Epidemiological and molecular updates on hookworm species in dogs from southern Italy.

BACKGROUND: The zoonotic hookworms Ancylostoma caninum and Uncinaria stenocephala are widespread soil-transmitted helminths in dogs in Europe. Given the veterinary and public health importance of hookworms in dogs and the recent changes in the molecular epidemiology of some species, there is a need to continuously monitor the epidemiological and molecular prevalence of these parasites also at the "local" level. The present study aimed to update the epidemiological scenario of hookworm infections in both owned and stray dogs in southern Italy and to discriminate between different hookworm species (A. caninum and U. stenocephala) through molecular analyses. For this purpose, a retrospective analysis was performed over 10 years (2011-2021), including a total of 7008 owned dogs and 5642 stray dogs referred to our laboratory for copromicroscopic examinations. Moreover, 72 faecal samples, from dogs naturally infected by hookworms, were used to discriminate between A. caninum and U. stenocephala using two PCR protocols. Prior to molecular analyses, a subsample of 40/72 positive faecal samples was used for morphometric investigations on hookworm eggs. RESULTS: The results of the ten-year retrospective analysis (2011-2021) showed an overall prevalence of hookworm infection of 9.16%, specifically 5.1% in owned dogs and 14.2% in stray dogs. Logistic regression showed a significant association between positivity to hookworms and the variable "puppies" both in stray (13.84%; OR = 2.4) and owned (7.07%; OR = 2.2) dogs. The results of molecular analyses showed that positivity was confirmed only in 21/72 samples, specifically, 6 samples using protocol A and 19 with protocol B. Sequencing revealed 15 samples positive to U. stenocephala and 6 to A. caninum. CONCLUSIONS: The findings of this study showed a high prevalence of hookworm infections in dogs in southern Italy, updating the epidemiological scenario of the last decade. Moreover, the results of the study revealed the first identification of hookworm species in dogs in Italy by molecular studies, highlighting that U. stenocephala is more prevalent than A. caninum.

Efficacy of topical administration of prallethrin-permethrin-piperonyl butoxide (Bronco® Equine Fly Spray) for the treatment and control of flies and other nuisance insects of horses.

Numerous biting and nuisance insects are a noted cause of discomfort and stress to horses. Pyrethrins and pyrethroids have been used for many years in numerous formulations for the control of insect pests in animals, humans and environment. There are, however, few studies reporting their field efficacy in horses. The aim of the present study was to evaluate the repellent activity of a spray formulation based on prallethrin and permethrin synergized with piperonyl butoxide (BRONCO® Equine Fly Spray, Farnam Companies, Inc., USA) against annoying and harmful insects for horses in field conditions. Nine horses of mixed breed were divided into 2 groups (treatment and control). Pre-treatment insect counts were compared to daily counts for 4 days post-treatment (pt). One minute after the administration of the product (day 0), all the horses were negative for the presence of insects. All counts up to the 6-h pt check remained negative for Hippobosca equina, tabanid flies and Simulium spp., showing 100% efficacy. This remained above 90% throughout the study. For the H. equina, the repellent efficacy remained > 99.7% for all 4 days pt, for tabanid flies > 93.3% and for Simulium spp. > 97.4%. The efficacy against Musca spp. decreased from 82.2% at day 0 to 62.2% at day 3. Treatment was well-tolerated. In conclusion, despite the low number of tested horses, Bronco® has demonstrated high insecticide and repellent efficacy and a good persistence, maintained for up to 4 days post-treatment, against the most common species of insects harmful for horses.

The Kubic FLOTAC microscope (KFM): a new compact digital microscope for helminth egg counts.

The Kubic FLOTAC microscope (KFM) is a compact, low-cost, versatile and portable digital microscope designed to analyse fecal specimens prepared with Mini-FLOTAC or FLOTAC, in both field and laboratory settings. In this paper, we present the characteristics of the KFM along with its first validation for fecal egg count (FEC) of gastrointestinal nematodes (GINs) in cattle. For this latter purpose, a study was performed on 30 fecal samples from cattle experimentally infected by GINs to compare the performance of Mini-FLOTAC either using a traditional optical microscope (OM) or the KFM. The results of the comparison showed a substantial agreement (concordance correlation coefficient = 0.999), with a very low discrepancy (−0.425 ± 7.370) between the two microscopes. Moreover, the KFM captured images comparable with the view provided by the traditional OM. Therefore, the combination of sensitive, accurate, precise and standardized FEC techniques, as the Mini-FLOTAC, with a reliable automated system, will permit the real-time observation and quantification of parasitic structures, thanks also to artificial intelligence software, that is under development. For these reasons, the KFM is a promising tool for an accurate and efficient FEC to improve parasite diagnosis and to assist new generations of operators in veterinary and public health.

Tick-borne pathogens in Ixodidae ticks collected from privately-owned dogs in Italy: a country-wide molecular survey.

BACKGROUND: Ticks and tick-borne diseases are increasingly recognized as a cause of disease in dogs worldwide. The epidemiology of ticks and tick-transmitted protozoa and bacteria has changed due to the spread of ticks to urban and peri-urban areas and the movement of infected animals, posing new risks for animals and humans. This countrywide study reports information on distribution and prevalence of pathogens in ticks collected from privately-owned dogs in Italy. We analyzed 2681 Ixodidae ticks, collected from 1454 pet dogs from Italy. Specific PCR protocols were used to detect i) Piroplasms of the genera Babesia and Theileria, ii) Gram-negative cocci of the family Anaplasmataceae and iii) Borrelia burgdorferi sensu lato. Sequencing of positive amplicons allowed for species identification. RESULTS: Babesia/Theileria spp. DNA was detected in 435 homogeneous tick-pools (Minimum Infection Rate (MIR) = 27.6%; 95% confidence interval (CI) = 25.4-29.8%) with higher prevalence in Ixodes ricinus and Rhipicephalus sanguneus group. The zoonotic B. venatorum was the most prevalent species (MIR = 7.5%; 95% CI = 6.3-9.0%). Anaplasma and Ehrlichia species were detected in 165 tick-pools (MIR = 10.5%; 95% CI = 9.3-11.8%) and specifically, A. phagocytophilum was identified with MIR = 5.1% (95% CI = 4.1-6.3%). Borrelia burgdorferi s.l. and B. afzelii were detected with MIR = 0.4% (95% CI = 0.2-0.8%) and MIR = 0.3% (95% CI 0.1-0.7%) respectively. CONCLUSIONS: Zoonotic pathogens B. venatorum and A. phagocytophilum were the most frequently detected in ticks collected from privately-owned dogs which might be used as markers of pathogens presence and distribution.

Efficacy of fluralaner spot-on solution for the treatment of Ctenocephalides felis and Otodectes cynotis mixed infestation in naturally infested cats.

BACKGROUND: Cats can be infested with several ectoparasite species, especially Ctenocephalides felis and Otodectes cynotis. The aim of this study was to evaluate the efficacy of a single topical application of fluralaner against C. felis and O. cynotis natural infestation in stray (study 1) and owned (study 2) cats in central and southern Italy. RESULTS: The number of live fleas found on each cat on Day 0 ranged from 1 to more than 30 (arithmetic mean live flea count = 11.9 in study 1; 14.6 in study 2) while no live fleas were found on days 7 and 84 post topical application of fluralaner. The number of live mites found on each cat on Day 0 ranged from 1 to 42 (arithmetic mean live mite count = 6.4 in study 1; 8.9 in study 2) while no live mites were found on days 7 and 84 post topical application of fluralaner. CONCLUSIONS: Topical fluralaner completely eliminated fleas and ear mites from infested cats and was 100% effective against both parasites up to 84 days after treatment.

Zoonotic ocular onchocercosis caused by Onchocerca lupi in dogs in Romania.

Onchocerca lupi is a filarial nematode, which infects the scleral conjunctival tissue of dogs, wolves and cats. Whilst adult nematodes localize in the conjunctive tissue of sclera or in the retrobulbar, microfilariae are found in the skin, and they are rarely diagnosed in asymptomatic animals. Since the first report of human ocular infection 5 years ago, up to 10 zoonotic cases have been identified in patients worldwide. We report, for the first time in Romania, three cases of canine ocular onchocercosis in dogs. Fragments of the harvested worms were characterized morphologically and molecularly. This article expands knowledge on the distribution of this parasite in Eastern Europe and sounds an alarm bell for ophthalmologists about the possible occurrence of human cases of O. lupi infection.

Dirofilaria immitis and Angiostrongylus vasorum: the contemporaneous detection in kennels.

BACKGROUND: The cardiopulmonary nematodes Dirofilaria immitis and Angiostrongylus vasorum are increasingly reported in dogs and are responsible for two diseases with overlapping endemic areas, especially in Europe: dirofilariosis and angiostrongylosis. The reasons for their apparent emergence are unknown, but several factors (e.g. increased disease awareness, better diagnostic tools, climatic changes, seasonal population dynamics and movements of animals) may play a role in the recent rise in reports of infection in the various countries of Europe. The aim of this study was to investigate the seroprevalence of D. immitis (by DiroCHECK® ELISA) and the fecal presence of first stage larvae (L1) of A. vasorum (by FLOTAC) in dogs from 68 kennels of the Campania region (southern Italy). The fecal samples were collected from pooled samples using the box as epidemiological unit. To the authors's knowledge, this is the first cross-sectional survey conducted at regional-scale in Italy and in Europe on the contemporaneous detection of D. immitis antigens and A. vasorum L1 in kennels. RESULTS: Antigens of D. immitis were detected in 24/537 (4.4%; 95% Confidence Interval = 3.0-6.7) dogs in 6 out of the 68 kennels (8.8%; 95% CI = 3.6-18.9). The 24 positive samples for D. immitis antigen were tested also with AngioDetect® and only 1 sample was seropositive for A. vasorum with a prevalence of 4.2%. A. vasorum L1 were detected in dogs from 9 out of the 68 kennels (13.2%; 95% CI = 21.8-44.9). Pooled fecal samples from 25 boxes out of the 1360 analyzed resulted positive to A. vasorum L1 (1.8%; 95% CI = 1.2-2.7). CONCLUSIONS: The present study indicates that cardiopulmonary nematodes are present in Campania region in symptomatic dogs as well as in asymptomatic ones. Therefore, regular parasitological surveillance, appropriate treatment strategies and high quality standard of hygiene are required to guarantee the health and welfare of kennel dogs.

Comparison of different molecular protocols for the detection of Uncinaria stenocephala infection in dogs.

The present study aims to assess the performance of different molecular targets using various matrices of samples for the detection of Uncinaria stenocephala (US) in hookworm infected dogs. To this end, the DNA extraction was performed on the following matrices of samples: (i) larvae of US obtained from experimentally infected dogs with US with different larvae counts per microliter (µl); (ii) pure US eggs suspension in distilled water with different egg counts per µl; (iii) spiked dog fecal samples with different US eggs per gram (EPG) of feces; (iv) feces from dogs naturally infected with hookworm eggs; (v) fecal suspension with hookworm eggs recovered from the FLOTAC apparatus. All the samples were tested with four different PCR protocols targeting specific regions for the detection of both hookworms US and AC as follows: Protocol A (ITS1, 5.8 S, ITS2) and Protocol B (18 S) for the detection of both species, Protocol C (ITS1) for the detection of AC and Protocol D (ITS1) for the detection of US. The best results were obtained with DNA extracted from US larvae matrix obtained from experimentally infected dogs, showing a detection limit of 3.5 larvae/ml for the protocols A, B and D. A moderate correlation was found between the FLOTAC technique and PCR protocols B and D with respect to fecal samples from dogs naturally infected with hookworms. Indeed, PCR protocols B (18 S) and D (ITS1) gave the best results for feces and fecal suspension from naturally infected dogs. However, all the PCR protocols used showed lower sensitivity than FLOTAC technique. Perhaps, isolating US eggs in advance could help to obtain better quality and quantity of DNA, avoiding some notable factors such as inhibitors present in faecal samples. However, a further study is needed to evaluate and standardise a protocol for the recovery of parasitic elements, that could be applied prior to DNA extraction. Therefore, this could lead to a better amplification of US eggs DNA. In conclusion, our results showed that the type of sample (sample-matrix) used for the DNA extraction samples is crucial, as this affects the diagnostic sensitivity of the technique.

Genetic variability of Dirofilaria repens isolates from humans and dogs in Italy.

Dirofilaria repens is a paradigmatic example of an emerging vector-borne pathogen (VBP) in both human and veterinary fields. The spatial expansion and the increasing zoonotic impact of this VBP can be related to several drivers including the genetic structure of parasite populations. Italy is one of the European countries traditionally endemic with the highest incidence of canine and human cases of subcutaneous dirofilariosis. The present study aimed to assess the genetic identity and variability of D. repens isolates of human and canine origin from areas of Central Italy, compared with those isolated from different areas of Europe by sequence analysis of mtDNA genes (i.e., 12 S rDNA and cox1). A total of twenty isolates of D. repens were obtained from biopsies of subcutaneous and ocular cases of dirofilariosis occurring in 10 dogs and 10 humans. The sequence analysis of 12 S rDNA showed that all the sequences obtained clustered as a monophyletic group with a strong nodal support, indicating that all sequence types represented D. repens. The cox1 and the 12 S sequence analysis did not show host-related polymorphisms between human and dog-derived specimens. The sequence analysis of cox1 was performed including 8 additional sequences previously obtained from human and canine isolates in the same areas. Out of the 28 sequences analyzed, 20 were grouped in a haplogroup comprising 15 haplotypes (i.e., DR1, DR2, DR4, DR5, DR7, DR8, DR10-DR18), 2 sequences matched to DR9, reported for the first time in Italy, and 6 showed peculiar polymorphisms that were not previously described. The results obtained have implications for a better understanding of the epidemiology and phylogeography of this emerging vector-borne zoonotic parasite.

How Italian practitioners manage dirofilariosis in dogs and cats? Data of a second national survey.

The zoonotic nematodes Dirofilaria immitis and Dirofilaria repens are endemic in many European countries and their distribution is changing. A correct diagnosis is the starting point to reduce their spread and veterinary practitioners represent the first line of defense against these two vector-borne diseases in pets and humans. With the aim to better understand current knowledge by veterinarians and to compare them with the previous survey, an online questionnaire was sent to Italian companion animal veterinary facilities. The overall response rate was 22.3%. Approximately 50% and 5% of facilities have diagnosed at least one case of Dirofilaria spp. in the last year in dogs and cats, respectively. In agreement with international guidelines, of the 665 facilities that responded to the questionnaire, 16.8% used Knott's test in combination with serology (Antigenic test-Ag-) to diagnose heartworm infection in dogs. For the diagnosis of D. repens infection, 13.8% used Knott's test in dogs and cats, and 10.5% used Knott's test only in dogs. Most facilities recommended beginning prevention of canine heartworm disease in April and ending in November. Injectable moxidectin and oral ivermectin were the two most commonly used preventives for D. immitis in dogs, while selamectin was most frequently used in cats. The drug most commonly used for heartworm adulticide treatment in dogs was melarsomine followed by the combined ivermectin-doxycycline treatment. The results of this study suggest that, on the one hand, veterinarians are aware of the recent developments in adulticide therapy, while on the other hand, diagnosis is not always made according to what is recommended by international guidelines and that the start and end times of prevention may not be optimal. Furthermore, the data show an increase in cases of coinfection compared to the previous survey.

Corrigendum: Emerging risk of Dirofilaria spp. infection in shelter dogs in southern Italy.

[This corrects the article DOI: 10.3389/fvets.2023.1112036.].

Emerging risk of Dirofilaria spp. infection in shelter dogs in southern Italy.

In southern Italy, the number of autochthonous cases of Dirofilaria immitis in dogs has increased considerably. This also occurs in the Campania region, particularly in coastal areas, where infections with D. immitis and Dirofilaria repens have been reported more frequently. Therefore the aim of the present study was to better investigate the occurrence of Dirofilaria spp. in a local dog shelter in Castel Volturno (Campania region, southern Italy). Briefly, a total of 260 blood samples were analysed for identification of microfilariae (mff) and detection of Dirofilaria immitis antigen. Dogs were classified according to their age (1-3 years; 4-6 years; 7-11 years; > 11 years) and length of stay in the shelter at the time of sampling (dogs that entered in the shelter in the last 4 months; dogs housed in the shelter for more than 4 months up to 2 years; dogs housed for more than 2 years). The modified Knott's test revealed that 195 dogs (75.0%) were positive for circulating mff of Dirofilaria spp. Specifically, 104/260 (40.0%) dogs were positive for D. immitis and 91/260 (35.0%) were positive for D. repens. In addition, 72/260 (27.7%) dogs had both D. immitis and D. repens mff. Antigen testing revealed that 78/260 (30.0%) dogs were positive for D. immitis. However, 26/104 (25.0%) of the dogs with D. immitis mff were antigen-negative. The overall k concordance between the modified Knott's test and the antigenic test was ≤0.2 (poor) (p = 0.000). The results of the logistic regression model showed a significant association between Dirofilaria exposure and the period of time the dogs had spent in the shelter at the time of sampling. Dogs housed in the shelter for 4 months (group 1) and between 4 months and 2 years (group 2) had higher Dirofilaria positivity than dogs in group 3 (housed for more than 2 years) (80.4% vs. 79.6% vs. 62.4%, respectively). Moreover, male dogs and older dogs (between 7 and 11 years of age) were more likely to be infected with Dirofilaria spp.

Comparison of Mini-FLOTAC, Flukefinder and sedimentation techniques for detection and quantification of Fasciola hepatica and Calicophoron daubneyi eggs using spiked and naturally infected bovine faecal samples.

BACKGROUND: Fasciolosis (Fasciola hepatica) and paramphistomosis (Calicophoron daubneyi) are two important infections of livestock. Calicophoron daubneyi is the predominant Paramphistomidae species in Europe, and its prevalence has increased in the last 10-15 years. In Italy, evidence suggests that the prevalence of F. hepatica in ruminants is low in the southern part, but C. daubneyi has been recently reported at high prevalence in the same area. Given the importance of reliable tools for liver and rumen fluke diagnosis in ruminants, this study evaluated the diagnostic performance of the Mini-FLOTAC (MF), Flukefinder(R) (FF) and sedimentation (SED) techniques to detect and quantify F. hepatica and C. daubneyi eggs using spiked and naturally infected cattle faecal samples. METHODS: Briefly, negative bovine faecal samples were artificially spiked with either F. hepatica or C. daubneyi eggs to achieve different egg count levels: 10, 50 and 100 eggs per gram (EPG) of faeces. Moreover, ten naturally infected cattle farms from southern Italy with either F. hepatica and/or C. daubneyi were selected. For each farm, the samples were analysed individually only with MF technique and as pools using MF, FF and SED techniques. Bayesian latent class analysis (LCA) was used to estimate sensitivity and accuracy of the predicted intensity of infection as well as the infection rate in the naturally infected farms. RESULTS: The outcome of this study showed that the highest number of eggs (F. hepatica and C. daubneyi) recovered was obtained with MF, followed by FF and SED in spiked infected samples at 50 and 100 EPG, while at lower infection levels of 10 EPG, FF gave the best results. Moreover, the sensitivity for all the techniques included in the study was estimated at > 90% at infection levels > 20 EPG for both F. hepatica and C. daubneyi eggs. However, MF was the most accurate of the three techniques evaluated to estimate fluke infection intensity. Nevertheless, all three techniques can potentially estimate infection rate at farm level accurately. CONCLUSIONS: Optimization and standardization of techniques are needed to improve the FEC of fluke eggs.

Efficacy of oral, topical and extended-release injectable formulations of moxidectin combined with doxycycline in Dirofilaria immitis naturally infected dogs.

BACKGROUND: Several studies in both experimentally and naturally infected dogs have reported the adulticide effect of a combination of macrocyclic lactones and doxycycline against Dirofilaria immitis, showing that these protocols can be used as an alternative to melarsomine. The present study evaluated the efficacy of oral, topical and extended-release injectable formulations of moxidectin when combined with doxycycline in dogs naturally infected with D. immitis from a shelter located in southern Italy. METHODS: Thirty dogs with naturally acquired D. immitis infection were divided in three groups (G) and treated with oral moxidectin (G1) once a month for 9 consecutive months, topical moxidectin (G2) once a month for 9 consecutive months or extended release moxidectin injectable (G3) at enrolment and again at 6 months (Day 180). All treatment groups received doxycycline for the first 30 days. Microfilarial concentrations in 1 ml (mff/ml) blood were determined monthly for 9 months with the modified Knott's test. A clinical scoring system was employed for each dog enrolled in the study based on thoracic radiography and cardiac ultrasound (CU) examinations performed at Day - 15 (before treatment) and at Day 180. RESULTS: Results from the present study suggest that the majority of dogs from all treatment groups became antigen negative, as evaluated at Day 270: 9/10 dogs (90.0%) from G1, 6/10 dogs (60.0%) from G2 and 8/10 dogs (80.0%) from G3. Improvement of radiographic alterations was observed in all treatment groups, and almost all dogs were cleared of pulmonary abnormalities by 6 months from the beginning of treatment (P = 0.000). Cardiac ultrasound examination showed a progressive improvement of cardiac function in a limited number of animals (4/30). CONCLUSIONS: The combination of doxycycline and three different formulations of moxidectin leads to antigen-negative status in naturally infected dogs.

New distribution patterns of Dirofilaria immitis in Italy.

In recent decades, the number of autochthonous cases and foci of Dirofilaria immitis in dogs from southern regions has increased considerably, suggesting that the distribution of the species is not limited to northern Italian regions. This epidemiological picture emerges from case reports or studies in specific locations where outbreaks of heartworm disease have occasionally been reported together with the presence of mosquito vectors. To obtain a more comprehensive picture of the current distribution of D. immitis in southern Italy, a multicenter cross-sectional survey of canine filariasis was conducted. Owned and sheltered dogs (n = 1,987) were included in the survey regardless their breed, attitude and/or sex. All included dogs were older than 1 year and had no history of chemoprophylactic treatment against filarioses. A blood sample was collected from enrolled dogs and screened by modified Knott's test and, when positive, tested using D. immitis specific ELISA rapid test (SNAP 4DX, IDEXX). The overall microfilaremia prevalence was 17% (n = 338) being single-species infection (92.6%) more common that mixed (7.4%). Remarkably, D. immitis was the most frequent species detected with an overall prevalence of 11.4% (n = 227), followed by Dirofilaria repens (n = 74; 3.7%), and Acanthocheilonema reconditum (n = 12; 0.6%). Sheltered dogs were significantly more infected by D. immitis, as well as mongrel dogs and animals housed in rural areas. Data here reported indicate that D. immitis is largely present in southern Italy, raising awareness about the necessity of proper screening and chemoprophylactic treatments in exposed animals.

Diversity and geographic distribution of haplotypes of Dirofilaria immitis across European endemic countries.

BACKGROUND: Dirofilaria immitis, also known as heartworm, is one of the most important parasitic nematodes of domestic dogs, causing a potentially serious disease, cardiopulmonary dirofilariosis, which can be lethal. This species seems to be less 'expansive' than its sister species Dirofilaria repens, and it is believed that climate change facilitates the spread of this parasite to new non-endemic regions. METHODS: In total, 122 heartworm isolates were analysed from nine endemic countries in Europe (Portugal, Spain, Italy, Greece, Hungary, Romania, Slovakia, and Ukraine) and a single isolate from Bangladesh by amplification and sequencing of two mitochondrial (mt) DNA markers: cytochrome c oxidase subunit 1 (COI) and dehydrogenase subunit 1 (NADH). The main aim of the current study was to determine the genetic diversity of D. immitis and compare it with D. repens haplotype diversity and distribution. DNA was extracted from adult heartworms or microfilariae in blood. Most isolates originated from dogs (Canis lupus familiaris) while 10 isolates originated from wildlife species from Romania, including eight isolates from golden jackals (Canis aureus), one isolate from a Eurasian otter (Lutra lutra) and one isolate from a red fox (Vulpes vulpes). RESULTS: Median spanning network analysis was based on the combined sequence (1721 bp) obtained from two mt markers and successfully delineated nine haplotypes (Di1-Di9). Haplotype Di1 was the dominant haplotype encompassing 91 out of the 122 sequences (75%) from all nine countries and four host species. Haplotype Di2 was the second most common haplotype, formed solely by 13 isolates from Italy. The remaining sequences were assigned to Di3-Di9 haplotypes, differing by 1-4 SNPs from the dominant Di1 haplotype. There was evidence for geographical segregation of haplotypes, with three unique haplotypes associated with Italy and four others associated with certain countries (Di4 and Di7 with Slovakia; Di8 with Greece; Di6 with Hungary). CONCLUSION: Diversity in D. immitis mt haplotypes was lower by half than in D. repens (9 vs. 18 haplotypes in D. immitis and D. repens, respectively), which may be associated with the slower expansion of heartworm in Central and NE Europe. NADH gene appears to be conserved in Dirofilaria sp. by showing lower genetic diversity than the analysed COI gene.

Haplotypes of Dirofilaria repens from Poland and selected countries of Central, North-Eastern Europe and the Middle East: An evaluation on the relation between the genetic diversity and the geographic distribution of the fast-spreading parasite.

Subcutaneous dirofilariosis is a fast-spreading infection of dogs, and occasionally of other carnivores and humans. Several factors contribute to its spread, including climate change, which facilitates development and survival of Dirofilaria repens in the mosquito vector. Movement/relocation of infected definitive hosts (dogs) from endemic regions to non-endemic regions is another possible cause of local emergence and the presence of a wide variety of wild reservoirs of the parasite may also contribute to its spread. The main aim of this study was to evaluate the genetic diversity of D. repens from different regions of Europe and to evaluate the spread of identified haplotypes and their geographic origin. A total of 95 D. repens isolates were obtained from Central and Eastern Europe (Poland, Belarus, Ukraine, Austria, Romania), NE Europe (Lithuania, Latvia, Estonia), Italy and Israel. All but two positive samples were obtained from the blood of dogs while one positive sample was obtained from an adult worm from a human case from the Lublin area in SE Poland and one sample was obtained from Anopheles plumbeus mosquito from Austria. Genetic diversity in D. repens isolates was evaluated by PCR amplification and sequencing of three genetic markers, including two mitochondrial genes (mtDNA): the cytochrome c oxidase subunit I (COI) and dehydrogenase subunit I (NADH). Additionally, the genomic marker, internal transcribed spacer 1 (ITS-1) was amplified and sequenced. Haplotypes were differentiated based on sequence alignments by identifying Single Nucleotide Polymorphism (SNPs) using DnaSP and Mega X. PopArt was used to construct a haplotype network including all identified haplotypes. Both mtDNA sequences (COI and NADH) were combined together for phylogenetic and network analyses. Altogether 18 haplotypes (DR1-DR18) were identified in combined mtDNA markers among 95 analysed samples. Haplotype DR1 was the most common encompassing 66 isolates: 42 isolates from Poland (41 from dogs and one from a human), 13 from Lithuania, 4 from Latvia, 2 from Ukraine and 5 from Romania. All other haplotypes grouped around haplotype DR1 separated by 1-5 SNPs, forming a star-like shape. Haplotype DR2 was the second most common haplotype, formed by six isolates from Romania. Interestingly, haplotype DR3 was represented only by four isolates from Israel. The remaining 15 haplotypes were represented by 1-4 isolates of different origins. Our study showed that only minor genetic diversity was found in D. repens since all isolates appear to have clustered in or branched out from haplotype DR1 with 1-5 SNP differences. The genetic diversity appears to be governed by geographic origin since isolates from neighbouring populations (countries) appear to share unique haplotypes while other populations that are geographically distant from individual haplotypes.

Single and Synergistic Effects of Fenbendazole and Metronidazole Against Subclinical Infection by Giardia duodenalis in Non-Human Primates in a Zoological Garden in Southern Italy.

The aim of this study was to assess the single and synergistic effects of fenbendazole (Fenb) and metronidazole (Metro) for the treatment of Giardia duodenalis infection in different species of non-human primates (NHPs) housed in a zoological garden of southern Italy. Moreover, the study also aimed to better define the circulation of G. duodenalis zoonotic assemblages in NHP and the potential occurrence of zoonotic transmission between the staff from the zoo and NHP. Briefly, six species that belonged to four families (Lemuridae, Cercopithecidae, Atelidae, and Hylobatidae) of NHP and housed in six cages (CG) were identified as Giardia positive and divided into two groups. Group F (N = 16 animals) was treated with Fenb (50 mg/kg, every 24 h for 5 consecutive days) and Group M (N = 7 animals) was treated with Metro (25 mg/kg, two times a day for 5 consecutive days). After the first round of therapy, all the animals were retreated for 5 days by inverting the drugs in each group. On each sampling day [study days (SDs) 3-24], the samples were tested for the presence of Giardia cysts using the FLOTAC technique. Multiple fecal tests for the antigen detection of Giardia, such as rapid ELISA and direct immunofluorescence (IFA), were performed at each sampling point only on samples that resulted in positive for Giardia cysts with FLOTAC. The efficacy of Fenb ranged from 30 to 67% and for Metro ranged from 82 to 96%. The results showed the synergistic effects of Metro and Fenb (98-100%) over the combination of Fenb and Metro (52-90%) against the infection by Giardia in NHPs. The overall k agreement between FLOTAC and IFA was reached 0.858 (p = 0.0001). In contrast, all the samples had a negative antigen result when using ELISA. At molecular analysis, six samples were confirmed positive for Giardia by nested PCR. Only two positive samples were successfully sequenced that showed 100% of identity with assemblage B. All the samples from the humans included in the study resulted in negative for Giardia cysts. Overall, the study emphasizes the need for regular monitoring of Giardia infections in NHP housed in zoos by traditional diagnostic tools combined with molecular characterization of the parasite.

Transporter gene expression and Wolbachia quantification in adults of Dirofilaria immitis treated in vitro with ivermectin or moxidectin alone or in combination with doxycycline for 12 h.

Due to their marked larvicidal activity, macrocyclic lactones (MLs) are used for the prevention of heartworm disease ( Dirofilaria immitis) in dogs. They have also been shown to eliminate adult parasites after long-term administration, with a so-called "slow-kill" effect. In addition, recent studies have established that a combination of doxycycline, which eliminates the endosymbiont Wolbachia, and MLs has superior adulticide effects when compared to MLs alone. It has been hypothesized that the apparent synergism between doxycycline/MLs may be due to interaction with drug efflux transport proteins. The aim of the present study was to evaluate gene expression of several transport proteins in D. immitis adults treated in vitro either with doxycycline alone, ivermectin alone, moxidectin alone, or a combination of ivermectin or moxidectin with doxycycline for 12 h. Quantitative PCR analysis showed a sex-dependent response to treatments. In female worms, Dim-pgp-10, Dim-haf-1 and Dim-haf-5 were upregulated compared to controls with doxycycline alone and when combined with ivermectin. Moxidectin did not induce any changes in gene expression. In males, moxidectin administered alone induced a slight increase in Dim-pgp-10, Dim-pgp-11and Di-avr-14, while ivermectin in combination with doxycycline produced significant upregulation of the ML receptor Di-avr-14. These results suggest possible synergism between the two drug classes and different susceptibility of males vs. females to adulticide effects.

A survey on canine leishmaniosis: Best practice and guideline awareness among Italian veterinary practitioners.

Canine leishmaniosis (CanL) is a severe, potentially life-threatening disease of dogs and is considered one of the most important vector-borne zoonoses in Europe, where there is evidence that it is currently spreading into previously unaffected areas. Veterinary practitioners represent the first-line defence against CanL and in recent years, several guidelines have been developed to assist veterinarians in the correct diagnosis, management and prevention of the disease. The present study was aimed at surveying Italian practitioners on how they manage CanL and compare results to recommendations from guidelines developed by the European Scientific Council for Companion Animal Parasites (ESCCAP®), by the Leishvet® organization and by the Canine Leishmaniasis Working Group (C.L.W.G.). Approximately 14.0 % of veterinarians contacted replied to the survey. Results suggest that Italian practitioners are aware of currently available guidelines for the management of CanL and adherence to recommendations is good. Interestingly, 16.0 % carry out year-long prevention, even though recent data suggest that transmission in Italy continues to be seasonal. In conclusion, the high awareness among practitioners reported here should be considered an excellent opportunity for guideline developers to continue updating their recommendations to include any new information that can support the correct management of CL.