A Canine Leptospirosis Clinical Case Due to Leptospira interrogans (Serogroup Icterohaemorrhagiae) in a Dog Kennel in Castelvetrano (Western Sicily, South Italy).

Leptospirosis is a worldwide widespread zoonosis caused by Leptospira genus. We report an acute leptospirosis case in a puppy housed at a municipal kennel and the subsequent diagnostic investigations carried out on all dogs housed in the kennel. Laboratory investigation included mainly a microagglutination test, real-time PCR, and multi-locus sequence typing (MLST) for Leptospira genus. Other agents of infection were excluded. The puppy resulted positive for Leptospira interrogans Icterohaemorrhagiae both with serological and molecular assays. All of the other 66 dogs in the kennel underwent clinical and laboratory investigations twice, 15 days apart. No other dog showed leptospirosis clinical signs. At the first sampling, eight dogs (12%) showed antibodies against Leptospira interrogans serogroup Icterohaemorragiae serovar Copenhageni. Real-time PCR on urine samples of seropositive dogs detected Leptospira spp. DNA in one sample, then identified as Leptospira interrogans serogroup Icterohaemorragiae by MLST. Fifteen days after, four of the previous seropositive dogs still showed antibodies against Leptospira spp. All urine samples collected from seropositive dogs were negative at real-time PCR. The study allowed the early confirmation of a Leptospirosis case and the identification of at least one asymptomatic carrier of pathogenic Leptospira spp. The prompt activation of all appropriate management measures allowed limiting and extinguishing the infection.

Serological and Molecular Evidence of Pathogenic Leptospira spp. in Stray Dogs and Cats of Sicily (South Italy), 2017-2021.

Leptospirosis is a zoonosis of public health concern. Its prevalence in stray animals in the South of Italy is unknown. This study aimed to investigate Leptospira spp. prevalence in 1009 stray animals. Out of them, 749 were alive animals, including 358 dogs (316 from Palermo and 42 from Ragusa) and 391 cats (359 from Palermo and 32 from Ragusa), and 260 were corpses (216 dogs and 44 cats) randomly collected in Sicily. Dogs and cats underwent a serological screening by Microscopic Agglutination Test and a molecular investigation by Real-Time PCR targeting lipL32. Corpses were subjected to Real-Time PCR. Serological analyses showed a prevalence of 1.12% (4/358) for dogs and 0.26% (1/391) for cats, with the only positive cat coming from Palermo. Serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae or Copenhageni, followed by Canicola and Bratislava, were the most spread among dogs, while the serological positive cat reacted with Hardjo serogroup. Two urine (2/32, 6.25%) and one blood (1/391, 0.26%) samples of cats, all from Ragusa, were positive at Real-Time PCR for pathogenic Leptospira spp. Sequencing analyses showed the presence of L. interrogans serogroup Icterohaemorrhagiae serovar Icterohaemorrhagiae or Copenhageni in one of the positive urine samples and in the positive blood sample. Analyses on corpses showed a prevalence of 1.85% (4/216) in Sicilian dog kidney samples, while all corpses of cats resulted in negative. Genotyping analysis showed a genetic relatedness between cat and human isolates. Results show Leptospira spp. circulation among Sicilian stray animals. The genetic relatedness between cat and human isolates suggests a possible common infection source.

Leptospira in Slaughtered Fattening Pigs in Southern Italy: Serological Survey and Molecular Typing.

Leptospirosis is a re-emerging zoonosis of worldwide significance; a wide spectrum of wild and domestic animal species act as natural or accidental hosts. Swine can act as maintenance or accidental hosts of pathogenic Leptospira spp. This study aimed at investigation of Leptospira spp. prevalence and diversity in slaughtered pigs in southern Italy (Sicily). In total, 55 samples of kidneys and blood were collected. Microscopic agglutination test and real-time PCR were performed to detect pathogenic and intermediately pathogenic Leptospira. Partial rpoB gene sequencing and multi-locus sequence typing (MLST) were performed to characterize Leptospira species. The analysis showed a seropositivity rate of 16.4%, with Australis representing the most frequently identified serogroup (63.64%); Pomona and Sejroe were detected with a prevalence of 27.27% and 9.09%, respectively. Pathogenic Leptospiral DNA was detected in 2 kidney samples (3.64%). Leptospira were identified through MLST as L. borgpetersenii serovar Tarassovi (serogroup Tarassovi). Obtained data confirmed the presence of Leptospira infection among pigs in southern Italy, suggesting that management of these animals may be considered an occupational risk for humans.

Leptospira interrogans Serogroup Pomona in a Dairy Cattle Farm in a Multi-Host Zootechnical System.

Bovine leptospirosis is an infectious zoonotic disease causing reproductive problems and economic losses in livestock. This work reports, for the first time in Sicily (South Italy), an outbreak of Leptospira interrogans serogroup Pomona that occurred in cattle farms within the Nebrodi Park and was mainly characterized by full-term abortion. Blood and urine samples were collected at different time points from animals of six different farms (Farms A-F) sharing the same grazing area. Research of antibodies against pathogenic Leptospira species in serum samples was carried out via Micro Agglutination Test (MAT). Urine samples were subjected to pathogen isolation and molecular analyses via TaqMan Real Time-PCR. Genotyping of Leptospira species was obtained by Multi-locus sequence typing. MAT detected antibodies against Leptospira interrogans serogroup Pomona in serum samples of all the farms. Pathogenic Leptospira spp. DNA and culture isolation was obtained from urine samples. Genotyping confirmed the excretion of L. interrogans serogroup Pomona. This study describes clinical manifestations, diagnostic implications and epidemiological characteristics of an outbreak in cattle due to L. interrogans Pomona in a protected multi-host area, where domestic and wild animals share the same habitat, suggesting a role of wild species in transmission and persistence of Pomona serogroup among cattle.

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.

Multispacer sequence typing of Coxiella burnetii from milk and hard tick samples from ruminant farms in Lebanon.

his study was carried out to detect and characterize Coxiella burnetii in ruminant milk samples and in different tick species from seropositive farms in four Lebanese regions. Milk and tick samples were screened for C. burnetii presence by quantitative real-time PCR (qPCR) targeting IS1111 region followed by multispacer sequence typing (MST). The overall positive percentages of 9.6% (27/282) and 95.45% (84/88) for C. burnetii were recorded in ruminant milk and tick samples, respectively. In detail, the C. burnetii DNA was recorded in 52/54 (96.3%) of Rhipicephalus annulatus, 20/21 (95.24%) of Rhipicephalus turanicus, 6/6 (100%) of Hyalomma anatolicum, 5/6 (83.3%) of Rhipicephalus sanguineus and 1/1 of Rhipicephalus bursa. After genotyping of some IS1111-positive samples (17/111), different MST genotypes were identified. Out of 15 positive ticks, 10 were infected with MST2 genotype, 4 were infected with MST7 genotype and 1 was infected with MST57. Moreover, genotypes MST20 and MST58 were found in one cow and one goat milk samples, respectively. The present study confirmed the high genetic diversity of C. burnetii in Lebanon.

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.

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.

A promising new ELISA diagnostic test for cattle babesiosis based on Babesia bigemina Apical Membrane Antigen-1.

Babesiosis due to Babesia bigemina is a relevant tick-borne disease, affecting cattle worldwide. Many surface proteins of the pathogen including the Apical Membrane Antigen 1 (AMA-1) - have been analysed for vaccine and diagnostic purposes. This study focused on B. bigemina AMA-1 and on its use for the assessment of diagnostic tests. After bioinformatic analyses, AMA-1 codifying region was amplified and cloned into an expression vector used to induce protein synthesis in Escherichia coli cells. AMA-1 was purified by affinity chromatography and used to set up the best condition for an ELISA protocol. Bovine field sera positive to B. bigemina were used to evaluate the presence of anti-AMA-1 antibodies. In order to verify the assay specificity, sera positive to Babesia bovis or to the piroplasm Theileria annulata were also included. Significant differences were obtained between sera negative to both B. bigemina and B. bovis and samples positive to B. bigemina, to B. bovis or to both pathogens. No significant reaction was observed with T. annulata positive sera. The results showed that AMA-1 protein is suitable to be used as antigen in diagnostic assays for babesiosis diagnosis in cattle, as it does not show any cross reaction with anti-T. annulata antibodies.

Development and validation of two PCR tests for the detection of and differentiation between Anaplasma ovis and Anaplasma marginale.

Anaplasma ovis and Anaplasma marginale are tick-transmitted bacteria that cause anaplasmosis in domestic and wild animals. Recent results show that some domestic and wild animals and ticks are susceptible to both A. ovis and A. marginale, thus supporting the need to differentiate between these species in hosts and ticks diagnosed with Anaplasma infection. However, although anaplasmosis is one of the most common diseases of grazing animals worldwide, rapid and effective tests are not available for the detection of and discrimination between these 2 Anaplasma species. The objective of this research was to develop an easy and reliable method to identify and discriminate between the closely related pathogens A. ovis and A. marginale. A. ovis and A. marginale major surface protein 4 (msp4) gene sequences were retrieved from different geographic strains and aligned to design 2 sets of primers in a region with significant differences between the 2 species, but completely conserved among strains. PCR reactions using these primers were 100% species-specific and detected all strains from each pathogen previously identified with other methods. The 2 sets of primers designed for the specific PCR amplification of A. ovis and A. marginale allow easy-to-detect and discriminate between the 2 pathogens, thus avoiding the time-consuming sequencing or multi-gene amplification procedures. This PCR provides a tool for the detection of A. ovis and A. marginale in ticks and in wildlife and domestic hosts.