Canine Dirofilaria immitis infection in a hyperenzootic area: examination by parasitologic findings at necropsy and by two serodiagnostic methods

A total of 174 dogs from an area hyperenzootic for Dirofilaria immitis were grouped into 4 age categories and necropsied; information was obtained on adult D immitis infections and on the presence of microfilariae. Serum samples from these dogs were examined by an enzyme-linked immunosorbent assay (ELISA) for antibody to adult D immitis and by an indirect fluorescent antibody test (IFAT) for antibody to microfilarial surface antigens. In dogs less than or equal to 5 months of age, necropsy demonstrated no evidence of infection; however, positive serologic results indicated that some of these dogs had prepatent infections. The percentage of dogs with ELISA titers (positive) increased with age, as did the percentage of dogs with adult D immitis infections. The IFAT results were positive in some dogs in each age category. Sera from all 29 dogs with occult infections were positive by ELISA. Sera from 6 of 20 dogs with occult dual-sex heartworm infections and 1 of 9 dogs with occult single-sex heartworm infections were positive by IFAT. For diagnosing occult dirofilariasis, the ELISA had a positive predictive value which increased with age of the dog to a maximum of 65.0% in dogs greater than or equal to 12 months of age; ELISA had a negative predictive value of 100% in all age groups. In contrast, positive and negative predictive values for the IFAT decreased with age of the dog to 60% and 37.5%, respectively, in dogs greater than or equal to 12 months of age.     

Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.     

Immune responses in vaccinated dogs with autoclaved Leishmania major promastigotes.

A comparative study was undertaken on the immunogen power of autoclaved Leishmania major promastigotes (ALM) vaccines given simultaneously with either BCG or saponin against canine leishmaniasis. The humoral immune response was assessed by ELISA and western blotting. The cellular immune response was evaluated by the lymphocyte transformation test. Dogs vaccinated simultaneously with ALM and saponin showed high antibody titres to crude L. infantum antigens after the first vaccine booster and reacted with several antigens, with molecular weights from 26 to 108 kDa by western blotting. However, the lymphocyte proliferation of these dogs to the crude L. infantum antigen was not significantly different from the control group. In contrast, in dogs vaccinated simultaneously with ALM and BCG, the antibody titres to crude antigen were low. Their sera reacted with the same proteins recognised by sera from dogs vaccinated simultaneously with ALM and saponin by western blotting. However, the 85-kDa protein was only identified by sera taken from dogs vaccinated simultaneously with ALM and BCG. These latter exhibited specific lymphocyte proliferation to the L. infantum antigen. This cell proliferation was observed for approximately 9 months after the first dose of the vaccine. This study indicates that a combination of ALM as the vaccine and BCG as the adjuvant, in the dog model, was successful in inducing a cell immune response, which is implicated in protection of dogs against a Leishmania infection.     

An evaluation of 4 commercially available ELISA kits for the diagnosis of Dirofilaria immitis infection in dogs

Serum samples from 100 pound dogs were used to evaluate 4 commercial ELISA kits available for the diagnosis of Dirofilaria immitis. The kits were assessed on sensitivity (the ability to identify infected dogs), specificity (the ability to identify uninfected dogs) and accuracy (sensitivity plus specificity). The kits varied in sensitivity from 36% to 86%, in specificity from 44% to 70%, and in accuracy from 53% to 65%. The sensitivity was not affected by the age of the dogs, nor by the number of circulating microfilariae. The kits were most specific when testing the youngest dogs (less than = 3 years). The problems associated with the serological diagnosis of D. immitis infection in practice are discussed.     

Immune response against Leishmania antigens in dogs naturally and experimentally infected with Leishmania infantum

Cell-mediated and humoral immune response in naturally and experimentally infected dogs was studied using crude and pure antigens. Both types of infections induced severe signs of visceral disease, but the symptoms observed in natural infections were more pronounced than in experimental infections. In addition, asymptomatic infections were not observed in experimentally infected animals. Disease evolution in laboratory infections was rapid and an increase in antibody titer to crude parasite antigen was correlated with the appearance and aggravation of clinical symptoms. Peripheral blood lymphocyte proliferation to crude antigen and pure gp63 was observed early following experimental infection, but was abolished once the infected dogs began to exhibit clinical signs. A similar pattern was observed in naturally infected dogs. Serum from all patent dogs showed high antibody titers to rK39 in enzyme-linked immunosorbent assays (ELISA), and reacted by western blotting with several antigens, 12 to 120 KDa, including gp63 and gp70. In the case of asymptomatic dogs. antibody titers to crude antigen were low and only a few antigens were identified by western blotting. None of the pure proteins examined, gp63, gp70, and rK39 were recognized by western blotting or ELISA. However, asymptomatic dogs exhibited specific lymphocyte proliferation to both crude antigen and the potential vaccine candidate gp63.     

Evaluation of a serological test system for the diagnosis of natural Echinococcus granulosus infection in dogs using E. granulosus protoscolex and oncosphere antigens

Serum antibody responses in feral or domesticated dogs naturally infected with Echinococcus granulosus or/and other common helminths were examined in an enzyme-linked immunosorbent assay (ELISA) using antigens prepared from E. granulosus protoscoleces or oncospheres. The ELISA using the protoscolex antigen was optimised with serums from experimental dogs monospecifically infected with E. granulosus or other helminth parasites, and helminth-free dogs. Anti-protoscolex antibody was detected in 16 of 22 (72.7%) serums from feral dogs with E. granulosus burdens ranging from 300 to 302,600 worms per dog. Seven serums from feral dogs which did not harbour E. granulosus at autopsy but which originated from an endemic hydatid region were tested using protoscolex antigen, and 1 serum gave a positive reaction. One hundred and two serums from dogs known never to have been infected with E. granulosus all gave negative reactions to protoscolex antigen. The sensitivity of the ELISA test proved to be superior to that which has been achieved by arecoline purging as a method of diagnosis for E. granulosus infection in dogs. For use of the assay in hydatid control or eradication campaigns, its sensitivity can be increased by choosing a lower absorbance discrimination value above which serums are regarded as having positive reactions. However, this does introduce positive reactions of some serums from dogs infected with helminths other than E. granulosus. In further development of the assay, use of defined recombinant antigens may improve both sensitivity and specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

DISTRIBUTION AND DIAGNOSIS OF DIROFILARIASIS AND TOXOCARIASIS IN AUSTRALIA

SUMMARY: The prevalence of Dirofilaria immitis infection in dogs continues to increase in the temperate east coast zone of Australia (and is extending further south into New South Wales and Victoria). However, the infection rate has not changed in the tropics over the past 10 years where it would appear that a maximum infection rate of 90% occurs in a given Australian dog population. Twelve percent of Brisbane dogs had occult dirofilariasis and it is suggested that the proportion of occult infections was probably higher in the tropics. Dogs of all breeds appear equally susceptible to D. immitis with infection being more common in older male dogs. The level of microfilaraemia was, occasionally, proportional to the number of heartwprms per dog. Toxocara canis was present in about 75% of dogs from all areas studied except in Central Australia where the level of infection was much lower.
Immunodiagnosis of D. immitis and T. canis with high specificity and sensitivity was achieved by cyanogen bromide indirect fluorescent antibody and cell-mediated immunity tests using parasite antigens purified by affinity chromatography. These tests enabled occult dirofilariasis to be differentiated from unrelated canine cardiac and pulmonary failure. Such immunodiagnosis can aid in the early diagnosis of dirofilariasis particularly in situations where no circulating microfilariae can be detected.
The prevalence of serum antibody in man to purified Dirofilaria and Toxocara antigens was proportional to the incidence of respective canine infections at each location.     

The occurrence of Dirofilaria immitis in dogs in South Australia

Heart, lung and samples of blood from 230 dogs were examined for infections of filarial parasites. Dirofilaria immitis worms and microfilariae were detected in one dog. Blood samples from a further 1428 dogs were examined for microfilariae and 22 were found to be infected. Eighteen dogs were infected with D immitis microfilariae and four with Dipetolonema reconditum microfilariae. The histories were available for 18 of the dogs infected with heartworm and only seven dogs had not travelled outside South Australia. It was concluded that heartworm infection was endemic in South Australia but the apparent prevalence was only about 1%.     

Specificity of scolex and oncosphere antigens for the serological diagnosis of taeniid cestode infections in dogs

Groups of dogs raised free of helminths were monospecifically infected with the common nematodes Toxocara canis, Ancylostoma caninum and Trichuris vulpis. Serums from these dogs, and a group of dogs of unknown history but infected with Dirofilaria immitis and Dipylidium caninum, had levels of antibody to their homologous nematode antigens readily detectable by ELISA. No cross-reactions were apparent when these serums were tested by ELISA using oncosphere antigens of Taenia hydatigena, T. pisiformis and T. ovis, scolex excretory/secretory antigens of T. hydatigena, T. pisiformis and Echinococcus granulosus or protoscolex antigen of E. granulosus.     

Antibody activity to Babesia canis in dogs in North Carolina

Sera from 600 dogs from 6 geographic regions of North Carolina were analyzed for antibody activity to Babesia canis, using an indirect fluorescent antibody test. Overall, 3.8% of the dogs had an antibody titer greater than or equal to 1:80, which was considered to be indicative of infection. Dogs were more likely to be seropositive if they were housed at humane facilities and were located within areas of the state with a milder climate than were pet animals living within colder regions of North Carolina.     

Seroprevalence of various infectious agents in dogs with suspected acute canine polyradiculoneuritis

Background: Acute canine polyradiculoneuritis (ACP) is considered to be an animal model of the acute axonal form of Guillain‐Barré syndrome (GBS) in humans. Various antecedent events have been associated with GBS, including bacterial or viral infection. The relationship between ACP and previous infection requires additional attention. Hypothesis: We hypothesized a relationship between ACP and serological evidence of exposure to Ehrlichia canis, Borrelia burgdorferi, Toxoplasma gondii, Neospora caninum, Campylobacter jejuni, and canine distemper virus (CDV). Animals: Eighty‐eight client‐owned dogs, 44 with ACP, 44 age‐matched controls. Methods: Retrospective study with stored serum samples. Serum antibodies against the target organisms were measured with commercially available assays. Sera from dogs with and without ACP that were positive for T. gondii IgG by ELISA were assayed by an IgG heavy chain‐specific, Western blot immunoassay. Results: Dogs with ACP (55.8%) were more likely to have T. gondii IgG serum antibody titers than dogs without ACP (11.4%). Serum antibodies from 8 affected dogs and 11 control dogs bound to T. gondii antigens with apparent molecular masses of 67, 61, 58, 45, 33, 24, 9, and 6 kDa. An antigen with an apparent molecular mass of 36 kDa was recognized by 2 dogs with ACP but none of the control dogs. Conclusions: Results of this study suggest that ACP in some dogs, like GBS in some humans, may be triggered by T. gondii and a prospective study should be performed to further evaluate this potential association.     

Anaplasma phagocytophilum in dogs in Germany

A total number of 111 dogs were included in the present prospective study investigating the prevalence of Anaplasma phagocytophilum in dogs in Germany. Dogs were divided into two groups. Dogs of group 1 (n = 49) showed clinical and/or haematological signs seen in infections with A. phagocytophilum, whereas those of group 2 (n = 62) did not have any evidence of anaplasmosis. For each dog, an A. phagocytophilum 16S rRNA-nested polymerase chain reaction (PCR) of ethylenediaminetetraacetic acid (EDTA)-anticoagulated whole blood analysis, a microscopic evaluation of a buffy coat and a serum indirect fluorescent antibody test (IFAT) were performed. Forty-eight seroreactive dogs were identified altogether, which amounts to an overall point prevalence of 43.2%. There was no significant difference between the seroreactivity to A. phagocytophilum antigens among group 1 (44.9%) and 2 (41.9%) (P > 0.5). Seven dogs (6.3%) had positive PCR results. All of them were seroreactive. Six belonged to group 1. Morulae in neutrophilic granulocytes were found in two dogs of group 1 but in none of group 2. Both dogs were seroreactive. Very high antibody titres (> or =1:1024) were detected significantly more frequently in dogs with clinical signs attributable to infection with A. phagocytophilum (group 1) than in those without (group 2) (P < 0.001). There was no significant correlation of overall positives or antibody titres to age, breed, sex, or whether the dogs were family or working dogs. Dogs with high tick infestation were significantly more often seroreactive to A. phagocytophilum than those with no or low tick infestation (P = 0.007). In conclusion, there seems to be a high risk of infection with A. phagocytophilum in Germany. Results of this study suggest that severe illness solely caused by A. phagocytophilum may be possible although definitive evidence does not exist. Very high antibody titres (>1:1024) may be associated with clinical anaplasmosis.     

Prevalence and epidemiological aspects of Dirofilaria immitis in dogs from Kayseri Province, Turkey

This study was conducted to determine the prevalence of Dirofilaria immitis infection and to investigate the risk factors related to heartworm disease in dogs from Kayseri, Turkey. Blood samples were collected from 280 dogs from May 2005 to March 2006 and were examined by membrane filtration-acid phosphatase histochemical staining and antigen Elisa techniques to detect circulating microfilariae and antigens of D. immitis, respectively. Of the total of 280 dogs, 27 were positive for D. immitis with a prevalence value of 9.6%. In addition 29.6% of positive dogs determined to have occult D. immitis infections. D. immitis was the only canine filarial parasite present in the study area. The mean number of microfilariae in infected dogs was 4730+/-5479 per ml of blood. The highest heartworm prevalence were observed in 7-10 age group (28.6%) followed by 4-6 (17.1%) and 0.5-3 (4.8%) age groups. The differences between 0.5-3 and other age groups were found significant, whereas no statistically significant difference was observed between 4-6 and 7-10 age groups. The infection was more prevalent in males, larger breeds and the dogs not on prophylaxis. No statistically significant difference was observed between stray and owned dogs. Our results suggest that heartworm treatment and prophylaxis should be considered in Kayseri Province.     

Prevalence of Leishmania spp. infection in domestic dogs in Chapare, Bolivia

Data on Leishmania spp. infection in dogs in Bolivia is scarce. Dogs from an area where 90% of human cutaneous leishmaniasis (CL) cases are due to Leishmania (Viannia) braziliensis were screened for Leishmania infection using established enzyme-linked immunosorbent antibody test (ELISA) protocols. Although none of the 51 dogs surveyed had clinical lesions indicative of CL, 6 out of 51 (11.8%) sampled dogs tested positive by ELISA.     

Antibody and lymphoblastogenic responses of dogs experimentally infected with Trypanosoma cruzi isolates from North American mammals

The humoral and cellular immune responses of dogs infected with either a non-pathogenic Trypanosoma cruzi isolated from a North American dog (Tc-D) or a pathogenic T. cruzi isolate from an opossum (Tc-O) were studied over a 240 day period. Antibody to T. cruzi epimastigote antigens prepared from Tc-O or Tc-D isolates were first detected by ELISA by Day 26 post infection (PI), peaked by day 175 PI and remained elevated throughout the experimental period in both Tc-O and Tc-D infected dogs. Differences in antibody levels between infected groups were not detected. Western blot analyses were performed using Tc-O and Tc-D epimastigote antigens probed with pooled sera and sera from individual Tc-O and Tc-D infected dogs prior to infection (Day 0), and during the acute (Day 16-35 PI), indeterminate (Day 50-135 PI) and chronic (Day 235 PI) stages of infection. Generally, the patterns, number of protein bands, and temporal appearance of the protein bands identified by pooled sera and sera from individual dogs within each antigen preparation were similar. However, similarities and differences were present in antibody responses between sera from Tc-O and Tc-D infected dogs. Blastogenic responses of peripheral blood mononuclear cells (PBMC) from Tc-O and Tc-D infected dogs to mitogens (concanavalin A, phytohemagglutinin and pokeweed) were not significantly different from controls at any time during the experimental period. The PBMC from both groups of dogs were unresponsive to epimastigote antigens during the acute stage of infection. Statistically significant differences (P less than 0.05) in PBMC responsiveness from controls were observed on Days 70 and 175 PI. Responses decreased to pre-infection levels by Day 240 PI. These studies demonstrate that although two North American T. cruzi isolates have markedly different virulence for dogs, some aspects of their cellular and humoral immune responses are similar while other responses, such as antibody recognition of specific T. cruzi antigens, vary.     

19s and 7s antibody response of Mastomys natalensis in experimental filarial (Litomosoides carinii, Acanthocheilonema viteae, Brugia malayi, B. pahangi) infections

Specific total antibody (ab), 19s and 7s ab levels in the serum of M. natalensis were investigated after infection with L. carinii, A. viteae, B. malayi and B. pahangi for period of about 500 days p.i., using ELISA (homologous adult antigen) and indirect immunofluorescence tests (IIFT: homologous adult and microfilariae antigen). Total ab levels in L. carinii infected animals rose moderately during prepatency Maximum levels occurred during patency. The response during prepatency was stronger in A. viteae and Brugia spp. infected hosts. Lateron ab levels increased continuously in Brugia infections; in A. viteae infection they decreased with decreasing parasitaemia. 19s abs were stimulated during prepatency and at the beginning of patency, or were found at moderate levels throughout to period of investigation (Brugia infections). 7s abs predominated beginning at the period of late prepatency (IIFT) or at the beginning of patency (ELISA). The time courses of 7s abs corresponded to those of total abs. As obvious by IIFT (adult worm antigen) total and 19s titres were higher against cuticle antigens, egg shell antigens and intrauterine amorphous material than against antigens located in the hypodermis and musculature. 7s abs showed best reactivity with cuticle antigens. Using microfilarial antigens 19s abs reacted predominantly with cuticle antigens whereas 7s abs often showed higher titres against antigens which were localized within the larvae than against cuticle antigens.     

Value of Western blotting in the clinical follow-up of canine leishmaniasis.

Specific serum antibody levels in Leishmania infantum-infected dogs treated with a combination of glucantime and allopurinol were estimated by indirect immunofluorescence and Western blotting. The sensitivity of Western blot was greater than that obtained with immunofluorescence titration. In general, both diagnostic methods concurred with the post-treatment clinical status of the animals. Clinical improvement of successfully treated dogs was related to lower immunofluorescence titers and simpler and/or less reactive immunodetection patterns in Western blotting. The recognition, by infected dogs, of certain low molecular weight antigens, particularly one of approximately 26 kDa, was restricted to pretreatment samples and a single animal in relapse thus apparently constituting an active infection marker.     

Anti-neosporal IgG and IgE antibodies in canine neosporosis

Neospora caninum infection provokes neurological disorders, recurrent abortion and death in dogs and cattle. Dogs are both intermediate and definitive host of N. caninum. Thus, the development of sensitive and specific immunoassays to diagnose canine neosporosis is essential to control this disease. This work investigated serum anti-neosporal IgG and IgE antibodies in 140 dogs represented by 30 healthy animals (group I), 11 dogs showing acute N. caninum infection (group II), 50 urban dogs with serological evidence of canine neosporosis in indirect fluorescent antibody test (IFAT) (group III) and 49 urban dogs without clinical and laboratory evidences of neosporosis (group IV). Enzyme-linked immunosorbent assay (ELISA) and western immunoblotting, both using a soluble N. caninum tachyzoite antigen (SNA), investigated these two isotypes of antibodies, while a Urea-ELISA measured the avidity of the IgG antibodies. Anti-Toxoplasma gondii IgG antibodies were also investigated in the animals. Anti-neosporal IgG was found in all animals from groups II and III, whereas 32.7% (16/49) of dogs from group IV were reactive. IgG antibodies of low avidity were demonstrated in dogs from group II (median 35.3%), while animals from groups III and IV had IgG antibodies of high avidity (medians of 61.5% and 61.7% respectively). IgE antibodies were found in four (13.3%) and five (16.6%) dogs from groups III and IV respectively. Dogs presenting acute infection (group II) or chronic infection (group III) had IgG antibodies to several neosporal antigens, mainly of 29-30 and 35 kDa, while 13 of 16 dogs from group IV recognized antigens from 14 to 170 kDa. Antibodies to T. gondii were detected in 36 of 50 (72%) sera from group III and 25 of 49 (51%) sera from group IV. We concluded that IgG-ELISA and Urea-ELISA with SNA may substitute for IFAT in both laboratory routine and epidemiological studies of canine neosporosis.     

Distinct antigen recognition pattern during zoonotic visceral leishmaniasis in humans and dogs

Leishmania infantum is a causative agent of endemic zoonotic visceral leishmaniasis (VL) in regions of South America and the Mediterranean. Dogs are the major reservoirs for L. infantum in these regions, and control of disease in dogs could have a significant impact on human disease. Although dogs share many symptoms of VL with humans as a result of L. infantum infection, they also show some unique clinical manifestations, which are often a combination of visceral and cutaneous leishmaniasis, suggesting different mechanisms of disease development in dogs and humans. Here, we compare antibody responses of dogs and humans with VL to various defined leishmanial antigens. Parasite lysate and K39, the two most commonly used antigens for serodiagnosis of VL, detected the highest levels of antibodies in both humans and dogs with VL, whereas the recognition patterns of these antigens were distinct between the hosts. Among other defined antigens tested, LmSTI1 and CPB detected higher levels of antibodies in dogs and humans, respectively. These results indicate there is a difference between humans and dogs in antigen recognition patterns during VL. We infer that different strategies may need to be used in development of vaccines and diagnostics for humans and for dogs. In addition, we show a correlation between antibody titers to several antigens and severity of clinical symptoms during canine VL.     

Incidence of coccidioides infection among dogs residing in a region in which the organism is endemic

OBJECTIVE: To determine the incidence of Coccidioides infection among dogs residing in a region in which the organism is endemic (Pima and Maricopa counties, Arizona) and estimate the rate of clinical illness. DESIGN: Community-based longitudinal and cross-sectional studies. ANIMALS: 124 healthy 4- to 6-month-old seronegative puppies (longitudinal study) and 381 4- to 18-month-old dogs with unknown serostatus (cross-sectional study). PROCEDURE: Dogs in the longitudinal study were tested at 6-month intervals for at least 1 year for anticoccidioidal antibodies. Dogs that became ill were evaluated for coccidioidomycosis. Dogs in the cross-sectional study were tested for anticoccidioidal antibodies once, and clinical abnormalities were recorded. RESULTS: 28 of the 104 (27%) dogs that completed the longitudinal study developed anticoccidioidal antibodies. Thirty-two of the 381 (8%) dogs in the cross-sectional study had anticoccidioidal antibodies. Five seropositive dogs in the longitudinal study and 13 seropositive dogs in the cross-sectional study had clinical signs of disease. The remaining seropositive dogs were otherwise healthy and were classified as subclinically infected. Survival analysis indicated that the cumulative probability of infection by 2 years of age was 28%, and the cumulative probability of clinical infection by 2 years of age was 6%. Titers for clinically and subclinically infected dogs overlapped. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that young dogs living in the study area had a high likelihood of becoming infected with Coccidioides spp, but few developed clinical illness. Serologic testing alone was insufficient for a diagnosis of clinical disease because of the overlap in titers between clinically and subclinically infected dogs.