This study was performed to investigate whethere there is cross-reactivity between Dirofilaria immitis and three intestinal nematodes of dogs. In ELISA, D. immitis-infected dog sera obtained at the 4th molting stage (9-11 weeks) and microfilaremic stage (25-30 weeks) were shown to be highly reactive with crude extract of T. canis. In immunoblotting, some antigenic fractions, 44, 57, 88, 100 kDa of crude extract of T. canis, were found to be positive reaction with sera of dogs infected with D. immitis. However, little or no cross-reaction were observed between sera of D. immitis-infected dogs and crude extract antigen of T. vulpis or A. caninum. These result suggest that there are partial cross reaction between sera of D. immitis-infected dogs and the antigen of T. canis.
Animals ; Antibodies, Helminth/*immunology ; Antigens, Helminth/*immunology ; Cross Reactions ; Dirofilaria immitis/*immunology ; Dirofilariasis/*immunology ; Dogs ; Enzyme-Linked Immunosorbent Assay ; Immunoblotting ; Toxocara canis/*immunology

Three dogs were experimentally infected with Dirofilaria immitis. All dogs were euthanised at 30, 36 and 37 weeks after inoculation of D. immitis for the recovery of adult worms. Three cases accounted to 42.91 % recovery of inoculated worms. Serum samples from dogs experimentally inoculated with D. immitis were analyzed by ELISA and immunoblotting methods. Antibody titers of dogs detected by ELISA peaked between 7 and 14 weeks then decreased between weeks 15 to 24 followed by another increase during weeks 25 to 30 and persisted throughout the remainde of the experiment period. Analysis of adult D. immitis protein stained with Coomassie brilliant blue R-250 indicated separately more than 10 bands, and the major bands were 22, 40, 46, 56, 70, 72 and 89 kDa. Antigenic identification of extracts antigens of adults D. immitis by immunoblotting analysis revealed several bands from pooled sera of patent infection (30 weeks after inoculation). The detected bands were 24, 70, 80 and 110 kDa, 22, 72 and 84 kDa, and 58 and 72 kDa in dogs 1, 2 and 3, respectively. Results of antibody titers reached high levels on the 4th molting stage after inoculation of infective larva (L3), and reinforced previous findings that high molecular weight regions are detected in young animals.
Animals ; Antibodies, Helminth/analysis ; Antigens, Helminth/analysis/chemistry ; Autopsy ; Dirofilaria immitis/chemistry/*immunology ; Dirofilariasis/*immunology/parasitology ; Dog Diseases/*immunology/*parasitology ; Dogs/*immunology/*parasitology ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Female ; Immunoblotting ; Male ; Time Factors

OBJECTIVETo evaluate the Anaplasma phagocytophilum (A. phagocytophilum), Ehrlichia canis (E. canis), Dirofilaria immitis (D. immitis) (canine heartworm), Borrelia burgdorferi (B. burgdorferi) infections in countryside dogs from Yunnan, Hainan and Anhui provinces.

METHODSSerum samples were collected from 26 dogs in Yunnan, Hainan and Anhui provinces. The samples were tested using a commercial ELISA rapid diagnostic assay kit (SNAP(®) 4Dx(®); IDEXX Laboratories, Inc. U.S.A.). Meanwhile, indirect immunofluorescence assay (IFA) recommended by WHO was conducted to detect IgG to A. phagocytophilum. Two methods were analyzed and compared.

RESULTSThe number of serologically positive dogs for IgG to A. phagocytophilum was only 2 which was from Hainan province and none of the 26 dogs responded positive for E. canis, D. immitis (canine heartworm), and B. burgdorferi by ELISA rapid diagnostic method. The number of serologically positive dogs for IgG to A. phagocytophilum was 13 (50%) by IFA method. Data of the two methods were analyzed by statistical software and the difference was statistically significant (P=0.002).

CONCLUSIONSIt can be concluded that IFA method was more sensitive than ELISA rapid diagnostic method. However, we need conduct further and intensive epidemiology survey on tick-born diseases pathogens including A. phagocytophilum, E. canis, D. immitis (canine heartworm), and B. burgdorferi which have public health significance.

Anaplasma phagocytophilum ; immunology ; Animals ; Borrelia burgdorferi ; immunology ; China ; epidemiology ; Dirofilaria immitis ; immunology ; Dirofilariasis ; blood ; epidemiology ; immunology ; Disease Vectors ; Dog Diseases ; epidemiology ; Dogs ; Ehrlichia canis ; immunology ; Ehrlichiosis ; blood ; epidemiology ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Fluorescent Antibody Technique, Indirect ; methods ; Immunoglobulin G ; blood ; Lyme Disease ; blood ; epidemiology ; immunology ; Tick-Borne Diseases ; epidemiology

Dirofilaria immitis (heartworm) infections affect domestic dogs, cats, and various wild mammals with increasing incidence in temperate and tropical areas. More sensitive antibody detection methodologies are required to diagnose asymptomatic dirofilariasis with low worm burdens. Applying current transcriptomic technologies would be useful to discover potential diagnostic markers for D. immitis infection. A filarial homologue of the mammalian translationally controlled tumor protein (TCTP) was initially identified by screening the assembled transcriptome of D. immitis (DiTCTP). A BLAST analysis suggested that the DiTCTP gene shared the highest similarity with TCTP from Loa loa at protein level (97%). A histidine-tagged recombinant DiTCTP protein (rDiTCTP) of 40 kDa expressed in Escherichia coli BL21 (DE3) showed immunoreactivity with serum from a dog experimentally infected with heartworms. Localization studies illustrated the ubiquitous presence of rDiTCTP protein in the lateral hypodermal chords, dorsal hypodermal chord, muscle, intestine, and uterus in female adult worms. Further studies on D. immitis-derived TCTP are warranted to assess whether this filarial protein could be used for a diagnostic purpose.
Animal Structures/chemistry ; Animals ; Antibodies, Helminth/blood ; Antigens, Helminth/chemistry/*genetics/immunology/*isolation & purification ; Cloning, Molecular ; Dirofilaria immitis/chemistry/*genetics/immunology ; Disease Models, Animal ; Dogs ; Escherichia coli/genetics ; Gene Expression ; Molecular Sequence Data ; Molecular Weight ; Recombinant Fusion Proteins/chemistry/genetics/immunology/isolation & purification ; Sequence Analysis, DNA ; Tumor Markers, Biological/chemistry/*genetics/immunology/*isolation & purification