Trichinella in pork: current knowledge on the suitability of freezing as a public health measure.
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Animals↗
Biomedical subjects
C M O Kapel
Publications and source records attributed to C M O Kapel.
Molecular epidemiology of Trichinella spp. in three Baltic countries: Lithuania, Latvia, and Estonia.
Meat of domestic pigs and wild boars has been the significant source of emerged human trichinellosis in Lithuania, Latvia, and Estonia over the past two decades. However, there is very little known on the occurrence of Trichinella spp. in main wildlife reservoirs and its transmission in domestic and sylvatic cycles in these countries. The present study demonstrated considerably higher endemicity of Trichinella spp. in main sylvatic reservoirs (28.9-42% in foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in all three countries than previously reported. Molecular identification of Trichinella larvae from more than 500 sylvatic and domestic animals revealed four Trichinella species (Trichinella spiralis, Trichinella britovi, Trichinella nativa, and Trichinella pseudospiralis) sympatric in a relatively small area and several as the first records for the respective countries. The nonencapsulating T. pseudospiralis is found for the first time in the Eastern Europe. Sylvatic T. britovi was found in domestic pigs in Lithuania and Latvia (16 and 57.1%, respectively) and only in these countries, domestic T. spiralis was detected in sylvatic animals in areas where domestic trichinellosis was registered. The study suggests that transmission of Trichinella between domestic and sylvatic cycles in Lithuania and Latvia is favored by improper human behavior, e.g., pig and slaughter waste management.
Animals↗
Helminths of red foxes (Vulpes vulpes) in Denmark.
An epidemiological study of helminths in 1040 red foxes collected from various localities in Denmark during 1997-2002, revealed 21 helminth species at autopsy, including nine nematode species: Capillaria plica (prevalence 80.5%), Capillaria aerophila (74.1%), Crenosoma vulpis (17.4%), Angiostrongylus vasorum (48.6% from Northern Zealand (endemic area)), Toxocara canis (59.4%), Toxascaris leonina (0.6%), Uncinaria stenocephala (68.6%), Ancylostoma caninum (0.6%), and Trichuris vulpis (0.5%); seven cestodes: Mesocestoides sp. (35.6%), a number of Taeniid species (Taenia pisiformis, T. hydatigena, T. taeniaeformis, T. crassiceps, and unidentified Taenia spp.) (22.8%), and Echinococcus multilocularis (0.3%); four trematodes: Alaria alata (15.4%), Cryptocotyle lingua (23.8%), Pseudamphystomum truncatum (3.6% from Northern Zealand), and Echinochasmus perfoliatus (2.4% from Northern Zealand); one acanthocephalan: Polymorphus sp. (1.2%). Significant difference in prevalence was found for T. canis and A. vasorum according to host sex, and for T. canis, U. stenocephala, Mesocestoides sp., Taenia spp., A. alata, A. vasorum, and Capillaria spp. according to age groups (adult, young or cub). Prevalence and average worm intensity for each helminth species varied considerably according to geographical locality, season, and year. Aggregated distribution was found for several helminth species. The two species E. multilocularis and E. perfoliatus are first records for Denmark.
Age Factors↗
Comparative development of Echinococcus multilocularis in its definitive hosts.
The comparative development of Echinococcus multilocularis was studied in its definitive hosts, the fox, dog, cat and raccoon dog, beyond the pre-patent period to 90 days post-infection. All host species, apart from cats were susceptible to infection and capable of supporting substantial worm burdens. Although worms in cats matured and produced thick-shelled eggs, their overall development was retarded compared to that in other species in which the parasite matured rapidly producing large populations of gravid worms. E. multilocularis matured rapidly in foxes and raccoon dogs and this was sustained in raccoon dogs but not in foxes in which maturation of worms declined during the later stages of infection, in contrast to that in both raccoon dogs and dogs. These populations were sustained for longer in raccoon dogs and dogs compared to foxes. Cats would appear to have only a minor role in the maintenance of E. multilocularis in endemic areas, and infections in cats may be of minimal public health significance. In contrast, foxes, dogs and the recently recognized definitive host the raccoon dog, are all capable of playing significant roles in the epidemiology of alveolar echinococcosis. This study also demonstrated that the developmental processes of growth, segmentation, proglottization and maturation in adult Echinococcus are independent and can be influenced by environmental factors thus confirming earlier in vitro observations.
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Meat inspection for Trichinella in pork, horsemeat and game within the EU: available technology and its present implementation.
A new EU directive relating to meat inspection for Trichinella, expected to come into force in 2006, imposes important modifications to current legislation. Nevertheless, several issues need more attention. Optimisation of methods, especially concerning sensitivity and digestibility of the meat to be inspected, along with further simplification of the legislation with regard to the number of techniques accepted, is recommended to guarantee that all member states of the EU will be given tools to perform inspection of consumer meat at the same high level. Additionally, there is a need for guidelines and protocols regarding optimal proficiency testing procedures. This paper presents an overview of the current methods for Trichinella meat inspection and their implementation in the EU, listing advantages and disadvantages for each method, including some suggestions for specific points of improvement.
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Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats.
A total of 15 red foxes, 15 raccoon dogs, 15 domestic dogs and 15 domestic cats were each infected with 20,000 protoscolices of Echinococcus multilocularis. At 35, 63, and 90 days post inoculation (dpi), five animals from each group were necropsied and the worm burdens determined. The highest worm burdens in foxes (mean of 16,792) and raccoon dogs (mean of 7930) were found at 35 dpi. These declined to a mean of just 331 worms in foxes and 3213 worms in raccoon dogs by day 63 with a further decline to 134 worms in foxes and 67 worms in raccoon dogs by day 90. In dogs, there was no significant difference between worm burdens recovered at days 35 (mean of 2466) and day 90 (mean of 1563), although reduced numbers were recovered on day 63 (mean of 899). In cats, worms were found in four animals 35 dpi (mean of 642), in three at 63 dpi (mean of 28) and in two at 90 dpi (mean of 57). Faecal egg counts were determined at 3 day intervals from 25 dpi. A mathematical model of egg excretion dynamics suggested that the mean biotic potential per infected animal was high in foxes (346,473 eggs); raccoon dogs (335,361 eggs) and dogs (279,910 eggs) but very low for cats (573 eggs). It also indicated that approximately 114, 42 and 27 eggs per worm were excreted in the faeces of dogs, raccoon dogs and foxes, respectively. The fecundity of worms in cats was low with an average of less than one egg per worm. The peak levels of coproantigen were detected earlier in foxes and raccoon dogs than in dogs. Eggs recovered from foxes, raccoon dogs and dogs resulted in massive infections in experimental mice. However, metacestodes did not develop from eggs originating from infected cats. It is concluded that foxes, raccoon dogs and dogs are good hosts of E. multilocularis. In contrast, the low worm establishment, the very few excreted eggs and the lack of infectivity of eggs strongly indicate that cats play an insignificant role in parasite transmission.
Animal Diseases↗
Molecular cloning of a cDNA encoding a putative cuticle collagen of Trichinella spiralis.
A 5-day-old adult stage-specific cDNA fragment from Trichinella spiralis was identified by suppression subtractive hybridization and was used as a probe to screen the cDNA library. The cDNA sequence coding for a putative T. spiralis cuticle collagen was isolated. The cDNA encoded an open reading frame of 343 amino acid residues with molecular weight of 35.1 k Da. The deduced protein contained an N-terminal signal peptide, a nematode cuticle collagen N-terminal domain and a collagen triple helix repeat domain. Searches in GenBank using BLASTP showed up to 47% identity to cuticle collagens from other nematodes. Southern blot analysis of genomic DNA indicated this gene was present as a single copy in T. spiralis genome.
Amino Acid Sequence↗
Effects of raw biles and their non-protein fractions from fox, pig, sheep and chicken on the survival of Trichinella spp. in vitro.
The in vitro differential effect of fox, pig, sheep and chicken bile and corresponding non-protein fractions at various concentrations on the motility of released muscle larvae of Trichinella spiralis, Trichinella nativa and Trichinella nelsoni was examined. In many cases, the percentages of motile (live) larvae of the three Trichinella species cultured in the presence of the non-protein fractions of bile from the study animals were significantly higher (p<0.001) compared to their respective control cultures. In addition, the percentages of motile (live) larvae of all Trichinella species cultured in the presence of the non-protein fraction of bile at every concentration from all study animals were significantly higher (p<0.001) compared to their respective cultures in the presence of raw bile. Not only did results with non-protein fractions of bile differ from those with raw bile, but also the non-protein fraction, with increased dilution, showed a decrease in the percentages of motile larvae while the opposite was true with the raw bile (p<0.001). These observations indicate that the non-protein fraction of bile prolongs the in vitro survival of larvae.
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Comparison of two antigens for demonstration of Trichinella spp. antibodies in blood and muscle fluid of foxes, pigs and wild boars.
For the surveillance of trichinellosis, the digestion method is reliable but also labour intensive. The serological methods for the detection of Trichinella-specific antibodies using ELISA offer a sensitive and relatively specific alternative. For serological studies, sera or plasma from blood samples are the most common source of antibodies, but although the concentration of antibodies is approximately 10-fold lower, muscle fluid can be a good alternative particularly for testing of wildlife samples. In the present study, an indirect ELISA technique was evaluated on both sera and muscle fluids from experimentally infected foxes, pigs, and wild boars using both excretory/secretory (E/S) antigens and a synthetic glycan antigen, beta-tyvelose. Although the synthetic antigen appears to be less sensitive than the E/S antigens, Trichinella-specific IgG antibodies were detected in both serum samples and muscle fluid samples from pigs, wild boars and foxes infected at levels which would be important for food safety or represent a significant reservoir for further transmission.
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Experimental studies in pigs on Trichinella detection in different diagnostic matrices.
A total of 72 specific pathogen-free (SPF) and Iberian pigs (three animals per group) were inoculated with 200, 1000 or 20,000 muscle larvae of T. spiralis, T. nativa, T. britovi and T. pseudospiralis. For each animal, the muscle larva burden was evaluated in nine muscle samples by digestion. The anti-Trichinella IgG kinetics in blood samples, taken twice prior and at days 5, 10, 15, 20, 25, 30, 40, 50 and 60 post-inoculation, and in muscle juice, obtained at necropsy, was evaluated by an ELISA using an excretory/secretory antigen. The mean larval recovery rate in SPF/Iberian pigs corresponded with the level of inoculum dose, and tongue, diaphragm and masseter were identified as predilection muscles. In SPF and Iberian pigs receiving 20,000 larvae of T. spiralis, an earlier seroconversion was detected from day 25 post-inoculation. At a 10-fold dilution, the muscle juice showed a good test agreement with blood serum.
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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.
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Serological evidence of Trichinellosis in local pigs of Nepal.
In Nepal, animal husbandry is a major source of income. Pig husbandry is practiced in rural, peri-urban, and urban communities. Free ranging "back yard" pigs and the practice of feeding offal is a very common management practice which potentially allows for the transmission of trichinellosis; however, this zoonosis has never been reported from this region. A total of 425 serum samples were collected from local pigs. These were initially screened by ELISA after which positive samples were examined by Western blot. This procedure identified two samples which had clear specific bands for Trichinella; however, muscle samples tested by HCL-pepsin digestion were found to be negative. If these highly specific serological analyses are confirmed, this would be the first report of trichinellosis in Nepal and a prevention program should be initiated to limit the access of pigs to open garbage dumps which exist both in towns and on farms.
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Changes in the EU legislation on Trichinella inspection--new challenges in the epidemiology.
The European Union (EU) countries are searching for new ways to certify meat free of Trichinella; however, with the expansion of the EU, the acceptance of a unilateral method is complicated by the variability of pig and human trichinellosis among EU countries, where significantly higher prevalence rates have been observed in the newly added eastern countries. Several attempts have been made to define Trichinella-free areas, but certification of Trichinella-free pig production farms appears to be the only feasible approach. The increasing prevalence of the non-encapsulating species, Trichinella pseudospiralis, in game, domestic pigs and humans has eliminated the compression technique from the new EU legislation to be enacted in 2006. Also, the observation that several species of Trichinella tolerate freezing in horse meat for up to 4 weeks has forced a change in legislation as well where freezing is no longer an option for certifying horse meat. Because current serological detection methods are not suited for meat inspection, classical direct detection methods and inactivation by freezing remain the methods of choice for pork. It has been proposed, therefore, to automate direct inspection methods as a cost effective alternative to certify pig farms free of Trichinella.
Animal Husbandry↗
Cloning and analysis of a novel cDNA from Trichinella spiralis encoding a protein with an FYVE zinc finger domain.
A cDNA library from Trichinella spiralis adults 3 days post-infection was screened with a cDNA probe, designated T 54, derived from a newborn larvae subtracted cDNA library. Sequence analysis showed that the positive clone contained a cDNA insert of 1464 bp in length with a single open reading frame of 1290 bp, which encoded a protein of 429 amino acids with a putative molecular mass of 49.9 k Da. Database analysis predicted the deduced protein had a leucine zipper motif and an FYVE zinc finger domain. The recombinant fusion protein was expressed and rabbit anti-recombinant protein sera reacted with a single peptide migrating at approximately 55 k Da in crude worm extract from muscle larvae, adults and newborn larvae stages.
Amino Acid Sequence↗
Experimental alveolar echinococcosis in pigs, lesion development and serological follow up.
Liver lesions were found in 6/6 pigs 7 months after oral inoculation with 5000 or 35,000 Echinococcus multilocularis eggs. However, lesion morphology differed considerably among the animals. The largest lesions (3-8mm in diameter) were found in a single pig and smaller lesions (1.5-3mm) in three pigs. These lesions were clearly circumscribed and had pronounced central necroses and dystrophic calcifications. In contrast, most of the smallest (usually <1.5mm in diameter) found in two other pigs, had small compact fibrotic areas and blurred borders with obvious fibrous infiltrations into the interlobular tissues. E. multilocularis specific DNA was detected by PCR in all lesion types, but metacestode viability, as assessed by in vivo intraperitoneal inoculations in jirds, could not be demonstrated. Within 1 month post inoculation, all pigs developed specific IgG antibody responses against a battery of different antigens (metacestode, cyst fluid, and protoscoleces-derived native E. multilocularis and E. granulosus antigens, affinity purified Em2G11 antigen, antigen B, recombinant Em II/3-10 antigen). Two different reaction patterns were recorded. In the two pigs with the small lesions, pronounced reactions against all crude antigens with peaks 3-5 months p.i. and clearly elevated levels until the end of the experiment were noted. In all other pigs, antibody reactions remained low in all cases. In conclusion, we demonstrated two types of E. multilocularis metacestode development in pigs with distinct immunological response patterns.
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Studies on vertical transmission of Trichinella spp. in experimentally infected ferrets (Mustela putorius furo), foxes (Vulpes vulpes), pigs, guinea pigs and mice.
Vertical transmission of Trichinella spiralis was evaluated in ferrets (n=21), foxes (n=11), pigs (n=12), guinea pigs (n=16), and mice (n=41). The placental barrier to be crossed by migratory Trichinella larvae varies structurally in different animal species. Ferrets and foxes have an endotheliochorial placenta structure, guinea pigs and mice a haemochorial, and pigs an epitheliochorial placenta. The non-encapsulating Trichinella pseudospiralis larvae have an extended muscle migration prior to entering a muscle cell. To evaluate if T. pseudospiralis was more likely to be transmitted to offspring, an additional group of foxes (n=11) infected with T. pseudospiralis was included. Two different dose levels were used for ferrets, pigs, guinea pigs, and mice. In pigs and guinea pigs, infection was given at different times of the gestation period. Vertical transmission, measured as recovery of muscle larvae in the offspring, was demonstrated in both ferrets groups, in all four guinea pig groups, and in the high dose mouse group, but not in any fox or pig groups.
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Trichinella spiralis: enteric mucin-related response to experimental infection in conventional and SPF pigs.
Duodenal and jejunal responses to infection with Trichinella spiralis were compared in weaned piglets with a "normal dirty" vs. a "clean SPF" gut flora. Histochemical staining of neutral, acidic, sialylated, and sulphated residues was used to assess biosynthetic responses in mucin-secreting goblet cells. Peanut and Ulex lectins were also used to assess responses within the intestinal glycocalyx. Histomorphometric analysis was undertaken to evaluate the distribution and staining patterns of goblet cells in villi and crypts. Our analysis showed that stored mucin within goblet cells increased more in the infected conventional animals than in the infected SPF group. This was accompanied by changes in the pattern of sulphation and sialylation in the duodenum and jejunum. The thickness of the glycocalyx was increased in both duodenum and jejunum in both infected groups. However, this effect was greater for the infected SPF animals than the infected conventional animals. No significant differences were observed between uninfected conventional and uninfected SPF pigs.
Animal Husbandry↗
Zoonotic risk of Toxocara canis infection through consumption of pig or poultry viscera.
The potential zoonotic risk of Toxocara canis infections from consumption of swine or poultry viscera containing larvae was assessed using a pig model. Two groups of six pigs were fed either fresh swine viscera (group FS) or poultry viscera (FP) containing around 3500 Toxocara larvae. Another two groups of six pigs were fed swine viscera (PS) or poultry viscera (PP) preserved at 4 degrees C for 1 week. All pigs were necropsied 14 days after the exposure. Liver white spots were counted and T. canis specific IgG antibodies were measured by ELISA. Larval burdens were assessed in the mesenteric lymph nodes, liver, lungs, brain, tongue, and eyes. All recipient pigs exhibited several white spots on the liver surface and detectable antibody levels. Larvae were recovered predominantly from the lungs, but also from the mesenteric lymph nodes and the liver, a few larvae were found in the brain and tongue of the pigs. Two larvae were found in the eyes of two pigs in group FS. Mean percentages of total larval recoveries in groups FS, FP, PS, and PP were 75.3, 63.6, 42.6, and 18.8%, respectively. Significantly higher numbers of larvae were recovered from pigs given swine viscera than pigs given poultry viscera. The preservation at 4 degrees C for 1 week caused a significant reduction in the larval infectivity overall, nevertheless, the recoveries remained substantial. The fact that larvae migrating in swine or poultry organs and tissues have high infectivity in pigs even after preservation at 4 degrees C for 1 week, suggests that human infection with T. canis might easily occur following consumption of raw or undercooked dishes, either fresh or refrigerated, prepared from swine or poultry organs and tissues harbouring T. canis larvae.
Animal Feed↗