Tissue S100/calgranulin expression and blood neutrophil-to-lymphocyte ratio (NLR) in prostatic disorders in dogs.

BACKGROUND: Prostatic carcinoma (PCA) is a rare but severe condition in dogs that is similar to the androgen-independent form of PCA in men. In contrast to humans, PCA is difficult to diagnose in dogs as reliable biomarkers, available for PCA screening in human medicine, are currently lacking in small animal oncology. Calprotectin (S100A8/A9) and S100A12 are Ca2+-binding proteins of the innate immune system with promising potential to distinguish malignant from benign urogenital tract conditions, similar to the blood neutrophil-to-lymphocyte-ratio (NLR). However, both have not yet been extensively investigated in dogs with PCA. Thus, this study aimed to evaluate the expression of the S100/calgranulins (calprotectin, S100A12, and their ratio [Cal-ratio]) in prostatic biopsies from nine dogs with PCA and compare them to those in dogs with benign prostatic lesions (eight dogs with prostatitis and ten dogs with benign prostatic hyperplasia [BPH]) as well as five healthy controls. In addition, blood NLRs were investigated in twelve dogs with PCA and 22 dogs with benign prostatic conditions. RESULTS: Tissue S100A8/A9+ cell counts did not differ significantly between tissue from PCA and prostatitis cases (P = 0.0659) but were significantly higher in dogs with prostatitis than BPH (P = 0.0013) or controls (P = 0.0033). S100A12+ cell counts were significantly lower in PCA tissues than in prostatitis tissue (P = 0.0458) but did not differ compared to BPH tissue (P = 0.6499) or tissue from controls (P = 0.0622). Cal-ratios did not differ significantly among the groups but were highest in prostatitis tissues and significantly higher in those dogs with poor prostatitis outcomes than in patients that were still alive at the end of the study (P = 0.0455). Blood NLR strongly correlated with prostatic tissue S100A8/A9+ cell counts in dogs with PCA (ρ = 0.81, P = 0.0499) but did not differ among the disease groups of dogs. CONCLUSIONS: This study suggests that the S100/calgranulins play a role in malignant (PCA) and benign (prostatic inflammation) prostatic conditions and supports previous results in lower urinary tract conditions in dogs. These molecules might be linked to the inflammatory environment with potential effects on the inflammasome. The blood NLR does not appear to aid in distinguishing prostatic conditions in dogs. Further investigation of the S100/calgranulin pathways and their role in modulation of tumor development, progression, and metastasis in PCA is warranted.

MOCOS-associated renal syndrome in a Brown Swiss cattle.

BACKGROUND: A recessive form of MOCOS-associated xanthinuria type II is described in Tyrolean grey cattle. A similar case was identified in a 5-month-old Brown Swiss calf with hoof overgrowth, rough coat, urine sediment, and pneumonia. HYPOTHESIS/OBJECTIVES: To characterize the disease phenotype, to evaluate its genetic etiology, and to determine the prevalence of the deleterious allele in the Brown Swiss population. ANIMALS: An affected calf, its parents, and 65 441 Swiss dairy cattle. METHODS: The affected animal was clinically examined and necropsied. Microarray genotyping was used to determine the genotypes and to assess the frequency of the MOCOS allele in a Brown Swiss control cohort. RESULTS: Ultrasonography revealed hyperechoic renal pyramids with multifocal distal shadowing and echogenic sediment in the urinary bladder. Necropsy revealed suppurative bronchopneumonia and urolithiasis. Histology revealed numerous nephroliths with multifocal chronic lymphohistiocytic interstitial infiltrates, fibrosis, tubular degeneration, chronic multifocal glomerulonephritis with sclerosis, and bilateral hydronephrosis. Dysplastic changes were observed in the corium of the claw and the cornea. Genetic testing identified the homozygous presence of a known MOCOS frameshift variant in the case. Both parents were heterozygous and the prevalence of carriers in genotyped Brown Swiss cattle was 1.4% (342/24337). CONCLUSIONS AND CLINICAL IMPORTANCE: The findings were consistent with the diagnosis of a recessive renal syndrome similar to xanthinuria type II described in Tyrolean grey cattle. The prevalence of the deleterious MOCOS allele is low in the Brown Swiss breed. However, mating of carriers should be avoided to prevent further losses.

Treatment of digital dermatitis using salicylic acid in European bison (Bison bonasus) reveals promising results.

Digital dermatitis (DD) associated with the presence of multiple Treponema spp. was recently described for the first time in European bison (Bison bonasus). DD is characterized by skin inflammation in the distal foot area in various ungulates. The objective of this proof of concept study was to test a treatment protocol adopted from cattle for its applicability in this wildlife species using five animals. Keratolytic salicylic acid paste was administered topically under bandages for seven days to enable removal of the affected skin. All interventions were performed under general anesthesia. To evaluate the treatment efficacy, photographs and biopsies were taken pre- and post-treatment. The biopsies were examined histologically, by PCR for the presence of different bacterial species, by Treponema-specific fluorescent in situ hybridization (FISH), and by transmission electron microscopy. Based on photographs, complete clinical healing of the 15 feet with macroscopical DD lesions was achieved. Histological examination showed mild to moderate dermatitis in 17/20 feet before, and in 12/20 feet after treatment. 17/20 feet were Treponema spp. PCR positive before, and none was positive after treatment. Dichelobacter nodosus, Fusobacterium necrophorum, and Porphyromonas levii could not be detected in any of the samples. By FISH and electron microscopy, Treponema spp. could be visualized in the stratum corneum before, but not after treatment. These results suggest that this treatment method can be applied as standard practice prior to transporting DD affected European bison to prevent the spread of this contagious disease.

"Non-healing" claw horn lesions in dairy cows: Clinical, histopathological and molecular biological characterization of four cases.

The increasing prevalence of bovine digital dermatitis (BDD) contributes to a higher occurrence of secondary infections of exposed corium with Treponema spp. in bovine claws. "Non-healing" claw horn lesions (NHL) clinically resemble BDD lesions. They are severe, cause chronic lameness, and may persist for several months. They poorly respond to standard treatments of BDD and represent a serious welfare issue. In this study, four cases of NHL were classified clinically either as BDD-associated axial horn fissures (BDD-HFA; n = 3) or BDD-associated sole ulcer (BDD-SU; n = 1). In all four cases, pronounced multifocal keratinolysis of the stratum corneum, ulceration, and severe chronic lymphoplasmacytic perivascular to interstitial dermatitis were observed. All lesional samples tested positive for Treponema spp., Fusobacterium (F.) necrophorum, and Porphyromonas (P.) levii by PCRs. BDD-HFA lesions contained Treponema pedis as revealed by genetic identities of 93, 99, and 100%. Treponemes in the BDD-SU lesion were 94% homologous to Treponema phylotype PT3. Fluorescent in situ hybridization (FISH) revealed extensive epidermal infiltration by treponemes that made up > 90% of the total bacterial population in all four lesions. FISH also tested positive for P. levii and negative for F. necrophorum in all four cases, whilst only one BDD-HFA contained Dichelobacter nodosus. Our data point to BDD-associated treponemes and P. levii constituting potential etiological agents in the development of "non-healing" claw horn lesions in cattle.

Tissue S100/calgranulin expression and blood neutrophil-to-lymphocyte ratio (NLR) in dogs with lower urinary tract urothelial carcinoma.

BACKGROUND: Urothelial carcinoma (UC) is the most common neoplasm of the canine lower urinary tract, affecting approximately 2% of dogs. Elderly female patients of certain breeds are predisposed, and clinical signs of UC can easily be confused with urinary tract infection or urolithiasis. Diagnosis and treatment are challenging given the lack of disease-specific markers and treatments. The S100A8/A9 complex and S100A12 protein are Ca2+-binding proteins expressed by cells of the innate immune system and have shown promise as urinary screening markers for UC. The neutrophil-to-lymphocyte ratio (NLR) can also aid in distinguishing certain neoplastic from inflammatory conditions. Our study aimed to evaluate the tissue expression of S100/calgranulins and the blood NLR in dogs with UC. Urinary bladder and/or urethral tissue samples from dogs with UC (n = 10), non-neoplastic inflammatory lesions (NNUTD; n = 6), and no histologic changes (n = 11) were evaluated using immunohistochemistry. Blood NLRs were analyzed in dogs with UC (n = 22) or NNUTD (n = 26). RESULTS: Tissue S100A12-positive cell counts were significantly higher in dogs with lower urinary tract disease than healthy controls (P = 0.0267 for UC, P = 0.0049 for NNUTD), with no significant difference between UC and NNUTD patients. Tissue S100A8/A9-positivity appeared to be higher with NNUTD than UC, but this difference did not reach statistical significance. The S100A8/A9+-to-S100A12+ ratio was significantly decreased in neoplastic and inflamed lower urinary tract tissue compared to histologically normal specimens (P = 0.0062 for UC, P = 0.0030 for NNUTD). NLRs were significantly higher in dogs with UC than in dogs with NNUTD, and a cut-off NLR of ≤ 2.83 distinguished UC from NNUTD with 41% sensitivity and 100% specificity. Higher NLRs were also associated with a poor overall survival time (P = 0.0417). CONCLUSIONS: These results confirm that the S100/calgranulins play a role in the immune response to inflammatory and neoplastic lower urinary tract diseases in dogs, but the tissue expression of these proteins appears to differ from their concentrations reported in urine samples. Further investigations of the S100/calgranulin pathways in UC and their potential as diagnostic or prognostic tools and potential therapeutic targets are warranted. The NLR as a routinely available marker might be a useful surrogate to distinguish UC from inflammatory conditions.

Intestinal S100/Calgranulin Expression in Cats with Chronic Inflammatory Enteropathy and Intestinal Lymphoma.

Diagnosing chronic inflammatory enteropathies (CIE) in cats and differentiation from intestinal lymphoma (IL) using currently available diagnostics is challenging. Intestinally expressed S100/calgranulins, measured in fecal samples, appear to be useful non-invasive biomarkers for canine CIE but have not been evaluated in cats. We hypothesized S100/calgranulins to play a role in the pathogenesis of feline chronic enteropathies (FCE) and to correlate with clinical and/or histologic disease severity. This retrospective case-control study included patient data and gastrointestinal (GI) tissues from 16 cats with CIE, 8 cats with IL, and 16 controls with no clinical signs of GI disease. GI tissue biopsies were immunohistochemically stained using polyclonal α-S100A8/A9 and α-S100A12 antibodies. S100A8/A9+ and S100A12+ cells were detected in all GI segments, with few significant differences between CIE, IL, and controls and no difference between diseased groups. Segmental inflammatory lesions were moderately to strongly correlated with increased S100/calgranulin-positive cell counts. Clinical disease severity correlated with S100A12+ cell counts in cats with IL (ρ = 0.69, p = 0.042) and more severe diarrhea with colonic lamina propria S100A12+ cells with CIE (ρ = 0.78, p = 0.021) and duodenal S100A8/A9+ cells with IL (ρ = 0.71, p = 0.032). These findings suggest a role of the S100/calgranulins in the pathogenesis of the spectrum of FCE, including CIE and IL.

Proof of an optimized salicylic acid paste-based treatment concept of ulcerative M2-stage digital dermatitis lesions in 21 dairy cows.

The efficacy of salicylic acid paste (SA) in the treatment of ulcerative bovine digital dermatitis (BDD) was assessed by combining clinical and histopathological analyses with molecular biological techniques. The latter were conducted in a blinded manner to reach maximum objectivity. Prior to treatment, M2-stage BDD lesions (n = 26, diagnosed in 21 dairy cows) exhibited ulceration, with severe perivascular, chronic, lymphoplasmacytic dermatitis and extensive keratinolysis being noted in most cases. Pretreatment biopsy samples (n = 12) followed by povidone-iodine ointment under bandage for one week before administration of SA paste were tested positive for Treponema spp. by blinded PCR and fluorescent in situ hybridization (FISH). Subsequent treatment consisted of application of SA and bandaging at weekly intervals until lesions had completely resolved. The treatment duration ranged between 2 and 4 weeks. Complete healing was achieved in 100% of cases, with 2/21 animals requiring a second round of treatment upon disease reoccurrence. Importantly, only 3/26 biopsies taken from previously affected sites still tested positive by Treponema PCR, and in another biopsy, the outermost layers of the stratum corneum scored weakly positive by Treponema-specific FISH. None of these Treponema DNA-positive biopsies showed signs of ulceration. One case exhibited focal keratinolysis. Positive PCR or FISH in these cases may have arisen from DNA traces of dead bacteria or environmental contamination during biopsy harvesting. To our knowledge, this is the first study on blinded molecular biological monitoring of the therapeutic efficacy of SA with respect to treponemal infection, and on complete BDD M2-stage remission in all animals achieved by SA treatment according to an optimized protocol. Although the etiology of BDD is considered as multifactorial, our data further support the concept that treponemes have a decisive role in BDD pathogenesis.

Establishment of well-differentiated camelid airway cultures to study Middle East respiratory syndrome coronavirus.

In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in Saudi Arabia and was mostly associated with severe respiratory illness in humans. Dromedary camels are the zoonotic reservoir for MERS-CoV. To investigate the biology of MERS-CoV in camelids, we developed a well-differentiated airway epithelial cell (AEC) culture model for Llama glama and Camelus bactrianus. Histological characterization revealed progressive epithelial cellular differentiation with well-resemblance to autologous ex vivo tissues. We demonstrate that MERS-CoV displays a divergent cell tropism and replication kinetics profile in both AEC models. Furthermore, we observed that in the camelid AEC models MERS-CoV replication can be inhibited by both type I and III interferons (IFNs). In conclusion, we successfully established camelid AEC cultures that recapitulate the in vivo airway epithelium and reflect MERS-CoV infection in vivo. In combination with human AEC cultures, this system allows detailed characterization of the molecular basis of MERS-CoV cross-species transmission in respiratory epithelium.

Dilated cardiomyopathy in a cat with congenital hyposomatotropism.

Case summary: A 7-month-old domestic shorthair cat was presented for evaluation of stunted growth, recurrent hypoglycaemia during the first months of its life and altered mentation. Complete blood count and biochemistry were unremarkable, except for mildly elevated serum creatinine concentration (despite low muscle mass) and concurrent isosthenuria. Hyposomatotropism was diagnosed based on persistent low circulating insulin-like growth factor 1 concentrations and a lack of response of circulating growth hormone (GH) concentration after the administration of GH-releasing hormone. Other endocrinopathies such as hypothyroidism and hypoadrenocorticism were excluded. MRI of the brain revealed a fluid-filled empty sella tursica, consistent with a pituitary cyst and atrophy/hypoplasia of the pituitary. Echocardiography was unremarkable at the time of diagnosis of hyposomatotropism. Three months later, ovariohysterectomy revealed immature ovaries, raising the suspicion of luteinising and follicle-stimulating hormone deficiency. At 1 year of age, the cat developed congestive heart failure secondarily to dilated cardiomyopathy (DCM) with severely reduced left ventricular systolic function and died a few days later. Pathology showed atrophy of the adenohypophysis, epithelial delineation of the pituitary cysts, mild cardiomegaly, multifocal fibrosis of the left ventricle and a mild, multifocal, chronic epicarditis. Relevance and novel information: GH deficiency is a very rare endocrinopathy in cats. This is the first case to describe the development of DCM with concurrent hyposomatotropism, which has previously been reported in human medicine. Other notable abnormalities that could be related to GH deficiency are juvenile self-limiting hypoglycaemia, behavioural changes and possible nephropathy.

Outbreak of equine coronavirus disease in adult horses, Switzerland 2021.

Coronaviruses are causing severe respiratory and enteric diseases in humans and animals. Here, we report an outbreak of equine coronavirus disease in adult horses, detected by a voluntary syndromic surveillance scheme for equine diseases in Switzerland. This scheme allowed a rapid concerted action to diagnose and contain the disease.

Computer-assisted mitotic count using a deep learning-based algorithm improves interobserver reproducibility and accuracy.

The mitotic count (MC) is an important histological parameter for prognostication of malignant neoplasms. However, it has inter- and intraobserver discrepancies due to difficulties in selecting the region of interest (MC-ROI) and in identifying or classifying mitotic figures (MFs). Recent progress in the field of artificial intelligence has allowed the development of high-performance algorithms that may improve standardization of the MC. As algorithmic predictions are not flawless, computer-assisted review by pathologists may ensure reliability. In the present study, we compared partial (MC-ROI preselection) and full (additional visualization of MF candidates and display of algorithmic confidence values) computer-assisted MC analysis to the routine (unaided) MC analysis by 23 pathologists for whole-slide images of 50 canine cutaneous mast cell tumors (ccMCTs). Algorithmic predictions aimed to assist pathologists in detecting mitotic hotspot locations, reducing omission of MFs, and improving classification against imposters. The interobserver consistency for the MC significantly increased with computer assistance (interobserver correlation coefficient, ICC = 0.92) compared to the unaided approach (ICC = 0.70). Classification into prognostic stratifications had a higher accuracy with computer assistance. The algorithmically preselected hotspot MC-ROIs had a consistently higher MCs than the manually selected MC-ROIs. Compared to a ground truth (developed with immunohistochemistry for phosphohistone H3), pathologist performance in detecting individual MF was augmented when using computer assistance (F1-score of 0.68 increased to 0.79) with a reduction in false negatives by 38%. The results of this study demonstrate that computer assistance may lead to more reproducible and accurate MCs in ccMCTs.

Krueppel-Like Factor 4 Expression in Phagocytes Regulates Early Inflammatory Response and Disease Severity in Pneumococcal Pneumonia.

The transcription factor Krueppel-like factor (KLF) 4 fosters the pro-inflammatory immune response in macrophages and polymorphonuclear neutrophils (PMNs) when stimulated with Streptococcus pneumoniae, the main causative pathogen of community-acquired pneumonia (CAP). Here, we investigated the impact of KLF4 expression in myeloid cells such as macrophages and PMNs on inflammatory response and disease severity in a pneumococcal pneumonia mouse model and in patients admitted to hospital with CAP. We found that mice with a myeloid-specific knockout of KLF4 mount an insufficient early immune response with reduced levels of pro-inflammatory cytokines and increased levels of the anti-inflammatory cytokine interleukin (IL) 10 in bronchoalveolar lavage fluid and plasma and an impaired bacterial clearance from the lungs 24 hours after infection with S. pneumoniae. This results in higher rates of bacteremia, increased lung tissue damage, more severe symptoms of infection and reduced survival. Higher KLF4 gene expression levels in the peripheral blood of patients with CAP at hospital admission correlate with a favourable clinical presentation (lower sequential organ failure assessment (SOFA) score), lower serum levels of IL-10 at admission, shorter hospital stay and lower mortality or requirement of intensive care unit treatment within 28 days after admission. Thus, KLF4 in myeloid cells such as macrophages and PMNs is an important regulator of the early pro-inflammatory immune response and, therefore, a potentially interesting target for therapeutic interventions in pneumococcal pneumonia.

Detection of treponemes in digital dermatitis lesions of captive European bison (Bison bonasus).

A newly-discovered foot disease of unknown origin in captive European Bison (Bison bonasus) was recently detected at Berne Animal Park. Dermatitis of the interdigital cleft of varying degrees of severity was diagnosed in all animals (n = 10). The aim of this study was to describe the gross and histological lesions of the interdigital cleft found in 10 captive European bison and to identify involved potential pathogens in affected feet using molecular-based methods for Treponema spp., Dichelobacter nodosus and Fusobacterium necrophorum. Lesions were scored according to the degree of gross pathology at limb level. In a single animal, the gross lesions were restricted to focal lesions on the dorsal aspect of the digital skin of each foot (score 1), whereas all other animals showed at least one foot with extended lesions including the interdigital cleft (score 2). The presence of viable spirochaetes was observed in all animals using dark field microscopy. Applying fluorescence in situ hybridisation (FISH) on biopsies, Treponema spp. were identified, infiltrating the skin lesions in varying numbers in nine animals. Nested PCRs for Treponema medium, Treponema phagedenis and Treponema pedis of swab samples showed three positive animals out of ten for the latter two, whereas pooled biopsy samples were positive in all ten animals for at least T. phagedenis (9/10) and/or T. pedis (7/10), while all samples were negative for T. medium. However, none of these Treponema species could be isolated and sequence analysis of the amplified products showed 100% match of 365 base pairs (bp) to Treponema phylotype PT3 and almost full match (530 of 532 bp, 99.6%) to Treponema phylotype PT13. The presence of T. phagedenis, PT3 and PT13 phylotypes was confirmed by FISH analyses. The phylotypes of T. phagedenis were present in all hybridized positive biopsies of Treponema spp., and PT13 and PT3 were less abundant. Neither D. nodosus nor F. necrophorum were detected. The histological Treponema score was mostly mild. Digital dermatitis in captive European Bison is contagious and differs from bovine digital dermatitis, concerning associated pathogens as well as gross appearance.

GWAS Hits for Bilateral Convergent Strabismus with Exophthalmos in Holstein Cattle Using Imputed Sequence Level Genotypes.

Bilateral convergent strabismus with exophthalmos (BCSE) is a malformation of the eyes and is recognized as a mild but progressive disorder that affects cattle in the first two years of life. This most likely inherited disorder is rarely described in cattle resembling autosomal dominantly inherited forms of human progressive external ophthalmoplegia (PEO). In German Braunvieh cattle, two linked genome regions were found that could be responsible for the development and/or progression of BCSE. The goal of this study was to phenotypically characterize BCSE in Holstein cattle from Germany and Switzerland as well as to identify associated genome regions by GWAS. The clinicopathological phenotype of 52 BCSE-affected Holstein cattle was in accordance with the phenotype described in German Braunvieh cattle, but in addition, signs of degeneration and cellular infiltration in the eye muscles were found. By using imputed sequence level genotype data, three genome-wide significant GWAS hits were revealed on different chromosomes that were not detected by initial GWAS based on high density SNP array data highlighting the usefulness of this approach for mapping studies. The associated genome regions include the ABCC4 gene as well as markers adjacent to the NCOR2 and DNAJC3 genes all illustrating possible functional candidate genes. Our results challenge a monogenic mode of inheritance and indicate a more complex inheritance of BCSE in Holstein cattle. Furthermore, in comparison to previous results from German Braunvieh cattle, it illustrates an obvious genetic heterogeneity causing BSCE in cattle. Subsequent whole genome sequencing (WGS)-based analyses might elucidate pathogenic variants in the future.

Causes of Abortions in South American Camelids in Switzerland-Cases and Questionnaire.

Over the last decade, South American camelids (SAC) have gained increasing popularity in Switzerland. They are used for several purposes such as fiber and meat production, as companion or guard animals and for trekking activities. The purpose of this study was to investigate the frequency and reasons for pregnancy loss and perinatal death in SAC herds. Within the scope of this study, early embryonic losses could not be identified, as pregnancy examinations by ultrasonography are not performed routinely. Aborted and stillborn fetuses were collected, necropsied and analyzed for infectious abortifacients. A nationwide survey among breeders was carried out. During a 1.5-year period, only eight cases of aborted or stillborn alpaca and llama (out of a population of 6550 animals) were reported by the breeders, and their causes were subsequently analyzed. In half of the cases, Coxiella burnetii was identified in the fetoplacental material. Abortions and stillbirths were reported to be rare in Swiss herds. As a conclusion, recording of embryonic losses through ultrasound training of veterinarians should be impaired and breeders motivated to have abortions and perinatal mortality examined. Special focus should be laid on C. burnetii due to its zoonotic risk.

Susceptibility of Well-Differentiated Airway Epithelial Cell Cultures from Domestic and Wild Animals to Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally, and the number of worldwide cases continues to rise. The zoonotic origins of SARS-CoV-2 and its intermediate and potential spillback host reservoirs, besides humans, remain largely unknown. Because of ethical and experimental constraints and more important, to reduce and refine animal experimentation, we used our repository of well-differentiated airway epithelial cell (AEC) cultures from various domesticated and wildlife animal species to assess their susceptibility to SARS-CoV-2. We observed that SARS-CoV-2 replicated efficiently only in monkey and cat AEC culture models. Whole-genome sequencing of progeny viruses revealed no obvious signs of nucleotide transitions required for SARS-CoV-2 to productively infect monkey and cat AEC cultures. Our findings, together with previous reports of human-to-animal spillover events, warrant close surveillance to determine the potential role of cats, monkeys, and closely related species as spillback reservoirs for SARS-CoV-2.

A filter-assisted culture method for isolation of Treponema spp. from bovine digital dermatitis and their identification by MALDI-TOF MS.

Digital dermatitis (DD) is a major infectious foot disease of cattle worldwide. Some DD stages are associated with lameness, and the disease has significant economic and animal welfare consequences. The pathogenesis of the disease is not yet fully understood, but Treponema spp. have been associated consistently with clinical cases. Isolation of these fastidious bacteria is difficult and cumbersome. We describe an improved method enabling the culturing of the 3 Treponema spp. (T. pedis, T. phagedenis, and T. medium) from bovine foot specimens derived from DD lesions, using a combination of membrane filtering and subsequent growth on selective agar media. The entire procedure from sampling to verification of individual Treponema spp. takes up to 24 d. In addition, we established a MALDI-TOF MS-based identification method to be applied for confirmation of the different Treponema spp. This scheme provides an unambiguous, simple, and straightforward identification procedure for DD-associated Treponema spp.

Fatal avian malaria in captive Atlantic puffins (Fratercula arctica) in Switzerland.

Avian malaria is a vector-borne disease caused by Plasmodium species, which may affect a broad spectrum of bird families worldwide. In most endemic and migratory birds, Plasmodium infections seem not to cause severe harm; however, non-indigenous species kept in human care such as penguins may experience high morbidity and mortality rates. Fatal avian malaria may also occur in other non-native seabirds such as puffins (Fratercula spp.), but reported cases are scarce. The aim of this study was to analyze seven cases of sudden death in captive Atlantic puffins (Fratercula arctica) at Berne Animal Park in Switzerland between 2010 and 2020, and to determine the involvement of haemosporidian parasites in the fatal outcome. In all cases, lymphoplasmacytic inflammation, necrotic lesions in several organs and presence of protozoan stages within tissues/erythrocytes or accumulation of iron-based pigment were observed histologically. A one-step multiplex PCR designed to simultaneously detect and discriminate haemosporidia belonging to the genera Plasmodium, Haemoproteus and Leucocytozoon, and a nested PCR detecting Plasmodium and Haemoproteus infections were performed on DNA extracted from formalin-fixed and paraffin-embedded (FFPE) or fresh liver and spleen tissues from five and two birds, respectively. Plasmodium spp. DNA was detected in the tissues from six of seven birds by the one-step multiplex PCR and in five of seven individuals by the nested PCR protocol. Direct sequencing of the amplification products allowed the molecular identification of Plasmodium relictum SGS1 as the involved species in three individuals and Plasmodium matutinum LINN1 in two of these fatal cases. In one bird, no haemosporidian DNA could be amplified from FFPE tissues despite of suggestive histopathological findings. These results indicate that avian malaria represents an important cause of death in captive puffins and it should be considered as a differential diagnosis in unclear or fatal cases in this threatened bird species.

Factors associated with cattle necropsy submissions in Switzerland, and their importance for surveillance.

Pathology data have been reported to be important for surveillance, as they are crucial for correctly recognizing and identifying new or re-emerging diseases in animal populations. However, there are no reports in the literature of necropsy data being compared or complemented with other data. In our study, we compared cattle necropsy reports extracted from 3 laboratories with the Swiss fallen stock data and clinical data collected by the association of Swiss Cattle Breeders. The objective was to assess the completeness, validity and representativeness of the necropsy data, as well as evaluate potential factors for necropsy submission and how they can benefit animal health surveillance. Our results showed that, on average, 1% of Swiss cattle that die are submitted for post-mortem examinations. However, different factors influence cattle necropsy submissions, such as the age of the animal, the geographical location and the number of sick and/or dead animals on the farm. There was a median of five animals reported sick and two animals reported dead within 30 days prior to a necropsy submission, providing quantitative evidence of a correlation between on farm morbidity/mortality and post-mortem examination. Our results also showed that necropsy data can help improve the accuracy and completeness of health data for surveillance systems. In this study, we were able to demonstrate the importance of veterinary pathology data for AHS by providing quantitative evidence that necropsied animals are indicative of farms with important disease problems and are therefore critically important for surveillance. Furthermore, thanks to the amount of information provided by combined data sources, the epidemiology (e.g. season, geographic region, risk factors) of potential diseases can be analysed more precisely and help supporting animal health surveillance systems.