A total of 319 Pasteurella multocida (Pm) strains isolated from pigs in Poland were examined. Phenotypic characterisation included: biochemical tests (to determine species, subspecies, and biovar), capsular typing, and antimicrobial susceptibility. Genotypic characterisation included detection of the toxA gene by PCR. All tested Pm strains were classified as Pm subsp. multocida: 87.2% biovar 3, 10.7%-2 and 0.9%-12. One strain was classified as biovar 1. Three strains of Pm did not suit any of the biovars. Using capsular typing methods, 77% of Pm strains isolated from nasal swabs belonged to type D and 33% to type A. Among Pm strains isolated from internal organs, 59.5% belonged to type A and 40.5% to type D. All the isolates showed a high susceptibility to β-lactams: ampicillin and amoxicilin with clavulonic acid (97.8%), penicillin (86.7%), doxicilline (100%), oxytetracycline (97.8%), and tetracycline (93.2%). It was found that all strains were susceptible to norfloxacin, 97.8% to enrofloxacin, and 95.6% to SxT. 24.4% and 15.6% of the strains were resistant to linco-spectin and tiamulin, respectively. The presence of toxA gene was confirmed by PCR in 20.8% of the strains isolated from nasal swabs and 29.1% of isolates from internal organs.
The objective of the study was to determine genotypic profiles of Bordetella bronchiseptica (Bbr) strains, based on the occurrence of genes encoding virulence factors, such as flagella (fla), dermonecrotoxin (dnt), and exogenous ferric siderophore receptor (bfrZ), using PCR. 209 tested Bbr strains were obtained from Polish swine herds with different health status (with progressive atrophic rhinitis - PAR, suspected for PAR, and unknown). In total, seven different Bbr genotypes were determined. In 39.2% of Bbr isolates all three genes were present. In 41.1% of the isolates only two genes were detected. The most common genotype dnt+bfrZ-fla+ was present in 60 (28.5%) Bbr strains, 65% of them were obtained from farms with PAR. Twenty five (12%) Bbr isolates were identified as dnt-bfrZ+fla+ genotype and, as above, they were more frequently isolated from clinical cases of disease (84%). Among 31 (14.8%) strains only fla gene was evident, and in nine (4.3%) only dnt gene was present. There were no Bbr strains with bfrZ gene only. These results confirm the heterogenicity among Bbr strains.
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, 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.
Andrzej Kowalczyk, Kinga Urbaniak, Iwona Markowska-Daniel and Zygmunt Pejsak
The aim of the study was to monitor genetic diversity and antigenic changes in the genome of influenza A(H1N1)pdm09 viral isolates detected during the post-pandemic period in Poland. Clinical specimens obtained from three suspected cases of influenza were analysed by sequencing. Among the differences identified in amino acids sequences, nine substitutions were located within the antigenic HA1 sites and in five residues forming receptor-binding pocket. The HA(D222G) mutation was shown in the isolate Swine/Poland/134312/12 obtained from a mild case of the disease. It must be emphasized that, in general, clinically mild cases are caused by the viruses in which that specific mutation, i.e. haemagglutinin (D222G), does not occur.
Andrzej Kowalczyk, Kinga Urbaniak and Iwona Markowska-Daniel
Phylogenetic analysis of the genes determining influenza virus subtype - haemagglutinin (HA) and neuraminidase (NA), was performed. The results showed that the Polish H1N2 isolate (A/Swine/Poland15817/2011) was reassortant of human-like swine H1N1 and human-like swine H3N2 origin. The novel isolate was presented to have a close phylogenic relationship with one of the latest European isolates of H1N2 (A/SW/Gent/102/2007 and A/SW/Hungary/13509/2007). Our evolutionary analyses also suggested that the HA and NA genes evolved in a significantly higher rate of synonymous substitutions after they were introduced from human to swine and established the European H1N2 swine lineage.
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.
Iwona Markowska-Daniel, Krzysztof Kwit, Kinga Urbaniak and Andrzej Kowalczyk
The aim of the study was to estimate the current epidemiological situation concerning swine influenza (SI) in Poland. The study was based on an annual passive survey of 11,770 fatteners’ sera from 584 herds, taken at slaughterhouses within the last 30 months (from January 2010 till June 2012), as well as, an active monitoring conducted in 2011 and 2012, in 25 farms, using 388 sera taken from life pigs of different age/technological groups. The analysis of simultaneous circulation of different swine influenza virus (SIV) subtypes was taken into a deep consideration. The wide spread of SIV in Poland, including the occurrence of multiple SIV infections was demonstrated. In 2010 and 2011, the domination of H1N1 subtype and the most frequently co-circulation of H1N1 and H1N2 viruses was evidenced, while in the first 6 months of 2012, the co-circulation of H1N1 and H3N2 viruses was detected more often. Based on the obtained results, it can be stated that the epidemiological situation concerning SI in Poland is dynamic and similar to that observed in other European regions with high pigs’ density; however, the prevalence of antibodies and the occurrence of mixed SIV infections is lower than in Western Europe.
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.
Iwona Markowska-Daniel, Karol Wierzchosławski, Kinga Urbaniak and Aandrzej Kowalczyk
The paper describes an outbreak of swine influenza, which was the first H1N2 outbreak confirmed in pig farm in Poland. The outbreak occurred in October 2011 in the farrows to finish farm, first in the fattening units, and subsequently in the reproduction and weaning sectors. Samples of the lungs taken from dead gilts were tested by the use of real time and multiplex PCR, sequencing, and virus isolation methods. The amplification of the genetic material extracted from the lungs confirmed the presence of the M1 gene sequence of type A influenza virus. Using multiplex PCR, the bands of 241 bp, typical for HA1 human-like type, and of 791 bp, typical for NA2, were demonstrated. The new isolates of swine influenza virus, named A/swine/Poland/15817/2011, were obtained in embryonated SPF chicken eggs and MDCK cells. Phylogenetic analysis of the HA and NA genes of the isolate revealed that the virus is of human swine linkage closely related to European human-like viruses. On the basis of such results, it can be stated that A/Swine/Poland/15817/2011 is a new swine influenza virus, representing the H1N2 subtype strain, which emerged in Poland.