Outbreaks of SARS-CoV-2 in naturally infected mink farms: Impact, transmission dynamics, genetic patterns, and environmental contamination.

SARS-CoV-2 infection outbreaks in minks have serious implications associated with animal health and welfare, and public health. In two naturally infected mink farms (A and B) located in Greece, we investigated the outbreaks and assessed parameters associated with virus transmission, immunity, pathology, and environmental contamination. Symptoms ranged from anorexia and mild depression to respiratory signs of varying intensity. Although the farms were at different breeding stages, mortality was similarly high (8.4% and 10.0%). The viral strains belonged to lineages B.1.1.218 and B.1.1.305, possessing the mink-specific S-Y453F substitution. Lung histopathology identified necrosis of smooth muscle and connective tissue elements of vascular walls, and vasculitis as the main early key events of the acute SARS-CoV-2-induced broncho-interstitial pneumonia. Molecular investigation in two dead minks indicated a consistently higher (0.3-1.3 log10 RNA copies/g) viral load in organs of the male mink compared to the female. In farm A, the infected farmers were responsible for the significant initial infection of 229 out of 1,000 handled minks, suggesting a very efficient human-to-mink transmission. Subsequent infections across the sheds wherein animals were being housed occurred due to airborne transmission. Based on a R0 of 2.90 and a growth rate equal to 0.293, the generation time was estimated to be 3.6 days, indicative of the massive SARS-CoV-2 dispersal among minks. After the end of the outbreaks, a similar percentage of animals were immune in the two farms (93.0% and 93.3%), preventing further virus transmission whereas, viral RNA was detected in samples collected from shed surfaces and air. Consequently, strict biosecurity is imperative during the occurrence of clinical signs. Environmental viral load monitoring, in conjunction with NGS should be adopted in mink farm surveillance. The minimum proportion of minks that need to be immunized to avoid outbreaks in farms was calculated at 65.5%, which is important for future vaccination campaigns.

Exposure Biomarkers and Histopathological Analysis in Pig Liver After Exposure to Mycotoxins Under Field Conditions: Special Report on Fumonisin B1.

Liver samples from finisher pigs were collected at the slaughterhouses for the analysis of zearalenone (ZEA), alfa-/beta-zearalenone (α-ZE, ß-ZE), zearalanone (ZA), alfa-/beta-ZA (α-ZA, ß-ZA), aflatoxin B1 (AFB1) and aflatoxin M1, fumonisin B1 (FB1), ochratoxin A (OTA) and ochratoxin B, deoxynivalenol and deepoxi-deoxynivalenol (DOM-1). For the analysis liquid chromatography-triple quadrupole coupled with mass spectrometry was applied. Liver samples with detected FB1 were further histopathologically evaluated after hematoxylin and eosin staining. Various levels of liver mycotoxins were detected in all farms. Pig livers with 2.91-8.30 µg/kg of FB1 were detected in three farms, estimate of 850-2400 µg/kg of FB1 intake, whereas 0.54 µg/kg of OTA was detected in one farm, estimate of 75 µg/kg of OTA intake. Moreover, pig livers with 0.30 µg/kg of ZEA, 1.87 µg/kg of α-ZE, and 0.63 µg/kg of ß-ZE were detected in one farm, estimate with of 300 µg/kg of ZEA intake. The histopathological analysis revealed that the lesions' grading and necrosis grading were analogously increased when FB1 concentration increased from 2.91 to 4.36-8.30 µg/kg. The severity of megalocytosis was analogously increased with FB1 detection levels and particularly in levels of 4.36-8.3 µg/kg. However, the increased FB1 detection levels did not show analogous behavior with the severity of hepatic cell vacuolization. Results showed that FB1 remained the most critical risk factor in the Greek pig industry, whereas ZEA and AFB1 were also prevalent. The OTA contamination in pig farms raised a high risk for animal and human health.

Diet replacement with whole insect larvae affects intestinal morphology and microbiota of broiler chickens.

Insect-based diets are gaining interest as potential ingredients in improving poultry gut health. This study assessed the dietary treatment with whole dried Tenebrio molitor larvae (TM) on broiler chickens' gut microbiota and morphology. 120 Ross-308 broilers received treated diets with 5% (TM5) and 10% (TM10) replacement ratio in a 35-day trial. Intestinal histomorphometry was assessed, as well as claudin-3 expression pattern and ileal and caecal digesta for microbial community diversity. Null hypothesis was tested with two-way ANOVA considering the intestinal segment and diet as main factors. The TM5 group presented higher villi in the duodenum and ileum compared to the other two (P < 0.001), while treated groups showed shallower crypts in the duodenum (P < 0.001) and deeper in the jejunum and ileum than the control (P < 0.001). Treatments increased the caecal Firmicutes/Bacteroidetes ratio and led to significant changes at the genus level. While Lactobacilli survived in the caecum, a significant reduction was evident in the ileum of both groups, mainly owed to L. aviarius. Staphylococci and Methanobrevibacter significantly increased in the ileum of the TM5 group. Results suggest that dietary supplementation with whole dried TM larvae has no adverse effect on the intestinal epithelium formation and positively affects bacterial population richness and diversity.

Effects of Dietary Supplementation of Essential Oils, Lysozyme, and Vitamins' Blend on Layer Hen Performance, Viral Vaccinal Response, and Egg Quality Characteristics.

Maintaining respiratory tract health is crucial for layers, impacting gut health, laying performance, and egg quality. Viral diseases and standard vaccinations can compromise tracheal epithelium function, leading to oxidative stress. This study assessed the impact of a blend of feed additives, predominantly lysozyme (L), essential oils (EO), and vitamins (VIT) (referred to as L + EO + VIT), on young layers during an oral vaccination schedule. The supplementation significantly enhanced antibody titers for Newcastle Disease Virus (NDV) and Infectious Bronchitis Virus (IBV) after vaccination, trachea functionality and intestinal health in the jejunum, increased egg production, and exhibited a trend toward higher egg weight. Although feed intake showed no significant difference, egg quality remained consistent across experimental groups. Moreover, L + EO + VIT supplementation elevated total phenolic content in eggs, improving oxidative stability in both fresh and stored eggs, particularly under iron-induced oxidation. Notably, it substantially reduced yolk lipid peroxidation and albumen protein carbonyls. In conclusion, water supplementation with L + EO + VIT may enhance humoral immune response to IBV and NDV, positively impacting hen productivity. These findings indicate improved tracheal function and enhanced oxidative stability, emphasizing the potential of this blend in promoting overall health and performance in layers.

Histopathological lesions of Actinobacillus pleuropneumoniae serotype 8 in infected pigs.

This study aimed to report, for the first time, histopathological lesions caused by an outbreak of acute Actinobacillus pleuropneumoniae serotype 8 infections in two farms in Cyprus. Lung tissue samples were collected from two different affected farms (a total of eight samples) for bacterial culture, multiplex polymerase chain reaction (PCR)-based serotyping and histopathological evaluation. Severe respiratory clinical signs, vomiting, anorexia, sudden deaths, a morbidity rate of around 25.00% and a mortality rate of over 60.00% in the fattening stage were reported. Macroscopic lesions included acute to subacute fibrotic, hemorrhagic and necrotizing pneumonia with occasionally encapsulated nodule-like abscesses and fibrous pleuritis. Histopathological evaluation revealed fibrous exudate between alveolar spaces and connective tissue, areas of necrosis mixed with alveolar macrophages, lymphocytes, plasma cells and necrotic leukocytes surrounding colonies of cocci. The bronchial and bronchiolar epithelia were degenerated and replaced by eosinophilic cell debris mixed with inflammatory cells. Several arteries and capillaries were clotted and/or infiltrated by inflammatory cells. In conclusion, these A. pleuropneumoniae serotype 8 cases were accompanied by acute illness, death and more pronounced bronchitis and bronchiolitis.

Histopathological Pulmonary Lesions in 1st-Day Newborn Piglets Derived from PRRSV-1 MLV Vaccinated Sows at the Last Stage of Gestation.

Modified live virus (MLV) vaccines for the control of porcine respiratory and reproductive syndrome virus (PRRSV) have been associated with the vertical and horizontal transmission of vaccine viruses. The present study aimed to describe pathological lung lesions in piglets born by gilts vaccinated with PRRSV-1 MLV. In total, 25 gilts were vaccinated at late gestation (100th day) and were divided into five groups according to the different vaccines (Vac) used: no vaccine-control group, Vac-1-strain DV, Vac-2-strain VP-046 BIS, Vac-3-strain 94881, Vac-4-strain 96V198. Within the first 0-9 h of the farrowing, blood samples were collected from all newborn piglets and lung samples were exanimated grossly, histopathologically and with scanning electron microscopy. PRRSV (RT-PCR-positive) and antibodies were detected in the serum of piglets from gilts vaccinated with Vac-2. In these piglets, moderate to severe interstitial pneumonia with thickened alveolar septa was noticed. Type II pneumocyte hyperplasia was also observed. The rest of the trial piglets showed unremarkable lung lesions. Phylogenetic analysis revealed the 98.7% similarity of the PRRSV field strain (GR 2019-1) to the PRRS MLV vaccine strain VP-046 BIS. In conclusion, the Vac-2 PRRSV vaccine strain can act as an infectious strain when vaccination is administrated at late gestation, causing lung lesions.

Effects of Enriched-in-Oleuropein Olive Leaf Extract Dietary Supplementation on Egg Quality and Antioxidant Parameters in Laying Hens.

The objective of the present study was to evaluate the effects of an olive leaf extract obtained with an up-to-date laboratory method, when supplemented at different levels in laying hens' diets, on egg quality, egg yolk antioxidant parameters, fatty acid content, and liver pathology characteristics. Thus, 96 laying hens of the ISA-Brown breed were allocated to 48 experimental cages with two hens in each cage, resulting in 12 replicates per treatment. Treatments were: T1 (Control: basal diet); T2 (1% olive leaf extract); T3 (2.5% olive leaf extract); T4 (Positive control: 0.1% encapsulated oregano oil). Eggshell weight and thickness were improved in all treatments compared to the control, with T2 being significantly higher till the end of the experiment (p < 0.001). Egg yolk MDA content was lower for the T2 and T4 groups, while total phenol content and Haugh units were greater in the T2. The most improved fatty acid profile was the one of T3 yolks. The α-tocopherol yolk content was higher in all groups compared to T1. No effect was observed on cholesterol content at any treatment. Based on the findings, it can be inferred that the inclusion of olive leaf extract at a concentration of 1% in the diet leads to enhancements in specific egg quality attributes, accompanied by an augmentation of the antioxidant capacity.

A Comprehensive Study of the Quality of Fat-Tailed Sheep Carcasses in Greece.

Sheep farming in Greece is focused on milk production. Meat is considered a by-product and consists mainly light carcasses of undefined quality. The main challenge of the sector is to ensure sustainability, and hence efforts are towards efficient use of available resources, including undervalued carcasses of local fat-tailed sheep. The objective here was twofold: (i) to assess the carcass quality of fat-tailed sheep slaughtered at different live weights and (ii) to compare them with carcasses from thin-tailed sheep. In total, 146 fat-tailed and 97 thin-tailed dairy sheep were used. They belonged to five live-weight categories (LWC), representing 25%, 35%, 50%, 70% and 100% of mature body weight. Carcass length/weight/yield/pH and wither height were recorded. Muscle fiber minimum Feret's diameter and meat color/tenderness/moisture/lipid and protein content were determined. Sex and LWC differences in fat-tailed sheep were assessed. Parametric and non-parametric tests were used to compare with thin-tailed sheep, considering the effects of LWC, sex and their interactions with sheep population (fat-tailed/thin-tailed). Most traits were significantly different (p < 0.05) between groups of fat-tailed sheep. Carcass yield of fat-tailed sheep was significantly higher compared to thin-tailed (p < 0.01). Interactions of sheep population with LWC or sex affected wither height, carcass pH, meat color and tenderness (p < 0.05). Fat-tailed sheep meat quality is equal or higher compared to thin-tailed. Finishing weights corresponding to 50 and 70% LWC may improve capitalization of fat-tailed carcasses.

The association of Curcuma and Scutellaria plant extracts improves laying hen thermal tolerance and egg oxidative stability and quality under heat stress conditions.

Chronic exposure to high ambient temperatures is detrimental to laying hen performance and egg quality. Plant secondary metabolites may alleviate effects, partly due to their antioxidant activities. Herein, we investigated the effects of dietary supplementation with a phytonutrient solution (PHYTO) consisting of a plant extract combination of Scutellaria baicalensis and Curcuma longa on young layers (25-32 wk of age) raised under naturally elevated temperature conditions. Four hundred, 24-wk-old Lohmann hens were allocated in 50 cages and, after a week of adaptation, were offered a diet either containing 2 g/kg of PHYTO or not, for 8 wk. Hen BW was measured at the beginning and end of the trial, and egg production and feed intake were recorded weekly. At week 32, four eggs per cage were collected to determine egg quality characteristics as well as the rate of lipid and albumen oxidation in fresh eggs. At the end of the trial, two hens per cage were blood sampled for assessment of biochemical markers, one of which was euthanized for histopathological evaluation of the liver and intestine and assessment of intestinal histomorphometry. The herbal mixture supplementation significantly increased egg production rate at weeks 28 and 29 and for the overall production period, and feed efficiency at weeks 26-29. In addition, the degree of liver necrosis and microvascular thrombosis was lower (P < 0.05) whereas intestinal villosity was greater in duodenal and jejunal segments (P < 0.05) in the PHYTO compared to the control group. Supplementation also reduced (P < 0.05) blood concentrations of corticosterone, alanine aminotransferase activity, and TBARS, and a reduction in catalase activity was observed. Egg quality characteristics were not affected, except for eggshell thickness, egg diameter, and eggshell breaking strength that were superior in the PHYTO group (P < 0.05). PHYTO supplementation significantly improved egg lipid oxidation status of fresh eggs. In conclusion, supplementation with PHYTO improved laying hen productivity and egg quality, which was associated with an improvement in laying hen thermotolerance.

Administration of a Multi-Genus Synbiotic to Broilers: Effects on Gut Health, Microbial Composition and Performance.

In recent years, the applicability of prebiotics, probiotics and their mixtures, defined as synbiotics, in poultry production has received considerable attention. Following the increasing regulation of antibiotic use, these nutraceuticals are seen as an alternative way to sustain production efficiency and resistance to pathogens and stressors by modulating birds' gut health. The aim of this study was to evaluate the benefits provided under field conditions by administering the multi-species synbiotic PoultryStar® sol to broilers in drinking water. To this purpose, three Ross 308 broiler flocks, representing separate progenies of a breeder flock which was treated with the same synbiotic, were housed in separate farms, divided into treatment and control groups, and followed throughout the productive cycle. Synbiotic administration was shown to improve gut health even in absence of a challenge, with limited changes in terms of macroscopic intestinal lesions and more overt differences related to histopathological scores and villi length. Synbiotic-fed chickens performed consistently better in terms of body weight gain, feed conversion ratio and survivability. Lastly, the evaluation of the caecal microbiome through next-generation sequencing highlighted the effects of synbiotic supplementation on the composition of the bacterial population, the implications of which will, however, require further studies to be better comprehended.

Efficacy of a Dietary Polyherbal Formula on the Performance and Gut Health in Broiler Chicks after Experimental Infection with Eimeria spp.

One-hundred and fifty, one-day-old Ross-308 female chicks were randomly allocated to five equal treatments: NCONTR negative control-not challenged; PCONTR positive control-challenged; PHERB1 and PHERB2 diets were supplemented with phytogenic formula (1 and 2 g/kg feed, respectively)-challenged; PSALIN diet was supplemented with salinomycin (60 mg/kg feed)-challenged. Challenge was made by oral inoculation with 3.5 × 104 E. acervulina, 7.0 × 103 E. maxima and 5.0 × 103 E. tenella oocysts, at 14 days of age. One week post inoculation, bloody diarrhea, oocysts numbers, and intestinal lesions were evaluated, along with intestinal microbiota, viscosity, and pH of digesta, and histopathology. PHERB2 had a comparable (p ≤ 0.001) growth performance and feed conversion ratio to PSALIN. PHERB1 and PHERB2 had similar (p ≤ 0.001) oocyst counts to PSALIN and lower than PCONTROL. PHERB2 and PSALIN had lower (p ≤ 0.001) jejunal, ileal, and cecal lesion scores compared to PCONTR. PHERB1 and PHERB2 had higher (p ≤ 0.001) jejunal and cecal lactobacilli and lower (p ≤ 0.001) coliform counts compared to other treatments. PCONTR had lower (p ≤ 0.001) jejunum villus height, height to crypt ratio, and villus goblet cells. Breast and thigh meat resistance to oxidation was improved (p ≤ 0.001) in PHERB1 and PHERB2 compared to the PCONTR. The polyherbal formula exerted a substantial improvement on growth performance and intestinal health of the Eimeria-challenged birds.

Quantitative proteomics of cerebrospinal fluid using tandem mass tags in dogs with recurrent epileptic seizures.

This prospective study included four dog groups (group A: healthy dogs, groups B: dogs with idiopathic epilepsy under antiepileptic medication (AEM), C: idiopathic epilepsy dogs without AEM administration, D: dogs with structural epilepsy). The purpose of the study was to compare the proteomic profile among the four groups. Samples were analyzed by a quantitative Tandem Mass Tags approach using a Q-Exactive-Plus mass-spectrometer. Identification and relative quantification were performed using Proteome Discoverer, and data were analyzed using R. Gene ontology terms were analyzed based on Canis lupus familiaris database. Data are available via ProteomeXchange with identifier PXD018893. Eighteen proteins were statistically significant among the four groups (P < 0.05). MMP2 and EFEMP2 appeared down-regulated whereas HP and APO-A1 were up-regulated (groups B, D). CLEC3B and PEBP4 were up-regulated whereas APO-A1 was down-regulated (group C). IGLL1 was down-regulated (groups B, C) and up-regulated (group D). EFEMP2 was the only protein detected among the four groups and PEBP4 was significantly different among the epileptic dogs. Western blot and SPARCL immunoassay were used to quantify HP abundance change, validating proteomic analysis. Both, showed good correlation with HP levels identified through proteomic analysis (r = 0.712 and r = 0.703, respectively). SIGNIFICANCE: The proteomic analysis from CSF of dogs with epileptic seizures could reflect that MMP2, HP and APO-A1 may contribute to a blood-brain barrier disruption through the seizure-induced inflammatory process in the brain. MMP2 change may indicate the activation of protective mechanisms within the brain tissue. Antiepileptic medication could influence several cellular responses and alter the CSF proteome composition.

Histopathological Lesions Accompanied with First-Time Isolation of a PRRSV-2 Strain in Greece.

Genotype 2 strains of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) have been reported sporadically in Europe. Even if, PRRSV-2 reported to be genetically homogenous in Europe due to the introduction of an MLV vaccine strain, independent introductions of PRRSV-2 field strains have been reported. The aim of the present study was to report the complete genome sequence and evaluate the histopathological lesions of a PRRSV-2 strain, isolated for the first time in Greece. During a routine blood sampling in a commercial pig farm, the results revealed positive samples in weaners of 40-60 days for the PRRSV-2, using real-time polymerase chain reaction. The clinical picture was characterized from respiratory symptoms in weaners, as well as coughing and poor performance at finishing stage and less than 3% mortality rate from weaning stage to finishing stage. The use of ORF5 for PRRSV phylogenetic analysis of the isolated PRRSV strain, named "x1544-1 strain", was successfully determined, belonging to the genotype PRRSV-2. Comparison of the obtained sequence revealed nucleotide sequence identity >98% with PRRSV-2 strain VR2332 and other related strains from Denmark and China. The histopathological evaluation revealed diffuse interstitial pneumonia, multifocal interstitial nephritis, while in the lymphoid organs, follicular and paracortical hyperplasia, coexisting with necrosis and depletion of germ cells were detected. The results of current study undersign the importance for veterinary practitioners to have up-to-date access to phylogenetic data linked to phenotypic information to follow-up the control and prevention strategies against PRRSV.

Amyloid-beta plasma and cerebrospinal fluid biomarkers in aged dogs with cognitive dysfunction syndrome.

BACKGROUND: Cognitive dysfunction syndrome (CDS) is a common progressive neurodegenerative disease that is poorly defined. Specific multitargeted protocols do not exist for setting the diagnosis and the prognosis of the syndrome. HYPOTHESIS/OBJECTIVES: To quantify Aß42 and Aß40 peptides in blood and cerebrospinal fluid (CSF) and to investigate their contribution to CCDS. ANIMALS: A total of 61 dogs from a hospital population. METHODS: Case-control study. Six young (YG: 0-4 years old), 8 middle-aged (4-8 years old), 17 cognitively unimpaired and aged (CU: 8-20 years old), and 30 cognitively impaired and aged (CI: 8-17 years). From the CI group, 10 dogs exhibited mild impairment (CI-MCI) and 20 exhibited severe impairment (CI-SCI). Cognitive status was assessed using a validated owner-based questionnaire. Direct and indirect Aß markers were determined in plasma fractions (total-TP, free-FP, bound to plasma components-CP) and CSF using commercial ELISA assays (AΒtest, Araclon Biotech). RESULTS: TPAß42/40 facilitated discrimination between CI-MCI and CU aged dogs with area under curve ≥ 0.79. CSFAß42 levels were higher (P = .09) in CU (1.25 ± 0.28 ng/mL) than in MCI (1.04 ± 0.32 ng/mL) dogs. CSF Aß42 levels were correlated with the CP fragment (CPAß40: P = .02, CPAß42: P = .02). CPAß42 was higher in the CI-MCI (23.03 ± 11.79 pg/µL) group compared to the other aged dogs (CU: 10.42 ± 7.18 pg/µL, P = .02, SCI: 11.40 ± 12.98 pg/µL, P = .26). CONCLUSION AND CLINICAL IMPORTANCE: The Aß should be determined in all of the 3 plasma fractions (TP, FP, CP). In the clinical approach, TPAß42/40 could be used as an efficient preselection tool for the aged canine population targeting dogs with mild cognitive impairment.

The Antiangiogenic Properties of Adipose-Derived Mesenchymal Stem/Stromal Cells in Corneal Neovascularization in a Rabbit Model.

The purpose was to study the anti-angiogenic effect of adipose-derived mesenchymal stem/stromal cells (ADMSCs) on experimentally induced corneal injuries. Corneal neovascularization (NV) was induced by incising and subsequently suturing the corneal surface in 32 New Zealand rabbits. Following suturing, the rabbits were randomly allocated into 2 groups, and received either phosphate-buffered saline (PBS) (control) or ADMSCs, both administered via three different routes. Digital images of the cornea were obtained two weeks post-incision to measure the area of neovascularized cornea. Tumor necrosis factor (TNF) was immunohistochemically assessed in the both groups. The corneal tissue was evaluated for vascular endothelial growth factor (VEGF). The extent of corneal NV in all eyes was assessed photographically by an independent observer. Fourteen days after the incisions, the degree of corneal NV was substantially decreased in the ADMSC-treated group (1.87 ± 0.9 mm2, 1.4 % ± 0.67 % of corneal surface) compared to the control and PBS-treated group (4.66 ± 1.74 mm2, 3.51 % ± 1.31 %, p < 0.001). ADMSCs significantly decreased injury-induced corneal NV in New Zealand rabbits two weeks post-treatment. This strategy has potential for use in the control of corneal NV in vivo.

Case Report of Transmissible Gastroenteritis Coronavirus Infection Associated with Small Intestine and Brain Lesions in Piglets.

This case study report describes a transmissible gastroenteritis coronavirus (TGEV) infection presented in a commercial pig herd. The clinical signs of infection appeared in newborn piglets, including medium morbidity and low mortality rates. Rectal swabs were collected from five different affected litters for laboratory examinations. Samples from two dead piglets and two euthanized affected piglets were collected for gross and histopathological examinations. All fecal samples were tested TGEV positive by real-time polymerase chain reaction (RT-PCR). Necropsy revealed nonspecific gross lesions. The histopathological examinations revealed villi fused with denuded tips and severe villus atrophy, leading to extensive epithelial flattening in middle and lower small intestine. The architecture pattern of villi presented columnar and cuboidal poorly differentiated enterocytes with mild subepithelial edema. In some enterocytes, pycnotic nuclei were detected. Microscopic examination of brain tissue revealed diffuse gliosis in the area of pia matter with mild congestion of the meningeal and parenchymal vessels and neuronal degeneration. In conclusion, this case study reported an epidemic TGEV infection in piglets, characterized by low mortality and medium morbidity rates accompanied by typical histopathological lesions in small intestine, as well as by coexisting brain lesions, that are described for the first time.