Jarosław Kaba

Seroprevalence of Toxoplasma gondii and Neospora caninum infections in goats in Poland

In 2007 a survey on herd-level seroprevalence of Toxoplasma gondii and Neospora caninum infections in goats in Poland was carried out. Sera were collected from all 49 breeding goat herds, scattered over the entire country, with the vast majority of them located in the western, central and northern provinces. Only adult females (≥12 months of age) were included in the study. A herd was recorded as infected if at least one seropositive female was detected. In each herd, simple random sampling was applied and sample size was determined in a way, which allowed to evaluate serological status of a herd at expected individual-level seroprevalence 10% and level of confidence 95%. In total, 1060 sera were tested using two commercial indirect ELISA kits. Sera positive to N. caninum were subsequently confirmed with IFAT. The true herd-level seroprevalence was 100% for T. gondii and 9.0% for N. caninum infection. Three herds positive to T. gondii infection were randomly selected and all adult goats were tested with an ELISA. Individual-level true seroprevalence in these herds ranged from 30.2% to 100%. This is the first time that antibodies to N. caninum have been detected in goats in Poland.

Development of an ELISA for the diagnosis of Corynebacterium pseudotuberculosis infections in goats

An ELISA was developed for the diagnosis of Corynebacterium pseudotuberculosis infections in goats. A bacterial whole cell extract was used as solid-phase antigen, and serum from a culture-positive animal served as the internal reference standard. The well-to-well and assay-to-assay variations were determined to be 12.7 and 33.0%, respectively. A cut off value was determined by parallel testing of 142 sera (112 ELISA-positive, 30 ELISA-negative) in a Western blot, and the correlation between both tests was highly significant (K=0, 93). In addition, the reliability of the ELISA for the detection of infected herds was proven in a double blind study testing 910 sera from 74 goat herds.

Modelling the spatial distribution of Fasciola hepatica in dairy cattle in Europe.

A harmonized sampling approach in combination with spatial modelling is required to update current knowledge of fasciolosis in dairy cattle in Europe. Within the scope of the EU project GLOWORM, samples from 3,359 randomly selected farms in 849 municipalities in Belgium, Germany, Ireland, Poland and Sweden were collected and their infection status assessed using an indirect bulk tank milk (BTM) enzyme-linked immunosorbent assay (ELISA). Dairy farms were considered exposed when the optical density ratio (ODR) exceeded the 0.3 cut-off. Two ensemble-modelling techniques, Random Forests (RF) and Boosted Regression Trees (BRT), were used to obtain the spatial distribution of the probability of exposure to Fasciola hepatica using remotely sensed environmental variables (1-km spatial resolution) and interpolated values from meteorological stations as predictors. The median ODRs amounted to 0.31, 0.12, 0.54, 0.25 and 0.44 for Belgium, Germany, Ireland, Poland and southern Sweden, respectively. Using the 0.3 threshold, 571 municipalities were categorized as positive and 429 as negative. RF was seen as capable of predicting the spatial distribution of exposure with an area under the receiver operation characteristic (ROC) curve (AUC) of 0.83 (0.96 for BRT). Both models identified rainfall and temperature as the most important factors for probability of exposure. Areas of high and low exposure were identified by both models, with BRT better at discriminating between low-probability and high-probability exposure; this model may therefore be more useful in practise. Given a harmonized sampling strategy, it should be possible to generate robust spatial models for fasciolosis in dairy cattle in Europe to be used as input for temporal models and for the detection of deviations in baseline probability. Further research is required for model output in areas outside the eco-climatic range investigated.

Twelve-year cohort study on the influence of caprine arthritis-encephalitis virus infection on milk yield and composition.

This long-term observational cohort study was carried out to evaluate the effect of caprine arthritis-encephalitis virus (CAEV) infection on the quantitative and qualitative characteristics of milk production in dairy goats. For this purpose, a dairy herd comprising both CAEV-infected and uninfected female goats was observed for 12 consecutive years. Records on daily milk yield, somatic cell count (SCC), and contents of the major milk components (fat, protein and lactose) were collected every month. In total, 3,042 records (1,114 from CAEV-positive and 1,928 from CAEV-negative animals) from 177 female goats were used for statistical analysis. The multi-trait repeatability test-day animal model using the derivative-free multivariate analysis package with the average information-REML method was applied to eliminate the influence of factors other than CAEV infection on milk production in goats. The statistical significance of the differences between estimates for seropositive and seronegative goats was evaluated using Students t-test. The effect of age of goats (parity) on their serological status was also estimated with the one-trait repeatability test-day model. The serological status of goats was linked to parity: the higher the parity, the greater the probability of CAEV infection. No significant differences between infected and uninfected goats with respect to daily milk yield and SCC were found. On the other hand, the milk of uninfected goats contained more total protein (3.40% vs. 3.35%), fat (3.69% vs. 3.54%), and lactose (4.30% vs. 4.25%) than the milk of infected goats. Even though these differences were highly significant, they were small when expressed numerically.

Schmallenberg virus antibodies detected in Poland.

Between 24 and 30 July 2012 230 adult goats from three western provinces of Poland bordering on Germany (Western Pomerania, Lubuskie and Lower Silesia) were blood-sampled and tested for antibodies to Schmallenberg virus (SBV) using indirect immunoenzymatic test (ID Screen® Schmallenberg virus indirect, IDvet Innovative Diagnostics). The ELISA test identified 21 seropositive goats - 15 in Western Pomerania (16% of all goats tested in this province), five in Lubuskie (6%) and one in Lower Silesia (2%). Our study demonstrates for the first time the presence of antibodies to SBV in Poland.

Seroprevalence of Toxoplasma gondii in wild boars, red deer and roe deer in Poland

Little is known about the prevalence of Toxoplasma gondii in wild life, particularly game animals in Poland. Meat juice collected during the 2009/2010 and 2010/2011 hunting seasons from 552 red deer (Cervus elaphus), 367 wild boars (Sus scrofa) and 92 roe deer (Capreolus capreolus) was tested for T. gondii antibodies using the multi-species ID Screen Toxoplasmosis Indirect kit (IDvet, Montpellier, France). Antibodies to T. gondii were detected in 24.1% of red deer (95% CI: 20.7%, 27.8%), 37.6% of wild boar (95% CI: 32.8%, 42.7%) and 30.4% of roe deer (95% CI: 22.0%, 40.5%). To the authors’ best knowledge, this is the first epidemiological report of T. gondii prevalence in red deer, roe deer and wild boars in Poland. T. gondii is present in wildlife animal tissues and consumption of the game may be a potential source of infection for humans.

Phenotypic characteristics and virulence genotypes of Trueperella (Arcanobacterium) pyogenes strains isolated from European bison (Bison bonasus)

Trueperella (Arcanobacterium) pyogenes is an opportunistic animal pathogen, which in European bison is associated with different suppurative infections mainly of the urogenital tract. Little is known about the virulence of this bacterium and about the pathogenesis of infections. The main objective of this study was to determine phenotypic properties and virulence genotypes of the twenty-five T. pyogenes strains isolated from lesions in various tissues of free-living European bison. Classical bacteriological methods were used for phenotypic characterization. Genes encoding seven known and putative virulence factors of T. pyogenes were detected by PCR technique. Analysis of 16S rDNA partial sequences was performed to establish phylogenetic relationships of the isolated strains. All isolates showed typical morphological features of T. pyogenes and variable biochemical activity. Most of them displayed a strong positive effect in synergistic CAMP test. For all isolates the 16S rRNA gene partial sequence was identical to that of the T. pyogenes reference strain. All isolates carried the plo and fimA genes, while the nanH, nanP, cbpA, fimC and fimG genes were present in 40, 44, 12, 88 and 24% of the isolates, respectively. The T. pyogenes strains isolated from European bison represented various phenotypes and virulence genotypes, but there was no association between the investigated properties of the bacteria and the type of anatomopathological lesions from which they were isolated. These results indicate that the studied virulence factors of T. pyogenes are not significant determinants of the localization and type of infection caused by this bacterium.

Risk factors associated with seropositivity to small ruminant lentiviruses in goat herds

Cross-sectional studies based on serological testing and questionnaires were conducted at 5-yr intervals (1996, 2002 and 2007) in goat breeding herds from Poland to determine true herd-level seroprevalence of caprine arthritis-encephalitis and the herd-level risk factors for seropositivity. Multivariable logistic regression models were developed for data from 1996 and the combined data from 2002 and 2007, separately. True herd-level seroprevalences in 1996, 2002 and 2007 were 30.8% (CI 95%: 20.5-41.0%), 65.7% and 71.9%, respectively. The import of goats from abroad was a risk factor only in 1996 (OR 13.6, CI 95%: 1.14-162). The presence of seropositive bucks in a herd was a risk factor in 1996 (OR 21, CI 95%: 1.89-233) and in 2002-2007 (OR 2.9, CI 95%: 1.04-8.4). Moreover, large herds (>30 does in 1996 or >100 does in 2002-2007) were more likely to be seropositive than smaller herds (OR=10.1, CI 95%: 2.17-46 in 1996 and OR 5.4, CI 95%: 1.11-26 in 2002-2007).

Serological evidence for BVDV-1 infection in goats in Poland — Short communication

A serological survey was conducted in 2007 in the breeding goat population in Poland to gain insights into the epidemiology of pestivirus infection. All breeding herds were included in the study and representative serum samples were taken in each herd to evaluate herd-level seroprevalence at 10% expected individual-level prevalence and 95% level of confidence. Altogether 1060 serum samples from 49 herds were tested with blocking ELISA and then the positive and inconclusive results were confirmed in a serum neutralisation test, which also allowed us to determine the pestivirus species responsible for seroconversion. Herd-level seroprevalence proved to be 10.2% and bovine viral diarrhoea virus type 1 (BVDV-1) was responsible for the seroconversion in seven out of eight cases. In the remaining serum sample the causative virus could not be identified due to a pronounced cross-neutralising activity possibly derived from multiple infections. This is the first report on the diagnosis of BVDV-1 infection in Polish goats.

The validation of housekeeping genes as a reference in quantitative Real Time PCR analysis: application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus.

The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expression in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N=13) and infected with CAEV (N=13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240days post-partum). The expression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of β-actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large subunit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were considered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of disease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and - according to the geNorm results - the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80°C was also proved.