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.     

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.     

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.     

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.     

Species-specific antibody responses to the recombinant 53-kilodalton excretory and secretory proteins in mice infected with Trichinella spp

The 53-kDa proteins in larval excretory and secretory (E-S) products were expressed from five Trichinella species (T. spiralis, T. britovi, T. nativa, T. pseudospiralis, and T. papuae), using the Escherichia coli expression system, and the antibody responses to the 53-kDa recombinant proteins in mice infected with Trichinella spp. were analyzed by Western blotting. The 53-kDa protein is conserved among the five Trichinella species, with >60% similarity in amino acid sequences. The 53-kDa recombinant proteins of T. spiralis and T. pseudospiralis reacted to sera from mice infected with T. spiralis and T. pseudospiralis at 8 days postinfection (p.i.), respectively. An antibody against the 53-kDa recombinant protein of T. spiralis recognized the 53-kDa protein in the crude extracts from adult worms and 30-day p.i. muscle larvae and E-S products from muscle larvae of T. spiralis but did not recognize any proteins from T. pseudospiralis. The sera from the mice infected with T. spiralis strongly reacted with the 53-kDa recombinant protein of T. spiralis but did not react with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, and T. papuae. Similarly, the sera from mice infected with T. britovi, T. nativa, T. pseudospiralis, or T. papuae strongly reacted with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, or T. papuae, respectively. These results showed that the 53-kDa recombinant proteins provide early and species-specific antibody responses in mice infected with Trichinella spp.     

Molecular identification and phylogenetic analysis of Trichinella isolates from different provinces in mainland China

Polymerase chain reaction (PCR) and sequencing are useful for species identification of Trichinella spp., especially when their morphological characteristics useful for identifying taxa are lacking. In the present study, nine Trichinella isolates from different provinces in mainland China were identified by the PCR-based method using the 5S ribosomal DNA intergene spacer region (5S ISR) and the mitochondrial large subunit ribosomal DNA genes as molecular markers. The results indicated that eight isolates originating from domestic pigs and one isolate originating from civet cat (Paguma larvata) showed identical DNA banding pattern to Trichinella spiralis. Sequence analysis of the 5S ISR gene further confirmed that the nine Trichinella isolates were T. spiralis and revealed the intraspecies genetic variation within T. spiralis.     

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     

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     

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 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.     

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.     

Trichinella sp. in red foxes (Vulpes vulpes) from Catalonia, NE Spain

European legislation allows the official recognition of Trichinella-free pig holdings, provided Trichinella sp. infection is absent from humans and prevalence of Trichinella sp. infection in red foxes (Vulpes vulpes) is below 0.1% in the area, region or country. Tibialis anterior muscle samples from 1,319 red foxes captured in Catalonia (NE Spain) between 1998 and 2007 were analyzed for Trichinella sp. using the digestion method. Four foxes resulted positive (one in 1999, one in 2002 and two in 2006), accounting for a low prevalence (0.3%). However, this prevalence was concentrated in mountain or rural areas with a low sample size, reaching high local prevalences. The two positive samples in 2006 were characterized as Trichinella britovi, and a sylvatic cycle of trichinellosis seems to occur, at least in the rural insufficiently sampled regions of Catalonia. Overall, the results obtained do not currently allow the establishment of Trichinella-free pig holdings in the study area, but further research is needed to better know the prevalence and cycle of Trichinella sp. in Catalonia.     

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     

Validation of a Western Blot for the detection of anti-<Emphasis Type="Italic">Trichinella</Emphasis> spp. antibodies in domestic pigs

Trichinellosis is a zoonotic disease in humans caused by Trichinella spp. According to international regulations and guidelines, serological surveillance can be used to demonstrate the absence of Trichinella spp. in a defined domestic pig population. Most enzyme-linked immunosorbent assay (ELISA) tests presently available do not yield 100% specificity, and therefore, a complementary test is needed to confirm the diagnosis of any initial ELISA seropositivity. The goal of the present study was to evaluate the sensitivity and specificity of a Western Blot assay based on somatic Trichinella spiralis muscle stage (L1) antigen using Bayesian modeling techniques. A total of 295 meat juice and serum samples from pigs negative for Trichinella larvae by artificial digestion, including 74 potentially cross-reactive sera of pigs with other nematode infections, and 93 meat juice samples from pigs infected with Trichinella larvae were included in the study. The diagnostic sensitivity and specificity of the Western Blot were ranged from 95.8% to 96.0% and from 99.5% to 99.6%, respectively. A sensitivity analysis showed that the model outcomes were hardly influenced by changes in the prior distributions, providing a high confidence in the outcomes of the models. This validation study demonstrated that the Western Blot is a suitable method to confirm samples that reacted positively in an initial ELISA. Frey and Schuppers contributed equally to this work. This work was funded by the Swiss Federal Veterinary Office, grant number 1.06.03.     

Reactivity and Specificity of Trichinella spiralis Fractions in Cutaneous and Serological Tests

Six diethylaminoethyl-cellulose fractions of a larval Trichinella spiralis extract, an Ascaris suum extract, and a nonrelated protein were used for cutaneous tests in guinea pigs with 8-, 14-, and 73-day-old T. spiralis infections, in guinea pigs with 13-day-old A. suum infections, and in normal guinea pigs. A selected T. spiralis fraction was used in hemagglutination (HA) tests with sera of 8 T. spiralis-infected rabbits, 41 sera of trichinellosis patients positive by bentonite agglutination tests, and 50 sera of clinically healthy persons. Immediate-type cutaneous reactions revealed extensive cross-reactivity between both parasites, although the establishment of conventional limits for considering a reaction positive allowed the specific diagnosis of acute or chronic trichinellosis with different fractions. Delayed-type reactions were specific with all fractions except one, and different fractions reacted during either the acute or the chronic phase of trichinellosis. HA detected anti-Trichinella antibodies in all the rabbits 9 to 10 days postinfection, in all trichinellosis patients, and in none of the healthy people. Correlation between HA and bentonite agglutination titers and other considerations suggest that HA with the selected fraction detects early antibodies. HA inhibition tests with A. suum extract suggest lack of HA cross-reactivity between the A. suum- and T. spiralis-selected fractions. The use of different fractions in diverse tests for clinical or epidemiological studies is suggested.     

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.     

Imported Human Trichinellosis: Sequential IgG4 Antibody Response to<Emphasis Type="Italic"> Trichinella spiralis</Emphasis>

Two out of five members of one household presented with clinical signs of trichinellosis after their return to the Netherlands. The family had consumed Trichinella-infected pork in Montenegro, formerly Yugoslavia. Serological tests were performed at 1, 2, 6, and 18 months after ingestion of the infected meat. Trichinella-specific IgM, IgG, and IgA antibodies measured in sera from symptomatic and asymptomatic patients remained positive up to 18 months after ingestion. The measured IgG4 antibody response directed to a 45 kDa Trichinella spiralis antigen also persisted 18 months after ingestion for three of the family members.     

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.     

Distribution of muscle larvae and antibody dynamics in goats experimentally infected with Trichinella spiralis

Herbivorous animals can play a very important role in spreading trichinellosis. In the study presented here, the susceptibility and distribution of Trichinella spiralis infection was examined in 16 goat kids. The goats were inoculated with 10,000 T. spiralis larvae isolated by artificial digestion methods. The animals were necropsied per two animals in weekly intervals, and the larval burdens in different muscle tissue and anti-Trichinella antibodies measured with the indirect enzyme-linked immunosorbent assay (ELISA) serological method using excretory–secretory (E/S) antigen for detecting anti-Trichinella antibodies were assessed during the experiment. T. spiralis larval burden was maximal at 6 weeks postinoculation (480–5,057 larvae/g according to locality), and the larvae were also found in the myocardium (0.77 larvae/g). In this paper, our next step was to compare the specificity and the time of seroconversion by means of ELISA based on E/S antigen prepared from T. spiralis. Antibody response was detected in all 16 goats. The ELISA test carried out showed the first increments in optical density 2 weeks postinfection (p.i.), reached their peak 4 weeks p.i., and remained elevated from that day until the end of the experiment (10 weeks p.i.). These results indicated that specific anti-Trichinella antibodies in goats persist for a relatively long time.     

Biochemical resolution of European and African isolates ofTrichinella nelsoni Britov and Boev, 1972

Isoenzyme analysis was used to characterize 47 isolates ofTrichinella nelsoni from Europe (EUR) and two from Kenya and Tanzania (AFR). In all, 27 isoenzymes showed polymorphism within the species. The EUR parasites, isolated from domestic, synanthropic, sylvatic animals and man, showed isoenzymatic profiles different from those exhibited by AFR parasites isolated from sylvatic animals. The EUR parasites showed polymorphism due to three isoenzymes, i.e. ME, MPI and PGM. Euclidean distance and a dendrogram were used to evaluate the relationships among isolates. The Euclidean values showed that EUR and AFR genotypes are distant from each other and from the reference strainsT. spiralis s. str. andT. pseudospiralis. The genetic data indicate the absence of gene flow between AFR and EUR isolates. The biochemical results suggest the presence of genetic heterogeneity inT. nelsoni and support the existence of a zoogeographical segregation of the two gene pools. The authors propose that as operational labels, code T3 be assigned to EUR isolates and code T7, to AFR isolates.