Anti-Trichinella antibodies detected in chronically infected horses by IFA and Western blot, but not by ELISA

In the Balkan countries, where trichinellosis is a re-emerging zoonosis, it is of great importance to determine Trichinella infection prevalence among the major hosts, including horses. One method for monitoring prevalence is serological surveillance; however, the validity of serological methods in horses is not well understood. The dynamics of anti-Trichinella IgG production and circulating excretory/secretory (ES) antigens were investigated in three horses experimentally-infected with Trichinella spiralis. Horses were slaughtered at 32 week post infection (p.i.). Low worm burdens were found in all three animals. Anti-Trichinella IgG was detected up to 32 weeks p.i. by an indirect immunofluorescence assay (IFA) and by Western blot (Wb), but not by ELISA. The ELISA test detected antibodies for only a short period of time (up to 18 weeks p.i. using ES antigen or up to 20 weeks p.i. using tyvelose-BSA antigen). The presence of circulating muscle larvae ES antigen in sera of infected horses was observed by dot blot from the 4th week p.i. up to the 32nd week p.i.     

New patterns of Trichinella infection

Human and animal trichinellosis should be considered as both an emerging and reemerging disease. The reemergence of the domestic cycle has been due to an increased prevalence of Trichinella spiralis, which has been primarily related to a breakdown of government veterinary services and state farms (e.g., in countries of the former USSR, Bulgaria, Romania), economic problems and war (e.g., in countries of the former Yugoslavia), resulting in a sharp increase in the occurrence of this infection in swine herds in the 1990s, with a prevalence of up to 50% in villages in Byelorussia, Croatia, Latvia, Lithuania, Romania, Russia, Serbia, and the Ukraine, among other countries. The prevalence has also increased following an increase in the number of small farms (Argentina, China, Mexico, etc.) and due to the general belief that trichinellosis was a problem only until the 1960s. The sylvatic cycle has been studied in depth at both the epidemiological and biological level, showing the existence of different etiological agents (Trichinella nativa, Trichinella britovi, Trichinella murrelli, Trichinella nelsoni) in different regions and the existence of "new" transmission patterns. Furthermore, the role of game animals as a source of infection for humans has greatly increased both in developed and developing countries (Bulgaria, Canada, Lithuania, some EU countries, Russia, USA, etc.). The new emerging patterns are related to non-encapsulated species of Trichinella (Trichinella pseudospiralis, Trichinella papuae, Trichinella sp.), infecting a wide spectrum of hosts (humans, mammals including marsupials, birds and crocodiles) and to encapsulated species (T. spiralis, T. britovi, and T. murrelli) infecting herbivores (mainly horses). The existence of non-encapsulated species infecting mammals, birds and crocodiles had probably remained unknown because of the difficulties in detecting larvae in muscle tissues and for the lack of knowledge on the role of birds and crocodiles as a reservoir of Trichinella. On the other hand, it is not known whether horse and crocodile infections existed in the past, and their occurrence has been related to improper human behavior in breeding. The problem of horse-meat trichinellosis is restricted to France and Italy, the only two countries where horse-meat is eaten raw, whereas mutton and beef have been found to be infected with Trichinella sp. only in China.     

Comparative assessment of a double antibody enzyme immunoassay test kit and a triple antibody enzyme immunoassay for the diagnosis of Trichinella spiralis spiralis and Trichinella spiralis nativa infections in swine.

Enzyme immunoassays using the triple antibody enzyme linked immunosorbent assay (ELISA) with both Trichinella spiralis spiralis and T. spiralis nativa excretory-secretory (ES) antigens and a commercial Trichinella spiralis enzyme immunoassay test kit were carried out on sera from pigs that were infected with light, moderate and high doses of infective T. spiralis spiralis and T. spiralis nativa respectively. Seroconversion occurred in all pigs given infective Trichinella larvae although no trichinae were recovered from pigs given T. spiralis nativa larvae and examined between days 92 and 99 postinfection by pepsin digestion. Anti-Trichinella antibodies were detected in pigs infected with T. spiralis spiralis and T. spiralis nativa by ELISA using either the homologous or heterologous ES antigen. The commercial Trichinella spiralis enzyme immunoassay test kit also detected anti-Trichinella antibodies in both the T. spiralis spiralis and T. spiralis nativa infected pigs. The commercial test kit did not appear to be as sensitive as the triple antibody ELISA since it usually took two to three days longer for seroconversion to be detected by the former procedure. Finally seroconversion occurred more rapidly in swine infected with T. spiralis spiralis than with pigs receiving comparable doses of T. spiralis nativa.     

Evaluation of different antigens in a seroepizootiological survey of trichinellosis by enzyme-linked immunosorbent assay

Antigen isolated from the large-particle fraction of the muscle Trichinella spiralis larvae (PAW), excretory/secretory (E/S) and crude worm extract (CWE) antigens were evaluated in a seroepizootiological survey of trichinellosis by the enzyme-linked immunosorbent assay (ELISA). The ELISA using PAW antigen yielded 16 positive animals (1.6%), E/S antigen revealed 21 (2.1%) positive, and the highest number of positive (23 or 2.3%) were obtained using CWE antigen. Parasitological post-mortem examination of all seropositive animals showed five and seven false-positive animals when E/S and CWE antigens were used, respectively.     

抗旋毛虫单克隆抗体细胞林的建立及其特性鉴定

为筛选旋毛虫保护性抗原,建立了4株分泌抗旋毛虫McAb的细胞株。经连续培养30余代,仍稳定地分泌抗体。其中,2G3和2C10的靶抗原为旋毛虫幼虫表膜。免疫球蛋白 型及亚 鉴定表明,2G3和2C10为IgG_1,1D5为IgG_3,1C9为IgM。除2C10和IC9对伊氏锥虫可溶性抗原呈现阳性反应外,未见对猪圆线虫、猪囊尾蚴、伊氏锥虫表膜抗原和正常猪肉反应。经ELISA测定,2G3、2C10、1D5、1C9均可与施毛虫幼虫可溶性抗原作用,其腹水效价分别为1:50000、1:10000、1:1000、1:800。各McAb均与旋毛虫幼虫排泄分泌物抗原(ES抗原)作用,经ELISA测定表明,2G3腹水效价达1:320000。     

Influence of methods for Trichinella detection in pigs from endemic and non-endemic European region

A total of 1401 German and 226 Croatian pigs raised either indoors or outdoors were tested for Trichinella infection by direct and indirect detection methods. A 10 g sample of diaphragm were examined for muscle larvae by the artificial digestion method; the species was determined by multiplex polymerase chain reaction (PCR). For detection of anti-Trichinella IgG, serum samples diluted 1:100, and meat juice samples diluted 1:10, were tested by enzyme-linked immunosorbent assay. All German pigs and those Croatian pigs raised indoors proved to be Trichinella-negative by all methods. Muscle larvae were detected in a total of eleven of the Croatian pigs, which were raised on small outdoor farms. For eight isolates, PCR results demonstrated that recovered larvae were Trichinella spiralis. Anti-Trichinella-IgG was detected in serum and meat juice of digestion positive animals when the worm burdens exceeded 0.38 larvae per gram of muscle. Positive results in Croatian pigs indicate a higher risk of infection for outdoor farming in areas where Trichinella is endemic. Results of direct and indirect detection were compared and are discussed with special regard to specificity and sensitivity of methods.     

Larval viability and serological response in horses with long-term Trichinella spiralis infection

The horse is considered an aberrant host for the nematode parasite Trichinella spiralis, and many aspects of the biology and epidemiology of Trichinella infection in the horse are poorly understood. It has been reported that experimentally-infected horses produce a transient serological response to infection and that muscle larvae are cleared more rapidly than in parasite-adapted hosts such as the pig and humans. However, limited numbers of animals have been studied, and both the longevity of larvae in horse musculature and the immune response to Trichinella larvae remain unclear. In this study, we infected 35 horses with 1000, 5000, or 10,000 T. spiralis muscle larvae and followed the course of infection for 1 year, assessing larval burdens in selected muscles, the condition and infectivity of recovered larvae, and the serological response of infected horses. The results demonstrated that T. spiralis establishes infection in horses in a dose dependent manner. Anti-Trichinella IgG antibodies peaked between weeks 6-10 post-inoculation. Viable, infective larvae persisted in horse musculature for the duration of the study (12 months), and exhibited no apparent reduction in muscle burdens over this period. Encapsulated larvae showed no obvious signs of degeneration in histological sections. Larval capsules were surrounded by infiltrates consisting of mature plasma cells and eosinophils. Macrophages were notably absent. Given the lack of a detectable serological response by 26 weeks p.i. and the persistence of infective muscle larvae for at least 1 year, parasite recovery methods are currently the only suitable detection assays for both meat inspection and epidemiological studies of Trichinella infection in the horse.     

Experimental trichinellosis in horses: biological and parasitological evaluation

Three groups of three horses each were, respectively, infected with 5000, 20,000 and 50,000 larvae of Trichinella spiralis. The strain used was isolated from a human biopsy during horsemeat-related outbreaks of trichinellosis in France. Transient muscular disorders were only observed in two of the horses infected with 50,000 larvae but none of the horses had fever. A significant increase in blood eosinophils was noticed in 5 horses. Serum LDH, aldolase and CPK peaked at the fifth week post-infection. Specific IgG assayed by indirect immunofluorescence and ELISA, appeared 2-5 weeks post-infection and disappeared between 16 and 40 weeks. The distribution of T. spiralis larvae was maximal in the tongue, masseters and diaphragm, but a large decrease in the number of larvae recovered from the muscles was noticed among the horses slaughtered at the beginning and end of the experiment. In muscular histological sections, larvae were observed in an intramyofibrillar position and were surrounded by a mild to severe inflammatory reaction.     

Spatial distribution of Trichinella britovi, T. pseudospiralis and T. spiralis in red foxes (Vulpes vulpes) in Hungary

The red fox (Vulpes vulpes) is considered one of the main reservoir of Trichinella spp. in Europe. As limited information on Trichinella infection in wildlife of Hungary is available, 2116 red foxes, representing more than 3% of the estimated fox population of the country, were screened to detect Trichinella larvae by a digestion method. Trichinella larvae from the 35 positive foxes were identified by a multiplex PCR as Trichinella britovi (30 isolates, 85.7%), Trichinella spiralis (4 isolates, 11.4%), and Trichinella pseudospiralis (1 isolate, 2.9%). The true mean intensity of T. britovi, T. spiralis and T. pseudospiralis larvae in lower forelimb muscles was 23.6, 3.5 and 13.5larvae/g, respectively. T. spiralis was detected only in the southern and eastern regions. The non-encapsulated T. pseudospiralis was recorded for the first time in Hungary. Although the overall true prevalence of Trichinella infection in foxes was only 1.8% (95% confidence interval, CI=1.5-2.1%), the spatial analysis reveals different risk regions. In the north-eastern counties bordering Slovakia and Ukraine (21% of the Hungarian territory), the true prevalence of Trichinella infection is significantly higher than that observed in other regions (6.0%, CI=4.8-7.1%). In the southern counties bordering Croatia, Serbia and Romania (41% of the Hungarian territory), the true prevalence of Trichinella infection is moderate (1.4%, CI=1.0-1.8%). In the north-western and central counties (38% of Hungarian territory), the prevalence of Trichinella infection is significantly lower (0.2%, CI=0.1-0.4%) than that of the other regions. Based on the statistical analysis and the evaluation of epidemiological data, none of the counties can be considered free of Trichinella infection. In the past decade, Trichinella infection has been detected only in few backyard pigs, and only few wild boar-related autochthonous infections in humans were described. Nevertheless, these results highlight the need of the maintenance of a strict monitoring and control programmes on Trichinella infection in farmed and hunted animals of Hungary.     

Influence of adjuvant formulation on the induced protection of mice immunized with total soluble antigen of Trichinella spiralis

Vaccination of pigs against the helminth nematode Trichinella could be a good alternative to prevent the risk of human infection. In order to develop an efficient and safe vaccine, the choice of the adjuvant is an important issue. In this study, two adjuvants were selected to prepare vaccines based on total soluble Trichinella spiralis muscle larvae (ML) antigen: Montanide ISA 70 water in oil emulsion and Montanide IMS nanoparticles. Aluminium hydroxide was used as a reference adjuvant. The immune response was checked by ELISA of parasite antigen specific IgG1 and IgE. Finally, protection induced in vaccinated mice was measured after a T. spiralis challenge by counting ML burdens. The results clearly showed an impact of adjuvants on the specific IgG1 and IgE antibody responses against T. spiralis. Differences were observed between the rates of protection induced according to the type of formulation, although the three adjuvants tested were able to enhance the humoral immune response. This work demonstrated the need to use an adjuvant to obtain a specific IgG1 and IgE responses directed against the total soluble extract of T. spiralis.     

青海省部分地区商品猪旋毛虫感染的血清学调查

以旋毛虫肌幼虫排泄分泌)抗原作为检测抗原,采用间接酶联免疫吸附试验(ELISA)法,分别对采集自青海省西宁市张氏生猪屠宰场、互助县屠宰场、平安县屠宰场的商品猪血清进行旋毛虫抗体检测,共检查猪血清1 065份.结果阳性血清为19份,阳性率为1.78％.可见在青海省商品猪中旋毛虫具有一定的感染率,动物卫生监督以及肉品检疫部...     

Murine model study of the practical implication of trypanosome-induced immunosuppression in vaccine-based disease control programmes

The relevance of trypanosome-induced immunosuppression in relation to the efficacy of vaccine-induced immunity was studied in mice. Mice were immunised with crude Trichinella spiralis muscle larvae homogenate vaccine and infected with T. spiralis and/or Trypanosoma brucei. Vaccination significantly decreased adult worm burden (p<0. 05) and accelerated worm expulsion in mice infected with T. spiralis only. T. brucei superinfection resulted in monocytosis, suppressed eosinophilia, significant decrease in PCV (p<0.001), higher numbers of adult worms (p<0.001) and failure to expel all adult worms by Day 12 post infection (p.i.). Regardless, they produced anti-Trichinella IgG(1) responses similar to those of the vaccinated non-T. brucei-infected group. T. brucei also suppressed the proliferative responses of spleen cells to stimulation with Con A and T. spiralis antigen, and induced strong production of interferon-gamma (IFN-gamma) in culture supernatants of antigen stimulated spleen and mesenteric lymph node cells. Interleukin-5 (IL-5) production was suppressed by T. brucei in supernatants of Con A- and antigen-stimulated spleen cells. It was concluded that trypanosome infections and the associated immunosuppression are of great practical significance in trypanosome endemic areas, especially with regards to disease control programmes involving vaccine-induced herd immunity.     

A Trichinella murrelli infection in a domestic dog in the United States

Trichinella murrelli infection was diagnosed in a naturally infected Beagle bitch from VA, USA, where encapsulated larvae were found in histological sections of several skeletal muscles. A laboratory reared dog fed infected muscles resulted in viable muscle larvae that were subsequently infective to Swiss-Webster mice. Multiplex PCR using larvae from the experimentally infected dog demonstrated two distinct bands migrating at 127 bp and 316 bp which together are diagnostic for T. murrelli; the isolate was assigned the ISS code: ISS1608 by the International Trichinella Reference Centre. This is the first report of T. murrelli infection in a companion animal.     

Detection of Trichinella spiralis nativa antibodies in porcine sera by ELISA using T. spiralis spiralis excretory-secretory antigen

Enzyme-linked immunosorbent assay examination of sera from pigs vaccinated with T. spiralis nativa infective larvae and/or challenged with T. spiralis spiralis larvae using a T. spiralis spiralis excretory-secretory antigen showed a significant cross-reaction between the two species of Trichinella. Eight of 12 pigs vaccinated with a high dose of T. spiralis nativa reacted positively 28 days postvaccination while the remaining four pigs had high but negative ELISA optical density readings. Five of six pigs challenged with the homologous species reacted positively 28 days postchallenge but the sixth pig remained negative despite having a muscle infection of 5.6 larvae/g of musculature.     

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.     

Detection of Trichinella murrelli in coyotes (Canis latrans) from Oklahoma and North Texas

We determined the prevalence and mean intensity of Trichinella sp. infection in coyotes from six counties in Oklahoma and one in northern Texas. Tongues from 77 coyotes were examined using histology and artificial tissue digestion. Histological examination showed a prevalence of 3.9% (3 of 77) whereas the prevalence was 6.5% (5 of 77) based on artificial digestion of 5.0 g of muscle from coyote tongues. One sample was positive for Trichinella sp. on histology but negative by artificial digestion. Combining data from both diagnostic techniques showed that six of 77 (7.8%) coyotes were infected with Trichinella spp. The mean intensity of Trichinella sp. larvae ranged from 0.2 to 66.2 with an average of 16.0 larvae per gram (LPG) of tongue. Genotyping results demonstrated that the coyotes were infected with Trichinella murrelli. This is the first report of T. murrelli infection in coyotes in Oklahoma. T. murrelli had previously been isolated from coyotes in Texas.     

旋毛虫排泄/分泌(ES)抗原致鼠胸腺淋巴细胞凋亡的研究

本试验首次以体外培养的小鼠胸腺淋巴细胞为实验对象,加入旋毛虫（Trichinella spiralis）肌幼虫ES抗原做刺激物,通过对鼠胸腺淋巴细胞DNA损伤、凋亡水平的检测,进而证明旋毛虫肌幼虫ES抗原能够诱导鼠胸腺淋巴细胞发生调亡。掌握这种免疫细胞凋亡（apoptosis）发生的过程,对分析免疫应答的特点和调控,以及探索旋毛虫病（Trichinosis）的发病机制和提供防治对策都具有重要意义。     

Evaluation of the ELISA for the serological diagnosis of trichinosis in Canadian swine

An ELISA using a Trichinella spiralis spiralis excretory-secretory antigen was evaluated as a procedure for the diagnosis of trichinosis in swine in Canada. Field and experimental trials were carried out using both indirect serological (ELISA) and direct parasitological (pepsin-digestion) methods concurrently on serum and musculature, respectively, from each animal. The ELISA is a sensitive and specific test for the detection of Trichinella antibodies in porcine sera when present. The development of Trichinella antibodies appears to be dependent on the magnitude of the infection established, age of the infection when the animal is tested and the immunocompetence or response to infection of individual animals. False negative reactions were recorded in both field and experimental trials. In the field study, five of the 1009 swine examined were parasitologically positive with light infections ranging from 0.01 to 0.046 larvae per gram (la/g) of musculature yet all were serologically negative. Experimentally it was shown that Trichinella antibodies develop slowly, at least two to three months postinfection, in pigs with very light infections. Even in pigs which developed infections of 33 to 55 la/g of musculature, seroconversion occurred greater than 23 and less than 30 days postinfection. The immunocompetence or response to infection of individual pigs was variable as illustrated by one pig inoculated with 3000 infective larvae which had consistently lower titers compared to others in the same group despite the establishment of a muscle infection of 8.5 la/g of musculature. One false positive reaction was recorded in the experimental trial in an animal which had received 100 larvae and seroconverted at about three months postinfection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors affecting the flow among domestic, synanthropic and sylvatic cycles of Trichinella

Nematodes of the genus Trichinella are maintained in nature by sylvatic or domestic cycles. The sylvatic cycle is widespread on all continents, from frigid to torrid zones, and it is maintained by cannibalism and scavenging behavior of carnivores. Trichinella is primarily a parasite of carnivorous mammals, although one non-encapsulated species, Trichinella pseudospiralis, has also been detected in birds. The anaerobic metabolism of larvae in nurse cells allows their survival in extremely decayed meat. Encapsulated larvae in the decomposing carcass function similarly to the species-dispersing population of eggs or larvae of other nematodes, suggesting that the natural cycle of Trichinella includes a free-living stage when the parasite is no longer protected by the homeothermy of the host. Consequently, environmental temperature and humidity play an important role in the transmission of Trichinella among wildlife. Of the 10 recognized genotypes of Trichinella, only Trichinella spiralis is transmitted and maintained in a domestic cycle, although it can be present also in wildlife. All other genotypes (Trichinella nativa, Trichinella britovi, T. pseudospiralis, Trichinella murrelli, Trichinella nelsoni and Trichinella papuae, Trichinella T6, T8, and T9) are transmitted and maintained only in a sylvatic cycle. This generalization does not preclude sylvatic species of Trichinella from invading the domestic habitat, and T. spiralis may return to this habitat when humans fail in the management of wildlife and domestic animals. However, the presence of sylvatic genotypes of Trichinella in the domestic habitat represents a "dead-end" for the sylvatic cycle. Synanthropic animals (rats, foxes, mustelids, cats, dogs, etc.) contribute to the flow of sylvatic Trichinella genotypes from wildlife to domestic animals and of T. spiralis from domestic to sylvatic animals. Furthermore, human behavior not only influences the transmission patterns of Trichinella genotypes in the domestic habitat, but also it can contribute to the transmission and spread of this infection among wildlife, for example by improper hunting practices.     

Identification and distribution of swine serum immunoglobins that react with Trichinella spiralis antigens and may interfere with the enzyme-labeled antibody test for trichinosis.

Sera from Trichinella spiralis digestion-negative swine contained variable amounts of two immunoglobins that reacted with T spiralis antigen in the indirect enzyme-labeled antibody test for trichinosis. One of these immunoglobins, detected by heavy chain-specific anti-swine immunoglobulin G (IgG) conjugate, was removed by absorption with T spiralis larvae. A second immunoglobin, detected by heavy chain-specific anti-swine IgM, was not removed by absorption with T spiralis larvae and increased in amount with the age of the animal. These two immunoglobins varied independently in individual animals and showed some specificity for the antigen; ie, they did not merely reflect changes in total serum IgG or IgM. In contrast to IgG anti-T spiralis antibody from experimentally infected animals, neither of these immunoglobins could be detected in double-diffusion tests against the antigen or by counter immunoelectrophoresis. Either of these immunoglobins could interfere with the indirect test for T spiralis antibodies, depending upon whether anti-swine IgG or IgM conjugate is used. The factors which initiate synthesis and control serum concentrations of these immunoglobins are not known.