Efficacy of afoxolaner (NexGard®) in preventing the transmission of Leishmania infantum and Dirofilaria immitis to sheltered dogs in a highly endemic area

Background: Leishmania infantum and Dirofilaria immitis are among the most important canine vector-borne pathogens (CVBPs) of zoonotic concern in Europe. In endemic areas for both of these CVBPs, the use of systemic ectoparasiticides, such as afoxolaner (NexGard®; Boehringer Ingelheim Animal Health), may have the potential for controlling these infections. The aim of this study was to assess, for the first time, the insecticidal efficacy of NexGard® in decreasing the transmission of D. immitis and L. infantum to sheltered dogs living in a hyperendemic area, compared to the year before treatment, as well as its impact on the abundance of mosquito and sand fly populations. Methods: All dogs (n = 179) enrolled in the study were divided into two groups based on their infection status at enrollment: a non-infected group (G1) and an infected group (G2; infected with D. immitis, L. infantum or both). The study was conducted from March 2020 to March 2021. In order to exclude all animals infected with L. infantum and D. immitis before March 2020 (sampling time: T0), dogs in G1 were sampled in June (T1; i.e. T0 + 90 days) and in October 2020 (T2; i.e. T0 + 210 days). From March to September 2020, all animals (G1 and G2) were weighed and treated monthly with NexGard®. Animals in G1 were tested for the last time in March 2021 (T3; i.e. T0 + 330 days) for assessing post-treatment incidence rate of infection and prevention efficacy. Results: The post-treatment incidence of D. immitis was 3.7% (1/27; 95% confidence interval [CI]: 0.2–18.1) and that of L. infantum was 3.6% (3/83; 95% CI: 1.0–10.1). Considering the annual incidence in 2019 and 2020, the protective efficacy against D. immitis and L. infantum infections was 94.2 and 64%, respectively. Of the female mosquitoes collected (n = 146), only one pool out of 50 tested positive for D. immitis DNA, whereas out of 1252 female Sergentomya minuta specimens collected, only four tested positive for L. infantum (0.3%). Conclusions: Afoxolaner is efficacious in decreasing the rate of transmission of both D. immitis and L. infantum; however, comparison of the pre- and post-treatment period demonstrated that there was a significant difference only in the seasonal incidences of D. immitis infection. Preventive measures are recommended throughout the year in endemic areas to reduce the risk of pathogen transmission to animals and humans. Graphical abstract: [Figure not available: see fulltext.]

Sequence based typing of Legionella pneumophila sg 1 isolated in nosocomial acquired infections in Apulia, Southern Italy

Objective. The present report aims to molecularly characterize seven clinical L. pneumophila (L. pn.) sg 1 isolated from noso- comial acquired infections in Apulia region, using the European Working Group on Legionella Infections (EWGLI), sequence- based typing (SBT) and amplified fragment length polymorphism (AFLP) protocols and to compare the identified sequence types (STs) with those available in the EWGLI database. Methods. In the period, January 2000 - December 2012, 151 cases (136 of community and 15 of nosocomial origin) of Legion- naires? disease were notified to the Regional Center for Epide- miology. With regard to nosocomial cases, 8 were confirmed by the isolation of Legionella spp. from respiratory secretions. These clinical isolates were characterized by amplified fragment length polymorphism (AFLP) and sequence-based typing (SBT), using the EWGLI standardized protocol. Results. The clinical isolates belong to ST42, ST23 and ST1. The AFLP confirms the SBT results. Comparing the STs herein detected with those already in the EWGLI SBT database, the 3 STs are frequent in other European countries. Conclusions. The molecular analysis demonstrates that the 3 STs are the most frequent in Italy and in Europe, supporting the hypothesis that some specific L. pn. sg 1 clones have gained widespread dissemination probably due to a common ecological niche. Further researches are required to investigate the potential changing incidence of STs and the fitness of emerging strains or clonal groups in environmental strains

Leishmania infantum and Dirofilaria immitis infections in Italy, 2009-2019: Changing distribution patterns

Background: For long time, canine leishmaniosis (CanL) was considered endemic in the southern, central, and insular regions of Italy, whereas heartworm disease (HW) caused by Dirofilaria immitis was considered endemic in the northern region and in the swampy Po Valley. Following the reports of new foci of both diseases, in this study we update the distribution patterns and occurrence of new foci of CanL and HW discussing the main drivers for the changes in the epidemiology of these two important zoonotic canine vector-borne diseases. Methods: Based on the statistical analyses of serological assays (n = 90,633) on L. infantum exposure and D. immitis infection performed by two reference diagnostic centres in Italy over a ten-year period (2009-2019) irrespective of the anamnesis of dogs. The distribution patterns of both parasites are herein presented along with the occurrence of new foci. Results: Results highlighted the changing distribution patterns of L. infantum vs D. immitis infection in Italy. CanL is endemic in some areas of northern regions and HW has endemic foci in central and southern regions and islands. Significant differences in L. infantum exposure and HW infection prevalence among the study macroareas were detected. The overall results of the positive tested samples were 28.2% in southern Italy and islands, 29.6% in central Italy and 21.6% in northern Italy for L. infantum and 2.83% in northern Italy, 7.75% in central Italy and 4.97% in southern Italy and islands for HW. HW positivity significantly varied over years (χ 2 = 108.401, df = 10, P < 0.0001), gradually increasing from 0.77% in 2009 to 8.47% in 2016-2017. Conclusions: New potential epidemiological scenarios are discussed according to a range of factors (e.g. environmental modifications, occurrence of competent insect vectors, transportation of infected animals to non-endemic areas, chemoprophylaxis or vector preventative measures), which may affect the current distribution. Overall, the results advocate for epidemiological surveillance programmes, more focussed preventative and control measures even in areas where few or no cases of both diseases have been diagnosed.[Figure not available: see fulltext.

Invasive fungal infections in Neonatal Intensive Care Units of Southern Italy: a multicentre regional active surveillance (AURORA Project)

Introduction. During the past years invasive fungal infections (IFIs) have become an increasingly important problem in infants hospitalized in the Neonatal Intensive Care Unit (NICU). Candida species is the third most-common agent of late-onset infections in critically ill neonates, with an estimated incidence of 2.6-10% in very low birth weight and 5.5-20% in extremely low birth weight infants. The aim of this observational study is to evaluate the epidemiology of IFIs among infants admitted to NICUs of one Italian region by a multicenter surveillance (Aurora Project). Methods. The IFIs surveillance was carried out prospectively in Apulia (Southern Italy) between February 2007 and August 2008. This report focuses on the results from 6 enrolled NICUs. Results. Twenty-one neonates developed IFIs: the overall incidence was 1.3% and crude mortality was 23.8%. Infants weighing ? 1500g (4.3%) showed a significantly higher incidence than those ? 2500g (0.2%). C.parapsilosis (61.9%) was the most frequent isolated species. The main potential risk factors were having a central venous catheter placed, length of stay in NICU > 7 days and total parenteral nutrition for > 5 days. The (1,3)-Ã-D glucan (BDG), mannan antigens and anti-Candida antibodies? evaluation was performed in 7 neonates. All neonates were positive to the BDG; the mannan antigen result was positive in 5 newborns, the anti-mannan antibodies were always negative. All isolates were amphotericin B and fluconazole-susceptible. Discussion. This first prospective study on neonatal fungal infection in one Italian region gives evidence of a preponderance of non-albicans Candida spp and indicates potential utility of BDG as an adjunct diagnostic test

Detection of Leishmania tarentolae in lizards, sand flies and dogs in southern Italy, where Leishmania infantum is endemic: hindrances and opportunities

Background: Leishmania tarentolae is a protozoan isolated from geckoes (Tarentola annularis, Tarentola mauritanica), which is considered non-pathogenic and is transmitted by herpetophilic Sergentomyia spp. sand flies. This species occurs in sympatry with Leishmania infantum in areas where canine leishmaniasis is endemic. In the present study, we investigated the circulation of L. tarentolae and L. infantum in sand flies, dogs and lizards in a dog shelter in southern Italy, where canine leishmaniasis by L. infantum is endemic. Methods: Sheltered dogs (n = 100) negative for Leishmania spp. (March 2020) were screened by immunofluorescence antibody test (IFAT) using promastigotes of both species at two time points (June 2020 and March 2021). Whole blood from dogs, tissues of Podarcis siculus lizards (n = 28) and sand flies (n = 2306) were also sampled and tested by a duplex real-time PCR (dqPCR). Host blood meal was assessed in sand flies by PCR. Results: Overall, 16 dogs became positive for L. infantum and/or L. tarentolae by IFAT at one or both sampling periods. One canine blood sample was positive for L. infantum, whilst two for L. tarentolae by dqPCR. At the cytology of lizard blood, Leishmania spp. amastigote-like forms were detected in erythrocytes. Twenty-two tissue samples, mostly lung (21.4%), scored molecularly positive for L. tarentolae, corresponding to 10 lizards (i.e., 35.7%). Of the female Sergentomyia minuta sampled (n = 1252), 158 scored positive for L. tarentolae, four for L. infantum, and one co-infected. Two Phlebotomus perniciosus (out of 29 females) were positive for L. tarentolae. Engorged S. minuta (n = 10) fed on humans, and one P. perniciosus, positive for L. tarentolae, on lagomorphs. Conclusions: Dogs and lacertid lizards (Podarcis siculus) were herein found for the first time infected by L. tarentolae. The detection of both L. tarentolae and L. infantum in S. minuta and P. perniciosus suggests their sympatric circulation, with a potential overlap in vertebrate hosts. The interactions between L. tarentolae and L. infantum should be further investigated in both vectors and vertebrate hosts to understand the potential implications for the diagnosis and control of canine leishmaniasis in endemic areas. Graphical abstract: [Figure not available: see fulltext.

Leishmania tarentolae and leishmania infantum in humans, dogs and cats in the pelagie archipelago, southern italy

Visceral leishmaniasis (VL) caused by Leishmania infantum is endemic in the Mediterranean basin with most of the infected human patients remaining asymptomatic. Recently, the saurian-associated Leishmania tarentolae was detected in human blood donors and in sheltered dogs. The circulation of L. infantum and L. tarentolae was investigated in humans, dogs and cats living in the Pelagie islands (Sicily, Italy) by multiple serological and molecular testing. Human serum samples (n = 346) were tested to assess the exposure to L. infantum by immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) and to L. tarentolae by IFAT. Meanwhile, sera from dogs (n = 149) and cats (n = 32) were tested for both Leishmania species by IFAT and all blood samples, including those of humans, by specific sets of real time-PCR for L. infantum and L. tarentolae. The agreement between serological tests performed for human samples, and between serological and molecular diagnostic techniques for both human and animal samples were also assessed. Overall, 41 human samples (11.8%, 95% CI: 8.9–15.7) were positive to L. infantum (5.2%, 95% CI: 3.3–8.1), L. tarentolae (5.2%, 95% CI: 3.3–8.1) and to both species (1.4%, 95% CI: 0.6–3.3) by serology and/or molecular tests. A good agreement among the serological tests was determined. Both Leishmania spp. were serologically and/or molecularly detected in 39.6% dogs and 43.7% cats. In addition to L. infantum, also L. tarentolae circulates in human and animal populations, raising relevant public health implications. Further studies should investigate the potential beneficial effects of L. tarentolae in the protection against L. infantum infection

Clinical, haematological and biochemical findings in tigers infected by leishmania infantum

Background: A large number of animal species are susceptible to Leishmania infantum (Kinetoplastida, Trypanosomatidae) in endemic areas, including domestic and wild felids such as tigers (Panthera tigris). Knowledge on the infection of this endangered species is still at its infancy, and therefore this study aims to identify clinical presentation and clinicopathological findings of tigers naturally infected by L. infantum. Results: Tigers either L. infantum-positive (group A) or -negative (group B) were apparently healthy or presented visceral leishmaniasis unrelated conditions, except for one animal in which a large non-healing cutaneous lesion was observed. However, histological exam and immunohistochemistry carried out on the lesion excluded the presence of L. infantum amastigotes. Biochemical analysis showed that the average concentration of total proteins, globulins and haptoglobin were significantly higher (p < 0.01, p = 0.01 and p = 0.02, respectively), while the albumin/globulin ratio significantly lower (p = 0.05) in group A compared with group B. The biochemical alterations were partially confirmed by the serum protein electrophoresis results revealing a significant increase in the total protein value (p = 0.01) and hypergammaglobulinemia (p = 0.03) but an unmodified albumin/globulin ratio in group A. Conclusions: In this study tigers infected by L. infantum have shown to be mainly asymptomatic. The absence of clinical signs may lead veterinarians to overlook leishmaniasis in animals kept in captivity. Therefore, diagnostic and screening tests as serology should be part of routinely surveillance programs to be performed on tigers in zoological gardens located in endemic areas. Though only few protein-related laboratory abnormalities were recorded in infected animals, they could provide diagnostic clues for a first suspicion of L. infantum infection in tigers. Indeed, considering the high risk of zoonotic transmission in heavily frequented environment as zoos, a prompt diagnosis of L. infantum infection is of pivotal importance

Ectoparasites of hedgehogs: From flea mite phoresy to their role as vectors of pathogens

Hedgehogs are synanthropic mammals, reservoirs of several vector-borne pathogens and hosts of ectoparasites. Arthropod-borne pathogens (i.e., Rickettsia spp., Borrelia spp., and Anaplasmataceae) were molecularly investigated in ectoparasites collected on hedgehogs (n = 213) from Iran (161 Hemiechinus auritus, 5 Erinaceus concolor) and Italy (47 Erinaceus europaeus). In Iran, most animals examined (n = 153; 92.2%) were infested by ticks (Rhipicephalus turanicus, Hyalomma dromedarii), and 7 (4.2%) by fleas (Archeopsylla erinacei, Ctenocephalides felis). Of the hedgehogs infested by arthropods in Italy (i.e., 44.7%), 18 (38.3%) were infested by fleas (Ar. erinacei), 7 (14.9%) by ticks (Haemaphysalis erinacei, Rh. turanicus, Rhipicephalus sanguineus sensu lato), and 6 (12.8%) by mites (Caparinia tripilis, Acarus nidicolous, Ornithonyssus spp.). Phoretic behavior of C. tripilis on Ar. erinacei was detected in two flea specimens from Italy. At the molecular analysis Rickettsia spp. was detected in 93.3% of the fleas of Italy. In Iran, Rickettsia spp. was detected in 8.0% out of 212 Rh. turanicus ticks, and in 85.7% of the Ar. erinacei fleas examined. The 16S rRNA gene for Ehrlichia/Anaplasma spp. was amplified in 4.2% of the 212 Rh. turanicus ticks. All sequences of Rickettsia spp. from fleas presented 100% nucleotide identity with Rickettsia asembonensis, whereas Rickettsia spp. from Rh. turanicus presented 99.84%–100% nucleotide identity with Rickettsia slovaca, except for one sequence, identical to Rickettsia massiliae. The sequences of the 16S rRNA gene revealed 99.57%–100% nucleotide identity with Anaplasma spp., except for one, identical to Ehrlichia spp. A new phoretic association between C. tripilis mites and Ar. erinacei fleas has been herein reported, which could be an important route for the spreading of this mite through hedgehog populations. Additionally, spotted fever group rickettsiae were herein detected in ticks and fleas, and Anaplasma/Ehrlichia spp. in ticks, suggesting that hedgehogs play a role as reservoirs for these vector-borne pathogens

Hyperendemic Dirofilaria immitis infection in a sheltered dog population: an expanding threat in the Mediterranean region

A study on the occurrence of Dirofilaria immitis and its vectors was carried out in order to assess the prevalence of the disease in dogs in previously non-endemic areas of southern Italy. Blood samples (n = 385) and mosquitoes (n = 1540) were collected in two dog shelters and analysed by Knott's test and duplex real-time PCR, respectively. Dirofilaria immitis was the most prevalent filarioid (44.2%), while Culex pipiens was the most prevalent mosquito species (68.8%). This high prevalence of D. immitis infection confirms this location as one of the most hyperendemic foci of dirofilariosis in Europe