Molecular identification of a Trichinella isolate from a naturally infected pig in Tibet, China

The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial large-subunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.     

Proteomic analysis of Trichinella spiralis proteins in intestinal epithelial cells after culture with their larvae by shotgun LC-MS/MS approach

Although it has been known for many years that Trichinella spiralis initiates infection by invading intestinal epithelium, the mechanisms by which the parasite invades the intestinal epithelium are unknown. The purpose of this study was to screen the invasion-related proteins among the increased proteins of intestinal epithelial cells after culture with T. spiralis and to study their molecular functions. The proteins of HCT-8 cells which cultured with T. spiralis infective larvae were analyzed by SDS-PAGE and Western blot. Results showed that compared with proteins of normal HCT-8 cells, four additional protein bands (115, 61, 35 and 24 kDa) of HCT-8 cells cultured with the infective larvae were recognized by sera of the mice infected with T. spiralis, which may be the invasion-related proteins released by the infective larvae. Three bands (61, 35 and 24 kDa) were studied employing shotgun LC-MS/MS. Total 64 proteins of T. spiralis were identified from T. spiralis protein database by using SEQUEST searches, of which 43 (67.2%) proteins were distributed in a range of 10-70 kDa, and 26 proteins (40.6%) were in the range of pI 5-6. Fifty-four proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Out of 54 annotated proteins, 43 proteins (79.6%) had binding activity and 23 proteins (42.6%) had catalytic activity (e.g. hydrolase, transferase, etc.), which might be related to the invasion of intestinal epithelial cells by T. spiralis. The protein profile provides a valuable basis for further studies of the invasion-related proteins of T. spiralis.     

Comparative efficacy of antigen and antibody detection tests for human trichinellosis

Sera collected from patients with suspected or confirmed exposure to Trichinella spiralis were tested for circulating parasite antigens and antiparasite antibodies. Using an immunoradiometric assay, excretory--secretory antigens from muscle-stage larvae of T. spiralis were detected in the sera of 47% of 62 patients with clinical trichinellosis and 13% of 39 patients without clinical signs but suspected of exposure to infected meat. In comparison, antibodies were detected using an indirect immunofluorescent test in the circulation of 100% of the 62 patients with clinical trichinellosis and 46% of the 39 patients with suspected exposure. The presence of antibodies specific to excretory-secretory products of T. spiralis muscle larvae was confirmed in the majority of the samples tested by a monoclonal antibody-based competitive inhibition assay. These results indicate that antibody detection is a more sensitive diagnostic method for human trichinellosis, but that antigen detection might be a useful confirmatory test because it is a direct demonstration of parasite products in the circulation.     

Epidemiological survey of Trichinella infection in domestic, synanthropic and sylvatic animals from Argentina

The presence of Trichinella larvae was investigated in 247 samples taken from domestic, synanthropic and sylvatic animals, collected during 1996 to 2005 in 12 endemic provinces of Trichinella infection in Argentina. Muscle larvae of Trichinella from 65 infected animals were identified at the species level by single larva nested polymerase chain reaction (PCR) technique based on the variability within the expansion segment V (ESV) region of the ribosomal DNA. Trichinella infections were found in 97 of 164 pigs, 38 of 56 pork products, two domestic dogs, one domestic cat, 7 of 11 armadillos and 3 of 9 synanthropic rats. All Trichinella isolates were identified as Trichinella spiralis by nested PCR. These findings add new data on the epidemiology of trichinellosis and should be considered when implementing new strategies to control this zoonosis.     

Trichinella spiralis as a modulator of Shope fibroma virus

After the works on the promoting effect of trichinellosis on some viral infections in rodents, many studies successively demonstrated that Trichinella spiralis confers resistance to many unrelated antigens including pathogens, such as Protozoa, Bacteria and tumour cells (B16 melanoma). Considering the above contradictory results, the present work was undertaken to study, in rabbits, T. spiralis as a modulator of Shope's fibroma virus, an oncogenic virus responsible for a benign neoplasia. Four groups of 6 rabbits each were used. The rabbits of group I, II and III were inoculated per os with 3000; 6000 and 12,000 T. spiralis larvae, respectively. The rabbits of group IV were used as controls. Thirty-five days after the inoculation, all the animals were injected at the fixed doses of 0.5 ml with dilutions (10(-1) to 10(-8] of Shope's fibroma virus given intradermally into 8 different points of the skin of each pretreated and untreated rabbits. After 9 days tumour lesions affecting the inoculating area were noticed and the DI 50/0.5 of Shope's fibroma virus was then determined for each of the 4 experimental groups. The rabbits pretreated with T. spiralis exhibited much lower virus titres than the controls, which was evidently related to a certain degree of aspecific immunity conferred by the parasite. The results indicated that T. spiralis produces, in rabbits, resistance to Shope's fibroma virus and its neoplastic effect.     

Detection of Trichinella spiralis, T. britovi and T. pseudospiralis in muscle tissue with real-time PCR

Infections caused by Trichinella species occur throughout the world in many wild and domestic animals resulting in trichinellosis in men. In Europe, domestic pigs are predominantly infected by three Trichinella species: T. spiralis, T. britovi and T. pseudospiralis. Present methods for detection of Trichinella spp. (compressorium method, artificial digestion) do not always sufficiently recognize Trichinella larvae and these techniques are labor-intensive, time consuming and do not differentiate isolates on the species level since there are no distinguishing morphological features. Additionally, conventional PCRs cannot quantify numbers of larvae in infectious material. In order to better meet these requirements, we developed a real-time PCR assay for the accurate, rapid and specific identification of the three common European species of the genus Trichinella. The assay targets the large subunit of the mitochondrial rRNA (rrnL) and enables sensitive determination and discrimination of larvae in muscle tissue samples. The real-time PCR assay was developed and validated using reference and field strains from T. spiralis, T. britovi and T. pseudospiralis. In the described real-time PCR assay, the melting points of specific amplificates were always discernable via the melting curve from melting points of unspecific amplificates. This is important for the methods workflow because only C(T) values connected with the additional melting curve analysis allow a distinction of the individual species with confidence. The sensitivity of the technique enabled detection down to 0.1 Trichinella larva per gram meat sample. High disruption levels of tissues by mincing generally resulted in higher sensitivities than protocols without mincing. With its short completion time as well as accurate and specific detection of selected species this assay could become a convenient tool for the fast detection of Trichinella larvae in meat.     

Trichinella spiralis in an agricultural ecosystem: transmission in the rat population

Four hundred forty-three Norway rats (Rattus norvegicus) were examined to determine their role in the transmission and maintenance of Trichinella spiralis on a pig farm. Rats, classified by sex and weight, were examined for trichinellosis by peptic digestion of muscle samples. Over a 25-mo period, 188 (42.4%) rats were found to be infected with T. spiralis. The mean intensity of infection was 293.2 larvae per gram (LPG) of muscle; 65 (34.6%) infected rats had intensities of infection greater than 100 LPG. Even in the absence of a known source of infected meat (garbage containing meat scraps or dead animals), the rat population maintained the infection, probably through cannibalism. Population reduction was an effective method for reducing the prevalence of infection within the rat population. Therefore, to reduce the likelihood of transmission of T. spiralis between rats and swine, it is essential that rat populations in a farmyard environment be controlled.     

Immunodiagnosis of human trichinellosis using excretory-secretory (ES) antigen.

Infective first stage larvae of Trichinella spiralis were recovered from muscles of laboratory infected mice by digesting the muscles with 1% HC1-1% pepsin and collecting the larvae by modified Baerman's method. The larvae were cultivated in a serum-free medium for 18 h. The ES antigen obtained from the culture medium was used in an enzyme-linked immunosorbent assay (ELISA) for detecting IgG antibodies to T. spiralis in serum samples collected from three groups of individuals. The individuals of the first group were parasitologically confirmed trichinellosis patients, while those of group 2 were patients with other helminthiasis and group 3 were healthy, parasite-free individuals. The specificity of the assay was 100%. The sensitivity of the test was also 100% when performed on sera of group 1 collected at days 57 and 120 after infection. Sera collected earlier (day 23) and those collected 700 days after infection had negligible reactivity. Thus IgG-ELISA using ES antigen of the L1 was useful not only for diagnosis but also in evaluation of cure. Western blot analysis revealed that specific antigens of T. spiralis were 94, 67, 63, and 39 kilodalton components.     

Parameters of intestinal inflammation in mice given graded infections of the nematode Trichinella spiralis

Four parameters of the intestinal inflammatory response (numbers of mucosal mast cells (MMC) and Paneth cells, villus:crypt ratios and mitotic figures) were measured in mice exposed to varying doses of infective larvae of Trichinella spiralis.The aim of the experiments was to determine whether generation of these components of inflammation required a threshold level of infection and whether, once triggered, inflammation became pan-mucosal. Near maximal MMC and Paneth cell responses were elicited even with infections as low as 35 larvae; changes in villus:crypt ratios and in mitotic indices also occurred at this level of infection, but were progressively greater with increasing levels of infection. In all infected mice, including those infected with 35 larvae, MMC and Paneth cell responses extended over most of the small intestine. These data are interpreted as showing: (i) that the intestinal mucosa is highly responsive to T. spiralis infection; (ii) that once triggered, components of the inflammatory response are amplified by T cell-dependent mechanisms, becoming pan-mucosal; and (iii) that MMC and Paneth cell responses, which require cell division and differentiation, become maximal at a lower infection threshold than changes in the villus:crypt ratio or in mitotic indices, which directly reflect increased rates of division in crypt cells.     

Sylvatic trichinellosis in southwestern Spain

The epidemiology of Trichinella spp. in their main sylvatic hosts, wild boar (Sus scrofa ferus and red fox (Vulpes vulpes), in Extremadura (southwestern Spain) was studied. We examined 88 Trichinella spp.-positive wild boar muscle-tissue samples from a total of 29,333 killed animals, referred to the Veterinary Parasitology Department (University of Extremadura, Spain) by the Extremadura Veterinary Service. Additionally, 227 red foxes killed during the hunting season and thus not subject to veterinary controls were examined for trichinellosis. Trichinella spp. larvae were found in six (3%) of the red foxes. All samples were examined using direct diagnostic techniques, including trichinoscopy and artificial digestion. The mean intensity of infection was 74.8 larvae/g (LPG) of muscle tissue in wild boars, compared to 30.6 LPG in foxes. Trichinella spiralis (sensu stricto) predominated over T. britovi in wild boars. Random amplified polymorphic DNA (RAPD) and alloenzyme typing showed that 74% of infected wild boars had only T. spiralis, 21% had only T. britovi, and 5% showed mixed infections. In contrast, 33% of infected foxes were infected only with T. spiralis, while 67% had T. britovi, suggesting a clear predominance of the latter in foxes. We suspect the existence of a paranthropic or sylvatic cycle in large areas of this region; given the ease of transfer between sylvatic and domestic or semi-domestic animals, this implies a high epidemiological risk.     

Immunoelectrotransfer blot assay in acute and chronic human trichinellosis

An immunoelectrotransfer blot assay (IETB) using excretory secretory products of muscle larvae of Trichinella spiralis (ML-ESP) and the avidin biotin system was developed in order to characterize reactivity against ML-ESP in sera from patients with acute and chronic trichinellosis. A complete pattern of up to 13 bands was developed by sera from individuals with trichinellosis where doublets, triplets, or single bands were shown to have molecular weights of roughly 66, 55, 45, 36, 29, 24, and 14 kDa. The bands at approximately 55, 36, 29, and 14 kDa proved specific for T. spiralis. The band at approximately 55 kDa was present in all trichinellosis sera, whereas the approximately 14-kDa band was present in only a small percentage of sera. The development of approximately 36- and 29-kDa bands suggests a modulation of the reactivity against ML-ESP over time. IETB proved more sensitive for the population of chronic trichinellosis under study than a conventional diagnostic enzyme-linked immunosorbent assay, allowing negative or borderline serum samples to be determined. Thus, this technique, when applied for human trichinellosis surveillance, should provide a useful tool in endemic areas.     

Spatial variation of Trichinella prevalence in rats in Finnish waste disposal sites

Trichinellosis is 1 of the most widespread parasitic zoonoses in the world and can be lethal to humans. Trichinella spp. are also parasites of considerable economic importance. Because rats may play a role in the transmission of trichinellosis to swine and farmed wild boar, 767 brown rats (Rattus norvegicus Berkenhout) from 13 Finnish waste disposal sites were examined for Trichinella spp. by a HCl-pepsin digestion method. Trichinella spp. were found to be a common parasite in trapped rats (overall prevalence, 19%) detected in 12 of 13 dumps. Significant differences were observed between sites in the prevalence (0-49%) of Trichinella spp. Female rats were more often and more heavily infected than males, but age was not shown to be a risk factor for trichinellosis. In addition, positive correlation was demonstrated between rat population density and prevalence. Trichinella spiralis was identified by multiplex polymerase chain reaction in 28 rats. The median density of infection was 42 (range, 0.5-6,925) larvae/ g of host tissue, but neither the occurrence nor the density of the parasite was related to the physical condition of the animal.     

Tolerance to low temperatures of domestic and sylvatic Trichinella spp. in rat muscle tissue

The present study was designed to investigate the tolerance to low temperatures of 9 Trichinella isolates in rat muscle tissue. Nine groups of 24 rats were infected with encapsulated Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella murrelli, Trichinella T6, Trichinella nelsoni, and 3 nonencapsulated Trichinella pseudospiralis strains. Six rats from each of the groups were necropsied at 5, 10, 20, and 40 wk postinfection (wpi). Muscle tissues containing Trichinella larvae were exposed to temperatures of -18, -5, and 5 C for 1 or 4 wk, and afterward the reproductive capacity index (RCI) in mice was determined for the 9 individual Trichinella isolates. Only T. nativa muscle larvae were infective after freezing at a temperature of -18 C. At 5 wpi all encapsulated isolates, except for the tropical species T. nelsoni, remained infective after exposure to a temperature of -5 C for both 1 and 4 wk, whereas nonencapsulated T. pseudospiralis survived only 1 wk of exposure. All Trichinella spp. remained infective after exposure to a temperature of 5 C. Muscle larvae for all investigated species remained infective as long as they persisted in live rats during the experiment. Analysis of variance showed a significant effect of age on the temperature tolerance of encapsulated T. spiralis and nonencapsulated T. pseudospiralis. In addition, significant interaction between age of muscle larvae and length of exposure was found. In general Trichinella muscle larvae of medium age (10 and 20 wpi) tolerated freezing better than early and late stages of infection (5 and 40 wpi). This is the first study to demonstrate such a relationship between age of infection and temperature tolerance of Trichinella spp. muscle larvae.     

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.     

The relationship between intestinal location and fecundity in adult Trichinella spiralis.

Adult female worms recovered from the jejunum of rats infected per os with 1000 larvae of Trichinella spiralis were significantly more fecund (peak jejunal fecundity = 35.7 +/- 3.1 newborn larvae per female) than females recovered from the terminal ileum (9.3 +/- 4.1 larvae per female) in the same infections. The majority of the adult worms were established in those sections of the small intestines that produced the most fecund females (r = 0.92; P less than 0.05). Worm fecundity is believed to be location-specific because adult females that were surgically implanted into the jejunum were significantly more fecund that were implanted into only the ileum. It is concluded that the physico-chemical conditions of the anterior small intestines are optimal for the parasites' reproductive fitness and this exerts a strong selective pressure on habitat selection behavior.     

Susceptibility of adult Heligmosomoides polygyrus to intestinal inflammatory responses induced by heterologous infection.

Adult H. polygyrus are capable of surviving for many months after primary exposure of mice to infective larvae, raising the possibility that worms of this species have inherent resistance to intestinal immune responses. Accordingly experiments were carried out to determine whether H. polygyrus are resistant to the inflammatory changes elicited during the acute phase of the intestinal response to Trichinella spiralis. Adult worms were expelled from mice when their presence coincided with the most intense phase of inflammation elicited by T. spiralis. The effect was dose-dependent with more intense T. spiralis challenge resulting in a correspondingly greater loss of H. polygyrus. Even the less pathogenic species T. pseudospiralis elicited a response of sufficient intensity in NIH mice to cause the expulsion of H. polygyrus from concurrently infected animals. Tissue larval stages of H. polygyrus were protected from expulsion by their location deep in the intestinal walls and the maximum detrimental effect against H. polygyrus was observed during the adult phase or during the establishment of L3 larvae. Acceleration of the response to T. spiralis in immune challenged mice resulted in earlier loss of H. polygyrus. When the expulsion of T. spiralis was delayed (e.g. from slow responder C57BL/10 mice) the loss of H. polygyrus took place correspondingly later. These experiments demonstrate unequivocally that mouse strains which normally tolerate chronic infections with H. polygyrus have the capacity to mount intestinal inflammatory responses of sufficient vigour to remove the worms but that this potential is not normally realized. However, the observation that some H. polygyrus always survived even when the response induced by T. spiralis was of the rapid secondary type suggests that the parasites are resilient in the face of the inflammatory response capable of removing most of the worms. It is suggested that in addition to the immunomodulatory strategy employed by adult worms to prevent the intestinal response being elicited, the worms have a second line of defence which is reflected in their resilience to responses which they have been unable to prevent.     

Trypanosoma musculi with Trichinella spiralis or Heligmosomoides polygyrus: concomitant infections in the mouse

Inbred mice infected with Trypanosoma musculi displayed wide variations in peak blood parasitemia. The most susceptible mice were C3H and A strain, while Balb/c, C57B1/6, and the related congenic B10 strains were the most resistant. The effect of an intestinal infection with either Trichinella spiralis or Heligmosomoides polygyrus on proliferation of T. musculi was investigated. T. spiralis infections given at the same time or up to 45 days before a T. musculi infection always caused an increase in blood parasitemia in C3H mice. Maximum increases were observed when T. spiralis infections preceded T. musculi by 5-10 days. In all mouse strains examined, dual infections increased maximum parasitemia by two- to four-fold, regardless of the degree of resistance of that mouse strain to either T. musculi or T. spiralis. This suggested that the immunological "cost" of a T. spiralis infection was the same for strains that were strong or weak responders to a primary infection with T. spiralis. In contrast, infection with H. polygyrus did not promote T. musculi parasitemia over the level of a single infection. The increase in blood parasitemia in T. spiralis-infected mice was largely due to the intestinal adult worm, but migratory larvae and mature muscle larvae also stimulated increased parasitemias. The increase in parasitemia was proportionate to the dose of T. spiralis, and the sex of the host did not affect the blood trypanosome level.     

Effect of aminoguanidine and albendazole on inducible nitric oxide synthase (iNOS) activity in T. spiralis-infected mice muscles

The aim of this study was to provide evidence for the expression of iNOS in the cells of inflammatory infiltrates around larvae in skeletal muscles of T. spiralis infected mice. The BALB/c mice (n = 8) divided into subgroups, received either aminoguanidine (AMG)--a specific iNOS inhibitor or albendazole (ALB)--an antiparasitic drug of choice in trichinellosis treatment. Control animals (n = 2 in each subgroup) were either uninfected and treated or uninfected and untreated. Frozen sections of hind leg muscles from mice sacrificed at various time intervals after infection were cut and subjected to immunohistochemistry, using monoclonal anti-iNOS antibody. The ALB-treated mice revealed stronger iNOS staining in the infiltrating cells around larvae than the infected and untreated animals. On the contrary, in the AMG-treated animals, the infiltrating cells did not show any specific iNOS reaction. These data confirm the specificity of iNOS staining in the cellular infiltrates around T. spiralis larvae and shed some light on the role of nitric oxide during ALB treatment in experimental trichinellosis.     

Characterization of a noncyst-forming isolate of Trichinella from a wild boar in Yugoslavia.

An isolate of Trichinella obtained from a wild boar in Yugoslavia did not form cysts in the musculature of its natural host. Subsequent inoculation into experimental hosts demonstrated that some larvae became encysted only after extended time periods, whereas others remained unencapsulated. Histological staining of larvae in the musculature demonstrated no deposition of collagen typically seen for Trichinella spiralis spiralis, Trichinella spiralis nativa, or Trichinella spiralis nelsoni. The Yugoslavian isolate, given the name of Zagreb isolate after the University where it was first studied, had low infectivity for pigs and mice. Isozyme analysis demonstrated greater homology with T. s. nelsoni than with other subspecies of Trichinella. Restriction fragment length polymorphisms and dot blot analyses further demonstrated the distinctive nature of this isolate. These results suggest that lack of cyst formation might be characteristic of isolates other than those designated Trichinella pseudospiralis and that this character might be important in the classification of Trichinella.     

Infectivity, persistence, and antibody response to domestic and sylvatic Trichinella spp. in experimentally infected pigs

Groups of pigs were inoculated with genotypes of Trichinella belonging to: Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis (from Caucasus), T. pseudospiralis (from USA), Trichinella murrelli, Trichinella sp. (from North America), and Trichinella nelsoni. The pigs were sacrificed between 5 and 40weeks p.i., and the number of muscle larvae per gram (l.p.g.) of tissue was determined as an average of 18 muscles. All Trichinella genotypes were infective for pigs, but both their infectivity and persistence varied: 5weeks p.i., T. spiralis muscle larvae were present in high numbers (mean=427l.p.g.), while T. britovi, T. nelsoni, and T. pseudospiralis larvae were present in moderate numbers (means=24-52l.p.g.); larvae of the remaining genotypes were recovered only in low numbers (means=0.05-5. 00l.p.g.). The total larval burden (live weight of pigxl.p.g.) was constant over time for T. spiralis, T. britovi, and T. nelsoni, but declined significantly (P<0.05) for the other genotypes. Antibody responses could be detected 3-4weeks p.i. by seven different Trichinella ES antigens, but the antibody levels and dynamics differed significantly among the experimental groups. In pigs inoculated with T. spiralis, T. britovi, or T. nelsoni, the antibody level increased rapidly between weeks 3 and 5 p.i. and was stable or increased slightly throughout the experimental period. In pigs inoculated with T. nativa, T. murrelli, or Trichinella (T6) (from North America), a rapid increase was detected between weeks 3 and 5 p.i., but for these genotypes a reduction in the antibody levels was seen thereafter. In the pigs inoculated with T. pseudospiralis, the antibody level increased more gradually over a period from week 3 p. i. to weeks 15-20 p.i., and decreased thereafter. In general, all species of Trichinella were detected by any of the seven ES antigens, which points to the potential use of one common antigen for surveillance and epidemiological studies on both domestic and sylvatic Trichinella in pigs. Homologous ES antigens were slightly more sensitive in detecting antibodies to the corresponding Trichinella species.