Identification of sylvatic Trichinella (T3) in foxes from France.

Thirty-three foxes (Vulpes vulpes) from a sample of 1912 collected in France were found to be infected with Trichinella spp. Four isolates were obtained for genetic identification. Isoenzymatic and biological analysis of these isolates revealed the presence of two distinct genetic types of Trichinella, Trichinella spiralis s.str. (T1) and Trichinella sp. (T3) (Trichinella nelsoni according to Soviet authors) in the fox population. The reproductive capacity index of these isolates in Wistar rats was high for T. spiralis and low for T3. This is the first report of T3 type from wild animals in France. The epidemiological implications are discussed.     

旋毛虫各隔离种对小鼠的感染性研究

比较研究了黑龙江省猪、犬旋毛虫及国际标准虫种旋毛形线虫(Trichinella spiralis)和本地毛形线虫(Trichinella nativa)对小鼠的感染性异同。结果表明，四者对小鼠的感染力存在着显著差异，猪旋毛虫和旋毛形线虫在小鼠体内的繁殖力指数(RCI)分别为121.01±7.80和149.86±7.47；而犬旋毛虫和本地毛形线虫的RCI分别为60.98±5.05和55.15±4.69。由实验结果可认为黑龙江省的猪旋毛虫相当于旋毛形线虫，而犬旋毛虫相当于本地毛形线虫。     

用DNA限制性片段长度多态性鉴定旋毛虫各隔离种

以旋毛虫国际标准虫种：旋毛形线虫（Trichinella spirlais）和本地长形线虫(Trichinella nativa）作对照,应用DNA限制性片段长度多态性（PELP）技术对黑龙江省猪、犬旋毛虫进行虫种鉴定。结果显示：猪旋毛虫和旋毛形线虫酶切图谱相同;犬旋毛虫和本地毛形线虫酶谱一致。结果提示,黑龙江猪旋毛虫为旋毛形线虫,犬旋毛虫为本地毛形线虫。     

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.     

猪、犬旋毛虫DNA限制性片段长度多态性分析

以旋毛虫国际标准虫种Ｔ．ｓｐｉｄａｉｓ和Ｔ．ｎａｔｉｖａ作对照,应用ＤＮＡ限制性片段长度多态性（ＲＦＬＰ）技术对黑龙江省猪、大旋毛虫进行虫种鉴定。结果显示：猪旋虫和Ｔ．ｓｐｉｒｌａｉｓ酶切图谱相同;犬旋毛虫和Ｔ．ｎａｔｉｖａ酶谱一致,结果提示,黑龙江猪旋毛虫为Ｔｓｐｉｒａｌｉｓ,犬旋毛虫为Ｔ．ｎａｔｉｖａ。     

旋毛虫各隔离种雌虫生殖能力的实验研究

本试验对旋毛虫各隔离种雌虫体外产新生幼虫能力进行了研究。结果显示，猪旋毛虫和旋毛形线虫(Trichinella spiralis)雌虫体外培养24h平均产新生幼虫数分别为66.00±7.34和76.20±7.57，而犬旋毛虫和本地毛形线虫(Trichinella nativa)分别是28.80±4.30和22.00±3.22，前者在雌虫体外产幼虫能力上明显高于后者。研究结果表明，黑龙江猪旋毛虫相当于旋毛虫形线虫，犬旋毛虫相当于本地毛形线虫。     

Characterisation of Trichinella isolates from Bulgaria by molecular typing and cross-breeding

Trichinella spp. larvae were collected from domestic and wild-life animals in association with 15 human trichinellosis outbreaks registered between 1999-2002 in Bulgaria. Furthermore, Trichinella spp. isolates were obtained from 62 naturally infected wild animals and of a rat. All isolates were subjected to speciation by both multiplex PCR and cross-breeding experiments. Epidemiological and clinical data were collected and analysed using standard protocols for epidemiological surveillance and control of outbreaks. Only two species were identified-Trichinella britovi and Trichinella spiralis. Results obtained by molecular typing fully matched those of cross-breeding. More specifically, parasite isolates obtained upon 15 epidemic outbreaks revealed the predominance of T. britovi (n = 10) when compared to T. spiralis (n = 5). With regard to host origin, the predominant species detected among wild boar was T. britovi (n = 4), and T. spiralis was identified in one wild boar sample only. Among the isolates obtained from domestic pig products, T. britovi was found in five cases and T. spiralis in four cases, respectively. In the naturally infected wild animals not related to epidemics, only T. britovi was demonstrated. The present results provide a strong indication that both T. britovi and T. spiralis operate within domestic and sylvatic cycles in Bulgaria. Geographically, the distribution of T. britovi appears to include Central, Southern, Eastern and Western parts of the country, and wildlife animals from the Mid Balkan Mountains and Mid Sredna Gora Mountains, T. spiralis was found in Western and Southwestern Bulgaria, only.     

旋毛虫病McAb快速ELISA诊断盒的应用研究

应用ＭｃＡｂ 快速ＥＬＩＳＡ 诊断盒,对感染了４ 个旋毛虫隔离种的猪定期检测其血清中抗体出现情况。结果,猪旋毛虫和旋毛形线虫（Ｔ．ｓｐｉｒａｌｉｓ）在感染２４ ｄ 后、犬旋毛虫和本地毛形线虫（ Ｔ．ｎａｔｉｖａ）３１ ｄ 后,可在猪血清中检出抗体;在抗体出现时间上前二者较后二者早７ ｄ 左右。     

Trichinella isolates from polar bears in Svalbard. Freeze resistance and infectivity in rats and swine

The author isolated Trichinella strains from five polar bears in Svalbard (the high arctic region of Norway). Based on infectivity experiments with white mice (BOM:NMR), the freeze resistance limits of the Trichinella strains are outlined. Further experiments showed that white rats (MOL:WIST) and pigs (sus scrofa domestica) are almost refractory to these Trichinella strains. The infectivity of Svalbard isolates in the above mentioned test animals was compared, in parallel experiments, with that of T. spiralis (Owen). The latter showed a very high infectivity to the same species of test animals. It is thus probable that the arctic Trichinella found in the polar bear is biologically distinct from both T. spinalis (Owen) and T. nativa ( Britov and Boev ).     

旋毛虫起源、进化及其宿主的感染来源与途径

毛形属在距今27 500万年前从毛形科中正式分化出来,各旋毛虫种间的遗传差异形成于距今的1 500万~2 000万年间。对亚洲和欧洲的共110个Trichinella spiralis地理隔离株的线粒体基因分析结果显示,欧洲地理株间仅存1个碱基差异,具有高度的一致性,而亚洲地理株间则具有8个碱基差异,存在明显的种内差异。依据线粒体累积变异速率计算,T.spiralis由亚洲传播至欧洲的时期大约在60 00年~16 000年以前。不同种旋毛虫对动物的感染具有一定的异嗜性,而宿主感染旋毛虫的途径则主要来源于水平传播,即食用感染有虫体的肉类;机械性传播,即经粪便、土壤、废水、食腐性昆虫的机械性传播;垂直传播,目前在人类、豚鼠中有检出报道。     

Genetic analysis of Trichinella populations by 'cold' single-strand conformation polymorphism analysis

A non-isotopic single-strand conformation polymorphism ('cold' SSCP) technique has been assessed for the analysis of sequence variability in the expansion segment 5 (ES5) of domain IV and the D3 domain of nuclear ribosomal DNA within and/or among isolates and individual muscle (first-stage) larvae representing all currently recognized species/genotypes of Trichinella. Data are consistent with the ability of cold SSCP to identify intra-specific as well as inter-specific variability among Trichinella genotypes. The cold SSCP approach should be applicable to a range of other genetic markers for comparative studies of Trichinella populations globally.     

First record of Trichinella pseudospiralis in the Slovak Republic found in domestic focus

Infection of Trichinella spp. is widespread among wildlife in Slovakia and the red fox (Vulpes vulpes) is the main reservoir of Trichinella britovi. Trichinella spiralis has been rarely documented in sylvatic and domestic animals of this country. During routine examination of domestic pigs at the slaughter, Trichinella larvae were detected by artificial digestion in a domestic pig of a large-scale breeding farm in Eastern Slovakia. The parasite has been identified by molecular (PCR) and biochemical (allozymes) analyses and by the morphology of the nurse cell as the non-encapsulated species Trichinella pseudospiralis infecting both mammals and birds. The epidemiological investigation carried out at the farm level revealed the presence of the same parasite species in other three pigs of 192 examined (2.1%), in 3 of 14 (21.4%) examined synanthropic rats (Rattus norvegicus) and in a domestic cat. The farm was characterized by inadequate sanitary conditions, insufficient nutrition, cannibalism and the presence of rat population. A different profile has been observed at the phosphoglucomutase locus in T. pseudospiralis isolates from Slovakia in comparison with the T. pseudospiralis reference isolate from the Palearctic region. This is the first documented focus of T. pseudospiralis from Central Europe. The detection in domestic pigs of a non-encapsulated parasite infecting both mammals and birds stresses the need to avoid the use of trichinelloscopy to detect this infection at the slaughterhouse.     

Autochthonous and imported Trichinella isolates in Germany

The study of Trichinella isolates from wildlife in Germany revealed the presence of Trichinella spiralis and Trichinella britovi in wild boars and foxes. T spiralis was detected in meat products imported from Spain, which is one of the two endemic areas of domestic trichinellosis in the European Union: It was also detected in meat from a grizzly bear marketed in Alaska, and Trichinella nativa was detected in a polar bear from the Berlin Zoo. These results stress the importance of examining for Trichinella live animals and meat products imported to Germany from both EU and non-EU countries. Furthermore, carnivores from Arctic regions that are born in the wild and placed in zoos can represent a risk for the introduction of the freeze-resistant species of Trichinella in a new region if, once the animal dies, the carcass is not properly destroyed.     

Species identification of Trichinella isolates from China

Two species of Trichinella were identified from China by means of PCR amplification of the mitochondrial small subunit ribosomal DNA and the expansion segment V region of the ribosomal DNA. Seven isolates originating from domestic pig and one isolate originating from dog showed identical DNA banding pattern to Trichinella spiralis, and two isolates from dog showed DNA banding pattern identical to Trichinella nativa. Sequence analysis of the 5S rDNA inter-gene spacer region from the ten Trichinella isolates confirmed the existence of only two Trichinella species and revealed the inner species genetic variation within T. spiralis and T. nativa.     

Prevalence of Trichinella spp. in North Spain wild fauna and new variety of Trichinella britovi identification

Trichinella spiralis and Trichinella britovi live in apparent sympatry among wild fauna of the Iberian Peninsula. In the present study 105 Trichinella isolates from wild mammals were typed by inter-sequence simple repeat PCR (ISSR-PCR). All isolates identified as T. spiralis were indistinguishable from the ISS48 reference strain. Among those belonging to T. britovi, four variations were clearly distinguishable; two of them, ISS11 C-76 and ISS86 MON, had been previously detected while the ISS2 reference strain and Trichinella Rioja 3, (MVUL/SP/02/R3) had not been reported before. The newly distinguished genotype of T. britovi was analyzed by ISSR-PCR, multiplex-PCR, UARR sequencing, and single larva cross-breeding with the other T. britovi genotypes including Trichinella T8 (ISS49). Among all of them, the ISS11 and ISS2 isolates were found to be the most frequent. The uniformity found within T. spiralis isolates is consistent with its recent introduction in Iberian Peninsula, whereas the presence of four variations within T. britovi suggests that this species is an endemic species. Orographical diversity of the West-End of Eurasian Region could act to preserve population diversity observed within T. britovi.     

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.     

Identification of Spanish Trichinella isolates by ISSR-PCR: Intra-specific variability of Trichinella britovi

In the present work, we investigated genetic variability of the Spanish Trichinella isolates by ISSR-PCR (inter-simple sequence repeat polymerase chain reaction), a technique that is being successfully used to study diversity among related populations. We recovered a total of 43 isolates from different host and geographic localization and identified them by molecular techniques (RAPD and multiplex-PCR) and by Western blot with monoclonal antibodies US5 and US9. Nineteen (44.2%) out of 43 were identified as Trichinella spiralis and 24 (55.8%) as Trichinella britovi. When these samples were analysed by the ISSR technique, all the T. spiralis isolates presented a pattern similar to the T. spiralis ISS116. By contrast, the ISSR-PCR analysis of the isolates identified as T. britovi, showed two different banding profiles compatible with the European T. britovi isolate pattern (ISS2), and the autochthonous Spanish T. britovi isolate (ISS11). Three of these 43 isolates were involved in human outbreaks; the three were identified as T. britovi and showed a pattern similar to the European isolate ISS2. As conclusion, we highlight that an intra-species variability within the Spanish T. britovi isolates analysed was observed, with a predominant group similar to T. britovi ISS2, while T. spiralis group isolates were more homogeneous. No correlations were found between the different ISSR-PCR T. britovi types and the host/geographical origin of the isolates.     

本地毛形线虫49 Ku ES蛋白结构基因的分子克隆及原核表达

提取Trichinella nativa(T.nativa)肌幼虫的总RNA,用RT-PCR方法扩增出了编码T.nativa 49 Ku ES蛋白的结构基因。基因克隆后测序,序列测定结果表明:目的基因TNPG长度为951 bp,核苷酸序列同已发表的Trichinella spiralis(T.spiralis)相应的序列P49同源性为97.68%,所推导的氨基酸序列同源性为95.24%。将目的基因TNPG插入到原核表达载体pET-30a的BamHⅠ酶切位点处,并转化到感受态表达菌中进行诱导表达。结果显示TNPG在原核表达菌BL-21中获得了高效表达,表达产物为40.8 Ku的融合蛋白,表达量达到菌体总蛋白的22.8%。通过Western blot分析,表达产物可以被小鼠T.nativa和T.spiralis阳性血清以及它们的中国地理株的小鼠血清特异性识别。     

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.     

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.