Hiding in plain sight: discovery and phylogeography of a cryptic species of Trichinella (Nematoda: Trichinellidae) in wolverine (Gulo gulo).

Understanding parasite diversity and distribution is essential in managing the potential impact of parasitic diseases in animals and people. Imperfect diagnostic methods, however, may conceal cryptic species. Here, we report the discovery and phylogeography of a previously unrecognized species of Trichinella in wolverine (Gulo gulo) from northwestern Canada that was indistinguishable from T. nativa using the standard multiplex PCR assay based on the expansion segment 5 (ESV) of ribosomal DNA. The novel genotype, designated as T13, was discovered when sequencing the mitochondrial genome. Phylogenetic analyses of the mitochondrial genome and of 15 concatenated single copy orthologs of nuclear DNA indicated a common ancestor for the encapsulated clade is shared by a subclade containing Trichinella spiralis and Trichinella nelsoni, and a subclade containing T13 and remaining taxa: T12 + (T2 + T6) + [(T5 + T9) + (T3 + T8)]. Of 95 individual hosts from 12 species of mammalian carnivores from northwestern Canada from which larvae were identified as T. nativa on multiplex PCR, only wolverines were infected with T13 (14 of 42 individuals). These infections were single or mixed with T. nativa and/or T6. Visual examination and motility testing confirmed that T13 is encapsulated and likely freeze-tolerant. We developed a new Polymerase Chain Reaction-Restriction Fragment Length Polymorphism which unequivocally distinguishes between T13 and T. nativa. We propose Trichinella chanchalensis n. sp. for T13, based on significant genetic divergence from other species of Trichinella and broad-based sampling of the Trichinella genome. Exploration of Alaskan and Siberian isolates may contribute to further resolution of a phylogeographically complex history for species of Trichinella across Beringia, including Trichinella chanchalensis n. sp. (T13).

A Simple and Rapid Staining Technique for Sex Determination of Trichinella Larvae Parasites by Confocal Laser Scanning Microscopy.

The roundworms of Trichinella genus are worldwide distributed and their prevalence in nature is high. Trichinella genus parasites are the causative agents of foodborne zoonosis trichinellosis. The main prevention and control of the infection are meat inspection by the magnetic stirrer method for the detection of Trichinella larvae in muscle samples. The treatment can be effective if the parasite is discovered early in the intestinal phase. Once the Trichinella larva has reached the muscle tissue, the parasite remains therein and there is no treatment for this life cycle stage. The Trichinella species is dioecious with separate male and female individuals. The developed staining technique that uses confocal laser scanning microscopy (CLSM) displays sufficient results for Trichinella larvae examination and this protocol is applicable to study the internal and external structures and for the sex determination of T. britovi and T. spiralis larvae samples. In the present study, a luminescent derivative was synthesized and used for staining of T. spiralis and T. britovi larvae samples for the examination by CLSM. Various fixatives, such as AFA, 70% ethanol, and Bouin's and Carnoy's solutions were tested for sample preparation. The synthesized luminescent compound demonstrates best visualization results for samples fixed in Bouin's fixative.

[Biological properties of Trichinella spp. isolated from the Central Region of the Russian Federation].

The biological properties of Trichinella isolated from the muscles of a common fox inhabiting in the Central Region of the Russian Federation have been studied. The paper presents the results of passaging this isolate of Trichinella on laboratory rats and mice, shows morphometric variability in relation to the change of Trichinella habitat. Evidence is provided that the shape and size of Trichinella capsules depend on the strain of the parasite and the species of an animal host the muscles of which contain these tissue helminths. The morphological data, biological properties, and high negative temperature resistance of the Trichinella that are present in the muscles of a fox shot in the Ryazan Region allow the above isolate to be classified as Trichinella nativa.

The first host record of trichinosis in a red fox, Vulpes vulpes japonica, from Aomori Prefecture, northern Honshu, Japan.

A case of wildlife trichinosis was found in a red fox (Vulpes vulpes japonica) captured at Rokkasho, Aomori Prefecture on November 27, 1998. Trichinella larvae were obtained from almost all of the muscle tissues except for the masseter. The highest number of larvae per gram of tissue was found in the muscles of the gluteal region and throat. The lowest number was found in the diaphragm and tongue. Trichina cysts within the muscle fibers had groups of fatty cells at the poles, and minimal tissue reaction was observed around the cyst. No calcification was found in the cyst. These morphological findings suggested that the considerable time had elapsed since the invasion. This is the first case of trichinosis in a red fox in Japan.

Trichinella murrelli n. sp: etiological agent of sylvatic trichinellosis in temperate areas of North America.

Trichinella T5, collected from sylvatic carnivores in North America, was identified previously as a different phenotype of Trichinella, with an uncertain taxonomic level due to the availability of only 2 isolates. Cross-breeding experiments carried out with single female and male larvae of 2 strains of Trichinella T5, with single female and male larvae of 2 strains of Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis, Trichinella nelsoni, and Trichinella T6, showed a reproductive isolation of Trichinella T5. Viable offspring were obtained only when a female of Trichinella T5 was crossed with a male of T. britovi, but not vice versa. Furthermore, the analysis of biological, biochemical, and molecular data of 32 isolates collected from sylvatic animals in the Nearctic region and identified as Trichinella T5 permitted its reassessment at the species level. Trichinella murrelli n. sp. is characterized by the following: distribution in temperate areas of the Nearctic region; newborn larvae production in vitro of 29-36/72 hr; nurse cell development time between 24 and 70 days postinfection; reproductive capacity index in Swiss mice 1.2-9.5, in wild mice 29.5-159.8, in rats 0.7-2.4, and in pigs 0.03-0.0004; no resistance to freezing; ribosomal DNA fragments of 7.2 kb and/or 11.4 kb, plus 2.2 kb and 1.8 kb present after Dra I digested DNA when probed with total T. spiralis RNA; a specific amplicon of 179 bp after polymerase chain reaction (PCR) amplification with the primer set SB147G; a specific fragment of 1,600 bp after PCR amplification with the primer set Ts43CA and Hhb I digestion; long incubation period; and moderate to severe pathogenicity for humans. The new species is most similar to T. britovi, though it differs from T. britovi in the pattern of 2 allozymes, in the patterns of major ribosomal DNA and PCR-restriction fragment length polymorphism fragments, and in geographical distribution.

Trichinella spiralis: modifications of the cuticle of the newborn larva during passage through the lung.

A scintigraphic method was developed to study the distribution of radioactivity after iv injection of 131I-labeled Trichinella spiralis newborn larvae into normal rats. It was found that the radioactivity was immediately retained in the lungs and thereafter slowly released, with a mean transit time in excess of 9 hr, as calculated by image analysis. At various times after iv injection of newborn larvae into normal mice, the lungs were removed and parasites were recovered and counted. Fifty to seventy percent of the larvae injected were recovered after 30 sec, between 10 and 30% after 1 min, and less than 4% at 15 min. These results indicate that during the very rapid passage of newborn larvae through the lungs, labeled components of the cuticle are detached and retained. It is suggested that the modifications produced in the cuticle of the newborn larva during its passage through the lung may increase its resistance to the nonspecific defense mechanisms of the host.

[Differentiation of the sex of Trichinella larvae collected in Changchun].

The length of the rectum in male Trichinella spiralis larvae is about twice that in female larvae, being 54.39 (50.4-63.0) microns in males and 28.05 (18.9-31.35) microns in females. The gonad rudiment of the female larvae has already differentiated obviously into seminal receptacle rudiment and uterus rudiment, while that of the male larvae remains undifferentiated. The seminal receptacle rudiment is a granular, small mass and dark brown in color, while the uterus rudiment is composed of a column of several cells, and the total length of these two rudiments is 62.18 (50.4-75.6) microns. Verifications through experimental infections proved that the length of rectum and the degree of differentiation of gonad rudiment might be used as indices to identify the sex of larvae. It was found that the sexual ratio of T. spiralis collected in Changchun was 1.82:1 (1.17-2.59:1).

[Intrastrain heterogeneity of Trichinella spiralis: a sedimentation analysis]. / Vnutrishtammovaia geterogennost' Trichinella spiralis: sedimentatsionnyi analiz.

Intrastrain heterogeneity of muscular larvae of trichinellids has been revealed in experiments of free sedimentation and isopyknic division in the density gradient of saccharose, the presence of which is confirmed by the study of morphological characters and infection activity. The presence of intrastrain heterogeneity indicates the necessity of introduction of standard technique for studies of biological characters of different strains of trichinellids.

A comparison of three geographical isolates of Trichinella spiralis from the mid-Atlantic United States.

Three isolates of Trichinella spiralis (black bear: 41o50'N, 79o00'W, 1982; grey fox: 41o00'N, 76o00'W, 1982; domestic pig: 39o80'N, 75o30'W, 1983) from the mid-Atlantic United States were characterized in Crl: COBS CFW (SW) mice during 10 successive 40-day generations. Over 10 generations, the isolates differed as follows: worm position of the ursine isolate was significantly more posteriad compared to the porcine isolate; sex ratio of the vulpine isolate was significantly lower compared to the ursine and porcine isolates; females of the porcine isolate produced significantly more newborn larvae in vitro than the sylvatic isolates; both the larvae per gram (LPG) and reproductive capacity index (RCI) were significantly higher for the porcine isolate; and male worms of the vulpine isolate were significantly smaller than those of the porcine or ursine isolates. No differences were observed among isolates for percentage of inoculum recovered, length of female worms, or uterine length. The fecundity of the isolates in hamsters, gerbils, multimammate rats, Sprague-Dawley rats, and Peromyscus sp. was examined also. Based upon RCI and LPG, the porcine isolate was the most fecund in all hosts except gerbils. This higher fecundity was often manifest as increased morbidity and mortality in hosts infected with this isolate. The relationship between the virulent nature of the porcine isolate and the enhanced transmission of T. spiralis are discussed.

Euparal as a permanent mounting medium for helminth eggs and proglottids.

The importance of euparal as a permanent mounting medium for eggs of parasites and the process of making permanent slides are analyzed and discussed in detail. Studies show that helminth eggs mounted in euparal exhibit excellent optical and drying properties. Euparal also can be used to identify proglottids of Taenia species and to examine for the presence of Trichinella spiralis in suspicious muscle biopsies.

[Specialization of the ovocytic endoplasmic reticulum near the contact of the hind gut in Trichinella spiralis (author's transl)]. / Spécialisation du réticulum endoplasmique au contact de l'intestin postérieur, chez les ovocytes de Trichinella spiralis (Nématode, Trichuroïde).

In Trichinella spiralis, the bigger ovocytes show contacts with the cuticula of the hind gut. Near these contacts, the ovocytic endoplasmic reticulum is hypertrophied. This device is described with details and compared to that one of the stichocytes; its possible role is discussed.

[Morpho-physiologic criteria for the taxonomic independence of the species Trichinella pseudospiralis Garkavi, 1972]. / Morfo-fiziologicheskie kriterii taksonomicheskoi samostoiatel'nosti vida Trichinella pseudospiralis Garkavi, 1972

A taxonomic status of a new species of Trichinella from Procyon lotor L. described by V. L. Garkavi in 1972 as T. pseudospiralis is discussed. Two-month invasional larvae resemble in their size and structure 19-20-day larvae of typical T. spiralis. However, the ratio between their organs points not only to the delay in development but also to significant structural morphological differences. Luminiscent microscopic analysis of muscular and intestinal forms of both species confirms this. Impossibility of crossing between both species is noted. Results of investigations show that T. pseudospiralis Garkavi can be accepted as a valid species.