“New insights in the etiopathogenesis of muscle inflammation and aging in animals”

Muscle pathology has unique relevance in many different fields of veterinary research, ranging from the diagnostics, to the comparative medicine involving animal models of human diseases, to the animal based food safety. The Laboratory of Comparative of Neuromuscular Disorders was established decades ago at the Department Veterinary Medicine of Naples in order to contribute to the knowledge of muscle pathology in domestic animals using modern techniques and undertaking international scientific collaborations. In this context, the project of this doctoral thesis had the main purpose to produce new scientific data in at least two interest area: the etiopathogenesis of inflammatory myopathies and skeletal muscle aging. Thus, the experimental results of four research project conducted in four different species are presented, including: 1) Inflammatory myopathy in horses affected by chronic piroplasmosis; 2) Lymphoplasmacytic myositis and expression of Major Histocompatibility Complex class I and II in ovine muscular sarcocystosis; 3) Age related skeletal muscle atrophy and upregulation of autophagy in dogs; 4) Expression and biochemical properties of cellular Prion protein in skeletal muscle of aged cows. In the first and second chapters of the thesis, two inflammatory myopathies associated with parasites having or not having a specific tropism for skeletal muscle (Sarcocystis tenella and Theileria equi -Babesia caballi, respectively) are explored. In the first study, an inflammatory myopathy characterized by a mainly CD8-CD4+ lymphocytic population and fiber degeneration is described in 16 horses serologically positive for equine piroplasms with clinical signs and serum biochemical evidence of a myopathy. To test the hypothesis of circulating autoantibodies directed against an unknown muscular antigen, the serum from affected horses was tested by indirect immunofluorescence to sections of normal equine muscle. In all cases, distinct sarcolemmal staining was detected in sections incubated with serum from affected horses, in contrast to sections incubated with phosphate-buffered saline or equine control sera. Furthermore, a significant increase of interferon-γ, interleukin-12, and tumor necrosis factor-α gene expression by compared to healthy controls was revealed by Reverse transcription polymerase chain reaction (RT-PCR). Thus, we concluded that inflammatory myopathy associated with equine piroplasmosis may involve an autoimmune pathogenesis with upregulation of inflammatory cytokines that may cause myofiber atrophy and degeneration. The aim of the second chapter, leading with muscle inflammation in ovine muscular sarcosytosis, was to investigate if parasitized muscle fibers could play a role in immune-stimulation, as sporadically described in accidental muscular sarcocystosis in definitive hosts. Skeletal muscle samples from 78 sheep presenting muscular sarcocysts revealed inflammatory changes in 69% of cases, consisting of an endomysial lymphoplasmacellular infiltrate, with attendant myofiber degeneration and necrosis. The predominant T cell populations were CD3+, CD8+ with lesser numbers of CD4+ and CD79α+ cells. Eosinophils were constantly absent. Notably, moderate to strong sarcolemmal labeling to Major histocompatibility complex (MHC) I and II was found both in biopsies with evident inflammatory infiltrate and in cases without inflammation. The wall of the cysts resulted strongly positive to MHC Iand II, and occasionally co-localized with the membrane associated protein Dystrophin. Our data suggest that muscle fibers respond to the presence of cysts by expression of MHC I and II that can play a role in stimulating and maintaining the lymphoplasmacellular inflammation. The possible role of MHC I on the cyst wall is discussed, but basically more research is needed in order to evaluate its possible involvement in inflammation. Moreover we speculated about the possibility that the abnormal expression of MHC I and II on muscle fibers is not only typical of myopathies having an immune-mediated pathogenesis but also of myositis associated with muscular parasites. The last two chapters of the thesis are dedicated to muscle aging, and in particular to the role of autophagy in canine sarcopenia and of cellular prion protein in bovine muscle aging. Sarcopenia, the age related loss of muscle mass and strength, is a multifactorial condition that occurs in a variety of species and represents a major healthcare concern in human medicine. We evaluated the expression of three markers of autophagy, Beclin 1, LC3 and p62, in muscle wasting of 25 geriatric dogs, to establish whether the levels of autophagy change with increasing age. Our results indicated a marked intracytoplasmic staining for Beclin 1 and LC3 in 80% of the muscle samples from the older dogs and a significantly greater expression of LC3 II and Beclin 1, by Western blotting. The results of the study suggest that enhanced autophagy might be one of the factors underlying muscle atrophy in canine age related muscle wasting. Finally, we explored the expression and biochemical properties of cellular prion protein (PrPC) in skeletal muscle of cows as well as their possible changes in geriatric animals. PrPC over expression has been described in many different human neuromuscular disorders but little is known about PrPC in health and disease in bovine muscle tissue. Skeletal muscle and brain samples of 12 aged and 8 young Podolica cows were collected and compared by histomorphological techniques as well as several experiments involving immunoblot and specifically analyzing the amount of PrPC, its glycosylation profile, proteinase resistance, solubility levels and tendency to aggregate. Histologically, muscle biopsies of aged cows showed typical myopathic features already described in bovine sarcopenia. Immunohistochemistry for PrP revealed a distinct granular positivity in intramuscular nerve branches and muscle spindles in all cases. Membrane immunopositivity was more prominent on angular atrophic fibers. Increased levels of positivity were found in degenerated fibers (desmin depleted) with a positive labeling of rimmed vacuoles. Prion protein positivity levels was also significantly increased in cases displaying inflammatory changes. No differences between muscles and brains of young and old animals were detected by quantitative immunoblot and Proteinase K titration. The glycoform profile and the molecular mass of PrPC in muscle samples appeared slightly different compared to the brain. Sucrose gradient velocity sedimentation gave variable results, with a generally lower amount of PrP in high density sucrose fractions in the muscle compared to the brain. After high speed centrifugation the majority of muscle PrPC was found in the soluble fraction, without significant differences between young and old animals. Our preliminary data suggest that: 1) the amount and the main biochemical properties of prion protein do not change with age, 2) PrPC is overexpressed in degenerated, vacuolated and angular muscle fibers; 3) The glycoform profile of PrPC differs between brain and muscle tissue. In conclusion, the main innovative aspects of this thesis are: 1) The first description in the literature of a likely immune-mediated myositis occurring in chronic equine piroplasmosis 2) The demonstration of high prevalence of lymphoplasmacellular myositis in ovine muscular sarcocystosis implying the overexpression of MHC I on muscle fibers as well as on the cyst wall. 3) The report of enhanced autophagy in age-related muscle atrophy in dogs. 4) The first detailed description of PrPC localization and biochemical properties in bovine muscle tissue and its possible involvement in neurogenic atrophy, muscle degeneration and chronic inflammation. Collectively, these results enhance the relevance of morphological studies as starting point to investigate the pathogenesis of muscle diseases in veterinary and comparative pathology

Angiostrongylus vasorum infection in red foxes (Vulpes vulpes) in southern Italy

Angiostrongylus vasorum (Nematoda: Angiostrongylidae) infection was detected at post-mortem examination in the pulmonary arteries and hearts of 34/102 (33,3%) of red foxes (Vulpes vulpes) from the Campania Region in southern Italy. Pathological changes consisted of granulomatous interstitial pneumonia caused by larvae and intravascular pulmonary adult nematodes. These changes confirm that angiostrongylosis infection in red foxes has a mainly chronic course, in which the infected host may disperse parasite larvae in the environment over its lifetime. Results suggest that the life cycle of A. vasorum is well established in the red fox in the Campania Region representing a potential infection risk for dogs

Extracorporeal shock waves alone or combined with raloxifene promote bone formation and suppress resorption in ovariectomized rats

Osteoporosis is a metabolic skeletal disease characterized by an imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. We examined the beneficial effect of shock waves (SW) alone or in combination with raloxifene (RAL) on bone loss in ovariectomized rats (OVX). Sixteen weeks after surgery, OVX were treated for five weeks with SW at the antero-lateral side of the right hind leg, one session weekly, at 3 Hz (EFD of 0.33 mJ/mm2), or with RAL (5 mg/kg/die, per os) or with SW+RAL. Sera, femurs, tibiae and vertebrae were sampled for following biochemical and histological analysis. SW, alone or combined with RAL, prevented femur weight reduction and the deterioration of trabecular microarchitecture both in femur and vertebrae. All treatments increased Speed of Sound (SoS) values, improving bone mineral density, altered by OVX. Serum parameters involved in bone remodeling (alkaline phosphatase, receptor activator of nuclear factor kappa-B ligand, osteoprotegerin) and osteoblast proliferation (PTH), altered by ovariectomy, were restored by SW and RAL alone or in combination. In tibiae, SW+RAL significantly reduced cathepsin k and TNF-α levels, indicating the inhibition of osteoclast activity, while all treatments significantly increased runt-related transcription factor 2 and bone morphogenetic-2 expression, suggesting an increase in osteoblastogenic activity. Finally, in bone marrow from tibiae, SW or RAL reduced PPARγ and adiponectin transcription, indicating a shift of mesenchymal cells toward osteoblastogenesis, without showing a synergistic effect. Our data indicate SW therapy, alone and in combination with raloxifene, as an innovative strategy to limit the hypoestrogenic bone loss, restoring the balance between bone formation and resorption

Evaluation of Ki-67 expression in feline non-ocular melanocytic tumours

Fifty tumours located in skin (n = 33) and mucosae (n = 17) were included. Forty-eight percent and 95% of amelanotic tumours (n = 21) stained positive for Melan A and S100, respectively. Most achromic tumours were mucosal (P  5, Ki-67 > 20% and lack of treatment administration. On multivariable analysis, only tumour histotype and treatment retained prognostic significance. Conclusions Although the majority of feline NOMs behave aggressively, Ki-67 index, together with other parameters, may contribute to prognostic assessment. Prospective studies on homogeneous populations are warranted to identify reliable threshold values for this marker

Hydroxytyrosol prevents metabolic impairment reducing hepatic inflammation and restoring duodenal integrity in a rat model of NAFLD.

The potential mechanisms of action of polyphenols in nonalcoholic fatty liver disease (NAFLD) are overlooked. Here, we evaluate the beneficial therapeutic effects of hydroxytyrosol (HT), the major metabolite of the oleuropein, in a nutritional model of insulin resistance (IR) and NAFLD by high-fat diet. Young male rats were divided into three groups receiving (1) standard diet (STD; 10.5% fat), (2) high-fat diet (HFD; 58.0% fat) and (3) HFD+HT (10 mg/kg/day by gavage). After 5 weeks, the oral glucose tolerance test was performed, and at 6th week, blood sample and tissues (liver and duodenum) were collected for following determinations. The HT-treated rats showed a marked reduction in serum AST, ALT and cholesterol and improved glucose tolerance and insulin sensitivity, reducing homeostasis model assessment index. HT significantly corrected the metabolic impairment induced by HFD, increasing hepatic peroxisome proliferator activated receptor PPAR-α and its downstream-regulated gene fibroblast growth factor 21, the phosphorylation of acetyl-CoA carboxylase and the mRNA carnitine palmitoyltransferase 1a. HT also reduced liver inflammation and nitrosative/oxidative stress decreasing the nitrosylation of proteins, reactive oxygen species production and lipid peroxidation. Moreover, HT restored intestinal barrier integrity and functions (fluorescein isothiocyanate-dextran permeability and mRNA zona occludens ZO-1). Our data demonstrate the beneficial effect of HT in the prevention of early inflammatory events responsible for the onset of IR and steatosis, reducing hepatic inflammation and nitrosative/oxidative stress and restoring glucose homeostasis and intestinal barrier integrity

First case of systemic fatal mycobacteriosis caused by Mycobacterium goodii in a pet Kenyan sand boa (Eryx colubrinus loveridgei)

Environmental nontuberculous mycobacteria species that are not members of the M. tuberculosis complex, are ordinary inhabitants of a wide variety of environmental reservoirs and their role in human and animal diseases has been fully recognized. Even if spontaneous mycobacterial infections have been reported in a wide variety of reptiles, this is the first report of systemic fatal mycobacteriosis sustained by Mycobacterium goodii in a pet reptile.  CASE PRESENTATION: An adult, wild caught (WC), male Kenyan sand boa (Eryx colubrinus loveridgei) age unknown, was presented for clinical examination due to decreased activity level, decreased appetite and diarrhea. Blood tests showed unreliable results. Coprologic exam showed a moderate to severe presence of flagellates. X rays and ultrasound showed moderate presence of air and faeces in the large intestine. The snake was hospitalized and oral metronidazole was chosen as antiprotozoal agent in association with subcutaneous warm fluids. The snake was discharged after 2 weeks therapy in good clinical condition. Faecal exam resulted negative. One month after, the snake was quickly hospitalized again because of a recrudescence of symptoms. Biochemistry showed severe increase of AST, ALT and biliary acids. Severe leucocytosis and moderate to severe anemia were highlighted. Ultrasound examination revealed a severe diffused alteration of the liver parenchyma and a fine needle aspiration was performed. The cytological diagnosis was mixed inflammation, with a numerous of unstained rod-shaped bacteria both inside macrophages and free in the sample. The snake's condition rapidly deteriorated and euthanasia was performed. The histology of the coelomic organs confirmed a systemic mycobacteriosis. Real-time PCR identified the mycobacteria as Mycobacterium goodii

Diagnosi ecografica transaddominale in un caso di teratoma maturo in un cavallo

This paper describes the finding of a testicular teratoma in an arabian horse with an abdominal undiscended testis. The neoplasm was identified by a preoperatory abdominal ultrasonography. It makes possible surgery approach planning and reduces the time of surgery and anesthesia. Also it was identified and described the typical aspect of testicular teratoma in according to the human ultrasonographic aspect of this lesion. The aim of this study was to suggest the preoperatory transabdominal and transrectal ultrasonography importance in criptorchyd horses for reduce the surgical complicance

Mature teratoma arising from an undescended testis in a horse: comparison between ultrasonographic and morphological features

Scant information is available on the ultrasonographic appearance of different testicular tumors in the stallion. Preoperative ultrasound imaging and gross and microscopic features of a testicular teratoma in a horse is described. An asymptomatic 4 years old cryptorchid Arabian horse was admitted for orchiectomy. Combined transabdominal and inguinal ultrasound examination revealed a large complex ovoid mass, containing both solid and cystic elements with internal echoes, located dorsally to the superficial inguinal ring. Two main hypoechoic cavities divided by a linear hyperechoic septum were evident, with a hyperechoic circular structure inside the lumen of the largest one. A tumor of the undescended testis was suspected. Ultrasound findings guided a provisional diagnosis of teratoma that was confirmed by histology following surgical removal of the tumor. Due to the correspondence between ultrasonographic and morphological features, trans-abdominal ultrasonography was conclusive in the preoperative planning