RESUMO
BACKGROUND Human trichinellosis is a foodborne parasitic zoonotic disease caused by ingestion of raw or undercooked meat infected with nematode larvae of the genus Trichinella. In the USA, sporadic cases and outbreaks caused by the consumption of wild game meat infected with Trichinella have been reported. The current methods for diagnosis such as serology and microscopy are not specific, may result in false negative results, and cannot differentiate encapsulated Trichinella larvae to species level. The molecular protocols currently available for the differentiation of all encapsulate Trichinella species prevalent in North America have some limitations such as the inability to identify and resolve the presence of several Trichinella species in a single test. OBJECTIVES/METHODS In this study we developed and evaluated a multiplex TaqMan quantitative real-time polymerase chain reaction (qPCR) assay, which can simultaneously detect, identify and differentiate all species of encapsulated Trichinella occurring in North America i.e., T. nativa, T. spiralis, T. murrelli and Trichinella T6, even in cases of multiple infection in a single sample. We investigated two human biopsies and 35 wild animal meat samples considered as having a high likelihood of harboring Trichinella larvae obtained from the United States during 2009-2017. FINDINGS Using the multiplex assay describe here, 22 (59%) samples that tested positive contained Trichinella spp., were identified as: T. nativa (n = 7, including a human biopsy), T. spiralis (n = 9, including a human biopsy), T. murrelli (n = 3), Trichinella T6 (n = 1). Results also included two rare mixed infection cases in bears, a T. nativa/T. spiralis from Alaska and a T. spiralis/Trichinella T6 from California. The species identifications were confirmed using a conventional PCR targeting the rRNA ITS1-ITS2 region, followed by DNA sequencing analysis. The estimated limit of detection (LOD) was approximately seven larvae per gram of meat. MAIN CONCLUSIONS Differentiation of Trichinella spp. is needed to improve efforts on identification of case, optimize food safety control and better understand the geographic distribution of Trichinella species. The Trichinella qPCR multiplex proved to be a robust, easy to perform assay and is presented as an improved technique for identification of all known encapsulated species occurring in North America continent.
Assuntos
Animais Selvagens/parasitologia , Trichinella/genética , Triquinelose/veterinária , Animais , Humanos , Reação em Cadeia da Polimerase Multiplex/veterinária , América do Norte/epidemiologia , Prevalência , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Trichinella/classificação , Trichinella/isolamento & purificação , Triquinelose/epidemiologia , Triquinelose/parasitologiaRESUMO
Trichinella patagoniensis, a new species of Trichinella, is widespread in Argentina. The success of parasite transmission depends, among other factors, on the resistance of L1 larvae present in the muscle tissue (ML) of dead hosts undergoing the decomposition process in different environmental conditions. The aim of the present work was to study the infectivity of T. patagoniensis muscle larvae in Cavia porcellus and the capability of the parasite to survive in decomposed muscle tissue of guinea pigs subjected to different environmental conditions. Thirty-two female Ssi:AL guinea pigs were orally inoculated with 2000 ML of T. patagoniensis (ISS2311). All the animals were sacrificed 42 days post-infection. Twenty-six animals were eviscerated, and carcasses were placed on the surface of soil inside plastic boxes that were exposed to environmental conditions in the summer 2014-2015 and autumn of 2015 in Buenos Aires, Argentina. Carcasses from six animals were placed into a plastic box inside the refrigerator at a temperature of 4 °C. The muscle tissue samples from the carcasses were examined weekly for the presence of larvae, and the infectivity of recovered ML was tested in BALB/c mice. Our results showed for the first time the ability of T. patagoniensis to complete its life cycle in guinea pigs, thus serving as a potential natural host. Also, larvae of T. patagoniensis remained infective in muscle tissue for several weeks while undergoing decomposition under different environmental conditions.
Assuntos
Músculos/parasitologia , Trichinella/classificação , Trichinella/fisiologia , Animais , Argentina , Feminino , Cobaias , Larva/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Temperatura , Triquinelose/parasitologiaRESUMO
Prior to this study, only encapsulated species of Trichinella had been found in South America, i.e., T. spiralis and T. patagoniensis. Here we report the molecular identification of a non-encapsulated isolate of Trichinella from a domestic pig in Argentina. The multiplex PCR technique and the analysis of mitochondrial and nuclear DNA sequences revealed that it belongs to T. pseudospiralis, which parasitises birds and mammals from Australian, Nearctic, and Palaearctic regions. Interestingly, the isolate is closely related to the Palaearctic population. This is the first report of a non-encapsulated species of Trichinella from the Neotropical region.
Assuntos
Doenças dos Suínos/parasitologia , Trichinella/classificação , Triquinelose/veterinária , Animais , Argentina/epidemiologia , Sequência de Bases , DNA de Protozoário/genética , Suínos , Doenças dos Suínos/epidemiologia , Triquinelose/epidemiologia , Triquinelose/parasitologiaRESUMO
Four different isolates of Trichinella spp. (Z1, Z2, Z3, and Z4) obtained from the skeletal muscle of street dogs in the state of Zacatecas, Mexico were serial passaged in Wistar rats; infective larvae from the skeletal muscle of the rats were collected and frozen in liquid nitrogen. After centrifugation, DNA was extracted and the 5SRNAr and IsRNAr genes were amplified. The isolates were identified by the size of the amplified products from the 5SRNAr and IsRNAr genes (750 and 290 bp, respectively). The amplicons obtained by PCR were sequenced, aligned, and compared to the reference strain Trichinella spiralis MSUS/MEX/91//EM isolated from pigs. Based on our results, we determined that the Trichinella isolates from canine (Z1-Z4) belonged to the T. spiralis species and had 83% identity with the reference strain. The phylogenetic tree constructed from the sequences showed differences between the isolates from pig and dog. These genetic differences may be related to the immune response of the host or the pathogenicity of the isolates. Therefore, these findings have important epidemiological and public health implications.
Assuntos
Doenças do Cão/parasitologia , Músculo Esquelético/parasitologia , Trichinella/genética , Triquinelose/veterinária , Animais , DNA de Helmintos/química , DNA Espaçador Ribossômico/química , Cães , Eletroforese em Gel de Ágar , México , Filogenia , RNA de Helmintos/genética , RNA Ribossômico 5S/genética , Ratos , Ratos Wistar , Alinhamento de Sequência , Trichinella/classificação , Trichinella/isolamento & purificação , Triquinelose/parasitologiaRESUMO
A bulk analysis of inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) provides a quick, reliable, and highly informative system for DNA banding patterns that permit species identification. The present study evaluates the applicability of this system to Trichinella species identification. After a single amplification carried out on a single larva with the primer 816([CA]nRY) under high stringency conditions, which provide high reproducibility, we were able to identify by consistent banding patterns 5 sibling species: Trichinella spiralis (ISS48), 2 Trichinella britovi isolates (ISS11 and ISS86), Trichinella murrelli (ISS35), Trichinella nativa (ISS71), Trichinella nelsoni (ISS29); 3 additional Trichinella genotypes: T8 (ISS149), T9 (ISS408 and ISS409), and T6 (ISS34); and the nonencapsulated species Trichinella pseudospiralis (ISS13). Moreover, 33 new Trichinella isolates from 2 zoogeographical regions were unequivocally identified. All Trichinella isolates have shown an identical pattern with those produced by the reference strain. According to these data, we have demonstrated that ISSR-PCR is a robust technique that emerges as a useful new application for the molecular identification of Trichinella isolates in epidemiological studies.
Assuntos
DNA de Helmintos/química , Reação em Cadeia da Polimerase/métodos , Trichinella/genética , Animais , Canidae , Primers do DNA/química , DNA de Helmintos/isolamento & purificação , Eletroforese em Gel de Ágar , Feminino , Genótipo , Masculino , Repetições de Microssatélites/genética , Sus scrofa , Trichinella/classificaçãoRESUMO
In 2000, two cases of human trichinellosis were detected in the Sierra Grande area of Rio Negro province, Argentina. As part of an investigation of the aetiology of these cases, 300 pigs slaughtered for consumption in the area between 2000 and 2002 were checked for Trichinella infection, by artificial digestion of a muscle sample. Twelve (5.6%) - four (7.3%) of the 55 checked in 2000, five (4.8%) of the 105 investigated in 2001, and three (2.1%) of the 140 investigated in 2002 - were found infected. Blood samples were collected from other pigs aged > 6 months old, so that sera could be tested, in ELISA and by western blotting, for anti- Trichinella antibodies. Of the 181 animals checked in the initial serological survey, 36 (19.9%) were found seropositive for Trichinella. When 35 of the seronegative pigs were re-checked 6 months later, three (8.6%) were found to have seroconverted. Four (15.4%) of 26 local rodents, caught in Sherman-type traps, were also found positive when checked for infection by artificial digestion. It appears that about 20% of pigs in the study area are infected each year, this high level of transmission being sustained by a high prevalence of infection in the local rodent populations.
Assuntos
Doenças dos Roedores/epidemiologia , Doenças dos Suínos/epidemiologia , Triquinelose/epidemiologia , Triquinelose/veterinária , Animais , Argentina/epidemiologia , Diafragma/parasitologia , Doenças Endêmicas/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Carne/parasitologia , Prevalência , Roedores , Estudos Soroepidemiológicos , Suínos , Trichinella/classificação , Trichinella/isolamento & purificaçãoRESUMO
An outbreak of trichinellosis caused by ingestion of pork infected with Trichinella britovi occurred in the province of Granada in southern Spain in April-May 2000. Thirty-eight people were affected and 15 of them were hospitalized at the University Hospital of San Cecilio (Granada). The probable source of infection was sausage made from uninspected wild boar meat and inspected pork. Ninety-two percent of the patients had myalgias, 47.6% had diarrhea and/or vomited, 78.6% had periorbital edema, and 76.0% had fever. Twenty-two patients (15 hospitalized and 7 nonhospitalized) were serologically studied. Eosinophil levels were less than 5% of the total leukocyte count in 86.7% of the patients. Levels of creatinine phosphokinase (range = 200-2,213 U/L) and lactate dehydrogenase (range = 560-7,558 U/L) were elevated in 85.7% and 78.6% of the patients, respectively. Sixteen (72.7%) and 20 (90.9%) patients were positive for T. britovi by indirect immunofluorescence and Western blot, respectively.