ABSTRACT: Lameness has been identified as a major welfare challenge in sheep farming production. The most common causes of lameness include several types of foot-related lesions. Accurate diagnosis of hoof lesions, particularly differentiating footrot from other foot disorders, is critical for implementing effective control strategies. This study assessed the ability of sheep farmers in Alentejo, Portugal, to recognise footrot lesions among other hoof disorders. A pictorial questionnaire depicting several hoof lesions, including footrot, interdigital dermatitis, contagious ovine digital dermatitis (CODD), toe granuloma, foot abscess, and shelly hoof, was applied to 61 Alentejo sheep farmers. Respondents identified which images represented footrot lesions and which of them they had previously observed in their flocks. Among the footrot images, the individual correct identification rate ranged from 95.1% to 100%. For other hoof disorders, the identification rate ranged between 88.5% (CODD) and 96.7% (shelly hoof). Eighty percent of the farmers (49/61) correctly answered all the eight questions. Farmers who reported no footrot in their flocks had a slightly higher accuracy (82.1%) compared to those who did (77.3%), though the difference was not statistically significant. In general, Alentejo sheep farmers evidenced a high level of accuracy in diagnosing footrot lesions, suggesting CODD does not look like to be an important confounding factor in Alentejo, as it mainly occurs in Great Britain.

Uterine health is paramount to fertility in broodmares and for the success of a breeding project, and the Lusitano breed is no exception. This study aimed to characterize the mare uterine microbiota using practical clinical evaluation methods. Mares were examined by transrectal palpation and ultrasonography, followed by the collection of samples by one of three different techniques: uterine lavage, biopsy, or swab. The results of cytology, histology, microbiology, and antimicrobial susceptibility testing were recorded, and statistical analyses were performed. Inflammation was present in 42.2% of the mares and positive culture in 65.4%. Escherichia coli and Streptococcus spp. were the most isolated microorganisms. The most efficient antimicrobials were gentamicin, trimethoprim-sulfamethoxazole, and enrofloxacin and resistance was detected mainly for doxycycline, penicillin, and ceftiofur. The phase of the cycle was significantly associated with the presence of inflammation (p = 0.0308). The isolation of Gram-positive or Gram-negative bacteria correlated to the microbiological isolation by primoculture/enrichment processes (p = 0.0183). This was a routine standard breeding evaluation of broodmares in the management of a stud farm, hence displaying the characteristics of a field study. The antimicrobial resistance findings reinforce the importance of performing microbiology and susceptibility tests, even under field conditions, to maximize targeted antimicrobial therapy efficiency and minimize the worldwide problem of antimicrobial resistance, promoting antimicrobial stewardship.

Background Ovine footrot caused by Dichelobacter nodosus ( D. nodosus ) is a contagious disease with serious economic and welfare impacts in sheep production systems worldwide. A better understanding of the host genetic architecture regarding footrot resistance/susceptibility is crucial to develop disease control strategies that efficiently reduce infection and its severity. A genome-wide association study was performed using a customized SNP array (47,779 SNPs in total) to identify genetic variants associated to footrot resistance/susceptibility in two Portuguese native breeds, i.e. Merino Branco and Merino Preto, and a population of crossbred animals. A cohort of 1375 sheep sampled across 17 flocks, located in the Alentejo region (southern Portugal), was included in the analyses. Results Phenotypes were scored from 0 (healthy) to 5 (severe footrot) based on visual inspection of feet lesions, following the Modified Egerton System. Using a linear mixed model approach, three SNPs located on chromosome 24 reached genome-wide significance after a Bonferroni correction ( p < 0.05). Additionally, six genome-wide suggestive SNPs were identified each on chromosomes 2, 4, 7, 8, 9 and 15. The annotation and KEGG pathway analyses showed that these SNPs are located within regions of candidate genes such as the nonsense mediated mRNA decay associated PI3K related kinase (SMG1) (chromosome 24) and the RALY RNA binding protein like (RALYL) (chromosome 9), both involved in immunity, and the heparan sulfate proteoglycan 2 (HSPG2) (chromosome 2) and the Thrombospodin 1 (THBS1) (chromosome 7) implicated in tissue repair and wound healing processes. Conclusion This is the first attempt to identify molecular markers associated with footrot in Portuguese Merino sheep. These findings provide relevant information on a likely genetic association underlying footrot resistance/susceptibility and the potential candidate genes affecting this trait. Genetic selection strategies assisted on the information obtained from this study could enhance Merino sheep-breeding programs, in combination with farm management strategies, for a more effective and sustainable long-term solution for footrot control.

In the Portuguese Alentejo region, Merino sheep breed is the most common breed, reared for the production of meat, dairy, and wool. Footrot is responsible for lameness, decreased animal welfare, and higher production losses, generating a negative economic impact. The disease is caused by Dichelobacter nodosus that interacts with the sheep foot microbiome, to date largely uncharacterized. In fact, Dichelobacter nodosus is not able to induce footrot by itself being required the presence of a second pathogen known as Fusobacterium necrophorum. To understand and characterize the footrot microbiome dynamics of different footrot lesion scores, a whole metagenome sequencing (WMGS) approach was used. Foot tissue samples were collected from 212 animals with different degrees of footrot lesion scores, ranging from 0 to 5. Distinct bacterial communities were associated with feet with different footrot scores identifying a total of 63 phyla and 504 families. As the severity of footrot infection increases the microorganisms’ diversity decreases triggering a shift in the composition of the microbiome from a dominant gram-positive in mild stages to a dominant gram-negative in the severe stages. Several species previously associated with footrot and other polymicrobial diseases affecting the epidermis and provoking inflammatory responses such as Treponema spp., Staphylococcus spp., Streptococcus spp. and Campylobacter spp. were identified proliferating along with the lesions’ severity. Although these bacteria are not able to initiate footrot, several evidences have been described supporting their association with the severity and incidence increase of footrot lesions caused by Dichelobacter nodosus and Fusobacterium necrophorum. Further investigation is required to establish the roles of particular taxa and identify which of them play a role in the disease process and which are opportunistic pathogens.

The animal cancer burden is essential for the translational value of companion animals in comparative oncology. The present work aims to describe, analyze, and compare frequencies and associations of tumors in dogs and cats based on the Animal Cancer Registry created by Vet-OncoNet. With 9079 registries, regarding 2019 and 2020, 81% (n = 7355) belonged to dogs. In comparison, cats have a general one-year right advance in the mean age of cancer diagnosis compared to dogs. The multivariate topography group analysis shows a distinct pattern between the two species: dogs have higher odds of cancer in the genito-urinary system, spleen, soft tissue tumors and skin, while cats show higher odds for tumors in the eyes, digestive organs, nasal cavity, lymph nodes, bones and mammary glands. Regarding morphologies, dogs are overrepresented in mast cell tumors (MCT), melanomas, and hemangiosarcomas. While cats are overrepresented in fibrosarcomas, lymphomas (T and B-cell), in malignant mammary tumors, and squamous cell carcinoma (SCC). Females have greater odds only in the mammary gland, with males having greater odds in six of twelve topographies. This study is the first outcome of continuous animal cancer registration studies in Portugal.

Leptin up-regulates the hypercapnic ventilatory response and upper airway patency during sleep acting on LEPR neurons in the DMH. Respiratory effects of leptin are likely mediated via projections of these neurons to the dorsal raphe.

The Mediterranean climate region of Alentejo in the Southern of Portugal is an important sheep production centre but little is known about the presence and characteristics of Dichelobacter nodosus in association with Fusobacterium necrophorum in the different footrot lesion scores. DNA from 261 interdigital biopsy samples, taken from 14 footrot affected flocks and from three non-affected flocks, were analysed for the presence of D. nodosus and F. necrophorum by real-time PCR. Both virulence and serogroup were determined for 132 and 53 D. nodosus positive biopsy samples, respectively. The co-infection with both bacteria was the commonest epidemiological finding associated with a greater disease severity. There was a statistically significant association (p = 0.002) between footrot-affected flocks and the presence of D. nodosus. Most D. nodosus positive samples were virulent (96.2 %) and belonged to serogroup B (90 %).

Analysis of canine and feline tumor malignancy data can help clinicians identify high-risk patients and make more accurate decisions. Based on a sample of 16,272 cancer records, including 3266 cats and 13,006 dogs, collected from January 2019 to December 2021 in the Vet-OncoNet Network database, this study aimed to compare the tumor malignancy profile between cats and dogs, considering animal-related factors (sex, age, and breed), topography, and geographic location using a mixed-effects logistic regression model. Cats had a higher proportion of malignant tumors (78.7%) than dogs (46.2%), and the malignancy profile was very different regarding tumors' topographies. The mean age of malignant tumors occurred eight months later than benign ones (9.1, SD = 3.4; 9.8, SD = 3.2), in general. Species (OR = 3.96, 95%CI 3.57: 4.39) and topography (MOR = 4.10) were the two most important determinants of malignancy risk. Female dogs had a higher risk than male dogs (OR = 1.19, 95%CI 1.08: 1.31), which does not appear to be the case in cats (OR = 0.98, 95%CI 0.77: 1.23). Breed contributed significantly to differences in malignancy risk in dogs (MOR = 1.56), particularly in pit bulls and boxers. District of residence was not so relevant in predicting malignancy risk (MOR = 1.14). In both species, the risk of malignancy increased by approximately 20% every three years. It could be hypothesized that species differences in genetic structure may contribute to tumor malignancy.

Skin is one of the organs most tested for toxicity and safety evaluation during the process of drug research and development and in the past has usually been performed in vivo using animals. Over the last few years, non-animal alternatives have been developed and validated epidermis models for human and rat skin are already available. Our goal was to develop a histotypical canine skin analog, suitable for non-animal biocompatibility and biosafety assessment. Canine keratinocytes were seeded in an air-lift culture using an adapted version of the CELLnTEC protocol. Corrosion and irritation protocols were adapted from human EpiSkinTM. For histological analysis, sample biopsies were fixed in neutral-buffered formalin, and paraffin slices were routinely processed and stained with hematoxylin and eosin. A canine multilayer and stratified epidermal-like tissue (cEpiderm), confirmed by histological analysis, was obtained. The cEpiderm tissue exhibited normal morphological and functional characteristics of epidermis, namely impermeability and an adequate response to stressors. The cEpiderm is a promising canine skin model for non-animal safety testing of veterinary pharmaceuticals and/or cosmetics, significantly contributing to reducing undesirable in vivo approaches. cEpiderm is therefore a valid canine skin model and may be made commercially available either as a service or as a product.

Canine cutaneous mast cell tumors (ccMCTs) are currently graded according to Patnaik and Kiupel grading schemes. The qualitative and semiquantitative parameters applied in these schemes may lead to inter- and intraobserver variability. This study investigates the prognostic value of volume-weighted mean nuclear volume (vv¯), a stereological estimation that provides information about nuclear size and its variability. vv¯ of 55 ccMCTs was estimated using the "point-sampled intercept" method and compared with histological grade and clinical outcome. The clinical history of dogs treated with surgical excision alone was available for 30 ccMCTs. Statistical differences in vv¯ were found between grade II (x¯ = 115 ± 29 µm3) and grade III ccMCTs (x ¯= 197 ± 63 µm3), as well as between low-grade (x ¯= 113 ± 28 µm3) and high-grade ccMCTs (x¯ = 184 ± 63 µm3). An optimal cutoff value of vv¯ ≥ 150 µm3 and vv¯ ≥ 140 µm3 was determined for grade III and high-grade ccMCTs, respectively. In terms of prognosis, vv¯ was not able to predict the clinical outcome in 42% of the cases; however, cases with vv¯ <125 µm3 had a favorable outcome. These results indicate that, despite having limited prognostic value when used as a solitary parameter, vv¯ is highly reproducible and is associated with histological grade as well as with benign behavior.

As key prey, the wild rabbit downsize constitutes a major drawback on the endangered Iberian lynx (Lynx pardinus) re-introduction in the Iberia. Several captive breeding units mostly located in Alentejo, endeavour the wild rabbit repopulation of depleted areas assigned for the lynx re-introduction. Here we report an RHDV2 outbreak that occurred in early 2016 in a wild rabbit captive breeding unit located in Barrancos municipality. The estimated mortality rate between March and April 2016 was approximately 8.67%. Anatomopathologic examination was carried out for 13 victimized rabbits. Molecular characterization was based on the complete vp60 capsid gene. The 13 rabbit carcasses investigated showed typical macroscopic RHD lesions testing positive to RHDV2- RNA. Comparison of the vp60 nucleotide sequences obtained from two specimens with others publically available disclosed similarities below 98.22% with RHDV2 strains originated in the Iberia and Azores and revealed that the two identical strains from Barrancos-2016 contain six unique single synonymous nucleotide polymorphisms. In the phylogenetic analysis performed, the Barrancos-2016 strains clustered apart from other known strains, meaning they may represent new evolutionary RHDV2 lineages. No clear epidemiological link could be traced for this outbreak where the mortalities were lower compared with previous years. Yet, network analysis suggested a possible connection between the missing intermediates from which the strains from Barrancos 2013, 2014 and 2016 have derived. It is therefore possible that RHDV2 has circulated endemically in the region since 2012, with periodic epizootic occurrences. Still, six years after its emergence in wild rabbits, RHDV2 continues to pose difficulties to the establishment of natural wild rabbit populations that are crucial for the self-sustainability of the local ecosystems.

Four cases of tumors in which cell internalization was frequently visualized are reported: one feline mammary carcinoma, one feline cutaneous squamous cell carcinoma, one canine pulmonary squamous cell carcinoma and one canine pleural mesothelioma. Cell internalization was observed by cytology in two of these cases (the feline mammary tumour and the pleural effusion in the canine mesothelioma) and by histopathology in all but the canine mesothelioma. Immunohistochemical staining for pancytokeratin was positive for both internalized and host cells, while E-cadherin expression was frequently absent, although internalized cells occasionally stained positive. This cell-to-cell interaction seems to be associated with tumors displaying a strong epithelial-mesenchymal transitional phenotype, in which cancer cells become engulfed by other cancer cells. Such event could be regarded as an important hallmark of very high malignancy.

In this article, we describe spontaneous melanotic lesions in the skin of axillary seabream, P agellus acarne ( R isso), from a defined area of the P ortuguese C oast, located in C abo da R oca and F oz do A relho. The lesions corresponded to the black pigmentation spots on the skin of the head, fins, lips and conjunctiva and, additionally, black nodules on the skin of the head and lips. In some specimens, the nodular formations in the head changed their anatomical conformation. Histologically, there were melanophores scattered along the basement membrane or forming aggregates in the dermis, infiltrating the subcutaneous tissue but not invading the adjacent muscle tissue. The aim of this study was to characterize the macroscopic and microscopic features of the pigmented lesions. These fish show sessile hyperpigmented lesions (spots) that correspond to proliferative lesions of melanophores in the dermis and nodular lesions that correspond to neoplastic lesions, melanophoromas. The melanophores in such lesions showed high concentration of melanin in the cytoplasm, moderate pleomorphism and compact distribution throughout all of the dermis.

The production of Alentejano breed pig started a recovery two decades ago due to increasing demand for gourmet products. These pigs are raised in rotational semi-extensive or extensive outdoor production systems in the "Montado" (green and cork oak forest), grazing and feeding acorns and other associated food resources. Bacteria of the genus Aeromonas are considered as emerging pathogens of importance for man and,animals, but its involvement in swine is not well documented. In the context of a study made at the University of Evora to assess the specific diseases of Alentejano swine, diseased piglets from two farms were submitted for pathological and bacteriological examinations. Pathological examinations revealed changes characteristic of septicemia, and Aeromonas hydrophila was isolated in pure culture from multiple organs of piglets from both farms. Antibiotic sensitivity tests showed that the isolates from one of the farms were susceptible to gentamicin, oxitetracycline, neomycin, enrofloxacin, colistin sulfate, trimethoprim, ceftiofur, and amoxicillin plus clavulanic acid. In contrast, the A. hydrophila isolated in the other farm was resistant to all drugs tested but enrofloxacin. This is the first report in the world showing the relationship between septicemia and A. hydrophila infection in piglets. The importance of this finding is further reinforced by the fact that these bacteria can be highly resistant to antimicrobial agents.

An outbreak of severe respiratory disease in a goat herd was associated with Mycoplasma ovipneumoniae, Mycoplasma arginini, Mannheimia haemolytica and Pasteurella multocida with mortality rates exceeding 20% in kids. Post mortem features in affected kids included severe pleuropneumonia, lung consolidation, large quantities of pleural fluid and pericarditis. This is the first report of atypical proliferative pneumonia in goats in Portugal.

A 10-year-old male mini Lop rabbit (Oryctolagus cuniculus) was presented with a 2-month history of progressive enlargement of the hemiscrotal sacs. On palpation, both testicles were enlarged, with the left being larger than the right. A bilateral orchiectomy was performed and the testicular tissue submitted for pathological examination. The histopathological diagnosis of the tissue revealed a right gonadal intratubular seminoma and diffuse seminoma of the left gonad. Although seminomas have been reported in several domestic and wild animals, including rabbits, this is, to the authors' knowledge, the first report of bilateral seminomas with dissimilar structural patterns in rabbits.

This study was carried out to investigate fifteen cases of acute lethal infection of calves (<or= 4 months of age) by the protozoan parasite Theileria (T.) annulata in the south of Portugal. Calves developed multifocal to coalescent nodular skin lesions, similar to multicentric malignant lymphoma. Infestation with ticks (genus Hyalomma) was intense. Theileria was seen in blood and lymph node smears, and T. annulata infection was confirmed by isolation of schizont-transformed cells and sequencing of hypervariable region 4 of the 18S rRNA gene. At necropsy, hemorrhagic nodules or nodules with a hemorrhagic halo were seen, particularly in the skin, subcutaneous tissue, skeletal and cardiac muscles, pharynx, trachea and intestinal serosa. Histologically, nodules were formed by large, round, lymphoblastoid neoplastic-like cells. Immunohistochemistry (IHC) identified these cells as mostly CD3 positive T lymphocytes and MAC387 positive macrophages. A marker for B lymphocytes (CD79alphacy) labeled very few cells. T. annulata infected cells in these nodules were also identified by IHC through the use of two monoclonal antibodies (1C7 and 1C12) which are diagnostic for the parasite. It was concluded that the pathological changes observed in the different organs and tissues were caused by proliferation of schizont-infected macrophages, which subsequently stimulate a severe uncontrolled proliferation of uninfected T lymphocytes.