Evaluation of the infectivity of Trichinella papuae and Trichinella zimbabwensis for equatorial freshwater fishes

The discovery of Trichinella species infecting poikilotherm vertebrates has opened new possibilities in the epidemiology of this parasite group. The aim of the present work was to investigate the infectivity of the two non-encapsulated species of Trichinella infecting both mammals and reptiles, Trichinella papuae and Trichinella zimbabwensis, for equatorial freshwater carnivore fishes. To this end, two species of piranhas, four Serrasalmus nattereri and four Serrasalmus rhombeus, were each inoculated per os with the two species of Trichinella larvae. Six days post infection (p.i.), one fish of each species inoculated with one of the two species of Trichinella was sacrificed. The intestines and celomatic cavities were searched for worms using dissection microscopy, and the presence of muscle larvae was evaluated by artificial digestion. The other 4 inoculated fish were sacrificed 60 days p.i. and similarly searched for the presence of worms. No larva or adult worms were detected in any organ or tissue at 6 or 60 days p.i. The lack of infectivity of T. papuae and T. zimbabwensis for fish suggests that the entozoic habitat of this animal does not represent a suitable environment for these two Trichinella species. More importantly, these data indicate that freshwater fishes, one of the food resources for crocodiles, caimans and alligators, are unlikely to play a role in the epidemiology of the known species of the genus Trichinella.     

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region.     

Survival of sylvatic Trichinella spiralis isolates in frozen tissue and processed meat products

The ability of Trichinella spiralis larvae to survive at subfreezing temperatures encysted in the musculature of wild carnivorous mammals was assessed by evaluating motility and infectivity (to rodents) of trichinae at various intervals after storage in frozen skeletal muscle. Fifty to 60% of the larvae in grizzly bear meat were alive after storage for 27 months at -6.5 to -20 C, and 30% to 50% were still alive at 34 months. However, none survived for 38 months, on the basis of infectivity in mice and larval motility. Trichinella larvae survived up to 4 months in frozen (-6.5 to -20 C) wolverine tissue. Viable larvae were not recovered from mountain lion or fisher muscle frozen for 1 month. The effect of postslaughter processing on Trichinella larvae encysted in bear meat was evaluated by use of a similar bioassay procedure. Viability of larvae recovered from black bear meat that had been processed into ham or jerky was not affected by dry curing with a commercial salt mixture. Trichinae from both preparations induced infections in mice (58 to 90 larvae/g of tissue). However, a combination of curing and smoking was consistently lethal to encysted larvae. Viable trichinae were not recovered from ground bear meat preparations (pepperoni, salami, or sausage) processed according to commercial standards.     

Differential detection of Trichinella papuae, T. spiralis and T. pseudospiralis by real-time fluorescence resonance energy transfer PCR and melting curve analysis

Trichinellosis caused by nematodes of Trichinella spp. is a zoonotic foodborne disease. Three Trichinella species of the parasite including Trichinella spiralis, Trichinella papuae and Trichinella pseudospiralis, have been etiologic agents of human trichinellosis in Thailand. Definite diagnosis of this helminthiasis is based on a finding of the Trichinella larva (e) in a muscle biopsy. The parasite species or genotype can be determined using molecular methods, e.g., polymerase chain reaction (PCR). This study has utilized real-time fluorescence resonance energy transfer PCR (real-time FRET PCR) and a melting curve analysis for the differential diagnosis of trichinellosis. Three common Trichinella species in Thailand were studied using one set of primers and fluorophore-labeled hybridization probes specific for the small subunit of the mitochondrial ribosomal RNA gene. Using fewer than 35 cycles as the cut-off for positivity and using different melting temperatures (T(m)), this assay detected T. spiralis, T. papuae and T. pseudospiralis in muscle tissue and found the mean T(m) ± SD values to be 51.79 ± 0.06, 66.09 ± 0.46 and 51.46 ± 0.09, respectively. The analytical sensitivity of the technique enabled the detection of a single Trichinella larva of each species, and the detection limit for the target DNA sequence was 16 copies of positive control plasmid. A test of the technique's analytical specificity showed no fluorescence signal for a panel of 19 non-Trichinella parasites or for human and mouse genomic DNA. Due to the sensitivity and specificity of the detection of these Trichinella species, as well as the fast and high-throughput nature of these tools, this method has application potential in differentiating non-encapsulated larvae of T. papuae from T. spiralis and T. pseudospiralis in tissues of infected humans and animals.     

Inappropriate feeding practice favors the transmission of Trichinella papuae from wild pigs to saltwater crocodiles in Papua New Guinea

The recent discovery of Trichinella zimbabwensis in farmed crocodiles (Crocodilus niloticus) of Zimbabwe and its ability to infect mammals, and the development of both T. zimbabwensis and Trichinella papuae in experimentally infected reptiles led to an investigation of Trichinella infection in saltwater crocodiles (Crocodylus porosus) and in wild pigs (Sus scrofa) of Papua New Guinea, to see if T. papuae also, is present in both cold- and warm-blooded animals. Of 222 crocodiles examined, 47 animals (21.2%), all from Kikori, Gulf Province, were positive for non-encapsulated larvae in the muscles. The greatest number of larvae was found usually in the biceps, with an average of 7 larvae/g. One isolate from a crocodile infected successfully both laboratory rats and mice. Of 81 wild pigs examined, 9 from Bensbach river area (Western Province) and 1 from Kikori area (Gulf Province) were positive for non-encapsulated larvae in the muscles. Trichinella larvae from both saltwater crocodiles and wild pigs have been identified by multiplex-PCR analysis as T. papuae. The sequence analysis of the region within the large subunit ribosomal DNA, known as the expansion segment V, has shown the presence of a molecular marker distinguishing T. papuae isolates of Bensbach river area from those of Kikori area. This marker could be useful to trace back the geographical origin of the infected animal. The epidemiological investigation carried out in the Kikori area has shown that local people catch young crocodiles in the wild and keep them in holding pens for several months, before sending them to the crocodile farm in Lae (Morobe Province). They feed the crocodiles primarily with wild pig meat bought at the local market and also with fish. These results stress the importance of using artificial digestion for routinely screening of swine and crocodiles, and of adopting measures for preventing the spread of infection, such as the proper disposal of carcasses and the adequate freezing of meat.     

First report of a Trichinella papuae infection in a wild pig (Sus scrofa) from an Australian island in the Torres Strait region

Multiple Trichinella species are reported from the Australasian region although mainland Australia has never confirmed an indigenous case of Trichinella infection in humans or animals. Wildlife surveys in high-risk regions are essential to truly determine the presence or absence of Trichinella, but in mainland Australia are largely lacking. In this study, a survey was conducted in wild pigs from mainland Australia's Cape York Peninsula and Torres Strait region for the presence of Trichinella, given the proximity of a Trichinella papuae reservoir in nearby PNG. We report the detection of a Trichinella infection in a pig from an Australian island in the Torres Strait, a narrow waterway that separates the islands of New Guinea and continental Australia. The larvae were characterised as T. papuae (Kikori strain) by PCR and sequence analysis. No Trichinella parasites were found in any pigs from the Cape York Peninsula. These results highlight the link the Torres Strait may play in providing a passage for introduction of Trichinella parasites from the Australasian region to the Australian mainland.     

Persistence of Trichinella spiralis in 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.     

Impeded establishment of the infective stage of Trichinella in the intestinal mucosa of mice by passive transfer of an IgA monoclonal antibody

Our previous study showed that the IgA monoclonal antibody (mAb) HUSM-Tb1 forms immunoprecipitates on the cuticular surface of infective larvae of Trichinella britovi, and that intraperitoneal injection of this mAb to mice 5 hr before challenge infection confers a high level of protection against intestinal T. britovi. The same treatment produced a similar effect in BALB/c mice inoculated orally with Trichinella pseudospiralis larvae, indicating that the effects may be seen upon most members of the genus Trichinella. Worms recovered from the intestinal mucosa at 1 hr after challenge infection with T. pseudospiralis was few in mice passively immunized with the mAb, whereas a substantial number of worms were recovered from the mucosa of control groups. These results suggest that the IgA mAb impedes establishment of infective Trichinella worms in the intestinal mucosa. Trichinella worms inoculated orally into BALB/c mice vaccinated with ultraviolet-irradiated muscle larvae 3 weeks earlier were expelled between days 4 and 7 after challenge infection. Although the mAb HUSM-Tb1 originated from the mesenteric lymph node cells of mice vaccinated repeatedly with such irradiated larvae, IgA-mediated expulsion does not seem to play an important role in this vaccination model.     

Viability and infectivity of Trichinella spiralis muscle larvae in frozen horse tissue

Many aspects of the biology and epidemiology of Trichinella infection in the horse are poorly understood, including survival of Trichinella spp in horse muscle. In this study, we have assessed the freeze tolerance of T. spiralis in horse meat stored at 5, -5, and -18 degrees C for 1 day to 24 weeks. Results demonstrate a steady reduction in the number of live ML recovered from the cold stored meat samples. On Day 1, recovery of live larvae had been reduced by 18.6%, 50.1%, and 37.2%, and by 4 weeks, recovery of larvae had been reduced by 65.4%, 66.5%, and 96.2% in samples stored at 5, -5, and -18 degrees C, respectively. Infectivity results (measured as reproductive capacity index (RCI)) from mice inoculated with larvae recovered from non-frozen meat samples at day 0 was 23.5. Following storage at -18 degrees C for one and two days, the RCIs were 2.09 and 0.99, respectively. Small numbers of infective larvae were still present in meat samples stored at -18 degrees C for 4 weeks. The RCI of ML recovered from meat samples stored at -5 degrees C was 14.99 and 6.36 at 2 weeks and 4 weeks respectively; the RCI of samples stored at 5 degrees C was 23.1 at 8 weeks, and fell rapidly thereafter (12 week RCI 1.33; 0 at 24 weeks). These data demonstrate that infective T. spiralis, a non-freeze tolerant species, can survive for at least 4 weeks in horse tissue frozen at -5 or -18 degrees C, and that the numbers of infective larvae decrease substantially by day 2 at -18 degrees C and by week 4 at -5 degrees C.     

Experimental trichinosis in sheep.

Trichinella spiralis spiralis infections were established in sheep by administering infective larvae via gavage or feeding infected musculature. Trichinella spiralis nativa infective larvae had a low infectivity for sheep although light infections may be established in some animals with large infective doses. For the most part, sheep were averse to ingesting musculature mixed in a grain ration unless it was camouflaged with molasses. The heaviest infections usually occurred in the masseter muscle. The fact that sheep are averse to ingesting muscle tissue may reduce the likelihood of trichinosis. Anti-Trichinella antibodies to both T: spiralis spiralis and T. spiralis nativa were produced as demonstrated by the enzyme-linked immunosorbent assay. Seroconversion occurred in several sheep challenged with T. spiralis nativa even though larvae were not recovered from the musculature by pepsin-digestion.     

Infectivity, reproductive capacity and distribution of Trichinella spiralis and T. pseudospiralis larvae in experimentally infected sheep

Twelve Merino sheep were experimentally shown to be susceptible to infection with Trichinella spiralis or T. pseudospiralis by feeding on infected carcasses of mice or by oral intubation with recovered muscle larvae. The larvae recovered from the sheep showed variable tissue distribution. The diaphragm and tongue were most affected. The viability of the recovered larvae was confirmed by successful passage in mice. The reproductive capacity of T. spiralis in sheep was higher than that of T. pseudospiralis, and also higher than its reproductive capacity in C57BL/6J mice. The reproductive capacity of T. pseudospiralis in sheep at a lower dose was higher than that observed in mice. However at higher doses, it was significantly lower than that in mice. Therefore, it may be concluded that the sheep may be considered a suitable host for both species of Trichinella.     

旋毛虫肌幼虫期胞外囊泡对小鼠免疫保护作用

本试验通过差速超速离心法获得旋毛虫肌幼虫期胞外囊泡(Trichinella spiralis muscle larvae extracellular vesicles,Ts-ML-EVs),经透射电镜观察、纳米颗粒追踪分析、流式细胞术和Western blot鉴定。选择6～8周龄的健康雌性BALB/c小鼠,随机分为4组,每组8只。试验设计为PBS对照组(PBS组)、佐剂对照组(PBS+佐剂组)、旋毛虫肌幼虫期排泄分泌产物免疫组(Ts-ML-ES+佐剂组)以及旋毛虫肌幼虫期胞外囊泡免疫组(Ts-ML-EVs+佐剂组),分别取相应抗原与佐剂等体积混合采用多点皮下注射方式免疫小鼠。首免后第2,4周各加强免疫1次,剂量不变。三免后2周,每只小鼠灌胃300条旋毛虫肌幼虫,灌胃后6 d每组取2只剖杀,统计旋毛虫成虫减虫结果;攻虫35 d后所有小鼠全部处死,计算肌幼虫减虫率。每次免疫前、攻虫前以及处死前眼眶采血收集血清并保存,统一进行ELISA分析。结果显示：成功获得Ts-ML-EVs,随着免疫次数增加,小鼠血清中特异性IgG、IgG1、IgG2a、IgA和IgE抗体水平均显著上升,在三免后2周达到...     

Larval viability and serological response in horses with long-term Trichinella spiralis infection

The horse is considered an aberrant host for the nematode parasite Trichinella spiralis, and many aspects of the biology and epidemiology of Trichinella infection in the horse are poorly understood. It has been reported that experimentally-infected horses produce a transient serological response to infection and that muscle larvae are cleared more rapidly than in parasite-adapted hosts such as the pig and humans. However, limited numbers of animals have been studied, and both the longevity of larvae in horse musculature and the immune response to Trichinella larvae remain unclear. In this study, we infected 35 horses with 1000, 5000, or 10,000 T. spiralis muscle larvae and followed the course of infection for 1 year, assessing larval burdens in selected muscles, the condition and infectivity of recovered larvae, and the serological response of infected horses. The results demonstrated that T. spiralis establishes infection in horses in a dose dependent manner. Anti-Trichinella IgG antibodies peaked between weeks 6-10 post-inoculation. Viable, infective larvae persisted in horse musculature for the duration of the study (12 months), and exhibited no apparent reduction in muscle burdens over this period. Encapsulated larvae showed no obvious signs of degeneration in histological sections. Larval capsules were surrounded by infiltrates consisting of mature plasma cells and eosinophils. Macrophages were notably absent. Given the lack of a detectable serological response by 26 weeks p.i. and the persistence of infective muscle larvae for at least 1 year, parasite recovery methods are currently the only suitable detection assays for both meat inspection and epidemiological studies of Trichinella infection in the horse.     

Peripheral blood mononuclear cell subsets during Trichinella spiralis infection in pigs

The immune response of 'Yugoslav meat breed' pigs inoculated with low doses of Trichinella spiralis muscle larvae was followed over two to nine weeks of primary infection, by analysing changes in peripheral blood mononuclear cell subsets, the development of a humoral antibody response and muscle larvae burden. During the course of the infection, infected animals showed a persistent elevation of both CD4+ and CD8+ T cell subsets from days 15 to 60 after the parasite exposure. During this time, the number of peripheral blood mononuclear cells expressing major histocompatibility complex class II antigens was also increased, while no significant differences were found in the number of circulating monocytes/macrophages and B cells over time. Humoral antibody responses to muscle larvae excretory-secretory products were evident as early as 41 days after infection, while the muscle larvae were recovered as early as 27 days after infection. The increased levels of CD4+ and CD8+ T cell subsets, as well as cells expressing major histocompatibility complex class II antigens in pigs exposed to T spiralis, may be indicative of some considerable alterations in cell subsets that are involved in the regulation of the swine immune response to this parasite.     

Factors affecting the flow among domestic, synanthropic and sylvatic cycles of Trichinella

Nematodes of the genus Trichinella are maintained in nature by sylvatic or domestic cycles. The sylvatic cycle is widespread on all continents, from frigid to torrid zones, and it is maintained by cannibalism and scavenging behavior of carnivores. Trichinella is primarily a parasite of carnivorous mammals, although one non-encapsulated species, Trichinella pseudospiralis, has also been detected in birds. The anaerobic metabolism of larvae in nurse cells allows their survival in extremely decayed meat. Encapsulated larvae in the decomposing carcass function similarly to the species-dispersing population of eggs or larvae of other nematodes, suggesting that the natural cycle of Trichinella includes a free-living stage when the parasite is no longer protected by the homeothermy of the host. Consequently, environmental temperature and humidity play an important role in the transmission of Trichinella among wildlife. Of the 10 recognized genotypes of Trichinella, only Trichinella spiralis is transmitted and maintained in a domestic cycle, although it can be present also in wildlife. All other genotypes (Trichinella nativa, Trichinella britovi, T. pseudospiralis, Trichinella murrelli, Trichinella nelsoni and Trichinella papuae, Trichinella T6, T8, and T9) are transmitted and maintained only in a sylvatic cycle. This generalization does not preclude sylvatic species of Trichinella from invading the domestic habitat, and T. spiralis may return to this habitat when humans fail in the management of wildlife and domestic animals. However, the presence of sylvatic genotypes of Trichinella in the domestic habitat represents a "dead-end" for the sylvatic cycle. Synanthropic animals (rats, foxes, mustelids, cats, dogs, etc.) contribute to the flow of sylvatic Trichinella genotypes from wildlife to domestic animals and of T. spiralis from domestic to sylvatic animals. Furthermore, human behavior not only influences the transmission patterns of Trichinella genotypes in the domestic habitat, but also it can contribute to the transmission and spread of this infection among wildlife, for example by improper hunting practices.     

Trichinellosis in Papua New Guinea

OBJECTIVES: To describe the discovery in a domestic pig of the first case of trichinellosis in Papua New Guinea, caused by a new taxon within the genus Trichinella (T papuae). Also, to establish if the disease occurred in the local wild pig population and in domestic pigs elsewhere in the country, and to test if the worm was infective to some other animals. PROCEDURE: Fresh and fixed tissue samples were examined by the digestion method and histologically, respectively, for the non-encapsulated larvae of T papuae. Feeding trials were conducted, using infected tissues and infective larvae, on animals under laboratory conditions. RESULTS: About 8.8% of a wild pig population in Western Province, adjacent to Irian Jaya, Indonesia, was found to be infected. Infection was not found in other local and feral animals or in domestic pigs from other parts of the country. Infection was experimentally established in cats, pigs and laboratory bred mice and rats. CONCLUSION: Trichinellosis is confined to one remote locality in PNG. Domestic pigs in the initial case became infected, probably, by eating infected wild pig meat.     

Inoculation of pigs against Trichinella spiralis, using larval excretory-secretory antigens

Pigs were inoculated with Trichinella spiralis excretory-secretory products derived from short-term in vitro maintenance of infective muscle larvae. Intraperitoneal administration of excretory-secretory products in Freund's complete adjuvant or aluminum hydroxide induced moderate, but variable, degrees of immunity to challenge exposure in a nondose-dependent manner; IM administration of products in Freund's incomplete adjuvant was less successful. Inoculated pigs harbored fewer adult worms, and the fecundity of female worms (numbers of newborn larvae shed in vitro) recovered after challenge exposure was significantly lower (alpha = 0.05) than the fecundity of females recovered from control pigs. The degree of resistance in inoculated pigs was directly related to serotiter against excretory-secretory antigens, as determined in an enzyme-linked immunosorbent assay.     

猪、犬旋毛虫新生蚴免疫原性试验

将猪、犬旋毛虫新生蚴制备成免疫原,在小鼠体内进行免疫试验,免疫１周后攻击感染肌幼虫,３５日蝗检查肌幼虫的减虫率。结果表明,猪旋毛虫新生蚴的同源免疫和其对犬旋毛虫的交叉免疫减虫率分别为８３．１１％和７９．３９％,犬旋毛虫新生蚴的为８１．３７％和８０．６９％。结果揭示,旋毛虫新生蚴具有良好的免疫原性,同源免疫的减虫率比交叉免疫的高,猪旋毛虫新生蚴抗原对犬旋毛虫的交叉免疫与犬旋毛虫新生蚴抗原的同源免疫在减虫率上差异不显著。     

Trichinella spiralis and Trichinella pseudospiralis mixed infection in a wild boar (Sus scrofa) of Germany

A wild boar (Sus scrofa) from the island Usedom in Mecklenburg-Western Pomerania (north-east Germany) was detected as Trichinella-positive during routine meat inspection. Encapsulated and non-encapsulated larvae were detected in the muscle tissue by trichinoscopy. In the diaphragm, 922 larvae per g were detected by artificial digestion. Muscle larvae displayed two different sizes of about 700 and 1100 microm. By a multiplex PCR analysis, larvae with a large size were identified as Trichinella spiralis, whereas those of a smaller size were identified as Trichinella pseudospiralis. This is the first finding of a mixed infection of T. spiralis and T. pseudospiralis in a naturally infected animal and it supports the tendency of more frequent detection of the non-encapsulated species T. pseudospiralis in Europe.     

New patterns of Trichinella infection

Human and animal trichinellosis should be considered as both an emerging and reemerging disease. The reemergence of the domestic cycle has been due to an increased prevalence of Trichinella spiralis, which has been primarily related to a breakdown of government veterinary services and state farms (e.g., in countries of the former USSR, Bulgaria, Romania), economic problems and war (e.g., in countries of the former Yugoslavia), resulting in a sharp increase in the occurrence of this infection in swine herds in the 1990s, with a prevalence of up to 50% in villages in Byelorussia, Croatia, Latvia, Lithuania, Romania, Russia, Serbia, and the Ukraine, among other countries. The prevalence has also increased following an increase in the number of small farms (Argentina, China, Mexico, etc.) and due to the general belief that trichinellosis was a problem only until the 1960s. The sylvatic cycle has been studied in depth at both the epidemiological and biological level, showing the existence of different etiological agents (Trichinella nativa, Trichinella britovi, Trichinella murrelli, Trichinella nelsoni) in different regions and the existence of "new" transmission patterns. Furthermore, the role of game animals as a source of infection for humans has greatly increased both in developed and developing countries (Bulgaria, Canada, Lithuania, some EU countries, Russia, USA, etc.). The new emerging patterns are related to non-encapsulated species of Trichinella (Trichinella pseudospiralis, Trichinella papuae, Trichinella sp.), infecting a wide spectrum of hosts (humans, mammals including marsupials, birds and crocodiles) and to encapsulated species (T. spiralis, T. britovi, and T. murrelli) infecting herbivores (mainly horses). The existence of non-encapsulated species infecting mammals, birds and crocodiles had probably remained unknown because of the difficulties in detecting larvae in muscle tissues and for the lack of knowledge on the role of birds and crocodiles as a reservoir of Trichinella. On the other hand, it is not known whether horse and crocodile infections existed in the past, and their occurrence has been related to improper human behavior in breeding. The problem of horse-meat trichinellosis is restricted to France and Italy, the only two countries where horse-meat is eaten raw, whereas mutton and beef have been found to be infected with Trichinella sp. only in China.