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Nodularin (NOD) is a cyclic peptide released by bloom-forming toxic cyanobacteria Nodularia spumigena commonly occurring in brackish waters throughout the world. Although its hepatotoxic effects are well known, other negative effects of NOD have not yet been completely elucidated. The present study aims were to evaluate and compare the cytotoxic and immunotoxic effects of the toxin on primary leukocytes (from head kidney [HK]) and stable fish leukocytes (carp leucocyte cell line [CLC] cells). The cells were incubated with the cyanotoxin at concentrations of 0.001, 0.01, 0.05, or 0.1 μg/ml. After 24 h of exposure, the concentrations ≥0.05 μg/ml of toxin resulted in cytotoxicity in the primary cells, while in CLC cells, the toxic effect was obtained only with the highest concentration. Similarly, depending on the concentration, exposure to NOD causes a significant inhibition of chemotaxis of the phagocytic abilities of primary leukocytes and a significant reduction in the proliferation of lymphocytes isolated from the HKs. Moreover, CLC cells and HK leukocytes incubated with this toxin at all the mentioned concentrations showed an increased production of reactive oxygen and nitrogen species. NOD also evidently influenced the expression of genes of cytokine TNF-α and IL-10 and, to a minor extent, IL-1β and TGF-β. Notably, the observed changes in the mRNA levels of cytokines in NOD-exposed cells were evident, but not clearly dose-dependent. Interestingly, NOD did not affect the production and release of IL-1β of the CLC cells. This study provides evidence that NOD may exert cytotoxicity and immune-toxicity effects depending on cell type and toxin concentration., (© 2021 John Wiley & Sons, Ltd.)

Numerous specific biomarkers with a prognostic and diagnostic value comparable to histopathological findings are now used for non-invasive diagnosis of prostate diseases in humans. Meanwhile, as far as dogs are concerned, the diagnosis of prostate disorders is based solely on clinical examination and ultrasound (USG). Therefore, the aim of the study was to assess the usefulness of two biomarkers, i.e. miRNA-129 and VEGF for the diagnosis of BPH in dogs. The study involved 40 dogs divided into three groups. Group I (n = 9) comprised healthy dogs up to the age of 5 years, Group II (n = 17) comprised dogs between the ages of 5-10 suffering from BPH as confirmed by the examination and Group III (n = 14) comprising dogs over 10 years of age, which also had BPH confirmed. The results demonstrated that dogs in group II and III exhibited a significant decrease in miRNA expression (P < 0.0001) and a significant increase in serum VEGF levels (P = 0.025) when compared to the dogs in group I. There was also a positive correlation between the prostate size and VEGF level. The findings led to the conclusion that the determination of miRNA-129 and VEGF can significantly contribute to the diagnosis of prostate disorders in dogs., Competing Interests: Declaration of competing interest All authors declare no conflicts of interest., (Copyright © 2021. Published by Elsevier Inc.)

Canine innate immune system role in cancer prevention and progression remains poorly understood. It has been revealed that innate immune cells could play a dual role in cancer immunology promoting or inhibiting tumor development and growth. Current immunotherapies target mainly the adaptive anti-tumor response and that may be a reason why they remain ineffective in a majority of patients. It is important to acquire detailed knowledge about innate immune mechanisms to broaden the diagnostic and therapeutic options and employ innate immune cells in anti-cancer therapies. In the present study, 21 female dogs of different breeds and types of spontaneous mammary tumors were investigated. The study aimed to find simple and cheap markers that can be used for preliminary diagnosis, prior to the surgical resection of the tumor. The differences in innate immune cell quantity and function were investigated between female dogs with malignant mammary tumors of epithelial and mesenchymal origin. Flow cytometry was used to evaluate the percentages of CD5+ lymphocytes including CD5 low lymphocytes, CD11b integrin expression on leukocytes, phagocytosis, and oxidative burst. The number of CD11b lymphocytes was increased in tumors with epithelial origin compared to the control group. No significant differences were found between the percentages of phagocytic cells neither for granulocytes nor for monocytes. However, the phagocytes of canine patients with tumors of epithelial origin showed increased phagocytosis compared to the control group. The percentages of granulocytes that produced reactive oxygen species (ROS) in response to E.coli and PMA were not altered in patients with malignant tumors compared to control. A statistically significant difference between the number of ROS produced by the single granulocyte was demonstrated only between the group of bitches with epithelial tumors and the control group in case of E. coli stimulation. The obtained results suggest that some innate immune cells may be involved in anti-tumor immune mechanisms and have the potential to be supportive diagnostic markers in canine mammary tumors.

Macular edema and its further complications due to the leakage from the choroidal neovascularization in course of the age-related macular degeneration (AMD) is a leading cause of blindness among elderly individuals in developed countries. Changes in tear film proteomic composition have been reported to occur in various ophthalmic and systemic diseases. There is an evidence that the acute form of neovascular AMD may be reflected in the tear film composition. Tear film was collected with Schirmer strips from patients with neovascular AMD and sex- and age-matched control patients. Two-dimensional electrophoresis was performed followed by MALDI-TOF mass spectrometry for identification of differentially expressed proteins. Quantitative analysis of the differential electrophoretic spots was performed with Delta2D software. Altogether, 11 significantly differentially expressed proteins were identified; of those, 8 were downregulated, and 3 were upregulated in the tear film of neovascular AMD patients. The differentially expressed proteins identified in tear film were involved in signaling pathways associated with impaired protein clearance, persistent inflammation, and neovascularization. Tear film protein analysis is a novel way to screen AMD-related biomarkers.

The purpose of the study was to carry out the molecular surveillance of piroplasmosis, granulocytic anaplasmosis and lyme borreliosis in horses which originated from Poland and exhibited symptoms raising the suspicion of the aforementioned disease units. The presence of Theileria equi genetic material was detected in 37 out of 512 examined horses (7.2%), and Anaplasma phagocytophilum in 9 (1.8%). The DNA of Borrelia burgdorferi was found in 11 out of 204 examined horses (5.4%). The above-cited results indicate that the problem of tick-borne diseases affecting horses in Poland is not as significant as in other parts of Europe, however they have to be considered in differential diagnosis of the diseases with lethargy, fever, anaemia and thrombocytopenia., (© 2021 The Authors Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Introduction: The aim of the study was to demonstrate a link between uncomplicated Babesia canis infection in dogs and blood concentrations of zinc and copper and erythrocytic antioxidant defence - activities of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT)., Material and Methods: The study was based on 15 naturally occurring cases of canine babesiosis with anorexia, pyrexia, depression, pale mucous membrane, splenomegaly and dark red urine. Microscopic examination of Giemsa-stained peripheral blood smears and the results of PCR confirmed B. canis infection. Seven apparently healthy dogs brought in for either a check-up or vaccination were used for comparison., Results: The levels of the erythrocytic antioxidant enzymes - SOD and CAT - were significantly higher in the infected dogs than in cytologically negative dogs. The levels of blood micronutrients were significantly lower in the infected dogs (0.478 μg of zinc per mL vs 1.241 μg/mL and 0.722 μg of copper per mL vs 1.392 μg/mL)., Conclusion: Oxidative stress can be posited as one of the mechanisms leading to anaemia in dogs with babesiosis, and therefore antioxidant biomarker and copper and zinc concentrations could be used as indicators of disease severity and prognostic markers., Competing Interests: Conflict of Interest Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2021 O. Teodorowski et al. published by Sciendo.)

Background: Anaplasma are obligate intracellular bacteria and aetiological agents of tick-borne diseases of both veterinary and medical interest. The genus Anaplasma comprises six species: Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Anaplasma phagocytophilum, Anaplasma bovis and Anaplasma platys. They can infect humans, carnivores, ruminants, rodents, insectivores, birds and reptiles. The aim of this study was to present the first clinical case of granulocytic anaplasmosis in a captive ring-tailed lemur in Poland., Case Presentation: A 4-year-old female lemur presented anorexia, epistaxis and tick infestation. The microscopic examination of a blood smear revealed morulae in neutrophils. Polymerase chain reaction test and sequencing of obtained PCR product confirmed infection by the GU183908 Anaplasma phagocytophilum strain. Therapeutic protocol included doxycycline (2.5 mg/kg p.o., b.i.d.) for 3 weeks and the lemur recovered within 24 h., Conclusions: This is the first report on granulocytic anaplasmosis in a ring-tailed lemur in Europe, indicating that A. phagocytophilum infection must also be considered in differential diagnosis in this animal species, especially in individuals with thrombocytopenia associated with Ixodes ricinus parasitism.

The aim of this study was to demonstrate a relationship between the occurrence of clinical signs of brain involvement in dogs with babesiosis and the concentration of manganese (Mn) in their serum. The study included seven dogs with early babesiosis (Group 1), seven dogs with cerebral babesiosis (Group 2) and seven healthy dogs (Group 3). Haematological and biochemical blood tests were performed in all dogs, and the results were analysed statistically. The Mann-Whitney rank test was used to demonstrate the differences in Mn concentrations, as well as other haematological and biochemical parameters between groups. In dogs in Group 2 with cerebral babesiosis, as compared to dogs in Groups 1 and 3, a statistically significant increase in serum Mn concentration was shown (P = 0.002 and P = 0.029) that may have been associated with the development of anaemia and/or impairment of liver function. Given the well-established neurotoxic effects of Mn in humans, experimental rodents and primates, additional studies on the role of Mn in the pathogenesis of the cerebral form of canine babesiosis are warranted.

Canine diabetes mellitus is a significant health burden, followed with numerous systemic complications, including diabetic cataracts and retinopathy, leading to blindness. Diabetes should be considered as a disease damaging all the body organs, including gastrointestinal tract, through a complex combination of vascular and metabolic pathologies, leading to impaired gut function. Tear film can be obtained in a non-invasive way, which makes it a feasible biomarker source. In this study we compared proteomic changes ongoing in tear film of diabetic dogs. The study group consisted of 15 diabetic dogs, and 13 dogs served as a control group. After obtaining tear film with Schirmer strips, we performed 2-dimensional electrophoresis, followed by Delta2D software analysis, which allowed to select statistically significant differentially expressed proteins. After their identification with MALDI-TOF (matrix assisted laser desorption and ionisation time of flight) spectrometry we found one up-regulated protein in tear film of diabetic dogs-SRC kinase signaling inhibitor 1 (SRCIN1). Eight proteins were down-regulated: phosphatidylinositol-4 kinase type 2 alpha (PI4KIIα), Pro-melanin concentrating hormone (Pro-MCH), Flotillin-1, Protein mono-ADP ribosyltransferase, GRIP and coiled coil domain containing protein 2, tetratricopeptide repeat protein 36, serpin, and Prelamin A/C. Identified proteins were analyzed by Panther Gene Ontology software, and their possible connections with diabetic etiopathology were discussed. We believe that this is the first study to target tear film proteome in canine diabetes. We believe that combined with traditional examination, the tear film proteomic analysis can be a new source of biomarkers both for clinical practice, and experimental research.

Canine babesiosis is a tickborne, protozoal, haemoparasitic disease. Babesia organisms are frequently classified as either large (B. canis) or small (B. gibsoni). The aim of this study was an attempt to detect B. gibsoni DNA in blood samples taken from dogs suspected of suffering from tick-borne diseases. 216 samples were tested using PCR, of which, in 99 of them B. canisDNA was detected, whereas in 3 of them B. gibsoni was detected. Positive PCR results for B. gibsoni were confirmed using a Qube MDx real-time analyzer. The results indicate that infec-tions with this B. gibsoni should be taken into account and included in the differential diagnosis of vector-borne diseases in dogs in Poland, and that the accurate identification of the species of parasite causing the infection is crucial for developing the correct treatment regimen and prognosis., (Copyright© by the Polish Academy of Sciences.)

Babesia canis is the predominant and clinically relevant canine Babesia species in Europe. Transmitted by vector ticks, the parasite enters red blood cells and induces a severe, potentially fatal hemolytic anemia. Here, we report on the antibabesial activities of three extracts of the West African tropical plant species Triphyophyllum peltatum (Dioncophyllaceae) and Ancistrocladus abbreviatus (Ancistrocladaceae) and of 13 genuine naphthylisoquinoline alkaloids isolated thereof. Two of the extracts and eight of the alkaloids were found to display strong activities against Babesia canis in vitro. Among the most potent compounds were the C,C-coupled dioncophyllines A (1a) and C (2) and the N,C-linked alkaloids ancistrocladium A (3) and B (4), with half-maximum inhibition concentration (IC 50 ) values of 0.48 μM for 1a, 0.85 μM for 2, 1.90 μM for 3, and 1.23 μM for 4. Structure-activity relationship (SAR) studies on a small library of related genuine analogs and non-natural synthetic derivatives of 1a and 2 revealed the likewise naturally occurring alkaloid N-methyl-7-epi-dioncophylline A (6b) to be the most potent (IC 50 , 0.14 μM) among the investigated compounds. Although none of the tested naphthylisoquinolines showed 100 % inhibition of parasite infection - as displayed by imidocarb dipropionate (IC 50 , 0.07 μM), which was used as a positive control - the antibabesial potential of the dioncophyllines A (1a) and C (2) and related compounds such as 6b, its atropo-diastereomer 6a (IC 50 , 1.45 μM), and 8-O-(p-nitrobenzyl)dioncophylline A (14) (IC 50 , 0.82 μM) is to be considered as high. The SAR results showed that N-methylation and axial chirality exert a strong impact on the antibabasial activities of the naphthylisoquinolines presented here, whereas dimerization, as in jozimine A 2 (5) (IC 50 , 140 μM), leads to a significant decrease of activity against B. canis. Alkaloids displaying good to high activities against B. canis like the dioncophyllines 1a, 2, 6a, and 6b were found to cause only a small degree of hemolysis (< 0.7 %), whereas compounds with moderate to weak antibabesial activities such as 6-O-methyl-4'-O-demethylancistrocladine (15a) (IC 50 , 14.0 μM) and its atropo-diastereomer 6-O-methyl-4'-O-demethylhamatine (15b) (IC 50 , 830 μM) caused a high degree of hemolysis (7.3 % for 15a and 11.2 % for 15b). In this respect, the most effective anti-Babesia naphthylisoquinolines are also the safest ones., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chitin, as one of nature's most abundant structural polysaccharides, possesses worldwide, high industrial potential and a functionality that is topically pertinent. Nowadays, the metallization of naturally predesigned, 3D chitinous scaffolds originating from marine sponges is drawing focused attention. These invertebrates represent a unique, renewable source of specialized chitin due to their ability to grow under marine farming conditions. In this study, the development of composite material in the form of 3D chitin-based skeletal scaffolds covered with silver nanoparticles (AgNPs) and Ag-bromide is described for the first time. Additionally, the antibacterial properties of the obtained materials and their possible applications as a water filtration system are also investigated.

Introduction: The prohibition of antibiotic use in edible snails obligates breeders to treat bacterial infections by different means, of which a common one is a bath in Gram-positive- and partially Gram-negative-bactericidal ethacridine lactate solution. The aim of the study was to determine the effect of bathing Cornu aspersum Müller snails in a 0.1% aqueous solution of ethacridine lactate on selected physiological parameters of haemolymph., Material and Methods: The study included 80 snails, divided into two equal groups (study and control). The study group was subjected to bathing in ethacridine lactate and the control group to bathing in tap water. Both groups were treated daily for seven days. The number of haemocytes in the haemolymph, the activity of alanine (ALT) and aspartate (AST) aminotransferases, and the concentration of urea were determined., Results: In the study group, after exposure to ethacridine lactate solution an increase in ALT activity, changes in the De Ritis ratio, an increase in the amount of haemocytes, and a decrease in body weight were found. No such changes were detected in the control group snails or in animals after the first bath., Conclusion: Multiple applications of a 0.1% ethacridine lactate bath may adversely affect Cornu aspersum Müller snails., Competing Interests: Conflict of Interest Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2020 J. Ziętek et al., published by Sciendo.)

Urban environments may negatively affect the development of organisms. In host-pathogen/parasite systems, this impact may lead to increased manifestations of pathogens that decrease the success of their hosts in urban environments compared to rural ones. We tested this hypothesis in the solitary bee Osmia bicornis L. We estimated the development of bees, their reproductive success and the manifestation of different pathogens and nest parasites along an urbanisation gradient. We conducted the experiment in an urbanisation gradient in sites representing three environments: urban, suburban and rural. First, we analysed the manifestation of bacterial and fungal microorganisms in pollen loads, within dried/mummified individuals, on the surface of cocoons and on the surface of diapausing adult individuals by using the MALDI-TOF MS technique. We also verified genetic samples from diapausing individuals for the presence of the parasitic Nosema apis (Zandler, 1909) and N. ceranae (Fries et al., 1996) species. Finally, we assessed the level of reproductive success and manifestations of brood parasites. Not any biological material from the nests was infected by pathogenic microorganisms. This result indicates that the nests are not a reservoir of the pathogenic bacteria and that O. bicornis offspring are not a source or vectors of these pathogens to the surrounding environment and indirectly to other bee species. In urban sites, there was a lower number of parasites than in suburban and rural environments. The presence of parasites was negatively correlated with the reproductive success and may be a limiting factor for O. bicornis populations. We also found that urban sites had the highest indices of reproductive success and the lowest number of breeding failures compared to suburban and rural sites. Moreover, bacterial and fungal transmission is not a serious threat in the studied region., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2018. Published by Elsevier B.V.)

Introduction: The aim of the study was to establish the prevalence of Bartonella spp. in cats in eastern Poland, and to determine the factors associated with the infection., Material and Methods: PCRs were performed to detect Bartonella DNA in the whole blood of 672 cats from four regions in eastern Poland (the Lublin, Podlasie, Masovian, and Subcarpathian provinces). The association between the previously selected variables and the dependent variable (presence of Bartonella DNA) was investigated using a logistic regression model., Results: The overall prevalence of infection was 40.48%. All PCR positive cats were infected with B. henselae . The living conditions of the animals (free outdoor roaming), mixed breed cats, Subcarpathian region, and absence of tick control were significant risk factors associated with Bartonella infection at a 95% confidence level., Conclusion: Cats in eastern Poland appear to be at risk of a bartonellosis epizootic. Factors which seem to impact the likelihood of infection in cats and factors which seem not to impact it have been suggested. We advocate additional research into the ways bartonellosis spreads, its geographical scope, and the factors that favour its development., Competing Interests: Conflict of Interest Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2020 Ł. Mazurek et al. published by Sciendo.)

Introduction: Dobermann dogs are reportedly predisposed to familial glomerulonephropathy. Proteinuria is a hallmark of canine familial glomerular diseases. The identification of glomerular abnormalities in breeds so predisposed is of great importance in improving breeding policy. Therefore, markers that allow the detection and localisation of renal damage are needed. The purpose of this study was to investigate the urinary concentrations of immunoglobulin G (uIgG), retinol-binding protein (uRBP), and Tamm-Horsfall protein (uTHP) in a family of Dobermanns with proteinuria and compare these concentrations with the corresponding values in healthy controls., Material and Methods: Ten dogs of the Dobermann breed with proteinuria (five with a urine protein-to-creatinine ratio (UPC) of 0.5-1 and five with a UPC >1) and twelve healthy dogs were enrolled. An ELISA was performed to measure uIgG, uRBP, and uTHP, and these proteins were quantified in relation to urinary creatinine (uCrea)., Results: uIgG/uCr and uRBP/uCr were significantly higher in the family of Dobermanns than in the healthy dogs. A significant difference in the uTHP/uCr value was found only in dogs with a UPC of >1., Conclusion: IgG seems to facilitate the diagnosis of primary hereditary glomerulopathy in Dobermanns. Moreover, in affected dogs, proteinuria characterisation seems to be a promising alternative option for the detection and localisation of renal lesions., Competing Interests: Conflict of Interest Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2020 D. Winiarczyk et al. published by Sciendo.)

The aim of this study was to recognise the etiological factor of a disease with symptoms of lameness and cardiac failure, which occurred in one dog 4 weeks after invasion by ticks. A serological examination as well as molecular examination (PCR) was done. In the sample of the serum, the presence of antibodies specific to Borrelia burgdorferi were detected. Antibiotic therapy with doxycycline did not cause significant improvement, so the owners of the dog decided about its euthanasia. During the necroscopy, a dilated heart was recognised. In the heart samples, the genetic material of Borrelia was detected. The results of serological and molecular examinations showed that in the discussed case, an etiological factor of the disease was spirochetes. In light of the research, veterinary practitioners should keep in mind the presence of Lyme disease in dogs in Poland and include it in differential diagnoses for lameness and cardiological problems.

The main aim of this paper was to show the variety of capsaicin's properties. Capsaicin is an active component of plants of the Capsicum genus, and is known for its pungency. Capsaicin is used in the food, pharmaceutical and cosmetic industries. Additional properties of capsaicin have been demonstrated, including pain relief, weight loss, body thermoregulation, and antioxidant, antimicrobial and anticancer activities. Studies of capsaicin's effects on the human and animal organism need to be continued, with special emphasis on new applications., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A 5-year-old Jack Russell Spaniel was presented in December 2017 to his veterinarian in Belgium for sudden weakness, reluctance to move, and pain. Blood analysis showed no deviations and serum increased levels of B. burgdorferi s.l. antibodies were detected. The dog recovered a few days after the onset of doxycycline treatment. This case illustrates the possible relationship between tick-borne diseases and orthopedic problems.

The aim of this paper was to analyse the amino acid sequences of the 18S rRNA gene of Babesia canis strains and the proteomic analysis of the serum of dogs infected with three various genotypes: 18S rRNA B. canis. Material for the research was DNA B. canis obtained from dogs with babesiosis. In total, 60 DNA tested samples were divided into three groups (20 samples each). The groups were formed by DNA samples of the sequences marked as 18S RNA-A (group 1), 18S RNA-B (group 2), and 18S RNA-C (group 3). The basis for the classification of protozoa to a specific group was the location of relevant nucleotides (GA, AG, or TT) in position 150-151 of the tested nucleotide sequence 18S rRNA. Nucleotide sequences were transcribed into amino acid sequences and then analysed using DNASTAR software. From all 60 infected and ten healthy dogs (control group), the serum was taken to make proteomic tests using MALDI-TOF mass spectrometer. It was demonstrated that the mutations found in position 150 and 151 of the nucleotide sequence, result in a change of amino acid sequences. Moreover, it was also demonstrated that the disease course in dogs infected with different strains of protozoa is different. Each of the analysed strains of protozoa induced in the serum of infected animals the appearance of a protein fraction of mass 51 kDa, which may then be treated as a nonspecific disease marker used for the diagnosis of this disease but not to differentiate the protozoa strains.

Rhodococcus equi (R. hoagii) is an opportunistic pathogen commonly found in foals up to 6 months old and animal environment. The R. equi genome contains genetically stable chromosomal DNA and an 80-90 kb plasmid containing vapA gene, responsible for virulence. Most reports from around the world focus on the determination of R. equi plasmid profiles. Few studies have attempted to determine differences in nucleotide sequences between virulent strains of R. equi isolated from foals and breeding environment. The aim of the study was to perform a molecular analysis of a fragment of the chromosomal gene encoding the 16S rRNA subunit and the vapA plasmid gene of virulent R. equi strains isolated on Polish studs from foals and from the breeding environment of horses. The sequencing method was used to compare the primary structure of fragments of the chromosomal and plasmid DNA of the virulent R. equi strains. The sequences of 22 clinical and 18 environmental R. equi isolates were compared with the sequences of the gene fragments of reference strains available in the NCBI GenBank database. All sequenced 16S rRNA amplicons of Polish field strains were identical and showed 99.5% similarity to the four randomly selected sequences of this gene fragment in the GenBank database. The results confirm that fragments of the 16S rRNA gene of R. equi strains are highly conserved and do not undergo variation in field conditions. Analysis of the sequencing results for the vapA gene fragment of the strains used in our study revealed two polymorphic variants and clear differences between the sequences of strains isolated from foals and from soil samples. Presumably, R. equi strains present in the breeding environment are more exposed than clinical strains to adverse external factors. This may result in changes in the DNA sequence due to natural selection., Competing Interests: The authors have declared that no competing interests exist.

Objective: Bovine piroplasmoses are tick-borne protozoan diseases caused by parasites of the genus Theileria and Babesia . The aim of this work was to study the epizootic situation of babesiosis in the cattle population in eastern Poland and possibly to determine which species of protozoa infects Polish cattle., Material, Methods and Results: Blood samples for molecular analysis (real time PCR) were collected from 192 dairy cattle from various farms located in eastern Poland. The infection was detected in 10.4 % of the samples. All animals were infected with Babesia occultans which sequence of the 18S RNA gene fragment showed a 92.8 % homology with the sequence of B. occultans EU 376017., Conclusion and Clinical Relevance: This is the first report about the detection of B. occultans DNA in asymptomatic cattle in eastern Poland. The results obtained indicate that the range of these parasites is increasing and that continuous monitoring of babesiosis in cattle in Europe and in Poland is necessary., Competing Interests: The authors declare that they have no competing interests. The authors certify that they have no affiliation with or financial involvement in any organization or entity with a direct financial interest in the subject matter or the materials discussed in the manuscript., (Georg Thieme Verlag KG Stuttgart · New York.)

Purpose: Age-related macular degeneration (AMD) is the main reason for blindness in elderly people in the developed countries. Current screening protocols have limitations in detecting the early signs of retinal degeneration. Therefore, it would be desirable to find novel biomarkers for early detection of AMD. Development of novel biomarkers would help in the prevention, diagnostics, and treatment of AMD. Proteomic analysis of tear film has shown promise in this research area. If an optimal set of biomarkers could be obtained from accessible body fluids, it would represent a reliable way to monitor disease progression and response to novel therapies., Methods: Tear films were collected on Schirmer strips from a total of 22 patients (8 with wet AMD, 6 with dry AMD, and 8 control individuals). 2D electrophoresis was used to separate tear film proteins prior to their identification with matrix-assisted laser desorption/ionization time of flight spectrometer (MALDI-TOF/TOF) and matching with functional databases., Results: A total of 342 proteins were identified. Most of them were previously described in various proteomic studies concerning AMD. Shootin-1, histatin-3, fidgetin-like protein 1, SRC kinase signaling inhibitor, Graves disease carrier protein, actin cytoplasmic 1, prolactin-inducible protein 1, and protein S100-A7A were upregulated in the tear film samples isolated from AMD patients and were not previously linked with this disease in any proteomic analysis., Conclusion: The upregulated proteins supplement our current knowledge of AMD pathogenesis, providing evidence that certain specific proteins are expressed into the tear film in AMD. As far we are aware, this is the first study to have undertaken a comprehensive in-depth analysis of the human tear film proteome in AMD patients.

Proteomic analyses are rapid and powerful tools that are used to increase the understanding of cancer pathogenesis, discover cancer biomarkers and predictive markers, and select and monitor novel targets for cancer therapy. Feline injection-site sarcomas (FISS) are aggressive skin tumours with high recurrence rates, despite treatment with surgery, radiotherapy, and chemotherapy. Doxorubicin is a drug of choice for soft tissue sarcomas, including FISS. However, multidrug resistance is one of the major causes of chemotherapy failure. The main aim of the present study was to identify proteins that differentiate doxorubicin-resistant from doxorubicin-sensitive FISS using two-dimensional gel electrophoresis (2DE), followed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Using the three-dimensional (3D) preclinical in ovo model, which resembles features of spontaneous fibrosarcomas, three significantly ( p ≤ 0.05) differentially expressed proteins were identified in tumours grown from doxorubicin-resistant fibrosarcoma cell lines (FFS1 and FFS3) in comparison to the doxorubicin-sensitive one (FFS5): Annexin A5 (ANXA5), Annexin A3 (ANXA3), and meiosis-specific nuclear structural protein 1 (MNS1). Moreover, nine other proteins were significantly differentially expressed in tumours grown from the high doxorubicin-resistant cell line (FFS1) in comparison to sensitive one (FFS5). This study may be the first proteomic fingerprinting of FISS reported, identifying potential candidates for specific predictive biomarkers and research targets for doxorubicin-resistant FISS., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. According to Polish law (The Act on the protection of animals used for scientific and educational purposes, which was passed in January 2015 and transposed Directive 2010/63/EU into current Polish legislation) the chick embryo studies do not need the approval of the Animal Ethic Commission. Nevertheless, all experiments were performed according to the animal welfare, minimizing the stress of chicken embryos according to American Veterinary Medical Association and Animal Care and Use Committee guidelines on the use of chicken embryos for scientific purposes.

Bartonellosis is a disease caused by Bartonella spp. microorganisms which belong to the Rickettsiales order. This disease is a zoonosis, B. henselae, whose primary reservoir is the cat, which in humans causes a cat-scratch disease. In infected cats, symptoms such as fever, lymphedema, reproduction disorders, myocarditis, rhinotracheitis, gingivitis, and arthritis may be observed. Bartonella appears to be transmitted among cats and dogs in vivo exclusively by arthropod vectors (excepting perinatal transmission), not by biting or scratching. In the absence of these vectors, the disease does not spread. On the other hand, the disease can be spread to humans by bites and scratches, and it is highly likely that it is spread by arthropod vectors as well. This review presents a potential role of ticks and fleas in the transmission of bartonellosis. Clinicians should be aware that a common illness, such as infection with Bartonella, can be transmitted by arthropod vectors, and that a history of animal scratches or bites is not necessary for disease transmission.

Objective: Canine babesiosis is a common and clinically significant tick-borne disease caused by haemoprotozoan parasites of the genus Babesia. Acute renal failure is considered to be one of the most prevalent complications of canine babesiosis. This complication leads to a decrease in the glomerular filtration rate and in consequence causes azotemia and uremia. The objective of this study was to assess the localization and extent of renal damage in dogs infected with Babesia canis using an urinary marker for glomerular (urinary immunoglobulin G, uIgG), proximal tubular dysfunction (urinary retinol binding protein, uRBP) and distal tubular dysfunction (urinary Tamm-Horsfal protein, uTHP). Material und methods: In 10 dogs naturally infected with B. canis and 10 healthy control dogs the levels of urinary biomarkers were measured using commercially available ELISA tests., Results: Higher concentrations of uIgG, uRBP and uTHP were found in the urine of all dogs with babesiosis than in those from the control group. This indicates that in the course of the disease, the glomeruli as well as the renal tubules become damaged., Conclusion and Clinical Relevance: The study results allow a better understanding of the pathogenesis of canine babesiosis. However, in order to fully determine the extent and the nature of the damage to the kidneys of the infected dogs, it is advisable to conduct additional histopathological examinations of these organs.

The aim of this study was to characterize multidrug resistant E. faecalis strains from pigs of local origin and to analyse the relationship between resistance and genotypic and proteomic profiles by amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI -TOF MS). From the total pool of Enterococcus spp. isolated from 90 pigs, we selected 36 multidrug resistant E. faecalis strains, which represented three different phenotypic resistance profiles. Phenotypic resistance to tetracycline, macrolides, phenicols, and lincomycin and high-level resistance to aminoglycosides were confirmed by the occurrence of at least one corresponding resistance gene in each strain. Based on the analysis of the genotypic and phenotypic resistance of the strains tested, five distinct resistance profiles were generated. As a complement of this analysis, profiles of virulence genes were determined and these profiles corresponded to the phenotypic resistance profiles. The demonstration of resistance to a wide panel of antimicrobials by the strains tested in this study indicates the need of typing to determine the spread of resistance also at the local level. It seems that in the case of E. faecalis, type and scope of resistance strongly determines the genotypic pattern obtained with the ADSRRS-fingerprinting method. The ADSRRS-fingerprinting analysis showed consistency of the genetic profiles with the resistance profiles, while analysis of data with the use of the MALDI- TOF MS method did not demonstrate direct reproduction of the clustering pattern obtained with this method. Our observations were confirmed by statistical analysis (Simpson's index of diversity, Rand and Wallace coefficients). Even though the MALDI -TOF MS method showed slightly higher discrimination power than ADSRRS-fingerprinting, only the latter method allowed reproduction of the clustering pattern of isolates based on phenotypic resistance and analysis of resistance and virulence genes (Wallace coefficient 1.0). This feature seems to be the most useful for epidemiological purposes and short-term analysis., Competing Interests: The authors have declared that no competing interests exist.

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.

In the pathology of poultry, infections caused by Staphylococcus spp. are taking on increasing significance. Although the Staphylococcus species most frequently isolated from these animals is Staphylococcus aureus, the literature data indicate that other species, both coagulase-positive and coagulase-negative, can also cause infections in birds. The aim of the study was to assess the frequency of occurrence of Staphylococcus infections in various poultry species in Western Poland and to test the susceptibility of isolated strains to selected antibiotics. The results obtained showed a relatively high rate of Staphylococcus infection in the poultry. From 2805 samples tested 302 strains (10.8%) of Staphylococcus were isolated. As many as 25 Staphylococcus species were distinguished among the strains isolated. S. cohnii (23.50%), S. aureus (15.89%) and S. lentus (13.90%) accounted for the highest percentages. Over half of the isolated staphylococci exhibited resistance to five of the antibiotics applied, with the highest percentage of resistant strains, 65%, noted for enrofloxacin.

The aim of the study was to establish the prevalence of Ehrlichia canis, Anaplasma phagocytophilum and Borrelia burgdorferi in dogs in eastern Poland and to determine the factors associated with exposure (seroposity) or infection (PCR). Anti-A. phagocytophilum, anti-B. burgdorferi and anti-E. canis antibodies were determined in 400 dogs, using the SNAP 4Dx ® test (IDEXX Laboratories). In addition, PCRs were performed for the detection of E. canis, A. phagocytophilum and B. burgdorferi DNA. In reference to the risk factor analysis, a regression logistic model was determined for each aetiological agent. The overall seroprevalence was highest for B. burgdorferi (11.0 %), followed by A. phagocytophilum (8.0 %) and E. canis (1.5 %). Eleven healthy dogs were found to be infected with A. phagocytophilum, as determined by PCR, while the remainder were seronegative. For B. burgdorferi, the DNA of the spirochetes was detected in the blood of 20 dogs, while the presence of anti-B. burgdorferi IgG was detected in the sera of ten of these. For E. canis, none of the dogs tested positive by PCR. Tick control was included as a protective factor for A. phagocytophilum and B. burgdorferi, while the origin (rural) was included as a risk factor for B. burgdorferi and A. phagocytophilum infection. In addition, breed (pure) was a risk factor for B. burgdorferi infection, and sex (female) was a risk factor for E. canis.

Anaplasma phagocytophilum is a tick-transmitted obligate-intracellular gram-negative bacteria that causes emerging human zoonosis. A. phagocytophilum is transmitted by Ixodid ticks. Recent studies suggest that wild animals may be reservoirs of A. phagocytophilum for humans. The organism infects and survives within neutrophils. The infection diagnosis is based on the detection of morulae within granulocytes of peripheral blood, results of serological tests and detection of the DNA of A. phagocytophilum using specific polymerase chain reaction assays (PCR). A. phagocytophilum in most cases is transmitted to people by tick bites, but sometimes direct contact with infected blood may cause human granulocytic anaplasmosis (HGA). The possibility of infection should be taken into consideration at each occurrence of heavy disease symptoms after people come into contact with ticks.

Unlabelled: The canine vector-borne diseases (CVBD) is a term, which describes a range of infectious or/and parasitic diseases whose etiological agents are transmitted by vectors. CVBD are becoming more widely in the world in relation to global warming and the increasing number of infected vectors. The aim of this study was to assess rapid mass spectrometry (MS) - based proteomics analyses for diagnosis of Babesia canis, Anaplasma phagocytophilum and Borrelia burgdorferi infections in dogs. The study was conducted on four groups of dogs - healthy dogs (group 1, n=10) and dogs infected with B. canis (group 2, n=20), A. phagocytophilum (group 3, n=20) and B. burgdorferi (group 4, n=20) which demonstrated symptoms of the diseases. The MALDI-TOF (Matrix Assisted Laser Desorption Ionization with Time of Flight detector) MS technique revealed the presence of specific protein fraction of 51-52 kDa only in the blood serum of all the animals infected with the B. canis protozoa. The proteins are suspected to be disease markers, whereas the MALDI-TOF technique itself has high specificity and sensitivity and can be applied in the diagnosis of canine babesiosis., Key Words: Anaplasma phagocytophilum, Babesia canis, Borrelia burgdorferi, MALDI-TOF, proteomics.

Cylindrospermopsin is a cyanotoxin with cytotoxic activity. It is released into water during and after cyanobacterial water blooms and thus poses a threat to the health of fish. There is very little information available concerning the effects of the toxin on fish immune cells. In this study, we assessed the potential impact of cylindrospermopsin on the basic functions of phagocytic cells from common carp (Cyprinus carpio L.), including phagocytosis, reactive oxygen and nitrogen species production, and the structure of microfilaments and selected cytokine expression. Phagocytic cells, isolated from fish head kidneys, were exposed to the toxin at concentrations of 0.05, 0.1, 0.5 or 1 µg ml(-1), for up to 24 h. Cytotoxicity, detected by lactate dehydrogenase release, was observed at the highest studied concentration. A decrease in phagocytic activity and changes in actin cytoskeletal structures were observed after the cell exposure to the toxin at 0.5 and 1 µg ml(-1). Moreover, at all tested concentrations, cylindrospermopsin increased the production of reactive oxygen and nitrogen species. It also evidently influenced the expression of genes of proinflammatory cytokines interleukin-1β and tumour necrosis factor-α and, to a minor extent, anti-inflammatory transforming growth factor-β, but had no effects on interleukin-10. The results indicated that the cyanotoxin cylindrospermopsin is able to modify basic features of carp phagocytic cells, which might result in adverse consequences for fish health., (Copyright © 2015 John Wiley & Sons, Ltd.)

The aim of this study was to use rapid mass spectrometry (MS)-based proteomics analyses for diagnosis of Babesia canis canis infections in dogs. The study was conducted on two groups of dogs--healthy dogs and dogs infected with B. canis canis which demonstrated symptoms of babesiosis. The matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS technique revealed the presence of a protein fraction of 51-52 kDa in the blood serum of all the animals infected with the protozoa, which was not found in the serum of healthy dogs. The proteins are suspected to be disease markers, whereas the MALDI-TOF technique itself has high specificity and sensitivity and can be applied in analytical laboratories in the diagnosis of canine babesiosis.

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.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Blooms of cyanobacteria producing very toxic secondary metabolites (especially microcystins) are potent environmental stressors, hazardous not only to aquatic animals but also to public health. The purpose of this study was to investigate the effects of an extract containing microcystins on immune cells isolated from the common carp (Cyprinus carpio L.). In the present study it has been found that the extract induced apoptosis and inhibited in vitro lymphocyte proliferation. In addition, the results indicated the possible role of oxidative stress in this cytotoxicity and apoptosis. The in vivo investigations showed that the extract containing microcystins had greater suppressive effects on the essential functions of immune cells (intracellular reactive oxygen species production and lymphocyte proliferation) than the pure toxin alone. Moreover, immersion of fish in the toxic extract caused changes in the mRNA levels of various pro- and anti-inflammatory cytokines in carp leukocytes, while after exposure to the pure toxin, only IL1-β expression was markedly up-regulated. The observed modulatory effects on immune cells could have important implications for the health of planktivorous fish, which feed more frequently on toxic cyanobacteria., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The aim of the study was to detect the presence of genetic material of equine piroplasmas and to determine the species isolates from apparently healthy equids, including horses, donkeys and mules, in southern Spain. Blood samples were collected from 135 animals to assess the presence of DNA from equine piroplasmas using PCR. Babesia (B.) caballi DNA was detected in blood samples of three horses and one donkey, while Theileria (T.) equi DNA was confirmed in blood of 19 horses, three mules and one donkey. All B. caballi isolates showed a 100% homology of the nucleotide sequence of the 18S RNA gene fragment with the Spanish isolate AY534883. T. equi isolates had a 99.8% homology with the Spanish isolate T. equi AY 534882 and a 98.2% homology with the sequence of the Spanish isolate T. equi DQ287951. The differences in the nucleotide sequence of 18S RNA gene between T. equi isolates in the present study and the above mentioned Spanish isolates suggest the circulation of different genotypes in this country. The presence of genetic material of these parasites in 20% of the examined animals indicates a widespread exposure to equine piroplasmosis in southern Spain, therefore, a monitoring program in this country is needed.

Cyanotoxins are toxic, secondary metabolites produced by different species of cyanobacteria that are present all over the world in aquatic environments. No data are available about the molecular mechanisms underlying the stress associated with exposure of fish immune cells to low concentrations of cyanotoxins. The purpose of this study was to determine whether the expression of cytokines that underlie immune regulation are changed after incubation of fish leukocytes with pure cyanotoxins: microcystin- LR (MC-LR), anatoxin-a (Antx-a), or an extract containing Antx-a. The study investigated the relative gene expression of four important cytokines, IL-1β, TNF-α, IL-10, and TGF-β, in carp head kidney and blood leukocytes exposed to toxins at concentrations of 0.01 or 0.1 μg/ml for 4 h. The data showed that pure toxins could induce dysregulation of pro-/anti-inflammatory cytokine expression. Expression of cytokine IL-1 β was highly upregulated following Antx-a exposure, whereas MC-LR induced merely moderate reactions. The expression of TNF-α mRNA was significantly suppressed in blood and head kidney cells incubated with toxins at the higher concentration. These results showed that pure toxins dysregulated the expression of pro-inflammatory cytokines IL-1β and TNF-α more promptly than the anti-inflammatory cytokines TGF-β and IL-10. In contrast, the studies demonstrated a clearly downward trend of pro-inflammatory cytokines and an upward trend of anti-inflammatory cytokines in leukocytes exposed to an extract containing defined concentrations of Antx-a. This study suggests that cyanotoxins present in aquatic environments may exert immunotoxic effects by altering the transcription of important mediators of the fish immune system., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The purpose of the studies was to determine electrolyte disturbances and blood pH changes in dogs with babesiosis and possibly show a connection between the Babesia (B.) canis strain causing the infection and the intensity of these irregularities. 40 animals (group 1) with early babesiosis and 40 healthy dogs (group 2) were studied and their blood pH and blood levels of potassium, chlorides; calcium and sodium were determined. At the same time, molecular typing of parasites was carried out to detect which B.canis strain (18S RNA-A or 185 RNA-B) had caused the disease in dogs of group 1. In group 1, four dogs were acidaemic, twelve had normal blood pH, and 24 were alkalaemic. Potassium concentration was below normal in 16 out of 40 dogs (40%) and normal in 24 dogs. Hypochloremia was present in 36 out of 40 dogs; chloride was normal in the remaining four animals. Serum sodium concentration was low in 16 of 40 dogs, normal in 20 of 40 dogs and high in four dogs. Calcium concentration was normal in all 40 dogs. In dogs of group 2 no abnormalities of haematological or blood biochemical parameters were observed. 29 out of the 40 dogs of group 1 were infected with the 18S RNA-A strain and eleven with the 18S RNA-B strain of Babesia canis canis. We did not observe any correlation between the type of strain causing the infection and the electrolyte disturbances in the serum of sick dogs. Hypocalaemia was observed in ten specimen infected with 18S RNA-A and six infected with 18S RNA-B. Additionally, in dogs infected with 18S RNA-A, hypochloraemia (28), hyponatraemia (10), hypernatraemia (2) were observed, as well as blood pH drop (4) or increase (14). The 18S RNA-B-infected dogs suffered from hypochloraemia (8), hyponatraemia (6), hypernatraemia (2) and increase in blood pH (10).The studies conducted did not answer the question of whether the type of electrolyte disturbances in dogs with babesiosis can be connected with the strain of the parasite that induced the disease, as happens in the case of other clinical parameters (Adaszek et al., 2009). Further studies in this respect, conducted on a larger group of animals, are necessary.

The aim of this paper was to determine the factors affecting the development of canine babesiosis in Poland. The studies included 800 animals from 16 polish voivodeships-districts (50 from each voivodeship) with suspected piroplasmosis. Hematological and molecular tests confirmed infections in 158 animals. Analysis of the genetic material using the Real-Time Sybr Green HRM technique showed that 96 dogs were infected with Babesia canis 18S RNA-A and 62 with B. canis 18S RNA-B. It was observed that the disease occurs more often in eastern Poland (OR: 8.91; CI: 5.7-13.8), in males (OR: 1.04; CI: 0.7-1.5) and especially in rural areas (OR: 1.7; CI: 1.2-2.4). More prone to infection were pure-bred dogs (OR: 2.24; CI: 1.6-3.2), young animals under 1 year old (OR: 0.73; CI: 0.4-1.5), animals that had suffered from babesiosis in the past (OR: 17.9; CI: 10.9-29.2) and those that had not received preventive measures against ectoparasites (OR: 0.32; CI: 0.2-0.5). Knowledge of the factors facilitating the development of canine babesiosis helps to understand the causal connection between the dogs' exposure to the pathogen and the development of the disease and to limit the occurrence of the infection in these animals, to diagnose it early and to start proper treatment before serious complications develop., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

The study was aimed at determining the cause of the diseases in three horses exhibiting symptoms of fever, ataxia, mucus membrane paleness, haematuria and thrombocytopenia. The PCR technique revealed the presence in the blood of 18S RNA Babesia/Theileria spp. genetic material. DNA amplification using primers RLB F2 and RLB R2 produced 430 bp size products. The sequences of these PCR products demonstrated a 95.6-97.5% similarity with the sequence of the fragment of 18S RNA Babesia equi, gene number DQ287951 in the GenBank. The treatment utilizing the subcutaneous application of the imidocarb resulted in gradual recovery of the diseased animals.

The aim of this study was to investigate short-term 4 and 14 weeks after fundectomy) and long-term (at the age of 8 months) postoperative effects of 3-hydroxy-3-methylbutyrate and/or alpha-ketoglutarate on selected serum biochemical markers in fundectomized pigs. Experimental fundectomy was performed in 30 castrated male pigs of the Puławska breed who received placebo or 3-hydroxy-3-methylbutyrate and/or alpha-ketoglutarate up to the age of 8 months. Plasma amino acid concentrations and selected blood parameters were analyzed. Main vital organs were weighed. Our study showed that the supplementations with alpha-ketoglutarate and/or 3-hydroxy-3-methylbutyrate to fundectomized pigs significantly prevented the reduction of stomach, liver and spleen weights. However, results of this study, either positive or negative, cannot categorically establish a beneficial effect of AKG and HMB nutritional support after fundectomy in pigs.

Babesia and Theileria are the tick-borne parasites belonging to the order Piroplasmida. Both of them cause a severe disease with symptoms of hemolytic anemia. The main etiological factors of canine piroplasmosis are protozoa Babesia. There are two species of this pathogen infective for dogs: Babesia canis and Babesia gibsoni. Based on the results of molecular biology techniques it is possible to distinguish them as well as many subspecies or strains. It is known since quite some time that there are substantial differences in vector specificity, cross-immunity and pathogenicity between isolates of this species. Also the results of molecular analysis of these protozoa indicate that their genetic structure is diversified. On the basis of analysis of Babesia DNA sequences, the new variants of protozoa in dogs were detected. It is possible that, these new variants of Babesia are characterized by high virulence for dogs and resistance to drugs used in babesiosis therapy.

The study was aimed at determining the cause of a disease in a horse exhibiting symptoms of fever, joint effusion, weakness, and extravasations on the mucous membranes. Blood was drawn from the animal for haematological and biochemical molecular tests. The PCR technique revealed the presence of 16S RNA Ehrlichia spp. genetic material in the blood samples. DNA amplification by means of primers EHR 521 and EHR 747 gave a product with a volume of 247 bp.The sequence of the PCR product obtained showed a 97.6% similarity with a sequence of a fragment of 16S RNA Ehrlichia phagocytophila, gene number EU 090186 in the GenBank. A microscopic examination of blood smears demonstrated the presence in the neutrophiles of intracytoplasmatic structures characteristic for Ehrlichia.The treatment involved the intravenous application of oxytetracycline at a dose of 8 mg/kg of body mass for 7 days, and resulted in a gradual recovery. The positive results of the molecular test and the positive reaction to the therapy are an indication of the first case of ehrlichiosis in a horse in Poland as confirmed in laboratory test.

In this study, we used RT-PCR to detect and characterize canine distemper virus isolated from 9 naturally infected foxes, 3 minks and 3 dogs in Poland by amplifying and sequencing a portion of the NP gene. A 293-bp fragment of the CDV NP gene was amplified by RT-PCR. Sequencing of the PCR products from the isolates led to the identification of 3 sequence variants. The mostly representative polymorphic variant No. 1 showed high homology with Chinese isolate of CDV with a accession number EF 375619. The sequences of all isolates from this polymorphic variants compared with the sequences of other polymorphic variants obtained in the study and with European and American isolates sequences from GenBank showed the conservative nucleotides changes in positions 57, 132, 143, 159 and 237. These mutations can indicate that in this part of Europe there are new variants of CDV.

The aim of the study was to trace the clinical course of babesiosis in 76 dogs infected with Babesia canis protozoa and to assess the usefulness of PCR method in the routine diagnosis of the disease. The investigations were conducted in three successive seasons of the biological activity of ticks on dogs displaying possible clinical signs of babesiosis, the latter assigned individual numbers from 001 to 076. All the animals underwent routine clinical examinations and blood was collected for haematological, biochemical, parasitological and molecular tests for babesiosis. The most frequent clinical signs observed in the course of the disease were changes in urine colour and xanthosis or paleness of mucous membranes, whereas in the haematological and biochemical examinations, the most frequent laboratory findings were thrombocytopenia, leucopoenia, anaemia and an increase in levels of urea and bilirubin. In all blood smears stained with the May-Grunwald and Giemsa methods, from the 76 dogs, the presence of Babesia canis protozoa was observed in erythrocytes, and their DNA was detected in 69 blood samples by means of PCR technique. The course of the disease and the results of molecular examinations suggested the haemolytic form of babesiosis. The previous genetic analysis of isolates of Babesia canis canis from the eastern areas of Poland helped to distinguish two specific groups, A and B, within the species (Adaszek and Winiarczyk 2008a). The present study revealed a certain interrelation between the intensification of thrombocytopenia and the fact that protozoa belong to either group A or B. The mean number of thrombocytes in dogs infected with protozoa from group A was 61.11 thousand/mm3, whereas the mean number of thrombocytes in the blood of dogs infected with protozoa from group B was 27.47 thousand/mm3. A strong correlation was also observed between the low level of thrombocytes and the increase in the internal body temperature (p = 0.02), accelerated pulse rate (p = 0.01) and discoloration of urine (p = 0.04). As a result of the treatment of dogs with imidocarb, recovery was observed in 73 out of the 76 dogs brought to the clinic.