Trichinella Isolates Research Articles

The epidemiology of animal trichinellosis in China

The epidemiology of animal trichinellosis in China based mainly upon original Chinese literature published between 1937 and 2004 is reviewed. The seroprevalence of Trichinella infection in herbivores was 0.7% (2/300) in cattle and 0.8% (4/500) in sheep. The prevalence of trichinellosis in naturally infected cattle was 1.2% (2/163). Trichinella larvae were detected in 1.4% (3/215) of sheep and in 2.1% (1/47) of beef cattle sold at markets. Canine trichinellosis was recorded in 13 Provinces, Autonomous Regions or Municipalities (P/A/M) and the average prevalence of the infection in dogs slaughtered in abattoirs was 16.2% (5654/34,983) ranging from 1.2% to 44.8%, with the highest prevalence located in northeast China. The prevalence in dog meat sold at markets was 3.5% (988/27,898) in 5 P/A. Feline Trichinella infection was reported in 10 P/A/M. The prevalence of Trichinella infection in rats varied from 1.1% (51/459) to 15.1% (50/332). Trichinella larvae were detected in 1.5% (9/587) of house rats ( Rattus norvegicus) as well as in 0.8% (3/369) of wild rats ( Apodemus chevrieri), and the infection was recorded also in other wildlife (foxes, bears, wild boar, weasels, raccoon dogs, muntjak and bamboo rats). Trichinella larvae were detected in 2.6% (4/156) of weasels ( Mustela sibirica), 1.5% (2/135) of shrews ( Tupaia belangeri) and 7.7% (1/13) of moles ( Parascapter leucurus). All Trichinella isolates from domestic pigs were identified as T. spiralis. Some Trichinella isolates from dogs in north-eastern China were identified as T. nativa, which has muscle larvae that are highly resistant to freezing. Twenty-seven outbreaks of human trichinellosis associated with mutton, dog and game meat occurred in China between 1964 and 2004, but the quarantine of Trichinella larvae in such meat is not mandatory in China at present.

Simple species identification of Trichinella isolates by amplification and sequencing of the 5S ribosomal DNA intergenic spacer region

We developed a PCR-based assay using a single primer pair to amplify the 5S ribosomal DNA intergenic spacer region to identify Trichinella isolates. In our method, amplified products are directly sequenced on both strands and compared to GenBank sequences. Using this method, we were able to identify Trichinella spiralis, T. britovi and T. nativa. This method permits rapid species identification of Trichinella isolates; however, further evaluation is required before recommending this approach for routine use.

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.

Molecular identification of Korean Trichinella isolates.

Muscle larvae of Trichinella isolates from two outbreaks in Korea were analyzed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiple-PCR. All of the muscle larvae showed a band similar to that of T. spiralis larvae of the reference strain. The two Korean Trichinella isolates (isolate code ISS623 and ISS1078) might be classifiable to Trichinella spiralis.

Epidemiological analysis of Trichinella spiralis infections of foxes in Brandenburg, Germany.

In a cross-sectional study conducted between March 1993 and February 1995, 7103 indiscriminately collected foxes were examined for Trichinella larvae. A total of 3295 serum samples were serologically investigated with an ELISA based on excretory-secretory antigen. The proportion of serologically positive animals ranged between 3.3% and 17.6% in random samples from individual counties or towns and resulted in an estimated overall prevalence of 7.7% (95% CI: 6.9-8.7%). Trichinella larvae were detected in the muscles of five foxes, corresponding to an estimated prevalence of 0.07% in the total sample (95% CI: 0.02-0.16%). The analysis of DNA of the Trichinella isolates by random amplification of polymorphic DNA (RAPD) lead to the identification of the isolates as Trichinella spiralis. The differences between serological and parasitological findings are discussed.

Trichinella nativa in sylvatic wild boars

Of 17 Trichinella isolates from domestic pigs and wild boars (Sus scrofa) in regions where Trichinella nativa is widespread among sylvatic animals, two wild boars from Estonia were found to be naturally infected with this Trichinella species. The other 15 animals were infected with Trichinella spiralis. Trichinella nativa is tolerant to freezing when in the muscles of carnivores. The biological characteristics and temperature tolerance of this species in swine need to be further investigated if pork is certified for consumption following freezing.

Trichinella nativa in sylvatic wild boars

Of 17 Trichinella isolates from domestic pigs and wild boars (Sus scrofa) in regions where Trichinella nativa is widespread among sylvatic animals, two wild boars from Estonia were found to be naturally infected with this Trichinella species. The other 15 animals were infected with Trichinella spiralis. Trichinella nativa is tolerant to freezing when in the muscles of carnivores. The biological characteristics and temperature tolerance of this species in swine need to be further investigated if pork is certified for consumption following freezing.

Evaluation of the infectivity of Trichinella spp. for reptiles (Caiman sclerops)

Experimental inoculation with nine well-characterised Trichinella isolates was performed on caimans ( Caiman sclerops) to determine their infectivity for reptiles belonging to the family Crocodilidae. As controls, the same larval batches of Trichinella isolates were inoculated into mice and guinea pigs. It was suggested that Trichinella pseudospiralis was more likely to infect reptiles than encapsulating species, but whereas all Trichinella species established in mice and guinea pigs, the caimans remained negative. The finding that caimans could not be experimentally infected contrasts with a recent report on infections in farmed crocodiles ( Crocodylus niloticus).

Polymerase chain reaction primers to identify Trichinella spiralis or T. pseudospiralis

Three sets of polymerase chain reaction (PCR) primers for Trichinella isolates were constructed by the following method. Random amplified polymorphic DNA (RAPD) of the corresponding isolates was produced and several fragments were sequenced. Primer sets SB4-2 and SB4-4 were designed from the resulting sequence that could amplify exclusively DNA from T. spiralis. These primers produced extra fragments in addition to the expected fragment, and the banding pattern differed among some strains of T. spiralis suggesting that the pattern can serve as a gene fingerprint of T. spiralis. Primer pairs SB5 amplified exclusively DNA from T. pseudospiralis. 1 1 The accession numbers for sequences SB4 and SB5 are U42045 and U37588, respectively.

Characterization of Spanish Trichinella isolates by random amplified polymorphic DNA (RAPD).

The random amplified polymorphic DNA (RAPD) assay was used to find molecular markers able to distinguish Trichinella spiralis from T. britovi, the two recognized Spanish Trichinella species. Fourteen Spanish Trichinella isolates, as well as reference Trichinella isolates representing the five species T. spiralis (T1), T. nativa (T2), T. britovi (T3), T. pseudospiralis (T4) and T. nelsoni (T7) and the three other taxa Trichinella T5, Trichinella T6 and Trichinella T8 of the genus, were characterized by RAPD using both purified and crude DNAs from infective muscle larvae (ML) and seven arbitrary primers. Three primers yielded diagnostic RAPD markers for the Spanish T. spiralis and T. britovi isolates as well as for the Trichinella reference isolates analysed, and in the case of crude DNAs the results were obtained in few hours. In addition, the species-specificity of the diagnostic RAPD markers from Spanish Trichinella isolates was studied by cross-hybridization assays. These assays confirmed that the selected diagnostic DNA fragments were not species-specific, but showed potential differences in the copy number among the examined Trichinella genetic clusters.

Characterization of Toxoplasma and Trichinella isolates from muscles of black bears in Pennsylvania

Summary During the hunting season of 1992, 322 black bears from Pennsylvania were examined for Toxoplasma gondii- and Trichinella spp-induced infections. Toxoplasma gondii antibodies were found in 79.8% of 322 bears–titer < 1:25 in 65 (20.2%), 1:25 in 18 (5.6%), 1:50 in 11 (34.5%) and 1:500 in 128 (38.7%) bears –by use of the modified agglutination test. Muscle tissues from 89 of these bears were bioassayed for T gondii parasites. Muscles from 64 bears, including heart from 1 bear, and heart alone from another bear, were digested in pepsin, and the digested samples were bioassayed in mice. Toxoplasma gondii was isolated from 5 bears; from the heart of 1, heart and skeletal muscles of 1, and skeletal muscles of 3. The T gondii antibody titers for the 5 bears with detectable T gondii were: ≥ 1:25 in all 5 bears by use of the modified agglutination test; < 1:10 (3 bears, considered Toxoplasma-negative), 1:20 and 1:320 by use of the Sabin-Feldman dye test; < 1:64 (3 bears, considered Toxoplasma-negative), 1:128, 1:512 by use of the indirect hemagglutination test, and < 1:16 (2 bears, considered Toxoplasma-negative), 1:32, 1:64, and 1:512 by use of the latex agglutination test. Toxoplasma gondii was not isolated from feces of 5 cats fed muscles from the remaining 25 bears with T gondii antibody titer < 1:25. Tissue cysts of the 4 T gondii isolates from bears were rendered noninfective by freezing at – 13 C. Antibodies against Trichinella spp were found in 6 (1.8%) of 319 bear sera; Trichinella spp larvae were detected in muscle digests of 2 of 63 bears, and in histologic sections of muscles from 3 of 162 bears. Genetic typing indicated that the 2 Trichinella isolates from bears were a sylvatic genotype and were not the species found in domestic pigs.

The differentiation of parasitic nematodes using random amplified polymorphic DNA.

DNA from species and races of plant parasitic nematodes (Meloidogyne, Globodera and Heterodera) and a human parasitic nematode (Trichinella) were subjected to polymerase chain reaction amplification using one arbitrary primer (M-10). This technique results in relatively simple DNA profiles that include polymorphic markers known as random amplified polymorphic DNA (RAPDs). The RAPD profiles of the plant nematode species of Meloidogyne made possible the identification of M. incognita and M. hapla, but no differences were found between the patterns of M. javanica, M. arenaria and M. graminicola. Moreover, the four races of M. incognita were indistinguishable by this primer. In contrast, when races of the plant nematode Globodera rostochiensis (Ro1 and Ro2/3) were studied under the same RAPDs conditions, a race specific profile allows these two most devastating races to be differentiated. When DNAs of eight Trichinella isolates were subjected to RAPD studies, four different patterns were identified, corresponding to the four Trichinella clusters previously defined by isozyme polymorphism.

Biochemical, Biological, and Genetic Characterization of a Sylvatic Isolate of Trichinella

Biological, biochemical, and genetic relationships of Trichinella isolates were assessed and compared from 3 species of Illinois fur-bearing mammals. Tongue muscle collected from 1987 through 1989 from 323 raccoons (Procyon lotor), 9 red fox (Vulpes fulva), and 1 coyote (Canis latrans) were digested and Trichinella muscle larval prevalences and mean intensities (larvae/g) determined. The prevalence and mean intensity of tongue muscle-stage larvae were 2.8% and 44.4% and 326 and 2 larvae/g for raccoon and red fox, respectively. The single coyote examined for muscle larvae was negative. Seven of 13 Trichinella isolates (5 raccoon, 2 red fox) were maintained and amplified in mice. Comparative analyses of DNA from larvae of these isolates and from other Trichinella isolates were performed by dot-blot hybridization using specific repetitive DNA probes. DNA from the 7 Illinois sylvatic isolates reacted with the Trichinella T5 isolate DNA probe pUPB-3.7 and did not cross-react with the Trichinella spiralis probe pBP-2. The sylvatic isolates of Trichinella were also analyzed using 27 allozymes. Their allozymic patterns were similar to Trichinella reference strain T5 from Pennsylvania, except for mannose phosphate isomerase. These results suggest that the Illinois wildlife isolates belong to the Trichinella T5 genotype according to the classification system established by the International Trichinella Reference Centre and as defined here by positive hybridization to the pUPB-3.7 probe. Results from Southern blot analyses using rRNA as a probe and allozyme patterns revealed some heterogeneity among geographical isolates of the T5 genotype.

Allozymic and Biological Characters of Trichinella pseudospiralis Isolates from Free-Ranging Animals

To evaluate biological and biochemical variability in nonencapsulated Trichinella isolates, biological and allozymic studies were conducted on isolates of Trichinella collected from a raptoral bird (Aquila rapax) and a fox (Vulpes corsac) in Kazakhstan and from a dasyurid marsupial (Dasyurus maculatus) on the island of Tasmania, Australia. Allozyme profiles of bird and marsupial isolates showed close similarity with the type isolate of Trichinella pseudospiralis. The avian and fox isolates successfully interbred with the type T. pseudospiralis isolate, but they failed to interbreed with 3 encapsulating species, Trichinella spiralis, Trichinella nativa, and Trichinella britovi. The reproductive index assessed in 4 inbred and 1 outbred strains of mice was lower for the avian isolate than for the marsupial and the type T. pseudospiralis isolates (P < 0.001).

Allozyme Analysis of Trichinella Isolates from Various Host Species and Geographical Regions

Allozyme analysis was carried out on 152 Trichinella isolates from synanthropic and wild animals and from humans; the isolates were collected from 5 continents. The analysis, involving 27 enzymes, revealed the presence of 8 distinct gene pools, termed T1-T8. Four of the genetic groups represent the 4 previously proposed species: Trichinella spiralis sensu stricto (T1), Trichinella nativa (T2), Trichinella nelsoni (T7), and Trichinella pseudospiralis (T4). The other 4, T3, T5, T6, and T8 are distinct from previously described species. The absence of allozymic hybrid patterns among even sympatric groups indicates a lack of gene flow among the groups. Principal component analysis and the unweighted pair group method of analysis were used to assemble allozyme patterns of the 152 isolates into discrete groups and to show their relative relationships. Both analyses indicated the presence of 8 primary clusters that correlated with the gene pools revealed by direct allozyme profile analysis. The absence of evidence of gene flow among the gene pools and the high level of allozymic differentiation between the cluster groups support the concept that the genus Trichinella is composed of several sibling species.

Taxonomic Revision of the Genus Trichinella

The analysis of genetic, biochemical, and biological data on about 300 Trichinella isolates, reported in the literature, allows a taxonomic revision of this genus. We propose the recognition of 5 sibling species, Trichinella spiralis (Owen, 1835) sensu stricto; Trichinella nativa Britov and Boev, 1972; Trichinella pseudospiralis Garkavi, 1972; Trichinella nelsoni Britov and Boev, 1972 sensu stricto; and Trichinella britovi n. sp., on the basis of biochemical and biological characteristics. Trichinella britovi n. sp. is characterized by distribution in the Palaearctic Region; newborn larvae (NBL) production in vitro of 35-55 NBL/72 hr; nurse cell development time (NC d.t.) between 24 and 42 days postinfection (d.p.i.); low reproductive capacity index (RCI) in mice, rats, and pigs; low resistance to freezing; 1 unique marker allozyme; and moderate pathogenicity for humans. The new species is most similar to Trichinella nativa but differs from it in 4 allozymes, in having less resistance to freezing, in having a different pattern of major ribosomal DNA fragments after endonuclease digestion, and in distribution area. Trichinella nativa is characterized by a holarctic distribution; hosts that are sylvatic mammals; NBL production in vitro 28-54/72 hr; NC d.t. between 20 and 30 d.p.i.; low RCI in mice, rats, and pigs; high resistance to freezing; 2 unique marker allozymes; and moderate to severe pathogenicity for humans. Trichinella spiralis sensu stricto is characterized by a cosmopolitan distribution in domestic pigs, associated wildlife, and humans; high NBL production in vitro (greater than 90 NBL/72 hr); NC d.t. between 16 and 37 d.p.i.; high RCI in mice, rats, and pigs; no resistance to freezing; 6 unique marker allozymes; and high pathogenicity for humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of Two Isolates of Trichinella Recovered from Humans in France

Two Trichinella isolates from humans in France were characterized using reproductive capacity indices and a combination of molecular methods. The isolate TRLL hybridized with the pig type-specific probe pPra and had pig type restriction profiles and rDNA patterns. It was therefore identified as a domestic or pig type isolate. The isolate CTRD-85 had similarities and differences in restriction profiles and rDNA patterns with both AF1 and Trichinella nelsoni and was identified as a sylvatic type. Pattern comparisons also show that T. nelsoni is similar to variants of the North American sylvatic type.

Molecular Characterization of Sylvatic Isolates of Trichinella spiralis

Genetic relationships of 20 Trichinella isolates from Indiana wildlife were assessed and compared to Trichinella isolated from an infected swine herd. Trichinella larvae were isolated from coyotes, mink, raccoons, and red foxes. The larvae were maintained and amplified in white mice (ICR) and wild mice (Peromyscus leucopus). Differences in phenotypic characters of sylvatic isolates in the 2 laboratory hosts included an approximately 10-30-fold increase in parasite fecundity in wild mice. DNA for each isolate was extracted from Trichinella larvae and analyzed by dot-blot hybridization using a repetitive DNA probe pBP2 that recognizes DNA sequences specific for swine Trichinella. The probe hybridized only to Trichinella from swine and a single coyote isolate. Restriction endonucleases were used to digest DNA and the resulting fragments were separated by gel electrophoresis. Based on the presence of repetitive DNA sequences in the Trichinella genome, distinctive banding patterns were seen among the isolates. Trichinella isolated from swine had a pattern distinct from all sylvatic isolates except 1 from a coyote. Because this coyote was from the same general locality as the swine Trichinella outbreak, it was concluded that the isolate represents transmission of swine trichinellosis to the wildlife population. Further analysis using the enzyme Cla I identified unique banding patterns for wild isolates, suggesting that the sylvatic group is a genetically heterogeneous complex.

Trichinella spiralis Infections of Inbred Mice: Immunologically Specific Responses Induced by Different Trichinella Isolates

The immune response of inbred mice was studied following infection with Trichinella spiralis var. pseudospiralis (TP) or with isolates of T. spiralis derived from a pig or from an arctic fox. Animals given a primary infection with 1 isolate of Trichinella and challenged 21 days later with the same or different isolates responded more quickly by expelling worms from the homologous challenge. In addition, although mesenteric lymph node cells from mice infected with each isolate of Trichinella would proliferate in vitro when cultured with antigen derived from each of the others, the strongest proliferation response always occurred when cells were cultured in the presence of antigen prepared from the specific isolate used to infect the mouse from which the cells were derived. In addition, it was possible to prepare monoclonal antibodies that recognized an antigen expressed by TP which was not shared by T. spiralis isolates and vice versa. Collectively, these data support the conclusion that the differences observed in the kinetics of immune responsiveness to different Trichinella isolates are referable, at least in part, to differences among the isolates in the expression of functionally relevant antigens.

Repetitive DNA as a tool for the identification and comparison of nematode variants: application to Trichinella isolates

DNA prepared from four isolates of Trichinella was compared by genomic DNA cross-hybridisation, by electrophoresis following restriction endonuclease digestion and by hybridisation studies using a cloned repetitive DNA sequence from T. spiralis. The DNA from T. spiralis, T. nelsoni and T. pseudospiralis isolates was distinct and the interrelationships of these isolates were inferred. In contrast to previous work on T. nativa and T. spiralis, our work suggests that these two isolates are very similar.