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Single muscle larvae (ML) of Trichinella belonging to 5 species and 2 genotypes collected from 126 experimentally infected pigs and preserved in 75% ethyl alcohol at room temperature were identified by random amplified polymorphic DNA (RAPD) analysis to evaluate the specificity and sensitivity of this assay. A double-blind protocol was used to ensure that the interpretation of electrophoretic patterns after RAPD amplification was not influenced by an expected result. RAPD analysis of one larva from each sample showed a sensitivity of 100% when only samples with undamaged DNA were considered, and the specificity resulted in an 88% match with the species or genotype that had been used to infect pigs, whereas the identification reached 100% specificity when an additional one to four ML were examined.

A method was developed to identify species and genotypes within the genus Trichinella using polymerase chain reaction (PCR) and specific primers. Enzymatic amplification of 2 partially conserved and repetitive genomic DNA sequences that have been shown to be variable in length within the different Trichinella genotypes form the basis of this test. Within these regions of the genome, 4 sets of primers were evaluated from which 2 were chosen for their ability to differentiate among the genotypes under stringent primer annealing conditions while maintaining high yields of amplification product. Differences in the size of PCR products from multiple isolates of each genotype indicate sufficient variation to identify 7 of the 8 parasite groups within this genus. One primer set can differentiate among some genotypes working from a single larva. Identification of Trichinella genotypes will assist in distinguishing between sylvatic and synanthropic life cycles. Such information will be critical in tracing sources of trichinellosis by easily and unambiguously identifying likely host reservoirs and will provide valuable information for instituting methods of control.

In the present study, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed to identify 5 species (Trichinella spiralis, Trichinella britovi, Trichinella nativa, Trichinella nelsoni and Trichinella pseudospiralis) and 3 phenotypes of uncertain taxonomic status (Trichinella T5, T6, and T8). Eleven restriction endonucleases were used to restrict 3 DNA fragments (1) a 2800 bp fragment of the 43 kDa excretory-secretory (E-S) protein gene, (2) a 1250 bp fragment amplified with the primer pair SB147A and (3) a 372 bp fragment amplified with the primer pair SB372A. This RFLP method allows the identification of the 8 Trichinella phenotypes as follows: T. spiralis by the HinfI or DdeI endonuclease restriction of the 2800 bp fragment; T. nativa by the RsaI restriction of the 2800 bp fragment, or by the AluI restriction of the 1250 bp fragment; T. britovi and Trichinella T8 by the AluI restriction of the 1250 bp fragments, and can be discriminated between them by the SspI restriction of the 2800 bp fragment; T. pseudospiralis by the MspI restriction of the 372 bp fragment; T. nelsoni by the HhaI or AluI restriction of the 2800 bp fragment; Trichinella T5 by the HhaI restriction of the 2800 bp fragment; Trichinella T6 by the AluI restriction of the 1250 bp fragment; and Trichinella T8 by the SspI or RsaI restriction of the 2800 bp fragment. This study reveals also an intraspecifies polymorphism in the 2800 bp and 1250 bp fragments for T. britovi, Trichinella T5 and T6.

An overall prevalence of 3.9% of Trichinella infection was observed in the fox population in The Netherlands. Random amplified polymorphic DNA analysis of individual muscle larvae demonstrated the presence of Trichinella britovi. This is the first report of T. britovi, etiological agent of sylvatic trichinellosis, in one of the most densely populated countries in Europe and, consequently, the occurrence of cannibalism and scavenger behavior of foxes in areas with a high human population density. The presence of T. britovi in the fox population in these areas appears to be without consequences for commercial pig farming.

Diagnostic PCR primers for Trichinella were constructed. Twelve pairs of primers were designed based on the sequences of random amplified polymorphic DNA, and 4 pairs of primers were designed based on the reported sequences of complementary DNA encoding excretory-secretory glycoproteins. With these primers, 31 samples of DNA from different strains of Trichinella including 5 species (Trichinella spiralis, Trichinella britovi, Trichinella nativa, Trichinella nelsoni and Trichinella pseudospiralis) and 3 phenotypes of uncertain taxonomic level (Trichinella T5, T6 and T8) were tested with PCR. Genus Trichinella can be identified by 4 different primer pairs (SB147D, SB372A, SB153, or Ts43). Trichinella spiralis can be identified by the presence of a 673 bp amplicon in PCR with the primer pair SB147B. Trichinella nelsoni can be identified using primer pair SB147F or by the presence of 673 bp and ca. 380 bp amplicon in PCR with the primer pair SB147B. Trichinella pseudospiralis can be identified by 2 primer pairs (SB147E or SB372B). Trichinella T5 can be identified by the primer pair SB147G. Trichinella T8 can be identified by its positivity by the primer pair SB147C and its negativity by the primer pair SB372C. A group of Trichinella species (T. britovi, T. nativa and Trichinella T6) can be identified by the primer pair SB372C.

DNA polymorphisms in two parasitic nematode species, Trichinella spiralis Oven, 1835, and Trichinella pseudospiralis Garkavi, 1972, were revealed via random amplification of polymorphic DNA by the polymerase chain reaction (RAPD PCR). The diagnostic value of seven 10-bp oligonucleotide primers was evaluated, and the extent of the homology between the genomes of the two species was estimated. The intraspecific variation of RAPD markers was revealed in larvae of both species isolated from experimentally infected white rats. The variation was higher in larvae from nonlinear rats than in larvae from linear rats. When animals were infected with both Trichinella species simultaneously, "hybrid" progeny were obtained that had capsule that somewhat differed in shape from one characteristic of the parental species, T. spiralis. In RAPD spectra, the hybrids showed higher similarity of T. spiralis than to T. pseudospiralis. Intra- and interspecific differentiation, genome divergence, and factors inducing the intraspecific variation in Trichinella species are discussed.

A survey on trichinellosis among sylvatic and domestic animals from Estonia revealed the presence of Trichinella nativa (Tn), Trichinella britovi (Tb), and Trichinella spiralis (Ts). Muscle samples were collected from 776 sylvatic and 1,086 domestic animals. Muscle larvae from 52 of the 74 positive samples were identified, using random-amplified polymorphic DNA analysis; 19 samples showed Tn, 27 samples Tb, and 4 samples Ts. A raccoon dog (Nyctereutes procyonoides) and a red fox (Vulpes vulpes) were infected with both Tn and Tb. Of the 19 animals infected with Tn, 16 (84%) were collected from the central-eastern regions of the country, east of the isotherm -5 C in January. Of the 27 animals infected with Tb, 22 (81%) were collected from the western regions of the country, west of the isotherm -4 C in January. Trichinella spiralis seemed to be present only in a focus (a fur-bearing animal farm) on Hiiumaa Island. These results can be used to support the hypothesis of a relationship between the distribution of Tn and Tb and the environmental temperature; they are also indicative of the importance of long-term survival of muscle larvae in host carcasses in the life cycle of these 2 species. In Estonia, the isotherms -4 and -6 C in January could be considered a thermic barrier for the distribution of Tn and Tb, respectively.

A polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis of the expansion segment 5 (domain IV) of the large subunit of ribosomal DNA was employed to characterize seven isolates of Trichinella from China (A-G), including six of pig origin from regions in Dengxian (A), Tianjin (B), Harbin (D), Baoshan (E), Xinye (F) and Xian (G), and one of canine origin from Changchun (C). Isolates A, D, E, F and G were classified as Trichinella spiralis based on SSCP patterns, while the patterns for isolates B and C were consistent with those of Trichinella nativa or Trichinella T6. The results were supported by random amplified polymorphic DNA (RAPD) analysis using five decamer primers and were in accordance with ecological information for the isolates. Single strand conformation polymorphism results also allowed the direct display of mutational sequence variation in the expansion segment among the five isolates of T. spiralis from China, indicating its usefulness for studying population variation within that species., (Copyright 1998 Academic Press Limited)

Reverse transcription-polymerase chain reaction (RT-PCR) was employed to amplify a cDNA encoding an excretory-secretory (ES) antigen with mol. wt 45-50 kDa by SDS-PAGE from T. spiralis muscle larvae. The PCR product was purified by electrophoresis and sequenced by thermal cycle sequencing with primer walking. The cDNA is 890 bp long and encodes a polypeptide of 255 amino acid (AA) residues. Using the same methods, we also recovered a corresponding cDNA from Trichinella T5, which is 891 bp long and encodes 255 AAs. Comparison of the 2 Trichinella species indicates approximately 2.6% and 2.4% differences between the 2 cDNA sequences and between the 2 deduced AA sequences, respectively.

We compared Trichinella T5 of bobcat (Lynx rufus) origin with Trichinella spiralis (T1) by using animal infectivity and molecular biology techniques. Swine, SD rats, and CF1 mice were highly resistant to infection with Trichinella T5 but sensitive to T. spiralis, whereas deer mice (peromyscus maniculatus) had similar sensitivity to both parasites. The fecundity of Trichinella T5 in deer mice was 10-35-fold higher in comparison to the fecundity in laboratory rodents (SD rats and CF1 mice). Fecundity of T. spiralis was approximately the same in both groups. A western blot, using excretory-secretory proteins (ESP) from first-stage larvae of T. spiralis as antigen, showed similar banding patterns in the pigs infected with either T. spiralis or Trichinella T5, however, the homologous reaction was stronger than the heterologous reaction. Antibodies were detectable in swine sera commencing 3 or 5 wk postinfection with T. spiralis or Trichinella T5, respectively. Complementary DNAs encoding the 46-, 49/43-, or 53-kDa ESP showed 3.54, 1.94, and 5.91% differences, respectively, between the 2 parasites. Deduced amino acid sequences of the 3 cDNAs were different at 7.20, 5.08, and 8.55%, respectively. All recombinant proteins of the 3 cDNAs from both parasites could detect antibodies in positive sera. The sequences of cDNAs encoding the 46-, 49/43-, or 53-kDa ESP from T. spiralis are also compared to the previously reported sequences, and the differences are discussed.

During a study on the epidemiology of trichinellosis in Spain, 91 animals and 9 samples of sausages homemade with pork were found positive for Trichinella. Parasite identification at the species level was carried out by the polymerase chain reaction with a random primer on single muscle larvae. Seventy-one animals harbored Trichinella spiralis (17 domestic pigs, 53 wild boars, 1 fox), and 17 were infected with Trichinella britovi (1 domestic pig, 13 wild boars, 3 foxes). Sausages were infected with T. spiralis. Three wild boars (3.3% of infected animals) harbored both species. The presence of both T. spiralis and T. britovi in the same host suggests that infections with 2 isolates of the same species can also occur, permitting the gene flow within the species.

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.

Although parasites of the genus Trichinella have been collected from wild animals in Japan, they have never been studied at species level. Muscle larvae of Trichinella collected from a black bear (Ursus thibetanus japonicus) and from a raccoon dog (Nyctereutes procyonoides viverrinus) in Japan have been identified as Trichinella britovi by random amplified polymorphic DNA analysis. The presence of T. britovi in Japan shows that this parasite has a wide distribution in the Palaearctic region, from western Europe to eastern Asia, and that the isotherm -6 C in January could be considered its northern border.

Enzymatic amplification of expansion segment 5 sequences within domain IV of the large subunit ribosomal DNA generated distinct results among geographical isolates of Trichinella pseudospiralis from Russia, North America, and Australia from both avian and mammalian hosts. Discrete, multiple DNA fragments ranging in approximate size from 285 to 360 bp were observed within and among each of the isolates tested. Polymerase chain reaction performed on individual adult parasites from each isolate resulted in multiple DNA fragments that were comparable to those generated from pooled genomic DNA. Sequence analysis of cloned, representative amplified fragments demonstrated that fragment length variation resulted primarily from the dinucleotide (TG)n and trinucleotide (TGC)n microsatellite repeats present within the expansion segment. Results are consistent with both population differences within the species as well as the presence of multiple alleles of the large subunit ribosomal RNA genes within individual parasites.

A 43-kDa secreted glycoprotein from the intracellular parasitic nematode Trichinella spiralis has been considered as a factor involved in the formation of the Nurse cell in infected muscle. The closely related intracellular parasitic nematode Trichinella pseudospiralis that also infects muscle cells, does not form Nurse cells and was thought not to secrete the 43-kDa glycoprotein. This implied a unique role for the 43-kDa glycoprotein in T. spiralis infection and supported the hypothesis of involvement of the 43-kDa glycoprotein in Nurse cell formation. Following cloning of a full length cDNA encoding the 43-kDa protein, antibodies were raised against several domains of the 43-kDa glycoprotein. Here we show that a protein related to the 43-kDa glycoprotein exists in T. pseudospiralis. Immunohistochemical studies reveal important similarities in the distribution of the 43-kDa glycoprotein and the related protein from T. pseudospiralis in muscle infections with either of the two parasites. The 43-kDa glycoprotein may therefore play a common role in the life cycles of these two parasites and probably is not involved in Nurse cell formation.

Heat shock proteins (HSPs) were documented for the first time in both somatic extracts and excretory/secretory (ES) products of the infective-stage larvae of Trichinella spiralis and T. pseudospiralis. Larvae recovered from muscles of infected mice were heat shocked at 37, 40, 43 and 45 degrees C in RPMI 1640 medium containing L(-)[35S]methionine. Somatic extracts and ES products of heat-shocked worms were then analysed by SDS-PAGE, autoradiography and laser densitometry. Prominent bands of HSPs were observed at 43 degrees C which is the optimal heat shock temperature. The major HSPs in somatic extracts of T. spiralis were 20, 47, 50, 70, 80 and 86 kDa. When the temperature was increased from 37 to 43 degrees C, the greatest increase in absorbance was observed in HSPs 70 and 86. In vitro translation of mRNA in a nuclease-treated rabbit reticulocyte lysate system showed an increase in the synthesis of the 80 kDa protein. This suggests that the production of HSP 80 is regulated at the transcriptional level. The major HSPs in the ES products were 11, 45, 53 and 64 kDa. In T. pseudospiralis, the major HSPs in the somatic extracts were 20, 26, 31, 50, 53, 70, 80 and 86 kDa, and in the ES products, 11, 35, 37, 41 and 64 kDa.

A human case of infection by Trichinella pseudospiralis has recently been described. Some morphologic anomalies of the muscle larvae, however, raise the possibility of an incorrect taxonomic attribution. A molecular taxonomic approach has therefore been applied for the identification of the parasite. Random amplified polymorphic DNAs were obtained from a single larva extracted from a muscle biopsy of the suspected case of T. pseudospiralis infection, and compared with those derived from 27 reference strains of Trichinella spp. Nearly identical amplification patterns were obtained from the suspected larva and from reference strains of T. pseudospiralis, thus supporting the original morphology-based identification. An enzyme-linked immunosorbent assay and Western blots carried out on pretreatment and post-treatment sera provided further confirmation.

Eight taxa have recently been proposed as being encompassed by the genus Trichinella on the basis of allozyme and biological data. In this paper we show that an analogous 8 taxon structure for this genus results from the random amplified polymorphic DNAs (RAPDs). Five 10-mer or 20-mer primers were used under different polymerase chain reaction (PCR) conditions to produce multiband RAPD fingerprints from muscle larvae of 40 isolates of Trichinella spp. The resulting RAPD data were analysed following the numerical taxonomic approach, and the resulting classification was compared to that derived from allozyme data. The agreement found between allozymes and RAPDs, while supporting the polyspecific structure of the genus Trichinella, confirms the potential of RAPDs as a tool for the detection of cryptic species. The selected primers were tested on individual muscle larvae in an attempt to standardize a RAPD assay for the routine identification of the 8 taxa of Trichinella. Only 1 of the 5 primers yielded reproducible fingerprints from the single larvae. Using this primer, the 5 species and the 3 other taxa of the genus Trichinella can be identified in a single assay without the need for massive in vivo parasite production.

Two restriction endonucleases (Cla I and Hpa II) produced polymorphic repetitive DNA profiles which were used in a clustering analysis to quantify the level of genetic variation among 14 North American sylvatic isolates of T5 Trichinella. Differences in genetic profiles reflected phenotypic differences in parasite reproductive success as measured by an isolate's reproductive capacity index in natural hosts. Two genetically distinct isolates of the T5 genotype and T. spiralis were used to infect white-footed mice Peromyscus leucopus for a behavioral analysis. Three behavioral characteristics were tabulated from videotapes: time to emerge from cage, time spent inside cage, and number of rears (index of exploratory activity). Mice infected with 400 T. spiralis larvae showed a significant decrease in exploratory activity and significant increases in time to emerge and time spent inside cage, whereas mice infected with 200 larvae displayed no differences in behavior. Mice infected with a T5 isolate from a raccoon (R9) also displayed decreased exploratory activity. In contrast, mice infected with the other T5 isolate from a coyote (C26) showed relatively constant rearing levels but spent less time inside their cages during the late period of the infection. Results suggest that different genetic strains of Trichinella induce varied types of behavioral modifications upon their hosts.

A new outbreak of trichinellosis occurred in France in December 1993 and involved around 550 patients. The authors report here how recent knowledge on Trichinella have been helpful to investigate this outbreak.

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.

The random amplified polymorphic DNA (RAPD) technique was successfully used to produce genetic fingerprints distinguishing between Trichinella spiralis and Trichinella britovi. The same patterns were obtained from purified and crude DNA preparations of pooled and single muscle larvae. RAPD fingerprinting was applied to muscle larvae preserved under different conditions and recovered from different hosts. Larvae recovered from fresh and frozen meat and stored at -20 degrees C for a long time or under 70% ethyl alcohol at room temperature for 30 d gave good and reproducible results. Single larvae recovered from a naturally infected wild boar and from a human biopsy gave fingerprints congruent to those obtained from T. britovi reference strains. The results prove that RAPD analysis is a quick method to distinguish between the autochthonous Trichinella species of Central-Southern Europe in less than 1 d after the detection of the infection. If necessary, the biological material can be frozen or stored under 70% ethyl alcohol at room temperature and sent to laboratories able to perform the RAPD analysis. The RAPD technique requires no prior knowledge of the molecular biology of the organism to be investigated and therefore appears to be a promising tool in parasitology for the identification of sibling species.

Recently, 5 sibling species and 3 other phenotypes were identified in the genus Trichinella. Single primers of arbitrary nucleotide sequence were used to produce random amplified polymorphic DNA starting from decreasing amounts of Trichinella spiralis and Trichinella britovi DNA. Reproducible amplification products from 30 pg of DNA were obtained using 1 of 6 examined primers. These fragments distinguish between 2 European Trichinella species, T. spiralis, showing a 1,350-bp band, and T. britovi, showing 400- and 1,100-bp bands. The developed procedure allows the characterization of crude DNA preparations of single muscle-stage larvae, avoiding time-consuming passages of parasites in laboratory animals.

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.

The taxonomic status of variants within the genus Trichinella is problematical. Some authors recognise no fewer than four species (Trichinella spiralis, T. pseudospiralis, T. nativa and T. nelsoni), others regard T. nativa and T. nelsoni as strains of T. spiralis (T. spiralis var nativa or sylvatica), while others consider the genus to be monospecific, with a variety of more or less well defined isolates. Much of the current evidence adduced to support these various positions is similar to that used pre-1983. It derives from studies of the incidence of Trichinella infections in wild and in domestic animals, comparisons of infectivity of different isolates in laboratory animals and studies of immunity. However, it has become clear that infectivity and epidemiological studies are unreliable tools for discriminating between isolates of Trichinella and it has been shown that differences in the elicitation of immune responses are as much a function of the host as of the parasite. The introduction of monoclonal antibody technology has, however, permitted the identification of specific antigens in different isolates. The information is as yet scant, and one antigen does not a species make. Isozyme analysis provides some support for separating the various isolates of Trichinella into distinct groups, but cannot of itself shed light on the species problem until certain conditions are met. These conditions are difficult to achieve even in organisms abundantly available and without the baggage of the parasitic habit. Isozyme analysis is probably best used to support the newer studies of genomic DNA. Recent analyses of DNA by restriction endonucleases and dot-blot hybridisation techniques show ample promise of insights into speciation, and a new technique for amplifying the DNA from a single larva by the polymerase chain reaction offers exciting prospects. However, the position yet remains as stated in the first section of this abstract.

Oligonucleotide primers were synthesized for the polymerase-chain-reaction amplification of target DNA from two sequences of Trichinella spiralis. Six strains belonging to T. spiralis, T. nativa, T. britovi, T. pseudospiralis, and T. nelsoni were tested. Amplification products were obtained with T. spiralis, T. britovi, and T. nelsoni DNA from a 53-kDa antigen cDNA sequence and with T. spiralis and T. 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 between T. spiralis and T. nelsoni, suggesting that the 1.6 kb repetitive DNA sequence would not be specific for T. spiralis. No amplification was detected for T. nativa or T. pseudospiralis DNA from the two sequences and for T. britovi DNA from the 1.6-kb repetitive DNA sequence.

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)

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.

A break was identified in the large subunit ribosomal RNA of Trichinella spiralis that results in its dissociation into 2 smaller fragments of approximately equal length. The approximate location of the break within the encoding gene was mapped from subcloned rDNA fragments by S1 protection experiments. The boundaries of the break were determined by cDNA primer extension and S1 nuclease protection assays. The excised fragment (gap sequence) was localized to expansion segment 5 within domain IV from which 86 bases are removed during the excision process. The gap region is flanked by the consensus sequence CGAAAG; however, comparison of expansion segment 5 sequences from T. spiralis, T. nativa, T. nelsoni and T. pseudospiralis, all of which undergo 'gap processing', demonstrates significant size and sequence heterogeneity and provides little evidence for additional consensus sequences which could be implicated in gap processing.

A method was developed to identify domestic isolates of Trichinella using the polymerase chain reaction. Oligonucleotide primers, based on the repetitive DNA sequence (pPRA) from the P1 isolate of Trichinella, were used to amplify genomic DNA from 13 domestic isolates and tested against sylvatic isolates of Trichinella. Pattern differences were observed among domestic isolates, indicating divergence of this repetitive sequence. The primers were specific for domestic Trichinella as no amplification was detected for sylvatic isolates or Trichinella pseudospiralis. It was possible to identify an isolate from a single larva following digestion or in situ in muscle tissue.

A rapid, sensitive and specific serologic test has been developed for the diagnosis of swine trichinellosis. The ELISA based test utilizes L1 stichosome antigens recovered as excretory-secretory (ES) products from in vitro cultivated muscle larvae. Field studies conducted with 20,000 commercial swine using crude ES antigen demonstrated that the test could detect 98% of the medically significant infections. The test had a false-positive rate of less than 3%. Because of difficulties in regulating the quality and quantity of ES antigen and the need to continually maintain infected laboratory animals for producing the diagnostic reagent, efforts have been made to clone and express the gene(s) encoding the immunodominant ES antigens. To date a cDNA sequence, designated TsA-12, which codes in part for a 53-kDa ES antigen, has been identified and expressed in bacteria. Results demonstrate that TsA-12 is recognized by immune sera and further suggest that the immunodominant 45-, 48- and 53-kDa ES proteins which share antigenic epitopes are distinct glycoproteins.

A partial genomic DNA library constructed in pUC 13 using DNA from a sylvatic isolate of Trichinella spiralis (T. spiralis T5) was differentially screened with radiolabeled homologous genomic DNA and with DNA from T. spiralis T1. One clone was identified and designated pUPB-3.7 which, by slot blot and Southern blot analyses, reacted specifically with T. spiralis T5 DNA and did not cross-react with DNA from any other T. spiralis genotype. The 482-bp repetitive sequence which is 70% rich in A and T residues, comprises at least 2.7% of the parasite genome and can detect as little as 0.4 ng of DNA. When used to assess the prevalence of T. spiralis T5 in Indiana wildlife, DNA from 19 of 20 independently obtained sylvatic isolates reacted positively with the pUPB-3.7 probe indicating that within this geographical locality, T. spiralis T5 is the predominating genotype in wild mammals.

Host antibody recognition of muscle larval (ML) antigens of Trichinella spiralis was examined. Monoclonal antibodies (MoAbs) to known host protective ML antigens have been produced in order to aid this examination. Eleven strains of mice with independent MHC haplotypes and seventeen T. spiralis infected human patients were all found to recognize the same three major antigens as the monoclonal antibodies; i.e., of mw 41, 46 and 55 kD. However all serum samples tested also recognized further ML antigens and this recognition varied with the individual or strain. This variation in antigen recognition also applied to the MoAb. Mutual inhibition studies demonstrated that even where the MoAb apparently recognized the same antigens, specific epitope recognition was disparate. Hence some of the major antigens recognized by hosts of T. spiralis, regardless of whether vaccinated or infected, correspond with antigens which have considerable host protective properties. There also appear to be a number of epitopes upon these antigens and the biological implications of this are discussed.

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.

Complementary DNA (cDNA) encoding a 49-kDa antigen of Trichinella spiralis was cloned, characterized, and expressed in Escherichia coli by recombinant DNA methods. As a first step, 29 residues of N-terminal amino acid sequence were determined by direct analysis of highly purified antigen. Mixed-sequence primers based on the amino acid sequence were then synthesized and used as primers to generate a partial cDNA by the polymerase chain reaction (PCR). A nearly full-length cDNA was then obtained by PCR using a specific 5'-end primer and a non-specific 3'-end primer. Complete sequence of the 1133-bp cDNA was determined. Translation of this sequence predicts an N-glycosylated polypeptide of 322 amino acids. The cDNA was expressed as a fusion protein in bacteria which could be recognized in Western blots both by sera from mice immunized with purified native 49-kDa antigen and by sera from swine infected with the parasite. These findings suggest that recombinant P49 is a potentially valuable antigen both for vaccine development and immunodiagnosis.

Isoenzyme analysis was used to characterize 47 isolates of Trichinella 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 strains T. spiralis s.str. and T. pseudospiralis. The genetic data indicate the absence of gene flow between AFR and EUR isolates. The biochemical results suggest the presence of genetic heterogeneity in T. 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.

A total of 13 Spanish Trichinella isolates were characterised by DNA analysis. Genomic DNA cross-hybridisation tests revealed two distinct groups that exhibited weak cross-reactions. Further diagnostic subdivision was attempted according to restriction-fragment polymorphism using a cloned T. spiralis DNA repetitive fragment as a probe, but no major difference between the two Trichinella groups was observed.

A Trichinella spiralis cDNA expression library was constructed in lambda gt11 from muscle larvae mRNA and immunologically screened to identify genes encoding previously described immunodiagnostic excretory-secretory (ES) antigens. Screening the library with T. spiralis infection serum from swine or rabbit antiserum to T. spiralis ES antigen identified one clone, designated TsA-12, that contains a cDNA transcript 539 bp in length and codes for an apparent 123-kDa beta-galactosidase fusion protein that does not cross-react with Trichuris suis or Ascaris suum infection serum. Western blots of T. spiralis extracts and immunoperoxidase staining of tissue sections from muscle larvae using antibodies to purified TsA-12 demonstrate homology between TsA-12 and the 53 kDa diagnostic antigen from ES products (designated Ts.53) and localize the homologous native antigen to the stichocyte cells of the parasite. ELISA tests using TsA-12 as antigen, detected antibodies to T. spiralis in experimentally-infected mice as early as 14 days post-inoculation with maximum antibody titers being reached at 28 days post-inoculation. The TsA-12 dscDNA hybridizes to mRNA sequences expressed in both the muscle larvae and adult stages; however, concomitant expression of the native antigen is not observed within adult ES products. Southern blots of homologous and heterologous genomic DNAs probed with 32P-labeled TsA-12 dscDNA fragments verify TsA-12 as a T. spiralis specific sequence that is present in multiple copies within the parasite genome.

DNA analysis is playing an increasingly important role in characterising and classifying nematode parasites. Though less emphasis has been placed on utilising DNA elements to study nematodes of veterinary importance, correct diagnosis is, nevertheless, critical to proper treatment and control. The genus Trichinella presents a particularly interesting problem since the level of classification within this genus remains unclear. Herein we discuss the application of DNA analysis to the diagnosis and speciation of the parasitic nematode Trichinella spiralis, where the study of cloned repetitive DNA elements and ribosomal RNA genes has led to significant advances in the understanding of the species-level systematics of this nematode. Despite advances in the recognition of multiple gene pools within the genus Trichinella, their taxonomic level is still uncertain because of insufficient knowledge regarding intraspecific variation within this genus. The utility of mitochondrial DNA and ribosomal DNA sequence analysis in studying the phylogeny and evolutionary history of parasites within this genus is also discussed.

The gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from T. spiralis infective larvae was ligated into phage vector lambda gt11 DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gt11 expression library was constructed. A cDNA clone encoding a 46 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing nearly full-length cDNA for a 46 kDa protein was isolated. The gene encoding this 46 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 46 kDa polypeptide. The antigenic polypeptide was excreted/secreted as a 46 kDa native antigen. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.

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.