Detection of Sarcocystis spp. and Toxoplasma gondii in swine and detection of DNA of these protozoa in tissues and sausages / Soroprevalência de Sarcocystis spp. e Toxoplasma gondii em suínos e detecção do DNA desses protozoários em tecidos e embutidos

Abstract The seroprevalence of Sarcocystis spp. and Toxoplasma gondii was researched in swine raised in Santa Maria, RS, Brazil. Serum samples from 84 pigs from 31 farms were tested using indirect immunofluorescence assay (IFA) for both agents. Additionally, 53 samples of pork sausages and tissues destined for human consumption, including: salami, sausage, black pudding, heart, tongue, brain, and rib muscle, were submitted to PCR to detect DNA for each agent. The frequency of anti-Sarcocystis spp. antibodies was 36.9% (31/84), with titers ranging from 32 to 1024, and 25% (21/84) for anti-T. gondii antibodies, with titers ranging from 64 to 2048. Sarcocystis spp. and T. gondii DNA were detected in 67.9% (36/53) and 13.2% (7/53) of samples, respectively. The presence of antibodies and the detection of DNA from Sarcocystis spp., and T. gondii suggests that the pigs were infected and may serve as an important reservoir for both parasites. The infection by these protozoa in the swine population is relevant to public health due to their zoonotic potential.

Resumo A soroprevalência de Sarcocystis spp. e Toxoplasma gondii foi pesquisada em suínos criados em Santa Maria, RS, Brasil. Amostras de soro de 84 suínos de 31 fazendas foram testadas pela reação deimunofluorescência indireta (IFA) para ambos os agentes. Adicionalmente, 53 amostras de embutidos suínos e tecidos cárneos destinados ao consumo humano, incluindo: salame, linguiça, morcela, coração, língua, cérebro e músculo da costela foram submetidas à PCR para detecção de DNA para cada agente. A frequência de anticorpos anti-Sarcocystis spp. foi de 36,9% (31/84), com títulos variando de 32 a 1.024; e 25% (21/84) para anticorpos anti-T. gondii, com títulos variando de 64 a 2048. A presença de DNA de Sarcocystis spp. e T. gondii foi detectada em 67,9% (36/53) e 13,2% (7/53) das amostras avaliadas, respectivamente. A detecção de anticorpos e DNA de Sarcocystis spp. e T. gondii sugere que os suínos foram infectados e podem servir como um importante reservatório de ambos os parasitas. A circulação desses agentes na população suína é relevante para a saúde pública devido ao seu potencial zoonótico.

Post mortem protozoan hemoparasites detection in wild mammals from Mato Grosso state, Midwestern Brazil / Detecção post mortem de hemoparasitas protozoários em mamíferos selvagens do estado de Mato Grosso, Centro-Oeste do Brasil

Abstract To a better insight into the epidemiology and genetic diversity of protozoan hemoparasites infections in wild mammals, this study aimed to the post mortem detection of DNA from species of the order Piroplasmida (Babesia sp., Cytauxzoon sp., and Theileria sp.) and suborder Adelorina (Hepatozoon sp.) using polymerase chain reaction based on the 18S rRNA gene followed by genetic sequencing of blood and spleen samples collected from carcasses of 164 free-ranging and captive wild mammals from Mato Grosso state. Among them, one Leopardus pardalis, three Panthera onca, two Puma concolor were positive for Cytauxzoon sp., and six Tapirus terrestris tested positive for Piroplasmida, while one L. pardalis was positive for Hepatozoon sp. Furthermore, an uncharacterized piroplasmid genetically related to Theileria sp. previously detected in cats from Brazil was described in lowland tapirs. Despite the controversy regarding the epidemiological threat of these protozoa, the detection of these tick-borne agents in wild free-living and captive mammals, even when asymptomatic, demonstrates the importance of monitoring, particularly in hotspots such as the state of Mato Grosso, to verify the circulation and genetic diversity, to anticipate the possible emergence of diseases, and even their consequences to other animals as well as humans.

Resumo Para uma melhor compreensão da epidemiologia e diversidade genética das infecções por hemoprotozoários em mamíferos selvagens, este estudo teve como objetivo a detecção post mortem de DNA de espécies da ordem Piroplasmida (Babesia sp., Cytauxzoon sp. e Theileria sp.) e subordem Adelorina (Hepatozoon sp.), utilizando-se a reação em cadeia pela polimerase, baseada no gene 18S rRNA, seguido de sequenciamento genético de amostras de sangue e baço, coletadas de 164 carcaças de mamíferos selvagens de vida livre e cativos do estado de Mato Grosso. Entre eles, um Leopardus pardalis, três Panthera onca, dois Puma concolor foram positivos para Cytauxzoon sp., e seis Tapirus terrestris testaram positivos para Piroplasmida, enquanto um L. pardalis foi positivo para Hepatozoon sp. Além disso, foi descrito em antas, um piroplasmídeo não caracterizado geneticamente, relacionado à Theileria sp., previamente detectado em gatos do Brasil. Apesar da controvérsia quanto à ameaça epidemiológica desses protozoários, a detecção desses agentes em mamíferos silvestres e cativos, mesmo quando assintomáticos, demonstra a importância do monitoramento, principalmente em hotspots, como no estado de Mato Grosso, para verificar a circulação e a diversidade genética, a fim de antecipar o possível surgimento de doenças e, até mesmo, suas consequências para outros animais, bem como os humanos.

Molecular detection of Apicomplexa protozoa in tissues from Alouatta guariba clamitans / Detecção molecular de protozoários Apicomplexa em tecidos de Alouatta guariba clamitans

The brown howler monkey (Alouatta guariba clamitans) is a primate species widely distributed in South America. Infections by protozoa are common in primates. However, studies on protozoa in primates in Brazil are scarce, so the goal of this study was to investigate DNA from the apicomplexan protozoa Neospora caninum, Sarcocystis spp. and Toxoplasma gondii in tissues of A. guariba clamitans. DNA extraction was performed on tissue samples from the heart, brain, liver, spleen, lung and intestine of six A. guariba clamitans from Santa Maria, Central Region of Rio Grande do Sul, Brazil. Conventional PCR was performed using 18S rRNA gene general primers for Apicomplexa and also specific primers to amplify Neosporaspp. and Toxoplasma gondii DNA. All animals were positive in the 18S PCR and the genetic sequencing confirmed the presence of Sarcocystis spp. DNA in the tissues of four animals belonging to at least two species (S. neurona and S. gigantea) and T. gondii DNA in the other two animals. One positive sample for T. gondii was genotypically characterized as atypical by the restriction fragment length polymorphism technique. N. caninum DNA was not detected in the tested samples. The presence of Apicomplexa protozoan DNA in the tissues of the six animals tested in this study highlights the importance of howler monkeys as maintainers of these pathogens in nature.(AU)

O bugio ruivo (Alouatta guariba clamitans) é uma espécie de primata amplamente distribuída na América do Sul. As infecções por protozoários são comuns em primatas. Entretanto, estudos sobre protozoários em primatas no Brasil são escassos, portanto o objetivo deste estudo foi pesquisar DNA dos protozoários Apicomplexa Neospora caninum, Sarcocystisspp. e Toxoplasma gondii em tecidos de A. guariba clamitans. A extração de DNA foi realizada em amostras de tecido do coração, cérebro, fígado, baço, pulmão e intestino de seis A. guariba clamitans oriundos de Santa Maria, Região Central do Rio Grande do Sul, Brasil. Foi realizada PCR convencional utilizando primers geral do gene 18S rRNA para Apicomplexa e também primers específicos para amplificação de DNA de Neospora spp.e Toxoplasma gondii. Todos os animais foram positivos no PCR geral para Apicomplexa e no sequenciamento genético confirmou-se a presença de DNA de Sarcocystis nos tecidos de quatro animais pertencentes a pelo menos duas espécies (S. neurona e S. gigantea), e DNA de T. gondii foi detectado nos outros dois animais. Uma amostra positiva para T. gondii foi caracterizada genotipicamente como atípico pela técnica de polimorfismo do comprimento do fragmento de restrição. Não foi detectado DNA de N. caninum nas amostras testadas. A presença de DNA de protozoários apicomplexa nos tecidos dos seis animais testados neste estudo destaca a importância dos bugios ruivos como mantenedores desses patógenos na natureza.(AU)

Copro-PCR in the detection and confirmation of Toxoplasma gondii oocysts in feces of stray and domiciled cats / Copro-PCR na detecção e confirmação de oocistos de Toxoplasma gondii em fezes de gatos errantes e domiciliados

Abstract Molecular methods such as Copro-PCR stand out in the diagnosis of T. gondii, because they are highly sensitive and specific, and can distinguish T. gondii from other morphologically similar coccids. The purpose was the detection of Toxoplasma gondii copro-prevalence by polymerase chain reaction in 149 fecal samples from stray and domiciled cats, using three distinct markers (B5-B6, 18S and 529bp RE). Oocysts of T. gondii/H. hammondi were detected in 15.4% by parasitology fecal tests (PFT), and 4% of these oocysts were positively identified as T. gondii by Copro-PCR. The presence of T. gondii genetic material was detected in 16.1%, but 12% of the samples that tested positive by Copro-PCR were negative in PFT. Samples with discordant results were subjected to a new Copro-PCR with 18S marker and a 529, and of the 17 samples, 9 contained T. gondii genetic material. A comparison of the PFT and the molecular methods showed the latter was more sensitive, since it detected 22.1% while the PFT detected 15.4%. Demonstrating the high sensitivity and specificity of the Copro-PCR, particularly with the association of primers (k=0.809), but also confirms the importance of using molecular techniques in laboratories, since Copro-PCR was able to detect samples considered negative by PFT.

Resumo Métodos moleculares como a Copro-PCR se destacam no diagnóstico de T. gondii por serem altamente sensíveis e específicos, podendo distinguir T. gondii de outros coccídeos morfologicamente semelhantes. O objetivo foi a detecção da coproprevalência de Toxoplasma gondii por reação em cadeia da polimerase, em 149 amostras fecais de gatos errantes e domiciliados, utilizando-se três marcadores distintos (B5-B6, 18S e 529pb RE). Oocistos de T. gondii/H. hammondi foram detectados em 15,4% pelos exames parasitológicos de fezes (EPF), e 4% desses oocistos foram identificados positivamente como T. gondii pela Copro-PCR. A presença de material genético de T. gondii foi detectada em 16,1%, mas 12% das amostras positivas pelo Copro-PCR foram negativas no EPF. As amostras com resultados discordantes foram submetidas a uma nova Copro-PCR com os marcadores 18S e 529 e, das 17 amostras, 9 continham material genético de T. gondii. A comparação do EPF com os métodos moleculares mostrou que esse último foi mais sensível, pois detectou 22,1%, enquanto o EPF detectou 15,4%. Isso demonstra a alta sensibilidade e especificidade da Copro-PCR, principalmente com a associação de marcadores (k = 0,809); mas também confirma a importância do uso de técnicas moleculares em laboratórios, uma vez que a Copro-PCR foi capaz de detectar amostras consideradas negativas pelo EPF.

Evaluation of cytochrome b sequence to identify Leishmania species and variants: the case of Panama

BACKGROUND The genetic heterogeneity of Leishmania parasites is a major factor responsible for the wide variety of Leishmania-associated manifestations. Consequently, understanding the genetic make-up of Leishmania species using suitable molecular markers is an important component of realising local and regional scale disease risk. The cytochrome b (cytb) is frequently used to type New World Leishmania species. However, its potential to discriminate Leishmania species and variants requires further evaluation. OBJECTIVES To explore the capacity of cytb gene to identify New World Leishmania species and variants and to develop an approach able to type local Leishmania species and variants. METHODS We retrieved 360 partial and complete Leishmania cytb gene sequences publicly available in GenBank database to study all single nucleotide polymorphisms (SNPs) across the cytb gene that differentiate New World Leishmania species. This information was used to develop an approach based upon the polymorphisms found in a DNA segment of 948bp. We also compared the typing results found with this technique with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiling obtained using HSP70 gene as target. One hundred Panamanian isolates were used to both typed Leishmania species and assess local genetic variability. FINDINGS We found complete agreement between our cytb approach and the PCR-RFLP profiling method based on HSP70 for Leishmania species identification. Ninety-two isolates were identified as L. panamensis, although other Viannia species were found circulating at a lower frequency. Three L. panamensis haplotypes were identified in Panamanian provinces. We also provide an initial report of L. guyanensis haplotypes circulating in Panama. MAIN CONCLUSIONS Cytb gene sequence encompasses key main SNPs that aid to identify Leishmania species. The cytb approach developed with this information was able to identify and assess genetic variability of local Leishmania species found in this study.

Current status of research regarding Blastocystis sp, an enigmatic protist, in Brazil

The present study aimed to evaluate the occurrence of Blastocystis sp. in Brazilian studies over a period of years (2000-2020), as well as point out relevant aspects of this enigmatic organism. We performed a literature search using six sources of international databases. The data were divided into diagnostic by parasitological and molecular techniques, and relevant aspects. After applying the inclusion and exclusion criteria, 52 studies were included in the final analysis. The occurrence of Blastocystis sp. in Brazil ranged from 0.5% to 86.6%, as determined using parasitological techniques. The highest occurrence was in the North (27.3%) and the lowest, in the Midwest region (13.4%). In Brazil, most studies have employed molecular techniques and are concentrated in the Southeast region. The Blastocystis sp. subtype ST3 had the highest average positivity, followed by ST1 and ST2. These findings represent a panorama that reflects the reality of Brazil; thus, we believe that the effectiveness of parasitological diagnosis should be considered with regard to making an appropriate choice of technique for detecting Blastocystis sp. Additionally, we emphasize the importance of further studies in the context of molecular epidemiology with regard to this genus. Blastocystis sp. is not well understood yet, and very little information regarding this genus is available; hence, further research regarding this genus is urgently needed.

Sarcocystidae in wild birds of southeastern Brazil / Sarcocystidae em aves silvestres do sudeste do Brasil

Abstract This study aimed to identify members of the Sarcocystidae family in naturally infected wild birds at a rescue center in the state of Minas Gerais, southeastern Brazil. The heart and brain of 44 wild birds were evaluated by bioassay in mice to detect T. gondii, and extracted DNA was used for nested PCR of the 18S ribosomal DNA gene to detect members of the Sarcocystidae family. The positive samples were sequenced, assembled, edited and compared with sequences deposited in GenBank. Toxoplasma gondii was isolated from six (13.6%) out of 44 birds. Toxoplasma gondii DNA was identified in 10/44 (22.7%) of the birds. The amplified sequences exhibited 100% similarity with the DNA of the ME49 strain of T. gondii. Sarcocystis DNA (99% similarity) was identified in 5/44 (11.4%) of the birds. T. gondii and Sarcocystis spp. are common in wild birds in Minas Gerais, Brazil.

Resumo O objetivo deste estudo foi identificar membros da família Sarcocystidae em aves silvestres de vida livre naturalmente infectadas e resgatadas no estado de Minas Gerais, Brasil. Coração e cérebro de 44 aves silvestres foram avaliados por bioensaio em camundongos para detecção de T. gondii e extração de DNA para Nested-PCR do gene 18S do DNA ribossomal de membros da família Sarcocystidae. As amostras positivas foram sequenciadas, analisadas, editadas e comparadas com sequências depositadas no GenBank. Toxoplasma gondii foi isolado de seis (13,6%) das 44 aves. DNA de T. gondii foi identificado em 10/44 (22,7%) das 44 aves. As sequências amplificadas exibiram 100% de similaridade com o DNA da cepa ME49 de T. gondii. DNA de Sarcocystis (99% de similaridade) foi identificado em 5/44 (11,4%) das 44 aves. T. gondii e Sarcocystis spp. são encontrados, comumente, em aves silvestres no estado de Minas Gerais, Brasil.

In silico and in vitro evaluation of primers for molecular differentiation of Leishmania species / Avaliação in silico e in vitro de iniciadores para a diferenciação molecular de espécies de Leishmania

Abstract Leishmaniasis is a zoonotic disease caused by over 20 species of protozoan parasites of the genus Leishmania. Infection is commonly spread by sandflies and produces a wide spectrum of clinical signs and symptoms. Therefore, from an epidemiological and therapeutic standpoint, it is important to detect and differentiate Leishmania spp. The objective of this study was to combinate in silico and in vitro strategies to evaluate the analytical specificity of primers previously described in the literature. According to electronic PCR (e-PCR) analysis, 23 out of 141 pairs of primers selected through literature search matched their previously reported analytical specificity. In vitro evaluation of nine of these primer pairs by quantitative PCR (qPCR) confirmed the analytical specificity of five of them at the level of Leishmania spp., L. mexicana complex or Leishmania and Viannia subgenera. Based on these findings, the combination of e-PCR and qPCR is suggested to be a valuable approach to maximize the specificity of new primer pairs for the laboratory diagnosis of infections with Leishmania spp.

Resumo As leishmanioses são zoonoses causadas por mais de 20 espécies de protozoários do gênero Leishmania. As infecções são comumente disseminadas por flebotomíneos e causam um amplo espectro de manifestações clínicas. Portanto, a detecção e diferenciação de espécies de Leishmania são importantes do ponto de vista epidemiológico e terapêutico. O objetivo deste estudo foi combinar estratégias in silico e in vitro para avaliar a especificidade analítica dos primers descritos anteriormente na literatura. De acordo com a PCR eletrônica (e-PCR), 23 dos 141 pares de primers selecionados por meio de pesquisa da literatura estavam de acordo com a especificidade analítica anteriormente relatada. A avaliação in vitro de nove desses pares de primers, por PCR quantitativa (qPCR), confirmou a especificidade analítica de cinco deles ao nível de espécie de Leishmania, do complexo L. mexicana ou dos subgêneros Leishmania e Viannia. Com base nos resultados, sugere-se que a combinação de e-PCR e qPCR é uma abordagem valiosa para a validação e maximização da especificidade de novos pares de primers para o diagnóstico laboratorial de infecções com Leishmania spp.

Filter paper performance in PCR for cutaneous leishmaniasis diagnosis

Abstract INTRODUCTION: The objective of this study was to evaluate the performance of filter paper (FP) for lesion scraping collection in a polymerase chain reaction (PCR) for cutaneous leishmaniasis (CL) diagnosis. METHODS: Lesion scrapings from 48 patients were collected and analyzed for PCR. RESULTS: PCR with FP detected up to three Leishmania braziliensis promastigotes. Considering the direct search by microscopy or PCR of samples collected in STE buffer as standards, the sensitivity of PCR with FP was 100%. CONCLUSIONS: FP can be useful for CL diagnosis in remote regions, allowing high sensitivity in the detection of the parasite by PCR.

Molecular characterization of Toxoplasma gondii and Sarcocystis spp. in raw kibbeh and other meat samples commercialized in Botucatu, Southeastern Brazil / Caracterização molecular de Toxoplasma gondii e Sarcocystis spp. em amostras de quibe cru e de outras carnes comercializadas em Botucatu, Região Sudeste do Brasil

Abstract Toxoplasmosis occurs worldwide causing economic losses to the animal production and problems to the public health. The study aimed to detect Toxoplasma gondii and Sarcocystis spp.in 141 meat products from commercial meat cuts of pork, beef, and kibbeh sold in commercial markets from Botucatu, SP, Brazil. Samples were bioassayed in mice to isolate the parasite, and the parasite DNA detected by PCR targeting the 529 base pairs repeat element region (PCR-529-bp). All samples resulted negative on bioassay, whereas PCR positive for 9 (6,38%), distributed as 5/48 beef, 3/49 pork, and 1/44 kibbeh. PCR-positive were investigated for the the parasite genotype using multiplex-, nested-, and RFLP-PCR for 11 markers (SAG1, 5'-3'SAG2, alt.SAG2, SAG3, B-TUB, GRA6, L358, c22-8, c29-6, PK1, Apico). Complete genotype was determined on just one PCR-positive sample that matched MAS, TgCkBr89 and TgCkBr147 isolates already identified. In addition, nested- and RFLP-PCR targeting 18S rRNA was run for all PCR-positive samples and, the products, sequenced and aligned to the GenBank at NCBI website. Four samples showed 100% homology with T. gondii (GenBank #L37415.1), three with Sarcocystis hominis (GenBank #AF006471.1), two Sarcocystis cruzi (GenBank #AF176934.1), and one Sarcocystis hirsuta (GenBank #AF006469.1), indicating the circulation of T. gondii and Sarcocystis spp.

Resumo A toxoplasmose está mundialmente distribuída e causa perdas na produção animal e problemas de saúde pública. Objetivou-se detectar Toxoplasma gondii e Sarcocystis spp. em 141 produtos cárneos de origem suína (49), bovina (48) e de quibe cru (44), comercializados em mercados de Botucatu, SP, Brazil. Realizou-se bioensaio das amostras em camundongos para isolamento do parasita, e detecção do DNA pela reação em cadeia pela polimerase, tendo como alvo a região do elemento repetitivo de 529 pares de bases (PCR-529-bp). Todas as amostras foram negativas ao bioensaio e 9 (6,38%) positivas à PCR, sendo 5/48 bovinas, 3/49 suínas e 1/44 quibe. Determinou-se a genotipagem das amostras positivas pela multiplex-, nested- e RFLP-PCR com 11 marcadores (SAG1, 5'-3'SAG2, alt.SAG2, SAG3, B-TUB, GRA6, L358, c22-8, c29-6, PK1, Apico). Obteve-se genótipo completo em uma amostra, semelhante a outros já identificados (MAS, TgCkBr89 e TgCkBr147). Nested- e RFLP-PCR do gene 18S rRNA das amostras positivas à PCR foram realizadas, e os produtos da nested-PCR, sequenciados e alinhados com dados do GenBank no NCBI. Quatro apresentaram 100% de homologia com T. gondii (L37415.1), duas Sarcocystis hominis (AF006471.1), duas Sarcocystis cruzi (AF176934.1), uma Sarcocystis hirsuta (AF006469.1), indicando a circulação de T. gondii e Sarcocystis spp.

Polymerase chain reaction using conjunctival swab samples for detecting Leishmania DNA in dogs / Reação em cadeia da polimerase usando amostras de Swab de conjuntiva para detecção de DNA de Leishmania em cães

Abstract The dog is the main domestic reservoir of Leishmania and font of infection for the vector, constituting an important host for the transmission of the parasite to humans. Non-invasive collection of swab samples for leishmaniasis diagnosis has been a promising alternative. This study analyzed the positivity of polymerase chain reaction (PCR) for the diagnosis of canine leishmaniasis in conjunctiva samples. DNA extraction was performed using SDS 20% and PCR was performed using 13A/13B primers that amplify 120-bp of Leishmania kDNA. Of the 77 dogs analyzed, 50 (64.93%) had ocular changes: 25 (32.47%) dogs had periocular lesion, 41 (53.25%) dogs had purulent eye discharge, and 17 (22.08%) dogs had both signals. PCR was positive in 35 dogs (45.45%), and there was no significant difference between dogs with and without ocular signals (p=0.4074). PCR positivity was significant higher in dogs without periocular injury (p=0.0018). Conjunctive PCR, a less invasive, fast, and painless collection technique, is indicated to complement the diagnosis, especially in dogs without periocular injury, independent of the presence of purulent eye discharge.

Resumo O cão é o principal reservatório doméstico de Leishmania e também fonte de infecção para o vetor, constituindo um importante hospedeiro para a transmissão do parasita ao homem. A coleta não invasiva de amostras em swab para diagnóstico das leishmanioses tem sido uma alternativa promissora. Este estudo analisou a positividade da reação em cadeia da polimerase (PCR) para o diagnóstico de leishmaniose canina em amostras de conjuntiva. A extração do DNA foi realizada com SDS 20%. A PCR foi realizada com primers 13A/13B que amplificam 120-pb do kDNA de Leishmania. Dos 77 cães analisados, 50 (64,93%) tiveram alterações oculares; 25 (32,47%) cães tiveram uma lesão periocular; 41 (53,25%) tiveram secreção ocular purulenta e 17 (22,08%) cães tiveram ambos os sinais. A PCR foi positiva em 35 cães (45,45%) e não houve diferença significativa em cães com e sem sinais oculares (p = 0,4074). A positividade da PCR foi significativamente maior em cães sem lesão periocular (p = 0,0018). PCR em conjuntiva, uma técnica de coleta menos invasiva, rápida e indolor, é indicada para complementar o diagnóstico, principalmente em cães sem lesão periocular, independentemente da presença de secreção ocular purulenta.

Evidence of a sylvatic enzootic cycle of Leishmania infantum in the State of Amapá, Brazil

Abstract INTRODUCTION: Leishmania infantum was considered to be absent from Amapá until 2017 when canine infection was detected. However, there is a lack of knowledge about which reservoir species are involved in transmission in this region. METHODS: Between 2014 and 2016, 86 samples from wild mammals and 74 from domestic dogs were collected in Wajãpi Indigenous Territory and were tested for the presence of deoxyribonucleic acid (DNA) of Leishmania. RESULTS: The DNA of Le. infantum was detected in two rodent samples, Dasyprocta sp. and Proechimys cuvieri. CONCLUSIONS: This is the first evidence characterizing a sylvatic transmission cycle of Le. infantum in the State of Amapá.

Efficacy of the Benznidazole+Posaconazole combination therapy in parasitemia reduction: An experimental murine model of acute Chagas

Abstract INTRODUCTION: Benznidazole (BZL) and Nifurtimox (NFX) are the pharmacological treatment for acute phase Chagas Disease (CD); however, therapy resistance and residual mortality development remain important unresolved issues. Posaconazole (POS) has shown a trypanocidal effect in vivo and in vitro. Thus, this study aimed at comparing the T. Cruzi parasitic load-reducing effect of the combination of BZL+POS against that of monotherapy with either, during acute phase CD, in an experimental murine model. METHODS Nineteen Wistar rats were randomly allocated to four groups and inoculated with the trypomastigotes of T. cruzi strain´s JChVcl1. The rats were administered anti-parasites from day 20-29 post-infection. The Pizzi and Brener method was used for parasitemia measurement. Longitudinal data analysis for the continuous outcome of repeated measures was performed using parasitemia as the outcome measured at days 20, 22, 24, 27, and 29 post-infection. RESULTS All four groups had similar parasitic loads (p=0.143) prior to therapy initiation. Among the three treatment groups, the BZL+POS (n=5) group showed the highest mean parasitic load reduction (p=0.000) compared with the control group. Likewise, the BZL+POS group rats showed an earlier therapeutic effect and were the only ones without parasites in their myocardial samples. CONCLUSIONS: Treatment of acute phase CD with BZL+POS was more efficacious at parasitemia and myocardial injury reduction, compared with monotherapy with either.

Clinical-dermatological, histological abnormalities and prevalence of Trypanosoma caninum and Leishmania infantum in dogs from Midwest region of Brazil / Anormalidades clínico-dermatológicas, histológicas e prevalência de Trypanosoma caninum e Leishmania infantum em cães da região Centro-Oeste do Brasil

Abstract Leishmania infantum is a trypanosomatid that causes parasitic dermatopathy in dogs. Trypanosoma caninum is another trypanosomatid, which infects the skin of dogs, although cutaneous abnormalities are absent. This study aimed to investigate the occurrence of T. caninum infection and its associated cutaneous and histological changes and compare it with the occurrence of L. infantum infection in dogs. The study included 150 dogs, of which T. caninum infection was identified in 3 (2%) and L. infantum infection in 15 (10%) of them, with no association (p>0.05) of these infections with the breed, gender, age, or cutaneous abnormalities. The cutaneous abnormalities were based on 1 (4.8%) and 12 (57.1%) dogs infected by T. caninum and L. infantum, respectively. The dermatohistopathological abnormalities in the dogs infected with T. caninum included mild perivascular lymphohistioplasmacytic infiltrates in the clinically asymptomatic ones, while in those with dermatological abnormalities, acanthosis, epidermal orthokeratotic hyperkeratosis, melanomacrophages, and co-infection with Microsporum sp. and Trichophyton sp. were observed. InL. infantum infected, the histopathological findings included chronic granulomatous inflammatory infiltrates and structures compatible with amastigotes. Despite the low frequency of T. caninum infection, our findings suggest that this trypanosomatid, unlike L. infantum, does not cause any macroscopic skin abnormalities.

Resumo Leishmania infantum é um tripanosomatídeo que causa dermatopatia parasitária em cães. Trypanosoma caninum é outro tripanosomatídeo, que infecta a pele de cães, embora anormalidades cutâneas sejam ausentes. Este estudo teve como objetivo investigar a ocorrência da infecção por T. caninum e suas alterações cutâneas e histológicas associadas e compará-las com a ocorrência da infecção por L. infantum em cães. O estudo incluiu 150 cães, dos quais a infecção por T. caninum foi identificada em 3 (2%) e a infecção por L. infantum em 15 (10%) deles, sem associação (p>0,05) dessas infecções com a raça, sexo, idade ou anormalidades cutâneas. As alterações cutâneas foram observadas em 1 (4,8%) e 12 (57,1%) cães infectados por T. caninum e L. infantum, respectivamente. As anormalidades dermato-histopatológicas nos cães infectados por T. caninum incluíram infiltrados linfo-histioplasmocitários perivasculares leves nos clinicamente assintomáticos, enquanto naqueles com anormalidades dermatológicas, foram observados acantose, hiperqueratose ortoqueratótica epidermal e melanomacrófagos e co-infecção por Microsporum sp. e Trichophyton sp. Nos cães infectados por L. infantum, os achados histopatológicos incluíram infiltrados inflamatórios granulomatosos crônicos e estruturas compatíveis com amastigotas. A despeito da baixa frequência da infecção por T. caninum, nossos achados sugerem que esse tripanosomatídeo, diferentemente de L. infantum, não causa anormalidades macroscópicas na pele.

Autochthonous canine visceral leishmaniasis cases occur in Paraná state since 2012: isolation and identification of Leishmania infantum / Leishmaniose visceral canina autóctone ocorre no estado do Paraná desde 2012: isolamento e identificação de Leishmania infantum

Abstract The aim of this study was to confirm the emergence of canine visceral leishmaniasis among dogs in Foz do Iguaçu. The disease was diagnosed through the isolation and molecular identification of Leishmania infantum. In the first sample collection stage (2012), three lymph node aspirates and 46 buffy coat samples were obtained mostly from the dogs that were seroreagents for leishmaniasis. In the second sample collection stage (2013), the buffy coat samples were collected from 376 dogs located close to Paraguay, Paraná river, center and peripheral parts of the city. The DNA from the six isolates, four from the first sampling stage (4/49) and two from the second sampling stage (2/376), was subjected to polymerase chain reaction using the K26F/R primers. The isolate was confirmed as L. infantum by sequencing. As none of the dogs had ever left the city, the isolates were confirmed as autochthonous. Further, the study confirmed the emergence of canine visceral leishmaniasis in Paraná through the identification of L. infantum among dogs in Foz do Iguaçu city. Hence, collaborative control measures should be designed and implemented by the public agencies and research institutions of Brazil, Argentina, and Paraguay to control the spread of visceral leishmaniasis.

Resumo O objetivo deste estudo foi confirmar a emergência da leishmaniose visceral canina em Foz do Iguaçu próximo à fronteira com a Argentina e ao Paraguai, por meio do isolamento e identificação molecular de Leishmania infantum. Em um primeiro estágio de coleta de animais (2012), três amostras de aspirados de linfonodos e 46 camadas leucocitárias foram obtidas de cães soropositivos para leishmaniose. Em um segundo estágio de coleta (2013), foram coletadas amostras de camada leucocitária de 376 cães de 20 localidades próximas à fronteira com o Paraguai, rio Paraná, centro e periferia da cidade. Seis isolados foram obtidos, quatro da primeira etapa (4/49) e dois da segunda etapa (2/376); estes isolados foram submetidos à amplificação com iniciadores K26F/R, e a análise de sua sequência confirmou a espécie como L. infantum. A autoctonia dos casos foi confirmada, pois 100% dos cães nunca haviam saído da cidade. O estudo confirma a emergência de leishmaniose visceral canina no Paraná com identificação de L. infantum em cães da cidade de Foz do Iguaçu. Assim, medidas de controle devem ser elaboradas e implementadas por órgãos públicos e instituições de pesquisa do Brasil, Argentina e Paraguai em parceria com o objetivo de controlar a disseminação de zoonoses e os casos humanos de LV.

Increasing putative vector importance of Trichophoromyia phlebotomines (Diptera: Psychodidae)

Despite some phlebotomines being well recognised as vectors of leishmaniasis agents, vector importance of those belonging to the genus Trichophoromyia has not been extensively studied. The present study provides evidence regarding the putative vector role played by some species of Trichophoromyia on leishmanine enzootics, based on literature reports and findings obtained from field experiments conducted in the ecotopes of Pará State, Brazil. The species Th. ubiquitalis, Th. velascoi, Th. auraensis, Th. ininii and Th. brachipyga possess minimal criteria to be included in the list of suspected leishmanine vectors. However, knowledge on man-biting behavior, substantiation of vector competence and determination of epidemiological implications are limited for all of the above mentioned species. Published studies together with present data draw attention to prioritize these phlebotomine species in entomological surveillance programs and studies on experimental susceptibility to Leishmania spp. infection.

An overview of the sandfly fauna (Diptera: Psychodidae) followed by the detection of Leishmania DNA and blood meal identification in the state of Acre, Amazonian Brazil

BACKGROUND In Acre state, Brazil, the dissemination of cutaneous leishmaniasis has increased in recent years, with limited knowledge of the potential Leishmania spp. vectors involved. OBJECTIVES Here, data concerning the sandfly fauna of Brasiléia municipality, Leishmania DNA-detection rates and the identification of blood meal sources of insects captured in 2013-2015 are presented. METHODS Parasite detection in female sandflies was performed individually by multiplex polymerase chain reaction (PCR) (Leishmania kDNA/sandfly cacophony-gene), with the identification of Leishmania spp. by hsp70-PCR and sequencing. The identification of blood gut-content from fed females was performed by cyt b-PCR and sequencing. FINDINGS A total of 4,473 sandflies were captured. A subgroup of 864 non-blood-fed females evaluated for the presence of Leishmania DNA showed 2.9% positivity for Leishmania (Viannia) braziliensis and L. (V.) guyanensis. The identification of blood meal sources was performed in 96 blood-fed females, allowing the identification of 13 vertebrate species. In nine/96 fed females, DNA from L. (V.) shawi, L. (V.) guyanensis, L. (V.) braziliensis and Endotrypanum sp. was detected. MAIN CONCLUSIONS In Brumptomyia sp. and Evandromyia termitophila, the first report of Leishmania DNA-detection is provided in Acre; Nyssomyia shawi is implicated as potential vector of L. (V.) braziliensis and L. (V.) guyanensis for the first time in Brazil.

Hepatozoon pyramidumi sp. n. (Apicomplexa: Adeleorina) from the blood of Echis pyramidum: morphology and SSU rDNA sequence / Hepatozoon pyramidumi sp. n. (Apicomplexa: Adeleorina) do sangue de Echis pyramidum: morfologia e sequência de SSU rDNA

Abstract Hepatozoon pyramidumi sp. n. is described from the blood of the Egyptian saw-scaled viper, Echis pyramidum, captured from Saudi Arabia. Five out of ten viper specimens examined (50%) were found infected with Hepatozoon pyramidumi sp. n. with parasitaemia level ranged from 20-30%. The infection was restricted only to the erythrocytes. Two morphologically different forms of intraerythrocytic stages were observed; small and mature gamonts. The small ganomt with average size of 10.7 × 3.5 μm. Mature gamont was sausage-shaped with recurved poles measuring 16.3 × 4.2 μm in average size. Infected erythrocytes were hypertrophied; their nuclei were deformed and sometimes displaced from their central position in the normal uninfected cell. Merogonic stages were observed in the lung endothelial cell and the liver parenchyma cells. Mature meront was 17.8 × 13.6 µm and contained banana-shaped merozoites with average size of ~15 × 2 µm. Phylogenetic analysis based on the SSU rDNA sequence clustered Hepatozoon pyramidumi sp. n with previously sequenced Hepatozoon spp., most of them infected reptilian hosts without geographic consideration. The morphological and molecular comparison with closely related species proved the taxonomic uniqueness and novelty of the present form.

Resumo Hepatozoon pyramidumi sp. n. é descrito a partir do sangue da víbora em escamas e quilhas serrilhadas, Echis pyramidum, capturada na Arábia Saudita. Cinco de dez espécimes de víbora examinadas (50%) foram encontradas infectadas com Hepatozoon pyramidumi sp. n. com nível de parasitemia de 20% a 30%. A infecção foi restrita apenas aos eritrócitos. Foram observadas duas formas morfologicamente diferentes de estágios intra-eritrocíticos: gamontes de tamanho pequeno e madura. As formas menores de gamontes apresentaram média de 10,7 × 3,5 μm. Os gamontes maduros apresentaram forma de salsicha, com pequenos polos recurvados, medindo 16,3 × 4,2 μm, em média. Os eritrócitos infectados estavam aumentados de tamanho; seus núcleos encontravam-se deformados e, algumas vezes, deslocados de sua posição central, quando comparados às células normais não-infectadas. Foram observados estágios merogônicos em células endoteliais pulmonares e nas células do parênquima hepático. Os merontes maduros apresentavam 17,8 × 13,6 µm e continham merozoítos em forma de banana com tamanho médio de ~ 15 × 2 µm. A análise filogenética baseada nas sequências SSU rDNA agrupou Hepatozoon pyramidumi sp. n com Hepatozoon spp. detectados em répteis de várias regiões geográficas. Por meio de análises morfológicas e moleculares com espécies intimamente relacionadas, demonstrou-se a singularidade dessa nova espécie de Hepatozoon.

Prevalence and molecular characterization of Cryptosporidium spp. in Père Davids deer (Elaphurus davidianus) in Jiangsu, China / Prevalência e caracterização molecular de Cryptosporidium spp. no cervo de Père David (Elaphurus davidianus) em Jiangsu, China

Abstract Cryptosporidium is a zoonotic parasite that causes diarrhea in a broad range of animals, including deer. Little is known about the prevalence and genotype of Cryptosporidium spp. in Père David's deer. In this study, 137 fecal samples from Père David's deer were collected between July 2017 and August 2018 in the Dafeng Reserve and analyzed for Cryptosporidium spp. by nested-PCR based on the small subunit ribosomal RNA (SSU rRNA) gene, followed by sequence analyses to determine the species. The 60 kDa glycoprotein (gp60) gene was used to characterize Cryptosporidium spp. Among 137 samples, 2 (1.46%) were positive for Cryptosporidium spp. according to SSU rRNA gene sequencing results. Both samples belonged to the Cryptosporidium deer genotype, with two nucleotide deletions and one nucleotide substitution. The prevalence data and molecular characterization of this study provide basic knowledge for controlling and preventing Cryptosporidium infections in Père David's deer in this area.

Resumo Cryptosporidium é um parasita zoonótico que causa diarreia em uma ampla gama de animais, incluindo veados. Pouco se sabe sobre a prevalência e o genótipo de Cryptosporidium spp. no cervo de Père David. Neste estudo, 137 amostras fecais do cervo de Père David foram coletadas entre julho de 2017 e agosto de 2018, na Reserva Dafeng, e analisadas para Cryptosporidium spp. por nested-PCR baseado no gene do RNA ribossômico da subunidade pequena (SSU rRNA), seguido de análises de sequências para determinar as espécies. O gene da glicoproteína de 60 kDa (gp60) foi utilizado para caracterizar Cryptosporidium spp. Dentre as 137 amostras, 2 (1,46%) foram positivas para Cryptosporidium spp. de acordo com os resultados do sequenciamento gênico de SSU rRNA. Ambas as amostras pertenciam ao genótipo do cervo Cryptosporidium, com duas deleções nucleotídicas e uma substituição nucleotídica. Os dados de prevalência e a caracterização molecular deste estudo fornecem conhecimentos básicos para controlar e prevenir infecções por Cryptosporidium nos cervos de Père David nessa.

Serological study of feline leishmaniasis and molecular detection of Leishmania infantum and Leishmania braziliensis in cats (Felis catus) / Pesquisa sorológica da leishmaniose felina e detecção molecular de Leishmania infantum e Leishmania braziliensis em gatos (Felis catus)

Abstract Blood samples and swabs from ocular conjunctiva and mouth were obtained from 64 cats. Of 64 serum samples, 19 were positive for Leishmania antibodies by ELISA (29.80%). Eight cats were positive by PCR (12.5%) in swab samples from mouth and/or ocular mucosa. Poor kappa agreement between serological and molecular results (k = 0.16) was obtained. From five positive PCR samples one was L. braziliensis and four were L. infantum. Phylogenetic analysis performed with the five isolates of Leishmania, showed that samples of L. infantum isolated from the cats were phylogenetically close to those isolated from domestic dogs in Brazil, while the L. braziliensis is very similar to the one described in humans in Venezuela. The study demonstrated that, despite high seropositivity for Leishmania in cats living in the study region, poor agreement between serological and molecular results indicate that positive serology is not indicative of Leishmania infection in cats. Parasite DNA can be detected in ocular conjunctiva and oral swabs from cats, indicating that such samples could be used for diagnosis. Results of phylogenetic analyzes show that L. infantum circulating in Brazil is capable of infecting different hosts, demonstrating the parasite's ability to overcome the interspecies barrier.

Resumo Amostras de sangue e swabs da conjuntiva ocular e oral foram obtidas de 64 gatos. Das 64 amostras de soro, 19 foram positivas para anticorpos contra Leishmania por ELISA (29,80%). Oito gatos foram positivos por PCR (12,5%) em amostras de swab da boca e / ou mucosa ocular. Demonstrou-se baixa concordância kappa entre os resultados sorológicos e moleculares (k = 0,16). Das cinco amostras positivas para PCR, uma era L. braziliensis e quatro eram L infantum. A análise filogenética realizada com os cinco isolados de Leishmania, mostrou que amostras de L. infantum, isoladas dos gatos, eram filogeneticamente próximas às isoladas de cães domésticos do Brasil enquanto L. braziliensis era muito semelhante ao descrito em humanos na Venezuela. O estudo demonstrou que, apesar da alta soropositividade para Leishmania, em gatos que vivem na região do estudo, pouca concordância entre os resultados sorológicos e moleculares indica que a sorologia positiva não é indicativa de infecção por Leishmania em gatos. O DNA do parasita pode ser detectado na conjuntiva ocular e nas zaragatoas orais de gatos, indicando que essas amostras podem ser usadas para o diagnóstico. . Resultados de análises filogenéticas mostram que L. infantum, circulando no Brasil, é capaz de infectar diferentes hospedeiros, demonstrando a capacidade do parasita de superar a barreira interespécies.