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.
Twenty pigs of similar genetics (PIC) were used. Pigs were randomly divided into two groups: experimental (ENRO, n = 10) and control (C, n = 10). From day 0 to day 4, pigs from ENRO group received enrofloxacin at the recommended therapeutic dose. Pigs from C group received PBS instead of enrofloxacin. Blood samples were collected on days 0 (before antibiotic administration), 2, 4 (during antibiotic therapy), 9, and 13 of the study (after enrofloxacin administration). Haematological examination and flow cytometry were used to establish the relative and absolute counts of various leukocyte subsets. Lymphocyte subpopulations were measured by fluorochrom-labelled antibodies according to following definitions: CD3+ (T cells), CD21+ (B cells), CD4+CD8- (helper T cells, Th), CD4-CD8+ (cytolytic T cells, CLT), CD4+CD8+ (cytolytic and memory T cells). The present study revealed the modulating effect of enrofloxacin on the composition of circulating lymphocytes in pigs. Concentration and percentage of CD8+ cells decreased significantly after treatment with enrofloxacin and as a result the absolute CD4/CD8 ratio increased significantly as compared to control group (P < 0.05).These findings should prompt further studies on the practical significance of the results obtained in terms of clinical implications. In view of the results, it cannot be excluded that enrofloxacin may also have immunomodulatory effects on host response to infection.
Ewelina Czyżewska-Dors, Krzysztof Kwit, Zygmunt Pejsak and Małgorzata Pomorska-Mól
Introduction: The aim of the study was to evaluate the effect of administration of therapeutic doses of ceftiofur and tulathromycin on the circulating lymphocyte subpopulations in healthy pigs. Material and Methods: The study was conducted on thirty healthy 7- to 10-week-old pigs, assigned to three groups: the TUL group, injected with tulathromycin (n = 10); the CEF group, injected with ceftiofur (n = 10); and the C group, the control with no antibiotic administration (n = 10). Blood samples were collected before, during, and after treatment with antimicrobials. Lymphocyte subpopulations circulating in the blood were determined by immunostaining and flow cytometry analyses. Results: Following administration of a therapeutic dose of tulathromycin, there were no changes in the lymphocyte subpopulations circulating in blood. In contrast, administration of ceftiofur at the recommended dose decreased the absolute number of CD3+, CD21+, CD4+CD8-, CD4-CD8+, and double positive CD4CD8 cells. Conclusion: Results from the study indicate that ceftiofur possesses the ability to modulate the immune system in healthy pigs by decreasing lymphocyte subpopulations circulating in blood.
Małgorzata Pomorska-Mól, Krzysztof Kwit and Iwona Markowska-Daniel
Age-related changes in serum concentrations of C-reactive protein (CRP), haptoglobin (Hp), serum amyloid A (SAA), and pig major acute phase protein (pig-MAP) were investigated in healthy pigs from birth to slaughter under field conditions. Repeated blood samples were obtained from 60 pigs at ages of 1-19 weeks. Concentrations of acute phase proteins (APP) were measured with the use of commercial ELISA kits. Concentrations of all APP increased with age (P<0.05) and positive correlations were evidenced between their concentrations and the age of pigs. Great variations in CRP, Hp, and SAA concentrations were found, as can been seen from standard deviation values. The minimal individual variability was found in regard to pig-MAP. A significant increase in all APP was observed in pigs’ serum after weaning, constituting an important characteristic of this period. The elevation of APP after weaning may be associated with stress induced by mixing animals after weaning or changes in the pattern of feed administration. The peak in APP may be also caused by the initiation of synthesis of these proteins by piglets. Because a significant association between age and APP concentrations exists, further studies are needed to decide whether the age may influence the diagnostic value of APP as a marker of infection. Additionally, studies are needed to estimate whether the APP response in infection is age-dependent to any clinical importance degree.
Małgorzata Pomorska-Mól, Iwona Markowska-Daniel, Krzysztof Kwit, Kinga Urbaniak and Zygmunt Pejsak
The kinetics of C-reactive protein (CRP), haptoglobin (Hp), serum amyloid A (SAA), and pig major acute protein (Pig-MAP) response in pigs co-infected with H3N2 swine influenza virus (SwH3N2) and Bordetella bronchiseptica (Bbr) was studied, with assessment of potential correlations between the concentration of acute phase proteins (APPs) in serum samples, lung lesions, and the clinical course of the disease in co-infected pigs. The standard bacteriological methods for detection of Bbr and PCR technique for identification of Bbr and SwH3N2 were used. The serum concentrations of APPs were measured using ELISA. The concentration of CRP, SAA, and Pig-MAP was significantly higher from 2 to 4 or 5 dpi. The concentration of Hp was elevated until the end of the study. Significant correlations were found between the serum concentration of SAA and Pig-MAP and clinical score, and between the concentration of SAA and lung score. Apart from their potential as biological markers for co-infections, SAA and Pig-MAP levels have additive value since they are related to the severity of infection. The results indicate that measurement of APP (i.e SAA) may prove valuable in assessing the severity of respiratory infection in pigs, and may be of supportive value in the clinical evaluation of animals and in the selection of more appropriate treatment.
Małgorzata Pomorska-Mól, Krzysztof Kwit, Ewelina Czyżewska and Iwona Markowska-Daniel
The aim of the study was to determine the effects of supplementation of sows’ and weaners’ diet with Stresomix, preparation containing extracts from Magnifera indica, Withania somnifera, Phyllanthus emblica, and Ocimum sanctum, on pig performance and immunity under field condition. The hypothesis was that anti-inflammatory, antistress, and immunomodulatory properties of the herbs would enhance production parameters and immune response, according to the manufacturer's claim. The study was performed on 16 sows and 160 piglets. The following parameters were recorded: concentration and proportion of white blood cells and their populations, concentration of serum immunoglobulins, specific humoral postvaccinal response after vaccination against swine influenza and swine erysipelas, and main production parameters. No significant differences among treatment groups were found with regard to concentrations of leukocyte subpopulations and immunoglobulins, as well as all investigated production parameters (P>0.05). In conclusion, the results of the study did not confirm that the investigated polyherbal product, administered at dose recommended by manufacturer, is able to significantly improve the performance and postvaccinal humoral response in clinically healthy pigs under field condition.
Małgorzata Pomorska-Mól, Iwona Markowska-Daniel and Jarosław Rachubik
Development of early immune response in piglets with subclinical swine influenza was investigated. Fourteen, seronegative piglets were used. Ten of them were infected intranasally with swine influenza virus (SIV) H1N1 subtype. Temperature and clinical signs were assessed daily. Leukocyte proportions and concentrations were analysed on a haematology analyser. Antibodies against SIVs were measured by haemagglutination inhibition assay. To measure influenza-specific cell-mediated immunity (CMI), the proliferation assay was performed. The real time reverse transcription PCR method was used for detection of SIV. No relevant respiratory or systemic clinical signs were observed. The presence of SIV RNA in nasal swabs from all infected piglets was confirmed between 2 and 5 dpi. The overall number of leukocytes did not differ during the study. The number of medium-sized cells (MID) was significantly higher on 2 and 4 dpi, as compared to day 0 level. The percentage of lymphocytes decreased from 74% on day 0 to 67.06% on 4 dpi, while the percentage of MID significantly increased at the same time. In control pigs no significant changes were observed. All infected pigs exhibited specific antibodies between 7 and 10 dpi. Specific CMI was observed before specific antibodies were present. Results of our research indicate that kinetics of the humoral and CMI response during subclinical infection is similar to that observed in clinical form of the disease.
Małgorzata Pomorska-Mól, Ewelina Czyżewska-Dors, Krzysztof Kwit and Zygmunt Pejsak
Introduction: The aim of the study was to explore the effect of enrofloxacin on production of selected cytokines by porcine peripheral blood mononuclear cells (PBMCs).
Material and Methods: Twenty pigs (10 control and 10 experimental) were used in this research. Pigs from experimental group received enrofloxacin at therapeutic doses. Blood samples were collected before, during, and after treatment with enrofloxacin. PBMCs were incubated with or without lipopolysaccharide (LPS). Production of IL-6, IL-10, INF-γ, and TNF-α were determined by ELISA.
Results: Administration of enrofloxacin to healthy pigs for 5 d induced a transient reduction of the PBMCs response to LPS in terms of IL-6 and TNF-α secretion. The concentration of IL-6 returned to the day 0 level shortly after treatment, while TNF-α production remained reduced for 10 d after the treatment. The production of IL-10 was not affected by enrofloxacin. The level of IFN-γ was below the detection limit of the tests.
Conclusion: The results indicate that enrofloxacin administered in vivo in therapeutic doses has an immunomodulatory effect through its capacity to inhibit secretion of IL-6 and TNF-α by porcine PBMC stimulated by LPS.
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, Krzysztof Kwit, Ewelina Stasiak, Jarosław Rachubik, Katarzyna Śliżewska and Małgorzata Pomorska-Mól
The aim of the study was to determine the effects of supplementation of sows’ and growing pigs’ diets with three newly developed synbiotic and two extant commercial probiotic products on selected immune parameters under field conditions.
Material and Methods
The study was performed on 30 sows and 48 piglets of the Danbred breed. Immune parameters such as concentration and proportion of white blood cells and their subpopulations, immunoglobulins amount in serum, and serum concentration of cytokines and acute phase proteins were recorded with the use of a haematology analyser and ELISA kits.
No significant differences between treatment groups and controls were found with regard to the immune parameters evaluated except for serum immunoglobulin concentration, which was significantly increased by synbiotic products B and C and probiotic product D.
The results of the study indicate that the synbiotic products B and C and probiotic product D are worthy of further investigation as promising candidates to improve the immune status of healthy sows and their offspring.