A molecular and morphological study of Ascaris suum in a human-pig contact scenario in northeastern Brazil / Estudo molecular e morfológico de Ascaris suum em um cenário de contato entre suínos e humanos no nordeste brasileiro

The aim of the present study was to assess morphologic and genetic data on ascariasis in swine (Sus scrofa domesticus) and humans in low-resource rural and periurban communities in the state of Piauí, Brazil. Our cross-sectional survey included 100 fecal samples obtained from swine and 682 samples from humans. Fifteen pigs were necropsied. Human and porcine fecal samples were examined to identify Ascaris eggs. Parasites obtained in the swine necropsies were studied using scanning electron microscopy (SEM), and the mitochondrial gene encoding the cytochrome oxidase 1 (cox1) enzyme was partially amplified and sequenced for molecular taxonomy and phylogenetic analyses. The overall prevalence of Ascaris eggs in the swine fecal samples was 16/100 (16%). No Ascaris eggs were identified in the human fecal samples. SEM of six worms recovered from pigs demonstrated morphological characteristics of A. suum. Cox1 sequences were compatible with A. suum reference sequences. Original and reference (GenBank) nucleotide sequences were organized into clusters that did not segregate the parasites by host species or and region. The largest haplogroups were dominated by haplotypes H01, H02 and H31. In the communities studied, there was no epidemiological evidence of the zoonotic transmission of ascariasis at the human-swine interface.(AU)

O presente estudo teve como objetivo acessar dados morfológicos e genéticos sobre a ascaridíase em suínos (Sus scrofa domesticus) e humanos, em comunidades rurais e periurbanas no estado do Piauí. O estudo transversal incluiu 100 amostras fecais de suínos e 682 amostras obtidas de humanos. Quinze suínos foram necropsiados. Amostras fecais suínas e humanas foram examinadas para detecção de ovos de Ascaris. Os parasitas adultos, obtidos nas necropsias, foram estudados através de microscopia eletrônica de varredura (MEV), e o gene mitocondrial codificante da enzima citocromo oxidase 1 (cox1) foi parcialmente amplificado e sequenciado para análises filogenéticas e de taxonomia molecular. A prevalência de Ascaris em amostras fecais de suínos foi 16/100 (16%), não sendo identificado nenhum caso de infecção por este parasita em humanos. A análise por MEV de parasitas recuperados de suínos demonstrou características morfológicas de Ascaris suum. As sequências nucleotídicas de cox1 foram compatíveis com A. suum. As sequências originais e de referência (obtidas no GeneBank) foram organizadas em clusters que não segregaram os parasitas por hospedeiro ou região geográfica. Os maiores haplogrupos foram dominados pelos haplótipos H01, H02 e H31. Nas comunidades estudadas, não foi evidenciada transmissão zoonótica de A. suum na interface suíno-humana.(AU)

Extract of Ascaris suum induces TGF-ß and early production of IgG1 in experimental autoimmune hepatitis / Extrato de Ascaris suum induz TGF-ß e produção precoce de IgG1 na hepatite autoimune experimental

Abstract In experimental autoimmune hepatitis (EAH) of Th1 profile, an extract of adult Ascaris suum worms (ASC) was previously found to deviate the immune response to a Th2/IL-10 pattern. Here, the effects of treatment with ASC on production of TGF-β and the anti-Ascaris isotypes IgG1 and IgG2a in EAH were evaluated. EAH was induced in BALB/c mice, intravenously with concanavalin A. Two hours later, these animals received ASC (EAH+ASC group) or PBS vehicle (EAH group). IgG1 and IgG2a were evaluated 8 h, 24 h and 7 d after induction. TGF-β was measured in a splenocyte culture at this last time. The isotype levels in the EAH group were low throughout the kinetics. In the EAH+ASC group, there was significant production of IgG1 at 24 h and 7 d, but of IgG2a only at 7 d. There was statistically greater production of TGF-β in the EAH+ASC group. The higher levels of IgG1 and TGF-β in this group suggest that an additional Th1 response control route exists in EAH, which needs to be investigated.

Resumo Na hepatite autoimune experimental (HAE) de perfil Th1, o extrato de vermes adultos Ascaris suum (ASC) desviou a resposta imune para um padrão Th2/IL-10. Neste trabalho, foram avaliados os efeitos do tratamento com ASC na produção TGF-β e dos isótipos de IgG1 e IgG2a anti-Ascaris na HAE. Esta foi induzida em camundongos BALB/c intravenosamente com Concanavalina A. Após duas horas, os animais receberam ASC (grupo HAE+ASC) ou veículo PBS (grupo HAE). IgG1 e IgG2a foram avaliados em 8 horas, 24 horas e 7 dias após indução. TGF-β foi mensurado em cultura de esplenócitos nesse último tempo. Os níveis dos isótipos no grupo HAE foram baixos durante toda a cinética. No grupo HAE+ASC, houve produção significativa de IgG1 em 24 horas e 7 dias, mas somente em 7 dias para IgG2a. A produção de TGF-β foi estatisticamente maior no grupo HAE+ASC. Níveis mais altos de IgG1 e TGF-β nesse grupo sugerem uma via adicional de controle da resposta Th1 na HAE que precisa ser investigada.

Absence of mutations associated with resistance to benzimidazole in the beta-tubulin gene of Ascaris suum

Abstract INTRODUCTION: Benzimidazoles are commonly used for the control of veterinary nematodes. Resistance to benzimidazoles has been associated with three single nucleotide polymorphisms in the β-tubulin gene of common nematodes. However, these mutations are infrequent in the genus Ascaris spp. METHODS: In order to determine mutations associated with benzimidazole resistance in Ascaris suum, worms were collected from slaughtered pigs and a partial region of the β-tubulin gene was sequenced. RESULTS: All parasites showed the wildtype genotype for codons 167, 198, and 200 of the β-tubulin gene. CONCLUSIONS: This is the first report of genetic sequences associated with benzimidazole resistance in A. suum.

Effect of endoparasites occurrence in sows from intensive production system / Efeito da ocorrência de endoparasitas em matrizes suínas de sistema de produção intensivo

Abstract The goal of this study was to assess the effect of farm size (FS) and farrowing order (FO) on the occurrence of endoparasites eggs in commercial sows housed in maternity and gestation areas during the period from May to July 2014. Forty-three piglet production units were classified by FS: small (100 to 250 sows), medium (251 to 510 sows), large (511 to 1,000 sows) and very large (more than 1,000 sows). Sows were classified by FO: up to two, three to five or more than five parturitions. Faecal samples were processed using the simple flotation technique in a hypersaturated salt solution (30-35% NaCl). The results revealed that the overall prevalence of gastrointestinal endoparasites obtained in this study was 12.47%, in that 4.64% were positive for Ascaris suum, 0.56% for Trichuris suis and 8.27% for coccidia oocysts. The prevalence of endoparasites obtained for small and medium size farm, and for large and very large farm was 34.58% and 15.52%, respectively. In conclusion, the study shows that more than half of the farms were positive for A. suum and coccidia oocysts, but mainly for younger females. In general, sows with up to two parturitions and small farms showed a higher endoparasites percentage.

Resumo O objetivo deste estudo foi o de avaliar o efeito de tamanho de granja (TG) e a ordem de parição (OP) sobre a ocorrência de ovos de endoparasitas em matrizes suínas comerciais alojadas na maternidade e gestação durante o período de maio a julho de 2014. Quarenta e três unidades produtoras de leitões foram classificadas por TG: pequena (100 a 250 porcas), média (251 a 510 porcas), grande (511 a 1.000 porcas) e muito grande (mais de 1.000 porcas). As porcas foram classificadas por OP: até dois, três a cinco e mais que cinco partos. As amostras fecais foram processadas usando a técnica de flotação em solução salina hipersaturada a 30-35%. Os resultados revelaram que a prevalência global de endoparasitas gastrointestinais obtidos neste estudo foi de 13,59%, em que 4,64% foram positivas para Ascaris suum, 0,56% para Trichuris suis e 8,27% para oocistos de coccídeos. A prevalência de endoparasitas obtidos para fazendas de pequeno e médio porte, e para fazendas grandes e muito grandes foi de 34,58% e 15,52%, respectivamente. Em conclusão, o estudo mostra que mais da metade das fazendas foram positivas para A. suum e oocistos de coccídeos, mas principalmente para as fêmeas mais jovens. Em geral, as porcas com até dois partos e pequenas propriedades mostraram uma porcentagem maior de endoparasitas.

Genetic Diversity of Ascaris in China Assessed Using Simple Sequence Repeat Markers

The giant roundworm Ascaris infects pigs and people worldwide and causes serious diseases. The taxonomic relationship between Ascaris suum and Ascaris lumbricoides is still unclear. The purpose of the present study was to investigate the genetic diversity and population genetic structure of 258 Ascaris specimens from humans and pigs from 6 sympatric regions in Ascaris-endemic regions of China using existing simple sequence repeat data. The microsatellite markers showed a high level of allelic richness and genetic diversity in the samples. Each of the populations demonstrated excess homozygosity (Ho 0). According to a genetic differentiation index (Fst=0.0593), there was a high-level of gene flow in the Ascaris populations. A hierarchical analysis on molecular variance revealed remarkably high levels of variation within the populations. Moreover, a population structure analysis indicated that Ascaris populations fell into 3 main genetic clusters, interpreted as A. suum, A. lumbricoides, and a hybrid of the species. We speculated that humans can be infected with A. lumbricoides, A. suum, and the hybrid, but pigs were mainly infected with A. suum. This study provided new information on the genetic diversity and population structure of Ascaris from human and pigs in China, which can be used for designing Ascaris control strategies. It can also be beneficial to understand the introgression of host affiliation.

Effects of Disinfectants on Larval Development of Ascaris suum Eggs

The objective of this study was to evaluate the effects of several different commercial disinfectants on the embryogenic development of Ascaris suum eggs. A 1-ml aliquot of each disinfectant was mixed with approximately 40,000 decorticated or intact A. suum eggs in sterile tubes. After each treatment time (at 0.5, 1, 5, 10, 30, and 60 min), disinfectants were washed away, and egg suspensions were incubated at 25℃ in distilled water for development of larvae inside. At 3 weeks of incubation after exposure, ethanol, methanol, and chlorohexidin treatments did not affect the larval development of A. suum eggs, regardless of their concentration and treatment time. Among disinfectants tested in this study, 3% cresol, 0.2% sodium hypochlorite and 0.02% sodium hypochlorite delayed but not inactivated the embryonation of decorticated eggs at 3 weeks of incubation, because at 6 weeks of incubation, undeveloped eggs completed embryonation regardless of exposure time, except for 10% povidone iodine. When the albumin layer of A. suum eggs remained intact, however, even the 10% povidone iodine solution took at least 5 min to reasonably inactivate most eggs, but never completely kill them with even 60 min of exposure. This study demonstrated that the treatment of A. suum eggs with many commercially available disinfectants does not affect the embryonation. Although some disinfectants may delay or stop the embryonation of A. suum eggs, they can hardly kill them completely.

Seropositivity for ascariosis and toxocariosis nd cytokine expression among the indigenous people in the Venezuelan delta region / Seropositividad para ascariosis y toxocariosis y expresión de citocinas entre la población indígena de la región del delta Venezolano

The present study aimed at measuring seropositivities for infection by Ascaris suum and Toxocara canis using the excretory/secretory (E/S) antigens from Ascaris suum (AES) and Toxocara canis (TES) within an indigenous population. In addition, quantification of cytokine expressions in peripheral blood cells was determined. A total of 50 Warao indigenous were included; of which 43 were adults and seven children. In adults, 44.1% were seropositive for both parasites; whereas children had only seropositivity to one or the other helminth. For ascariosis, the percentage of AES seropositivity in adults and children was high; 23.3% and 57.1%, respectively. While that for toxocariosis, the percentage of TES seropositivity in adults and children was low; 9.3% and 14.3%, respectively. The percentage of seronegativity was comparable for AES and TES antigens in adults (27.9%) and children (28.6%). When positive sera were analyzed by Western blotting technique using AES antigens; three bands of 97.2, 193.6 and 200.2 kDas were mostly recognized. When the TES antigens were used, nine major bands were mostly identified; 47.4, 52.2, 84.9, 98.2, 119.1, 131.3, 175.6, 184.4 and 193.6 kDas. Stool examinations showed that Blastocystis hominis, Hymenolepis nana and Entamoeba coli were the most commonly observed intestinal parasites. Quantification of cytokines IFN-γ, IL-2, IL-6, TGF-β, TNF-α, IL-10 and IL-4 expressions showed that there was only a significant increased expression of IL-4 in indigenous with TES seropositivity (p < 0.002). Ascaris and Toxocara seropositivity was prevalent among Warao indigenous.

El objetivo del presente estudio fue determinar la seropositividad de infección por Ascaris suum y Toxocara canis, utilizando antígenos de excreción/secreción (E/S) de Ascaris suum (AES) y Toxocara canis (TES) en una población indígena. Adicionalmente, se cuantificó la expresión de citocinas a partir de células de sangre periférica. Un total de 50 indígenas Warao se incluyeron en el estudio; 43 fueron adultos y 7 niños. Entre los adultos, 44,1% fueron seropositivos para ambos parásitos; mientras que los niños sólo mostraron seropositividad a uno u otro de los helmintos. Para ascariosis, el porcentaje de seropositividad para los antígenos AES fue alto tanto en adultos como en niños; 23,3% y 57,1%, respectivamente. Para toxocariosis, el porcentaje de seropositividad para los antígenos TES fue bajo en adultos así como en niños; 9,3% y 14,3%, respectivamente. El porcentaje de seronegatividad fue similar tanto para los antígenos AES como para TES en adultos (27,9%) y niños (28,6%). Cuando la seropositividad fue analizada a través de la técnica de Western blotting utilizando los antígenos AES; 3 bandas de 97,2, 193,6 y 200,2 kDas fueron principalmente reconocidas. Para los antígenos TES, 9 bandas fueron mayormente identificadas; 47,4, 52,2, 84,9, 98,2, 119,1, 131,3, 175,6, 184,4 y 193,6 kDas. Los análisis coproparasitológicos mostraron que los parásitos Blastocystis hominis, Hymenolepis nana y Entamoeba coli fueron los parásitos intestinales más comúnmente observados. La cuantificación de la expresión de las citocinas IFN-γ, IL-2, IL-6, TGF-β, TNF-α, IL-10 e IL-4 mostró que hubo un significante incremento de la expresión de IL-4 entre los indígenas con seropositividad para los antígenos TES (p < 0.002). La seropositividad para Ascaris y Toxocara fue prevalente entre los indígenas Warao.

Ascaris suum in pigs of the Zona da Mata, Minas Gerais State, Brazil / Ascaris suum em suínos da Zona da Mata, Estado de Minas Gerais, Brasil

Among the parasites that affect pigs, Ascaris suum stands out for causing the greatest losses to livestock production systems. This parasite can be monitored during the slaughter of animals through the identification of milk spots or white patches on the liver caused by its larval migration. However, infection in the herd is usually subclinical, which is why the presence of this parasite in industrial pig production has been overlooked. The aim of the study was therefore to evaluate the occurrence of milk spots on the liver of animals slaughtered in the micro-region of Ponte Nova in the Zona da Mata - Minas Gerais, Brazil, and to associate these lesions with the time of year, herd size and source of origin of the animals. An evaluation was made of 1,069 lots, totaling 108,073 animals, based on data extracted from the Federal Inspection Service. The animals were slaughtered during the period of January 2011 to June 2013. Out of the total number of slaughtered animals, 10,535 (9.75%) tested positive for these lesions. Therefore, veterinarians and producers should be warned about the inefficiency of the deworming protocols that are used, and the need to develop and/or review control strategies for this parasite in production systems.

Dentre os parasitas que acometem os suínos, Ascaris suumdestaca-se como o mais impactante nos sistemas de criação. Seu monitoramento pode ser realizado durante o abate dos animais, por meio da identificação de milk spots ou manchas de leite presentes no fígado, decorrentes da sua migração larval. Entretanto devido ao fato da infecção ocorrer no rebanho normalmente de forma subclínica, a presença desse parasita na produção industrial de suínos vem sendo negligenciada. O objetivo do estudo foi avaliar a ocorrência de manchas de leite no fígado de animais abatidos na micro-região de Ponte Nova, na Zona da Mata de Minas Gerais - Brasil, e associar tais lesões com a época do ano, tamanho do rebanho e local de origem dos animais. Foram avaliados 1.069 lotes, totalizando 108.073 animais. Os dados foram extraídos do Serviço de Inspeção Federal, e eram referentes aos animais abatidos, durante o período de janeiro de 2011 a junho de 2013. Do total de animais abatidos, 10.535 (9,75%) foram positivos para a lesão. Portanto, veterinários e produtores devem ser alertados quanto à ineficiência dos protocolos de vermifugação utilizados e a necessidade do desenvolvimento e/ou revisão de estratégias de controle para este parasita na produção.

Effects of Some Pesticides on Development of Ascaris suum Eggs

To evaluate the effects of pesticides to parasite eggs, Ascaris suum eggs were incubated with 5 different pesticides (1:1,500-1:2,000 dilutions of 2% emamectin benzoate, 5% spinetoram, 5% indoxacarb, 1% deltamethrin, and 5% flufenoxuron; all v/v) at 20degrees C for 6 weeks, and microscopically evaluated the egg survival and development on a weekly basis. The survival rate of A. suum eggs incubated in normal saline (control eggs) was 90+/-3% at 6 weeks. However, the survival rates of eggs treated with pesticides were 75-85% at this time, thus significantly lower than the control value. Larval development in control eggs commenced at 3 weeks, and 73+/-3% of eggs had internal larvae at 6 weeks. Larvae were evident in pesticide-treated eggs at 3-4 weeks, and the proportions of eggs carrying larvae at 6 weeks (36+/-3%-54+/-3%) were significantly lower than that of the control group. Thus, pesticides tested at levels similar to those used in agricultural practices exhibited low-level ovicidal activity and delayed embryogenesis of A. suum eggs, although some differences were evident among the tested pesticides.

Cytosolic Ca(2+) as a multifunctional modulator is required for spermiogenesis in Ascaris suum

The dynamic polar polymers actin filaments and microtubules are usually employed to provide the structural basis for establishing cell polarity in most eukaryotic cells. Radially round and immotile spermatids from nematodes contain almost no actin or tubulin, but still have the ability to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein (MSP) during spermiogenesis (sperm activation). However, the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly understood. Here we show that Ca(2+) oscillations induced by the Ca(2+) release from intracellular Ca(2+) store through inositol (1,4,5)-trisphosphate receptor are required for Ascaris suum sperm activation. The chelation of cytosolic Ca(2+) suppresses the generation of a functional pseudopod, and this suppression can be relieved by introducing exogenous Ca(2+) into sperm cells. Ca(2+) promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly. On the other hand, Ca(2+) promotes MSP disassembly by activating Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase calcineurin. In addition, Ca(2+)/camodulin activity is required for the fusion of sperm-specifi c membranous organelle with the plasma membrane, a regulated exocytosis required for sperm motility. Thus, Ca(2+) plays multifunctional roles during sperm activation in Ascaris suum.

Effect of Temperature on Embryonation of Ascaris suum Eggs in an Environmental Chamber

The influence of temperature on the development and embryonation of Ascaris suum eggs was studied using coarse sand medium in an environmental chamber with 50% humidity. The time required for development and embryonation of eggs was examined under 3 different temperature conditions, 5degrees C, 25degrees C, and 35degrees C. A. suum eggs did not develop over 1 month at the temperature of 5degrees C. However, other temperature conditions, 25degrees C and 35degrees C, induced egg development to the 8-cell-stage at days 5-6 after incubation. All eggs examined developed to the 8-cell stage at day 6 after incubation in the sand medium at 25degrees C. The higher temperature, 35degrees C, slightly accelerated the A. suum egg development compared to 25degrees C, and the development to the 8-cell stage occurred within day 5 after incubation. The formation of larvae in A. suum eggs at temperatures of 35degrees C and 25degrees C appeared at days 17 and 19 after incubation, respectively. These findings show that 35degrees C condition shortens the time for the development of A. suum eggs to the 8-cell-stage in comparison to 25degrees C, and suggest the possibility of accelerated transmission of this parasite, resulting from global warming and ecosystem changes.

Quantitative Evaluation of Viability- and Apoptosis-Related Genes in Ascaris suum Eggs under Different Culture-Temperature Conditions

Ascaris suum eggs are inactivated by composting conditions; however, it is difficult to find functional changes in heat-treated A. suum eggs. Here, unembryonated A. suum eggs were incubated at 20degrees C, 50degrees C, and 70degrees C in vitro, and the gene expression levels related to viability, such as eukaryotic translation initiation factor 4E (IF4E), phosphofructokinase 1 (PFK1), and thioredoxin 1 (TRX1), and to apoptosis, such as apoptosis-inducing factor 1 (AIF1) and cell death protein 6 (CDP6), were evaluated by real-time quantitative RT-PCR. No prominent morphological alterations were noted in the eggs at 20degrees C until day 10. In contrast, the eggs developed rapidly, and embryonated eggs and hatched larvae began to die, starting on day 2 at 50degrees C and day 1 at 70degrees C. At 20degrees C, IF4E, PFK1, and TRX1 mRNA expression was significantly increased from days 2-4; however, AIF1 and CDP6 mRNA expression was not changed significantly. IF4E, PFK1, and TRX1 mRNA expression was markedly decreased from day 2 at 50degrees C and 70degrees C, whereas AIF1 and CDP6 mRNA expression was significantly increased. The expressions of HSP70 and HSP90 were detected for 9-10 days at 20degrees C, for 3-5 days at 50degrees C, and for 2 days at 70degrees C. Taken together, incremental heat increases were associated with the rapid development of A. suum eggs, decreased expression of genes related to viability, and earlier expression of apoptosis-related genes, and finally these changes of viability- and apoptosis-related genes of A. suum eggs were associated with survival of the eggs under temperature stress.

Caracterização bioquímica e funcional de PAS-1, proteína imunossupressora de Ascaris suum / Biochemical and functional characterization of PAS-1, the immunossupressive protein from Ascaris suum

Os efeitos de infecções parasitárias na responsividade do sistema imune do hospedeiro têm sido amplamente estudados. Muitos destes resultados sugerem que esta imunomodulação possa ser um dos mecanismos utilizados para promover a sobrevivência do parasita. Neste sentido, as infecções crônicas gastrointestinais por nematóides são particularmente intrigantes, pois os mecanismos que os parasitas utilizam para se evadir da imunidade do hospedeiro são eficazes e de natureza diversa. Com relação ao Ascaris suum, apesar de produzir proteínas que são potentes alérgenos, tanto a infecção experimental como extratos de vermes adultos suprimem substancialmente a resposta imune do hospedeiro. Em nosso laboratório isolamos uma proteína denominada PAS-1(Proteína Ascaris suum), presente no extrato bruto de vermes adultos de Ascaris suum, que possui uma intensa atividade supressora sobre a resposta imune humoral e celular. Assim, esta proteína PAS-1 e o anticorpo monoclonal contra ela dirigido (MAIP-1) são ferramentas importantes para os estudos sobre a imunomodulação induzida por Ascaris suum. Neste trabalho investigamos a presença de PAS-1 no sobrenadante de cultura de ovos embrionados e no fluído pseudocelomático de vermes adultos, e comprovamos que PAS-1 é uma proteína secretada e excretada por larvas em diferentes estágios precoce do ciclo vital do parasita e também por vermes adultos. Nossos resultados demonstram que a proteína PAS-1 possui aproximadamente 200 KDa, com N-terminal His-His-Phe-Thr-Leu-Glu-Ser-Ser-Leu-Asp-Thr, caracterizando uma homologia com uma proteína de Ascaris lumbricóides (ABA-1). Por outro lado, PAS-1 suprime intensamente a resposta inflamatória aguda induzida por LPS, inibindo a produção de citocinas pro-inflamatórias e estimulando a secreção de IL-10. Além disso, a atividade anti-inflamatória de PAS-1 foi totalmente revertida pelo...

Effects of Kimchi Extract and Temperature on Embryostasis of Ascaris suum Eggs

To determine the effects of kimchi extracts at different temperatures on larval development, Ascaris suum eggs were mixed with soluble part of 7 different brands of commercially available kimchi and preserved at either 5degrees C or 25degrees C for up to 60 days. A. suum eggs incubated at 25degrees C showed marked differences in larval development between kimchi extract and control group. While all eggs in the control group completed embryonation by day 21, only 30% of the eggs in the kimchi extract group became embryonated by day 36 and about 25% never became larvated even at day 60. At 5degrees C, however, none of the eggs showed larval development regardless of the incubation period or type of mixture group. To determine the survival rate of A. suum eggs that showed no embryonation after being preserved at 5degrees C, eggs preserved in kimchi extracts for 14, 28, and 60 at 5degrees C were re-incubated at 25degrees C for 3 weeks in distilled water. While all eggs in the control group became larvated, eggs in the kimchi extract group showed differences in their embryonation rates by the incubation period; 87.4 % and 41.7% of the eggs became embryonated after being refrigerated for 14 days and 28 days, respectively. When refrigerated for 60 days, however, no eggs mixed in kimchi extract showed larval development. Our results indicate that embryogenesis of A. suum eggs in kimchi extract was affected by duration of refrigeration, and that all eggs stopped larval development completely in kimchi kept at 5degrees C for up to 60 days.

Ocorrência de Ascaris suum em suínos abatidos na Zona da Mata, Minas Gerais / Occurence of the Ascaris suum in slaughtered swine in Zona da Mata region, Minas Gerais State

Os suínos são acometidos por diversas enfermidades e as verminoses se constituem como as principais causas de nãoganho de peso, competição e diminuição da conversão alimentar, descarte de vísceras ao abate e perdas financeiras. Onematoide Ascaris suum se constitui na principal parasitose na suinocultura nacional. Esse trabalho teve como objetivoavaliar o nível de infestação por A. suum em suínos em idade de abate provenientes de granjas tecnificadas localizadasna região do município de Piranga, região oeste da Zona da Mata, Minas Gerais, latitude 20º45’45’’S, longitude43º18’10’’W. Os animais foram abatidos em três matadouros localizados no município de Piranga, e foram observadosos tratos gastrintestinais dos suínos abatidos quanto à presença desse parasito. Foi observada a presença do nematoideem animais provenientes de 90% das granjas e a ocorrência total foi 22,4%, média de todo o período experimental.Também foi observado que, durante o período experimental, a ocorrência de A. suum no lume intestinal dos animaisnão apresentou diminuição e os protocolos de everminação não foram eficazes para a diminuição da ocorrência desseshelmintos nas propriedades onde foram empregados.

Swine are affected by several diseases and worms constitute the main causes of absence of weight gain, competitionand decreased feed conversion, discards of slaughter viscera and financial losses. The nematode Ascaris suum remainsa major parasitic disease in national livestock pigs. The objective of this work was to evaluate the level of infestationby A. suum in pigs at slaughter age provenient from intensive production systems located in Zona da Mata region,latitude 20º45’45’’S, longitude 43°18’10’’W, in tropical Southeastern, Brazil. The animals were slaughtered at threeslaughterhouses located in the municipality of Piranga, and their gastrointestinal tracts were observed in order toidentify the presence of those helminthes. We observed the presence of nematode in animals from 90% of the farms andthe total occurrence was 22.4%, media of all the experimental period. It was also observed that, during the experimentalperiod, the occurrence of A. suum in the gastrointestinal tube did not present reduction and the protocol to administeranthelmintic were not efficient to decrease the occurrence this helminthes in proprieties in which they were applied...

Boosting the suppressive effects of Ascaris suum components in IFN-γ-deficient mice / Potencialização do efeito supressor de componentes do Ascaris suum em camundongos deficientes em IFN-γ

High molecular weight components from Ascaris suum extract suppress ovalbumin-specific immunity in mice. In IFN-γ-deficient mice, ovalbumin-specific delayed-type hypersensitivity reactions are more strongly downregulated by these suppressive components. Here, the cellularity of the delayed-type hypersensitivity reaction in IFN-γ-deficient mice and the increased downregulation induced by Ascaris suum components were analyzed. IL-12p40-dependent neutrophilic influx was predominant. Suboptimal doses of the suppressive fraction from this nematode completely inhibited the hypersensitivity reaction, thus indicating intensification of the immunosuppression under conditions of intense recruitment of IFN-γ-independent neutrophils.

Componentes de alto peso molecular do extrato de Ascaris suum suprimem a imunidade específica à ovalbumina em camundongos. Em camundongos geneticamente deficientes de IFN-γ a reação de hipersensibilidade tardia específica para ovalbumina foi mais fortemente prejudicada por estes componentes supressivos. Aqui, a celularidade da reação de hipersensibilidade tardia em camundongos deficientes de IFN-γ e o incremento na supressão induzida por componentes do Ascaris suum foram analisados. Influxo neutrofílico, dependente de IL-12p40, foi predominante. Dose sub-ótima da fração supressiva do nematódeo inibiu completamente a reação de hipersensibilidade, indicando uma intensificação da imunossupressão em condições de recrutamento intenso de neutrófilos independente de IFN-γ.

Antígenos de larvas pulmonares de Ascaris suum reconocidos por anticuerpos producidos en Oryctolagus cuniculus / Ascaris suum lung larvae antigens recognized by antibodies produced in Oryctolagus cuniculus

El presente trabajo tuvo como objetivo determinar mediante la técnica de Western Blot los antígenos de larvas pulmonares de Ascaris suum que son detectados por anticuerpos producidos en Oryctolagus cuniculus inmunizado experimentalmente. Las larvas pulmonares (L3 y L4) fueron obtenidas en 120 ejemplares de Mus musculus cepa BALB/c ratón infectados experimentalmente por vía oral con huevos infectivos de A. suum. Parte de estas larvas fueron cultivadas en el medio Eagle (MEM) para la obtención de antígenos de excreción/secreción y la otra fue sonificada para la obtención de antígenos somáticos, los cuales sirvieron para inmunizar dos ejemplares de O. cuniculus, utilizando Adyuvante Completo e Incompleto de Freund. A las 5 semanas de inmunización se obtuvo sangre de los conejos por punción cardiaca a fin de recuperar el suero, parte del cual fue purificado parcialmente por precipitación salina y diálisis. Mediante la técnica de electroinmuno-transferencia (Western Blot) y usando sueros de los conejos inmunizados se detectaron en los antígenos de excreción/secreción de 20 horas de cultivo reducidos con dithiothreitol, 12 bandas antigénicas de 100, 72.4, 56.2, 42.7, 39.8, 34.6, 31.6, 30.2, 19.5, 16.9, 15.5 y 14.9 KDa, siendo las más reactivas las de 100, 72.4, 16.9, 15.5 y 14.9 KDa. En los antígenos somáticos bajo condiciones de reducción, se detectaron solamente seis bandas de 42.7, 39.8, 34.6, 30.2, 28 y 25.2 KDa de poca reactividad. Estos resultados permiten afirmar que los antígenos de excreción/secreción de A. suum de 20 horas de incubación en el medio MEM inducen la producción de un mayor número de anticuerpos de tipo IgG en conejos inmunizados experimen-talmente.

Excretory/secretory antigens (E/SAg) and somatic antigens (SAg) of Ascaris suum lung larvae that induce the immunoglobulin G antibodies production in Oryctolagus cuniculus experimentally immunized was determined. For this purposes, specimens of Mus musculus BALB/c were inoculated orally with infective eggs of A. suum obtained from pigs naturally parasitized in order to obtain the lung larvae. Part of these larvae was cultured in Minimum Essential Medium (MEM) to obtain E/SAg and another part was sonicated to obtain the SAg too. Both, E/SAg and SAg mixed with complete and incomplete Freund's adjuvant were used for rabbits immunization. Five weeks after the immunization, the rabbits were bled by cardiac puncture obtaining the immunosera by centrifugation, which was purified partially by saline precipitation and dialysis. By using an Western blot technique with purified immunoserum and E/SAg obtained to 20 hours and reduced with dithiothreitol, fourteen antigens bands of 100, 72.4, 56.2, 42.7, 39.8, 34.6, 31.6, 30.2, 19.5, 16.9, 15.5 y 14.9 KD, were detected. The bands of 100, 72.4, 16.9, 15.5 and 14.9 KDa were been the most reactives. Thereby the SAg, also reduced with dithiothreitol, seven bands of 42.7, 39.8, 34.6, 30.2, 28.0 and 25.2 KDa were detected. They were a bit clear. In conclusion, the E/SAg induce the highest production of immunoglobulin G antibodies in rabbits experimentally immunized.

Dinámica de la infección por ascaris suum en un granja porcina del Municipio Carlos Arvelo, Parroquia Güigüe del estado Carabobo Venezuela / Dynamic of ascaris suum on a swine farm in the Carlos Arvelo Municipality in Carabobo state Venezuela

Un estudio sobre dinámica de infección por Ascaris suum se realizó en una granja porcina del municipio Carlos Arvelo del estado Carabobo, con antecedentes de decomisos de hígados por lesiones características del parásito. Se muestrearon 525 animales para análisis coprológico, mediante la técnica McMaster y 40 tractos intestinales en matadero, para determinar cantidad de vermes y análisis coprológico (Técnicas McMaster y Wisconsin). La prevalencia observada fue 22,66 por ciento por coprología y 68,29 por ciento por recuperación de vermes. La disposición espacial de huevos en heces y vermes en tractos intestinales fue en agregados. Los casos de infección se observaron en las etapas; desarrollo (31,43 por ciento), hembras de reemplazo (75,3 por ciento) y engorde (73,3 por ciento), evdenciándose recuentos más altos de huevos por gramo de heces (HPG) en engorde. La intensidad de infección en las etapas desarrollo y engorde fue, en la mayoría de los casos, leve y en poco moderada e independiente del sexo. Se observó una correlación positiva y significativa (P menor o igual 0,05) entre: el grado de infección establecido por HPG y la cantidad de vermes recuperados, demostrándose que la coprología continúa siendo una alternativa diagnóstica válida para evaluar y clasificar esta infección. Igualmente, se demostró asociación significativa (P menor o igual 0,05) entre: carga en vermes hembras y vermes machos; la carga en vermes hembras y el HPG, así como también entre carga vermes machos y HPG. Se demostró presencia de animales falsos positivos atribuidos a la condición de coprofagia. La variación mensual de hígados decomisados por A. suum fue calculada a través del I.V.M durante el período 1996-1999. Sus valores indicaron decomisos durante todo el año, sugieriendo la existencia de condiciones climáticas favorables para el establecimiento de esta infección

Comparison of different monoclonal antibodies against immunosuppressive proteins of Ascaris suum

The extract of Ascaris suum suppresses the humoral and cellular immune responses to unrelated antigens in the mouse. In order to further characterize the suppressive components of A. suum, we produced specific monoclonal antibodies which can provide an important tool for the identification of these proteins. The A. suum immunosuppressive fractions isolated by gel filtration from an extract of adult worms were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells and the cloned hybrid cells obtained were screened to determine the specificity of secreted antibodies. Three monoclonal antibodies named MAIP-1, MAIP-2 and MAIP-3 were selected and were shown to react with different epitopes of high molecular weight proteins from the A. suum extract. All antibody molecules have kappa-type light chains but differ in heavy chain isotype. MAIP-1 is a mouse IgM, MAIP-2 is an IgA immunoglobulin and MAIP-3 is an IgG1 immunoglobulin and they recognize the antigen with affinity constants of 1.3 x 10(10) M-1, 7.1 x 10(9) M-1 and 3.8 x 10(7) M-1, respectively. The proteins recognized by these monoclonal antibodies (PAS-1, PAS-2 and PAS-3) were purified from the crude extract by affinity chromatography and injected with ovalbumin in BALB/c mice in order to determine their suppressive activity on heterologous antibody production. It was demonstrated that these three proteins are able to significantly suppress anti-ovalbumin antibody secretion, with PAS-1 being more efficient than the others.

Evolution of ascariasis in humans and pigs: a multi-disciplinary approach

The nematode parasite Ascaris lumbricoides infects the digestive tracts of over 1.4 billion people worldwide, and its sister species, Ascaris suum, has infected a countless number of domesticated and feral pigs. It is generally thought that the putative ancestor to these worms infected either humans or pigs, but with the advent of domestication, they had ample opportunity to jump to a new host and subsequently specialize and evolve into a new species. While nuclear DNA markers decisively separate the two populations, mitochondrial sequences reveal that three major haplotypes are found in A. suum and in A. lumbricoides, indicating either occasional hybridization, causing introgression of gene trees, or retention of polymorphism dating back to the original ancestral species. This article provides an illustration of the combined contribution of parasitology, archaeoparasitology, genetics and paleogenetics to the history of ascariasis. We specifically investigate the molecular history of ascariasis in humans by sequencing DNA from the eggs of Ascaris found among ancient archeological remains. The findings of this paleogenetic survey will explain whether the three mitochondrial haplotypes result from recent hybridization and introgression, due to intensive human-pig interaction, or whether their co-occurrence predates pig husbandry, perhaps dating back to the common ancestor. We hope to show how human-pig interaction has shaped the recent evolutionary history of this disease, perhaps revealing the identity of the ancestral host