Urinary proteome of dogs with kidney injury during babesiosis.

BACKGROUND: Acute kidney injury is the most frequent complication of babesiosis in dogs and may provide a natural model for identifying early and specific markers of kidney injury in this species. There are limited data on urine proteomics in dogs, and none of the effect of babesiosis on the urine proteome. This study aimed to identify urinary proteins of dogs with kidney injury during the natural course of babesiosis caused by Babesia canis, and to compare them with proteins in a control group to reveal any potential biomarkers predicting renal injury before the presence of azotemia. Urine samples were collected from 10 dogs of various breeds and sex with naturally occurring babesiosis, and 10 healthy dogs. Pooled urine samples from both groups were separated by 2D (two-dimensional) electrophoresis, followed by protein identification using MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometry. RESULTS: In total, 176 proteins were identified in the urine samples from healthy dogs, and 403 proteins were identified in the urine samples from dogs with babesiosis. Of the 176 proteins, 146 were assigned exclusively to healthy dogs, and 373 of the 403 proteins were assigned exclusively to dogs with babesiosis; 30 proteins were common for both groups. Characteristic analysis of 373 proteins found in dogs with babesiosis led to the isolation of 8 proteins associated with 10 metabolic pathways involved in immune and inflammatory responses. CONCLUSIONS: It was hypothesized that epithelial-mesenchymal transition might play an important role in the mechanisms underlying pathological changes in renal tissue during babesiosis, as indicated by a causal relationship network built by combining 5 of the 10 selected metabolic pathways, and 4 of the 8 proteins associated with these pathways; this network included cadherins, gonadotropin releasing hormone receptors, inflammatory responses mediated by chemokine and cytokine signalling pathways, integrins, interleukins, and TGF-ß (transforming growth factor ß) pathways. Those pathways were linked by interleukin-13, bone morphogenetic protein 7, α2(1) collagen, and tyrosine protein kinase Fer, which are potential biomarkers of damage during babesiosis in dogs, that might indicate early renal injury.

Seroprevalence and risk factors associated with Brucella seropositivity in dairy and mixed cattle herds from Ecuador.

An extensive cross-sectional study to determine the seroprevalence of and associated risk factors for Brucella infection was performed in dairy and mixed (dairy-beef) cattle herds in Ecuador. A total of 2666 serum samples from 386 farms were analyzed using Rose Bengal test and a blocking ELISA test. In addition, a questionnaire with 57 variables related to management, feeding, facilities, biosecurity, and animal health was filled in every cattle farm. A Generalized Estimating Equations model was used to determine the factors associated with Brucella seropositivity. The true prevalence of Brucella seropositivity in dairy and mixed cattle from Ecuador reached 17.0% (CI95% 15.6-18.4%). The herd prevalence was 45.1% (174/386) (CI95% 40.1-50.1%), and the within-herd prevalence ranged from 10 to 100% (mean 38.9%; Q1 14.3%, Q2 26.8%, Q3 52.5%). Seven factors were included in the GEE model for Brucella seropositivity: the nominal variables sex (OR 2.03; CI95% 1.32-3.13), herd type (dairy) (OR 1.79; CI95% 1.11-2.87), closed facilities in the farm (OR 1.80; CI95% 1.19-2.74), and ad libitum feeding (OR: 0.32; CI95%: 0.19-0.54), and the quantitative variables age (OR 1.005; CI95% 1.001-1.009), average slope in the farm (%) (OR 1.013; CI95% 1.002-1.024), and annual abortion rate (OR 1.016; CI95% 1.002-1.031). This study remarks the high spread of Brucella infection in cattle farms from Ecuador. In addition, it reports the risk factors associated to this infection in the predominant extensive system existent in this country.

Molecular identification of Babesia spp isolated from Polish cattle with asymptomatic protozoa infections.

The aim of the paper was to study the epizootic situation of babesiosis in the cattle population in eastern Poland and possibly to determine what species of protozoa infects Polish cattle. Blood samples for molecular analysis (real time PCR) were collected from 192 dairy cows 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 93.1%, homology with the sequence of B. occultans EU 376017. This is the first report about the detection of B. occultans DNA in asymptomatic cattle in eastern Poland.

Study on establishing normal ranges of chosen biochemical parameters of haemolymph of Cornu aspersum maxima and Cepaea nemoralis gastropods.

The aim of the study was to establish normal ranges for chosen biochemical parameters of haemolymph of snails (Gasropoda: Mollusca), in the light of the use of these animals as experi- mental models in various types of studies. The study was conducted on 100 specimens of Cornu aspersum maxima (CAM) and 100 specimens of Cepaea nemoralis (CN). The haemolymph col- lected from the animals was analysed using colorimetry to assay aspartate transaminase (AST) activity, alanine transaminase (ALT) activity, amylase activity and the concentrations of urea and triglycerides. In the further part of the study, the influence of administering doxycycline with feed on the change of AST and ALT activity in snail haemolymph has been studied. The normal values established for CAM are as follow: AST activity: 26-38 u/l, ALT activity: 0-11 u/l, amylase activity 9-16 u/l, concentration of urea: 3-6 mg/dl, concentration of triglycerides: 16-20 mg/dl. For CN, the following data have been obtained: AST activity: 30-80 u/l, ALT activity: 0-15 u/l, amylase activity 12-15 u/l, concentration of urea: 5-8 mg/dl, concentration of triglycerides: 18-24 mg/dl. It has been shown that doxycycline presents a high workload on the hepatopancreas of snails, which is reflected by a statistically significant (p ⟨ 0.05) increase of AST and ALT activity in the haemolymph of the specimens which obtained doxycycline in feed, as compared to the groups with antibiotic-free feed. The haemolymph activity of both studied parameters increased together with study time and tetracycline administration time.

Cardiac disorders in dogs infected with Babesia canis.

The aim of this study was to assess cardiac disorders in dogs infected with B. canis. The study included 50 dogs with babesiosis and 20 healthy control animals. All the animals had haematological tests, ECG, echocardiography and serum troponin I and CK-MB levels checked. The haematology in the group of dogs with babesiosis confirmed thrombocytopaenia in 100% of dogs, decreased haematocrit in 52% and anaemia in 46%. The most common abnormalities in ECG and echocardiography in dogs infected with protozoa included: change in appearance and/or amplitude of the T-wave (34%), increased fractional shortening (24%), an increased sinus rhythm (14%) and heart axis deviation (10%). In 19 of the 50 dogs with babesiosis, the level of serum troponin I was elevated. In 2 dogs that died from babesiosis, the troponin level I was very high. The ECG confirmed sinus tachycardia and interpolated ventricular beat in these animals. In all dogs with babesiosis that were used in the study, the serum CK-MB was high or very high and was within limits of 23.17 U/L - 369.62 U/L. The highest kinase concentration (367.33 U/L and 369.62 U/L) was observed in dogs that died due to the disease. The presented results prove that cardiac changes are common in canine babesiosis, but that most changes are nonspecific and appear to have little clinical significance. Cardiovascular assessment should be based on the assessment of the level of troponin I and CK-MB in the serum of sick animals. High concentrations of these factors might be indicators of poor prognosis.

Factors responsible for subclinical mastitis in cows caused by Staphylococcus chromogenes and its susceptibility to antibiotics based on bap, fnbA, eno, mecA, tetK, and ermA genes.

The aim of this study was to recognize selected factors of virulence determining the adhesion of Staphylococcus chromogenes to cows' udder tissues in subclinical mastitis and to evaluate the susceptibility of this pathogen to antibiotics. The subjects of the study were 38 isolates of Staph. chromogenes from 335 samples of milk from cows with subclinical coagulase-negative staphylococci mastitis. Somatic cell count ranged between 216,000 and 568,000/mL of milk (average 356,000/mL of milk). We confirmed the ability to produce slime in 24 isolates (63.2%), and the ability to produce protease in 29 isolates (76.3%). In each slime-producing isolate, the bap gene was not found, and the fnbA and eno genes were not detected. In vitro tests showed that ceftiofur had the highest effectiveness against Staph. chromogenes (89.5% of susceptible isolates). Minimum inhibitory concentrations ranged from 0.06 to 2µg/mL for susceptible isolates. The minimum concentrations required to inhibit growth of 90 and 50% of the isolates for ceftiofur were at or below the cutoffs recommended by the Clinical and Laboratory Standards Institute (2 and 0.06µg/mL, respectively). A significant percentage of the isolates were susceptible to other ß-lactam antibiotics: amoxicillin with clavulanic acid (84.2%) and ampicillin (81.6%). The lowest effectiveness among ß-lactams was for penicillin (73.7% of susceptible isolates), and the minimum inhibitory concentration for penicillin ranged from <0.06 to 8µg/mL. None of the examined isolates had the mecA gene, but ß-lactamase was detected in 4 isolates (10.5%). Erythromycin and oxytetracycline exhibited the lowest activity against Staph. chromogenes (71.1 and 63.2% of susceptible isolates, respectively). The genes tetK (6 isolates) and ermA (1 isolate) were also detected.

Occurence of Bordetella bronchiseptica in domestic cats with upper respiratory tract infections.

Bordetella bronchiseptica is a widespread Gram-negative pathogen occurring in different mammal species. It is known to play a role in the etiology of infectious atrophic rhinitis of swine, canine kennel cough, respiratory syndromes of cats, rabbits and guinea pigs, and sporadic human cases have also been reported. The aim of this article is to present the occurrence of infections caused by these bacteria in domestic cats with respiratory symptoms, as well as to conduct a molecular analysis of the flaA gene B. bronchiseptica for the purpose of ascertaining whether cats become infected with one or more bacteria strains. B. bronchiseptica was isolated from the respiratory system of 16 out of 35 domestic cats with symptoms of respiratory tract infections. Polymorphism analysis of polymerase chain reaction products of B. bronchiseptica flaA was performed to reveal the possible differences in nucleotide sequences of the flagellin gene. The phylogenetic analysis of nucleotide sequences obtained during PCR indicated that the isolates of bacteria from our own studies are characterised by 100% homology of the analysed fragment of the flaA gene, which suggests maintenance of a single genotype of these microorganisms in the cat population. Moreover, the bacteria revealed full homology with reference strain B. bronchiseptica ATCC 4617, and 99.4% homology with strain B. parapertussis ATCC 15311. This indicates that the PCR optimised for the Bordetella spp. flaA gene, combined with sequencing of amplicons obtained in PCR, is an effective diagnostic method allowing differentiation of Bordetella spp. type microorganisms.

Application of Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry for Identification of Coagulase-negative Staphylococci Isolated from Milk of Cows with Subclinical Mastitis.

The aim of this study was to use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of coagulase-negative staphylococci (CNS) isolated from the milk of cows with subclinical mastitis. The study material consisted of 33 isolates of CNS, identified by the results of API Staph tests, obtained from the milk of cows with subclinical mastitis. Based on the spectra analyses, MALDI-TOF MS tests of 33 bacterial samples allowed identification of the microorganisms in 27 cases (81.8%). The most frequent cause of subclinical mastitis was found to be Staphylococcus sciuri (39%), while S. vitulinus was detected in 15% of the milk samples. The results obtained indicate that MALDI-TOF MS can be used for the identification of CNS isolated from bovine mastitis as a method supplementary to biochemical tests.

Identification of new 18S rRNA strains of Babesia canis isolated from dogs with subclinical babesiosis.

In this study, we used PCR to detect and characterize B. canis from naturally infected dogs in Poland with subclinical babesiosis by amplifying and sequencing a portion of the 18S ribosomal RNA (rRNA) gene. Venous blood samples were collected from ten dogs with subclinical babesiosis. A 559-bp fragment of the B. canis 18S rRNA gene was amplified by PCR. Sequencing of the PCR products led to the identification of a new variant of Babesia canis, differing from the previously detected protozoa genotypes (18S rRNA-A and 18S rRNA-B) with nucleotide substitutions in positions 150 and 151 of the tested gene fragment. The results indicate the emergence within the Polish territory of a new, previously unencountered Babesia canis genotype responsible for the development of subclinical babesiosis.

Detection of the bovine viral diarrhoea virus (BVDV) in young beef cattle in eastern and south-eastern regions of Poland.

In view of the scarcity of information concerning viral diarrhoea virus (BVDV) infections in beef cattle in Poland, the aim of this study was to evaluate the presence of the BVDV in young beef cattle from selected herds in eastern and south-eastern regions of Poland. The material consisted of 78 sera obtained from beef cattle from 15 farms, aged 6-12 months. The anti-BVDV antibody level in the sera was estimated with an ELISA kit, and detection of the BVDV was carried out by standard PCR and one step Real-Time RT-PCR. The ELISA results showed a high degree (80%) of positivity in 5 of the 78 samples. In 7 samples the degree of positivity was in the very low range: < 40%. Of the 78 cDNA samples, the presence of genetic material with a length of 288 bp was found by standard PCR in 3 sera. The genetic material of BVDV was also found in the sera of the same three calves by Real-Time HRM PCR. BVDV infection in young beef cattle in south-eastern Poland is not a significant problem. This was confirmed by the positive ELISA results for 6.4% of the animals and the positive PCR results for 3.9%. The percentage of positive beef herds was about 8.6%. However, due to the severe nature of the disease and rapid transmission of the virus, regular monitoring of BVDV should be carried out.

Diagnosis of the Encephalitozoon cuniculi infections in pet rabbits with neurological symptoms.

The purpose of the study was the in vivo diagnosing of E. cuniculi invasions in pet rabbits with neurological symptoms using the Real-Time PCR, and determination of the rate of invasion, in this group of animals. The study involved 103 pet rabbits with neurological symptoms. Parasitic invasions were diagnosed using Real-Time PCR. The DNA of the parasites for molecular tests was isolated from the urine of the diseased animals. Out of the 103 tested DNA samples, the presence of the E. cuniculi genetic material was detected in 27 samples (26.21%). The melting temperature (Tm) of all products was 77.5 degrees C. The presence of parasitic DNA in the urine of 26.21% of examined animals indicates that E. cuniculi infections occur widely in pet rabbits in Poland and are a significant cause of neurological disorders in those animals.

Detection of Borrelia burgdorferi sensu lato DNA in the blood of wild bison from Bialowieza primeval forest in eastern Poland.

The aim of the present study was to investigate the occurrence of Borrelia burgdorferi sensu lato DNA in a group of 120 wild bison (Bison bonasus) from the Bialowieza Primeval Forest in eastern Poland. The PCR technique revealed the presence of 16S RNA of Borrelia burgdorferi sensu lato in the blood of 16 out of 120 examined animals. DNA amplification by means of primers SC1 and SC2 gave a product with a size of 300-bp. The sequences of the PCR products obtained showed 100% homology with each other and 100% homology with B. burgdorferi s.1. 16S RNA gene DQ111061. Results of this study suggest that wild bison are important in maintaining agents of Lyme borreliosis, and that studies of reservoir competence of this species are indicated.

Applicability of 2D gel electrophoresis and liquid chromatography in proteomic analysis of urine using mass spectrometry MALDI-TOF.

Proteomics including the studies of the structure, function and dependences between proteins is more and more extensively applied in human medicine and veterinary medicine. The analysis of protein profiles of tissues and body fluid from healthy and ill individuals allows to identify diagnostic, prognostic and predictive markers in various pathological states in people and animals. This paper presents preparation of urine samples for analysis in the mass spectrometer MALDI-TOF (Ultraflextreme, Bruker, Bremen, Germany) by means of two methods: liquid chromatography based on the system Nano-LC (PROTEINER FC II, Bruker Daltonics, Bremen Germany). and two-direction electrophoresis 2DE (GE Healthcare, United Kingdom). Both methods enable separation of the mixture under consideration into individual fractions of high purity indispensable for obtaining readable mass spectra. The purpose of this paper is to determine applicability of these methods in analysis of protein composition of urine samples.

The loop-mediated isothermal amplification assay for rapid diagnosis of Babesia canis canis infections in dogs.

The aim of this study was to use a rapid and easy DNA-based test, the loop-mediated isothermal amplification (LAMP), for diagnosis of Babesia canis canis infections in dogs. 10 DNA samples of 18S RNA-A and 10 DNA samples of 18S RNA-B of B. canis canis were used in the study. LAMP method could successfully detect DNA in all examined samples down to 0.1 pg dilution. Obtained results suggest that this method has high specificity and sensitivity and can be applied in analytical laboratories in diagnosis of canine babesiosis.

Comparison of serum protein profiles of Borrelia burgdorferi-positive Bernese Mountain Dogs and dogs of other breeds using MALDI-TOF technique.

The aim of the study was to compare the serum protein profile of Bernese Mountain Dogs (BMDs) reacting positive for Bb in snap testing with the serum protein profile of dogs of other breeds (healthy and with clinical borreliosis) using the MALDI time-of-flight (MALDI-TOF) technique. The observations included five groups of dogs. BMDs reacting positively to Bb in snap serological testing and showing symptoms of borreliosis (group 1), BMDs for which no borreliosis symptoms were determined but with seropositivity for Bb determined with snap serological tests (group 2), clinically healthy BMDs with no antibodies for Bb found in the serum (group 3), five dogs of different breeds, reacting positively in serological testing, in which borreliosis symptoms were observed (group 4), clinically healthy dogs of different breeds with negative reaction in tests towards Bb (group 5). A proteomic analysis demonstrated the presence of five identical protein fractions among all five groups. An additional two protein fractions of approximately 7.630 and 15.260 kDa were found in all the serum samples obtained from the dogs positive for borrelia in a snap test, both in those exhibiting symptoms of borreliosis, and seropositive BMDs not presenting symptoms of the disease. These two additional protein fractions may be used to differentiate between seropositive and seronegative B. burgdorferi dogs and may be considered a seropositivity marker, however, it cannot be used to differentiate between animals with the clinical form of the disease and those that are only seropositive.

The identification of Anaplasma spp. isolated from fallow deer (Dama dama) on a free-range farm in eastern Poland.

The aim of the present study was to investigate the occurrence of Anaplasma spp. in group of 50 fallow deer (Dama dama) from free-range farm in eastern Poland and determine what species of Anaplasma could infect these animals based on PCR gene sequencing. The PCR technique revealed the presence of 16S RNA Anaplasma spp. genetic material in the blood of 7 out of 50 examined animals. The sequences of the PCR products obtained showed a 100% homology with each other and 100% homology with GU 183908 sequence of A. phagocytophilum, isolated in our earlier study from a horse with clinical form of anaplasmosis. Here, we report the first molecular evidence of Anaplasma spp. among naturally infected fallow deer in eastern Poland.

Molecular analysis of a fragment of gene E1B 19K of canine adenovirus 2 (CAV-2) isolated from dogs with symptoms of cough.

The aim of this study was to perform molecular analysis of canine adenovirus 2 (CAV-2) E1B 19K gene fragment isolated from 20 dogs of various breeds (12 males and 8 females aged 1-9 years), with clinical symptoms of upper respiratory tract infections, from the Lubelszczyzna region. Nasal swabs were taken from dogs. DNA of CAV-2 was detected using the PCR method in 16 swabs. All PCR products were sequenced, and the obtained sequences were compared with each other and with the sequence of the E1B 19K gene of the CAV-2 strain from an online database of NCBI GenBank: AC 000003. Based on analysis of the obtained sequences, three polymorphic variants of CAV-2 (No. 1-3) with homology of 78 - 100% were distinguished. The nucleotide and amino acid sequences of the most frequently represented polymorphic variant, No. 1, differed from the sequences of polymorphic variant No. 2 with one substitution. The nucleotide and amino acid sequence of the E1B 19K gene of CAV-2 AC 000003 differed from the analogous sequences of representatives of variant No. 1 with 44 nucleotide and 19 amino acid substitutions. The small number of nucleotide differences in the E1B 19K CAV-2 gene among the examined own isolates, compared with AC 000003, suggest that the infections in dogs were caused by a relatively genetically stable virus which occurs in eastern

Lyme disease in Bernese Mountain Dogs. Is it a real problem?

Borreliosis is the most frequently diagnosed tick-borne disease caused by spirochete bacteria belonging to the genus Borreliae - Borrelia burgdorferi sensu stricto (s.s.), Borrelia afzelii and Borrelia garinii. Clinical manifestations in dogs include fever, lameness, polyarthritis and glomerulonephritis. Diagnosis is mainly serological and is based on an immunoenzymatic test followed by a Western blot confirmatory test. Early treatment with antibiotics such as doxycycline or amoxicillin, for four weeks, usually reduces the risk of chronic disease. Tick control, including tick repellents, is highly reliable in preventing transmission. Vaccines are available to reduce transmission and the clinical manifestations of infection in dogs. Bernese Mountain Dogs are a breed that often test positive for antibodies against B. burgdorferi without showing any clinical symptoms of the disease. Quantitative determination of the immunoglobulin level for spirochetes has indicated that Bernese Mountain Dogs may have an increased susceptibility to Borrelia spp. infections of a hereditary nature.

Anti-babesial potential and chemical composition of essential oil from yarrow Achillea millefolium.

Essential oils from plants used in traditional medicine are known as a rich source of chemically diverse compounds with specific biological activities. Achillea millefolium essential oil (AEO) was screened for in vitro activity against Babesia canis. The AEO was obtained by hydrodistillation and analysed by gas chromatography coupled to mass spectrometry (GC-MS). GC-MS revealed the presence of 47 compounds in the essential oil. Those present in the highest concentrations were chamazulene (34.45%), ß-caryophyllene (8.93%), (E)-germacrene D (7.55%), patchoulene (7.27%), ß-guaiene (4.62%), α-humulene (4.59%), santolina epoxide (4.41%), ethyl iso-allocholate (2.97%), aromadendrene (2.62%), and neoclovenoxid-alkohol (2.46%). AEO was found to be active in vitro against B. canis, with 50% inhibitory concentration (IC50) values of 0.06 mg/mL, as compared to imidocarb, with IC50 = 0.007 mg/mL. The study confirms that essential oil from A. millefolium has anti-babesial properties in vitro.