Genotyping USA laboratory-maintained isolates and European clinical isolates of Dirofilaria immitis to assess macrocyclic lactone susceptibility or resistance at predictive SNP sites using droplet digital PCR.

Dirofilaria immitis is a parasitic nematode that causes cardiovascular dirofilariosis ("heartworm disease") primarily in canids. The principal approach for mitigating heartworm infection involves the use of macrocyclic lactone (ML) for prophylaxis. Recent research has substantiated the emergence of D. immitis displaying resistance to MLs in the USA. Numerous factors, such as the mobility of companion animals and competent vectors could impact the spread of drug resistance. Genomic analysis has unveiled that isolates resistant to ML exhibit unique genetic profiles when compared to their wild-type (susceptible) counterparts. Out of the ten single nucleotide polymorphism (SNP) markers validated in clinical samples of D. immitis from the USA, four have demonstrated their effectiveness in distinguishing between isolates with varying ML efficacy phenotypes. This study explores the potential of these confirmed SNPs for conducting surveillance studies. Genotypic analysis using SNP markers emerges as a valuable tool for carrying out surveys and evaluating individual clinical isolates. Two USA laboratory-maintained isolates (Berkeley, WildCat) and twenty-five random European clinical samples of either adult worms or microfilariae (mf) pools isolated from domestic dogs, were tested by droplet digital PCR (ddPCR)-based duplex assay. This approach elucidates genetic evidence pertaining to the development of drug resistance and provides baseline data on resistance related genotypes in Europe. The data on these clinical samples suggests genotypes consistent with the continued efficacy of ML treatment regimens in Europe. In addition, this assay can be significant in discriminating cases of drug-resistance from those possibly due to non-compliance to the recommended preventive protocols.

Efficacy of Bravecto® Plus spot-on solution for cats (280 mg/ml fluralaner and 14 mg/ml moxidectin) in the prevention of feline Aelurostrongylus abstrusus infection evaluated in a multi-diagnostic approach.

BACKGROUND: Aelurostrongylus abstrusus is one of the most important respiratory nematodes of felines. Infections may lead to respiratory clinical signs with varying severity or even death, emphasizing the need for preventive treatment of cats with outdoor access to circumvent patent infections. METHODS: Therefore, the preventive efficacy of a spot-on formulation of 280 mg/ml fluralaner and 14 mg/ml moxidectin (Bravecto® Plus spot-on solution for cats, MSD) against A. abstrusus was evaluated in a negative controlled, randomized and partially blinded efficacy study with 28 purpose-bred cats in a non-terminal design. In three different treatment regimes, the minimum recommended dose of 40 mg fluralaner and 2.0 mg moxidectin/kg bodyweight (BW) was administered once at 12, 8 or 4 weeks (study group G1, G2 and G3, respectively) prior to experimental infection with 300 third-stage A. abstrusus larvae, while G4 served as placebo-treated control. RESULTS: From 30 to 46 days post infection (dpi; SD 114 to 130), faeces were sampled to monitor first-stage larvae (L1) excretion for efficacy determination. Secondary efficacy criteria, including respiratory parameters, serological antibody levels and computed tomography (CT) findings, were assessed once before enrolment (SD -7 to -1) and before infection (SD 75 to 83). After infection, CT evaluation was performed once at 47-50 dpi (SD 131 to 134), and respiratory parameters and antibody levels were regularly assessed twice or once a week, respectively (1 up to 78 dpi, SD 85 up to 162). All animals in the control group excreted L1 by 33-37 dpi and remained positive throughout the study period from 41 to 46 dpi (SD 125 to 130). In the treatment groups, only one animal each of G1 and G2 excreted L1 at two consecutive days, and four cats of G1, two of G2 and three of G3 were positive on single occasions. While the geometric mean (GM) of the maximum number of excreted L1 per 5 g of faeces was 7380.89 in the control group (G4), GMs were significantly lower in the treatment groups with 1.63 in G1, 1.37 in G2 and 0.79 in G3. Thus, based on GMs, the reduction in excreted L1 exceeded 99.9% in all three treatment groups. Based on CT severity scores, all lungs of the animals of the control group showed severe pulmonary changes post infection, whereas lungs of the cats of the treatment groups were either unaltered (4 animals), mildly (11 animals), or moderately altered (5 animals). Moreover, seroconversion was observed in all cats of the control group, but not in those of the treatment groups. CONCLUSIONS: The combination of diagnostic methods used in this non-terminal study yielded coherent and reliable results. A single administration of Bravecto® Plus spot-on solution for cats was well tolerated and effective in the prevention of aelurostrongylosis for at least 12 weeks.

Efficacy of a topical formulation containing eprinomectin, esafoxolaner and praziquantel (NexGard® Combo) in the treatment of natural respiratory capillariosis of cats.

Feline pulmonary capillariosis is a significant disorder due to its distribution and clinical impact. This study evaluated the safety and efficacy of two administrations 28 days apart of a topical solution containing esafoxolaner, eprinomectin and praziquantel (NexGard® Combo) in treating Eucoleus aerophilus (syn. Capillaria aerophila) infection in naturally infected cats. Cats were allocated to two groups: G1 cats (n = 23) received two treatments at study days (SDs) 0 and 28 (±2) and were evaluated for 6 weeks, and G2 cats (n = 17) served as a negative control for 6 weeks and were then treated twice on SDs 42 (±2) and 70 (±2), allowing for an additional 6-week assessment of efficacy. Each cat was subjected to McMaster coproscopy at SDs -7/0, 28 (±2) and 42 (±2) for both groups, 70 (±2) and 84 (±2) only for G2. Clinical examination and chest radiographic images were performed at SDs 0, 28 (±2) and 42 (±2) for G1 and G2, 70 (±2) and 84 (±2) only for G2. The comparison of EPG (eggs per gram of feces), clinical (CS), and radiographic scores (RS) at each time-point was used as a criterion. The efficacy based on the EPG reduction was 99.5% (G1) and 100% (G2) after two administrations of NexGard® Combo 2 weeks apart. At SD 0, no significant differences for CS and RS were recorded between G1 and G2, while a significant reduction (p < 0.05) was observed post-treatment for CS, RS, oculo-nasal discharge, auscultation noises, and cough. Two doses of NexGard® Combo 28 days apart stopped egg shedding and significantly improved clinical alterations in cats infected by E. aerophilus.

Title: Efficacité d'une formulation topique contenant de l'éprinomectine, de l'esafoxolaner et du praziquantel (NexGard® Combo) dans le traitement de la capillariose respiratoire naturelle du chat. Abstract: La capillariose pulmonaire féline est un trouble important, de par sa répartition et son impact clinique. Cette étude a évalué l'innocuité et l'efficacité de deux administrations à 28 jours d'intervalle d'une solution topique contenant de l'esafoxolaner, de l'éprinomectine et du praziquantel (NexGard® Combo) dans le traitement de l'infection à Eucoleus aerophilus (syn. Capillaria aerophila) chez des chats naturellement infectés. Les chats ont été répartis en deux groupes : les chats G1 (n = 23) ont reçu deux traitements aux jours d'étude (JE) 0 et 28 (±2) et ont été évalués pendant 6 semaines et les chats G2 (n = 17) ont servi de contrôle négatif pendant 6 semaines, puis ont été traités deux fois aux JE 42 (±2) et 70 (±2), permettant une évaluation supplémentaire de l'efficacité sur 6 semaines. Chaque chat a été soumis à une coproscopie McMaster aux JE −7/0, 28 (±2) et 42 (±2) pour les deux groupes, 70 (±2) et 84 (±2) uniquement pour G2. L'examen clinique et les images radiographiques thoraciques ont été réalisés aux JE 0, 28 (±2) et 42 (±2) pour G1 et G2, 70 (±2) et 84 (±2) uniquement pour G2. La comparaison des nombres d'œufs par gramme de matières fécales (OPG), score clinique (SC) et score radiographique (SR) à chaque point ont été utilisées comme critères d'efficacité. L'efficacité basée sur la réduction de OPG était de 99,5 % (G1) et de 100 % (G2) après deux administrations de NexGard Combo à deux semaines d'intervalle. À JE 0, aucune différence significative pour SC et SR n'a été enregistrée entre G1 et G2, tandis qu'une réduction significative (p < 0,05) a été observée après le traitement pour SC, SR, écoulements oculo-nasaux, bruits d'auscultation et toux. Deux doses de NexGard® Combo à 28 jours d'intervalle arrêtent l'excrétion des œufs et améliorent considérablement les altérations cliniques chez les chats infectés par E. aerophilus.

Efficacy of a combination of esafoxolaner, eprinomectin and praziquantel (NexGard® Combo) against Thelazia callipaeda in naturally infected cats.

This clinical study assessed the efficacy of a topical combination of esafoxolaner, eprinomectin and praziquantel (NexGard® Combo) in treating cats naturally infected with the eyeworm Thelazia callipaeda (Nematoda, Thelaziidae). On Study Day (SD) 0, sixteen client-owned cats with eyeworm infection were allocated to an untreated control group (G1, 8 cats) or to a NexGard® Combo treated group (G2, 8 cats) and subjected to ocular examination. Cats in G2 received the treatment as per label recommendations. On SD 7 and 14 (±1), cats were examined for the presence of eyeworms and clinical signs. On SD 14, eyeworms were collected and counted. On SD 7 and 14, all cats in G1 were still infected with eyeworms, while G2 cats were free from eyeworms on SD 7 and 14, demonstrating 100% efficacy (p < 0.0001). All collected eyeworms were morphologically and molecularly confirmed to be T. callipaeda. On SD 0, fifteen out of the sixteen cats (7 in G1 and 8 in G2) displayed inflammatory ocular signs. On SD 7, all eight untreated cats and seven treated cats displayed inflammatory ocular signs. On SD 14, five out of eight G2 treated cats had recovered, while the eight untreated cats still displayed inflammatory ocular signs. The treatment significantly reduced lacrimation and conjunctivitis (p = 0.0001). No adverse reactions occurred. This clinical study provides evidence that NexGard® Combo is highly safe and effective for the treatment of T. callipaeda infection in cats under field conditions.

Title: Efficacité d'une association d'esafoxolaner, d'éprinomectine et de praziquantel (NexGard® Combo) contre Thelazia callipaeda chez le chat naturellement infecté. Abstract: Cette étude clinique a évalué l'efficacité d'une association topique d'esafoxolaner, d'éprinomectine et de praziquantel (NexGard® Combo) dans le traitement des chats naturellement infectés par le ver oculaire Thelazia callipaeda (Nematoda, Thelaziidae). Au jour d'étude (JE) 0, seize chats appartenant à des clients et atteints d'une infection par le ver oculaire ont été attribués à un groupe témoin non traité (G1, 8 chats) ou à un groupe traité NexGard® Combo (G2, 8 chats) et soumis à un examen oculaire. Les chats du groupe G2 ont reçu le traitement conformément aux recommandations de l'étiquette. Aux JE 7 et 14 (±1), les chats ont été examinés pour détecter la présence de vers oculaires et de signes cliniques. Au JE 14, les vers oculaires ont été collectés et comptés. Aux JE 7 et 14, tous les chats du G1 étaient toujours infectés par des vers oculaires, tandis que les chats du G2 étaient exempts de vers oculaires aux JE 7 et 14, démontrant une efficacité de 100 % (p < 0,0001). Tous les vers oculaires collectés ont été confirmés morphologiquement et moléculairement comme étant T. callipaeda. Au JE 0, quinze chats sur seize (7 en G1 et 8 en G2) présentaient des signes oculaires inflammatoires. Au JE 7, les huit chats non traités et les sept chats traités présentaient des signes oculaires inflammatoires. Au JE 14, cinq des huit chats traités par G2 s'étaient rétablis tandis que les huit chats non traités présentaient toujours des signes oculaires inflammatoires. Le traitement a réduit de manière significative le larmoiement et la conjonctivite (p = 0,0001). Aucun effet indésirable n'est survenu. Cette étude clinique indique que NexGard® Combo est hautement sûr et efficace pour le traitement de l'infection à T. callipaeda chez les chats dans des conditions de terrain.

Efficacy of a spot-on formulation containing 280 mg/ml fluralaner and 14 mg/ml moxidectin (Bravecto® Plus) in the prevention of cat aelurostrongylosis under field conditions.

Aelurostrongylus abstrusus is the most important respiratory nematode of domestic cats. Effective control options are crucial to protect health and welfare of cats and to reduce the spread of aelurostrongylosis in both enzootic and free regions. The present study evaluated the efficacy of a spot-on formulation containing 280 mg/ml fluralaner and 14 mg/ml moxidectin (Bravecto® Plus, MSD) in the prevention of aelurostrongylosis in cats under field conditions. One hundred and fifty-two cats from Italy, Hungary and Bulgaria, were randomly divided in two groups, one treated with Bravecto® Plus on Study Days (SDs) 0 and 84 (74 cats, IVP Group) and one left untreated (78 cats, control group). Faecal samples were collected from all animals on SDs 42 ± 4, 84, 126 ± 4 and 168 ± 4 and subjected to the Baermann's technique and species-specific PCR for A. abstrusus. Each cat was subjected to a clinical examination on SDs 0, 84 and 168 ± 4 to check health condition and possible adverse events. The results of the faecal analysis were statistically analyzed for treatment group differences in the percentage of cats negative to the Baermann's test and PCR and percentage of reduction of fecal larvae counts as the primary and secondary efficacy criteria, respectively. The percentage of negative cats was higher in the IVP group compared to the control group and the percentage of reduction of fecal larvae counts in the IVP group compared to the control group was 100%. These results show that two administrations of Bravecto Plus® spot-on 12 weeks apart were safe and effective in the prevention of aelurostrongylosis for a period of almost 6 months.

Use of In-Clinic Diagnostic Kits for the Detection of Seropositivity to Leishmania infantum and Other Major Vector-Borne Pathogens in Healthy Dogs.

Canine Vector-Borne Diseases (CVBDs) are widespread in Europe and enzootic in many other countries. Though severe illnesses may occur, dogs living in enzootic areas often show vague or no clinical signs of CVBDs. Undiagnosed infections/co-infections in subclinically infected animals favor the spread of CVBDs and increase the risk of transmission to other animals and, in some cases, humans. This study has evaluated the exposure of dogs living in key enzootic countries, i.e., Italy and Greece, to major CVBDs via the use of in-clinic diagnostic kits. Overall, 300 privately owned dogs without/with single mild clinical signs living in different regions of Italy (n. 150) and Greece (n. 150) were included in the study. As part of a clinical examination, a blood sample was collected from each dog and subjected to two serological rapid tests, i.e., the SNAP® 4Dx®Plus (IDEXX Laboratories Inc.) for the detection of antibodies against Ehrlichia spp., Anaplasma spp., Borrelia burgdorferi s.l. and Dirofilaria immitis antigen and the SNAP®Leishmania (IDEXX Laboratories Inc.) for the detection of antibodies against Leishmania infantum. In all, 51 dogs (17%; 95% CI 12.9-21.7) were seropositive to at least 1 pathogen, i.e., 4 in Italy (2.7%; 95% CI 1.4-13.1) and 47 in Greece (31.3%; 95% CI 24-39.4). Dirofilaria immitis antigens were found in 39 dogs (13%; 95% CI 9.4-17.3), while antibodies against Ehrlichia, Anaplasma and Leishmania were detected in 25 (8.3%; 95% CI 5.5-12.1), 8 (2.7%; 95% CI 1.2-5.2) and 5 (1.7%; 95% CI 0.5-3.8) dogs, respectively. None of the dogs tested seropositive for B. burgdorferi s.l. Statistical analyses were performed to evaluate associations between exposure to CVBDs and possible risk factors. The present results indicate that dogs living in enzootic areas may be seropositive for one or more CVBDs in absence of clinical signs. Rapid kits are among first line tools for the detection of CVBDs in clinical settings, as they are cost-effective, straightforward and quick to use. Also, in-clinic tests used herein allowed detection of co-exposure to CVBDs investigated.

Seroexposure to Zoonotic Anaplasma and Borrelia in Dogs and Horses That Are in Contact with Vulnerable People in Italy.

Equine and canine anaplasmosis and borreliosis are major tick-borne zoonotic diseases caused by Anaplasma phagocytophilum and various species of Borrelia (the most important being Borrelia burgdorferi s.l.), respectively. This study evaluated the seroexposure to Anaplasma and Borrelia in dogs and horses used in Animal-Assisted Interventions or living in contact with children, elderly people or immunocompromised persons. A total of 150 horses and 150 dogs living in Italy were equally divided into clinically healthy animals and animals with at least one clinical sign compatible with borreliosis and/or anaplasmosis (present at clinical examination or reported in the medical history). Serum samples were tested with ELISA and immunoblot for the presence of antibodies against A. phagocytophilum and B. burgdorferi s.l., and the association between seropositivity and possible risk factors was analyzed using multivariate and univariate tests. Overall, 13 dogs (8.7%) and 19 horses (12.7%) were positive for at least one of the two pathogens. In addition, 1 dog (0.7%) and 12 horses (8%) were positive for antibodies against A. phagocytophilum, while 12 dogs (8.0%) and 10 horses (6.7%) had antibodies against B. burgdorferi s.l. Tick infestation in the medical history of the dogs was significantly associated with seropositivity to at least one pathogen (p = 0.027; OR 7.398). These results indicate that, in Italy, ticks infected with A. phagocytophilum and/or B. burgdorferi circulate in places where horses and dogs are in contact with people at risk of developing severe diseases. Awareness should be increased, and adequate control plans need to be developed to protect human and animal health, especially where vulnerable, at-risk individuals are concerned.

Ticks, Fleas, and Harboured Pathogens from Dogs and Cats in Cyprus.

Ticks and fleas are blood-sucking ectoparasites that cause irritation and anaemia to their hosts and act as vectors of pathogens (vector-borne pathogens, VBPs) of relevance for animal and human health. In the present study, tick and flea species in dogs and cats from Cyprus were recorded and VBPs were detected in the collected specimens. Ectoparasites were collected from 220 animals (161 dogs and 59 cats), and a questionnaire including demographic, clinical, and other information was filled out for each animal. The ectoparasites were morphologically identified and the detection of VBPs was performed by PCR-coupled sequencing. Rhipicephalus sanguineus sensu lato was found on 108 dogs and 13 cats, and Ixodes gibbosus on 2 dogs. Ctenocephalides felis was the predominant flea species (on 62 dogs and 45 cats), while one dog and one cat were infested by Ctenocephalides canis and Echidnophaga gallinacea, respectively. The VBPs in ticks were Anaplasma platys, Rickettsia massiliae, Rickettsia conorii, Rickettsia felis, Hepatozoon felis and Hepatozoon canis, while Rickettsia felis, Rickettsia sp., Bartonella koehlerae, Bartonella clarridgeiae, and Bartonella henselae were recorded in fleas. Statistical analysis (chi-square test and multiple univariate generalized linear model) showed that animals up to 6 months of age were less likely to be infested with ticks than older animals, but more likely to be infested with fleas. Ticks were more prevalent in sheltered than in owned animals, while the odds ratio of flea presence was higher in owned animals than those living in shelters. The present study is the first investigation on the occurrence of ticks and fleas in dogs and cats from Cyprus, showing the presence of different VBPs in these important ectoparasites. The results point out the importance of systematic ectoparasite control in dogs and cats.

World Association for the Advancement of Veterinary Parasitology (WAAVP): Second edition of guidelines for evaluating the efficacy of anthelmintics for dogs and cats.

These revised guidelines have been developed to assist in the design, execution, and interpretation of studies to assess the efficacy of anthelmintic drugs against internal parasites in dogs and cats. The design and execution of studies are outlined and discussed. Considerations for specific targeted parasites are included. Information is provided on the principles of selection of animals, procedures for randomization, housing, feeding, necropsy procedures, and record keeping for dose determination dose confirmation studies and field studies. Complementary to the WAAVP general anthelmintic guidelines, these species-specific guidelines should assist investigators in the evaluation of anthelmintic drugs in dogs and cats by using comparable and standardized procedures in studies with appropriate numbers of animals.

Dirofilaria immitis: Genotyping Randomly Selected European Clinical Samples and USA Laboratory Isolates with Molecular Markers Associated with Macrocyclic Lactone Susceptibility and Resistance.

Dirofilaria immitis is a parasitic nematode and causes dirofilariosis, a potentially fatal pulmonary infection which primarily infects canids. Dirofilariosis infections are controlled via prophylactic macrocyclic lactone (ML) regimens. Recent evidence has confirmed the development of ML-resistant isolates in the USA, which are genetically distinct from wildtype populations. Single nucleotide polymorphisms (SNP) associated with ML-resistant phenotypes were clinically validated in USA populations. In this study, 3 USA laboratory-maintained isolates (Berkeley, Georgia II, and WildCat) and 11 randomly selected European clinical samples from 7 hosts were analyzed. The samples tested were fresh microfilaria (mf) in blood or adult worms preserved in ethanol. The samples underwent MiSeq sequencing of the top 9 SNP associated with ML resistance. The results provide the first genotypic analysis of the three USA laboratory-maintained isolates and any European samples. The European clinical samples show no genomic evidence of ML resistance based on the 9 SNP. The early adoption of genotyping of clinical D. immitis samples could provide an early indication of the potential development of ML resistance and aid to distinguish clinical cases of heartworm infection due to ML resistance from those due to a lack compliance with the recommended treatments, as has been seen in North America.

Multicenter randomized, and blinded European field study evaluating the efficacy and safety of Felpreva®, a novel spot-on formulation containing tigolaner, emodepside and praziquantel, in treating cats with mixed infection with intestinal nematodes, cestodes and/or lungworms.

This paper describes a multicentric field study which has evaluated the safety and efficacy of a novel spot on formulation containing emodepside 2.04% w/v, praziquantel 8.14% w/v and tigolaner 9.79% w/v (Felpreva®, Vetoquinol) when administered at the intended commercial dose of 0.15 ml/kg body weight to privately owned cats infected with major intestinal nematodes (Toxocara cati, Toxascaris leonina, Ancylostoma tubaeforme, Uncinaria stenocephala) and/or cestodes (Dipylidium caninum, Taenia taeniaeformis) and/or lungworms (Aelurostrongylus abstrusus, Troglostrongylus brevior). A total of 219 cats from 26 veterinary clinics located in Albania, Greece, Hungary, Italy and Portugal were included in the study. Feces from the cats were examined on a single occasion between Study Day -7 and Day 0 (baseline) and post-treatment (i) twice between Day 7 and Day 14 (± 2) (for intestinal helminths) or (ii) twice between Day 21 (± 2) and Day 28 (± 2) (for lungworms). Cats were allocated into two groups at a ratio of 2:1 (Felpreva®: Profender®, i.e. a commercial control product containing emodepside and praziquantel). Cats infected with intestinal helminths were treated once on Day 0 (i) with Felpreva® (Group 1) or (ii) with Profender® (Group 2). Animals infected with lungworms received a second treatment with Profender® on Day 14 (± 2) regardless of group allocation. Faecal egg or larval count reduction for Felpreva® was 97.47% for intestinal nematodes and 96.80% for lungworms. No cats infected with cestodes at baseline resulted positive after treatment with Felpreva®. However, the low number of cats (n = 10) did not allow for a statistical analysis to be performed. Non-inferiority of Felpreva® compared to Profender® was statistically demonstrated for all target intestinal and respiratory parasites. No adverse events nor application site reactions were observed. These results show that the new topical combination product Felpreva® is highly safe and efficacious in treating infections caused by major species of feline intestinal nematodes, cestodes and lungworms under field conditions.

Efficacy of two topical combinations containing emodepside plus praziquantel, and emodepside plus praziquantel plus tigolaner, for the treatment of troglostrongylosis in experimentally infected cats.

Feline troglostrongylosis caused by Troglostrongylus brevior is increasingly reported in European countries. Although the disease can be severe and potentially life-threatening, especially in kittens and young cats, effective treatment options are still limited. Two administrations of emodepside 2 weeks apart have shown promising results for the treatment of T. brevior infection in single cases and in a field trial. Therefore, the present study has been conducted to evaluate the efficacy of two spot-on combinations containing emodepside (i.e. 2.14% w/v emodepside and 8.58% w/v praziquantel - Profender®, and 2.04% w/v emodepside, 8.14% w/v praziquantel and 9.79% w/v tigolaner - Felpreva®) in the treatment of troglostrongylosis under experimental conditions. Twenty-four cats were experimentally infected with T. brevior and randomly assigned to one of three groups of eight cats each, i.e. (i) Group 1 (G1) left untreated, (ii) Group 2 (G2) receiving Profender® on Days 28 and 44, and (iii) Group 3 (G3) receiving Felpreva® on Day 28 and Profender® on Day 44. Doses corresponded to the minimum effective dose of 0.140 and 0.148 ml/kg body weight, for Profender® and Felpreva®, respectively. The primary efficacy criterion was the number of viable adult T. brevior counted at necropsy conducted between Days 69 and 72. The fecal shedding of first-stage larvae (L1) was also assessed. L1 of T. brevior were detected in samples from all cats within 20 days post-infection. At necropsy, 4 of 8 G1 cats harbored adult T. brevior, while no adult T. brevior worms or other development stages were recovered from any of the G2 and G3 cats. The primary efficacy criterion was not evaluated as the worm counts in G1 did not meet VICH guideline requirements. After the first treatment (Day 28), most G2 and G3 cats were negative at the Baermann examination. After the second treatment (Day 44), L1 were found in two cats from G2 on Day 49 and in one G3 cat on Day 51. No adverse events occurred in G2 and G3 cats. These results indicate that two applications of emodepside spot-on given 2 weeks apart represent a safe and efficacious treatment regime against troglostrongylosis.

A Comparison of Copromicroscopic and Molecular Methods for the Diagnosis of Cat Aelurostrongylosis.

The gold standard method for the diagnosis of cat aelurostrongylosis is the detection of Aelurostrongylus abstrusus first stage larvae with the Baermann's examination. Nevertheless, molecular assays have shown higher diagnostic performances compared to copromicroscopy. This study evaluated the usefulness of an A. abstrusus species-specific PCR on different biological samples collected in clinical settings from 100 privately-owned cats in Italy (n. 60) and Greece (n. 40). A fecal sample was collected from each animal and a pharyngeal swab was also obtained for cats from Italy. All stool samples were subjected to flotation and Baermann's test. The cats were categorized in three groups based on the results of copromicroscopy, i.e., Group A (n. 50 cats with A. abstrusus infection regardless of positivity for other helminths), Group B (n. 25 cats negative for A. abstrusus but positive for at least one of any other helminth), Group C (n. 25 cats negative for any helminth). DNA was extracted from individual aliquots of feces, flotation supernatant, Baermann's sediment and the pharyngeal swab and then subjected to a PCR specific for A. abstrusus. At least one fecal aliquot or the pharyngeal swab scored positive by the A. abstrusus-specific PCR for 48/50 (96%) cats enrolled in Group A; in particular, 38/50 (76%), 35/50 (70%), 41/50 (82%) and 21/25 (84%) DNA extracts from feces, flotation supernatant, Baermann's sediment and pharyngeal swabs were positive by PCR. These results confirm that molecular tools are highly sensitive and specific and indicate that pharyngeal swabs are the most suitable sample for molecular analysis in clinical settings.

Zoonotic intestinal helminthes diagnosed in a 6-year period (2015-2020) in privately owned dogs of sub-urban and urban areas of Italy.

Intestinal helminthes of dogs have a relevant role for health and welfare of canine populations and many of them are zoonotic. Thus, a constant surveillance is pivotal to safeguard companion dogs and human health. Data from faecal examinations of pet dogs obtained in the last six years during the routine activity of the laboratory of Parasitology and Parasitic diseases of the University Teaching Veterinary Hospital of the University of Teramo, Italy, are here reported. From 2015 to 2020 a total of 1083 individual faecal samples from privately owned dogs were examined using conventional copro-microscopic techniques. Endoparasite elements were found in 278 (25.7%) dogs, and among them, eggs of intestinal zoonotic helminthes were detected in 184 (17%) samples. The most common zoonotic parasites were Toxocara canis and hookworms (8.9% positivity rate for both). Most dogs (88%) were positive for a single zoonotic parasite while 12% of the positive animals harbored two or more zoonotic helminthes. A statistical analysis showed a significant association between dogs with less than one year of age and positivity to at least one intestinal zoonotic helminth, and that infections by T. canis are significantly more prevalent in dogs under one year of age. Despite being well known in canine clinical practice, these results indicate that zoonotic helminthes are still frequent in owned dogs. Thus, increased awareness of veterinary practitioners and pet owners is necessary to implement current diagnostic and control strategies to minimize the risk of zoonotic transmission of intestinal helminthes.

Efficacy of a spot-on combination containing 10% w/v imidacloprid and 1% w/v moxidectin for the treatment of troglostrongylosis in experimentally infected cats.

BACKGROUND: Parasitic bronchopneumonia in domestic cats in Europe, which can manifest with moderate to severe clinical signs, is frequently caused by Troglostrongylus brevior. Data on epizootiological and clinical relevance of cat troglostrongylosis have been published in the last decade but treatment options are still limited. Promising effectiveness data have been generated from clinical cases and field trials for a spot-on formulation containing 1% w/v moxidectin and 10% w/v imidacloprid (Advocate®, Elanco Animal Health). Therefore, two studies have been conducted to confirm under experimental conditions the efficacy of moxidectin 1% contained in Advocate® for the treatment of cat troglostrongylosis. METHODS: Sixteen and 20 cats experimentally infected with T. brevior were included in two separate studies, i.e., Study 1 and 2, respectively. Cats were infected with T. brevior third-stage larvae via gastric tube. In both studies cats were randomized to untreated (control, Group 1) and treatment (Group 2) groups. In Study 1 and Study 2, the two groups comprised eight and 10 cats each. Treated cats received Advocate® spot-on twice at a 4-week interval. The primary efficacy criterion was the number of viable adult T. brevior counted at necropsy. Throughout the trial, the fecal shedding of first-stage larvae (L1) was assessed in treated and untreated control cats. RESULTS: The experimental model was successful in both studies, as all cats started shedding T. brevior L1 within 25 days post-infection. At necropsy, T. brevior adults were found in 4/8 and 4/10 cats of the control groups in Study 1 and 2, respectively, while none of the treated cats harbored adult worms. The necropsy worm counts in controls did not meet relevant guideline requirements for adequacy of infection, with fewer than six infected cats in the control groups, thus limiting conclusions on treatment efficacy. The fact that 6/8 and 8/10 control cats in Study 1 and 2, respectively, shed L1 up to necropsy while larval shedding ceased in all treated animals after the first treatment provides supporting evidence on the level of efficacy. No remarkable adverse events were recorded in the two studies. CONCLUSION: These results indicate that Advocate® spot-on is a safe and effective option for treating cats infected by T. brevior.

Hemorrhagic Meningoencephalomyelitis Due to Ectopic Localization of Aelurostrongylus abstrusus in a Cat: First Case Report.

The lungworm Aelurostrongylus abstrusus is one of the main causes of respiratory diseases in cats worldwide. This report describes the unusual case of a kitten infected with A. abstrusus and presented to a veterinary clinic in Brazil with lethargy, dysphagia, non-ambulatory tetraplegia, and pelvic limbs bilateral myoclonus. The clinical picture of the kitten worsened with generalized flaccid tetraplegia and death a few days after hospitalization. At necropsy, hemorrhagic necrosis and subarachnoid hemorrhages were detected in several areas of the central nervous system. Nematode stages were found at post-mortem histological examinations in lungs, cerebellum, subarachnoid space of the brain and spinal cord. Microscopic and molecular (PCRs-coupled-sequencing protocols) examination showed the presence of A. abstrusus in histological samples. This study describes the first neurological aelurostrongylosis due to ectopic localization of adult worms in the central nervous system of a cat, causing acute hemorrhagic multifocal meningoencephalomyelitis. Further studies are necessary to elucidate whether unusual localizations and the migration of A. abstrusus are more frequent than expected.

An Uncommon and Severe Clinical Case of Sarcoptes scabiei Infestation in a Cat.

The burrowing mite Sarcoptes scabiei causes sarcoptic mange in dogs and other mammals, including humans. Despite S. scabiei infests several animals, little is known about the epidemiology, clinical features, and treatment of sarcoptic mange in cats. A few reports have shown that clinical signs of S. scabiei infestation in cats may vary from non-pruritic crusted lesions to itchy and mild lesions, while severe signs are very infrequent. The present report describes a severe case of S. scabiei infestation in a stray cat from Italy, characterized by bilateral alopecia, diffuse and multifocal hyperpigmented, crusted, erythematous, scaled, and exfoliated lesions. The cat was FIV-positive and also infected by the cestode Dipylidium caninum. After treatment with a broad-spectrum parasiticide and an antibiotic, the cat showed an almost complete clinical recovery in 4 weeks. Unfortunately, no further clinical examinations were performed due to the lack of compliance of the owner and to the death of the cat for causes unrelated to sarcoptic mange. This clinical case indicates that under certain circumstances, S. scabiei can reproduce and cause severe clinical signs in cats which are usually considered non-permissive hosts for this mite, and in which the disease is usually self-limiting. Clinic-pathological, epidemiological, and zoonotic implications are discussed.

Highly Variable Clinical Pictures in Dogs Naturally Infected with Angiostrongylus vasorum.

Canine angiostrongylosis by Angiostrongylus vasorum is increasingly reported in both enzootic and previously free areas. The complex pathogenesis of the disease makes the clinical workup challenging. Infected dogs show highly variable clinical pictures, characterized by subclinical to life-threatening general, cardio-respiratory, neurological and/or gastrointestinal signs. The present study reports the high variability of clinical pictures from 36 dogs across central and southern Italy that were naturally infected by A. vasorum. Of them, 23 (63.9%) presented at least one clinical sign, while 13 (36.1%) were subclinically infected and apparently healthy. Overall, 19 dogs (52.8%) showed cardiorespiratory signs, 14 (38.9%) had non-specific abnormalities, 2 (5.6%) presented coagulation disorders and 1 (2.8%) had a severe neurological condition. Importantly, four dogs presenting with clinical signs had neither cough nor dyspnea. These results underline that angiostrongylosis should be included in the differential diagnosis, even when dogs display only non-specific clinical signs. The proportion of apparently healthy dogs highlights the relevance of routine copromicroscopic and/or antigenic tests in enzootic areas to avoid the sudden onset of potentially life-threatening signs.

Angiostrongylosis in Animals and Humans in Europe.

Lungworms in the genus Angiostrongylus cause disease in animals and humans. The spread of Angiostrongylus vasorum within Europe and the recent establishment of Angiostrongylus cantonensis increase the relevance of these species to veterinary and medical practitioners, and to researchers in parasitology, epidemiology, veterinary science and ecology. This review introduces the key members of the genus present in Europe and their impacts on health, and updates the current epidemiological situation. Expansion of A. vasorum from localized pockets to wide distribution across the continent has been confirmed by a rising prevalence in foxes and increasing reports of infection and disease in dogs, while the list of carnivore and mustelid definitive hosts continues to grow. The tropically distributed rat lungworm A. cantonensis, meanwhile, has been recorded on islands south of Europe, previously the Canary Islands, and now also the Balearic Islands, although so far with limited evidence of zoonotic disease. Other members of the genus, namely, A. chabaudi, A. daskalovi and A. dujardini, are native to Europe and mainly infect wildlife, with unknown consequences for populations, although spill-over can occur into domestic animals and those in zoological collections. The epidemiology of angiostrongylosis is complex, and further research is needed on parasite maintenance in sylvatic hosts, and on the roles of ecology, behaviour and genetics in disease emergence. Improved surveillance in animals and humans is also required to support risk assessments and management.

Efficacy of fipronil/(S)-methoprene/eprinomectin/praziquantel (Broadline®) against Thelazia callipaeda in naturally infected cats.

BACKGROUND: The present clinical field trial was conducted to assess the efficacy of a broad-spectrum parasiticide spot-on formulation containing eprinomectin (Broadline®) against Thelazia callipaeda eyeworm in naturally infected cats. METHODS: Fifteen privately owned cats harboring at least one live adult T. callipaeda were included in the study. Cats were randomly allocated to an untreated control group of seven cats or to a Broadline®-treated group of eight cats. Cats were treated on Day 0; ocular examinations were performed at inclusion and on Days 7 and 14; eyeworms were recovered and counted on Day 14. The primary efficacy assessment was based on group comparison of number of T. callipaeda on Day 14. RESULTS: Seven days after treatment, six of eight treated cats were negative for eyeworm infection per visual examination, and on Day 14 no eyeworms were found in the treated cats while the seven untreated cats were still infected (geometric mean: 1.97). All cats had inflammatory ocular signs at inclusion; on Day 14, five of eight treated cats had recovered while all untreated control cats were still symptomatic. All collected parasites were confirmed to be T. callipaeda by morphology and molecular characterization. CONCLUSIONS: A single treatment with Broadline® provided 100% efficacy against feline thelaziosis and improved related ocular inflammation signs.