer SAG-1) was then used to investigate any presence of protozoa. This molecular technique demonstrated the presence of DNA of T. gondii in 2.5% of the pools of oysters (n = 2/80): these oysters were exclusively from farms. The results from this study allow the conclusion that oysters of the genus Crassostrea that are farmed in the state of Maranhão are capable of filtering oocysts of T. gondii and maintaining them in their tissues. They are therefore potential sources of contamination for humans and other animals.