Synergistic effects of using sodium hypochlorite (bleach) and desiccation in surface inactivation for Toxocara spp.

Toxocara cati and T. canis are parasitic nematodes found in the intestines of cats and dogs respectively, with a cosmopolitan distribution, and the potential for anthropozoonotic transmission, resulting in human toxocariasis. Spread of Toxocara spp. is primarily through the ingestion of embryonated eggs contaminating surfaces or uncooked food, or through the ingestion of a paratenic host containing a third-stage larva. The Toxocara spp. eggshell is composed of a lipid layer providing a permeability barrier, a chitinous layer providing structural strength, and thin vitelline and uterine layers, which combined create a biologically resistant structure, making the Toxocara spp. egg very hardy, and capable of surviving for years in the natural environment. The use of sodium hypochlorite, household bleach, as a disinfectant for Toxocara spp. eggs has been reported, with results varying from ineffective to limited effectiveness depending on parameters including contact time, concentration, and temperature. Desiccation or humidity levels have also been reported to have an impact on larval development and/or survival of Toxocara spp. eggs. However, to date, after a thorough search of the literature, no relevant publications have been found that evaluated the use of sodium hypochlorite and desiccation in combination. These experiments aim to assess the effects of using a combination of desiccation and 10% bleach solution (0.6% sodium hypochlorite) on fertilized or embryonated eggs of T. cati, T. canis, and T. vitulorum. Results of these experiments highlight the synergistic effects of desiccation and bleach, and demonstrate a relatively simple method for surface inactivation, resulting in a decrease in viability or destruction of T. cati, T. canis and T. vitulorum eggs. Implications for these findings may apply to larger scale elimination of ascarid eggs from both research, veterinary, and farming facilities to mitigate transmission.

Comparison of extracellular vesicle isolation methods for the study of exosome cargo within Toxocara canis and Toxocara cati excretory secretory (TES) products.

Toxocara is a genus of nematodes, which infects a variety of hosts, principally dogs and cats, with potential zoonotic risks to humans. Toxocara spp. larvae are capable of migrating throughout the host tissues, eliciting eosinophilic and granulomatous reactions, while surviving for extended periods of time, unchanged, in the host. It is postulated that larvae are capable of altering the host's immune response through the release of excretory-secretory products, containing both proteins and extracellular vesicles (EVs). The study of EVs has increased exponentially in recent years, largely due to their potential use as a diagnostic tool, and in molecular therapy. To this end, there have been multiple isolation methods described for the study of EVs. Here, we use nanoparticle tracking to compare the yield, size distribution, and % labelling of EV samples acquired through various reported methods, from larval cultures of Toxocara canis and T. cati containing Toxocara excretory-secretory products (TES). The methods tested include ultracentrifugation, polymer precipitation, magnetic immunoprecipitation, size exclusion chromatography, and ultrafiltration. Based on these findings, ultrafiltration produces the best results in terms of yield, expected particle size, and % labelling of sample. Transmission electron microscopy confirmed the presence of EVs with characteristic cup-shaped morphology. These findings can serve as a guide for those investigating EVs, particularly those released from multicellular organisms, such as helminths, for which few comparative analyses have been performed.

Elimination of probable praziquantel-resistant Dipylidium caninum with nitroscanate in a mixed-breed dog: a case report.

BACKGROUND: Praziquantel is the drug of choice for treating the tapeworm Dipylidium caninum in dogs; however, resistance is possible, and regular, non-targeted administration of praziquantel may select for anthelminthic-resistant populations. METHODS: The zinc sulfate fecal floatation procedure was conducted. Gross visualization was used to identify Dipylidium spp. segments, and capsule endoscopy was used to visualize adult tapeworms within the intestinal tract. RESULTS: An 18-month-old spayed female terrier mix was presented due to diarrhea, hematochezia and weight loss. The dog received appropriate anthelmintic therapy for Giardia spp., Ancylostoma spp. and Dipylidium spp. The dog's clinical signs resolved, and elimination of Ancylostoma spp. was confirmed by subsequent fecal analysis. However, Dipylidium spp. segments were repeatedly present in the stool. Observation of the segments confirmed the presence of adult Dipylidium spp in feces. Treatment with praziquantel and epsiprantel were unsuccessful in eliminating the organism but was apparently successful in flea prevention. A single dose of nitrosconate was administered and eliminated Dipylidium spp. infection in the dog. CONCLUSIONS: Nitrosconate can be an effective treatment for praziquantel-resistant dipylidiasis in dogs. The novel application of capsule endoscopy confirmed the anthelmintic efficacy of this treatment.

Acanthamoeba sclerokeratitis in a cat.

CASE DESCRIPTION: A 12-year-old neutered male domestic shorthair cat with chronic anterior uveitis and secondary glaucoma of the right eye was examined for persistent blepharospasm 2 weeks after corneal debridement and grid keratotomy for nonhealing superficial ulcerative keratitis. CLINICAL FINDINGS: Examination of the right eye revealed a central superficial corneal ulcer associated with corneal epithelial and subepithelial infiltrates and mild aqueous flare. Structures consistent with amoeboid cysts and trophozoites were detected in the cornea by in vivo confocal microscopy. Suppurative keratitis was identified cytologically. An Acanthamoeba spp was isolated through culture and identified by a PCR assay of corneal specimens. TREATMENT AND OUTCOME: Symptomatic and antiamoebic (polyhexamethylene biguanide 0.02% ophthalmic solution) treatments were instituted. Over the following 6 weeks, the cat lost vision in the affected eye and lesions progressed to nonulcerative stromal keratitis associated with a dense paracentral corneal stroma ring infiltrate and anterior lens luxation. The globe was enucleated, and lymphoplasmacytic sclerokeratitis, anterior uveitis, and retinal detachment were noted. Acanthamoeba organisms were detected within the corneal stroma and anterior sclera with histologic and immunohistochemical stains. The amoebae were classified to the Acanthamoeba T4 genotype by DNA sequencing. The cat had no medical problems attributed to Acanthamoeba infection over 36 months after enucleation, until the cat was lost to follow-up. CLINICAL RELEVANCE: Naturally acquired Acanthamoeba sclerokeratitis is described in a cat for the first time. Acanthamoeba infection should be considered for cats with superficial corneal disease refractory to appropriate treatments and especially occurring after ocular trauma, including keratotomy.

Current time-temperature relationships for thermal inactivation of Ascaris eggs at mesophilic temperatures are too conservative and may hamper development of simple, but effective sanitation.

Ascaris eggs are commonly used as indicators for pathogen inactivation during the treatment of fecal sludge and wastewater due to their highly resistant lipid membrane and ability to survive in the environment for long periods of time. Current guidelines suggest that thermal treatment alone cannot inactivate Ascaris eggs at temperatures below 45 °C, although some evidence in the literature suggests this to be incorrect. Here, we performed a controlled experiment to test the effect of mesophilic temperatures on Ascaris inactivation. We exposed Ascaris suum eggs to a temperature gradient between 34°C and 45 °C under anaerobic and aerobic conditions to observe the required exposure times for a 3-log reduction. Indeed, we found that temperatures lower than 45 °C did inactivate these eggs, and the required exposure times were up to two orders of magnitude shorter than suggested by current guidelines. Results from the anaerobic exposures were used to develop a time-temperature relationship that is appropriate for Ascaris inactivation at mesophilic temperatures. Data from the literature demonstrated that our relationship is conservative, with faster inactivation occurring under environmental conditions when Ascaris eggs were suspended in fecal sludge or manure. A specific aerobic relationship was not developed, but we demonstrated that aerobic conditions cause faster inactivation than anaerobic conditions. Therefore, the anaerobic relationship provides a conservative guideline for both conditions. We demonstrate that relatively low temperatures can considerably impact Ascaris viability and suggest that mesophilic temperatures can be used in waste treatment processes to inactivate pathogens. The development of safe, low-input, mesophilic treatment processes is particularly valuable for ensuring universal access to safe sanitation and excreta management.

Isolation, genotyping and subtyping of single Cryptosporidium oocysts from calves with special reference to zoonotic significance.

The ability of the small-subunit ribosomal RNA (SSU rRNA) based nested PCR and Restriction Fragment Length Polymorphism (PCR-RFLP) to identify and genotype a single Cryptosporidium oocyst isolated from bovine faecal samples was evaluated in this study. In addition, subtyping was carried out by sequencing the 60 kDa glycoprotein (gp60) gene from the same single oocyst. Faecal samples were collected from 40 pre-weaned calves (5-20 days old) from 7 dairy farms located in 3 different counties within the Finger Lakes region of Upstate New York. All the samples were microscopically positive for Cryptosporidium spp. A total of 400 Cryptosporidium oocysts (10 single oocysts from each calf sample) were individually isolated and analyzed using a nested PCR targeting the SSU rRNA gene. The SSU rRNA gene was amplified in 324 (81%) individual oocysts. All SSU rRNA amplified individual oocysts DNA was genotyped using PCR-RFLP. C. parvum was the only identified species; 107 single oocysts generated PCR products from the A gene, 18 generated PCR products from the B gene and 199 generated PCR products from both. Sequence analysis of the gp60 gene in 99 individual oocysts revealed the presence of only subtype IIaA15G2R1 with 99.4-100% and 99.1-100% identity of nucleotides and amino acids, respectively. These sequences were identical (100%) in oocysts from 35 calves and exhibited mutations in the non-repeat region of the gp60 gene in those of 5 other calves. The examination of DNA from individual oocysts with genotyping and subtyping tools provides methodology to more clearly define the genetic characteristics of Cryptosporidium spp. on farms and within individual animals.

Efficacy and safety of a new topical formulation of selamectin plus sarolaner in the treatment and control of natural infections of Ancylostoma tubaeforme and Toxocara cati in cats presented as veterinary patients in the United States.

A new topical formulation of selamectin plus sarolaner (Revolution® Plus/Stronghold® Plus, Zoetis) was evaluated in the treatment and control of naturally occurring infections of Ancylostoma tubaeforme and Toxocara cati in cats presented as veterinary patients in the United States. Three thousand three hundred three (3303) cats were screened in 25 veterinary practices in 15 states and 153 hookworm-positive cats (A. tubaeforme and/or A. braziliense), mainly from Alabama, Mississippi, Texas, and Hawaii, were identified; 135 cats met all the criteria for enrollment and were included on study. The cats were randomly assigned to treatment with Revolution® (at the label dosage, to provide a minimum dosage of 6 mg/kg selamectin) or selamectin plus sarolaner (at a dosage of 6-12 mg/kg plus 1-2 mg/kg, respectively). Treatments were administered at the time of enrollment and repeated 30 days later. Fecal samples were collected for differential fecal egg count prior to the first treatment (Day 0), prior to the second treatment (Day 30), and approximately 30 days later (Day 60). Efficacy was based on the percentage reductions in geometric mean fecal egg count for A. tubaeforme on Day 30 and Day 60 compared with Day 0. Where cats were co-infected with T. cati, efficacy against this species was also evaluated. Efficacy data were evaluated for A. tubaeforme for 40 cats on both Day 30 and Day 60 for the group treated with the selamectin/sarolaner combination and reductions in geometric mean fecal egg counts of 99.4% and 99.7% were demonstrated for Day 30 and Day 60, respectively. For the group treated with selamectin alone, 44 and 40 cats were evaluated and percent reductions for Day 30 and Day 60 were 99.5% and 99.9%, respectively. For T. cati, 14 cats were evaluated in the selamectin/sarolaner-treated group for Day 30 and for Day 60, and the reduction in geometric mean fecal egg count was 100% for both days. There were 11 and 9 cats evaluated for Day 30 and Day 60, respectively, for the selamectin-treated group and the reduction was again 100% for both days. The geometric mean fecal egg counts post-treatment were significantly lower than pre-treatment for both A. tubaeforme and T. cati, for both treatments, and for both periods of interest (P < 0.0001). No serious adverse events related to treatment with either product occurred during the study. Thus, both selamectin alone and the combination product of selamectin/sarolaner were safe and effective when administered on a monthly basis for the treatment and control of natural infections of A. tubaeforme and T. cati. The addition of sarolaner to the formulation did not interfere with the efficacy of selamectin against these nematodes.

Comparative performance of reference laboratory tests and in-clinic tests for Giardia in canine feces.

BACKGROUND: We examined the performance of four in-clinic Giardia diagnostic tests by comparing results to three laboratory methods for detection of Giardia. A set of 177 fecal samples originally submitted to a commercial laboratory by veterinarians for routine ova and parasite (O&P) testing was used. Specimens were examined by direct immunofluorescence assay (DFA) for presence of Giardia cysts which served as the gold standard. Fecal samples were tested using a Giardia-specific cyst wall antigen microtiter plate format enzyme-linked immunosorbent assay (ELISA) and each of the in-clinic assays adhering to the package insert for each kit. RESULTS: Evaluated were four in-clinic antigen test kits: VetScan® Canine Giardia Rapid Test (Abaxis), Anigen® Rapid CPV-CCV-Giardia Antigen Test (BioNote), SNAP® Giardia Test (IDEXX) and Witness® Giardia Test (Zoetis). In the comparison of the in-clinic tests to the DFA standard test sensitivity ranged between 70.0-87.1%, and specificity ranged between 71.1-93.4%. CONCLUSION: Of the tests evaluated here, the SNAP test had the highest sensitivity and specificity. The SNAP test had the highest percent positive and percent negative agreement when compared to the microtiter plate format ELISA and the O&P assay.

Inactivation of Ascaris Eggs in Human Fecal Material Through In Situ Production of Carboxylic Acids.

Discovering new ways to inactivate pathogens in human waste is critical for the improvement of worldwide access to sanitation and for the reduction of the environmental impact of conventional waste treatment processes. Here, we utilized the carboxylate platform and chain elongation to produce n-butyric acid and n-caproic acid via the anaerobic fermentation of human fecal material. Then, we inactivated Ascaris eggs through exposure to these carboxylic acids. Using batch experiments with human fecal material as substrate, we accumulated n-butyric acid and n-caproic acid at total concentrations (uncharged acid plus conjugate base) of 257 and 27.1 mM, respectively. We then showed that carboxylic acids at these concentrations inactivated Ascaris eggs when the pH was below the pKa for the acids, causing them to exist primarily in the uncharged forms. We observed that uncharged carboxylic acids affected viability rather than the pH itself or conjugate bases. In addition, we modeled the viability of Ascaris eggs as a function of uncharged carboxylic acid concentration for n-butyric acid and n-caproic acid at exposure times of 2, 6, 12, and 20 days. The results presented here indicate that in situ biological production of carboxylic acids in HFM provides a promising method of pathogen inactivation and may lead to new developments in sanitation technology and treatment of fecal sludge.

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.

Use of filter papers to determine seroprevalence of Toxoplasma gondii among hunted ungulates in remote Peruvian Amazon.

Toxoplasmosis is a zoonosis caused by the protozoan Toxoplasma gondii, and it is found worldwide. To determine whether ungulates are reservoirs of T. gondii in an isolated and remote region of the northeastern Peruvian Amazon, antibodies to T. gondii were determined in 5 species of ungulates by the modified agglutination test (MAT). These animals were hunted by subsistence hunters along the Yavarí-Mirín River, in the northeastern Peruvian Amazon. Blood samples were collected by hunters on filter papers. For determination of T. gondii antibodies, blood was eluted from filter papers, and a titer of 1:25 was considered indicative of exposure to T. gondii. Antibodies to T. gondii were found in 26 (31.0%) peccaries (Pecari tajacu, Tayassu pecari), six (17.1%) brocket deer (Mazama americana, Mazama gouazoubira), and four (40.0%) lowland tapir (Tapirus terrestris). We also introduced a modification to the MAT protocol that allows the extraction of fluid samples from several types of laboratory-grade filter paper, thus enabling researchers to easily adapt their approaches to the materials presented to them.

Enzyme-linked immunosorbent assay using a crude antigen extract to measure Cuterebra-specific immunoglobulin G in sera of cats with systemic infections.

In cats, larvae of the dipteran fly, Cuterebra, sometimes cause severe disease by their migration through the tissues of the larynx, pharynx, nasal sinuses, brain, and spinal cord; such infected cats may die without the maggots ever reaching the subcutaneous tissues where they would typically mature. The current study examines the ability of an indirect enzyme-linked immunosorbent assay (ELISA) using crude Cuterebra antigen from maggots to detect parasite-specific immunoglobulin (Ig)G in cats with known (n = 42), suspected (n = 25), or no known exposure to the infection (n = 68). The probability of a given optical density (OD) predicting the infection status of a given animal was determined using logistic regression, and both 1:20 and 1:80 serum dilutions were highly predictive of the potential of a cat being infected with a larval Cuterebra. In 5 cases where 2 samples were collected 1-2 weeks apart, there was a mean OD increase in the second sample for both the 1:20 and 1:80 dilutions, but it was significant (P = 0.044) only at the 1:20 dilution. Sex of the sampled cat was not a significant contributor to the ability of the OD to predict the presence of a larva, but the age of the cat added significantly to the predictive value of the generated curves, with the only exception being with the 1:20 serum dilution with the curve being generated only using the cats known to be positive for larval presence. This ELISA should aid in ruling cuterebriasis in or out in suspect systemic and, specifically, neurologic cases and provide information on kinetics of antibody presence postexposure.

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.

Description of fecal shedding of Cryptosporidium parvum oocysts in experimentally challenged dairy calves.

The objective was to describe the probability of Cryptosporidium parvum fecal oocyst shedding at different magnitudes of exposure, the pattern of fecal shedding over time, and factors affecting fecal shedding in dairy calves. Within the first 24 h of life, 36 calves were experimentally challenged with C. parvum oocysts at one of four possible magnitudes of oral exposure (1 × 10(3), 1 × 10(4), 1 × 10(5), and 1 × 10(6) oocysts), and 7 control calves were sham dosed. Fecal shedding occurred in 33 (91.7 %) experimentally challenged calves and in none of the control calves. There was a difference in the log-total number of oocysts counted per gram of feces dry weight among the four exposure groups; calves with the lowest magnitude of exposure (1 × 10(3) oocysts) shed less than the other three groups. At higher magnitudes of exposure, there was more variability in the range of fecal oocyst shedding. There was an inverse relationship between the log-total amount of oocysts counted per gram of feces dry weight and the number of days to the onset of fecal shedding per calf, i.e., the more time that elapsed to the onset of fecal shedding, the fewer oocysts that were shed. The pattern of fecal shedding over time for all calves shedding oocysts was curvilinear; the number of oocysts increased with time, reached a peak, and declined. Therefore, the dynamics of oocyst shedding can be influenced in part by limiting exposure among calves and delaying the onset of fecal oocyst shedding.

Ineffectiveness of a quaternary ammonium salt and povidone-iodine for the inactivation of Ascaris suum eggs.

Two commonly used disinfectants, a quaternary ammonium salt and povidone-iodine, were tested for effectiveness against unembryonated Ascaris suum eggs. The quaternary ammonium salt (alkyl dimethyl benzyl ammonium chloride) had no effect on the Ascaris eggs (10 minutes and 22°C) when compared with the controls in water with egg viabilities of 88.8% ± 3.3% and 86.9% ± 6.2%, respectively. An additional quaternary ammonium salt, 2.5% benzethonium chloride, also had no effect. Phenol (5%) and cresol (3%) completely inactivated the eggs. Povidone-iodine at 100%, 50%, 10%, and 1% had no effect on the eggs at exposures of 5, 15, 30, 60, or 120 minutes (22°C) compared with the water controls.

Differentiation of the microfilariae of Dirofilaria immitis and Dirofilaria repens in stained blood films.

An examination was made of Giemsa-stained microfilariae in thin blood films from (n = 9) dogs naturally or experimentally infected with Dirofilaria immitis or Dirofilaria repens. Morphological measurements (total length, length of cephalic space, anterior end to nerve ring and last body nucleus, and nucleus-free tail tip) were made on 2-6 microfilariae from each dog with the use of digitally captured images and imaging software. The microfilariae of D. repens were significantly greater (P < 0.001) in all measured dimensions except for the length of the cephalic space, which was significantly shorter (P < 0.001) than that of D. immitis. The cephalic space of D. repens was characterized by being short and routinely being terminated by a distinct pair of nuclei that were separate from the remaining somatic nuclei of the microfilaria. The cephalic space of the smaller microfilaria of D. immitis was longer and did not have the distinct nuclei separated from the somatic column nuclei near the anterior end. The character of the cephalic space seems to be a criterion that could be routinely used for the easy differentiation of these 2 microfilariae in stained blood films.

Inactivation kinetics of Cryptosporidium parvum oocysts in a swine waste lagoon and spray field.

Because of outbreaks of cryptosporidiosis in humans, some Cryptosporidium spp. have become a public health concern. Commercial swine operations can be a source of this protozoan parasite. Although the species distribution of Cryptosporidium is likely dominated by Cryptosporidium suis , a fraction may be comprised of other Cryptosporidium species infectious to humans such as Cryptosporidium parvum . To better understand the survival dynamics of Cryptosporidium spp., oocysts associated with swine operations, 2 experiments were performed to determine die-off rates of C. parvum oocysts in a swine waste lagoon (2009 and 2010) and its spray field (2010 and 2011). Sentinel chambers containing a lagoon effluent suspension of C. parvum oocysts were submerged in the lagoon, and triplicate chambers were removed over time; oocysts were extracted and assayed for viability. For comparative purposes, inactivation rates of Ascaris suum eggs contained in sentinel chambers were also determined. For 2 spray field experiments, air-dried and sieved surface soil was placed in sentinel chambers, hydrated, and inoculated with a lagoon effluent suspension of C. parvum oocysts. Sentinel chambers and control oocysts in PBS contained in microcentrifuge tubes were buried 1.5 cm below the soil surface in 3 blocks. Triplicate chambers and controls were removed over time; oocysts were extracted and assayed for viability. Based on the first order decay equation, days to reach 99% die-off (T(99)) were determined. T(99)-values determined for the 2 lagoon experiments were 13.1 and 20.1 wk, respectively. A T(99)-value for C. parvum in the spray field was significantly longer at 38.0 wk than the control oocysts in PBS at 29.0 wk. The waste lagoon and spray field system of manure management at this large-scale farrowing operation appeared to reduce the load of C. parvum oocysts before they can be hydrologically transported off the operation and reduces their likelihood of contaminating surface waters and threatening public health.

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.