Detection of Trichinella murrelli in coyotes (Canis latrans) from Oklahoma and North Texas

We determined the prevalence and mean intensity of Trichinella sp. infection in coyotes from six counties in Oklahoma and one in northern Texas. Tongues from 77 coyotes were examined using histology and artificial tissue digestion. Histological examination showed a prevalence of 3.9% (3 of 77) whereas the prevalence was 6.5% (5 of 77) based on artificial digestion of 5.0 g of muscle from coyote tongues. One sample was positive for Trichinella sp. on histology but negative by artificial digestion. Combining data from both diagnostic techniques showed that six of 77 (7.8%) coyotes were infected with Trichinella spp. The mean intensity of Trichinella sp. larvae ranged from 0.2 to 66.2 with an average of 16.0 larvae per gram (LPG) of tongue. Genotyping results demonstrated that the coyotes were infected with Trichinella murrelli. This is the first report of T. murrelli infection in coyotes in Oklahoma. T. murrelli had previously been isolated from coyotes in Texas.     

Predilection muscles and physical condition of raccoon dogs (Nyctereutes procyonoides) experimentally infected with Trichinella spiralis and Trichinella nativa

The predilection muscles of Trichinella spiralis and T. nativa were studied in 2 experimental groups of 6 raccoon dogs (Nyctereutes procyonoides), the third group serving as a control for clinical signs. The infection dose for both parasites was 1 larva/g body weight. After 12 weeks, the animals were euthanized and 13 sampling sites were analysed by the digestion method. Larvae were found in all sampled skeleton muscles of the infected animals, but not in the specimens from the heart or intestinal musculature. Both parasite species reproduced equally well in the raccoon dog. The median density of infection in positive tissues was 353 larvae per gram (lpg) with T. spiralis and 343 lpg with T. nativa. All the infected animals had the highest larvae numbers in the carpal flexors (M. flexor carpi ulnaris). Also tongue and eye muscles had high infection levels. There were no significant differences in the predilection sites between these 2 parasite species. Trichinellosis increased the relative amount of fat, but not the body weight in the captive raccoon dogs. Thus, Trichinella as a muscle parasite might have catabolic effect on these animals.     

Evaluation of ELISA and Western Blot Analysis using three antigens to detect anti-Trichinella IgG in horses

We assessed a serological method for detecting Trichinella infection in horses, specifically, an ELISA using three antigens to detect anti-Trichinella IgG (i.e. a synthetic tyvelose glycan-BSA (stg-BSA) antigen, an excretory/secretory (ES) antigen, and a crude worm extract (CWE) antigen). Serum samples were collected from 2502 horses (433 live horses from Romania and 2069 horses slaughtered in Italy and originating from Italy, Poland, Romania, and Serbia). Serum samples were also taken from horses experimentally infected with different doses of T. spiralis and T. murrelli larvae, as controls. The cut-off value of ELISA was determined on serum samples from 330 horses from Trichinella-free regions of Italy, which were also examined by artificial digestion of preferential-muscle samples. In the experimentally infected horses, the stg-BSA and ES antigens were less sensitive than the CWE antigen. Trichinella spiralis showed a higher immunogenicity than T. murrelli, and the IgG immunoresponse was dose-dependent. The kinetics of anti-Trichinella IgG were similar among all experimentally infected horses. No circulating antibodies were detected 4-5 months after experimental infection, although these horses still harbored infective larvae. Depending on the antigen used, for 4-7 of the 330 horses from Trichinella-free areas, the optical density (OD) of the serum sample was higher than the cut-off value, yet these samples were negative when subjected to Western Blot. Similar results were obtained for the 1739 horses slaughtered in Italy (originating from Italy, Poland, Romania, and Serbia) and the 433 live Romanian horses. Of the 4 horses with muscle larvae, only one was positive by ELISA and Western Blot. Because the anti-Trichinella IgG remain circulating for only a short period of time, whereas the larvae remain infective for longer periods, serology cannot be used for either diagnosing Trichinella infection in horses or estimating the prevalence of infection. Artificial digestion of at least 5 g of preferential-muscle tissue continues to be the method of choice at the slaughterhouse for preventing equine-borne trichinellosis in humans.     

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。     

PCR检测感染早期小鼠血液中旋毛虫幼虫的研究

为了研究PCR检测感染小鼠血液中旋毛虫DNA的敏感性,应用旋毛虫1.6 kb重复序列为扩增靶序列对旋毛虫(T1)、乡土旋毛虫(T2)、布氏旋毛虫(T3)、伪旋毛虫(T4)和南方旋毛虫(T7)肌幼虫DNA进行PCR扩增,并检测小鼠感染20、100、300条T1肌幼虫后不同时间的外周血.结果表明,T1、T4和T7肌幼虫可扩增出特异性目的条带(510 bp),而T2和T3无扩增产物;1、0.04和0.02条T1、T4和T7肌幼虫均能扩增到清晰的目的条带(510 bp).20条幼虫感染小鼠后5 d～6 d,PCR阳性率均为7.69%;100条幼虫感染小鼠后5 d～12 d可检出旋毛虫DNA,其中感染后5 d～7 d的阳性率分别为30.77%、38.46%及30.77%;300条幼虫感染小鼠后5 d～15 d可检出旋毛虫DNA,感染后7 d的阳性率为61.54%,感染后6 d与8 d～10 d的阳性率均为53.85%. 3组旋毛虫感染小鼠PCR阳性率间的差异有统计学意义(p<0.01),PCR阳性率随感染剂量的增加而升高(p<0.01),100条与300条感染小鼠感染后不同时间的PCR阳性率与检测时间有相关性(p<0.01).以上实验结果表明PCR检测感染小鼠血液中旋毛虫DNA的敏感性与感染程度和检测时间有关,对感染早期旋毛虫抗体阴性宿主有一定诊断价值.     

猪、犬旋毛虫成虫免疫原性试验

通过提取猪、犬旋毛虫成虫抗原，制备成虫油佐剂免疫原，在小鼠体内进行同源特异性免疫试验和交叉免疫试验。试验得到猪旋毛虫成虫抗原同源免疫减虫率为５５．８０％，其对犬旋毛虫的交叉免疫减虫率是３５．１１％；犬旋毛虫成虫抗原同源免疫的减虫率为３９．００％，其对猪旋毛虫的交叉免疫减虫率是２４．９６％。试验结果揭示，猪、犬旋毛虫成虫具有一定的免疫原性，其交叉免疫减虫率低于同源特异性免疫。猪旋毛虫成虫抗原对犬旋毛虫的交叉免疫与犬旋毛虫自身的同源特异性免疫差异不显著。     

The epidemiology of animal trichinellosis in China

The epidemiology of animal trichinellosis in China based mainly upon original Chinese literature published between 1937 and 2004 is reviewed. The seroprevalence of Trichinella infection in herbivores was 0.7% (2/300) in cattle and 0.8% (4/500) in sheep. The prevalence of trichinellosis in naturally infected cattle was 1.2% (2/163). Trichinella larvae were detected in 1.4% (3/215) of sheep and in 2.1% (1/47) of beef cattle sold at markets. Canine trichinellosis was recorded in 13 Provinces, Autonomous Regions or Municipalities (P/A/M) and the average prevalence of the infection in dogs slaughtered in abattoirs was 16.2% (5654/34,983) ranging from 1.2% to 44.8%, with the highest prevalence located in northeast China. The prevalence in dog meat sold at markets was 3.5% (988/27,898) in 5 P/A. Feline Trichinella infection was reported in 10 P/A/M. The prevalence of Trichinella infection in rats varied from 1.1% (51/459) to 15.1% (50/332). Trichinella larvae were detected in 1.5% (9/587) of house rats (Rattus norvegicus) as well as in 0.8% (3/369) of wild rats (Apodemus chevrieri), and the infection was recorded also in other wildlife (foxes, bears, wild boar, weasels, raccoon dogs, muntjak and bamboo rats). Trichinella larvae were detected in 2.6% (4/156) of weasels (Mustela sibirica), 1.5% (2/135) of shrews (Tupaia belangeri) and 7.7% (1/13) of moles (Parascapter leucurus). All Trichinella isolates from domestic pigs were identified as T. spiralis. Some Trichinella isolates from dogs in north-eastern China were identified as T. nativa, which has muscle larvae that are highly resistant to freezing. Twenty-seven outbreaks of human trichinellosis associated with mutton, dog and game meat occurred in China between 1964 and 2004, but the quarantine of Trichinella larvae in such meat is not mandatory in China at present.     

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.     

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.     

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.     

Clinical features of experimental trichinellosis in the raccoon dog (Nyctereutes procyonoides)

Three groups of six raccoon dogs (Nyctereutes procyonoides) were provided for the experiment: the first group was infected with pig-origin Trichinella spiralis, the second with raccoon dog-origin Trichinella nativa, and the third served as controls. Infection dose for both parasite species was 1000 larvae/kg of body weight, which led to intense final infection. Clinical signs, haematology and serum biochemistry with repeated blood samples were monitored up to 12 weeks post-infection. The most significant findings were a short-term eosinophilia in peripheral blood from the end of the first week post-infection until the end of the third week, loss of weight, and mild anaemia. In the early phase of the infection, the animals had gastrointestinal signs, loss of appetite and diarrhoea. No specific differences in clinical findings could be noticed between the groups infected with T. nativa and T. spiralis. In contrast to the symptoms reported in human outbreaks, fever was not observed in any of the infected animals and serum levels of muscle-specific enzymes did not change. No acute-phase response was observed in the enteral or parental phase of the infection. These findings indicate that because Trichinella spp. are very well adapted to the raccoon dog, it thus, could serve as the most crucial reservoir animal for sylvatic trichinellosis in Finland.     

各旋毛虫隔种对猪的感染性试验

用消化法所得的猪旋毛虫、犬旋毛虫、旋毛形线虫 (Trichinellaspiralis)和本地毛形线虫 (Trichinellanativa)分别感染健康猪。结果表明 :4个旋毛虫隔离种对猪的感染性存在着明显差异 ,猪旋毛虫和T .spiralis对猪易感 ,其繁殖力指数 (RCI)分别为 385 .6 8± 41.5 1和 30 0 .5 5± 12 .45 ;而犬旋毛虫和T .nativa对猪不易感 ,RCI分别是 0 .0 6 4± 0 .0 31和 0 .0 33± 0 .0 33。结果揭示黑龙江省猪旋毛虫相当于T .spiralis,犬旋毛虫相当于T .nativa ;犬旋毛虫和T .nativa很难通过猪的感染而对人体健康构成威胁。     

Spatial distribution of Trichinella britovi, T. pseudospiralis and T. spiralis in red foxes (Vulpes vulpes) in Hungary

The red fox (Vulpes vulpes) is considered one of the main reservoir of Trichinella spp. in Europe. As limited information on Trichinella infection in wildlife of Hungary is available, 2116 red foxes, representing more than 3% of the estimated fox population of the country, were screened to detect Trichinella larvae by a digestion method. Trichinella larvae from the 35 positive foxes were identified by a multiplex PCR as Trichinella britovi (30 isolates, 85.7%), Trichinella spiralis (4 isolates, 11.4%), and Trichinella pseudospiralis (1 isolate, 2.9%). The true mean intensity of T. britovi, T. spiralis and T. pseudospiralis larvae in lower forelimb muscles was 23.6, 3.5 and 13.5larvae/g, respectively. T. spiralis was detected only in the southern and eastern regions. The non-encapsulated T. pseudospiralis was recorded for the first time in Hungary. Although the overall true prevalence of Trichinella infection in foxes was only 1.8% (95% confidence interval, CI=1.5-2.1%), the spatial analysis reveals different risk regions. In the north-eastern counties bordering Slovakia and Ukraine (21% of the Hungarian territory), the true prevalence of Trichinella infection is significantly higher than that observed in other regions (6.0%, CI=4.8-7.1%). In the southern counties bordering Croatia, Serbia and Romania (41% of the Hungarian territory), the true prevalence of Trichinella infection is moderate (1.4%, CI=1.0-1.8%). In the north-western and central counties (38% of Hungarian territory), the prevalence of Trichinella infection is significantly lower (0.2%, CI=0.1-0.4%) than that of the other regions. Based on the statistical analysis and the evaluation of epidemiological data, none of the counties can be considered free of Trichinella infection. In the past decade, Trichinella infection has been detected only in few backyard pigs, and only few wild boar-related autochthonous infections in humans were described. Nevertheless, these results highlight the need of the maintenance of a strict monitoring and control programmes on Trichinella infection in farmed and hunted animals of Hungary.     

Experimental Trichinella spiralis infection in sheep.

During susceptibility studies of non-specific hosts, three merino sheep were infected with 3000, 5000 or 7000 Trichinella spiralis larvae by gavage. Clinical, physiological and serological parameters were assessed during the experiment. On the 152nd day p.i., animals were necropsied and, using artificial digestion methods, numbers of Trichinella larvae in muscle tissues were determined. The most infected parts were masseters with 3122 larvae g-1 muscle, 5526 larvae g-1 muscle and 4058 larvae g-1 muscle and diaphragms with 2778 larvae g-1 muscle, 2725 larvae g-1 muscle and 2320 larvae g-1 muscle, for the 3000, 5000 and 7000 infection levels, respectively. A positive correlation between infective rate and circulating antibodies was observed using ELISA and latex agglutination (LA) test methods. Trichinella larvae from sheep applied by gavage to ICR mice developed to the muscle stage. No significant changes were found in the clinical and physiological parameters of infected animals. Our results confirm the high susceptibility of merino sheep to T. spiralis infection.     

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.     

Trichinella pseudospiralis foci in Sweden

In Sweden, the prevalence of Trichinella infection in domestic pigs has greatly decreased since the 1970s, with no reports in the past 4 years. However, infected wild animals continue to be found. The objective of the present study was to identify the species of Trichinella present in animals of Sweden, so as to contribute to the knowledge on the distribution area and hosts useful for the prevention and control of this zoonosis. In the period 1985-2003, Trichinella larvae were detected in the muscles of 81/1800 (4.5%) red foxes (Vulpes vulpes), 1/6 (16.7%) arctic fox (Alopex lagopus), 1/7 (14.3%) wolf (Canis lupus), 10/200 (5.0%) lynxes (Lynx lynx), 4/8000 (0.05%) wild boars (Sus scrofa), and 27/66 x 10(6) (0.000041%) domestic pigs. All four Trichinella species previously found in Europe were detected (Trichinella spiralis, T. nativa, T. britovi and T. pseudospiralis). The non-encapsulated species T. pseudospiralis was detected in three wild boars from Holo (Stockholm area) and in one lynx from Froso (Ostersund area), suggesting that this species is widespread in Sweden. These findings are consistent with those of a study from Finland, both for the unexpected presence of T. pseudospiralis infection and the presence of the same four Trichinella species, suggesting that this epidemiological situation is present in the entire Scandinavian region. The widespread diffusion of T. pseudospiralis in the Scandinavian region is also important in terms of it potential impact on public health, given that human infection can occur and the difficulties to detect it by the trichinelloscopic examination.     

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.     

Epizootiological survey of Trichinella spp. infection in carnivores, rodents and insectivores in Hokkaido, Japan

In order to evaluate the present epidemiological situation of Trichinella infection in wild animals in Hokkaido, Japan, red foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) , brown bears (Ursus arctos) , martens (Martes melampus), rodents and insectivores captured in Hokkaido were examined for muscle larvae by the artificial digestion method from 2000 to 2006. Foxes (44/319, 13.8%), raccoon dogs (6/77, 7.8%) and brown bears (4/126, 3.2%) were found to be infected with Trichinella larvae and all other animal species evaluated were negative. Multiplex PCR and DNA sequencing revealed that larvae from a fox captured in Otofuke, in south-eastern Hokkaido, were T. nativa, and larvae from 27 animals including 21 foxes, 2 raccoon dogs and 4 brown bears captured in western Hokkaido were Trichinella T9.     

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.     

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.