Expellency, anti-feeding and speed of kill of a dinotefuran-permethrin-pyriproxyfen spot-on (Vectra®3D) in dogs weekly challenged with adult fleas (Ctenocephalides felis) for 1 month-comparison to a spinosad tablet (Comfortis®).

This study was designed to compare the efficacy of two ectoparasiticides against adult fleas on dogs: a topical (DPP, dinotefuran-permethrin-pyriproxyfen) and a systemic (S, spinosad). Dogs (n = 48; 10.21-22.86 kg BW) were allocated to six groups of eight dogs each (C1, C4, DPP1, DPP4, S1, S4). Dogs in the treated groups were administered a topical (3.6 mL of DPP) or a tablet (665 or 1040 mg of S) on day 0. Infestations with 100 unfed fleas (Ctenocephalides felis) occurred on days -6, -1, 2, 7, 14, 21 and 28. An additional untreated group (QC, n = 6) was involved to evaluate the flea-anti-feeding efficacy. These dogs were infested once with 150 fleas prior to combing of at least 50 live fleas from each dog 5 or 10 min after infestation. In the treated group, dislodged dead and moribund fleas were collected from dogs 5, 10, 15 and 60 min (DPP1, S1) or 5, 10, 30 and 240 min (DPP4, S4) post-treatment and subsequent flea infestations on pans placed underneath the cages. Fleas were counted and removed from dogs by combing 1 (C1, DPP1, S1) or 4 h (C4, DPP4, S4) post-treatment and subsequent infestations. Quantitative PCR analysis of the canine cytochrome b gene was conducted on dislodged fleas collected from treated and control (QC) dogs 5 and 10 min after post-treatment infestations. The number of gene copies was used as a marker of blood volume ingested by fleas. Dislodgeability and insecticidal efficacy were calculated using arithmetic means. A rapid onset of killing was observed for DPP with 12.7 % of dead and moribund fleas being dislodged in average from dogs as soon as 5 min after infestation. DPP exhibited a significantly higher and sustained speed of kill than S. The average insecticidal efficacy was 86 ± 8.8 and 95.3 ± 2.1 % with DPP, whereas it was only 33.7 ± 19.9 and 57.6 ± 18.6 % with S at respectively 1 and 4 h after weekly reinfestations. The DPP combination significantly inhibited the feeding of fleas (89 % reduction) up to onset of flea mortality for 1-month post-treatment.

Increasing temperature denatures canine IgG reducing its ability to inhibit heartworm antigen detection.

BACKGROUND: Immune complexing of target antigen to high affinity host antibody is recognized to impact the sensitivity of commercial heartworm antigen tests. Published information describing the effect of heat on interfering canine host antibodies is lacking. Immune complex dissociation (ICD) by heat treatment of serum for samples initially testing negative for heartworm antigen increases sensitivity of commercial antigen tests, particularly for single sex or low adult infection intensities. In this study the stability and nature of the targeted epitope and mechanism of heat ICD were examined. METHODS: Canine IgG was isolated using protein-A columns from serum originating from four dogs evaluated after necropsy: one dog with evidence of previously cleared infection and three dogs with confirmed heartworm infections. These dogs were expected to have an excess of antibodies based on negative antigen test and to have no or low antigen optical density, respectively, following heat treatment. Interference of antigen detection on (non-heated) positive serum was evaluated, following 1:1 mixing of antibody/PBS solutions previously heated at 25 °C, 65 °C, 75 °C, 85 °C, 95 °C and 104 °C, compared to positive serum/PBS control measured by optical density using a commercial heartworm antigen ELISA and protein quantification. Live heartworms incubated in media for 72 h provided excretory/secretory antigen for antigen stability studies following heat, endopeptidase digestion and disulfide bond reduction. RESULTS: Mixing antigen-positive heartworm serum with antibody solutions demonstrated a significant inhibition of antigen detection for antibody solutions previously heated at 25 °C and 65 °C relative to positive serum/PBS control. Antigen detection optical density was restored at or above the control when positive serum was mixed with solutions previously heated at 75 °C, 85 °C, 95 °C and 104 °C. Significant changes occurred in protein levels for antibody solutions heated at 75 °C, 85 °C, 95 °C and 104 °C. Relative stability of antigen from live heartworms in culture was demonstrated following heat, chemical and enzymatic treatment. CONCLUSIONS: Significant changes in protein levels and antigen binding ability occurred in IgG solutions heated above 65 °C. The findings confirm heat denaturation of antibodies as the suspected mechanism of heat ICD at 104 °C for antigen diagnosis of heartworm. No significant change occurred in antigen detection following heat, chemical or enzymatic digestions supporting a heat-stable linear nature of the epitope.

Detection of heartworm antigen without cross-reactivity to helminths and protozoa following heat treatment of canine serum.

BACKGROUND: Detection of Dirofilaria immitis, or heartworm, through antigen in sera is the primary means of diagnosing infections in dogs. In recent years, the practice of heat-treating serum prior to antigen testing has demonstrated improved detection of heartworm infection. While the practice of heat-treating serum has resulted in earlier detection and improved sensitivity for heartworm infections, it has been suggested that heat treatment may cause cross reactivity with A. reconditum and intestinal helminth infections of dogs. No studies have assessed the potential cross-reactivity of these parasites with heartworm tests before and after heat treatment using blood products and an appropriate gold standard reference. METHODS: Canine sera (n=163) was used to evaluate a heartworm antigen-ELISA (DiroCHEK®) and potential cross-reactivity with common parasitic infections. The heartworm status and additional parasite infections were confirmed by necropsy and adult helminth species verified morphologically or by PCR, and feces evaluated by centrifugal fecal flotation. RESULTS: Intestinal parasites were confirmed in 140 of the dogs by necropsy, and 130 by fecal flotation. Acanthocheilonema reconditum microfilariae were confirmed in 22 dogs. Prevalence of heartworm infection confirmed by necropsy was 35.6% (58/163). In the 105 dogs without heartworms, specificity remained unchanged at 100% both before and after heat treatment despite confirmed infections with A. reconditum, Ancylostoma caninum, Ancylostoma brasiliense, Trichuris vulpis, Toxocara canis, Dipylidium caninum, Spirometra mansonoides, Macracanthorynchus ingens, Cystoisospora sp., Giardia sp., and Sarcocystis sp. CONCLUSIONS: These findings suggest that the use of heat treatment improves sensitivity of heartworm tests and is unlikely to cause false positive antigen results due to Acanthocheilonema reconditum, intestinal helminths, and protozoal parasites in dogs.

Canine heartworm and heat treatment: An evaluation using a well based enzyme-linked immunosorbent assay (ELISA) and canine sera with confirmed heartworm infection status.

Heat treatment of serum has demonstrated improved detection of Dirofilaria immitis antigen in sera of sheltered dogs without knowing the true infection status of the animals and in dogs confirmed experimentally to be infected with heartworm. Utilizing archived sera with necropsy confirmed heartworm infection status (n = 665) and a micro-titer well based ELISA antigen assay, this study evaluated how the composition of heartworm infections affects antigen test results pre- and post-heat treatment, determined subsequent changes to the antigen test sensitivity and specificity, and application of optical density values. The composition of heartworm infections present in dogs with sera initially testing antigen negative consisted of infections by dead 1/34 (2.9 %), immature 10/34 (29.4 %), male only 7/34 (20.6 %), female only 5/34 (14.7 %), and mixed sex infections 11/34 (32.4 %) with 2-62 heartworms of which 6 were microfilaremic. The composition of heartworm infections remaining antigen negative post-heat treatment consisted of infections by dead 1/14 (7.1 %), immature 9/14 (64.3 %), male only 2/14 (14.3 %), and mixed sex infections 2/14 (14.3 %) with 6 and 62 heartworms of which 1 was microfilaremic. The overall sensitivity for all infections, mature heartworms, and mature females before heat treatment were 86.9 %, 90.7 %, and 93.3 % and after heat treatment sensitivity increased to 94.6 %, 98.4 %, and 99.2 % respectively. A decrease in specificity from 97.8%-96.1% was observed following heat treatment of heartworm negative sera. Optical density values for the varying infection intensities present in this study clearly indicate that result intensity is not reflective of the number of heartworms present. This study provides additional context for interpreting post-heat antigen results for dogs originating from animal shelters, demonstrates diagnostic utility of optical density, and highlights the need for improved heartworm diagnostics.

Tritrichomonas foetus infections in surveyed pet cats.

Tritrichomonas foetus is the causative agent of feline trichomoniasis, a large-bowel disease resulting in chronic diarrhea. Feline trichomoniasis has been reported in cats of both pure and mixed breeds and in both males and females. In order to estimate the prevalence of trichomoniasis in the pet cat population, we requested fecal samples, via veterinarians throughout the United States, from cats with or without clinical signs of trichomoniasis. Of the 173 feline fecal samples received from veterinarians, 17 were culture and PCR positive for T. foetus. Our results suggested no correlation between breed or sex and infection with T. foetus. All cats that were infected with T. foetus had diarrhea at the time the fecal sample was taken. Other enteric pathogens were present in nine of the 17 positive cats. Our results support that trichomoniasis is a disease of younger male and female cats of all breeds.

Diagnosis of canine Hepatozoon spp. infection by quantitative PCR.

Hepatozoon (H.) americanum and H. canis are the etiological agents of canine hepatozoonosis, a disease that is found worldwide and is also prevalent in the southeastern United States. Current laboratory diagnosis of canine hepatozoonosis caused by H. americanum is usually dependent on visual identification of Hepatozoon "onion skin cysts" in muscle biopsies, an approach that requires invasive sampling and can result in false negatives. We have developed a diagnostic method for detection of Hepatozoon spp. DNA that integrates nucleic acid extraction with extensive agitation to maximize DNA extraction efficiency. The DNA extracted from canine EDTA-whole blood is subjected to real-time PCR, and fluorescence resonance energy transfer (FRET) probes detect a signature polymorphism in the amplified DNA. This PCR method amplifies a fragment of the Hepatozoon 18S rDNA gene, detects as few as 7 genomic copies of Hepatozoon spp. per ml of blood with high specificity, and differentiates between H. americanum and H. canis amplicons. A surprising 300-fold increase of H. americanum 18S rDNA targets occurred during 3-0 days of storage of positive blood specimens. Examination of 614 EDTA-blood samples submitted mostly from the southeastern Unites States from dogs with suspected hepatozoonosis identified H. americanum in 167 samples (27.2%). An additional 14 samples (2.3%) were positive for H. canis, and 14 samples (2.3%) were positive for both H. americanum and H. canis. These results suggest that the Hepatozoon spp. 18S rDNA quantitative PCR may be a valuable tool that can improve diagnosis and therapy of canine hepatozoonosis.

Experimental infection of cats (Felis catus) with Tritrichomonas foetus isolated from cattle.

Tritrichomonas foetus is recognized as the causative agent of venereal trichomoniasis in cattle. It is characterized by embryonic and early fetal death and post-coital pyometra, and feline trichomoniasis, manifest as chronic, large bowel diarrhea. Many of the infected cats are less than 2 years old and specific routes of transmission remain unknown. We recently demonstrated that feline isolates of T. foetus can successfully infect heifers, resulting in pathologic changes similar, but not identical to those previously reported as representative of bovine trichomoniasis. In this study, we experimentally infected six cats less than 1 year of age with a bovine (D-1) isolate of T. foetus and one cat with a feline (AUTf-1) isolate of T. foetus. Within 2 weeks, the cat infected with the feline (AUTf-1) isolate was culture positive for trichomonads in weekly fecal samples. At the end of 5 weeks, only one cat infected with the bovine (D-1) isolate was fecal culture positive for trichomonads. At necropsy, the intestine of each cat was removed and divided into five sections (ileum, cecum, anterior, medial and posterior colon). Contents from each section were collected and cultured. The cat infected with the feline (AUTf-1) isolate was culture positive in the ileum, cecum, medial and posterior colon. Two cats infected with the bovine (D-1) isolate were culture positive in the cecum only. Additionally, each intestinal section was submitted to a pathologist for histopathological examination. The combined results indicate that there are demonstrable differences between the feline (AUTf-1) and bovine (D-1) isolates regarding their infectivity in cats.

Proliferative sparganosis in a dog.

CASE DESCRIPTION: A 21-month-old spayed female Border Collie was examined because of progressive right forelimb lameness, signs of pain, and subcutaneous edema. The dog lived in a fenced yard in Tampa, Fla, that contained a small area of marshy terrain. CLINICAL FINDINGS: The subcutis and intermuscular fascia contained multiple cystic cavities filled with larval cestodes (plerocercoids or spargana) and cloudy red fluid. Parasites were identified morphologically and by DNA sequence analysis as pseudophyllidean cestodes, most likely Sparganum proliferum. The dog developed a progressively worsening fever, dyspnea, mature neutrophilia, and hypoproteinemia. Septic pleuritis and peritonitis complicated the later stages of the disease. TREATMENT AND OUTCOME: Treatment with praziquantel, fenbendazole, and nitazoxanide failed to control the proliferation and dissemination of larval cestodes. The dog was euthanatized after 133 days of treatment. At necropsy, numerous parasitic tissue cysts were present in the subcutis and intermuscular fascia; these cysts were most abundant in the soft tissues of the forelimbs and cervical musculature. The pleural and peritoneal cavities contained multiple larval cestodes and were characterized by neutrophilic inflammation and secondary bacterial infection. CLINICAL RELEVANCE: Findings indicated that clinical signs associated with proliferative sparganosis in dogs may be rapidly progressive and that the condition may be refractory to antiparasitic treatment. Veterinarians should be aware of this zoonotic, water-borne agent.

Prevalence of intestinal parasites in companion animals in Ontario and Quebec, Canada, during the winter months.

Veterinarians in Ontario and Quebec, Canada, typically prescribe monthly heartworm prophylactic and anthelmintic medications for use during the warm months of the year. In many patients, the use of dewormers is discontinued during the winter because of the perception that intestinal parasite infections and shedding of nematode eggs are unlikely when the weather is cold and the ground is frozen or covered with snow. This study examined fecal samples obtained from 96 shelter dogs and cats during the winter in Ontario and Quebec. Intestinal parasites were identified in 34% of submitted samples. These findings support the recommendation that veterinarians should advise pet owners to continue administration of broad-spectrum parasiticides to companion animals during the winter months.

Prevalence of Anaplasma phagocytophilum in domestic felines in the United States.

Anaplasma phagocytophilum is among the more common tick-borne disease agents in the United States. It is of veterinary and public health significance as dogs, cats, and human beings are known to be susceptible. A. phagocytophilum is transmitted trans-stadially by either nymphs or adults of either the black-legged tick (Ixodes scapularis) or the western black-legged tick (Ixodes pacificus). Little information is available regarding either the prevalence of this agent in cats or the dynamics of vector transmission. Four hundred and sixty feline blood samples from sites throughout the United States were assayed for antibodies to A. phagocytophilum using an indirect immunofluorescence assay (IFA). Results of the prevalence study showed that 20 samples (4.3%) were positive for A. phagocytophilum antibodies by IFA at a 1:50 dilution, however these results could not be confirmed by PCR analysis. PCR analysis for other cross-reacting Ehrlichia/Anaplasma spp. was also negative. These results demonstrate that natural infection of A. phagocytophilum in cats is uncommon.

Comparative evaluation of commercially available point-of-care heartworm antigen tests using well-characterized canine plasma samples.

BACKGROUND: Dirofilaria immitis is a worldwide parasite that is endemic in many parts of the United States. There are many commercial assays available for the detection of D. immitis antigen, one of which was modified and has reentered the market. Our objective was to compare the recently reintroduced Witness® Heartworm (HW) Antigen test Kit (Zoetis, Florham Park, NJ) and the SNAP® Heartworm RT (IDEXX Laboratories, Inc., Westbrook, ME) to the well-based ELISA DiroChek® Heartworm Antigen Test Kit (Zoetis, Florham Park, NJ). METHODS: Canine plasma samples were either received at the Auburn Diagnostic Parasitology Laboratory from veterinarians submitting samples for additional heartworm testing (n = 100) from 2008 to 2016 or purchased from purpose-bred beagles (n = 50, presumed negative) in 2016. Samples were categorized as "positive," "borderline" or "negative" using our established spectrophotometric cutoff value with the DiroChek® assay when a sample was initially received and processed. Three commercially available heartworm antigen tests (DiroChek®, Witness® HW, and SNAP® RT) were utilized for simultaneous testing of the 150 samples in random order as per their package insert with the addition of spectrophotometric optical density (OD) readings of the DiroChek® assay. Any samples yielding discordant test results between assays were further evaluated by heat treatment of plasma and retesting. Chi-square tests for the equality of proportions were utilized for statistical analyses. RESULTS: Concordant results occurred in 140/150 (93.3%) samples. Discrepant results occurred in 10/150 samples tested (6.6%): 9/10 occurring in the borderline heartworm (HW) category and 1/10 occurring in the negative HW category. The sensitivity and specificity of each test compared to the DiroChek® read by spectrophotometer was similar to what has been reported previously (Witness®: sensitivity 97.0% [94.1-99.4%], specificity 96.4% [95.5-100.0%]; SNAP® RT: sensitivity 90.9% [78.0-100.0%], specificity 98.8% [96.0-100.0%]). There were significant differences detected when comparing the sensitivities of the SNAP® RT and the Witness® HW to the DiroChek® among the 150 total samples (p = 0.003) and the 50 "borderline" samples (p = 0.001). CONCLUSIONS: In this study, the sensitivity of the Witness® HW was higher than the sensitivity of the SNAP® RT when compared with the DiroChek® test results prior to heat treatment of samples.

Increased detection of Dirofilaria immitis antigen in cats after heat pretreatment of samples.

Objectives To determine whether pretreating diagnostic samples with heat increases the detection of Dirofilaria immitis antigen in adult cats, we evaluated feline serum and plasma samples collected in heartworm-endemic areas of the southern United States. Methods Commercial microtiter well assays for detection of D immitis antigen were used to evaluate serum or plasma samples from 385 shelter and free-roaming cats from the southcentral and southeastern United States before and after heat treatment; commercial antibody tests were performed on a subset of samples. Results Prior to sample heat treatment, 1/220 (0.5%) shelter cats and 4/165 (2.4%) free-roaming cats had detectable D immitis antigen. After heat pretreatment, the detection rate increased to 13/220 (5.9%) and 13/165 (7.9%), respectively. Antibody reactive to D immitis was significantly more common ( P <0.001) in the serum of cats that were antigen positive after heat treatment (10/13; 76.9%) than serum from cats that remained antigen negative after heat treatment (22/163; 13.5%). Conclusions and relevance Heat pretreatment of feline samples increased antigen detection by commercial assays for D immitis and improved overall concordance of antigen and antibody test results in antigen-positive samples in this population. Although further work to investigate the specificity of D immitis antigen assays when using pre-treated samples is warranted, this approach may be useful in the diagnosis of heartworm infection in individual cats and may increase the accuracy of surveys based on antigen detection.

Evaluation of the larval migration inhibition assay for detecting macrocyclic lactone resistance in Dirofilaria immitis.

Anthelmintics of the macrocyclic lactone (ML) drug class are widely used as preventives against the canine heartworm (Dirofilaria immitis). Over the past several years, however, reports of ML lack of efficacy (LOE) have emerged, in which dogs develop mature heartworm infection despite the administration of monthly prophylactics. More recently, isolates from LOE cases have been used to infect laboratory dogs and the resistant phenotype has been confirmed by the establishment of adult worms in the face of ML treatment at normally preventive dosages. Testing for and monitoring resistance in D. immitis requires a validated biological or molecular diagnostic assay. In this study, we assessed a larval migration inhibition assay (LMIA) that we previously optimized for use with D. immitis third-stage larvae (L3). We used this assay to measure the in vitro ML susceptibilities of a known-susceptible laboratory strain of D. immitis and three highly suspected ML-resistant isolates originating from three separate LOE cases; progeny from two of these isolates have been confirmed ML-resistant by treatment of an infected dog in a controlled setting. A nonlinear regression model was fit to the dose-response data, from which IC50 values were calculated. The D. immitis LMIA yielded consistent and reproducible dose-response data; however, no statistically significant differences in drug susceptibility were observed between control and LOE parasites. Additionally, the drug concentrations needed to paralyze the L3 were much higher than those third- and fourth-stage larvae would experience in vivo. IC50 values ranged from 1.57 to 5.56µM (p≥0.19). These data could suggest that ML resistance in this parasite is not mediated through a reduced susceptibility of L3 to the paralytic effects of ML drugs, and therefore motility-based assays are likely not appropriate for measuring the effects of MLs against D. immitis in this target stage.

Effect of heartworm disease and heartworm-associated respiratory disease (HARD) on the right ventricle of cats.

BACKGROUND: Dirofilaria immitis infection occurs in dogs and cats, both of which species are clinically affected by mature adult infections. Cats are uniquely affected by immature-adult infections with an inflammatory pulmonary disease called Heartworm-Associated Respiratory Disease (HARD). D. immitis infection causes pulmonary parenchymal and vascular pathology in the dog and cat. Dogs develop pulmonary hypertension and cor pulmonale, whereas the development of pulmonary hypertension is rare in the cat. D. immitis infection in the dog causes alteration of the right ventricular (RV) extracellular matrix, including a decrease in myocardial collagen. In this study, the RV myocardial changes of cats infected with adult and immature-adult D. immitis were assessed. METHODS: The cardiopulmonary systems of six groups of SPF cats (n = 9-10 per group) were examined 8 or 18 months after infection with L3 D. immitis. Two groups were untreated and allowed to develop adult HW; two groups were treated with ivermectin starting 3 months post infection, thus allowing HARD but no mature adult heartworms; and two groups were treated with selamectin beginning 1 month post infection, preventing development of L5 or adult heartworms. A group of specific pathogen free (SPF) normal cats was utilized as a negative control (n = 12). Lung pathologic lesions were objectively assessed, and both RV and left ventricular (LV) weights were obtained to calculate an RV/LV ratio. Intramural RV myocardial collagen content was quantitatively assessed. RESULTS: RV/LV weight ratios were not different between groups. Negative control cats had significantly greater RV collagen content than all other affected groups (P = 0.032). Analysis of the RV/LV ratios and collagen content revealed no significant relationship (r = 0.03, P = 0.723, respectively). Collagen content had a modest, but significant, negative correlation, however, with both pulmonary vascular pathology (r = -0.25, P = 0.032) as well as the total pulmonary parenchymal and vascular pathology (r = -0.26, P = 0.025). CONCLUSIONS: Cats infected with mature and immature D. immitis did not develop RV hypertrophy but did demonstrate loss of RV myocardial collagen content. The collagen loss was present at 8 and 18 months after infection in all infected cats. This loss of RV myocardial collagen was correlated with the severity of pulmonary parenchymal and vascular pathology.

Heartworm-associated respiratory disease (HARD) induced by immature adult Dirofilaria immitis in cats.

BACKGROUND: A controlled, blind research study was conducted to define the initial inflammatory response and lung damage associated with the death of immature adult Dirofilaria immitis in cats as compared with cats developing adult heartworm infections and cats on preventive medication. METHODS: Three groups of cats were utilized, 10 per group. All cats were infected with 100 third-stage (L3) larvae by subcutaneous injection. Group A cats were treated topically with selamectin (Revolution®; Zoetis) per label directions at 28 days post infection (PI) and once monthly for 8 months. Group B cats were treated orally with ivermectin (Ivomec®; Merial) at 150 µg/kg at 70 days PI, then every 2 weeks for 5 months. Group C cats were untreated PI. At baseline (Day 0) and on Days 70, 110, 168, and 240 PI, peripheral blood, serum, bronchial lavage, and thoracic radiographic images were collected on all cats. Upon completion of the study (Day 245), cats were euthanized and necropsies were conducted. RESULTS: Results were analyzed statistically between groups by ANOVA and by paired sample T testing for changes within the group over time. The selamectin-treated cats (Group A) did not develop radiographically evident changes throughout the study and were free of adult heartworms or worm fragments at necropsy. The heartworm life cycle was abbreviated with oral doses of ivermectin (Group B), shown by the absence of adult heartworms or worm fragments at necropsy. The early stage of immature adult worm in Group B cats, however, did induce severe pulmonary airway, interstitial, and arterial lung lesions, revealing that the abbreviated infection is a significant cause of respiratory pathology in cats. Cats in Groups B and C could not be differentiated based on radiographic changes, serologic antibody titers, complete blood count, or bronchoalveolar lavage cytology at any time point throughout the study. Eighty percent of cats in Group A and 100% of cats in Groups B and C became heartworm antibody positive at some time point post infection. CONCLUSIONS: The clinical implications of this study are that cats that become infected with immature adult heartworms may not develop fully mature heartworms and are only transiently heartworm antibody positive, but do develop Heartworm-Associated Respiratory Disease (HARD).

New methods and strategies for monitoring susceptibility of fleas to current flea control products.

A flea larval bioassay was developed by an international team of scientists to monitor the susceptibility of fleas (Ctenocephalides felis) to imidacloprid (Advantage, Bayer HealthCare). The assay was validated using laboratory and field isolates of C. felis. Flea eggs representing different field isolates of C. felis were collected by veterinarians in the United States, United Kingdom, and Germany. Of the 972 flea isolates obtained during the 5-year study, 768 contained sufficient numbers of eggs to conduct the larval bioassay. Greater than 5% survival occurred for only six of the field isolates evaluated. Further evaluation and analysis of these isolates demonstrated that they did not differ significantly in their susceptibility to imidacloprid from the reference strains used to develop the assay. Collections of field flea isolates will continue in an attempt to detect and document any change in the susceptibility of field flea populations to imidacloprid.

Efficacy of four commercially available heartworm preventive products against the JYD-34 laboratory strain of Dirofilaria immitis.

BACKGROUND: Heartworm disease in dogs can be severe and life threatening. Resistance to available heartworm preventives was considered among potential causes of increased reports of failed heartworm prevention in dogs. The objective of the present study was to compare the efficacy of four commercially available heartworm disease preventives against the JYD-34 strain of D. immitis. METHODS: Forty laboratory-reared dogs approximately 6 months old were used. Each dog was infected with fifty, third-stage heartworm larvae on study day (SD) -30. On SD-1, the dogs were randomized to five groups of eight dogs each. On SD-0, dogs in groups 1-4 were treated as follows: Group 1: ivermectin/pyrantel pamoate chewable tablets; Group 2: milbemycin oxime/spinosad tablets; Group 3: selamectin topical solution; and Group 4: imidacloprid/moxidectin topical solution. Dogs in Group 5 were not treated and served as controls. The dogs were treated according to their current body weights and labelled dose banding for each product. Groups 1, 2, and 3 were retreated with their respective products and current body weights on SD 31 and 60. On SDs 124-126 the dogs were euthanized and necropsied for recovery of adult heartworms. RESULTS: Adult heartworms were recovered at necropsy from each of the dogs in the control group (13-32 worms/dog, geometric mean (GM) = 18.4 worms/dog). Adult heartworms and/or worm fragments were also recovered from each of the dogs treated with ivermectin/pyrantel pamoate, milbemycin oxime/spinosad or selamectin. Geometric means of worms recovered from dogs in each of these groups were 13.1, 8.8, and 13.1, resulting in efficacies compared to controls of 29.0, 52.2, and 28.8 %, respectively. All dogs in Group 4 (imidacloprid/moxidectin) were free of adult heartworms (100 % efficacy). CONCLUSIONS: The combination of imidacloprid/moxidectin was 100 % effective in this study in preventing development of JYD-34 laboratory strain of D. immitis in dogs following a single treatment, while three monthlytreatments of the three other commercial products provided less than 100 % efficacy. The high efficacy achieved with imidacloprid/moxidectin was likely due to the unique pharmacokinetic properties of the topical formulation delivering greater and sustained drug concentrations necessary to prevent development of D. immitis larvae.

Neospora caninum: adoptive transfer of immune lymphocytes precipitates disease in BALB/c mice.

Neospora caninum is a recently described apicomplexan parasite first isolated from a dog in 1988 and has subsequently been shown to infect a wide range of mammals. In mice, Neospora can cause primary pneumonia, myositis, encephalitis, radiculoneuritis, and pancreatitis. Whereas, certain aspects of the host immune response to Toxoplasma gondii have been well studied, not as much is known about the full immune response to Neospora. This paper examines whether or not immune splenocytes are able to adoptively transfer protection against N. caninum infection in BALB/c mice. Mice receiving immune enriched CD8+ cells had severe neurological signs by 19 days post infection. Mice receiving immune enriched CD4+ cells had mild neurological signs on day 22 post infection. It would appear that additional immune cells can precipitate disease in the presence of circulating lymphocytes.

Moxidectin steady state prior to inoculation protects cats from subsequent, repeated infection with Dirofilaria immitis.

BACKGROUND: Infection of cats with Dirofilaria immitis causes seroconversion on antibody tests and pulmonary pathology, often without subsequent development of adult heartworms. Consistent administration of topical 10% imidacloprid-1% moxidectin has been shown to result in sustained plasma levels of moxidectin in cats after three to five treatments, a pharmacokinetic behavior known as "steady state". METHODS: To evaluate the ability of moxidectin at "steady state" to protect cats from subsequent infection with D. immitis, cats (n = 10) were treated with the labeled dose of topical 10% imidacloprid-1% moxidectin for four monthly treatments. Each cat was inoculated with 25 third-stage larvae of D. immitis 7, 14, 21, and 28 days after the last treatment; non-treated cats (n = 9) were inoculated on the same days, serving as infection controls. Blood samples were collected from each cat from 1 month prior to treatment until 7 months after the final inoculation and tested for antibody to, and antigen and microfilaria of, D. immitis. RESULTS: Measurement of serum levels of moxidectin confirmed steady state in treated cats. Cats treated with topical 10% imidacloprid-1% moxidectin prior to trickle inoculation of D. immitis L3 larvae throughout the 28 day post-treatment period remained negative on antibody and antigen tests throughout the study and did not develop gross or histologic lesions characteristic of heartworm infection. A majority of non-treated cats tested antibody positive by 3-4 months post infection (6/9) and, after heat treatment, tested antigen positive by 6-7 months post-infection (5/9). Histologic lesions characteristic of D. immitis infection, including intimal and medial thickening of the pulmonary artery, were present in every cat with D. immitis antibodies (6/6), although adult D. immitis were confirmed in only 5/6 antibody-positive cats at necropsy. Microfilariae were not detected at any time. CONCLUSIONS: Taken together, these data indicate that prior treatment with 10% imidacloprid-1% moxidectin protected cats from subsequent infection with D. immitis for 28 days, preventing both formation of a detectable antibody response and development of pulmonary lesions by either immature stages of D. immitis or young adult heartworms.

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.