Serodiagnosis of infections of pigs with Stephanurus dentatus

A serological survey of 135 pigs over 7 months old, some of which had liver and renal lesions in the presence ofStrephanurus dentatus, was undertaken for the presence of antibodies to S. dentatus. Only one sample gave a positive gel immunodiffusion reaction with crude whole worm extracts and none with immunoelectrophoresis. Oral inoculation of pigs with 1200 or 2000 L3 resulted in a specific reaction of anodal lines on immunoelectrophoresis from Day 37, and with double diffusion up to 4 specific lines were produced beginning from Day 43–53 using adult and juvenile antigens. Juvenile antigens gave more intense lines. Serological responses remained positive until 76–80 days post-infection. At necropsy infected pigs had fibrotic lesions in livers and portal thrombi containing larval S. dentatus even after 270 days. It is concluded that immunodiffusion and immunoelectrophoresis have limited usefulness in serodiagnosis of naturally occurring stephanuriasis.     

Serodiagnosis of bovine besnoitiosis by ELISA and immunofluorescence tests

Sera from non-infected cattle and cattle infected with Anaplasma, Babesia, Theileria and Sarcocystis were tested for antibodies to Besnoitia in ELISA and immunofluorescence tests (IFT) with Besnoitia besnoiti of blue wildebeest origin as antigen. Only 2 out of 86 sera gave false positive reactions in ELISA and none in the IFT, indicating a high specificity for the tests. Three-hundred-and-three bovine sera from 3 farms in an area endemic for besnoitiosis were similarly tested and the results were correlated with clinical findings based on visual inspection for typical symptoms and the presence of cysts in the scleral conjunctiva. Most of the positive tests were observed in cattle older than 1 year. Of the cases with scleral cysts, 68,7% were positive in the ELISA and 81,74% in the IFT. However, 45,74% (ELISA) and 49,47% (IFT) of the clinically negative cattle were clinically positive, indicating a high incidence of clinically inapparent infection. These results indicate a relatively low sensitivity for these serological tests. An unexpected finding was that the ELISA remained negative for at least 60 days after experimental infection of the cattle, the maximum period for which tests were done, whereas the IFT became positive. No antibodies against B. besnoiti could be found in human sera. Besnoitia jellisoni antigen gave positive results with B. besnoiti antibodies in ELISA, but not in the IFT.     

Serodiagnosis of Dermatophilus congolensis infection by counterimmunoelectrophoresis

Sixty-one sera from animals that had contact with Dermatophilus congolensis were examined by comparing three serological methods; counterimmunoelectrophoresis, passive haemagglutination, and agar gel diffusion, and by using four different antigenic extracts of D congolensis. The counterimmunoelectrophoresis was the most satisfactory of the methods having been found to be specific and sensitive, easy to perform and suitable for screening large numbers of samples. It was also found to have a higher antibody detection rate (82.2 per cent) than the other methods thus making it suitable for seroepidemiological surveys. It was found to be capable of detecting multiple antibodies and also revealed dissimilarities among the different antigenic extracts. The cellular antigens of D congolensis were found to detect antibody in more sera than the extracellular antigen; the cell wall extract proved to be the most satisfactory of all, detecting antibody from the largest number of sera compared to the other extracts in all the three serological tests.     

Rapid diagnosis of Aujeszky's disease in pigs by immunofluorescence

Direct immunofluorescence on impression smears of brain and pharynx was compared with virus isolation in cell culture for the diagnosis of Aujeszky's disease in experimentally and naturally infected pigs. Pharyngeal impression smears were more sensitive than virus isolation in two pigs killed 10 and 12 days after experimental infection. Both methods were of similar sensitivity in the detection of virus from field cases of disease. Smears of brain and pharynx were more sensitive than virus isolation for tissue which had been stored at room temperature (approximately 20 degrees C) for up to 48 hours. Some reduction in the amounts of virus recovered from tissues and the intensity of fluorescent staining occurred in these samples.     

Ecological studies of Yersinia enterocolitica. II. Experimental infection with Y. enterocolitica in pigs

Fattening pigs were inoculated with the human pathogenic strains of Yersinia enterocolitica, biovar 4 serovar 3 phagovar 8 and biovar 2 serovar 5.27. Each pig received 2.6 × 10⁹ organisms by catheter into the stomach and thereafter 10% sodium bicarbonate solution, by the same route. The serovar 3 strain became established in the intestines of four out of six 11-week-old pigs and in all three 24-week-old pigs. Serovar 5.27 established in the intestines of all four 10-week-old pigs. In these pigs, both serovars were excreted in the feces at 10²?10⁶ cells g^?1 for a few weeks. However, the serovar 5.27 was excreted in the feces at less than 10^3·3 cells g^?1 for 2 weeks by 24-week-old pigs. Horizontal transmission with the serovar 5.27 strain was observed on the 5th–11th day, and the bacteria were present in the intestines during the 2nd week. However, the serum titers to O-agglutinin were 1/10 and less, but in one pig, the titer was 1/40 against serovar 5.27 strain.     

Serodiagnosis of Fasciola gigantica infection in buffaloes

The agar gel precipitation test (AGPT), counter immunoelectrophoresis (CIEP) and indirect haemagglutination (IHA) were evaluated for the diagnosis of fascioliasis due to Fasciola gigantica in buffaloes. The sensitivity of these tests varied with the intensity of infection; and was greatest when the fluke burden in liver exceeded 100. CIEP detected 76.06% of infected sera and was most sensitive, followed by IHA which detected 68.37% of the infected sera. The AGPT was found to be least sensitive, detecting only 57.4% of the infected sera. Although these tests were limited by the occurrence of false-positive reactions, their use may be an aid for effective diagnosis of fascioliasis in buffaloes.     

Blastocystis sp. infections in pigs

A species of Blastocytis has been identified and isolated from pigs in England. A limited survey was carried out, which showed that the organism was present on each of five pig farms visited in Berkshire and East Anglia. Sixty percent of pigs sampled harboured Blastocystis, though usually in low numbers. Higher numbers were seen only in those pigs suffering diarrhoea or dysentery but there is no evidence as yet to suggest that Blastocystis has a pathogenic role.     

An immunofluorescence technique for the detection of Toxocara canis antibodies

An immunofluorescent (IF) test for the serodiagnosis of Toxocara canis infections in puppies is described. Frozen sections of male adult T. canis worms were used as antigen.A group of seven puppies, 6 weeks of age, was infected orally with 10 000 embryonated T. canis eggs each. In the sera of all animals IF antibodies could be detected from approximately 4 weeks after infection onwards. Titers were detectable until the end of the observation period (22 weeks).Two puppies of the same age were infected with 30 000 or 50 000 embryonated T. canis eggs respectively. Positive IF results were also obtained in the sera of these pups from week 4 post infection (p.i.) onwards. No correlation between titer and initial number of egges administered was observed. Furthermore, no correlation was noticed between titer and number of adult worms recovered from the dogs. For comparison all sera were tested with the complement fixation (CF) test, using cuticle material of adult worms as antigen. Complement fixing antibodies could be detected in none of the serum samples.     

Serodiagnosis of leptospirosis in pigs using an axial filament enzyme-linked immunosorbent assay.

The axial filament (AF) from Leptospira interrogans serovar canicola was isolated by cesium chloride density gradient centrifugation of 2% sarcosyl treated whole cells. Isolation of AF was confirmed by electron microscopic examination, by protein-A immunogold labelling, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting. Analysis by SDS-PAGE of the purified preparation showed relatively weak bands of molecular size 41 kDa and 21 kDa, and strong bands of 35 kDa and 34.5 kDa. Immunoblot analysis using antiserum to the AF against sonicated leptospires of a variety of serovars showed prominent reaction against the 41, 35, and 34.5 kDa protein bands, as well as against minor bands of molecular weight 43, 39, and 37 kDa. Antisera prepared against leptospiral serovars also identified minor bands at 33 and 32 kDa. Immunoblots with antiserum to whole cells of serovar bratislava detected the 35 and 34.5 kDa AF bands of Borrelia burgdorferi moderately and of Treponema hyodysenteriae only slightly in comparison to leptospiral AF. Antibody to B. burgdorferi did not detect the leptospiral AF antigen. Immunoblots with antiserum to T. hyodysenteriae showed a marked reaction with a 41 kDa band of B. burgdorferi but only a very minor reaction with leptospiral AF. The AF was tested in an AF-ELISA against sera from 260 pigs, many of which reacted in the microscopic agglutination test (MAT) against one or more leptospiral serovars. A sensitivity of 97.1% and a specificity of 93.1% was determined in comparison to the MAT. Only moderate correlation was observed between titres detected in the AF-ELISA and the MAT (r = 0.4). When sonicated whole cells (WC) of serovar canicola were used in an ELISA (WC-ELISA), high correlation was observed between AF-ELISA and WC-ELISA (r = 0.97). These findings show that the AF-ELISA can be used effectively as a species-specific antigen for the serological diagnosis of leptospirosis in swine and that sonicated whole cells can substitute excellently for purified AF as the antigen source. These findings may be extrapolated to the use of AF in immunodiagnosis of leptospirosis in other species.     

Ecological studies of Yersinia enterocolitica I. Dissemination of Y. enterocolitica in pigs

Pigs were examined on five farms for carrier status of Yersinia enterocolitica. Yersinia enterocolitica biovar 4, serovar 3, phagovar VIII was isolated consistently from the feces of fattening pigs on one farm and sporadically from those of similar pigs on the other farms and of sows on all five farms, during a one-year period of weekly surveys. Seasonal variation was not a feature of fattening pigs on a highly contaminated farm. In other pigs, however, the organisms were not isolated during the summer months. On a highly contaminated farm, the organisms were excreted in the feces of 8 to 15-week-old pigs within 1–3 weeks of entering pens which were thought to be contaminated with the organisms. On a detailed observation of natural infection of Y. enterocolitica in eight pigs, the organism appeared in the pigs' feces within 2–7 weeks of them being moved to a pen which had been washed thoroughly after becoming contaminated, by a previous group of pigs, with feces containing 10⁵ viable organisms per g. Thus, Y. enterocolitica is apparently transmitted from infected feces or picked up from the floor of a contaminated pen, and the regular schedule of pig movement among the pens is an important factor in the spread of Y. enterocolitica within a piggery. Intestinal colonization continues for a long time and does not occur by re-infection. The organisms were not isolated from eight pigs at the time of slaughter, and their serum O-agglutinin titers were $\text{1}\text{40}$ or less. Thus, circulating antibody may not inhibit intestinal colonization by Y. enterocolitica.     

Serodiagnosis of infection withTrypanosoma evansi in camels in the Sudan

Summary Five diagnostic tests for infection withTrypanosoma evansi have been compared in groups of camels experimentally infected or exposed to natural infection in the Sudan. The correlation of positive results obtained by assays of IgM levels, the mercuric chloride test and the formol gel test with the presence of active infection was unsatisfactory, but there was a good correlation between results obtained using IFAT and ELISA and proven infection. Sera from a high proportion of apparently uninfected camels from endemic areas gave positive reactions with all 5 tests, possibly indicating inadequate parasitological diagnosis or persistence of antibody after unsatisfactory chemotherapy. It was concluded that serological tests using trypanosomal antigens to detect antibodies were more sensitive for diagnosis than indirect tests based on raised euglobulin levels. Serodiagnostic tests may therefore have a place in future programmes for surveillance and control ofT. evansi infections in camels.

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Serodiagnostico De La Infeccion PorTrypanosoma Evansi En Camellos En Sudan

Resumen Se compararon cinco pruebas diagnósticas para infecciones porTrypanosoma evansi en grupos de camellos infectados experimentalmente o expuestos a la infección natural. La correlación de los resultados positivos utilizando pruebas de difusión radial aisladas, le niveles de IgM, la prueba de cloruro de mercúrio, y la prueba de agar formol con la presencia de infección activa, fue insatisfactória. Hubo buena correlación entre los resultados obtenidos con IFAT y ELISA con infección comprobada. El suero de una proporción alta de camellos aparentemente sanos, provenientes de áreas endémicas, presentaron reacciones positivas con las cinco pruebas, indicando posiblemente un diagnóstico parasitológico inadecuado, o la persistencia de anticuerpos despues de una quimioterápia inadecuada. Se concluyó, que las pruebas serológicas usando antígenos preparados de tripanosomas para detectar anticuerpos, fueron más sensitovos que las pruebas indirectas basadas en el aumento de euglobulinas. Las pruebas serodiagnósticas posiblemente tendran un lugar en programas futuros de reconocimiento y control deT. evansi en camellos.

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Serodiagnostic De La Trypanosomose AT. Evansi Chez Le Chameau Du Soudan

Résumé Cinq méthodes de diagnostic de la trypanosomose àT. evansi chez le chameau ont été comparées en utilisant des chameaux expérimentalement et naturellement infectés, au Soudan. La corrélation des résultats positifs obtenus par utilisation de la diffusion radiale des Igm, par le test au chlorure mercurique et par le test de la formol gélification à l'occasion des infections en évolution, n'a pas été satisfaisante alors qu'il y a eu bonne corrélation entre les résultats obtenus par IFAT et ELISA et des infections véritables.Les sérums d'une importante proportion de chameaux apparemment non infectés, en provenance de régions à maladie endémique, ont donné des réactions positives aux cinq tests, ce qui peut indiquer soit une insuffisance dans le diagnostic parasitologique soit la persistance d'anticorps résultant d'une chimiothérapie insuffisánte.Les auteurs concluent que les tests sérologiques utilisant des trypanosomes comme antigènes pour mettre les anticorps en évidence sont plus sensibles, en matière de diagnostic, que les tests indirects basés sur la détection des niveaux des IgM. C'est pourquoi les tests faisant appel aux méthodes de sérodiagnostic doivent avoir leur place dans les programmes à venir concernant tant la surveillance que la lutte contre les infections du chameau àT. evansi.

     

Naturally acquired Lawsonia intracellularis infection in pigs studied from weaning to slaughter by indirect immunofluorescence antibody test and polymerase chain reaction on faeces

The course of naturally acquired Lawsonia intracellularis infection was studied in 41 pigs by testing blood and faeces samples collected four to seven times from before weaning to slaughter 5 months old. At slaughter, a sample of ileum was taken for histopathology. In the first sampling when the pigs were 2-4 weeks old maternally derived IgG against L. intracellularis was demonstrated by immunofluorescence antibody test in nine pigs whereas the bacterium was detected by PCR in faeces from six pigs. The maternally derived antibodies did not prevent pigs from becoming infected as seven pigs later on shed and/or were seropositive for L. intracellularis. The lowest prevalence of L. intracellularis was observed in 6-13 weeks old pigs and it seemed as though L. intracellularis in early infected pigs only activates a minor antibody response. At slaughter 66% of the pigs were found positive by immunofluorescence antibody test compared to 24% by immunohistochemistry on ileal samples. Thus, applied at the time of slaughter the antibody test appeared to be a highly sensitive ante-mortem diagnostic tool for identifying L. intracellularis exposed pigs with or without current proliferative enteropathy.     

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.     

Rapid diagnosis of TGEV-like coronavirus in fattened pigs by indirect immunofluorescence labelling in nasal cells

Diagnosis of TGEV-like coronavirus was made by an immunofluorescence technique using monoclonal antibodies directed against TGEV. Six, 15 weeks-old pigs were inoculated intratracheally with the strain P 6008 ST 4. No clinical sign was observed during the course of the infection. Viral antigens were detected in the cytoplasm of nasal cells obtained from swab in five animals as early as one day p.i. and during several days. The sensitivity and specificity of this method appeared to be comparable to virus isolation of this cell-adapted TGEV-like strain.     

Enhanced protective immune responses against Salmonella Enteritidis infection by Salmonella secreting an Escherichia coli heat-labile enterotoxin B subunit protein

Escherichia coli heat-labile enterotoxin B subunit (LTB) protein is a potent mucosal adjuvant. In this study, the effect of an attenuated Salmonella secreting LTB protein as an adjuvant strain (JOL1228) for a live Salmonella Enteritidis (SE) vaccine candidate (JOL919) was evaluated. In a single immunization experiment, chickens immunized with a mixture of JOL919 (5 parts) and JOL1228 (1 part) showed enhanced mucosal and cellular immune responses and efficient protection against salmonellosis as compared to those unimmunized control chickens. In further analysis, chickens were primed at one day of age and were boosted at the fifth week of age to prolong immune responses and to maximize the protection efficacy against salmonellosis. The immunized groups B (prime and booster with JOL919), C (prime with JOL919-JOL1228 mixture and booster with JOL919), and D (prime and booster with JOL919-JOL1228 mixture) showed significantly higher humoral and cellular immune responses as compared to those in the unimmunized control group A. In addition, immunized groups C and D showed fewer gross lesions in the liver and spleen and a lower number of SE-positive organs, with the lowest bacterial counts in the SE challenge strain as compared to the control group. These results indicate that SE vaccination with the LTB strain can have an adjuvant effect on the vaccine candidate by enhancing immune responses, and that a prime-boost strategy with the addition of the adjuvant strain can efficiently protect birds against salmonellosis.