Spirocerca lupi draft genome, vaccine and anthelmintic targets.

Spirocerca lupi is a parasitic nematode affecting predominantly domestic dogs. It causes spirocercosis, a disease that is often fatal. The assembled draft genome of S. lupi consists of 13,627 predicted protein-coding genes and is approximately 150 Mb in length. Several known anthelmintic gene targets such as for ß-Tubulin, glutamate, and GABA receptors as well as known vaccine gene targets such as cysteine protease inhibitor and cytokines were identified in S. lupi by comparing orthologs of C. elegans anthelmintic gene targets as well as orthologs to known vaccine candidates. New anthelmintic targets were predicted through an inclusion-exclusion strategy and new vaccine targets were predicted through an immunoinformatics approach. New anthelminthic targets include DNA-directed RNA polymerases, chitin synthase, polymerases, and other enzymes. New vaccine targets include cuticle collagens. These gene targets provide a starting platform for new drug identification and vaccine design.

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.

Thelazia rhodesi in a dairy farm in Romania and successful treatment using eprinomectin.

Bovine thelaziosis can be caused by Thelazia rhodesi Desmarest 1828, Thelazia gulosa Railliet & Henry 1910 and Thelazia skrjabini Erschow 1928 which are known to be transmitted by species of Muscidae. Therapeutic measures in cattle include mechanical removal of the adult parasites, followed by irrigation of the conjunctival sac with different solutions such as levamisole (1%) or administration of ivermectin (0.2 mg/kg SC), doramectin (0.2 mg/kg SC or pour-on) both with high efficacy, however, the use of such macrocyclic lactones can be problematic in lactating cattle. Nematodes harboured in the eyes of some cattle from Romania were morphologically identified as Thelazia rhodesi and the PCR analysis confirmed the diagnosis with a 98.0% nucleotide similarity for ITS1 sequence, with other sequences available in GenBank. The present paper reports the presence of T. rhodesi in cattle from a dairy farm in Romania and the recovery after treatment with eprinomectin. To the best of our knowledge this is the first report on the efficacy of eprinomectin against Thelazia spp. and the first molecular confirmation of T. rhodesi in Romania.

Prevention of canine ocular thelaziosis (Thelazia callipaeda) with a combination of milbemycin oxime and afoxolaner (Nexgard Spectra®) in endemic areas in France and Spain.

In the past decade, canine thelaziosis due to Thelazia callipaeda has been diagnosed in an increasing number of European countries, with endemic areas being identified. A multi-center field trial was conducted in endemic areas in France and Spain to evaluate the efficacy of monthly administrations of the oral milbemycin oxime/afoxolaner combination (NexGard Spectra®) for the prevention of T. callipaeda infection in at-risk dogs. A total of 79 dogs negative for T. callipaeda and with a clinical history of eyeworm infection in the past two years completed the study. Dogs were randomly allocated either to a negative control group (42 dogs) or to the NexGard Spectra® treated group (37 dogs). All dogs were followed up for a 6-month period and assessed monthly for the presence of nematodes on the eyes and for the signs of ocular thelaziosis (e.g., conjunctivitis, keratitis, and ocular discharge). When the presence of nematodes was confirmed, the conjunctival fornix was flushed with a saline solution for parasite recovery and counting, and the dogs were treated appropriately. Recovered parasites were stored in 70% alcohol for subsequent morphological identification. During the course of the study, 57.1% (24/42) of the control dogs were diagnosed positive for Thelazia infection, which illustrates a high incidence rate of parasite infection. Conversely, no eyeworm was recovered from any of the 37 dogs that received NexGard Spectra®. All parasites sampled were confirmed to be T. callipaeda. This clinical field study demonstrated that monthly administrations of NexGard Spectra® provided 100% preventive efficacy against canine thelaziosis.

Evaluation of the efficacy of monthly oral administration of afoxolaner plus milbemycin oxime (NexGard Spectra(®), Merial) in the prevention of adult Spirocerca lupi establishment in experimentally infected dogs.

The nematode Spirocerca lupi (Rudolphi, 1809) is widely distributed but mostly occurs sporadically with stable populations only in certain geographic areas. This helminth mainly infects dogs and wild canids. Primary pathology relates to migration of third stage larvae (L3) damaging the thoracic aorta and establishment of adults in nodules in the oesophagus. The objective of the present study was to evaluate the efficacy of milbemycin oxime in combination with afoxolaner (NexGard Spectra(®), Merial), administered monthly, in preventing establishment of adult worms after experimental infection. Two groups consisting of eight animals each were experimentally infected with 15 L3 on Days -28, -14 and -2, respectively (45 L3 per animal in total). Group 1 dogs served as untreated (negative) control, whereas animals in group 2 were treated with NexGard Spectra(®) at a minimum dose of 0.5mg/kg milbemycin oxime on Day 0 and from then onwards every 28 days up to Day 140 (six treatment occasions). Endoscopy was performed on Day 112 and for some animals also Day 140. Necropsy for worm recovery and nodule/lesion scoring was performed on Day 168. All eight animals in the control group (group 1) presented with 1-3 nodules and worm counts ranging from 9 to 41. Six animals in the NexGard Spectra(®) group presented with 1-4 nodules and worm counts ranging from 1 to 5. Significantly (p<0.05) fewer worms were collected from treated animals in the treated group (geometric mean 1.7) versus the negative control group (geometric mean 22.0) with 92.3% efficacy calculated. There was no significant (p>0.05) difference between groups with reference to number of nodules in the oesophagus. However, nodules in the control group were significantly (p<0.05) larger than those in the treated group. Number and size of lesions in the dorsal aorta did not differ statistically between groups 1 and 2. Because NexGard Spectra(®) was administered 28 days after onset of inoculation, migrating and developing L3 caused damage to the aorta wall of animals in the treated group. Milbemycin oxime (administered as NexGard Spectra(®)) demonstrated effectiveness in reducing infection with adult Spirocerca lupi worms in the oesophagus.

Comparative evaluation of the prophylactic activity of a slow-release insecticide collar and a moxidectin spot-on formulation against Thelazia callipaeda infection in naturally exposed dogs in France.

BACKGROUND: The relative efficacy of a collar containing 10% imidacloprid and 4.5% flumethrin (Seresto, Bayer HealthCare Animal Health) and a spot on formulation containing 10% imidacloprid and 2.5% moxidectin (Advocate, Bayer HealthCare Animal Health) was evaluated as a control measure to prevent canine thelaziosis in dogs in an endemic area of France. FINDINGS: Ninety-six privately-owned dogs were enrolled in the multicentre, controlled study. Before summer (the period of transmission by fruit flies), dogs were allocated to one of three groups: Group A (n = 36)- treated once with a collar containing 10% imidacloprid and 4.5% flumethrin; Group B (n = 33)- treated every month for 8 months with a spot-on containing imidacloprid 10% and moxidectin 2.5%; and Group C (n = 27)- untreated control animals. Dogs were regularly subjected to ocular examination in order to assess Thelazia callipaeda infection. During the trial, T. callipaeda nematodes were detected in 12 (33%) collared dogs (group A) whereas no eyeworm could be found in dogs who received a monthly spot on application of moxidectin (group B). In the control group, 8 (30%) dogs became infected. CONCLUSIONS: The monthly application of a spot on formulation containing 10% imidacloprid and 2.5% moxidectin was shown to be highly effective in preventing T. callipaeda infection in a population of dogs living in an endemic area in France. On the contrary, the slow-release collar tested in this study did not display any protection against canine thelaziosis.

Resolution of canine ocular thelaziosis in avermectin-sensitive Border Collies from Spain.

Ocular thelaziosis by Thelazia callipaeda is an emerging disease that affects primarily dogs, but also cats, foxes and other wild carnivores, as well as humans. Three clinical cases of unilateral conjunctivitis caused by Thelazia nematodes were detected in Border Collie, a dog breed intolerant to the macrocyclic lactones. Animals came from southwestern Spain, on the border with Portugal. Eight worms were collected and identified molecularly as T. callipaeda by amplification and sequencing of partial cytochrome c oxidase subunit 1 gene. Oral treatment with mebendazole 20mg/kg (Telmin(®)) was effective in curing the infection.

Successful resolution of esophageal granulomas in a dog infected with Spirocerca lupi.

A 13-year-old female Labrador Retriever presented with chronic regurgitation. Radiography and computed tomography (CT) revealed nodules in the caudal esophagus. Upper gastrointestinal endoscopy followed by histopathological examination revealed esophageal granulomas caused by Spirocerca lupi. The infection was treated with milbemycin oxime. The therapy was successful, and a remarkable reduction in granulomas was observed. This case report describes the diagnostic imaging findings and treatment outcome of a dog with S. lupi infection in Japan.

The efficacy of a topically applied imidacloprid 10 % / moxidectin 2.5 % formulation (Advocate®, Advantage® Multi, Bayer) against Immature and Adult Spirocerca lupi worms in experimentally infected dogs.

This study investigated the efficacy and safety of an imidacloprid 10 %/moxidectin 2.5 % spot-on combination (Advocate®, Advantage® Multi, Bayer) against immature and mature stages of Spirocerca lupi in experimentally infected dogs. 24 dogs were allocated to 3 groups and infected with approximately 10 L3 larvae of S. lupi orally on study day (SD) +2, +14, +28 and +42. Group 1 remained as untreated control group. Group 2 dogs were treated on SD ­28, 0, and thereafter monthly until Day 280 (12 treatments). Group 3 dogs were treated weekly on 19 occasions starting on SD +170. The dosage for all treatments was the licensed dose of 10­25 mg imidacloprid/2.5­6.25 mg moxidectin per kg body weight. All dogs were examined on SD +169 or +176 by endoscopy. Group 3 dogs were additionally examined approximately every two weeks up to Day 296. On Day +308 or +310, all dogs were necropsied to recover S. lupi worms and to quantify lesions in the thoracic aorta and oesophagus. Dogs in the control group were adequately infected with S. lupi, demonstrated by the extensive damage to the thoracic aorta, the nodules in the oesophagus and the large numbers of worms recovered. In total 144 worms were collected (geometric mean of 16.8 worms per dog). Dogs in group 2 had no or very slight damage to the thoracic aorta and no nodules or worms in the oesophagus, indicating 100 % efficacy of the monthly treatments. Dogs in group 3 were also adequately infected, showing nodules in the oesophagus before initiation of weekly treatment, and at necropsy extensive damage was seen in the thoracic aorta. After treatment, three dogs of 8 still had a few nodules and in total three worms (GM of 0.25 per dog) were recovered, demonstrating an efficacy of 98.5 % against adult S. lupi. All dogs tolerated the treatment well and no treatment- related adverse events occurred.

Successful resolution of oesophageal spirocercosis in 20 dogs following daily treatment with oral doramectin.

The purpose of this study was to evaluate the effect of a daily oral dose of doramectin in dogs with spirocercosis. Twenty naturally infected dogs were treated with 0.5 mg/kg doramectin administered orally once daily for 42 days. In 13 of the dogs there was resolution of the nodules after 42 days. Nodules were eliminated in five of the remaining seven dogs following treatment for an additional 42 days. In the remaining two dogs, treatment continued for a further 42 days (total 126 days), resulting in complete resolution. No adverse events associated with treatment were observed. This study concluded that doramectin at 0.5 mg/kg once a day is effective in the elimination of Spirocerca lupi nodules in dogs.

Treatment of dog thelaziosis caused by Thelazia callipaeda (Spirurida, Thelaziidae) using a topical formulation of imidacloprid 10% and moxidectin 2.5%.

Canine thelaziosis caused by Thelazia callipaeda infects dogs, cats, foxes, rabbits, and humans resulting in conjunctivitis, pain and excessive lacrimation. T. callipaeda live in the eyes under the nictitating membrane and females release first stage larvae which are ingested by flies which act as intermediate hosts. Control of canine thelaziosis is currently based on the removal of nematodes directly from the eyes of affected dogs or on the local instillation of antiparasitic drugs. With the aim of evaluating the efficacy against T. callipaeda of an association of imidacloprid 10% and moxidectin 2.5% by spot-on formulation administered via dermal application, three groups of naturally infected animals were selected: i.e. group A (21 dogs) received a single dose of imidacloprid 10% and moxidectin 2.5% by spot-on; group B (21 dogs) received a single dose of imidacloprid 10% by spot-on and group C (20 dogs) were left untreated. The efficacy of treatments was established by eye inspection and parasite viability and vitality after 1, 5 and 9 days after animal treatments (groups A and B) and after 9 days only in untreated animals (group C). Imidacloprid 10% and moxidectin 2.5% in spot-on formulation showed to be effective with regards the control of dog thelaziosis within 5 (90.47%) to 9 (95.23%) days after treatment. Only one dog from group A presented nematodes after treatment. The presence of parasites in the eyes of dogs from groups B (imidacloprid 10%) and C confirm that the anthelmintic efficacy against T. callipaeda in animal from group A (imidacloprid 10% and moxidectin 2.5%) was most likely attributable to moxidectin 2.5%. The spot-on formulation containing imidacloprid 10% and moxidectin 2.5% is very easy to apply and helps overcome problems linked to the mechanical removal of parasites or to the restraining of the animals for the local instillation of drugs in the eyes.

Evaluation of doramectin for the treatment of experimental canine spirocercosis.

The nematode Spirocerca lupi is primarily a parasite of dogs, which causes typical lesions of esophageal nodular granulomas, aortic aneurysms and spondylitis. In order to evaluate the therapeutic effect of doramectin on experimental canine spirocercosis, seven beagle dogs experimentally infected with 40 infectious S. lupi larvae (L(3)) were treated with doramectin. Treatment was commenced following endoscopic visualization of esophageal granulomas, and typical S. lupi eggs were detected in the feces. The treatment protocol included six treatments of doramectin (400 microg/kg subcutaneously) at 2 weeks intervals, followed by monthly injections until the disappearance of the esophageal granulomas or the end of the study (768 days post-inoculation). Eggs could not be found on fecal examinations 3-10 days after the first or second doramectin treatment. In addition, a gradual decrease in size of granulomas was noticed in all seven dogs during the course of the study. Esophageal granulomas had completely resolved in six of the seven dogs between day 35 and day 544 post-initial doramectin treatment, by day 35 in one dog (after three treatments), by day 43 in two dogs (after four treatments), by day 98 in one dog (after seven treatments), by day 460 in one dog (after 18 treatments) and by day 544 in another dog (after 21 treatments). In one dog, remnants of S. lupi granulomas could still be seen 544 days post-initiation of treatment with doramectin. Multiple subcutaneous injections of doramectin (400 microg/kg) were shown to be effective and safe in the treatment of canine spirocercosis.

Spirocerca lupi esophageal granulomas in 7 dogs: resolution after treatment with doramectin.

Seven dogs with Spirocerca lupi esophageal granulomas were identified based on the site of involvement (ie, distal esophagus) and characteristic endoscopic appearance. Six dogs presented with signs of esophageal disease and 1 dog was asymptomatic. Ova were only identified in the feces of 2 dogs. On thoracic radiographs, 4 dogs had evidence of a caudodorsal mediastinal mass, and 2 of these dogs had spondylitis of midthoracic vertebrae. On endoscopy, single esophageal nodules were observed in 5 dogs, 1 dog had 3 nodules, and 1 dog had 6 nodules. All 7 dogs were treated with doramectin at a dosage of 200 microg/kg SC at 14-day intervals for 3 treatments. Dogs had physical and endoscopic examinations at 2, 4, and 6 weeks after treatment. By 6 weeks, clinical signs had resolved in 6 dogs (1 dog was asymptomatic), and the esophageal nodules had completely resolved in 4 dogs, and incompletely resolved in 3 dogs. Two dogs with incomplete resolution were treated again with doramectin at 500 microg/kg PO daily for an additional 6 weeks. Complete resolution of the esophageal nodules was confirmed by endoscopy in all dogs. Nodules had resolved in 4 dogs by 6 weeks, in 2 dogs by 12 weeks (after 6 weeks additional daily oral therapy), and in 1 dog 22 months after the initial 200 microg/kg treatment regimen. No dog experienced adverse effects to the drug, and all symptomatic dogs have been free of disease for periods ranging from 3 to 4 years.

The effect of treating beef cattle on pasture with ivermectin on the prevalence and intensity of Thelazia spp. (Nematoda: Thelazioidea) in the vector, Musca autumnalis (Diptera: Muscidae).

Trihedron sticky traps were used to monitor the prevalence and intensity of Thelazia spp. in face flies associated with 2 groups of cattle. Traps were set for 24 hr once a week beginning 2 wk before cattle were turned out to pasture following winter housing and continued until flies reached diapause in October. The 50 heifers in group 1 received 1 treatment with ivermectin (Ivomec Pour-On for cattle, 500 micrograms/kg body weight) on the day of turnout onto pasture. The 50 cows and their calves in group 2 were not treated. Treating cattle with ivermectin delayed the onset of infection in Musca autumnalis by 20 days and reduced the prevalence to 2.2% compared to 26.9% in flies from the control pasture. Although residues of ivermectin eradicate many fly larvae developing in cattle feces, there was no significant difference in the number of flies counted on the faces of cattle or in the number of flies caught on sticky traps on the 2 pastures (P > 0.05).

The efficacy of ivermectin against Thelazia rhodesii (Desmarest, 1828) in the eyes of cattle.

One hundred cattle with confirmed natural infections of Thelazia rhodesii were included in three studies to evaluate the efficacy of ivermectin for the treatment of eyeworm infection. Thelazia rhodesii were counted in situ in each eye of each animal on Day 0 (prior to treatment) and the cattle were paired by the number of worms, within categories of sex, age or number of infected eyes. Within each pair, one animal was randomly assigned to serve as an untreated control, while the other was treated subcutaneously with ivermectin at 200 micrograms kg-1 body weight. Eight days later, parasites were recovered from each eye separately, identified and counted. In each study, significantly (P less than 0.01) fewer T. rhodesii were collected on Day 8 from ivermectin-treated cattle than from controls. Overall, the reduction in numbers of T. rhodesii collected from treated cattle was greater than 99% (P less than 0.05) relative to controls, with reductions of 100% being recorded in two of the three studies.

Immunization of cattle using varying infective doses of Theileria parva lawrencei sporozoites derived from an African buffalo (Syncerus caffer) and treatment with buparvaquone.

A Theileria parva lawrencei isolate in the form of a sporozoite stabilate, derived by feeding clean Rhipicephalus appendiculatus nymphal ticks on an African buffalo (Syncerus caffer) captured in the Laikipia District, Kenya, was inoculated into groups of cattle at dilutions between 10(0) and 10(-3). Groups of 3 cattle infected with 1 ml inocula at 10(0), 10(-1) and 10(-2) dilutions were treated with 2.5 mg/kg body weight of buparvaquone on day 0 and similar groups were left untreated to act as controls. An additional group, given 10(0) dilution of the stabilate, was treated with buparvaquone on day 8 post-inoculation. It was found that all control cattle inoculated with the stabilate at dilutions between 10(0) and 10(-2) became infected, but only 2 out of 3 cattle developed patent infections at 10(-3) dilution. All 3 control cattle receiving 10(0) dilution died of theileriosis, 2 at 10(-1) and 10(-2) dilutions, and 1 at 10(-3) dilution died. Buparvaquone treatment on day 0 at 10(0) dilution resulted in the survival of 2 of 3 cattle and all the cattle at 10(-1) and 10(-2) dilutions. All the surviving cattle eventually developed a significant serological response against T. parva in the indirect fluorescent antibody test, except 1 in the 10(-3) dilution group, and were immune to homologous challenge when tested 3 months later with a lethal inoculum of stabilate, except 2 cattle in the 10(-3) dilution group. As a result of a theileriosis problem at about day 60 after inoculation in 2 cattle given 10(-2) dilution of stabilate and buparvaquone treatment on day 0, an additional 5 cattle were given 10(-2) dilution of stabilate and developed a good immunity after buparaquone treatment. None was shown to develop the carrier state. Treatment with buparvaquone on day 8 after infection with 10(0) dilution of stabilate was not successful since 2 died. The stabilate used was shown to produce reproducible infection in cattle at different dilutions.

Apparent inactivity of several antiparasitic compounds against the eyeworm Thelazia lacrymalis in equids.

Activity of 15 compounds, given alone or in mixtures [butamisole, cambendazole, caviphos, febantel (alone or with trichlorfon), fenbendazole, ivermectin, levamisole-piperazine, oxfendazole, oxibendazole, pyrantel pamoate (alone or with piperazine-carbon disulfide complex), thiabendazole (alone or with piperazine or with trichlorfon), tioxidazole, and trichlorfon], against Thelazia lacrymalis was evaluated in 102 equids. Determination of activity was based on comparison of infection rate in treated animals examined at necropsy with infection rate of dead equids in our contemporary surveys. None of the compounds appeared to be active against T lacrymalis.