Molecular based confirmation of puma meat sausages implicated in trichinellosis outbreaks in Argentina.

Trichinellosis is a zoonotic disease caused by Trichinella, with the main source of infection being the consumption of pork and pork-derived products. However, it can also be acquired from eating the meat from wild animals targeted for sport hunting. The objectives of this study were: 1) to develop and implement a molecular method for the identification of Sus scrofa (pig and wild boar) and Puma concolor (Puma) meat in sausages eaten raw, which were linked to trichinellosis outbreaks occurring in Córdoba, Buenos Aires and La Pampa provinces, Argentina, in 2010, 2021, and 2022, respectively; and 2) to identify the Trichinella species present in the food. Specific primers were designed for PCR amplification and nucleotide sequencing of a region of the mitochondrial cytochrome b gene from both host species. Samples from the mentioned outbreaks were analysed, and the molecular identification of Trichinella spp. larvae was also performed. Results of the species identification system revealed that sausages from Córdoba and Buenos Aires had a mixed composition of pork and puma meat, while those from La Pampa contained puma meat only. Trichinella spiralis was implicated in all three outbreaks. The species identification system developed and implemented in this study revealed two host species of Trichinella related to human cases, and alerts about the risk of zoonotic transmission to humans through infected puma meat.

Serology survey of Ascaris suum and Trichinella spiralis in rural pigs in Southwestern Mexico.

BACKGROUND: Parasitic diseases of pigs are a public and veterinary health problem. Helminths influence pork production, whereas backyard pigs can transmit these parasites. OBJECTIVES: This work aimed to investigate the prevalence of antibodies against Ascaris suum and Trichinella spiralis in backyard pigs from Jamiltepec, Region de la Costa, Oaxaca, in Southwestern Mexico. METHODS: Six hundred sixty-four serum samples were obtained from backyard pigs from 23 rural villages distributed in 5 municipalities; samples were taken in a non-probabilistic manner with the owner's consent. The presence of serum antibodies against a total extract of A. suum adult worm was determined by ELISA. In contrast, antibodies to the excretion-secretion products of the T. spiralis muscle larva were determined by Western blot. RESULTS: The global seroprevalence for A. suum was 5.12% and 2.41% for T. spiralis; however, antibodies were only found in 8 villages and distributed in 3 municipalities. The highest frequency of positivity for Ascaris was found in the municipality of Santa Catarina Mechoacán (13.01%), whereas, in Santa María Huazalotitlán, the highest frequency of positivity for Trichinella was found (5.75%). In San Andrés, frequencies were 7.23% and 4.82%, respectively. No statistical differences were observed between populations. CONCLUSIONS: Our data suggest that helminth transmission is restricted by locality. However, further studies must be conducted to understand the factors limiting this transmission to promote pork meat production in parasite-free zones.

Trichinella spiralis Infecting Wild Boars in Southern Chile: Evidence of an Underrated Risk.

Trichinellosis in Chilean wild animals has scarcely been documented. The introduction of wild boars into the wild environment represents a viable new host with a potential risk of infection for human health. The aim of this study was to determine the presence and prevalence of Trichinella in wild boars. Two hundred seventy eight wild boars from of the Southern Chile were examined by compression and artificial digestion techniques. The larvae in the positive samples were collected for taxonomic analysis through polymerase chain reaction-inter-simple sequence repeats and to calculate the parasitic burden. A prevalence of 1.8% (5/278) of infected animals and an average parasitic burden of 6.8 ± 2.1 larvae per gram were estimated. The only species identified by molecular techniques was Trichinella spiralis. Prevalence of T. spiralis in wild boars was similar to those described around the world. T. spiralis infection rate and parasite burden detected in Chilean wild boars represent a certain food-borne risk for human population.

Trichinella spiralis in a South American sea lion (Otaria flavescens) from Patagonia, Argentina.

Trichinella spp. from a sylvatic cycle has been found in several animal species such as pumas (Puma concolor), armadillos (Chaetophractus villosus), rats (Rattus norvegicus), and wild boars (Sus scrofa) in Argentina. Moreover, Trichinella infection has been detected in a wide range of marine mammals around the world, including polar bears (Ursus maritimus) and walruses (Odobenus rosmarus). Until the present time, Trichinella spp. infection has not been detected in marine mammals of South America. Samples from four South American sea lions (Otaria flavescens) found dead in Rio Negro, Argentina, were analyzed by artificial digestion, and in the case of one animal, Trichinella larvae were identified at the species level by nested multiplex PCR as Trichinella spiralis. This is the first report of a Trichinella species infecting marine mammals from South America.

Análisis proteómico de los productos de excreción-secreción de cuatro aislados de Trichinella spiralis obtenidos de hospederos accidentales / Proteomic analysis of the excretion-secretion products of four Trichinella spiralis isolates obtained from accidental hosts

Resumen: Introducción: Trichinella spiralis es un nemátodo tisular que se aloja en el músculo esquelético de humanos y otros mamíferos y causa una serie de alteraciones fisiológicas. Las proteínas de los productos de excreción-secreción de T. spiralis juegan un papel importante en la aparición y regulación de estas alteraciones. Sin embargo, aún no se conoce el efecto de estos productos en la infección e invasión del parásito al hospedero. Métodos: Mediante un análisis electroforético en una dimensión, Western blot y espectrometría de masas, se evaluaron las diferencias y similitudes entre proteínas antigénicas y de superficie de cuatro aislados de T. spiralis obtenidos de hospederos accidentales (perros) y la cepa de referencia aislada de cerdos (MSUS/MEX/91/CM). Resultados: Utilizando ontología de genes, se encontraron cinco proteínas exclusivas de los hospederos accidentales. Después del análisis, se encontró que estas proteínas forman parte de la matriz extracelular del parásito, cuentan con actividad catalítica y están implicadas en la adhesión a las células del hospedero. La actividad antigénica de las cuatro cepas aisladas de hospederos accidentales es idéntica a la reportada para T. spiralis, visualizándose el triplete antigénico característico de 43, 45 y 47 kDa. Conclusiones: Las proteínas exclusivas de los hospederos accidentales proveen información para entender el mecanismo de acción de este parásito para penetrar el músculo y evadir la respuesta inmune en el hospedero.

Abstract: Background: Trichinella spiralis is an intestinal and tissue nematode specific for mammalian skeletal muscle, causing a series of physiological alterations. T. spiralis excretory-secretion products play an important role in the appearance and regulation of these alterations. However, the effect of these products on the infection and invasion of the parasite to the host is unknown. Methods: Differences and similarities between antigenic proteins and surface proteins of four accidental hosts isolates (dogs) of T. spiralis and the reference strain isolated from pigs (MSUS/MEX/91/CM) were assessed by electrophoresis, western blot and mass spectrometry. Results: Using gene ontology, five proteins exclusive to the accidental hosts were analyzed. The results showed that these proteins are part of the extracellular matrix of the parasite, present catalytic activity, and bind to host cells. The antigenic activity the four strains showed the antigenic triplet characteristic of T. spiralis of 43, 45 and 47 kDa. Conclusions: Five proteins exclusive to dog isolates provided information to understand the mechanism of action of this parasite to penetrate the muscle and evade the immune response in the host.

Avaliação da presença de Trichinella spiralis em equinos abatidos em Araguari, MG / Evaluation of the presence of Trichinella spiralis in horses slaughtered in Araguari, MG

A larva do nematódeo Trichinella spiralis é responsável por infectar animais como porcos, javalis e cavalos e por causar grave doença em seres humanos. Surtos devido ao consumo da carne de cavalo são mais comuns na Europa, principalmente na França e Itália, onde existe a cultura de ingeri-la crua. Apesar de no Brasil não ter relatos sobre a infecção desse parasita em animais e seres humanos, os abatedouros exportadores para alguns mercados, como Europa e Estados Unidos, são obrigados a pesquisar o parasita. No presente estudo, a presença de larvas de Trichinella spiralis foi avaliada em 14.852 cavalos abatidos entre dezembro de 2014 a julho de 2016 em um frigorífico de Araguari sob inspeção federal. Os animais eram procedentes dos estados de Minas Gerais, Goiás e Bahia. A técnica empregada foi a de digestão artificial, em que se utilizou o músculo masseter, seguindo as legislações da União Europeia: Diretiva 91/497/CEE, Diretiva 77/96/CEE, Regulamento (CE) N° 2075/2005 e suas alterações. A larva não foi encontrada em nenhuma amostra analisada.

[Proteomic analysis of the excretion-secretion products of four Trichinella spiralis isolates obtained from accidental hosts]. / Análisis proteómico de los productos de excreción-secreción de cuatro aislados de Trichinella spiralis obtenidos de hospederos accidentales.

BACKGROUND: Trichinella spiralis is an intestinal and tissue nematode specific for mammalian skeletal muscle, causing a series of physiological alterations. T. spiralis excretory-secretion products play an important role in the appearance and regulation of these alterations. However, the effect of these products on the infection and invasion of the parasite to the host is unknown. METHODS: Differences and similarities between antigenic proteins and surface proteins of four accidental hosts isolates (dogs) of T. spiralis and the reference strain isolated from pigs (MSUS/MEX/91/CM) were assessed by electrophoresis, western blot and mass spectrometry. RESULTS: Using gene ontology, five proteins exclusive to the accidental hosts were analyzed. The results showed that these proteins are part of the extracellular matrix of the parasite, present catalytic activity, and bind to host cells. The antigenic activity the four strains showed the antigenic triplet characteristic of T. spiralis of 43, 45 and 47 kDa. CONCLUSIONS: Five proteins exclusive to dog isolates provided information to understand the mechanism of action of this parasite to penetrate the muscle and evade the immune response in the host.

Parasitic zoonoses present some risks with low-temperature cooking of pork.

A recent article in this journal documents enhanced sensory qualities of pork cooked to low temperatures. The aim of this letter is to point out that the incidence of Trichinella spiralis in many countries and the more widespread incidence of Toxoplasma gondii present a concern for the adoption of low temperature cooking of pork unless extended cooking times are used.

Quantitative Risk Assessment of Human Trichinellosis Caused by Consumption of Pork Meat Sausages in Argentina.

In Argentina, there are three known species of genus Trichinella; however, Trichinella spiralis is most commonly associated with domestic pigs and it is recognized as the main cause of human trichinellosis by the consumption of products made with raw or insufficiently cooked pork meat. In some areas of Argentina, this disease is endemic and it is thus necessary to develop a more effective programme of prevention and control. Here, we developed a quantitative risk assessment of human trichinellosis following pork meat sausage consumption, which may be used to identify the stages with greater impact on the probability of acquiring the disease. The quantitative model was designed to describe the conditions in which the meat is produced, processed, transported, stored, sold and consumed in Argentina. The model predicted a risk of human trichinellosis of 4.88 × 10(-6) and an estimated annual number of trichinellosis cases of 109. The risk of human trichinellosis was sensitive to the number of Trichinella larvae that effectively survived the storage period (r = 0.89), the average probability of infection (PPinf ) (r = 0.44) and the storage time (Storage) (r = 0.08). This model allowed assessing the impact of different factors influencing the risk of acquiring trichinellosis. The model may thus help to select possible strategies to reduce the risk in the chain of by-products of pork production.

Feasibility study for the detection of Trichinella spiralis in a murine model using mid-Fourier transform infrared spectroscopy (MID-FTIR) with attenuated total reflectance (ATR) and soft independent modelling of class analogies (SIMCA).

Fourier transform infrared (FTIR) spectroscopy with attenuated total reflectance (ATR) and soft independent modelling by class analogies (SIMCA) was used to assess the feasibility of detecting Trichinella spiralis in a murine model. The selected FTIR wavenumber range was 1700-900 cm(-1) and the first derivative of the spectra was subjected to SIMCA analysis. The SIMCA model developed for rat meat spiked with T. spiralis larvae was successfully apply to classify non-infected from infected rat meat with a limit of detection of 3 larvae/10 g rat meat and no false positives with 99% confidence limit. To avoid false positives arising from the presence of other parasites, another chemometric model was developed to demonstrate the capacity of the model to discriminate between Ascaris suum, Taenia solium and T. spiralis. Results confirmed that this method could correctly distinguish these parasites. Additional studies are needed to prove the effectiveness of this technique for other types of muscle meats, including those relevant to human consumption.

Persistence of Trichinella spiralis muscle larvae in natural decaying mice.

The influence of natural weather conditions on the viability and reproductive capability of Trichinella spiralis muscle larvae in mouse corpses exposed to summer and winter conditions in the Buenos Aires Province, Argentina, was studied. For this purpose, a total of 49 mouse corpses harbouring muscle larvae of T. spiralis were exposed for a period of 1, 2, 4 and 6 weeks in each of the seasons. Control corpses maintained at 8°C were also included. In summer, T. spiralis muscle larvae were recovered from corpses exposed up to 1 week only. The viability of these larvae was 54.2%, and the reproductive capability index in mice (RCI) was 13.1 and significantly lower than the control (p<0.0005). Morphologic deterioration and reduction in the glycogen content of cysts and larvae were observed at the second week of exposition. By week 4, larval stages of Dermestes maculatus were observed inside corpses, and 22 live muscle larvae of T. spiralis were obtained by artificial digestion of their bodies. In winter, T. spiralis muscle larvae were always recovered, the viability being almost 100% except for a significant reduction by week 6 of exposition (p<0.0001). For this season, the RCI were 50.5, 46.9, 59.7 and 45.2 for the periods of 1, 2, 4 and 6 weeks of exposition, respectively. The morphology of cysts and larvae did not show alterations, and no variations were observed as well in glycogen reserves during the 6-week period of exposition. RCI of non-exposed muscle larvae were always significantly higher that any of those recorded from muscle larvae that belonged to exposed corpses (p=0.0005). The present results demonstrate that muscle larvae of T. spiralis are able to survive in nature and keep infective for a 1-week period in summer and at least for 6 weeks in winter, becoming an important source of infection for scavengers. In summer, larvae stages of D. maculatus, and probably other insects, may play an important role in the survival and transmission of T. spiralis in the sylvatic cycle.

Trichinella infection in wild animals from endemic regions of Argentina.

Natural infection with Trichinella has been described in more than 150 mammalian species. However, few reports of Trichinella infection in wild animals have come from Argentina. In this study, muscle tissue was obtained from wild animals in Argentina with the aim of evaluating the presence of Trichinella. A total of 169 muscle samples were collected to determine the presence of Trichinella larvae by artificial digestion. The 169 muscle samples originated from 12 species including 36 opossums (Didelphis albiventris), 19 armadillos (Chaetophractus villosus), 9 capybaras (Hydrocaeris hydrocaeris), 1 puma (Puma concolor), 3 grey fox (Lycalopex gymnocercus), 6 coypus (Myocastor coypus), 6 skunks (Conepatus chinga), 2 ferrets (Galictis cuja), 66 rats (Rattus norvegicus), 6 mice (Mus musculus), 12 wild boars (Sus scrofa), and 3 wild cats (Felis geoffroyi). Trichinella infection was detected in 1 puma [2 larvae per gram (LPG)], 3 wild boars (8-420 LPG), 3 armadillos (0.04-0.08 LPG), and 9 rats (0.1 to 150 LPG). Only 3 Trichinella isolates, of 1 rat and 2 wild boars from Neuquén, were identified as Trichinella spiralis by nested PCR. The presence of Trichinella infection among wild animal populations suggests a sylvatic cycle of transmission in Argentina, which can serve as a reservoir for humans and domestic animals. Further, evidence of high prevalence in rats emphasizes the need to improve pig management, mainly in small individual farms without adequate technology, to enhance the quality of feeds, and to improve veterinary services to avoid exposure of pigs to Trichinella.

Trichinella infection in wild boars (Sus scrofa) from a protected area of Argentina and its relationship with the presence of humans.

In Argentina, Trichinella infection has been documented in humans and animals of several provinces since 1930. This zoonotic parasite infection has been recently detected in humans and pigs of a region historically considered as Trichinella-free, suggesting the spread of these pathogens. The aim of the present work was to investigate the presence of Trichinella infection in wild boars (Sus scrofa) and in the human population living in a protected area. Trichinella infection has been investigated by serology (in humans and wild boars) and by artificial digestion of wild boar muscles. The isolated Trichinella larvae have been identified at the species level by multiplex PCR. A geographical information system has been used to collect environmental data. The results showed the circulation of Trichinella spiralis in wild boars with a low parasite burden, and suggest the influence of human behavior on the transmission. The transplacental passage of parasite is postulated. It follows that the declaration of region as Trichinella-free should be carefully established by means of extensive monitoring programs, not only in humans and domestic animals but also in wildlife.

Clinical, haematological, biochemical and economic impacts of Trichinella spiralis infection in pigs.

The purpose of this work was to assess the clinical, haematological and biochemical responses of pigs experimentally inoculated with Trichinella spiralis. Groups of three pigs were inoculated per os with 100, 500 and 5000 T. spiralis muscle larvae, two pigs were used as control. Clinical evaluation of disease in pigs included daily examination, rectal temperature measurements and cardiac and respiration rates. Haematological studies included: hematocrit (%), hemoglobin (g/dl), and white cell, neutrophil, lymphocyte and eosinophil counts. Blood biochemistry included: bun (mg/dl), creatinine (mg/dl), AST (UI/l), ALT (UI/l), CPK (UI/l) and ALP (UI/l). No significant differences were observed in rectal temperature and in cardiac and respiration rates between inoculated animals and the control group (p> or =0.05). Significant differences were detected (p< or =0.05) in the values of % hemoglobin, and eosinophils, as well as in the values of CK, ALP, AST and ALT. The variations observed in some cases were related to the number of T. spiralis larvae inoculated and varied with the number of days post-infection. Inoculated pigs showed significant differences (p< or =0.05) in weight gain when compared with uninoculated controls. This study has clinical, haematological, and enzyme alterations in Trichinella infected pigs provides a better understanding of acute and chronic trichinellosis in pigs.

Application of Giemsa stain for easy detection of Trichinella spiralis muscle larvae.

The application of Giemsa technique to stain compressed diaphragm samples obtained from rodents experimentally infected with Trichinella spiralis is described. Diaphragm samples from rats heavily infected with 20 muscle larvae per gram of body weight (20 ML/gbw) were cut into several pieces and stained with Giemsa; on the other hand, whole diaphragms from slightly infected mice (1 ML/gbw) were also stained with Giemsa. Besides, muscle samples were also stained with Giemsa. Observation at 10 x magnification revealed that both ML and nurse cells (NC) look as bluish structures clearly contrasting with the pinkish color of the non-infected muscle fibers. NC in the diaphragms of mice could be easily observed at naked eye as blue points contrasting with the pink surrounding areas formed by the non-infected muscle fibers. Among NC observed in the diaphragms of rats infected with 20 ML/gbw, 4.4% was multiple infection. These findings were confirmed in sectioned and hematoxylin-eosin stained specimens. This data could be usefulness for a rapid diagnosis of trichinellosis in post-mortem mammals without magnification procedures.

First record of human trichinosis in Chile associated with consumption of wild boar (Sus scrofa).

The first South American case of human trichinosis, resulting from the consumption of roast wild boar (Sus scrofa) is reported in Chile. The patient presented fever, diarrhea, myalgias, facial edema, sub-conjunctival reddening, photophobia, eosinophilia, and elevated glutamic oxalacetic transaminase. The diagnosis was confirmed by two immunoenzymatic tests (ELISA) using somatic and excretion-secretion antigens.

First record of human trichinosis in Chile associated with consumption of wild boar (Sus scrofa)

The first South American case of human trichinosis, resulting from the consumption of roast wild boar (Sus scrofa) is reported in Chile. The patient presented fever, diarrhea, myalgias, facial edema, sub-conjunctival reddening, photophobia, eosinophilia, and elevated glutamic oxalacetic transaminase. The diagnosis was confirmed by two immunoenzymatic tests (ELISA) using somatic and excretion-secretion antigens.

Detection of circulating and fecal Trichinella spiralis antigens during experimental infection using monoclonal antibodies against the new born larvae.

Different assays to detect antigens of Trichinella spiralis during current infection have been standardized, although sensitivity values have been the main limitation to use them as routine diagnostic test. We report the production and use of monoclonal antibodies (MAbs) against the new born larvae which recognize both somatic and metabolic antigens from adult and muscular larvae (ML). We used two IgG3 MAbs (4B1, 4B2) and two IgG2a (2D3, 2D4) to detect antigens during experimental infection. All MAbs detect fecal antigens starting the second to third week post infection (wpi), although it was less clear with MAbs 4B1 and 4B2, while circulating antigens were detected from third to fourth wpi. Thus, the recognition of shared antigens among T. spiralis developmental stages can be used for early diagnosis of trichinellosis.

Localization of Trichinella spiralis in muscles of commercial and parasitologic interest in pork.

Trichinellosis is widespread around the world with different representatives of the genus Trichinella found in almost every continent. In Argentina the main source of transmission for the disease to humans is pig meat infected with Trichinella spiralis. The object of this work was to determine the distribution of Trichinella larvae in fresh meat cuts which are sold for human consumption and in the muscles traditionally used for the disease diagnosis at meat-packing plants. Cranial muscles to the last rib showed more Trichinella spiralis larvae than those with a caudal location (p < 0.01). No significant differences were found (p > 0.05) between bilateral left and right muscles. Significant larval concentrations were found in the neck muscles, even in carcasses with a low parasitic load; these muscles are used to prepare cold meats (boston butt). Commercial cuts of meat had a substantial larval burdens in animals experimentally infected with 500 to 5,000 Trichinella spiralis larvae, with parasite burdens similar to infection levels in muscles evaluated at the meat packing plant.

Cytotoxicity-blocking antibodies in human chronic trichinellosis.

Antisurface newborn larva (NBL) antibodies (Abs) were found in sera from individuals chronically infected with Trichinella spiralis. These Abs were incapable of inducing NBL death by activation of normal human leukocytes of peripheral blood as determined by in vitro assays of antibody-dependent cell cytotoxicity (ADCC). Besides, such sera blocked the cytotoxic reaction mediated by Abs produced a few weeks after infection. The blocking activity could not be attributed to any particular isotype by the indirect immunofluorescence technique. Purified antisurface NBL Abs obtained from sera from chronically infected patients recognized antigens of muscle-larva excretory-secretory products (ML-ESP) in an enzyme-linked immunosorbent assay (ELISA) and an immunoelectrotransfer blot assay. Likewise, as did chronic sera, a monoclonal Ab raised against ML-ESP blocked NBL death in ADCC assays. These results suggest that during the course of an infection by T. spiralis, Abs related to ML-ESP provide an immunoevasive mechanism for avoidance by NBL of an important anti-NBL host effector mechanism.