Systematic analysis of different degrees of haemolysis on miRNA levels in serum and serum-derived extracellular vesicles from dogs.

BACKGROUND: Circulating microRNAs (miRNAs) are described as promising non-invasive biomarkers for diagnostics and therapeutics. Human studies have shown that haemolysis occurring during blood collection or due to improper sample processing/storage significantly alters the miRNA content in plasma and serum. Nevertheless, no similar research has been performed in dogs so far. We therefore investigated the effects of different degrees of haemolysis on the levels of selected miRNAs in serum and serum-derived extracellular vesicles (EVs) from dogs, by inducing a controlled in vitro haemolysis experiment. RESULTS: The abundance of miR-16, miR-92a, miR-191, miR-451 and miR-486 was significantly sensitive to haemolysis in serum and serum-derived EVs, while other selected miRNAs were not influenced by haemolysis. Furthermore, we found that the abundance of some canine miRNAs differs from data reported in the human system. CONCLUSIONS: Our results describe for the first time the impact of haemolysis on circulating miRNAs not only in whole serum, but also in serum-derived EVs from dogs. Hence, we provide novel data for further analyses in the discovery of canine circulating biomarkers. Our findings suggest that haemolysis should be carefully assessed to assure accuracy when investigating circulating miRNA in serum or plasma-based tests.

Effects of dietary cellobiose on the intestinal microbiota and excretion of nitrogen metabolites in healthy adult dogs.

In order to evaluate the potential prebiotic effects of cellobiose, 10 healthy adult research beagle dogs received a complete diet containing 0, 0.5 and 1 g cellobiose/kg bodyweight (BW)/day. At the end of each feeding period, faeces, urine and blood of the dogs were collected. The results demonstrated a significant increase of faecal lactate concentrations, indicating a bacterial fermentation of cellobiose in the canine intestine. Along with this, a dose-dependent linear increase of the relative abundance of Lactobacillaceae in the faeces of the dogs was observed (p = 0.014). In addition, a dose-dependent increase (p < 0.05) of Alloprevotella, Bacteroides and Prevotella, and a linear decrease for unidentified Lachnospiraceae (p = 0.011) was observed when cellobiose was added to the diet, although the relative abundance of these genera was low (<1%) among all groups. The faecal pH was not affected by dietary cellobiose. Cellobiose seemed to modulate the excretion of nitrogen metabolites, as lower concentrations of phenol (p = 0.034) and 4-ethylphenol (p = 0.002) in the plasma of the dogs were measured during the supplementation periods. Urinary phenols and indoles, however, were not affected by the dietary supplementation of cellobiose. In conclusion, cellobiose seems to be fermented by the intestinal microbiota of dogs. Although no effect on the faecal pH was detected, the observed increase of microbial lactate production might lower the pH in the large intestine and consecutively modulate the intestinal absorption of nitrogen metabolites. Also, the observed changes of some bacterial genera might have been mediated by increased intestinal lactate concentrations or a higher relative abundance of lactobacilli. Whether these results could be considered as a prebiotic effect and used as a dietetic strategy in diseased animals to improve gut function or hepatic and renal nitrogen metabolism should be evaluated in future studies.

Canine Leishmaniasis Control in the Context of One Health.

Dogs are the main reservoir of Leishmania infantum and in some countries have been regularly culled as part of government policy to control visceral leishmaniasis. At the 13th Symposium of the Companion Vector-Borne Diseases World Forum in Windsor, UK, March 19-22, 2018, we consolidated a consensus statement regarding the usefulness of dog culling as a means of controlling visceral leishmaniasis. The statement highlighted the futility of culling infected dogs, whether healthy or sick, as a measure to control the domestic reservoir of L. infantum and reduce the risk for visceral leishmaniasis.

Blepharitis in dogs: a clinical evaluation in 102 dogs.

BACKGROUND: Blepharitis is a common finding in many dogs with various skin diseases. OBJECTIVES: To establish a definition for canine blepharitis versus periocular dermatitis (POD), to evaluate the clinical findings and underlying skin diseases of blepharitis, and to document the effects of blepharitis on tear production in dogs. ANIMALS: One hundred and two privately owned dogs with clinical signs of blepharitis and a definitive diagnosis of skin disease. METHODS AND MATERIALS: Prospective evaluation of clinical signs and underlying diseases in dogs with blepharitis alone compared to dogs with blepharitis and POD. RESULTS: Brachycephalic dogs were significantly more likely to present with blepharitis than other breeds. Twenty five dogs had blepharitis alone [three dogs (2.9%) without and 22 dogs (21.5%) with cutaneous lesion beyond the periocular skin]. Seventy one of 102 (69%) presented with POD in addition to blepharitis. In six cases a differentiation between blepharitis and POD was not possible. Typical lesions included alopecia/hypotrichosis (97%), erythema (93%), eyelid oedema (50%) and crusts (39.2%). Allergic skin disease (52%) was the most common underlying cause of blepharitis, followed by infectious/parasitic diseases (21.5%), autoimmune disorders (17.7%) and neoplasia (4.9%). Four dogs could not be allocated to any of these disease groups. A diagnosis of parasitism was always accompanied by POD. Reduced tear production was detected in ten of the 53 dogs tested (18.8%). CONCLUSION: Blepharitis occurring in the absence of other skin lesions is rare. The most common underlying cause of blepharitis is allergic dermatitis. Measurement of tear production should be recommended in all cases of blepharitis.

Influence of protein concentration and quality in a canned diet on urine composition, apparent nutrient digestibility and energy supply in adult cats.

BACKGROUND: Protein concentration and quality in cat food can vary considerably, and the impact on feline urine composition and nutrient supply is of high practical relevance. In the present study, 6 canned diets with varying protein concentrations and qualities were fed to 10 healthy adult cats. Protein quality in the diet differed depending on the amount of collagen-rich ingredients. Hydroxyproline concentrations were 2.56-4.45 g/kg dry matter in the high quality and 3.76-9.44 g/kg dry matter in the low quality diets. Protein levels were 36.2, 43.3 and 54.9% in the high quality and 36.7, 45.0 and 56.1% in the low quality groups. Each diet was fed for 6 weeks, using a randomized cross-over design. In the last 2 weeks of each feeding period, urine and faeces of the cats were collected. RESULTS: Renal calcium (Ca), oxalate (Ox) and citrate excretion were unaffected by the dietary protein concentration, possibly mediated by a high urine volume (24.2-34.2 ml/kg bodyweight (BW)/day) in all groups. However, renal Ox excretion was lower when the high quality diets were fed (P = 0.013). Urinary relative supersaturation (RSS) with calcium oxalate (CaOx) was low in general, but reduced in the high quality groups (P = 0.031). Urinary RSS values for magnesium ammonium phosphate (MAP) were high (2.64-5.00) among all groups. Apparent digestibility of crude protein and most minerals was unaffected by the different diets. Feed intake was higher in the low quality groups (P = 0.026), but BW of the cats did not differ depending on dietary protein quality. BW of the cats increased with increasing dietary protein concentrations (P = 0.003). CONCLUSION: In conclusion, a high protein canned diet might not be a specific risk factor for CaOx urolith formation in cats. In contrast, all diets resulted in high RSS MAP values, which might be critical concerning MAP crystallization. Protein quality had a minor, but significant impact on urine composition, necessitating further research on this subject. A lower energy supply when feeding a low protein quality can be assumed. Changes in BW were only small and require a careful interpretation.

Exploration of serum- and cell culture-derived exosomes from dogs.

BACKGROUND: Exosomes are defined as extracellular membrane vesicles, 30-150 nm in diameter, derived from all types of cells. They originate via endocytosis and then they are released through exocytosis to the extracellular space, being found in various biological fluids as well as in cell culture medium. In the last few years, exosomes have gained considerable scientific interest due to their potential use as biomarkers, especially in the field of cancer research. This report describes a method to isolate, quantify and identify serum- and cell culture-derived exosomes from dog samples, using small volumes (100 µL and 1 mL, respectively). RESULTS: Quantification and sizing of exosomes contained in serum and cell culture samples were assessed by utilizing nanoparticle tracking analysis, transmission electron microscopy and immunoelectron microscopy. Detected particles showed the normal size (30-150 nm) and morphology described for exosomes, as well as presence of the transmembrane protein CD63 known as exosomal marker. CONCLUSIONS: Based on a validated rapid isolation procedure of nanoparticles from small volumes of different types of dog samples, a characterization and exploration of intact exosomes, as well as facilitation for their analysis in downstream applications was introduced.

Treatment of 5 dogs with immune-mediated thrombocytopenia using Romiplostim.

BACKGROUND: Immune thrombocytopenia (ITP) in dogs is analogous to that in humans. Romiplostim, a novel thrombopoietin receptor (TPO-R) agonist, is currently used for the treatment of refractory ITP in humans, but not in dogs. Here, we describe the response to romiplostim in five dogs with refractory ITP. Five dogs with severe and refractory ITP (three primary and two secondary) received romiplostim subcutaneously. Four dogs were administered 3-5 µg/kg and one dog received 10-13 µg/kg body weight once weekly. RESULTS: Romiplostim was well-tolerated and administration was associated with an increase in platelet counts in all five dogs. Four of the five dogs entered remission and relapses were not observed over a follow-up period of 3-10 months. CONCLUSIONS: Romiplostim is effective in the treatment of ITP in dogs at least as well as in humans. This finding may help to develop and use new therapeutics for ITP in dogs and humans.

Energy intake, growth rate and body composition of young Labrador Retrievers and Miniature Schnauzers fed different dietary levels of vitamin A.

Research in rodents has shown that dietary vitamin A reduces body fat by enhancing fat mobilisation and energy utilisation; however, their effects in growing dogs remain unclear. In the present study, we evaluated the development of body weight and body composition and compared observed energy intake with predicted energy intake in forty-nine puppies from two breeds (twenty-four Labrador Retriever (LAB) and twenty-five Miniature Schnauzer (MS)). A total of four different diets with increasing vitamin A content between 5·24 and 104·80 µmol retinol (5000-100 000 IU vitamin A)/4184 kJ (1000 kcal) metabolisable energy were fed from the age of 8 weeks up to 52 (MS) and 78 weeks (LAB). The daily energy intake was recorded throughout the experimental period. The body condition score was evaluated weekly using a seven-category system, and food allowances were adjusted to maintain optimal body condition. Body composition was assessed at the age of 26 and 52 weeks for both breeds and at the age of 78 weeks for the LAB breed only using dual-energy X-ray absorptiometry. The growth curves of the dogs followed a breed-specific pattern. However, data on energy intake showed considerable variability between the two breeds as well as when compared with predicted energy intake. In conclusion, the data show that energy intakes of puppies particularly during early growth are highly variable; however, the growth pattern and body composition of the LAB and MS breeds are not affected by the intake of vitamin A at levels up to 104·80 µmol retinol (100 000 IU vitamin A)/4184 kJ (1000 kcal).

Repeated imidocarb treatment failure suggesting emerging resistance of Babesia canis in a new endemic area in north-eastern Germany.

Canine babesiosis has been increasingly diagnosed in various regions of Germany such as north-eastern Germany in recent years. A dog with several relapses of Babesia canis infection after treatment with imidocarb is described. A 9-year-old male Magyar Viszla with B. canis infection was referred after two treatments with imidocarb (dosage 2.1 mg/kg SC) because of lethargy, fever and pancytopenia (additional treatments with prednisolone and doxycycline). Merozoites were detected in the blood smear and imidocarb treatment was repeated. Clinical signs, pancytopenia and a positive B. canis PCR occurred after the 3rd (6 mg/kg SC), 4th (7.7 mg/kg SC) and 5th (7.5 mg/kg SC and doxycycline for 4 weeks in addition) imidocarb injection and thorough tick prevention with isoxazoline and permethrin products. 12 days after the 5th injection, the PCR was negative for the first time. The dog was again presented with fever 35 days after the 5th injection. The B. canis PCR was positive and laboratory examination revealed pancytopenia. Treatment with atovaquone/azithromycin for 18 days was performed and no further relapse occurred for 32 weeks. In the case of suspected imidocarb resistance in B. canis infection, treatment with atovaquone/azithromycin can be an alternative.

ACVIM consensus statement on the diagnosis of immune thrombocytopenia in dogs and cats.

Immune thrombocytopenia (ITP) is the most common acquired primary hemostatic disorder in dogs. Immune thrombocytopenia less commonly affects cats but is an important cause of mortality and treatment-associated morbidity in both species. Immune thrombocytopenia remains a diagnosis of exclusion for which diagnostic guidelines are lacking. Primary, or non-associative, ITP refers to autoimmune platelet destruction. Secondary, or associative, ITP arises in response to an underlying disease trigger. However, evidence for which comorbidities serve as ITP triggers has not been systematically evaluated. To identify key diagnostic steps for ITP and important comorbidities associated with secondary ITP, we developed 12 Population Evaluation/Exposure Comparison Outcome (PECO) format questions. These questions were addressed by evidence evaluators utilizing a literature pool of 287 articles identified by the panelists using a structured search strategy. Evidence evaluators, using panel-designed templates and data extraction tools, summarized evidence and created guideline recommendations that then were integrated by diagnosis and comorbidity domain chairs. The revised PECO responses underwent a Delphi survey process to reach consensus on final guidelines. A combination of panel expertise and PECO responses were employed to develop algorithms for diagnosis of ITP in dogs and cats, which also underwent 4 iterations of Delphi review. Comorbidity evidence evaluators employed an integrated measure of evidence (IME) tool to determine evidence quality for each comorbidity; IME values combined with evidence summaries for each comorbidity were integrated to develop ITP screening recommendations, which also were subjected to Delphi review. Commentary was solicited from multiple relevant professional organizations before finalizing the consensus. The final consensus statement provides clinical guidelines for the diagnosis of, and underlying disease screening for, ITP in dogs and cats. The systematic consensus process identified numerous knowledge gaps that should guide future studies. This statement is a companion manuscript to the ACVIM Consensus Statement on the Treatment of Immune Thrombocytopenia.

ACVIM consensus statement on the treatment of immune thrombocytopenia in dogs and cats.

Management of immune thrombocytopenia (ITP) in dogs and cats is evolving, but there are no evidence-based guidelines to assist clinicians with treatment decisions. Likewise, the overall goals for treatment of ITP have not been established. Immunosuppressive doses of glucocorticoids are the first line treatment, but optimal treatment regimens beyond glucocorticoids remain uncertain. Additional options include secondary immunosuppressive drugs such as azathioprine, modified cyclosporine, and mycophenolate mofetil, usually selected based on clinician preference. Vincristine, human IV immunoglobulin (hIVIg), and transfusion of platelet or red blood cell-containing products are often used in more severe cases. Splenectomy and thrombopoietin receptor agonists are usually reserved for refractory cases, but when and in which patient these modalities should be employed is under debate. To develop evidence-based guidelines for individualized treatment of ITP patients, we asked 20 Population Intervention Comparison Outcome (PICO) format questions. These were addressed by 17 evidence evaluators using a literature pool of 288 articles identified by a structured search strategy. Evidence evaluators, using panel-designed templates and data extraction tools, summarized evidence and created guideline recommendations. These were integrated by treatment domain chairs and then refined by iterative Delphi survey review to reach consensus on the final guidelines. In addition, 19 non-PICO questions covering scenarios in which evidence was lacking or of low quality were answered by expert opinion using iterative Delphi surveys with panelist integration and refinement. Commentary was solicited from multiple relevant professional organizations before finalizing the consensus. The rigorous consensus process identified few comparative treatment studies, highlighting many areas of ITP treatment requiring additional studies. This statement is a companion manuscript to the ACVIM Consensus Statement on the Diagnosis of Immune Thrombocytopenia in Dogs and Cats.

A novel high-resolution melt PCR assay discriminates Anaplasma phagocytophilum and "Candidatus Neoehrlichia mikurensis".

"Candidatus Neoehrlichia mikurensis" (Anaplasmataceae) is an emerging pathogen transmitted by Ixodes ticks. Conventional PCR and the newly developed high-resolution melt PCR were used to detect and discriminate "Candidatus Neoehrlichia mikurensis" and Anaplasma phagocytophilum. Both bacterial species were frequently found in Ixodes ricinus and Ixodes hexagonus but virtually absent from Dermacentor reticulatus. In rodents, "Candidatus N. mikurensis" was significantly more prevalent than A. phagocytophilum, whereas in cats, only A. phagocytophilum was found.

Molecular and Serological Detection of Anaplasma phagocytophilum in Dogs from Germany (2008-2020).

Anaplasma phagocytophilum is an obligate intracellular bacterium that causes granulocytic anaplasmosis in domestic animals, wildlife, and humans and is primarily transmitted by ticks of the Ixodes persulcatus complex. This retrospective study aims to determine the percentages of dogs that tested positive for A. phagocytophilum in Germany. It included the results of direct (polymerase chain reaction [PCR]) and indirect (immunofluorescence antibody test [IFAT], antibody-enzyme-linked immunosorbent assay [ELISA]) detection methods performed in the laboratory LABOKLIN on canine samples provided by German veterinarians from 2008 to 2020. Out of a total of 27,368 dogs tested by PCR, 1332 (4.9%) tested positive, while 24,720 (27.4%) of the 90,376 dogs tested by IFAT/ELISA had positive serology. High rates of positive PCR results were observed in months with known peaks in vector activity, showing that the dynamics of A. phagocytophilum infections in dogs in Germany are consistent with vector activity. In dogs with a positive PCR result, peaks in serology could be observed four weeks after initial testing. Male and senior dogs had higher rates of positive serology. A possible impact of environmental factors such as changes in climate should be investigated further. Overall, the upward trend in positive test results over the years indicates that canine granulocytic anaplasmosis will continue to become increasingly important for veterinary medicine.

Thyroid hormones in canine pregnancy and lactation.

It is believed that thyroid function has a significant effect on fertility and fetal development in mammals. So far, however, only few studies have been published about potential effects of the reproductive cycle stage on thyroid hormone concentrations in dogs. Therefore, over the course of 122 pregnant and non-pregnant cycles in healthy bitches, Thyroid stimulating hormone (TSH), free Thyroxine (fT4), total Thyroxine (tT4) and Progesterone (P4) were measured six times to assess the influence of the cycle stage and pregnancy on hormone concentrations. The aim was to evaluate established reference intervals for the thyroid hormones in a female study population. Of the 122 bitches, 98 became pregnant. Blood samples were collected during estrus, three times in pregnancy, during lactation and after weaning, or at equivalent times during and after estrus in non-pregnant dogs. No differences between pregnant and non-pregnant animals in any of the thyroid hormones were found. Hormone concentrations, however, differed significantly between the six samplings (p < .01). TSH initially declined during pregnancy, then rose again. The mean concentration of all dogs exceeded the overall upper reference limit of 0.70 ng/mL during lactation. Concentrations of tT4 and ft4 increased during the first third of pregnancy and then subsequently declined. The overall reference limits for tT4 were 0.47-3.20 µg/dL, and for fT4 4.86-29.60 pmol/L, but the reference intervals varied between the sampling dates. The observed patterns may reflect that maternal tT4 and fT4 seem to have important effects during early pregnancy, including a pronounced negative feedback effect on TSH. The initial increase and subsequent decline of tT4 and fT4 concentrations during the course of pregnancy is in accordance with findings in humans and may support the development of fetal thyroid function. The observed peak of TSH concentrations during lactation suggests that the demand for thyroid hormones in this phase is largest. Even if the underlying causes and mechanisms of thyroid regulation are not fully understood, the results of this study show relevant changes of hormone concentrations in the course of the sexual cycle and pregnancy. In that regard, cycle stage needs to be considered when assessing thyroid function in bitches.

Autochthonous Babesia canis infections in 49 dogs in Germany.

BACKGROUND: Vector-borne diseases are of increasing importance in Germany. Since 2015, autochthonous cases have been increasingly documented in Berlin/Brandenburg. OBJECTIVES: Describe autochthonous Babesia canis infection in the Berlin/Brandenburg region. ANIMALS: Forty-nine dogs with autochthonous B. canis infection. METHODS: Evaluation of history, clinical signs, laboratory abnormalities, treatment, and outcome. RESULTS: Dogs were presented between March and August (9) and September and January (40) in the years 2015-2021. Historical and clinical findings were lethargy (100%), pale mucous membranes (63%), fever (50%), and pigmenturia (52%). Common clinicopathological findings were thrombocytopenia (100%), anemia (85%), intravascular hemolysis (52%), pancytopenia (41%), and systemic inflammatory response syndrome (SIRS; 37%). Babesia detection was based on blood smear evaluation (n = 40) and PCR targeting the 18S rRNA gene of piroplasms (n = 49). Sequencing indicated 99.47% to 100% identity to B. canis sequences from GenBank. All dogs were treated with imidocarb (2.4-6.3 mg/kg; median, 5 mg/kg); 8 dogs received 1, 35 received 2, and 1 dog each received 3, 4, or 5 injections, respectively. Continued PCR-positive results were detected in 7 dogs after the 1st, in 5 after the 2nd, in 2 after the 3rd, and in 1 28 days after the 4th injection. Four dogs were euthanized and 3 dogs died. CONCLUSIONS AND CLINICAL IMPORTANCE: Autochthonous B. canis infections in Berlin/Brandenburg were associated with severe clinicopathological changes, SIRS, and multiorgan involvement. Testing by PCR during and after treatment is advisable to monitor treatment success. Screening of blood donors in high-risk areas and year-round tick protection is strongly recommended.

Limiting Factors in Treatment Success of Biofilm-Forming Streptococci in the Case of Canine Infective Endocarditis Caused by Streptococcus canis.

An 8-year-old male Rhodesian Ridgeback was presented with fever and severe thrombocytopenia. Clinical and laboratory examination, echocardiography, blood culture, and pathohistology revealed evidence of infective endocarditis, ischemic renal infarcts, and septic encephalitis. Treatment was started immediately but the dog's condition worsened, and the dog had to be euthanized. The causative Streptococcus canis strain was detected by blood culture and MALDI-TOF MS and analyzed using whole-genome sequencing and multilocus sequence typing. Antibiotic susceptibility testing did not detect any resistance. The affected heart valve was analyzed using FISH imaging, which showed a streptococcal biofilm on the heart valve. Bacteria in biofilms are recalcitrant to antibiotic treatment. Early diagnosis could be beneficial to treatment outcome. Treatment of endocarditis could be improved by researching the optimal dosage of antibiotics in conjunction with the use of biofilm-active drugs.

Metabolomics during canine pregnancy and lactation.

During pregnancy and parturition, female dogs have to cope with various challenges such as providing nutrients for the growth of the fetuses, hormonal changes, whelping, nursing, milk production, and uterine involution. Metabolomic research has been used to characterize the influence of several factors on metabolism such as inter- and intra-individual factors, feeding, aging, inter-breed differences, drug action, behavior, exercise, genetic factors, neuter status, and pathologic processes. Aim of this study was to identify metabolites showing specific changes in blood serum at the different phases of pregnancy and lactation. In total, 27 privately owned female dogs of 21 different breeds were sampled at six time points: during heat, in early, mid and late pregnancy, at the suspected peak of lactation and after weaning. A validated and highly automated canine-specific NMR metabolomics technology was utilized to quantitate 123 measurands. It was evaluated which metabolite concentrations showed significant changes between the different time points. Metabolites were then grouped into five clusters based on concentration patterns and biochemical relationships between the metabolites: high in mid-pregnancy, low in mid-pregnancy, high in late pregnancy, high in lactation, and low in lactation. Several metabolites such as albumin, glycoprotein acetyls, fatty acids, lipoproteins, glucose, and some amino acids show similar patterns during pregnancy and lactation as shown in humans. The patterns of some other parameters such as branched-chain amino acids, alanine and histidine seem to differ between these species. For most metabolites, it is yet unstudied whether the observed changes arise from modified resorption from the intestines, modified production, or metabolism in the maternal or fetal tissues. Hence, further species-specific metabolomic research may support a broader understanding of the physiological changes caused by pregnancy that are likely to be key for the normal fetal growth and development. Our findings provide a baseline of normal metabolic changes during healthy canine pregnancy and parturition. Combined with future metabolomics findings, they may help monitor vital functions of pre-, intra-, and post-partum bitches and may allow early detection of illness.

Molecular detection of Babesia spp. in dogs in Germany (2007-2020) and identification of potential risk factors for infection.

BACKGROUND: In Europe, canine babesiosis is most frequently caused by Babesia canis and Babesia vogeli, and occasionally by Babesia gibsoni.. In Germany, B. canis is recognized as endemic. The aims of this study were to assess how often Babesia spp. infections were diagnosed in a commercial laboratory in samples from dogs from Germany, and to evaluate potential risk factors for infection. METHODS: The database of the LABOKLIN laboratory was screened for Babesia spp.-positive polymerase chain reaction (PCR) tests for dogs for the period January 2007-December 2020. Sequencing was performed for positive tests from 2018 and 2019. Binary logistic regression analysis was performed to determine the effects of sex, season, and year of testing. Questionnaires were sent to the submitting veterinarians to obtain information on travel abroad, tick infestation, and ectoparasite prophylaxis of the respective dogs. Fisher's exact test was used to calculate statistical significance and P < 0.05 was considered statistically significant. RESULTS: In total, 659 out of 20,914 dogs (3.2%) tested positive for Babesia spp. by PCR. Of 172 sequenced samples, B. canis was identified in 156, B. vogeli in nine, B. gibsoni in five, and B. vulpes in two. Season had a statistically significant impact on test results when summer/winter (1.6% tested positive) was compared to spring/autumn (4.7%), with peaks in April (5.2%) and October (7.4%) [P < 0.001, odds ratio (OR) = 3.16]. Sex (male 3.5%, female 2.8%; P = 0.012, OR = 1.49) and age (< 7 years old 4.0%, ≥ 7 years old 2.3%; P < 0.001, OR = 1.76) of the tested dogs also had a statistically significant effect. A statistically significant impact was demonstrated for observed tick attachment (P < 0.001, OR = 7.62) and lack of ectoparasite prophylaxis (P = 0.001, OR = 3.03). The frequency of positive Babesia spp. tests did not significantly differ between the 659 dogs that had never left Germany and the 1506 dogs with known stays abroad (P = 0.088). CONCLUSIONS: The possibility of canine infection with B. canis needs to be especially taken into consideration in spring and autumn in Germany as the activity of the tick Dermacentor reticulatus, a potential vector for canine babesiosis, is highest in these seasons. Travel and importation of dogs are considered major factors associated with canine babesiosis in Germany. However, autochthonous Babesia spp. infections also occur in a considerable number of dogs in Germany.

Host-pathogen associations revealed by genotyping of European strains of Anaplasma phagocytophilum to describe natural endemic cycles.

BACKGROUND: The zoonotic intracellular alpha-proteobacterium Anaplasma phagocytophilum is a tick-transmitted pathogen. The associations between vertebrate reservoirs and vectors are described as wide-ranging, and it was previously shown that the pathogenicity of A. phagocytophilum differs depending on the combination of pathogen variant and infected host species. This leads to the question of whether there are variations in particular gene loci associated with different virulence. Therefore, this study aims at clarifying existing host-variant combinations and detecting possible reservoir hosts. To understand these interactions, a complex toolset for molecular epidemiology, phylogeny and network theory was applied. METHODS: Sequences of up to four gene loci (msp4, msp2, groEL and 16S rRNA) were evaluated for different isolates from variable host species, including, for example, dogs, cattle and deer. Variant typing was conducted for each gene locus individually, and combinations of different gene loci were analysed to gain more detailed information about the genetic plasticity of A. phagocytophilum. Results were displayed as minimum spanning nets and correlation nets. RESULTS: The highest diversity of variants for all gene loci was observed in roe deer. In cattle, a reduced number of variants for 16S rRNA [only 16S-20(W) and 16S-22(Y)] but multiple variants of msp4 and groEL were found. For dogs, two msp4 variants [m4-20 and m4-2(B/C)] were found to be linked to different variants of the other three gene loci, creating two main combinations of gene loci variants. Cattle are placed centrally in the minimum spanning net analyses, indicating a crucial role in the transmission cycles by possibly bridging the vector-wildlife cycle to infections of humans and domestic animals. The minimum spanning nets confirmed previously described epidemiological cycles of the bacterium in Europe, showing separation of variants originating from wildlife animals only and a set of variants shared by wild and domestic animals. CONCLUSIONS: In this comprehensive study of 1280 sequences, we found a high number of gene variants only occurring in specific hosts. Additionally, different hosts show unique but also shared variant combinations. The use of our four gene loci expand the knowledge of host-pathogen interactions and may be a starting point to predict future spread and infection risks of A. phagocytophilum in Europe.

MRSA variant in companion animals.

Methicillin-resistant Staphylocoocus aureus (MRSA) harboring mecA(LGA251) has been isolated from humans and ruminants. Database screening identified this MRSA variant in cats, dogs, and a guinea pig in Germany during 2008-2011. The novel MRSA variant is not restricted to ruminants or humans, and contact with companion animals might pose a zoonotic risk.