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.

Angiotensin-converting enzyme 2 in dogs with Dirofilaria immitis.

BACKGROUND: Infection by the canine heartworm, Dirofilaria immitis, causes significant cardiopulmonary disease, with progression impacted by increasing parasite numbers and duration of infection. The renin-angiotensin-aldosterone system (RAAS) is an important mediator of cardiac and pulmonary disease. Angiotensin-converting enzyme 2 (ACE2) mitigates the maladaptive effects of angiotensin II by converting it to angiotensin (1-7). We hypothesized that circulating ACE2 activity would be altered in dogs with high heartworm infection intensities relative to dogs without heartworms. METHODS: Frozen serum samples (-80 °C) from 30 dogs euthanized at Florida shelters were analyzed for ACE2 activity using liquid chromatography-mass spectrometry/mass spectroscopy and a kinetics approach with and without an ACE2 inhibitor. A convenience sample of 15 dogs without heartworms (HW0) and 15 dogs with > 50 heartworms (HW>50) was included. Heartworm number and microfilariae presence were determined at necropsy. The effects of heartworm status, body weight, and sex on ACE2 were evaluated using regression analysis. Values of P < 0.05 were considered significant. RESULTS: All HW0 dogs were D. immitis microfilariae-negative and all HW>50 dogs were D. immitis microfilariae-positive with a median adult worm count of 74 (minimum = 63, maximum = 137). The ACE2 activity of HW>50 dogs (median = 28.2 ng/ml; minimum = 13.6, maximum = 76.2) was not different from HW0 dogs (median 31.9 ng/ml; minimum = 14.1, maximum = 139.1; P = 0.53). The ACE2 activity was higher in dogs with high body weight (median 34.2 ng/ml minimum = 14.1, maximum = 76.2) than in dogs with low weight (median 27.5 ng/ml; minimum = 16.4, maximum = 139.1; P = .044). CONCLUSIONS: Heartworm infection did not impact ACE2 activity in shelter dogs with or without heartworms, but heavier dogs had higher ACE2 activity compared to lighter dogs. Comprehensive RAAS evaluation and additional clinical information would aid in understanding how ACE2 activity relates to the entire cascade and clinical status in dogs with heartworm disease.

Cutaneous filarioid nematodes of dogs in the United States: Are they emerging, neglected, or underdiagnosed parasites?

Filarioid nematodes, which are vector-borne parasites of cosmopolitan distribution, of dogs are medically important. They are represented by species in which microfilariae were found to be circulating in the bloodstream (e.g., Dirofilaria sp., Acanthocheilonema sp., and Brugia sp.) or skin-dwelling (e.g., Cercopithifilaria sp. and Onchocerca sp.). Those species whose microfilariae are detected in blood have been extensively studied, especially Dirofilaria immitis, due to their clinical importance. In recent decades, there has been an increased interest by the scientific community in filarioid nematodes whose microfilariae are detected in the skin because of the zoonotic aspect of Onchocerca lupi. In the United States (US), although D. immitis has been considered the main filarioid infecting dogs, the intense animal movement and global canine filarioid diversity may indicate that the likely presence of cutaneous filarioid nematodes is more common than previously expected. Hence, a question remains: Are these canine filarioid nematodes emerging, neglected, or simply underdiagnosed in the US? In this review, we provide an overview of pertinent information that briefly summarizes the biology of the different canine filarioid nematode species, clinical signs associated with infections, and currently available diagnostic tools using molecular and microscopy-based methods and highlight knowledge gaps where research and surveillance efforts remain necessary. The data herein presented serve as an alert to the scientific community about the importance of filarioid nematodes infecting dogs other than D. immitis. Additionally, the zoonotic potential of several filarioid species reinforces the necessity of a proper diagnosis and the need for broader surveillance to understand their diversity and distribution, to highlight the potential introduction of certain species, and mitigate their establishment in the country and new animal and human cases.

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.

Evaluation of oral fluralaner (Bravecto®) for efficacy against nymphs of Amblyomma americanum and Rhipicephalus sanguineus (sensu lato).

BACKGROUND: Amblyomma americanum and Rhipicephalus sanguineus (sensu lato) nymphs commonly feed on and transmit pathogens to dogs (Canis familiaris). Control of immature and adult tick life stages is necessary to fully protect animals. We evaluated efficacy of oral fluralaner (Bravecto®) against induced infestations with A. americanum and R. sanguineus (s.l.) nymphs on dogs in two experiments. METHODS: In each experiment, 10 dogs were administered oral fluralaner chewable tablets one time on Day 0 at a targeted minimum dose of 25 mg/kg body weight and 10 dogs remained non-treated controls. Dogs were infested with two groups of 50 A. americanum nymphs and two groups of 50 R. sanguineus (s.l.) nymphs on Days -1, 6, 28, 56 and 84. At 48 h and 72 h post-infestation, nymphs were collected from dogs, assessed as live or dead, and enumerated into categories defining attachment and engorgement status. Fluralaner efficacy was determined in separate analyses against all live nymphs and against live-fed nymphs, i.e. live nymphs that were attached to dogs at the time of collection and/or were engorged. Fluralaner was considered effective when mean numbers of live ticks were reduced in fluralaner-treated dogs by ≥ 90%. RESULTS: Fluralaner efficacy against all live and live-fed A. americanum nymphs in the first experiment was > 94% on all collection days. Efficacy against all live R. sanguineus (s.l.) nymphs in the first experiment was > 96% on all collection days  excluding the 48 h counts for infestations on Days 28 (83.7%), 56 (82.9%) and 84 (86.7%); efficacy against live-fed R. sanguineus (s.l.) nymphs was > 95% on all 48 h/72 h count days. Fluralaner efficacy against all live A. americanum nymphs in the second experiment was > 93% on all collection days for 8 weeks excluding the 48 h count for infestation on Day 56 (87.8%); efficacy against live-fed A. americanum nymphs was > 91% on all count days for 8 weeks. Efficacy against all live R. sanguineus (s.l.) nymphs in the  second experiment was > 91% on all 72 h collection days  except for infestations on Days 28 (76.8%) and 56 (86.3%); efficacy against live-fed R. sanguineus (s.l.) nymphs was 100% on all 72 h count days. CONCLUSIONS: A single administration of oral fluralaner to dogs is effective against A. americanum and R. sanguineus (s.l.) nymphs for up to 12 weeks.

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.

Active surveillance to update county scale distribution of four tick species of medical and veterinary importance in Oklahoma.

The incidence of tick-borne disease continues to increase in humans and companion animals in the United States, yet distribution maps for several tick vectors in Oklahoma, including Dermacentor variabilis, Dermacentor albipictus, Ixodes scapularis, and Amblyomma maculatum, are not available or are outdated. To address this issue, county-scale tick records from peer-reviewed literature and passive collections were reviewed for Oklahoma. Additionally, dry ice traps, tick drags, and harvested deer were utilized to actively collect adult ticks throughout the state. Through these methods, D. variabilis, D. albipictus, I. scapularis, and A. maculatum were identified in 88% (68/77), 45.4% (35/77), 66.2% (51/77), and 64.9% (50/77) of the counties in Oklahoma, respectively. Baseline maps were developed for the distribution of D. variabilis and D. albipictus and distribution maps were updated for I. scapularis and A. maculatum. This data confirms that these four species of ticks continue to be widespread within Oklahoma with a western expansion of the range of I. scapularis within the state. These results assist efforts to better understand the epidemiology of the different diseases caused by pathogens transmitted by these tick species within the Great Plains region.

Temperate and tropical lineages of brown dog ticks in North America.

Recent studies document that brown dog ticks, previously considered as Rhipicephalus sanguineus, are actually comprised of multiple taxonomic units now referred to as Rhipicephalus sanguineus sensu lato (Rssl); two lineages of Rssl have been described in the Americas to date - tropical and temperate. To identify the lineage of Rssl from dogs or premises at multiple sites in the United States and the Caribbean, we evaluated ticks (n=191) collected from several geographic locations (n=21), including Arizona, California, Florida, Hawaii, Illinois, Oklahoma, and Texas in the United States, and from Haiti. All ticks were identified as brown dog ticks by morphologic examination and comparison to standard keys. Sequence analysis of 12S rRNA mitochondrial gene fragments confirmed the presence of both lineages, with the Rssl tropical lineage predominating in Florida, Haiti, Hawaii, and far southern Texas (n=9 locations) and the Rssl temperate lineage present in California, Oklahoma, and Texas (n=12 locations). Mixed populations were not identified although the temperate lineage appeared to separate into two distinct clades. Analysis of additional brown dog tick specimens from the region will allow more complete understanding of the full extent of diversity in the R. sanguineus complex and likely has important implications for disease transmission, including zoonotic risk.

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.

County Scale Distribution of Amblyomma americanum (Ixodida: Ixodidae) in Oklahoma: Addressing Local Deficits in Tick Maps Based on Passive Reporting.

Geographic distribution records for the lone star tick [Amblyomma americanum (L.)] in the peer-reviewed literature are incomplete for Oklahoma, preventing accurate disease risk assessments. To address this issue and document the presence of A. americanum in available habitats throughout the state, county-scale tick records published in U.S. Department of Agriculture-Cooperative Economic Insect Reports and specimens maintained at the K.C. Emerson Entomology Museum, Oklahoma State University, were reviewed. In addition, dry ice traps and tick drags were used to collect adult and nymphal A. americanum from throughout the state. Review of published USDA reports and the local museum collection documented A. americanum in 49 total counties (35 and 35, respectively). Active surveillance efforts confirmed the presence of this tick in 50 counties from which this species had not been previously reported to be established, documenting A. americanum is established in 68 of the 77 (88.3%) counties in Oklahoma. Taken together, these data verify that A. americanum ticks are much more widespread in Oklahoma than reflected in the literature, a phenomenon likely repeated throughout the geographic range of this tick in the eastern half of North America.

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.

False negative antigen tests in dogs infected with heartworm and placed on macrocyclic lactone preventives.

BACKGROUND: Dogs with chronic inflammation, including those with heartworm being managed with macrocyclic lactones and doxycycline (slow kill, SK), may develop immune complexes that block detection of Dirofilaria immitis antigen on commercial tests. METHODS: To determine if SK could result in development of false-negative antigen tests, we collected serum samples from dogs that had been diagnosed with heartworm by antigen detection, with or without confirmation by detection of D. immitis microfilariae, placed on monthly macrocyclic lactones and doxycycline, and that later tested negative on an antigen test, and then tested them for antigen of D. immitis before and after treatment to disrupt immune complexes. RESULTS: Serum samples from a total of 15 dogs managed with SK were negative for antigen prior to heating on commercial assay (DiroCHEK, Zoetis) by colorimetric detection and spectrophotometry, but after heat treatment, 8/15 (53.3%) samples converted to positive. Review of the medical records of each dog indicated that, after the heartworm diagnosis, only 7/15 (46.7%) dogs appeared to receive preventive monthly as prescribed, including 3 dogs that had detectable antigen after heating the sample and 4 dogs that did not have detectable antigen after heating. Whole blood was available from 9 dogs; microfilariae of D. immitis were detected in 1 sample. CONCLUSIONS: These data suggest that immune complex formation in dogs infected with heartworm and managed with SK can induce false negative antigen test results, misleading veterinarians and owners about the efficacy of this approach. Moreover, compliance with preventive administration appears poor, even after a heartworm diagnosis. The presence of persistent microfilaremia in at least one dog has implications for resistance selection.

Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure.

Dogs exposed to ticks in the southern US may become infected with multiple species of Ehrlichia. To better define infection risk, blood samples collected from 10 dogs infested with ticks via a natural infestation model were evaluated by blood smear examination, PCR, patient-side ELISAs (SNAP® 4Dx® and SNAP® 4Dx® Plus), IFA, and peptide based ELISA for evidence of infection with Ehrlichia canis, E. chaffeensis, and/or E. ewingii. Although morulae were rarely identified in blood smears, every dog (10/10) became infected with Ehrlichia spp. as evidenced by nested PCR detection of E. chaffeensis (7/10) and E. ewingii DNA (10/10); real-time PCR detection of E. chaffeensis (0/10) and E. ewingii (9/10); seroconversion on two different patient-side ELISAs (4/10 or 10/10); seroconversion on IFA to E. canis (10/10, maximum inverse titer=128-4096, GMTMAX=548.7) and E. chaffeensis (10/10, maximum inverse titer=1024-32,768, GMTMAX=4096); and seroconversion on peptide specific ELISA to E. chaffeensis VLPT (7/10) and E. ewingii p28 (9/10). Rickettsemia with E. chaffeensis and E. ewingii, as determined by nested PCR, persisted in dogs for an average of 3.2 or 30.5 days, respectively. Ehrlichia canis was not detected in any dog by any method, and no dogs developed signs of clinical disease. Our data suggest that in areas where ticks are common, dogs are at high risk of infection with Ehrlichia spp., particularly E. ewingii and E. chaffeensis, and can serve as a sentinel for monitoring for the presence of these zoonotic 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.