Porcine Reproductive and Respiratory Syndrome (PRRS) and CD163 Resistance Polymorphic Markers: What Is the Scenario in Naturally Infected Pig Livestock in Central Italy?

Porcine Reproductive and Respiratory Syndrome (PRRS) caused by the PRRS virus affects farmed pigs worldwide, causing direct and indirect losses. The most severe manifestations of PRRS infection are observed in piglets and pregnant sows. The clinical outcome of the infection depends on the PRRSV strain's virulence, the pregnancy state of the female, environmental factors, the presence of protective antibodies due to previous infections, and the host's genetic susceptibility. The latter aspect was investigated in this study, in particular, evaluating the most significant polymorphisms (SNPs) of the CD163 gene in slaughtered pigs reared in Central Italy. Total RNAs were extracted from 377 swine samples and subjected to RT-PCR targeted to the CD163 gene, followed by sequencing analysis. Contextually, the viral RNA was detected by RT-qPCR in order to phenotypically categorize animals into infected and not infected. In particular, 36 haplotypes were found, and their frequencies ranged from 0.13% to 35.15%. There were 62 resulting genotypes, three of which were associated with a putative resistance to the disease. Both the haplotypes and genotypes were inferred by PHASE v.2.1 software. To the best of our knowledge, this type of investigation was conducted for the first time on pig livestock distributed in different regions of Central Italy. Thus, the obtained findings may be considered very important since they add useful information about swine genetic background in relation to PRRS infection, from the perspective of adopting Marker-Assisted Selection (MAS) as a possible and alternative strategy to control this still widespread disease.

Genetic Characterization of Small Ruminant Lentiviruses (SRLVs) Circulating in Naturally Infected Sheep in Central Italy.

Small ruminant lentiviruses (SRLVs) represent a very heterogeneous group of ss-RNA viruses that infect sheep and goats worldwide. They cause important, deleterious effects on animal production and limit the animal trade. SRLVs show a high genetic variability due to high mutation rate and frequent recombination events. Indeed, five genotypes (A-E) and several subtypes have been detected. The aim of this work was to genetically characterize SRLVs circulating in central Italy. On this basis, a phylogenetic study on the gag-pol genetic region of 133 sheep, collected from 19 naturally infected flocks, was conducted. In addition, to evaluate the frequency of mutation and the selective pressure on this region, a WebLogo 3 analysis was performed, and the dN/dS ratio was computed. The results showed that 26 samples out of 133 were clustered in genotype A and 106 samples belonged to genotype B, as follows: A9 (n = 8), A11 (n = 10), A24 (n = 7), B1 (n = 2), B2 (n = 59), and B3 (n = 45). No recombination events were found. Mutations were localized mainly in the VR-2 region, and the dN/dS ratio of 0.028 indicated the existence of purifying selection. Since the genetic diversity of SRLVs could make serological identification difficult, it is important to perform molecular characterization to ensure a more reliable diagnosis, to maintain flock health status, and for the application of local and national control programs.

First Survey of SNPs in TMEM154, TLR9, MYD88 and CCR5 Genes in Sheep Reared in Italy and Their Association with Resistance to SRLVs Infection.

Maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV), referred to as small ruminant lentiviruses (SRLVs), belong to the genus Lentivirus of the Retroviridae family. SRLVs infect both sheep and goats, causing significant economic losses and animal welfare damage. Recent findings suggest an association between serological status and allelic variants of different genes such as TMEM154, TLR9, MYD88 and CCR5. The aim of this work was to investigate the role of specific polymorphisms of these genes in SRLVs infection in some sheep flocks in Italy. In addition to those already known, novel variants in the TMEM154 (P7H, I74V, I105V) gene were detected in this study. The risk of infection was determined finding an association between the serological status and polymorphisms P7H, E35K, N70I, I74V, I105V of TMEM154, R447Q, A462S and G520R in TLR9 gene, H176H* and K190K* in MYD88 genes, while no statistical association was observed for the 4-bp deletion of the CCR5 gene. Since no vaccines or treatments have been developed, a genetically based approach could be an innovative strategy to prevent and to control SRLVs infection. Our findings are an important starting point in order to define the genetic resistance profile towards SRLVs infection.

Frequencies Evaluation of ß-Casein Gene Polymorphisms in Dairy Cows Reared in Central Italy.

The majority of proteins in cow's milk are caseins, which occur in four groups (α-s1, α-s2, ß, and k) encoded by different genes (CSN1S1, CSN1S2, CSN2, and CSN3, respectively). In this study, we focused on the ß-casein allele variants A1 and A2 due to their influence on milk's technological characteristics and human health. Digestion of the ß-casein variant A1 leads to the formation of ß-casomorphin 7 (BCM-7), a bioactive peptide that has been suggested to be a possible cause of various human diseases and associated with low milk digestibility. The potential negative role of the ß-casein variant A1 in human health has stimulated the planning of cattle breeding programs based on genetic selection to increase the frequency of the A2 variant, which is associated with increased milk digestibility. The aim of this work was to evaluate the frequencies of the different ß-casein variants in Italian Holstein Friesian dairy cows from cattle farms located in central Italy to select a population of A2 homozygous animals. ß-casein genotypes were identified by evaluating the presence of single nucleotide polymorphisms (SNPs) of the CSN2 gene using PCR and sequencing analysis. The frequency of the desirable ß-casein variant A2 in the studied bovine population was 0.61. The frequency of the undesirable A1 variant in the studied bovine population was 0.30. The frequency of the A2 allele was higher than expected for the breed; therefore, genetic selection for the A2 variant in these animals could be achieved in a fairly short time using A2 homozygous bulls.

Microbial, chemical-physical, rheological and organoleptic characterisation of roe deer (Capreolus capreolus) salami.

Game meat and related products are important in the promotion of local economies and rural areas. Microbiological, chemical-physical, rheological and sensory characteristics of fermented meat products (salami) made by different percentages of pork and hunted roe-deer (Capreolus capreolus) meat were evaluated. The microbiological determination indicated that the products are safe to eat, as neither Listeria monocytogenes nor Salmonella spp. was isolated from the samples. The hygienic adequacy of the process was guaranteed, as there was below 3 log CFU/g of Enterobacteriaceae level in the final products. The proximal composition analyses showed lower lipid levels in comparison to pork salami. The difference in chemical composition affects the rheological and sensory traits of the final products; the products were harder and with higher gumminess when 50% of roe-deer meat was used. Game meat flavour and odour increased with the increasing percentage of roe-deer meat.

Advanced therapies in patients with congenital heart disease-related pulmonary arterial hypertension: results from a long-term, single center, real-world follow-up.

Pulmonary arterial hypertension (PAH) is a common finding in patients with congenital heart disease (CHD), and has relevant prognostic implications. The recent introduction of advanced therapies (AT) considerably improved the clinical outcome of these patients, but real-world data are still lacking. We aimed at reporting the results of a long-term follow-up of CHD patients with PAH undergoing AT, followed at a tertiary Center during the two last decades. The study population included a total of 34 patients with an established diagnosis of CHD-related PAH. In addition to conventional treatment, 97% of patients started AT during the follow-up. Over a median follow-up of 9 [3-31] years, 11 (32.4%) patients died: 7 of them were affected by Eisenmenger syndrome and the majority of patients were in NYHA class ≥3 at the time of death. Among the 23 patients who were alive at the last follow-up, the majority were in NYHA class I-II. Oxygen saturation and 6-min walking distance improved in all subjects within the first 6 months after starting of AT. One patient with ventricular septum defect and high pulmonary resistances was successfully treated with AT to lower resistances and underwent defect closure. A good clinical outcome was also observed in the subset (n = 8) with Down syndrome. The results of this real-world experience suggest that, despite a relatively high mortality rate mostly related to late commencement of AT, the clinical outcome of subjects with CHD-related PAH undergoing AT are characterized by a good quality of life and clinical improvement in most patients.

Risk of recurrence in patients with pulmonary embolism: predictive role of D-dimer and of residual perfusion defects on lung scintigraphy.

The stratification of recurrence risk after a first episode of venous thromboembolism (VTE) is an important topic of research, especially in patients with pulmonary embolism (PE). Elevated D-dimer levels and residual vein obstruction (RVO) at compression ultrasonography have been studied as predictors of recurrence after withdrawing oral anticoagulant treatment (OAT). It is still unknown if residual perfusion defects (PD) on lung scintigraphy are related to recurrent PE. In the present study, we evaluated the association of PD with PE recurrence. The relationship between PD, elevated D-dimer levels, and RVO was also investigated. We prospectively followed 236 consecutive patients who survived a first episode of objectively confirmed PE, with or without deep-vein thrombosis. After at least three months of OAT, treatment was withdrawn in 139 patients. D-dimer levels were evaluated at one month of OAT withdrawal, RVO was measured, and perfusion lung scan (P-scan) was performed to evaluate PD. During follow-up, 20 patients experienced a recurrent episode of VTE. Elevated D-dimer levels were significantly associated with VTE recurrence, (p=0.003). RVO was present in 22% of the patients with recurrence and in 7.5% of those without (p=0.07). No significant association was found between PD >10% and VTE recurrence, D-dimer, or RVO. In conclusion, we confirmed the positive predictive value of elevated D-dimer levels for recurrent VTE. Residual PD on lung scintigraphy are neither predictive of recurrence nor related to D-dimer levels or RVO.

Prevalence and clinical characteristics of adult patients with congenital heart disease in Tuscany.

AIMS: The clinical features of the adult population with congenital heart disease (CHD) are still not well characterized, particularly in the subset with more severe lesions. We report the data collected in the National Association of Hospital Cardiologists Toscana grown-up CHD (GUCH) registry over its first 8-month enrolment period. METHODS: The Registry included consecutive patients aged more than 16 years with a documented diagnosis of CHD, enrolled in seven different Tuscan hospitals using a web-based electronic form. Severe CHD was defined as cyanotic CHD, or acyanotic lesion with significant haemodynamic impact requiring surgical and/or percutaneous correction. RESULTS: Between November 2009 and June 2010 a total of 1641 patients (mean age 41.8 ±â€Š19.3 years, 52.2% women) were enrolled. Atrial septal defect was the most common lesion, accounting for more than one-third of cases. Atrial and ventricular septal defects together accounted for about half of all CHDs. Nearly one-third of patients had New York Heart Association (NYHA) class 2 or more. A history of recurrent arrhythmias was reported in 15% of cases, and 12% of patients were on oral anticoagulants at the time of enrolment. The prevalence of pulmonary hypertension was 6%, and the prevalence of Eisenmenger syndrome was 1.2%. Severe CHD was present in 42% of patients. Younger age, higher NYHA class, male sex, and the need for oral anticoagulants were the only independent predictors of severe CHD. CONCLUSION: Information about the clinical characteristics and the CHD type distribution of a sample of Tuscan GUCH population was provided. Severe CHD accounts for about 40% of all CHDs in this population. CHD severity is associated with younger age, male gender, worse NYHA class, and need for oral anticoagulation.

Incidence of recurrent venous thromboembolism and of chronic thromboembolic pulmonary hypertension in patients after a first episode of pulmonary embolism.

After a first episode of pulmonary embolism (PE), two major problems need to be considered: risk of recurrence when anticoagulation is stopped, and risk of chronic thromboembolic pulmonary hypertension (CTPH). We followed prospectively consecutive patients who survived a first episode of PE, with or without deep vein thrombosis, to assess the incidence of venous thromboembolism (VTE) recurrences and of symptomatic and asymptomatic CTPH. After 3-6 months of oral anticoagulant therapy (OAT) patients underwent transthoracic echocardiography for measuring transtricuspid (rV-rA) gradient. When rV-rA gradient was >35 mmHg further evaluations were performed to rule in or out CTPH. During follow-up patients who developed persistent dyspnea were re-evaluated. In patients who underwent OAT withdrawal D-dimer (DD), prothrombin fragment 1 + 2 (F1 + 2), and thrombophilia were evaluated one month after warfarin discontinuation. Overall, 239 patients, 118 males, median age 59(16-89) years, were followed up for a median time of 36(9-192) months. Nine patients had rV-rA gradient >30 mmHg and ≤35 mmHg, and one of 37 mmHg. Among patients with normal rV-rA gradient, one developed persistent dyspnea 55 months after the first event and CPTH was confirmed. Among 206 patients who stopped OAT, 23(11.2%) had VTE recurrence, 11 PE(48%). Elevated DD and F1 + 2 levels after stopping OAT were significantly associated with recurrence. None of patients with recurrent VTE had elevated rV-rA gradient. In our series the incidence of CTPH after a first episode of PE was 0.4%. VTE recurrence and elevated DD and F1 + 2 levels seemed not to be related to the development of CTPH.

Fibrin resistance to lysis in patients with pulmonary hypertension other than thromboembolic.

RATIONALE: Reportedly, fibrin isolated from patients with chronic thromboembolic pulmonary hypertension (CTEPH) is resistant to lysis. Persistence of regions within the fibrin beta chain, which mediate cell signaling and migration, could trigger the organization of pulmonary thromboemboli into chronic intravascular scars. OBJECTIVES: Ascertain whether fibrin resistance to lysis occurs in patients with pulmonary hypertension (PAH) other than CTEPH, and in those with prior pulmonary embolism (PE) and no pulmonary hypertension. METHODS: Fibrinogen was purified from 96 subjects (17 with CTEPH, 14 with PAH, 39 with prior PE, and 26 healthy control subjects) and exposed to thrombin to obtain fibrin clots. Plasmin-mediated cleavage of fibrin beta chain was assessed hourly over a 6-hour period by polyacrylamide gel electrophoresis. Fibrin band intensity was measured by densitometry of stained gels. Data were normalized to the band intensity of the undigested protein. MEASUREMENTS AND MAIN RESULTS: By 1 hour of digestion, fibrin band intensity had decreased by a median of 25% (interquartile range [IQR], 20 to 27%) in control subjects, and by 15% (IQR, 11 to 18%) in patients with prior PE (P < 0.0001). The 1-hour median reduction in band intensity was 2% (IQR, 1 to 3%) in CTEPH, and 4% (IQR, 2 to 7%) in PAH (P < 0.0001 vs. control subjects and PE). The decline in fibrin band intensity remained significantly different among the four groups up to 6 hours (P < 0.0001). CONCLUSIONS: Fibrin resistance to lysis occurs in pulmonary hypertension other than CTEPH and, to a smaller extent, in patients with prior PE and no pulmonary hypertension.

Rheolytic thrombectomy in patients with massive and submassive acute pulmonary embolism.

OBJECTIVES: To appraise the impact of AngioJet rheolytic thrombectomy (RT) on angiographic and clinical endpoints in patients with acute pulmonary embolism (PE). BACKGROUND: The management of patients with acute PE and hemodynamic compromise, based mainly on anticoagulant and thrombolytic therapies, is challenging and still suboptimal in many patients. In such a setting, mechanical removal of thrombus from pulmonary circulation holds the promise of significant clinical benefits, albeit remains under debate. METHODS: We retrospectively report on 51 patients referred to our catheterization laboratory and treated with AngioJet RT. Patients were classified according to the degree of hemodynamic compromise (shock, hypotension, and right ventricular dysfunction) to explore thoroughly the degree of angiographic pulmonary involvement (angiographic massive PE was defined as the presence of a Miller index >or= 17) and the impact on angiographic (obstruction, perfusion, and Miller indexes) and clinical (all-cause death, recurrence of PE, bleeding, renal failure, and severe thrombocytopenia) endpoints of AngioJet RT. RESULTS: Angiographic massive PE was present in all patients with shock, whereas patients with right ventricular dysfunction and hypotension showed a similar substantial pulmonary vascular bed involvement. Technical success was obtained in 92.2% of patients, with a significant improvement in obstruction, perfusion and Miller indexes in each subgroup (all P < 0.0001). Four patients reported major bleedings and eight (15.7%) died in-hospital. Laboratory experience was significantly associated to a lower rate of major bleedings. All survivors were alive at long-term follow-up (35.5 +/- 21.7 months) except three who expired due to cancer and acute myocardial infarction. CONCLUSIONS: In experienced hands AngioJet RT can be operated safely and effectively in most patients with acute PE, either massive or submassive, and substantial involvement of pulmonary vascular bed.

Exercise Doppler echocardiography identifies preclinic asymptomatic pulmonary hypertension in systemic sclerosis.

In systemic sclerosis (SSc), the involvement of the interstitium or vascular system of the lung may lead to pulmonary arterial hypertension (PAH). PAH is often asymptomatic or oligosymptomatic in early SSc and, when it becomes symptomatic, pulmonary vascular system is already damaged. Exercise echocardiography (ex-echo), measuring pulmonary artery pressure (PAP) during exercise and allowing to differentiate physiologic from altered PAP responses, may identify subclinical PAH. Our aims were (a) to evaluate by ex-echo the change of PAP in patients with SSc without lung involvement; and (b) to correlate PAP during exercise (ex-PAP) values to clinical and biohumoral parameters of PAH. Twenty-seven patients with limited SSc (ISSc) without interstitial lung involvement were studied. Patients underwent rest and exercise two-dimensional and Doppler echocardiography by supine cycloergometer. Systolic PAP was calculated using the maximum systolic velocity of the tricuspid regurgitant jet at rest and during exercise values of systolic PAP exceeding 40 mmHg at ex-echo were considered as abnormal, and biohumoral markers potentially related to PAH were assessed. Eighteen of 27 SSc patients presented an ex-PAP > 40 mmHg, while in 9 of 27 patients ex-PAP values remained < 40 mmHg (48.8 +/- 4.5 mmHg versus 36.2 +/- 3.1 mmHg; P < 0.001). Other echocardiographic and ergometric parameters, clinical tests, and biohumoral markers were not different in the two groups. Ex-PAP significantly correlated with D-dimer (P = 0.0125; r2 = 0.2029). Ex-echo identifies a cluster of SSc patients with subclinical PAH that may develop PAH. This group should be followed up and may be considered for specific therapies to prevent disease evolution.