Forensic findings in urban red fox mortality in the metropolitan area of Madrid, 2014-2022.

The expanding presence of red foxes (Vulpes vulpes) in urban and suburban regions could potentially lead to increased instances of human aggression towards this species. We studied 10 deceased red foxes that were submitted by law enforcement agencies in the metropolitan area of Madrid in 2014-2022 because of suspected abuse. Forensic autopsies were performed to establish the cause and manner of death. In 4 of the 10 cases, the cause of death was deemed unnatural, involving blunt-force trauma (n = 2), asphyxia resulting from hanging (n = 1), and firearm injury (n = 1). Among the remaining cases, most had succumbed to natural causes (n = 4), often marked by severe emaciation and a high burden of parasites, primarily Sarcoptes scabiei. In 2 cases, death was undetermined given the poor preservation of the carcass. The growing prevalence of wildlife species in urban areas, particularly red foxes, may require forensic veterinary investigation of deaths potentially related to abuse.

Forensic investigations of suspected livestock depredation by vultures: scientific tools for compensation programmes.

Human-wildlife conflicts may have devastating consequences for fauna due to targeting by humans of wildlife populations suspected to have predated livestock. Suspicion of depredation of extensively raised livestock by vultures in Europe has triggered public administration-led forensic investigations intended to distinguish between predation and scavenging in order to compensate farmers for attacks on their livestock. In this study, gross and histological analyses were carried out on suspected cases of domestic animal depredation by griffon vultures (Gyps fulvus) over a 1-year period. Fifty-eight animals were affected (n = 41, suspected depredation), including domestic cattle (75.9%), sheep (22.4%) and a goat (1.7%). All the adults affected were female and most cases of suspected depredation occurred during the peripartum period (56%). Histological investigations distinguished between post-mortem (84% of diagnostic samples) and ante-mortem (16% of diagnostic samples) cases, and gross examinations revealed significant differences (P ≤ 0.002) between ante-mortem, post-mortem and non-diagnostic samples. This study highlights the need to optimize sampling protocols to increase the success of forensic studies. The forensic investigations presented here may be applied to resolve human-wildlife conflicts involving not only vultures but other endangered carnivores and contribute to human-wildlife coexistence in rural areas by protecting the interests of both the livestock sector and endangered species.

Forensic investigations of religious rituals involving poultry: a case report.

Animal victims of human cruelty are receiving increasing attention from the press and society. Veterinary pathologists and civic authorities have a duty not only to elucidate the cause, method and manner of death but also to address the motivation behind a case. Poultry are commonly used as offering to gods in Santería rituals (ebós). Only a few cases have been reported in scientific journals, in contrast with the number of cases described in the media. Here, a hen and a cockerel (Gallus domesticus) were submitted to the VISAVET Health Surveillance Centre for autopsy, which comprised macroscopic and histological studies and microbiological testing for Chlamydia psittaci, avian orthoavulavirus 1 (Newcastle disease virus), West Nile virus and avian influenza virus. The carcasses were in an active decay stage of decomposition, containing larvae of Lucilia eximia. Both animals had been decapitated and haemorrhages in the cutting region were observed histologically. Post-mortem burns were also observed. To the best of the authors' knowledge, this is the first post-mortem description of lesions in a domestic avian species due to religious rituals. European Union and Spanish forensic practitioners should be aware that these ritual sacrifices occur in some European countries. Moreover, future European and national legislation updates should consider this type of animal abuse.

An Outbreak of Aeromonas salmonicida in Juvenile Siberian Sturgeons (Acipenser baerii).

Aeromonas salmonicida is one of the major threats to world aquaculture, causing fish furunculosis and high mortality rates in cultured fish, particularly salmonids. Although Aeromonas spp. is a thoroughly studied pathogen, little is known regarding aeromoniasis in sturgeons. After a mortality outbreak, four juvenile sturgeons (Acipenser baerii) were submitted for autopsy and tissue samples were collected for histopathological and microbiological studies. The external examination revealed size heterogenicity, skin hyperpigmentation and reduced body condition of sturgeons. Within the abdominal cavity, mild hepatomegaly and splenomegaly were observed, as well as generalized organic congestion. Histology revealed severe multifocal haemorrhagic and ulcerative dermatitis, mainly localized in the dorsal and latero-ventral areas of fish. The histological study also showed moderate to severe inflammation of gills and organic lesions compatible with septicaemia. Bacterial isolates were identified as Aeromonas salmonicida subsp. salmonicida using MALDI-TOF MS and PCR. Overall, the lesions first described here are consistent with those previously reported in other cultured fish species and contribute to a better understanding of the pathogenesis of Aeromonas salmonicida subsp. salmonicida in the Siberian sturgeon, aside from providing new diagnostic tools for bacterial diseases impacting the fast-growing industry of caviar.

Primary Feline Tauopathy: Clinical, Morphological, Immunohistochemical, and Genetic Studies.

Tauopathies are a group of neurodegenerative diseases characterized by the pathological aggregation of hyperphosphorylated tau in neurons and glia. Primary tauopathies are not uncommon in humans but exceptional in other species. We evaluate the clinical, neuropathological, and genetic alterations related to tau pathology in 16 cats aged from 1 to 21 years with different clinical backgrounds. Interestingly, a 10-year-old female cat presented a six-year progressive history of mental status and gait abnormalities. The imaging study revealed generalized cortical atrophy. Due to the poor prognosis, the cat was euthanatized at the age of ten. Neuropathological lesions were characterized by massive neuronal loss with marked spongiosis and associated moderate reactive gliosis in the parietal cortex, being less severe in other areas of the cerebral cortex, and the loss of Purkinje cells of the cerebellum. Immunohistochemical methods revealed a 4R-tauopathy with granular pre-tangles in neurons and coiled bodies in oligodendrocytes. Deposits were recognized with several phospho-site antibodies (4Rtau, tau5, AT8, PFH, tau-P Thr181, tau-P-Ser 262, tau-P Ser 422) and associated with increased granular expression of active tau kinases (p38-P Thr180/Tyr182 and SAPK/JNK-P Thr138/Thr185). The genetic study revealed well-preserved coding regions of MAPT. No similar alterations related to tau pathology were found in the other 15 cats processed in parallel. To our knowledge, this is the first case reporting a primary 4R-tauopathy with severe cerebral and Purkinje cell degeneration in an adult cat with neurological signs starting at a young age.

Soil C dynamics after deforestation and subsequent conversion of arable cropland to grassland in humid temperate areas.

Land use and plant-soil management influence soil organic C stocks and soil properties. This study aimed to identify the main mechanisms by which these factors alter soil organic matter (SOM) dynamics and stocks. Changes in the organic C pools and biochemical quality in different OM compartments were assessed: a) after deforestation and intensive cultivation (SOM loss) and then, b) after the conversion of cropland to grassland (SOM replenishment) in a chronosequence of recovery (1-45 years). Topsoil samples were subjected to physical fractionation to assess the distribution of free particulate OM (POM) and mineral associated OM (MAOM). SOM quality was characterized by 13C NMR spectroscopy, thermal analysis (DSC/TG), and microbial activity was monitored by isothermal microcalorimetry. Deforestation and intensive cultivation led to the loss of 80 % of the C stored in the upper mineral soil (up to 30-35 cm). The POM was almost depleted, MAOM underwent significant losses (>40 %) and all OM compounds, including the aromatic C, were affected. The large and unexpected loss of MAOM can be attributed to the low specific surface soil area and also to the labile (biodegradable) nature of the OM in this fraction. After 45 years, conversion of cropland to grassland recovered 68 % of the C lost in the mineral soil (mainly as MAOM), at an annual rate of 1.25 Mg C ha-1. The present findings showed that the persistence of long-term OM depends on how strongly organic compounds are adsorbed onto mineral surfaces (i.e., the specific surface area) and the biochemical nature of OM compounds. Adequate plant-soil management favoured the replenishment of the MAOM under these experimental conditions, and this fraction was an active pool in terms of C storage and biochemical quality. This study served to test current theories about changes in soil C fractions due to land use changes and soil-plant management.

Immunopathology of early and advanced epididymis lesions caused by Brucella ovis in rams.

Ovine brucellosis is an infectious disease that causes alterations in the reproductive tract in ram and abortion in ewes. Their negative economic impact in ovine production warrants a thorough understanding the interactions between B. ovis and the host. Here, epididymis lesions of rams infected by B. ovis were histopathologically staged into early and advanced. Expression by immunohistochemistry of Brucella antigens, inflammatory cell markers (CD3, CD79αcy) and cytokines (IFN-γ, TNF-α, TGF-ß1) was assessed in both stages. Early lesions were characterized by epithelial changes, interstitial inflammation, and mild fibrosis; whereas advanced lesions displayed caseous granulomas containing numerous macrophages, multinucleated giant cells, lymphocytes, and plasma cells. Expression of Brucella antigens were observed in both stages. The cellular response in B. ovis lesions were predominantly of T-cells (CD3+) whereas low numbers of B-cells and plasma cells (CD79αcy+) were present in both early and advanced lesions. IFN-γ was expressed by lymphocytes in early lesions suggesting that the adaptive immune response against B. ovis is initiated by Th1 cells, this response was also preserved in advanced stages. Expression of TNF-α was observed in neutrophils of epithelial microabscesses and intraepithelial T-cells of early lesions suggesting a promotion of neutrophil phagocytosis triggered by TNF-α. On the other hand, advanced lesions showed a reduction of TNF-α expression which may permit B. ovis persistence in granulomas. Lastly, TGF-ß1 expression (fibroblast, macrophages and less in lymphocytes) were increased with time, suggesting that B. ovis promotes TGF-ß1 secretion promoting chronicity of the lesions.

First detection of Flavobacterium psychrophilum in juvenile Siberian sturgeons (Acipenser baerii) and description of the pathological findings.

Flavobacterium psychrophilum affects many cultured fish species and is considered one of the most important bacterial pathogens causing substantial economic losses in salmonid aquaculture worldwide. Here, F. psychrophilum was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nested PCR as the aetiological agent causing mortality in diseased juvenile Siberian sturgeons (Acipenser baerii) reared on a freshwater fish farm. Diseased sturgeons were lethargic and displayed dark skin pigmentation, increased mucus production and the presence of skin ulcerations and haemorrhages specially on the ventral side and the base of fins. The histological examination of fish revealed proliferative branchitis, ulcerative and necrotizing dermatitis and myositis, lymphoid tissue atrophy, liver and kidney degeneration and thrombosis. To the best of our knowledge, this is the first report describing the infection of Siberian sturgeons by F. psychrophilum. The detection of F. psychrophilum in diseased Siberian sturgeons and the description of the pathological findings observed during the outbreak may contribute to a better understanding of the bacterium pathogenicity and the range of fish species susceptible to infection.

Histopathologic and immunohistochemical findings in the placentas and fetuses of domestic swine naturally infected with Brucella suis biovar 2.

Porcine brucellosis, which is caused by Brucella suis biovar (bv) 2, is a re-emerging disease that causes reproductive problems in pigs in Europe. The pathogenesis and lesions of B. suis intrauterine infection are poorly characterized; characterization could facilitate the diagnosis and investigation of porcine brucellosis. We collected samples of placentas and fetuses for histologic and microbiologic studies during an outbreak of abortions on a pig-breeding farm in Spain. Brucella was cultured from the vaginal swabs obtained from sows that had aborted, some placentas, and fetal tissues (spleen, liver, lung, gastric content); molecular testing confirmed B. suis bv 2 infection. Histologically, there was necrotizing and hemorrhagic placentitis; suppurative hepatitis; lymphoid depletion and sinusoidal histiocytosis in the spleen, lymph nodes, and thymus; and bronchointerstitial pneumonia. Hemorrhages were observed in the umbilical cord, heart, kidneys, and brain. We detected Brucella by immunohistochemistry (IHC) in all of the placentas and fetal organs studied, specifically in the trophoblasts of the chorionic epithelium, in the cytoplasm of macrophages in the chorionic stroma, and extracellularly in necrotic debris. Furthermore, we assessed the lymphocyte population in the placentas through the use of IHC (anti-CD3, anti-Pax5 antibodies), revealing that the lymphocytic response was composed of T cells but not B cells.

Engineered live bacteria suppress Pseudomonas aeruginosa infection in mouse lung and dissolve endotracheal-tube biofilms.

Engineered live bacteria could provide a new modality for treating lung infections, a major cause of mortality worldwide. In the present study, we engineered a genome-reduced human lung bacterium, Mycoplasma pneumoniae, to treat ventilator-associated pneumonia, a disease with high hospital mortality when associated with Pseudomonas aeruginosa biofilms. After validating the biosafety of an attenuated M. pneumoniae chassis in mice, we introduced four transgenes into the chromosome by transposition to implement bactericidal and biofilm degradation activities. We show that this engineered strain has high efficacy against an acute P. aeruginosa lung infection in a mouse model. In addition, we demonstrated that the engineered strain could dissolve biofilms formed in endotracheal tubes of patients with ventilator-associated pneumonia and be combined with antibiotics targeting the peptidoglycan layer to increase efficacy against Gram-positive and Gram-negative bacteria. We expect our M. pneumoniae-engineered strain to be able to treat biofilm-associated infections in the respiratory tract.

Intestinal T-cell lymphoma in an Asian small-clawed otter: case report and literature review of lymphoma in the subfamily Lutrinae.

The Asian small-clawed otter (Aonyx cinereus) is an endangered species that is common in zoologic collections. A 17-y-old female Asian small-clawed otter under human care, with a clinical history of chronic renal disease, was euthanized because of deteriorating health. Histologically, the jejunal wall was infiltrated by a monomorphic population of small neoplastic lymphocytes that expanded the lamina propria of the villi and crypts, and on rare occasions invaded the submucosa. The tumor was composed of T cells (CD3+) with a proliferation index of 16%. Based on the World Health Organization (WHO) Classification of Hematopoietic Neoplasms in Domestic Animals, this lymphoma was classified as an enteropathy-associated T-cell lymphoma (EATL) type 2. We also present here a review of the literature on intestinal lymphoma in the subfamily Lutrinae (otters).

Extracellular matrix proteins (fibronectin, collagen III, and collagen I) immunoexpression in goat tuberculous granulomas (Mycobacterium caprae).

The lesion resulting from the interaction between Mycobacterium and the host immune response is the tuberculous granuloma. Tuberculous granulomas, except in incipient stages, are partially or totally encapsulated by connective tissue. The aim of this study was to assess the immunoexpression of the extracellular matrix proteins fibronectin, collagen III, and collagen I in granulomas caused by Mycobacterium caprae in goats (Capra aegagrus hircus) to understand capsule development at different granuloma stages. For this purpose, a retrospective study of 56 samples of tuberculous granulomas in lung (n = 30) and mediastinal lymph node (n = 26) from 17 goats naturally infected with M. caprae in stages I (n = 15), II (n = 14) and III (n = 27) was carried out. Fibronectin immunoreaction was extracellular, fibrillar-reticular in the center of stage I, II and III granulomas and peripheral in stages II and III granulomas. Collagen III immunoexpression was extracellular and fibrillar in the center of stages I, II and III tuberculous granulomas in lung and mediastinal lymph node, and progressive expression was observed in the periphery of stages II and III granulomas. Finally, collagen I immunoexpression was extracellular and fibrillar, showing a progressive loss of central expression and an increase in peripheral expression in stage III granulomas compared to stage I granulomas. Immunoexpression of these extracellular matrix proteins could help understand fibrogenesis and dating in tuberculous granuloma in both animal models and humans.

Environment and Offspring Surveillance in Porcine Brucellosis.

Porcine brucellosis, caused by Brucella suis (B. suis), is a notifiable disease causing significant economic losses in production systems. Most infected pigs may act as carriers and shed B. suis even if asymptomatic. This can contribute to environmental persistence, thus hindering control efforts. Here, the environment and the offspring were investigated during and after a B. suis outbreak at a sow breeding farm. The diagnosis of B. suis in sows (n = 1,140) was performed by culture and polymerase chain reaction (PCR) from vaginal swabs, indirect enzyme-linked immunosorbent assay (I-ELISA) from sera, and brucellin skin test (BST). B. suis diagnosis in post-weaning pigs (n = 899) was performed by I-ELISA in sera and BST. The environmental surveillance programme was implemented by placing gauze sponges (n = 175) pre-hydrated in a surfactant and inactivating liquid for Brucella DNA detection by PCR in different farm areas. Our results showed that the offspring of infected sows reacted to in vivo techniques for B. suis. Furthermore, the offspring born during the outbreak displayed higher seropositivity (I-ELISA) and reactivity (BST) than those pigs born after. Brucella DNA was detected in pregnant sow areas, boxes, boots, and post-weaning pig areas. In addition, Brucella DNA environmental detection was higher during the B. suis outbreak than the post B. suis outbreak. The environmental approach has proven to be a simple, practical, valuable, and safe method to detect and monitor B. suis. These results suggest a role of the environment and the offspring that should be considered in porcine brucellosis surveillance and control programmes.

Feline Gastrointestinal Eosinophilic Sclerosing Fibroplasia-Extracellular Matrix Proteins and TGF-ß1 Immunoexpression.

Feline gastrointestinal eosinophilic sclerosing fibroplasia (FGESF) has been described as an inflammatory disorder with an eosinophilic component with etiopathogenesis that is still unknown. Sixteen intestinal samples from two veterinary diagnostic services (2014-2017) were included in the study. A histopathological criterion classified the cases into three grades (mild, moderate, and severe) according to the distribution of the lesions and the course. An immunohistochemical study of collagen I, collagen III, fibronectin, and transforming growth factor ß1 (TGF-ß1) was performed in each case. An immunohistochemical study of mild grades shows greater collagen III immunoexpression, compared to collagen I and fibronectin, which suggests an "early" stage of fibrosis. In more intense grades, an increased immunoexpression of collagen I, compared to collagen III, suggests a "late" stage of fibrosis. Otherwise, the highest expression of TGF-ß1 was observed in the moderate phase, due to the high proliferation of reactive fibroblast and intense inflammation. The results suggest that the inflammatory infiltrate is the trigger for the elevation in TGF-ß1, altering the collagen type III:I ratio. In conclusion, immunohistochemical studies can be a very useful method in diagnosing cases of FGESF of mild grades and could help to apply a differential diagnosis regarding feline eosinophilic chronic enteritis (CEE) in the context of inflammatory bowel disease (IBD).

Bcl-2 Immunoexpression in Feline Epitheliotropic Intestinal T-Cell Lymphomas.

Lymphoma is the most common malignant hematopoietic neoplasm in domestic felines. Twenty-two cases of feline epitheliotropic duodenal T-cell lymphoma were characterized morphologically and immunohistochemically (CD3, Pax5, Ki-67), and Bcl-2 immunoexpression was established. Most cases were in domestic shorthair cats (88.2%), with a mean age of 11.2 years. All lymphomas were CD3+, with a low-to-moderate expression of Ki-67 (<30%). A correlation between the tumoral pattern of infiltration in the lamina propria and the intraepithelial distribution of the neoplastic lymphocytes was established (p = 0.0155). Intraepithelial nests of neoplastic lymphocytes were predominantly observed in lymphomas with a patchy distribution in the lamina propria, whereas intraepithelial plaques were seen in lymphomas with an obliteration pattern. Bcl-2 was expressed in neoplastic cells in all cases, and a higher expression was associated with increased villous stunting (p = 0.0221), and tended to be present in those cases with increased epithelial damage. The expression of Bcl-2 and the degree of epitheliotropism were correlated with neoplastic progression in epitheliotropic intestinal T-cell lymphomas; those displaying high Bcl-2 immunoexpression showed increased villous stunting and epithelial damage, suggesting that Bcl-2 is overexpressed in advanced tumor stages, and may be used as a predictor of tumoral behavior in feline epitheliotropic intestinal T-cell lymphomas. This entity showed many similarities with human MEITL, so the latter entity should be considered in further lymphoma classifications of domestic animals.

Colistin Selection of the Mcr-1 Gene in Broiler Chicken Intestinal Microbiota.

Colistin has a long story of safe use in animals for the treatment and prevention of certain bacterial diseases. Nevertheless, the first description of the mcr-1 gene showed that colistin resistance can spread by horizontal gene transfer and changed the landscape. This study aimed to assess the effect of colistin administration on the dispersion of resistance in the microbiota of day-old broiler chicks and how the presence of mcr-1 genes influences the spread of colistin resistance determinants. In this study, 100 one-day-old chicks were divided into four groups of 25 animals (G1, G2, G3, and G4). Animals from G3/G4 were challenged with mcr-1-carrying Salmonella (day 7), while colistin (600 mg/L) was administered daily to G2/G4 animals through drinking water (from day 8 to day 15). Two quantitative PCR assays were performed to compare the amount of Salmonella and mcr-1 that were present in the caecal samples. We observed that levels of mcr-1 were higher in G3/G4 animals, especially G4, due to the spread of mcr-1-carrying Salmonella. On day 21, Salmonella levels decreased in G4, reaching similar values as those for G3, but mcr-1 levels remained significantly higher, suggesting that colistin may accelerate the spreading process when mcr-1-carrying bacteria reach the gut.

Effect of Biscuit Flour and Fermented Defatted "Alperujo" Co-Administration on Intestinal Mucosa Morphology and Productive Performance in Laying Hens.

In this study, the effects of co-administration with biscuit flour and fermented defatted "alperujo" (FDA) on gut health were evaluated in a batch of laying hens (Hy-Line 2015) on a commercial farm. Animals were divided into two groups: control group and treatment group; and histological and morphometric analyses of all sections of the intestine (duodenum, jejunum, ileum, cecum and rectum) were performed at 10, 18, 25, 50 and 75 weeks of age. During the whole productive period, a decrease in the mortality rate (p = 0.01) was observed in treated hens, as well as an increase in the number of eggs produced (p < 0.001), their size (p < 0.025), and weight (p < 0.024). In the early and late stages of production (10, 18 and 50 weeks), a significant increase (p < 0.001) in the height and depth of the intestinal villi was observed in the treatment group. Villi height was also significantly higher (p < 0.001) in the treatment group up to week 50 in the cecum, and at weeks 18 and 50 in the rectum. We concluded that an economical and sustainable feeding system with less environmental impact, such as co-supplementation with biscuit flour and FDA, could maintain gut health without negatively impacting laying hens' productive performance.

Dietary supplementation with fermented defatted "alperujo" induces modifications of the intestinal mucosa and cecal microbiota of broiler chickens.

Nutraceuticals are not only nutritionally beneficial for animals but also their use as feed supplements may reduce environmental contamination. The effect of fermented defatted "alperujo," an olive oil by-product, supplementation on the intestinal health of broiler chickens was assessed by analyzing the intestinal mucosal morphology of the duodenum and the cecum. The microbiota of the cecum was also characterized by analyzing the V3-V4 region of the 16S rRNA gene on days 7, 14, 21, 28, 35, and 42. Supplemented broilers from 14 to 35 D of age showed an increase in villus height in the duodenum. This increase likely improved digestibility and absorption capacity during growth, leading to the observed increase in BW at day 35 of life. A progressive increase in crypt depth in both the duodenum and the cecum was also observed. This modification likely enhanced epithelial renewal, thus safeguarding the turnover capacity of the intestinal mucosa. Our molecular analysis of cecal microbiota suggests that this dietary supplement may favor the growth of certain bacteria and may control the spread of pathogenic bacteria by means of competitive exclusion.