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The present paper reports the case of a 3 years old, female Bearded Dragon (Pogona vitticeps) presenting lethargy, anorexia, weight loss, and anemia and marked leukocytosis at CBC. The majority of leukocytes were lymphocytic/lymphoblastic cells (97%). Immunocytochemical staining of blood smears marked for CD3 (neg) and CD79a (pos) suggested immunophenotype B. The patient died after one month from diagnosis. Histology evidenced lymphoid infiltration in the heart, spleen, liver, kidneys and gut. In addition, in the bone marrow a massive infiltration of lymphoid cells confirmed the diagnosis of leukemia. Immunohistochemistry confirmed the CD79a positivity of a large part of infiltrating lymphoid cells indicating a B cells immunophenotype of the neoplastic population. The presence of lymphocytosis and multiorgan infiltration supported the diagnosis of lymphocytic leukemia. Finally, a revision of the literature has also been made.

The interest for in ovo feeding has grown in the last decades mainly concerning probiotics, live microorganisms that can actively interact with the embryo. The aim of this study was to evaluate the effects of a multi-strain probiotic diluted in Marek's disease vaccine (MDV) on zootechnical performances, intestinal morphology and Eimeria spp. infection. One hundred and twenty eggs of Ross 308 broiler chickens were incubated until 18 d, when 105 fertilised and vital eggs were randomly allocated into three groups. A control group (C) was inoculated with MDV; two treated groups (P1 and P2) were inoculated with MDV and different concentrations of probiotics: 1 × 10 5 CFU/100 μL in P1 and 1 × 10 6 CFU/100 μL in P2. After hatching, chickens were separated into three replicates (10/replicate). Zootechnical parameters were determined. At the end of the cycle (35 d), chickens were slaughtered, and the intestine was collected for morphological analysis from nine chickens per group (three/replicate). Eimeria spp. oocyst shedding was determined weekly, and parasitic lesions were analysed on the histological sample. In ovo treatment with probiotic did not influence hatching rate but significantly improved body weight and positively influenced intestinal morphometric data compared to C. Oocyst shedding in faeces resulted in an increase in C, with significant differences at sampling performed at 14, 21 and 28 d of age. These results suggest that the tested probiotic compound is safe for in ovo supplementation and effectively improves zootechnical performances and coccidian resistance.

Background: In this study, a probiotic mixture (Honeybeeotic) consisting of seven bacterial strains isolated from a unique population of honeybees (Apis mellifera ligustica) was used. That honeybee population was located in the Roti Abbey locality of the Marche Region in Italy, an area isolated from human activities, and genetic contamination from other honeybee populations. The aim was to investigate the effects of this probiotic mixture on the innate immunity and intestinal microbiome of healthy common honeybees in two hives of the same apiary. Hive A received a diet of 50% glucose syrup, while hive B received the same syrup supplemented with the probiotics, both administered daily for 1 month. To determine whether the probiotic altered the immune response, phenoloxidase activity and hemolymph cellular subtype count were investigated. Additionally, metagenomic approaches were used to analyze the effects on gut microbiota composition and function, considering the critical role the gut microbiota plays in modulating host physiology., Results: The results revealed differences in hemocyte populations between the two hives, as hive A exhibited higher counts of oenocytoids and granulocytes. These findings indicated that the dietary supplementation with the probiotic mixture was safe and well-tolerated. Furthermore, phenoloxidase activity significantly decreased in hive B (1.75 ± 0.19 U/mg) compared to hive A (3.62 ± 0.44 U/mg, p < 0.005), suggesting an improved state of well-being in the honeybees, as they did not require activation of immune defense mechanisms. Regarding the microbiome composition, the probiotic modulated the gut microbiota in hive B compared to the control, retaining core microbiota components while causing both positive and negative variations. Notably, several genes, particularly KEGG genes involved in amino acid metabolism, carbohydrate metabolism, and branched-chain amino acid (BCAA) transport, were more abundant in the probiotic-fed group, suggesting an effective nutritional supplement for the host., Conclusions: This study advocated that feeding with this probiotic mixture induces beneficial immunological effects and promoted a balanced gut microbiota with enhanced metabolic activities related to digestion. The use of highly selected probiotics was shown to contribute to the overall well-being of the honeybees, improving their immune response and gut health., (© 2024. The Author(s).)

Background: In recent years, the field of transfusion medicine for dogs has advanced significantly, becoming a crucial aspect of veterinary clinical practice. Nowadays, blood still remains a fundamental biological source and the welfare and health status of eligible species-specific blood donors are essential for veterinary transfusion medicine., Aim: This study focused on evaluating two shelters in Central Italy (Marche region), located in Tolentino (TS) and in Camerino (CS), in order to assess the potential of the shelter dogs as canine blood donors., Methods: We evaluated a total of 45 dogs from these shelters based on physical (age and size), clinical, behavioural, and blood analysis criteria described in the Italian Ministerial Guideline for Veterinary Transfusion Medicine (2016)., Results: At the TS shelter, out of 206 resident dogs, 125 met the donation criteria (60.68%), with 28 (13.59%) selected for the study due to the impossibility to collect the samplings or other exclusion causes. In the CS shelter, of the 149 dogs, 17 (11.41%) were identified as potential blood donors and included in the study. Among these, seven dogs (25%) from TS and five dogs (29.41%) from CS were found to have DEA1 negative blood group. High percentages (TS = 25.24%, CS = 40.27%) of dogs were excluded for seniority and 29.53% in CS for behavioural issues. Notable findings included reduced erythrocyte mean corpuscular volume [mean TS = 63.93 fl; CS = 64.00 fl] and Reticulocyte Hemoglobin [mean TS = 22.39 pg; CS = 21.38 pg]. Additionally, in both shelters' dogs showed a modest increase in eosinophils levels [mean TS = 1.59 K/μl; CS = 1.02 K/μl]., Conclusion: Shelter dogs can fulfill the blood donation requirements set by the Italian Ministerial Guideline on Veterinary Transfusion Medicine. They are generally in good health and present a low risk of transmitting parasitic diseases; however, many are ineligible for donation due to behavioural pathologies or temperament issues and seniority., Competing Interests: The authors declare that there is no conflict of interest.

Diets rich in readily fermentable carbohydrates primarily impact microbial composition and activity, but can also impair the ruminal epithelium barrier function. By combining microbiota, metabolome, and gene expression analysis, we evaluated the impact of feeding a 65% concentrate diet for 4 weeks, with or without a phytogenic feed additive (PFA), on the rumen ecosystem of cattle. The breaking point for rumen health seemed to be the second week of high grain (HG) diet, with a dysbiosis characterized by reduced alpha diversity. While we did not find changes in histological evaluations, genes related with epithelial proliferation (IGF-1, IGF-1R, EGFR, and TBP) and ZO-1 were affected by the HG feeding. Integrative analyses allowed us to define the main drivers of difference for the rumen ecosystem in response to a HG diet, identified as ZO-1, MyD88, and genus Prevotella 1. PFA supplementation reduced the concentration of potentially harmful compounds in the rumen (e.g. dopamine and 5-aminovaleric acid) and increased the tolerance of the epithelium toward the microbiota by altering the expression of TLR-2, IL-6, and IL-10. The particle-associated rumen liquid microbiota showed a quicker adaptation potential to prolonged HG feeding compared to the other microenvironments investigated, especially by the end of the experiment., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The Polichete worms of the genus Polydora are considered very destructive for oysters, excavating channels in their shell and inducing oysters to create mud blisters in response to the irritation, interfering with their physiology and ethology. The parasite also causes important economic damage for oyster farmers, as products with a high degree of infestation cannot be commercialized. The present study aims to evaluate whether two non-invasive advanced diagnostic techniques, computed tomography scans (CT) and magnetic resonance imaging (MRI), are suitable to show the alterations induced by this parasite on live Crassostrea gigas and Ostrea edulis oyster broodstocks. A CT scan is also able to identify small lesions in the shell during the first stage of infection. MRI allows for the visualization of the advanced status of the lesions when blisters occupy the inner surface of the shell and can impact the health status and the economic value of the mollusk. Both techniques resulted in satisfactory spatial resolution, and no motion artifacts were reported, thus enabling the authors to faithfully visualize in vivo the damage caused by the parasite.

Goji berry (GB) shows beneficial effects on human health, although its effects on the male rabbit have been little investigated. This study examines the impact of GB dietary supplementation on the semen traits, antioxidant capacity of seminal plasma, and histological features of the reproductive tract of rabbit buck. Eighteen rabbits were distributed into two dietary groups: one receiving a commercial feed (Control), and the other a feed supplemented with 1% of GB (Goji). After a nutritional adaptation period of 60 days, the animals were subjected to semen collection every 15 days. The semen traits, libido, antioxidant, and inflammatory parameters were collected and analyzed. The rabbits were sacrificed after 60 days, and tissues of the genital tract were analyzed. Compared to the Control group, the Goji group showed higher spermatozoa concentration, motility, and vitality ( p < 0.05), as well as fewer abnormal spermatozoa and a higher libido ( p < 0.1). Histological features such as functional activity and hyperplasia were improved by GB and correlated with some semen traits ( p < 0.05). Conversely, antioxidant and anti-inflammatory parameters were unaffected by the diet. These findings suggest that GB acts on the tissues of the reproductive tract positively influencing semen quality, although further studies are needed to understand the effect on oxidative stress.

This study assessed the influence of supplementing the rabbit semen extender with various concentrations of glutathione (GSH) and taurine at 24, 48, and 72 h postchilling at 5°C. Semen samples were collected from 20 New Zealand bucks, and ejaculates with standard color, motility (>85%), about 0.5 mL volume, and ∼400 × 10 6 /mL concentration were used and diluted with extenders supplemented with 0.5, 1, and 2 mM of GSH and 1, 5, and 10 mM of taurine and chilled at 5°C. Nonsupplemented samples were used as a control. Sperm's progressive motility, acrosome reaction, and extracellular oxidative stress biomarkers such as MDA contents and GPx, SOD, and CAT concentrations and intracellular transcriptomic levels of SOD and CAT genes were assessed. GSH and taurine supplementation improved the sperm's kinetics by reducing cooling-associated stress, which was ascertained by lowering MDA concentration and increasing SOD, CAT, and GPx concentrations ( P  < 0.05). Increasing the levels of antioxidant enzymes in the extender was due to the increasing mRNA copies of the SOD and CAT genes ( P  < 0.05). Furthermore, GSH and taurine maintained the fructose levels in the extender and lowered the GPT levels, which implies sperm membrane stability is maintained through GSH and taurine supplementation. GSH and taurine supplementation to the extender had protective influences on the in vitro rabbit semen quality during chilled storage for up to 72 h, which were remarkable with increasing supplementation dose and cooling time at 5°C., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Mohamed F. F. Bayomy et al.)

Octopus vulgaris is a commercially valuable species. It is overexploited in the natural environment and is considered to be an innovative species for aquaculture. However, large-scale farming is generally designed only based on economic requirements, disregarding any form of enrichment that induces the natural behavior of aquatic species. Although many studies have shown the influence of environmental enrichment on terrestrial vertebrates, fish, and cephalopod mollusks, information on the effect of environmental enrichment on the body patterns of O. vulgaris is limited. Therefore, in this study, we assessed how different environmental conditions (Basic vs. Enriched) affect sub-adults of O. vulgaris kept in recirculation systems, through qualitative-quantitative studies of the main body patterns and their potential application in the commercial production of this species. The results indicated that octopuses kept in the enriched environment showed several body patterns and gained a significantly higher weight than those kept in the basic environment. The body patterns displayed by the individuals kept in the basic environment were similar to those exhibited under situations of hostility and inter/intra-specific conflict. Hence, the environment of octopuses needs to be enriched, especially for the large-scale production of this species., Competing Interests: The authors declare no conflict of interest.

This study assessed the use of feeders equipped with light-emitting diodes and their effects on the productivity of broiler chickens under productive conditions. A total of 87,200 ROSS 308 chickens, 1-day old, were housed in two poultry houses (CONTROL, F-LED). In CONTROL, 20,000 females (mean body weight 41.12 ± 3 g) and 25,000 males (mean body weight 41.56 ± 3 g) were housed, while 19,200 females and 23,000 males of the same genetic make-up and mean body weight were housed in F-LED under the same environmental conditions. In F-LED, to encourage chickens to feed and to redistribute more feed down the feeding line, a feeder equipped with a LED light has been installed at the end of each line. In CONTROL, no light was located on the feeders. At the end of the cycle, the average body weight never showed significant differences both for females (1345 g in CONTROL; 1359 g in F-LED) and for males (2771 g in CONTROL; 2793 g in F-LED). Uniformity improved in F-LED, at 75.2% in females and 54.1% in males, compared to CONTROL, at 65.7% and 48.5%, respectively, for females and males. The feed conversion ratio followed the same trend, being more favorable in chickens reared in F-LED (1.567) compared to those raised in CONTROL (1.608). The application of a single F-LED at the end of each feeding line demonstrated its utility in improving size uniformity and feed conversion.

Mycoplasmopsis agassizii causes the Upper Respiratory Tract Disease (URTD) in tortoises. The severity of the disease usually ranges from mild to severe respiratory signs. Animals can recover, die, or become asymptomatic carriers and are source of infection for other tortoises. This study describes (i) the clinical history and the results obtained in ten years of diagnostic PCR activity for detecting M. agassizii in different species of captive tortoises in Italy, and (ii) the phylogenetic analysis of the 16S rRNA gene sequences of M. agassizii . A total of 26.0% out of 169 samples resulted positive by PCR and 32 out of 75 (42.7%) animals with symptoms were positive. Sequences ob-tained from the PCR products were conserved, differed from the sequence of the M. agassizii type strain PS6, and were identical to many M. agassizii sequences deposited in databases. In particular, the sequences were identical or very similar to sequences obtained previously from tortoises in It-aly. Since samples collected from different anatomical sites resulted positive, it is suggested that pools of conjunctival, nasal and oral swabs are tested for diagnostic purpose in both symptomatic and asymptomatic animals.

The current study aimed to investigate the dietary supplementation effect of corn silk (CS) on performance and blood chemistry of brown and white-feathered quails during the grower and layer periods. Japanese quails of brown and white-feathered color (180 birds/variety at 2 wks old) were randomly allotted into three groups with 3 replicates each (n = 20 birds/replicate). Corn silk powder (CS) was supplemented to the basal diet at 0, 1, and 2% of the diet for each quail variety for 1-month growing period, then continued for another 6-wk laying period to assess the egg production and egg quality characteristics. CS supplementation at 1% and 2% for brown and white-feathered quails respectively improved their growth performance (body weight and weight gain), carcass yield, and intestinal villi length with increasing feed consumption but without changes in feed conversion ratio. In both quail varieties, CS addition had a hypolipidemic effect, confirmed by lowering serum triglyceride (TG), cholesterol (CHO), and low density lipoprotein (LDL) while increased high density lipoprotein (HDL) concentrations (P < 0.05) with a clear response observed in white quails than the brown ones. Besides, CS supplementation increased (P = 0.002) hen day egg production in brown feathered quails, while reducing it in the white-feathered quails compared with the CS-free diet. The increased egg production was not significantly (P > 0.05) correlated with lower content of TG and CHO, while significantly increased the antioxidant content in both quail varieties (P < 0.05). Moreover, CS dietary supplementation significantly enhanced (P = 0.003) the yolk color, especially in brown-feathered quail. In conclusion, CS can be safely supplemented to the Japanese quail diet (1% and 2% for brown-feathered and white-feathered quails respectively) to improve growth performance, and egg quality characteristics., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

To histologically evaluate the gastric compartments of Risso’s (Grampus griseus) and bottlenose dolphins (Tursiops truncatus) and provide suggestions for the diet of Risso’s dolphins in captivity, we examined 12 stomachs from both species. While slight differences in keratinization were observed in the forestomach, significant differences came to light in the second stomach’s mucosa. At this level, in Risso’s dolphin, the principal cells are markedly reduced in size and located externally to the parietal cells, not interspersed between them, compared to bottlenose dolphins; differences were also observed in the structure and concentration of the parietal and principal cells of the gastric body glands (p < 0.0001). The quantitative results of G- and D-cell counts in the gastric mucosa show a clear difference, with a higher concentration of G cells in the mucosa of Risso’s dolphin (t = 7.334; p < 0.0001) and a higher level of D cells in bottlenose dolphin mucosa (t = 3.123; p = 0.0049). These results suggest that parietal cells undergo greater stimulation by gastrin produced by G cells, with greater acid secretion in G. griseus. Further studies are needed to understand whether an inappropriate diet could lead to severe clinical signs due to gastric acidity in Risso’s dolphin.

The aim of this study was to test the effect of mixing doses of glutamate (Glu) and glutamine (Gln) on the growth, health and gut health of post-weaning piglets. One hundred twenty weaned piglets (24 ± 2 days of age) were assigned to 6 dietary groups: (1) standard diet (CO); (2) CO plus Glu (6 kg/Ton): 100Glu; (3) CO plus 75Glu + 25Gln; (4) CO plus 50Glu + 50Gln; (5) CO plus 25Glu + 75Gln and (6) CO plus 100Gln. At days 8 and 21, blood was collected for haematological and reactive oxygen metabolite analysis, intestinal mucosa for morphological and gene expression analysis, and caecal content for microbial analysis. Data were fitted using a Generalised Linear Model (GLM). Piglet growth increased linearly with an increase in Gln from d7 to d14. The Glu:Gln ratio had a quadratic effect on faecal consistency and days of diarrhoea, neutrophil% and lymphocyte%, and a positive linear effect on monocyte% in the blood at d8. The amino acids (AAs) reduced the intraepithelial lymphocytes in the jejunum, and 100Gln improved intestinal barrier integrity at d8. The caecal microbiota did not differ. Overall, this study suggested a favourable effect of mixing Glu and Gln (25 + 75-50 + 50) as a dietary supplementation in post-weaning piglets to benefit the immune and barrier function of the gut, resulting in an increase in faecal consistency and improvement of growth during the first 2 weeks post-weaning., (© 2022. The Author(s).)

The purpose of this study was to estimate the safety, feasibility, and efficacy of the intra-articular treatment of autologous microfragmented adipose tissue in dogs with spontaneous osteoarthritis (OA) in comparison with hyaluronic acid (HA), the standard intra-articular treatment. Specifically, it clinically evaluated pain and lameness, the radiographic progression of osteoarthritis, and synovial fluid inflammation. This was a prospective, single-center, parallel-group, randomized, controlled, in vivo clinical study. Participants ( n = 40) received either a single intra-articular injection of microfragmented adipose tissue or a single intra-articular injection of HA (1:1). Clinical outcomes were determined using a specialistic clinician assessment obtained by the completion of a specific clinical form based on the Vesseur modified lameness classification system, a pain evaluation using the Visual Analogue Scale (VAS), the measurement of the range of motion (ROM) of the affected joint, limb circumference, and the owners' score evaluation using the Canine Brief Pain Inventory (CBPI) for up to 6 months after the time of injection. Patients underwent a radiographic examination to establish the degree of OA in the affected joint, and synovial fluid samples were collected to assess the biochemical environment of the joint and evaluate and quantify the cellular population and the presence of three specific inflammation biomarkers for up to 60 days. The results of this study suggest that microfragmented autologous adipose tissue is safe and can effectively relieve pain and improve function in dogs with spontaneous articular OA. This one-step procedure is simple, timesaving, cost-effective, minimally invasive, and eliminates the need for complex and time-intensive cell culture processing. Furthermore, the clinical evidence and cytological results suggest better long-term pain control, resulting in an improvement in joint function, compared to HA treatment. The canine spontaneous OA model could play a key role in developing successful treatments for human medicine.

A retrospective study was conducted on parrots submitted from necropsy to the Department of Veterinary Pathology, School of Biosciences and Veterinary, University of Camerino, Italy, from 2007 to 2018. From a total of 2,153 parrots examined at post-mortem, four cases were diagnosed with atypical mycosis and were considered for determination of the fungus species by PCR. A Fischer's lovebird ( Agapornis fischeri ), Peach-faced lovebirds ( Agapornis roseicollis ), and two Blue and Gold Macaws ( Ara ararauna ) from four different aviaries died after some days of lethargy and ruffled feathers. Records of gross necropsy and histopathological exams (H&E, PAS, and Grocott stain) were described and biomolecular analyses were carried out. No specific gross lesions were appreciated at necropsy, while histopathology evidenced a systemic mycosis in several organs, particularly in the lungs. In affected organs, broad and non-septate hyphae, suggestive of mycoses, were observed. Molecularly, Mucor racemosus (Fischer's lovebird) and M. circinelloides (Peach-faced lovebirds) were identified from formalin-fixed and paraffin-embedded (FFPE) lung and liver tissue. In addition, Alternaria alternata and Fusicladium spp. (respectively in male and female Blue and Gold macaws) were identified in FFPE tissue from several organs; whereas the role of Mucor spp. as true pathogens is well-demonstrated, and the behavior of A. alternata and Fusicladium spp. in macaws as opportunistic pathogens have been discussed. To our knowledge, this report is the first one reporting mucormycosis caused by M. racemosus and M. circinelloides in lovebirds, and A. alternata and Fusicladium spp. in macaws., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Galosi, Falcaro, Danesi, Zanardello, Berardi, Biagini, Attili and Rossi.)

Duckweed (Lemna minor) meal was included in the formulation of three experimental feeds (L1, L2, L3) for rainbow trout at 10%, 20%, 28% of the protein source, respectively. Increasing the duckweed inclusion, the other protein sources were adjusted to get isonitrogenous (41%) and isolipidic (20%) diets, as the control diet (LC). 540 fish (mean body weight 124.5 ± 0.7 g) were randomly allocated in 12 tanks divided equally among the four different diets. After 90 days, fish were weighed and the most important productive performances, fillet quality and fatty acid profile were determined. The final body weight in L1 (340.53 g) and L2 (339.42 g) was not different from LC (348.80 g); L3 trout significantly (p < 0.05) exhibited the lowest one (302.16 g). Similar trends were found in final mean length, weight gain, specific growth rate, food conversion rate. Somatic indices were affected by duckweed inclusion. Diets had not significant effects on the proximate composition and fatty acids of the fillet in L1, L2, L3 respect to LC. Based on this study, duckweed meal derived from Lemna minor can be included in the feed for the rainbow trout without negative effects on the growth performances at 20% of the protein substitution.

The rapid decline of cheetah ( Acinonyx jubatus ) throughout their range and long-term studies of captive breeding has increased conservation action for this species including the study of chronic diseases. Gastritis is one of the captive diseases that leads to high mortality presented with symptoms including vomiting, diarrhea, anorexia, and weight loss. The disease presents different histological lesions in the gastrointestinal tract that are characterized by inconstant and different clinical appearance in captive and free-range cheetahs. The aim of this review is to summarize the causes of chronic gastritis in the cheetah. Factors including diet, living conditions, infections with gastric Helicobacter -like organisms (GHLOs), the lack of genetic polymorphism and the cheetah's specific-immunocompetence are analyzed. All studies on gastroenteric cheetah pathologies, conducted between 1991 (to the best of our knowledge, the first report on online databases) and 2021, are included in this review, highlighting the possible correlation between stress-related captive conditions and chronic gastric pathology.

In poultry, severe gastrointestinal diseases are caused by bacteria and coccidia, with important economic losses in the poultry industry and requirement of treatments which, for years, were based on the use of antibiotics and chemotherapies. Furthermore, Salmonella spp., Clostridium perfringens , and Campylobacter jejuni can cause serious foodborne diseases in people, resulting from consumption of poultry meat, eggs, and derived products. With the spread of antibiotic resistance, which affects both animals and humans, the restriction of antibiotic use in livestock production and the identification of a list of "critically important antimicrobials" became necessary. For this reason, researchers focused on natural compounds and effective alternatives to prevent gastrointestinal disease in poultry. This review summarizes the results of several studies published in the last decade, describing the use of different nutraceutical or phytonutrients in poultry industry. The results of the use of these products are not always encouraging. While some of the alternatives have proven to be very promising, further studies will be needed to verify the efficacy and practical applicability of other compounds.

Cheetahs ( Acinonyx jubatus ) are classified as "vulnerable" species due to the low numbers persisting in the wild. Gastrointestinal diseases are very common in this species when they are kept in captivity, in particular gastritis. Clinical signs are predominantly characterized by vomiting, diarrhea, weight loss and anorexia. In this study, we evaluated the efficacy of a multi-strain probiotic in two groups of cheetahs: Group A ( n = 4)-rescued cheetahs housed at the Cheetah Conservation Fund (Otjiwarongo, Namibia); Group B ( n = 9)-captive cheetahs housed in Italian zoos. Animals showed gastrointestinal signs of different severity, and were positive for Helicobacter spp., detected by PCR in stool samples. Two sachets of probiotic formulation were administered to all cheetahs once a day for 21 consecutive days. Clinical conditions (appetite loss, vomiting, stool consistency and Body Condition Score) before (T0) and after 21 days of probiotic administration (T1) were then compared using a simplified Feline Chronic Enteropathy Activity Index (FCEAI) score. A slight but not significant improvement in the scores was observed in Group A, which had mild intestinal symptoms, while a significant decrease in vomiting and stool consistency (** p < 0.01) scores was observed in Group B, which had more pronounced symptoms. Results suggest that high concentrations of live probiotics can be of help in managing gastrointestinal signs in cheetahs.

The knowledge of raptor pathogens and associated lesions may be extremely important to enhancing raptor conservation efforts and reducing pathogen spillover to humans and domesticated animals and vice versa. Parasite infections of the digestive system and associated bacteria and pathological changes were evaluated in deceased diurnal and nocturnal raptors in central Italy. Overall, the prevalence of parasites (nematodes, cestodes, trematodes, acanthocephalans, and protozoa) identified in the examined birds was 72.41%, and most of the positive raptors (71.42%) showed multiple parasite infections. Among bacteria, Salmonella typhimurium , Salmonella enterica subspecies diarizonae , Escherichia coli , Clostridium perfringens , Yersinia enterocolitica, and Pasteurella multocida were identified. The results obtained showed that both parasites and bacteria may cause severe lesions in the digestive system of diurnal and nocturnal raptors; parasites and bacteria may concur in causing these lesions; most severe lesions are caused by the interaction of multiple pathogens, both parasites and bacteria; and the same pathogen taxa are frequently associated with the same pathological changes. This study is the first report of S. typhimurium and S. enterica subspecies diarizonae in Buteo buteo , while Andracantha mergi , Spirocerca spp., Sarcocystis dispersa, &nbsp; Sarcocystis columbae, and Eumonospora spp. were recorded for the first time in Italy.

In Europe, approximatively 100 000 to 500 000 tendon repairs are performed every year. These procedures are associated with a considerable rate of postoperative complications (from 6% to 11%). Autologous micro-grafts (AAMG) and stromal vascular fraction (SVF) have been shown to improve tendon healing in 60% to 70% of treated rodents. The purpose of this study was to evaluate the effects of AAMG in a sheep model with tendinopathy. We used sheep models because, as a large animal, they are more comparable to humans. The hypothesis was that SVF injection would improve tendon healing compared with the control group, reducing inflammatory and matrix degrading, while increasing anti-inflammatory expression and collagen synthesis in the early stage of tendon injury. Sixteen Apennine sheep aged 2 to 5 years underwent 500 UI type I collagenase injection into both common calcaneal tendons (CCT) to induce tendinopathy. After 15 days (T0), one CCT in every ovine underwent randomly to 2.5 mL of AAMG obtained by mechanical disruption and the contralateral CCTs received no treatment. Clinical, ecographic, and sonographic evaluations were performed after 4 weeks (T1) and 8 weeks (T2). Histological, immunohistochemical, real-time polymerase chain reaction (RT-PCR), and biomechanical evaluations were performed at T2. At T2, the treated group showed a final tendon diameter (9.1 ± 1.4 mm) and a hardness expression (62%) that were similar to the original healthy tendon (8.1 ± 1.1 mm; 100%), with a significant recovery compared with the control group (9.5 ± 1.7 mm; 39%). Moreover, histological analysis of the treated group revealed an improvement in the fiber orientation score, fiber edema score, infiltrative-inflammatory process, and necrosis score (4.3 ± 3.3) compared with control group (8.8 ± 2.9). Immunohistochemically, the treated group showed high expression of collagen 1, Factor VIII and significantly low expression of collagen 3. These data were confirmed by RT-PCR analysis. The study findings suggested that AAMGs obtained through mechanical disruption present a safe, efficient, and reliable technique, enhancing tendon healing., (© 2021 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

To understand the effectiveness of a probiotic mixture on intestinal morphology, mucus layer composition, and cecal microbiota diversity, 40 10-day-old Guinea fowls ( Numida meleagris ) were assigned to two groups: the control group (C), receiving drinking water, and the treated group (P), receiving water plus a commercial multi-strain probiotic (Slab51®, 2 × 10 11 CFU/L). Birds were slaughtered after 4 months, and the intestines were collected. Samples from the duodenum, ileum, and cecum were processed for morphological and morphometric studies, and conventional glycohistochemistry. Cecal samples were also used to assess the microbiota by 16S metataxonomic approach. Group P showed significant increase in the villus height ( p < 0.001 in the duodenum and p < 0.05 in the ileum and cecum), villus width ( p < 0.05 in all investigated tracts), depth of crypts ( p < 0.001 in the duodenum and cecum; p < 0.05 in the ileum), and goblet cells per villus ( p < 0.001 in all investigated tracts) compared with group C. Cecal microbiota of the birds varied considerably and comparing the relative abundance of the main observational taxonomic units (OTUs), a positive enrichment of several beneficial taxa, such as Oscillospira, Eubacterium, Prevotella , and members of the Ruminococcaceae , was observed. The enrichment of those taxa can improve microbiota stability and resilience facing environmental stresses, enhancing its resistance against invading pathogens. Ruminococcaceae , which represent the most important taxon in both groups, and Prevotella have a key role in the gut physiology due to the production of short-chain fatty acids (SCFAs), which are a vital energy source for enterocytes, improve glucose metabolism, and exert an overall anti-inflammatory effect. Probiotic administration enriches the presence of Coprococcus, Oscillospira , and Eubacterium taxa that produce butyrate, which exerts a beneficial effect on growth performance, structure of villi, and pathogen control and has anti-inflammatory properties too. This study indicates that Slab51® supplementation positively affects the morphology and microbiota diversity of the guinea fowl intestine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Galosi, Desantis, Roncarati, Robino, Bellato, Nebbia, Ferrocino, Santamaria, Biagini, Filoni, Attili and Rossi.)

Canine intestinal lymphangiectasia (IL) is a condition characterized by variably severe gastrointestinal signs, frequently associated with laboratory abnormalities; the research for markers allowing a better understanding of the severity degree and/or obtaining an early diagnosis and/or monitoring is continuously progressing. In the present study, we investigated possible new diagnostic/follow-up markers in IL dogs, namely, serum C-reactive protein, serum bacterial lipopolysaccharide, serum cleaved cytokeratin 18, serum citrulline, and zonulin (in both serum and feces). A fecal proteomic study looking for possible confirmation and/or new marker candidates was also performed. All markers in both substrates, with the exception of serum citrulline, significantly differed between diseased and control dogs. Fecal proteomics allowed the retrieval of three proteins in IL dogs (Fc fragment of IgG-binding protein; transthyretin; proproteinase E) that were not previously found in clinically healthy subjects. Although further studies are needed, C-reactive protein, bacterial lipopolysaccharide, cleaved cytokeratin 18, and zonulin (in both serum and feces) resulted as promising markers for canine IL; similarly, fecal proteomics represents a road worthy of being pursued in the search for candidate biomarkers.

This in vitro study was carried out to evaluate the potential antibacterial properties of canine non-transfusional hemo-components. Therapeutic formulations commonly used for regenerative medicine purposes (platelet-rich plasma, platelet gel, platelet lysate, fibrin glue), considering both leukocyte-rich and leukocyte-poor formulations, but also platelet-poor plasma and activating substances (thrombin, calcium gluconate), were tested to detect elements with potential antimicrobial properties. The antibacterial effect was tested on different bacterial strains ( Staphylococcus aureus subspecies aureus, Staphylococcus cohnii subspecies cohnii, Escherichia coli, Pseudomonas aeruginosa , and Klebsiella pneumoniae subspecies pneumoniae ) isolated from canine wounds and classified as susceptible, multidrug-, extensively, and pandrug-resistant bacteria toward a known panel of human and veterinary antibiotics. The evaluation was carried out by agar gel diffusion method (Kirby-Bauer) and micro-inhibition in broth using microplates and spectrophotometer reading. The study findings confirmed the hypothesized antibacterial properties of canine non-transfusional hemo-components. A more effective bacteriostatic effect was found against Gram-negative bacteria, drug-resistant too. The presence of leukocytes or platelets does not appear to be essential for the antibacterial effect. Further studies should be conducted to evaluate the exact mechanism of action of the antimicrobial activity. However, non-transfusional hemo-components could be a useful natural aid in controlling bacterial infections in dogs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Attili, Iacoucci, Serri, Cuteri, Cantalamessa, Linardi, Rifici, Mazzullo, Rossi, Galosi and Tambella.)

Tuberculosis is a worldwide zoonosis involving a wide range of hosts among domestic and wild animals. We describe tuberculosis caused by Mycobacterium bovis in a wild crested porcupine (Hystrix cristata) found dead in the district of Macerata, Marche Region, Italy in 2019., (© Wildlife Disease Association 2021.)

Sexing of birds is indispensable for scientific, breeding and conservation programs but is difficult in many species and is particularly problematic in the case of nestlings showing no sexual dimorphism. Most useful and efficient methods of sex determination are based on unique features of the Z and W sex chromosomes detected via PCR to distinguish males (ZZ) and females (ZW). During the last twenty-five years researchers searched for the universal marker capable of sexing a maximally wide spectrum of species in a single PCR assay. We screened the phylogenetically representative set of 135 Psittaciformes species including 59 species sexed for the first time. Two known (P2P8, CHD1iA) PCR markers and four additional W/Z polymorphisms (CHD1iE, CHD1i16, CHD1i9 and NIPBLi16) located within the Chromo Helicase DNA binding CHD1 or the Nipped-B homolog NIPBL genes were applied. We present the electrophoretic patterns obtained for the PCR products of the analyzed markers including most typical and atypical patterns allowing sex determination, as well as those obtained when the given marker failed in sexing. Technical aspects of molecular sex determination are discussed: the optimization of amplification conditions, direct PCR and potential misinterpretations. A truly universal marker has not been found, and therefore, we propose a sexing strategy based on multiple CHD1i16, NIPBLi16, CHD1i9 and CHD1iE markers. This new strategy confirms the sex of a given bird with at least two markers detecting independent Z/W polymorphisms, reduces the number of necessary PCR reactions and minimizes the risk of sex misidentification.

Background: In free-ranging reptile populations, bacterial, fungal, viral and parasitic pathogens may affect hosts through impairment in movements, thermoregulation, reproduction, survival, and population dynamics. The speckled dwarf tortoise (Chersobius [Homopus] signatus) is a threatened species that is mostly restricted to the Succulent Karoo biome in South Africa, and little information on pathogens of this species is available yet. We derived baseline parameters for five males and five females that were captured to genetically enhance a conservation breeding program in Europe. Upon collection of the tortoises, ticks were removed and identified. Immediately upon arrival in Europe, ocular, nasal, oral and cloacal swabs were taken for viral, bacteriological and mycological examinations. Fecal samples were collected before and 1 month after fenbendazole treatment, and analyzed for parasites. A panel of PCR, aiming to detect herpesviruses, adenoviruses and iridoviruses, was carried out., Results: Samples were negative for viruses, while bacteriological examination yielded detectable growth in 82.5% of the swabs with a mean load of 16 × 10 7  ± 61 × 10 8 colony forming units (CFU) per swab, representing 34 bacterial species. Cloacal and oral swabs yielded higher detectable growth loads than nasal and ocular swabs, but no differences between sexes were observed. Fungi and yeasts (mean load 5 × 10 3  ± 13 × 10 3 CFU/swab) were detected in 25% of the swabs. All pre-treatment fecal samples were positive for oxyurid eggs, ranging from 200 to 2400 eggs per gram of feces, whereas after the treatment a significantly reduced egg count (90-100% reduction) was found in seven out of 10 individuals. One remaining individual showed 29% reduction, and two others had increased egg counts. In five tortoises, Nycthocterus spp. and coccidian oocysts were also identified. Soft ticks were identified as Ornithodoros savignyi., Conclusions: Our baseline data from clinically healthy individuals will help future studies to interpret prevalences of microorganisms in speckled dwarf tortoise populations. The study population did not appear immediately threatened by current parasite presence.

Laboratory tests can be altered in cardiovascular diseases and the investigation of specific tests or biomarkers may provide additional information about myocardial damage. Traditional laboratory tests, such as cell blood count, serum biochemistry, and coagulation, can be useful in investigating patients, but are not specific. However, markers like Troponin and Natriuretic Peptides may possibly furnish further data on myocardium damage and can be used in both studying and monitoring cats with cardiac disease. Moreover, the evaluation of the thyroid profile is very important as hyperthyroid cats concomitant cardiovascular diseases are very common and they can also be a direct consequence of endocrinopathy. The purpose of this manuscript is to provide the widest possible overview of what is present in the literature about the feline clinical pathology of heart diseases through a rational division of the main alterations of traditional tests and biomarkers.

Probiotics have become highly recognized as supplements for poultry.Since gut health can be considered synonymous withanimal health, the effects of probiotic Slab51 ® on the morphology and the glycan composition of guineafowlintestine were examined. The probiotics were added in drinking water (2 × 10 11 UFC/L) throughout the grow-out cycle.Birds were individually weighed andslaughtered after four months. Samples from the duodenum, ileum and caecum were collected and processed for morphological, morphometric, conventional and lectin glycohistochemical studies.The results were analyzed for statistical significance by Student's t test. Compared with control samples, probiotic group revealed (1) significant increase in villus height ( p < 0.001 in duodenum and ileum; p < 0.05 in caecum), crypt depth ( p < 0.001 in duodenum and caecum; p < 0.05 in ileum) and goblet cells (GCs) per villus ( p < 0.001) in all investigated tracts; (2) increase in galactoseβl,3N-acetylgalacyosamine(Galβl,3GalNAc)terminating O-glycans and αl,2-fucosylated glycans secretory GCs in the duodenum; (3) increase in α2,6-sialoglycans and high-mannose N-linked glycans secretory GCs but reduction in GCs-secreting sulfoglycans in the ileum; (4) increase in Galβl,3GalNAc and high-mannose N-linked glycans secretory GCs and decrease in GCs-producing sulfomucins in the caecum; (5) increase in the numbers of crypt cells containing sulfate and non-sulfated acidic glycans. Overall, dietary Slab51 ® induces morphological and region-specific changes in glycoprotein composition of guinea fowl intestine, promoting gut health.

The breeding of meat rabbits is an important sector in the livestock industry in Italy. The focus of this study was to describe the antibiotic resistance profile distribution among the Methicillin-sensitive Staphylococcus aureus isolated in a rabbit farm. From 400 animals of different ages and three farm workers, 96 randomly selected strains isolated from various anatomical sites and lesions were analysed. According to spa typing and the resistance profiles towards veterinary and human antibiotics, 26 pathotypes were identified. The highest resistance was observed against Tetracyclines (92.3%) and Macrolides (80.8%), while almost all were susceptible to Penicillins, according to the limited use of β-lactams on the farm. In total, 92.3% of pathotypes were multidrug resistant (MDRs). Two MDR pathotypes belonging to the t2802 spa type were isolated from both farmers and rabbits. Age categories harboured significantly different pathotypes ( p = 0.019), while no association was found between pathotypes and lesions ( p = 0.128) or sampling sites ( p = 0.491). The antibiotic resistance was observed to increase with the time spent in the farm environment (age category). The selective pressure exerted by antibiotic use acted by giving advantage to more resistant strains rather than by lowering susceptibility to various drug categories within strains.

The gut microbiota plays a crucial role in several physiologic functions of the host. In humans and animals, manipulation of the intestinal microbiota by oral administration of probiotic lactic acid bacteria plays a significant role in modulating the immune system. The aim of this study was to evaluate the safety of the probiotic mixture Slab51® and the capacity of this mixture to stimulate immune function in healthy dogs. Twenty dogs were divided in two groups and received a control diet or the same diet supplemented with a dose of 400 billion cfu of lyophilized bacteria for a period of 60 days. Body weight, food intake, body condition score (BCS), fecal score (FSS), fecal immunoglobulin IgA concentration, plasma IgG concentration, and fecal microbiota composition were monitored. Weight, food intake, BCS, FSS, and biochemical parameters remained unchanged during the treatment in both groups of animals. The fecal microbiota showed a significant decrease in the abundance of Clostridium perfringens and a significant increase in the abundance of beneficial Bifidobacterium and Lactobacillus organisms ( p < 0.05). Fecal IgA and plasma IgG levels were significantly higher in the group receiving the probiotic compared to healthy controls. These data show that dietary supplementation with the probiotic mixture Slab51® is safe and well-tolerated, modulating the composition of the intestinal microbiota, and enhancing specific immune functions in healthy dogs., (Copyright © 2020 Rossi, Pengo, Galosi, Berardi, Tambella, Attili, Gavazza, Cerquetella, Jergens, Guard, Lidbury, Stainer, Crovace and Suchodolski.)

Alzheimer's disease (AD) is a progressive neurodegeneration characterized by neuron death ending in memory and cognitive decline. A major concern in AD research is the identification of new therapeutics that could prevent or delay the onset of the disorder, with current treatments being effective only in reducing symptoms. In this perspective, the use of engineered probiotics as therapeutic tools for the delivery of molecules to eukaryotic cells is finding application in several disorders. This work introduces a new strategy for AD treatment based on the use of a Lactobacillus lactis strain carrying one plasmid (pExu) that contains a eukaryotic expression cassette encoding the human p62 protein. 3xTg-AD mice orally administered with these bacteria for two months showed an increased expression of endogenous p62 in the brain, with a protein delivery mechanism involving both lymphatic vessels and neural terminations, and positive effects on the major AD hallmarks. Mice showed ameliorated memory, modulation of the ubiquitin-proteasome system and autophagy, reduced levels of amyloid peptides, and diminished neuronal oxidative and inflammatory processes. Globally, we demonstrate that these extremely safe, non-pathogenic and non-invasive bacteria used as delivery vehicles for the p62 protein represent an innovative and realistic therapeutic approach in AD.

Periodontal disease (PD) is a common inflammatory condition in dogs; in severe stages, dental extraction is frequently required. Platelet-rich fibrin (PRF) has been used in human oral surgical procedures and has been experimentally tested on post-extraction sockets in healthy dogs. This is the first split-mouth, randomized, controlled trial designed to compare post-extractive alveolar socket healing with and without topical application of PRF in canine spontaneous PD. Clinical evaluation, radiographic density, and histological scores for inflammation and regeneration were assessed at recruitment (T0) and after a three-week follow up (T1) on 12 dogs, for a total of 31 pairs of sockets. No complications or clinically evident differences between the treated sites and the control sites were observed. Comparing the radiographic densities of the extraction sites measured at T0 and T1, a significant enhancement was observed within the PRF group, but not within control group. The histological score decreased significantly from T0 to T1 within group PRF, but not within the control group; at T1, the PRF group showed a significantly lower histological score than the control group. These findings suggest that PRF could be able to stimulate the natural process of tissue healing and regeneration of post-extraction sites in dogs with spontaneous periodontal disease (PD).

A study in an intensive Italian rabbit farm was carried out to assess the effect of age category and anatomical site on detection of S. aureus and to describe the diversity of spa-types within farm, including workers. On 400 rabbits of different age, 2066 samples from the ear, nose, axilla, groin, perineum and lesions were analyzed and 595 S. aureus were isolated. In total, 284 (71.0%) were colonized by S. aureus in at least one anatomical site and 35 animals (8.8%) had lesions. S. aureus prevalence was higher in adults than other age categories. Ear (29.4%) and nose (27.2%) were the most frequently colonized sites. The presence of lesions due to S. aureus was strongly associated with the colonization of at least one body site and the risk of lesions was proportionate to the number of sites colonized by S. aureus . In addition, a genotypic characterization was performed for 96 S. aureus strains randomly selected. All isolates resulted as low-virulence methicillin-sensitive- Staphylococcus aureus (LV-MSSA). Five different clonal lineages were found: spa-t2802, the most frequently detected (53.1%), also in human samples; spa-t491 (38.5%); spa-t094; t605; and spa-t2036. Strains t094, t491 and t2036 have not been isolated in Italy before.

Canine fiber responsive diarrhea is a form of chronic colitis that improves clinically after adding fiber to the diet. In the present study, we investigated the effect of a combination of a high-fiber, highly digestible, hypoallergenic diet with a probiotic mixture in 30 dogs with chronic colitis that were unresponsive to various dietary and/or pharmacological interventions. Fecal scores, canine chronic enteropathy clinical activity index (CCECAI) scores, the dysbiosis index (DI), and histologic images of colonic biopsies were evaluated. At baseline (day 0; T0) and after 30 days of treatment (T1), all variables evaluated in our patients (i.e., fecal and CCECAI scores and histopathology) improved significantly at T1, with the exception of DI. However, there was a numerical shift from a state of dysbiosis to one of normobiosis. The combination of the diet and the probiotic used in the present study induced the resolution of clinical signs in a mean of 8.5 days (maximum 15 days) and did not necessitate any other treatments or the further addition of alimentary fiber., Competing Interests: Dr. Graziano Pengo worked with the R&D Department at SANYpet SpA to design the composition of the diet used in the present study: Intestinal Colitis Phase 1 (currently Intestinal Colon Phase 1; SANYpet SpA - Forza10, Bagnoli di Sopra (PD), Italy). He is being compensated based on the commercial sales of this diet. No other conflicts of interest are being disclosed for the other authors of the present study.

Macrorhabdus ornithogaster is an opportunistic yeast that colonizes the gastric mucosa of many avian species. Until now, no studies have focused on the influence of a gastric infection on the balance of the intestinal microbiota of birds. In this study, 44 faecal samples from individual canaries, with and without M. ornithogaster infection, were analysed. The detection of the yeast was evaluated by 18S rRNA PCR. In order to evaluate the impact of the Macrorhabdus infection on the bacterial communities, culture-independent methods, by the use of amplicon-based sequencing as well as 16S rRNA-DGGE, were adopted. The different health status of animals affected the relative abundance of the main OTUs, with a greater diversification of the gut microbiota in healthy animals compared to the infected. In particular, Lactococcus, Pseudomonas, Acinetobacter, Lachnospiraceae, Propionibacterium and Weissella were found to be characteristic of uninfected animals (FDR < 0.05), while Lactobacillus and Candidatus Arthromitus were characteristic of infected animals (FDR < 0.05). Both these taxa have been reported as immunostimulatory, involved in immunological disorders. In infected animals the inferred metagenome assessed by PICRUST clearly showed a positive correlation between the presence of M. ornithogaster and KEGG genes related to ether lipid metabolism, already reported to be immunostimulatory by activation of macrophages and to play a pathophysiological role in several immunological disorders. Finally, our results show an interaction between infection of the digestive tract and intestinal microbiota of pet birds and provide insight into the changing of the complex enteric bacterial community. HIGHLIGHTS Macrorabdus ornithogaster is a gastric yeast that colonizes a wide range of birds. Differences were found between infected and healthy animals in gut microbiota. Candidatus Arthromitus was closely associated with infected birds. M. ornithogaster can affect intestinal microbiota composition of canaries.

Necropsy of two free-ranging common blackbirds ( Turdus merula ) found dead in central Italy revealed the presence of a high number of cyclocoelid flukes in the coelomatic cavity. Cyclocoelid flukes primarily infect avian respiratory system. Histologically, air sac walls were covered with a fibrinous exudate containing degenerate heterophils, many trematodes and some colonies of Gram-positive cocci. The superficial bronchi and parabronchi were markedly distended, and the adjacent pulmonary parenchyma was congested and collapsed. Trematodes, surrounded by a mild suppurative to pyogranulomatous inflammatory reaction, were also observed on the pericardial, intestinal, kidney and hepatic serosal surfaces. The death of the two examined birds was likely due to the high parasite load and associated severe lesions. At parasitological examination, flukes showed a tongue-shaped elongate body, tapered anteriorly and rounded posteriorly, of 2,088-2,314 μm in width and 8,268-11,830 μm in length. The mouth was slightly oval and sub-terminal, with a small oral sucker. The oval pharynx measured 250-309 μm, and the two caeca joined posteriorly. Two large (550-702 μm × 450-520 μm) globular testes were situated obliquely to each other, whereas an oval (250 × 300 μm in mean) or round (about 334 μm in diameter) intertesticular ovary was placed in a longitudinal straight line with the testes. The ootype was about 110 μm in diameter, while the brown-yellow eggs measured 131.5 × 73.9 μm in mean. The genital pore was post-pharyngeal, while the symmetrically arranged vitelline glands were not confluent posteriorly. Morphoflogical diagnosis led to the identification of Morishitium polonicum , a cyclocoelid fluke species that typically inhabits the air sacs of blackbirds. The morphological diagnosis was corroborated by molecular phylogenetic analysis of the mitochondrial (CO1, ND1) DNA loci. The present study provides the first report of pathological lesions and death caused by M. polonicum in birds.

The effects on Giardia duodenalis of Slab51 probiotic supernatants were evaluated in vitro and ex vivo. In vitro, Slab51 (101 UFC) was cultured and the obtained supernatant was filtered, adjusted at pH 7, and added (100μl/ml) as such (Slab51 FS) or after heat-treatment, to G. duodenalis cultures to evaluate its effects on G. duodenalis trophozoites growth and adherence. For comparison, negative and metronidazole (20μg/ml) treated controls were used. The morphological and ultrastructural alterations of G. duodenals trophozoites following treatment with Slab51 FS supernatant were investigated by transmission electron microscopy. Ex vivo, mice duodenal portions were cultivated in standard conditions with 5x105 G. duodenalis trophozoites/ml, while to further five duodenal portions similarly cultured and infected, Slab51 FS 200μl was added. After 12 and 18h, samples were fixed in 10% buffered formalin and histologically processed to score Giardia infection and cell damage. Cell proliferation/apoptosis was scored by Ki67, TUNEL and Caspase-3 tests. All experiments were conducted in triplicate throughout the study. All data were statistically evaluated (P< 0.05). Results showed that Slab51 FS significantly reduced Giardia growth and adherence respect to negative controls, but its efficacy was overall lower than that of metronidazole. Moreover, the effects of Slab51 FS were significantly lowered by heat-treatment and this reduction was statistically higher at 90°C than at 56°C, indicating a heat-sensitive nature of active Slab51 FS compounds. At the ultrastructural level, Slab51 FS treated Giardia trophozoites were swelling, increased in size and showed alterations of their cellular membrane and vacuole patterns, loss of the nuclear envelope and nuclear architecture. In ex vivo trials, viable G. duodenalis trophozoites and enterocyte TUNEL+ and Caspase-3 expression were significantly reduced in intestinal sections added with Slab51 FS, while enterocyte Ki67 expression was significantly increased, confirming the anti-G. duodenalis activity of Slab51 FS observed in vitro. In conclusion, results from this study showed that the fresh culture supernatant of the commercial probiotic Slab51 has anti-G. duodenalis properties both in vitro and ex vivo in a mouse model., Competing Interests: The authors declare that this study was partly supported by Actial Farmaceutica Lda, Praça Severino Ferraz, 258 082 Funchal, Madeira, Portugal. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript and no authors received a salary from this funder. This does not alter our adherence to PLOS ONE policies on sharing data and materials.