Assessment of African Swine Fever Diagnostic Techniques as a Response to the Epidemic Outbreaks in Eastern European Union Countries: How To Improve Surveillance and Control Programs.

This study represents a complete comparative analysis of the most widely used African swine fever (ASF) diagnostic techniques in the European Union (EU) using field and experimental samples from animals infected with genotype II ASF virus (ASFV) isolates circulating in Europe. To detect ASFV, three different PCRs were evaluated in parallel using 785 field and experimental samples. The results showed almost perfect agreement between the Universal ProbeLibrary (UPL-PCR) and the real-time (κ = 0.94 [95% confidence interval {CI}, 0.91 to 0.97]) and conventional (κ = 0.88 [95% CI, 0.83 to 0.92]) World Organisation for Animal Health (OIE)-prescribed PCRs. The UPL-PCR had greater diagnostic sensitivity for detecting survivors and allows earlier detection of the disease. Compared to the commercial antigen enzyme-linked immunosorbent assay (ELISA), good-to-moderate agreement (κ = 0.67 [95% CI, 0.58 to 0.76]) was obtained, with a sensitivity of 77.2% in the commercial test. For ASF antibody detection, five serological methods were tested, including three commercial ELISAs, the OIE-ELISA, and the confirmatory immunoperoxidase test (IPT). Greater sensitivity was obtained with the IPT than with the ELISAs, since the IPT was able to detect ASF antibodies at an earlier point in the serological response, when few antibodies are present. The analysis of the exudate tissues from dead wild boars showed that IPT might be a useful serological tool for determining whether or not animals had been exposed to virus infection, regardless of whether antibodies were present. In conclusion, the UPL-PCR in combination with the IPT was the most trustworthy method for detecting ASF during the epidemic outbreaks affecting EU countries in 2014. The use of the most appropriate diagnostic tools is critical when implementing effective control programs.

Phenotypic and genotypic characterization of Bordetella bronchiseptica strains isolated from pigs in Poland.

A total of 209 Bordetella bronchiseptica (Bbr) strains isolated from pigs were examined. Phenotypic study included: biochemical characterization (motility, catalase, oxidase, urease activity, nitrate reduction and growth on MacConkey agar) and antimicrobial susceptibility (disc diffusion method). Genotypic studies based on detection of three genes encoded virulence factors, such as: flagella (fla), dermonecrotoxin (dnt), and exogenous ferric siderophore receptor (bfrZ), using PCR. Most of the Bbr strains tested had a homogeneous biochemical profile. 97.6% of them provided suitable results in biochemical tests. All Bbr isolates tested showed high resistance to penicillin (100%), linco-spectin (100%) and ceftiofur (97.9%). Over 57% and 43% of Bbr strains were resistant to ampicillin and amoxicillin, respectively. All Bbr isolates showed high sensitivity to most chemotherapeutics used such as enrofloxacin (97.9%), tetracycline (97.9%), oxytetracycline (97.9%), amoxicillin with clavulonic acid (95.8%), florfenicol (90.4%), and gentamicine (77.6%). Over of 94% of Bbr strains were moderately susceptible to norfloxacine. Molecular analysis confirmed that almost all evaluated Bbr strains (94.7%) possessed the fla gene. A lower percentage of isolates had the dnt gene (72.7%) and the lowest percentage of strains (51.7%), had the bfrZ gene.

Epidemiology of African Swine Fever in Poland since the detection of the first case.

The purpose of this paper is to provide characteristics of the spread of African Swine Fever (ASF) in Poland from February to August, 2014. The samples from dead wild boar and domestic pigs were submitted to the National Veterinary Research Institute, National Reference Laboratory for ASF in Pulawy, Poland, for testing by PCR and ELISA methods. In the studied period, fourteen cases of ASF in wild boar and two outbreaks in backyard pigs were confirmed. In addition to the results of laboratory tests performed in 2014, the article describes the ASF surveillance programme in wild boar and pigs in Poland carried out in 2011-2013. The spread of ASF in Poland is compared with the epidemiological situation in Lithuania, Latvia, Belarus and the Russian Federation.

Antibodies to parainfluenza virus type 3 in goat population in Poland.

Respiratory diseases constitute a major health problem in small ruminant herds around the world, and parainfluenza virus type 3 (PIV-3) has been shown to play a vital role in their etiology. This cross-sectional study describes the serological status of the non-vaccinated dairy goat popu- lation in Poland with respect to PIV-3 infection and investigates the relationship between the presence of antibodies to PIV-3 and some basic herd-level and animal-level factors, including small ruminant lentivirus (SRLV) infection. Serum samples from 1188 goats from 48 herds were tested for the concentration of antibodies to PIV-3 using a quantitative immunoenzymatic assay. Specific antibodies were detected in all tested goats from all herds. The concentration of PIV-3 antibodies varied from 8.4 to >240 ng/ml (median 95.9 ng/ml) and was significantly higher in goats from larger herds and from these herds in which cough was often observed by farmers. Moreover, it was noted that female goats had higher antibody concentrations than males. On the other hand, the concentration of PIV-3 antibodies did not prove to be significantly linked to the presence of SRLV infection. This study shows that PIV-3 infection in the Polish goat population is widespread and appears to contribute to the occurrence of respiratory diseases in goat herds.

Phylogenetic analysis of swine influenza viruses isolated in Poland.

Swine influenza virus (SIV) of H1N1 and H3N2 subtypes are dominated in European pigs population. "Classical swine" H1N1 subtype was replaced by "avian-like" H1N1 subtype. It co-circulates with H3N2 reassortant possessing "avian" genes. In the present study, 41 SIV strains isolated from pigs with pneumonia, raised in 20 Polish farms, were identified and characterised. Since it was evidenced that isolates from the same geographic district and the same year of isolation are in 100% similar, 15 strains representing different district and different year of isolation were chosen to construct phylogenetic trees. Two genes, conservative matrix 1 (M1) and the most variable, haemagglutynin (HA), were sequenced and subjected into phylogenetic analysis. The results of the analysis confirmed that "avian-like" swine H1N1 strains evolved faster than classical SIV strains. HA gene of these isolates have been derived from contemporary strains of "avian-like" SIV. In contrast, the M1 gene segment may have originated from avian influenza viruses. H3N2 strain is located in swine cluster, in the main prevalent European group of H3N2 isolates called A/Port Chalmers/1/73-like Eurasian swine H3N2 lineage, which has evolved separately from the human H3N2 virus lineage around 1973.

Phylogenetic evolution of swine-origin human influenza virus: a pandemic H1N1 2009.

The knowledge of the genome constellation in pandemic influenza A virus H1N1 2009 from different countries and different hosts is valuable for monitoring and understanding of the evolution and migration of these strains. The complete genome sequences of selected worldwide distributed influenza A viruses are publicly available and there have been few longitudinal genome studies of human, avian and swine influenza A viruses. All possible to download SIV sequences of influenza A viruses available at GISAID Platform (Global Initiative on Sharing Avian Influenza Data) were analyzed firstly through the web servers of the Influenza Virus Resource in NCBI. Phylogenetic study of circulating human pandemic H1N1 virus indicated that the new variant possesses a distinctive evolutionary trait. There is no one way the pandemic H1N1 have acquired new genes from other distinguishable viruses circulating recently in local human, pig or domestic poultry populations from various geographic regions. The extensive genetic diversity among whole segments present in pandemic H1N1 genome suggests that multiple introduction of virus have taken place during the period 1999-2009. The initial interspecies transmission could have occurred in the long-range past and after it the reassortants steps lead to three lineages: classical SIV prevalent in the North America, avian-like SIV in Europe and avian-like related SIV in Asia. This analysis contributes to the evidence that pigs are not the only hosts playing the role of "mixing vessel", as it was suggested for many years.

Dynamic changes of immunoglobulin concentrations in pig colostrum and serum around parturition.

The aim of the study was the determination of IgA, IgM and IgG concentrations in porcine serum and colostrum, in order to evaluate their variations in the perinatal period, as well as to clarify whether there is a correlation between colostrum intake, initial level of immunoglobulins (Ig) in piglet serum and development of their own immunity. The mean IgA, IgM and IgG concentrations in sow serum 10 days before parturition were 1.58, 6.12 and 39.56 mg/ml, respectively. Seven days later only the IgG level was insignificantly lower (34.94 mg/ml, p = 0.55), while concentrations of IgA and IgM increased to 2.25 and 7.25 mg/ml, respectively (p = 0.23 and 0.62, respectively). The mean initial IgG concentration in colostrum at farrowing was 118.5 mg/ml and differed between sows. The average value of IgA in colostrum at birth was 23.8 mg/ml and decreased to 7.85 mg/ml at 6 hours (h) and to 4.59 mg/ml at 24 h after the onset of farrowing. IgM concentration at birth was 12.1 mg/ml and decreased to 4.23 mg/ml at 24 h postpartum. Positive relationships were found between concentrations of IgM and IgA in serum of piglets at 14 and 56 days of life (r = 0.41 and 0.80, respectively, p < or = 0.05) as well as for IgG concentration in the piglets serum at 7 days and 56 days of age (r = 0.48, p < or = 0.05). The above observations suggest that there is a correlation between the level of Ig in piglet serum in the first days of life and improvement of their own immunity.

Antibiotic susceptibility of bacteria isolated from respiratory tract of pigs in Poland between 2004 and 2008.

Antibiotic susceptibility of bacteria isolated from nasal swabs and lungs of pigs, to 16 commonly used antibiotics, was determined by disc diffusion test. beta-lactams showed the best activity against Streptococcus suis (S. suis) (> 99% of susceptible strains). The lowest sensitivity of S. suis was evidenced to: tylosin, tetracycline and neomycin (50%, 40% and 25%, respectively). Isolates of Escherichia coli (E. coli) demonstrated the highest susceptibility to cephalosporin (85% strains), gentamicin and norfloxacin (over 74%). The lowest susceptibility of E. coli was demonstrated to tiamulin and penicillin (11.3% and 1.9%, respectively). Over 80% of Actinobacillus pleuropneumoniae (App) strains were susceptible to all antibiotics tested. The highest resistance of App, but demonstrated by below 20% of tested isolates only, was evidenced to neomycin and LxS. Isolates of Pasteurella multocida (Pm), Haemophilus parasuis (Hps) and Arcanobacterium pyogenes (A. pyogenes) were highly susceptible to the most antibiotics included in the analysis. The comparison of the in vitro susceptibility of pathogens to the chemotherapeutics used on Polish farms for the therapy of bacterial infection of pigs within the last five years and the last 10 years, showed an increasing percent of E. coli and S. suis strains resistant to commonly used antibiotics. It is also shown that Pm, Hps, App and A. pyogenes isolates were continuously susceptible to the most chemotherapeutics applied.

Randomised, placebo-controlled trial of a live vaccine against porcine reproductive and respiratory syndrome virus in sows on infected farms.

A randomised, double-blinded, placebo-controlled study into the effectiveness and safety of a vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) was carried out on three farms with a history compatible with chronic PRRSV infection; representative groups of sows and gilts were injected with a live vaccine against PRRSV, and during the next six weeks no side effects were observed. The remaining sows and gilts on the three farms were then vaccinated with the same vaccine. Again, no side effects were observed. There were significant reductions in abortion, reproductive disease, returns to oestrus and the numbers of stillborn pigs per sow, and significant increases in the numbers of liveborn pigs per sow and weaned pigs per sow among the vaccinated animals.

Clinical signs and economic losses caused by porcine reproductive and respiratory syndrome virus in a large breeding farm.

In July of 1994 an acute onset of maternal reproductive failure occurred in a 2,330 sow farrow-to-finish farm. Clinical signs observed in the affected sows were typical for porcine reproductive and respiratory syndrome (PRRS). During the first 6 weeks of the epizootic 1,117 sows farrowed; 216 (19.33%) farrowed before the 110th day of gestation. The majority of piglets born before term died within a few days of birth and the mortality rate for term piglets increased to a maximum of 75.56% during the 5th week of the epizootic when 1,562 out of 2,067 piglets were either born dead or died prior to weaning. Preweaning mortality rates gradually returned to normal values within 16 weeks. The incidence of respiratory disease in the weaned and fattening pigs increased during this time. Although specific prophylactics against respiratory diseases were administered, the death rate doubled for the weaned and fattening pigs.

Losses due to porcine reproductive and respiratory syndrome in a large swine farm.

The aim of the presented studies was to demonstrate losses and economical consequences of Porcine Reproductive and Respiratory Syndrome (PRRS) outbreak in a large swine farm in Poland. Prior to the occurrence of PRRS the piglets' mortality rate did not exceed 6%, losses among weaners and fatteners were lower than 3.5% and the percentage of sows that farrowed before term was 1.4%. During the first month after onset of the outbreak 25.6% of sows farrowed before 110 days of pregnancy, the percentage of mummies was 21.7%, the percentage of piglets that died before weaning was 43.3%, losses among fatteners and weaners were four times greater, and average production of weaned piglets per sow per year dropped from 21.1 to 18.1. Farrowing rate dropped from 80.5 to 47.7% and even 12 months after onset of the outbreak did not reach the level found before the outbreak. Expenses dealing with preventing and treating secondary infections, during the 12 months after the outbreak were on average 60% higher than, those found, during the previous year.

Field evaluation of thirteen regimens for the control of progressive atrophic rhinitis.

The purpose of the study was to evaluate, under field conditions, the effect of prophylactic programs for the control of progressive atrophic rhinitis (PAR)--recommended in different countries. The investigations were carried out on 280 pregnant sows divided into 14 equal groups and 50 slaughter pigs randomly selected from the litters produced by the sows of each of the groups. Efficacy of all programs was recognized on the basis of comparative evaluation of the average daily gain (ADG), morphometric examination of turbinate bones and computer conchal morphometry (TPR). An increase in ADG was noted in 12 of 13 experimental groups when compared to the control group. Usefulness of the evaluated programs differed significantly. Results of TPR relate only partially to the results of visual morphometry and results of ADG.

Evaluation of different methods (ELISA, IF, EM, PAGE) for the diagnosis of rotavirus infection in piglets.

The study on the prevalence of rotaviral infections among diarrhoetic piglets in 11 commercial farms in Poland was carried out on 531 faecal samples tested using an ELISA to screen for rotavirus. Of these, 169 were found to contain rotavirus antigen (32% of all cases of diarrhoea tested). Those pig herds managed using the Bisprol system had a lower prevalence of rotavirus in pigs with diarrhoea than those faecal samples obtained from Gi-Gi or Agrokomplex Agard farms. To further establish the presence of rotavirus antigen, 28 of those positive in ELISA samples were taken for isolation of the virus using tissue culture; 18 (64%) isolates were successfully adapted into MA-104 cells and the presence of rotavirus confirmed by immunofluorescence (IF) and electron microscopy (EM). In addition, an analysis of the band patterns visualised on PAGE showed 9 distinct electropherotypes for rotaviral dsRNA among the tested specimens. These findings suggest that rotavirus may represent an important contribution to the incidence of diarrhoea in Polish pig herds. The use of ELISA technology provided an efficient and effective means of evaluating the presence of rotavirus antigen in faecal samples and indicates that this procedure is a very useful tool in epidemiological studies, but that other techniques are required to confirm the presence of virus.

Phenotypic analysis of peripheral leukocytes in piglets infected with classical swine fever virus.

The phenotypic changes in circulating leukocytes in swine fever influenced by classical swine fever virus (CSFV) infection with different strain virulence was studied in piglets. The phenotypic differences were measured by monoclonal antibodies specific for porcine differentiation antigens. The pattern of phenotypic change varied with the virulence of CSFV. Infection with virulent, but not the attenuated strain of CSFV resulted in the dramatic early loss of CD8-bearing T lymphocytes from the circulation. A similar trend was also seen in the gammadelta T-cell compartment following infection with the highly virulent strain, Washington. The loss of circulating B-lymphocytes was consistent with the failure to generate neutralising antibody. These observations contrasted the finding that the number of leukocytes expressing the CD4 surface antigen increased in piglets infected with CSFV. These data provide preliminary information on the potential range of leukocyte changes produced in piglets following infection with CSFV.

Preliminary study of the influence of plasma proteins on immunological and production parameters in pigs.

The health and productivity of pigs is important to farmers worldwide. One potential method of improving animal health is by providing functional proteins in the diet, for example spray-dried animal plasma (SDAP). These proteins, particularly plasma IgG, can improve health in many species, including humans. By unspecific activation of the immune system, SDAP can reduce morbidity and mortality as well as improve growth and efficiency what results in reduced costs of production. In this study, the influence of SDAP on expression of T-lymphocyte markers and production parameters in weaned pigs was evaluated. The results showed a significant increase in the percentage of T-lymphocytes, especially the CD8+ subpopulation, as well as a positive effect on body weight gain.

Efficacy of a combination of amoxicillin and clavulanic acid in the treatment of pneumonia of pigs.

Amoksiklav was used in the therapy of mixed respiratory tract infections in weaned pigs under field conditions. Positive effects of therapy with Amoxicillin and Clavulanic acid were observed in the majority of treated pigs. The production losses due to pneumonia in pigs treated with this combination were lower than among control pigs treated with Oxytetracycline, also a significantly lower ratio of death was observed among experimental weaners in comparison to the controls. Thirty days after the end of the therapy it was found that the body weight gain (b. w. g) of the experimental animals was on average 800 g higher after this period and the experimental piglets grew daily in average 20 g more than the controls. Experimental pigs were slaughtered 3 days earlier than the controls and the average weight gain at slaughter was highest by 1.1 kg in this group. Average daily b. w. g. of experimental pigs during the period from birth to slaughter was 13 g higher in comparison to the controls.

Prophylactic application of Propionibacterium avidum KP-40 in swine with acute experimental infections. I. Viral infections--Aujeszky's disease and classical swine fever.

The usefulness of the prophylactic application of Propionibacterium avidum KP-40 (PA), a potent stimulator of the macrophage-monocyte system and inducer of endogenous interferons, was demonstrated in swine infected experimentally with Aujeszky's disease or classical swine fever viruses. Some of the infected animals were preimmunized with respective vaccines containing live, attenuated viruses. In vaccinated and non-vaccinated swine infected with Aujeszky's disease virus, pretreatment with PA lowered the morbidity rate, shortened the period of fever and fastened the recovery. Infection with classical swine fever virus resulted in 100% mortality of PA-pretreated non-vaccinated swine, but the length survival of the animals was significantly longer (p < 0.05).

Prophylactic application of Propionibacterium avidum KP-40 in swine with acute experimental infections. II. Bacterial infections: pleuropneumonia and swine erysipelas.

The potent immunomodifier Propionibacterium avidum KP-40 (PA) demonstrated prophylactic potency in swine infected experimentally with Haemophilus (Actinobacillus) pleuropneumoniae or Erysipelothrix rhusiopathiae. Animals received PA either together with the respective vaccine or PA only; 3 resp. 4 weeks later all animals were inoculated with virulent pathogens. Eight of 10 swine immunized with inactivated pleuropneumonia vaccine developed mild-moderate forms of infection with temporary stagnation of body weight; application of the vaccine together with PA lowered the morbidity rate to 1 of 10 (p < 0.05). Also in non-vaccinated swine infected with pleuropneumonia or erysipelas PA application resulted in milder clinical symptoms, faster recovery and a larger gain of body weight.

Prophylactic and therapeutic application of Propionibacterium avidum KP-40 in swine and calves with acute enzootic bronchopneumonia.

The usefulness of the prophylactic or therapeutic application of Propionibacterium avidum KP-40, a potent stimulator of the monocyte-macrophage-system, was demonstrated in piglets and calves. After a 3-month-period of observation PA-treated piglets showed a significantly improved development (decreased number of infections, gain of body weight). In piglets and calves the therapeutic use of PA together with oxytetracycline proved to be superior in the treatment of acute endemic enzootic bronchopneumonia (AEB) as compared to groups of animals receiving PA or oxytetracycline alone.

Local and systemic immune response in pigs during subclinical and clinical swine influenza infection.

Local and systemic immune responses in pigs intranasally (IN) and intratracheally (IT) inoculated with swine influenza virus (SIV) were studied. No clinical signs were observed in IN-inoculated pigs, while IT-inoculated pigs developed typical signs of influenza. Significantly higher titres of specific antibodies and changes of haematological parameters were found only in IT-inoculated pigs. Because positive correlations between viral titre, local cytokine concentration, and lung pathology have been observed, we hypothesise that both viral load and the local secretion of cytokines play a role in the induction of lung lesions. It could be that a higher replication of SIV stimulates immune cells to secrete higher amounts of cytokines. The results of the present study indicate that pathogenesis of SIV is dependent on both, the damage caused to the lung parenchyma directly by virus, and the effects on the cells of the host's immune system.