Molecular identification of<Emphasis Type="Italic"> Trichinella spiralis</Emphasis> and<Emphasis Type="Italic"> Trichinella britovi</Emphasis> by diagnostic multiprimer large mitochondrial rRNA amplification

Trichinella parasites with different epidemiological features still occur in Europe and four species of genus Trichinella have been identified: T. spiralis, T. britovi , T. nativa and T. pseudospiralis. Until now, two of them, T. spiralis and T. britovi, have been identified in Poland. In our studies we selected sequence coding for large mitochondrial rRNA (mt LrDNA) as a genetic marker and developed a sensitive LrDNA multiprimer PCR assay allowing for rapid identification of T. spiralis and T. britovi, parasites present in wild and domestic animals in Poland.     

Differentiation ofTrichinella isolates by polymerase chain reaction

Oligonucleotide primers were synthesized for the polymerase-chain-reaction amplification of target DNA from two sequences ofTrichinella spiralis. Six strains belonging toT. spiralis, T. nativa, T. britovi, T. pseudospiralis, andT. nelsoni were tested. Amplification products were obtained withT. spiralis, T. britovi, andT. nelsoni DNA from a 53-kDa antigen cDNA sequence and withT. spiralis andT. nelsoni DNA from a 1.6-kb repetitive DNA sequence. Differences in the length of the amplification products obtained from the repetitive sequence would enable a differentiation betweenT. spiralis andT. nelsoni, suggesting that the 1.6 kb repetitive DNA sequence would not be specific forT. spiralis. No amplification was detected forT. nativa orT. pseudospiralis DNA from the two sequences and forT. britovi DNA from the 1.6-kb repetitive DNA sequence.     

Molecular epidemiology of <Emphasis Type="Italic">Trichinella</Emphasis> spp. in three Baltic countries: Lithuania,Latvia, and Estonia

Meat of domestic pigs and wild boars has been the significant source of emerged human trichinellosis in Lithuania, Latvia, and Estonia over the past two decades. However, there is very little known on the occurrence of Trichinella spp. in main wildlife reservoirs and its transmission in domestic and sylvatic cycles in these countries. The present study demonstrated considerably higher endemicity of Trichinella spp. in main sylvatic reservoirs (28.9–42% in foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in all three countries than previously reported. Molecular identification of Trichinella larvae from more than 500 sylvatic and domestic animals revealed four Trichinella species (Trichinella spiralis, Trichinella britovi, Trichinella nativa, and Trichinella pseudospiralis) sympatric in a relatively small area and several as the first records for the respective countries. The nonencapsulating T. pseudospiralis is found for the first time in the Eastern Europe. Sylvatic T. britovi was found in domestic pigs in Lithuania and Latvia (16 and 57.1%, respectively) and only in these countries, domestic T. spiralis was detected in sylvatic animals in areas where domestic trichinellosis was registered. The study suggests that transmission of Trichinella between domestic and sylvatic cycles in Lithuania and Latvia is favored by improper human behavior, e.g., pig and slaughter waste management.     

First report of Trichinella pseudospiralis in Poland, in red foxes (Vulpes vulpes)

Nematode worms of the genus Trichinella are one of the most widespread zoonotic pathogens. Natural transmission between hosts can only occur through the ingestion of infected meat. To date, two Trichinella species are known to be etiological agents of disease among domestic animals and wildlife in Poland: T. spiralis and T. britovi. In the last decades, since the administration of an oral vaccination against rabies, the red fox population in Poland has increased exponentially. The study area covers the Nowy Targ region: a mountainous area (585–1138 m above the sea) in southern Poland. Of 24 red foxes examined in the study, four were infected with Trichinella isolates: three were identified as T. britovi and one as T. pseudospiralis. The muscle of red foxes infected with T. britovi harboured 2.75, 3.11, 4.4 LPG and with T. pseudospiralis 0.36 LPG. Trichinella larvae were identified at species level by genomic and mitochondrial multiplex PCR, the products of which were sequenced for comparison with other sequences available in GenBank. The sequences obtained from the Polish T. pseudospiralis isolate, deposited in GenBank under the accession numbers JQ809660.1 and JQ809661.1, matched sequences already published in GenBank. Sequence comparison showed a 100% match with the large subunit ribosomal RNA gene of T. pseudospiralis isolate ISS 013, and a 96–95% match with those of T. pseudospiralis isolates ISS 141 and ISS 470. This is the first report of the identification of T. pseudospiralis larvae from red fox in Poland.     

Development of cellular immune response of mice to infection with low doses of Trichinella spiralis, Trichinella britovi and Trichinella pseudospiralis larvae

The murine cellular immune response to the infection with ten larvae of encapsulating (Trichinella spiralis, Trichinella britovi) and non-encapsulating species (Trichinella pseudospiralis) was studied. Both T. spiralis and T. britovi stimulated the proliferation of splenic T and B lymphocytes during the intestinal phase of infection, but T. spiralis activated the proliferative response also at the muscle phase, particularly in B cells. Non-encapsulating T. pseudospiralis stimulated the proliferation of T and B cells only on day 10 post-infection (p.i.) and later at the muscle phase. The numbers of splenic CD4 and CD8 T cells of T. spiralis infected mice were significantly increased till day 10 p.i., i.e., at the intestinal phase, and then at the late muscle phase, on day 60 p.i. T. britovi infection increased the CD4 and CD8 T cell numbers only on day 30 p.i. Decreased numbers of CD4 and CD8 T cells after T. pseudospiralis infection suggest a suppression of cellular immunity. Both encapsulating Trichinella species induced the Th2 response (cytokines interleukin-5 (IL-5) and interleukin-10) at the intestinal phase and the Th2 dominant response at the advanced muscle phase. Interferon-γ (IFN-γ) production (Th1 type) started to increase with migrating newborn larvae from day 15 p.i. till the end of the experiment. IL-5 production was suppressed during the intestinal phase of T. pseudospiralis infection. The immune response to T. pseudospiralis was directed more to the Th1 response at the muscle phase, the high IFN-γ production was found on day 10 p.i. and it peaked on days 45 and 60 p.i.     

Infectivity, predilection sites, and freeze tolerance of Trichinella spp. in experimentally infected sheep

A total of 36 sheep in groups of 4 were inoculated with 9 isolates of Trichinella and euthanized after 10 weeks. Thereafter, numbers of muscle larvae were determined in 13 different muscles/muscle groups. Muscle larvae were found in high numbers in all four sheep inoculated with T. spiralis, in lower numbers in two sheep inoculated with T. pseudospiralis (USA isolate), and in very low numbers in one sheep inoculated with T. pseudospiralis (USSR isolate) and one inoculated with T. britovi. In infections of high and moderate larval intensity, predilection sites of T. spiralis were the masseter muscles, the tongue, and the diaphragm and those of T. pseudospiralis were the masseter muscle and the neck. In low-intensity infections, muscle larvae were detected only in the diaphragm or in pooled muscle samples. For evaluation of the freeze tolerance of the different Trichinella species in sheep-muscle tissue, samples taken from the filet were stored at +5°, −5°, and −18 °C, respectively. After exposure for 1 and 4 weeks the tissue was digested and the released larvae were inoculated into mice for determination of the reproductive capacity index (RCI). Larvae of both T. spiralis and T. pseudospiralis survived freezing at −5° and −18 °C for 4 weeks. Received: 10 September 1999 / Accepted: 22 October 1999     

The karyotype of four Trichinella species

Karyological studies ofTrichinella spiralis, T. pseudospiralis, T. nativa andT. nelsoni were undertaken. Comparison of the karyotypes of theseTrichinella species showed that the chromosome number of all four species is 2n=6 for female specimens and 2n=5 for males. The differences found in the relative chromosome lengths of the individualTrichinella species are not significant.Centromeric index data indicate thatT. nativa andT. spiralis have similar centromere dispositions and differ from the other two species by the disposition of the centromere of the first submetacentric chromosome pair. InT. nativa andT. nelsoni the univalent sex chromosome is the second in size. It is a slightly submetacentric chromosome, while inT. spiralis andT. pseudospiralis it is the third metacentric chromosome. The data from the karyological investigations may be used as additional karyosystematic characteristics when differentiating theTrichinella species studied.     

Trichinella spiralis,T. britovi,and T. nativa : infectivity,larval distribution in muscle,and antibody response after experimental infection of pigs

The infectivity of Trichinella spiralis, T. nativa, and T. britovi was experimentally compared in pigs. Blood sampling was performed weekly, and muscle juices were obtained at slaughter 10 weeks after inoculation. Muscle larvae were found in all of four pigs inoculated with T. spiralis [mean 190 larvae per gram (lpg)] and in three of four pigs inoculated with T. britovi (mean 7 lpg). No larvae were found in pigs inoculated with T. nativa. For T. spiralis and T. britovi, the neck muscle (m. splenius) appears to be a predilection site in addition to the tongue, the diaphragm, and the jaw. High antibody responses were found in all experimental groups, independent of the antigen used, and even in pigs in which no muscle larvae were recovered. The strong and consistent antibody response found with meat juice indicates the usefulness of this material where a blood sample is not obtainable, e.g. meat samples from wild animals. Immunoblotting (Western blots) on slaughter sera revealed no species specificity when comparing homologous versus heterologous staining. Received: 30 July 1997 / Accepted: 25 September 1997     

First report of <Emphasis Type="Italic">Trichinella britovi</Emphasis> in Serbia

In Europe, Serbia ranks among countries with a high prevalence of Trichinella infection in pigs, which continues to be a serious human health problem. While in some Balkan countries, more than one Trichinella species/genotype has been described in both the sylvatic and domestic cycles, these data are lacking for Serbia. To date, only a few Serbian isolates of Trichinella have been genetically specified, and all were classified as T. spiralis. Although transmission of Trichinella from domestic pigs to wildlife could be assumed, neither the infection status nor the species of Trichinella circulating among wildlife in Serbia has been investigated. This study shows the presence of two Trichinella species, T. spiralis and T. britovi, in wild animals originating from five districts in Serbia, where Trichinella infections in domestic pigs and humans have been recorded. Trichinella spiralis was detected in jackals (n = 3), red foxes (n = 2) and a wild cat (n = 1). We also established that wolves (n = 4) and red foxes (n = 2) serve as sylvatic reservoirs for T. britovi. This is the first report on the presence of T. britovi in Serbia.     

Analysis of a novel cathepsin B circulating antigen and its response to drug treatment in Trichinella-infected mice

In this paper, we cloned a novel full-length cDNA that encodes a Trichinella spiralis cathepsin B-like protease gene (TsCPB) using 3'-RACE PCR. The recombinant mature TsCPB protein (rTsCPB) was then expressed in an Escherichia coli expression system and purified with Ni-affinity chromatography. Real-time quantitative PCR revealed that TsCPB was expressed across all development stages of the parasite but had the highest expression level during the adult stage. Furthermore, rTsCPB was detected in Trichinella excretory–secretory products with anti-rTsCPB rabbit polyclonal antibodies. Interestingly, rTsCPB was strongly recognized by the T. spiralis-infected sera in Western blotting, implying that TsCPB protein appeared in the peripheral blood of Trichinella-infected mice as circulating antigens (CAg). We then analyzed the dynamic levels of TsCPB CAg and its antibodies in T. spiralis-infected sera by using an improved double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) and indirect ELISA, respectively. The results showed that TsCPB CAg can be detected much earlier compared to antibody detection in Trichinella-infected mice. In addition, we monitored the effects of albendazole drug therapy (a dosage of 370 mg/kg body weight, twice a day) on T. spiralis-infected mice by detecting the levels of TsCPB CAg and its antibody in the sera of drug-treated mice. The results showed that the levels of CAg dramatically decreased after successful drug treatment, while the antibody level remained unchanged. Overall, the novel Trichinella antigen TsCPB could be a promising novel circulating antigen molecule for the detection of Trichinella infection and for monitoring the efficacy of drug treatment of trichinellosis.     

A novel cDNA clone encoding a specific excretory/secretory antigen of larval Trichinella pseudospiralis

A cDNA library for Trichinella pseudospiralis was constructed to study the expression of specific antigens. Four positive clones were identified using antibodies against the excretory/secretory (ES) products of the nematode as probe. Sequence analysis showed that they contained identical cDNA inserts of 606 bp, including a 5′ non-translated region of 96 bp, a core translated segment of 408 bp and a poly(A)⁺ 3′ terminus. It encoded a novel 136-amino-acid polypeptide. Southern blot analysis indicated that the cDNA did not cross-hybridize to the genomic digests of T. spiralis, mouse, or rat. A single copy only of its complementary sequence was found in the genome of T. pseudospiralis. Using the lambda ZAP expression system, the cDNA was induced to express a 23-kDa β-galactosidase-fusion protein which did not cross-react with polyclonal and monoclonal antibodies against T. spiralis, heat shock proteins, or four heterologous species of nematodes. The antiserum against the fusion protein recognized a 15-kDa band from the ES products of T. pseudospiralis in immunoblotting. Immunocytolocalization demonstrated that the anti-fusion protein serum only recognized an epitope in the stichosome of T. pseudospiralis and not in T. spiralis. The protein can therefore serve as a specific antigen for the differential diagnosis of trichinellosis. Received: 22 December 1998 / Accepted: 17 February 1999     

Biochemical analysis of encapsulated and non-encapsulated species of<Emphasis Type="Italic"> Trichinella</Emphasis> (Nematoda,Trichinellidae) from cold- and warm-blooded animals reveals a high genetic divergence in the genus

Multilocus enzyme electrophoresis was used to analyse genetic variation in the genus Trichinella. Twenty-eight isolates belonging to eight species and six genotypes were analysed for 12 enzyme systems, producing 19 different phenotypes. According to Jaccards similarity index, the isolates clustered into two main groups, specifically, encapsulated species/genotypes and non-encapsulated species/genotypes. Furthermore, the non-encapsulated species clustered into two other groups: the species infecting mammals and birds (Trichinella pseudospiralis) and those infecting mammals and reptiles (Trichinella papuae and Trichinella zimbabwensis). The encapsulated species/genotypes, which only infect mammals, clustered into four main groups: the cosmopolitan species Trichinella spiralis, the species/genotypes of the temperate regions (Trichinella britovi, Trichinella murrelli , Trichinella T8, and Trichinella T9), the species/genotype of the arctic region (Trichinella nativa and Trichinella T6), and the equatorial species Trichinella nelsoni. These results are consistent with biological, epidemiological, and molecular data, which show a high genetic divergence in this genus.     

Antigenic differences betweenTrichinella spiralis andT. pseudospiralis detected by monoclonal antibodies

Antigenic differences betweenTrichinella spiralis andT. pseudospiralis were established using two monoclonal antibodies (mAbs) that show different specificities to muscle larvae of the two variants. Enzymelinked immunosorbent assay (ELISA) revealed that mAb 3G6 reacts positively againstT. spiralis, T. nelsoni, T. nativa andT. pseudospiralis, whereas mAb 3E10 does not react withT. pseudospiralis under the same experimental conditions. These antigenic differences were confirmed after preabsorption of the antibodies with serial dilutions of extracts ofT. spiralis orT. pseudospiralis muscle larvae. The indirect immunofluorescence technique showed that the antigen corresponding to mAb 3G6 is located in the stichosomes and the cuticle surface of bothT. spiralis andT. pseudospiralis. In contrast, mAb 3E10 positively stained cryostat sections ofT. spiralis, forming a dense reaction product on the surface of the whole larvae and the surrounding capsule. This antibody can be quite useful as a specific probe for distinguishingT. spiralis fromT. pseudospiralis in taxonomic studies. Using an avidin-biotin system, we could prove that mAb 3G6 recognizes an excretory/secretory-type antigen.     

Construction and use of a Trichinella spiralis phage display library to identify the interactions between parasite and host enterocytes

Although it has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine, the mechanisms by which T. spiralis infective larvae recognize and invade the intestinal epithelial cells (IECs) are unknown. It is speculated that the molecular interactions between the parasite and host enterocytes may mediate the recognition and invasion of IECs by T. spiralis. However, no Trichinella proteins that interact with the enterocytes have been identified previously. The aim of this study was to identify Trichinella proteins that bind to IECs by screening a T7 phage display cDNA library constructed using messenger RNA from T. spiralis intestinal infective larvae. Following five rounds of biopanning, sequencing, and bioinformatics analysis, ten T. spiralis proteins (Tsp1–Tsp10) with significant binding to normal mouse IECs were identified. The results of the protein classification showed that six proteins (Tsp1, calcium-transporting ATPase 2 protein; Tsp4, ovochymase-1; Tsp6, T-complex protein 1 subunit eta; Tsp7, glycosyl hydrolase family 47; Tsp8, DNA replication licensing factor MCM3; and Tsp10, nudix hydrolase) of these T. spiralis proteins were annotated with putative molecular functions. Out of the six proteins, five have catalytic activity, four have binding activity, and one has transporter activity. Anti-Tsp10 antibodies prevented the in vitro partial invasion of IECs by infective larvae and the mice immunized with the recombinant phage T7-Tsp10 showed a 62.8 % reduction in adult worms following challenge with T. spiralis muscle larvae. Although their biological functions are not yet fully known, these proteins might be related to the larval invasion of host enterocytes. Future experiments will be necessary to ascertain whether these proteins play important roles in the recognition and invasion of host enterocytes. The construction and biopanning of Trichinella phage display libraries provide a novel approach for searching for candidate genes that are related to invasion and for identifying protein interactions between parasite and host.     

Cloning and characterization of Rab and Ran/TC4 cDNA clones in Trichinella spp.

The cloning and characterization of seven Rab and three Ran/TC4 partial cDNA sequences in both cystic (Trichinella spiralis and T. britovi) and noncystic species (T. pseudospiralis) are reported. These molecules were cloned by rapid amplification of cDNA ends via polymerase chain reaction (RACE-PCR), using cDNA from the aforementioned Trichinella spp. coupled to the AP1 adaptor. As primers, AP1 and 5B (derived from the WDTAGQE sequence of region 2 specific for Rab and Ran proteins) sequences were included in the PCR. The cloned cDNAs were sequenced and characterized by both Southern-blot and Northern-blot analysis. Trichinella spp. Rab- and Ran-like molecules showed divergences in both the nucleotide and the deduced amino acid sequences as compared with the corresponding homologues previously described in other organisms. In addition, differences were observed among the Trichinella species, mainly between the cystic and the noncystic species, in both DNA restriction-enzyme polymorphism and expression of the six GTPases isolated. Received: 12 November 1998 / Accepted: 3 February 1999     

Specific IgG4 response directed against the 45-kDa glycoprotein in trichinellosis: a re-evaluation of patients 15 years after infection

The aim of this study was to evaluate the humoral immune response in late human trichinellosis with particular attention to the presence of IgG4 antibodies directed against the Trichinella-45-kDa glycoprotein (gp). This study re-evaluates subjects 15 years after they were involved in a trichinellosis outbreak that occurred in Central Italy following the consumption of raw boar meat infected with Trichinella britovi. The results show that ELISA tests using the E/S antigen identified five IgM- and eight IgG-positive patients and no IgA-positive patients. Tests using immunoblot (IB) with E/S antigens identified three IgM-, five IgA-, seven- IgG1- and three IgG4-positive sera. When the purified 45-kDa gp was used as an antigen, the IB revealed that six of the ten sera tested were positive for IgG4. Sera were also evaluated with a commercial kit, revealing that 11 of 12 patients had a highly sensitive reactivity against Trichinella proteins (64 and 44–43 kDa). In conclusion, humoral immune response against Trichinella is still present in these patients 15 years after the initial infection, including an IgG4 response directed to the 45-kDa gp.     

Development and Evaluation of a Western Blot Kit for Diagnosis of Human Trichinellosis

We evaluated industrially prepared Western blot strips designed to avoid the cross-reactions observed with indirect immunofluorescence and enzyme-linked immunosorbent assays used for the serodiagnosis of trichinellosis. The antigen preparations were crude extracts of Trichinella spiralis. The Western blot profile characteristic of trichinellosis was characterized by comparing 60 sera from patients infected by Trichinella to 11 sera from healthy subjects, 51 sera from patients with other proven parasitic diseases (cysticercosis, schistosomiasis, strongyloidosis, fascioliasis, toxocariasis, liver amebiasis, anisakiasis, filariasis, toxoplasmosis, hydatidosis, or malaria), and 23 sera from patients with autoantibodies. Specific 43- to 44-kDa and 64-kDa bands were obtained with all of the sera from 51 patients with acute trichinellosis, in 4 out of 9 patients at the early stages of the disease, and in only 1 control patient, who had suspected anisakiasis and in whom trichinellosis could not be ruled out by muscle biopsy.     

Molecular cloning and functional expression of enolase from Trichinella spiralis

The cDNA library was constructed from muscle larvae of Trichinella spiralis, and immunoscreened with sera from mice infected with T. spiralis. A cDNA clone, designated as TsENO, encoded 2-phospho-D-glycerate hydro-lyase (enolase) that catalyzed a reversible conversion of 2-phospho-D-glyceric acid (2PGA) to phosphoenolpyruvate (PEP) in the glycolytic pathway. The recombinant TsENO protein was produced in an Escherichia coli expression system. The recombinant full-length TsENO protein had no activity in the conversion of 2PGA to PEP, but gained enolase activity after cutting off the signal peptide from the full-length protein. There was no meaningful difference in the expression level of TsENO gene at three distinct stages of T. spiralis. Also, antibody against the recombinant TsENO protein reacted with crude extract of muscle larvae, but not with the excretory and secretory products.This revised version was published online in June 2005 with corrections to Figure 1.     

Cloning and Expression of a Helicobacter bilis Immunoreactive Protein

In an effort to identify immunoreactive Helicobacter bilis antigens with potential for serodiagnosis, sera from mice experimentally infected with H. bilis were used to screen an H. bilis genomic DNA expression library. Among 17 immunoreactive clones, several contained sequences that encoded a predicted 167-kDa protein (P167). Five overlapping P167 peptides (P167A to P167E) of approximately 40 kDa each were generated and tested. Immune sera reacted with fragments P167C and P167D at dilutions of 1:1,600 and 1:6,400, respectively, and reacted with an H. bilis membrane extract at a dilution of 1:800 in an enzyme-linked immunosorbent assay. Sera from mice experimentally infected with H. hepaticus did not react with P167C and P167D. Sera from mice naturally infected with H. bilis but not sera from mice naturally infected with H. hepaticus reacted with P167C and P167D. Hyperimmune sera against P167C peptide reacted with recombinant P167C and with a 120-kDa band in H. bilis lysates but did not react with a protein of the same size on immunoblots prepared from H. hepaticus, H. muridarum, or unrelated Borrelia burgdorferi and Campylobacter jejuni whole-cell lysates. Nevertheless, the P167A, P167B, P167C, and P167D primers, but not the P167E primers, amplified DNA from H. hepaticus, and all five primer sets amplified DNA from H. muridarum. These results suggest that P167 is an immunodominant, H. bilis-specific antigen that may have potential for use in serodiagnosis.     

Evaluation of baculovirus-derived recombinant 53-kDa protein of <Emphasis Type="Italic">Trichinella spiralis</Emphasis> for detection of <Emphasis Type="Italic">Trichinella</Emphasis>-specific antibodies in domestic pigs by ELISA

The complete gene encoding the 53-kDa protein derived from Trichinella spiralis was cloned and expressed using a baculovirus-based system. Characterization of a purified fusion protein consisting of the 53-kDa protein and the glutathione S-transferase protein showed unspecific reactivity with swine pre-immune serum in both enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Subsequently, a purified C-terminal 6×His-tagged 53-kDa protein was used in an ELISA. The evaluation of the test using a negative serum panel showed a high specificity for the ELISA. Serum panels of pigs infected with T. spiralis of two independent experiments showed that pigs of one experiment were tested positive by the ELISA, whereas all sera of the second experiment were negative, indicating a low sensitivity of the ELISA. Furthermore, experimental evidence was found by using mass spectroscopy and Western blot analysis that the 53-kDa protein was not part of the excretory/secretory antigen of T. spiralis as shown in this study.