Maria Elena Turba

Use of combined conventional and real-time PCR to determine the epidemiology of feline haemoplasma infections in northern Italy.

Although knowledge of feline haemotropic mycoplasmas (haemoplasmas) has dramatically improved in recent years, some issues still remain to be elucidated. The aim of the current study was to evaluate the prevalence of feline haemoplasma infections in blood samples collected from cats in northern Italy. A convenience-sample of 307 cats (40 anaemic; 258 non-anaemic; nine with unknown haematocrit [HCT]) was investigated using polymerase chain reaction assays. Furthermore, the date of blood collection, signalment and clinicopathological data were retrospectively evaluated to assess predictors and risk factors for infection. Haemoplasma infections were highly prevalent in the sample investigated with an overall prevalence of 18.9% (95% confidence interval: 14.5–23.3%). The prevalence for the three feline haemoplasmas was 17.3% for ‘Candidatus Mycoplasma haemominutum’ (CMhm), 5.9% for Mycoplasma haemofelis (Mhf) and 1.3% for ‘Candidatus Mycoplasma turicensis’ (CMt). Feline immunodeficiency virus-positive status represented a risk factor for infection with an odds ratio of 4.19 (P=0.02). Moreover, a higher prevalence was observed in summer (odds ratio 1.78; P=0.04) which may be consistent with arthropod-borne disease transmission. Cats infected with Mhf showed significantly lower HCT (P=0.03), haemoglobin values (P=0.02) and red blood cell counts (P=0.04), lower mean corpuscular haemoglobin concentration (P<0.01) and higher white blood cell counts (P<0.01) when compared with non-infected cats.

Heat shock protein 70, heat shock protein 32, and vascular endothelial growth factor production and their effects on lipopolysaccharide-induced apoptosis in porcine aortic endothelial cells

Abstract Lipopolysaccharide (LPS) is a highly proactive molecule that causes in vivo a systemic inflammatory response syndrome and activates in vitro the inflammatory pathway in different cellular types, including endothelial cells (EC). Because the proinflammatory status could lead to EC injury and apoptosis, the expression of proinflammatory genes must be finely regulated through the induction of protective genes. This study aimed at determining whether an LPS exposure is effective in inducing apoptosis in primary cultures of porcine aortic endothelial cells and in stimulating heat shock protein (Hsp)70 and Hsp32 production as well as vascular endothelial growth factor (VEGF) secretion. Cells between third and eighth passage were exposed to 10 μg/mL LPS for 1, 7, 15, and 24 hours (time-course experiments) or to 1, 10, and 100 μg/mL LPS for 7 and 15 hours (dose-response experiments). Apoptosis was not affected by 1 μg/mL LPS but significantly increased in a dose-dependent manner with the highest LPS doses. Furthermore, apoptosis rate increased only till 15 hours of LPS exposure. LPS stimulated VEGF secretion in a dose-dependent manner; its effect became significant after 7 hours and reached a plateau after 15 hours. Both Hsp70 and Hsp32 expressions were induced by LPS in a dose-dependent manner after 7 hours. Subsequent studies were addressed to evaluate the protective role of Hsp32, Hsp70, and VEGF. Hemin, an Hsp32 inducer (5, 20, 50 μM), and recombinant VEGF (100 and 200 ng/mL), were added to the culture 2 hours before LPS (10 μg/mL for 24 hours); to induce Hsp70 expression, cells were heat shocked (42°C for 1 hour) 15 hours before LPS (10 μg/mL for 24 hours). Hemin exposure upregulated Hsp32 expression in a dose-dependent manner and protected cells against LPS-induced apoptosis. Heat shock (HS) stimulated Hsp70 expression but failed to reduce LPS-induced apoptosis; VEGF addition did not protect cells against LPS-induced apoptosis at any dose tested. Nevertheless, when treatments were associated, a reduction of LPS-induced apoptosis was always observed; the reduction was maximal when all the treatments (HS + Hemin + VEGF) were associated. In conclusion, this study demonstrates that LPS is effective in evoking “the heat shock response” with an increase of nonspecific protective molecules (namely Hsp70 and Hsp32) and of VEGF, a specific EC growth factor. The protective role of Hsp32 was also demonstrated. Further investigations are required to clarify the synergic effect of Hsp32, Hsp70, and VEGF, thus elucidating the possible interaction between these molecules.

Intravitreal NGF administration counteracts retina degeneration after permanent carotid artery occlusion in rat

BackgroundThe neurotrophin nerve growth factor (NGF) is produced by different cell types in the anterior and posterior eye, exerting a neuroprotective role in the adult life. The visual system is highly sensitive to NGF and the retina and optic nerve provides suitable subjects for the study of central nervous system degeneration. The model of bilateral carotid occlusion (two-vessel occlusion, 2VO) is a well-established model for chronic brain hypoperfusion leading to brain capillary pathology, to retina and optic nerve degeneration. In order to study if a single intravitreal injection of NGF protects the retina and the optic nerve from degeneration during systemic circulatory diseases, we investigated morphological and molecular changes occurring in the retina and optic nerve of adult rats at different time-points (8, 30 and 75 days) after bilateral carotid occlusion.ResultsWe demonstrated that a single intravitreal injection of NGF (5 μg/3 μl performed 24 hours after 2VO ligation) has a long-lasting protective effect on retina and optic nerve degeneration. NGF counteracts retinal ganglion cells degeneration by early affecting Bax/Bcl-2 balance- and c-jun- expression (at 8 days after 2VO). A single intravitreal NGF injection regulates the demyelination/remyelination balance after ischemic injury in the optic nerve toward remyelination (at 75 days after 2VO), as indicated by the MBP expression regulation, thus preventing optic nerve atrophy and ganglion cells degeneration. At 8 days, NGF does not modify 2VO-induced alteration in VEFG and related receptors mRNA expression.ConclusionThe protective effect of exogenous NGF during this systemic circulatory disease seems to occur also by strengthening the effect of endogenous NGF, the synthesis of which is increased by vascular defect and also by the mechanical lesion associated with NGF or even vehicle intraocular delivery.

Gelatinases, endonuclease and Vascular Endothelial Growth Factor during development and regression of swine luteal tissue

BackgroundThe development and regression of corpus luteum (CL) is characterized by an intense angiogenesis and angioregression accompanied by luteal tissue and extracellular matrix (ECM) remodelling. Vascular Endothelial Growth Factor (VEGF) is the main regulator of angiogenesis, promoting endothelial cell mitosis and differentiation. After the formation of neovascular tubes, the remodelling of ECM is essential for the correct development of CL, particularly by the action of specific class of proteolytic enzymes known as matrix metalloproteinases (MMPs). During luteal regression, characterized by an apoptotic process and successively by an intense ECM and luteal degradation, the activation of Ca++/Mg++-dependent endonucleases and MMPs activity are required. The levels of expression and activity of VEGF, MMP-2 and -9, and Ca++/Mg++-dependent endonucleases throughout the oestrous cycle and at pregnancy were analyzed.ResultsDifferent patterns of VEGF, MMPs and Ca++/Mg++-dependent endonuclease were observed in swine CL during different luteal phases and at pregnancy. Immediately after ovulation, the highest levels of VEGF mRNA/protein and MMP-9 activity were detected. On days 5–14 after ovulation, VEGF expression and MMP-2 and -9 activities are at basal levels, while Ca++/Mg++-dependent endonuclease levels increased significantly in relation to day 1. Only at luteolysis (day 17), Ca++/Mg++-dependent endonuclease and MMP-2 spontaneous activity increased significantly. At pregnancy, high levels of MMP-9 and VEGF were observed.ConclusionOur findings, obtained from a precisely controlled in vivo model of CL development and regression, allow us to determine relationships among VEGF, MMPs and endonucleases during angiogenesis and angioregression. Thus, CL provides a very interesting model for studying factors involved in vascular remodelling.

Prognostic significance of Kit receptor tyrosine kinase dysregulations in feline cutaneous mast cell tumors.

Feline cutaneous mast cell tumors (FeCMCTs) are characterized by variable biological behavior. Development of multiple nodules and potential visceral involvement, along with inconsistency of conventional prognostic aids, justify uncertainty in differentiating benign from malignant forms. c-Kit proto-oncogene activating mutations have been reported in feline mast cell tumors (MCTs), but their prognostic relevance was not investigated. This study was performed on FeCMCTs with variable clinical outcome to assess whether Kit cytoplasmic immunohistochemical labeling can be regarded as indicative of c-Kit mutations and to evaluate the relationship between Kit dysregulation and survival. Twenty-four cats diagnosed with a primary cutaneous MCT were enrolled. Kit immunohistochemical pattern and c-Kit (exons 8, 9, 11) mutational status were assessed in 34 tumor samples. Risk factors affecting survival were a number of mitoses greater than 5 per 10 HPFs (P = .017) and cytoplasmic Kit labeling (P = .045). Increased mitotic activity was associated with Kit cytoplasmic expression (P = .01). c-Kit encoding mutations were present in 19 (56%) tumors (exon 8, 19%; exon 9, 71%; exon 11, 10%), however, they were not significantly related to protein expression and they had no influence on prognosis. Additionally, in 6 of 9 (67%) cats, multiple nodules from the same cat had different mutational statuses. Mutations in the fifth immunoglobulin-like domain of Kit occur frequently in FeCMCT, but they are variably associated with aberrant protein expression and do not appear to be strictly correlated with biological behavior. These findings need to be confirmed in larger series, and exploration of further genomic regions of c-Kit is warranted.

Detection of Wolbachia DNA in Blood for Diagnosing Filaria-Associated Syndromes in Cats

ABSTRACT A fundamental role for the endosymbiotic bacteria Wolbachia pipientis in the pathogenesis of Dirofilaria immitis infections has emerged in recent years. Diagnostic opportunities arising from this breakthrough have not yet been fully exploited. This study was aimed at developing conventional and real-time PCR assays to carry out a molecular survey in a convenience sample of cats living in an area where D. immitis is endemic and to evaluate the detection of bacterial DNA in blood as a surrogate assay for diagnosing filaria-associated syndromes in cats. COI and FtsZ loci were used as targets for D. immitis and Wolbachia PCR assays, respectively, and real-time TaqMan PCR assays were used only for Wolbachia. A convenience sample of 307 disease-affected or healthy cats examined at a University facility were PCR tested, and their medical records were investigated. Conventional nested PCR for Wolbachia amplified the endosymbionts of both D. immitis and D. repens, while real-time PCR was highly specific only for the former. Observed prevalences of 0.3 and 10.4% were found using conventional nested PCR assays for D. immitis and real-time PCR for Wolbachia, respectively. Similar prevalences were established using the Wolbachia nested PCR (98% concordance with real-time PCR). The group of Wolbachia-positive samples had a significantly higher proportion of subjects with respiratory signs (29.0% versus 9.7%; P = 0.002). The findings of this study indicate that a highly sensitive PCR assay can be used to detect the Wolbachia organism in the peripheral blood of cats with respiratory signs.

Reduced diversity of immunoglobulin and T-cell receptor gene rearrangements in chronic inflammatory gastrointestinal diseases in dogs

Inflammatory bowel disease has a multifactorial etiology in dogs as it does in humans. Evidence has been accumulated showing an abnormal response of the immune system, mostly represented by lymphocyte infiltration in the lamina propria of the gastrointestinal tract and in the epithelium, likely driven by chronic antigenic stimulation against luminal microorganisms. A relevant role is also ascribed to the genetic predisposition typical of some canine breeds. The role of chronic antigenic stimulation is still under debate. It may be responsible for selective pressure on the lymphoid population, favouring the emergence of some lymphocyte clones. This cross-sectional study is aimed at investigating the immunoglobulin and T-cell receptor gene rearrangements in a group of dogs affected by inflammatory bowel disease. The database of a referral Veterinary Laboratory was investigated. Based upon the histological evaluation of the bioptic samples collected during endoscopy, 54 canine cases met the WSAVA criteria for diagnosing IBD and were included in the study. The histological slides were retrieved and the gDNA was purified using protocols for formalin-fixed tissue. The gDNA was PCR amplified using fluorescent-labelled primers specific for canine immunoglobulin and T-cell receptor gene rearrangements; the PCR products were analysed with fragment analysis by means of capillary electrophoresis on an automatic sequencer (GeneScanning). In 47/54 (87.3%) cases, it was possible to amplify the gDNA. Twenty-one patients out of 47 (44.7%) showed polyclonal patterns in both the immunoglobulin and the T-cell receptors, 18/47 (38.3%) showed at least one oligoclonal pattern without monoclonal ones while 8/47 (17.0%) cases showed an Ig (7/47; 14.9%) or TCR (1/47: 2.1%) monoclonal pattern. These findings indicate that reduced diversity of the immunoglobulin and T-cell receptor repertoire occurs in canine inflammatory bowel disease. The reduced diversity correlated significantly with the severity of the histological lesions and carried a significantly increased risk of death. Beside its possible role as a reliable ancillary assay, immunoglobulin and T-cell receptor GeneScanning analysis points to the possible role of aberrant chronic antigenic stimulation, leading to clonal expansion of certain lymphocyte subsets in the pathogenesis of canine IBD.

Cytological, immunohistochemical and mutational analysis of a gastric gastrointestinal stromal tumour in a cat.

Gastrointestinal stromal tumours (GISTs) represent a distinctive group of primary mesenchymal tumours of the gastrointestinal tract identified immunohistochemically by expression of CD117. A 10-year-old neutered female domestic shorthair cat with a history of recurrent vomiting was examined. The presence of a gastric mass was recognized and a laparotomy was performed. Cytological examination was consistent with a low-grade malignant mesenchymal tumour and histopathological investigation suggested myogenic differentiation of tumour cells. The diagnosis of GIST was confirmed by immunohistochemical expression of CD117. Sequence analysis of the KIT gene identified a deletion in exon 11. The same mutation is found often in human GISTs.

Recruited leukocytes and local synthesis account for increased matrix metalloproteinase-9 activity in cerebrospinal fluid of dogs with central nervous system neoplasm

Matrix metalloproteinases (MMP) 2 and 9 are enzymes known to degrade several protein components of the extracellular matrix. In humans, increased concentrations of these enzymes have been demonstrated in the cerebrospinal fluid (CSF) of subjects affected by many neurological conditions including brain tumours; nevertheless comparative data in dogs are completely lacking. Aim of this study was to investigate these molecules in CSF of dogs diagnosed with CNS neurological diseases. Higher activity of MMP 2 and 9 was revealed in dogs with space occupying lesions of likely neoplastic origin in comparison to dogs with idiopathic epilepsy. Statistical modelling reveals that increased MMP 9 activity is mainly due to leukocytes recruitment and local synthesis.

Real-time detection of the mutation responsible for progressive rod―cone degeneration in Labrador Retriever dogs using locked nucleic acid TaqMan probes

Progressive rod-cone degeneration (prcd) is a late onset, autosomal recessive, inherited disease in dogs caused by a G > A substitution in the PRCD locus, which has been reported in more than 18 breeds, including Labrador Retriever dogs. In this study, a real-time polymerase chain reaction (PCR) assay, exploiting the features of locked nucleic acid (LNA) fluorescent-labeled probes, was developed to genotype the sequence variants responsible for the disease. Two Labrador Retrievers were diagnosed with prcd by ophthalmological examination performed by a panelist of the Italian hereditary eye disease control program. The 2 dogs, as well as 8 related and 14 unrelated Labrador Retrievers, were genotyped with both direct sequencing of the disease locus and real-time LNA TaqMan PCR assay. Even though the region surrounding the mutation was predicted to be highly structured, making probe annealing difficult, the real-time PCR assay allowed researchers to correctly genotype the dogs in all cases with a sensitivity threshold of 4 ng/reaction of genomic DNA. A real-time PCR assay will allow a high-throughput analysis of a larger cohort of dogs, thereby enabling researchers to investigate the prevalence of the mutated allele in the affected breeds.