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.

Caracterização molecular de Sarcocystis spp. em amostras de carne / Molecular characterization of Sarcocystis spp. in samples of meat

A sarcocistose é uma doença distribuída mundialmente, podendo acometer aves, répteis e diversos mamíferos, incluindo o homem. O objetivo desse trabalho foi detectar a presença de Sarcocystis spp. e caracterizar as espécies encontradas em 375 amostras de produtos cárneos (filé mignon bovino, carne moída bovina e salame colonial). Para isso, foi realizada a detecção do parasita através da técnica de PCR para amplificação parcial do gene 18S rRNA e sua caracterização molecular utilizando o polimorfismo no comprimento do fragmento de restrição (RFLP) com as enzimas de restrição Bcl I, Rsa I e Alu I. A ocorrência de Sarcocystis spp. foi de 17% (64/375) do total de amostras testadas pelo PCR. Entre os produtos cárneos avaliados, 5,6% (7/125) das amostras de filé mignon, 12,8% (16/125) de carne moída e 32,8% (41/125) de embutido colonial, foram positivas para presença do DNA do Sarcocystis spp. Entre estas amostras positivas, as espécies caracterizadas foram Sarcocystis hirsuta e Sarcocystis hominis com prevalências de 93,7% (60/64) e 6,3% (4/64), respectivamente. Considerando à relevância da sarcocistose na área da saúde pública, a ocorrência de S. hominis encontrado neste estudo, pode ser um fator de risco para a contaminação humana. Porém, a presença do DNA deste protozoário não significa necessariamente potencial de infecção aos humanos, pois cuidados nos processos de fabricação podem reduzir a viabilidade dos cistos.(AU)

The sarcocystosis is a worldwide spread disease and can affect birds, reptiles and many mammals, including man. The aim of this study was to detect the presence of Sarcocystis spp. and characterize the species found in 375 samples of meat products (filet mignon, ground beef and colonial salami). For this, we carried out the detection of the parasite by PCR for the amplification of the partial 18S rRNA gene and molecular characterization using the restriction fragment length polymorphism (RFLP) with restriction enzymes Bcl I, Alu I and Rsa I. The occurrence of Sarcocystis spp. was 17% (64/375) of all samples. Among the meat products evaluated, the filet mignon samples were positive in 5.6% (7/125), the ground beef in 12.8% (16/125) and the colonial salami in 32.8% (41/125). Of the positive samples, Sarcocystis hirsuta and Sarcocystis hominis were detected, with prevalence of 93.7% (60/64) and 6.3% (4/64), respectively. Considering the relevance of sarcocystosis in public health, the occurrence of S. hominis found may be a risk factor to human contamination. However, the presence of DNA of this parasite does not necessarily mean potential of infection to humans, because good practices in the manufacturing processes can reduce the viability of the cysts.(AU)

Detección molecular de Sarcocystis aucheniae en sangre de llamas de Argentina / Molecular detection of Sarcocystis aucheniae in the blood of llamas from Argentina

Sarcocystis aucheniae are apicomplexan protozoa that infect South American camelids (SACs), giving rise to macroscopic cysts similar to rice grains in skeletal muscles. Visual detection of macrocysts in slaughtered animals hampers commercialization of SAC meat, a highly relevant economic exploitation for Andean rural families. Importantly, the consumption of undercooked S. aucheniae-infested meat causes gastroenteritis. A carnivore definitive host, possibly the dog, acquires the parasite when feeding on infected SAC meat, and later eliminates infective oocysts in its feces. The parasite cycle is completed when SACs ingest contaminated water or pastures. We hypothesized that parasite DNA can be detected in SAC blood using molecular methods. In order to test this hypothesis, a seminested PCR format was specifically designed to target the hypervariable 18S rRNA gene region of S. aucheniae. PCR conditions were optimized using genomic DNA extracted from macrocyst bradyzoites. A detection limit of up to 1 parasite in 10 μl of llama blood was established based on DNA samples extracted from aliquots of S. aucheniae bradyzoite-spiked non-infected llama blood. The seminested PCR allowed to detect natural infections of S. aucheniae in llama blood samples originating in the Andean flatlands of Argentina. Specific amplification of S. aucheniae DNA was corroborated by amplicon sequencing. This is the first report of S. aucheniae detection in llama blood, which provides a valuable diagnostic tool for epidemiological studies and for the evaluation of the efficacy of control measures for this parasitosis.

Sarcocystis aucheniae es un protozoo apicomplexa que infecta a camélidos sudamericanos (CS), dando lugar a la formación de quistes macroscópicos similares a granos de arroz en los músculos esqueléticos. La detección visual de macroquistes en animales faenados dificulta la comercialización de la carne de CS, una explotación de gran relevancia para la economía de las familias rurales andinas. Es importante destacar que el consumo de carne infectada con S. aucheniae no suficientemente cocida causa gastroenteritis. Un hospedador definitivo carnívoro, posiblemente el perro, adquiere el parásito cuando se alimenta de carne de CS infectada y luego elimina ooquistes infectivos en las heces. El ciclo del parásito se completa cuando un CS ingiere agua o pasturas contaminadas. Hemos hipotetizado que es posible detectar ADN del parásito en la sangre de CS usando métodos moleculares. Para poner a prueba esta hipótesis, se diseñó una PCR semianidada que utiliza como blanco una región del gen 18S ARNr específica para S. aucheniae. Se optimizaron las condiciones de la PCR usando ADN genómico extraído de bradizoítos presentes en macroquistes. Se estableció un límite de detección de un parásito en 10 μl de sangre de llama, basado en muestras de ADN extraído de alícuotas de sangre de llama no infectada a las que se agregaron cantidades conocidas de bradizoítos de S. aucheniae. Más aún, la PCR semianidada permitió la detección de infecciones naturales por este parásito en muestras de sangre de llama de la Puna argentina. La amplificación específica de ADN de S. aucheniae fue corroborada por secuenciación de los productos de amplificación. Este es el primer reporte de la detección de S. aucheniae en sangre de llama. Además, este estudio contribuye una herramienta diagnóstica valiosa para estudios epidemiológicos y para la evaluación de la efectividad de medidas de control para esta parasitosis.

Natural Sarcocystis gigantea infection in sheep from Southern Brazil / Infecção natural por Sarcocystis gigantea em ovinos no Rio Grande do Sul

ABSTRACT: Protozoal diseases caused by species of Sarcocystiscan cause serious damage in sheep flocks, inducing decreased growth conversion rates and partial or complete loss of carcasses at the slaughterhouse. This article describes an outbreak of Sarcocystis giganteainfection in sheep slaughtered in a farm in Rio Grande do Sul State, Brazil. Between July and September 2013, three sheep showed multiple nodules in the esophagus that were microscopically characterized as encapsulated cysts filled with elongated, basophilic, nucleated structures morphologically consistent with S. giganteabradyzoites. Diagnosis was made based on the epidemiological, macroscopic, and microscopic findings. This is the first report of this infection in sheep in Rio Grande do Sul and should be recognized by veterinarians, especially during meat inspection.

RESUMO: Doenças causadas por protozoários do gênero Sarcocystispodem causar sérios prejuízos em rebanhos de ovinos, por induzirem retardo no crescimento e condenação parcial ou total das carcaças em abatedouro. O presente trabalho descreve a infecção natural por Sarcocystis giganteaem ovinos abatidos em uma propriedade rural no Rio grande do Sul. Entre julho e setembro de 2013, três ovinos apresentaram múltiplos nódulos no esôfago, os quais, microscopicamente, corresponderam a cistos circundados por cápsula rugosa, repletos de estruturas alongadas, basofílicas e nucleadas, morfologicamente compatíveis com bradizoítos de S. gigantea.Os dados epidemiológicos unidos aos achados macroscópicos e microscópicos permitiram o diagnóstico conclusivo. Esse foi o primeiro relato dessa infecção no Rio Grande Sul e deve ser reconhecida por veterinários de campo e especialmente no momento da inspeção das carcaças no abatedouro.

Human and animal sarcocystosis in Malaysia:A review

Sarcocystosis is a zoonotic disease caused by a coccidian intracellular protozoan parasite of the genus Sarcocystis. More than 200 Sarcocystis species have been recorded and the parasites are found in mammals, birds and reptiles. They require two hosts to complete their life cycle. In Malaysia, sarcocystosis was reported as a potential emerging food and water-borne disease after a series of large outbreak of human infections. There was not enough attention given before even though it was reported in both humans and animals. The first human case of invasive muscular sarcocystosis among local Malaysian was reported in 1975. Besides, a retrospective autopsy examination on 100 tongues revealed 21% positive cases. On top of that, a sero-epidemiological survey conducted in 243 subjects in West Malaysia showed that 19.7% had Sarcocystis antibodies. The clinical symptoms of muscular sarcocystosis were first described comprehensively in 1999. Meanwhile, many types of animals including livestock were found harbor the sarcocysts in their tissue. The first case of human intestinal sarcocystosis was reported in 2014. This review indicates that human sarcocystosis is currently endemic in Malaysia and parallel to that reported in animals. However, more studies and investigations need to be conducted since the source of human infection remains unknown.

Human and animal sarcocystosis in Malaysia: A review

Sarcocystosis is a zoonotic disease caused by a coccidian intracellular protozoan parasite of the genus Sarcocystis. More than 200 Sarcocystis species have been recorded and the parasites are found in mammals, birds and reptiles. They require two hosts to complete their life cycle. In Malaysia, sarcocystosis was reported as a potential emerging food and water-borne disease after a series of large outbreak of human infections. There was not enough attention given before even though it was reported in both humans and animals. The first human case of invasive muscular sarcocystosis among local Malaysian was reported in 1975. Besides, a retrospective autopsy examination on 100 tongues revealed 21% positive cases. On top of that, a sero-epidemiological survey conducted in 243 subjects in West Malaysia showed that 19.7% had Sarcocystis antibodies. The clinical symptoms of muscular sarcocystosis were first described comprehensively in 1999. Meanwhile, many types of animals including livestock were found harbor the sarcocysts in their tissue. The first case of human intestinal sarcocystosis was reported in 2014. This review indicates that human sarcocystosis is currently endemic in Malaysia and parallel to that reported in animals. However, more studies and investigations need to be conducted since the source of human infection remains unknown.

Surveillance For Sarcocystosis In Tioman Island, Malaysia

In October 2011, the National International Health Regulations (IHR) 2005 Focal Point for Malaysia received notification from the United States’ Centers for Disease Control and Prevention (CDC) of a probable Sarcocystis outbreak amongst 23 travellers from six countries who had vacationed on Tioman Island between June and August 2011. The Ministry of Health, Malaysia (MOH) in collaboration with the Department of Veterinary Services, Malaysia (DVS) conducted a cross sectional study in November 2011 to determine the presence of Sarcocystosis among humans, animals and in the environment in Tioman Island. Epidemiological investigations conducted involved a community health survey of 44 residents in Kampung Salang, Tioman and review of outpatient attendance cards for suspected or confirmed cases of Sarcocystosis. Twenty-eight fresh stool samples were collected and sent to the National Public Health Laboratory (NPHL) for detection of Sarcocystis oocysts using fluorescence microscopy. Water samples taken from 27 water sampling points around the island were processed and analysed under the fluorescence microscope using ultraviolet (UV) light at the Institute for Medical Research (IMR) to detect the presence of Sarcocystis sporocyst. DVS collected 84 faecal samples from four types of domesticated animals and then analysed them at the Veterinary Services Centre in Tioman Island for Sarcocystis oocysts and other parasitic ova and cysts using qualitative Floatation Technique. The results showed that Sarcocystis was not present in humans, animals and in the environment in Tioman Island during the study period. Further surveillance among humans, wildlife and the environment is needed to determine Sarcocystis endemicity in Tioman Island.

Sarcocystosis among Wild Captive and Zoo Animals in Malaysia

Sarcocystis sp. infection was investigated in 20 necropsied captive wild mammals and 20 birds in 2 petting zoos in Malaysia. The gross post-mortem lesions in mammals showed marbling of the liver with uniform congestion of the intestine, and for birds, there was atrophy of the sternal muscles with hemorrhage and edema of the lungs in 2 birds. Naked eye examination was used for detection of macroscopic sarcocysts, and muscle squash for microscopic type. Only microscopically visible cysts were detected in 8 animals and species identification was not possible. Histological examination of the sections of infected skeletal muscles showed more than 5 sarcocysts in each specimen. No leukocytic infiltration was seen in affected organs. The shape of the cysts was elongated or circular, and the mean size reached 254 x 24.5 micrometer and the thickness of the wall up to 2.5 micrometer. Two stages were recognized in the cysts, the peripheral metrocytes and large numbers of crescent shaped merozoites. Out of 40 animals examined, 3 mammals and 5 birds were positive (20%). The infection rate was 15% and 25% in mammals and birds, respectively. Regarding the organs, the infection rate was 50% in the skeletal muscles followed by tongue and heart (37.5%), diaphragm (25%), and esophagus (12.5%). Further ultrastructural studies are required to identify the species of Sarcocystis that infect captive wild animals and their possible role in zoonosis.