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Dirofilaria immitis and D. repens are globally distributed mosquito-borne parasitic filarial nematodes. Data on the prevalence of Dirofilaria spp. is not aggregated or publicly available at the national level for countries in North Africa and the Middle East. A systematic review and meta-analysis of publications describing cases of D. immitis and D. repens in 21 countries in North Africa and the Middle East was performed following PRISMA guidelines to estimate the prevalence of Dirofilaria spp. where national and regional estimates don’t exist. In total, 460 publications were reviewed, and 34 met all inclusion criteria for the meta-analysis model. This analysis found that the combined prevalence of Dirofilaria spp. in the included countries was 2.4% (95% CI: 1.6–3.6%; I2 = 81.7%, 95% CI: 78.6–84.3%). Moderator analysis showed a statistically significant difference in the prevalence estimate between diagnostic test methods used. The model detected a high degree of heterogeneity among studies and publication bias. Removal of model identified outliers reduced the estimated prevalence from 2.4% to 1.0%, whereas the trim-and-fill analysis suggested a higher adjusted prevalence (12%). Despite these findings, Dirofilaria spp. prevalence is likely dynamic due to seasonal variations in mosquito vector populations and differences in local mosquito control practices. Additional studies from the countries in and surrounding this region are needed to better identify key risk factors for Dirofilaria spp. in domestic canids and other species (including humans) to inform prevention and control decisions to limit further transmission.

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. Graphical Abstract

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. Graphical Abstract

Background Heartworms, Dirofilaria immitis , are known to be widespread in dogs and cats in the USA, but there have been no country-wide prevalence studies performed to date. There have also been no large-scale studies to determine whether the closely related species, Dirofilaria repens , occurs in the USA. Methods To provide this large-scale data, we examined whole blood samples ( n  = 2334) submitted from around the USA to the Molecular Diagnostic Laboratory at Auburn University between 2016 and 2022. Quantitative PCRs for D. immitis (targeting 16S rRNA) and D. repens (targeting cytochrome c oxidase subunit 1 gene) were performed to determine the presence of Dirofilaria DNA. DNA sequencing was performed to confirm the results. Results Dirofilaria immitis DNA was found in 6.3% (68/1080) of the dogs from 17/39 states, and 0.3% (4/1254) of the cats from 4/42 states. None of the dogs or cats were positive for D. repens . The average 16S rRNA copy number of D. immitis in the dogs was 1,809,604 in 200 µl whole blood, while only a single copy was found in each of the four D. immitis -positive cats. The prevalence of D. immitis in dogs of different ages, sexes, and breeds did not differ significantly, but the prevalence in Southern states (7.5%, 60/803) was significantly higher than in the Western (1.7%, 1/58), Midwest (3.3%, 4/120), and Northeastern states (3.1%, 3/98) ( P  < 0.05). Dogs positive for D. immitis were identified in each study year (2016: 4.2%, 2/48; 2017: 9.8%, 4/41; 2018: 5.1%, 8/156; 2019: 4.9%, 15/306; 2020: 9.8%, 26/265; 2021: 4.9%, 13/264). Interestingly, dogs infected with Hepatozoon spp. (11.8%, 37/313) were significantly more likely to also be positive for D. immitis than dogs without evidence of Hepatozoon infection (3.9%, 30/760) ( P  < 0.0001). Conclusions To our knowledge, this is the first nationwide molecular survey of Dirofilaria spp. in dogs and cats in the USA, and the largest molecular survey of canine and feline dirofilariosis worldwide. Further studies are warranted to combine PCR with standard heartworm diagnostics to better understand the prevalence of Dirofilaria spp. and aid in determining the risks posed to dogs and cats in the USA. Graphical Abstract

Background Feline heartworm disease (HWD) is a complex and often misdiagnosed disease in cats, caused by the filarial nematode Dirofilaria immitis . Despite its significant impact, studies reporting the prevalence of D. immitis in apparently healthy pet cats in the USA are lacking. Methods To investigate feline heartworm seroprevalence in apparently healthy pet cats in the USA, serum samples ( n  = 2165) collected from cats across 47 states and Washington District of Columbia were analyzed for D. immitis antibody (Heska Corp.) and antigen (DiroCHEK®; Zoetis Inc.) with and without acid treatment of the samples. Results Antibodies to D. immitis antibodies were identified in 3.5% (76/2165) of cats from 26 states, with a significantly higher prevalence in cats from the westernmost US states (West region; 5.4%, 23/429) compared to those from the South (3.8%, 32/847), Midwest (2.7%, 9/338) and Northeast regions (2.2%, 12/551) ( P  < 0.04). Antigen from D. immitis was detected in 0.3% (6/2165) of cats, which was significantly lower than the antibody detection ( P  < 10 –4 ), and no samples were positive for both antibody and antigen. Conclusions This is the largest antibody-based, nationwide serosurvey of feline heartworm in an apparently healthy cat population, and the results suggest that cats in the USA have a high risk of exposure to D. immitis -infected mosquitoes. The high nationwide prevalence (3.5%) indicates that the true prevalence of cats infected with D. immitis in the USA may be significantly underestimated. Our findings emphasize the need for increased awareness and routine testing of cats for heartworm infection, especially in non-endemic areas of the USA. Clinicians should consider appropriate use of broad-spectrum veterinary-approved parasiticides and lifestyle management in feline patients to reduce the risk of infection. Future studies should focus on evaluating the D. immitis infection status in healthy cats and developing better diagnostic assays to detect this complex infection. Graphical Abstract

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).

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.

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.

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.

Ticks are obligate hematophagous arthropods and act as vectors for a great variety of pathogens, including viruses, bacteria, protozoa, and helminths. Some tick-borne viruses, such as Powassan virus and tick-borne encephalitis virus, are transmissible within 15–60 min after tick attachment. However, a minimum of 3–24 h of tick attachment is necessary to effectively transmit bacterial agents such as Ehrlichia spp., Anaplasma spp., and Rickettsia spp. to a new host. Longer transmission periods were reported for Borrelia spp. and protozoans such as Babesia spp., which require a minimum duration of 24–48 h of tick attachment for maturation and migration of the pathogen. Laboratory observations indicate that the probability of transmission of tick-borne pathogens increases with the duration an infected tick is allowed to remain attached to the host. However, the transmission time may be shortened when partially fed infected ticks detach from their initial host and reattach to a new host, on which they complete their engorgement. For example, early transmission of tick-borne pathogens (e.g., Rickettsia rickettsii, Borrelia burgdorferi, and Babesia canis) and a significantly shorter transmission time were demonstrated in laboratory experiments by interrupted blood feeding. The relevance of such situations under field conditions remains poorly documented. In this review, we explore parameters of, and causes leading to, spontaneous interrupted feeding in nature, as well as the effects of this behavior on the minimum time required for transmission of tick-borne pathogens.