The epidemiological investigation of Trichinella infection in brown rats (Rattus norvegicus) and domestic pigs in Croatia suggests that rats are not a reservoir at the farm level

Whether the brown rat (Rattus norvegicus) is a reservoir of Trichinella spp. infection or merely an accidental host, which may be vector of Trichinella spp., continues to be debated. We estimated the prevalence of Trichinella sp. infection in brown rat populations and in domestic pigs in 2 villages in Croatia, where Trichinella sp. infection in pigs has been endemic in the past 10 yr. Trichinella spiralis larvae, identified by a multiplex polymerase chain reaction analyses, were the only species detected in both rats and pigs. In 2001 and 2002, 2,287 rats were collected on 60 farms with different levels of sanitation and with, or without, T. spiralis-infected pigs. The prevalence of infection in rats ranged from 0.2 to 10.7%. Infected rats were detected only on farms with T. spiralis-positive pigs and low sanitation or formerly with low sanitation (P = 0.007, Fisher's exact test), yet no infected rat was detected on farms with T. spiralis-negative pigs. The finding that no infected rat was found on farms with T. spiralis-negative pigs suggests that, in the investigated area, the brown rat is not a reservoir but only a victim of improper pig slaughtering.     

Trichinella spiralis in an agricultural ecosystem: transmission in the rat population

Four hundred forty-three Norway rats (Rattus norvegicus) were examined to determine their role in the transmission and maintenance of Trichinella spiralis on a pig farm. Rats, classified by sex and weight, were examined for trichinellosis by peptic digestion of muscle samples. Over a 25-mo period, 188 (42.4%) rats were found to be infected with T. spiralis. The mean intensity of infection was 293.2 larvae per gram (LPG) of muscle; 65 (34.6%) infected rats had intensities of infection greater than 100 LPG. Even in the absence of a known source of infected meat (garbage containing meat scraps or dead animals), the rat population maintained the infection, probably through cannibalism. Population reduction was an effective method for reducing the prevalence of infection within the rat population. Therefore, to reduce the likelihood of transmission of T. spiralis between rats and swine, it is essential that rat populations in a farmyard environment be controlled.     

Spatial variation of Trichinella prevalence in rats in Finnish waste disposal sites

Trichinellosis is 1 of the most widespread parasitic zoonoses in the world and can be lethal to humans. Trichinella spp. are also parasites of considerable economic importance. Because rats may play a role in the transmission of trichinellosis to swine and farmed wild boar, 767 brown rats (Rattus norvegicus Berkenhout) from 13 Finnish waste disposal sites were examined for Trichinella spp. by a HCl-pepsin digestion method. Trichinella spp. were found to be a common parasite in trapped rats (overall prevalence, 19%) detected in 12 of 13 dumps. Significant differences were observed between sites in the prevalence (0-49%) of Trichinella spp. Female rats were more often and more heavily infected than males, but age was not shown to be a risk factor for trichinellosis. In addition, positive correlation was demonstrated between rat population density and prevalence. Trichinella spiralis was identified by multiplex polymerase chain reaction in 28 rats. The median density of infection was 42 (range, 0.5-6,925) larvae/ g of host tissue, but neither the occurrence nor the density of the parasite was related to the physical condition of the animal.     

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.     

Suppression of multiple sclerosis in the rat during infection with Trichinella pseudospiralis

Injection of the rat with guinea pig myelin basic protein (MBP) induces an inflammatory demyelination that leads to development of a condition mimicking human multiple sclerosis (MS), including severe depressions in mobility, coordination, and strength in the affected animal. This model was used to observe and compare the antiinflammatory effects of the intestinal and late migratory phases of infection with Trichinella pseudospiralis on development of MBP-induced, MS-like debilitation in rats. Animal performance was measured in an activity monitor and in a series of physical tests designed to assess animal coordination and strength. Uninfected animals injected with MBP showed declines in mobility, coordination, and strength typical for this model. These changes were similar in rats infected so that the intestinal phase of infection coincided with the peak of MBP-induced debilitation. Rats infected so that the late migratory phase of infection occurred during the period of peak MBP-induced debilitation showed significantly higher performance scores in mobility, coordination and strength compared to the latter 2 groups. These finding demonstrate the potency of the anti-inflammatory effects of elevations in host corticosteroids seen during the migratory phase of infection with T. pseudospiralis.     

A longitudinal study of porcine serological responses to experimental infections with T-1 and T-3 Spanish Trichinella isolates.

Comparison of antibody response and antigen recognition was made by ELISA and western-blot analysis in pig experimental infections by T-1 and T-3 Spanish Trichinella isolates. Two groups of Iberian pigs were experimentally infected with 150 larvae/kg body weight of GM-1 and C-76 Spanish Trichinella isolates as representatives of T-1 and T-3 gene pools respectively. Antibody levels and antigen recognition were measured on days -14, 0, 6, 16, 20, 27, 34, 49, 63 and 82 after infection by ELISA and western-blotting assays. Antibody response against C-76 infection was significantly delayed and lower than against GM-1. The two Trichinella isolates were indistinguishable, however, by western blotting analysis, although recognition of larval antigens was quantitatively higher than adult ones. Interestingly, the principle larval antigenic components recognized by pigs were those recognized by the monoclonal anti-sera NIM-M1. Finally, there were no serological patterns indicative of the stage of infection ("antibody windows") discriminating, for example between early versus late infections.     

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.     

Epidemiological survey of Trichinella infection in domestic, synanthropic and sylvatic animals from Argentina

The presence of Trichinella larvae was investigated in 247 samples taken from domestic, synanthropic and sylvatic animals, collected during 1996 to 2005 in 12 endemic provinces of Trichinella infection in Argentina. Muscle larvae of Trichinella from 65 infected animals were identified at the species level by single larva nested polymerase chain reaction (PCR) technique based on the variability within the expansion segment V (ESV) region of the ribosomal DNA. Trichinella infections were found in 97 of 164 pigs, 38 of 56 pork products, two domestic dogs, one domestic cat, 7 of 11 armadillos and 3 of 9 synanthropic rats. All Trichinella isolates were identified as Trichinella spiralis by nested PCR. These findings add new data on the epidemiology of trichinellosis and should be considered when implementing new strategies to control this zoonosis.     

Trichinella nativa in grey seal Halichoerus grypus: spill-over from a highly endemic terrestrial ecosystem

Trichinella spp. infections of marine mammals pose a human health risk in Arctic regions where game meat is an important part of the diet. In the last decade, the grey seal population of the sub-arctic Baltic Sea has reached a level at which hunting is possible and seal meat is now available for food. We conducted a survey on the occurrence of Trichinella spp. in grey seals Halichoerus grypus (n = 171) and ringed seals Phoca hispida botnica (n = 56) of the Baltic Sea in the coastal waters of Finland, a highly Trichinella sp. endemic area. Muscle samples were examined by a mechanically assisted digestion method during 2006-2010. One grey seal was positive for Trichinella nativa , while all samples from ringed seals were negative. Even though just 1 grey seal was infected, the finding here emphasizes the importance of proper meat inspection of seals intended for human consumption, especially in areas with high infection pressure.     

Conservation of diagnostic antigen epitopes among biologically diverse isolates of Trichinella spiralis

Crude and immunoaffinity-purified excretory-secretory antigens derived from a domestic pig isolate of Trichinella spiralis were used in an enzyme-linked immunosorbent assay to test serum from mice infected with 25 different pig and wild animal isolates of T. spiralis sspp. All of the sera were found positive by ELISA using either of the antigen preparations, indicating all isolates shared certain antigen epitopes. Excretory-secretory antigens were prepared from 3 distinct isolates of T. spiralis sspp.--Trichinella spiralis spiralis (pig isolate), Trichinella spiralis nativa (polar bear isolate), and Trichinella spiralis pseudospiralis--and compared by electrophoresis and monoclonal antibody binding. While protein profiles varied among the isolates, a monoclonal antibody recognizing a major immunodiagnostic antigen epitope bound all 3 antigen preparations. However, this antigen epitope occurred on different molecular weight excretory-secretory proteins from the different isolates.     

Molecular identification of a Trichinella isolate from a naturally infected pig in Tibet, China

The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial large-subunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.     

Importance of the wild boar in the epidemiology of wild trichinellosis in Piedmont and Liguria

Muscle samples from 1,508 wild boars (Sus scrofa) harvested in Piedmont and Liguria between 1987 and 1990 were examined for larvae of Trichinella sp. by trichinelloscopy and peptic digestion. No wild boar was infected, though trichinellosis was present in foxes (Vulpes vulpes) from the same areas (14 positives out of 608) with the highest prevalence in the mountain range (25.0%). Infection was also not found in 40 stone martens, 5 badgers and 3 stoats. Five vulpine isolates were identified as belonging to T3 zymodeme. These results show that free-ranging wild boars, although being a source of trichinellosis infection for man in Italy (9.2% of the 584 cases diagnosed since 1961), have no significance in maintaining the sylvatic cycle of this parasite in the study area. The data agree with the low infectivity of Italian strains of fox origin for swine seen in the experimental infections carried out so far.     

Helminth parasitism in martens (Martes americana) and ermines (Mustela erminea) from Washington, with comments on the distribution of Trichinella spiralis

Helminths are reported for the first time from ermines (Mustela erminea) and martens (Martes americana) in Washington (USA). Among 22 adult ermines, 41% were infected by one or more of five species (Taenia mustelae, Alaria mustelae, Molineus patens, M. mustelae and Trichinella spiralis). Among 78 adult martens from three geographic localities, the prevalence was 83%. Nine species were identified (Mesocestoides sp., T. mustelae and T. martis americana, Euryhelmis squamula, M. patens, Baylisascaris devosi, Physaloptera sp., Soboliphyme baturini and T. spiralis). Trichinella spiralis occurred with a maximum prevalence of 50% in martens, but only occurred in 9% of ermines. Compression and digestion techniques provided a similar estimate of prevalence of T. spiralis, yet neither was entirely accurate in identifying all infected hosts. The species richness of the helminth community of martens in Washington was greater than that reported from other regions of North America.     

Application of Giemsa stain for easy detection of Trichinella spiralis muscle larvae

The application of Giemsa technique to stain compressed diaphragm samples obtained from rodents experimentally infected with Trichinella spiralis is described. Diaphragm samples from rats heavily infected with 20 muscle larvae per gram of body weight (20 ML/gbw) were cut into several pieces and stained with Giemsa; on the other hand, whole diaphragms from slightly infected mice (1 ML/gbw) were also stained with Giemsa. Besides, muscle samples were also stained with Giemsa. Observation at 10 x magnification revealed that both ML and nurse cells (NC) look as bluish structures clearly contrasting with the pinkish color of the non-infected muscle fibers. NC in the diaphragms of mice could be easily observed at naked eye as blue points contrasting with the pink surrounding areas formed by the non-infected muscle fibers. Among NC observed in the diaphragms of rats infected with 20 ML/gbw, 4.4% was multiple infection. These findings were confirmed in sectioned and hematoxylin-eosin stained specimens. This data could be usefulness for a rapid diagnosis of trichinellosis in post-mortem mammals without magnification procedures.     

Trichinella spiralis: modulation of experimental autoimmune encephalomyelitis in DA rats

Helminth infection has a potent systemic immunomodulatory effect on the host immune response, which also affects the development of autoimmune diseases. We investigated the dose-dependent influence of Trichinella spiralis infection on experimental autoimmune encephalomyelitis (EAE). Our model of concomitant T. spiralis infection and EAE demonstrates that established infection of Dark Agouti (DA) rats with the parasite causes amelioration of the clinical course of induced EAE in a dose-dependent way. Infection with T. spiralis L1 stage muscle larvae (TSL1) reduced the severity of the autoimmune disease as judged by lower maximal clinical score, cumulative index, duration of illness and degree of mononuclear cell infiltration in T. spiralis infected animals compared to control, EAE-induced group. This study provides a valuable model of worm infection to investigate helminth-induced regulatory mechanisms for optimal benefit to the host.     

Persistence of Trichinella spiralisin Rat Carcasses Experimentally Mixed in Different Feed

Trichinella spiralis infected rat carcasses were incubated for 6 weeks in several animal feeds to assess how long Trichinella can present a risk for an outbreak in contaminated feeds. In groups of 6, 24 infected target rats were placed in silage, grained barley, propionic acid-preserved feed, and also into simulated pasture conditions. Test environments were sampled after one-, 2-, 4-, and 6-week-incubations. Trichinella larvae were recovered by digestion, and their infectivity was evaluated in rats. A two-week incubation reduced the number of recovered larvae, but still after 6 weeks low numbers were isolated from all feeds except from the experimental group simulating pasture conditions. After 2 weeks storage, the larvae were infective in all storage environments. However, up to 4 weeks, they survived only in the propionic acid-fermented feed and there in small numbers with reduced reproductive capability. This indicates the possibility of farm animals to get infection from rats or other infected material being hazardously mixed with hay or other feed. If silage is stored for at least one month before use, however, the risk from this forage appears to be minimized.     

Trichinella spiralis: antifecundity and antinewborn larvae immunity in swine

The development of antifecundity and antinewborn larvae immunity in swine infected with Trichinella spiralis was investigated. In primary infections, adult female worm fecundity dropped sharply after 3 weeks, although adults could be recovered from the small intestine for at least 7 weeks after infection. In challenge infections of pigs infected previously, adult female worm fecundity was depressed up to 51% and the adults were expelled within 3 weeks. Since immune pigs are almost completely resistant to the secondary establishment of muscle larvae, this suggested the existence of immune effector mechanisms also acting on the newborn larvae. This was supported by observations, using an indirect fluorescent antibody assay, that pig antibody bound to the surface of the newborn larvae. Passive transfer of immune pig serum resulted in a large reduction in muscle larvae burden in both infected pig and rat recipients. Adult female worm fecundity in such immune serum recipients was reduced only by 20% and worm survival in the intestine was unaffected. These results indicate that immunity to the newborn larvae, in addition to antifecundity effects, are responsible for the high levels of acquired resistance to T. spiralis in swine.     

Trichinella britovi etiological agent of sylvatic trichinellosis in the Republic of Guinea (West Africa) and a re-evaluation of geographical distribution for encapsulated species in Africa

In West Africa, Trichinella infection was documented in humans and animals from Senegal in the 1960s, and the biological characters of one isolate showed a lower infectivity to domestic pigs and rodents when compared with that of a Trichinella spiralis pig isolate from Europe. To identify the Trichinella species present in West Africa, a survey was conducted in a total of 160 wild animals in the Republic of Guinea. Three Viverridae, one true civet (Viverra civetta) and two African palm civets (Nandinia binotata) from the Fouta Djallon Massif, Pilimini Subprefecture, were found positive by artificial digestion of muscle samples. Trichinella larvae from these three viverrids were identified as Trichinella britovi and no difference was detected in three examined sequences from these African isolates and the reference strain of T. britovi from Europe, indicating common ancestry, an historically continuous geographic distribution, and recent isolation for African and European populations. The detection of T. britovi in West Africa modifies our knowledge about the distribution of encapsulated species of Trichinella in Africa. Thus, Trichinella nelsoni is now considered to have a distribution limited to the Eastern part of the Afrotropical region from Kenya to South Africa. This provides a plausible explanation for the presence of Trichinella T8 in Namibia and South Africa, and further suggests that T. britovi could be the Trichinella species circulating among wild animals of Northern Africa.     

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)     

Inhibition of leukocyte function by serum from patients with trichinellosis

Modification of leukocytic function has been reported in only a few human parasitic diseases. In this study we evaluated the effects of the sera from patients infected with Trichinella on chemotactic and phagocytic responses in leukocytes. Leukocyte chemotaxis was tested by the agarose method and phagocytosis by the technique of Yamamura, modified for Saccharomyces cerevisiae. Sera were acquired from patients during a trichinellosis outbreak that occurred in northern Italy in 1986. The parasite was isolated from 1 patient and isoenzymatically typed as Trichinella sp. 3, a new taxon, previously considered Trichinella nelsoni. The results indicated that sera from Trichinella-infected humans inhibited both chemotaxis and phagocytic responses in leukocytes. These findings suggest the existence of serum factor(s) in trichinellosis patients that modify host leukocytic functions. The source and nature of active serum components and the mechanism by which they modulate leukocyte function remain to be clarified.