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.     

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.     

Integrating genomics and phylogenetics in understanding the history of Trichinella species

In 2004, funding was received by Washington University's Genome Sequencing Center through NHGRI, to completely sequence several nematode genomes as part of a holistic effort to advance our understanding of the human genome and evolution within the Metazoa. Trichinella spiralis was among this group of worms because of its strategic location at the base of the phylum Nematoda, and the belief that extant species represented an ancient divergent event that occurred as early as the Paleozoic. At the same time, a concerted effort was put forth to solidify the phylogeny of extant species of Trichinella based upon molecular analyses of a multi-gene system to understand the history of the genus and thereby enhance utilization of the forthcoming sequence data. Since the inception of this research, several findings have emerged: (1) the size of T. spiralis genome estimated by flow cytometry (71.3 Mb) is substantially smaller than originally predicted (270 Mb); (2) to date, a subset of the total of 3,534,683 sequences have been assembled into a 59.3 Mb unique sequence; (3) 19% of the assembled sequence is comprised of repetitive elements; and (4) sequence data are predicated upon extant T. spiralis which probably diverged as little as 20 million years ago. Thus, the utility of the T. spiralis genome as representative of an archaic species must be tempered with the knowledge that encapsulated and non-encapsulated clades probably separated during the mid-Miocene as temperate ecosystems changed.     

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.     

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.     

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.     

Effect of phytohaemagglutinin-P on apoptosis and necrosis in Trichinella spiralis infected mice

Flow cytometry analyses were used to evaluate the contribution of apoptotic and necrotic lymphocytes in the selected organs of Trichinella spiralis infected mice treated with phytohaemagglutinin-P (PHA-P). The Tunnel method was used to examine apoptosis in a cryostat section from the jejunum and masseter muscle. CFW mice (Groups I and II) were infected with 200 larvae of T. spiralis. PHA-P was administered intravenously at a dose of 10 mg/kg 24 h prior to infection in Group II mice only. Group III mice were treated with PHA-P without T. spiralis infection, and Group IV mice were untreated controls. The lymphocytes obtained from the spleen, mesenteric lymph nodes (MLN) and muscular inflammatory infiltration on 7, 14, 21, 28, 35, 42 and 60 days post infection (DPI) were incubated with the Annexin-V-Fluos Staining Kit (Roche). The cryostat preparation made from the jejunum and masseter muscle was evaluated using a fluorescence microscope. PHA-P administration stimulated apoptosis in the jejunal mucosa and in the muscular inflammatory infiltration. In Group I mice, infected with T. spiralis only, the highest percentage of apoptotic cells was found on 7 DPI in the spleen and in MLN, and on 14 DPI among the cells of the muscular inflammatory infiltration. The peak of the necrotic lymphocytes was found on 7 DPI in the spleen, on 28 DPI in MLN, and on 21 DPI in the cells of muscular inflammatory infiltration. In Group II mice, infected with T. spiralis and treated with PHA-P, the peak in apoptotic cells occurred on 7 DPI in the spleen and in the muscular inflammatory infiltration. The highest level of necrotic lymphocytes was observed only on 7 DPI in the muscular inflammatory infiltration. Percentage of necrotic lymphocytes in the spleen was the same and in MLN it was lower than in Group I (T. spiralis only). Moreover, the number of muscle larvae in mice treated with PHA-P (Group II) was lower than in Group I (T. spiralis only).     

Species identification of Trichinella isolates from China

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

A Study on the Predilection Sites of Trichinella spiralis Muscle Larvae in Experimentally Infected Foxes (Alopex lagopus,Vulpes vulpes)

Kapel, Chr. M., Sv. Aa. Henriksen, H. H. Dietz, P. Henriksen, P. Nansen: A study on the predilection sites of Trichinella spiralis muscle larvae in experimentally infected foxes (Alopex lagopus, Vulpes vulpes). Acta vet. scand. 1994, 35, 125-132.–Studies were carried out on the predilection sites of Trichinella spiralis muscle larvae in experimentally infected arctic foxes (Alopex lagopus) and silver foxes (Vulpes vulpes) reared in cages. The highest number of larvae per gramme tissue was found in the muscles of the legs, eyes, diaphragm, and tongue. The 2 fox species showed no significant differences with regard to predilection sites.     

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.     

Immunity to Trichinella spiralis muscle infection

Trichinella spiralis larvae establish chronic infections in skeletal muscles of immunocompetent hosts. Muscle infection is crucial to transmission and survival of the parasite in nature. Chronic infections by this highly immunogenic parasite are associated with modulation or escape from potentially destructive immune responses. This review summarizes our current knowledge of immunity to muscle infection with T. spiralis.     

Evaluation of a Direct Agglutination Test for Detection of Antibodies Against Toxoplasma gondii in Cat,Pig and Sheep Sera

The protozoan parasite Toxoplasma gondii is the causative agent of the zoonosis toxoplasmosis. In sheep and goats, it is one of the most prevalent causes of infectious abortion. Also in pregnant women, a primary infection can result in miscarriage. Humans acquire the infection either by ingestion of oocysts excreted by cats, the definitive host of the parasite, or by eating raw or undercooked meat from latently infected animals (Dubey & Beattie 1988). In Sweden, toxoplasmosis is a notifiable disease, and cases of clinical disease in humans as well as animals must be reported. In both veterinary and human medicine serological assays based on detecting the humoral antibody response of the host against the parasite are used as diagnostic tools. So far, solid phase assays, such as the indirect fluorescent antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA), have been widely used to diagnose T. gondii infection in many species including cats, pigs and sheep (Dubey & Beattie 1988). However, both IFAT and ELISA require appropriate anti-species specific immunoglobulins (Ig) that must be carefully evaluated for each species prior to use. This makes these assays complicated and time consuming. Consequently, alternative, simpler methods that do not require specific antisera would be of great value. The direct agglutination test (DA), which is based on the principle that formalin-treated organisms agglutinate in the presence of specific IgG antibodies, is such an assay (Fulton & Turk 1959). The DA-test is widely used in human medicine as a screening test for T gondii infection but it has not yet been thoroughly evaluated for use in veterinary medicine (Uggla & Buxton 1990).     

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.     

The Growth Precipitation Test As a Diagnostic Method for Differentiation of Mycoplasma and Acholeplasma Species

The purpose of this paper is to evaluate the growth precipitation (GP) test for routine identification. The test was performed as described by Heitmann & Kirchhoff (1978) which is a modification of the method of Krogsgaard-Jensen (1972). On the basis of examination of 82 strains, using indirect immunofluorescence (IMF) and growth inhibition (GI) as well as GP tests it is concluded that the GP test seems to be very useful for species identification in the genus Acholeplasma, as this method displayed fewer cross-reactions between species than the other 2 tests. When applied to the genus Mycoplasma, however, the GP test is not species-specific, due to cross reactions observed within the group of arginine positive and within the group of glucose and scrum digestion positive species. In the genus Mycoplasma the method can only be used as a screening tool, and final identification is in general based on growth inhibition and immunofluorescence.     

Isolation of Yersinia Enterocolitica from a Dog with Chronic Enteritis: A Case Report

During recent years, Yersinia enterocolitica has been isolated from humans and various animal species in connection with intestinal disorders, such as acute ileitis and appendicitis. Cases of septicaemia, polyarthritis and erythema nodosum have also been described (Mollaret & Destombes 1964, Nilehn 1969, Winblad 1969, Lassen 1972, Langford 1972). Y. enterocolitica has been isolated most frequently from chinchillas and hares, but sporadic isolations from deer, cow, horse, rabbit, goat and dog have been reported (Langford, Krogstad et al. 1972). In Norway, an outbreak of the disease in a goat herd is the only described case of yersiniosis among animal species (Krogstad et al.). A case of chronic enteritis in a dog from which Y. enterocolitica was isolated is presented in the following.     

A Field Outbreak Caused by Bovine Respiratory Syncytial Virus

Bovine respiratory syncytial virus (BRSV) caused a large epizootic of acute respiratory disease in Japan in 1968—69 (Inaba et al. 1970, Inaba et al. 1972). A much smaller outbreak occurred in Switzerland (Paccaud & Jacquier 1970). In Belgium the virus has been isolated from an outbreak of respiratory disease (Wellemans et al. 1970). BRSV has later been proved an important causal agent of respiratory disorders in the same country (Wellemans & Leiinen 1975). In England and USA the virus has caused and been isolated from outbreaks of acute respiratory disease in calves (Jacobs & Edington 1971, Rosenquist 1974, Smith et al. 1974). In Denmark BRSV has sporadically been isolated from pneumonic calf lungs (Bitsch et al. 1976).     

The influence of concurrent infection with Oesophagostomum species on the interpretation of the Elisa test for trichinosis in pigs

The effects of simulatenous infection of pigs with Oesophagostomum spp. and Trichinella spiralis on the interpretation of the Enzyme-Linked Immunosorbent Assay (ELISA) for trichinosis were examined. Extinction values were observed from four groups of pigs. The first group acted as uninfected controls, the second was infected only with Oesophagostomum spp., the third with T. spiralis alone and the fourth by both nematodes.It was found that the pigs infected with T. spiralis could be differentiated from the others, but that those infected with both species had lower extinction values than the group with T. spiralis alone. The differences may be related to the numbers of T. spiralis larvae able to establish and develop into adults in the small intestine of the host. Those infected with Oesophagostomum spp. alone showed no rise in extinction values, and it was concluded that there was no cross-reaction in the ELISA between thos pecies and T. spiralis antigen.     

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.