Preliminary Study on the Host Response to Bivalent and Monovalent Autogenous Vaccines against Mycoplasma agalactiae in Dairy Sheep.

In Italy, dairy sheep farming represents a vital agro-industry sector, but it is still challenged by contagious agalactia (CA), which is endemic there, and vaccination is the most economical and sustainable tool for control. This study aimed to evaluate the combined Mycoplasma agalactiae (Ma)-Staphylococcus aureus (Sa) vaccine (Ma-Sa) against the Ma monovalent vaccine in ewes. Twelve primiparous Ma-free ewes were randomly grouped into three equal groups: first, the control group injected with placebo, second, the group vaccinated with the Ma monovalent vaccine, and third, the group vaccinated with Ma-Sa combined vaccine, with two S/C doses at 45-day intervals. The animals were examined for serological, hematological, and somatic cell count (SCC) changes for 17 successive weeks. A significant increase in anti-Ma antibody mean titers, leukocytes, and platelets was observed in the vaccinated animals, with the highest values in those who received the combined vaccine. Neutrophils were high only in the animals who received the combined vaccine. SCC was lower in the vaccinated animals during the first six weeks. This study concludes that the combined Ma-Sa vaccines enhance immune response and potentiate its efficacy against Ma. This improvement might be attributed to the sensitization/activation effect of S. aureus on platelets, which are recoded to act as a key regulator for the coordination of all components of the innate immune system. Even though this study included a small number of animals, its findings about the potentialities of this inactivated vaccine in the control of CA are strongly encouraging. Further confirmation might be needed through additional replicates and a challenge study is needed before proceeding with widespread use.

Study on the Canine Adenovirus Type 1 (CAdV-1) Infection in Domestic Dogs in Southern Italy.

Canine adenovirus type 1 (CAdV-1) is the causative agent of a systemic and potentially fatal viral disease of domestic and wild canids. In Italy, CAdV-1 infection has also been occasionally described in dogs, but information on the epidemiology and its genomic features is still limited. A study was conducted on 291 dogs suspected of infectious gastrointestinal disease. Samples collected from dogs in southern Italy between 2017 and 2020 were analyzed. Virological and histopathological assays were carried out. The presence of CAdVs and other canine viral enteropathogens was investigated, and sequence and phylogenetic analyses were performed. CAdV-1 was detected in six (2.1%) dead stray dogs alone or in mixed infections with other viruses. Gross lesions and histopathological findings referred to CAdV infection were observed, also involving the central nervous system tissues. All inoculated samples were successfully isolated. Sequence analysis evidenced divergences with the circulating strains previously described in Italy and a closer relation with older CAdV-1 strains collected from other countries, suggesting a genetic heterogeneity of CAdV-1 in Italy. The evidence of the circulation of CAdV-1 and its genomic features allows us to have more in-depth knowledge of the epidemiology and evolution of the CAdV-1 genomic variants.

First Molecular Survey to Detect Mycoplasma gallisepticum and Mycoplasma synoviae in Poultry Farms in a Strategic Production District of Sicily (South-Italy).

Mycoplasmas are recognized as avian pathogens, which may cause both respiratory disease and synovial infections in poultry, resulting in severe economic losses. Our study aims to determine the occurrence of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) among commercial and rural laying hens located in Ragusa province (South Italy), using a duplex real time PCR. Four hundred tracheal swabs were collected from seven commercial (200 swabs) and 25 rural (200 swabs) farms without any clinical disease history. Out of 400 swabs collected, 50 (12.5%) and 93 (23.25%) were positive for MG and MS, respectively. In particular, 9 (18%) and 22 (23.65%) positive swabs for MG and MS, respectively, originated from commercial farms, compared to 41 (82%) and 71 (76.34%) obtained from rural farms. Data obtained show a lower prevalence of MG than MS in the studied farms. Moreover, both pathogens were spread in rural and commercial farms. PCR could be concluded as a rapid and sensitive method for the identification of MG and MS in areas where commercial farms that are declared Mycoplasma-free and rural flocks coexist. These data highlight the importance of surveillance also in rural poultry to monitoring the occurrence of mycoplasmas strains in strategic productive districts.

Diagnostic Findings in a Confirmed Outbreak of Brucella ovis Infection in a Traditional Sheep Farm in Sicily (South-Italy).

Aim of this study is to report a laboratory investigation performed following the isolation of Brucella ovis, causing ovine epididymitis, in a traditional sheep farm in Sicily (South Italy). This disease represents a newly emerging risk for Italian livestock and is listed among diseases of EU priority (EU Reg 2016/429). Blood samples from 56 rams and 143 ewes were analyzed by both Enzyme-Linked Immunosorbent Assay (ELISA) and Complement Fixation Test (CFT). Genital swabs from all rams and 15 lactating ewes were collected to perform real-time PCR. Eighteen serologically positive rams were slaughtered and postmortem-inspected. Samples of testicle, epididymis, lymph nodes, and urine were also collected in order to perform microbiological, molecular, and histopathological analysis. Twelve slaughtered rams showed anatomo-pathological lesions. Real-time PCR for B. ovis BOV_A0504 gene was positive for 13 testicles and epididymis and 11 urine while B. ovis was isolated from epididymis and testicles of 7 slaughtered rams. This is the first exhaustive laboratory report of a microbiological, molecular, and serological pattern of the disease in sheep in Italy. Despite the impact on health and animal welfare, the epidemiology of B. ovis infection is still unknown, particularly in our country where the disease is considered endemic.

The NUPR1/p73 axis contributes to sorafenib resistance in hepatocellular carcinoma.

The multikinase inhibitor sorafenib was the first drug approved by the FDA for treating patients with advanced hepatocellular carcinoma (HCC). However, sorafenib resistance remains a major challenge for improving the effectiveness of HCC treatment. Previously, we identified several genes modulated after sorafenib treatment of human HCC cells, including the stress-inducible nuclear protein 1 (NUPR1) gene. Multiple studies have shown that NUPR1 regulates autophagy, apoptosis, and chemoresistance. Here, we demonstrate that treatment of HCC cells with sorafenib resulted in the activation of autophagic flux. NUPR1 knock-down (KD) in HCC cells was associated with increased p62 expression, suggesting an impairment of autophagic flux, and with a significant increase of cell sensitivity to sorafenib. In NUPR1 KD cells, reduced levels of NUPR1 were associated with the increased expression of p73 as well as its downstream transcription targets PUMA, NOXA, and p21. Simultaneous silencing of p73 and NUPR1 in HCC cells resulted in increased resistance to sorafenib, as compared to the single KD of either gene. Conversely, pharmacological activation of p73, via the novel p73 small molecule activator NSC59984, determined synergistic anti-tumor effects in sorafenib-treated HCC cells. The combination of NSC59984 and sorafenib, when compared to either treatment alone, synergistically suppressed tumor growth of HCC cells in vivo. Our data suggest that the activation of the p73 pathway achieved by NUPR1 KD potentiates sorafenib-induced anti-tumor effects in HCC cells. Moreover, combined pharmacological therapy with the p73 activator NSC59984 and sorafenib could represent a novel approach for HCC treatment.

A hyaluronic acid/cyclodextrin based injectable hydrogel for local doxorubicin delivery to solid tumors.

Localized delivery of anticancer drugs is often the most useful therapeutic approach for the treatment of solid tumors. The use of injectable polymeric systems that maximize drug concentration in the proximal area of the tumor represents an extremely advantageous therapeutic strategy. Here, the development of an injectable in situ forming hydrogel was accomplished by exploiting the azo-type Michael reaction between an amine derivative of hyaluronic and vinylsulfone functionalized ß-cyclodextrins complexing doxorubicin. This injectable system can be easily prepared and administered with timelines compatible with normal operating room procedures, as demonstrated by rheological tests. In vitro experiments revealed that the peculiar physicochemical properties of the hydrogel guarantee a sustained release of the anticancer drug that blocks the growth of colorectal carcinoma micromasses cultured in 3D conditions. In vivo studies have confirmed that the medicated hydrogel can drastically reduce the tumor mass in the animal model without causing cytotoxic side effects in other areas of the body such as the heart. Overall, the proposed system has shown promising characteristics that make it an interesting useful device for localized chemotherapy of solid tumors.

Morphological and Immunohistochemical Examination of Lymphoproliferative Lesions Caused by Marek's Disease Virus in Breeder Chickens.

Marek's disease is widely controlled by vaccination programs; however, chickens are not totally protected, especially immediately after the vaccination when a strong challenge could interfere with the effectiveness of vaccination in the absence of proper biosecurity practice. This case report describes the occurrence of Marek's disease (MD) observed in a breeder chicken flock reared southeast of Sicily. MD outbreak occurred from 32 to 47 weeks with an increase in weekly mortality rate (+0.4-0.6%). Overall, mortality rate related to Marek's disease was about 6% at the end of the cycle. Carcasses of chickens found during the occurrence of disease underwent necropsy, and tissues were collected to confirm the infection. Gizzard, cecal tonsil, intestine, spleen and tumor mass were collected and analyzed from a carcass of one hen, 32 weeks old and apparently asymptomatic. Multiplex real-time PCR performed on spleen tissues detected the presence of MD virus pathogenic strain. Macroscopic and microscopic evaluation of the rest of the samples confirmed the neoplastic disease. Moreover, the immunophenotype of the tumor cells was identified as CD3 positive by immunohistochemical (IHC) staining. The vaccinated flock had become rapidly infected with the MD virus, which proves that the challenge of the MD virus was too strong in the rearing house at the beginning of the cycle, causing the outbreak.

Effects of Microencapsulated Blends of Organics Acids (OA) and Essential Oils (EO) as a Feed Additive for Broiler Chicken. A Focus on Growth Performance, Gut Morphology and Microbiology.

The goal of the trial was testing the effects of a blend of organic acids and essential oils dietary supplementation on growth performance and gut healthiness in broiler chickens. In total, 420 male Ross 308 chicks (1-day old) were randomly assigned to two dietary treatments: basal (BD) and organic acids and essential oils (OA&EO) diets (three replicates/treatment; 70 broilers/replicate). BD group received commercial diets whereas OA&EO group basal diets + 5 g/kg of microencapsulated organic acids and essential oils. OA&EO treatment improved the average daily gain (p < 0.01) and feed conversion ratio at 37-47 days compared to BD treatment. OA&EO treatment improved gut morphology mostly at ileum and duodenum levels in terms of villi height, crypt depth, number of villi, mucosa thickness and villi area at 24 and 34 sampling days. A certain selective action against Clostridium perfringens in ileum of OA&EO group was shown at 33 (p = 0.053) and 46 days (p = 0.09) together with lower median values for Enterobacteriaceae, Enterococci, Mesophilic bacteria and Clostridium perfringens at ceca level. Overall, organic acids and essential oils supplementation improved growth performance in the final growth stage and some morphological gut traits and reduced to a certain extent Clostridium perfringens count in ileum.

Shear stress reverses dome formation in confluent renal tubular cells.

BACKGROUND/AIMS: It has been shown that MDCK cells, a cell line derived from canine renal tubules, develop cell domes due to fluid pumped under cell monolayer and focal detachment from the adhesion surface. In vitro studies have shown that primary cilia of kidney tubular epithelial cells act as mechanosensors, increasing intracellular calcium within seconds upon changes in fluid shear stress (SS) on cell membrane. We then studied the effect of prolonged SS exposure on cell dome formation in confluent MDCK cell monolayers. METHODS: A parallel plate flow chamber was used to apply laminar SS at 2 dynes/cm(2) to confluent cell monolayers for 6 hours. Control MDCK cell monolayers were maintained in static condition. The effects of Ca(2+) blockade and cell deciliation on SS exposure were also investigated. RESULTS: Seven days after reaching confluence, static cultures developed liquid filled domes, elevating from culture plate. Exposure to SS induced almost complete disappearance of cell domes (0.4±0.8 vs. 11.4±2.8 domes/mm(2), p < 0.01, n=14). SS induced dome disappearance took place within minutes to hours, as shown by time-lapse videomicroscopy. Exposure to SS importantly affected cell cytoskeleton altering actin stress fibers expression and organization, and the distribution of tight junction protein ZO-1. Dome disappearance induced by flow was completely prevented in the presence of EGTA or after cell deciliation. CONCLUSIONS: These data indicate that kidney tubular cells are sensitive to apical flow and that these effects are mediated by primary cilia by regulation of Ca(2+) entry in to the cell. SS induced Ca(2+) entry provokes contraction of cortical actin ring that tenses cell-cell borders and decreases basal stress fibers. These processes may increase paracellular permeability and decrease basal adhesion making dome disappear. Elucidation of the effects of apical fluid flow on tubular cell function may open new insights on the pathophysiology of kidney diseases associated with cilia dysfunction.

Podocyte repopulation contributes to regression of glomerular injury induced by ACE inhibition.

Angiotensin-converting enzyme (ACE) inhibition induces glomerular repair in the Munich Wistar Frömter (MWF) rat, a model of spontaneous glomerular injury. In this study, we investigated whether this effect is related to changes in glomerular cell number, particularly of podocytes, which are progressively lost with age. MWF rats with advanced nephropathy were studied at both 40 weeks and after 20 weeks of observation either with or without treatment with the ACE inhibitor lisinopril. Forty-week-old Wistar rats were used as controls. In untreated MWF rats, proteinuria, hypertension, glomerulosclerosis, and renal function worsened, while lisinopril induced regression of both functional and structural changes. Despite glomerular hypercellularity in untreated MWF rats, the number of endothelial cells per glomerulus did not change, and podocyte number even decreased. ACE inhibition halted the progressive increase in glomerular cell number and enhanced endothelial cell volume density. Surprisingly, lisinopril not only halted age-related podocyte loss but also increased the number of glomerular podocytes above baseline, which was associated with an increased number of proliferating Wilms tumor 1-positive cells, loss of cyclin-dependent kinase inhibitor p27 expression, and increased number of parietal podocytes. These data indicate that ACE inhibition restructures glomerular capillary, primarily by restoring the podocyte population in this model of glomerular injury. Increased parietal podocyte number in lisinopril-treated MWF rats suggests that the remodeling of Bowman's capsule epithelial cells contributes to this effect.