Arkadiusz Dors, Andrzej Kowalczyk and Małgorzata Pomorska-Mól
Introduction: Porcine pleuropneumonia inflicts important economic losses on most commercial herds. Detection of subclinical or chronic infection in animals still remains a challenge, as isolation and identification of A. pleuropneumoniae serotypes is difficult and quantification of the bacteria on agar plates is often almost impossible. The aim of the study was to develop and evaluate a serotype-specific quantitative TaqMan probe-based PCR for detection of serotype 2 in pig lungs, tonsils, and nasal swabs.
Material and Methods: The primers were designed from the capsular polysaccharide biosynthesis genes of A. pleuropneumoniae serotype 2. PCR specificity and sensitivity were evaluated using reference strains and several other bacterial species commonly isolated from pigs.
Results: The real-time qPCR for detection of A. pleuropneumoniae serotype 2 was highly specific and gave no false positives with other serotypes or different bacterial species of pig origin. The detection limit for pure culture was 1.2 × 104 CFU/mL, for lung tissue and nasal swabs it was 1.2 × 105 CFU/mL, and for tonsils - 1.2 × 105 CFU/mL.
Conclusion: The method can be used to serotype A. pleuropneumoniae isolates obtained during cultivation and to detect and identify A. pleuropneumoniae serotype 2 directly in nasal swabs and tonsil scrapings obtained from live pigs or lung tissue and tonsils collected post-mortem.
Ewelina Czyżewska-Dors, Arkadiusz Dors, Krzysztof Kwit, Ewelina Stasiak and Małgorzata Pomorska-Mól
Introduction: The aim of this study was to evaluate and compare the local innate immune response to the swine influenza virus (SIV) and Actinobacillus pleuropneumoniae (App) infection in pigs. Material and Methods: The study was performed on 37 seven-week-old pigs, divided into four groups: App-infected (n=11), App+SIV-infected (n=11), SIV-infected (n=11), and control (n=4). Lung samples were collected, following euthanasia, on the 2nd and 4th dpi (three piglets per inoculated group) and on the 10th dpi (remaining inoculated and control pigs). Lung concentrations of IL-1β, IL-6, IL-8, TNF-α, IL-10, IFN-α, and IFN-γ were analysed with the use of commercial porcine cytokine ELISA kits. Results: Lung concentrations of IL-1β, IL-6, IL-8, TNF-α, IFN-α, and IFN-γ were induced in SIV-infected and App+SIV-infected pigs. In the lung tissue of App-infected pigs, only concentrations of IL-1β, IL-6, IL-8, and IFN-γ were elevated. Additionally, in App+SIV-infected pigs, significantly greater concentrations of IL-1β, IL-8, and IFN-α were found when compared with pigs infected with either SIV or App alone. In each tested group, the lung concentration of IL-10 remained unchanged during the entire study. Conclusion: The results of the study indicate that the experimental infection of pigs with SIV or App alone and co-infection with both pathogens induced a local lung inflammatory response. However, the local cytokine response was considerably higher in co-infected pigs compared to singleinfected pigs
Ewelina Czyżewska-Dors, Arkadiusz Dors, Krzysztof Kwit, Zygmunt Pejsak and Małgorzata Pomorska-Mól
Introduction: The aim of this study was to assess the seroprevalence of swine influenza A virus (SIV) in Polish farrow-to-finish pig herds.
Material and Methods: Serum samples collected from 5,952 pigs, from 145 farrow-to-finish herds were tested for the presence of antibodies against H1N1, H1N1pdm09, H1N2, and H3N2 SIV subtypes using haemagglutination inhibition (HI) test. Samples with HI titres equal or higher than 20 were considered positive.
Results: HI antibodies to at least one of the analysed SIV subtypes were detected in 129 (89%) herds and in 2,263 (38%) serum samples. Antibodies to multiple SIV subtypes were detected in 104 (71.7%) herds and in 996 (16.7%) serum samples. Concerning the seroprevalence rate, according to age category, the highest prevalence of the antibodies was detected in weaners, with regard to the H1N1, H1N2, and H3N2, and in sows, with regard to the H1N1pdm09. The lowest seroprevalence for all evaluated SIV subtypes was detected in finishers.
Conclusion: The study indicates that antibodies against single and multiple SIV subtypes are circulating in Polish farrow-to-finish herds and highlights the importance of conducting a molecular surveillance programme in future studies.
Maciej Kochanowski, Jacek Karamon, Joanna Dąbrowska, Arkadiusz Dors, Ewelina Czyżewska-Dors and Tomasz Cencek
Introduction: The aim of study was to estimate the prevalence and intensity of intestinal parasite infections in pigs in Poland and evaluate the influence of factors related to the production system on the infection intensity.
Material and Methods: A total of 70 pig farms of all Polish provinces, differing in the herd size and production system, were selected for the study. Fresh faecal samples were collected from all age groups: suckling piglets, weaners, fatteners, and lactating sows. Moreover, data were obtained regarding the size of the herd, the use of paddock and all-in/all-out system, the presence of diarrhoea, and the type of flooring.
Results: Parasite eggs or oocysts were detected in 57 of the 70 examined pig farms. Oesphagostomum spp. eggs were found in the largest number of farms (68.6%). Moreover, coccidia (42.9%), Ascaris suum (28.6%), Trichuris suis (21.4%), and Strongyloides spp. (11.4%) were detected. The highest prevalence of coccidia and Strongyloides spp. was found in suckling piglets, A. suum and T. suis in fatteners, and Oesphagostomum spp. in sows. Higher prevalence of parasites was detected in small farms than in medium and large farms, except the prevalence of coccidia, which was the highest in medium farms. Simultaneous infection with several parasites was more often detected than with one parasite. Odds ratio of parasites occurrence was higher in farms with paddock and litter floor and in farms which do not use all-in/all-out system.
Conclusion: Relatively high prevalence of intestinal parasites was found in pigs in Poland. Moreover, specific distribution of parasites in different age groups and farms of different size was observed. Influence of breeding factors on parasite prevalence was identified.
Ewelina Czyżewska-Dors, Małgorzata Pomorska-Mól, Arkadiusz Dors, Aneta Pluta, Katarzyna Podgórska, Krzysztof Kwit, Ewelina Stasiak and Anna Łukomska
The study evaluated the patterns of local innate immune response in bronchoalveolar lavage fluid (BALF) cells of pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) alone or co-infected with swine influenza virus (SIV).
Material and Methods
The study was performed on 26 seven-week-old pigs in three groups: PRRSV-infected (n = 11), PRRSV and SIV-infected (n = 11), and control (n = 4). BALF was collected post euthanasia at 2 and 4 dpi (three piglets per inoculated group) and at 21 dpi (all remaining pigs). Expression of IFN-α, IFN-γ, IL-1β, IL-6, IL-8, and IL-10 mRNA was quantified in BALF cells. PRRSV RNA was quantified in BALF samples using a commercial real-time RT-PCR kit.
The three cytokines IFN-α, IFN-γ, and IL-1β presented significant expression changes in all experimental pigs. In PRRSV-infected animals IL-8 also did, but in co-infected subjects IL-6 and IL-10 were the additional upregulated cytokines. The highest number of differentially expressed genes was observed at 4 dpi, and significant differences in cytokine gene expression did not occur between the experimental groups at any other time point. The mean PRRSV load in the BALF of PRRSV-infected pigs was higher than that of co-infected pigs at each time point, having statistical significance only at 4 dpi.
The results of the study indicate that infection with PRRSV alone as well as with SIV interferes with innate and adaptive immune response in the infected host. They also showed that co-infection demonstrates additive effects on IL-6 and IL-10 mRNA expression levels.
Artur Jabłoński, Dominika Borowska, Sylwia Zębek, Andrzej Kowalczyk, Arkadiusz Dors, Jacek Żmudzki, Agnieszka Nowak and Zygmunt Pejsak
The aim of the study was to develop and validate a real-time PCR method, using a TaqMan probe, for quantification of Mycoplasma suis in porcine blood. No PCR signals with closely related non-haemotrophic mycoplasmas were obtained. The detection limit of PCR for plasmid combined with blood DNA was determined to be 103/reaction (5 μL of DNA) (1.2x105 target copies in 1 mL of blood). The linearity of real-time PCR (near 1) indicates its use as a quantitative method. Real-time and quantitative PCR were sensitive and specific for the detection and quantification of M. suis in the blood of animals with acute and chronic form of eperythrozoonosis. Developed quantitative PCR cannot be used to detect carrier animals with a small amount of M. suis in their blood. The validity of real-time PCR used in the studies was confirmed by the low inter- and intra-assay coefficients of variation. This fact confirms the applicability of the assay in other laboratories.