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.

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).

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.

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.

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.

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.

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.

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.

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.

Quantitative factors proposed to influence the prevalence of canine tick-borne disease agents in the United States.

The Companion Animal Parasite Council hosted a meeting to identify quantifiable factors that can influence the prevalence of tick-borne disease agents among dogs in North America. This report summarizes the approach used and the factors identified for further analysis with mathematical models of canine exposure to tick-borne pathogens.

Increased prevalence of Dirofilaria immitis antigen in canine samples after heat treatment.

Canine serum samples may contain factors that prevent detection of antigen of Dirofilaria immitis on commercial assays, precluding accurate diagnosis. To determine the degree to which the presence of blocking antibodies or other inhibitors of antigen detection may interfere with our ability to detect circulating antigen in canine samples, archived plasma and serum samples (n=165) collected from dogs in animal shelters were tested for D. immitis antigen before and after heat treatment. Negative samples were also evaluated for their ability to block detection of D. immitis antigen in a sample from a positive dog. All 165 samples were negative prior to heating, but 11/154 (7.1%) became positive after heat treatment, a conversion that was documented and quantified on spectrophotometric plate assays, and 7/165 (4.2%) samples decreased detection of antigen when mixed with a known positive sample, suggesting some blocking ability was present. An additional 103 plasma and serum samples that tested positive prior to heating also were evaluated; the optical density of 14/101 (13.9%) increased by ≥50%, and one sample by as much as 15-fold, after heat treatment. Our results suggest that canine serum and plasma samples from dogs in the southeastern United States can contain inhibitors of D. immitis antigen detection, and that prevalence estimates of heartworm infection based on these assays would benefit from heat treatment of samples prior to testing.

Heat treatment prior to testing allows detection of antigen of Dirofilaria immitis in feline serum.

BACKGROUND: Diagnosis of Dirofilaria immitis infection in cats is complicated by the difficulty associated with reliable detection of antigen in feline blood and serum samples. METHODS: To determine if antigen-antibody complex formation may interfere with detection of antigen in feline samples, we evaluated the performance of four different commercially available heartworm tests using serum samples from six cats experimentally infected with D. immitis and confirmed to harbor a low number of adult worms (mean = 2.0). Sera collected 168 (n = 6), 196 (n = 6), and 224 (n = 6) days post infection were tested both directly and following heat treatment. RESULTS: Antigen was detected in serum samples from 0 or 1 of 6 infected cats using the assays according to manufacturer's directions, but after heat treatment of serum samples, as many as 5 of 6 cats had detectable antigen 6-8 months post infection. Antibodies to D. immitis were detected in all six infected cats by commercial in-clinic assay and at a reference laboratory. CONCLUSIONS: These results indicate that heat treatment of samples prior to testing can improve the sensitivity of antigen assays in feline patients, supporting more accurate diagnosis of this infection in cats. Surveys conducted by antigen testing without prior heat treatment of samples likely underestimate the true prevalence of infection in cats.

Real-time PCR of the mammalian hydroxymethylbilane synthase (HMBS) gene for analysis of flea (Ctenocephalides felis) feeding patterns on dogs.

BACKGROUND: Precise data on quantitative kinetics of blood feeding of fleas, particularly immediately after contact with the host, are essential for understanding dynamics of flea-borne disease transmission and for evaluating flea control strategies. Standard methods used are inadequate for studies that simulate early events after real-life flea access to the host. METHODS: Here, we developed a novel quantitative polymerase chain reaction targeting mammalian DNA within fleas to quantify blood consumption with high sensitivity and specificity. We used primers and fluorescent probes that amplify the hydroxymethylbilane synthase (HMBS) gene, an evolutionary divergent gene that is unlikely to be detected in insects by mammalian-specific primers and probes. To validate this assay, fleas were placed on dogs, allowed to distribute in the hair, and removed at specific time points with single-use combs. Fleas were then immediately homogenized by vigorous shaking with ceramic beads in guanidinium-based DNA preservation buffer for DNA extraction. RESULTS: The specificity of this assay was ascertained by amplification of canine, feline and equine blood with differential product melting temperatures (Tm), and lack of amplification of bovine and porcine blood and of adult fleas reared from larvae fed with bovine blood. Sensitivity of the assay was established by limiting dilution and detection of single copies of HMBS DNA equivalent to 0.043 nL blood. Application of the assay indicated that after 15 minutes on a dog, male and female fleas had ingested low, but similar amounts of approximately 1.1. nL blood. Saturation uptake of 118 and 100 nL blood per flea was found at 30 and 60 min on the dog, respectively. CONCLUSIONS: The HMBS PCR method developed here offers the advantages of both exquisite sensitivity and specificity that make it superior to other approaches for quantification of blood ingested by fleas. The capability to detect minute quantities of blood in single fleas, particularly immediately after colonization of the host, will provide a superior tool for studying flea-host interactions, flea-borne disease transmission, and flea control strategies.

Survival of a feline isolate of Tritrichomonas foetus in water, cat urine, cat food and cat litter.

Feline intestinal trichomoniasis caused by Tritrichomonas foetus is associated with large bowel diarrhea in cats from many parts of the world. It has long been recognized as an economically important sexually transmitted disease that causes early abortion in cattle. Isolates of T. foetus from cattle are infectious for the large intestine of cats and isolates of T. foetus from cats are infectious for the reproductive system of cattle. The parasite is maintained by fecal-oral transmission in cats. The present study was conducted to examine the survival of a feline isolate of T. foetus, AUTf-12, under various conditions that are relevant to fecal-oral transmission in cats. Trophozoites were grown in TYM medium and then exposed to water, cat urine, dry cat food, canned cat food, clumping cat litter, or filter paper for various lengths of time and then re-cultured in TYM medium. Trophozoites survived exposure to distilled or tap water for 30 but not 60 min, while they survived for at least 180 min in urine. Trophozoites survived for 30 min on dry cat food but survived for 120-180 min in canned cat food. No survival of trophozoites was observed on cat litter but trophozoites survived for 15 min when placed on filter paper. Our results indicate that T. foetus can survive and be potentially infectious in water, urine, dry cat food and canned cat food.

Lyme borreliosis in dogs and humans in the USA.

Borrelia burgdorferi sensu stricto is the only established etiologic agent of Lyme borreliosis in dogs and in humans in North America. Lyme borreliosis differs in dogs and humans in terms of clinical outcome following infection, diagnostic approaches, prevention strategies and treatment recommendations. Nonetheless, serologic evidence of exposure of dogs to B. burgdorferi agrees with the geographical distribution of autochthonous transmission of the agent of Lyme borreliosis, and continued monitoring of exposure rates in dogs might allow early recognition of geographic expansion of endemic areas as well as identify hyperendemic areas where both humans and dogs are at increased risk of infection.

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.

Flea-associated zoonotic diseases of cats in the USA: bartonellosis, flea-borne rickettsioses, and plague.

Cat-scratch disease, flea-borne typhus, and plague are three flea-associated zoonoses of cats of concern in the USA. Although flea concentrations may be heaviest in coastal and temperate climates, fleas and flea-borne disease agents can occur almost anywhere in the USA. Understanding flea-borne pathogens, and the associated risks for owners and veterinarians, is important to reduce the likelihood of zoonotic infection.