Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Effect of meat processing on viability of Toxoplasma gondii: Towards replacement of mouse bioassay by in vitro testing
Opsteegh, M. ; Dam-Deisz, C. ; Boer, P. de; Fare, A. ; Hengeveld, P. ; Luiten, R. ; Smits, C.B. ; Verkleij, T. ; Giessen, Judith van der; Wisselink, H.J. - \ 2019
Felines are the definitive hosts of T. gondii and primary infection results in fecal shedding of infectious oocysts. Infected intermediate hosts will develop tissue cysts, which are infective to both cats and intermediate hosts. Meat containing viable tissue cysts is considered one of the main sources of human infection. In contrast to fresh meat, raw meat products usually undergo processing, including salting and mixing in additives such as acetate and lactate, which affects the viability of T. gondii. However, the experiments currently described in literature, are not always performed in line with the processing methods applied in industry. Therefore we aimed to study the effect of salting and additives according to the recipes used by commercial producers. Mouse or cat bioassay is the gold standard to demonstrate the presence of viable T. gondii. However, it is costly, time consuming and for ethical reasons not preferred for large-scale studies. Therefore, our second aim was to develop an alternative for mouse bioassay that can be used to determine the effect of processing on the viability of T. gondii tissue cysts. We focused on a tissue culture method to determine the parasite's ability to multiply, and a PMA-based assay to selectively detect DNA from live cells. Results with the PMA-based method were inconsistent and did not sufficiently discriminate between live and dead parasites. The tissue culture method showed promising results, but further optimization is needed before it can replace or reduce the number of mouse bioassays needed. Small scale experiments with minced meat incubated for 20h with low concentrations of salt, lactate and acetate showed a large but incomplete reduction of the number of infected mice. In future, in vitro methods are needed to allow more extensive testing of product-specific processing methods, thereby providing a better indication of the risk of T. gondii infection for consumers.
A risk based surveillance programme for Toxoplasma gondii in pigs using a combination of farm auditing and serological screening
Wisselink, H.J. ; Swanenburg, M. ; Gonzales Rojas, Jose ; Asseldonk, M.A.P.M. van; Wagenberg, Coen van; Giessen, J. van der; Meerburg, B.G. ; Krijger, Inge ; Eppink, D.M. ; Bouwknegt, M. ; Oorburg, D. - \ 2019
Toxoplasma gondii is recognized as one of the major foodborne pathogens with a high human disease burden. In the Netherlands, pork contributes to about 11 % of the meatborne T. gondii infections. To control T. gondii infections in pigs, EFSA has advised to perform serological testing of pigs and audits of pig farms on risk factors for T. gondii infection. In the Netherlands, a program was started to translate the EFSA advice into a practical risk based surveillance system. In first instance, a large scale serological monitoring of fattening pigs was started and seroprevalence over time was determined. Next, the association between within-herd seroprevalence and risk factors for T. gondii on fattening pig farms in the Netherlands was determined. For this, a questionnaire for auditing farms for the presence of risk factors of T. gondii was developed and used on 25 case and 50 control farms. Results show that there is a significant association between seroprevalence and risk factors as cats present on farms, use of unheated feed products and feeding wet feed. Moreover, on-farm presence of rats and mice also increases Toxoplasma transmission risks. Subsequently, a study was started on farms to quantify the effectiveness of interventions on farms. A cross-over clinical trial was set up in which case farms were their own control and the cross-over moment is the implementation of interventions on risk factors to change farm management. Farms with a high within-herd seroprevalence were followed for at least during a year and monitored periodically for seroprevalence and implementation of interventions to eventually reduce the disease burden. The break-even point was calculated for which the intervention cost at fattening pig farms equal averted human disease burden and averted cost-of-illness minus cost of the surveillance program. The results shows favourable economic perspectives for interventions to control pig meat-born transmission of T. gondii.
A European inter-laboratory trial to evaluate the performance of three serological methods for diagnosis of Mycoplasma bovis infection in cattle using latent class analysis
Andersson, Anna Maria ; Aspán, Anna ; Wisselink, Henk J. ; Smid, Bregtje ; Ridley, Anne ; Pelkonen, Sinikka ; Autio, Tiina ; Lauritsen, Klara Tølbøll ; Kensø, Jane ; Gaurivaud, Patrice ; Tardy, Florence - \ 2019
BMC Veterinary Research 15 (2019)1. - ISSN 1746-6148 - 1 p.
ELISA - Inter-laboratory trial - Latent class analysis - Mycoplasma bovis cattle - Western blot

BACKGROUND: Mycoplasma bovis (M. bovis) is an emerging bovine pathogen, leading to significant economic losses in the livestock industry worldwide. Infection can result in a variety of clinical signs, such as arthritis, pneumonia, mastitis and keratoconjunctivitis, none of which are M. bovis-specific. Laboratory diagnosis is therefore important. Serological tests to detect M. bovis antibodies is considered an effective indicator of infection in a herd and often used as a herd test. Combined with clinical judgement, it can also be used to implement control strategies and/or to estimate the disease prevalence within a country. However, due to lack of harmonisation of approaches to testing, and serological tests used by different laboratories, comparisons of prevalence data between countries is often difficult. A network of researchers from six European countries designed and participated in an inter-laboratory trial, with the aim of evaluating the sensitivity (Se) and specificity (Sp) of two commercially available ELISA tests (ID Screen® ELISA (IDvet) and BIO K302 ELISA (BIO-X Diagnostics)) for diagnosis of M. bovis infection. Each laboratory received a blinded panel of bovine sera and tested independently, according to manufacturer's instructions. Western blot analyses (WB) performed by one of the participating laboratories was used as a third diagnostic test in the statistical evaluation of Se and Sp values using latent class analysis. RESULTS: The Se of WB, the ID Screen® ELISA and the BIO K302 ELISA were determined to be 91.8, 93.5 and 49.1% respectively, and corresponding Sp of the three tests were 99.6, 98.6 and 89.6%, respectively. CONCLUSIONS: The present study is, to our knowledge, the first to present an inter-laboratory comparison of the BIO K302 ELISA and the ID Screen® ELISA. Based on our results, the ID Screen® ELISA showed high consistency with WB and performed with higher precision and accuracy than the BIO K302 ELISA.

Clonal expansion of a virulent Streptococcus suis serotype 9 lineage distinguishable from carriage subpopulations
Willemse, Niels ; Ark, Kees C.H. van der; Stockhofe-Zurwieden, Norbert ; Smith, Hilde ; Picavet, Daisy I. ; Solt-Smits, Conny van; Wisselink, Henk J. ; Schultsz, Constance ; Greeff, Astrid de - \ 2019
Scientific Reports 9 (2019)1. - ISSN 2045-2322

Streptococcus suis is a porcine pathogen, causing severe invasive infections. S. suis serotype 9 is increasingly causing disease in Dutch and Chinese pig herds, but it is unknown whether all serotype 9 isolates are equally virulent and markers that can identify virulent strains are not available. Therefore, discrimination between virulent isolates and carriage isolates typically not associated with disease, is currently not possible. We collected tonsillar S. suis isolates from 6 herds not previously diagnosed with S. suis infections, and clinical S. suis isolates of previously diseased pigs. We confirmed the virulence of a virulent type strain and one representative clinical isolate, and the lack of virulence of two carriage isolates, in a pig infection model. Phylogenetic analysis of whole genome sequences of 124 isolates resulted in 10 groups, of which two were almost uniquely populated by clinical isolates. The population structure of S. suis serotype 9 appears highly diverse. However, analysis of the capsule loci sequences showed variation in a single region which fully correlated with a virulent genotype. Transmission electron microscopy suggested differences in capsule thickness between carriage and clinical genotypes. In conclusion, we found that that the S. suis serotype 9 population in the Netherlands is diverse. A distinct virulence-associated lineage was identified and could be discriminated based on the capsule locus sequence. Whilst the difference in virulence cannot be directly attributed to the DNA sequence, the correlation of capsule locus sequence with virulence could be used in the development of diagnostic tests to identify potential virulent S. suis serotype 9 in pigs.

Salt inactivation of classical swine fever virus and African swine fever virus in porcine intestines confirms the existing in vitro casings model
Jelsma, Tinka ; Wijnker, Joris J. ; Smid, Bregtje ; Verheij, Eline ; Poel, Wim H.M. van der; Wisselink, Henk J. - \ 2019
Veterinary Microbiology 238 (2019). - ISSN 0378-1135
3D collagen matrix model - African swine fever - Classical swine fever - D-values - Intestine - Virus inactivation

Natural casings, to be used as sausage containers, are being traded worldwide and may be contaminated with contagious viruses. Standard processing of such natural casings is by salt treatment with a duration of 30 days before shipment. Since information is lacking about the efficacy of these virus inactivation procedures, an in vitro 3D collagen matrix model, mimicking natural casings, was developed previously to determine the efficacy of salt to inactivate specific viruses. To validate this model, a comparison in vivo experiment was performed using intestines of pigs experimentally infected with African swine fever virus (ASFV) and classical swine fever virus (CSFV). Decimal reduction (D) values, were determined at 4 °C, 12 °C, 20 °C and 25 °C. The standard salt processing procedure showed an efficient inactivation of ASFV and CSFV over time in a temperature dependent way. Dintestine values of both viruses, treated with the standard salt treatment, were in line with the Dcollagen values. It was concluded that these results underline the suitability of the 3D collagen matrix model to determine virus inactivation and to replace animal experiments. Furthermore, an increase in storage time for standard salt processed casings derived from CSFV endemic regions is highly recommended for an efficient inactivation of CSFV.

Identification of potential risk factors for Toxoplasma gondii in fattening pigs in the Netherlands using a Bayesian approach
Eppink, D.M. ; Bouwknegt, M. ; Oorburg, D. ; Urlings, H.A.P. ; Asseldonk, Marcel van; Wagenberg, Coen van; Krijger, Inge ; Giessen, J.W.P. van der; Swanenburg, M. ; Wisselink, H.J. - \ 2019
Introduction
Toxoplasma gondii is a relevant foodborne pathogen,it is estimated that up to one third of the worldpopulation has been exposed to the parasite (Tenteret al. 2000). In the Netherlands toxoplasmosis rankssecond on a list of prioritized emerging zoonosis(Havelaar et al. 2010) and also second in disease burden among 14 foodborne diseases (Mangen et al.2017). Data suggest that ingesting improperly cooked meat containing T. gondii is one of the major sources of infection in Europe and North America (Crotta et al. 2017; Guo et al. 2015). The contribution of pork to meatborne T. gondii infections is estimated to be11 % in the Netherlands (Opsteegh 2011) and is seen as an important possible source of human T. gondii infections (Foroutan et al. 2019). The European Food Safety Authority (EFSA) advised to perform serological testing of pigs and on farm audits on risk factors (EFSA 2011). To that end, a serological monitoring program was developed in a slaughterhouse in the Netherlands. In this study, the objective is to determine the association between within herd seroprevalence, corrected for misclassification of samples through Bayesian analyses, and risk factors for T. gondii on fattening pig farms in TheNetherlands.
Materials and MethodsFrom 2015 to 2018, HACCP based audits were performedon 75 fattening pig farms in The Netherlands to identify the presence of potential T. gondii risk factors. All farms were conventional pig farms, with 15 farms being farrow to finish. As overall seroprevalence of T. gondii in pigs in the Netherlands is low, estimated at 5 % (1-12 % 95 % CI) by Foroutanet al. 2019, approached farms were chosen with the knowledge of previous serology data. In this way there would be farms with positive serum samples and farms without them included in the study. The audits were based on an updated version of the questionnaire from Mul et al. (2015) and covered the following topics: outdoor access, farm biosecurity, rodent control, presence of cats, feed and watersupply. In addition, serum samples (n=6272) from fattening pigs were obtained at slaughter throughout the year before the audit on the farm was performed. These samples were used for antibody testing bya PrioCHECK™ Toxoplasma Antibody ELISA. Data were analysed using Bayesian statistics, with the within farm T. gondii prevalence as dependent variable and potential risk factors as independent variables. As always with serology, misclassification due to false-positive or false-negative results can occur. Statistical methods have been developed to account for such misclassification, based on frequentistic as well as Bayesian approaches (Hui & Walter 1980; Josephet al. 1995). First, all independent variables wereanalysed in a univariate logistic model, and variables with a probability ≤0.25 that zero is included in the 95 % interval were analysed in a multivariable model. The multivariate logistic model was fitted using backward elimination until all remaining variables showed a probability ≤0.05 that zero is included in the 95 % interval. Two-way interaction terms were evaluated similarly to the main variables regarding statistical significance.
ResultsDescriptive results showed that 50 out of the 75 farms had 1 or more positive serum samplein the year before the audit was performed. In total 438 samples were positive out of the 6272 samples. Final Bayesian analyses are currently being conducted. However, preliminary results from data analysis using frequentistic logistic multivariate regression identified two significant risk factors: the accessibility of pig feed for cats and theprovision of well water as drinking water for the pigs (Table 1).
Discussion and ConclusionsThe use of serological testing seems to be a valuable guide and monitoring tool for the control of T.gondii in pork production. In a preliminary analysis, a higher within-herd T. gondii seroprevalence on fattening pig farms in the Netherlands was associated with the accessibility of pig feed for cats and the provision of well water as drinking water for the pigs. Improvements in farm management on fattening pig farms will likely contribute to reduction of the human disease burden and is presently studied.
Large-scale serological screening of slaughter pigs for Toxoplasma gondii infections in The Netherlands during five years (2012-2016): Trends in seroprevalence over years, seasons, regions and farming systems
Swanenburg, M. ; Gonzales Rojas, Jose ; Bouwknegt, M. ; Boender, G.J. ; Oorburg, D. ; Heres, L. ; Wisselink, H.J. - \ 2019
Veterinary Parasitology 2 (2019). - ISSN 0304-4017
Toxoplasma gondii is the causative agent of the parasitic disease toxoplasmosis, which is an important foodborne zoonosis. Eating undercooked meat of infected animals, including pigs, has been considered the major transmission route of T. gondii to humans. Therefore, it is urgent to develop and implement intervention measures in the pork meat chain to reduce risks of acquiring a T. gondii infection. Proposed measures for control of T. gondii in pigs include serological testing of pigs and audits of pig farms on risk factors for T. gondii infection. So far, these ideas have not been tested in practice. In order to generate knowledge about the epidemiology and seroprevalence of T. gondii, as a basis for developing a surveillance system, we studied the long term seroprevalence over years, farming systems and regions, and seasonal patterns of T. gondii seroprevalence in Dutch slaughter pigs. During a five year study period from 2012 to 2016, serum samples were routinely collected in five Dutch pig slaughterhouses. The sera were tested in an ELISA for the presence of antibodies against Toxoplasma. In total 226,340 serum samples were collected and tested during the study period. The observed seroprevalence varied over years, with the highest overall seroprevalence in 2014 (2.8%) and the lowest in 2016 (1.4%). A higher seroprevalence was observed in pigs from organic farms compared to pigs from conventional farms. The overall risk of infection was on average 2.63 times significantly (p < 0.001) higher for organically raised pigs than for conventionally raised pigs. A seasonal pattern in seroprevalence was observed: the results showed a dominant annual periodicity with a seroprevalence peak in winter around week 1 and a minimum seroprevalence in summer around week 27. To our knowledge, this is the first large scale study on the seroprevalence of T. gondii in slaughter pigs. In comparison to other European serological studies, the observed seroprevalence seems to be relatively low. However, care is needed when comparing the results with other studies because of differences in test setup, the number of samples and time period of sampling. The results can be used as a starting point for developing a surveillance system for T. gondii, and for implementation of intervention measures.
The relationship between the presence of antibodies and direct detection of Toxoplasma gondii in slaughtered calves and cattle in four European countries
Opsteegh, M. ; Spano, F. ; Aubert, D. ; Balea, A. ; Burrells, A. ; Cherchi, S. ; Cornelissen, J.B.W.J. ; Dam-Deisz, C. ; Guitian, J. ; Györke, A. ; Innes, E.A. ; Katzer, F. ; Limon, G. ; Possenti, A. ; Pozio, E. ; Schares, G. ; Villena, I. ; Wisselink, H.J. ; Giessen, J.W.B. van der - \ 2019
International Journal for Parasitology 49 (2019)7. - ISSN 0020-7519 - p. 515 - 522.
Cattle - Detection - Mouse bioassay - PCR - Serology - Toxoplasma gondii

In cattle, antibodies to Toxoplasma gondii infection are frequently detected, but evidence for the presence of T. gondii tissue cysts in cattle is limited. To study the concordance between the presence of anti-T. gondii IgG and viable tissue cysts of T. gondii in cattle, serum, liver and diaphragm samples of 167 veal calves and 235 adult cattle were collected in Italy, the Netherlands, Romania and the United Kingdom. Serum samples were tested for anti-T. gondii IgG by the modified agglutination test and p30 immunoblot. Samples from liver were analyzed by mouse bioassay and PCR after trypsin digestion. In addition, all diaphragms of cattle that had tested T. gondii-positive (either in bioassay, by PCR on trypsin-digested liver or serologically by MAT) and a selection of diaphragms from cattle that had tested negative were analyzed by magnetic capture quantitative PCR (MC-PCR). Overall, 13 animals were considered positive by a direct detection method: seven out of 151 (4.6%) by MC-PCR and six out of 385 (1.6%) by bioassay, indicating the presence of viable parasites. As cattle that tested positive in the bioassay tested negative by MC-PCR and vice-versa, these results demonstrate a lack of concordance between the presence of viable parasites in liver and the detection of T. gondii DNA in diaphragm. In addition, the probability to detect T. gondii parasites or DNA in seropositive and seronegative cattle was comparable, demonstrating that serological testing by MAT or p30 immunoblot does not provide information about the presence of T. gondii parasites or DNA in cattle and therefore is not a reliable indicator of the risk for consumers.

CoVetLab: Working together to strengthen collaboration on Mycoplasma bovis and compare available diagnostic tools
Ridley, A. ; Tardy, Florence ; Wisselink, H.J. ; Pelkonen, Sinikka ; Lauritsen, Klara T. ; Aspan, A. - \ 2019
In: European Mycoplasma Conference London 2019. - - p. 74 - 74.
Background: Different clinical presentations of disease caused by Mycoplasma bovis predominate in European countries with significant economic and welfare impacts. M. bovis disease control relies on good husbandry and early diagnosis, but lack of standardised approaches and diagnostic methods applied make comparisons of disease prevalence between countries difficult. Five CoVetLab National Veterinary institutes (SVA, WVBR, VET-DTU, APHA,ANSES) together with RUOKAVIRASTO joined forces to develop a network of scientists and share tools and expertise. Objectives included developing ring trials to evaluate available serological and PCR-based diagnostic tests.Methods: The sensitivity and specificity of two commercial ELISA systems (ID screen® ELISA (IDvet) and BIO K302ELISA (BIO-X Diagnostics)) for serodiagnosis of M. bovis in cattle serum were assessed by inter-laboratory comparison using a randomly blinded panel of bovine sera. Inclusion of Western blot analysis enabled statistical evaluation by latent class analysis. In addition, analytical specificity, sensitivity and comparability of seven different PCR methods used to identify presence of M. bovis organisms were assessed.Results: The ID Screen ELISA showed high agreement with Western blot analysis and performed with higher precision and accuracy than the Bio K302 ELISA, also showing higher sensivity and specificity. In contrast the analytical specificity and overall performance of the different PCR methods was comparable, although limits of detection varied from 10 to 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively.Conclusions: CoVetLab has facilitated improved collaboration between veterinary institutes in Europe undertaking M.bovis diagnostics. We believe this inter-laboratory comparison of these ELISAs is the first to include the ID Screen. The comparison of PCR tests has provided reassurance regarding the quality of diagnosis, despite the multiplicity of the methods.
A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis
Wisselink, H.J. ; Smid, B. ; Plater, J. ; Ridley, A. ; Andersson, Anna-Maria ; Aspán, A. ; Pohjanvirta, T. ; Vähänikkilä, Nella ; Larsen, Helena ; Hogberg, Jonas ; Colin, A. ; Tardy, Florence - \ 2019
BMC Veterinary Research 15 (2019). - ISSN 1746-6148
Background: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity
of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national
veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis.
Results: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one
commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories.
Conclusion: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.
An Interlaboratory trial to evaluate the reliability of PCR methods for Mycoplasma bovis diagnosis in six european countries
Wisselink, H.J. ; Smid, B. ; Plater, J. ; Ridley, A. ; Andersson, A.M. ; Aspán, A. ; Pohjanvirta, T. ; Vähänikkilä, N. ; Larsen, H. ; Hogberg, J. ; Colin, A. ; Tardy, F. - \ 2018
In: 5th congress of the European Association of Veterinary Laboratory Diagnosticians (EAVLD). - Brussels, Belgium : European Association of Veterinary Laboratory Diagnosticians - p. 36 - 36.
Association between within-herd seroprevalence and risk factors for Toxoplasma gondii in fattening pigs in the Netherlands
Eppink, D.M. ; Bouwknegt, M. ; Oorburg, D. ; Urlings, H.A.P. ; Asseldonk, Marcel van; Wagenberg, Coen van; Krijger, I.M. ; Giessen, J.W.P. Van der; Swanenburg, M. ; Wisselink, H.J. - \ 2018
In: Annual General Meeting of the European College of Veterinary Public Health, "Fading of the HACCP after four decades: new trends in VPH for food safety ", Perugia, 17th-19th October 2018. - European College of Veterinary Public Health (ECVPH) - p. 40 - 40.
Serological screening of Dutch slaughter pigs to estimate the seroprevalence of Toxoplasma gondii infections at farms
Swanenburg, M. ; Rojas, J.G. ; Bouwknegt, M. ; Oorburg, D. ; Giessen, Joke van der; Wisselink, H.J. - \ 2018
In: The 15th international symposium of veterinary epidemiology and economics. - Chang Mai, Thailand : The World Organisation for Animal Health (OIE) - p. 252 - 252.
The Animal by-products legislation; complex legal requirements that sometimes collide with established contained use regimes
Custers, René ; Bohne, J. ; Müller-Doblies, Uwe ; Sülli, Tamás ; Meulen, Karen van der; Wisselink, H.J. - \ 2018
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A European interlaboratory evaluation of PCR and ELISA methods for Mycoplasma bovis diagnostics
Aspan, A. ; Wisselink, H.J. ; Ridely, Anne ; Andersson, Anna-Maria ; Pohjanvirta, Tarja ; Pelkonen, Sinikka ; Lauritsen, Klara T. ; Kenso, Jane ; Larsen, Helena ; Hogberg, Jonas ; Tardy, Florence - \ 2018
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Exploring target-specific primer extension in combination with a bead-based suspension array for multiplexed detection and typing using Streptococcus suis as a model pathogen
Wal, F.J. van der; Achterberg, R.P. ; Smits, C.B. ; Bergervoet, J.H.W. ; Weerdt, M. de; Wisselink, H.J. - \ 2018
Journal of Veterinary Diagnostic Investigation 30 (2018)1. - ISSN 1040-6387 - p. 71 - 77.
We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [gdh], and the gene encoding the extracellular protein factor [epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population.
Using serological monitoring, internet-based feedback and on-farm auditing to improve Toxoplasma gondii control at Dutch pig farms
Oorburg, D. ; Eppink, Dorien ; Heijltjes, Janneke ; Bouwknegt, Martijn ; Urlings, Bert ; Giessen, Joke van der; Krijger, Inge ; Mul, Monique ; Swanenburg, M. ; Wisselink, H.J. - \ 2017
In: 12th International Symposium on the Epidemiology and Control of Biological,Chemical and Physical Hazards in Pigs and Pork - Proceedings Book, Foz doIguaçu, august 21-24, 2017. - Embrapa - p. 201 - 201.
Toxoplasma gondii is a relevant foodborne pathogen due to its human disease burden. In the Netherlands, pork is estimated to contribute to 11% of the meatborne T. gondii infections. The European Food Safety Authority advised to perform serological testing of pigs and on farm audits on risk factors for T. gondii infection.
Toxoplasma gondii at pig farms in the Netherlands: a case-control study
Heijltjes, Janneke ; Oorburg, D. ; Wisselink, H.J. - \ 2017
Lelystad : Central Veterinary Institute
Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid
Wisselink, H.J. ; Cornelissen, J.B.W.J. ; Wal, F.J. van der; Kooi, Engbert A. ; Koene, M.G.J. ; Bossers, A. ; Smid, B. ; Bree, F.M. de; Antonis, A.F.G. - \ 2017
BMC Veterinary Research 13 (2017)1. - ISSN 1746-6148
Background
Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents.
Results
The analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10−1 to 2 × 100 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni.
Conclusion
It was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.
Antibiotics in 16-day-old broilers temporarily affect microbial and immune parameters in the gut
Wisselink, H.J. ; Cornelissen, J.B.W.J. ; Mevius, D.J. ; Smits, M.A. ; Smidt, H. ; Rebel, Johanna M.J. - \ 2017
Poultry Science 96 (2017)9. - ISSN 0032-5791 - p. 3068 - 3078.
Animal health benefits from a stable intestinal homeostasis, for which proper development and functioning of the intestinal microbiota and immune system are essential. It has been established that changes in microbial colonization in early life (the first 2 wk post hatch) impacts the functioning of the adult gut and the associated crosstalk between microbiota and intestinal mucosal cells. The aim of the present study was to study the effect of the administration of antibiotics later in life (d 15 to 20 post hatch) on microbiota and immune parameters. For this purpose, chickens received from 15 d post hatch during 5 d amoxicillin or enrofloxacin through their drinking water. Before and at 6, 16, and 27 d after start of the administration of antibiotics, the composition of the microbiota in the jejunum was determined using a 16S ribosomal RNA gene-targeted DNA microarray, the CHICKChip. At 6 d after the start of the administration of the antibiotics, the composition and diversity of the microbiota were affected significantly (P < 0.05), but this change was small and observed only temporarily since differences disappeared at 16 d after initiating treatment with amoxillin and at 27 d after starting treatment with enrofloxacin. Intestinal morphology and development were not visibly affected since there were no differences between villus/crypt ratios and numbers of PAS+ and PCNA+ cells in the duodenum and jejunum at any time point. At 16 d after the start of antibiotic administration, the number of CD4+ T-cells and CD8+ T-cells in the duodenum was lower compared to the control animals; however, this difference was not significant. At some time points, significant differences (P < 0.05) were observed among the groups to locally expressed IL-8, IL-1β, IFN-γ, IL-2, and IL-4 mRNA. However, this effect was not long lasting, as differences that were observed at 16 d after starting the treatment had disappeared at 27 d after treatment was started. The results of this study indicate that later in the broiler's life, antibiotics only temporarily affect intestinal microbial and immune parameters.
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