Diagnostic tests for Trichinella spiralis infection in pigs. A comparative study of ELISA for specific antibody and histamine release from blood cells in experimental infections.

A study on the histamine release test (HR) for the demonstration of infections with Trichinella spiralis in pigs was carried out on 18 pigs, six infected with 200 larvae, six infected with 5000 larvae and six non-infected (control group). The results obtained by HR during a 7 week infection were compared with those of the enzyme-linked immunosorbent assay (ELISA). All inoculated pigs were found to be positive on Day 40 post-inoculation (p.i.) by necropsy examination of selected muscle groups, with mean recoveries of 7.9 and 225 larvae g-1 of tissue in the low- and high-dose group, respectively. At this time, all animals of the high-dose group and five out of six animals of the low-dose group were antibody positive in ELISA with any of three coating antigens employed (a crude muscle larva extract, an excretory/secretory (ES) antigen and a purified 45 kDa antigen). HR performed on whole blood was positive in four out of six pigs of the high-dose group and one out of six pigs of the low-dose group. The earliest ELISA seroconversions took place at Day 15 p.i. with crude and ES antigens. The earliest measurable reaction in HR performed on whole blood was found on Day 19 p.i. There was considerable individual variation regarding which test was the most sensitive for the early detection of infection. Washing of the blood cells prior to antigen provocation led to a markedly improved sensitivity of HR, all animals of the high-dose and three out of six animals of the low-dose group being positive by Day 40 p.i. The time course of the development of ELISA titres and HR reactivity indicated that this effect is due to the removal of blocking antibodies.     

犬细粒棘球绦虫粪抗原夹心ELISA检测方法的建立

为建立特异性和敏感性高的检验犬细粒棘球绦虫感染的方法。用细粒棘球绦虫(简称,Eg)成虫抗原分别免疫兔和绵羊,收集高免血清,纯化的高免抗体。依据抗体夹心ELISA工作原理,以兔抗体包被,检测感染Eg、不同犬带科绦虫的实验犬和空白犬粪样,绵羊抗体扑捉抗原,HRP标记兔抗绵羊IgG(1∶8 000)催化显色,用酶标仪测定OD 405nm吸光度,用以确定其特异性和敏感性。试验结果表明,敏感性为82.69%(43/52),特异性为85.88%(140/163);粪抗原在感染细粒棘球绦虫16d后可检出,最低抗原浓度为9.7ng/mL即犬感染5条成虫时可检测出阳性。该检测方法具有较好的特异性和灵敏性,为进一步研制检测细粒棘球绦虫虫体抗原ELISA检测试剂盒奠定了基础。     

Four-layer enzyme immunoassay (EIA) detection of differences in IgG, IgM and IgA antibody response to Aujeszky's disease virus in infected and vaccinated pigs

The use of the four-layer enzyme immunoassay (EIA) for the detection of IgG, IgM and IgA antibodies against Aujeszky's disease virus in blood and oropharyngeal swabs of infected and vaccinated pigs is described. Mean antibody titres obtained using the four-layer EIA were 6.1 and 3829 times higher compared with the indirect enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) test, respectively. The VN test detected mainly IgG antibodies, while the IgM antibodies did not react. Using the EIA, the first antiviral antibodies in sera were demonstrated on Days 5-7 after infection or vaccination. Up to the 7th day, demonstrable antibodies were almost exclusively of the IgM class. In infected pigs high titres of IgM antibodies were still detected on Day 18, while in vaccinated animals they were absent by this time. Antibodies of the IgG class appeared in infected pigs sooner (Day 7) than in vaccinated pigs (Day 10) and reached higher mean titres. Antibodies of the IgA class were demonstrable from Day 10 only in samples from infected pigs. Similar antibody dynamics and distribution were detected in oropharyngeal swabs, except that the IgG and IgM titres were roughly 100 times lower than in sera. However, titres of IgA antibodies in oropharyngeal swabs were two times higher than in sera. The greatest differences between both groups of animals were recorded on Day 18; in the infected pigs, IgG, IgM and IgA antibodies were present in sera and oropharyngeal swabs at that time, while in vaccinated pigs only IgG antibodies were demonstrable. The effect of infection and vaccination on the pattern of the immune response as well as the importance of the detection of individual immunoglobulin classes for the specificity of the enzyme immunoassay are discussed.     

Detection of Taenia hydatigena copro-antigens by ELISA in dogs

A sandwich-ELISA was developed for the detection of soluble Taenia hydatigena antigens in fecal samples of dogs. Affinity-purified polyclonal catching antibodies and alkaline phosphatase-conjugated detecting antibodies were employed, which had been obtained from rabbits hyperimmunized with excretory/secretory antigens derived from in vitro maintained adult Taenia hydatigena. The assay allowed the detection of 800 ng T. hydatigena antigen g-1 of feces as a lower limit. Six helminth-free dogs were each infected with 10 T. hydatigena cysticerci isolated from Swiss sheep. After prepatent periods ranging from 57 to 71 days, the dogs started to excrete Taenia eggs and/or proglottids. The ELISA detected Taenia antigens in all six dogs during the prepatent period starting individually between Day 18 and 45 post-infection (p.i.). Anthelmintic treatment of three dogs at Day 95 p.i. resulted in elimination of the cestodes and within the 5 following days in the disappearance of Taenia antigens from feces. The specificity of the assay was evaluated by testing crude antigens derived from helminths or bacteria. Four Taenia species showed cross-reactivity at concentrations of 5 micrograms protein ml-1. Conversely, no cross-reactions occurred with various antigen batches derived from Echinococcus granulosus, E. multilocularis, Dipylidium caninum, Mesocestoides corti, Diphyllobothrium sp., Toxocara canis and bacterial antigens (Salmonella and Escherichia). Moreover, fecal samples from dogs naturally infected with T. canis (n: 13), hookworms (n: 2), Trichuris vulpis (n: 13) and of 10 dogs with mixed infections with these three nematode groups were tested, and results confirmed the high degree of specificity. The Taenia antigens detectable by this ELISA remained immunologically stable in native feces stored at +25 degrees, +4 degrees or at -20 degrees C for at least 5 days.     

Preliminary evaluation of diagnostic tests using horses experimentally infected with trypanosoma evansi

Seven surra negative horses were intravenously inoculated with 3 x 10(6)Trypanosoma evansi parasites derived from a camel. One horse was maintained as an uninfected negative control. Three antigen and three antibody detection tests were evaluated for diagnosis of infection in horses. The microhaematocrit centrifugation test (MHCT) was the most sensitive, first detecting parasites between one and three days (x 2.4) post infection (p.i.). The antigen (ag)-ELISA detected antigen between three and ten days (x 6.6) p.i. The latex agglutination test (LAT) first gave positive results on day 3 (x 3.0) p.i. Following the treatment of horses with trypanocidal drugs, the MCHT and the mouse inoculation test (MIT) became negative. Antigen levels using LAT declined and reached pre-infection levels in five out of six horses during the period of observation (92-279 days). Antigen levels using the ag-ELISA declined as well but did not reach pre-infection levels in any of the six horses.Three antibody detection techniques, ab-ELISA, card agglutination test (CATT), and immunofluorescent antibody test (IFAT) detected antibodies in the blood of all seven infected horses but not in the uninfected control. However, the ab-ELISA did not discriminate clearly between sera from infected and uninfected horses because unacceptably high ELISA background readings were detected in 15% of the surra negative horses shipped to the UAE from the UK. The ELISA antibody increased above pre-infection levels in the six horses experimentally infected, but not in one horse. In this horse the ELISA antibody level exceeded the cut-off level only after the reoccurrence of the T. evansi infection. The IFAT detected antibodies 15.7 days p.i. in all infected horses.     

Haematological and serological data from dogs raised worm-free and monospecifically infected with helminths

Pups free from helminth infection were obtained by treating the bitch prior to parturition, and the bitch and her pups after whelping, with oxfendazole and praziquantel. They were then housed in an area free from contamination with helminth eggs. Freedom from infection was confirmed by necropsy examinations and by serological tests using the Enzyme Linked Immunosorbent Assay (ELISA). Groups of pups were infected with Toxocara canis, Echinococcus granulosus, Taenia ovis, T. pisiformis and T. hydatigena and bled at 5-day intervals. Haematological examinations and measurement of serum proteins were performed on blood samples obtained throughout 30 days of infection. Levels of serum proteins were not significantly altered in any infection. Pups infected with T. canis showed a marked increase in numbers of circulating eosinophils during the phase of larval migration through the liver and lungs; they also showed marked antibody responses to T. canis larval ES antigen. Haematological values were not significantly altered in any other pups.     

Detection of antibodies against porcine parvovirus nonstructural protein NS1 may distinguish between vaccinated and infected pigs

The humoral antibody response against the nonstructural protein NS1 and the structural protein VP2 of porcine parvovirus (PPV) was evaluated by immuno-peroxidase test (IPT) and enzyme linked immuno sorbent assay (ELISA) using recombinant PPV antigens. The coding sequence for NS1 and VP2 was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) genome resulting in two recombinant baculoviruses AcNPV-NS1 and AcNPV-VP2, respectively. Sf9 cells (Spodoptora frugidiperda) inoculated with AcNPV-NS1 producing recombinant nonstructural protein (rNS1) and AcNPV-VP2 producing recombinant virion protein (rVP2) were used in IPT and ELISA to analyse serum antibodies. Pigs vaccinated with an inactivated whole virus vaccine and experimentally infected pigs were studied. Significant titers against rVP2 were obtained in both vaccinated and infected pigs. Specific antibodies against rNS1 could only be detected in infected pigs and NS1 may in this way allow the specific detection of infected animals. Analysis of serum samples collected up to 18 days post infection (p.i.) from four pigs experimentally infected with PPV showed that antibodies against rNS1 and rVP2 could in all cases be detected on day 9 p.i. Two individual pigs were inoculated twice with PPV and the antibody response was followed 89 days after second inoculation. Serum antibodies against borth rVP2 and rNS1 could be detected for this period of time.     

Detection of IgM antibodies against Babesia bovis in cattle

The diagnosis of acute babesiosis by direct examination of blood smears has some limitations and the indirect serological methods currently in use are designed for detection of IgG, which may not be detectable at an early stage of infection. There is a need, therefore, for rapid and reliable procedures to diagnose acute infections. An ELISA system using a crude antigenic preparation of Babesia bovis was standardized for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Serum samples of cattle imported from tick-free areas collected before and during an immunization process were used to validate the tests. The specificity was 94% and sensitivity 100%. Specific IgM antibodies against B. bovis first appeared on the 11th day post-inoculation (p.i.) in animals infested with Boophilus microplus ticks and on the 19th day p.i. in animals which had been inoculated with infected blood. Antibody titers decreased after Day 33; however, all animals remained positive until the end of the experiment (124 days).     

Serological diagnosis of Echinococcus granulosus infection in sheep using cyst fluid antigen processed by antibody affinity chromatography

Serum antibody responses in sheep naturally or experimentally infected with Echinococcus granulosus and/or other larval cestodes were examined using an enzyme-linked immunosorbent assay (ELISA) with various antigens prepared from sheep hydatid cyst fluid ( SHCF ). Serum donors included: sheep experimentally infected with E. granulosus and their age-matched non-infected controls; sheep experimentally infected with other helminth parasites; sheep naturally infected with E. granulosus both from Tasmania and the Australian mainland; sheep from Tasmania naturally infected with larval cestodes other than E. granulosus; and naturally reared sheep completely free from infection with larval cestodes. Attempts were made to eliminate serological reactions which were not specific for E. granulosus by using a series of antibody affinity chromatography steps to deplete crude SHCF antigen; these included adsorption with a monoclonal antibody, 3EgH 29-2, removal of host IgG using rabbit anti-sheep IgG antibody, and removal of antigens which bound non-specifically to normal sheep immunoglobulin. The final affinity-depleted antigen product was designated AD SHCF . Specific serological reactivity in infected sheep was very low. Affinity depletion of SHCF using 3EgH 29-2 did not appear to increase the specificity of serological diagnosis of E. granulosus infection when experimentally infected sheep were compared with their non-infected controls provided the latter were age-matched with experimental animals. The other affinity adsorption steps significantly reduced non-specific background binding to antigen by normal sheep serum. Despite this reduction in background in the ELISA, only low levels of antibody could be detected in naturally-infected sheep.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of acute feline herpesvirus-1 and chronic feline immunodeficiency virus infections in experimentally infected specific pathogen free cats.

Cats with or without chronic feline immunodeficiency virus (FIV) infection were exposed to feline herpesvirus, type 1 (FHV-1). FIV infected cats became sicker than non-FIV infected cats and required more supportive treatment. However, there were no differences in the length of their illness or in the levels and duration of FHV-1 shedding. FHV-1 infection caused a transient neutrophilia at Day 7 with a rapid return to preinfection levels. The neutrophilia coincided with a transient lymphopenia that was accompanied by a decline in both CD4+ and CD8+ T-lymphocytes. A brief decrease in the CD4+/CD8+ T-lymphocyte ratio occurred at Day 14 in both FIV infected and non-infected cats. This decrease was mainly the result of an absolute and transient increase in CD8+ T-lymphocytes. CD4+ and CD8+ T-lymphocyte numbers and CD4+/CD8+ T-lymphocyte ratios returned to baseline within 4-8 weeks in both FIV infected and non-infected cats. FIV infected cats produced less FHV-1 neutralizing antibodies during the first 3 weeks of infection than non-FIV infected animals. The IgM FHV-1 antibody response was depressed in FIV infected cats whereas the IgG antibody response was unaffected. FHV-1 infection evoked a comparable transient loss of lymphocyte blastogenic responses to concanavalin A and pokeweed mitogen in both FIV infected and non-infected cats. However, response to pokeweed mitogen took longer to return to normal in FIV infected animals. Lymphocytes from FIV infected cats had a greater and more sustained proliferative response to FHV-1 antigen than non-FIV infected cats. The ongoing IgG antibody response to FIV was not affected by FHV-1 infection.     

A serosurvey of Borna disease virus infection in wild rats by a capture ELISA

For a serological diagnostic test for Borna disease (BD), we developed a capture ELISA with specificity and sensitivity based on detection of antibodies against BD virus (BDV) p40 protein. Using our capture ELISA system, the antibody response of rats inoculated intracerebrally with BDV at 4 weeks after birth showed a sharp increase from 1 to 4 weeks postinoculation (p.i.) and a steady level after 5 weeks p.i. To investigate prevalence of BDV infection among wild rats, we examined sera of Rattus norvegicus in Kami-iso town, Oshima district, Hokkaido, suggesting that rats in this area had not been infected by BDV.     

Evaluation of a serological test system for the diagnosis of natural Echinococcus granulosus infection in dogs using E. granulosus protoscolex and oncosphere antigens

Serum antibody responses in feral or domesticated dogs naturally infected with Echinococcus granulosus or/and other common helminths were examined in an enzyme-linked immunosorbent assay (ELISA) using antigens prepared from E. granulosus protoscoleces or oncospheres. The ELISA using the protoscolex antigen was optimised with serums from experimental dogs monospecifically infected with E. granulosus or other helminth parasites, and helminth-free dogs. Anti-protoscolex antibody was detected in 16 of 22 (72.7%) serums from feral dogs with E. granulosus burdens ranging from 300 to 302,600 worms per dog. Seven serums from feral dogs which did not harbour E. granulosus at autopsy but which originated from an endemic hydatid region were tested using protoscolex antigen, and 1 serum gave a positive reaction. One hundred and two serums from dogs known never to have been infected with E. granulosus all gave negative reactions to protoscolex antigen. The sensitivity of the ELISA test proved to be superior to that which has been achieved by arecoline purging as a method of diagnosis for E. granulosus infection in dogs. For use of the assay in hydatid control or eradication campaigns, its sensitivity can be increased by choosing a lower absorbance discrimination value above which serums are regarded as having positive reactions. However, this does introduce positive reactions of some serums from dogs infected with helminths other than E. granulosus. In further development of the assay, use of defined recombinant antigens may improve both sensitivity and specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental inoculation of beagle dogs with Ehrlichia species detected from Ixodes ovatus

Three beagle dogs were inoculated with mice spleen/liver homogenate infected with Ehrlichia species detected from Ixodes ovatus (EIO) and one dog was used as a control. All three infected dogs did not show clinical signs of disease except for mild pyrexia throughout the 41-day study period. Splenomegaly was observed from Day 7 post-inoculation (p.i.) in two of the dogs. Hematological and biochemical abnormalities included mild thrombocytopenia, hypoproteinaemia, hypoalbuminaemia and increased C-reactive protein values. One of the dogs' splenic aspirate sample was PCR-positive for Ehrlichia Day 7 p.i. and another dogs' blood and bone marrow aspirate sample was PCR-positive Day 41 p.i. Sequence analysis of the PCR products showed 100% homology with the 16SrRNA partial gene sequence of Ehrlichia sp. HF565. Antibody titers to EIO were observed in all three experimentally infected dogs starting from the first week p.i. and cross-reactivity with Ehrlichia canis was detectable in one of the dogs starting Day 7 p.i. These data suggest that infection of dogs with EIO is possible, though is probably of low pathogenic importance. Cross-reactivity of EIO infected dog serum with E. canis raises the likelihood of false E. canis seropositive dogs.     

Experimental infection of possums with macropodid herpesvirus 1

AIM: To determine whether macropodid herpesvirus 1 (MaHV-1) could infect brushtail possums (Trichosurus vulpecula) and whether such infection would lead to latency in possums. METHODS: Possums (n=9) were instilled with 1.9 x 10(8) TCID50 of MaHV-1 into the conjunctivae and nostrils, while controls (n=3) were administrated with sterile saline. For virus isolation, all possums were swabbed from the conjunctivae and nasal cavities prior to inoculation, and on Days 1, 3, 7, 10 and 14 post-inoculation (p.i.), and then at weekly intervals for up to a further 6 weeks. Sera were collected on Day 0 immediately prior to inoculation and fortnightly thereafter, for the measurement of antibody. Corticosteroids were administrated to 4/9 virus-inoculated possums on Days 36 and 41 p.i. to determine whether latent MaHV-1 infection could be reactivated. Trigeminal ganglia and other tissues were collected at necropsy for viral detection. A nested polymerase chain reaction (PCR) targeting the DNA-dependent DNA polymerase (DNA pol) gene of the herpesviruses using degenerate primers was conducted on DNA samples isolated from the ganglia and livers from the possums, and blind-passaged cell cultures, for viral detection. RESULTS: A mild conjunctivitis was observed in the majority of the virus-inoculated possums between Days 3 and 21 p.i. Virus was recovered from the conjunctiva and/or nasal swabs from 8/9 virus-inoculated possums on Days 1 and 3 p.i. and from 3/9 on Day 7 p.i., but not following administration of corticosteroids. No virus was recovered from the trigeminal ganglia of the virus inoculated possums and possum DNA samples were negative for viral DNA following nested PCR. All of the virus-inoculated possums produced a virus neutralisation antibody response by Day 28 p.i., and five had a titre >/=512. The sequence of a 133 base pair (bp) segment of the MaHV-1 DNA pol gene was considerably different from that of other published data. CONCLUSION: This study demonstrated that MaHV-1 might have established a transient infection at the inoculation sites, and the inoculation of MaHV-1 via intranasal and conjunctival routes could elicit MaHV-1-specific antibody responses in possums. However, systemic and latent infection of MaHV-1 in possums was not demonstrated.     

Some studies on the growth and development of Echinococcus granulosus, camel origin in experimentally infected dogs.

The development of Echinococcus granulosus of camel subspecies in 35 experimentally infected dogs was studied at 3, 7, 10, 13, 18, 23, 35 and 56 days post-infection (d.p.i.). The morphological characteristics of each developmental stage is studied and discussed. Morphological characters included number, total length, shape, arrangement of rosteller hooks and blade length to total length percentage (B1/T1%). In addition, total worm length, scolex, suckers, rostellum, neck and segments were measured and described. Other useful characters are considered to be the position of common genital pore, number and distribution of testes, uterus, shape of ovary and vitelline glands. Generally, the results indicated that the segmentation first appears at 18 d.p.i. Moreover, male and female genitalia could be detected at the same period. However, completely developed genitalia appeared at 56 d.p.i. Histological examination of the small intestine of experimentally infected dogs revealed that the parasites were found in distended and thin wall crypts of Liberkuhn at the periods of 3.7 and 10 d.p.i, while the parasite scoleces were found embeded in the mucosa at the periods of 13, 18 and 23 d.p.i. No significant pathological changes were encountered in both infected and control dogs. The data presented here are the first report of strobilar development of E. granulosus of camel origin in experimentally infected dogs.     

Use of two polysaccharide antigens in ELISA for the detection of antibodies to Echinococcus granulosus in sheep sera

Polysaccharide antigens were obtained from either the secretions produced during in vitro cultivation of Echinococcus granulosus protoscoleces or from mouse hydatid cyst membranes by phenol extraction. When either of these antigens was used in an enzyme-linked immunosorbent assay antibody activities were detected in sera from sheep infected 27 or more weeks earlier with at least 100 E granulosus eggs. These antibody responses were significantly higher (P less than 0.05) than those of sheep infected with Taenia hydatigena or T ovis and tested with the E granulosus antigens. Very high cross-reacting antibody responses in sera from sheep recently infected with T hydatigena were only detected with the protoscoleces secretions antigen. Neither antigen was sufficiently sensitive or specific for serodiagnostic use. However, when sera were first tested with one antigen and then with the other, and only sera that were positive in both tests were regarded as positive, the overall sensitivity and specificity of this two antigen method increased to about 80 per cent.     

鸭源鸡杆菌对SPF雏鸡的感染特性研究

为研究鸭源鸡杆菌的流行病学特性,本研究采用从自然病例分离得到的鸭源鸡杆菌人工感染4日龄SPF雏鸡,通过形态学、PCR、荧光定量PCR和组织学等方法分别对感染后SPF雏鸡的组织脏器进行了细菌分离、检测和鉴定,用ELISA对其血清抗体水平进行检测.结果表明,人工感染鸡无临床症状,组织学检查感染鸡的肝脏、气管和肺脏组织均有损伤.人工感染后第3d和同居感染第2d鸡体内可分离出鸭源鸡杆菌,人工感染后第96d仍能够分离到鸡杆菌.ELISA方法证实人工感染后47 d和同居后32 d出现抗体高峰,持续2～3周,人工感染组和同居组抗体水平均高于对照组(p＜0.05);荧光定量PCR检测病料结果显示人工感染组和同居组的气管组织中细菌含量最高.本研究表明雏鸡在4日龄即可感染鸭源鸡杆菌,并能通过同居传播,长期带菌、排菌;感染后抗体产生缓慢、持续期短.本研究为鸭源鸡杆菌的流行病学、致病机理研究及防治措施的制定等提供了参考依据.     

Immune response to Encephalitozoon cuniculi infection in laboratory mice.

The study was performed to determine the immune response to Encephalitozoon cuniculi infection in immunocompetent mice during 120 days of experiment. Mice infected with E. cuniculi had an increased number of CD4+ T cells up to Day 20 post infection (p.i.), but counts of CD8+ T lymphocytes were significantly lower up to Day 90 p.i. in peripheral blood. Blood monocytes were significantly increased on the Day 60 and Day 120 of infection. A lack of significant decrease of CD4+ T cells may be considered as an important event in the immune response to E. cuniculi infection in immunocompetent mice.