MANAGEMENT OF A FATAL OUTBREAK OF STRONGYLOIDIASIS IN A CAPTIVE POPULATION OF PANTHER CHAMELEONS (FURCIFER PARDALIS) WITH IVERMECTIN.

An outbreak of the nematode Strongyloides sp. occurred in a population of 18 male and 29 female panther chameleons (Furcifer pardalis) at the Singapore Zoo. The parasite was first detected in one individual during routine microscopic examination of feces using the direct examination and magnesium sulfate flotation methods. The parasite was later found to have a closest match (98.96%) with Strongyloides sp. Okayama by DNA sequencing. Over a period of 6 mon, 97.9% (46/47) of the panther chameleons tested positive for the parasite, and 25.5% (12/47) of the animals died due to the disease. All the animals that died were female. Of the positive tests, magnesium sulfate flotation identified the parasite 98.1% (105/107) of the time, compared to direct fecal microscopy, which identified the parasite only 43.9% (47/107) of the time. Parasite eggs were detected in 100% (105/105) of the positive magnesium sulfate flotation tests but only 66.0% (31/47) of the positive direct fecal microscopy tests. Parasite larvae were detected in 61.7% (29/47) of the positive direct fecal microscopy tests but only 9.5% (10/105) of the magnesium sulfate flotation tests. Treatments with fenbendazole and pyrantel pamoate at published doses were ineffective at eliminating the parasite. Ivermectin (0.2 mg/kg PO q2wk for two doses) was successful at treating the parasite, with all animals testing negative for the parasite at the end of the treatment course without any observed adverse reactions. However, complete eradication of the parasite could not be achieved, as Strongyloides sp. could still be detected in the population on routine coproscopy intermittently over 3 yr. There were no further mortalities due to the disease with prompt treatment with ivermectin. Strongyloidiasis may cause high morbidity in panther chameleons, but severe disease leading to mortality can be prevented with the use of ivermectin.

Multipathogen infections and multifactorial pathogenesis involved in noble pen shell (Pinna nobilis) mass mortality events: Background and current pathologic approaches.

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell (Pinna nobilis), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae, although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae. Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei. The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis. The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

The long-distance relationship between Dirofilaria and the UK: case report and literature review.

Over the last two decades, vector-borne pathogens (VBPs) have changed their distribution across the globe as a consequence of a variety of environmental, socioeconomic and geopolitical factors. Dirofilaria immitis and Dirofilaria repens are perfect exemplars of European VBPs of One Health concern that have undergone profound changes in their distribution, with new hotspots of infection appearing in previously non-endemic countries. Some areas, such as the United Kingdom, are still considered non-endemic. However, a combination of climate change and the potential spread of invasive mosquito species may change this scenario, exposing the country to the risk of outbreaks of filarial infections. Only a limited number of non-autochthonous cases have been recorded in the United Kingdom to date. These infections remain a diagnostic challenge for clinicians unfamiliar with these "exotic" parasites, which in turn complicates the approach to treatment and management. Therefore, this review aims to (i) describe the first case of D. repens infection in a dog currently resident in Scotland, (ii) summarise the available literature on Dirofilaria spp. infections in both humans and animals in the United Kingdom and (iii) assess the suitability of the United Kingdom for the establishment of these new VBPs.

Molecular characterisation of Giardia duodenalis from human and companion animal sources in the United Kingdom using an improved triosephosphate isomerase molecular marker.

Giardia duodenalis is a protozoan parasite known for its ability to cause gastrointestinal disease in human and non-human mammals. In the UK, the full impact of this parasite has yet to be fully explored, due to the limited testing which has been undertaken in humans and the low-resolution assemblage-typing methods currently available. Rather than being primarily a travel-associated condition, a recent study has highlighted that an endemic Giardia cycle is present in the UK, although the source of human disease is unclear in the majority of cases. This study focussed on the improvement of one of the commonly used assemblage-typing assays, a nested topoisomerase phosphate (tpi) PCR, to increase the amplification success rate across both human and companion animal samples. After comparing published primers to full Giardia reference genomes, this marker protocol was optimised and then deployed to test a substantial number of human (n â€‹= â€‹79) and companion animal (n â€‹= â€‹174) samples to gain an insight into the molecular epidemiology of Giardia in the UK. One assemblage A1 and eleven assemblage A2 genotypes were detected in humans, along with and 25 assemblage B genotypes. Assemblage A1 genotypes, known to be human-infective, were found in three feline and one canine sample, while one feline sample contained assemblage A2. Additionally, four feline samples contained assemblage B, which is recognised as potentially human-infective. This study demonstrates the presence of potentially human-infective Giardia genotypes circulating in the companion animal population, notably with 17.4% (8/46) of feline-derived Giardia strains being potentially zoonotic. Using a modified tpi-based genotyping assay, this work highlights the potential for domestic pets to be involved in the endemic transmission of giardiasis in the UK and underlines the need for appropriate hygiene measures to be observed when interacting with both symptomatic and asymptomatic animals. It also serves to underline the requirement for further studies to assess the zoonotic risk of Giardia associated with companion animals in high-income countries.

Dirofilaria immitis infection in the Pelagie archipelago: The southernmost hyperendemic focus in Europe.

Dirofilaria immitis and Dirofilaria repens are mosquito-borne filarioids of zoonotic concern, which primarily cause canine heartworm disease (HWD) and subcutaneous dirofilariosis, respectively. The geographical distribution of these nematodes is constantly changing, due to many factors such as the colonization by new invasive mosquito species, the increased movement of animals and the absence of chemoprophylactic strategies in non-endemic regions. Although HWD has always been considered endemic in northern Italy, an increase in the prevalence of this disease has been recorded in the last decades in central and southern regions. We describe the southernmost hyperendemic European focus of heartworm disease in the Pelagie archipelagos. From June to November 2020, 157 dogs and 46 cats were sampled in Linosa and Lampedusa islands for the detection of Dirofilaria immitis and Dirofilaria repens by modified Knott's, rapid antigen and molecular tests. Mosquitoes were collected using CDC light and BG sentinel-2 traps and aspirators, and tested for host blood meal and Dirofilaria spp. DNA. Out of 56 dogs in Linosa and 101 in Lampedusa, 33 (i.e. 58.9%) and 8 (i.e. 7.9%) were positive to D. immitis and D. repens, respectively. Three cats scored positive to D. immitis (i.e. 17.6%) in Linosa. Six mosquito species were identified, and the abdomen of a non-engorged Aedes albopictus was positive for D. immitis and human DNA. The results suggest that D. immitis infection could spread to new previously non-endemic territories in southern Europe, representing a real threat to animal and human health.

Incidence of Dirofilaria immitis and Leishmania infantum infections in sheltered dogs from Southern Italy.

Leishmania infantum and Dirofilaria immitis are among the most important vector-borne pathogens in Europe, affecting animal and human health. In endemic areas, the epidemiology of both infections is conditioned by abundance of vectors and chemoprophylaxis measures. However, knowledge on the incidence of heartworm (HW) and Leishmania infections occurring in sympatry is still scant. Thus, this study aimed to evaluate the incidence of both infections in two dog shelters from southern Italy, which represent hotspots for these two diseases. In June and in October 2020, all dogs that previously scored negative for L. infantum (n = 111, site 1; n = 70, site 2) and D. immitis (n = 58, site 1; n = 61, site 2) in 2019 were tested for the estimation of the incidence of both infections. Anti-L. infantum IgG was detected by immunofluorescence antibody test, whereas D. immitis infection was diagnosed by modified Knott's test, SNAP 4Dx Plus test and real-time PCR. The overall D. immitis and L. infantum infection incidence values were both higher in site 2 (i.e. 63.9% and 10%, respectively) than site 1 (i.e. 39.7% and 1.8%, respectively). The dog shelter in site 2 was shown to be more suitable for the development of the mosquito/sand fly populations and, consequently, for the spreading of both parasites representing a potential threat for animal and human health. The high incidence of both infections recorded in this study suggests the need for chemoprophylaxis measures and vector monitoring and control to minimize the risk for animals and humans living in shelters or in their neighbourhoods.

Efficacy of afoxolaner for the treatment of ear mite infestation under field conditions.

Otodectes cynotis is a commonly occurring surface mite that can be easily transmitted between suitable hosts, including dogs, causing otocariosis. The activity of the systemic insecticide afoxolaner against O. cynotis has been tested once under experimental conditions, showing a high efficacy. The present study aimed to i) assess the efficacy of two consecutive monthly oral administrations of afoxolaner (NexGard®) against O. cynotis in naturally infested dogs under field conditions and ii) evaluate its impact in reducing bacteria or fungal secondary infections. Dogs, positive for O. cynotis (n = 20), were included in the study and allocated in two groups of ten animals each (G1, control group, and G2, treated group). The first group of ear mite-infested dogs was treated with a placebo, while afoxolaner was administered orally to the second group of dogs at Day 0 (D0) and Day 30 (D30), following label instructions. Otoscopic assessments, deep-swab method and swab samples were performed on all dogs (Days 0, 30, 42) to evaluate the presence or absence of live mites and their number throughout the study, as well as to conduct bacterial and fungal assessments. No adverse events likely related were recorded throughout the study. By Day 42 (D42), all dog's ears were flushed to recover ear mites. All treated dogs became negative, as well as two dogs of the control group. The treatment efficacy of afoxolaner was 100 % based on the arithmetic means of the live mite counts. The clinical scores did not change significantly in the control group, whereas they significantly improved in the treated one from D0 to D30 (p-value = 5.47 10-5). No live mites were present in the afoxolaner-treated group at D42 (p-value = 0.00073). In this field study, two oral administrations of afoxolaner at the recommended dose allowed a complete cure of the infestation. Bacterial and Malassezia pachydermatis infections were detected in both groups, although no significant trend was associated to the ear mite treatment.

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.

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.

Dirofilarioses in two cats in southern Italy.

Two cats infected by Dirofilaria immitis and Dirofilaria repens, respectively, were taken to two different private practitioners for a clinical examination. The analyses conducted on the first cat revealed a microfilaraemia due to D. repens of 66 mfs/mL by a modified Knott's test. No clinical signs of D. repens infection were observed in the cat. The animal was euthanised because of a lymphoma condition, and two adult females of D. repens were found in the subcutaneous tissue of the lumbar and left scapular regions at the post-mortem examination. The second cat showed severe abnormalities in the white blood cells, including eosinophil count. Microfilariae of D. immitis were detected in the blood smear, with an average length (n = 2) of 296.2 µm. These clinical cases represent the first reports of feline dirofilarioses in southern Italy and are indicative of a common occurrence of dirofilarial infection in the local canine population.

Comparison of Diagnostic Tools for the Detection of Dirofilaria immitis Infection in Dogs.

In the last two decades, reports of canine heartworm (HW) infection have increased even in non-endemic areas, with a large variability in prevalence data due to the diagnostic strategy employed. This study evaluated the relative performance of two microtiter plate ELISA methods for the detection of HW antigen in determining the occurrence of Dirofilaria immitis in a dog population previously tested by the modified Knott's test and SNAP 4Dx Plus test. The prevalence of this infection in the sheltered dog population (n = 363) from a high-risk area for HW infection was 44.4% according to the modified Knott's test and 58.1% according to a point-of-care antigen ELISA. All serum samples were then evaluated by a microtiter plate ELISA test performed with and without immune complex dissociation (ICD). The prevalence increased from 56.5% to 79.6% following ICD, indicating a high proportion of samples with immune complexing. Comparing these results to that of the modified Knott's test, the samples negative for microfilariae (mfs) and those positive only for D. repens mfs demonstrated the greatest increase in the proportion of positive results for D. immitis by ELISA following ICD. While the ICD method is not recommended for routine screening, it may be a valuable secondary strategy for identifying HW infections in dogs.

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.

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 (χ2 = 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.

Haplosporidium pinnae associated with mass mortality in endangered Pinna nobilis (Linnaeus 1758) fan mussels.

The fan mussel, Pinna nobilis (Linnaeus 1758), is an endemic bivalve of the Mediterranean basin, protected by international legislation as an endangered species. In the early summer of 2018, a mass mortality event (MME) of P. nobilis was recorded in the Gulf of Taranto (Southern Italy, Ionian Sea). Moribund specimens of P. nobilis were collected by scuba divers and processed by bacteriological, parasitological, histopathological and molecular analyses to investigate the causes of this MME. Different developmental stages (i.e., plasmodia, spores and sporocysts) of a presumptive haplosporidian parasite were observed during the histological analysis in the epithelium and in the lumen of the digestive tubules, where mature spores occurred either free or in sporocysts. The spores presented an operculum and an ovoid shape measuring 4.4 µm (±0.232) in length and 3.6 µm (±0.233) in width. BLAST analysis of an 18SrRNA sequence revealed a high nucleotide similarity (99%) with the reference sequence of Haplosporidium pinnae available in GenBank database. Phylogenetic analysis clustered the sequence of the pathogen in a paraphyletic clade with the reference sequence of H. pinnae, excluding other haplosporidians (i.e., Bonamia and Minchinia genera). Based on data reported, H. pinnae was the causative agent of MME in the populations of P. nobilis sampled in the Ionian Sea, where the conservation of this endangered species is heavily threatened by such a protozoan infection. Further investigations should contribute to knowledge about the life cycle of H. pinnae in order to reduce spread of the pathogen and to mitigate the burden of the disease where P. nobilis is facing the risk of extinction.

Shedding of feline lungworm larvae and their infectivity to snail intermediate hosts after anthelmintic treatment.

Aelurostrongylus abstrusus and Troglostrongylus brevior are snail-transmitted helminths causing respiratory diseases in infected cats. The shedding of feline lungworm L1s and their infectivity to the snail intermediate host, after administration of anthelminthic products to cats, are poorly documented. To assess the efficacy of 8.3% fipronil, 10% (S)-methoprene, 0.4% eprinomectin and 8.3% praziquantel (i.e. eprinomectin formulation) and 10% imidacloprid/1% moxidectin (i.e. moxidectin formulation) against these nematodes and to determine the number of days post-treatment until viable L1s are released in the faeces, 384 animals were screened by faecal examination. Of the 54 positive animals (i.e., 14.1%; 7.3% A. abstrusus, 6.2% T. brevior and 0.5% coinfected), 36 were randomly allocated to four groups. Groups A and B were composed of cats positive for T. brevior and treated with the eprinomectin and with the moxidectin formulations, respectively, whereas cats in groups C and D were positive to A. abstrusus and treated with the eprinomectin and the moxidectin formulations, respectively. Prior to and every day after treatment, faecal samples were analysed by the Baermann technique and the number of larvae per gram of faeces determined, and again four weeks after treatment, to assess the efficacy of a single administration of the products. In addition, to evaluate the pre- and post-treatment infectivity of L1s to snail intermediate hosts, one/two snails per cat were infected with 100 L1s collected from the faeces of enrolled animals and then digested 28 days p.i. Based on L1s faecal counts, the efficacy of the eprinomectin and the moxidectin formulations at 28 days was 100% for both A. abstrusus and T. brevior, with a mean number of days of 7.9 ±â€¯1.2 in group A, 7.8 ±â€¯1.9 in B, 6.9 ±â€¯1.6 in C and 8.9 ±â€¯2.0 in D to become negative. Following the artificial digestion, active L3s of T. brevior and A. abstrusus were found in 160 (87.4%) experimentally infected snails. The results of this study demonstrate that a single administration of the two formulations is effective in the treatment of A. abstrusus and T. brevior infections and that during the post-treatment period live L1s are shed for up to 8.9 ±â€¯2.0 days. L1s of both lungworm species released in the faeces after drug administration are still able to reach the infective larval stage in the infected snails. Hence, preventative measures after the treatment of infected animals should include keeping cats indoors and disposal of their faeces for approximately 10 days to avoid environmental contamination and infection of gastropod intermediate hosts.