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.     

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.     

Infectivity of Canadian isolates of Trichinella spiralis nativa for swine, rats and carnivores.

The infectivity of Trichinella spiralis nativa isolates from widely separated geographic areas of Canada was determined by feeding infected musculature to swine, laboratory rats and carnivores (cats, foxes, ferrets). Low infectivity for swine and rats and high infectivity for carnivores were observed. Light infections were established in four of 16 swine examined between 25 and 53 days postinfection. Feeding of infected porcine musculature to ferrets demonstrated that Trichinella spiralis nativa can be passaged through swine even though the infectivity rate is low.     

Factors affecting preconditioning of Trichinella spiralis nativa larvae in musculature to low temperatures.

The influence of preconditioning temperature, length of the preconditioning period, host and age of the infection on the survival of Trichinella spiralis nativa larvae in musculature to low temperature refrigeration was investigated. Dogs, foxes, ferrets, mink and guinea pigs were infected with a T. spiralis nativa isolate, killed at various times postinfection, preconditioned at temperature of -10 degrees C, -15 degrees C or -20 degrees C for varying periods of time prior to low temperature refrigeration and subsequent pepsin digestion to determine survival of larvae. The preconditioning temperature played an important role in the subsequent survival of larvae in musculature at low refrigeration temperatures. Under the conditions of this study, survival of larvae was greater as the preconditioning temperature became lower. The minimum period of preconditioning required had an inverse relationship with the refrigeration temperature. Preconditioning of the T. spiralis nativa isolate used occurred in the musculature of guinea pigs, foxes, ferrets, mink and dogs with larvae surviving longer in vulpine and canine musculature than in the other hosts studied. Age of the infection was not a major factor in the survival of preconditioned larvae in musculature at low refrigeration temperatures although survival was slightly longer in older infections.     

Experimental Trichinella infections in ponies.

Light Trichinella infections were established in three ponies given 1000, 5000 and 25000 T. spiralis spiralis infective larvae respectively by stomach tube. A predilection site of infection in all ponies was the tongue. Anti-Trichinella antibodies were detected in all ponies by the enzyme-linked immunosorbent assay using a T. spiralis spiralis excretory-secretory antigen. The ponies given 5000 and 25000 infective larvae reacted positively on days 26 and 24 postinfection, respectively, using a criterion of greater than or equal to 5 X mean optical density readings of preinfection sera as positive. The pony given 1000 larvae did not react positively although antibodies were present as indicated by 3 X to 4 X mean optical density readings of preinfection sera. The results of this limited study indicate that the enzyme-linked immunosorbent assay cannot be used to certify horsemeat free of Trichinella since the presence of detectable antibody levels appears to be related to the magnitude of the infection and duration of the infection when the animal is tested.     

旋毛虫病McAb快速ELISA诊断盒的应用研究

应用ＭｃＡｂ 快速ＥＬＩＳＡ 诊断盒,对感染了４ 个旋毛虫隔离种的猪定期检测其血清中抗体出现情况。结果,猪旋毛虫和旋毛形线虫（Ｔ．ｓｐｉｒａｌｉｓ）在感染２４ ｄ 后、犬旋毛虫和本地毛形线虫（ Ｔ．ｎａｔｉｖａ）３１ ｄ 后,可在猪血清中检出抗体;在抗体出现时间上前二者较后二者早７ ｄ 左右。     

Vaccination of rats and pigs against Trichinella spiralis spiralis using the subspecies, T. spiralis nativa.

Rats and pigs were vaccinated against Trichinella spiralis spiralis either by feeding infective larvae of the subspecies, Trichinella spiralis nativa in musculature or by gavage. The number of larvae established in the musculature of vaccinated nonchallenged and vaccinated challenged rats and pigs were negligible and statistically comparable, while highly significant infections were established in the nonvaccinated challenged rats and pigs. High vaccination doses of T. spiralis nativa gave virtually complete protection to challenge with T. spiralis spiralis in pigs. The results of one trial in rats with a lower vaccination dose of larvae suggest that there is a minimal vaccination dose of larvae required to elicit marked resistance to challenge. The low numbers of muscle larvae established due to the high vaccination doses of larvae confirm the low infectivity of the subspecies, T. spiralis nativa in rats and pigs.     

Comparison of monoclonal antibody-based competitive and indirect enzyme-linked immunosorbent assays for the diagnosis of swine trichinosis

A competitive enzyme-linked immunosorbent assay (ELISA) for the detection of swine trichinosis has been developed using a biotinylated monoclonal antibody and an avidin-enzyme conjugate. The assay is based on competitive binding between swine serum antibodies and a monoclonal antibody specific for an antigenic determinant present on proteins from Trichinella spiralis excretory-secretory products with molecular weights of 45,000, 49,000, and 53,000. The competitive ELISA reliably detected pigs infected experimentally with T. spiralis and eliminated false-positive reactions in pigs infected with other swine nematodes, particularly Trichurus suis. When the competitive ELISA and an indirect ELISA using affinity-isolated antigen were compared using serum from pigs with naturally-acquired infections of T. spiralis, both tests were highly effective in detecting infected animals.     

Experimental trichinosis in sheep.

Trichinella spiralis spiralis infections were established in sheep by administering infective larvae via gavage or feeding infected musculature. Trichinella spiralis nativa infective larvae had a low infectivity for sheep although light infections may be established in some animals with large infective doses. For the most part, sheep were averse to ingesting musculature mixed in a grain ration unless it was camouflaged with molasses. The heaviest infections usually occurred in the masseter muscle. The fact that sheep are averse to ingesting muscle tissue may reduce the likelihood of trichinosis. Anti-Trichinella antibodies to both T: spiralis spiralis and T. spiralis nativa were produced as demonstrated by the enzyme-linked immunosorbent assay. Seroconversion occurred in several sheep challenged with T. spiralis nativa even though larvae were not recovered from the musculature by pepsin-digestion.     

A tongue biopsy technique for the detection of trichinosis in swine

A tongue biopsy technique developed for the detection of Trichinella spiralis infection in swine involves taking a deep core biopsy of the tongue musculature, and examination of the sample by digestion. Using this procedure, 31 of 52 (60%) swine from an Indiana herd were found to be infected with T. spiralis. The average biopsy weighed 0.42 g, and the intensity of infection averaged 180 larvae per gram (range 2-1157). The biopsy was quick and easy to perform and the tongues healed well following the procedure. This technique may have applicability for Trichinella detection in epidemiological, control and research studies on swine and other animals.     

Comparison of pepsin-digestion and enzyme-linked immunosorbent assay for the diagnosis of trichinosis in swine.

Comparison of parasitological and serological diagnosis of trichinosis in swine was carried out on 36 pigs given 15,400 infective larvae each by gavage. Circulating eosinophil levels were determined and sera were examined by enzyme-linked immunosorbent assay for anti-Trichinella antibodies. Two pigs were killed per day from days 15 to 29 postinfection. Muscle was examined by pepsin-digestion and comparable tissue was fed to a rat. Eosinophil counts increased at about day 6 and reached peak levels about day 25 postinfection and returned to approximate preinfection levels about two months postinfection in those pigs still in the study. Infective larvae were recovered from all pigs killed at greater than or equal to 18 days postinfection. Using the criterion of 5 x mean optical density readings of negative sera as positive, seroconversion occurred between days 19 and 26 postinfection. Use of a lower criterion of 3 x mean optical density readings of negative sera resulted in only three of 30 pigs killed greater than or equal to 18 days postinfection seroconverting less than or equal to 18 days postinfection, when infective larvae were first recovered in the musculature. In pigs, even in those heavily infected, there is a lag between the period that trichinae in musculature become infective and development of antibodies as detected by enzyme-linked immunosorbent assay which results in false negative reactions in many animals. This study demonstrated that the enzyme-linked immunosorbent assay using an excretory-secretory antigen should not be used to certify pork or pork products free of infective Trichinella larvae or safe for human consumption.     

Immunity in swine inoculated with larvae or extracts of a pig isolate and a sylvatic isolate of Trichinella spiralis

Inoculation of swine with a sylvatic isolate of Trichinella spiralis, designated T s nativa, resulted in low numbers of muscle larvae, compared with muscle larvae accumulation in swine inoculated with a pig type of T s spiralis. Despite low infectivity of T s nativa for swine, primary inoculation resulted in high levels of immunity against challenge infection with T s spiralis. This immunity was expressed in accelerated expulsion of challenge adults from the intestine and reduced numbers of muscle larvae. Pigs inoculated with T s nativa developed cellular and humoral responses similar to those in pigs inoculated with T s spiralis. However, in immunoblots, sera from pigs inoculated with T s nativa recognized additional proteins in muscle larvae excretory-secretory (ES) products, compared with sera from pigs inoculated with T s spiralis. Active immunization of pigs with ES products from T s nativa resulted in numerically higher, but not significantly different levels of immunity, compared with pigs immunized with ES from T s spiralis. The highest levels of immunity were obtained in pigs immunized with a T s spiralis newborn larval extract. The combination of ES products and newborn larval extract did not result in additive levels of immunity. These results indicate that the major immune effector response to Trichinella sp in pigs is against the newborn larvae, regardless of the genetic type of Trichinella sp.     

Concurrent infection with Trichinella spiralis and other helminths in pigs

The aim of this study was to investigate possible influence of different helmintosis in the development of Trichinella spiralis in experimental infected pigs. Forty-two Iberian pigs were allocated to six groups. Three groups were single inoculated with Ascaris suum, Metastrongylus apri or T. spiralis, respectively. Two groups were co-infected with T. spiralis and A. suum or T. spiralis and M. apri, respectively, while the last group included uninfected control pigs. Clinical signs were only observed in pigs with single or concurrent M. apri infections, with more severe respiratory symptoms in pigs with mixed M. apri infection. The number of A. suum and M. apri lung larvae, intestinal larvae of A. suum and adult M. apri were reduced in pigs with mixed Trichinella infections compared to pigs with single infections. In contrast, the number of liver white spots was higher in pigs with mixed infections. While T. spiralis muscular larval burdens were increased in pigs concomitantly infected with M. apri, they were reduced in pigs concomitantly infected with A. suum, compared to pigs receiving single infections with either of these helminths. Pigs with single or mixed A. suum infections showed higher eosinophil levels compared to the remaining groups. IgGt, IgG1, IgG2 and IgM against T. spiralis antigen could not be detected in pigs with single Ascaris or Metastrongylus infections, indicating that no cross-antibodies were produced. IgGt, IgG1 and IgM antibodies were detected earlier and generally at higher levels in mixed T. spiralis infections compared to single T. spiralis infections. The results suggest that T. spiralis had a low synergistic interaction with M. apri in concomitantly infected pigs, and an antagonistic interaction in concurrent infection with A. suum.     

Husbandry, parasitic and other diseases as factors in the reliability of the enzyme linked immunosorbent assay (ELISA) for detection of trichinellosis in pigs

Serum samples were obtained from pigs originating from specified pathogen-free farms, large industrialised farms and small conventional farms. All animals proved to be free of Trichinella spiralis by a pooled sample digestion method. Careful meat inspection studies on parasitic infections other than trichinellosis, and other inflammatory reactions were recorded and used for subdivision of the animals in different groups. It was concluded that animal husbandry, parasitic infections, or inflammatory reactions have not influenced the mean extinction values of an ELISA for the detection of T. spiralis antibodies, however, the specified pathogen free animals yielded somewhat lower results.     

Evaluation of an enzyme-linked immunosorbent assay for diagnosis of trichinellosis in swine.

Experimental and field trials were conducted to evaluate an ELISA for its ability to detect Trichinella-infected domestic swine and to compare ELISA results with muscle-digestion test results. The ELISA used was a commercial double-antibody kit, containing an excretory-secretory antigen, and was evaluated principally for epidemiologic use. Experimentally induced infection in swine (4 groups of 3 pigs each; inoculated with 0, 50, 500 or 5,000 larvae) was detected as early as postinoculation week 4, with seroconversion of all inoculated swine by postinoculation week 8. The rate of seroconversion appeared to be affected by initial larval dose, time after inoculation, and immunocompetence of the individual host. Determination of antibody kinetics generally revealed rapidly increasing antibody titer, followed by its steady decrease in most pigs. Once seropositive, however, all pigs remained seropositive for the duration of the 10-week study. Presence of muscle larvae was confirmed in all infected pigs at termination of the study. We recognize that the experimental conditions may not be truly representative of those under which natural infection develops in pigs; however, the ELISA detected an infected pig with muscle larval density of 0.87 larvae/g of tissue. Results of a field trial (n = 310) indicated no muscle digestion test-positive pigs (35 g of diaphragm muscle digested/pig), but 3 samples tested positive by ELISA for specificity of 99.0%.     

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...     

Prevalence of trichinosis in southern Louisiana swine

After an outbreak of human trichinosis in Louisiana involving 45 cases and 1 death in 1979 and 1980, a survey of pigs killed in 21 selected small slaughterhouses in southwestern Louisiana was conducted from November 1980 to September 1981. The sera from 1,225 pigs were examined for trichinella antibodies using an enzyme-linked immunosorbent assay (ELISA); 1,223 diaphragms were subjected to peptic digestion and examined for the presence of Trichinella spiralis larvae. One diaphragm (0.08%) was found to contain T spiralis (26 larvae/g of muscle) and 4 of the slaughterhouse sera were positive (0.33% seroprevalence). Pigs in 52 herds throughout the state were also tested for ELISA antibodies. The ELISA-positive pigs were not found among the 267 pigs tested from the 52 herds.     

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)     

Pathogenesis and serodiagnosis of experimental Trichinella spiralis spiralis and Trichinella spiralis nativa infections in cattle.

Trichinella spiralis spiralis infections were established in cattle by gavage and by feeding infected musculature in the ration. Trichinae were present in greatest numbers in masseter, tongue and diaphragm. Trichinella spiralis nativa had a low infectivity to cattle although a light infection was established in one cow by a heavy challenge. Cattle had an aversion to eating musculature unless it was camouflaged with molasses. Clinical signs of reluctance to eat and masticate were observed between 10 and 30 days postinfection. Eosinophil counts started to increase at seven days and peaked at about 30 days postinfection. By day 60 eosinophil counts returned to near preinfection levels but in animals examined greater than 90 days postinfection, the counts were variable. Focal lesions of eosinophilic myositis were observed up to about 90 days postinfection. Little cellular reaction was observed surrounding trichinae after muscle invasion and cyst development was completed except for cysts undergoing disintegration. Seroconversion occurred in all cattle examined between 7 and 14 days postinfection. Seroconversion was associated with IgG1 and IgG2 immunoglobulins. Peak levels of antibody occurred between 30 and 60 days. Cattle examined at 182 and 369 days postinfection showed a gradual decrease in antibody levels over time.     

五种旋毛虫抗原对猪的免疫保护作用研究

本实验研究了旋毛虫肌幼虫可溶性粗抗原、排泄分泌抗原（ＥＳ）、表面抗原（ＳＡ）及成虫ＥＳ、ＳＡ５种抗原对猪的免疫保护作用。结果５种抗原对猪均具有一定程度的免疫原性，可诱导猪体产生对攻击感染的抵抗力（减虫率），其中肌幼虫粗抗原为５５．２０％；肌幼虫ＥＳ为４２．５６％，肌幼虫ＳＡ为７２．２１％；成虫ＥＳ为３２．９２％；成虫ＳＡ为４２．１７％。免疫５种抗原后用肌幼虫“Ｂ”抗原、新生幼虫可溶性抗原及成虫可溶性抗原进行ＥＬＩＳＡ检测，均可测出血清抗体应答反应，其中以相应抗原测出的抗体应答较强烈。免疫５种抗原后猪外周血液中Ｂ淋巴细胞减少，Ｔｈ及Ｔｓ增加，Ｔｈ／Ｔｓ比值降低，呈暂时的细胞免疫抑制现象。