Association between vector-borne pathogen seroprevalence in shelter-housed and owned dog populations in the contiguous United States of America.

Domestic dogs are susceptible to numerous vector-borne pathogens that are of significant importance for their health. In addition to being of veterinary importance, many of these pathogens are zoonotic and thus may pose a risk to human health. In the USA, owned dogs are commonly screened for exposure to or infection with several canine vector-borne pathogens. Although the screening data are widely available to show areas where infections are being diagnosed, testing of owned dogs is expected to underestimate the actual prevalence in dogs that have no access to veterinary care. The goal of this study was to measure the association between the widely available data from a perceived low-risk population with temporally and spatially collected data from shelter-housed dog populations. These data were then used to extrapolate the prevalence in dogs that generally lack veterinary care. The focus pathogens included Dirofilaria immitis, Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi. There was a linear association between the prevalence of selected vector-borne pathogens in shelter-housed and owned dog populations and, generally, the data suggested that prevalence of heartworm (D. immitis) infection and seroprevalence of Ehrlichia spp. and B. burgdorferi are higher in shelter-housed dogs, regardless of their location, compared with the owned population. The seroprevalence of Anaplasma spp. was predicted to be higher in areas that have very low to low seroprevalence, but unexpectedly, in areas of higher seroprevalence within the owned population, the seroprevalence was expected to be lower in the shelter-housed dog population. If shelters and veterinarians make decisions to not screen dogs based on the known seroprevalence of the owned group, they are likely underestimating the risk of exposure. This is especially true for heartworm. With this new estimate of the seroprevalence in shelter-housed dogs throughout the USA, shelters and veterinarians can make evidence-based informed decisions on whether testing and screening for these pathogens is appropriate for their local dog population. This work represents an important step in understanding the relationships in the seroprevalences of vector-borne pathogens between shelter-housed and owned dogs, and provides valuable data on the risk of vector-borne diseases in dogs.

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.

Human IgE mAbs identify major antigens of parasitic worm infection.

BACKGROUND: Much of our understanding of the targets of IgE comes from studies of allergy, though little is known about the natural immunogenic targets seen after parasitic worm infections. OBJECTIVE: We used human monoclonal antibodies (mAbs) for an unbiased and comprehensive characterization of the immunodominant antigens targeted by IgE in conditions like allergy or helminth infection that are associated with elevated levels of IgE. METHODS: Using human hybridoma technology to immortalize IgE encoding B-cells from peripheral blood of subjects with filarial infections and elevated IgE, we generated naturally occurring human IgE mAbs. B-cell cultures were screened in an unbiased manner for IgE production without regard to specificity. Isolated IgE mAbs were then tested for binding to Brugia malayi somatic extracts using ImmunoCAP, immunoblot, and ELISA. Immunoprecipitation followed by mass spectrometry proteomics was used to identify helminth antigens that were then expressed in Escherichia coli for IgE binding characterization. RESULTS: We isolated 56 discrete IgE mAbs from 7 individuals with filarial infections. From these mAbs, we were able to definitively identify 19 filarial antigens. All IgE mAbs targeted filarial excreted/secretory proteins, including a family of previously uncharacterized proteins. Interestingly, the transthyretin-related antigens acted as the dominant inducer of the filaria-specific IgE antibody response. These filaria-specific IgE mAbs were potent inducers of anaphylaxis when passively administered to human FcεRI-expressing mice. CONCLUSIONS: We generated human hybridomas secreting naturally occurring helminth-specific IgE mAbs from filarial-infected subjects. This work provides much-needed insight into the ontogeny of helminth-induced immune response and IgE antibody response.

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.

HELMINTH FAUNA OF ICE SEALS IN THE ALASKAN BERING AND CHUKCHI SEAS, 2006-15.

Climate warming may affect the distribution of helminth parasites, allowing endemic species to increase in prevalence and new species to appear. We analyzed tissues from 141 ice-associated seals collected in the Alaskan (US) Bering and Chukchi seas during 2006-15 for internal helminth parasites and compared results with past studies. Specimens were collected from: ringed seals (Pusa hispida), bearded seals (Erignathus barbatus), spotted seals (Phoca largha), and ribbon seals (Histriophoca fasciata). Helminths were present in 94% (133/141) of the seals sampled. Nematodes were most prevalent in bearded (97%, 72/74) and spotted seals (93%, 13/14). Cestodes were most prevalent in bearded seals (82%, 61/74) and absent in ribbon seals, trematodes were only found in bearded (64%, 47/74) and ringed (5%, 2/44) seals, and acanthocephalans were mostly found in ringed (61%, 27/44) and spotted (64%, 9/14) seals. Although no helminths were new to the Bering-Chukchi Seas region, this study found a previously unreported host record for the lungworm Parafilaroides (Filaroides) gymnurus in a ribbon seal. We also found the lungworm Otostrongylus circumlitus in a ribbon seal and P. (F.) gymnurus in bearded seals, representing location records previously unreported from the Bering-Chukchi Seas region (although they have been reported from the Sea of Okhotsk). We found the cestode genus Pyramicocephalus in bearded seals (3%, 2/74) at a lower prevalence than was reported previously for Pyramicocephalus phocarum (44-100%) in the Bering-Chukchi Seas region. We found no species of the acanthocephalan genus Bolbosoma, although the genus was previously identified in ringed, spotted, and ribbon seals. This study yielded no new helminths and no increases in the prevalence of endemic parasites in these seal species.

Canine vector-borne disease: mapping and the accuracy of forecasting using big data from the veterinary community.

Diagnosis, treatment, and prevention of vector-borne disease (VBD) in pets is one cornerstone of companion animal practices. Veterinarians are facing new challenges associated with the emergence, reemergence, and rising incidence of VBD, including heartworm disease, Lyme disease, anaplasmosis, and ehrlichiosis. Increases in the observed prevalence of these diseases have been attributed to a multitude of factors, including diagnostic tests with improved sensitivity, expanded annual testing practices, climatologic and ecological changes enhancing vector survival and expansion, emergence or recognition of novel pathogens, and increased movement of pets as travel companions. Veterinarians have the additional responsibility of providing information about zoonotic pathogen transmission from pets, especially to vulnerable human populations: the immunocompromised, children, and the elderly. Hindering efforts to protect pets and people is the dynamic and ever-changing nature of VBD prevalence and distribution. To address this deficit in understanding, the Companion Animal Parasite Council (CAPC) began efforts to annually forecast VBD prevalence in 2011. These forecasts provide veterinarians and pet owners with expected disease prevalence in advance of potential changes. This review summarizes the fidelity of VBD forecasts and illustrates the practical use of CAPC pathogen prevalence maps and forecast data in the practice of veterinary medicine and client education.

PREVALENCE OF PARELAPHOSTRONGYLUS ANDERSONI IN WHITE-TAILED DEER, OTHER CERVIDS, AND BOVIDS IN NORTHERN FLORIDA.

Parelaphostrongylus andersoni, the muscleworm, commonly infects white-tailed deer (Odocoileus virginianus) and also infects caribou (Rangifer tarandus [R.t.] groenlanicus, R.t. grantii, R.t. tarandus, and R.t. caribou). Heavy infection with P. andersoni leads to weakness in the hindquarters, abnormal gait, and pulmonary lesions. The geographical range and full host spectrum of this parasite are not fully known. This study aims to understand host specificity better, especially in nonnative cervids and bovids. This study involved the collection of 140 fecal samples from native and nonnative cervid and bovid species, and 34 snail specimens. With the use of real-time PCR, we found 4/47 (8.5%) O. virginianus fecal samples were positive for P. andersoni. No previously undocumented species of cervids or bovids were found to be infected. Further research is warranted to understand P. andersoni range, host distribution, and potential impact on host health.

Burden of exposure to infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus, Mycoplasma gallisepticum, and intestinal parasites in introduced broiler chickens on the Galapagos.

Diseases in introduced broilers can possibly spill over to wild birds on the Galapagos. Knowledge about the current burden of exposure to pathogens in broilers on the Galapagos is very limited. The objective of the study reported here was to measure the burden of exposure to infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (MG), and intestinal parasites in a sample of broiler chickens on 13 farms on Santa Cruz Island and San Cristobal Island in July 2017. Blood serum samples were tested for detection of antibodies to IBDV, IBV, NDV, and MG by using an IDEXX Enzyme-linked Immunosorbent Assay. In addition, fecal samples and pen bedding environmental samples were processed and analyzed for diagnosis of intestinal parasite eggs under a compound light microscope. The frequency of seropositive broilers to IBDV was 74/130 or 56% (95% CI = 48, 65%), to IBV was 27/130 or 20% (14, 28%), and to NDV was 1/130 or 0.7% (0.1, 4%). All broilers tested negative to MG antibodies. Eimeria spp. infection was common in study broilers. Finally, we observed interaction between broiler chickens and wild birds (finches) inside broiler pens, as well as the presence of backyard chickens inside property limits of study farms. This study produced evidence that exposure to IBDV, IBV, and intestinal parasites in broilers on Santa Cruz Island and San Cristobal Island is important. Study results are relevant because (i) they provide new baseline data on the burden of exposure to avian pathogens in broiler farms, (ii) justify the need to verify standard operating procedures in hatcheries that supply (non-vaccinated) day-old chicks to the Galapagos and (iii) to implement enhanced biosecurity standards on broiler chicken farms to mitigate risk of disease transmission between broilers, backyard poultry, and wild birds on the Galapagos.

Dog overpopulation and diagnosis of intestinal parasites on Santa Cruz Island, Galapagos 2016.

Dog overpopulation and diseases are hazards to native island species and humans on the Galapagos. The main objective of the study reported here was to estimate the observed human:dog ratio on Santa Cruz Island, Galapagos in September 2016. In addition, dog demographic data were used to model the expected annual dog population growth in the next 10 years. A secondary objective was to measure the burden of dogs infected with intestinal parasites. The observed human:dog ratio was 964:202 (or 4.77:1), which extrapolates to 3290 dogs; an increase of 31% in the dog population on Santa Cruz from 2014 to 2016. Study results show that current spay-neuter efforts (about 300 dogs per year; 60% females, 40% males) are not enough to keep the population stable (i.e., current baseline of 3290 dogs). The frequency of dogs infected with Ancylostoma spp., an intestinal parasite in dogs that can cause cutaneous larval migrans in humans, was 18/44 or 41% (95% CI = 27%, 55%). These results provide the most complete assessment of the dog overpopulation on the Galapagos to date.

Comparison of six commercial antigen kits for detection of Dirofilaria immitis infections in canines with necropsy-confirmed heartworm status.

Patient-side test kits for detecting antigenemia in dogs associated with sexually mature female heartworms (Dirofilaria immitis) have been available for three decades, and these tests are continually updated and improved. To define the sensitivity (Se) and specificity (Sp) of contemporary antigen detection tests against cardiopulmonary D. immitis burden, we evaluated five patient-side kits-Anigen Rapid One Step® (Bio note), SNAP® 4Dx Plus Test Kit (IDEXX), WITNESS® Heartworm Canine Heartworm Antigen Test Kit (Zoetis), VetScan® Canine Heartworm Rapid Test (Abaxis), and Solo Step® CH Canine Heartworm Antigen Test (Heska), and one microplate ELISA (DiroCHEK®; Zoetis), using archived canine sera divided into five subclasses of female worms (0, 1-5, 6-20, 21-40, and >40). The patient-side tests were performed once, side-by-side according to each manufacturer's protocol by personnel blinded to the D. immitis status of each dog. The overall Se and Sp of the patient-side kits was ≥97.5 and =94.0%, respectively. For samples from dogs with 1-5, 6-20, and 21-40 D. immitis, the Se was between 96 and 100%, with a slight increase in Se in dogs with ≥41 worms. The agreement between tests for all subclasses of D. immitis burden was between 99 and 100%. The Se and Sp for the ELISA compared with the necropsy results of dogs was 99 and 96%, respectively. Agreement between each patient-side test and the ELISA was between 97 and 100%. All commercially available tests can give practitioners excellent patient-side information, allowing them to make informed decisions on the need for additional diagnostic work-up before instituting new or continuing D. immitis prophylaxis.

Dog overpopulation and burden of exposure to canine distemper virus and other pathogens on Santa Cruz Island, Galapagos.

Dog overpopulation and diseases are hazards to native island species and humans on the Galapagos. Vaccination and importation of dogs are prohibited on the Galapagos. Risk management of these hazards requires the use of science-based risk assessment and risk communication. The objectives of the study reported here were (i) to estimate the human:dog ratio and (ii) the prevalence of and identify exposure factors associated with positive antibody titers to canine distemper virus (CDV) and other pathogens, as well as infection with intestinal parasites in owned dogs on Santa Cruz Island, Galapagos in September 2014. The observed human:dog ratio was 6.148:1 which extrapolates to 2503 dogs (two times more than a recent dog count conducted by Galapagos Biosecurity Agency in March 2014). The proportion of spayed female dogs (50%) was higher, compared to neutered male dogs (30%) (p=0.04). Prevalence of dogs with positive antibody titers to CDV was 36% (95% CI=26, 46%), to canine parvovirus was 89% (95% CI=82, 95%), and to canine adenovirus was 40% (95% CI=30, 51%). The frequency of seropositive dogs to CDV was lower in urban dogs (26%), compared to rural dogs (53%) (p<0.05). A positive interaction effect between rural residence and spay/neuter status on seropositivity to CDV was observed, which we discuss in this report. Because vaccination is prohibited, the dog population on Santa Cruz is susceptible to an outbreak of CDV (particularly among urban dogs) with potential spill over to marine mammals. Dog's age (1-2 or 3-14 years old, compared to younger dogs), and residence (rural, urban) were associated with positive antibody titers to parvovirus, adenovirus, Ehrlichia spp., or Anaplasma spp., as well as infection with Ancylostoma spp., an intestinal parasite in dogs that can be transmitted to humans, particularly children. These results provide the most comprehensive assessment of dog overpopulation and exposure to CDV and other pathogens on the Galapagos to date.