Trichinella species circulating among wild and domestic animals in Romania

Trichinellosis is one of the most important zoonotic diseases in Romania. Even though the disease is a serious public health concern, only a limited number of Trichinella isolates have been identified at the species level; in the past, all larvae were assumed to be Trichinella spiralis. The present study was conducted to identify Trichinella spp. circulating among wild and domestic animals in Romania, using PCR-based methods. Trichinella spp. larvae originating from 54 wild and 23 domestic mammals were examined. No Trichinella spp. larvae were detected in muscle samples of 182 birds. T. spiralis and Trichinella britovi were the only two species identified in the 40 isolates that yielded a positive PCR result. Overall, T. britovi was more prevalent (n = 26; 65%) than T. spiralis (n = 14; 35%). T. spiralis was the predominant species found in domestic animals (n = 9; 75%), while T. britovi was more prevalent in wildlife (n = 24; 86%). No mixed infections were found. The highest prevalence of Trichinella infection was detected in wolves (11/35; 31%), in European wild cats (4/28; 14%), and in red foxes (5/71; 7%). The distribution of Trichinella spp. in Romania does not show a species-specific clustering; both of the two species found were present over the entire range of counties studied.     

Trend analysis of Trichinella in a red fox population from a low endemic area using a validated artificial digestion and sequential sieving technique

Freezing of fox carcasses to minimize professional hazard of infection with Echinococcus multilocularis is recommended in endemic areas, but this could influence the detection of Trichinella larvae in the same host species. A method based on artificial digestion of frozen fox muscle, combined with larva isolation by a sequential sieving method (SSM), was validated using naturally infected foxes from Latvia. The validated SSM was used to detect dead Trichinella muscle larvae (ML) in frozen muscle samples of 369 red foxes from the Netherlands, of which one fox was positive (0.067 larvae per gram). This result was compared with historical Trichinella findings in Dutch red foxes. Molecular analysis using 5S PCR showed that both T. britovi and T. nativa were present in the Latvian foxes, without mixed infections. Of 96 non-frozen T. britovi ML, 94% was successfully sequenced, whereas this was the case for only 8.3% of 72 frozen T. britovi ML. The single Trichinella sp. larva that was recovered from the positive Dutch fox did not yield PCR product, probably due to severe freeze-damage. In conclusion, the SSM presented in this study is a fast and effective method to detect dead Trichinella larvae in frozen meat. We showed that the Trichinella prevalence in Dutch red fox was 0.27% (95% CI 0.065-1.5%), in contrast to 3.9% in the same study area fifteen years ago. Moreover, this study demonstrated that the efficacy of 5S PCR for identification of Trichinella britovi single larvae from frozen meat is not more than 8.3%.     

Evaluation of the risk of transmission of Trichinella in pork production systems in Argentina

Recently, there has been interest in programs that certify pork production practices that minimize the risk of exposure of pigs to Trichinella spiralis. Certification might be useful for reducing the risk of human trichinellosis from pork in Argentina, but more information is needed on pig production practices and sources of Trichinella infection in Argentinian pigs. In this study, 21 pig farms were assessed for Trichinella infection including some farms using total and partial confinement management, and others with pigs raised exclusively outdoors. A total of 3224 muscle samples were collected from pigs raised on these farms and tested to determine the presence of T. spiralis larvae by artificial digestion. Serum samples from the same 3224 pigs were tested for antibodies to T. spiralis by ELISA. For each farm, a questionnaire was completed summarizing information about management factors and this information was used to assess risk factors for exposure of T. spiralis. Based on the results, pigs raised outdoors were more likely to be infected than pigs raised in total or partial confinement (p ≤ 0.05). Pigs fed waste products containing meat were 12.5 times more likely to be infected than pigs not fed waste containing meat (p < 0.01). The role played by rats in transmission of Trichinella is unclear; however, on farms with evidence of wild animals and access of pigs to wildlife carcasses, the prevalence of Trichinella infection was significantly higher. All pigs raised under good hygienic and sanitary conditions were negative for Trichinella infection by both artificial digestion and ELISA.     

Long-term survey on Trichinella prevalence in wildlife of Slovakia

In Slovakia, monitoring the prevalence of Trichinella spp. in wildlife was performed since 2000 in the main reservoir animals, the red fox (Vulpes vulpes) and wild boar (Sus scrofa), using artificial digestion method as recommended by International Commission on Trichinellosis. The results of investigation performed in 5270 red foxes showed that Trichinella infection is widespread across Slovakia and prevalence increased significantly from 4.9% in 2000 to 20.5% in 2007. Recently, a higher Trichinella prevalence (0.11%) in wild boars was also demonstrated. The results indicate that foxes and wild boars are involved in the spread of Trichinella, although the latter host species seems to play a secondary role in the maintenance of the sylvatic cycle in Slovakia. Trichinella britovi is the predominant species circulating in Slovakia, both in foxes and wild boars, and Trichinella spiralis occurs only sporadically. Mixed infections of T. britovi and Trichinella pseudospiralis were recorded in 2005 in one wild boar from Eastern Slovakia and in 2006 in one red fox from the same region. These findings are important with respect to an outbreak caused by T. pseudospiralis in a pig farm in the same district 3 years ago. This study provides a complex picture on Trichinella occurrence in all regions of Slovakia and may be a good basis for evaluating the risk of parasite transmission to the domestic cycle and human beings.     

Detection of trichinellosis in a historically Trichinella-free area of Argentina

The aim of the present work was to determine the presence of human and porcine trichinellosis in an area of Argentina historically regarded as Trichinella-free. Human blood donors (n = 216) and swine destined for consumption (n = 57) were evaluated by serological techniques (ELISA, immunofluorescence, and/or Western Blot). Muscle tissues from 26 of the pigs were evaluated for the presence of Trichinella larvae by the artificial digestion method. A questionnaire was used to collect and evaluate data on eating habits of the human population under study and on swine-raising conditions. The survey showed that 98.1% of the individuals (n = 212) were regular consumers of pork in the form of stuffed products such as sausages produced by local butchers. The seroprevalence (positive sera by at least two of the three methods) was 8.3% (n = 18) for human trichinellosis and 24.5% (n = 14) for porcine trichinellosis. Trichinella spiralis larvae were found in 2 of the 26 pigs (7.7%) with parasite loads of 0.33 and 2.4 muscle larvae per gram. Twelve swine found positive by serological and/or parasitological tests were raised under poor sanitary conditions (presence of rubbish in the surroundings, with cannibalism and scavenging behaviors, presence of rodents, etc.). Our study confirms the existence of porcine trichinellosis in an area regarded as Trichinella-free, provides supporting serological evidence of human infection in this area, and indicates that failure to report cases of trichinellosis based on inadequate surveillance can result in incorrect prevalence classification of an area.     

Complement membrane attack complex formation and infectivity of Trichinella spiralis and T. nativa in rats

Rats readily become infected with Trichinella spiralis but are more resistant to T. nativa. We infected complement factor C6-deficient (C6?) rats and control (C6+) rats with T. spiralis and T. nativa to compare the effects of membrane attack complex on these parasites in vivo. The 2000 larvae infection dose per rat yielded 652 lpg (larvae per gram) in the C6? group and 608 lpg in the C6+ group with T. spiralis, whereas with T. nativa the corresponding figures were only 1.05 and 1.87 lpg. The difference between the Trichinella species was evident, but the infection intensity was unaffected by the C6 deficiency. When newborn larvae were incubated in C6-deficient and control rat sera for 24 h in vitro, no changes in viability were observed. Immunohistochemistry revealed that the musculature of cross-sectioned adults and certain stichocytes bound human complement factors C3, C8 and C9, but not C1q. Interestingly, the outermost layer of the cuticle and the newborn larvae did not show any binding activity. Similar findings were obtained with immunofluorescence microscopy of intact newborn larvae. These results indicate that both T. spiralis and T. nativa have efficient mechanisms to protect themselves against complement attack. The difference in infectivity for rats between the two species, however, is not due to a differential resistance to complement membrane attack complex.     

Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa)

Trichinella surveillance in wildlife relies on muscle digestion of large samples which are logistically difficult to store and transport in remote and tropical regions as well as labour-intensive to process. Serological methods such as enzyme-linked immunosorbent assays (ELISAs) offer rapid, cost-effective alternatives for surveillance but should be paired with additional tests because of the high false-positive rates encountered in wildlife. We investigated the utility of ELISAs coupled with Western blot (WB) in providing evidence of Trichinella exposure or infection in wild boar. Serum samples were collected from 673 wild boar from a high- and low-risk region for Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were examined using both an ‘in-house’ and a commercially available indirect-ELISA that used excretory–secretory (E/S) antigens. Cut-off values for positive results were determined using sera from the low-risk population. All wild boar from the high-risk region (352) and 139/321 (43.3%) of the wild boar from the low-risk region were tested by artificial digestion. Testing by Western blot using E/S antigens, and a Trichinella-specific real-time PCR was also carried out on all ELISA-positive samples. The two ELISAs correctly classified all positive controls as well as one naturally infected wild boar from Gabba Island in the Torres Strait. In both the high- and low-risk populations, the ELISA results showed substantial agreement (k-value = 0.66) that increased to very good (k-value = 0.82) when WB-positive only samples were compared. The results of testing sera collected from the Australian mainland showed the Trichinella seroprevalence was 3.5% (95% C.I. 0.0–8.0) and 2.3% (95% C.I. 0.0–5.6) using the in-house and commercial ELISA coupled with WB respectively. These estimates were significantly higher (P < 0.05) than the artificial digestion estimate of 0.0% (95% C.I. 0.0–1.1). Real-time PCR testing of muscle from seropositive animals did not detect Trichinella DNA in any mainland animals, but did reveal the presence of a second larvae-positive wild boar on Gabba Island, supporting its utility as an alternative, highly sensitive method in muscle examination. The serology results suggest Australian wildlife may have been exposed to Trichinella parasites. However, because of the possibility of non-specific reactions with other parasitic infections, more work using well-defined cohorts of positive and negative samples is required. Even if the specificity of the ELISAs is proven to be low, their ability to correctly classify the small number of true positive sera in this study indicates utility in screening wild boar populations for reactive sera which can be followed up with additional testing.     

Trichinella pseudospiralis in pig from Croatia

Trichinella spiralis and Trichinella britovi are species that are frequently found in domestic pigs and various sylvatic animals in Croatia. During routine trichinoscopy, non-encapsulated larvae were detected in the muscle tissue of a domestic pig. Artificial digestion revealed a larvae burden of 602 muscle larvae per gram of tissue. Tissue section analysis confirmed the presence of non-encapsulated larvae. Multiplex PCR identified the larvae as T. pseudospiralis. This observation is consistent with the reports of a local veterinary inspector who described the presence of non-encapsulated Trichinella in four individual cases over the last 2 years. This is the first report of T. pseudospiralis in Croatia and one of very few cases of T. pseudospiralis infection described in domestic pigs. The detection of non-encapsulated larvae stresses the need for implementation of artificial digestion instead of trichinoscopy for the detection and identification of Trichinella infections.     

Emerging Trichinella britovi infections in free ranging pigs of Greece

Trichinella infections in humans and pigs have been documented in Greece since 1945 and a high prevalence of infection in pigs occurred in the 1950s. Up to 1984 only sporadic infections in humans were documented, and this zoonosis was not considered as a public health problem until 2009 when a human outbreak caused by the consumption of pork from an organic pig farm occurred. In the present study, we describe the re-emergence of Trichinella spp. infections in free-ranging pigs from organic farms of 3 counties (Dramas, Evros and Kavala) in Northern-Eastern Greece during the period 2009–2012. Totally 37 out of 12,717 (0.29%) free-ranging pigs which were tested during the period in question, were positive for Trichinella spp. larvae. The etiological agent was identified as Trichinella britovi. The average larval burden was 13.7 in the masseter, 6.2 in the foreleg muscles and 7.5 in the diaphragm. The 37 positive animals originated from seven free range pig farms. The practice of organic pig production systems in Greece has grown in popularity over the last years due to the increasing interest of consumers for products considered as traditional. However, this type of pig production increases the risk for Trichinella spp. infections, since animals can acquire the infection by feeding on carcasses or the offal of hunted or dead wild animals. The awareness and education of hunters and farmers is extremely important to reduce the transmission among free ranging pigs and the risk for humans.     

Gasterophilus spp. infections in horses from northern and central Kazakhstan

A cross-sectional survey was performed to obtain current data on the gastrointestinal myiasis of horses in the provinces of Kostanay, Akmola and Karagandy, northern and central Kazakhstan. The stomach, small intestine and rectum of 148 slaughter horses were examined for Gasterophilus spp. larvae during a 26-month study period. All horses were infected with 2nd and 3rd stage larvae (mean intensity: 803 ± 350), and 22% of them harboured >1000 Gasterophilus spp. larvae each. Four species were identified: G. intestinalis (prevalence: 100%; mean intensity: 361 ± 240 larvae), G. haemorrhoidalis (100%; 353 ± 191), G. nasalis (100%; 73 ± 36) and G. pecorum (91.2%; 18 ± 10). Horses aged < 2 years were higher infected with Gasterophilus larvae than 2–4 years old animals. Both the prevalence and extremely high intensity of Gasterophilus infections of horses in these Kazakh regions suggest respective control measurements to improve the health and performance of the animals and to increase the economic income of horse owners.     

Humoral immune response of mice infected with low doses of Trichinella spiralis muscle larvae

Serological techniques are frequently used to detect parasite status and to monitor epidemiology and disease prevalence in important reservoir hosts of zoonotic diseases. Small mammals present the most important link in the epidemiological chain in the spread of trichinellosis. In experimental studies, high infective doses are used to provoke strong immune response of laboratory animals. Wild animals, however, could be infected with very low numbers of Trichinella larvae. The aim of this work was to reveal the size of infective doses that can evoke an adequate immune response with detectable level of specific antibodies in mice. Sixty inbred (Balb/c) mice were infected with 50 L1 and 60 outbred (ICR) mice were infected with 5 L1 T. spiralis. The total larval burdens (TLB) in the intestinal and muscle phases, reproductive capacity index (RCI), and the kinetics of development of specific antibodies by iELISA with different conjugates were determined. In the first 10 days post infection (dpi), more adults were found in the intestines of inbred mice. In both mice strains, the first muscle larvae were observed at 20 dpi. The RCI was significantly higher in outbred mice. Sero-conversion of IgM antibodies was detected at 30 dpi. The IgG antibodies appeared at 40 dpi in inbred mice, and at 50 dpi in outbred mice. Using a polyvalent conjugate, the earliest sero-conversion was recorded at 30 dpi. Antibody levels increased until the end of the experiment (80 dpi). Our results support the suitability of ELISA in large epidemiological surveys to detect low-level infection in naturally infected small mammals, and are useful in epidemiological studies of the sylvatic circulation of trichinellosis to determine likely modes of transmission.     

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.     

Evaluation of three immunoserological techniques in the detection of porcine trichinellosis

Three immunoserological tests (IST) used for the detection of porcine trichinellosis, immunofluorescence (IF), enzyme-inmunoanalysis (EIA), and Western blot (WB), were compared. Three groups of animals were analyzed: Group 1, animals naturally infected with parasite burdens (PB) of <1 muscle larvae (ML)/g (n = 18); Group 2, animals naturally infected with PB of ≥2 ML/g (n = 23); Group 3, animals raised and home-slaughtered on farms in Argentina (n = 59). Animals from Groups 1 and 2 were identified in outbreaks and were analyzed by individual artificial digestion (AD) of ≥30 g of muscle. Animals in Group 3 were subjected to AD of 5 g of muscle.The detection percentages in sera of swine with the lower PB were 100% for IF, 72% for EIA, and 50% for WB. Eighty-three percent of the animals were serologically positive by two or three techniques. In pigs with the higher PB, the detection percentage was similar for IF and EIA (100% vs. 91%, respectively), and was lower for the WB (61%). Ninety-six percent of the animals were serologically positive by two or three techniques. Group 3 animals had similar detection percentages for the three techniques (IF, 30%; EIA, 29%; WB, 42%). Twenty-five percent of the animals were serologically positive by two or three techniques. Two animals were positive by AD with PB of 0.33 and 2.4 ML/g, and were positive for IF and WB, or IF, EIA, and WB. Results indicate that the sensitivity of each technique depends on the PB, and always ranked in sensitivity as IF > EIA > WB. For the lower PB, the decrease in the sensitivity is more pronounced for the EIA. Although the WB has a low sensitivity, the detection of the specific bands for Trichinella spiralis makes it a useful confirmatory tool. Considering that more than 83% of the parasitologically positive animals had 2 or 3 positive serological results using the techniques tested here, for the diagnosis of porcine trichinellosis, pigs positive by two of these serological techniques must be regarded as truly infected pigs.     

Micro-environmental conditions modulate protein secretion and infectivity of the Trichinella spiralis L1 larva

After digestion of infected meat the free L1 of Trichinella spp. penetrate the intestinal mucosa where they moult to the mature adult stage. We have used proteomics to identify changes in protein secretion during in vitro culture of free T. spiralis muscle larvae under different environmental conditions, and to correlate these changes with their infectivity in mice. Muscle larvae were cultured in different media (RPMI-1640, C-199 and HBSS) under conditions of anaerobiosis, microaerobiosis and in 5% CO₂ at 37 °C. Following incubation the larval excretory/secretory proteins were analysed by two-dimensional gel electrophoresis and the larvae were used to orally infect naïve CD1 mice. For all culture media tested, infectivity of the L1 was preserved following incubation in anaerobic conditions. In contrast, the infectivity of worms cultured in nutrient-rich media was almost completely abolished in both microaerobiosis and in the presence of 5% CO₂. Some infectivity was retained in poor or reduced culture media. Comparative analysis of larval infectivity and protein secretion showed that loss of infectivity correlated with the appearance of non-tyvelosylated proteins that in turn may be related to the onset of moulting.     

Report of Trichinella spiralis in a red fox (Vulpes vulpes) in Northern Ireland

No systematic studies of the occurrence of Trichinella in wildlife have been carried out in Northern Ireland (NI) in recent years, and the last reports of trichinellosis in livestock and human outbreaks in NI date back to 1979 and 1945, respectively. In this study, covering the period 2003/2004 and 2007/2008, a total of 443 red foxes (Vulpes vulpes) were collected throughout the country and screened for trichinellosis using a modified muscle digest method. One examined animal was found to be infected with larvae from Trichinella spiralis, indicating a national prevalence in NI of Trichinella in foxes of 0.2%. This prevalence compares well to the findings reported from the bordering Republic of Ireland [Rafter, P., Marucci, G., Brangan, P., Pozio, E., 2005. Rediscovery of Trichinella spiralis in red foxes (Vulpes vulpes) in Ireland after 30 years of oblivion. J. Infect. 50, 61–65] and could be a further indication for a sylvatic Trichinella life cycle existing independently from the domestic cycle.     

Trichinellosis Outbreak Caused by Meat from a Wild Boar Hunted in an Italian Region Considered to be at Negligible Risk for Trichinella

The wild boar is an important source of trichinellosis for people in European countries as a large number of hunted animals escape veterinary control. In November 2012, uncooked sausages made with meat from wild boar were consumed by 38 persons in a village of the Lucca province (Tuscany region, Italy). Of them, 34 were serologically positive, 32 developed clinical signs and symptoms of trichinellosis, and two were asymptomatic. Trichinella britovi larvae were detected in vacuum‐packed sausages made with the same batch of sausages consumed raw which had been prepared with meat from wild boar hunted in the Lucca province. As no case of trichinellosis had been reported in this region during the last 20 years, the regional public health authority considered the risk for this zoonosis to be negligible and put in place a surveillance programme on Trichinella spp. in indicator animals (mainly foxes and including wild boar for private consumption), by testing only a percentage of heads. The experience from this outbreak shows that the definition of a region with a negligible risk for Trichinella infection is not applicable to wild boar and stresses the need to test all Trichinella‐susceptible wild animals intended for human consumption and to implement risk communication to consumers and hunters.     

Strain difference in susceptibility to Trichinella spiralis infection between dark agouti and albino oxford rats

The influence of host genetics on the susceptibility to primary Trichinella spiralis infection has been extensively studied in a mouse model, but has not been clarified for rats. Analyses of interstrain and intrastrain genetic variation in response to infectious agents could be beneficial not only for elucidating the genetic basis of host resistance/susceptibility, but for revealing immune response mechanisms as well. The aim of this study was to analyse interstrain differences in worm burdens and cytokine production between Albino Oxford (AO) and Dark Agouti (DA) rats in muscle phase of T. spiralis infection. Clear strain-dependent variation was observed in the number of T. spiralis larvae per gram (lpg) of muscle tissue where values for DA rats (626.7 ± 171 lpg) vastly exceeded those found in AO rats (49.8 ± 25.9 lpg, p < 0.001). Differences between the strains were also noticed in key cytokine levels. In infected AO rats, the cytokine production remained in favor of Th1 type response, while infected DA rats showed a shift towards a Th2 type response. The level of regulatory IL-10 was significantly increased only in T. spiralis infected DA rats. Our results provide evidence that DA rats express higher susceptibility to T. spiralis infection in comparison to AO rats with respect to muscle larvae burden. The infection in DA rats was accompanied by the production of anti-inflammatory cytokines, while the response of AO rats was characterized by a proinflammatory type of immune response.     

Second outbreak of Trichinella pseudospiralis in Europe: clinical patterns,epidemiological investigation and identification of the etiological agent based on the western blot patterns of the patients' serum

Trichinellosis is a zoonotic disease due to the ingestion of raw or undercooked meat from animals infected with the larvae of nematodes belonging to the genus Trichinella. In January–February 2015, an outbreak of trichinellosis occurred in Genoa, Northern Italy. The epidemiological link was traced back to a dinner served at an agritourism farm on 31 December 2014, where a majority of the 52 guests had consumed the ‘beef’ steak tartare. The source of infection was not traced; however, it was noted that the amount of beef purchased officially for providing at the dinner did not correspond with that served, suggesting that meat of a different origin had been added to the beef to prepare the steak tartare. Clinical and laboratory data of 30 individuals out of the 52 (57.7%), of which four were hospitalized, were consistent with that of the case definition of trichinellosis. Western blot patterns of the sera from patients with confirmed trichinellosis were similar to the diagnostic pattern identified for the reference sera of Trichinella pseudospiralis but different from those of the control sera tested for patients infected with Trichinella spiralis and Trichinella britovi. Identification of T. pseudospiralis as the aetiological agent responsible for the outbreak of trichinellosis using an indirect tool represents an advancement in the epidemiological investigation of this zoonotic disease.     

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.     

Trichinella spiralis—A potential anti-tumor agent

Murine forestomach carcinoma (cell line MFC), ascitic hepatoma (cell line H22) and sarcoma (cell line S180) solid tumor models were used to test the anti-tumor effect of Trichinella spiralis in vivo. Mice previously infected by oral administration of 400 viable T. spiralis larvae per mouse for 7 days were grafted with various solid tumor cell lines. Other groups of tumor-bearing mice were given caudal vein injection of crude extracts of adult and newborn larvae at 17.5, 35.0 or 70.0 mg kg^?1. These treatments to inhibit tumor growth were dose-dependent (p < 0.05). The anti-proliferative activity of crude T. spiralis extract was examined in vitro at 0.035, 0.070 or 0.140 mg ml^?1 using MFC, H22, S180, human chronic myeloid leukemia cell line (K562) and hepatoma cell line (H7402), tumor cell proliferation in vitro was measured by methyl thiazolium stain and was inhibited in dose-dependent manner (p < 0.05). At the same doses, crude T. spiralis extracts induced apoptosis of K562 and H7402 as detected by DNA fragmentation. Cell cycle analysis indicated that crude T. spiralis extracts, at 0.140 mg ml^?1, arrested the cell cycle of K562 and H7402 in G1 or S phase. It is concluded that T. spiralis contains anti-tumor active agent.