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.     

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.     

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.     

Epidemiology of trichinellosis in Asia and the Pacific Rim

The epidemiology of trichinellosis, species of Trichinella present and the food and eating habits of people affected in Asia and the Pacific Rim are reviewed with emphasis on Japan, China and Thailand. Trichinella seems to be prevalent throughout this region although outbreaks of trichinellosis have not been reported in some areas. Major outbreaks of the disease have been reported primarily in China and Thailand. This is the result of three factors: (1) China and Thailand are highly endemic areas for this parasite; (2) the two countries are well-organized and there is a public health system that enables precise reporting of disease outbreaks and (3) culinary habits provide many opportunities to eat undercooked meats. Trichinella found in Asia and the Pacific Rim includes both encapsulated species (Trichinella spiralis, Trichinella britovi, Trichinella nativa) and noncapsulated species (Trichinella pseudospiralis, Trichinella papuae). T. britovi, isolated in Japan, is a different genotype from the European strain. Therefore, the Japanese strain of T. britovi is designated Trichinella T9. Human trichinellosis caused by T. pseudospiralis has occurred in New Zealand and Thailand. Tasmania has had animal cases of T. pseudospiralis infection and animals with T. papuae infection have been found in Papua New Guinea. Economic losses due to Trichinella infection are not negligible in China, where there have been more than 500 outbreaks of human trichinellosis, affecting more than 20,000 people and causing more than 200 deaths. In Thailand, over the past 27 years, 120 outbreaks were reported involving nearly 6700 patients and 97 deaths. Japan has had fewer outbreaks and some sporadic cases have been attributed to imported infection.     

Spatial distribution of Trichinella britovi, T. spiralis and T. pseudospiralis of domestic pigs and wild boars (Sus scrofa) in Hungary

Trichinellosis is a foodborne disease caused by the consumption of raw meat and raw meat-derived products from swine, horse and some game animals infected with nematode worms of the genus Trichinella. Between June 2006 and February 2011, 16 million domestic pigs and 0.22 million wild boars (Sus scrofa) were tested for Trichinella sp. in Hungary. Trichinella infection was not found in any pigs slaughtered for public consumption. Nevertheless, Trichinella spiralis was detected in four backyard pigs when trace back was done following a family outbreak. Trichinella infection was demonstrated in 17 wild boars (0.0077%). Larvae from wild boars were identified as Trichinella britovi (64.7%), T. spiralis (29.4%) and Trichinella pseudospiralis (5.9%). Although the prevalence of Trichinella sp. infection in wild boars and domestic pigs is very low, the spatial analysis reveals that the level of risk differs by region in Hungary. Most of the T. britovi infected wild boars (63.6%) were shot in the north-eastern mountain area of Hungary; whereas domestic pigs and wild boars infected with T. spiralis were detected only in the southern counties bordering Croatia and Romania. In the north-western and central counties, the prevalence of Trichinella infection seems to be negligible.     

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.     

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.     

Muscle distribution of sylvatic and domestic Trichinella larvae in production animals and wildlife

Only a few studies have compared the muscle distribution of the different Trichinella genotypes. In this study, data were obtained from a series of experimental infections in pigs, wild boars, foxes and horses, with the aim of evaluating the predilection sites of nine well-defined genotypes of Trichinella. Necropsy was performed at 5, 10, 20 and 40 weeks post inoculation. From all host species, corresponding muscles/muscle groups were examined by artificial digestion. In foxes where all Trichinella species established in high numbers, the encapsulating species were found primarily in the tongue, extremities and diaphragm, whereas the non-encapsulating species were found primarily in the diaphragm. In pigs and wild boars, only Trichinella spiralis, Trichinella pseudospiralis and Trichinella nelsoni showed extended persistency of muscle larvae (ML), but for all genotypes the tongue and the diaphragm were found to be predilection sites. This tendency was most obvious in light infections. In the horses, T. spiralis, Trichinella britovi, and T. pseudospiralis all established at high levels with predilection sites in the tongue, the masseter and the diaphragm. For all host species, high ML burdens appeared to be more evenly distributed with less obvious predilection than in light infections; predilection site muscles harbored a relatively higher percent of the larval burden in light infections than in heavy infections. This probably reflects increasing occupation of available muscle fibers as larger numbers of worms accumulate. Predilection sites appear to be influenced primarily by host species and secondarily by the age and level of infection.     

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.     

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.     

Vaccination of rats and pigs against Trichinella spiralis spiralis using the subspecies, T. spiralis nativa.

Rats and pigs were vaccinated against Trichinella spiralis spiralis either by feeding infective larvae of the subspecies, Trichinella spiralis nativa in musculature or by gavage. The number of larvae established in the musculature of vaccinated nonchallenged and vaccinated challenged rats and pigs were negligible and statistically comparable, while highly significant infections were established in the nonvaccinated challenged rats and pigs. High vaccination doses of T. spiralis nativa gave virtually complete protection to challenge with T. spiralis spiralis in pigs. The results of one trial in rats with a lower vaccination dose of larvae suggest that there is a minimal vaccination dose of larvae required to elicit marked resistance to challenge. The low numbers of muscle larvae established due to the high vaccination doses of larvae confirm the low infectivity of the subspecies, T. spiralis nativa in rats and pigs.     

Infectivity of Trichinella papuae for experimentally infected red foxes (Vulpes vulpes)

To evaluate infectivity for carnivores as well as other biological characteristics of the newly described Trichinella papuae, eight red foxes were experimentally infected with the parasite. Five weeks after inoculation, T. papuae larvae were recovered from nine different muscle types. The larvae recovered from muscle tissue were shown to be infective to mice, to have a very low tolerance to freezing, and to survive longer than the other Trichinella genotypes in decaying tissue up to 5 weeks after infection.     

Detection of Trichinella spiralis nativa antibodies in porcine sera by ELISA using T. spiralis spiralis excretory-secretory antigen

Enzyme-linked immunosorbent assay examination of sera from pigs vaccinated with T. spiralis nativa infective larvae and/or challenged with T. spiralis spiralis larvae using a T. spiralis spiralis excretory-secretory antigen showed a significant cross-reaction between the two species of Trichinella. Eight of 12 pigs vaccinated with a high dose of T. spiralis nativa reacted positively 28 days postvaccination while the remaining four pigs had high but negative ELISA optical density readings. Five of six pigs challenged with the homologous species reacted positively 28 days postchallenge but the sixth pig remained negative despite having a muscle infection of 5.6 larvae/g of musculature.     

Distribution and pathogenicity during development of Camelostrongylus mentulatus in the abomasum of sheep

Fifteen lambs were each inoculated orally with 150 000 infective Camelostrongylus mentalutus larvae. Lambs were killed 3, 4, 8, 12, 16, 21 and 28 days after infection (DAI). Feed consumption and plasma total protein levels of the lambs killed 21 and 28 DAI did not differ significantly from those of uninfected controls. Three and 8 DAI most larvae were recovered from the fundic mocusa, but the intensity of establishment of larvae per gram of tissue was greatest at the cardia and diminished gradually toward the pylorus. Emergence of worms from the cardia and fundus occurred between 8 and 12 DAI, and a proportion of the lume-dwelling population shifted into the antral area, while the number of worms in the antral mucosa remained stable. Nodularity of the mucosa, mucous metaplasia of fundic epithelium, and vascular permeability as indicated by colloidal carbon label, were evident 3–4 DAI, and were most severe 8–12 DAI, when erosion of and inflammatory cell effusion from heavily infected mucosa was marked. pH and Na⁺ concentration in abomasal content were elevated at all times after infection, with peak values 12 DAI. Plasma pepsinogen levels in infected lambs were elevated beginning 3–12 DAI.     

Intestinal establishment and reproduction of adult Trichinella spp. in single and mixed species infections in foxes (Vulpes vulpes)

Intestinal establishment and reproduction of adult Trichinella spiralis, Trichinella nativa, Trichinella britovi and Trichinella pseudospiralis were examined as single species or mixed species infections in foxes. This is the first study of intestinal dynamics of Trichinella spp. in a carnivore model and the results suggest that the intestinal phase is relatively short as only very few worms were recovered 10 days post-inoculation (dpi). In mixed species infection with equal doses of T. nativa and T. spiralis, molecular typing demonstrated that 64% of the intestinal worms and 78% of the muscle larvae were T. nativa. Conversely, T. spiralis dominated in the mixed species infections with T. pseudospiralis, constituting 66% of the intestinal worms and 94% of the muscle larvae. Although, the individual recoveries of intestinal worms were only up to 5.6% on day 1, and up to 1.5% on day 4 post-infection, the muscle larvae establishment was comparable to other fox studies. Infectivity, measured as muscle larvae burden did not differ among the four species of Trichinella, which is in contrast to other models with mice, rats, pigs or herbivores. Although statistically significant differences in intestinal worm burdens were found for some days, no distinct species were recovered in consistently higher numbers than the others.     

旋毛形线虫分类的研究进展

概述了旋毛形线虫属种分类研究的现状及虫体杂交试验、同工酶酶谱分析、分子生物学及分子遗传学试验等旋毛虫分类方法的研究进展,指出目前国际上已将毛形属分为8个隔离种（即T．spiralis,T1;T．nativa,T2;T．britovi,T3;T．pseudospiralis ,T4;T．murrelli,T5;T．nelsoni,T7;T．papuae,T10：Lzimbabwensis,T11）和3个分类地位尚未确定的基因型（即T6、T8和T9）。     

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.     

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.     

First isolation of Trichinella britovi from a wild boar (Sus scrofa) in Belgium

Since 1992, when the European Union Council Directive requires that wild boars (Sus scrofa) hunted in EU for commercial purpose should be examined for Trichinella, the infection has not been detected in wild boars from Belgium, despite serological evidence of the presence of anti-Trichinella antibodies in wildlife and previous reports of Trichinella larvae in this host species. In November 2004, Trichinella larvae were detected in a wild boar hunted near Mettet, Namur province (Southern Belgium). Larvae were identified as Trichinella britovi by polymerase chain reaction methods. This is the first report of the identification of Trichinella larvae from Belgium at the species level. The detection of T. britovi in wildlife in Belgium is consistent with findings of this parasite in other European countries and confirms the need to test game meat for Trichinella to prevent its transmission to humans.     

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.