Enzyme-linked Immunosorbent Assay (ELISA) as a diagnostic method for Trichinella spiralis infections in pigs

The application of the enzyme-linked immunosorbent assay (ELISA) in the detection of Trichinella spiralis infections in pigs is presented. Two experiments using conventionally raised pigs infected with various numbers of T. spiralis larvae are described. Blood samples were collected for serological examination, prior to and at various days post infection (pi). At slaughter, on the 28th day pi, samples from the diaphragm were collected for isolation of muscle larvae by means of the digestion method. The results from these sera were compared with those from non-infected conventionally raised pigs. At day 28 pi, 21 out of 33 infected pigs showed positive ELISA results. In only two of those serologically positive animals were no larvae detected at slaughter. Of the 12 infected pigs with a negative ELISA result, only two harboured more than 3 larvae/g (the detection limit of trichinoscopy). Of the nine non-infected control animals, one had a false positive ELISA result. The significance of these findings in relation to slaughterhouse control is discussed. ELISA, therefore, presents an alternative to other detection methods for the control of T. spiralis infections in pigs.     

Comparison of the enzyme-linked immunosorbent assay (ELISA) with three other methods for the detection of Trichinella spiralis infections in pigs

Four methods employed in the diagnosis of trichinellosis [trichinoscopy, digestion method, immunofluorescence techniques and Enzyme-Linked Immunosorbent Assay (ELISA)] were compared by laboratories in eight countries of the European Economic Community. Material from 24 pigs infected with 10 000, 5000, 500, 150 and 0 T. spiralis larvae was examined during a period from 17 days to 12 weeks post infection. ELISA was more sensitive than immunofluorescence during the onser of the infection in groups in infected with higher numberts of larvae (1500, 5000 and 10 000 larvae). In general, however, results of both ELISA and immunofluorescence were comparable with regard to reliability. In pigs infected with a lower number of T. spiralis larvae both serological assays were more sensitive than the direct methods (trichinoscopy and digestion method).It was concluded that not enough evidence was available to suggest ELISA as an alternative to the direct methods for slaughterhouse control. Both the ELISA and the immunofluorescence technique may prove to be applicable for epidemiological surveys.     

Swine trichinosis in New England slaughterhouses

Diaphragms of 5,315 slaughter hogs from the New England states were examined for Trichinella spiralis infection between June 22 and Dec 22, 1983. Thirty-nine hogs (0.73%) were infected. The infections were light, none exceeding 30 larvae/g of tissue, with an arithmetic mean of 4.7 larvae/g. Infected hogs originated from 5 of 6 New England states. The widespread occurrence of porcine trichinosis in New England indicates that the small or part-time hog farming operations frequently found in this region may be especially vulnerable to the introduction and maintenance of the parasite.     

Increased resistance in the rat to Nippostrongylus brasiliensis following immunization against Trichinella spiralis

Rats which were immunized with increasing doses of Trichinella spiralis muscle larvae per os and inoculated with 2 000 Nippostrongylus brasiliensis third-stage larvae 11 day after the last immunizing dose harbored significantly fewer N. brasiliensis adults at necropsy than did unimmunized controls. Immunized rats were solidly immune to homologous infection with T. spiralis. Some N. brasiliensis infective larvae incubated in vitro at 37°C in T. spiralis antiserum developed mild oral precipitates. They developed strong oral, excretory pore and occasionally anal precipitates in homologous antiglobulins, and none in normal rat sera or globulins. T. spiralis larvae developed strong oral precipitates in homologous antisera, but none in N. brasiliensis antiglobulins or normal rat sera or globulins. Inflammation, altered intestinal conditions and delayed hypersensitivity following T. spiralis infection may be contributing to the cross-resistance observed.     

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.     

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.     

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.     

Field evaluation of the enzyme-linked immunosorbent assay for swine trichinosis: efficacy of the excretory-secretory antigen

A field evaluation of an enzyme-linked immunosorbent assay (ELISA) for swine trichinosis was done with sera obtained from 5 herds experiencing ongoing transmission of Trichinella spiralis. Epizootiologic studies conducted on these herds offered an opportunity to evaluate the accuracy of an ELISA, using larval T spiralis excretory-secretory antigens. Sera from 162 infected pigs and 143 serum samples from noninfected pigs originating from the same farms were tested. The infection status of the pigs was determined by digestion of diaphragm or tongue muscle samples. Two criteria were established to classify the ELISA optical density (OD) readings: Criterion I stated that an OD greater than or equal to 5 times the mean OD of several normal swine sera pools was positive; criterion II stated that a OD greater than or equal to 4 times the normal sera values was positive. The results obtained did not reveal obvious serologic variations among infected herds located in the 4 states involved. Overall, the test detected 93% (criterion I) and 96% (criterion II) of infected pigs. The majority of false-negative sera was from hogs that had less than 5 larvae/g of muscle; 1 hog had 73.8 larvae/g of diaphragm muscle. The false-positive rates were 8% for criterion I and 9% for criterion II. The actual rate for these false-positive samples may have been overestimated, because generally, only small tissue samples (0.4 to 10 g) were digested; larger sample sizes might have altered the results. The relevance of this qualification is that these pigs originated from herds with prevalence rates greater than 50%. Other factors that may account for occasional false-positive sera or false-negative sera in the swine trichinosis ELISA are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trichinellosis in wolves from Croatia

The aim of the present study was to investigate the prevalence of Trichinella infection in wolves (Canis lupus) in a 17,468 km² area in Croatia. Muscle samples were collected from 67 wolves between 1996 and 2007 and analyzed by artificial digestion. Muscle larvae were detected in 21 wolves (31%) and genotyped by multiplex PCR. Trichinella britovi was the predominant species confirmed in 90% (19 wolves) while Trichinella spiralis was detected in 9% (2 wolves). The presence of the so called “domestic” Trichinella species was a surprise since, to date, only T. britovi had been reported in wild animals in this region. The larval burdens in infected animals ranged from 0.3 to 45.9 larvae per gram. The prevalence of infected animals varied by geographic region; infected animals were found in the region of Gorski Kotar (20%) which has very similar environment to the region of Lika, where almost all wolves were found infected. Interestingly, this is the first report of infected wolves in Dalmatia.     

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).     

Toxocara cati larva migrans in domestic pigs - detected at slaughterhouse control in Norway

Routine Trichinella meat inspection at the slaughterhouse detected one larva in a pooled batch of 100 pig samples. The larva was sent to the Norwegian Veterinary Institute (NVI) for species identification.Morphological examination revealed that the larva was not Trichinella spp. Molecular analysis was performed. PCR and sequencing of 5S/ITS identified the larva as Toxocara cati. A second round of digests was carried out at the meat inspection laboratory, in smaller batches to try to identify the infected animal. No further larvae were detected and it was not possible to identify which of the 100 animals the larva had come from. This is the first time that Toxocara cati has been reported in slaughterhouse pigs in Norway.Although the infected individual could not be identified, the meat originated from one of six potential farms. A small survey regarding rodent control and cats was sent to each of these farms. Cats had restricted access to food storage areas (two farms reported that cats had access) whilst none of the farms allowed cats into the production housing. Cats were, however, present on all the farms (mostly stray cats of unknown health status). Half of the farms also reported seeing rodents in the pig housing during the previous six months and half reported finding rodents in the feed and straw storage areas. We were unable to narrow down the source of infection – however contamination of food or bedding material, with cat faeces or infected rodents, in addition to the presence of infected rodents in pig housing remain potential routes of infection.     

Antibody-dependent mechanisms of immunity against migrating larvae of Trichinella spiralis

Normal rat peritoneal exudate cells adhere to new-born Trichinella spiralis larvae in the presence of specific antibodies produced during the course of an oral infection. After 24 hours incubation with the peritoneal cells and antibodies most of the larvae were killed. Heated immune serum had less activity. With normal serum no adherence was observed. In order to investigate the influence of antibodies on new-born larvae in vivo, larvae were pre-treated in vitro with immune serum and then inoculated intravenously into normal mice. After 30 days the yield of normal larvae was 50% of the number recovered in a control group infected with non-treated new-born larvae.     

Retrospective study of four years of carcass condemnation rates for malignant lymphoma in California cows

A retrospective study was undertaken to examine monthly rates of carcass condemnation for bovine malignant lymphoma in adult dairy cows slaughtered between January 1979 and December 1982, in 2 plants (A and B) in the southern San Joaquin Valley of California. Extremes (mean) of monthly rates for slaughterhouse A were 41 to 100 (65.9) per 10,000 slaughtered and for slaughterhouse B, 39 to 113 (94.1) per 10,000. The overall monthly mean rate was 80 per 10,000 slaughtered. Equations for long-term trend lines for condemnation rates for slaughterhouses A and B were T = 0.398 + 0.0176t - 0.0002t2 and T = 0.314 + 0.0378t - 0.0007t2, where t = month. A difference in patterns of trends was not apparent, although slaughterhouse A tended to have lower rates of condemnation than did slaughterhouse B. Seasonal components of trend lines, estimated by time-series analysis, were not consistent between plants, except for the month of August, during which time seasonally adjusted rates were low for both slaughterhouses. For slaughterhouse A, 2 classic cyclical components were identified--between January 1981 and September 1981 and between September 1981 and September 1982. For slaughterhouse B, the cyclical components were between November 1979 and July 1981 and between July 1981 and August 1982. The later cycle resembled the second cycle of rates from slaughterhouse A. Rates remained stable through 1979 and then increased steadily for 2.5 years. During the last 6 months of 1982, rates leveled off and perhaps began to decline.     

Ascariasis, respiratory diseases and production indices in selected Prince Edward Island swine herds.

The levels of production, ascarid burden and respiratory disease were measured on 15 purposively selected swine herds, and the relationships between the various measures of ascarid burden were examined. On each farm 30 randomly selected pigs were weighed and rectal fecal samples were collected at approximately 11, 15, 19 and 22 weeks of age, and at slaughter. Fecal ascarid-egg counts and duration of infection were combined to calculate a composite measure of ascarid burden called "lifetime burden". At the abattoir the carcass weight and levels of anteroventral pneumonia, atrophic rhinitis, and liver lesions were recorded for each hog. The number of ascarids in the small intestines were counted. Study hogs were marketed at an average of 189 +/- 22 days. The average dressed carcass weight was 77.0 +/- 5.9 kg and the mean average daily gain was 0.519 +/- 0.071 kg/day. The percent of hogs with ascariasis varied widely among farms, no matter what measure of ascariasis was used; the percent with intestinal ascarids at slaughter ranged from 0% to 96%, the percent that shed ascarid eggs during their lifetime ranged from 0% to 100%, and the range for hogs with liver lesions ranged from 27% to 100%. Of the hogs slaughtered, 82% had milk spot lesions, 32% shed ascarid eggs during their lifetime and 35% had intestinal ascarids. The latter had an average of 12 intestinal ascarids. Anteroventral pneumonia occurred in 55% of the slaughtered hogs and 9% had atrophic rhinitis scores of five. The percent of hogs per farm with pneumonia ranged from 17% to 96%. The percent of hogs per farm with atrophic rhinitis scores of five ranged from 0% to 57%.     

Why must we rush to bury our dead (pigs): The option of excarnation by exposure

The accepted paradigm of foreign animal disease preparedness in Canada, the emergency for which to prepare, starts with identification of the exotic viral agent in a Canadian farm animal population. This narrative focuses on the containment of the infectious agent, within diseased animals, on infected premises. Framing the emergency as a disease incursion limits rational imagination to only one version of one potential animal emergency. This framing of the problem directs the carcass disposal solutions to consider only methods to dispose of viral infected material. However, in all documented responses to catastrophic swine diseases in the past three decades, the number of uninfected animals caught up in movement control zones and killed greatly exceeds the number of infected animals killed. The temporary closures of slaughterhouses in spring 2020 due to COVID-19 transmission resulted in thousands of healthy market hogs surplus to market; an unanticipated emergency of healthy pigs. This paper proposes an alternate carcass disposal option for material from uninfected farms. “Excarnation by exposure” is a natural process of debulking and dehydrating carcasses by blow fly larvae, mitigating financial costs of final disposal. Excarnation by exposure is a reasonable and possibly necessary additional option for the management of uninfected carcasses in a catastrophic emergency response in commercial pigs.     

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.     

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.     

Slaughterhouse effluent discharges into rivers not responsible for environmental occurrence of enteroaggregative Escherichia coli

Enteroaggregative Shiga-toxin-producing Escherichia coli strains were responsible for a massive outbreak in Europe in 2011, and had been previously isolated from French patients. The objective of this study was to investigate the presence of enteroaggregative E. coli (EAEC) in slaughterhouse effluents (wastewater, slurry, sludge and effluents), and in river waters near these slaughterhouses. A total of 10,618 E. coli isolates were screened by PCR for the presence of EAEC-associated genetic markers (aggR, aap and aatA). None of these markers was detected in E. coli isolated from slaughterhouse samples. A unique enteroaggregative E. coli (EAEC) O126:H8 was detected in river water sampled upstream from slaughterhouse effluent discharge. These results confirmed that animals might not be reservoirs of EAEC, and that further studies are required to evaluate the role of the environment in the transmission of EAEC to humans.     

Evaluation of ELISA and Western Blot Analysis using three antigens to detect anti-Trichinella IgG in horses

We assessed a serological method for detecting Trichinella infection in horses, specifically, an ELISA using three antigens to detect anti-Trichinella IgG (i.e. a synthetic tyvelose glycan-BSA (stg-BSA) antigen, an excretory/secretory (ES) antigen, and a crude worm extract (CWE) antigen). Serum samples were collected from 2502 horses (433 live horses from Romania and 2069 horses slaughtered in Italy and originating from Italy, Poland, Romania, and Serbia). Serum samples were also taken from horses experimentally infected with different doses of T. spiralis and T. murrelli larvae, as controls. The cut-off value of ELISA was determined on serum samples from 330 horses from Trichinella-free regions of Italy, which were also examined by artificial digestion of preferential-muscle samples. In the experimentally infected horses, the stg-BSA and ES antigens were less sensitive than the CWE antigen. Trichinella spiralis showed a higher immunogenicity than T. murrelli, and the IgG immunoresponse was dose-dependent. The kinetics of anti-Trichinella IgG were similar among all experimentally infected horses. No circulating antibodies were detected 4-5 months after experimental infection, although these horses still harbored infective larvae. Depending on the antigen used, for 4-7 of the 330 horses from Trichinella-free areas, the optical density (OD) of the serum sample was higher than the cut-off value, yet these samples were negative when subjected to Western Blot. Similar results were obtained for the 1739 horses slaughtered in Italy (originating from Italy, Poland, Romania, and Serbia) and the 433 live Romanian horses. Of the 4 horses with muscle larvae, only one was positive by ELISA and Western Blot. Because the anti-Trichinella IgG remain circulating for only a short period of time, whereas the larvae remain infective for longer periods, serology cannot be used for either diagnosing Trichinella infection in horses or estimating the prevalence of infection. Artificial digestion of at least 5 g of preferential-muscle tissue continues to be the method of choice at the slaughterhouse for preventing equine-borne trichinellosis in humans.     

Hepatitis E virus in swine and effluent samples from slaughterhouses in Brazil

Hepatitis E is an infectious disease which virus (HEV) is highly disseminated in swine herd populations. Sporadic acute human hepatitis E cases have been associated to genotype 3 and 4 strains of HEV also reported in swine populations of endemic and non-endemic areas. With the aim to evaluate the incidence of animals with current infection of HEV, 115 bile samples were collected from three slaughterhouses under inspection by Animal Sanitary Protection Agency of Rio de Janeiro, Brazil. In parallel, effluent samples were collected from six sewage pipe exit sites of two slaughterhouses. HEV RNA was detected in 11 out of 115 (9.6%) bile samples collected and three waste samples from one slaughterhouse. Viral loads observed for bile samples varied from 10(1)-10(5) genome copies/mL and for effluent samples mean load was 10(2) genome copies/mL. Sequencing and phylogenetic analysis classified samples within genotype 3 subtype 3b closely related to the sample obtained from the first reported autochthonous human case and samples from swine of commercial herds in Brazil. Our data demonstrates that although most animals achieve slaughter age (around 20 weeks old) already immune to HEV, a significant number of animals are with current infection at commercial age. Further studies should be addressed to consider risk analysis and possible evaluation of inspection regulations considering food safety measures regarding hepatitis E zoonotic aspect in Brazil.