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Abstract Metal azides are highly energetic materials that release a large amount of gas upon detonation. They also release metal particles, generating an aerosol. The most common azide is sodium azide (NaN3), which is used nowadays in car airbags. If the decomposition is not complete, harmful azide particles might be inhaled. Heavy metal azides find application as a primary explosive (primer) in ammunition. Public health officials have raised concerns about heavy metal particles released during training in shooting ranges. We identify a lack of knowledge on airborne metal particles properties released from azide detonation and on the analytical methods applied to characterize them. As a case study, we detonated milligram amounts of silver azide, copper azide, and a mixture of them in a glove box. We then analyse the airborne particles with an ensemble analytical setup, able to measure real-time their particle size distribution and chemical composition. We detected spherical metal nanoparticles in the range of 2–500 nm. These findings and the developed analytical tools may allow identifying airborne nanoparticles the passenger compartments of vehicles after airbag activation as well as in indoor shooting ranges, contributing to the evaluation of public health risks.

Background: Besides immunoglobulins which are essential for the calf to prevent Failure of Transfer of Passive Immunity (FTPI) colostrum contains multiple other constituents such microminerals, macrominerals and metals. The concentration of the minerals seems to be higher in colostrum than in normal milk. The aims of this study were to describe macromineral, micromineral and metal concentration of bovine colostrum samples from Salzburg, Austria and to see if there are differences in the concentrations between different cow feeding strategies and farm locations. Methods: Therefore, 1050 individual colostrum samples were collected from 72 dairy farms from different districts of Salzburg. All colostrum samples were analysed for macromineral (K, Mg, Na), micromineral (Co, Cu, Fe, I, Mg, Se, Zn) andmetal (As, Ba, Cd, Li, Mo, Ni, Pb, Sr, Tl, U) concentrations by inductive coupled plasma mass spectrometry. Results: The mean K, Mg and Na levels were 1218.71 mg l-1, 245.44 mg l-1 and 358.62 mg l-1, respectively. The micromineral concentrations of I, Se, Co and Zn were significantly higher in colostrum from dairy cows feeding a Total-Mixed-Ration during the lactation period in comparison to cows receiving microminerals by hand feeding, transponder feeding or via a licking bucket or licking stone. The metal analysis showed that in three farms, most of the colostrum samples showed Pb levels above the limit of 5 µg l-1. Implications: Herd-level factors need to be addressed to elevate micromineral concentrations and to reduce metal concentrations in colostrum from dairy cows from Salzburg, Austria. Conclusion: The results show that the macrominerals K, Mg and Na are within the range of published concentrations. For the microminerals, Co, Cu, Fe, I, Mg, Se and Zn the concentrations were significantly different depending on the mineral feeding strategies during the lactation and dry period. Metal concentrations of As, Ba, Cd, Li, Mo, Ni, Pb, Sr, Tl and U differed between the districts of Salzburg.

Knowledge about potential risk factors for animal health is crucial to achieve animal welfare. The aim of this study was to provide practical guidance for farmers to improve the health status of their youngstock by identifying and eliminating risk factors for omphalitis in neonatal calves. A cross-sectional study including 3,445 dairy calves from 567 farms located in three structurally different regions of Germany was performed from December 2016 to July 2019. On each farm calves aged five to 21 days underwent clinical examinations with special consideration of the umbilicus for signs of inflammation. Information regarding animal health, hygiene, and management was obtained via interviews with the farmers. Rearing conditions were recorded following visual inspection using written standard operating procedures. Multifactorial statistical analyses were performed to identify potential risk factors for omphalitis on animal and farm level. The overall omphalitis prevalence in calves aged five to 21 days was 30.9%. Across all regions and farms, every fourth calf per farm exhibited signs of omphalitis (median farm prevalence: 25.0%, interquartile range: 0.0–50.0%). According to the farmers, however, only 4.5% of the calves had been treated for omphalitis in the preceding 12 months. Risk factors for omphalitis identified included the dampness of the lying area in the first 2 weeks of life, a body condition score of the dam after calving outside the optimal range, and the time that calves spent with their dam after birth. Calves on farms providing dry lying areas in the first 2 weeks of life had 0.77 times the risk of omphalitis compared to calves on farms with predominantly damp bedding. When the dams were judged under- or over-conditioned after calving, their offspring had a 1.4 times higher omphalitis risk, respectively, compared to calves from dams optimal conditioned. Calves from farms separating calf and dam beyond 12 h after birth were 0.75 times as likely to develop omphalitis than calves from farms performing immediate separation. These results highlight the complexity of multifactorial diseases such as umbilical infection. The evidence presented can help to establish guidelines for dairy farmers to improve the umbilical health of their calves.

S100A9, a multifunctional protein mainly expressed by neutrophils and monocytes, poses an immunological paradox. In virus infections or sterile inflammation, it functions as an alarmin attracting innate immune cells, as well as mediating proinflammatory effects through TLR4 signaling. However, in cancer, S100A9 levels have been shown to associate with poor prognosis and lack of response to immunotherapy. Its expression by myeloid cells has been related to an immune suppressive phenotype, the so-called myeloid derived suppressor cells (MDSCs). Targeting S100A9 in cancer has therefore been proposed as a potential way to relieve myeloid-mediated immune suppression. Surprisingly, we found that blocking the extracellular TLR4 signaling from S100A9 using the inhibitor Paquinimod, resulted in increased tumor growth and a detrimental effect on anti-PD-L1 efficacy in the CT26 tumor model. This effect was caused by a reduction in the tumor immune infiltration to about half of untreated controls, and the reduction was made up of a 5-fold decrease in Ly6Chigh monocytic cells. The suppressive Ly6G+ myeloid cells compartment was not reduced by Paquinimod treatment, suggesting alternative mechanisms by which S100A9 contributes to myeloid-mediated suppression. Intratumoral injection of recombinant S100A9 early after mice inoculation with CT26 cells had an anti-tumor effect. These findings indicate an important yet understudied role of S100A9 as an alarmin and immune stimulatory signal in cancer settings, and highlight the potential to exploit such signals to promote beneficial anti-tumor responses.

ABSTRACT: The aim of the present study was to evaluate the associations between milk recording data, body condition score (BCS), housing factors, management factors, and lameness in freestall-housed dairy cows in 3 structurally different regions in Germany. These regions substantially vary regarding herd size, breeds, access to pasture, farm management (family run or company owned), and percentage of organic farms. The data used was collected in a large cross-sectional study from 2016 to 2019. A total of 58,144 cows from 651 farms in 3 regions of Germany (North, East, and South) was scored for locomotion and body condition. Additionally, data on milk yield, milk composition, breed, age, as well as information on housing and management were retrieved. One mixed-logistic regression model was fitted per region to evaluate the association of the data with the target variable “lame” and to allow for a comprehensive reflection across different kinds of farming types. In all regions, undercondition (BCS lower than recommended for the lactation stage; North: odds ratio [OR] 2.15, CI 1.96–2.34; East: OR 2.66, CI 2.45–2.88; South: OR 2.45, CI 2.01–2.98) and mid-lactation stage (102–204 d in milk; North: OR 1.15, CI 1.05–1.27; East: OR 1.24, CI 1.17–1.32; South: OR 1.38, CI 1.18–1.62) were associated with higher odds for lameness, whereas overcondition (BCS higher than recommended for the lactation stage; North: OR 0.51, CI 0.44–0.60; East: OR 0.51, CI 0.48–0.54; South: OR 0.65, CI 0.54–0.77) and parity of 1 or 2 was associated with lower odds (parity 1 = North: OR 0.32, CI 0.29–0.35; East: OR 0.19, CI 0.18–0.20; South: OR 0.28, CI 0.24–0.33; parity 2 = North: OR 0.51, CI 0.47–0.46; East: OR 0.41, CI 0.39–0.44; South: OR 0.49, CI 0.42–0.57), irrespective of the regional production characteristics. Low energy-corrected milk yield was associated with higher odds for lameness in South and North (North: OR 1.16, CI 1.05–1.27; South: OR 1.43, CI 1.22–1.69). Further factors such as pasture access for cows (North: OR 0.64, CI 0.50–0.82; and South: OR 0.65, CI 0.47–0.88), milk protein content (high milk protein content = North: OR 1.34, CI 1.18–1.52; East: OR 1.17, CI 1.08–1.28; low milk protein content = North: OR 0.79, CI 0.71–0.88; East: OR 0.84, CI 0.79–0.90), and breed (lower odds for “other” [other breeds than German Simmental and German Holstein] in East [OR 0.47, CI 0.42–0.53] and lower odds both for German Holstein and “other” in South [German Holstein: OR 0.62, CI 0.43–0.90; other: OR 0.46, CI 0.34 – 0.62]) were associated with lameness in 2 regions, respectively. The risk of ketosis (higher odds in North: OR 1.11, CI 1.01–1.22) and somatic cell count (higher odds in East: increased (>39.9 cells × 1,000/mL): OR 1.10; CI 1.03–1.17; high (>198.5 cells × 1,000/mL): OR 1.08; CI 1.01–1.06) altered the odds for lameness in 1 region, respectively. Cows from organic farms had lower odds for lameness in all 3 regions (North: OR 0.18, CI 0.11–0.32; East: OR 0.39, CI 0.28–0.56; South: OR 0.45, CI 0.29–0.68). As the dairy production systems differed substantially between the different regions, the results of this study can be viewed as representative for a wide variety of loose-housed dairy systems in Europe and North America. The consistent association between low BCS and lameness in all regions aligns with the previous literature. Our study also suggests that risk factors for lameness can differ between geographically regions, potentially due to differences in which dairy production system is predominantly used and that region-specific characteristics should be taken into account in comparable future projects.

Abstract 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

Abstract Background In Europe, feline vector-borne infections are gaining importance because of the changing climate, expanding habitats of potential vectors and expanding pathogen reservoirs. The main objective of this study was to assess the prevalence of vector-borne pathogens (VBPs) in stray cats in Zaragoza, Spain, and to investigate potential risk factors for infection, including feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV). Methods Blood samples from stray cats presented to the veterinary faculty in Zaragoza between February 2020 and 2022 were tested by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum, Anaplasma platys, Bartonella henselae, Ehrlichia canis, Rickettsia spp., haemotropic Mycoplasma spp., Hepatozoon spp., Leishmania infantum, piroplasms and microfilariae at the LABOKLIN laboratory. The cats were also tested for FeLV and FIV by PCR. Results Nearly half of the cats (158/332, 47.6%) were positive for at least one VBP. Hepatozoon spp. were detected in 25.6%, haemotropic Mycoplasma spp. in 22.9%, B. henselae in 9.3% and L. infantum in 2.1% of the cats. Male sex had a statistically significant association with test results for haemotropic Mycoplasma spp. (odds ratio 1.38 [1.21;1.57]); regionality with Hepatozoon spp., B. henseale and FIV; and seasonality with Hepatozoon spp., haemotropic Mycoplasma spp., L. infantum and FeLV (P ≤ 0.05 each). A strong positive correlation was reported for the amount of rainfall and the number of cats that tested positive for Hepatozoon spp. (ρ = 753, P = 0.05). None of the cats tested positive for A. phagocytophilum, A. platys, E. canis, Rickettsia spp., piroplasms, or microfilariae. Co-infections with multiple VBPs were detected in 56 out of 332 cats (16.9%). Thirty-one of the 332 cats included in the study (9.3%) tested positive for FeLV (6.9%) and for FIV (3.6%). In 20/31 cats (64.5%) that tested positive for FeLV/FIV, coinfections with VBP were detected (P = 0.048, OR 2.15 [0.99; 4.64]). Conclusions VBPs were frequently detected in stray cats in Zaragoza. In particular, regionality and seasonality had a statistically significant association with PCR results for most VBPs included in the study. Graphical Abstract

Abstract Background Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks of the Ixodes ricinus complex and causes febrile illness called granulocytic anaplasmosis primarily in humans, horses, dogs, sheep, cattle and goats. In comparison, clinically apparent disease has been described rarely in cats especially compared to dogs and horses. It is currently unknown whether cats are less susceptible to A. phagocytophilum or whether granulocytic anaplasmosis might be underdiagnosed in cats. Methods To address this question, we examined clinical signs and laboratory findings in seven A. phagocytophilum infected cats from Germany and Switzerland. We then genetically characterized feline A. phagocytophilum strains and compared them to those from other hosts showing clinically apparent disease. For this purpose, ankA-based, groEL-based and multilocus sequence typing (MLST) were applied. Furthermore, the concordance between these typing methods was assessed. Results Fever, lethargy and anorexia were the most common clinical signs in cats suffering from granulocytic anaplasmosis. The most frequent laboratory finding was thrombocytopenia. All three typing methods consistently indicated that the A. phagocytophilum strains found infecting cats are the same as those that cause disease in humans, dogs and horses. In general, the three typing methods applied exhibited high concordance. Conclusions The genetic characterization of the feline A. phagocytophilum strains indicates that strain divergence is not the explanation for the fact that granulocytic anaplasmosis is much less frequently diagnosed in cats than in dogs and horses. Otherwise, it may be possible that cats are less susceptible to the same strains than dogs and horse are. However, due to the unspecific clinical signs, it should be considered that granulocytic anaplasmosis may be under-diagnosed in cats. Graphical Abstract

Mammary tumors are one of the most common neoplasms in female dogs, and cytology represents a non-invasive diagnostic method. The protozoal pathogen Leishmania spp. was previously demonstrated in canine mammary glands. An eight-year-old, female-spayed Doberman was imported from Crete, Greece, three years before the first presentation. The dog was presented due to a mammary tumor two years after adoption. The clinical examination revealed fever and weight loss. Smears of the mammary secretion were investigated cytologically. Multiple atypical epithelial cells with moderate to marked criteria of malignancy were detected. Furthermore, amastigotes were visible intra- and extracellularly. The diagnosis of L. infantum infection was based on a positive PCR out of the cytological smear, and a positive serology. Mammary carcinoma and granulomatous inflammation with amastigotes were confirmed by histopathology. We aimed to provide a complete report of cytological, histopathological, hematological, and biochemistry findings in a dog with L. infantum in the mammary glands with focus on trans-mammary pathogen transmission as a potential alternative way of spreading Leishmania infections. Canine leishmaniasis should be a potential differential diagnosis in dogs with lesions and/or inflammation in the mammary glands and a history of presence in areas endemic for L. infantum, especially the Mediterranean in Europe.

Six hundred fifty-nine farms in three regions of Germany (North: n = 240, East: n = 247, and South: n = 172) were included in the study, which aims at determining the association of management-related risk factors with farm-level lameness in German dairy herds. For each risk factor, a generalised linear regression model with negative binomial distribution and logit link was built. Results showed that cows housed in deep-bedded cubicles had a lower risk of being lame than cows housed in other cubicle types. A larger cubicle width was associated with a lower risk of being lame. Feeding a total mixed ration was associated with lower lameness prevalence (compared to feeding a partial mixed ration or single components). For first lactation cows, lameness assessment performed daily (compared to less than daily) and during other work tasks (compared to lameness assessment as a separate work task) were associated with lower risk for lameness. Finally, the present study provided evidence for crucial associations of management-related risk factors with lameness in German dairy cows, especially in the fields of cubicle design, feeding management, and lameness assessment.

Lameness in dairy cows is an expression of pain most likely originating from a claw disorder, causing impaired animal wellbeing and substantial economic losses for farmers. The aim of this study was to investigate the effects of access to pasture, time spent on pasture, and season on farm level lameness prevalence. The survey was part of a cross-sectional observational study, in which farms in three regions of Germany (North, East and South) were visited by study veterinarians. On each farm (total: 659, N: 240, E: 247, S: 172), management data were recorded, and cows were scored for locomotion, according to Sprecher. Median farm-level lameness prevalence (Score 3/5 or higher) was 29.4% (IQR: 18.7% - 42.0%), and 8.2% (IQR: 3.7% - 14.0%) for severe lameness (Score 4/5 or higher). Farm-level lameness prevalence continuously decreased with increasing time spent on pasture (up to approximately 10 hours per cow per day). On farms that did not offer their cows access to pasture lameness prevalence did not show a seasonal variation. On farms where cows had pasture access for up to three hours per day lameness prevalence peaked in autumn. In contrast, on farms offering their cows access to pasture beyond three hours per day the peak of lameness was observed in spring. Our results revealed that even short periods of pasture access of at least two hours per cow and day (on average per year) are beneficial for the locomotion of dairy cows.

Abstract Background In Europe, feline leishmaniosis is commonly caused by Leishmania infantum. There is little knowledge regarding pathogenesis, ocular manifestations and long-term follow-ups in cats with leishmaniosis. Findings A 6-year-old female, spayed European Shorthair cat was imported from Spain to Germany 2 years prior to its first clinical presentation. The cat showed lethargy, weight loss, ulcerative lesions on the front limbs and high-grade chronic uveitis. The diagnosis of L. infantum infection was based on the cytological finding of amastigotes in skin lesions, positive qPCR of EDTA-blood and positive PCR of a cyto-brush sample from the conjunctiva. Supportive findings included positive serology by IFAT, serum protein capillary electrophoresis with peaks in alpha2- and gamma-globulin sections and marked elevation of SAA. Enucleation had to be performed on day 288 on both eyes because of blindness, glaucoma and high-grade uveitis. Histologically, high numbers of Leishmania spp. amastigotes were found in histiocytes. IFAT and PCR were positive in the aqueous humor in both eyes, respectively. Feline leukemia virus antigen and feline immunodeficiency virus antibody testings were positive. Hematological and biochemical results revealed mild leukocytosis with lymphocytosis, monocytosis and eosinopenia as well as marked elevation of SAA and hyperglobulinemia. The cat was treated with allopurinol, responded well and was still alive at follow-up on day 288 after first presentation. However, enucleation was necessary because of refractory glaucoma and uveitis. Conclusion For the first time, ocular evidence of Leishmania IgG antibodies was demonstrated in the aqueous humor of both eyes in cats. There is limited knowledge about the pathogenesis, treatment options and outcomes in cats infected with L. infantum. This case report supports the hypothesis that immunosuppression increases the risk of clinical signs of leishmaniasis in cats. Alpha2- and gamma-globulin peaks in serum protein capillary electrophoresis are supportive criteria for the diagnosis of L. infantum infection. SAA is valuable for monitoring. Regarding ophthalmology, uveitis and glaucoma may have a poor prognosis. Graphical abstract

Fibroblast growth factor (FGF)-23 is a phosphaturic hormone. An association between increasing FGF-23 levels and progression of chronic kidney disease (CKD) was documented in cats, dogs, and humans. The information regarding reference intervals (RIs) of FGF-23 in cats is limited. We aimed to establish RIs in a large cohort of clinically healthy cats and to investigate correlations with sex and age. A total of 118 cats with unremarkable complete blood count and serum chemistry profile were included. Clinically sick cats, cats with concurrent diseases, suspicion of CKD, or receiving renal diets were excluded. FGF-23 concentrations were measured with the FGF-23 ELISA Kit. RIs were calculated using the reference interval advisor software 2.1 (Microsoft Excel). FGF-23 concentrations were correlated with sex and age. The RI for FGF-23 concentrations spanned 85.8 to 387.0 pg/mL (90% confidence interval: lower limit 40.5 to 103.9 pg/mL, upper limit: 354.6 to 425.0 pg/mL). No significant relationships (r2 = 0.044) were detected with age (p = 0.081) or sex (p = 0.191). Other studies of the same diagnostic assay calculated RIs of 56 to 700 pg/mL in 79 cats and

From 2016 to 2020, the “PraeRi” study, conducted by three German veterinary universities, was aimed at enhancing animal health and welfare in dairy farms. With 765 dairy farms visited and 101,307 animals examined, this study provided a basis for improving animal health and welfare. The study population comprised three different regions representing a broad variety of characteristics. To ensure representative estimates, a sample size of 250 farms was determined for each region, employing a stratified sampling plan based on farm size. According to the information provided by the farmers, the most commonly occurring disease in their herds was mastitis without general disorder (14.2% to 16.3% of the herd—depending on the region). For most disorders, prevalence data were lowest for the region South compared with the two remaining regions. Multivariable regression analyses were performed to identify risk factors for various target variables, and the results were communicated through individual reports and benchmarking flyers to participating farmers. The authors encountered challenges in management and communication due to the project’s size in terms of personnel, data, and farms examined. Harmonizing data management and hypothesis testing across all involved parties added complexity.

Equine piroplasmosis (EP) is caused by Theileria (T.) equi and/or Babesia (B.) caballi. The aim was to assess the percentage of positive test results for EP in horses in Europe and to identify risk factors for pathogen contact/infection. This study included results from PCR and competitive enzyme-linked immunosorbent assay testing requested by European veterinarians between 2008 and 2021. Binary bivariate logistic regression was used to analyze risk factors. A total of 4060 horses were included. PCR testing was positive in 9.7% (154/1589), serology for T. equi in 15.2% (393/2591) and for B. caballi in 6.8% (175/2578). The odds of positive serology increased by 6.8% (B. caballi, p = 0.008) and 9.5% (T. equi, p < 0.001) each year. Regionality had a statistically significant impact on PCR (Eastern p = 0.047/OR = 1.605; Southern p = 0.029/OR = 1.451; Central p = 0.007/OR = 0.617) and serological testing for T. equi (Southern p < 0.001/OR = 2.521; Central p < 0.001/OR = 0.537; Northern p = 0.003/OR = 0.462), as well as breeds on seroprevalence of B. caballi (heavy horses: p = 0.016/OR = 2.239) and T. equi (ponies: p = 0.007/OR = 0.340; warmbloods: p = 0.025/OR = 1.602). In conclusion, there was a significant geographical impact on the results of PCR and serology, consistent with known vector habitats. The rising numbers of horses tested serologically positive highlights the importance of surveillance.

Abstract Reducing precious metal loading in the anodic catalyst layer (CL) is indispensable for lowering capital costs and enabling the widespread adoption of polymer electrolyte water electrolysis. This work presents the first three-dimensional reconstruction of a TiO2-supported IrO2 based core shell CL (3 mgIrO2/cm2), using high-resolution X-ray ptychographic tomography at cryogenic temperature of 90 K. The high data quality and phase sensitivity of the technique have allowed the reconstruction of all four phases namely pore space, IrO2, TiO2 support matrix and the ionomer network, the latter of which has proven to be a challenge in the past. Results show that the IrO2 forms thin nanoporous shells around the TiO2 particles and that the ionomer has a non-uniform thickness and partially covers the catalyst. The TiO2 particles do not form a percolating network while all other phases have high connectivity. The analysis of the CL ionic and electronic conductivity shows that for a dry CL, the ionic conductivity is orders of magnitudes lower than the electronic conductivity. Varying the electronic conductivity of the support phase by simulations, reveals that the conductivity of the support does not have a considerable impact on the overall CL electrical conductivity.

Abstract 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.

Abstract Hypokalemia in dairy cows, which is characterized by too low serum potassium levels, is a severe mineral disorder that can be life threatening. In this paper, we explore different originating conditions of hypokalemia—reduced potassium intake, increased excretion, acid-base disturbances, and increased insulin—by using a dynamic mathematical model for potassium balance in non-lactating and lactating cows. The simulations confirm observations described in literature. They illustrate, for example, that changes in dietary intake or excretion highly effect intracellular potassium levels, whereas extracellular levels vary only slightly. Simulations also show that the higher the potassium content in the diet, the more potassium is excreted with urine. Application of the mathematical model assists in experimental planning and therefore contributes to the 3R strategy: reduction, refinement and replacement of animal experiments.

The acquisition speed and spatial resolution of X-ray nanotomography have continuously improved over the last decades. Coherent diffraction-based techniques breach the 10 nm resolution barrier frequently and thus pose stringent demands on sample positioning accuracy and stability. At the same time there is an increasing desire to accommodate in situ or operando measurements. Here, an environmental control system for X-ray nanotomography is introduced to regulate the temperature of a sample from room temperature up to 850°C in a controlled atmospheric composition. The system allows for a 360° sample rotation, permitting tomographic studies in situ or operando free of missing wedge constraints. The system is implemented and available at the flOMNI microscope at the Swiss Light Source. In addition to the environmental control system itself, the related modifications of flOMNI are described. Tomographic measurements of a nanoporous gold sample at 50°C and 600°C at a resolution of sub-20 nm demonstrate the performance of the device.

Abstract Background Hepatozoon canis is a protozoal agent that is known to be transmitted by oral uptake of H. canis-infected Rhipicephalus sanguineus sensu lato ticks in dogs. Vertical transmission of H. canis has only been described once in a study evaluating dogs from Japan. The aim of this study was to investigate the parasitological status of puppies from a bitch that had tested positive for Hepatozoon spp. prior to giving birth. Findings A 4-year-old, female, pregnant dog imported from Italy (Sardinia) to Germany showed clinical signs of lethargy and tachypnoea and tested positive for H. canis by PCR. The dog gave birth to eight puppies, one of which was stillborn and another that had to be reanimated. Haematology, buffy coat analysis and a biochemistry profile were performed for each dog. EDTA-blood of the surviving seven puppies and bone marrow, liver, spleen, amniotic fluid, and umbilical cord of the stillborn puppy was tested for the presence of Hepatozoon spp. by PCR. The mother and the seven surviving puppies tested positive for H. canis by PCR at day 62 post-partum. Gamonts were detected in all dogs by buffy coat evaluation. Haematological and biochemistry results revealed mild abnormalities. In the stillborn puppy, spleen, umbilical cord, and amniotic fluid were positive for H. canis. Conclusion The results confirm that vertical transmission is a possible route of H. canis infection in dogs, demonstrated by molecular detection of the pathogen in the stillborn puppy. In the seven surviving puppies, vertical transmission was the most likely transmission route. A potential impact of the level of parasitaemia on the health of puppies, as well as its pathogenesis, should be investigated further. Graphical Abstract

IntroductionAs part of the EU Joint Action on Antimicrobial Resistance (AMR) and Healthcare-Associated Infections, an initiative has been launched to build the European AMR Surveillance network in veterinary medicine (EARS-Vet). So far, activities included mapping national systems for AMR surveillance in animal bacterial pathogens, and defining the EARS-Vet objectives, scope, and standards. Drawing on these milestones, this study aimed to pilot test EARS-Vet surveillance, namely to (i) assess available data, (ii) perform cross-country analyses, and (iii) identify potential challenges and develop recommendations to improve future data collection and analysis.MethodsEleven partners from nine EU/EEA countries participated and shared available data for the period 2016–2020, representing a total of 140,110 bacterial isolates and 1,302,389 entries (isolate-antibiotic agent combinations).ResultsCollected data were highly diverse and fragmented. Using a standardized approach and interpretation with epidemiological cut-offs, we were able to jointly analyze AMR trends of 53 combinations of animal host-bacteria–antibiotic categories of interest to EARS-Vet. This work demonstrated substantial variations of resistance levels, both among and within countries (e.g., between animal host species).DiscussionKey issues at this stage include the lack of harmonization of antimicrobial susceptibility testing methods used in European surveillance systems and veterinary diagnostic laboratories, the absence of interpretation criteria for many bacteria–antibiotic combinations of interest, and the lack of data from a lot of EU/EEA countries where little or even surveillance currently exists. Still, this pilot study provides a proof-of-concept of what EARS-Vet can achieve. Results form an important basis to shape future systematic data collection and analysis.

Fibroblast growth factor-23 (FGF-23) is a phosphaturic hormone used to monitor chronic kidney disease (CKD) in humans. The aims of this study were (1) to determine the intra- and interassay precision of the FGF-23 concentrations in dogs as measured via the Kainos ELISA FGF-23 kit, (2) to calculate a reference interval, and (3) to assess the correlation of the FGF-23 concentration with the hematological and biochemical parameters. The coefficient of variation was below 15% for both the intra- and interassay precision, indicating good reproducibility. The reference interval ranged between 95.8 (90% confidence interval: 44.6; 139.2) and 695.1 pg/mL (598.7; 799.1) based on 136 clinically healthy dogs, classified as such according to the information of treating veterinarians as well as the unremarkable results of hematology and biochemistry. The FGF-23 concentration differed significantly between dogs aged p = 0.045). Four groups of 10 dogs each were retrospectively formed based on the creatinine concentration classification according to the IRIS staging. Correlation was the strongest for the renal parameters. Statistically significant differences in the FGF-23 concentration were demonstrated between the study groups I and III (p < 0.001), I and IV (p < 0.001), and II and IV (p = 0.005). There was a trend for a rising FGF-23 concentration in older dogs. Due to the wide reference interval, diagnostic cut-offs and/or subject-based FGF-23 reference values in each dog are needed for monitoring and clinical interpretation.

Abstract Hepatopathies can cause major metabolic abnormalities in humans and animals. This study examined differences in serum metabolomic parameters and patterns in left-over serum samples from dogs with either congenital portosystemic shunts (cPSS, n = 24) or high serum liver enzyme activities (HLEA, n = 25) compared to control dogs (n = 64). A validated targeted proton nuclear magnetic resonance spectroscopy platform was used to assess 123 parameters. Principal component analysis of the serum metabolome demonstrated distinct clustering among individuals in each group, with the cluster of HLEA being broader compared to the other groups, presumably due to the wider spectrum of hepatic diseases represented in these samples. While younger and older adult control dogs had very similar metabolomic patterns and clusters, there were changes in many metabolites in the hepatopathy groups. Higher phenylalanine and tyrosine concentrations, lower branched-chained amino acids (BCAAs) concentrations, and altered fatty acid parameters were seen in cPSS dogs compared to controls. In contrast, dogs with HLEA had increased concentrations of BCAAs, phenylalanine, and various lipoproteins. Machine learning based solely on the metabolomics data showed excellent group classification, potentially identifying a novel tool to differentiate hepatopathies. The observed changes in metabolic parameters could provide invaluable insight into the pathophysiology, diagnosis, and prognosis of hepatopathies.

We conducted a severe acute respiratory syndrome coronavirus 2 antibody seroprevalence study among >2,000 domestic cats from 4 countries during the first coronavirus disease wave in Europe. We found 4.4% seroprevalence using a virus neutralization test and 4.3% using a receptor-binding domain ELISA, demonstrating probable human-to-cat transmission.

Abstract Background A 2019 ACVIM consensus statement on diagnostics for immune-mediated hemolytic anemia (IMHA) in dogs made testing recommendations. As data on the performance of immunohematological tests was lacking, we undertook a comparative analysis. Material and methods Anticoagulated blood samples from 126 dogs suspected of having IMHA submitted to a diagnostic veterinary laboratory for a routine direct antiglobulin test (DAT) and from 28 healthy control dogs were evaluated for spherocytosis and autoagglutination before and after three saline washes. Samples were also subjected to different DATs: a gel minitube and an immunochromatographic strip kit used in clinics; neutral gel column cards, microtiter plates (at 4°, 22°, and 37°C), capillary tubes, and flow cytometry used in laboratories. Results Samples from healthy dogs yielded negative results with all immunodiagnostic tests. Among the 126 samples submitted for DAT 67 were positive by a DAT utilizing microtiter plates with goat anti-dog antiglobulin DAT at 22°C. Notably, DAT results were comparable and consistent across all evaluated methods regardless of antiglobulin and temperature used. DAT+ dogs were more severely anemic and more likely to have erythroid regeneration compared to DAT- dogs. Macroscopic agglutination in tubes or on slides was observed in 48 samples after 1:1 and 1:4 blood to saline dilution, but only persisted in four samples after washing. Among the DAT+ samples, 57% had agglutination, 87% had spherocytosis, and 45% had both. There was good correlation between spherocytosis and DAT results from the six DAT techniques, but the correlation with autoagglutination was only fair. Clinical follow-up was available for 42 dogs. Of the sample from 12 DAT+ dogs collected during treatment, 10 remained DAT+ when tested 1–24 weeks after initial assessment. Conclusions Based upon this comparative prospective survey, all in-clinic and laboratory DAT techniques produced similar results when performed by trained personnel and can therefore be recommended for detection of antibody-coated erythrocytes and immunohematological diagnosis. In addition, use of these tests for monitoring response of IMHA dogs to treatment might be valuable.

Bacterial, protozoal, and viral vector-borne pathogens (VBPs) can cause infections in cats. There is little information on feline VBP prevalence in Germany. Stray cats are frequently exposed to vectors but receive no veterinary care. The aim of this study was to determine the prevalence of selected VBPs in stray cats. EDTA blood and serum samples were taken from apparently healthy stray cats during a spay/neuter campaign in the federal state of Thuringia. Overall, 11/50 (22%) and 32/50 (64%) cats tested positive for at least one VBP by direct and indirect detection methods, respectively. PCR testing of EDTA blood detected hemotropic Mycoplasma spp. in 12% of cats, Hepatozoon spp. in 10%, and Anaplasma phagocytophilum in 4%. PCR testing for Rickettsia spp. and piroplasms was negative. IFAT on serum samples showed 46% of cats had detectable antibodies for Bartonella spp., 30% for Rickettsia spp., and 16% for A. phagocytophilum. The cats were additionally tested for feline coronavirus, FIV, and FeLV to identify potential risk factors for pathogen contact and/or infections. No correlation between FIV and FeLV status and VBP positivity was detected. Anaplasma phagocytophilum, Rickettsia spp., and Bartonella spp. have zoonotic potential, and surveillance is recommended in the context of the One Health approach.

Good calf health is crucial for a successfully operating farm business and animal welfare on dairy farms. To evaluate calf health on farms and to identify potential problem areas, benchmarking tools can be used by farmers, herd managers, veterinarians, and other advisory persons in the field. However, for calves, benchmarking tools are not yet widely established in practice. This study provides hands-on application for on-farm benchmarking of calf health. Reference values were generated from a large dataset of the “PraeRi” study, including 730 dairy farms with a total of 13,658 examined preweaned dairy calves. At herd level, omphalitis (O, median 15.9%) was the most common disorder, followed by diarrhea (D, 15.4%) and respiratory disease (RD, 2.9%). Abnormal weight bearing (AWB) was rarely detected (median, 0.0%). Calves with symptoms of more than one disorder at the same time (multimorbidity, M) were observed with a prevalence of 2.3%. The enrolled farms varied in herd size, farm operating systems, and management practices and thus represented a wide diversity in dairy farming, enabling a comparison with similar managed farms in Germany and beyond. To ensure comparability of the data in practice, the reference values were calculated for the whole data set, clustered according to farm size (1–40 dairy cows (n = 130), 41–60 dairy cows (n = 99), 61–120 dairy cows (n = 180), 121–240 dairy cows (n = 119) and farms with more than 240 dairy cows (n = 138), farm operating systems (conventional (n = 666), organic (n = 64)) and month of the year of the farm visit. There was a slight tendency for smaller farms to have a lower prevalence of disorders. A statistically significant herd-size effect was detected for RD (p = 0.008) and D (p < 0.001). For practical application of these reference values, tables, diagrams, and an Excel® (Microsoft®) based calf health calculator were developed as tools for on-farm benchmarking (https://doi.org/10.6084/m9.figshare.c.6172753). In addition, this study provides a detailed description of the colostrum, feeding and housing management of preweaned calves in German dairy farms of different herd sizes and farm type (e.g., conventional and organic).

The immunodiagnostic assessment of dogs suspected of having immune-mediated hemolytic anemia (IMHA) is based on persistent autoagglutination of erythrocytes (after three saline washes), marked spherocytosis, and a positive direct antiglobulin (Coombs’) test (DAT). However, the value of using the indirect antiglobulin test (IAT) for the detection of anti-erythrocytic autoantibodies in serum from dogs suspected of having IMHA is unclear. To evaluate the IAT, leftover serum samples from a large cohort of 94 dogs suspected of having IMHA and for which DAT results were known were incubated with DAT− erythrocytes, and five IAT techniques were performed (in part with different reagents and temperatures): microtiter plate (MICRO), microcapillary, laboratory gel column, gel minitube kit (GEL KIT), and immunochromatographic strip kit. Two IAT techniques (MICRO at 37 °C and GEL KIT with rabbit anti-dog polyvalent reagent) detected autoantibodies against erythrocytes in serum from 53% and 57% of DAT+ dogs, respectively, while other IATs performed less well. Moreover, while the analytic specificity of the IAT methods compared to the DAT ranged from 96–100%, the sensitivity range was only 9–57%. Thus, we still recommend DAT for diagnosis and monitoring of IMHA in dogs but conclude that a positive IAT result may aid diagnostically when serum is available, but fresh red blood cells are not.

Fibroblast growth factor-23 (FGF-23) is a phosphaturic hormone used to monitor chronic kidney disease (CKD) in humans. The aim of this pilot study was to compare three diagnostic assays and to assess how the results correlate with parameters of renal dysfunction in cats. Four groups of 10 cats each were formed retrospectively according to creatinine, based on IRIS staging. FGF-23 was measured using two different ELISAs (MyBioSource and Kainos ELISA FGF-23 Kit) and an automated assay on the DiaSorin Liaison platform. Measurements were performed in 40 cats. Spearman’s rank correlation coefficient showed a strong correlation between the Kainos and DiaSorin assays (ρ = 0.742/p < 0.001) and a low correlation (ρ = 0.443/p = 0.005) between the Kainos and MyBioSource assays. The measurements with the Kainos assay strongly correlated with urea (ρ = 0.835/p < 0.001) and creatinine (ρ = 0.764/p < 0.001), and moderately correlated with SDMA (ρ = 0.580/p < 0.001) and phosphorus (ρ = 0.532/p < 0.001). The results of the MyBioSource and DiaSorin assays only showed a moderate correlation with urea (ρ = 0.624/0.572) and creatinine (ρ = 0.622/0.510) concentrations (p = 0.001 each). The Kainos assay showed the strongest correlation (ρ = 0.806) with the various creatinine concentrations according to the IRIS, followed by the MyBioSource (ρ = 0.663/p < 0.001) and DiaSorin assays (ρ = 0.580/p < 0.001). Overall, the Kainos assay demonstrated the best correlations with both biomarkers and various creatinine concentrations according to the IRIS. Individual assay-based reference values should be established to make a reliable interpretation of FGF-23 levels possible to diagnose or monitor feline CKD.

Abstract Background Blood-feeding arthropods can transmit parasitic, bacterial, or viral pathogens to domestic animals and wildlife. Vector-borne infections are gaining significance because of increasing travel and import of pets from abroad as well as the changing climate in Europe. The main objective of this study was to assess the percentage of cats with positive test results for selected vector-borne pathogens in Germany and explore any possible association of such results with time spent abroad. Methods This retrospective study included test results from cats included in the “Feline Travel Profile” established by the LABOKLIN laboratory at the request of veterinarians in Germany between April 2012 and March 2020. This diagnostic panel includes the direct detection of Hepatozoon spp. and Dirofilaria spp. via PCR as well as indirect detection assays (IFAT) for Ehrlichia spp. and Leishmania spp. The panel was expanded to include an IFAT for Rickettsia spp. from July 2015 onwards. Results A total of 624 cats were tested using the “Feline Travel Profile.” Serum for indirect detection assays was available for all 624 cats; EDTA samples for direct detection methods were available from 618 cats. Positive test results were as follows: Ehrlichia spp. IFAT 73 out of 624 (12%), Leishmania spp. IFAT 22 out of 624 (4%), Hepatozoon spp. PCR 53 out of 618 (9%), Dirofilaria spp. PCR 1 out of 618 cats (0.2%), and Rickettsia spp. IFAT 52 out of 467 cats (11%) tested from July 2015 onwards. Three cats had positive test results for more than one pathogen before 2015. After testing for Rickettsia spp. was included in 2015, 19 cats had positive test results for more than one pathogen (Rickettsia spp. were involved in 14 out of these 19 cats). Conclusions At least one pathogen could be detected in 175 out of 624 cats (28%) via indirect and/or direct detection methods. Four percent had positive test results for more than one pathogen. These data emphasize the importance of considering the above-mentioned vector-borne infections as potential differential diagnoses in clinically symptomatic cats.

German dairy farming has intensified markedly in recent years, and the demand for Veterinary Herd Health Management (VHHM) is rising. To protect farms from epidemics, ensure food safety, and prevent developing of antibiotic resistance, VHHM has been anchored in EU law since April 2021. Via an online survey, distributed by different farmers' organizations, dairy farmers were asked about the cooperation with their veterinarian. The aim was to evaluate farm performance as a function of participation in VHHM. From 216 analyzed questionnaires, 106 respondents participated in VHHM. Results showed that farmers who make use of VHHM and consult their veterinarian in decision-making frequently have the highest 305-day milk yield (305dMY), the lowest bulk tank somatic cell counts, and the lowest age at first calving (AFC). However, these farmers tended to have higher replacement rates and a higher mortality of cows in the period up to 60 days in milk (MORT60DIM). Furthermore, respondents who defined VHHM as “evaluation of herd data, strategic planning” had the highest 305dMY compared with those who defined VHHM through one of the different options given (“pregnancy checks and support in reproduction”/“problem solving”). In the multifactorial regression model, VHHM participating farms had a 660-kg higher 305dMY and 1 month less in AFC, compared with farms not participating in VHHM. However, within the VHHM participants, no association between VHHM practices and performance parameters was found. Further research is needed, to find out if tailored advice of the VHHM approach may show effect herein.

Abstract Background Diagnosis of pancreatic diseases in dogs is still challenging because of variable clinical signs, which do not always correspond with clinical pathology and histopathological findings. Objectives To characterize inflammatory and neoplastic pancreatic diseases of dogs and to correlate these findings with clinical findings and canine pancreatic lipase immunoreactivity (cPLI) results. Animals Tissue specimens and corresponding blood samples from 72 dogs submitted for routine diagnostic testing. Methods Four groups were defined histologically: (1) normal pancreas (n = 40), (2) mild pancreatitis (n = 8), (3) moderate or severe pancreatitis (acute, n = 11; chronic, n = 1), and (4) pancreatic neoplasms (n = 12). An in‐house cPLI ELISA (310 μg/L, pancreatitis) was performed. Results In dogs with normal pancreas, 92.5% of serum cPLI results were within the reference range and significantly lower than in dogs with mild acute pancreatitis, moderate or severe acute pancreatitis and pancreatic tumors. In dogs with moderate or severe acute pancreatitis, cPLI sensitivity was 90.9% (95% confidence interval [CI], 58.7%‐99.8%). Most dogs (9/12) with pancreatic tumors (group 4) had additional pancreatic inflammation and cPLI results were increased in 10 dogs. Conclusions and Clinical Importance High cPLI indicates serious acute pancreatitis but underlying pancreatic neoplasms should also be taken into consideration. This study confirms the relevance of histopathology in the diagnostic evaluation of pancreatic diseases.

Lameness is one of the most challenging problems in the dairy industry. Control is impeded because farmers often underestimate the number of lame cows. The objectives of this study were to assess German farmers' awareness of lameness in their herds and to determine the associations between farmers' awareness and their management practices, farm characteristics as well as with farmers' education, personality traits and attitudes. As a part of a large cross-sectional study, veterinarians visited farms in three structurally different regions of Germany: north (n = 253), east (n = 252), and south (n = 260). The cows (n = 84,998) were scored for locomotion and farmers were asked to estimate the number of cows that were lame or did not walk soundly. The ratio of farmers' estimated prevalence and the veterinarians' observed prevalence (Farmer's Detection Index; FDI) was calculated. The median lameness prevalence assessed by the veterinarians was 23.1, 39.1, and 23.2%, and the median prevalence of lame cows estimated by the farmers was 9.5, 9.5, and 7.1% in the north, east, and south, respectively. On average, farmers were conscious of only 45.3% (north), 24.0% (east), and 30.0% (south) of their lame cows. Farmers managing their herds according to organic principles had a higher FDI than farmers who managed their herds conventionally. Surprisingly, no significant associations between FDI and factors concerning claw health management could be detected. Therefore, increased awareness did not seem to be necessarily linked to improved management. Moreover, the FDI was not significantly associated with farmers' education or herd size. In the south, more extraverted farmers had a lower FDI. Those farmers who totally agreed with the statement, “I am satisfied with my herd's health,” had a lower FDI than farmers who disagreed or were undecided. Moreover, farmers who disagreed or were undecided with the statement, “It affects me to see a cow in pain” had a higher FDI than those farmers who agreed to the statement. The results indicate that poor awareness of lameness was linked to the farmers' attitude and personality. Therefore, new approaches concerning the consultation regarding lameness control, such as the use of Motivational Interviewing, might be useful in the future.

Veterinary Herd Health Management plays an important role in veterinary medicine on dairy farms and has also been mandatory at the European Union level since April 21, 2021. Despite the increasing importance of VHHM, little is known about the extent of utilization of VHHM by dairy farmers and their view on this type of collaboration. Therefore, this cross-sectional study aimed to determine the status quo of the currently practiced VHHM in Germany. For this purpose, an online survey was conducted among dairy farmers in November and December 2020. From 216 analyzed questionnaires, about half (n = 106) of the surveyed dairy farmers used VHHM at different scopes. However, regardless of the group, the term “veterinary herd health management” generally was given most relative importance by the participants as a veterinary service for herd fertility improvement, rather than the actual definition of a holistic approach. In contrast to this, the actual motivation of the VHHM participants, to take part in such a program was primarily based on the desire to safeguard animal health by employing preventive measures, that is, to avoid the occurrence of diseases via improved management and to improve farm performance (and profitability). Dairy farmers who opted for VHHM tended to manage larger higher yielding herds than those who did not. Additionally, the farmers in latter farms were more likely to make joint animal health decisions with their veterinarians. Using a latent class analysis, two groups of farmers among farms that were not currently using VHHM were identified, one of which expressed great interest in using VHHM while the other did not. Since the new legal basis makes the topic even more relevant than before, dairy farmers, animals, and veterinarians might benefit from the study to exploit hidden opportunities for VHHM collaboration.

(1) Background: Devriesea (D.) agamarum is a potential cause of dermatitis and cheilitis in lizards. The aim of this study was to establish a real-time PCR assay for the detection of D. agamarum. (2) Methods: Primers and probe were selected targeting the 16S rRNA gene, using sequences of 16S rRNA genes of D. agamarum as well as of other bacterial species derived from GenBank. The PCR assay was tested with 14 positive controls of different D. agamarum cultures as well as with 34 negative controls of various non-D. agamarum bacterial cultures. Additionally, samples of 38 lizards, mostly Uromastyx spp. and Pogona spp., submitted to a commercial veterinary laboratory were tested for the presence of D. agamarum using the established protocol. (3) Results: Concentrations of as low as 2 × 104 colonies per mL were detectable using dilutions of bacterial cell culture (corresponding to approximately 200 CFU per PCR). The assay resulted in an intraassay percent of coefficient of variation (CV) of 1.31% and an interassay CV of 1.80%. (4) Conclusions: The presented assay is able to detect D. agamarum in clinical samples, decreasing laboratory turn-around time in comparison to conventional culture-based detection methods.

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.

Herpesviruses are important pathogens in tortoises and turtles, yet little is known about the epidemiology of these viruses. We analyzed herpesviruses detected by PCR in samples from captive chelonians in Europe according to virus strain, host species, year and season in which the animal was tested, and country in which the animal was kept. A total of 4,797 samples submitted to a diagnostic laboratory in Europe between January 2016 and December 2020 were evaluated. Of these, 312 (6.50%) were positive for herpesviruses. The types most commonly found were testudinid herpesvirus (TeHV)1 (143 positive, 45.83%) and TeHV3 (153 positive, 49.04%), but also included TeHV2 (1 positive, 0.32%), TeHV4 (3 positive, 0.96%), Terrapene herpesvirus 1 (7 positive, 2.24%), Trachemys herpesvirus 1 (2 positive, 0.64%), and three previously undescribed herpesviruses (0.96%). Herpesviruses were detected in chelonians in the families Testudinidae, Emydidae, Geoemydidae, and in the suborder Pleurodira. Among the species for which 100 samples or more were available, the highest proportions of positive samples (positivity rates) were found in samples from Horsfield's tortoises (Testudo horsfieldii) (14.96%), and radiated tortoises (Astrochelys radiata) (14.05%). Among tortoises (Testudinidae), viruses were most often detected in the spring, while in emydid turtles (Emydidae) they were most often detected in the summer. A comparison of the positivity rates according to country showed significant differences, with the highest rate in samples from Italy (16.01%). This study indicated possible differences in herpesvirus positivity rates depending on host species, virus strain, year of sampling, season, and country of origin. It provides useful information in further understanding fluctuations in infection rates as well as in helping to guide decision making for herpesvirus diagnostics in chelonian patients. It also provides evidence for the international dispersal of herpesviruses with their hosts through international trade.

Abstract Background Ferlaviruses are important pathogens in snakes often associated with respiratory and neurological disease. The detection of ferlaviral RNA by PCR is considered to be the most reliable method for the diagnosis of infection. The PCRs that have been used most commonly for this purpose have not been properly assessed to determine their sensitivity, specificity and ability to detect the known genetic diversity of this group of viruses. The aim of this study was to compare three published PCR protocols so that a single method could be recommended to laboratories that perform this testing. Results Comparisons were carried out using cell culture isolates and tissues from snakes infected with specific virus genotypes. A single round PCR targeting a short segment of the viral polymerase (L) gene provided the highest sensitivity and specificity, and detected isolated ferlaviruses from all four described genogroups, as well as from tissues of infected snakes. Conclusion A broadly-reactive PCR for the detection of all known ferlaviruses was found to provide a good combination of detection limit, specificity and speed. Based on these criteria, this method is recommended for the diagnosis of ferlavirus infections.

Abstract Background Canine vector-borne infections have gained importance in Germany due to growing tourist traffic and an increased import of dogs from abroad. Endemic regions for pathogens such as Leishmania infantum, Hepatozoon canis, Ehrlichia canis, Anaplasma platys and Dirofilaria spp. are the Mediterranean area and southeastern Europe. Babesia species and Anaplasma phagocytophilum are present all over Europe. The objective of this retrospective study was to evaluate the prevalence of vector-borne infections in dogs imported from defined endemic countries in the Mediterranean area and southeastern Europe. Methods Medical records and laboratory test results of 345 dogs that were imported to Germany from 17 endemic countries and that were presented to the Small Animal Clinic at Freie Universität Berlin between 2007 and 2015 were retrospectively reviewed. A total of 1368 test results from external laboratories were descriptively analysed including 576 and 792 test results of direct and indirect detection methods, respectively. Results Overall, 35% (122/345 dogs) were positive for at least one pathogen. Concurrent infections with two to four pathogens were detected in 8% of the dogs (27/345). The positive results were: L. infantum 21% (66/314 dogs; methods: PCR 20/79, IFAT or ELISA 63/308 dogs), E. canis 16% (45/278 dogs; methods: PCR 8/68, IFAT 43/257 dogs), H. canis 11% (3/28 dogs; method: PCR), Babesia spp. 10% (25/251 dogs; methods: Babesia spp. PCR 3/98, B. canis/vogeli IFAT or ELISA 22/214 and B. gibsoni IFAT 0/13 dogs), Dirofilaria spp. 7% (13/178 dogs; methods: D. immitis Ag-ELISA 8/156, Knott’s test 7/95, microfilariae PCR 5/23 dogs) and A. platys 5% (1/21 dogs; method: PCR). None of 8 tested dogs were positive in a combined Babesia spp./Hepatozoon spp. PCR test. Conclusions Dogs, which are imported from countries which are endemic for vector-borne infections should be thoroughly tested using direct and indirect detection methods. Potential owners of imported dogs should be informed about the diseases, risks and incubation periods.

Background: Kratom is a psychoactive plant preparation originating from Southeast Asia. It has been used as a recreational and performance drug in Southeast Asia, and is now increasingly used in Europe and the U.S. Case report: We describe the case of a 63-year-old man who presented for treatment after his long-term Kratom use failed as a self-management for persistent major depression (ICD 10: F33.2) and a generalized anxiety disorder (ICD-10: F41.1). The failure coincided with emerging stress at the beginning of the COVID-19 pandemic. The patient suffered from childhood on from ruminative thinking and depressive mood, which was treated in several settings during his life. He started to use alcohol to control his depression, but developed an alcohol addiction. This was successfully treated and the patient remained abstinent for more than 25 years afterwards. About 7 years ago, he started to use Kratom 3–4 times daily on a regular, but constant rate. Kratom use worked efficiently as a self-management of his depression with no escalation of dosing. It was also very effective in reducing Morbus Menière symptoms of tinnitus and sudden hearing loss, which eventually allowed regular performance as a caregiver in a demanding job on shift-work. During recently increased stress load in the work environment and the COVID-19 pandemic, the established Kratom doses failed to control hyperarousal and mental nervousness. The patient was treatment seeking and subsequently detoxified from Kratom. Anxiety- and depression management was shifted to treatment attempts with Lorazepam, Venlafaxine, Opipramol, Mirtazapine and psychotherapy. Conclusion: Kratom instrumentalization for self-management of depression and anxiety may effectively work without causing escalation of drug use and addiction, but may be limited by a temporary increase in psychological stress load and a relapse into major depression and generalized anxiety disorder.

The adrenal glands play a major role in metabolic processes, and both excess and insufficient serum cortisol concentrations can lead to serious metabolic consequences. Hyper- and hypoadrenocorticism represent a diagnostic and therapeutic challenge. Serum samples from dogs with untreated hyperadrenocorticism (n = 27), hyperadrenocorticism undergoing treatment (n = 28), as well as with untreated (n = 35) and treated hypoadrenocorticism (n = 23) were analyzed and compared to apparently healthy dogs (n = 40). A validated targeted proton nuclear magnetic resonance (1H NMR) platform was used to quantify 123 parameters. Principal component analysis separated the untreated endocrinopathies. The serum samples of dogs with untreated endocrinopathies showed various metabolic abnormalities with often contrasting results particularly in serum concentrations of fatty acids, and high- and low-density lipoproteins and their constituents, which were predominantly increased in hyperadrenocorticism and decreased in hypoadrenocorticism, while amino acid concentrations changed in various directions. Many observed serum metabolic abnormalities tended to normalize with medical treatment, but normalization was incomplete when compared to levels in apparently healthy dogs. Application of machine learning models based on the metabolomics data showed good classification, with misclassifications primarily observed in treated groups. Characterization of metabolic changes enhances our understanding of these endocrinopathies. Further assessment of the recognized incomplete reversal of metabolic alterations during medical treatment may improve disease management.

Lameness affects dairy cows worldwide and is usually associated with pain. Behavioral differences in lame compared to non-lame tie-stall-housed dairy cows might be less pronounced than in free-stall-housed, since the principle demands to a cow's locomotor system and thus the impact of lameness on behavior seem to be lower in tie stalls. Behavioral differences between lame and non-lame cows might be used to estimate the impact of lameness on the well-being of tied dairy cows. In the current study, lame cows were categorized as locomotion scoring between 2.25 and 3.25 on a 1–5 scale. The aim was to compare the eating, rumination and lying behavior of lame cows against non-lame tied dairy cows, in order to draw conclusions on the association of lameness, behavior and well-being in tied dairy cows. The eating and rumination behavior of 26, the lying behavior of 30, and the relative upright and lying activities of 25 matched case-control pairs were analyzed, considering the matching criteria farm, breed-type, and parity-group. Lame cows had fewer [mean of the pairwise differences (case–control) (meandiff) = −2.6 bouts, CI95% (−3.8–−1.4) bouts, p = 0.001], but longer lying bouts [meandiff = 26.7 min per bout, CI95% (10.1–43.4) min per bout, p = 0.006]. The lying time was shorter [meandiff = −64.7 min, CI95% (−104.4–−24.9) min, p = 0.006] in lame cows compared to their non-lame controls. Lame cows had a shorter eating time [meandiff = −27.7 min, CI95% (−51.5–−4.0) min, p = 0.042] and spent a larger proportion of their upright time ruminating [meandiff = 7.2%, CI95% (3.2–11.1)%, p = 0.001] instead of eating. The results of the current study indicate that the eating, rumination, and lying behavior of lame tied dairy cows is altered. These findings indicate that slight and moderate lameness (locomotion score between 2.25 and 3.25 on a 1–5 scale) are likely to be associated with an impaired well-being in affected tied dairy cows. This underlines the need to continuously reduce the lameness prevalence and severity in tied dairy herds.

Background: Kratom is a Southeast Asian plant, which is widely used in this region, and making an increasing appearance in Europe and the US. Case report: We present the case of a 26-year-old man in Substitol-assisted treatment of excessive Kratom and Tilidin use expressing the wish for a drug-free management of a chronic pain condition. After an accidental calcaneus impression fracture, the patient was suffering from severe chronic pain and anxiety of further accidents. This was managed initially with Tilidin. Resulting from the wish to self-manage the pain condition in a way that permitted continuation of a job, the patient searched for a ‘natural’ treatment alternative obtained from an Internet vendor. He successfully instrumentalized Kratom for 3 years with daily consumption intermixed with occasional Tilidin for pain management. However, the dose of Kratom was increased considerably up to a level of effect reversal, when no analgesic and behaviorally activating effects occurred any more, but only intense drowsiness. The patient was treatment seeking and subsequently detoxified from Kratom and Tilidin. Pain management was shifted to retarded morphine. Conclusion: Kratom instrumentalization for pain management might appear to be more problematic for addiction development than when its use is established for other consumption motives.

More than twelve blood group systems have been described in dogs, but little is known about their distribution frequencies within breed populations. Here, we report on an extensive typing survey carried out using available reagents and either established or new clinical kits in purebred dogs from Germany. Leftover anticoagulated blood samples were examined using an immunochromatographic strip method for DEA 1, a gel column technique for Dal and Kai 1/2, and new card agglutination tests for DEA 4 and DEA 5 (which were partially compared with the gel column technique). Monoclonal antibodies were used for DEA 1 and Kai 1/2 typing, and polyclonal antibodies were used for all other types. Among the 206 dogs, 59.2% were DEA 1+, 100% DEA 4+, 9% (Card)/11% (Gel) DEA 5+, 89.3% Dal+, 96.6% Kai 1+, and 2.9% Kai 2+. None of the dogs were Kai 1+/2+, and only one was Kai 1–/2–. Dal– dogs were found in several breeds. Erythrocytes from most DEA 1+ dogs bound strongly on the strips. The agglutination reactions for DEA 5 on the new card tests were generally less than those on the gel column. The blood group pattern DEA 4+, DEA 5–, Dal+, Kai 1+/2– and either DEA 1+ or DEA 1– was found among 80% of the dogs. In this first extensive blood typing survey of purebred dogs from Europe, the proportions of positive and negative blood types were similar to those found in the United States and, for DEA 1, were also similar to those from other European countries, with considerable breed variation in blood types. The newer typing techniques seem to work well and will likely be useful for detecting and preventing specific blood type incompatibilities in the clinic.