Fluralaner (Bravecto®) treatment kills Aedes aegypti after feeding on Dirofilaria immitis-infected dogs.

BACKGROUND: Transmission of canine heartworm (Dirofilaria immitis) from infected to naïve dogs is dependent on successful mosquito feeding and survival. METHODS: To determine whether treating heartworm-infected dogs with fluralaner (Bravecto®) limits the survival of infected mosquitoes, and potentially the transmission of D. immitis, we allowed female mosquitoes to feed on microfilaremic dogs and evaluated mosquito survival and infection with D. immitis. Eight dogs were experimentally infected with D. immitis. On day 0 (~ 11 months post-infection), four microfilaremic dogs were treated with fluralaner according to label directions while the other four were non-treated controls. Mosquitoes (Aedes aegypti Liverpool) were allowed to feed on each dog on days -7, 2, 30, 56, and 84. Fed mosquitoes were collected, and the number of live mosquitoes determined at 6 h, 24 h, 48 h, and 72 h post-feeding. Surviving mosquitoes held for 2 weeks were dissected to confirm third-stage D. immitis larvae; PCR (12S rRNA gene) was performed post-dissection to identify D. immitis in mosquitoes. RESULTS: Prior to treatment, 98.4%, 85.1%, 60.7%, and 40.3% of mosquitoes fed on microfilaremic dogs were alive at 6 h, 24 h, 48 h, and 72 h post-feeding, respectively. Similarly, mosquitoes fed on microfilaremic, non-treated dogs were alive 6 h post-feeding (98.5-100%) throughout the study. In contrast, mosquitoes fed on fluralaner-treated dogs 2 days after treatment were dead or severely moribund by 6 h post-feeding. At 30 and 56 days post-treatment, > 99% of mosquitoes fed on treated dogs were dead by 24 h. At 84 days post-treatment, 98.4% of mosquitoes fed on treated dogs were dead by 24 h. Before treatment, third-stage larvae of D. immitis were recovered from 15.5% of Ae. aegypti 2 weeks after feeding, and 72.4% were positive for D. immitis by PCR. Similarly, 17.7% of mosquitoes fed on non-treated dogs had D. immitis third-stage larvae 2 weeks after feeding, and 88.2% were positive by PCR. Five mosquitoes fed on fluralaner-treated dogs survived 2 weeks post-feeding, and 4/5 were from day 84. None had third-stage larvae at dissection, and all were PCR-negative. CONCLUSION: The data indicate that fluralaner treatment of dogs kills mosquitoes and thus would be expected to reduce transmission of heartworm in the surrounding community.

Coproantigen Detection Augments Diagnosis of Common Nematode Infections in Dogs.

Microscopic methods which employ active or passive flotation have been used to detect parasite diagnostic stages in the feces of companion animals for many years. More recently, coproantigen ELISAs for the detection of excretory/secretory products from intestinal nematodes have been introduced. These assays can identify the presence of parasites when eggs are not recovered by flotation (e.g. prepatent infection or intermittent egg shedding). The study was designed to assess the added benefit of these coproantigen tests in canine fecal diagnostics. The work was performed at 3 separate sites where canine fecal samples were each independently evaluated by both centrifugal flotation with an expert examiner (CFE) and passive flotation with a less experienced examiner. All samples were also tested using coproantigen ELISA to detect ascarid, hookworm, or whipworm antigen (IDEXX Laboratories, Inc, Westbrook, Maine). A total of 1202 samples were collected; 626 were from shelter dogs and 576 were from pet dogs. CFE recovered ascarid eggs in 58 samples, hookworm eggs in 229 samples, and whipworm eggs in 95 samples. Of the positive samples identified by CFE, the PFE and ELISA identified 40 and 51 ascarid samples, 188 and 203 hookworm samples, and 65 and 67 whipworm positive samples, respectively. The coproantigen ELISA identified 8 ascarid, 82 hookworm, and 22 whipworm positive samples that were not detected by CFE. The combined results of passive flotation and the coproantigen ELISA improved the percent agreement with centrifugal flotation, suggesting that greater sensitivity of detection may be achieved through the use of complementary diagnostic methods. However, errors of misidentification and poor recovery apparently introduced by less experienced examiners using an inferior flotation method remained. A diagnostic approach that combines coproantigen assays with centrifugal flotation and examination by an expert allows detection of more ascarid, hookworm, and whipworm infections.

Ticks from cats in the United States: Patterns of infestation and infection with pathogens.

Ticks are an important but under recognized parasitic threat to cats in many areas of the United States. To characterize the species and stages of ticks most commonly recovered from cats and determine the prevalence of disease agents in the ticks, we conducted a survey of ticks removed from cats at veterinary practices in 18 states from April 2016-June 2017. A total of 796 ticks were submitted from 332 cats from 41 different veterinary practices. A single tick was submitted from the majority of cats, with a mean infestation intensity of 2.4 (range 1-46). The most common tick was Ixodes scapularis, accounting for 422/796 (53.0%) ticks submitted, followed by Amblyomma americanum (224/796; 28.1%) and Dermacentor variabilis (131/796; 16.5%); a few I. pacificus, I. banksi, D. occidentalis, A. maculatum, Rhipicephalus sanguineus, and Otobius megnini were also submitted. A majority of ticks were adults (593/796; 74.5%); females predominated in all adult tick submissions including I. scapularis (277/327; 84.7% female), A. americanum (66/128; 51.6% female), and D. variabilis (75/126; 59.5% female). Immature ticks included 186 nymphs and 17 larvae and were primarily I. scapularis and A. americanum. Adult I. scapularis were most reported to be attached to the dorsal head and neck; A. americanum to the abdomen and perianal region; and D. variabilis to the back and ear. Ticks were collected in every month; the largest number of submissions were in May and June (42.5% of ticks) and October and November (35.9% of ticks). Adults of I. scapularis were most commonly submitted October through December, A. americanum March through June, and D. variabilis May through July. Cats with ticks were predominantly male (58.8%) and altered (76.2%), and most reportedly spent >30% of time outdoors, although 64/294 (21.8%) for which lifestyle estimates were provided were reported to live primarily (≤30% of time outside; n = 54) or entirely (100%; n = 10) indoors. Assay of ticks removed from cats revealed I. scapularis were infected with Borrelia burgdorferi (25.7%) and Anaplasma phagocytophilum (4.4%); A. americanum were infected with Ehrlichia chaffeensis (1.3%); and D. variabilis were infected with spotted fever group Rickettsia spp. (3.1%). No ticks in this study tested positive for Cytauxzoon felis. Pet cats, including those that live primarily indoors, are at risk of tick infestation, potentially exposed to tick-borne disease agents, and would benefit from routine tick control.

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.

Vector-Borne Infections in Tornado-Displaced and Owner-Relinquished Dogs in Oklahoma, USA.

To determine the prevalence of infection with vector-borne agents in a cross-section of dogs from Oklahoma, where canine vector-borne diseases are common, blood samples were evaluated through serology and molecular analysis. Antibodies reactive to Ehrlichia spp., Rickettsia rickettsii, R. montanensis, and "R. amblyommii" were detected in 10.5% (11/105), 74.3% (78/105), 58.1% (61/105), and 55.2% (58/105) of dogs, respectively. Presence of spotted fever group Rickettsia spp. DNA was identified in 13.1% (8/61) of shelter dogs but not in any pet dogs (0/44). DNA of "R. amblyommii" was confirmed by sequencing, constituting the first report of this agent in a naturally infected dog. Antigen of Dirofilaria immitis was detected in 10.5% (11/105) and 16.2% (17/105) of samples before and after heat treatment, respectively. In total, 87.6% (92/105) of the dogs had evidence of infection with at least one vector-borne disease agent, confirming high risk of exposure to multiple vector-borne disease agents, several of which are zoonotic.

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.

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.

Prevalence of antibodies to spotted fever group Rickettsia spp. and Ehrlichia spp. in coyotes (Canis latrans) in Oklahoma and Texas, USA.

Coyotes (Canis latrans) are commonly infested with ticks, including Amblyomma americanum, the predominant vector of Ehrlichia chaffeensis and Ehrlichia ewingii; Dermacentor variabilis, an important vector of Rickettsia rickettsii; and Amblyomma maculatum, a major vector of Rickettsia parkeri, a spotted fever group (SFG) Rickettsia. To determine the degree to which coyotes are infected with or exposed to tick-borne bacterial disease agents, serum samples collected from coyotes in Oklahoma and Texas were tested for antibodies reactive to R. rickettsii, Ehrlichia canis, E. chaffeensis, E. ewingii, Borrelia burgdorferi, and Anaplasma phagocytophilum by indirect fluorescent antibody (IFA) testing or enzyme-linked immunosorbent assay (ELISA). Of the coyotes tested, 60% (46/77) and 64% (47/74) had antibodies reactive to R. rickettsii and E. chaffeensis, respectively, on IFA. Additionally, 5% (4/77) had antibodies reactive to E. canis, but not B. burgdorferi or A. phagocytophilum, on SNAP(®) 4Dx(®) ELISA; subsequent serologic analysis by plate ELISA using species-specific peptides revealed antibodies to E. ewingii, E. canis, and E. chaffeensis in 46% (23/50), 18% (9/50), and 4% (2/50) of serum samples, respectively. Taken together, these data indicate that coyotes in this region are commonly exposed to SFG Rickettsia and E. ewingii and that further consideration of coyotes as a component of the maintenance cycle for these pathogens may be warranted.