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Hemorrhagic bowel syndrome (HBS) is characterized by a dissecting intramucosal hematoma at the small bowel, causing obstruction and severe hemorrhage in dairy cattle. Recent investigation revealed the presence of early-stage lesions in cows affected by HBS. These are presumed to be the initial stage of the hematoma, as both share unique dissection of the lamina muscularis mucosae (LMM) as histological hallmark. Early-stage lesions of HBS have not been characterized in greater detail, and neither has the hypothesis of mucosal abrasion as etiology been explored. Therefore, the first objective of the present study was to characterize the morphology of early-stage lesions, by gross examination, histochemistry, immunohistochemistry and transmission electron microscopy. The second objective was to determine the effect of mucosal abrasion to the small intestine in an ex vivo model. A total of 86 early-stage lesions from 10 cows with HBS were characterized. No underlying alterations at the LMM were evident which could explain their occurrence. However, degeneration at the ultrastructural level of the LMM smooth muscle cells was present in 3 of 4 lesions, it is however unclear whether this is primary or secondary. Bacteriological examination did not reveal any association with a specific bacterium. Experimental-induced and early-stage lesions were gross and histologically evaluated and scored in three cows with HBS and seven controls. Experimentally induced lesions in both affected cows and controls, were histologically very similar to the naturally occurring early-stage lesions. Altogether, the results are suggestive for mucosal trauma to play a role in the pathogenesis of HBS.

IntroductionThe increasing prevalence of infertility in male dogs in clinical practice mirrors current trends seen in human medicine. Acquired infertility is notably more common in dogs compared to congenital causes, with conditions such as testicular degeneration leading to irreversible loss of fertility. Current diagnostic methods for testicular degeneration, such as histopathological and cytological examinations, rely on testicular biopsy or fine needle aspiration, making them less feasible for routine use. Anti-Müllerian hormone (AMH), produced by Sertoli cells, has emerged as a potential alternative biomarker for testicular health, which can be measured in serum. This study evaluates AMH as a potential marker for testicular degeneration, using cryptorchid dogs as models for impaired fertility and altered testicular histology.MethodsThe relationship between serum AMH levels and AMH tissue expression with impaired spermatogenesis and altered histology was investigated. Serum AMH levels were determined in intact, cryptorchid, and castrated individuals using an immuno-enzymatic ELISA kit and compared between subgroups based on testicular location. Tissue AMH immuno-expression was differentially quantified in two regions of interest (ROIs), the interstitial space and the seminiferous tubule, in both descended and retained gonads. Furthermore, testicles were analyzed using histomorphometric analysis in seminiferous tubules, while spermatogenesis was evaluated using the Johnsen score.ResultsSerum AMH levels were positively correlated with AMH expression assessed in both interstitial space (ρ = 0.494, p ≤ 0.01) and seminiferous tubules (ρ = 0.610, p ≤ 0.001). Conversely, serum AMH levels showed a negative correlation with the seminiferous tubule area (ρ = −0.435, p ≤ 0.05). Smaller seminiferous tubule areas were linked to increased AMH reactivity in both seminiferous tubules (ρ = −0.774, p ≤ 0.001) and interstitial space (ρ = −0.725, p ≤ 0.001). Additionally, lower Johnsen scores were associated with higher serum AMH levels (ρ = −0.537, p ≤ 0.01) and elevated AMH expression in both seminiferous tubules (ρ = −0.756, p ≤ 0.001) and interstitial space (ρ = −0.679, p ≤ 0.001).DiscussionOur results suggest that higher serum levels and tissue expression of AMH are linked to smaller seminiferous tubules and poorer Johnsen scores, reflecting degenerative changes and Sertoli cell dysfunction in retained testicles. Given the similarities in the mechanisms that increase AMH levels in both cryptorchid and non-cryptorchid testicles affected by testicular degeneration, this study recommends using AMH as a marker for diagnosing testicular degeneration in dogs.

Abstract This study investigated the role of causative infectious agents in ulceration of the non-glandular part of the porcine stomach (pars oesophagea). In total, 150 stomachs from slaughter pigs were included, 75 from pigs that received a meal feed, 75 from pigs that received an equivalent pelleted feed with a smaller particle size. The pars oesophagea was macroscopically examined after slaughter. (q)PCR assays for H. suis, F. gastrosuis and H. pylori-like organisms were performed, as well as 16S rRNA sequencing for pars oesophagea microbiome analyses. All 150 pig stomachs showed lesions. F. gastrosuis was detected in 115 cases (77%) and H. suis in 117 cases (78%), with 92 cases (61%) of co-infection; H. pylori-like organisms were detected in one case. Higher infectious loads of H. suis increased the odds of severe gastric lesions (OR = 1.14, p = 0.038), while the presence of H. suis infection in the pyloric gland zone increased the probability of pars oesophageal erosions [16.4% (95% CI 0.6–32.2%)]. The causal effect of H. suis was mediated by decreased pars oesophageal microbiome diversity [−1.9% (95% CI − 5.0–1.2%)], increased abundances of Veillonella and Campylobacter spp., and decreased abundances of Lactobacillus, Escherichia-Shigella, and Enterobacteriaceae spp. Higher infectious loads of F. gastrosuis in the pars oesophagea decreased the odds of severe gastric lesions (OR = 0.8, p = 0.0014). Feed pelleting had no significant impact on the prevalence of severe gastric lesions (OR = 1.72, p = 0.28). H. suis infections are a risk factor for ulceration of the porcine pars oesophagea, probably mediated through alterations in pars oesophageal microbiome diversity and composition.

Abstract Vaccines that protect against any H1N1 influenza A virus strain would be advantageous for use in pigs and humans. Here, we try to induce a pan-H1N1 antibody response in pigs by sequential vaccination with antigenically divergent H1N1 strains. Adjuvanted whole inactivated vaccines are given intramuscularly in various two- and three-dose regimens. Three doses of heterologous monovalent H1N1 vaccine result in seroprotective neutralizing antibodies against 71% of a diverse panel of human and swine H1 strains, detectable antibodies against 88% of strains, and sterile cross-clade immunity against two heterologous challenge strains. This strategy outperforms any two-dose regimen and is as good or better than giving three doses of matched trivalent vaccine. Neutralizing antibodies are H1-specific, and the second heterologous booster enhances reactivity with conserved epitopes in the HA head. We show that even the most traditional influenza vaccines can offer surprisingly broad protection if they are administered in an alternative way.

Abstract While seven gastric non-Helicobacter pylori Helicobacter (NHPH) species are known to commonly colonize the stomach of cats and dogs, the potential of H. pylori and H. pylori-like organisms to infect animals remains controversial and was investigated in this study using gastric samples of 20 cats and 27 dogs. A Helicobacter genus-specific 16 S rRNA PCR assay, H. pylori-specific ureAB and glmM PCR assays and a nested PCR detecting 23 S rRNA in a Helicobacter genus-specific manner in a first round of PCR and a H. pylori-specific manner in a second round, were performed in combination with sequencing. Histopathological and anti-Helicobacter immunohistochemical evaluations were also performed. Based on 16 S rRNA sequence analysis, 39/47 animals (83%) appeared infected with canine/feline gastric NHPHs in the corpus and/or antrum. H. pylori-specific ureAB amplicons were obtained in samples of 22 stomachs (47%). One canine antrum sample positive in the ureAB assay was also positive in the H. pylori-specific glmM assay. While 36/47 (77%) animals had a positive sample in the first round of the nested 23 S rRNA PCR assay, all samples were negative in the second round. Sequence analysis of obtained amplicons and immunohistochemistry point towards the presence of unidentified H. pylori-like organisms in cats and dogs. Histopathological examination suggests a low pathogenic significance of the gastric Helicobacter spp. present in these animals. In conclusion, cats and dogs may be (co-)infected with gastric Helicobacter organisms other than the known gastric NHPHs. Culture and isolation should be performed to confirm this hypothesis.

Abstract Background As current therapies for canine osteoarthritis (OA) provide mainly symptomatic improvement and fail to address the complex pathology of the disease, mesenchymal stem cells (MSCs) offer a promising biological approach to address both aspects of OA through their immunomodulatory properties. Methods This study aimed to investigate the safety and efficacy of xenogeneic MSCs in dogs with OA at different dose levels after intravenous injection. OA was surgically induced in the right stifle joint. Thirty-two male and female dogs were divided into three treatment groups and a control group. Regular general physical examinations; lameness, joint, radiographic, and animal caretaker assessments; pressure plate analyses; and blood analyses were performed over 42 days. At study end, joint tissues were evaluated regarding gross pathology, histopathology, and immunohistochemistry. In a follow-up study, the biodistribution of intravenously injected 99mTc-labeled equine peripheral blood-derived MSCs was evaluated over 24h in three dogs after the cruciate ligament section. Results The dose determination study showed the systemic administration of ePB-MSCs in a canine OA model resulted in an analgesic, anti-inflammatory, and joint tissue protective effect associated with improved clinical signs and improved cartilage structure, as well as a good safety profile. Furthermore, a clear dose effect was found with 0.3 × 106 ePB-MSCs as the most effective dose. In addition, this treatment was demonstrated to home specifically towards the injury zone in a biodistribution study. Conclusion This model-based study is the first to confirm the efficacy and safety of systemically administered xenogeneic MSCs in dogs with OA. The systemic administration of a low dose of xenogeneic MSCs could offer a widely accessible, safe, and efficacious treatment to address the complex pathology of canine OA and potentially slow down the disease progression by its joint tissue protective effect.

Abstract Porcine ear necrosis (PEN) is characterized by ulcerative lesions of the ear auricle. To investigate that problem, three farms with PEN in nursery pigs were included, and the study aim was to characterize PEN and the potential role of pathogens and mycotoxins. Within each farm, one batch of weaned piglets was included and the prevalence and severity of PEN were monitored for 6–7 weeks. Within each batch, 30 PEN-affected/non-affected animals were randomly selected. Blood samples were taken from these animals, to assess the systemic presence of pathogens and mycotoxins, as well as punch biopsies from the ear auricle for histopathological examination. From 10 animals, scrapings and swabs from the lesions were subjected to nanopore metagenomic sequencing and bacteriological cultivation, respectively. In all three farms, lesions appeared within 3–4 weeks post-weaning. The prevalence at the end of the nursery was 33%, 24%, and 46% for farms A, B, and C, respectively. Most affected pigs had mild to moderate lesions. Blood samples revealed low to very low levels of pathogens and mycotoxins. Different bacteria such as Staphylococcus, Streptococcus, Fusobacterium, Mycoplasma, and Clostridium species were identified by sequencing in the scrapings. The first two pathogens were also most often identified in bacterial cultures. Mycoplasma hyopharyngis was only found in PEN-affected pigs. Histopathological changes were primarily observed in the outer layer of the epidermis. The results suggest that PEN lesions develop by damage to the outer part of the skin e.g. by ear suckling or biting, followed by multiplication of opportunistic pathogens.

IntroductionIn this proof-of-concept study, the post-mortem feasibility of dual-energy computed tomography (DECT) in the detection of bone edema-like lesions in the equine foot is described in agreement with the gold standard imaging technique, which is magnetic resonance imaging (MRI).MethodsA total of five equine cadaver feet were studied, of which two were pathological and three were within normal limits and served as references. A low-field MRI of each foot was performed, followed by a DECT acquisition. Multiplanar reformations of DECT virtual non-calcium images were compared with MRI for the detection of bone edema-like lesions. A gross post-mortem was performed, and histopathologic samples were obtained of the navicular and/or distal phalanx of the two feet selected based on pathology and one reference foot.ResultsOn DECT virtual non-calcium imaging, the two pathological feet showed diffuse increased attenuation corresponding with bone edema-like lesions, whereas the three reference feet were considered normal. These findings were in agreement with the findings on the MRI. Histopathology of the two pathologic feet showed abnormalities in line with bone edema-like lesions. Histopathology of the reference foot was normal.ConclusionDECT virtual non-calcium imaging can be a valuable diagnostic tool in the diagnosis of bone edema-like lesions in the equine foot. Further examination of DECT in equine diagnostic imaging is warranted in a larger cohort, different locations, and alive animals.

IntroductionThe effect of dietary fiber on pig production has been extensively evaluated. Inspired by observations of the diet of wild, young piglets, this study aimed to examine the possibility of feeding grass hay to suckling piglets besides concentrated creep feed.MethodsThe sow-nursed piglets in this study were divided into two groups based on balanced sow parities. The control group (CON, n = 7 sows) only received a regular, concentrated creep feed, while the treatment piglets (GH, n = 8 sows) were also provided with chopped grass hay from 2 days of age until weaning (28 days). At weaning, one piglet with a median weight was selected from each litter for post-mortem evaluation. Subsequently, six pigs around median weight per sow were grouped into nursery pens and monitored for their feed intake and body weight gain until 9 weeks of age.Results and discussionPiglets in GH consumed, on average, 57 g of grass hay per piglet during the entire lactation period. The emptied weight of the small and large intestine was significantly greater in GH (280 vs. 228 g, 88.8 vs. 79.3 g, respectively, p < 0.05), and the length of the large intestine was stimulated by the grass hay (164 vs. 150 cm, p < 0.05). Morphologically, the villus height in the jejunum was higher in GH (p < 0.05). In the large intestine, the crypt depth of the mid-colon was lower in GH. Moreover, the short-chain fatty acid (SCFA) concentrations in the cecum were increased in GH compared to CON (1,179 vs. 948 µmol/g dry matter, p < 0.05), whereas in the colon, SCFA concentrations were lower in CON (341 vs. 278 µmol/g dry matter, p < 0.05). There was no major impact of grass hay inclusion on the colonic microbiota composition. Only a trend was observed for a lower inverse of the classical Simpson (InvSimpon) index and a higher abundance of Lactobacillus genera in GH. After weaning, no significant differences in feed intake and body weight gain were observed. In conclusion, supplementing the grass hay to suckling piglets led to alterations in intestinal morphology, increased SCFA fermentation in proximal sections of large intestine, stimulation of gastrointestinal tract growth, and subtle modifications in colonic microbiota.

ABSTRACT: In order to minimize animal loss and economical loss, industrial poultry is heavily vaccinated against infectious agents. mRNA vaccination is an effective vaccination platform, yet little to no comprehensive, comparative studies in avians can be found. Nevertheless, poultry mRNA vaccination could prove to be very interesting due to the relatively low production cost, especially true when using self-amplifying mRNA (saRNA), and their extreme adaptability to new pathogens. The latter could be particularly useful when new pathogens join the stage or new variants arise. As a first step toward the investigation of saRNA vaccines in poultry, this study evaluates a luciferase-encoding saRNA in avian tracheal explants, conjunctival explants, primary chicken cecal cells and 18-day embryonated eggs. Naked saRNA in combination with RNase inhibitor and 2 different lipid-based formulations, that is, ionizable lipid nanoparticles (LNPs) and Lipofectamine Messenger Max, were evaluated. The saRNA-LNP formulation led to the highest bioluminescent signal in the tracheal explants, conjunctival explants and cecal cell cultures. A dose-response experiment with these saRNA-LNPs (33–900 ng/well) in these avian organoids and cells showed a nonlinear dose-response relationship. After in ovo administration, the highest dose of the saRNA-LNPs (5 µg) resulted in a visual expression as a weak bioluminescence signal could be seen. The other delivery approaches did not lead to a visual saRNA expression in the embryos. In conclusion, effective entry of saRNA encapsulated in LNPs followed by successful saRNA translation in poultry was established. Hence, mRNA vaccination in poultry could be possible, but further in vivo testing is needed.

Current study evaluated the effect of a fine and coarsely ground insoluble dietary fibre source on the gastrointestinal development of suckling pigs. Oat hulls (OH) were selected as a model feedstuff, rich in cellulose, lignin, and insoluble dietary fibre. Three experimental supplemental diets were formulated: a finely ground, low fibre and nutrient dense diet served as control (CON). For the 2 high fibre diets, 15% heat-treated starch in CON was exchanged with OH, either finely (OH-f) or coarsely ground (OH-c). Litters of 10 primi- and multiparous sows (mean litter size 14.6 ± 0.84) were used. Within a litter, experimental diets were allotted to triplets of 4 piglets. From approximately 12 d of age, piglets’ individual feed intakes were recorded 2 times per day when separated from their dam for 70 min. Piglets could suckle with their dam for the remainder of the day. On d 24 and 25, from the total pool of 120 piglets, seven healthy well-eating piglets per treatment were selected for post-mortem evaluation, resulting in 14 replicates per treatment. Consumption of OH-c and OH-f did not impede clinical health and production performance of piglets. The full stomach weights tended to be greater for OH-c compared to OH-f whereas CON was intermediate (P = 0.083). Supplementing OH significantly increased ileal villus height and caecal dry matter concentration (P

Abstract In 2019 a low pathogenic H3N1 avian influenza virus (AIV) caused an outbreak in Belgian poultry farms, characterized by an unusually high mortality in chickens. Influenza A viruses of the H1 and H3 subtype can infect pigs and become established in swine populations. Therefore, the H3N1 epizootic raised concern about AIV transmission to pigs and from pigs to humans. Here, we assessed the replication efficiency of this virus in explants of the porcine respiratory tract and in pigs, using virus titration and/or RT-qPCR. We also examined transmission from directly, intranasally inoculated pigs to contact pigs. The H3N1 AIV replicated to moderate titers in explants of the bronchioles and lungs, but not in the nasal mucosa or trachea. In the pig infection study, infectious virus was only detected in a few lung samples collected between 1 and 3 days post-inoculation. Virus titers were between 1.7 and 4.8 log10 TCID50. In line with the ex vivo experiment, no virus was isolated from the upper respiratory tract of pigs. In the transmission experiment, we could not detect virus transmission from directly inoculated to contact pigs. An increase in serum antibody titers was observed only in the inoculated pigs. We conclude that the porcine respiratory tract tissue explants can be a useful tool to assess the replication efficiency of AIVs in pigs. The H3N1 AIV examined here is unlikely to pose a risk to swine populations. However, continuous risk assessment studies of emerging AIVs in pigs are necessary, since different virus strains will have different genotypic and phenotypic traits.

Abstract Background Flatfish live in a diverse marine ecosystem that is changing due to natural variations and anthropogenic influences. These changes can evoke a stress response mainly resulting in production of the glucocorticoid cortisol, which mediates effects on various levels of biological organization. The finding that cortisol accumulates in fish scales, offering a retrospective view on cortisol production, provides opportunities to use this matrix for chronic stress assessment. The present study is the first to gather information on scale cortisol concentration in wild-caught common dab (Limanda limanda), based on a two-pronged approach using (1) field measurements and (2) a laboratory in vivo-study where wild-caught dab were fed by cortisol-spiked feed during 30 or 90 days to demonstrate the possible accumulation of cortisol in the scales and to evaluate its impact on fish health. Results Based on the field measurements, the average scale cortisol concentration in wild-caught fish was 0.0034 ± 0.0046 µg kg−1 scale (n = 67). This indicates that wild common dab is indeed able to incorporate cortisol in the scales. Based on the experimental data, the cortisol-fed fish showed an increased plasma cortisol concentration (80.16 ± 82.58 µg L−1) compared to the control group (4.54 ± 9.57 µg L−1) after 30 days of cortisol feeding. The increase in plasma cortisol concentration was positively correlated with an increased cortisol concentration in the scale after 30 days of cortisol-spiked feeding. This correlation was, however, no longer observed after 90 days of cortisol-spiked feeding. Interestingly, cortisol concentration of the scales on the pigmented side was significantly higher compared to the non-pigmented side. Some health parameters such as epidermal thickness, body condition and Ichthyobodo sp. parasitic infection showed a correlation with scale cortisol concentration after 30 days. Conclusions We have demonstrated that common dab is able to accumulate cortisol in its scales. This seems to occur proportionally to circulating concentrations of plasma cortisol in fish fed with cortisol supplemented feed after 30 days.

Hemorrhagic bowel syndrome (HBS) is a poorly understood, sporadic and often fatal disease in cattle. Although, HBS is considered an important disease in dairy cattle, epidemiological data is largely lacking. This study describes the epidemiology of HBS in Belgium and the Netherlands, based on necropsy records from 2009 to 2022, and reports characteristics from 27 cows and 35 dairy operations with HBS, gathered through a survey. The annual incidence of HBS has a significantly increasing trend both at cow and herd level, with incidence above 3.2% in necropsied mature dairy cattle in the most recent years. Estimated herd-level incidence in the Netherlands was double the estimated incidence in Belgium, which might be explained by higher herd size in the Netherlands. Occurrence of HBS was most prevalent in fall, being 40.1% higher than the average of the other seasons. In 35 Flemish (Belgian) surveyed dairy herds with HBS, manifestation of HBS was mostly as solitary cases, and if multiple cases occurred, the time interval was highly variable. In addition, the majority of cows with HBS (61.1%; 16/26) were in more than 100 days lactation. In conclusion, HBS is an important and possibly emerging disease in dairy cattle in Belgium and the Netherlands.

Abstract Heterologous prime-boost vaccination with experimental or commercial influenza vaccines has been successful in various animal species. In this study, we have examined the efficacy of alternating 3 different European commercial swine influenza A virus (swIAV) vaccines: the trivalent Respiporc® FLU3 (TIV), the bivalent GRIPORK® (BIV) and the monovalent Respiporc® FLUpan H1N1 (MOV). Five groups of 6 pigs each received 3 vaccinations at 4–6 week intervals in a homologous or heterologous prime-boost regimen. A sixth group served as a mock-vaccinated challenge control. Four weeks after the last vaccination, pigs were challenged intranasally with a European avian-like H1N1 (1C.2.1) swIAV, which was antigenically distinct from the vaccine strains. One heterologous prime-boost group (TIV–BIV–MOV) had higher hemagglutination inhibition (HI) and neuraminidase inhibition antibody responses against a panel of antigenically distinct H1N1, H1N2 and H3N2 IAVs than the other heterologous prime-boost group (BIV–TIV–MOV) and the homologous prime-boost groups (3xTIV; 3xBIV; 3xMOV). Group TIV–BIV–MOV had seroprotective HI titers (≥ 40) against 56% of the tested viruses compared to 33% in group BIV–TIV–MOV and 22–39% in the homologous prime-boost groups. Post-challenge, group TIV–BIV–MOV was the single group with significantly reduced virus titers in all respiratory samples compared to the challenge control group. Our results suggest that the use of different commercial swIAV vaccines for successive vaccinations may result in broader antibody responses and protection than the traditional, homologous prime-boost vaccination regimens. In addition, the order in which the different vaccines are administered seems to affect the breadth of the antibody response and protection.

Abstract This article focuses on the pathogenic significance of Helicobacter species naturally colonizing the stomach of dogs, cats and pigs. These gastric “non-Helicobacter (H.) pylori Helicobacter species” (NHPH) are less well-known than the human adapted H. pylori. Helicobacter suis has been associated with gastritis and decreased daily weight gain in pigs. Several studies also attribute a role to this pathogen in the development of hyperkeratosis and ulceration of the non-glandular stratified squamous epithelium of the pars oesophagea of the porcine stomach. The stomach of dogs and cats can be colonized by several Helicobacter species but their pathogenic significance for these animals is probably low. Helicobacter suis as well as several canine and feline gastric Helicobacter species may also infect humans, resulting in gastritis, peptic and duodenal ulcers, and low-grade mucosa-associated lymphoid tissue lymphoma. These agents may be transmitted to humans most likely through direct or indirect contact with dogs, cats and pigs. Additional possible transmission routes include consumption of water and, for H. suis, also consumption of contaminated pork. It has been described that standard H. pylori eradication therapy is usually also effective to eradicate the NHPH in human patients, although acquired antimicrobial resistance may occasionally occur and porcine H. suis strains are intrinsically less susceptible to aminopenicillins than non-human primate H. suis strains and other gastric Helicobacter species. Virulence factors of H. suis and the canine and feline gastric Helicobacter species include urease activity, motility, chemotaxis, adhesins and gamma-glutamyl transpeptidase. These NHPH, however, lack orthologs of cytotoxin-associated gene pathogenicity island and vacuolating cytotoxin A, which are major virulence factors in H. pylori. It can be concluded that besides H. pylori, gastric Helicobacter species associated with dogs, cats and pigs are also clinically relevant in humans. Although recent research has provided better insights regarding pathogenic mechanisms and treatment strategies, a lot remains to be investigated, including true prevalence rates, exact modes of transmission and molecular pathways underlying disease development and progression.

Abstract Background Scientific knowledge regarding alpaca dentistry is relatively limited despite its clinical implications. The present gap in available supportive data leads to limited treatment options for dental pathology in alpacas in comparison to other species. The main goal of this study was to gain novel insights into the general and pulp morphology of maxillary cheek teeth to allow development of more advanced treatment strategies in the future. Also, the risk of causing pulp exposure when floating maxillary cheek teeth was of particular interest. Concurent research focusing on the anatomy of mandibular cheek teeth has been performed accordingly. The results obtained in mandibular teeth are expected to be non-extrapolatable because of the structural differences between mandibular and maxillary teeth. Results Pulp morphology of maxillary cheek teeth showed great variation. A common pulp chamber was identified in 46/83 (55.4%) teeth with a mean dental age of 2 years and 7 months (± 2 years and 5 months). Pulpal segmentation was more commonly observed in teeth of increasing age. Full columnar segmentation was seen in 33/69 teeth (47.8%), whereas within-column segmentation was observed in 36/83 teeth (43.4%). Age and degree of segmentation of the pulpal tissue varied greatly according to Triadan position. Physical contact between roots of adjacent teeth was found in the majority of examined molars (range 82–94%) which resulted in morphological adaptations at the level of the root tips. The measured sub-occlusal dentinal thickness was as low as 0.46 mm above pulp horn 2 in a 14 years and 11 months old Triadan 09, emphasizing the risk of pulp exposure attributed to dental floating. Conclusion This study offers an objective description of age-dependent maxillary cheek teeth pulp morphology in alpacas. Current findings are of great value to provide a basis for the development of tooth-saving techniques as a treatment for dental disease in this species. Observed physical contact between the roots of different examined molars may be a facilitating factor in the spread of apical infection in chronically diseased cases. Finally, a conservative approach regarding dental floating is recommended in order to avoid iatrogenic damage to pulp tissue.

Injuries to equine tendons and ligaments are career-compromising, causing reduced performance and premature retirement. Promising treatment alternatives have been investigated in the field of mesenchymal stem cells (MSCs). In this study, the tissue adherence and protein expression of tenogenic primed mesenchymal stem cells (tpMSCs) after administration to ex vivo tendon and ligament explants is investigated. First, collagen type I (COL I) and smooth muscle actin (SMA) expression was assessed in cytospins prepared from native MSCs and tpMSCs. Second, equine superficial digital flexor tendon and suspensory ligament explants were cultivated, and a lesion was treated with both cell types. Subsequently, cell adhesion to the explants and the amount of COL I and SMA positive cells was evaluated. The cytospins revealed a significantly higher COL I and lower SMA expression in tpMSCs compared to native MSCs. In the explants, tpMSCs showed a significantly higher tendon and ligament adherence. Furthermore, a significantly higher percentage of COL I positive and a lower percentage of SMA positive cells were observed in the lesions treated with tpMSCs. The results of these explant co-cultures may demonstrate at least a part of the mechanism of action and functional properties of tpMSCs in restoring function to tendons and ligaments.

Toxin-producing Corynebacterium ulcerans, a causative agent of diphtheria in humans, was isolated from 53 hedgehogs in Belgium during the spring of 2020. Isolates showed low levels of acquired antimicrobial drug resistance. Strain diversity suggests emergence from an endemic situation. These findings stress the need for raising public awareness and improved wildlife disease surveillance.

Abstract Background Despite dental disease being a common health concern in alpacas, important dental pathology including apical infection, remains poorly understood. Treatment options are limited compared to veterinary dentistry techniques in other species. The primary goal of this study was to increase understanding of the external and internal anatomy of mandibular cheek teeth to enable the development of tooth sparing techniques in this species. Also, an objective evaluation of the sub-occlusal dentinal thickness in normal mandibular cheek teeth is warranted to understand the risks associated with reduction of overgrown teeth. Results Overall pulp anatomy was variably characterized by the presence of a common pulp chamber in younger teeth, and segmentation of pulp cavities into multiple separate pulp entities within the same tooth with increasing age. A common pulp chamber was identified in 55.3% (26/47) of teeth with a mean dental age of 1 year and 11 months (± 1 year and 8 months). Columnar segmentation was recorded in the remaining teeth with a mean dental age of 6 years and 5 months (± 3 years and 11 months). Age of segmentation of the common pulp chamber into multiple separate pulp entities shows wide variation and is dependent of the specific Triadan position. The present study illustrates the presence of disto-mesial root contacts between adjacent tooth roots, often leading to morphological adaptations, most frequently observed between Triadan 09–10s (80%) and 10–11s (67%). The measured sub-occlusal dentinal thickness was as low as 1.11 mm over some pulp horns. The sub-occlusal dentinal thickness was lower than 2, 3, and 4 mm in 13.1, 38.1 and 61.4% of performed measurements, respectively. Conclusion This study provides detailed information on age-dependent mandibular cheek teeth anatomy in alpacas, which may support the use and development of advanced dental treatments in this species such as endodontics and tooth sectioning techniques. Apical morphological adaptations caused by disto-mesial root contact between adjacent mandibular cheek teeth are clearly illustrated. The limited amount of sub-occlusal secondary dentin warrants a cautious approach with regards to dental floating in alpacas.

The skin disease chytridiomycosis is linked to global amphibian declines but effective mitigation measures require improved understanding of the mechanisms underpinning the disease ecology. This study identifies key mediators of interactions between the fungal pathogen and amphibian skin, providing a marker of host colonization that can predict susceptibility between amphibian species.

Scientific literature on veterinary dentistry in alpacas has historically focused on the description of tooth root abscesses. However, recent studies have shown a variety of other, sometimes preceding dental conditions to be widespread in this species. To allow the development and finetuning of treatment strategies in this species, a more thorough understanding of the underlying etiopathogenesis of dental disease is required. Histological studies focusing on normal dental and surrounding tissues might serve as a basis for this purpose. Nine teeth, extracted from seven alpacas were collected. All samples were retrieved from animals that died or were euthanized for non-dental reasons. Histological sections were prepared at three different levels in each tooth and examined using light microscopy focusing on the assessment of pulp tissue, dentin, cementum, periodontal tissues and the apical region. The histological appearance of the investigated dental tissues in alpacas showed great similarities with other hypsodont species. However, a rather rare type of dentin called “vasodentin” could be identified in all examined cheek teeth. Another species-specific finding was the extremely close proximity of varying neighboring tooth roots that seemed to be responsible for inducing massive resorptive lesions. The results of this study might contribute to a better understanding of the etiopathogenesis of some dental diseases in the alpaca.

Mesenchymal stem cells (MSCs) are a promising candidate for both human and veterinary regenerative medicine applications because of their abundance and ability to differentiate into several lineages. Mesenchymal stem cells are however a heterogeneous cell population and as such, it is imperative that they are unequivocally characterized to acquire reproducible results in clinical trials. Although the tri-lineage differentiation potential of MSCs is reported in most veterinary studies, a qualitative evaluation of representative histological images does not always unambiguously confirm tri-lineage differentiation. Moreover, potential differences in differentiation capacity are not identified. Therefore, quantification of tri-lineage differentiation would greatly enhance proper characterization of MSCs. In this study, a method to quantify the tri-lineage differentiation potential of MSCs is described using digital image analysis, based on the color deconvolution plug-in (ImageJ). Mesenchymal stem cells from three species, i.e., bovine, equine, and porcine, were differentiated toward adipocytes, chondrocytes, and osteocytes. Subsequently, differentiated MSCs were stained with Oil Red O, Alcian Blue, and Alizarin Red S, respectively. Next, a differentiation ratio (DR) was obtained by dividing the area % of the differentiation signal by the area % of the nuclear signal. Although MSCs isolated from all donors in all species were capable of tri-lineage differentiation, differences were demonstrated between donors using this quantitative DR. Our straightforward, simple but robust method represents an elegant approach to determine the degree of MSC tri-lineage differentiation across species. As such, differences in differentiation potential within the heterogeneous MSC population and between different MSC sources can easily be identified, which will support further optimization of regenerative therapies.

Chronic progressive lymphedema (CPL) in draft horses is characterized by increased dermal thickness and fibrosis, with the development of skinfolds and nodules, hyperkeratosis, and ulcerations on the distal limbs of affected horses. Secondary bacterial, fungal, or parasitic infections frequently complicate and aggravate the lesions, as well as the progression of this disease. CPL has a particularly high prevalence of up to 85.86% in the Belgian draft horse breed. Due to the disease’s progressive and incurable nature, affected horses are often euthanized prematurely. The treatment options are solely symptomatic, aimed at improving the horse’s quality of life. Despite the severity of this condition, many uncertainties about its etiology and pathogenesis still remain to date. The established scientific research on CPL is rather limited, although there is an urgent need for strategies to tackle this disease. This review summarizes the available knowledge, serving as a guideline for practitioners, and provides perspectives for future research programs.

In the present study, the histological characteristics of squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) obtained from 22 squamate and 13 chelonian species were retrospectively evaluated. While the examined tissues were originally diagnosed as 28 SCCs and 7 BCCs based on histological evaluation by a specialty diagnostic service, eight SCCs could be re-classified as BCCs and three SCCs proved to be non-neoplastic lesions. In addition, all SCCs and BCCs were classified into distinct histological variants. The SCCs could be categorized as one SCC in situ, three moderately differentiated SCCs, seven well-differentiated SCCs, and six keratoacanthomas. BCCs were classified as five solid BCCs, four infiltrating BCCs, five keratotic BCCs, and one basosquamous cell carcinoma. In addition, the present study reports the occurrence of BCCs in seven reptile species for the first time. In contrast to what has been documented in humans, IHC staining with the commercially available epithelial membrane antigen and epithelial antigen clone Ber-EP4 does not allow differentiation of SCCs from BCCs in reptiles, while cyclooxygenase-2 and E-cadherin staining seem to have discriminating potential. Although the gross pathological features of the examined SCCs and BCCs were highly similar, each tumor could be unequivocally assigned to a distinct histological variant according to the observed histological characteristics. Based on the results of this study, a histopathological classification for SCCs and BCCs is proposed, allowing accurate identification and differentiation of SCCs and BCCs and their histological variants in the examined reptile species. Presumably, BCCs are severely underdiagnosed in squamates and chelonians.

Abstract Psoroptic mange is a common disease of livestock, caused by Psoroptes ovis. Compared to Holstein–Friesian (HF) cattle, the Belgian Blue (BB) cattle breed is highly susceptible to the infestation. However, the mechanism for this difference is still unclear. To determine the factors responsible for this breed susceptibility, the immune response to P. ovis was studied in experimentally infested BB and HF cattle, using clinical signs, histology, immunohistochemical profiling and gene expression analysis of skin biopsies. The mite numbers and lesion area of BB cattle were greater than in HF during the whole study period. Significant influxes of eosinophils in the epidermis and dermis were detected in comparison with the pre-infestation samples in both breeds, with significantly higher eosinophils in BB at 6 weeks post infestation (wpi). Mast cell numbers were unaffected at all stages of infestation in HF, but were significantly elevated relative to pre-infestation in BB cattle at 2 and 6 wpi. The more pronounced cutaneous eosinophilia and higher IL-4 levels at 6 wpi in BB cattle suggest that a Th2-type immune response is underlying the higher susceptibility of the BB breed. In naturally infested BB cattle, development of the psoroptic mange lesions and eosinophils and CD3+ T cell areas were severely depressed after anti-inflammatory treatment with dexamethasone. Together, these results suggest that a stronger Th2-type immune response to P. ovis causes the skin lesions in psoroptic mange in BB cattle and that local anti-inflammatory treatment could potentially be an alternative to control the pathology caused by this parasite.

The use of mesenchymal stem cells (MSCs) for the treatment of equine tendon disease is widely investigated because of their regenerative and immunomodulatory potential. However, questions have been raised concerning the immunogenic properties of allogeneic MSCs. Therefore, two studies were conducted to assess the safety of equine allogeneic peripheral blood-derived tenogenic primed MSCs (tpMSCs). The objective was to evaluate if a single and repeated tpMSC administration induced a cellular and humoral immune response in horses suffering from tendon injuries. Horses enrolled in the first study (n = 8) had a surgically induced superficial digital flexor tendon core lesion and were treated intralesionally with tpMSCs. Before and after treatment the cellular immunogenicity was assessed by modified mixed lymphocyte reactions. The humoral immune response was investigated using a crossmatch assay. Presence of anti-bovine serum albumin (BSA) antibodies was detected via ELISA. Horses enrolled in the second study (n = 6) suffered from a naturally occurring tendon injury and were treated twice with tpMSCs. Blood was collected after the second treatment for the same immunological assays. No cellular immune response was found in any of the horses. One out of eight horses in the first study and none of the horses in the second study had anti-tpMSC antibodies. This particular horse had an equine sarcoid and further investigation revealed presence of antibodies against sarcoid cells and epithelial-like stem cells before treatment, which increased after treatment. Additionally, formation of antibodies against BSA was observed. These findings might indicate a non-specific immune response generated after treatment. Serum from the other horses revealed no such antibody formation. These two studies showed that the administration of tpMSCs did not induce a cellular or humoral immune response following an intralesional single or repeated (two consecutive) allogeneic tpMSC treatment in horses with tendon injury, except for one horse. Therefore, a larger field study should confirm these findings and support the safe use of tpMSCs as a therapeutic for horses suffering from tendon injuries.

Abstract This case report describes the clinical presentation, the necropsy findings, and genetic results of a 13‐year‐old Warmblood mare presented with colic and a bilaterally loud, holosystolic murmur. Echocardiographic examination revealed the presence of a thoracic aortic aneurysm, an aortic pseudoaneurysm, a periaortic hematoma (circumferential cuffing by perivascular hemorrhage), and aortopulmonary fistulation. A supravalvular aortic stenosis (SVAS) was visible during echocardiography. Necropsy confirmed that the thoracic aortic aneurysm had ruptured and connected to the pseudoaneurysm, which fistulated into the pulmonary artery. Histologically, the aneurysm wall revealed chronic lesions such as fibrosis, mucin depositions, mineralizations, and elastin fragmentation. The mid abdominal aorta showed lesions suggestive of a systemic elastin arteriopathy. Molecular analysis, however, could not attribute this disease to a variant in the elastin gene, the most common causative gene for SVAS. To the authors' knowledge, this case report describes a case of aortopulmonary fistulation in a Warmblood horse associated with the presence of SVAS and an aortic aneurysm.

Abstract Background Aortic rupture is more common in Friesians compared to Warmbloods, which might be related to differences in arterial wall composition and, as such, arterial wall stiffness (AWS). Currently, nothing is known about differences in AWS between these breeds. Objectives Comparison of AWS parameters and noninvasive blood pressure between Friesians and Warmbloods. Animals One hundred one healthy Friesians and 101 age‐matched healthy Warmbloods. Methods Two‐dimensional and pulsed‐wave Doppler ultrasound examination was performed of the aorta, common carotid artery, and external iliac artery to define local and regional AWS parameters. Regional aortic AWS was estimated using aortic‐to‐external iliac artery pulse wave velocity (PWVa‐e) and carotid‐to‐external iliac artery pulse wave velocity (PWVc‐e). Noninvasive blood pressure and heart rate were recorded simultaneously. Results Systolic, diastolic, and mean arterial blood pressure and pulse pressure were significantly higher in Friesians compared to Warmbloods. No significant difference in heart rate was found. Most local AWS parameters (diameter change, compliance coefficient, distensibility coefficient) were significantly lower in Friesians compared to Warmbloods, indicating a stiffer aorta in Friesians. This difference could be confirmed by the regional stiffness parameters. A higher PWVa‐e and PWVc‐e was found in Friesians. For the cranial and caudal common carotid artery and external iliac artery, most local AWS parameters were not significantly different. Conclusions and clinical importance Results indicate that aortic AWS differs between Friesian and Warmblood horses. Friesians seem to have a stiffer aorta, which might be related to the higher incidence of aortic rupture in Friesians.

The present study describes the clinical behavior as well as the histopathologic and immunohistochemical characteristics of keratoacanthomas (Kas) in three different saurian species. While Kas presented as two dermal lesions in a bearded dragon (Pogona vitticeps), multicentric Kas were observed in three panther chameleons (Furcifer pardalis) and a veiled chameleon (Chamaeleo calyptratus). Macroscopically, Kas presented as dome-shaped skin tumors with a centralized keratinous pearl and a diameter ranging from 0.1–1.5 cm. In all lizards, Kas were predominantly located at the dorsolateral body wall, and KA of the eyelid was additionally observed in three out of four chameleons. Histologically, KAs presented as relatively well-defined, circumscribed epidermal proliferations that consisted of a crateriform lesion containing a central keratinous pearl with minimally infiltrating borders. In all KAs, a consistent immunohistochemical pattern was observed, with the expression of cyclooxygenase-2, E-cadherin, and pan-cytokeratin. A follow-up period of one to two years was established in all lizards. While no recurrence was observed in the panther chameleons, recurrence of a single keratoacanthoma was observed in the bearded dragon after one year, and in the veiled chameleon, multicentric keratoacanthomas reappeared during a follow-up period of two years. We describe KA as a previously unrecognized neoplastic entity in lizards that constitutes a low-grade, non-invasive but rapidly growing skin tumor that may show a multicentric appearance, especially in chameleons. As previously postulated for dermal squamous cell carcinomas (SCC), artificial ultraviolet lighting may play an important role in the oncogenesis of KAs in lizards. Although dermal SCCs in lizards show similar predilection sites and gross pathologic features, our results suggest that KA should be considered as a histologic variant of SCC that represents a rather benign squamous proliferation in comparison to conventional SCCs. Early diagnosis of KA and reliable discrimination from SCCs are essential for the prognosis of this neoplastic entity in lizards.

Two adult female hippos in Zoo Antwerp who were naturally infected with SARS-CoV-2 showed nasal discharge for a few days. Virus was detected by immunocytochemistry and PCR in nasal swab samples and by PCR in faeces and pool water. Serology was also positive. No treatment was necessary.

In a previous vaccination study in pigs, heterologous prime-boost vaccination with whole-inactivated H1N1 virus vaccines (WIV) induced superior antibody responses and protection compared to homologous prime-boost vaccination. However, no pan-H1 antibody response was induced. Therefore, to stimulate both local and systemic immune responses, we first vaccinated pigs intranasally with a pseudorabies vector vaccine expressing the pH1N1 hemagglutinin (prvCA09) followed by a homologous or heterologous WIV booster vaccine. Homologous and heterologous WIV–WIV vaccinated groups and mock-vaccinated or prvCA09 single-vaccinated pigs served as control groups. Five weeks after the second vaccination, pigs were challenged with a homologous pH1N1 or one of two heterologous H1N2 swine influenza A virus strains. A single prvCA09 vaccination resulted in complete protection against homologous challenge, and vector–WIV vaccinated groups were significantly better protected against heterologous challenge compared to the challenge control group or WIV–WIV vaccinated groups. Furthermore, vector–WIV vaccination resulted in broader hemagglutination inhibition antibody responses compared to WIV–WIV vaccination and higher numbers of antibody-secreting cells in peripheral blood, draining lymph nodes and nasal mucosa. However, even though vector–WIV vaccination induced stronger antibody responses and protection, we still failed to induce a pan-H1 antibody response.

Abstract Recently, Vibrio tapetis was isolated for the first time from skin ulcerations in wild-caught common dab (Limanda limanda). To further examine its role in the development of these skin lesions, an in vivo experiment was performed. The significance of the skin barrier and in addition the difference between pigmented and non-pigmented side were investigated. Hence, the skin of common dab was treated in three different ways on both the pigmented and non-pigmented side. On a first “treatment zone”, the scales and overlying epidermal tissue were removed whereas in a second zone only the mucus was discarded. The third zone served as a non-treated zone. Thereafter, fish were challenged with V. tapetis. The control group was sham treated. Mortality, clinical signs, severity and size of the developing lesions were recorded. All animals were sacrificed and sampled 21 days post-inoculation. Significantly more fish of the group challenged with V. tapetis died compared to the control group with the highest incidence occurring 4 days post-inoculation. Fish challenged with V. tapetis developed more severe skin ulcerations. In zones where scales and epidermal tissue were removed, the ulcerations were more severe compared to zones where only mucus was eliminated. Ulcerations occurred more frequently, were more severe and larger on the pigmented side. Our data represents prove of V. tapetis as causative agent of ulcerative skin lesions although prior damage of the skin seems to be a major contributing factor. Furthermore, the pigmented side seemed predisposed to the development of skin ulcerations.

Background: Tendon injuries are very common in horses and jeopardize the athletic performance, and due to the high risk of reinjury may lead to early retirement. The use of mesenchymal stem cells for the treatment of equine tendon disease is widely investigated because of their regenerative potential. The objective of this study is to investigate the safety and efficacy of equine allogeneic tenogenic primed mesenchymal stem cells (tpMSCs) for the management of tendinitis in horses.Methods: A core lesion was surgically induced in the superficial digital flexor tendon of both forelimbs of eight horses. After 7 days, one forelimb was treated with tpMSCs, while the contralateral forelimb served as an intra-individual control and was treated with saline. A prescribed exercise program was started. All horses underwent a daily clinical evaluation throughout the entire study period of 112 days. Blood samples were taken at different time points for hematological and biochemical analysis. Tendon assessment, lameness examination, ultrasound assessment and ultrasound tissue characterization (UTC) were performed at regular time intervals. At the end of the study period, the superficial digital flexor tendons were evaluated macroscopically and histologically.Results: No suspected or serious adverse events occurred during the entire study period. There was no difference in local effects including heat and pain to pressure between a single intralesional injection of allogeneic tpMSCs and a single intralesional injection with saline. A transient moderate local swelling was noted in the tpMSC treated limbs, which dissipated by day 11. Starting at a different time point depending on the parameter, a significant improvement was observed in the tpMSC treated limbs compared to the placebo for echogenicity score, fiber alignment score, anterior-posterior thickness of the tendon and echo type by UTC assessment. Immunohistochemistry 112 days post-injection revealed that the amount of collagen type I and Von Willebrand factor were significantly higher in the tendon tissue of the tpMSC group, while the amount of collagen type III and smooth muscle actin was significantly lower.Conclusion: Equine allogeneic tenogenic primed mesenchymal stem cells were shown to be well-tolerated and may be effective for the management of tendon injuries.

Arterial rupture is a well-recognized cause of sudden death in horses, which mainly affects older horses. The arterial wall is known to stiffen with age, although the underlying age-related histological and biomechanical changes remain unclear. The purpose of this study was to investigate the effect of aging by histological analysis of the arterial wall and examination of the arterial wall biomechanical properties using an inflation-extension test. Entire circular samples of the proximal and distal aorta, cranial and caudal common carotid, external iliac, femoral and median artery were collected from 6 young (6 years) and 14 old horses (≥15 years). Samples of all arteries were histologically examined and intima media thickness as well as area % of elastin, smooth muscle actin and collagen type I and III were determined. Older horses had a significantly larger intima media thickness and a significantly higher area % of smooth muscle actin compared to young horses. Samples of the proximal and distal aorta, the caudal common carotid and the external iliac artery were mechanically assessed using an in-house developed inflation-extension device with ultrasound analysis. Rupture occurred in a minority of arteries (8/78) at high pressures (between 250-300 mmHg), and mostly occurred in older horses (7/8). Pressure-area, pressure-compliance and pressure-distensibility curves were constructed. A significant difference in the pressure-area curves of the distal aorta, common carotid artery and external iliac artery, the pressure-compliance curves of the proximal aorta and carotid artery and the pressure-distensibility curve of the proximal aorta was observed between young and old horses. Results demonstrate an effect of age on the histological and biomechanical properties of the arterial wall, which might explain why arterial rupture occurs more often in older horses.

The current study was designed to evaluate the pathogenesis, pathology and immune response of female genital tract infection with Chlamydia trachomatis L2c, the most recently discovered lymphogranuloma venereum strain, using a porcine model of sexually transmitted infections. Pigs were mock infected, infected once or infected and re-infected intravaginally, and samples were obtained for chlamydial culture, gross and microscopic pathology, and humoral and cell-mediated immunity. Intravaginal inoculation of pigs with this bacterium resulted in an infection that was confined to the urogenital tract, where inflammation and pathology were caused that resembled what is seen in human infection. Re-infection resulted in more severe gross pathology than primary infection, and chlamydial colonization of the urogenital tract was similar for primary infected and re-infected pigs. This indicates that primary infection failed to induce protective immune responses against re-infection. Indeed, the proliferative responses of mononuclear cells from blood and lymphoid tissues to C. trachomatis strain L2c were never statistically different among groups, suggesting that C. trachomatis-specific lymphocytes were not generated following infection or re-infection. Nevertheless, anti-chlamydial antibodies were elicited in sera and vaginal secretions after primary infection and re-infection, clearly resulting in a secondary systemic and mucosal antibody response. While primary infection did not protect against reinfection, the porcine model is relevant for evaluating immune and pathogenic responses for emerging and known C. trachomatis strains to advance drug and/or vaccine development in humans.

Abstract The chytrid fungus Batrachochytrium dendrobatidis has caused the most prominent loss of vertebrate diversity ever recorded, which peaked in the 1980s. Recent incursion by its sister species B. salamandrivorans in Europe raised the alarm for a new wave of declines and extinctions in western Palearctic urodeles. The European Commission has responded by restricting amphibian trade. However, private amphibian collections, the main end consumers, were exempted from the European legislation. Here, we report how invasion by a released, exotic newt coincided with B. salamandrivorans invasion at over 1000 km from the nearest natural outbreak site, causing mass mortality in indigenous marbled newts (Triturus marmoratus), and posing an acute threat to the survival of nearby populations of the most critically endangered European newt species (Montseny brook newt, Calotriton arnoldi). Disease management was initiated shortly after detection in a close collaboration between policy and science and included drastic on site measures and intensive disease surveillance. Despite these efforts, the disease is considered temporarily contained but not eradicated and continued efforts will be necessary to minimize the probability of further pathogen dispersal. This precedent demonstrates the importance of tackling wildlife diseases at an early stage using an integrated approach, involving all stakeholders and closing loopholes in existing regulations.

Abstract Background Friesian horses are known for their high inbreeding rate resulting in several genetic diseases such as hydrocephaly and dwarfism. This last decade, several studies focused on two other presumed hereditary traits in Friesian horses: megaoesophagus and aortic rupture. The pathogenesis of these diseases remains obscure but an important role of collagen has been hypothesized. The purpose of this study was to examine possible breed-related differences in collagen catabolism. Urinary specimens from Friesian (n = 17, median age 10 years old) and Warmblood horses (n = 17, median age 10 years old) were assessed for mature collagen cross-links, i.e. pyridinoline (PYD) (=hydroxylysylpyridinoline/HP) and deoxypyridinoline (DPD) (lysylpyridinoline /LP). Solid-phase extraction was performed, followed by reversed-phase ion-paired liquid chromatography prior to tandem mass spectrometry (MS/MS) detection. Results Mean urinary concentrations of free PYD, expressed as fPYD/creatinine ratio, were significantly higher in Friesian horses compared to Warmblood horses (28.5 ± 5.2 versus 22.2 ± 9.6 nmol/mmol, p = 0.02) while mean fDPD/creatinine ratios were similar in both horse breeds (3.0 ± 0.7 versus 4.6 ± 3.7 nmol/mmol, p = 0.09). Conclusions Since DPD is considered a specific bone degradation marker and PYD is more widely distributed in connective tissues, the significant elevation in the mean PYD/DPD ratio in Friesian versus Warmblood horses (9.6 ± 1.6 versus 5.7 ± 1.8, p

The genus Macavirus, subfamily Gammaherpesvirinae, comprises ungulate viruses that infect domestic and wild ruminants and swine. They cause asymptomatic latent infections in reservoir hosts and malignant catarrhal fever in susceptible species. Lung, spleen, bronchial lymph node, and tongue were collected from 448 cattle (348 necropsied, 100 slaughtered) in Switzerland, United Kingdom, Finland, Belgium, and Germany to determine their infection with bovine herpesvirus-6 (BoHV-6) and gammaherpesviruses of other ruminants, i.e., ovine herpesvirus-1 and -2, caprine herpesvirus-2, and bison lymphotropic herpesvirus, using quantitative PCR. Only BoHV-6 was detected, with an overall frequency of 32%, ranging between 22% and 42% in the different countries. Infection was detected across all ages, from one day after birth, and was positively correlated with age. There was no evidence of an association with specific disease processes. In positive animals, BoHV-6 was detected in all organs with high frequency, consistently in the lungs or spleen. Viral loads varied substantially. In BoHV-6-positive gravid cows, organs of fetuses tested negative for infection, indicating that the virus is not vertically transmitted. Our results confirm previous data indicating that BoHV-6 is a commensal of domestic cattle not associated with disease processes and confirm that infections with other macaviruses are rare and sporadic.

Pediatric drug development, especially in disease areas that only affect children, can be stimulated by using juvenile animal models not only for general safety studies, but also to gain knowledge on the pharmacokinetic and pharmacodynamic properties of the drug. Recently, the conventional growing piglet has been suggested as juvenile animal model. However, more studies with different classes of drugs are warranted to make a thorough evaluation whether the juvenile pig might be a suitable preclinical animal model. Ibuprofen is one of the most widely used non-steroidal anti-inflammatory drugs in human. The present study determined the PK parameters, gastro-intestinal and renal safety of 5 mg/kg BW ibuprofen after single intravenous, single oral and multiple oral administration to each time eight pigs (four males, four females) aging 1, 4, 8 weeks and 6–7 months. Oral administration was performed via a gastrostomy button. A jugular catheter was used for intravenous administration and blood sampling. To assess NSAID induced renal toxicity, renal function was evaluated using iohexol and p-aminohippuric acid as markers for glomerular filtration rate and renal plasma flow, respectively. After the trial, necropsy and histology was performed to evaluate macroscopic and microscopic gastro-intestinal as well as renal lesions. Both enantiomers, R-ibuprofen and S-ibuprofen, were determined in plasma using an in-house developed and validated UHPLC-MS/MS method. Pharmacokinetic parameters were estimated using compartmental analysis. Clearance and volume of distribution of total ibuprofen and both enantiomers increased with age as was observed in human. The rate of stereochemical conversion decreased with age. Multiple oral dosing decreased the absolute oral bioavailability and maximum plasma concentration of R-ibuprofen and food consumption did not influence drug absorption. Based on the limited available pediatric literature, the current study might suggest the conventional pig as suitable animal model to evaluate NSAIDs for pediatric use.

In a previous study in influenza-naïve pigs, heterologous prime-boost vaccination with monovalent, adjuvanted whole inactivated vaccines (WIV) based on the European swine influenza A virus (SwIAV) strain, A/swine/Gent/172/2008 (G08), followed by the US SwIAV strain, A/swine/Pennsylvania/A01076777/2010 (PA10), was shown to induce broadly cross-reactive hemagglutination inhibition (HI) antibodies against 12 out of 15 antigenically distinct H3N2 influenza strains. Here, we used the pig model to examine the efficacy of that particular heterologous prime-boost vaccination regimen, in individuals with pre-existing infection-immunity. Pigs were first inoculated intranasally with the human H3N2 strain, A/Nanchang/933/1995. Seven weeks later, they were vaccinated intramuscularly with G08 followed by PA10 or vice versa. We examined serum antibody responses against the hemagglutinin and neuraminidase, and antibody-secreting cell (ASC) responses in peripheral blood, draining lymph nodes, and nasal mucosa (NMC), in ELISPOT assays. Vaccination induced up to 10-fold higher HI antibody titers than in naïve pigs, with broader cross-reactivity, and protection against challenge with an antigenically distant H3N2 strain. It also boosted ASC responses in lymph nodes and NMC. Our results show that intramuscular administration of WIV can lead to enhanced antibody responses and cross-reactivity in pre-immune subjects, and recall of ASC responses in lymph nodes and NMC.

Background: Clinical results of regenerative treatments for osteoarthritis are becoming increasingly significant. However, several questions remain unanswered concerning mesenchymal stem cell (MSC) adhesion and incorporation into cartilage. Methods: To this end, peripheral blood (PB) MSCs were chondrogenically induced and/or stimulated with pulsed electromagnetic fields (PEMFs) for a brief period of time just sufficient to prime differentiation. In an organ culture study, PKH26 labelled MSCs were added at two different cell densities (0.5 x106 vs 1.0 x106). In total, 180 explants of six horses (30 per horse) were divided into five groups: no lesion (i), lesion alone (ii), lesion with naïve MSCs (iii), lesion with chondrogenically-induced MSCs (iv) and lesion with chondrogenically-induced and PEMF-stimulated MSCs (v). Half of the explants were mechanically loaded and compared with the unloaded equivalents. Within each circumstance, six explants were histologically evaluated at different time points (day 1, 5 and 14). Results: COMP expression was selectively increased by chondrogenic induction (p = 0.0488). PEMF stimulation (1mT for 10 minutes) further augmented COL II expression over induced values (p = 0.0405). On the other hand, MSC markers remained constant over time after induction, indicating a largely predifferentiated state. In the unloaded group, MSCs adhered to the surface in 92.6% of the explants and penetrated into 40.7% of the lesions. On the other hand, physiological loading significantly reduced surface adherence (1.9%) and lesion filling (3.7%) in all the different conditions (p Conclusion: The present study demonstrates that primed chondrogenic induction of MSCs at a lower cell density without loading results in significantly enhanced and homogenous MSC adhesion and incorporation into equine cartilage.

Mammal skin has a crucial function in several life-preserving processes such as hydration, protection against chemicals and pathogens, initialization of vitamin D synthesis, excretion and heat regulation. Severe damage of the skin may therefore be life-threatening. Skin wound repair is a multiphased, yet well-orchestrated process including the interaction of various cell types, growth factors and cytokines aiming at closure of the skin and preferably resulting in tissue repair. Regardless various therapeutic modalities targeting at enhancing wound healing, the development of novel approaches for this pathology remains a clinical challenge. The time-consuming conservative wound management is mainly restricted to wound repair rather than restitution of the tissue integrity (the so-called “restitutio ad integrum”). Therefore, there is a continued search towards more efficacious wound therapies to reduce health care burden, provide patients with long-term relief and ultimately scarless wound healing. Recent in vivo and in vitro studies on the use of skin wound regenerative therapies provide encouraging results, but more protracted studies will have to determine whether the effect of observed effects are clinically significant and whether regeneration rather than repair can be achieved. For all the aforementioned reasons, this article reviews the emerging field of regenerative skin wound healing in mammals with particular emphasis on growth factor- and stem cell-based therapies.

Background: Mammal skin plays a pivotal role in several life preserving processes and extensive damage may therefore be life threatening. Physiological skin regeneration is achieved through ongoing somatic stem cell differentiation within the epidermis and the hair follicle. However, in severe pathological cases, such as burn wounds, chronic wounds, and ulcers, the endogenous repair mechanisms might be insufficient. For this reason, exogenous purification and multiplication of epithelial-like stem/progenitor cells (EpSCs) might be useful in the treatment of these skin diseases. However, only few reports are available on the isolation, purification and characterization of EpSCs using suspension cultures. Methods: In the present study, skin was harvested from 6 mares and EpSCs were isolated and purified. In addition to their characterization based on phenotypic and functional properties, sphere formation was assessed upon isolation, i.e. at passage 0 (P₀), and at early (P4) and late (P10) passages using different culture conditions. Results: On average 0.53 ± 0.28% of these primary skin-derived cells showed the capacity to form spheres and hence possessed stem cell properties. Moreover, significantly more spheres were observed in EpSC medium versus differentiation medium, corroborating the EpSCs' privileged ability to survive in suspension. Furthermore, the number of cells per sphere significantly increased over time as well as with subsequent passaging. Upon immunophenotyping, the presumed EpSCs were found to co-express cytokeratin (CK) 14, Casein kinase 2 beta and Major Histocompatibility Complex (MHC) I and expressed no pan CK and wide CK. Only a few cells expressed MHC II. Their differentiation towards keratinocytes (at P4 and P10) was confirmed based on co-expression of CK 14, Casein kinase 2 beta, pan CK and wide CK. In one of six isolates, a non-EpSC cell type was noticed in adherent culture. Although morphological features and immunohistochemistry (IHC) confirmed a keratinocyte phenotype, this culture could be purified by seeding the cells in suspension at ultralow clonal densities (1 and 10 cells/cm2), yet with a significantly lower sphere forming efficiency in comparison to pure EpSCs (P = 0.0012). Conclusion: The present study demonstrated sphere formation as a valuable tool to purify EpSCs upon their isolation and assessed its effectiveness at different clonal seeding densities for eliminating a cellular contamination.

Degenerative joint disease (DJD) is a major cause of reduced athletic function and retirement in equine performers. For this reason, regenerative therapies for DJD have gained increasing interest. Platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) were isolated from a 6-year-old donor horse. MSCs were either used in their native state or after chondrogenic induction. In an initial study, 20 horses with naturally occurring DJD in the fetlock joint were divided in 4 groups and injected with the following: 1) PRP; 2) MSCs; 3) MSCs and PRP; or 4) chondrogenic induced MSCs and PRP. The horses were then evaluated by means of a clinical scoring system after 6 weeks (T1), 12 weeks (T2), 6 months (T3) and 12 months (T4) post injection. In a second study, 30 horses with the same medical background were randomly assigned to one of the two combination therapies and evaluated at T1. The protein expression profile of native MSCs was found to be negative for major histocompatibility (MHC) II and p63, low in MHC I and positive for Ki67, collagen type II (Col II) and Vimentin. Chondrogenic induction resulted in increased mRNA expression of aggrecan, Col II and cartilage oligomeric matrix protein (COMP) as well as in increased protein expression of p63 and glycosaminoglycan, but in decreased protein expression of Ki67. The combined use of PRP and MSCs significantly improved the functionality and sustainability of damaged joints from 6 weeks until 12 months after treatment, compared to PRP treatment alone. The highest short-term clinical evolution scores were obtained with chondrogenic induced MSCs and PRP. This study reports successful in vitro chondrogenic induction of equine MSCs. In vivo application of (induced) MSCs together with PRP in horses suffering from DJD in the fetlock joint resulted in a significant clinical improvement until 12 months after treatment.

Broiler chickens are rather resistant to deoxynivalenol and thus, clinical signs are rarely seen. However, effects of subclinical concentrations of deoxynivalenol on both the intestine and the liver are less frequently studied at the molecular level. During our study, we investigated the effects of three weeks of feeding deoxynivalenol on the gut wall morphology, intestinal barrier function and inflammation in broiler chickens. In addition, oxidative stress was evaluated in both the liver and intestine. Besides, the effect of a clay-based mycotoxin adsorbing agent on these different aspects was also studied. Our results show that feeding deoxynivalenol affects the gut wall morphology both in duodenum and jejenum of broiler chickens. A qRT-PCR analysis revealed that deoxynivalenol acts in a very specific way on the intestinal barrier, since only an up-regulation in mRNA expression of claudin 5 in jejunum was observed, while no effects were seen on claudin 1, zona occludens 1 and 2. Addition of an adsorbing agent resulted in an up-regulation of all the investigated genes coding for the intestinal barrier in the ileum. Up-regulation of Toll-like receptor 4 and two markers of oxidative stress (heme-oxigenase or HMOX and xanthine oxidoreductase or XOR) were mainly seen in the jejunum and to a lesser extent in the ileum in response to deoxynivalenol, while in combination with an adsorbing agent main effect was seen in the ileum. These results suggest that an adsorbing agent may lead to higher concentrations of deoxynivalenol in the more distal parts of the small intestine. In the liver, XOR was up-regulated due to DON exposure. HMOX and HIF-1α (hypoxia-inducible factor 1α) were down-regulated due to feeding DON but also due to feeding the adsorbing agent alone or in combination with DON.

Ascaris lumbricoides remains the most common endoparasite in humans, yet there is still very little information available about the immunological principles of protection, especially those directed against larval stages. Due to the natural host-parasite relationship, pigs infected with A. suum make an excellent model to study the mechanisms of protection against this nematode. In pigs, a self-cure reaction eliminates most larvae from the small intestine between 14 and 21 days post infection. In this study, we investigated the mucosal immune response leading to the expulsion of A. suum and the contribution of the hepato-tracheal migration. Self-cure was independent of previous passage through the liver or lungs, as infection with lung stage larvae did not impair self-cure. When animals were infected with 14-day-old intestinal larvae, the larvae were being driven distally in the small intestine around 7 days post infection but by 18 days post infection they re-inhabited the proximal part of the small intestine, indicating that more developed larvae can counter the expulsion mechanism. Self-cure was consistently associated with eosinophilia and intra-epithelial T cells in the jejunum. Furthermore, we identified increased gut movement as a possible mechanism of self-cure as the small intestinal transit time was markedly decreased at the time of expulsion of the worms. Taken together, these results shed new light on the mechanisms of self-cure that occur during A. suum infections.

The aim of this study was to explore the mechanisms of resistance against invading Ascaris suum larvae in pigs. Pigs received a low dose of 100 A. suum eggs daily for 14 weeks. This resulted in a >99% reduction in the number of larvae that could migrate through the host after a challenge infection of 5000 A. suum eggs, compared to naïve pigs. Histological analysis at the site of parasite entry, i.e. the caecum, identified eosinophilia, mastocytosis and goblet cell hyperplasia. Increased local transcription levels of genes for IL5, IL13, eosinophil peroxidase and eotaxin further supported the observed eosinophil influx. Further analysis showed that eosinophils degranulated in vitro in response to contact with infective Ascaris larvae in the presence of serum from both immune and naïve animals. This effect was diminished with heat-inactivated serum, indicating a complement dependent mechanism. Furthermore, eosinophils were efficient in killing the larvae in vitro when incubated together with serum from immune animals, suggesting that A. suum specific antibodies are required for efficient elimination of the larvae. Together, these results indicate an important role for eosinophils in the intestinal defense against invading A. suum larvae.

Despite Giardia duodenalis being one of the most commonly found intestinal pathogens in humans and animals, little is known about the host-parasite interactions in its natural hosts. Therefore, the objective of this study was to investigate the intestinal response in calves following a G. duodenalis infection, using a bovine high-density oligo microarray to analyze global gene expression in the small intestine. The resulting microarray data suggested a decrease in inflammation, immune response, and immune cell migration in infected animals. These findings were examined in more detail by histological analyses combined with quantitative real-time PCR on a panel of cytokines. The transcription levels of IL-6, IL-8, IL-13, IL-17, and IFN-γ showed a trend of being downregulated in the jejunum of infected animals compared to the negative controls. No immune cell recruitment could be seen after infection, and no intestinal pathologies, such as villus shortening or increased levels of apoptosis. Possible regulators of this intestinal response are the nuclear peroxisome proliferator-activated receptors alpha (PPARα), and gamma (PPARγ) and the enzyme adenosine deaminase (ADA), all for which an upregulated expression was found in the microarray and qRT-PCR analyses.

Background"Helicobacter (H.) heilmannii" type 1 is the most prevalent gastric non-H. pylori Helicobacter species in humans suffering from gastric disease. It has been shown to be identical to H. suis, a bacterium which is mainly associated with pigs. To obtain better insights into the long-term pathogenesis of infections with this micro-organism, experimental infections were carried out in different rodent models.Methodology/principal findingsMongolian gerbils and mice of two strains (BALB/c and C57BL/6) were infected with H. suis and sacrificed at 3 weeks, 9 weeks and 8 months after infection. Gastric tissue samples were collected for PCR analysis, histological and ultrastructural examination. In gerbils, bacteria mainly colonized the antrum and a narrow zone in the fundus near the forestomach/stomach transition zone. In both mice strains, bacteria colonized the entire glandular stomach. Colonization with H. suis was associated with necrosis of parietal cells in all three animal strains. From 9 weeks after infection onwards, an increased proliferation rate of mucosal epithelial cells was detected in the stomach regions colonized with H. suis. Most gerbils showed a marked lymphocytic infiltration in the antrum and in the forestomach/stomach transition zone, becoming more pronounced in the course of time. At 8 months post infection, severe destruction of the normal antral architecture at the inflamed sites and development of mucosa-associated lymphoid tissue (MALT) lymphoma-like lesions were observed in some gerbils. In mice, the inflammatory response was less pronounced than in gerbils, consisting mainly of mononuclear cell infiltration and being most severe in the fundus.Conclusions/significanceH. suis causes death of parietal cells, epithelial cell hyperproliferation and severe inflammation in mice and Mongolian gerbil models of human gastric disease. Moreover, MALT lymphoma-like lesions were induced in H. suis-infected Mongolian gerbils. Therefore, the possible involvement of this micro-organism in human gastric disease should not be neglected.