Evaluation of a recombinant enzyme-linked immunosorbent assay for detecting Chlamydophila psittaci antibodies in turkey sera

Chlamydophila psittaci (formerly Chlamydia psittaci) is one of the major pathogens associated with turkey respiratory disease. Devastating outbreaks with high mortality rates, similar to those of 1950 to 1970 in the USA occasionally occur, but respiratory signs without or with low mortality mostly characterize outbreaks now a day. Accurate diagnostic methods should be made available. The present study examined the sensitivity and specificity of a recombinant ELISA (rMOMP ELISA) for detecting Cp. psittaci major outer membrane specific antibodies in turkey sera. Test results were compared to those of immunoblotting and of a competitive ELISA (Chlamydia-psittaci-AK-EIA, R?hm Pharma, Germany) and an indirect ELISA (LPS/LGP) detecting antibodies to the lipopolysaccharide/lipoglycoprotein complex. The rMOMP ELISA was most sensitive as determined on serial dilutions of positive control sera originating from experimentally infected SPF turkeys. The competitive ELISA gave false positives since three negative controls reacted positive. For conventional sera, the sensitivities of the competitive ELISA, immunoblotting and the indirect ELISA were found to be 99.4, 93.1 and 82.2%, respectively, as compared to the rMOMP ELISA (100%). The specificities of the rMOMP ELISA, immunoblotting and the indirect ELISA were found to be 100% while the specificity of the competitive ELISA was only 2.7%. The rMOMP ELISA was chosen to compare the prevalence of chlamydiosis in 2002 with the one from 1992. In 2002, 188 on 200 (94%) turkey sera reacted positive compared to 175 on 200 (87.5%) in 1992 and like 10 years ago all examined farms were seropositive at slaughter. Interestingly, Belgian as well as French farms were seropositive.     

用固定细胞阻断酶联免疫吸附试验鉴别诊断猪传染性胃肠炎病毒和猪呼吸道冠状病毒的感染

用固定细胞阻断酶联免疫吸附试验（ＥＬＩＳＡ）对来自丹麦猪的１８０份血清进行了猪传染性胃肠炎病毒（ＴＧＥＶ）和猪呼吸道冠状病毒（ＰＲＣＶ）感染的鉴别诊断，共检出了ＰＲＣＶ抗体阳性血清１０７份（５９．４％），ＴＧＥＶ抗体阳性血清０份。同时也检测了一些来自国内不同ＴＧＥＶ感染类型的猪场血清。该鉴别诊断方法在我国的建立和应用为从ＰＲＣＶ阳性国家进口猪的ＴＧＥ的检疫提供了一条有效途径。     

The prevalence of Chlamydia psittaci infections in Belgian commercial turkey poults

The prevalence of Chlamydia psittaci infections in Belgian commericial turkey poults was examined and a follow-up study of one Belgian turkey flock was performed. Sera were examined for the presence of anti-chlamydia antibodies by immunoblotting. Cloacal and conjunctival swab smears and lung impression smears were examined for the presence of chlamydial antigen using the IMAGEN Chlamydia immunofluorescence test. Anti-chlamydia antibodies were found in 90 of 100 sera collected at slaughter from turkeys raised during the summer of 1992. The following winter, 73 of 100 sera reacted positively. On all twenty farms examined during 1992, turkeys were positive for anti-chlamydial antibodies. During 1993, chlamydial antigen was detected in swabs from 20 of 40 slaughterhouse turkeys tested. Antigen was found more often in the cloaca than in the conjunctiva. Chlamydial antigen was detected in samples from each of the 4 farms examined. The follow-up study on a turkey farm, sampling the birds at weekly intervals from one week old until 12 weeks of age, revealed that chlamydial antigen and anti-chlamydial antibodies were present during the whole period. During 1994, chlamydial antigen was detected in 45 of 60 lungs from slaughterhouse turkeys from all of 6 farms. During 1995, chlamydial antigen was detected in 41 of 54 lungs of 6 week old commercial turkey poults. The results of the present study indicate that Chlamydia psittaci infections are highly prevalent amongst Belgian commercial turkey poults with apparently little seasonal or year-to-year variation and that turkeys can contract the infection at an early age.     

Key role of Chlamydophila psittaci on Belgian turkey farms in association with other respiratory pathogens

Two hundred turkey sera from eight Belgian and two French farms were tested for the presence of antibodies against avian pneumovirus (APV), Ornithobacterium rhinotracheale (ORT), Mycoplasma gallisepticum, Mycoplasma meleagridis and Chlamydophila psittaci. At slaughter, C. psittaci, APV and ORT antibodies were detected in 94, 34 and 6.5% of the turkeys, respectively. No antibodies against M. gallisepticum or M. meleagridis were present. Additionally, turkeys on three Belgian farms were examined from production onset until slaughter using both serology and antigen or gene detection. All farms experienced two C. psittaci infection waves, at 3-6 and 8-12 weeks of age. Each first infection wave was closely followed by an ORT infection starting at the age of 6-8 weeks, which was still detectable when the second C. psittaci infection waves started. Animals on farm A were not vaccinated against APV leading to an APV subtype B outbreak accompanying the first C. psittaci infection wave. Despite subtype A APV vaccination on farms B and C, the second C. psittaci infection waves were accompanied (farm B) or followed (farm C) by a subtype B APV infection. On all farms respiratory signs always appeared together with a proven C. psittaci, APV and/or ORT infection. This study suggests an association between C. psittaci, APV and ORT, and indicates the multi-factorial aetiology of respiratory infections in commercial turkeys. All three pathogens should be considered when developing prevention strategies for respiratory disease.     

Laboratory decision-making during the classical swine fever epidemic of 1997-1998 in The Netherlands

The National Reference Laboratory for classical swine fever (CSF) virus in the Netherlands examined more than two million samples for CSF virus or serum antibody during the CSF epizootic of 1997–1998. The immense amount of samples and the prevalence of border disease (BD) virus and bovine viral diarrhoea (BVD) virus infections in Dutch pig herds necessitated the diagnostic efforts of the laboratory to be focused on generating CSF specific test results throughout the eradication campaign.

Detection of 82% of the 429 outbreaks was achieved through the combined use of a direct immunofluorescence and peroxidase assay (FAT/IPA) with samples (tonsils) collected from clinically-suspected pigs. This suggests that in the majority of the outbreaks, the pigs had clinical signs that were recognised by the farmer and/or veterinarians, indicating the presence of CSF virus in a pig herd. A positive diagnosis of 74% of all the tissue samples (tonsils) collected at infected pig holdings was established by FAT. More than 140,000 heparinised blood samples were examined by virus isolation, resulting in the detection of 4.5% of the infected herds. CSF virus was isolated in approximately 29% of all the blood samples collected from pigs at infected or suspected farms.

Several serological surveys — each done within a different framework — led to the detection of 13.5% of the total number of outbreaks. The detection of CSF virus antibody in serum was carried out by semi-automated blocking ELISA. Approximately 28.5% of the sera which reacted in the ELISA were classified as CSF virus-neutralising antibody positive and 26.5% as positive for other pestiviruses following the virus neutralisation test (VNT).

We concluded that two of the CSF laboratory diagnostic methods described were determinative in the eradication campaign: first, the FAT for the screening of diseased pigs; and second, the ELISA and VNT when millions of predominantly healthy pigs needed to be screened for the presence of CSF serum antibody. Decision-making on the basis of results generated by either method can, however, be seriously hindered when samples are examined from pig herds with a high prevalence of non-CSF pestiviruses.     

2016年-2017年新疆规模化猪场伪狂犬病抗体检测

为了解新疆地区不同规模化猪场伪狂犬病病毒(PRV)野毒感染情况,采用ELISA对2016年4月-2017年12月来自67个不同规模化猪场的2 516份血清进行了PRV-gE抗体血清学调查。结果显示,血清总阳性率为34.02%,2016年和2017年阳性率分别为17.77%和39.52%;哺乳仔猪、保育猪、育肥猪、后备母猪、妊娠母猪、哺乳母猪、公猪总阳性率分别为26.07%、22.35%、35.38%、32.70%、33.13%、31.06%和31.71%;猪场总阳性率为82.09%,北疆、南疆和东疆猪场阳性率分别为78.57%、100.00%和100.00%。结果表明,2016年-2017年新疆地区猪群中PRV感染率明显上升,南疆与东疆地区阳性率显著高于北疆地区,各个阶段猪群阳性率均显著升高,母猪与公猪感染加重是伪狂犬病持续存在的重要原因,对其进行防控与净化刻不容缓。     

规模化猪场蓝耳病血清学调查

蓝耳病是影响规模化猪场繁殖性能的主要传染性疾病,笔者在江苏省6个年产万头商品猪的规模化猪场猪群中随机采集297头种猪及仔猪血清进行蓝耳病血清学调查,使用IDEXX酶联免疫吸附诊断试剂盒,检测蓝耳病病毒抗体。结果表明,2个未免疫接种老猪场猪群抗体阳性率52%,断奶仔猪群阳性率39.1%;2个免疫接种老猪场猪群抗体阳性率94%,断奶仔猪群阳性率89.8%。而较新猪场的种猪群阳性率28.6%,断奶仔猪群阳性率5%。     

大庆市及周边地区非免疫猪群猪圆环病毒感染情况调查

为了解大庆市及周边地区猪群中PCV的感染情况,应用ELISA和PCR技术对采自大庆市5区4县及周边地区55个村屯/猪场未免疫PCV2疫苗猪血清样本649份,5家病猪场和6家屠宰场组织样本448套,进行血清学和分子生物学检查,并对检测结果进行了比较分析。结果显示,大庆市及周边地区未免疫PCV2疫苗猪血清样品PCV2抗体平均阳性率为54.70%,其中断奶仔猪样品阳性率为50.68%,育肥猪样品为70.00%,繁育母猪样品为54.70%。组织样品PCV1阳性率为4.46%,PCV2阳性率为22.32%。调查结果可为确定地区流行毒株序列提供原始材料和制定科学防控措施提供依据。     

Evaluation of a serodiagnostic test using Ascaris suum haemoglobin for the detection of roundworm infections in pig populations

The significant economical consequences of infections with Ascaris suum in pigs are already well documented. However, due to the subclinical nature of the disease and the lack of practical diagnostic means, ascariasis often remains undiagnosed. Here we describe the development and evaluation of a novel indirect ELISA using the purified A. suum haemoglobin (AsHb) molecule as an antigen. Initial validation using sera from 190 pigs experimentally infected twice a week with A. suum and Trichuris suis (25 and 5eggskg(-1)day(-1) respectively) demonstrated that the AsHb ELISA is able to detect long-term exposure to A. suum with a high sensitivity and specificity (99.5% and 100.0% respectively). Furthermore, this serological technique proved to be more sensitive than faecal examination on week 7 and 14 of the experiment (99.5% and 100% compared to 59.5% and 68.4% respectively). Cross-reactivity caused by T. suis infection was shown to be limited after analysing sera from pigs with an experimental T. suis mono-infection. Seroconversion was shown to occur from week 6 onwards in pigs receiving 100 A. suum eggs 5 times a week. Preliminary testing of the ELISA on six randomly selected farms confirmed the results obtained in the artificial infection trials, showing a higher sensitivity of the serologic method compared to faecal examination. Finally, the ELISA was used to investigate Ascaris infection rates on 101 conventional Flemish pig farms. The results showed that on 38.6% of the farms less than 20% of the tested samples were seropositive, while in 19.8% of the farms 80-100% of all pigs were seropositive. The results of this study suggest that the AsHb ELISA could provide pig farmers and veterinarians with an easier and more sensitive way to estimate the overall prevalence of A. suum on their farm.     

Serological response to pgp3 protein in animal and human chlamydial infections

Specific antibodies to plasmid-encoded protein pgp3 are known to be encountered in human Chlamydia (C.) trachomatis infections. In order to verify whether antibodies to this protein could be developed in animals infected with plasmid-carrying chlamydial strains, 454 animal sera were examined using a home-made pgp3 protein ELISA and Western blots (WB) of recombinant pgp3 protein from Chlamydophila (Cp.) psittaci. Likewise, 50 human sera were tested by ELISA and WB of recombinant pgp3 from C. trachomatis. The reactivity against pgp3 protein was compared to the reactivity against chlamydial elementary bodies (EBs) detected by microimmunofluorescence (MIF) test. The presence of pgp3-specific antibodies was demonstrated in most ducks and pigeons with Cp. psittaci infection detected by MIF, as well as in the majority of symptomatic cats and pigs infected with Cp. felis and C. suis, respectively, which reacted at high titres to Cp. felis and C. suis EBs by MIF. Moreover, most of the sera collected from patients with C. trachomatis culture-confirmed infection and seropositive to C. trachomatis by MIF, presented antibodies specific to C. trachomatis pgp3 recombinant protein. Therefore, pgp3 protein could be a useful marker of chlamydial infections in animals, as well as in humans.     

Chlamydiaceae infections in pig

ABSTRACT: Chlamydiaceae are Gram-negative obligate intracellular bacteria. They are responsible for a broad range of diseases in animals and humans. In pigs, Chlamydia suis, Chlamydia abortus, Chlamydia pecorum and Chlamydia psittaci have been isolated. Chlamydiaceae infections in pigs are associated with different pathologies such as conjunctivitis, pneumonia, pericarditis, polyarthritis, polyserositis, pseudo-membranous or necrotizing enteritis, periparturient dysgalactiae syndrome, vaginal discharge, return to oestrus, abortion, mummification, delivery of weak piglets, increased perinatal and neonatal mortality and inferior semen quality, orchitis, epididymitis and urethritis in boars. However, Chlamydiaceae are still considered as non-important pathogens because reports of porcine chlamydiosis are rare. Furthermore, Chlamydiaceae infections are often unnoticed because tests for Chlamydiaceae are not routinely performed in all veterinary diagnostic laboratories and Chlamydiaceae are often found in association with other pathogens, which are sometimes more easily to detect. However, recent studies have demonstrated that Chlamydiaceae infections in breeding sows, boars and piglets occur more often than thought and are economically important. This paper presents an overview on: the taxonomy of Chlamydiaceae occurring in pigs, diagnostic considerations, epidemiology and pathology of infections with Chlamydiaceae in pigs, public health significance and finally on prevention and treatment of Chlamydiaceae infections in pigs.     

Development and evaluation of an immunochromatographic strip for detection of Streptococcus suis type 2 antibody.

In this study, an immunochromatographic strip (ICS) was developed for the detection of antibody against Streptococcus suis serotype 2 (SS2). Colloidal gold particles labeled with staphylococcal protein A (SPA), which can bind to the F(C) fragment of mammalian immunoglobulin, were used as the detector reagent. The capsular polysaccharide (CPS) of SS2 and affinity-purified IgG from a healthy naive pig were immobilized on test and control regions of a nitrocellulose membrane, respectively. The ICS was used to 1) detect anti-CPS antibody in 14 sera taken from 4 SS2-infected pigs, 24 sera from pigs hyperimmunized with SS2, and 68 sera from pigs inoculated or infected with bacteria other than SS2; 2) determine anti-CPS antibody titers of 20 positive sera for comparison with enzyme-linked immunosorbent assay (ELISA); and 3) detect anti-CPS antibody in 226 clinical sera taken from diseased pigs also for comparison with ELISA. An ELISA used as a reference test determined the specificity and sensitivity of the ICS to be 97.1% and 86.3%, respectively. There was excellent agreement between the results obtained by ELISA and the ICS (kappa = 0.843). Additionally, there was strong agreement between the results of bacterial isolation from pig tonsils and ICS test (kappa = 0.658). Because it is rapid and easy to use, the test is suitable for the serological surveillance of SS2 at farms.     

Evaluation of recombinant Bhlp29.7 as an ELISA antigen for detecting pig herds with swine dysentery

Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Diagnosis of SD traditionally has relied on detecting the spirochaete in the faeces of acutely affected pigs. To date simple and reliable serological assays that can be applied as a diagnostic tool at the herd level have not been available. In the current study a recombinant histidine tagged 29.7 kDa lipoprotein of B. hyodysenteriae (His6-Bhlp29.7) was used as an ELISA plate-coating antigen. Sera (n=1121) from slaughter-aged pigs on 19 farms were tested in this ELISA. Following optimization of the ELISA conditions using hyperimmune control sera, a set of 464 sera from slaughter-aged pigs from five herds where SD did not occur was tested. From these results a suitable cut-off value for herd negativity was defined as the mean optical density reading plus three standard deviations. Testing of 337 pig sera from six farms with SD then showed that the sensitivity of the test at the herd level was 100%, with all six farms having one or more serum samples exceeding the cut-off value for negativity. Finally, 320 sera from eight herds suspected of having SD were examined. Four of these herds were shown to have pigs with titres consistent with SD. The true health status of the other four herds that were serologically negative could not be confirmed. In conclusion, when used on sets of 40 sera from slaughter-aged pigs the His6-Bhlp29.7 ELISA as established proved to be a useful adjunct to the diagnosis of SD at the herd level.     

Seroprevalence of F4+ enterotoxigenic Escherichia coli in regions with different pig farm densities.

Sera of young sows from 135 closed Belgian pig breeding farms were examined for the presence of serum antibodies specific for the F4 fimbrial antigen of enterotoxigenic Escherichia coli (ETEC). Since 80% of all pig farms in Belgium are located in the provinces West-Vlaanderen (44%, approximately 18 farms/10 km2), Oost-Vlaanderen (20%, approximately 9 farms/10 km2), Antwerpen (10%, approximately 5 farms/10 km2) and Vlaams-Brabant (6%, approximately 3 farms/10 km2), the farms examined were randomly selected in these four regions. On 68% of all tested farms, sows were not vaccinated against enteric colibacillosis. In general, 65% of these non-vaccinated farms were F4-seropositive (mean optical density [OD405] >0.5) of which 38% were weakly (mean OD405 = 0.5-1.0), 21% moderately (mean OD405 = 1.0-2.0) and 6% strongly positive (mean OD405 > or =2.0). These percentages showed major differences between the four provinces. In Vlaams-Brabant, only 31% of non-vaccinated farms were seropositive, of which most were regarded as weakly positive. In Antwerpen, Oost- and West-Vlaanderen however, this percentage was clearly higher and respectively 68, 71, and 79% of non-vaccinated farms were seropositive, of which most were weakly positive. These observations indicate that F4+ ETEC is widely spread and highly prevalent on non-vaccinated pig breeding farms in Belgium. Moreover, an increasing F4-seropositivity with increasing pig farm density suggests a possible influence of the swine density on the prevalence of F4+ ETEC infections.     

Comparison of Different Commercial Serological Tests for the Detection of Toxoplasma gondii Antibodies in Serum of Naturally Exposed Pigs

Toxoplasma gondii is the aetiological agent of the zoonotic disease toxoplasmosis and transmitted among other ways by chemically and physically untreated, that is, raw pork to humans. The detection of Toxoplasma gondii is impossible by currently practiced meat inspection, but serological tests can be used to detect Toxoplasma gondii antibodies in pig herds and can consequently be helpful to identify potentially contaminated pork. Therefore, appropriate serological tests are required. In this study, serum samples of 1368 naturally exposed slaughter pigs from 73 Austrian farms were collected. Serum samples of at least 16 slaughter pigs per farm were tested. The prevalence of Toxoplasma gondii antibodies in serum was measured by a commercial available modified agglutination test (MAT) and compared to three different commercial available enzyme‐linked immunosorbent assays (ELISA). The MAT detected 6.5%, ELISA I 6.7%, ELISA II 4.8% and ELISA III 4.3% of the pigs as Toxoplasma gondii antibody positive. The agreement, according to the kappa coefficient (κ), was substantial between the MAT and ELISA I (κ = 0.62), II (κ = 0.64) and III (κ = 0.67). A better agreement was determined between ELISA I and II (κ = 0.715), ELISA I and III (κ = 0.747) and ELISA II and III (κ = 0.865). At least one pig per farm was detected Toxoplasma gondii antibody positive in 17 (23.3%) farms by the MAT, 26 (35.6%) farms by ELISA I, 16 (21.9%) farms by ELISA II and 11 (15.1%) farms by ELISA III. Pig farms with a high number of Toxoplasma gondii antibody‐positive pigs or high antibody titres were identified by all of the four used serological tests. Concerning the occurrence of Toxoplasma gondii antibodies in Austrian pig farms, a monitoring and surveillance programme would be reasonable to find high‐risk farms.     

High prevalence of Hepatitis E virus in French domestic pigs

The importance of the domestic pig reservoir for Hepatitis E virus (HEV) was assessed by estimating the seroprevalence and prevalence of HEV contaminated livers in French slaughter-aged pigs. 6565 sera and 3715 livers were randomly sampled from 186 pig farms throughout the country. Taking the sampling design into account, the farm-level seroprevalence was 65% (95% CI 57–74) and 31% (95% CI 24–38) of the slaughter-aged pigs had antibodies against HEV. The individual prevalence of HEV RNA positive livers was 4% (95% CI 2–6) and 24% (95% CI 17–31) of the farms had at least 1 positive liver. Most isolates were of genotype 3f (76.7%) with smaller amounts of 3c (18.6%) and 3e (4.6%). The high prevalence of HEV in pigs and the similarities between HEV subtypes from pigs and humans corroborates the possible zoonotic origin of some HEV autochthonous infections.     

规模化猪场猪圆环病毒2型的血清流行病学调查

为了解规模化猪场猪圆环病毒2型(PCV2)血清学流行情况,采用ELISA进行PCV2抗体检测。结果表明,运城地区规模化猪场广泛存在PCV2感染,未免疫接种猪圆环病毒疫苗的外观健康猪血清样品中PCV2抗体平均阳性率为49.66%,其中种猪感染率达67%,哺乳仔猪感染率4.55%,保育猪感染率43%,育肥猪感染率64.71%;外观健康猪群检测结果提示,随着年龄的增长PCV2感染有升高趋势。为了进一步研究猪圆环病毒感染各症候群在规模化场发病特点及流行规律,采集临床疑似断奶仔猪多系统衰竭综合征(PMWS)、仔猪先天性震颤(Congenital tremors,CT)、猪肾炎皮炎综合征(PDNS)、猪的繁殖障碍(Reproductive failure)、PCV2相关性肺炎等症状猪血清400份进行PCV2抗体检测。检测结果显示,疑似PMWS症状PCV2抗体阳性率最高,达86.25%,然后依次是疑似PCV2相关性肺炎阳性率56.25%,疑似PDNS阳性率46.25%,疑似繁殖障碍阳性率26.25%,最低的是疑似CT阳性率11.25%。研究结果表明,运城地区规模化猪场PCV2感染以PMWS和PCV2相关性肺炎为主,其次是PDNS和繁殖障碍,仔猪先天性震颤感染率最低。     

A serological survey of porcine reproductive and respiratory syndrome virus in wild boar in Gifu Prefecture,Japan

Porcine reproductive and respiratory syndrome (PRRS) is an infectious swine disease caused by the PRRS virus (PRRSV) that results in economic loss to the pig-rearing industry. To study PRRSV infection in wild boars and pigs, we conducted a serological survey in Gifu Prefecture, Japan, from 2020 to 2021. Three out of 453 (0.7%) wild boar sera were positive for PRRSV antibodies in a commercial ELISA. However, given that PRRSV RNA was not detected in these three wild boars and the specificity and sensitivity of the test kit, these are considered as false positives. Although seropositive pigs were found in multiple pig farms in the study area, the role of wild boars as a source of PRRS to pig farms appeared to be minimal.