Stanisław Winiarczyk

Molecular characterization of Babesia canis canis isolates from naturally infected dogs in Poland.

Babesia canis has generally been considered the only large Babesia to infect dogs. In this study, we used PCR to detect and characterize B. canis canis isolated from naturally infected dogs in Poland by amplifying and sequencing a portion of the 18S ribosomal RNA (rRNA) gene. Venous blood samples were collected from 76 Babesia-symptomatic dogs. A 559-bp fragment of the B. canis canis 18S rRNA gene was amplified by PCR. The PCR products were then digested with HincII restriction enzyme, and isolates were classified according to whether they were cut (group A) or not (group B) by this endonuclease. Sequencing of the PCR products from the isolates led to the identification of seven sequence variants (four in group A, and three in group B). Sequences were compared with GenBank sequences, and alignments showed that all B. canis canis isolates from Europe may be classified into groups A or B as defined in our study.

Three clinical cases of Anaplasma phagocytophilum infection in cats in Poland

The purpose of this study was to describe Anaplasma phagocytophilum infection of three cats in Poland showing signs of fever, swollen and painful joints, pale mucous membranes and epistaxis. Morulae consistent with A phagocytophilum were present within the neutrophils of two of the cats. A polymerase chain reaction (PCR) was found targeting the 16S rRNA gene amplified DNA consistent with A phagocytophilum in the blood of all three cats. The sequence of the PCR product obtained showed 99.6–100% homology with the sequence of A phagocytophilum, gene number EU 090186 from Genbank. Applied therapy (including administration of tetracyclines for 3 weeks) resulted in a gradual clinical recovery.

Equine granulocytic anaplasmosis.

Anaplasma phagocytophilum is an emerging pathogen of horses that is transmitted by Ixodid ticks. Recent studies suggest that multiple strains of A. phagocytophilum may be circulating in wild and domestic animal populations, and these strains may have differential host tropisms and pathogenicity. The organism infects and survives within neutrophils. Co-infections with other tick-borne pathogens may occur, especially Borrelia burgdorferi. A. phagocytophilum causes an acute febrile illness in horses with lethargy, inappetence, lameness and hemorrhages. Diagnosis is based on finding morulae within granulocytes in the peripheral blood, and detection of the DNA of A. phagocytophilum using specific polymerase chain reaction assays. Most reports suggesting that anaplasmosis is a self-limiting disease that responds well to a tetracycline therapy.

Prevalence and antibiotic resistance of Enterococcus strains isolated from poultry

The aim of this study was to evaluate the frequency of occurrence of bacteria of the genus Enterococcus in poultry, to identify them by means of matrixassisted laser desorption/ionisation time-of-flight mass spectrometry (MALDITOF MS), and to analyse the antimicrobial susceptibility of the isolated strains to the drugs most frequently used in poultry. The material for the bacteriological tests was obtained mainly from the heart (97%) of the birds investigated. Of a total of 2,970 samples tested, 911 (30.7%) tested positive for Enterococcus spp. Enterococci were detected in broilers (88.1%), laying hens (5.3%), turkeys (3.9%), breeding hens (2.2%), and geese (0.4%). The most commonly identified species were Enterococcus (E.) faecalis (74.7%), E. faecium (10.1%), E. gallinarum (5.5%), E. hirae (4.6%), and E. cecorum (4.1%). The most frequent resistance properties were resistance to sulphamethoxazole/trimethoprim (88%), tylosin (71.4%), enrofloxacin (69.4%), doxycycline (67.3%), and lincomycin/spectinomycin (56.1%). Only one vancomycin-resistant Enterococcus, E. cecorum from a broiler, was found.

Application of the SYBR Green real-time HRM PCR technique in the differentiation of the Babesia canis canis protozoa isolated in the areas of eastern Poland

The aim of this study was to determine the usefulness of the real-time polymerised chain reaction (PCR) high-resolution melting (HRM) method in the differentiation of the Babesia canis canis protozoa isolated from dogs in the areas of eastern Poland. The studies involved 20 isolates of B. canis canis qualified depending on the analysis of the 18S RNA gene sequence to group A (EU 622792) and 20 isolates qualified to group B (EU 622793). It was proven with the real-time PCR technique that the melting temperature (Tm) of the obtained products of amplification was 78°C for the representatives of group A and 81°C for the representatives of group B, which proves that the real-time SYBR Green HRM PCR method is a technique allowing for the differentiation of the B. canis isolates which are slightly different with respect to the genetic structure, without the necessity to carry out time-consuming studies, i.e., sequencing and restriction fragment length polymorphism.

Dog Tear Film Proteome In-Depth Analysis.

In this study, mass spectrometry was used to explore the canine tear proteome. Tear samples were obtained from six healthy dogs, and one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1D SDS-PAGE) was used as a first step to separate intact proteins into 17 bands. Each fraction was then trypsin digested and analysed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) to characterize the protein components in each fraction. In total, 125 tear proteins were identified, with MCA (Major Canine Allergen), Serum albumin, UPF0557 protein C10orf119 homolog, Collagen alpha-2(I) chain, Tyrosine -protein kinase Fer, Keratine type II cytoskeletal, Beta-crystallin B2, Interleukin-6 and Desmin occuring as the most confident ones with the highest scores. The results showed that the proteomic strategy used in this study was successful in the analysis of the dog tear proteome. To the best of our knowledge, this study is the first to report the comprehensive proteome profile of tears from healthy dogs by 1D SDS PAGE and MALDI-TOF. Data are available via ProteomeXchange with identifier PXD003124.

Prevalence of Babesia canis, Borrelia burgdorferi sensu lato, and Anaplasma phagocytophilum in hard ticks collected from meadows of Lubelskie Voivodship (eastern Poland)

Abstract The aim of the study was to assess the distribution of Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, and Babesia canis in adult females and males of Ixodes ricinus and Dermacentor reticulatus ticks, inhabiting meadows near large forest complexes throughout the Lubelskie Voivodship (eastern region of Poland). Ticks were collected using the flagging method. Among 720 ticks collected, 506 were identified as D. reticulatus, and 214 as I. ricinus. DNA of B. canis and B. burgdorferi s.l. was detected in 21.3% and 0.6% of D. reticulatus ticks, respectively. In I. ricinus ticks, DNA specific to B. burgdorferi s.l. and A. phagocytophilum was detected in 5.6% and 10.3%, respectively. Co-infections of B. burgdorferi s.l. and A. phagocytophilum were found in two I. ricinus ticks. These results indicate that the Lublin region is an area at risk of tick-borne diseases of humans and animals, which must be considered in clinical practice.

In vitro cultivation of Babesia canis canis parasites isolated from dogs in Poland

The aim of this study was to perform in vitro cultivation of Babesia canis protozoa isolated from dogs with clinical babesiosis. A primary culture initiated in RPMI-1640 medium supplemented with 40% canine serum supported parasite growth in vitro in 5% carbon dioxide in air atmosphere. Subsequent subcultures into HL-1 medium with 40% dog serum or EMEM with 40% foetal bovine serum also supported parasite propagation. The parasites have been continuously cultured through six passages, although the parasitemias are low, ranging from 0.56% to 0.59%. The partial small subunit ribosomal rRNA gene sequence was identical in blood-derived and culture-derived Babesia. The parasites from 17 cultures were classified as EU622792, and from 13 cultures as EU622793. These data show that an efficient in vitro cultivation of B. canis could serve as a starting point to obtain a protozoan antigen used for immunisation of the dogs against piroplasmosis

Phase I/II Clinical Trial of Encapsulated, Cytochrome P450 Expressing Cells as Local Activators of Cyclophosphamide to Treat Spontaneous Canine Tumours

Based upon promising preclinical studies, a clinical trial was performed in which encapsulated cells overexpressing cytochrome P450 enzyme isoform 2B1 were implanted around malignant mammary tumours arising spontaneously in dogs. The dogs were then given cyclophosphamide, one of the standard chemotherapeutic agents used for the treatment of mammary tumours. The dogs were assessed for a number of clinical parameters as well as for reduction in tumour size. The treatment was well tolerated with no evidence of adverse reactions or side effects being associated with the administration of the encapsulated cells. Reductions in tumour size of more than 50% were observed for 6 out of the 11 tumours analysed while 5 tumours showing minor responses, i.e. stable disease. In contrast, the tumours that received cyclophosphamide alone showed only stable disease. Taken together, this data suggests that encapsulated cytochrome P450 expressing cells combined with chemotherapy may be useful in the local treatment of a number of dog mammary tumours and support the performance of further clinical studies to evaluate this new treatment.

Comparative analysis of ORF5 nucleotide sequences and amino acid sequences of the GP5 protein of equine arteritis virus (EAV) detected in the semen of stallions from Eastern Poland.

The purpose of this study was to conduct a comparative analysis of the ORF5 gene fragment nucleotide sequences and the GP5 protein amino acid sequences formed on this matrix, for the equine arteritis virus (EAV) strains isolated from the semen of infected stallions from Eastern Poland. The study covered 41 stallions whose blood serum tested positive for antigens specific to the EAV. The presence of EAV genetic material was shown in material from 5 horses, in one of which permanent presence of viral RNA was detected over the entire 4-year study period (the material was sampled four times at yearly intervals). The mutual similarity among the ORF5 nucleotide sequences of EAV obtained in our own studies was 90.7-99%, whereas their similarity to a sequence of an isolate of the PL1 virus, determined in Polish horses previously, was 76.6-83%. A comparison of the primary structure of capsid glycoprotein encoded by the analysed section of ORF5 showed that amino acid substitution happens most frequently in region V1 of GP5, between positions 61 and 121. A phylogenetic analysis of our own isolates with sequences of viruses isolated from horses from the USA, Europe and New Zealand (available in the gene bank), made it possible to determine that the majority of the detected strains of the pathogen can be classified into the European group, with the Austrian strain of EAV as its protoplast.