Neuropathological lesions in intravenous BCG-stimulated K18-hACE2 mice challenged with SARS-CoV-2.

In the wake of the COVID-19 pandemic caused by SARS-CoV-2, questions emerged about the potential effects of Bacillus Calmette-Guérin (BCG) vaccine on the immune response to SARS-CoV-2 infection, including the neurodegenerative diseases it may contribute to. To explore this, an experimental study was carried out in BCG-stimulated and non-stimulated k18-hACE2 mice challenged with SARS-CoV-2. Viral loads in tissues determined by RT-qPCR, histopathology in brain and lungs, immunohistochemical study in brain (IHC) as well as mortality rates, clinical signs and plasma inflammatory and coagulation biomarkers were assessed. Our results showed BCG-SARS-CoV-2 challenged mice presented higher viral loads in the brain and an increased frequency of neuroinvasion, with the greatest differences observed between groups at 3-4 days post-infection (dpi). Histopathological examination showed a higher severity of brain lesions in BCG-SARS-CoV-2 challenged mice, mainly consisting of neuroinflammation, increased glial cell population and neuronal degeneration, from 5 dpi onwards. This group also presented higher interstitial pneumonia and vascular thrombosis in lungs (3-4 dpi), BCG-SARS-CoV-2 mice showed higher values for TNF-α and D-dimer values, while iNOS values were higher in SARS-CoV-2 mice at 3-4 dpi. Results presented in this study indicate that BCG stimulation could have intensified the inflammatory and neurodegenerative lesions promoting virus neuroinvasion and dissemination in this experimental model. Although k18-hACE2 mice show higher hACE2 expression and neurodissemination, this study suggests that, although the benefits of BCG on enhancing heterologous protection against pathogens and tumour cells have been broadly demonstrated, potential adverse outcomes due to the non-specific effects of BCG should be considered.

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.

Tertiary lymphoid organs in wild boar exposed to a low-virulent isolate of African swine fever virus.

Despite the great interest in the development of a vaccine against African swine fever (ASF) in wild boar, the immunological mechanisms that induce animal protection are still unknown. For this purpose, tertiary lymphoid organs (TLOs) of wild boar were characterised and compared with mucosa-associated lymphoid tissues (MALTs) by histopathology, histomorphometry and immunohistochemistry (CD3, CD79, PAX5, LYVE1, fibronectin). In addition, real-time polymerase chain reaction (qPCR) and immunohistochemistry (p72) were used to evaluate the presence of ASF virus (ASFV) in blood and tissues samples, respectively. TLOs were observed in animals infected with a low-virulent ASFV isolate (LVI), animals co-infected with low and high-virulent ASFV isolates (LVI-HVI) and animals infected only with the high virulence isolate (HVI). TLOs in LVI and LVI-HVI groups were located adjacent to the mucosa and presented a similar structure to MALT. Immunoexpresion of p72 observed in the inflammatory cells adjacent to TLOs/MALTs confirmed its development and reactivity generated by ASF attenuated isolates. Immunohistochemical evaluation, based on cellular composition (T and B lymphocytes), and histomorphometrical study revealed a more pronounced maturation of TLOs/MALTs in the LVI-HVI group. It is currently unclear whether these formations play a protective role by contributing to local immunity in chronic inflammatory diseases. However, the structural similarities between TLOs and MALTs and the location of TLOs close to the mucosa suggest that they may perform a similar function, facilitating a local protective response. Nevertheless, further investigations are warranted to assess the cellular and humoral dynamics of these lymphoid organs induced by attenuated isolates.

Intrapancreatic accessory spleens in African swine fever infection of wild boar (Sus scrofa).

Intrapancreatic accessory spleen (IPAS) is one of the most frequent congenital splenic anomalies in humans; however, studies in veterinary medicine are scarce. This study aimed to describe the macroscopic, histopathological and immunohistochemical features of 11 suspected cases of IPAS in wild boar piglets of 3-4 months old. Seven of the 11 animals were immunised with a low virulence isolate of African swine fever virus (ASFV) and subsequently challenged with a highly virulent ASFV isolate (LVI-HVI group). The remaining four animals were exclusively infected with a highly virulent isolate of ASFV (HVI group). Grossly, lesions comprised focal or multifocal reddish areas of variable shape, located on the surface of the pancreatic tail or within the parenchyma. Histological and immunohistochemical studies (anti-CD79 and CD3) confirmed the presence of IPAS in eight of the 11 cases. IPAS shared the same histological structure and alterations as those observed in the original spleen. The immunohistochemical study against ASFV revealed the presence of VP72+ cells in both the spleen and IPAS of seven of the eight piglets. The results of this study describe for the first time the presence of IPAS in ASFV infection of wild boar (Sus scrofa) regardless the isolate and suggest that the infection may induce the development of ectopic splenic tissue due to an increased demand for phagocytic cells from the reticuloendothelial system. However, further studies are needed to understand the immunological mechanisms that trigger the formation of these accessory organs.

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.

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.

Safety of African Swine Fever Vaccine Candidate Lv17/WB/Rie1 in Wild Boar: Overdose and Repeated Doses.

African swine fever (ASF) is a highly lethal infectious disease that affects domestic pigs and wild boar. Outbreaks of ASF have grown considerably in the last decade causing important economic consequences for the swine industry. Its control is hampered by the lack of an effective treatment or vaccine. In Europe, the wild boar is a key wild reservoir for ASF. The results of the oral vaccination trial of wild boar with Lv17/WB/Rie1 are hope for this problem. However, this vaccine candidate has certain safety concerns, since it is a naturally attenuated vaccine. Therefore, the current study aims to evaluate the safety of this vaccine candidate in terms of overdose (high dose) and repeated doses (revaccination) in wild boar. Low-dose orally vaccinated animals developed only a slight transient fever after vaccination and revaccination. This was also the case for most of the high-dose vaccinated wild boar, except for one of them which succumbed after revaccination. Although this fatality was related to hierarchical fights between animals, we consider that further studies are required for clarification. Considering these new results and the current epidemiological situation of ASF in wild boar, this vaccine prototype is a promising tool for the control of the disease in these wild populations, although further studies are needed.

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.

Effects on Intestinal Mucosal Morphology, Productive Parameters and Microbiota Composition after Supplementation with Fermented Defatted Alperujo (FDA) in Laying Hens.

The olive oil sector is currently adapting its traditional function to also become a supplier of high-value by-products that possess antioxidant, anti-inflammatory and antimicrobial properties. In this study, we evaluated the effect of the fermented defatted alperujo (FDA) on the intestinal health of laying hens. The morphology of the duodenal and cecal mucosa, the composition of the intestinal microbiota and the productivity of a batch of laying hens were evaluated after FDA supplementation. At early life stages, significant differences (p < 0.001) were observed in duodenal villi height and in crypt depth of both the duodenum and the cecum in the FDA-supplemented group, indicating improved intestinal health in this group. Microbiota composition in the hatchery group supplemented with FDA had a higher abundance of Actinobacteria, Firmicutes and Proteobacteria, and higher bacterial diversity. During the production period, significant differences (p < 0.05) were observed in the number of broken eggs from the supplemented group. We conclude that FDA supplementation improves the absorption capacity of the intestinal mucosa and modifies the intestinal microbiota to favor a greater immune response, leading to an increase in egg production.