Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS) technology is currently increasingly used in diagnostic laboratories as a cost effective, rapid and reliable routine technique for the identification and typing of microorganisms. In this study, we used MALDI-TOF MS to analyze a collection of 160 strains belonging to the Bacillus cereus group (57 B. anthracis, 49 B. cereus, 1 B. mycoides, 18 B. wiedmannii, 27 B. thuringiensis, 7 B. toyonensis and 1 B. weihenstephanensis) and to detect specific biomarkers which would allow an unequivocal identification. The Main Spectra Profiles (MSPs) were added to an in-house reference library, expanding the current commercial library which does not include B. toyonensis and B. wiedmannii mass spectra. The obtained mass spectra were statistically compared by Principal Component Analysis (PCA) that revealed seven different clusters. Moreover, for the identification purpose, were generated dedicate algorithms for a rapid and automatic detection of characteristic ion peaks after the mass spectra acquisition. The presence of specific biomarkers can be used to differentiate strains within the B. cereus group and to make a reliable identification of Bacillus anthracis, etiologic agent of anthrax, which is the most pathogenic and feared bacterium of the group. This could offer a critical time advantage for the diagnosis and for the clinical management of human anthrax even in case of bioterror attacks.

The poultry red mite (PRM), Dermanyssus gallinae, is a nonburrowing haematophagous nest-dwelling ectoparasite of birds; occasionally it bites humans, inducing dermatitis. The possibility that this parasite may also be involved in transmission of pathogens is an additional concern. We investigated the presence of zoonotic agents in PRMs from bird nests and pets, and related them to urban outbreaks of dermatitis. A total of 98 PRMs from 12 outbreaks of PRM dermatitis that occurred in Italian cities from 2001 to 2017 were molecularly investigated for detection of Coxiella spp. (16S rRNA), Chlamydophila spp. (16S rRNA), Rickettsia spp. (17 kDa protein-encoding gene), Borrelia burgdorferi sensu lato (groEL gene) and Bartonella spp. (16S-23S rRNA intergenic spacer). Of the 12 tested mite pools, one was positive for Coxiella burnetii (100% identity) and two for B. burgdorferi sensu lato (99% with Borrelia afzelii). For the first time, the presence of B. burgdorferi sensu lato and C. burnetii is reported in PRMs from urban areas. Birds, mainly pigeons, can harbour both pathogens. Therefore, birds and their nest-dwelling PRMs may play a role in the epidemiology of these infections.

ORF virus (Poxviridae) is the causative agent of contagious ecthyma (soremouth), a disease primarily affecting sheep and goats worldwide, but also humans exposed to disease-ridden animals. Pathogens are shed with scabs, and infection mainly occurs by direct contact. Although the disease is relatively benign and self-limiting, the morbidity rate is high in livestock with subsequent significant financial and economic impact. The aim of the study was to experimentally investigate the potential for the housefly, Musca domestica, to act as a mechanical vector of the virus. Homogenate of crusted scabs from ORFV-positive sheep (Italy, Apulia) were used to infect laboratory-reared flies. Flies walking on viral mixture and flies inoculated on their wings were individually placed in Falcon tubes and the ORFV DNA was searched by PCR on tube walls; flies were fed on the same homogenized crusts and their crop and spots (vomit and feces) molecularly examined for ORF DNA at 2, 4, and 6 h. All of the flies (100%) used in the experiments were able to pick up and transmit the viral genome to contact surfaces; 60% were found ORF virus (DNA)-positive in both spots and crop. These results suggest that M. domestica could play a role as potential mechanical vector and/or reservoir in the epidemiology of the ORF virus infection. Thus, house fly management should be considered in the measures to control the disease in ovine–caprine farms.

Ixodidae ticks are vectors and reservoirs of several species of rickettsiae, and tick‐borne rickettsioses are reported worldwide. This study was aimed to verify the distribution of spotted fever group rickettsiae associated with ticks in a wild environment, the National Park of Gargano, where there is proximity between wild and domestic animals, and which is within an endemic area for rickettsiosis. Ticks were collected from animals or vegetation, morphologically identified and tested by a PCR targeting the 17kDa gene, and by a loop‐mediated isothermal amplification ( LAMP ) targeting ompB gene. Out of 34 tested tick pools, 2 from Dermacentor marginatus, 1 from Ixodes ricinus, and 1 from Rhipicephalus turanicus resulted positive. Nucleotide sequences of amplicons showed high similarity with sequences from Rickettsia slovaca , Rickettsia raoultii , Rickettsia helvetica, and Rickettsia felis . The overall calculated infection rate was 26.19 per 1,000, while it rose up to 107.77 when only D. marginatus was considered. The results highlight the association among Ri. slovaca , Ri. raoultii , D. marginatus and wild boars from which infected ticks were collected. Finally, the study shows the low efficacy of the previously described LAMP method for the detection of Rickettsia spp., when compared to PCR , making urgent the development of most effective LAMP protocols.

Background: Anopheles sacharovi, a member of the Anopheles maculipennis complex, was a historical malaria vector in Italy, no longer found since the last report at the end of 1960s. In September 2022, within the Surveillance Project for the residual anophelism, a single specimen of An. maculipennis sensu lato collected in Lecce municipality (Apulia region) was molecularly identified as An. sacharovi. This record led to implement a targeted entomological survey in September 2023. Methods: Investigation was conducted in the areas around the first discovery, focusing on animal farms, riding stables and potential breeding sites. Adult and immature mosquitoes were collected, using active search or traps, in several natural and rural sites. Mosquitoes belonging to An. maculipennis complex were identified morphologically and molecularly by a home-made routine quantitative polymerase chain reaction (qPCR) assay, developed specifically for the rapid identification of An. labranchiae, and, when necessary, by amplification and sequencing of the ITS-2 molecular marker. Results: Out of the 11 sites investigated, 6 were positive for Anopheles presence. All 20 An. maculipennis s.l. (7 adults, 10 larvae and 3 pupae) collected in the areas were identified as An. sacharovi by ITS-2 sequencing. Conclusions: The discovery of An. sacharovi, considered to have disappeared from Italy for over 50 years, has a strong health relevance and impact, highlighting an increase in the receptivity of the southern areas. As imported malaria cases in European countries are reported every year, the risk of Plasmodium introduction by gametocyte carriers among travellers from endemic countries should be taken into greater consideration. Our findings allow rethinking and building new models for the prediction and expansion of introduced malaria. Furthermore, to prevent the risk of reintroduction of the disease, the need to strengthen the surveillance of residual anophelism throughout the South should be considered. Graphical

Dirofilariasis by Dirofilaria repens is an important mosquito vector borne parasitosis, and the dog represents the natural host and reservoir of the parasite. This filarial nematode can also induce disease in humans, and in the last decades an increasing number of cases have been being reported. The present study describes the first loop mediated isothermal amplification (LAMP) assay to detect D. repens DNA in blood and mosquitoes. Two versions of the technique have been developed and described: in the first, the amplification is followed point by point through a real time PCR instrument (ReT-LAMP); in the second, the amplification is visualized by checking UV fluorescence of the reaction mixture after addition of propidium iodide (PI-LAMP). The two variants use the same set of 4 primers targeting the D. repens cytochrome oxidase subunit I (COI) gene. To assess the specificity of the method, reactions were carried out by using DNA from the major zoonotic parasites of the family of Onchocercidae, and no amplification was observed. The lower limit of detection of the ReT-LAMP assay was 0.15 fg/μl (corresponding to about 50 copy of COI gene per μl). Results suggest that the described assay is specific, and its sensitivity is higher than the conventional PCR based on the same gene. It is also provide a rapid and cost-effective molecular detection of D. repens, mainly when PI-LAMP is applied, and it should be performed in areas where this emerging parasitosis is endemic. Dirofilaria repens is a filarial nematode which mainly infests the dog, but humans may be occasionally infested, too. The spread of the parasite is mediated by a number of mosquitoes species, which are well recognized as vectors of D. repens. The majority of reports of the disease come from the European Countries, especially those along the Mediterranean basin, but in the last decade several cases have been recorded also from Asian and African Countries, and this led the scientific community to consider such parasitosis an emerging disease. To date, diagnosis is based the morphologic analysis of microfilariae, isolated from the blood of infected hosts, but this may be time-consuming and the identification of parasite requires specialized parasitologists. The here described approach, based on the loop-mediated isothermal amplification (LAMP), allows the detection of D. repens genomic DNA directly from the biological samples, and it may be easily and rapidly performed, producing unequivocal results in less than a hour. We also presented two versions of the assays. The first, a real-time LAMP, is characterized by a very high sensitivity but it requires an expensive real time PCR instrument, while the second, performed with the addition of propidium iodide, does not need such equipment, therefore being very affordable. This makes it suitable to be carried out in field and whenever expensive equipment or specialized personnel lacks.