Mixed infection, Trichinella spiralis and Trichinella britovi, in a wild boar hunted in the Province of Cáceres (Spain)

The etiological agents of human trichinellosis are distributed worldwide in domestic and wild animals. In Spain, two morphologically indistinguishable Trichinella species have been described—Trichinella spiralis and Trichinella britovi—that are perpetuated in both domestic and sylvatic cycles. The present work reports a double natural infection involving these species in a wild boar killed by hunters in the Province of Cáceres, Spain. After artificial digestion of the boar’s muscles, nine larvae/g were collected. These were characterized by multiplex-PCR and Western-blotting using the Trichinella-specific monoclonal antibodies US5 and US9, and both T. spiralis and T. britovi were detected. The mechanism by which this wild boar came to acquire a mixed infection remains unclear.     

Susceptibility of Laboratory Rodents to Trichinella papuae

Members of the genus Trichinella are small nematodes that can infect a wide range of animal hosts. However, their infectivity varies depending on the parasite and host species combination. In this study, we examined the susceptibility of 4 species of laboratory rodents, i.e., mice, rats, hamsters, and gerbils to Trichinella papuae, an emerging non-encapsulated Trichinella species. Trichinella spiralis and Trichinella pseudospiralis were also included in this study for comparison. Fifteen animals of each rodent species were infected orally with 100 muscle larvae of each Trichinella species. Intestinal worm burden was determined at day 6 and 10 post-inoculation (PI). The numbers of muscle larvae were examined at day 45 PI. The reproductive capacity index (RCI) of the 3 Trichinella species in different rodent hosts was determined. By day 6 PI, 33.2-69.6% of the inoculated larvae of the 3 Trichinella species became adult worms in the small intestines of the host animals. However, in rats, more than 96% of adult worms of all 3 Trichinella species were expelled from the gut by day 10 PI. In gerbils, only 4.8-18.1% of adult worms were expelled by day 10 PI. In accordance with the intestinal worm burden and the persistence of adults, the RCI was the highest in gerbils with values of 241.5±41.0 for T. papuae, 432.6±48 for T. pseudospiralis, and 528.6±20.6 for T. spiralis. Hamsters ranked second and mice ranked third in susceptibility in terms of the RCI, Rats yielded the lowest parasite RCI for all 3 Trichinella species. Gerbils may be an alternative laboratory animal for isolation and maintenance of Trichinella spp.     

Within-host dynamics of Trichinella spiralis predict persistent parasite transmission in rat populations

Trichinella spiralis is transmitted and maintained in both a domestic and sylvatic cycle, whereby rats contribute to the spread of T. spiralis from domestic to sylvatic animals and vice versa. As a model for T. spiralis transmission in wildlife, we studied the potential of rats to act as a reservoir species for T. spiralis, by assessing experimentally its within-host infection dynamics, and simulating the between-host dynamics by a Monte Carlo approach. The distribution of parasite burden in individual rats is mathematically defined by roots of the dose response equation intersecting with the diagonal. In simulated between-host dynamics, up to 10⁴ events of uninterrupted parasite transmission were observed. Histograms of parasite burdens per individual rat matched closely with the mixture of two gamma distributions, which were derived from the within-host infection dynamics. In conclusion, T. spiralis transmission persists in a population of rats when they cannibalize their own species. Rats should be included in the minimal set of wildlife species that maintain the life cycle of T. spiralis.     

Hypoglycaemia induced by Trichinella infection is due to the increase of glucose uptake in infected muscle cells

The present study investigates how Trichinella infection induces host hypoglycaemia and explores a potential relationship between infection and the insulin signalling pathway. The results showed that mice infected with Trichinella spiralis or Trichinella pseudospiralis exhibited a temporary decrease in blood glucose level between 8 and 28 days p.i. and the kinetics of the glucose levels corresponded to the process of muscle larval growth and development. Histochemical results showed that glycogen accumulation increased in infected muscle cells during the period of hypoglycaemia. Analysis of gene expression profiles with quantitative PCR demonstrated that insulin signalling pathway-related genes, such as insulin receptor (IR), insulin receptor substance 1 (IRS-1), IRS-2, phosphatidylinositol 3-kinase (PI3-K) and V-akt murine thymoma viral oncogene homologue 2 (Akt2) were up-regulated in infected muscle cells during infection and these expression changes correlated with the kinetics of blood glucose level, glycogen accumulation and the process of larval growth and development in infected muscle cells. Western blot analysis clarified that the expression of IR and Akt2 proteins increased in muscle tissues infected with both species of Trichinella. This study suggests that hypoglycaemia induced by Trichinella infection is the result of an increase in glucose uptake by infected muscle cells via up-regulation of insulin signalling pathway factors.     

Early Trichinella spiralis and Trichinella nativa infections induce similar gene expression profiles in rat jejunal mucosa

Trichinella spiralis causes a significantly higher parasite burden in rat muscle than Trichinella nativa. To assess whether the difference in infectivity is due to the early intestinal response, we analyzed gene expression changes in the rat jejunum during Trichinella infection with a whole-genome microarray. The rats were euthanized on day five of infection, and their jejunal mucosa was sampled for microarray analysis. In addition, intestinal histology and hematology were examined. Against our expectations, the gene expression changes were similar in both T.nativa- and T. spiralis-infected groups. The two groups were hence pooled, and in the combined Trichinella-infected group, 551 genes were overexpressed and 427 underexpressed when compared to controls (false discovery rate ?0.001 and fold change at least 2 in either direction). Pathway analysis identified seven pathways significantly associated with Trichinella infection (p < 0.05). The microarray data suggested nonspecific damage and an inflammatory response in the jejunal mucosa. Histological findings, including hyperemia, hemorrhage and a marked infiltration of inflammatory cells, supported the microarray data. Trichinella infection caused complex gene expression changes that indicate a host response to tissue damage in the mucosa of the jejunum, but the changes were not notably dependent on the studied species of Trichinella.     

Molecular identification of three Trichinella isolates from Heilongjiang Province, People's Republic of China

DNAs of Trichinella dog isolate (HC), Trichinella swine isolate (HH) and Trichinella cat isolate (SW), obtained from Heilongjiang Province, were amplified by the fragments of 18S rDNA and ITS2. Two reference strains, Trichinella spiralis (ISS3) and Trichinella nativa (ISS10) were used for sequence comparison. Sequence and dendrogram analysis indicated that HC belonged to T.nativa and HH together with SW belonged to T.spiralis. This method permits rapid species identification of Trichinella isolates, although further evaluation is required before precisely identification.     

Identification and characterization of a full-length cDNA encoding paramyosin of Trichinella spiralis

A full-length cDNA encoding Trichinella spiralis paramyosin (Ts-Pmy) was cloned by immunoscreening a cDNA library of the adult T. spiralis worm. Ts-Pmy cDNA consists of 2655 bp that encode 885 amino acids. The recombinant protein (rTs-Pmy) was expressed and purified by Ni-affinity chromatography. Western blot analysis showed that rTs-Pmy could be recognized by sera from T. spiralis-infected humans, swine, rabbits, and mice. Immunolocalization demonstrated that Ts-Pmy was abundant on the surface of T. spiralis larvae. BALB/c mice vaccinated with rTs-Pmy demonstrated 36.2% reduction in muscle larvae burden following T. spiralis larvae challenge. Vaccination of the mice with rTs-Pmy resulted in a high level of specific anti-Ts-Pmy IgG antibodies and generated a Th1/Th2 mixed type of immune response, with Th2 predominant. These studies showed that rTs-Pmy induced protective immunity in mice and could be considered as a potential vaccine candidate for trichinellosis.     

An Outbreak of Trichinosis with Molecular Identification of Trichinella sp. in Vietnam

The 5th outbreak of trichinosis occurred in a mountainous area of North Vietnam in 2012, involving 24 patients among 27 people who consumed raw pork together. Six of these patients visited several hospitals in Hanoi for treatment. Similar clinical symptoms appeared in these patients within 5-8 days after eating infected raw pork, which consisted of fever, muscle pain, difficult moving, edema, difficult swallowing, and difficult breathing. ELISA revealed all (6/6) positive reactions against Trichinella spiralis antigen and all cases showed positive biopsy results for Trichinella sp. larvae in the muscle. The larvae detected in the patients were identified as T. spiralis (Vietnamese strain) by the molecular analysis of the mitochondrial cytochrome c oxidase subunit III (cox3) gene.     

The genetic analysis of F1 hybrid larvae between female Trichinella spiralis and male Trichinella britovi

Hybrids between female Trichinella spiralis and male Trichinella britovi were constructed. Then, hybrid genotype was characterized by DNA markers including mitochondrial cytochrome c oxidase subunit I (CO I) gene, the gene encoding the 43-kDa excretory–secretory (ES) protein, and genomic DNA fragments specific for T. spiralis and T. britovi identified from random amplified polymorphism DNA (RAPD). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial CO I gene revealed that all hybrids carried a T. spiralis pattern. The same analysis of the gene encoding the 43-kDa ES protein showed that each hybrid carried both T. spiralis and T. britovi gene type simultaneously. In the analysis of genomic DNA using RAPD-derived PCR primers, some hybrids carried T. spiralis and T. britovi-specific RAPD markers, while others carried the RAPD marker of T. spiralis only.     

Trichinella britovi etiological agent of sylvatic trichinellosis in the Republic of Guinea (West Africa) and a re-evaluation of geographical distribution for encapsulated species in Africa

In West Africa, Trichinella infection was documented in humans and animals from Senegal in the 1960s, and the biological characters of one isolate showed a lower infectivity to domestic pigs and rodents when compared with that of a Trichinella spiralis pig isolate from Europe. To identify the Trichinella species present in West Africa, a survey was conducted in a total of 160 wild animals in the Republic of Guinea. Three Viverridae, one true civet (Viverra civetta) and two African palm civets (Nandinia binotata) from the Fouta Djallon Massif, Pilimini Subprefecture, were found positive by artificial digestion of muscle samples. Trichinella larvae from these three viverrids were identified as Trichinella britovi and no difference was detected in three examined sequences from these African isolates and the reference strain of T. britovi from Europe, indicating common ancestry, an historically continuous geographic distribution, and recent isolation for African and European populations. The detection of T. britovi in West Africa modifies our knowledge about the distribution of encapsulated species of Trichinella in Africa. Thus, Trichinella nelsoni is now considered to have a distribution limited to the Eastern part of the Afrotropical region from Kenya to South Africa. This provides a plausible explanation for the presence of Trichinella T8 in Namibia and South Africa, and further suggests that T. britovi could be the Trichinella species circulating among wild animals of Northern Africa.     

Trichinella spiralis: intranasal immunization with attenuated Salmonella enterica carrying a gp43 antigen-derived 30mer epitope elicits protection in BALB/c mice

Trichinellosis is a public health problem and is considered an emergent/re-emergent disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects domestic animals such as pigs and horses, as well as wild animals and humans. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Attenuated Salmonella strains are especially attractive live vectors because they elicit mucosal immunity, which is known to be important for the control of Trichinella spiralis infection at the intestinal level and can be administered by oral or intranasal routes. In this study, the autotransporter ShdA was used to display, on the surface of the Salmonella enterica serovar Typhimurium SL3261, the 210–239 amino acid epitope, (designated as Ag30) derived from the 43 kDa glycoprotein of T. spiralis muscle larvae. The fusion protein elicited antibodies in BALB/c mice that were able to recognize the native epitope on the surface of T. spiralis muscle larvae. Mice immunized by intranasal route with the recombinant Salmonella induced a protective immune response against the T. spiralis challenge, reducing by 61.83% the adult burden at day eight postinfection. This immune response was characterized by the induction of antigen-specific IgG1 and of IL-5 production. This study demonstrates the usefulness of Salmonella as a carrier of nematode epitopes providing a surface display system for intestinal parasite vaccine applications.     

Trichinella spiralis: comparison of stages in host intestine with those of an Arctic Trichinella sp

The intestinal phase of Trichinella spiralis and of Trichinella sp. isolated in the Arctic were compared in experimental animals. Reproductive capacity, pathogenicity, distribution, and persistence of adults in the small intestine, morphological measurements, and release of newborn larvae in vitro were examined. Numerous passages of 40 days each for T. spiralis and the Trichinella sp. isolate in mice did not affect reproductive capacity, distribution of adults in the small intestine, and size of worms. Reproductive capacity index for T. spiralis, I = 151.27 ± 27.30 was significantly higher compared to the Trichinella sp. isolate index, I = 63.46 ± 19.34. The Trichinella sp. isolate was more pathogenic to mice and wild rodents compared to T. spiralis during the intestinal phase. Both parasites were located in the anterior part of the small intestine but the position of T. spiralis adults (P = 17.08) differed from position of the Trichinella sp. isolate adults (P = 23.46) in the small intestine. Intestinal phase of T. spiralis was longer (20 days) compared to the Trichinella sp. isolate (15 days) but sex ratios (:♂) were similar for both parasites. T. spiralis females released significantly higher numbers of larvae in vitro/24 hr compared to the Trichinella sp. isolate. Release of larvae was continuous during the intestinal phase and the average fecundity for T. spiralis was 335 larvae/female and for the Trichinella sp. isolate 114 larvae/female. Adults of T. spiralis and the Trichinella sp. isolate were morphologically indistinguishable and did not differ in size. A comparative index for the intestinal phase is proposed for comparison of any Trichinella spp. isolates and standard T. spiralis.     

Therapeutic Potential of Myrrh and Ivermectin against Experimental Trichinella spiralis Infection in Mice

Trichinosis is a parasitic zoonosis caused by the nematode Trichinella spiralis. Anthelmintics are used to eliminate intestinal adults as well as tissue-migrating and encysted larvae. This study aimed to investigate the effects of ivermectin and myrrh obtained from the aloe-gum resin of Commiphora molmol on experimental trichinosis. Ninety albino mice were orally infected with 300 T. spiralis larvae. Drugs were tested against adult worms at day 0 and day 5 and against encysted larvae on day 15 and day 35 post-infection (PI). Mature worms and encysted larvae were counted in addition to histopathological examination of muscle specimens. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, albumin, globulin, urea, and creatinine values were estimated. Significant reductions in mean worm numbers were detected in ivermectin treated mice at day 0 and day 5 PI achieving efficacies of 98.5% and 80.0%, while efficacies of myrrh in treated mice were 80.7% and 51.5%, respectively. At days 15 and 35 post-infection, ivermectin induced significant reduction in encysted larval counts achieving efficacies of 76.5% and 54.0%, respectively, while myrrh efficacies were 76.6% and 35.0%, respectively. AST, ALT, urea, and creatinine levels were reduced, while total proteins were increased in response to both treatments compared to their values in the infected non-treated mice. Ivermectin use for controlling T. spiralis could be continued. Myrrh was effective and could be a promising drug against the Egyptian strains of T. spiralis with results nearly comparable to ivermectin.     

Immune Correlates of Resistance to Trichinella spiralis Reinfection in Mice

The immune correlate of host resistance induced by reinfection of Trichinella spiralis remains unclear. In this study, we investigated immune correlates between the resistance and serum IgG antibody level, CD23⁺ IgM⁺ B cells, and eosinophil responses induced by T. spiralis reinfection. Mice were primarily infected with 10 or 100 T. spiralis larvae (10 TS, 100 TS), respectively, and after 4 weeks, they were challenge infected with 100 T. spiralis larvae (10–100 TS, 100-100 TS). Upon challenge infections, 10–100 TS mice induced significantly higher levels of T. spiralis-specific total IgG antibody responses in sera and antibody secreting cell responses in spleens compared to 100-100 TS mice, resulting in significantly reduced worm burdens in 10–100 TS mice (60% and 70% reductions for adult and larvae, respectively). Higher levels of eosinophils were found in mice primarily infected with 10 TS compared to those of 100 TS at week 8 upon challenge. CD23⁺ IgM⁺ B cells were found to be increased significantly in mice primarily infected with 10 TS. These results indicate that primary infection of 10 larvae of T. spiralis, rather than 100 larvae, induces significant resistance against reinfection which closely correlated with T. spiralis-specific IgG, eosinophil, and CD23⁺ IgM⁺ B cell responses.     

An evolutionary 'intermediate state' of mitochondrial translation systems found in Trichinella species of parasitic nematodes: co-evolution of tRNA and EF-Tu

EF-Tu delivers aminoacyl-tRNAs to ribosomes in the translation system. However, unusual truncations found in some animal mitochondrial tRNAs seem to prevent recognition by a canonical EF-Tu. We showed previously that the chromadorean nematode has two distinct EF-Tus, one of which (EF-Tu1) binds only to T-armless aminoacyl-tRNAs and the other (EF-Tu2) binds to D-armless Ser-tRNAs. Neither of the EF-Tus can bind to canonical cloverleaf tRNAs. In this study, by analyzing the translation system of enoplean nematode Trichinella species, we address how EF-Tus and tRNAs have evolved from the canonical structures toward those of the chromadorean translation system. Trichinella mitochondria possess three types of tRNAs: cloverleaf tRNAs, which do not exist in chromadorean nematode mitochondria; T-armless tRNAs; and D-armless tRNAs. We found two mitochondrial EF-Tu species, EF-Tu1 and EF-Tu2, in Trichinella britovi. T.britovi EF-Tu2 could bind to only D-armless Ser-tRNA, as Caenorhabditis elegans EF-Tu2 does. In contrast to the case of C.elegans EF-Tu1, however, T.britovi EF-Tu1 bound to all three types of tRNA present in Trichinella mitochondria. These results suggest that Trichinella mitochondrial translation system, and particularly the tRNA-binding specificity of EF-Tu1, could be an intermediate state between the canonical system and the chromadorean nematode mitochondrial system.     

Trichinella: Differential expression of angiogenic factors in macrophages stimulated with antigens from encapsulated and non-encapsulated species

The newborn larval stage of Trichinella spiralis enters the host striated skeletal muscle cell resulting in the formation of the nurse cell. Vascular Endothelial Growth Factor (VEGF) was detected in cells in the area immediately surrounding the nurse cells. However, no data are available on the antigens involved, the role of other angiogenic factors or the relationship of angiogenesis with Nitric Oxide (NO) production.Using macrophage cell culture we study the effect of different Trichinella L1 antigens from one encapsulated (T. spiralis) and one non-encapsulated (Trichinellapseudospiralis) on the expression of VEGF and basic Fibroblast Growth Factor (FGF2). Also, we investigate the relationship between the production of NO and angiogenic mediators. The results show that encapsulated and non-encapsulated Trichinella species are different in their capacity to stimulate the expression of VEGF and FGF2 from host macrophages. Finally, there is no relationship between angiogenic factors and NO production by T. spiralis antigen.     

Characterisation of novel protein families secreted by muscle stage larvae of Trichinella spiralis

Proteins secreted by Trichinella spiralis have a potential role in remodelling host skeletal muscle. However, whilst many parasite-secreted proteins have been identified, it has rarely been demonstrated that these are secreted into the nurse cell. Using an informatics-based analysis, we have searched the T. spiralis expressed sequence tag (EST) datasets for cDNAs encoding potential secreted proteins. Here we describe the characterisation of three of the top candidates isolated from our analysis, termed secreted from muscle stage larvae (SML)-1, -2 and -3. All three proteins were demonstrated to be secreted by muscle stage larvae, and immunohistochemical analysis established that SML-1 and -2 are secreted into developing nurse cells. We also show that SML-2 is processed from a precursor into smaller peptides by a metalloprotease contained within T. spiralis-secreted products. With the identification of these and other secreted proteins, we now have molecules to test in functional assays designed to dissect molecular features of the developing nurse cell.     

Evaluation of the PrioCHECK™ Trichinella AAD kit to detect Trichinella spiralis,T. britovi,and T. pseudospiralis larvae in pork using the automated digestion method Trichomatic-35

Trichinellosis is a potentially deadly parasitic zoonosis that is contracted by consuming undercooked infected meat. Reliable detection of infectious Trichinella spp. larvae in meat is therefore pivotal to ensure consumer's safety. The recently authorised PrioCHECK™ Trichinella Alternative Artificial Digestion (AAD) test kit appears promising when used with the standard magnetic stirrer method, but evaluation with other apparatus types is lacking.In this study, the performance of the AAD kit in an adapted Trichomatic-35 (TM35) instrument was evaluated, first, at the Swiss National Reference Laboratory for trichinellosis (NRL); second, in a ring trial involving four Swiss official laboratories. Proficiency pork samples spiked with larvae of Trichinella spiralis, T. britovi, or T. pseudospiralis were tested with the AAD kit and with the reference pepsin-HCl digestion method in TM35 instruments.At the NRL, both methods yielded identical qualitative and similar quantitative results independently of the Trichinella species. In the ring trial, satisfactory results were obtained for 47/50 (94.0%) (AAD) and 62/67 (92.5%) (reference method) of the analysed samples. Technical problems impairing analysis were more frequently observed with the AAD kit (n = 22) than with the reference method (n = 5) and were mainly (16/22) reported by one of the external labs. When no technical issues were recorded, the performance of both methods was comparable, in agreement with the observations at the NRL; however, these results suggest a need for further training with the kit and standardisation of the adapted TM35 instruments.     

Patterns and Risks of Trichinella Infection in Humans and Pigs in Northern Laos

Several outbreaks of trichinellosis associated with the consumption of raw pork have occurred in Laos since 2004. This cross-sectional study was conducted in four provinces of northern Laos to investigate the seroepidemiology of trichinellosis in the human population and determine the prevalence and species of Trichinella infection in the domestic pig population. Serum samples and questionnaire data were obtained from 1419 individuals. Serum samples were tested for Trichinella antibodies by ELISA using larval excretory–secretory (ES) antigens and a subset of 68 positive samples were tested by western blot. The seroprevalence of Trichinella antibodies was 19.1% (95% confidence interval (CI) = 17.1–21.1%). The risk of having antibodies detected by ELISA using ES antigens increased with age, being of Lao-Tai ethnicity, living in Oudomxay province and being male. Tongue and diaphragm muscle samples were collected from 728 pigs and tested for Trichinella larvae by the artificial digestion method. Trichinella larvae were isolated from 15 pigs (2.1%) of which 13 were identified as T. spiralis by molecular typing; the species of the two remaining isolates could not be determined due to DNA degradation. Trichinella spp. are endemic in the domestic environment of northern Laos and targeted preventative health measures should be initiated to reduce the risk of further outbreaks occurring.