Pig Serum Samples Research Articles

Expression and purification of virus like particles (VLPs) of foot-and-mouth disease virus in Eri silkworm (Samia cynthia ricini) larvae.

Foot-and-mouth disease (FMD) is a highly contagious viral disease, which causes severe economic loss to livestock. Virus like particles (VLPs) produced by recombinant DNA technology are gaining importance because of their immunogenic properties and safety in developing a new vaccine for FMD. In the present study, a practical and economically feasible approach of expression, purification and characterization of VLPs of FMDV in Eri silkworm (Samia cynthia ricini) larvae was described. Although three lepidopteran insect larvae (Helicoverpa armigera, Spodoptera litura and Samia cynthia ricini) were tested for production of VLPs, expression was obtained only in Eri silkworm larvae. High titred recombinant baculovirus encoding the polyprotein P1-2A-3C of FMDV was prepared in Sf9 cells. Injection of recombinant baculovirus into hemocoel of Eri silkworm larvae resulted in increasing levels of expression of VLPs in the hemolymph from 3 to 7days post infection (dpi) compared to low level expression by oral feeding. The VLPs reacted in Sandwich ELISA with serum raised against whole virus particles of FMDV type O/IND/R2/75 and protein banding pattern of 26, 37 and 47kDa in Western blotting demonstrated their antigenic resemblance to native virus. Sucrose density gradient purified VLPs were used for immunization of rabbits and guinea pigs for assessing immunogenicity. Further, the reactivity of serum samples of rabbits and guinea pigs in Indirect-ELISA with titres (1.30-2.81 Log10) indicated that the VLPs were antigenic and immunogenic in nature. We demonstrate that Eri silkworm larvae could be used for production of VLPs of FMDV type O/IND/R2/75 for the first time. This approach could be useful for large scale production of recombinant VLPs for vaccine or diagnostic use in FMD control programme.

Evidence of infection with hepatitis B virus-like agent in pigs

Human hepatitis B virus (HBV) infection remains a serious public health problem worldwide, as it is one of the main risk factors for hepatocellular carcinoma (HCC). Cross-species transmission of HBV has been reported in non-human primates, and pigs may also be infected with HBV or an HBV-like agent. The purpose of this study was to demonstrate the presence of HBV antigens and anti-HBV antibodies in pig sera, providing further support for the existence of HBV or an HBV-like agent in pig populations. The HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in pig serum samples were detected using HBsAg and HBeAg ELISA Kits, respectively. Antibodies to HBsAg and the Hepatitis B core antigen (HBcAg) in serum samples were also detected using anti-HBsAg and anti-HBcAg antibody ELISA Kits, respectively. HBsAg and HBeAg were detected in 7 of 442 (1.6%) and 7 of 184 (3.8%) pig serum samples, respectively. Furthermore, antibodies specific to HBsAg and HBcAg were identified in 45 of 442 (10.2%) and 39 of 434 (9.0%) pig serum samples, respectively. However, neither HBV DNA nor antibodies to HBeAg were detected in 409 and 298 pig serum samples, respectively. HBV antigens and anti-HBV antibodies were both present in a considerable number of pig serum samples, suggesting that pigs could be infected with a variant HBV or an HBV-like agent. Further studies will be necessary to confirm cross-species infection of pigs with HBV.

Seroprevalence of Trichinella Spp. in Pigs and Knowledge, Attitude and Practices of Pig Farmers of Eastern and Midwestern Regions of Nepal

A cross-sectional study was conducted on four major pig raising districts of eastern and mid-western region of Nepal from February to May 2014 to find out the seroprevalence of Trichinella spp. A total of 184 serum samples of pigs were collected and antibodies against trichinella were detected using ID screen trichinella indirect multi- species ELISA kit. The study revealed prevalence rate of 3.8% and difference in prevalence rate according to age, sex, breed, rearing system, ecozone, region & district were statistically insignificant (P>0.05) as analysed by Chi-square test using PHStat version 2.5 and Fisher’s exact test. This study confirms that antibodies of Trichinella spp. are circulating in pigs of Nepal. Further, the knowledge, attitude and practices survey of meat borne helminthic zoonoses was conducted among 50 pig raisers and pork consumers by face to face interview using a semi-structure questionnaire. This survey concludes that although there were significant portion of the respondents aware of meat borne helminthic zoonoses but there were still a noticeable proportion of respondents who didn’t have a proper knowledge that upsurge public health risks. Moreover, present-day situation of their pig raising practices & pork consumption system possess them to a menace of public health zoonoses. be done for enhancing the potential of isolates. Int J Appl Sci Biotechnol, Vol 3(3): 402-407

Epizootiology, laboratory and virulence analyses during the emergency phase of the African swine fever eradication program in Brazil in 1978: a historic account

Após a ocorrência do primeiro surto de peste suína Africana (PSA) no Brasil, o laboratório oficial de diagnóstico de PSA (LDPSA) foi implantado. O trabalho atual revisa o esforço da equipe de laboratório para estabelecer o diagnóstico de PSA na fase emergencial do programa de erradicação. De Junho a Dezembro de 1978, 3803 amostras de tecido, sangue e soro de suínos foram analisadas. O primeiro isolamento do vírus da peste suína africana (VPSA) realizado pelo LDPSA foi feito em amostras de suínos oriundas da cidade de Teresópolis no Estado do Rio de Janeiro, Brasil. Em todo país, nos primeiros dois meses, de 320 amostras analisadas, 130 (40.62%) foram positivas pelo teste de hemadsorção o que sugere que a infecção com VPSA alcançou suínos em 96 dos 214 municípios analisados no período. A distribuição dos isolamentos do vírus no país mostrou uma possível rota de dispersão viral. Devido à rapidez das ações de eliminação dos focos, o número de amostras positivas caiu de 48,36% em Junho para 33,53% em Julho até nenhuma em Agosto, de 1978. Paralelamente, a detecção de anticorpos aumentou de 17,89% a 52,04% de Junho para Agosto de 1978. Os isolamentos do vírus pela técnica de hemadsorção (HAD) quando comparados com a descrição da doença e o forte declínio na taxa de mortalidade no Estado do Paraná sugeriram a ocorrência de cepas virais de VPSA de baixa a moderada virulência. A implantação do LDPSA foi crucial para o programa que assegurou a erradicação do VPSA em todo país após seis anos do primeiro surto. Os avanços na biologia celular e molecular corroboraram as suposições dos estudos pioneiros sobre a virulência viral e alertam para a importância de se estabelecer e manter medidas de segurança para prevenir a reintrodução do VPSA no país. A informação sobre heterogeneidade na virulência das populações virais nos surtos fornece uma ferramenta fundamental para a adoção de melhores medidas de erradicação.

Toxoplasmosis and trichinellosis: an epidemiological survey of pig population in Madagascar

Besides cysticercosis, scarce information is available on the other meat-borne zoonotic parasitoses in the domestic ani­mal population of Madagascar. Trichinella is an intracellular parasitic nematode of mammalian skeletal muscle, causing a serious zoonotic disease. Trichinella is transmitted to humans by consumption of raw or undercooked meat from pig, wild boar and other sensitive species (1). Toxoplasma gondii is a protozoan parasite that can probably infect all warm-blooded animals (mammals and birds) and humans. Humans become infected postnatal by ingesting tissue cysts from undercooked meat (various species), consuming food or drink contaminated with oocysts, or by accidentally ingesting oocysts from the envi­ronment (2). The present study aimed at investigating the extent of two major parasitic diseases, namely toxoplasmosis and trich­inellosis, within the Malagasy pig population.Two hundred and fifty pig serum samples were collected dur­ing 2010 in the four major slaughterhouses of Antananarivo, the Malagasy capital. Sampled pigs were raised in 11 different regions (of a total of 22 regions) and transported by traders before slaughtering. Samples were stored at -80°C and sent for analysis to the Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail (ANSES, Maisons-Alfort, France). Serological investigations were conducted for both pathogens using ELISA technique with commercial test kits: ID Screen® Toxoplasmosis Indirect ELISA kit (IdVet, France) and PrioCHECK® Trichinella Ab (Prionics, Switzerland), according to the manufacturer’s instructions.Results on seroprevalence of T. gondii in pigs confirm that the zoonotic parasite is present in Madagascar. Preliminary results show a seroprevalence of 22.8% (57/250) for toxoplasmosis. All regions are endemic for toxoplasmosis with a frequency of posi­tive pigs ranging from 12% (n = 8) in the southern province of Toliara, to 21% in Antananarivo (n = 69) or Fianarantsoa (n = 79) in central uplands, and up to 33% (n = 69) in Mahajanga in the coastal northwestern region; no region of the island can be considered free of toxoplasmosis. No correlations were found with sex, breed or age of pigs. From a public health perspective these results emphasize the need to inform the public on the importance of proper meat handling and preparation.Regarding Trichinella, two pigs presented positive values by ELISA based on excretory/secretory antigens but those results were not confirmed in western blot. Even if the serological test for the detection of Trichinella provides a high degree of sensitiv­ity and specificity, false negative results occur during the early stages of infection, especially for light to moderate infection. Further investigations correlating serology and muscle analy­sis need to be conducted to conclude on Trichinella status in Madagascar.National field investigations in pig and human populations will have to be carried out to confirm the present results as well as to correlate the observed prevalence with on-farm risk factors, and with the transmission of trichinellosis and toxoplasmosis to Malagasy consumers, especially to pregnant women.

Simultaneous LC-MS/MS determination of JWH-210, RCS-4, ∆(9)-tetrahydrocannabinol, and their main metabolites in pig and human serum, whole blood, and urine for comparing pharmacokinetic data.

A series of new synthetic cannabinoids (SC) has been consumed without any toxicological testing. For example, pharmacokinetic data have to be collected from forensic toxicological case work and/or animal studies. To develop a corresponding model for assessing such data, samples of controlled pig studies with two selected SC (JWH-210, RCS-4) and, as reference, ∆(9)-tetrahydrocannabinol (THC) should be analyzed as well as those of human cases. Therefore, a method for determination of JWH-210, RCS-4, THC, and their main metabolites in pig and human serum, whole blood, and urine samples is presented. Specimens were analyzed by liquid-chromatography tandem mass spectrometry and multiple-reaction monitoring with three transitions per compound. Full validation was carried out for the pig specimens and cross-validation for the human specimens concerning precision and bias. For the pig studies, the limits of detection were between 0.05 and 0.50 ng/mL in serum and whole blood and between 0.05 and 1.0 ng/mL in urine, the lower limits of quantification between 0.25 and 1.0 ng/mL in serum and 0.50 and 2.0 ng/mL in whole blood and urine, and the intra- and interday precision values lower than 15% and bias values within ±15%. The applicability was tested with samples taken from a pharmacokinetic pilot study with pigs following intravenous administration of a mixture of 200 μg/kg body mass dose each of JWH-210, RCS-4, and THC. The cross-validation data for human serum, whole blood, and urine showed that this approach should also be suitable for human specimens, e.g., of clinical or forensic cases.

Detection of Trichinella Antibodies in Slaughtered Pigs and Risk Factors Associated with Trichinellosis in Pig Farms in Kaduna Metropolis, Nigeria

An experiment was conducted to detect antibodies against Trichinella in slaughtered pigs and to identify farm management practices and risk factors associated with trichinellosis in pigs and pig farms in Kaduna Metropolis, Nigeria. A total of 286 pigs, 186 from slaughterhouses and 100 from pig farms, were tested in this study. Serum samples collected from the slaughtered pigs were tested serologically for antibodies for Trichinella by ELISA using excretory-secretory (E/S) antigen. Out of the 186 pig serum samples collected from slaughtered pigs at the main pig abattoir in Kaduna Metropolis, 56 were from juvenile pigs (10.7%) and 130 from adult pigs (14.6%). Male pigs had a prevalence rate of 12.6%, which was slightly lower than that of the females (14.1%). An overall prevalence rate of 13.4% was observed in this study. The age and sex of the animals were not associated with the infection. Also, a survey of 100 slaughtered pigs from 100 pig farms was carried out. From the structured questionnaire administered, 90% of the pig farmers had scavenging pigs, 98% had seen rodents around and within their piggeries, 85% had no rodents control program, 70% feed their pigs with food leftovers, 4% had seen their pigs interact with stray pigs, 16% feed their pigs with dead animals, and 10% had seen snakes around their piggeries. Only 2% were aware of trichinellosis while 30% knew of pork-borne diseases. Significant association existed between all the risk factors studied in the backyard pig farms visited with trichinellosis except for factors such as rodents around and within pig pens. The result also classified the distribution of pig owners based on farm management practices into categories of extensive (55%), semi-intensive (40%), and intensive (5%) with prevalence rates of 96.4%, 70.0% and 0.0% respectively. The survey established serological evidence of trichinellosis in slaughtered pigs in Kaduna Metropolis, Nigeria.

Assessing the ability of two specific, immunodominant Taenia solium antigens to differentiate non-infected from cysticercotic subjects

Event Abstract Back to Event Assessing the ability of two specific, immunodominant Taenia solium antigens to differentiate non-infected from cysticercotic subjects Yuliet M. Diaz1, Gladis Fragoso1, Edda Sciutto1, Julio C. Carrero1, Ada N. Martínez2, Juan Pedro Laclette1 and Raúl J. Bobes1* 1 Universidad Nacional Autónoma de México, Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Mexico 2 Universidad Nacional Autónoma de México, Departamento de Patología, Facultad de Medicina Veterinaria y Zootecnia, Mexico INTRODUCTION: Human and porcine cysticercosis are infectious diseases associated to poverty and poor sanitary conditions. Cysticercosis is a severe public health issue and causes great economic losses at the rustic pork rearing industry in Mexico and other developing countries. Many variables can influence the current Immunodiagnostic methods and alter the assay sensitivity and specificity. This has prevented the development of a suitable diagnostic test for a highly specific diagnosis. OBJECTIVE: This work is aimed to find recombinant antigens or derivative peptides suitable to develop a more sensitive and specific diagnostic test for human and porcine cysticercosis. METHODS: Based on the amino acid sequence of two antigens identified in a previous study as specific for porcine cysticercosis (annexin B1 and cAMP-dependent kinase), peptides with the highest antigenicity rates were identified by bioinformatic tools. These peptides were evaluated for their suitability as antigen source in a cysticercosis ELISA, using serum samples from cysticercotic and non-cysticercotic pigs as antibody source. Additionally, the coding sequences for the selected antigens were cloned in a prokaryotic system to evaluate the diagnostic usefulness of the entire protein. RESULTS: Seven peptides with the highest antigenic index for annexin B1 and 12 peptides for cAMP-dependent kinase were selected. A series of ELISA experiments led us to identify those peptides that were most recognized by antibodies in pig serum samples. For annexin B1, 3 peptides out of 7 showed higher recognition, while 5 out of 12 peptides showed higher recognition for cAMP-dependent kinase. The cloning, expression, and purification of the recombinant proteins annexin B1 and cAMP-dependent kinase were also performed. CONCLUSIONS: The antigens evaluated in this work, annexin B1 and cAMP-dependent kinase include linear peptides which might be good candidates to be used in a diagnostic test for cysticercosis. Acknowledgements This work is supported in part by grants from PAPIIT-UNAM: IN228609-3 and CONACyT: 61334. Keywords: Cysticercosis, diagnosis, Antigenicity, Specific antigens, Taenia solium Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Poster Presentation Topic: Infectious and parasitic diseases Citation: Diaz YM, Fragoso G, Sciutto E, Carrero JC, Martínez AN, Laclette J and Bobes RJ (2015). Assessing the ability of two specific, immunodominant Taenia solium antigens to differentiate non-infected from cysticercotic subjects. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00191 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Apr 2015; Published Online: 14 Sep 2015. * Correspondence: PhD. Raúl J Bobes, Universidad Nacional Autónoma de México, Departamento de Inmunología, Instituto de Investigaciones Biomédicas, México, D. F., Mexico, rbobes@biomedicas.unam.mx Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Yuliet M Diaz Gladis Fragoso Edda Sciutto Julio C Carrero Ada N Martínez Juan Pedro Laclette Raúl J Bobes Google Yuliet M Diaz Gladis Fragoso Edda Sciutto Julio C Carrero Ada N Martínez Juan Pedro Laclette Raúl J Bobes Google Scholar Yuliet M Diaz Gladis Fragoso Edda Sciutto Julio C Carrero Ada N Martínez Juan Pedro Laclette Raúl J Bobes PubMed Yuliet M Diaz Gladis Fragoso Edda Sciutto Julio C Carrero Ada N Martínez Juan Pedro Laclette Raúl J Bobes Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Correlation between antibodies against porcine reproductive and respiratory syndrome virus and pathological-anatomical organ findings in slaughter pigs at farm level

The porcine reproductive and respiratory syndrome (PRRS) worldwide causes important economic losses in pig production. Its causative agent, the PRRS virus (PRRSV), is one of the most frequently detected infectious agents in relation to respiratory diseases in pigs in Austria. We investigated the correlation between the PRRSV status of pig farms, determined by detection of PRRSV antibodies in the serum of slaughter pigs, and the prevalence of pathological-anatomical lung lesions in slaughter pigs of the respective farms. Between December 1, 2011 and April 16, 2012, a total of 1056 serum samples of slaughter pigs from 66 pig farms were collected at an Austrian abattoir. The presence of PRRSV antibodies was tested by enzyme-linked immunosorbent assay in each sample and the PRRSV status of the respective farms was determined. No PRRSV vaccination was performed on any of the farms. In addition, the pathological-anatomical lung lesions of all slaughter pigs of the 66 farms that were slaughtered between September 1, 2011 and December 11, 2012 were recorded by authorized veterinarians at the abattoir. The prevalence of lung lesions and pleuritis in PRRSV-positive and unsuspected farms was compared and statistically interpreted. Slaughter pigs of PRRSV positive farms had a significantly higher prevalence of severe lung lesions and pleuritis visceralis and parietalis than slaughter pigs of PRRSV unsuspected farms. Pigs of combined farms (nursery and fattening unit at the same location) displayed a tendency for more moderate and severe lung lesions than pigs of exclusive fattening farms. In the present study, the PRRSV status of pig farms displayed a significant influence on the prevalence of lung lesions in the slaughter pigs. Findings untypical for PRRS, including pleuritis, were also found significantly more often on those farms. This leads to the conclusion that other primary and/or secondary infections are involved, which can be exacerbated by the immunosuppressive effect of the PRRSV. There was a tendency for combined farms to be more severely affected than fattening farms.

Bayesian Estimation of the True Prevalence and of the Diagnostic Test Sensitivity and Specificity of Enteropathogenic Yersinia in Finnish Pig Serum Samples.

Bayesian analysis was used to estimate the pig's and herd's true prevalence of enteropathogenic Yersinia in serum samples collected from Finnish pig farms. The sensitivity and specificity of the diagnostic test were also estimated for the commercially available ELISA which is used for antibody detection against enteropathogenic Yersinia. The Bayesian analysis was performed in two steps; the first step estimated the prior true prevalence of enteropathogenic Yersinia with data obtained from a systematic review of the literature. In the second step, data of the apparent prevalence (cross-sectional study data), prior true prevalence (first step), and estimated sensitivity and specificity of the diagnostic methods were used for building the Bayesian model. The true prevalence of Yersinia in slaughter-age pigs was 67.5% (95% PI 63.2–70.9). The true prevalence of Yersinia in sows was 74.0% (95% PI 57.3–82.4). The estimates of sensitivity and specificity values of the ELISA were 79.5% and 96.9%.

Development of an AGID based on baculovirus expressed Pseudorabies virus glycoprotein B

Development of an AGID based on baculovirus expressed Pseudorabies virus glycoprotein B

Development and Evaluation of an Antigen Capture Enzyme-Linked Immunosorbent Assay (AC-ELISA) for the Diagnosis of African Swine fever

African swine fever (ASF) is a viral haemorrhagic fever of pigs with devastating impact on pig production and household income in Africa. Lack of a vaccine and treatment for ASF has increased the dependence on accurate diagnosis as basis for control and possible eradication of the disease. The aim of this study therefore, was to develop and evaluate an in-house sandwich enzyme linked immune sorbent assay (ELISA) using antibodies raised against African swine fever virus (ASFv) isolates from Uganda for diagnosis of ASF. The ASFv was grown in pig alveolar macrophages, the infected cells harvested, lysed and the virus precipitated using polyethylene glycol 6000. The virus was purified on Sepadex G-200 column equilibrated with 50mM Tris-HCl PH 7.2 containing 0.15M NaCl and viral proteins separated by SDS-PAGE. The target protein (vp73), was quantified and used to immunize rabbits to produce polyclonal antibodies against it. The purified immunoglobulin IgG (rabbit anti ASF-vp73) was used for antigen capture in sandwich ELISA. Eighty eight (88) known positive and 176 known negative pig serum samples were used to evaluate assay performance. The diagnostic sensitivity of the ELISA was 82.95% (95% CI, 78-100%), diagnostic specificity was 96.59% (95% CI, 90–100%). Positive and negative predictive values were 92.4% and 91%, respectively. The inter samples coefficient of variation of raw optical density values for known positive samples in different runs was <10% (range 1.1-7.8), while intra sample coefficient of variation ranged from 0.6-5.5% between runs. The developed antigen capture ELISA has a high diagnostic sensitivity and specificity and is therefore good for detection of active ASF infection. However, the developed assay should be further validated using larger sample size under different laboratory conditions and referenced serum samples from different ASF endemic countries.

Preliminary evaluation of Community-Led Total Sanitation for the control of Taenia solium cysticercosis in Katete District of Zambia

Taenia solium taeniasis/cysticercosis is a zoonotic disease endemic in sub-Saharan Africa. It is associated with poor sanitary practices, free-range pig husbandry and lack of disease awareness in endemic communities. A comparative research was conducted with pre and post-intervention assessments in nine villages to evaluate Community-Led Total Sanitation (CLTS) as an intervention measure for the control of porcine cysticercosis in Katete District in the Eastern Province of Zambia. Blood samples were collected from pigs for circulating antigen detection and a questionnaire focused on the household was administered to a total of 153 respondents whose pigs were examined (64 pre-intervention, 89 post-intervention), in order to obtain information on general demographic characteristics, pig husbandry practices, sanitation practices and associated knowledge and awareness of T. solium infections. The first sampling was conducted prior to the implementation of the CLTS and second sampling eight months after triggering of CLTS in the selected villages. A total of 379 pig serum samples were examined using the B158/B60 Ag-ELISA to detect T. solium cysticercosis, 104 pre-intervention and 275 post-intervention, of which 14 (13.5%) and 45 (16.4%) were positive, respectively. Wald test p-values were computed to assess significant differences in the variables of interest mentioned above for the pre and post CLTS. The research revealed that CLTS as a control measure did not significantly improve T. solium infections in pigs. The research also revealed that the sanitation practices and awareness of cysticercosis did not change. It is recommended that a longer term evaluation be undertaken when the villages have been declared open defaecation free. In addition, the research recommends that health education, mass drug treatment and pig vaccination be incorporated, as an essential component of prevention and control programmes for T. solium infections.

High prevalence of co-infection with multiple Torque teno sus virus species in Italian pig herds.

Torque teno viruses (TTVs) are a large group of vertebrate-infecting small viruses with circular single-stranded DNA, classified in the Anelloviridae family. In swine, two genetically distinct species, Torque teno sus virus 1a (TTSuV1a) and 1b (TTSuV1b) are currently grouped into the genus Iotatorquevirus. More recently, a novel Torque teno sus virus species, named Torque teno sus virus k2b (TTSuVk2b), has been included with Torque teno sus virus k2a (TTSuVk2a) into the genus Kappatorquevirus. In the present study, TTSuV1 (TTSuV1a and TTSuV1b), TTSuVk2a and TTSuVk2b prevalence was evaluated in 721 serum samples of healthy pigs from Sardinian farms, insular Italy. This is the largest study to date on the presence of TTSuV in healthy pigs in Italy. The global prevalence of infection was 83.2% (600/721), being 62.3% (449/721), 60.6% (437/721), and 11.5% (83/721) the prevalence of TTSuV1, TTSuVk2a and TTSuVk2b, respectively. The rate of co-infection with two and/or three species was also calculated, and data show that co-infections were significantly more frequent than infections with single species, and that TTSuV1+TTSuVk2a double infection was the prevalent combination (35.4%). Quantitative results obtained using species-specific real time-qPCR evidenced the highest mean levels of viremia in the TTSuV1 subgroup, and the lowest in the TTSuVk2b subgroup. Interestingly, multiple infections with distinct TTSuV species seemed to significantly affect the DNA load and specifically, data highlighted that double infection with TTSuVk2a increased the viral titers of TTSuV1, likewise the co-infection with TTSuVk2b increased the titers of TTSuVk2a.

An indirect ELISA established using the capsid protein of PCV2 for detection of antibodies to porcine circovirus 2

To establish an indirect enzyme-linked immunosorbent assay(ELISA) for detection of antibodies to PCV2,the cap gene without nuclear localization signal peptide sequence of PCV2 was cloned into the pET 28a(+) vector,and the recombinant Cap⊿41 protein was expressed in soluable form in Escherichia coli(E.coli).The optimum working conditions of the indirect ELISA were determined using Cap⊿41 protein as diagnostic antigen,and 394 pig serum samples from 4 pig farms were tested.The results showed the diagnostic sensitivity and specificity of the established indirect ELISA were 93.14% and 95.17%,respectively,compared with indirect immunofluorescence(IIF).And the inter-assay and intra-assay variations of the ELISA were low.Besides,the seroprofiles of 4 pig farms revealed by the testing results were in accordance with clinical results,indicating the established indirect ELISA is reliable and can be used for detection of antibodies to PCV2.

A new multi-host species indirect ELISA using protein A/G conjugate for detection of anti-Toxoplasma gondii IgG antibodies with comparison to ELISA-IgG, agglutination assay and Western blot

Toxoplasma gondii is a zoonotic protozoan parasite which can cause significant disease and losses in livestock and wild animals. It is increasingly recognized as an important foodborne pathogen in a broad range of food animals and products. Effective control strategies require rapid, reliable and cost-effective detection methods for large scale surveys and diagnostic applications in a broad range of warm-blooded animals. To overcome one or more of these shortcomings in the currently available detection methods for T. gondii infection a non-species-specific protein A/G conjugate was used in the development of an indirect ELISA (ELISA-A/G) for the detection of IgG antibodies in serum samples obtained from experimentally infected pigs. The performance of the assay was evaluated using serum samples from pigs, cats, mice and seals with known positive or negative status for T. gondii infection. Results of the ELISA-A/G obtained with pig serum samples were compared with those generated by traditional ELISA using host specific IgG conjugate (ELISA-IgG), modified agglutination test (MAT) and Western blot analysis (WB). Using protein A/G conjugate, comparative analysis of results from 77 samples obtained from T. gondii infected pigs showed excellent agreement between the ELISA-A/G and in-house ELISA-IgG (0.917κ). Similar agreements were also observed when these samples were tested by a commercial ELISA kit (0.816κ), MAT (0.816κ) and WB (0.79κ). A total of 86 serum samples obtained from cats, mice and seals experimentally infected with T. gondii and tested by the ELISA-A/G as well as MAT for the presence of anti-Toxoplasma IgG antibodies yielded Kappa value of 1.0 for cats and mice and 0.79 for seals. These results show that the ELISA-A/G is a suitable method for serological detection of T. gondii infection in multiple host species and has the potential for testing samples from a broad range of domestic, wild, and aquatic mammalian host species. Simultaneous testing of samples from multiple host species on the same ELISA plate, and the use of multiple plates in a single run for large scale screening will enhance the cost effectiveness and speed of the test in the control and management of toxoplasmosis. This study also shows the effectiveness of the protein A/G conjugate in a modified WB assay for confirmation of T. gondii infection in mammalian hosts. Appropriate validation studies using field samples from various host species to validate the performance of ELISA-A/G is recommended prior to its application for diagnostic and surveillance programs.

Pig α1-acid glycoprotein: characterization and first description in any species as a negative acute phase protein.

The serum protein α1-acid glycoprotein (AGP), also known as orosomucoid, is generally described as an archetypical positive acute phase protein. Here, porcine AGP was identified, purified and characterized from pooled pig serum. It was found to circulate as a single chain glycoprotein having an apparent molecular weight of 43 kDa by SDS-PAGE under reducing conditions, of which approximately 17 kDa were accounted for by N-bound oligosaccharides. Those data correspond well with the properties of the protein predicted from the single porcine AGP gene (ORM1, Q29014 (UniProt)), containing 5 putative glycosylation sites. A monoclonal antibody (MAb) was produced and shown to quantitatively and specifically react with all microheterogenous forms of pig AGP as analyzed by 2-D electrophoresis. This MAb was used to develop an immunoassay (ELISA) for quantification of AGP in pig serum samples. The adult serum concentrations of pig AGP were in the range of 1–3 mg/ml in a number of conventional pig breeds while it was lower in Göttingen and Ossabaw minipigs (in the 0.3 to 0.6 mg/ml range) and higher in young (2–5 days old) conventional pigs (mean: 6.6 mg/ml). Surprisingly, pig AGP was found to behave as a negative acute phase protein during a range of experimental infections and aseptic inflammation with significant decreases in serum concentration and in hepatic ORM1 expression during the acute phase response. To our knowledge this is the first description in any species of AGP being a negative acute phase protein.

First indigenous transmission of Japanese Encephalitis in urban areas of National Capital Territory of Delhi, India

Until 2010, no Japanese encephalitis (JE) had been reported from Delhi. Upon report of four confirmed cases of JE in September 2011, detailed investigations were carried out to determine whether the cases were imported or indigenous. Entomological surveys were carried out and all mosquito pools were tested for the detection of JE virus by ELISA method using specific monoclonal antibody. Human blood samples from contacts of the patients were tested by IgM-captured ELISA method. Pig's blood samples were also tested for the detection of JE virus. Culex tritaeniorhynchus, Culex vishnui and Culex pseudovishnui mosquitoes were found. In contrast to rural areas, their breeding habitats were different in the city. 19 pools were tested. JE virus was detected in two pools of Cx.tritaeniorhynchus females reared from field-collected larvae, indicating vertical transmission. One pool of Cx.vishnui was also positive. This is the first report for the detection of JE virus in mosquitoes from Delhi. JE IgM antibodies in five contacts/residents indicate recent infection. JE virus was also detected in pigs. Present analysis shows that of four reported JE cases, three were confirmed indigenous, indicating that the virus is multiplying in the city. Mapping of infected JE vector mosquitoes in the cities is required for preventive measures to contain further spread of the disease.

Assessment of diagnostic accuracy of a commercial ELISA for the detection of Toxoplasma gondii infection in pigs compared with IFAT, TgSAG1-ELISA and Western blot, using a Bayesian latent class approach

Serological methods are the most commonly used diagnostic tools to detect Toxoplasma gondii infections in pigs. In the absence of a readily available ‘gold standard’, an estimation of diagnostic accuracy is difficult to assess. A commercial ELISA (PrioCHECK® Toxoplasma Ab porcine ELISA, Prionics, Schlieren, Switzerland) for the diagnosis of T. gondii infection in pigs was evaluated in naturally infected animals from two distinct populations; indoor and outdoor living animals. An assessment of diagnostic accuracy, using a Bayesian latent class approach with adjustment for within indoor and outdoor farm clustering using random effects, was performed. Tests used for comparison were: IFAT; ELISA using native affinity-purified P30 (SAG1) T. gondii tachyzoite surface antigen (TgSAG1-ELISA); and Western blot with T. gondii tachyzoites lysate. The data set comprised 297 pig serum samples across outdoor (n=149) and indoor (n=148) farms in Argentina. The estimated sensitivity and specificity for the commercial ELISA were 98.9% (95% credible interval: 96.2; 100) and 92.7% (95% credible interval: 87.7; 96.6), respectively. The analysis of sera and plasma from pigs (n=6) experimentally inoculated with 5,000 T. gondii oocysts revealed a pronounced antibody response beginning 2weeks p.i. until the end of the observation period (11weeks p.i.) in all animals. Meat juice obtained from inoculated animals after euthanasia also tested positive. These results suggest that the PrioCHECK® Toxoplasma Ab porcine ELISA may be a useful tool to perform serological diagnosis of T. gondii infections in pigs to control Toxoplasma infection in pigs and humans.

The use of acute phase proteins for monitoring animal health and welfare in the pig production chain: The validation of an immunochromatographic method for the detection of elevated levels of pig-MAP

The serum concentration of acute phase proteins (APPs) increases in the presence of disease or stress, which makes APPs notable parameters for the global assessment of animal health and welfare. A rapid, immunochromatographic test (ICT) for the detection of elevated levels of pig Major Acute-phase Protein (pig-MAP), one of the main APPs in pigs, was evaluated in more than 1400 pig serum samples obtained from commercial farms. The ICT showed a good performance with a relative sensitivity (Sn) and specificity (Sp) of 94 and 97%, respectively, for a threshold of 1.5mg/mL (comparison with ELISA). Differences in the pig-MAP levels and the number of positive samples with the ICT were observed within the season of sampling, farms, and age groups at one farm, according to the presence of disease or lesions. The ICT was also evaluated in blood samples obtained at slaughter in association with the carcase inspection. The results from this study indicate that the ICT may be used for the evaluation of groups of pigs, after analysing one sub-sample of these pigs, and might be a useful tool in routine health and welfare monitoring programmes aimed to improve the quality of pig production.