Haplotypic analysis of cox1 from Toxocara canis demonstrates five distinct clades that are not geographically defined.

BACKGROUND: Toxocara canis is a cosmopolitan parasite of dogs that is transmitted transplacentally to puppies resulting in widespread shedding of eggs in the environment. However, it is not clear if there are dominant parasite genotypes that are more common, pathogenic, or likely to be zoonotic. METHODS/PRINCIPLE FINDINGS: Sequences of mitochondrial cox1 gene from adult worms were used to compare parasites from the United States with submitted sequences from parasites isolated from dogs in different countries. Our analysis revealed at least 55 haplotypes. While we expected the North American worms to form a distinct cluster, we found haplotypes of T. canis reported elsewhere existing in this population. Interestingly, combining the sequence data from our study with the available GenBank data, analysis of cox1 sequences results in five distinct clades that are not geographically defined. CONCLUSIONS: The five clades of T. canis revealed in this study potentially have unique life histories, traits, or host preferences. Additional investigation is needed to see if these distinct clades represent cryptic species with clinically useful attributes or genotypes with taxonomic value. Evaluation of common mitochondrial genes may reveal distinct populations of zoonotic T. canis.

Bacillus thuringiensis Cry5B protein as a new pan-hookworm cure.

Hookworms are intestinal nematode parasites that infect nearly half a billion people and are globally one of the most important contributors to iron-deficiency anemia. These parasites have significant impacts in developing children, pregnant women and working adults. Of all the soil-transmitted helminths or nematodes (STNs), hookworms are by far the most important, with disease burdens conservatively estimated at four million DALYs (Disability-Adjusted Life Years) and with productivity losses of up to US$139 billion annually. To date, mainly one drug, albendazole is used for hookworm therapy in mass drug administration, which has on average ∼80% cure rate that is lower (<40%) in some places. Given the massive numbers of people needing treatment, the threat of parasite resistance, and the inadequacy of current treatments, new and better cures against hookworms are urgently needed. Cry5B, a pore-forming protein produced by the soil bacterium Bacillus thuringiensis (Bt) has demonstrated good efficacy against Ancylostoma ceylanicum hookworm infections in hamsters. Here we broaden studies of Cry5B to include tests against infections of Ancylostoma caninum hookworms in dogs and against infections of the dominant human hookworm, Necator americanus, in hamsters. We show that Cry5B is highly effective against all hookworm parasites tested in all models. Neutralization of stomach acid improves Cry5B efficacy, which will aid in practical application of Cry5B significantly. Importantly, we also demonstrate that the anti-nematode therapeutic efficacy of Cry5B is independent of the host immune system and is not itself negated by repeated dosing. This study indicates that Bt Cry5B is a pan-hookworm anthelmintic with excellent properties for use in humans and other animals.

Determination of anthelmintic efficacy against Toxocara canis in dogs by use of capsule endoscopy.

Industry guidelines for anthelmintic testing call for postmortem inspection of animals to verify treatment efficacy. A previous study showed that capsule endoscopy (CE) can be performed on dogs in vivo to quantify hookworms in the small intestine. Adoption of a minimally invasive procedure such as this could reduce the need for necropsy in efficacy trials. The present study employed CE to enumerate Toxocara canis in dogs, with two main goals: to determine if multiple capsule examinations improves the accuracy of worm counts compared to a single examination, and to establish if the efficacy of an anthelmintic compound is the same whether calculated using CE or necropsy data. To avoid needless animal sacrifice, the study was carried out on beagle dogs already in a product development trial with a planned terminal endpoint. Dogs were infected by oral inoculation with T. canis eggs. Untreated control dogs (n=8) were evaluated by CE three times while dogs treated with test compounds (3 groups of 4) were examined only once. Utilizing either the average count or just the last complete capsule examination, a robust correlation was found between CE and postmortem numbers (r=0.94, p<0.001). Calculated anthelmintic efficacy was essentially identical for the two enumeration methods, ranging from 94% to 100% for the three research compounds. CE may therefore be a viable alternative to necropsy for T. canis parasiticide trials.

Macrocyclic lactone resistance in Dirofilaria immitis: Failure of heartworm preventives and investigation of genetic markers for resistance.

Macrocyclic lactone (ML) endectocides are used as chemoprophylaxis for heartworm infection (Dirofilaria immitis) in dogs and cats. Claims of loss of efficacy (LOE) of ML heartworm preventives have become common in some locations in the USA. We directly tested whether resistance to MLs exists in LOE isolates of D. immitis and identified genetic markers that are correlated with, and therefore can predict ML resistance. ML controlled studies showed that LOE strains of D. immitis established infections in dogs despite chemoprophylaxis with oral ivermectin or injectable moxidectin. A whole genome approach was used to search for loci associated with the resistance phenotype. Many loci showed highly significant differences between pools of susceptible and LOE D. immitis. Based on 186 potential marker loci, Sequenom(®) SNP frequency analyses were conducted on 663 individual parasites (adult worms and microfilariae) which were phenotypically characterized as susceptible (SUS), confirmed ML treatment survivors/resistant (RES), or suspected resistant/loss of efficacy (LOE) parasites. There was a subset of SNP loci which appears to be promising markers for predicting ML resistance, including SNPs in some genes that have been associated with ML resistance in other parasites. These data provide unequivocal proof of ML resistance in D. immitis and identify genetic markers that could be used to monitor for ML resistance in heartworms.

Assessing the speed of kill of hookworms, Ancylostoma caninum, by Advantage Multi ® for Dogs using endoscopic methods.

Endoscopic capsules and endoscopy were used to assess the speed of kill and the clearance of hookworms in dogs experimentally infected with Ancylostoma caninum. A total of four adult dogs were inoculated in two separate cohorts comprised of two 4-year-old females and two 7-year-old males. Dogs were treated topically with Advantage Multi(®) for Dogs 13 days (Cohort 1) or 16 days (Cohort 2) after infection. Endoscopic imaging of the small intestine was carried out both pre- and post-treatment. Examination of the first cohort revealed that the worms had been cleared and the hookworm-induced lacerations were markedly diminished within 48 h of treatment. In the second cohort, endoscopic capsules were given the day of, the day after, and two days after treatment; within 24h of product administration, the worms had been removed with a concurrent reduction in observed lesions. Topical application of Advantage Multi(®) for Dogs rapidly removed worms from the small intestine of the dogs in this study as early as 24h post-treatment, with a marked reduction in the number of mucosal lesions seen.

Detecting Aelurostrongylus abstrusus-specific IgG antibody using an immunofluorescence assay.

Diagnosis of feline lungworm, Aelurostrongylus abstrusus, is typically achieved by identifying larvae in feces following concentration through flotation or using the Baermann technique. This work presents observations on the usefulness of an indirect immunofluorescence antibody assay for detection of antibodies to this parasite in the sera of infected cats. Using first-stage larvae of A abstrusus and sera from both experimentally and naturally infected cats, it was determined that the test was fairly sensitive and did not cross-react with serum from an Ancylostoma braziliense (hookworm)-infected cat.

Comparison of Ancylostoma caninum worm counts acquired by endoscopy and necropsy.

Many regulatory agencies require that the efficacy of veterinary anthelmintic medications be evaluated by enumerating parasites in treated and untreated animals after necropsy. Current ethical considerations, i.e., the 3 Rs of research, call for the replacement of this method with less invasive techniques that would not require animal sacrifice. This study tested standard gastrointestinal endoscopy as an in vivo method of quantifying the intestinal hookworm, Ancylostoma caninum. Worm counts were compared with those from gold standard necropsy. Thirteen dogs inoculated with third-stage A. caninum larvae underwent endoscopy 4-6 weeks post-infection, just prior to necropsy. Two-thirds of the adult hookworms were located in the middle section of the small intestine that could not be reached for endoscopic examination. Not surprisingly, the total worm counts obtained by endoscopy did not correlate with those from necropsy (R(2)=0.05, p=0.464). One method to increase small intestinal access would be to use specialized balloon or spiral endoscopes developed for this purpose in human gastroenterology. Based on the results of this study, standard endoscopy alone is unsuitable for quantification of A. caninum in the small intestine. Parasites in more accessible sites, such as whipworms in the cecum and colon, might be more appropriate targets for endoscopic counting.

Serological survey of Toxoplasma gondii, Dirofilaria immitis, Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) infections in pet cats in Bangkok and vicinities, Thailand.

The seroprevalence of Toxoplasma gondii, Dirofilaria immitis (heartworm), feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections was examined using serum or plasma samples from 746 pet cats collected between May and July 2009 from clinics and hospitals located in and around Bangkok, Thailand. The samples were tested for heartworm, FIV, and FeLV using a commercial ELISA. Of the 746 samples, 4.6% (34/746) were positive for heartworm antigen, 24.5% (183/746) had circulating FeLV antigen, and 20.1% (150/746) had antibodies against FIV. In addition, the first 348 submitted samples were tested for T. gondii antibodies using a modified agglutination test (MAT, cut off 1:25); 10.1% (35/348) were seropositive. Of the 348 cats sampled for all four pathogens, 11, 10, and 1 were positive for T. gondii antibodies and FIV antibodies, FeLV antigen, or D. immitis antigen, respectively. Of the 35 T. gondii-seropositive cats, 42.9% (15/35) were co-infected with at least one of the other three pathogens. The presence of antibodies to FIV was significantly associated with both age and gender, while FeLV antigen presence was only associated with age. In the case of FIV, males were twice as likely to be infected as females, and cats over 10 years of age were 13.5 times more likely to be infected than cats less than 1 year of age. FeLV antigen was more common in younger cats, with cats over 10 years of age being 10 times less likely to be FeLV positive than cats under 1 year of age. This is the first survey for these four pathogens affecting feline health in Thailand.

Obtaining an isolate of Ancylostoma braziliense from dogs without the need for necropsy.

Isolation of a specific Ancylostoma species typically requires death of the source animal, or holding an animal long enough to collect feces after treatment, for worm recovery and identification. The reason for collecting worms is that the eggs are not easy to distinguish morphologically. In keeping with the 3 Rs of laboratory animal research (reduction, refinement, replacement), the objective of this study was to obtain an isolate of Ancylostoma braziliense from 1-time field-collected samples of canine feces without the need for killing the host. During a collection trip to Florida, fecal samples (n  =  148) were collected and identified as containing eggs of Ancylostoma species (n  =  64) using centrifugal sugar flotation. Eggs from hookworm-positive slides were washed into tubes, DNA was extracted, and 2 samples were identified as A. braziliense using restriction fragment length polymorphism (RFLP) with Hinf1. Larval cultures were initiated from these samples, and larvae from the cultures were returned to New York and used to inoculate a purpose-bred kitten with the goal of inhibiting the growth of any contaminating Ancylostoma caninum that might be present in the culture. The infection was patent at 15 days, and eggs were identified as A. braziliense by RFLP and DNA sequencing. Using forceps during endoscopy, 2 adult worms (1 male, 1 female) were recovered from the cat and identified morphologically as A. braziliense . Larvae were cultured from the feces of this cat and used to infect a laboratory-reared beagle dog. Additionally, worms recovered from the feces of the cat post-treatment were confirmed to be A. braziliense , except for 1 female A. caninum containing infertile eggs. The dog (patent 14 days post-infection) was also infected with A. braziliense as determined by RFLP and DNA sequencing of eggs and cultured larvae. Both the cat and dog were treated, verified to be no longer shedding eggs, and then placed into adoptive homes.

Obtaining an isolate of Ancylostoma braziliense from cats without the need for necropsy.

Because the eggs of Ancylostoma species of dogs and cats are difficult to readily distinguish morphologically, isolation of a certain species often requires the humane death of the source animal or holding an animal after treatment to obtain worms for specific identification or to harvest ex utero eggs. The objective of this study was to obtain an isolate of Ancylostoma braziliense from 1-time, field-collected samples of feline feces without the need for the killing of any animals. During a collection trip to Florida, fecal samples (n  =  40) were collected and identified as containing A. braziliense eggs (n  =  26) using centrifugal sugar flotation. Eggs from hookworm-positive slides were washed into tubes, DNA was extracted, and 10 samples were identified as containing A. braziliense using restriction fragment length polymorphism (RFLP) with Hinf1. Six of these samples also contained DNA of Ancylostoma tubaeforme and, thus, only 4 samples were from cats infected only with A. braziliense. Larvae cultured from two of the latter samples were used to subcutaneously inoculate a purpose-bred puppy with the intention to inhibit the growth of any potentially contaminating A. tubaeforme larvae in the culture. The infection was patent at 14 days after inoculation, and the eggs were identified as A. braziliense by RFLP and DNA sequencing. Larvae were cultured from the feces of this dog and used to infect a laboratory-reared, specific-pathogen-free cat; the eggs and larvae produced by the cat were also identified molecularly as those of A. braziliense. The larvae from this cat were used to infect other cats to maintain the isolate for further research. Both the puppy and the first cat used in this study were treated to clear their infections and have since been adopted by new owners.

Utility of capsule endoscopy for evaluating anthelmintic efficacy in fully conscious dogs.

The current accepted standard for evaluating the efficacy of gastrointestinal anthelmintic drugs is necropsy of infected animals followed by a comparison of worm counts between treated and non-treated groups. In this study capsule endoscopy, a minimally invasive method of imaging the small intestine of humans, is evaluated as a possible alternative to necropsy for the purposes of worm quantification in dogs. Eighteen Beagle dogs were included in this study. These dogs were part of a separate trial intended to determine the efficacy of various candidate parasiticides against Ancylostoma caninum via the necropsy standard. Dogs were inoculated with A. caninum L3s 4 weeks prior to treatment with one of the candidate compounds; a control group (n=8) received no treatment. Capsule endoscopy was performed 6-14 days post-treatment, followed by necropsy the following day. Seventeen dogs had complete examinations, i.e. the capsule traversed the small intestine and reached the colon within the battery life of the capsule. A strong correlation (r(s)=0.87, P<0.0001) was observed between the worm counts acquired by capsule endoscopy and necropsy. There was no clear relationship between the ability of the capsule endoscope to detect hookworms and either visibility of the intestinal lumen or small intestinal transit time. Generation of a virtual spatial record of hookworm location from the capsule endoscopy data revealed a temporal trend, with the majority of worms present in the proximal small intestine in the morning versus the central to distal small intestine in the afternoon. Worm distribution as determined by capsule endoscopy closely resembled post-mortem findings. In conclusion, capsule endoscopy shows promise as an alternative to necropsy for the enumeration of A. caninum in the canine small intestine, although further work is required to improve completion rates and optimise intestinal examination.

Evaluation of a new in-clinic method for the detection of canine heartworm antigen.

Canine heartworm is endemic in many parts of the world, and veterinarians rely on rapid in-clinic antigen tests to screen for this infection. Recently, an in-clinic, instrument-based rotor employing a colloidal gold agglutination immunoassay was launched in the marketplace (VetScan VS2(®) Canine Heartworm (HW) Antigen Test Kit; Abaxis, Inc.). Because of the widespread use of heartworm prevention and possible false negative test results in dogs with low heartworm burdens, the performance of the VetScan VS2(®) HW test and a commercially available in-clinic, membrane-based ELISA test (SNAP(®) Heartworm RT Test; IDEXX Laboratories) was compared using samples from dogs with low heartworm burdens and/or low levels of circulating antigen. Ninety serum samples were evaluated using the two methods. Testing was performed according to the manufacturer's product insert by personnel blinded to sample status. The samples were derived from two populations: dogs with necropsy-confirmed heartworm status (40 with 1-4 female worms, 30 with no worms), and field dogs (20) confirmed positive for antigen by microtiter plate ELISA (PetChek(®) Heartworm PF Antigen Test; IDEXX Laboratories). All 40 dogs with heartworms on necropsy were also confirmed to have circulating antigen by the PetChek HW ELISA. In necropsy-negative dogs (n=30), neither the VetScan VS2 HW nor SNAP HW tests detected heartworm antigen. Of the samples testing positive for antigen by PetChek HW (n=60), the VetScan VS2 HW and SNAP HW tests detected antigen in 15 and 56 samples, respectively. Percent agreement (plus 95% confidence interval) for each test relative to the PetChek HW qualitative result was 50% (40-60%) for VetScan VS2 HW and 96% (89-98%) for SNAP HW. Relative to the presence or absence of female worms at necropsy, agreement was 61% (50-72%) for VetScan VS2 HW and 99% (92-99.6%) for SNAP HW tests. It is clinically important that dogs with low heartworm burdens and/or low levels of circulating heartworm antigen be correctly identified by veterinarians in order to ensure prompt treatment, and the VetScan(®) VS2 HW test does not appear to be as accurate as the SNAP HW or PetChek HW tests when performed on this subset of patients.

Understanding feline heartworm infection: disease, diagnosis, and treatment.

Feline heartworm disease is a very different clinical entity from canine heartworm disease. In cats, the arrival and death of immature heartworms in the pulmonary arteries can cause coughing and dyspnea as early as 3 months postinfection. Adult heartworms suppress the function of pulmonary intravascular macrophages and thus reduce clinical disease in chronic feline heartworm infection. Approximately 80% of asymptomatic cats self-cure. Median survival time for symptomatic cats is 1.5 years, or 4 years if only cats living beyond the day of presentation are considered. Aberrant worm migration is more frequent than it is in dogs, and sudden death can occur with no prior clinical signs. The bacterial endosymbiont Wolbachia likely contributes to the inflammatory pathology of heartworm disease, but its role is not yet fully clear. Unfortunately, the diagnosis, treatment, and management of feline heartworm disease are far from simple. Antemortem diagnosis is hampered by low worm burdens, the frequency of all-male infections, and nonspecific radiographic lesions. It is up to the veterinarian to determine the correct index of suspicion and choose the right combination of diagnostic tests to achieve an answer. Treatment is symptomatic because adulticide therapy is risky and does not increase survival time. Despite the dangers of feline heartworm disease, less than 5% of cats in the United States are on chemoprophylaxis. It is important for veterinarians to take a proactive preventive stance because heartworm infection in cats is a multisystemic disease that has no easy cure.

Public health issues concerning the widespread distribution of canine heartworm disease.

Heartworms can cause serious cardiopulmonary disease in their canid hosts. Canine heartworm has become widespread in many parts of the world, and its range continues to expand. Wildlife reservoirs play a role in perpetuation and transmission of this parasite to dogs. Human heartworm infection is incidental and is typically not associated with severe clinical disease; however, because no serological test is readily available, patients must undergo invasive procedures to differentiate heartworm from other more serious diseases. Human cases have been reported mainly in areas of high canine prevalence, highlighting the importance of heartworm testing and chemoprophylaxis in all dogs to reduce transmission. Future efforts should focus on the development of a non-invasive diagnostic test for people, and on epidemiological surveys for both animals and people.

Epidemiologic and zoonotic aspects of ascarid infections in dogs and cats.

Toxocaracanis and Toxocara cati of dogs and cats, respectively, can cause significant disease in people. Human seroprevalence for Toxocara antibodies varies with factors such as geographic location, socio-economic status, and dietary habits. Risk factors for infection include geophagia and low-level education. Toxocara canis is better recognized as a cause of human toxocariasis, but Toxocara cati should not be overlooked. In addition, patent infections with Baylisascaris procyonis, the raccoon ascarid, have been increasingly recognized in dogs. Pet owners need to be properly educated about zoonotic risks, and veterinarians should institute regular parasite screening and treatment for all pets. Establishment of national surveillance programs to determine the incidence and specific etiological agent in human larva migrans patients would aid in the development of targeted intervention strategies.

Mammomonogamus auris infection in the middle ear of a domestic cat in Saipan, Northern Mariana Islands, USA.

A 2-year-old female domestic shorthair cat on the island of Saipan was presented to a local veterinarian for headshaking. Otoscopic examination showed mild erythema of the right tympanic membrane, but was otherwise unremarkable. Headshaking resolved with topical gentamicin/betamethasone/clotrimazole therapy; however, erythema persisted. Further otoscopy revealed movement of the erythematous region, which was in fact the red-colored strongylid nematode, Mammomonogamus auris, residing within the middle ear. Myringotomy and a saline flush were performed under heavy sedation. A silastic tube was inserted into the incision and the worms were retrieved by applying negative pressure. Follow-up treatment included topical thiabendazole/dexamethasone/neomycin ointment as well as selamectin. Mammomonogamus auris has previously been documented only three times, once each in China, Sri Lanka and Japan. This is the first report of M auris in cats from Saipan.