Isolation and molecular characterization of Toxoplasma gondii from placental tissues of pregnant women who received toxoplasmosis treatment during an outbreak in southern Brazil.

Toxoplasma gondii is a protozoan that has great genetic diversity and is prevalent worldwide. In 2018, an outbreak of toxoplasmosis occurred in Santa Maria, Brazil, which was considered the largest outbreak ever described in the world. This paper describes the isolation and molecular characterization of Toxoplasma gondii from the placenta of two pregnant women with acute toxoplasmosis who had live births and were receiving treatment for toxoplasmosis during the outbreak. For this, placental tissue samples from two patients underwent isolation by mice bioassay, conventional PCR and genotyping using PCR-RFLP with twelve markers. Both samples were positive in isolation in mice. The isolate was lethal to mice, suggesting high virulence. In addition, the samples were positive in conventional PCR and isolates submitted to PCR-RFLP genotyping presented an atypical genotype, which had never been described before. This research contributes to the elucidation of this great outbreak in Brazil.

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.

Autochthonous canine visceral leishmaniasis cases occur in Paraná state since 2012: isolation and identification of Leishmania infantum.

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.

Genotyping of Toxoplasma gondii isolated from pigs for human consumption.

The present study aimed to isolate and genotype strains of T. gondii from pigs slaughtered for human consumption in South Brazil. Blood and tissues (heart, diaphragm, liver, tongue, and masseter) from 400 animals were collected at two slaughterhouses. Sera were obtained, and antibodies against T. gondii were detected by both indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT). The tissues of animals that tested positive in MAT, IFAT, or both (cut-off ≥ 64) were bioassayed. Twenty-six (6.5%) of the 400 animals were positive by serology. A total of 18 (69.2%) out of those 26 were positive in the mouse bioassay. The isolates were characterized by using 10 PCR-RFLP genetic markers. Fourteen isolates were fully genotyped, and four isolates were genotyped using nine of the 10 markers. All isolates belonged to ToxoDB PCR-RFLP genotype #206. The present study reports on genotype #206 in pigs for the first time, and it confirms the atypical nature of the Brazilian T. gondii isolates. Additionally, even with low levels of antibodies detected in pig herds, pork presents a T. gondii infection risk for humans.

The effect of water source and soil supplementation on parasite contamination in organic vegetable gardens.

The objective of this study was to determine factors associated with vegetable contamination with zoonotic protozoan. Samples of water, soil and vegetables were collected from July/2014 to May/2016, totaling 83 samples, 21 properties of Londrina region, Paraná, Brazil. DNA amplification of Toxoplasma gondii, Cryptosporidium spp. and Giardia intestinalis in the samples was conducted using polymerase chain reaction (PCR). The PCR results were positive for T. gondii in 12.9% (8/62), Cryptosporidium spp. in 11.3% (7/62) and G. intestinalis in 25.8% (16/62) of the samples. DNA sequencing identified C. parvum in five samples and G. intestinalis Assemblage E in three. The statistical associations demonstrated greater probability of positive samples for T. gondii and for at least one of the three protozoa when the source of irrigation water was the river; a greater chance of positive samples for Cryptosporidium spp. when deer were present on the property; and a smaller chance of positive samples for at least one of the three etiologic agents when soil was supplemented with limestone. The results expose some critical contamination points, providing support for training farmers on good management practices during the production process.

Variables associated with the prevalence of anti-Leishmania spp. antibodies in dogs on the tri-border of Foz do Iguaçu, Paraná, Brazil.

The aim of this study was to investigate the occurrence of anti-Leishmania spp. antibodies in dogs from localities in the city of Foz do Iguaçu, Paraná state, Brazil, on the border with Argentina and Paraguay. Blood samples dogs were collected to perform the following serologic tests: immunochromatographic DPP® rapid test, indirect immunoenzymatic assay (ELISA) and indirect immunofluorescence assay (IFA). In 2012, 285 dogs were analyzed on Argentina border, and in 2013, serum samples from 396 dogs on the border of Paraguay were collected. Using ELISA for screening and IFA for the confirmatory test, the results showed that the antibody prevalence was 1.8% (5/285) on the border of Argentina and 3.0% (12/396) on Paraguay border. When using the DPP® for screening and ELISA as a confirmatory analysis, we observed a seroreagent prevalence in dogs of 2.5% (7/285) on Argentina border and 5.1% (20/396) on Paraguay border. The non-public collection of domestic waste (p= 0.0004) was shown to be associated with leishmaniasis. This study shows the presence of leishmaniasis and suggest the emergence of canine visceral leishmaniasis in state of Paraná due to the confirmed occurrence of seroreactive dogs on Argentina and Paraguay border, which has environmental and geographical characteristics that favor the spread of the parasite.

Variables associated with the prevalence of anti-Leishmania spp. antibodies in dogs on the tri-border of Foz do Iguaçu, Paraná, Brazil / Variáveis associadas à prevalência de anticorpos anti- Leishmania spp. em cães na tríplice fronteira de Foz do Iguaçu, Paraná, Brasil

Abstract The aim of this study was to investigate the occurrence of anti-Leishmania spp. antibodies in dogs from localities in the city of Foz do Iguaçu, Paraná state, Brazil, on the border with Argentina and Paraguay. Blood samples dogs were collected to perform the following serologic tests: immunochromatographic DPP® rapid test, indirect immunoenzymatic assay (ELISA) and indirect immunofluorescence assay (IFA). In 2012, 285 dogs were analyzed on Argentina border, and in 2013, serum samples from 396 dogs on the border of Paraguay were collected. Using ELISA for screening and IFA for the confirmatory test, the results showed that the antibody prevalence was 1.8% (5/285) on the border of Argentina and 3.0% (12/396) on Paraguay border. When using the DPP® for screening and ELISA as a confirmatory analysis, we observed a seroreagent prevalence in dogs of 2.5% (7/285) on Argentina border and 5.1% (20/396) on Paraguay border. The non-public collection of domestic waste (p= 0.0004) was shown to be associated with leishmaniasis. This study shows the presence of leishmaniasis and suggest the emergence of canine visceral leishmaniasis in state of Paraná due to the confirmed occurrence of seroreactive dogs on Argentina and Paraguay border, which has environmental and geographical characteristics that favor the spread of the parasite.

Resumo O objetivo deste estudo foi investigar a ocorrência de anticorpos anti- Leishmania spp. em cães da cidade de Foz do Iguaçu, estado do Paraná, Brasil, fronteira com a Argentina e o Paraguai. Amostras de sangue de cães foram coletadas para realização dos seguintes testes sorológicos: teste rápido imunocromatográfico DPP®, ensaio imunoenzimático indireto (ELISA) e ensaio de imunofluorescência indireta (IFI). Em 2012, 285 cães foram analisados na fronteira com Argentina e, em 2013, amostras de soro de 396 cães na fronteira com o Paraguai. Utilizando ELISA para triagem e IFA para o teste confirmatório, os resultados mostraram uma prevalência de anticorpos de 1,8% (5/285) na fronteira da Argentina e 3,0% (12/396) na fronteira com o Paraguai. Ao usar o DPP® para triagem e ELISA como uma análise confirmatória, observou-se uma prevalência de cães sororreagentes de 2,5% (7/285) na fronteira com a Argentina e 5,1% (20/396) na fronteira com o Paraguai. A não coleta pública de lixo doméstico (p = 0,0004) mostrou-se associada à leishmaniose. Este estudo demonstra a presença de leishmaniose e sugere a emergência da leishmaniose visceral canina no estado do Paraná devido à ocorrência de cães sororreagentes na fronteira Argentina e Paraguai, que possuem características ambientais e geográficas que favorecem a disseminação do parasito.

The effect of water source and soil supplementation on parasite contamination in organic vegetable gardens / O efeito da fonte de água e suplementação de solo na contaminação parasitária em hortas orgânicas

Abstract The objective of this study was to determine factors associated with vegetable contamination with zoonotic protozoan. Samples of water, soil and vegetables were collected from July/2014 to May/2016, totaling 83 samples, 21 properties of Londrina region, Paraná, Brazil. DNA amplification of Toxoplasma gondii, Cryptosporidium spp. and Giardia intestinalis in the samples was conducted using polymerase chain reaction (PCR). The PCR results were positive for T. gondii in 12.9% (8/62), Cryptosporidium spp. in 11.3% (7/62) and G. intestinalis in 25.8% (16/62) of the samples. DNA sequencing identified C. parvum in five samples and G. intestinalis Assemblage E in three. The statistical associations demonstrated greater probability of positive samples for T. gondii and for at least one of the three protozoa when the source of irrigation water was the river; a greater chance of positive samples for Cryptosporidium spp. when deer were present on the property; and a smaller chance of positive samples for at least one of the three etiologic agents when soil was supplemented with limestone. The results expose some critical contamination points, providing support for training farmers on good management practices during the production process.

Resumo O trabalho teve como objetivo determinar os fatores associados à contaminação de vegetais por protozoários zoonóticos. Amostras de água, solo e vegetais foram coletadas de julho/2014 a maio/2016, totalizando 83 amostras de 21 propriedades da região de Londrina, Paraná, Brasil. A amplificação de fragmentos de DNA de T. gondii, Cryptosporidium spp. e Giardia intestinalis foi realizada por meio da reação em cadeia da polimerase (PCR). Os resultados da PCR foram positivos para T. gondii em 12,9% (8/62), Cryptosporidium spp. em 11,3% (7/62) e G. intestinalis. em 25,8% (16/62) das amostras. O sequenciamento de DNA identificou C. parvum em cinco amostras e G. intestinalis, Assemblage E em três amostras. As associações estatísticas evidenciaram maior probabilidade de amostras serem positivas para T. gondii ou para pelo menos um dos três protozoários quando a fonte de água de irrigação era o rio; uma maior chance de amostras positivas para Cryptosporidium spp. quando havia cervos na propriedade; e uma menor chance das amostras serem positivas para pelo menos um dos três agentes etiológicos quando o solo era suplementado com calcário. Os resultados expõem alguns pontos críticos de contaminação, fornecendo suporte para capacitar os agricultores em boas práticas de gestão durante o processo de produção.

Antibodies anti-trypanosomatides in domestic cats in Paraná: who is at highest risk of infection? / Anticorpos anti-tripanossomatídeos em gatos domésticos no Paraná: quem está em maior risco de infecção?

Abstract The aim of this study were to detect antibodies anti-Leishmania spp. and anti-Trypanosoma cruzi in two different populations of domestic cats (Felis catus domesticus) from North Paraná referred for surgical castration and to determine which characteristics of the animals studied may be associated with seropositivity. Serum samples from 679 cats were analyzed using enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody test (IFAT) in series. Associations between age, sex, race, year of care and animal group were verified using the simple logistic regression. Percentage of 8.5% (58/679) of cats were positive for Leishmania spp. and 7.6% (51/673) for T. cruzi by the tests ELISA and IFAT. Animals collected by non-governmental animal protection organizations presented more seropositivity for Leishmania spp. (p<0.0001). Results shown that Leishmania spp. and T. cruzi are present in domestic cats in the northern part of the state of Paraná, as well as, owners of non-governmental animal protection organizations may be more exposed to leishmaniasis when compared to other animal owners evaluated in the present study.

Resumo O objetivo desse estudo foi detectar a presença de anticorpos IgG anti-Leishmania spp. e anti-Trypanosoma cruzi em duas populações de gatos domésticos (Felis catus domesticus) do Norte do Paraná encaminhados para castração cirúrgica e determinar quais as características dos animais estudados que podem estar associadas à soropositividade. Amostras de soro de 679 gatos foram analisadas utilizando-se os testes imunoenzimático (ELISA) e a reação de imunofluorescência indireta (RIFI), em série. Associações entre idade, sexo, raça, ano de atendimento e grupo animal ​​foram verificadas usando regressão logística simples. Um percentual de 8,5% (58/679) dos gatos apresentou positividade para Leishmania spp. e 7,6% (51/673) para T. cruzi pelos testes ELISA e RIFI. Gatos mantidos em organizações não governamentais de proteção animal apresentaram maior sororeatividade para Leishmania spp. (p<0.0001). As sorologias reativas para Leishmania spp. e Trypanosoma cruzi mostram que esses agentes estão presentes em gatos domésticos na parte norte do estado do Paraná, bem como, os proprietários de organizações não governamentais de proteção animal podem estar mais expostos à leishmaniose quando comparados com outros proprietários de animais avaliados no presente estudo.

Antibodies anti-trypanosomatides in domestic cats in Paraná: who is at highest risk of infection?

The aim of this study were to detect antibodies anti-Leishmania spp. and anti-Trypanosoma cruzi in two different populations of domestic cats (Felis catus domesticus) from North Paraná referred for surgical castration and to determine which characteristics of the animals studied may be associated with seropositivity. Serum samples from 679 cats were analyzed using enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody test (IFAT) in series. Associations between age, sex, race, year of care and animal group were verified using the simple logistic regression. Percentage of 8.5% (58/679) of cats were positive for Leishmania spp. and 7.6% (51/673) for T. cruzi by the tests ELISA and IFAT. Animals collected by non-governmental animal protection organizations presented more seropositivity for Leishmania spp. (p<0.0001). Results shown that Leishmania spp. and T. cruzi are present in domestic cats in the northern part of the state of Paraná, as well as, owners of non-governmental animal protection organizations may be more exposed to leishmaniasis when compared to other animal owners evaluated in the present study.

Spatial and simultaneous seroepidemiology of anti-Leishmania spp. antibodies in dog owners and their dogs from randomly selected households in a major city of southern Brazil.

Although leishmaniasis has been described as a classic example of a zoonosis requiring a comprehensive approach for control, to date, no study has been conducted on the spatial distribution of simultaneous Leishmania spp. seroprevalence in dog owners and dogs from randomly selected households in urban settings. Accordingly, the present study aimed to simultaneously identify the seroprevalence, spatial distribution and associated factors of infection with Leishmania spp. in dog owners and their dogs in the city of Londrina, a county seat in southern Brazil with a population of half a million people and ranked 18th in population and 145th in the human development index (HDI) out of 5570 Brazilian cities. Overall, 564 households were surveyed and included 597 homeowners and their 729 dogs. Anti-Leishmania spp. antibodies were detected by ELISA in 9/597 (1.50%) dog owners and in 32/729 (4.38%) dogs, with significantly higher prevalence (p = 0.0042) in dogs. Spatial analysis revealed associations between seropositive dogs and households located up to 500 m from the local railway. No clusters were found for either owner or dog case distributions. In summary, the seroepidemiological and spatial results collectively show a lack of association of the factors for infection, and the results demonstrated higher exposure for dogs than their owners. However, railway areas may provide favorable conditions for the maintenance of infected phlebotomines, thereby causing infection in nearby domiciled dogs. In such an urban scenario, local sanitary barriers should be focused on the terrestrial routes of people and surrounding areas, particularly railways, via continuous vector surveillance and identification of phlebotomines infected by Leishmania spp.

Correction: Spatial and simultaneous representative seroprevalence of anti-Toxoplasma gondii antibodies in owners and their domiciled dogs in a major city of southern Brazil.

[This corrects the article DOI: 10.1371/journal.pone.0180906.].

An outbreak of caprine toxoplasmosis - investigation and case report / Surto de toxoplasmose caprina - investigação e relato de caso

ABSTRACT: The present study aimed to investigate an abortion outbreak in a dairy goat herd in the municipality of Arapoti, Parana, Brazil. At the beginning of the outbreak, blood samples were collected from 33 goats with clinical signs; later, of the whole goat herd, two cats and two dogs. Milk samples were collected from 78 lactating goats. Four environmental soil samples and four samples of feed residue from goat feeders were collected too. Immunofluorescence antibody test (IFA) was used for serodiagnosis, the molecular analysis was conducted by means of the polymerase chain reaction (PCR), for the isolation of the etiological agent the bioassay was used. The results of the IFA revealed that 76.53% (137/179) of the goats, two dogs and two cats were seropositive for Toxoplasma gondii. Bioassay revealed one buffy coat and two milk sample having viable T. gondii. In the PCR, 11 whole blood samples, eight milk, three feeder troughs, and all soil samples were positive. The findings of the present study confirmed an outbreak caused by environmental contamination (of soil and feed) with T. gondii oocysts that could have been shed by kittens that lived on the farm and had access to the stock of goat food, facilitating this contamination, which reinforces the need for veterinary assistance and good management practices on farms.

RESUMO: O presente estudo teve como objetivo investigar um surto de aborto em um rebanho de cabras leiteiras no município de Arapoti, Paraná, Brasil. No início do surto, foram coletadas amostras de sangue de 33 cabras com sinais clínicos; mais tarde, de todo o rebanho caprino, dois gatos e dois cachorros. Foram obtidas amostras de leite das 78 cabras em lactação. Quatro amostras ambientais de solo e quatro de resíduos de comedouro também foram coletadas. O teste de imunofluorescência (IFI) foi utilizado para o sorodiagnóstico, a análise molecular foi conduzida por meio da reação em cadeia da polimerase (PCR), para isolamento do agente etiológico utilizou-se o bioensaio. Os resultados da IFI revelaram que 76,53% (137/179) das cabras, todos os cães e gatos eram soropositivos para Toxoplasma gondii. O bioensaio revelou uma amostra de camada leucocitária e duas de leite contaminadas com T. gondii viável. Na PCR, 11 amostras de sangue total, oito de leite, três resíduos alimentares e todas as amostras de solo foram positivas. Os resultados do presente estudo confirmaram um surto causado por contaminação ambiental (de solo e alimentos) com oocistos de T. gondii que, provavelmente, foram eliminados por gatos que permaneceram na fazenda e tinham acesso ao estoque de alimento dos caprinos, reforçando a necessidade de assistência técnica veterinária e boas práticas de manejo.

Spatial and simultaneous representative seroprevalence of anti-Toxoplasma gondii antibodies in owners and their domiciled dogs in a major city of southern Brazil.

Toxoplasmosis, caused by Toxoplasma gondii, has traditionally been considered an important water and foodborne protozoonosis with important public health considerations. Although felids play a well-established role as definitive hosts, canine epidemiological involvement in the parasite's life cycle remains questionable and controversial. The increasing closeness of the human-dog bond, particularly seen in urban settings, has been recognized as a historically unprecedented worldwide movement. Sharing daily lives in the same households, dogs may be exposed to similar associated risks of T. gondii infection as their owners. Thus, epidemiological assessment of the intra-domiciled environment, especially among socio-economically different human populations, may provide novel information regarding the actual role of dogs in animal and human toxoplasmosis. Despite spatial approaches being recently used for other water and foodborne diseases, no study has been conducted on the simultaneous spatial seroprevalence of both human and animal IgG anti-T. gondii antibodies in urban areas of major cities. Accordingly, the aim of the present study was to assess the seroprevalence and associated variables of Toxoplasma infection in owners and their domiciled dogs in Londrina, southern Brazil. Human and canine seroprevalence rates and variables associated with seroprevalence were investigated through representative random sampling among 564 households, which included 597 owners and 729 dogs. Overall, statistically significant differences between the seroprevalence of human and dog anti-T. gondii antibodies were found by Immunofluorescence Antibody Testing in 248/597 (41.54%) owners and 119/729 (16.32%) dogs. Through multiple analysis, significant concomitant variables for seropositivity of household individuals (people and dogs) were determined, including public sewer service, yard cleaning frequency, and having a dirty yard. Although no statistically significant multiple logistic model was observed among owners, univariate analysis detected associations with monthly income, soil contact, and occupation. Among dogs, the absence of other dogs and the absence of a dirty yard were concomitant significantly protective associated factors. Age differences between seropositive and seronegative individuals was significant only for human beings, with the median age of negative individuals significantly higher than positive individuals. Although no spatial clusters were identified for humans or residences, a significant cluster was identified for dogs. In conclusion, characteristics of urban toxoplasmosis may include significantly higher owner seroprevalence than their owned dogs, with canine seroprevalence directly associated with having more dogs and a dirty backyard, and spatial differences in both human and dog exposures. Although not a good indicator for human foodborne diseases, dogs may be a reliable sentinel for environmental infection. Moreover, such a holistic approach may provide crucial information for more focused prevention and monitoring programs, particularly in households with multiple pets and trash-filled backyards.

Cryptosporidium spp. and Giardia spp. in feces and water and the associated exposure factors on dairy farms.

The aims of this study were to verify the prevalence of Cryptosporidium spp. and Giardia spp. in animal feces and drinking water on dairy farms and to identify a possible relation between the exposure factors and the presence of these parasites. Fecal samples from cattle and humans and water samples were collected on dairy farms in Paraná, Brazil. Analysis of (oo)cysts in the feces was performed by the modified Ziehl-Neelsen staining and centrifugal flotation in zinc sulfate. Test-positive samples were subjected to nested PCR amplification of the 18SSU ribosomal RNA gene for identification of Cryptosporidium and Giardia and of the gp60 gene for subtyping of Cryptosporidium. Microbiological analysis of water was carried out by the multiple-tube method and by means of a chromogenic substrate, and parasitological analysis was performed on 31 samples by direct immunofluorescence and nested PCR of the genes mentioned above. Identification of the species of Cryptosporidium was performed by sequencing and PCR with analysis of restriction fragment length polymorphisms. The prevalence of Giardia and Cryptosporidium was higher in calves than in adults. Among the samples of cattle feces, Cryptosporidium parvum was identified in 41 (64%), C. ryanae in eight (12.5%), C. bovis in four (6.3%), C. andersoni in five (7.8%), and a mixed infection in 20 samples (31.3%). These parasites were not identified in the samples of human feces. Thermotolerant coliform bacteria were identified in 25 samples of water (45.5%). Giardia duodenalis and C. parvum were identified in three water samples. The gp60 gene analysis of C. parvum isolates revealed the presence of two strains (IIaA20G1R1 and IIaA17G2R2) in the fecal samples and one (IIaA17G2R1) in the water samples. The presence of coliforms was associated with the water source, structure and degradation of springs, rain, and turbidity. The prevalence of protozoa was higher in calves up to six months of age. C. parvum and G. duodenalis were identified in the water of dairy farms, as were thermotolerant coliforms; these findings point to the need for guidance on handling of animals, preservation of water sources, and water treatment.

Leishmania in synanthropic rodents (Rattus rattus): new evidence for the urbanization of Leishmania (Leishmania) amazonensis / Leishmania em roedores sinantrópicos (Rattus rattus): uma nova evidência da urbanização de Leishmania (Leishmania) amazonensis

Abstract This study aimed to detect parasites from Leishmania genus, to determine the prevalence of anti-Leishmania spp. antibodies, to identify circulating species of the parasite, and to determine epidemiological variables associated with infection in rats caught in urban area of Londrina, Paraná, Brazil. Animal capture was carried out from May to December 2006, serological and molecular methods were performed. DNA was extracted from total blood, and nested-PCR, targeting SSu rRNA from Leishmania genus, was performed in triplicate. The positive samples were sequenced twice by Sanger method to species determination. In total, 181 rodents were captured, all were identified as Rattus rattus and none showed clinical alterations. Forty-one of the 176 (23.3%) animals were positive for Leishmania by ELISA and 6/181 (3.3%) were positive by IFAT. Nine of 127 tested animals (7.1%) were positive by PCR; seven were identified as L. (L.) amazonensis, one as L. (L.) infantum. Four rats were positive using more than one test. This was the first description of synanthropic rodents naturally infected by L. (L.) amazonensis (in the world) and by L. (L.) infantum (in South Brazil). Regarding L. (L.) amazonensis, this finding provides new evidence of the urbanization of this etiological agent.

Resumo Esse estudo objetivou detectar parasitos do gênero Leishmania, determinar a prevalência de anticorpos anti-Leishmania spp., identificar as espécies circulantes do parasito e determinar variáveis epidemiológicas associadas com a infecção em ratos capturados em área urbana de Londrina, Paraná, Brazil. A captura dos animais ocorreu de maio a dezembro de 2006, métodos sorológicos e moleculares foram realizados. O DNA foi extraído do sangue total, uma nested-PCR cujo alvo foi o gene SSu rRNA do gênero Leishmania, foi realizado em triplicata. As amostras positivas foram sequenciadas duas vezes pelo método de Sanger para a determinação da espécie. No total, 181 roedores foram capturados, todos foram identificados como Rattus rattus e nenhum apresentou alterações clínicas. Quarenta e um dos 176 (23,3%) animais foram positivos no ELISA para Leishmania e 6/181 (3,3%) foram positivos na RIFI. Nove dos 127 animais testados (7,1%) foram positivos na PCR; sete foram identificadas como L. (L.) amazonensis, um como L. (L.) infantum. Quatro ratos foram positivos em mais de um teste. Essa é a primeira descrição de roedores sinantrópicos naturalmente infectados por L. (L.) amazonensis (no mundo) e por L. (L.) infantum (no Sul do Brasil). Com relação a L. (L.) amazonensis, esse resultado é uma nova evidência da urbanização desse agente etiológico.

Leishmania in synanthropic rodents (Rattus rattus): new evidence for the urbanization of Leishmania (Leishmania) amazonensis.

This study aimed to detect parasites from Leishmania genus, to determine the prevalence of anti-Leishmania spp. antibodies, to identify circulating species of the parasite, and to determine epidemiological variables associated with infection in rats caught in urban area of Londrina, Paraná, Brazil. Animal capture was carried out from May to December 2006, serological and molecular methods were performed. DNA was extracted from total blood, and nested-PCR, targeting SSu rRNA from Leishmania genus, was performed in triplicate. The positive samples were sequenced twice by Sanger method to species determination. In total, 181 rodents were captured, all were identified as Rattus rattus and none showed clinical alterations. Forty-one of the 176 (23.3%) animals were positive for Leishmania by ELISA and 6/181 (3.3%) were positive by IFAT. Nine of 127 tested animals (7.1%) were positive by PCR; seven were identified as L. (L.) amazonensis, one as L. (L.) infantum. Four rats were positive using more than one test. This was the first description of synanthropic rodents naturally infected by L. (L.) amazonensis (in the world) and by L. (L.) infantum (in South Brazil). Regarding L. (L.) amazonensis, this finding provides new evidence of the urbanization of this etiological agent.

Evaluation of the Western blotting method for the diagnosis of congenital toxoplasmosis / Avaliação do método Western Blotting para diagnóstico de toxoplasmose congênita

Abstract Objective: To evaluate the Western blotting method for the detection of IgG anti-Toxoplasma gondii (T. gondii) (IgG-WB) in the serum of children with suspected congenital toxoplasmosis. Methods: We accompanied 47 mothers with acquired toxoplasmosis in pregnancy and their children, between June of 2011 and June of 2014. The IgG-WB was done in house and the test was considered positive if the child had antibodies that recognized at least one band on IgG blots different from the mother's or with greater intensity than the corresponding maternal band, during the first three months of life. Results: 15 children (15.1%) met the criteria for congenital toxoplasmosis and 32 (32.3%) had the diagnosis excluded. The symptoms were observed in 12 (80.0%) children and the most frequent were cerebral calcification in 9 (60.0%), chorioretinitis in 8 (53.3%), and hydrocephalus in 4 (26.6%). IgM antibodies anti-T. gondii detected by chemiluminescence (CL) were found in 6 (40.0%) children and the polymerase chain reaction (PCR) for detection of T. gondii DNA was positive in 5 of 7 performed (71.4%). The sensitivity of IgG-WB was of 60.0% [95% confidence interval (CI) 32.3-83.7%] and specificity 43.7% (95% CI 26.7-62.3%). The sensitivity of IgG-WB increased to 76.0 and 89.1% when associated to the research of IgM anti-T. gondii or PCR, respectively. Conclusions: The IgG-WB showed greater sensitivity than the detection of IgM anti-T. gondii; therefore, it can be used for the diagnosis of congenital toxoplasmosis in association with other congenital infection markers.

Resumo Objetivo: Avaliar o método Western Blotting para detecção de IgG anti-Toxoplasma gondii (T. gondii) (IgG-WB) no soro de crianças com suspeita de toxoplasmose congênita. Métodos: Acompanhamos 47 mães com toxoplasmose adquirida na gravidez e seus filhos, entre junho de 2011 e junho de 2014. O IgG-WB foi feito internamente e o teste foi considerado positivo quando a criança apresentava anticorpos que reconheciam pelo menos uma banda nas manchas de IgG diferente das bandas da mãe ou com maior intensidade do que a banda materna correspondente, durante os primeiros 3 meses de vida. Resultados: Atenderam aos critérios para diagnóstico de toxoplasmose congênita 15 crianças (15,1%) e 32 (32,3%) tiveram o diagnóstico excluído. Os sintomas foram observados em 12 crianças (80%) e os mais frequentes foram calcificação cerebral em nove (60%), coriorretinite em oito (53,3%) e hidrocefalia em quatro (26,6%). Os anticorpos IgM anti-T. gondii detectados por quimiluminescência (QL) foram encontrados em seis crianças (40%) e a reação em cadeia da polimerase (RCP) para detecção do DNA de T. gondii foi positiva em cinco de sete reações (71,4%). A sensibilidade do IgG-WB foi de 60% [intervalo de confiança (IC) de 95%, 32,3 a 83,7%] e a especificidade foi de 43,7% (IC de 95%, 26,7 a 62,3%). A sensibilidade do IgG-WB aumentou para 76 e 89,1% quando relacionada à pesquisa de IgM anti-T. gondii ou à RCP, respectivamente. Conclusões: O IgG-WB mostrou maior sensibilidade do que a detecção de IgM anti-T. gondii; portanto, pode ser usado para o diagnóstico de toxoplasmose congênita em associação com outros marcadores de infecção congênita.

Evaluation of the Western blotting method for the diagnosis of congenital toxoplasmosis.

OBJECTIVE: To evaluate the Western blotting method for the detection of IgG anti-Toxoplasma gondii (T. gondii) (IgG-WB) in the serum of children with suspected congenital toxoplasmosis. METHODS: We accompanied 47 mothers with acquired toxoplasmosis in pregnancy and their children, between June of 2011 and June of 2014. The IgG-WB was done in house and the test was considered positive if the child had antibodies that recognized at least one band on IgG blots different from the mother's or with greater intensity than the corresponding maternal band, during the first three months of life. RESULTS: 15 children (15.1%) met the criteria for congenital toxoplasmosis and 32 (32.3%) had the diagnosis excluded. The symptoms were observed in 12 (80.0%) children and the most frequent were cerebral calcification in 9 (60.0%), chorioretinitis in 8 (53.3%), and hydrocephalus in 4 (26.6%). IgM antibodies anti-T. gondii detected by chemiluminescence (CL) were found in 6 (40.0%) children and the polymerase chain reaction (PCR) for detection of T. gondii DNA was positive in 5 of 7 performed (71.4%). The sensitivity of IgG-WB was of 60.0% [95% confidence interval (CI) 32.3-83.7%] and specificity 43.7% (95% CI 26.7-62.3%). The sensitivity of IgG-WB increased to 76.0 and 89.1% when associated to the research of IgM anti-T. gondii or PCR, respectively. CONCLUSIONS: The IgG-WB showed greater sensitivity than the detection of IgM anti-T. gondii; therefore, it can be used for the diagnosis of congenital toxoplasmosis in association with other congenital infection markers.

Abordagem da saúde única na ocorrência de enteroparasitas em humanos de área urbana no norte do Paraná / Single health approach in the occurrence of enteroparasites in humans in the urban area in northern Paraná / Enfoque de la salud única en ocurrencia de enteroparásitos en humanos de la zona urbana en el norte de Paraná

As enteroparasitoses causadas por helmintos e protozoários infectam humanos e animais no mundo todo, e sua prevalência está relacionada à falta de hábitos de higiene pessoal em consequência do seu ciclo oral-fecal. No Brasil, como não são doenças de notificação obrigatória, é possível que sua prevalência esteja subnotificada. Em Londrina, não existem dados sobre a prevalência dessas parasitoses na população urbana adulta, que ofereçam suporte para delinear programas de saúde preventiva na comunidade. Foram coletadas amostras de fezes de 187 humanos residentes na área urbana de Londrina para pesquisa de enteroparasitas. Dados epidemiológicos foram coletados por meio de um questionário epidemiológico aplicado a todos os participantes. As variáveis associadas ao risco foram analisadas conforme foram relatadas pelos indivíduos parasitados. Foram discutidos: gênero, idade, renda familiar mensal, lavagem das mãos antes da alimentação e após utilizar o banheiro, água de consumo, ocorrência de diarreia nos últimos sete dias, abastecimento de água e rede de esgoto. Foram detectados ovos ou cistos de parasitas em 8/187 (4,27%) amostras investigadas. A ocorrência do protozoário Giardia spp. foi verificada em 7/187 (3,74%) amostras e o helminto Enterobius vermiculares em 1/187 (0,53%). Devido ao ciclo destes parasitas envolver a saúde humana, animal e do ambiente, é sugerido que a ocorrência das enteroparasitoses reduza por meio da abordagem de Saúde Única na área urbana de Londrina, PR.

Intestinal parasitic infections are caused by helminthes and protozoa infecting humans and animals worldwide. Its prevalence is related to lack of personal hygiene habits due to its oral-fecal cycle. In Brazil, they do not require compulsory notification and, therefore, it is possible that the prevalence is underreported. In Londrina, there are no data on the prevalence of these parasites in the adult urban population to support the designing of preventive health programs in the community. Stool samples were collected from 187 human residents in the urban area of Londrina in order to research for enteroparasites. Parasite eggs or cysts were detected in 8/187 (4.27%) samples investigated. The occurrence of Giardia spp. was seen in 7/187 (3.74%) samples, while Enterobius vermicularis was observed in 1/187 (0.53%) sample. Epidemiological data were collected using an epidemiological questionnaire applied to all participants. The variables associated with the risk were analyzed and reported by infected individuals, such as: gender, age, family income, washing hands before eating and after using the bathroom, water consumption, occurrence of diarrhea in the past seven days, water supply and sewage system. Since the parasite cycle involves human, animal and environmental health, it is suggested that the occurrence of intestinal parasites be reduce through the Single Health approach in the urban area of Londrina, PR.

Las enteroparasitosis causadas por helmintos y protozoarios infectan humanos y animales en todo el mundo, y su prevalencia está relacionada con la falta de hábitos de higiene personal, como resultado de su ciclo fecal-oral. En Brasil, no son enfermedades de declaración obligatoria, es posible que su prevalencia no se denuncie. En Londrina, no hay datos sobre la prevalencia de esos parásitos en la población adulta urbana, que apoyen el diseño de programas preventivos de salud en la comunidad. Se recogieron muestras de heces de 187 residentes humanos en el área urbana de Londrina para investigación de enteroparásitos. Datos epidemiológicos se han recogido mediante un cuestionario epidemiológico administrado a todos los participantes. Las variables asociadas al riesgo se analizaron conforme relatado por los individuos infectados. Se discutieron: género, edad, renta familiar mensual, lavarse las manos antes de comer y después de ir al baño, agua de consumo, la aparición de diarrea en los últimos siete días, el suministro de agua y alcantarillado. Se ha detectado huevos o quistes de parásitos en 8/187 (4,27%) muestras investigadas. La aparición del protozoário Giardia spp. se observó en 7/187 (3,74%) muestras y el helminto Enterobius vermicularis en 1/187 (0,53%). Debido al ciclo de estos parásitos involucrar la salud humana, animal y el medio ambiente, se sugiere que la aparición de enteroparasitosis se reduzca a través del enfoque de Salud Única en la zona urbana de Londrina, PR.