Usutu virus in blackbirds (Turdus merula) with clinical signs, a case study from northern Italy

Usutu virus (USUV) is a mosquito-borne virus belonging to the family Flaviviridae, genus Flavivirus. Natural transmission cycle of USUV involves mosquitoes and birds, so humans and other mammals are considered incidental hosts. In this study, USUV infection was diagnosed in all wild blackbirds, collected from July to September 2018 in a wildlife recovery center in the province of Bologna, in the Emilia-Romagna region, northern Italy. All blackbirds showed neurological clinical signs, such as overturning, pedaling, and incoordination. Moreover, the subjects died shortly after arriving at the hospitalization center. Virological investigations were performed by real-time PCR on frozen samples of the spleen, kidney, myocardium, and brain for the detection of Usutu (USUV) and West Nile (WNV) viruses. The small and large intestine were used as a matrix for the detection of Newcastle disease virus (NDV). All 56 subjects with neurological clinical signs were positive for USUV, only one subject (1.8%) tested positive for WNV, and no subject was positive for NDV. The most represented age class was class 1 J (58.9%), followed by class 3 (25.0%), and lastly from class 4 (16.1%). Most of the blackbirds before dying were in good (51.8%) and fair (39.3%) nutritional status, while only five subjects (8.9%) were cachectic. The USUV genomes detected in the blackbirds of this study fall within the sub-clade already called EU2 that has been detected since 2009 in the Emilia-Romagna region. Neurological clinical signs in USUV-affected blackbirds are still widely discussed and there are few works in the literature. Although our results require further studies, we believe them to be useful for understanding the clinical signs of Usutu virus in blackbirds, helping to increase the knowledge of this zoonotic agent in wild species and to understand its effect on the ecosystem. The goal of this study was to report—in the context of the regional passive surveillance program—the detection of USUV RNA in its most important amplifying host, the common blackbird, when showing clinical signs before death

Isolation and molecular typing of Leishmania infantum from Phlebotomus perfiliewi in a reemerging focus of leishmaniasis, Northeastern Italy

Visceral leishmaniasis (VL) caused by Leishmania (L.) infantum is a public health threat in the Emilia-Romagna region, northeastern Italy, but its epidemiology has not been fully elucidated in this area. The objective of this study was to characterize Leishmania infection in sand flies collected in a re-emerging focus of VL in the Bologna province. During the summer of 2016, 6114 sand flies were collected, identified, and tested for Leishmania detection. Of the identified sand flies, 96.5% were Phlebotomus (P.) perfiliewi and 3.5% were P. perniciosus. Detected parasites were characterized by biomolecular methods (multilocus microsatellite typing and characterization of repetitive region on chromosome 31), and quantified by real-time PCR. The prevalence of Leishmania infection in individually-tested P. perfiliewi sand flies varied from 6% to 10% with an increasing trend during the season. Promastigotes of L. infantum were isolated by dissection in one P. perfiliewi female; the isolated strain (Lein-pw) were closely related to Leishmania parasites from VL cases in northeastern Italy, but differed from strains isolated in dogs from the same area. Our findings strongly support the vector status of P. perfiliewi for human VL in the study area

Fate of Salmonella enterica Typhimurium and Listeria monocytogenes in Black Soldier Fly (Hermetia illucens) Larvae Reared on Two Artificial Diets

Ensuring food security is one of the main challenges facing the world over the next 30 years. There is, thus, an urgent need to significantly increase the supply of sustainable protein that can be transformed into animal feed. Proteins from insects offer a valuable alternative. This article presents the results of challenge tests conducted to investigate the dynamics of the microbial load of Salmonella enterica Typhimurium and Listeria monocytogenes in black soldier fly (Hermetia illucens) larvae grown on contaminated substrates. Four separate challenge tests were performed on two substrates: the Gainesville diet and a homemade diet. The challenge test procedure was carried out in accordance with ISO/DIS 20976-2 (under development). The results of this study show that, when grown on contaminated substrates, BSF larvae do not eliminate Salmonella Typhimurium or L. monocytogenes, but can reduce their microbial load. Sanitation processes downstream of the breeding of BSF larvae are, however, required to reduce the microbiological risks of this novel food

Biofortification of selenium in black soldier fly (Hermetia illucens) prepupae reared on seaweed or selenium enriched substrates

Selenium (Se) is an essential trace element for livestock. The element can be supplemented to feed in inorganic or organic Se forms, where the chemical form in the diet affects the accumulation of Se in animal tissues. Insects are known to be natural bioaccumulators of different nutrients, but no studies have so far looked up on the capacity of insects to be biofortified with Se, as a potential future source for Se in feed. In this study, black soldier fly (BSF, Hermetia illucens) larvae were reared on three different substrates: (1) a control substrate (CTR diet) of plant-ingredients (Gainesville diet); (2) Ascophyllum nodosum diet (AN30%), with 30% substitution of the alfalfa meal with the brown algae; and (3) a Se diet, where the CTR diet was fortified with 0.3 mg/kg of Se. All experiments were carried out under dark condition, at 25 °C with 70% relative humidity for two weeks, and the final BSF prepupae were analysed for Se, metals and minerals, and Se species. The mean total Se content in the prepupae reared on Se dietary group was over five times higher compared to the CTR group, whereas lower Se levels were detected in AN30% dietary group. Se speciation analyses showed that organic selenomethionine (SeMet) was the major Se species present, both in CTR and AN30% group. For the Se group, SeMet was accounting for 54% of the total Se. The levels of SeMet were higher in the Se group compared to the CTR group, indicating that BSF prepupae are able to convert inorganic Se to organic Se. The overall results show that the substrates hereby studied affect the total Se and Se species, as well as the levels of other elements, in the BSF prepupae.publishedVersio

Longitudinal study of Salmonella infection in four Italian farrow-to finish swine herds

A longitudinal study of Salmonella enterica infection was carried out in 4 Italian farrow-to-finish swine herds. In each herd 5 litters were randomly selected and in each litter 6 piglets ear tagged. Thus, on each farm 30 pigs were included in the study. Individual blood samples were collected for serologic examination at weaning from all piglets and in the same day from all sows 1n the farrowing unit. Piglets were bled again at approximately 60, 90, 150, 210 and 270 days of life with the last blood sample collected at slaughtering. In one herd, in which the duration of productive cycle was about 12 months, the last blood samples was collected at 350 days of life. 5 pen pooled faecal samples were collected from each herd for bacteriological examination with the same time schedule of blood samples. At slaughtering mesenteric lymph nodes were collected from each ear tagged pig. Sera-prevalence (cutoff S/P ratio 0,25) in sows varied from 93,8% to 100%. In all herds sera-prevalence in piglets showed a similar profile with complete decline of maternal antibodies at day 60 and clear sera-conversion between day 90 and day 150. The peak of sera-prevalence was observed between day 210 and day 270. Sera-prevalence at slaughtering varied from 66% to 100%. Salmonella was isolated from faecal samples in 3 out of 4 herds. No Salmonella was isolated from mesenteric lymph nodes at slaughter in 2 herds. Culture prevalence from mesenteric lymph nodes in the other herds was respectively 3,3% and 30%. This longitudinal study provides original information about epidemiological dynamics of Salmonella enterica infection in Italian swine herds in consideration of the typical longer fattening cycles

Antimicrobial Resistance Profile and ExPEC Virulence Potential in Commensal <i>Escherichia coli</i> of Multiple Sources.

We recently described the genetic antimicrobial resistance and virulence profile of a collection of 279 commensal E. coli of food-producing animal (FPA), pet, wildlife and human origin. Phenotypic antimicrobial resistance (AMR) and the role of commensal E. coli as reservoir of extra-intestinal pathogenic Escherichia coli (ExPEC) virulence-associated genes (VAGs) or as potential ExPEC pathogens were evaluated. The most common phenotypic resistance was to tetracycline (76/279, 27.24%), sulfamethoxazole/trimethoprim (73/279, 26.16%), streptomycin and sulfisoxazole (71/279, 25.45% both) among the overall collection. Poultry and rabbit were the sources mostly associated to AMR, with a significant resistance rate (p > 0.01) to quinolones, streptomycin, sulphonamides, tetracycline and, only for poultry, to ampicillin and chloramphenicol. Finally, rabbit was the source mostly associated to colistin resistance. Different pandemic (ST69/69*, ST95, ST131) and emerging (ST10/ST10*, ST23, ST58, ST117, ST405, ST648) ExPEC sequence types (STs) were identified among the collection, especially in poultry source. Both ST groups carried high number of ExPEC VAGs (pandemic ExPEC STs, mean = 8.92; emerging ExPEC STs, mean = 6.43) and showed phenotypic resistance to different antimicrobials (pandemic ExPEC STs, mean = 2.23; emerging ExPEC STs, mean = 2.43), suggesting their role as potential ExPEC pathogens. Variable phenotypic resistance and ExPEC VAG distribution was also observed in uncommon ExPEC lineages, suggesting commensal flora as a potential reservoir of virulence (mean = 3.80) and antimicrobial resistance (mean = 1.69) determinants

Effect of production process and high-pressure processing on viability of Listeria innocua in traditional Italian dry-cured coppa

In this study the effect of the application of High Pressure Treatment (HPP) combined with four different manufacturing processes on the inactivation of Listeria innocua, used as a surrogate for L. monocytogenes, in artificially contaminated coppa samples was evaluated in order to verify the most suitable strategy to meet the Listeria inactivation requirements needed for the exportation of dry-cured meat in the U.S. Fresh anatomical cuts intended for coppa production were supplied by four different delicatessen factories located in Northern Italy. Raw meat underwent experimental contamination with Listeria innocua using a mixture of 5 strains. Surface contamination of the fresh anatomical cuts was carried out by immersion into inoculum containing Listeria spp. The conditions of the HPP treatment were: pressure 593 MPa, time 290 seconds, water treatment temperature 14\ub0C. Listeria innocua was enumerated on surface and deep samples post contamination, resting, ripening and HPP treatment. The results of this study show how the reduction of the microbial load on coppa during the production process did not vary among three companies (P&gt;0.05) ranging from 3.73 to 4.30 log CFU/g, while it was significantly different (P&lt;0.01) for the fourth company (0.92 log CFU/g). HPP treatment resulted in a significant (P&lt;0.01) deep decrease of L. innocua count with values ranging between 1.63-3.54 log CFU/g with no significant differences between companies. Regarding superficial contamination, HPP treatment resulted significant (P&lt;0.01) only in Coppa produced by two companies. The results highlight that there were processes less effective to inhibit the pathogen; in particular for company D an increase of L. innocua count was shown during processing and HPP alone cannot be able to in reaching the Listeria inactivation requirements needed for exportation of dry-cured meat in the U.S. According to the data reported in this paper, HPP treatment increases the ability of the manufacturing process of coppa in reducing Listeria count with the objective of a lethality treatment

Effect of production process and high-pressure processing on viability of Salmonella spp. in traditional Italian dry-cured coppa

The aim of the study was to investigate the combined effect of the manufacturing process followed by HPP treatment on the inactivation of Salmonella spp. in artificially contaminated coppa samples, in order to verify the ability of the combined processes to achieve the objective of a 5-log reduction of Salmonella spp. needed for exportation to the U.S. Fresh anatomical cuts intended for coppa production were supplied by four different delicatessen factories located in Northern Italy. Raw meat underwent experimental contamination with Salmonella spp. using a mixture of 3 strains. Surface contamination of the fresh anatomical cuts was carried out by immersion into inoculum containing Salmonella spp. The conditions of the HPP treatment were: pressure 593 MPa, time 290 seconds, water treatment temperature 14\ub0C. Surface and deep samples were performed post contamination (T0), end of the cold phase (T1), end of process (Tend), and after HPP treatment (postHPP) and Salmonella spp. Enumerated. The results of this study show a significant reduction of Salmonella spp. all through the production process (P&lt;0.01) for all companies, followed by an additional reduction of bacterial counts due to HPP treatment (P&lt;0.01), both in superficial and deep contaminations (P&lt;0.01). The superficial overall reduction resulted of 1.58 to 5.04 log CFU/g during the production process. HPP treatment resulted in a significant (P&lt;0.01) superficial and deep decrease in Salmonella spp. enumeration varying from 0.61 to 4.01 log and from 1.49 to 4.13 log. According to the data presented in this study, only the combined approach of coppa manufacturing process followed by HPP treatment always led to a 5-log reduction of Salmonella spp. required by USDA/FSIS guidelines