Species-specific antibody responses to the recombinant 53-kilodalton excretory and secretory proteins in mice infected with Trichinella spp

The 53-kDa proteins in larval excretory and secretory (E-S) products were expressed from five Trichinella species (T. spiralis, T. britovi, T. nativa, T. pseudospiralis, and T. papuae), using the Escherichia coli expression system, and the antibody responses to the 53-kDa recombinant proteins in mice infected with Trichinella spp. were analyzed by Western blotting. The 53-kDa protein is conserved among the five Trichinella species, with >60% similarity in amino acid sequences. The 53-kDa recombinant proteins of T. spiralis and T. pseudospiralis reacted to sera from mice infected with T. spiralis and T. pseudospiralis at 8 days postinfection (p.i.), respectively. An antibody against the 53-kDa recombinant protein of T. spiralis recognized the 53-kDa protein in the crude extracts from adult worms and 30-day p.i. muscle larvae and E-S products from muscle larvae of T. spiralis but did not recognize any proteins from T. pseudospiralis. The sera from the mice infected with T. spiralis strongly reacted with the 53-kDa recombinant protein of T. spiralis but did not react with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, and T. papuae. Similarly, the sera from mice infected with T. britovi, T. nativa, T. pseudospiralis, or T. papuae strongly reacted with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, or T. papuae, respectively. These results showed that the 53-kDa recombinant proteins provide early and species-specific antibody responses in mice infected with Trichinella spp.     

Molecular identification of Trichinella britovi in martens (Martes martes) and badgers (Meles meles); new host records in Poland

Trichinella larvae were detected in a marten (Martes martes) and a badger (Meles meles) in Poland. The animals were found dead following car accidents. All examined animals derived from the Mazurian Lake district, north-east Poland, near the village Kosewo Górne where Trichinella infection were earlier confirmed in wildlife; red foxes and wild boars. The muscle samples were examined by artificial pepsin-HCl digestion method. The parasites were identified as Trichinella britovi by multiplex polymerase chain reaction method. Larvae were found in two out of three martens and one out of seven examined badgers. This is the first report of the identification of Trichinella britovi larvae from martens and badgers in Poland.     

Molecular identification of<Emphasis Type="Italic"> Trichinella spiralis</Emphasis> and<Emphasis Type="Italic"> Trichinella britovi</Emphasis> by diagnostic multiprimer large mitochondrial rRNA amplification

Trichinella parasites with different epidemiological features still occur in Europe and four species of genus Trichinella have been identified: T. spiralis, T. britovi , T. nativa and T. pseudospiralis. Until now, two of them, T. spiralis and T. britovi, have been identified in Poland. In our studies we selected sequence coding for large mitochondrial rRNA (mt LrDNA) as a genetic marker and developed a sensitive LrDNA multiprimer PCR assay allowing for rapid identification of T. spiralis and T. britovi, parasites present in wild and domestic animals in Poland.     

Molecular epidemiology of <Emphasis Type="Italic">Trichinella</Emphasis> 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.     

First report of <Emphasis Type="Italic">Trichinella britovi</Emphasis> in Serbia

In Europe, Serbia ranks among countries with a high prevalence of Trichinella infection in pigs, which continues to be a serious human health problem. While in some Balkan countries, more than one Trichinella species/genotype has been described in both the sylvatic and domestic cycles, these data are lacking for Serbia. To date, only a few Serbian isolates of Trichinella have been genetically specified, and all were classified as T. spiralis. Although transmission of Trichinella from domestic pigs to wildlife could be assumed, neither the infection status nor the species of Trichinella circulating among wildlife in Serbia has been investigated. This study shows the presence of two Trichinella species, T. spiralis and T. britovi, in wild animals originating from five districts in Serbia, where Trichinella infections in domestic pigs and humans have been recorded. Trichinella spiralis was detected in jackals (n = 3), red foxes (n = 2) and a wild cat (n = 1). We also established that wolves (n = 4) and red foxes (n = 2) serve as sylvatic reservoirs for T. britovi. This is the first report on the presence of T. britovi in Serbia.     

Trichinella sp. in red foxes (Vulpes vulpes) from Catalonia, NE Spain

European legislation allows the official recognition of Trichinella-free pig holdings, provided Trichinella sp. infection is absent from humans and prevalence of Trichinella sp. infection in red foxes (Vulpes vulpes) is below 0.1% in the area, region or country. Tibialis anterior muscle samples from 1,319 red foxes captured in Catalonia (NE Spain) between 1998 and 2007 were analyzed for Trichinella sp. using the digestion method. Four foxes resulted positive (one in 1999, one in 2002 and two in 2006), accounting for a low prevalence (0.3%). However, this prevalence was concentrated in mountain or rural areas with a low sample size, reaching high local prevalences. The two positive samples in 2006 were characterized as Trichinella britovi, and a sylvatic cycle of trichinellosis seems to occur, at least in the rural insufficiently sampled regions of Catalonia. Overall, the results obtained do not currently allow the establishment of Trichinella-free pig holdings in the study area, but further research is needed to better know the prevalence and cycle of Trichinella sp. in Catalonia.     

Development of cellular immune response of mice to infection with low doses of Trichinella spiralis, Trichinella britovi and Trichinella pseudospiralis larvae

The murine cellular immune response to the infection with ten larvae of encapsulating (Trichinella spiralis, Trichinella britovi) and non-encapsulating species (Trichinella pseudospiralis) was studied. Both T. spiralis and T. britovi stimulated the proliferation of splenic T and B lymphocytes during the intestinal phase of infection, but T. spiralis activated the proliferative response also at the muscle phase, particularly in B cells. Non-encapsulating T. pseudospiralis stimulated the proliferation of T and B cells only on day 10 post-infection (p.i.) and later at the muscle phase. The numbers of splenic CD4 and CD8 T cells of T. spiralis infected mice were significantly increased till day 10 p.i., i.e., at the intestinal phase, and then at the late muscle phase, on day 60 p.i. T. britovi infection increased the CD4 and CD8 T cell numbers only on day 30 p.i. Decreased numbers of CD4 and CD8 T cells after T. pseudospiralis infection suggest a suppression of cellular immunity. Both encapsulating Trichinella species induced the Th2 response (cytokines interleukin-5 (IL-5) and interleukin-10) at the intestinal phase and the Th2 dominant response at the advanced muscle phase. Interferon-γ (IFN-γ) production (Th1 type) started to increase with migrating newborn larvae from day 15 p.i. till the end of the experiment. IL-5 production was suppressed during the intestinal phase of T. pseudospiralis infection. The immune response to T. pseudospiralis was directed more to the Th1 response at the muscle phase, the high IFN-γ production was found on day 10 p.i. and it peaked on days 45 and 60 p.i.     

Infectivity, predilection sites, and freeze tolerance of Trichinella spp. in experimentally infected sheep

A total of 36 sheep in groups of 4 were inoculated with 9 isolates of Trichinella and euthanized after 10 weeks. Thereafter, numbers of muscle larvae were determined in 13 different muscles/muscle groups. Muscle larvae were found in high numbers in all four sheep inoculated with T. spiralis, in lower numbers in two sheep inoculated with T. pseudospiralis (USA isolate), and in very low numbers in one sheep inoculated with T. pseudospiralis (USSR isolate) and one inoculated with T. britovi. In infections of high and moderate larval intensity, predilection sites of T. spiralis were the masseter muscles, the tongue, and the diaphragm and those of T. pseudospiralis were the masseter muscle and the neck. In low-intensity infections, muscle larvae were detected only in the diaphragm or in pooled muscle samples. For evaluation of the freeze tolerance of the different Trichinella species in sheep-muscle tissue, samples taken from the filet were stored at +5°, −5°, and −18 °C, respectively. After exposure for 1 and 4 weeks the tissue was digested and the released larvae were inoculated into mice for determination of the reproductive capacity index (RCI). Larvae of both T. spiralis and T. pseudospiralis survived freezing at −5° and −18 °C for 4 weeks. Received: 10 September 1999 / Accepted: 22 October 1999     

Apicomplexan parasites of red foxes (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>) in northeastern Poland

Molecular detection of apicomplexan parasites in splenic samples of red foxes collected from northeastern Poland was conducted by PCR amplification of a fragment of the 18S rRNA spanning the V4 gene region of Apicomplexa. Positive PCR products were further analysed by restriction fragment length polymorphism (RFLP) and sequencing to identify species. One hundred and eleven red foxes (Vulpes vulpes) were acquired from 15 localities in the Mazovian Province and 27 foxes were acquired from the Mazurian Lakeland. Apicomplexan 18S rDNA was detected in 15.9% of 138 fox spleens examined. Three apicomplexan species were identified: Hepatozoon canis was detected in 11.6% of the spleen samples, Toxoplasma gondii was detected in 3.6% of the spleen samples and a Babesia sp. was sequenced from 1 sample (0.7%). This data represent the first record of H. canis, T. gondii and a B. sp. from naturally infected red foxes in Poland. Infected foxes may act as sylvatic reservoirs of these apicomplexan parasites as well as serving as a source of infection for arthropod definitive hosts and vectors.     

Red fox (Vulpes vulpes) cannibalistic behaviour and the prevalence of Trichinella britovi in NW Italian Alps

Food habits of the red fox (Vulpes vulpes) were studied in the Aosta Valley region (NW Italian Alps) and were related to the prevalence of Trichinella infection in the red fox itself and in two Mustelid species (the stone marten (Martes foina) and the badger (Meles meles)). The search of Trichinella by the automatic digestion of muscles samples led us to determine a prevalence of 3.5±1.2% in red foxes, 7.9±4.3% in stone martens and 1.9±1.8% in badgers, with no significant differences among the species. All larvae were identified as Trichinella britovi. The fox diet was assessed through the analysis of both faeces (n=180) and the stomach contents of road-killed animals (n=109). Our results confirmed the opportunistic feeding behaviour of the red fox, which is able to use various trophic resources, both of animal and vegetal origin: e.g. wild and cultivated fruits (F%=47.1; V%=67.3), rodents (F%=22.8; V%=64.8) and carrion (F%=15.6; V%=78.6) formed the bulk of the foxs diet. The frequency of occurrence of potential events of cannibalism was 1.0%, even if the complete absence of undigested remains, other than hairs, suggested the possibility of confusing cannibalism with coat-cleaning. We suggest that intra-specific necrophagy could not represent the unique way of transmission of the nematode in natural conditions.     

Cloning and characterization of Rab and Ran/TC4 cDNA clones in Trichinella spp.

The cloning and characterization of seven Rab and three Ran/TC4 partial cDNA sequences in both cystic (Trichinella spiralis and T. britovi) and noncystic species (T. pseudospiralis) are reported. These molecules were cloned by rapid amplification of cDNA ends via polymerase chain reaction (RACE-PCR), using cDNA from the aforementioned Trichinella spp. coupled to the AP1 adaptor. As primers, AP1 and 5B (derived from the WDTAGQE sequence of region 2 specific for Rab and Ran proteins) sequences were included in the PCR. The cloned cDNAs were sequenced and characterized by both Southern-blot and Northern-blot analysis. Trichinella spp. Rab- and Ran-like molecules showed divergences in both the nucleotide and the deduced amino acid sequences as compared with the corresponding homologues previously described in other organisms. In addition, differences were observed among the Trichinella species, mainly between the cystic and the noncystic species, in both DNA restriction-enzyme polymorphism and expression of the six GTPases isolated. Received: 12 November 1998 / Accepted: 3 February 1999     

Biochemical analysis of encapsulated and non-encapsulated species of<Emphasis Type="Italic"> Trichinella</Emphasis> (Nematoda,Trichinellidae) from cold- and warm-blooded animals reveals a high genetic divergence in the genus

Multilocus enzyme electrophoresis was used to analyse genetic variation in the genus Trichinella. Twenty-eight isolates belonging to eight species and six genotypes were analysed for 12 enzyme systems, producing 19 different phenotypes. According to Jaccards similarity index, the isolates clustered into two main groups, specifically, encapsulated species/genotypes and non-encapsulated species/genotypes. Furthermore, the non-encapsulated species clustered into two other groups: the species infecting mammals and birds (Trichinella pseudospiralis) and those infecting mammals and reptiles (Trichinella papuae and Trichinella zimbabwensis). The encapsulated species/genotypes, which only infect mammals, clustered into four main groups: the cosmopolitan species Trichinella spiralis, the species/genotypes of the temperate regions (Trichinella britovi, Trichinella murrelli , Trichinella T8, and Trichinella T9), the species/genotype of the arctic region (Trichinella nativa and Trichinella T6), and the equatorial species Trichinella nelsoni. These results are consistent with biological, epidemiological, and molecular data, which show a high genetic divergence in this genus.     

Trichinella infection in wild animals from endemic regions of Argentina

Natural infection with Trichinella has been described in more than 150 mammalian species. However, few reports of Trichinella infection in wild animals have come from Argentina. In this study, muscle tissue was obtained from wild animals in Argentina with the aim of evaluating the presence of Trichinella. A total of 169 muscle samples were collected to determine the presence of Trichinella larvae by artificial digestion. The 169 muscle samples originated from 12 species including 36 opossums (Didelphis albiventris), 19 armadillos (Chaetophractus villosus), 9 capybaras (Hydrocaeris hydrocaeris), 1 puma (Puma concolor), 3 grey fox (Lycalopex gymnocercus), 6 coypus (Myocastor coypus), 6 skunks (Conepatus chinga), 2 ferrets (Galictis cuja), 66 rats (Rattus norvegicus), 6 mice (Mus musculus), 12 wild boars (Sus scrofa), and 3 wild cats (Felis geoffroyi). Trichinella infection was detected in 1 puma [2 larvae per gram (LPG)], 3 wild boars (8–420 LPG), 3 armadillos (0.04–0.08 LPG), and 9 rats (0.1 to 150 LPG). Only 3 Trichinella isolates, of 1 rat and 2 wild boars from Neuquén, were identified as Trichinella spiralis by nested PCR. The presence of Trichinella infection among wild animal populations suggests a sylvatic cycle of transmission in Argentina, which can serve as a reservoir for humans and domestic animals. Further, evidence of high prevalence in rats emphasizes the need to improve pig management, mainly in small individual farms without adequate technology, to enhance the quality of feeds, and to improve veterinary services to avoid exposure of pigs to Trichinella.     

The first report of Angiostrongylus vasorum (Nematoda; Metastrongyloidea) in Poland,in red foxes (Vulpes vulpes)

Angiostrongylus vasorum belongs to the superfamily of Metastrongyloidea. This nematode occurs in foxes, dogs and other predators. The Nematode A. vasorum place themselves in the pulmonary artery and its branches, and in the right ventricle and atrium of the heart. Numerous species of land snails are the intermediate hosts of the parasite. In 2013, lungs and hearts of 76 foxes shot in the Forest District G??boki Bród in Augustowska Primeval Forest were parasitologically necropsied. Four of the examined foxes were infected with the nematode A. vasorum, a prevalence of 5.2%. In one fox pericardium there were 6 male and 6 female nematodes. In the remaining three foxes nematodes were localized in the pulmonary artery. In two foxes 2 specimens of nematodes were detected (male and female, and two females) while 1 female was detected in the other fox. This is the first report of the presence of the nematode A. vasorum in fox in Poland.     

Differentiation ofTrichinella isolates by polymerase chain reaction

Oligonucleotide primers were synthesized for the polymerase-chain-reaction amplification of target DNA from two sequences ofTrichinella spiralis. Six strains belonging toT. spiralis, T. nativa, T. britovi, T. pseudospiralis, andT. nelsoni were tested. Amplification products were obtained withT. spiralis, T. britovi, andT. nelsoni DNA from a 53-kDa antigen cDNA sequence and withT. spiralis andT. nelsoni DNA from a 1.6-kb repetitive DNA sequence. Differences in the length of the amplification products obtained from the repetitive sequence would enable a differentiation betweenT. spiralis andT. nelsoni, suggesting that the 1.6 kb repetitive DNA sequence would not be specific forT. spiralis. No amplification was detected forT. nativa orT. pseudospiralis DNA from the two sequences and forT. britovi DNA from the 1.6-kb repetitive DNA sequence.     

Infections with cardiopulmonary and intestinal helminths and sarcoptic mange in red foxes from two different localities in Denmark

Monitoring parasitic infections in the red fox is essential for obtaining baseline knowledge on the spread of diseases of veterinary and medical importance. In this study, screening for cardiopulmonary and intestinal helminths and sarcoptic mange (Sarcoptes scabiei) was done on 118 foxes originating from two distinct localities in Denmark, (Copenhagen) greater area and southern Jutland. Fifteen parasite species were recorded in 116 foxes (98.3%), nine parasitic species are of zoonotic potential. Parasite diversity was greater in foxes of Copenhagen in terms of overall parasite species richness and species richness of all helminth groups individually: trematodes; cestodes; and nematodes. Six parasite species were recovered from foxes of Copenhagen, but not from foxes of Southern Jutland: Echinochasmus perfoliatus; Echinostoma sp.; Pseudamphistomum truncatum; Dipylidium caninum; Angiostrongylus vasorum; and Sarcoptes scabiei, but Toxascaris leonina was only recorded in foxes of southern Jutland. A high prevalence and abundance of A. vasorum in foxes of Copenhagen was observed. The prevalence of four nematode species; Eucoleus (Capillaria) aerophilus, Uncinaria stenocephala, Toxocara canis, and Crenosoma vulpis, in foxes of both localities were comparable and ranging from 22.9% to 89%. The prevalence of Mesocestoides sp. was significantly higher in foxes of Copenhagen. Taenia spp. were detected using morphological and molecular analysis, which revealed the dominance of T. polyacantha in foxes of both localities. Infections with sarcoptic mange were evident only among foxes of Copenhagen (44.9%), which significantly affected the average weight of the infected animals. Further remarks on the zoonotic and veterinary implications of the parasites recovered are given.     

Epidemiology, histpathology, and muscle distribution of Trichinella T9 in feral raccoons (Procyon lotor) and wildlife of Japan

The prevalences of Trichinella T9 in trapped raccoons (Procyon lotor) and several other potential mammalian reservoirs in Hokkaido, Wakayama, and Nagasaki Prefectures were investigated. Muscle samples were collected from 2003 to 2006 from 1,080 raccoons, 113 raccoon dogs including 2 species (Nyctereutes procyonoides albus and N. p. viverrinus), 41 wild boars (Sus scrofa leucomystax), 14 martens (Martes melampus), 10 badgers (Meles meles), 5 Siberian weasels (Martes sibirica coreana), 7 mink (Mustela vison), and 1 red fox (Vulpes vulpes japonica). The samples were digested, and the prevalence and mean intensity of infection with the Trichinella muscle larvae were determined. The prevalence and intensity of the muscle larvae were 0.9% and 93.3 larvae/g (range 0.4–201.8) in raccoons, and 1.6% and 61.6 larvae/g in raccoon dogs, respectively. The infected animals were captured in different areas in Hokkaido Prefecture. These results confirmed that raccoons, which have been introduced from North America since the 1970s, are involved in the sylvatic cycle of Trichinella in Japan. In raccoons, the muscle density of Trichinella T9 larvae was highest in the tongue, and larvae were not found in the heart muscle or diaphragm. This is the first report of Trichinella T9 infection of feral raccoons in Japan.     

Pathological findings of Angiostrongylus vasorum infection in red foxes (Vulpes vulpes) from Central Italy,with the first report of a disseminated infection in this host species

In Europe, the red fox (Vulpes vulpes) is considered the reservoir of Angiostrongylus vasorum, nematode residing in the pulmonary arteries and right heart of dogs and many species of wild carnivores. Italy is considered one of the European countries where this nematode is actually spreading. Between May 2007 and November 2013, 62 foxes found dead in Central Italy were necropsied. Right heart and pulmonary arteries were opened and checked for the presence of adult parasites. Impression smears from sectioned lungs were examined for the presence of first-stage larvae, and samples of lungs were processed for histological examination. In order to detect eventual disseminated infections, samples of heart, pulmonary lymph nodes, liver, kidneys, and brain of foxes positive for A. vasorum at necropsy or lungs histological examination were processed for histological examination. An overall prevalence of 43.5 % was recorded. Light, mild, and severe lung lesions were detected in 33.3, 22.2, and 25.9 % of infected animals, respectively. Severe lesions were more frequent in animals younger than 12 months. In five infected foxes (18.5 %), no gross lesions were observed, while for three animals, angiostrongylosis was considered the cause of death. A case of disseminated angiostrongylosis was detected and another one was suspected. This is the firs report of disseminated angiostrongylosis in the fox.     

Infection of red foxes (Vulpes vulpes) with Echinococcus multilocularis during the years 2001–2004 in Poland

During the period of 2001-2004, 1,514 red foxes from the north and the southeast of Poland were sampled and examined parasitologically for infections with Echinococcus multilocularis. A mean 23.8% were found infected in the whole study area. The highest prevalence (50.0-62.9%) was found in some counties in the northeastern (Warmia-Mazuria) and the southeastern (Carpathian Foothill) part of the country. During the study period, significant differences in the prevalence were found on a regional level. Based on the results obtained to date, the paper presents an overview of the current epidemiological situation of E. multilocularis in the fox population in Poland.     

The prevalence of Echinococcus multilocularis in red foxes in Poland—current results (2009–2013)

The aim of the study was to determine the prevalence of Echinococcus multilocularis in red foxes (Vulpes vulpes) in Poland. Overall, 1,546 intestinal samples from 15 of the 16 provinces in Poland were examined by the sedimentation and counting technique (SCT). The mean prevalence of E. multilocularis in Poland was 16.5 % and was found in 14 of the 15 examined provinces. The mean intensity of infection was 2,807 tapeworms per intestine. Distinct differences in prevalence were observed between regions. In some provinces of eastern and southern Poland, the level of prevalence was 50.0 % (Warmińsko-Mazurskie), 47.2 % (Podkarpackie), 30.4 % (Podlaskie) and 28.6 % (Ma?opolskie), while in other provinces (west and south-west), only a few percent was found: 2.0 % (Dolno?l?skie), 2.5 % (Wielkopolskie) and 0.0 % (in Opolskie). The border between areas with higher and lower prevalence seems to coincide with a north–south line running through the middle of Poland, with prevalence from 17.5 to 50.0 % in the eastern half and from 0.0 to 11.8 % in the western half. The dynamic situation observed in the prevalence of this tapeworm indicated the necessity of continuing to monitor the situation concerning E. multilocularis in red foxes in Poland.