Sylvatic and domestic Trichinella spp. in wild boars; infectivity, muscle larvae distribution, and antibody response

Thirty-six wild boars were inoculated with Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis (USSR), T. pseudospiralis (USA), T. pseudospiralis (AUST), Trichinella murrelli, Trichinella T6, and Trichinella nelsoni. The wild boars were killed at 5 and 10 wk postinoculation (PI), and the number of muscle larvae per g (lpg) of tissue was determined for 18 muscles or muscle groups. Five weeks PI, all Trichinella genotypes had established as muscle larvae, but their infectivity varied widely: T. spiralis established in high numbers (mean = 296 lpg), T. britovi, T. nelsoni, and 1 of the T. pseudospiralis genotypes (AUST) in moderate numbers (mean = 53-74 lpg), whereas the remaining genotypes were poorly infective (mean 2-16 lpg). Because of considerable weight gain of the wild boars, an estimated total larval burden (live weight x lpg) was calculated for each animal. The total larval burden did not change significantly over time for T. spiralis, T. murrelli, T. britovi, T. nelsoni, and T. pseudospiralis (USA and USSR), whereas a significant reduction could be demonstrated for T. nativa, Trichinella T6, and T. pseudospiralis (AUST). Diaphragm and tongue were predilection sites in wild boars, independent of Trichinella genotype and infection level. At low infection levels, a greater percentage of larvae were found in diaphragm and tongue at 10 wk than 5 wk PI. Antibody responses increased rapidly between weeks 3 and 5 PI. For T. spiralis and T. nelsoni, the high antibody level persisted throughout the experimental period, but for T. nativa, T. britovi, T. murrelli, or Trichinella T6, the levels declined. For T. pseudospiralis, the antibody response increased more gradually between weeks 3 to 10 PI. Infection with all genotypes of Trichinella were detected using any of 7 excretory-secretory antigens, which points to the potential use of 1 common antigen for epidemiological studies on Trichinella in wild boars. In conclusion, T. spiralis is highly infective to wild boars, T. britovi, T. nelsoni, T. pseudospiralis (USA), and T. pseudospiralis (USSR) are moderately infective, and T. nativa, T. murrelli, T. pseudospiralis (AUST), and Trichinella T6 are poorly adapted to this host species.     

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.     

Piperine modulation of carcinogen induced oxidative stress in intestinal mucosa

The plasma-borne long-chain free fatty acids (FFA) enter skeletal muscle cells. Upon entering they are oxidized or esterified and a fraction remains free (non-esterified). The data on free fatty acids in skeletal muscles remain highly controversial. Furthermore, the composition of individual fatty acids in various lipid fractions including free fatty acids, monoglyceride and diglyceride in muscles has not been characterized. Also data on the composition of fatty acids esterified into muscle triglycerides and phospholipids are incomplete. The present study was undertaken to examine a composition of fatty acids in lipid fractions of different skeletal muscle types. For this purpose, samples of the rat soleus, red and white portions of gastrocnemius were excised, trimmed of visible fat and fascias and immediately frozen in liquid nitrogen. Samples were then pulverized and, lipids were extracted and fractionated by thin-layer chromatography. Individual long-chain fatty acids in different fractions were identified, characterized and quantitated by gas-liquid chromatography. FFA composition in the plasma was also determined. The total FFA content in the soleus, red and white gastrocnemius was 69.1 ± 10.8, 49.0 ± 13.6 and 22.7 ± 8.6 nmol/g, respectively. Palmitic and oleic acids were the major fatty acids in the muscles FFA fraction. Monoglyceride fraction of each muscle contained palmitic, stearic and linoleic acid as the major fatty acids, Diglyceride fraction contained mostly palmitic and oleic acid whereas triglyceride fraction mostly palmitic and linoleic acid.. The fraction of phospholipids was composed mostly of palmitic and linoleic acid but contained also considerable percentage of archidonic acid. Total plasma FFA/muscle FFA ratio depended on a muscle type and was: 2.4 in the soleus, 3.5 in the red and 7.4 in the white gastrocnemius. This assured transport of FFA to the myocytes. However, there were great differences in the ratio between particular FFA within the same muscle as well between the muscles. It indicates that individual FFA are either selectively transported from the plasma to the muscles or selectively used within the myocytes or both.     

Infectivity, persistence, and antibody response to domestic and sylvatic Trichinella spp. in experimentally infected pigs

Groups of pigs were inoculated with genotypes of Trichinella belonging to: Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis (from Caucasus), T. pseudospiralis (from USA), Trichinella murrelli, Trichinella sp. (from North America), and Trichinella nelsoni. The pigs were sacrificed between 5 and 40weeks p.i., and the number of muscle larvae per gram (l.p.g.) of tissue was determined as an average of 18 muscles. All Trichinella genotypes were infective for pigs, but both their infectivity and persistence varied: 5weeks p.i., T. spiralis muscle larvae were present in high numbers (mean=427l.p.g.), while T. britovi, T. nelsoni, and T. pseudospiralis larvae were present in moderate numbers (means=24-52l.p.g.); larvae of the remaining genotypes were recovered only in low numbers (means=0.05-5. 00l.p.g.). The total larval burden (live weight of pigxl.p.g.) was constant over time for T. spiralis, T. britovi, and T. nelsoni, but declined significantly (P<0.05) for the other genotypes. Antibody responses could be detected 3-4weeks p.i. by seven different Trichinella ES antigens, but the antibody levels and dynamics differed significantly among the experimental groups. In pigs inoculated with T. spiralis, T. britovi, or T. nelsoni, the antibody level increased rapidly between weeks 3 and 5 p.i. and was stable or increased slightly throughout the experimental period. In pigs inoculated with T. nativa, T. murrelli, or Trichinella (T6) (from North America), a rapid increase was detected between weeks 3 and 5 p.i., but for these genotypes a reduction in the antibody levels was seen thereafter. In the pigs inoculated with T. pseudospiralis, the antibody level increased more gradually over a period from week 3 p. i. to weeks 15-20 p.i., and decreased thereafter. In general, all species of Trichinella were detected by any of the seven ES antigens, which points to the potential use of one common antigen for surveillance and epidemiological studies on both domestic and sylvatic Trichinella in pigs. Homologous ES antigens were slightly more sensitive in detecting antibodies to the corresponding Trichinella species.     

Ribo HRM--detection of inter- and intra-species polymorphisms within ribosomal DNA by high resolution melting analysis supported by application of artificial allelic standards

Ribo HRM, a single-tube PCR and high resolution melting (HRM) assay for detection of polymorphisms in the large subunit ribosomal DNA expansion segment V, was developed on a Trichinella model. Four Trichinella species: T. spiralis (isolates ISS3 and ISS160), T. nativa (isolates ISS10 and ISS70), T. britovi (isolates ISS2 and ISS392) and T. pseudospiralis (isolates ISS13 and ISS1348) were genotyped. Cloned allelic variants of the expansion segment V were used as standards to prepare reference HRM curves characteristic for single sequences and mixtures of several cloned sequences imitating allelic composition detected in Trichinella isolates. Using the primer pair Tsr1 and Trich1bi, it was possible to amplify a fragment of the ESV and detect PCR products obtained from the genomic DNA of pools of larvae belonging to the four investigated species: T. pseudospiralis, T. spiralis, T. britovi and T. nativa, in a single tube Real-Time PCR reaction. Differences in the shape of the HRM curves of Trichinella isolates suggested the presence of differences between examined isolates of T. nativa, T. britovi and T. pseudospiralis species. No differences were observed between T. spiralis isolates. The presence of polymorphisms within the amplified ESV sequence fragment of T. nativa T. britovi and T. pseudospiralis was confirmed by sequencing of the cloned PCR products. Novel sequences were discovered and deposited in GenBank (GenBank IDs: JN971020-JN971027, JN120902.1, JN120903.1, JN120904.1, JN120906.1, JN120905.1). Screening the ESV region of Trichinella for polymorphism is possible using the genotyping assay Ribo HRM at the current state of its development. The Ribo HRM assay could be useful in phylogenetic studies of the Trichinella genus.     

Characterization of glucosylceramides in the Polygonaceae, Rumex obtusifolius L. injurious weed

Rumex obtusifolius L., a member of Polygonaceae, is one of the world's worst weeds. We characterized the glucosylceramide molecular species in leaves of R. obtusifolius by liquid chromatography/tandem mass spectrometry. 4,8-Sphingadienines were principally paired with 2-hydroxy palmitic acids. In contrast, 4-hydroxy-8-sphingenines were chiefly attached to 2-hydroxy fatty acids with 22 to 26 carbon-chain length. A unique characteristic of the 2-hydroxy fatty acid composition of R. obtusifolius was the high content of n-9 monoenoic 2-hydroxy fatty acids with 22 and 24 carbon-chain length. The levels of the Z and E stereoisomers of the 8-unsaturated long-chain bases were reliably distinguished from those in other plant families in ten species of Polygonaceae.     

Molecular identification of natural hybrids between Trichinella nativa and Trichinella T6 provides evidence of gene flow and ongoing genetic divergence

To date, there are no data available on the population genetics of Trichinella due to the lack of genetic markers and the difficulty of working with such small parasites. In the Arctic region of North America and along the Rocky Mountains, there exist two genotypes of Trichinella, Trichinella nativa and Trichinella T6, respectively, which are well differentiated by biochemical and molecular characters. However, both are resistant to freezing, show other common biological characters (e.g. low or no infectivity to rodents and swine) and produce fertile F1 offspring upon interbreeding. To data, these two genotypes have been considered allopatric. In this study, we detected both genotypes in wolves of the same wolf packs in Alaska, suggesting sympatry. A single GTT trinucleotide present in the ITS-2 sequence of T. nativa but not in Trichinella T6 was used as a genetic marker to study gene flow for this character in both a murine infection model and in larvae from naturally-infected Alaskan wolves. Only F1 larvae originating from a cross between T. nativa male and Trichinella T6 female were able to produce F2 offspring. Larvae (F1) originating from a cross between Trichinella T6 male and T. nativa female were not reproductively viable. As expected, all F1 larvae showed a heterozygote pattern for the GTT character upon heteroduplex analysis; however, within the F2 population, the number of observed heterozygotes (n=52) was substantially higher than expected (n=39.08), as supported by the F(is) index, and was not in the Hardy-Weinberg equilibrium. Larvae from two of the 16 Trichinella positive Alaskan wolves, showed the Trichinella T6 pattern or the T. nativa/Trichinella T6 hybrid pattern. Our data demonstrate that T. nativa and Trichinella T6 live in sympatry at least in Alaskan wolves, where T. nativa occurs more frequently (69%) than Trichinella T6 (31%). One explanation for this phenomenon is that glacial periods may have caused a geographical relocation, colonisation and independent evolution of T. nativa within the Rocky Mountains, resulting in a bifurcation of the freeze-resistant genotype. Additional studies will be required to test this hypothesis.     

Prevalence of Trichinella spp. in black bears, grizzly bears, and wolves in the Dehcho Region, Northwest Territories, Canada, including the first report of T. nativa in a grizzly bear from Canada

Samples of muscle from 120 black bears (Ursus americanus), 11 grizzly bears (Ursus arctos), and 27 wolves (Canis lupus) collected in the Dehcho Region of the Northwest Territories from 2001 to 2010 were examined for the presence of Trichinella spp. larvae using a pepsin-HCl digestion assay. Trichinella spp. larvae were found in eight of 11 (73%) grizzly bears, 14 of 27 (52%) wolves, and seven of 120 (5.8%) black bears. The average age of positive grizzly bears, black bears, and wolves was 13.5, 9.9, and approximately 4 yr, respectively. Larvae from 11 wolves, six black bears, and seven grizzly bears were genotyped. Six wolves were infected with T. nativa and five with Trichinella T6, four black bears were infected with T. nativa and two with Trichinella T6, and all seven grizzly bears were infected with Trichinella T6 and one of them had a coinfection with T. nativa. This is the first report of T. nativa in a grizzly bear from Canada. Bears have been linked to trichinellosis outbreaks in humans in Canada, and black bears are a subsistence food source for residents of the Dehcho region. In order to assess food safety risk it is important to monitor the prevalence of Trichinella spp. in both species of bear and their cohabiting mammalian food sources.     

Biological characterization of Trichinella isolates from various host species and geographical regions.

Forty isolates of Trichinella collected from 5 continents were compared for 7 biological characters: newborn larvae produced per female worm cultured in vitro at the seventh, eighth, and ninth day postinfection, host muscle nurse cell development time, reproductive capacity index in rats and chickens, and resistance of muscle larvae to freezing. The isolates also were compared by analyses of an environmental character of the location from which they were isolated: the isotherms for January and July. By factorial analysis of correspondence of the biological and environmental data, the 40 isolates were grouped into 8 gene pools (T1-T8). The environmental temperature-related distribution was more evident for the sylvatic isolates (T2, T3, T5, T6, T7, T8), than for T1, which was isolated from domestic pigs, and for T4, a bird-adapted, nonencapsulating genetic type. The 8 biological groups correlated closely with the 8 gene pools previously identified on the basis of allozyme analysis. These results support the concept that the genus Trichinella is composed of at least 5 distinct gene pools or sibling species: Trichinella spiralis sensu stricto (T1), Trichinella nativa (T2), Trichinella sp. (T3), Trichinella pseudospiralis (T4), and Trichinella nelsoni (T7), and 3 other groups of uncertain taxonomic status (i.e., T5, T6, and T8).     

Biological variation in Trichinella species and genotypes

At present, the genus Trichinella comprises seven species of which five have encapsulated muscle larvae (T. spiralis, T. nativa, T. britovi, T. nelsoni and T. murrelli) and two do not (T. pseudospiralis and T. papuae) plus three genotypes of non-specific status (T6, T8 and T9). The diagnostic characteristics of these species are based on biological, biochemical and genetic criteria. Of biological significance is variation observed among species and isolates in parameters such as infectivity and immunogenicity. Infectivity of Trichinella species or isolates is determined, among other considerations, by the immune status of the host in response to species- or isolate-specific antigens. Common and particular antigens determine the extent of protective responses against homologous or heterologous challenge. The kinetics of isotype, cytokine and inflammatory responses against T. spiralis infections are isolate-dependent. Trichinella spiralis and T. pseudospiralis induce different dose-dependent T-cell polarizations in the early host response, with T. spiralis initially preferentially promoting Th1-type responses before switching to Th2 and T. pseudospiralis driving Th2-type responses from the outset.     

Characterization of a noncyst-forming isolate of Trichinella from a wild boar in Yugoslavia.

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.     

Experimental Trichinella infection in seals

The susceptibility of seals to infection with Trichinella nativa and the cold tolerant characteristics of muscle larvae in seal meat were evaluated. Two grey seals, Halichoerus grypus, were inoculated with 5000 (100 larvae/kg) T. nativa larvae and two grey seals with 50000 (1000 larvae/kg). One seal from each dose group and two control seals were killed at 5 and 10 weeks post-inoculation (p.i.). At 5 weeks p.i., infection was established in both low and high dose seals with mean larval densities of 68 and 472 larvae per gram (lpg), respectively, using eight different muscles for analyses. At 10 weeks p.i., mean larval densities were 531 and 2649 lpg, respectively, suggesting an extended persistence of intestinal worms. In seals with high larval density infections, the distribution of larvae in various muscles was uniform, but in one seal with a low larval density infection, predilection sites of larvae included muscle groups with a relative high blood flow, i.e. diaphragm, intercostal and rear flipper muscles. Trichinella-specific antibody levels, as measured by ELISA, increased during the 10 week experimental period. Infected seal muscle was stored at 5, -5 and -18 degrees C for 1, 4 and 8 weeks. Muscle larvae released from stored seal muscle by artificial digestion were inoculated into mice to assess viability and infectivity. Larvae from seal muscle 10 weeks p.i. tolerated -18 degrees C for 8 weeks but larvae from seal muscle 5 weeks p.i. tolerated only 1 week at -18 degrees C, supporting the hypothesis that freeze tolerance increases with the age of the host-parasite tissue complex. The expressed susceptibility to infection, extended production of larvae, antibody response and freeze tolerance of T. nativa in seals are new findings from the first experimental Trichinella infection in any marine mammal and suggest that pinnipeds (phocids, otariiids or walrus) may acquire Trichinella infection by scavenging even small amounts of infected tissue left by hunters or predators.     

Effects of selenium deficiency on fatty acid metabolism in rats fed fish oil-enriched diets.

The hepatic fatty acid metabolism was investigated in rats stressed by selenium deficiency and enhanced fish oil intake. Changes in the composition of lipids, peroxides, and fatty acids were studied in the liver of rats fed either a Sedeficient (8 microg Se/kg) or a Se-adequate (300 microg Se/kg) diet, both rich in n-3 fatty acid-containing fish oil (100 g/kg diet) and vitamin E (146 mg alpha-tocopherol/kg diet). The two diets were identical except for their Se content. Se deficiency led to a decrease in hair coat density and quality as well as to changes in liver lipids, individual lipid fractions and phospholipid fatty acid composition of the liver. The low Se status did reduce total and reduced glutathione in the liver but did not affect the hepatic malondialdehyde level. In liver phospholipids (PL), Se deficiency significantly reduced levels of palmitic acid [16:0], fatty acids of the n-3 series such as DHA [22:6 n-3], and other long-chain polyunsaturates C-20-C-22, but increased n-6 fatty acids such as linoleic acid (LA) [18:2 n-6]. Thus, the conversion of LA to arachidonic acid was reduced and the ratio of n-6/n-3 fatty acids was increased. As in liver PL, an increase in the n-6/n-3 ratio was also observed in the mucosal total fatty acids of the small intestine. These results suggest that in rats with adequate vitamin E and enhanced fish oil intake, Se deficiency affects the lipid concentration and fatty acid composition in the liver. The changes may be related to the decreased levels of selenoenzymes with antioxidative functions. Possible effects of Se on absorption, storage and desaturation of fatty acids were also discussed.     

Fatty acid specificity of acyl-CoA synthetase in rat glomeruli

The fatty acid specificity of acyl-CoA synthetase in rat glomeruli for physiologically and pathologically important long-chain fatty acids was studied. The apparent Michaelis constants (Km) for substrate fatty acids increased in the order, linolenic less than linoleic less than eicosapentaenoic less than arachidonic less than oleic less than palmitic acid. The maximum velocities with these fatty acids decreased in the order, oleic greater than linoleic greater than palmitic (approximately equal to) linolenic greater than arachidonic greater than eicosapentaenoic acid. The syntheses of radioactive arachidonyl-CoA and palmitoyl-CoA from radioactive arachidonic and palmitic acid, respectively, were both inhibited by all fatty acids mentioned above including the substrate fatty acids, their inhibitory effects being inversely correlated with their apparent Km values. These results suggest that the enzyme in glomeruli has a unique specificity for fatty acids and that there is no arachidonic acid-specific acyl-CoA synthetase in glomeruli. The possible contribution of the glomerular enzyme with this specificity to the abnormal fatty acid levels in diabetic animals is discussed.     

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)     

Trichinella papuae n.sp. (Nematoda), a new non-encapsulated species from domestic and sylvatic swine of Papua New Guinea

Encapsulated and non-encapsulated species of the genus Trichinella are widespread in sylvatic animals in almost all zoogeographical regions. In sylvatic animals from Tasmania (Australian region), only the non-encapsulated species Trichinella pseudospiralis has been reported. Between 1988 and 1998, non-encapsulated larvae of Trichinella were detected in five domestic pigs and six wild boars from a remote area of Papua New Guinea. Morphological, biological, and molecular studies carried out on one strain isolated from a wild boar in 1997 suggest that these parasites belong to a new species, which has been named Trichinella papuae n.sp. This species can be identified by the morphology of muscle larvae, which lack a nurse cell in host muscles, and whose total length is one-third greater than that of the other non-encapsulated species, T. pseudospiralis. Adults of T. papuae do not cross with adults of the other species and genotypes. Muscle larvae of T. papuae are unable to infect birds, whereas those of T. pseudospiralis do. The expansion segment V of the large subunit of the ribosomal DNA differs from that of the other species and genotypes. All of these features allow for the easy identification of T. papuae, even in poorly equipped laboratories. The discovery and identification of a second non-encapsulated species in the Australian region strongly supports the existence of two evolutionary lines in the genus Trichinella, which differ in terms of the capacity of larvae to induce a modification of the muscle cell into a nurse cell.     

Experimental trichinellosis in reindeer

Six female reindeer calves were inoculated intraruminally with various doses of Trichinella muscle larvae. Four calves were inoculated with T. nativa, receiving 15,000 (n = 1), 5,000 (1), and 2,500 (2) larvae each. Two calves were inoculated with 5,000 T. spiralis larvae each. Blood samples were collected twice per week for total white blood cell (WBC) and differential counts and for serology using enzyme-linked immunosorbent assay (ELISA) based on T. spiralis excretory-secretory antigen. On day 56, the calves were slaughtered and muscle samples were examined according to the standard digestion method for Trichinella larvae. Blood samples were also collected twice a week from 4 uninoculated, but otherwise similar, reindeer calves corralled separately. Both the total WBC and eosinophil counts of the inoculated animals were, on average, higher during the experimental period. All the inoculated calves seroconverted, showing an increase in the optical density (OD) in the ELISA starting between day 23 and day 27 postinoculation. Very few muscle larvae (<0.08 larvae/g [lpg]) were to be found from the animals inoculated with T. nativa, but about 4 and 6 lpg were recovered from the masseter muscles of those inoculated with T. spiralis.     

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 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.     

Effects of cold adaptation and re-adaptation upon the mitochondrial phospholipids of brown adipose tissue.

1. A study of the mitochondrial phospholipids, phospholipid fatty acid patterns and enzyme activities was investigated in brown tissue (B.A.T.) from rats chronically exposed to cold and/or treated with thyroxine. 2. The total activities of the oxidative enzymes were increased after cold exposure, but not after thyroxine treatment. 3. Cold exposure increased the amount of phosphatidylethanolamine, phosphatidylcholine, cardiolipin and lysophospholipids, the effect being greatest for phosphatidylethanolamine. At the same time, there were marked alterations in the fatty acid composition of the mitochondrial phospholipids (decrease of palmitic, palmitoleic and oleic acids ; increase of stearic, linoleic and arachidonic acids). 4. All these cold-induced alterations were reversed by re-adaptation of the animal to a normal temperature range. 5. The alterations of the fatty acid composition of phospholipids could be explained by changes in the rate of individual fatty acid biosynthesis.