Rapid detection and quantification of plasmid-mediated colistin resistance genes (mcr-1 to mcr-5) by real-time PCR in bacterial and environmental samples.

AIM: The aim of the study was to validate a rapid method to detect and quantify colistin resistance genes (mcr-1 to mcr-5) by real-time polymerase chain reaction (RT-PCR) in diverse matrices. METHODS AND RESULTS: The detection limit of two newly designed SYBR Green real-time PCR assays for mcr-4 and mcr-5 and of previously published protocols for mcr-1 to mcr-3 was assessed using serial dilutions of reference strains. The assays could detect all five mcr genes with the lower limit of 102 copy numbers. Escherichia coli isolates (n = 1062) and environmental samples (n = 93) were tested for the presence of mcr genes. The assays enabled the detection of colistin resistance genes both in bacterial isolates and in complex environmental samples. CONCLUSIONS: This method represents a set of sensitive, rapid and effective assays for the screening of colistin resistance directly from the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Colistin is an antimicrobial commonly used in animals and has recently emerged as a last-resort treatment in humans. Plasmid-mediated mcr genes confer resistance to colistin and represent a major threat for public health since they can be easily disseminated through horizontal gene transfer. The rapid and sensitive detection of mcr genes is of utmost necessity.

At-vessel mortality of skates (Rajidae) taken in coastal fisheries and evidence of longer-term survival.

Data on the vigour and at-vessel mortality (AVM) of 6798 skates (comprising Raja clavata n = 6295; R. brachyura n = 208; R. undulata n = 185, R. montagui n = 98 and R. microocellata n = 12) captured by commercial fishing vessels in the inshore waters of the southern North Sea and English Channel were recorded. AVM in longline fisheries averaged 0·44% across five vessels (0-1·47%), although skates were usually unhooked manually and did not usually pass through a bait-stripper. AVM in otter trawls averaged 0·76% (0-2·35%), from four vessels fishing with tow durations of <1·5 h (southern North Sea) or 1-4 h (English Channel). No AVM was noted for skates taken as a by-catch in drift trammel nets (soak times <4 h). Anchored tangle nets resulted in an overall AVM of 2·0-2·7%, but increased from 1·47% (13-28 h soak time) to 6·16% (42-53 h soak time). There were significant differences in the vigour of skates between gears, with R. clavata caught by longline and tangle nets in better condition than those captured by otter trawl or drift trammel net. Similarly, R. undulata caught by tangle net were in better condition than those caught by otter trawl. The vigour of R. undulata was also found to be higher than other skate species for both trawl and tangle net. In total, 5283 skates were tagged with Petersen discs and released, with recapture rates for the various combinations of vessel and gear ranging up to 24·8% for R. clavata. Whilst confirming a degree of post-release survival, quantitative estimates of post-release mortality for skates remain unknown.

Antimicrobial resistance and class 1 and 2 integrons in Escherichia coli from meat turkeys in Northern Italy.

This study is aimed at determining the antimicrobial resistance (AMR) and the presence of class 1 and 2 integrons in 48 avian pathogenic Escherichia coli (APEC) strains isolated from meat turkeys during three sequential production cycles. Thirty avian faecal E. coli (AFEC) strains from the first cycle were also analysed. Strains were tested for AMR against 25 antimicrobials by disk diffusion test and were screened for the presence of integrons and associated gene cassettes by polymerase chain reaction followed by sequencing. Genetic relatedness of isolates was established by pulsed-field gel electrophoresis. High levels of resistance were detected to tetracyclines, penicillins and sulphonamides in APEC and AFEC. Resistance to aminoglycosides, fluoroquinolones, cephalosporins and phenicols was variable, based on the antimicrobial drug and the isolate (APEC vs. AFEC). Full susceptibility to colistin was detected. Multidrug resistance of up to seven antimicrobial classes was exhibited by APEC (93.8%) and AFEC (100%). Nearly 44% of strains tested positive for class 1 and/or class 2 integrons containing the dfrA, aadA and sat2 genes, alone or in combination, coding for streptomycin/spectinomycin, trimethoprim and streptothricin resistance, respectively. The estX and orfF genes of unknown function were also detected. A significant association was found between the presence of integrons and the resistance to aminoglycosides and potentiated sulphonamides. The results of this study showed that AMR, multidrug resistance and class 1 and 2 integrons are widespread among pathogenic and commensal E. coli from Italian turkeys. More attention should be addressed to limit the use of antimicrobials in turkeys and the AMR of turkey E. coli.

Enrofloxacin against Escherichia coli in turkeys: which treatment scheme is effective?

The efficacy of enrofloxacin (ENRO) was evaluated against multidrug-resistant avian pathogenic Escherichia coli correlating the minimum inhibitory concentrations (MIC) of 235 E. coli field strains with its pharmacokinetics (PK) in 50 healthy turkeys (5 groups) with a PK/pharmacodynamic approach. The treatments were as follows: a) single oral gavage and b) single subcutaneous (SC) treatment at the recommended dose of 10 mg/kg; c) single oral gavage, d) 5 d of 10-h pulsed water medication, and e) 5 d of 24-h continuous water medication at the doubled dose of 20 mg/kg. Blood samples were collected at established times over 24 h. Plasma was analyzed using a liquid chromatography tandem mass spectrometry method that was validated in house. A monocompartmental and a noncompartmental model were applied to the data to obtain the PK results. After gavage administration, the mean maximum concentration Cmax/MIC50 and area under the curve AUC0-24/MIC50 ratios were, respectively, 3.07 ± 0.62 and 7.01 ± 1.03 and 25.48 ± 3.04 and 57.2 ± 3.73 for the 10 and 20 mg/kg doses, respectively. After SC administration of 10 mg/kg, Cmax/MIC50 and AUC0-24/MIC50 ratios were 3.45 ± 0.75 and 33.96 ± 7.46, respectively. After the administration of 10-h pulsed or 24-h continuous medicated water at 20 mg/kg, lower values of Cmax/MIC50 (10-h pulsed: 3.45 ± 0.7; 24-h continuous: 3.05 ± 0.48) and AUC0-24/MIC50 (10-h pulsed: 42.42 ± 6.17; 24-h continuous: 53.32 ± 5.55) were obtained. Based on these results, the European Union-recommended dosage of 10 mg/kg seems ineffective to achieve adequate drug plasma concentrations and even the 20 mg/kg by 10 h pulsed or continuous medicated water administration did not reach completely efficacious concentrations in plasma against colibacillosis. Although the results obtained were not completely encouraging, the medicated water should preferably be provided continuously. To conclude about the efficacy of ENRO treatment against colibacillosis, target tissue concentration should be extensively considered.

Pharmacokinetic/pharmacodynamic evaluation of the efficacy of flumequine in treating colibacillosis in turkeys.

Flumequine (FLU) is used in the treatment of systemic bacterial infections in poultry, including colibacillosis, which is a common disease in turkeys. The pharmacokinetic (PK) behavior of FLU administered to 32 healthy turkeys as an oral bolus via gavage or as 10-h pulsed administration in drinking water were compared, using the authorized dose of 15 mg/kg and the double dose of 30 mg/kg. The minimum inhibitory concentrations (MIC) of 235 Escherichia coli field strains isolated from poultry were determined for pharmacodynamics (PD) to develop a PK/PD model. Blood samples were collected at established times over 24 h, and the obtained plasma was analyzed using a liquid chromatography tandem mass spectrometry method that was validated in-house. A monocompartmental model and a noncompartmental model were applied to the data to obtain the PK results. For both types of administration and both dosages, the ratios of the maximum concentration (Cmax)/MIC50 and the area under the plasma concentration-time curve (AUC)/MIC50 achieved were considerably lower than the fluoroquinolone breakpoints usually adopted for efficacy. The Cmax/MIC50 and AUC0-24/MIC50 ratios were, respectively, 0.67 ± 0.09 and 4.76 ± 0.48 and 1.18 ± 0.35 and 7.05 ± 2.40 for the 15 and 30 mg/kg bolus doses, respectively. After 10-h pulsed administration of 15 mg/kg, values of Cmax/MIC50, 0.19 ± 0.02 on d 1 and 0.30 ± 0.08 on d 5 of therapy were obtained, the AUC/MIC50 ratios were 2.09 ± 0.29 and 3.22 ± 0.93 on d 1 and 5, respectively. Higher values were obtained with the doubled dose of 30 mg/kg: the Cmax/MIC50 ratios were 0.49 ± 0.11 on d 1 and 0.69 ± 0.18 on d 5; the AUC/MIC50 ratios were 5.15 ± 1.15 and 6.57 ± 1.92 on d 1 and 5, respectively. Based on these results, FLU administration should be adopted when specific diagnostic findings indicate its efficacy, and revising the dosage scheme to comply with the prudent and responsible use of antimicrobials in veterinary medicine is advisable.

[Methodological aspects of clinical and economic impact of vaccine interventions and HTA. Focus on HPV vaccination]. / Aspetti metodologici della valutazione di impatto clinico ed economico negli interventi vaccinali in HTA. Focus sulla vaccinazione anti-HPV.

The aim of the present study is to analyze the methodological and technical aspects of Health Technology Assessment (HTA) as a tool for the clinical and economic impact of vaccine interventions, describe and comment the main studies at the national level, with a particular focus on HPV vaccination. The work was conducted in 3 phases: a) revision of the scientific literature, strictly linked to methodologies adopted in different studies on economic evaluations on HPV vaccines and analysis of Guidelines for building models for the economic assessment; b) analysis of the peculiarities and critical elements of economic evaluations in the field of vaccinology, from the clinical and epidemiological point of view, as well as the recognition of lack of knowledge on HPV infection dynamics; c) a comparative analysis of the two italian studies and of the results coming from them. Many differences between studies were found. Nevertheless, there is a general agreement on the economic profile of HPV vaccination for adolescent girls, if compared with the actual practice on the prevention of cervical carcinoma (pap-test screening). All the models showed a significant impact in terms of reduction of the incidence of cervical carcinoma and related mortality, in the long run, as well as a reduction of pre-cancer lesions and abnormal Pap tests. HTA approach has been recently recognized as a tool for decision making in vaccinology, and its methodologies and procedures are currently debated by public health experts. There is a strong need to continue the work in improving the model techniques of economic evaluations concerning HPV vaccination, as well as the adoption of homogeneous methods and standards, with the aim of helping the decision process in the field of Public Health.

Anti-inflammatory activity of citrus pectin on chicken monocytes' immune response.

Pectin is a dietary fibre composed of galacturonic acid, primarily found in the citrus fruits' cell walls. Citrus pectin (CP) has demonstrated antioxidative, anticancer, and anti-inflammatory properties in humans and animals. In broilers, CP supplementation improves energy utilization and nutrient digestibility, but limited information on its effects on chicken immunity is available so far. This study aimed to assess the in vitro impact of CP on chicken monocytes' immune response. Cells were purified from whole blood of healthy chickens and incubated with increasing concentrations (0, 0.25, 0.5, 0.75, 1 mg/mL) of CP to determine CP working concentration. The effects of different CP concentrations on cells' apoptosis and viability were assessed by measuring caspase-3 and -7 and the cells' metabolic activity (MTT assay), respectively. CP had no dose-dependent effect on monocyte apoptosis and viability.Then, the effects of CP (0.5 mg/mL) on chicken monocytes' chemotaxis and phagocytosis were assessed by measuring transwell migration and fluorescein-labelled E. coli incorporation, respectively. CP inhibited both monocytes' chemotaxis and phagocytosis.These data demonstrate that CP exerts an immunomodulatory role in chicken monocytes, supporting its integration in nutrition strategies that might be beneficial for the animal's immunity and health.

Containing a measles outbreak in Minnesota, 2017: methods and challenges.

AIMS: We report on a measles outbreak largely occurring in Minnesota's under-vaccinated Somali community in the spring of 2017. The outbreak was already into its third generation when the first two cases were confirmed, and rapid public health actions were needed. The aim of our response was to quickly end transmission and contain the outbreak. METHODS: The state public health department performed laboratory testing on suspect cases and activated an Incident Command staffed by subject matter experts that was operational within 2 h of case confirmation. Epidemiologic interviews identified exposures in settings where risk of transmission was high, that is, healthcare, childcare, and school settings. Vaccination status of exposed persons was assessed, and postexposure prophylaxis (PEP) was offered, if applicable. Exposed persons who did not receive PEP were excluded from childcare centers or schools for 21 days. An accelerated statewide measles, mumps, and rubella (MMR) recommendation was made for Somali Minnesota children and children in affected outbreak counties. Partnerships with the Somali Minnesota community were deepened, building off outreach work done with the community since 2008. RESULTS: Public health identified 75 measles cases from 30 March to 25 August 2017: 43% were female, 81% Somali Minnesotan, 91% unvaccinated, and 28% hospitalized. The median age of cases was 2 years (range: 3 months-57 years). Most transmission (78%) occurred in childcare centers and households. A secondary attack rate of 91% was calculated for unvaccinated household contacts. Over 51,000 doses of MMR were administered during the outbreak above expected baseline. At least 8490 individuals were exposed to measles; 155 individuals received PEP; and over 500 persons were excluded from childcare and school. State and key public health partners spent an estimated $2.3 million on response. CONCLUSION: This outbreak demonstrates the necessity of immediate, targeted disease control actions and strong public health, healthcare, and community partnerships to end a measles outbreak.

Early activation of the hypothalamic-pituitary-adrenal-axis in very-low-birth-weight infants with small thymus at birth.

BACKGROUND: An acute thymic involution in human fetuses and newborns has been described in very-low-birth-weight (VLBW) infants with histological chorioamnionitis. However, the mechanisms of thymic involution remain to be clarified. Here, we tested the hypothesis that an activation of the hypothalamic-pituitary-adrenal (HPA) axis occurs in VLBW infants with acute thymic involution at birth. METHODS: A total of 180 randomly selected VLBW newborns (28.8 +/- 3.15 wk gestation; 1093 +/- 305 g) entered the study. Thymic size was measured on standard chest radiographs at birth, and expressed as the ratio between the transverse diameter of the cardiothymic image at the level of the carina (CT) and that of the thorax (T). CT/T < 0.28 was considered to indicate a small thymic size. Plasma cortisol and adrenocorticotropic hormone (ACTH) concentrations were determined on days 1 (d-1) and 7 (d-7), and at 1 month (mo-1). Results. A total of 66 (36.7%) newborns had CT/T < 0.28. Infants with small thymus had significantly increased cortisol on d-1 ( approximately 5.2-folds) [median: 18.95 (95% CI: 11.20-39.4) microg/dl vs. 3.66 (1.94-6.82) microg/dl, p < 0.0001)] and d-7( approximately 1.7-folds) [12.0 (4.39-22.97) microg/dl vs. 7.8 (3.63-12.8) microg/dl, p = 0.0384)], as compared with those with normal thymic size, together with higher adrenocorticotropic hormone (ACTH) concentrations on d-1 ( approximately 1.9-folds) [28 (15.6-61.07) pg/ml vs. 14.9 (9.0-23.42) pg/ml, p = 0.0005)], while no significant differences for cortisol at mo-1 or ACTH concentrations on d-7 and mo-1 were evidenced (p > 0.50). From a multivariate logistic regression analysis, a small thymus at birth was a significant independent predictor of plasma cortisol concentrations in the top-quartile (OR = 14.4; 95% CI: 6.079-34.11), and plasma ACTH concentrations in the top-quartile (OR = 4.40 (95% CI: 1.99-9.74) on d-1 (results adjusted for variables significant at univariate analysis). CONCLUSIONS: Our data indicated the presence of a previously unrecognized, early activation of the HPA axis in VLBW newborns with a small thymus at birth.

Retraction notice to "Fungal diversity is negatively affected by habitat fragmentation: a meta-analysis" [Current Opinion in Microbiology 37 (2017) 61-66].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article that has already been published in , http://dx.doi.org/10.1016/j.mib.2017.03.015 has been withdrawn at the request of the editor and publisher. The publisher regrets that an error occurred which led to the premature publication of this paper. This error bears no reflection on the article or its authors. The publisher apologizes to the authors and the readers for this unfortunate error.

Fungal diversity at fragmented landscapes: synthesis and future perspectives.

Fungi are organisms with important roles in ecosystem functioning and services, but knowledge about how habitat fragmentation affect fungal diversity is biased by experimental approaches and it is spread in different trophic groups. We analyzed the empirical evidences of fungal diversity in fragmented landscapes, and proposed future perspectives for the study of these organisms under land use changes. Fungal diversity might be negatively affected by habitat fragmentation; however, this trend may differ in magnitude depending on fungal groups and their nutritional habits. In addition, due to the fact that fungal diversity at fragmented landscapes has been studied mainly through few indicators (e.g. isolation, area and edge effect); we propose incorporating the landscape structure and accurate spatio-temporal scales to the study of fungal diversity responses to fragmented landscapes. Together, this methodological refinement may allow improving knowledge on fungi when designing proper strategies for landscape management.

WITHDRAWN: Fungal diversity is negatively affected by habitat fragmentation: a meta-analysis.

This article that has already been published in , 10.1016/j.mib.2017.03.015 has been withdrawn at the request of the editor and publisher. The publisher regrets that an error occurred which led to the premature publication of this paper. This error bears no reflection on the article or its authors. The publisher apologizes to the authors and the readers for this unfortunate error". The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Spillover Events of Infection of Brown Hares (Lepus europaeus) with Rabbit Haemorrhagic Disease Type 2 Virus (RHDV2) Caused Sporadic Cases of an European Brown Hare Syndrome-Like Disease in Italy and Spain.

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80-90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV-related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS-like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.

Immunophenotyping of Rabbit Testicular Germ and Sertoli Cells Across Maturational Stages.

During testicular maturation, both Sertoli cells (SCs) and germ cells (GCs) switch from an immature to a mature immunophenotype. The reexpression of markers of immaturity in adults has been reported in cancer and in other testicular pathologies, in men as well as in animal species. Naturally affected with testicular cancer, rabbits have long been used in human reproductive research, but reports on the expression of testicular cell markers in this species are few and data about the immunophenotype of normal postnatal SCs and GCs are lacking. The aim of this study was to investigate the immunophenotype of SCs and GCs in the rabbit, from neonatal to adult age, using the antibodies anti-Müllerian hormone (AMH), vimentin (VIM), CKAE1/AE3 (cytokeratins [CKs]), desmin (DES), inhibin alpha (INH-α), placental alkaline phosphatase (PLAP), and periodic acid-Schiff (PAS) staining. In SCs, VIM was constantly expressed, and AMH and CKs expression was limited to neonatal and prepubertal age, whereas DES, INH-α, PLAP, and PAS were constantly negative. GCs were negatively stained for PLAP, PAS, and for the other markers. Results revealed analogies with human testicular immunophenotype, suggesting that rabbits could represent a potential experimental model for the study of human testicular pathology.

[Paraneoplastic syndromes: a review]. / Le sindromi paraneoplastiche: revisione critica della letteratura.

Modern oncology often obtains good results against earlier neoplasms, whilst it's still in difficulties against the advanced ones. The knowledge of paraneoplastic syndromes is crucial both to cure patients and to do an earlier diagnosis. When we recognize a paraneoplastic syndrome that comes before the clinic beginning of a neoplasm, perhaps we save a life. This review discusses all the main paraneoplastic syndromes, focusing mainly on their clinical aspect and reminding the most commonly associated cancers.

Collection and cryopreservation of mononuclear blood leukocytes and of CFU-C in man.

For bone marrow reconstitution after hemopoietic failure as a consequence of the action of a variety of etiological factors, hemopoietic stem cells are needed. These have been derived in the past mainly from bone marrow. This report describes studies and their results that indicate that granulocytic progenitor cells, measured in cell culture systems as "colony forming units in culture - CFU-C", can be collected in large quantities from the peripheral blood of human blood donors by continuous flow leukapheresis. They can be stored at ultra-low temperatures. Their recovery rate after thawing and washing is better than 85%. In a canine model, evidence was obtained that the presence of CFU-C in a suspension of mononuclear blood leukocytes is also indicative for the presence of pluripotent hemopoietic stem-cells. Therefore it is suggested that stem cells can also be collected from human blood as an alternative source for bone marrow reconstitution.

Performance and mortality of farmed hares.

Performance and mortality of hares were evaluated for 2 consecutive years in a large farm in Veneto Region (Northern Italy). On average, fertile reproductive pairs (n=318) gave birth 4.8 times and produced 11.4 live leverets, weaned 8.4 leverets and produced 7.0 growing hares (60 days) every year. Mean mortality was 3.6%, 22.9%, 9.7% and 2.5% in newborn (0 to 2 days of age), suckling (3 to 25 days), growing (26 to 60 days) and sub-adult (61 days until sale) hares, respectively. The main causes of mortality were enteric diseases (75.5%, 75.9% and 12.1% in suckling, growing and sub-adult hares, respectively), followed by respiratory diseases (3.4%, 8.0% and 36.2% in suckling, growing and sub-adult hares, respectively), starvation (11.3% and 8.8% in suckling and growing hares, respectively) and trauma (7.1%, 2.3% and 30.2% in suckling, growing and sub-adult hares, respectively). In reproducing hares, mortality was 24.7% and 15.4% in 2010 and 2011, respectively. Respiratory diseases (34.8%) and ulcerative pododermatitis (18.9%) were the most common pathological changes detected in reproducing hares. Farmed hares seem to be affected by diseases resembling those of rabbits reared under intensive conditions. It seems necessary to improve the husbandry of hares to reach satisfactory technical standards and to preserve their health.

Childhood Ataxia with Cerebral Hypomyelination Syndrome: a Variant of Patient with Early Childhood Onset Related to EIF2B3 Mutation. A Case Report.

Childhood ataxia with central nervous system hypomyelination (CACH) syndrome is an autosomal recessive transmitted leukodystrophy characterised by early childhood onset and acute deterioration following febrile illnesses or head trauma. We describe the case of a child with early onset of CACH syndrome. He presented with cerebellar ataxia beginning around two years of age with mild mental retardation. MRI showed diffuse white matter signal changes with thinning of the corpus callosum.