Scolopax rusticola Carrying Enterobacterales Harboring Antibiotic Resistance Genes.

The Eurasian woodcock (Scolopax rusticola) belongs to those bird species that make systematic migratory flights in spring and autumn in search of favorable breeding and wintering areas. These specimens arrive in the Mediterranean Area from northeastern European countries during the autumn season. The purpose of this study was to assess whether woodcocks can carry antibiotic resistance genes (ARGs) along their migratory routes. Although the role of migratory birds in the spread of some zoonotic diseases (of viral and bacterial etiology) has been elucidated, the role of these animals in the spread of antibiotic resistance has not yet been clarified. In this study, we analyzed the presence of beta-lactam antibiotic resistance genes. The study was conducted on 69 strains from 60 cloacal swabs belonging to an equal number of animals shot during the 2022-2023 hunting season in Sicily, Italy. An antibiogram was performed on all strains using the microdilution method (MIC) and beta-lactam resistance genes were investigated. The strains tested showed no phenotypic resistance to any of the 13 antibiotics tested; however, four isolates of Enterobacter cloacae and three of Klebsiella oxytoca were found to carry the blaIMP-70, blaVIM-35, blaNDM-5 and blaOXA-1 genes. Our results confirm the importance of monitoring antimicrobial resistance among migratory animals capable of long-distance bacteria spread.

FeliLeish: An Update on Feline Leishmaniosis and Factors Associated with Infection in Different Feline Populations from Italy.

Feline leishmaniosis is a worldwide infection caused by the parasite of the genus Leishmania transmitted by sandflies. Based on the complexity of epidemiology and diagnosis of this infection, the role of cats in the epidemiology and clinical impact of disease is still under debate. By using serological and molecular methods, this study aimed to update the epidemiology of the infection in different feline populations from various areas of Italy and to study factors associated with the infection. Of 1490 cats tested, 124 (8.3%, 95% CI 6.9-9.9) were infected, 96 had only specific L. infantum IgG, 18 were only positive for parasite DNA and 10 were both IFAT and qPCR positive. Risk factors for infection were sampling in the winter season (OR = 3.2, 95% CI 2.2-4.8), originating from the Sicily region (OR = 2.0, 95% CI 1.3-3.0), male gender (OR = 1.8, 95% CI 1.1-3.2), outdoor lifestyle (OR = 2.3, 95% CI 0.9-5.6) and seropositivity for FIV antibodies (OR = 2.2, 95% CI 1.2-4.2), while sampling in the spring (OR = 0.5, 95% CI 0.3-0.7) and summer (OR = 0.3, 95% CI 0.1-0.7), and originating from the Lazio region (OR = 0.1, 95% CI 0.05-0.4) were protective factors for infection. In endemic areas, Leishmania infection should be investigated by using both serological and molecular methods and cats should be protected from sandfly bites, particularly if they are FIV infected.

Cultivation of Protozoa Parasites In Vitro: Growth Potential in Conventional Culture Media versus RPMI-PY Medium.

The in vitro cultivation of Leishmania and Trypanosoma parasites plays an important role in the diagnosis and treatment of parasitic diseases. Although Evans's modified Tobie and Novy-MacNeal-Nicolle media, for Leishmania spp. and Trypanosoma cruzi, respectively, are the two commonly used media for both isolation and maintenance of strains in vitro, their preparation is expensive and laborious and requires fresh rabbit blood from housed animals. The purpose of this study was to evaluate the in vitro growth of both parasites with an alternative monophasic, blood-free, easy, and affordable medium called RPMI-PY, which was previously demonstrated suitable for the in vitro growth of Leishmania infantum. The potential growth of different Leishmania species and Trypanosoma cruzi was evaluated in traditional culture media versus RPMI-PY medium, and we recorded the protozoa parasites' morphology via orange acridine-ethidium bromide staining. The results of our study show that RPMI-PY medium can be used for Trypanosoma cruzi, Leishmania amazonensis, Leishmania major, and Leishmania tropica species since in all the species except Leishmania braziliensis, the exponential growth of the parasite was observed, in many cases higher than conventional media. The staining confirmed not only their growth during the 72 h investigation but also the optimal morphology and viability of the protozoa in the RPMI-PY medium.

Retrospective Analysis of Leishmaniasis in Sicily (Italy) from 2013 to 2021: One-Health Impact and Future Control Strategies.

Leishmaniasis is an important vector-borne disease that represents a serious public health problem, including in Sicily (Italy), which is considered an endemic area. We collected canine, feline and human data from 2013 to 2021 in Sicily, while entomological surveys were conducted only in 2013 and 2021. Overall, 23,794/74,349 (34.4%) of dogs and 274/4774 (11.8%) of cats were positive in one or more diagnostic tests. A total of 467 cases of human Leishmaniasis were reported, with 71% showing cutaneous and 29% visceral involvement. The provinces with the largest number of patients were Agrigento (45.4%) and Palermo (37%). In 2013, Phlebotomus perfiliewi was the dominant sandfly species in Sicily (68.7%), followed by Phlebotomus perniciosus (17.2%) and Sergentomya minuta (14%). In 2021, Phlebotomusperfiliewi was confirmed as the most common species (61.6%), followed by Phlebotomusperniciosus (33.1%) and Sergentomyaminuta (4.7%). Of particular interest was the identification of Phlebotomus papatasi (0.41%) in Agrigento. Our retrospective study can inform health authorities for the development of appropriate screening, treatment and control strategies to reduce Leishmania incidence rate. This study examined the present state of Leishmaniasis control, surveillance, and prevention in Sicily, but also highlighted deficiencies that could be addressed through the application of One-Health principles.

Clinical Case of Feline Leishmaniosis: Therapeutic Approach and Long-Term Follow-Up.

The response to allopurinol treatment and survival time of a case of feline leishmaniosis in a FIV co-infected cat is herein reported. In May 2019, a 13-year-old neutered European Shorthair male was referred due to weight loss and exfoliative dermatitis. Lymphadenomegaly and splenomegaly were detected upon clinical inspection, while the presence of several Leishmania infantum amastigotes were detected on splenic and lymphonodal fine needle aspiration (FNA). Allopurinol (10 mg/kg PO q 12 h) was administered. After two months, the cat's clinical symptoms disappeared, and the owners decided to interrupt the therapy. In February, two reddish nodular fleshy neoformations appeared in both eyes, and amastigotes of Leishmania were detected by cytology on conjunctival swabs. Allopurinol treatment was re-started at the same rate; the ocular lesions regressed in two weeks, and the owner again decided to interrupt the therapy. In July, the patient had a new relapse, but the owner, tired of continuous relapses, refused further treatment of the disease. The cat's health condition continued to worsen: in October 2021, the ocular lesions appeared again, and in November the patient died. This case underlines the effectiveness of allopurinol and highlights how interruption of treatment frequently leads to relapse, impairing the animal's health condition and prognosis.

Detection of Mycoplasma agalactiae in Ticks (Rhipicephalus bursa) Collected by Sheep and Goats in Sicily (South-Italy), Endemic Area for Contagious Agalactia.

The aim of this preliminary study was to investigate the presence of Mycoplasma agalactiae (Ma) or other Contagious Agalactia (CA) causative organisms, in hard ticks infesting milking sheep and goats in endemic areas for CA in Sicily (South-Italy). Although there is accumulating evidence to support the role of ticks in the transmission of blood-borne haemoplasmas, information regarding their role in the transmission of CA, remains scarce. Ticks (n = 152) were collected from 25 lactating sheep and goats from three farms with previous outbreaks of CA. Microbiological and biomolecular, as well as serological analysis were performed on milk, tick, and serum samples, respectively. Rhipicephalus bursa species predominated, comprising 84.8% of the sampled ticks. Mycoplasma-like colonies were isolated from 5/56 (8.9%) tick pools and were identified as Ma by specific PCR and 16S rRNA gene sequencing. Unexpectedly, the organism was isolated from R. bursa ticks recovered only from animals whose milk tested negative for the pathogen. This preliminary demonstration suggests the potential role for ticks to act as a reservoir for the organisms, with potential involvement in the spread and maintenance of CA. Further work is required to determine the location of the organisms within the body of the ticks and to assess transmission potential.

First neuroinvasive human case of West Nile Disease in Southern Italy: Results of the 'One Health' approach.

BACKGROUND: West Nile Disease (WND) is a zoonotic mosquito-borne infection involving viral pathogens, human and animal hosts, vectors and environment. Cooperation among medical, veterinary and entomological fields has been promoted by the Italian Public Health Authorities, and an integrated West Nile Virus (WNV) Surveillance Plan has been in force in Italy since 2016 to prevent the transmission risk of WND to humans through an early detection of viral circulation by animal and entomological surveillance. This managing model is unique in Europe. OBJECTIVES: This survey aimed at presenting the 'One Health' approach applied in 2016 to the first autochthonous human case of West Nile Neuroinvasive Disease (WNND) in Sicily (Southern Italy). METHODS: Serological (anti-WNV IgM and IgG ELISA, anti-WNV neutralizing antibodies) and molecular tests were conducted on blood, liquor and urine of a 38-year-old man with encephalitis and meningitis. Overall, 2704 adult culicides from 160 mosquito catches were morphologically identified. Female mosquitoes were analysed in pools for WNV RNA detection. Serological (anti-WNV IgM and IgG ELISA) and molecular analyses for WNV were carried out in 11 horses, 271 chickens and two dogs sampled in farms around the man's residence. RESULTS AND CONCLUSIONS: WNND was confirmed by serological analysis on patient's liquor and serum. Collected mosquito species included Culex pipiens (93.56%, CI95% 92.64%-94.49%), Aedes albopictus (5.25%, CI95% 4.41%-6.09%), Culex hortensis (0.59%, CI95% 0.30%-0.88%), Culiseta longiareolata (0.55%, CI95% 0.27%-0.83%) and Anopheles maculipennis s.l. (0.04%, CI95% -0.04% to 0.11%). Mosquito pools were negative for WNV RNA. Two dogs (100%) and two horses (18.18%, CI95% -4.61 to 40.97%) resulted positive for anti-WNV specific antibodies. The 'One Health' approach allowed to report the first human neuroinvasive WND in Sicily and to confirm the local circulation of WNV in animals of the same area where the clinical case occurred, defining the autochthonous origin of the infection.

Molecular Investigation on Tick-Borne Hemoparasites and Coxiella burnetii in Dromedary Camels (Camelusdromedarius) in Al Dhafra Region of Abu Dhabi, UAE.

Camels represent an important resource for inhabitants of the most arid regions of the world and their survival is mainly related to environment conditions including the risk of parasitic diseases, which may represent a significant cause of losses in livestock production of these areas. Camels may be parasitized by several hematophagous arthropods, which can be vectors of several diseases including zoonosis. This study aimed to investigate in dromedary camels and their ticks the importance of tick-borne hemoparasites that might be responsible for a recent and obscure morbidity of camels in Al Dhafra region of Abu Dhabi, UAE. Blood samples and ticks from 93 naturally infected camels belonging to 36 herds, affected by variable acute clinical syndromes lasting from 3 to 5 days, were analyzed through molecular techniques for specific DNA presence of different blood pathogens: Anaplasmamarginale/Anaplasmaovis, Anaplasma phagocytophilum, Coxiella burnetii,Babesia spp., and Theileria spp. DNA. All the 72 ticks collected belonged to the Hyalomma dromedarii species and were negative for blood pathogens. n = 15 camels (16.1%) were found positive to the following tick-borne hemoparasites: A. phagocytophilum 11 (11.8%), Coxiella burnetii 3 (3.2%), and Babesia/Theileria spp. 2 (2.1%). One singular camel showed coinfection of C. burnetii and A. phagocytophiulm. Genetic profile of C. burnetii showed a high phylogenetic relatedness to European, Asian and African C. burnetii strains. This is the first laboratory investigation on tick-borne pathogens in camels in UAE, and the first report of A. phagocytophilum and C. burnetii. Moreover, since the detected pathogens are recognized pathogens for humans, this study highlights the zoonotic risk for humans working in camel husbandry.

Serological Survey and Molecular Characterization of Theileria annulata in Sicilian Cattle.

Tropical theileriosis is a tick-borne disease caused by hemoprotozoan parasites with considerable veterinary and economic impact worldwide. Ticks transmitting the disease belong to the Haemaphysalis, Rhipicephalus, and Hyalomma genera. The Hyalomma genus is very common in Sicily (Italy) and represents the main Theileria annulata vector in the island. Data concerning the molecular epidemiology of this pathogen are missing in the region. In 2018-2019, blood and serum samples were collected from 480 cows in seven Sicilian farms from four different provinces. Seroprevalence in the farms ranged from 22% to 71%. Three farms were selected for molecular analysis consisting of real-time PCR targeting the almost complete 18S ribosomal RNA (rRNA). Four amplicons per farm were sequenced and phylogenetic analyses were carried out. The four sequences were identical within each farm and showed 92-99% identity with the other farms and with sequences from Genbank. According to the phylogenetic analysis, these three sequences and an additional one from a laboratory-cultured Theileria annulata strain obtained in 1999 belonged to a single T. annulata clade with good bootstrap support with other sequences from Italy, India, and Iran, indicating limited geographical and temporal genetic variability of the parasite. This study represents the first phylogenetic analysis of T. annulata in Sicily, which will be useful to improve the strategies for theileriosis control and prevention.

Immune Response to Tick-Borne Hemoparasites: Host Adaptive Immune Response Mechanisms as Potential Targets for Therapies and Vaccines.

Tick-transmitted pathogens cause infectious diseases in both humans and animals. Different types of adaptive immune mechanisms could be induced in hosts by these microorganisms, triggered either directly by pathogen antigens or indirectly through soluble factors, such as cytokines and/or chemokines, secreted by host cells as response. Adaptive immunity effectors, such as antibody secretion and cytotoxic and/or T helper cell responses, are mainly involved in the late and long-lasting protective immune response. Proteins and/or epitopes derived from pathogens and tick vectors have been isolated and characterized for the immune response induced in different hosts. This review was focused on the interactions between tick-borne pathogenic hemoparasites and different host effector mechanisms of T- and/or B cell-mediated adaptive immunity, describing the efforts to define immunodominant proteins or epitopes for vaccine development and/or immunotherapeutic purposes. A better understanding of these mechanisms of host immunity could lead to the assessment of possible new immunotherapies for these pathogens as well as to the prediction of possible new candidate vaccine antigens.

New Real-Time PCRs to Differentiate Rickettsia spp. and Rickettsia conorii.

Rickettsia species are an important cause of emerging infectious diseases in people and animals, and rickettsiosis is one of the oldest known vector-borne diseases. Laboratory diagnosis of Rickettsia is complex and time-consuming. This study was aimed at developing two quantitative real-time PCRs targeting ompB and ompA genes for the detection, respectively, of Rickettsia spp. and R. conorii DNA. Primers were designed following an analysis of Rickettsia gene sequences. The assays were optimized using SYBR Green and TaqMan methods and tested for sensitivity and specificity. This study allowed the development of powerful diagnostic methods, able to detect and quantify Rickettsia spp. DNA and differentiate R. conorii species.

Insecticide Resistance Associated with kdr Mutations in Aedes albopictus: An Update on Worldwide Evidences.

Insecticide resistance is an increasing problem worldwide that limits the efficacy of control methods against several pests of health interest. Among them, Aedes albopictus mosquitoes are efficient vectors of relevant pathogens causing animal and human diseases worldwide, including yellow fever, chikungunya, dengue, and Zika. Different mechanisms are associated in conferring resistance to chemical insecticides. One of the most widespread and analysed mechanisms is the knockdown resistance (kdr) causing resistance to DDT and pyrethroids. The mechanism is associated with mutations in the voltage sensitive sodium channel, which is involved in beginning and propagation of action potentials in nervous cells. The mechanism was originally discovered in the housefly and then it was found in a large number of arthropods. In 2011, a kdr associated mutation was evidenced for the first time in A. albopictus and afterward several evidences were reported in the different areas of the world, including China, USA, Brazil, India, and Mediterranean Countries. This review aims to update and summarize current evidences on kdr in A. albopictus, in order to stimulate further researches to analyse in depth A. albopictus resistance status across the world, especially in countries where the presence of this vector is still an emerging issue. Such information is currently needed given the well-known vector role of A. albopictus in the transmission of severe infectious diseases. Furthermore, the widespread use of chemical insecticides for control strategies against A. albopictus progressively lead to pressure selection inducing the rise of insecticide resistance-related mutations in the species. Such event is especially evident in some countries as China, often related to a history of uncontrolled use of chemical insecticides. Thus, a careful picture on the diffusion of kdr mutations worldwide represents a milestone for the implementation of control plans and the triggering of novel research on alternative strategies for mosquito-borne infections.

A Geographical Information System Based Approach for Integrated Strategies of Tick Surveillance and Control in the Peri-Urban Natural Reserve of Monte Pellegrino (Palermo, Southern Italy).

Ticks (Acari: Ixodidae) are bloodsucking arthropods involved in pathogen transmission in animals and humans. Tick activity depends on various ecological factors such as vegetation, hosts, and temperature. The aim of this study was to analyse the spatial/temporal distribution of ticks in six sites within a peri-urban area of Palermo (Natural Reserve of Monte Pellegrino) and correlate it with field data using Geographical Information System (GIS) data. A total of 3092 ticks were gathered via dragging method from June 2012 to May 2014. The species collected were: Ixodes ventalloi (46.09%), Hyalomma lusitanicum (19.99%), Rhipicephalus sanguineus (17.34%), Rhipicephalus pusillus (16.11%), Haemaphisalis sulcata (0.36%), Dermacentor marginatus (0.10%), and Rhipicephalus turanicus (0.03%). GIS analysis revealed environmental characteristics of each site, and abundance of each tick species was analysed in relation to time (monthly trend) and space (site-specific abundance). A relevant presence of I. ventalloi in site 2 and H. lusitanicum in site 5 was observed, suggesting the possible exposure of animals and humans to tick-borne pathogens. Our study shows the importance of surveillance of ticks in peri-urban areas and the useful implementation of GIS analysis in vector ecology; studies on temporal and spatial distribution of ticks correlated to GIS-based ecological analysis represent an integrated strategy for decision support in public health.

Geo-statistical analysis of Culicoides spp. distribution and abundance in Sicily, Italy.

BACKGROUND: Biting midges belonging to Culicoides imicola, Culicoides obsoletus complex and Culicoides pulicaris complex (Diptera: Ceratopogonidae) are increasingly implicated as vectors of bluetongue virus in Palaearctic regions. Culicoides obsoletus complex includes C. obsoletus (sensu stricto), C. scoticus, C. dewulfi and C. chiopterus. Culicoides pulicaris and C. lupicaris belong to the Culicoides pulicaris complex. The aim of this study was a geo-statistical analysis of the abundance and spatial distribution of Culicoides spp. involved in bluetongue virus transmission. As part of the national bluetongue surveillance plan 7081 catches were collected in 897 Sicilian farms from 2000 to 2013. METHODS: Onderstepoort-type blacklight traps were used for sample collection and each catch was analysed for the presence of Culicoides spp. and for the presence and abundance of Culicoides vector species (C. imicola, C. pulicaris / C. obsoletus complexes). A geo-statistical analysis was carried out monthly via the interpolation of measured values based on the Inverse Distance Weighted method, using a GIS tool. Raster maps were reclassified into seven classes according to the presence and abundance of Culicoides, in order to obtain suitable maps for Map Algebra operations. RESULTS: Sicilian provinces showing a very high abundance of Culicoides vector species were Messina (80% of the whole area), Palermo (20%) and Catania (12%). A total of 5654 farms fell within the very high risk area for bluetongue (21% of the 26,676 farms active in Sicily); of these, 3483 farms were in Messina, 1567 in Palermo and 604 in Catania. Culicoides imicola was prevalent in Palermo, C. pulicaris in Messina and C. obsoletus complex was very abundant over the whole island with the highest abundance value in Messina. CONCLUSIONS: Our study reports the results of a geo-statistical analysis concerning the abundance and spatial distribution of Culicoides spp. in Sicily throughout the fourteen year study. It provides useful decision support in the field of epidemiology, allowing the identification of areas to be monitored as bases for improved surveillance plans. Moreover, this knowledge can become a tool for the evaluation of virus transmission risks, especially if related to vector competence.

First record of the invasive mosquito species Aedes (Stegomyia) albopictus (Diptera: Culicidae) on the southernmost Mediterranean islands of Italy and Europe.

BACKGROUND: Aedes albopictus, a known worldwide vector of several mosquito-borne disease pathogens including dengue, chikungunya and Zika viruses, was introduced into Europe in the late 1970s through global trade. First recorded in northern Italy in 1990, this mosquito species has rapidly spread throughout the country, where it was responsible for an outbreak of chikungunya in 2007 that affected more than 200 people. As part of the VectorNet project, which is aimed at improving preparedness and responsiveness for animal and human vector-borne diseases in Europe, a mosquito targeted study was carried out on the three southernmost Italian islands. The objective was to verify the current European southern distribution limits of Ae. albopictus and the potential occurrence of other invasive mosquito species, in the light of the introduction of high risk for vector-borne disease pathogens into Europe via migration flows. RESULTS: In the summer 2015, six surveys for container-breeding mosquitoes were carried out by setting up a network of oviposition traps and BG Sentinel traps in selected areas on the islands of Pantelleria, Lampedusa and Linosa. Aedes albopictus was found on all three islands under investigation. The consequences on public health with regard to the presence of this mosquito vector and the migrant people entering the country from Africa and the Middle East are also discussed here. CONCLUSIONS: The detection of the Asian tiger mosquito on these islands, which represent the last European strip of land facing Africa, has important implications for public health policy and should prompt the national authorities to implement tailored surveillance activities and reinforce plans for preparedness strategies in such contexts.

Diversity and distribution of ticks from domestic ruminants in Lebanon.

Ticks (Acari: Ixodidae) are ectoparasites infesting livestock in every geographic area in the world and they are vectors of several viral, bacterial, and protozoan pathogens to animals and humans worldwide. A deep knowledge of the geographical distribution of these arthropods would have a key role in the control of tick-borne diseases. Few data are available about tick presence in domestic ruminants in Lebanon. The study aimed at providing an analysis of tick presence and distribution in Lebanon. Ticks were collected from cattle, sheep, and goats farms distributed in 6 Lebanese provinces between June and September 2014. A total of 272 adult hard ticks were randomly collected from domestic ruminants (cattle, sheep, and goats) located at 37 Lebanese farms, distributed among 30 villages. Ticks belonged to 4 Ixodidae genera: Rhipicephalus (72.4%), Haemaphysalis (11.4%), Dermacentor (8.1%), and Hyalomma (8.1%). They included the following species: Rhipicephalus annulatus (50.7%), Rhipicephalus turanicus (18.8%), Hyalomma anatolicum (8.1%), Haemaphylasis punctata (11.4%), Dermacentor marginatus (8.1%), Rhipicephalus sanguineus (2.5%), and Rhipicephalus bursa (0.4%). Rhipicephalus turanicus and H. anatolicum were found on cattle, sheep, and goats, R. annulatus on cattle and sheep, R. sanguineus, D. marginatus and Hea. punctata on sheep and goats, while R. bursa was collected only on sheep. Tick species involved in pathogen transmission were found and some of the identi ed species were recorded in Lebanon for the rst time.

A retrospective study of the characterization of Rickettsia species in ticks collected from humans.

Rickettsiae (family Rickettsiaceae, order Rickettsiales) are obligate intracellular bacteria transmitted by arthropod vectors. Several Rickettsia species causing vector-borne rickettsioses belong to the spotted fever group (SFG). Traditionally, Rickettsia conorii has been considered as the main etiologic agent of Mediterranean spotted fever. However, the molecular characterization of rickettsiae allowed identifying other species involved in spotted fever in the Mediterranean region. In this study, 42 ticks collected from humans were subjected to morphological identification and molecular characterization of Rickettsia species potentially involved in human rickettsiosis in Sicily. Fourteen ticks positive to at least two Rickettsia spp. molecular markers were used in the study. Identified Rickettsia spp. included R. conorii, found in Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus, Rickettsia aeschlimannii found in Hyalomma marginatum, Hyalomma lusitanicum, Dermacentor marginatus and Ixodes ricinus, Rickettsia massiliae found in R. turanicus and R. sanguineus s.l., and Rickettsia slovaca found in D. marginatus and R. sanguineus s.l. Our results showed a great variety of zoonotic Rickettsia spp. in ticks collected from humans in Sicily. The Rickettsia spp. reported in this study were identified in previously recognized or new potential tick vectors in Europe, highlighting the risk of infection by different Rickettsia spp. for humans bitten by ticks in Sicily.