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.     

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.     

Human adenovirus antibody in sera of normal and tumour-bearing dogs.

Serum specimens from 42 normal dogs and 42 with untreated malignant tumours were assayed for the presence of antibodies to human adenovirus types 5, 21 and 31 and to infectious canine hepatitis (ICH) virus. Radioimmunoassays using human adenovirus antigens showed that 71 per cent (30/42) of all dogs with tumours, but only 19 per cent (8/42) of all normal dogs, were positive for human adenovirus antibody. Canine sera reactive with antigens of one human adenovirus type in radioimmunoassays were also reactive with antigens of the other two types. Dogs bearing malignant lymphoma or squamous cell carcinoma tumours had higher levels of antibody against adenovirus type 5 antigens. Human adenovirus type 5 was neutralised by sera from four tumour-bearing and two normal dogs, while sera from two normal and five tumour-bearing dogs were positive in immunodiffusion tests with human adenovirus antigens. Levels of ICH antibody in sera of normal adult dogs and adult dogs with tumours were not markedly different when measured by radioimmunoassays. Likewise, sera from these two groups of dogs had similar ranges of ICH neutralising antibody titres. In contrast, levels of ICH antibody detected by the serological assays in sera from non-pet, non-vaccinated pups were either markedly low or absent. Possible explanations for the observed increased levels of human adenovirus antibody in sera of tumour-bearing canine pets are discussed.     

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.     

Counterimmunoelectrophoresis (immunoelectroosmosis) and serum electrophoretic pattern in serologic diagnosis of canine blastomycosis

Counterimmunoelectrophoresis (CIEP) with blastomyces and histoplasma antigens was used in a serologic study of 181 dogs clinically suspected of having blastomycosis and of 8 dogs with confirmed blastomycosis or histoplasmosis. Thirteen of the 181 dogs, positive by CIEP, were euthanatized, and the diagnosis was confirmed by cultivation and/or microscopic detection of Blastomyces dermatitidis. Additional CIEP-positive dogs were confirmed by staining of aspirates collected in vivo. Radiographic support for the diagnosis was reported in 4 other dogs in which histoplasmosis was excluded by a negative CIEP with histoplasma antigen. The precipitating antibody may disappear during the course of the disease, as it did in 1 dog treated with amphotericin B, but not cured. This dog reverted from CIEP-positive to CIEP-negative within 17 months of treatment (with a weak reaction after 10 months of treatment). The CIEP-detectable antibody was present only in 1 dog without a confirmation by histopathologic findings or cultivation among 24 well-documented cases and 181 total tested sera. The CIEP was more sensitive and specific than was the gel-diffusion precipitin test, eliminated the problems of anticomplementarity that often affected the results of complement-fixation tests with canine sera, and served well in detecting dogs with blastomycosis. Electrophoretic pattern of sera from CIEP-positive dogs with blastomycosis showed a decrease in albumin and an increase in alpha 2- and often in beta- and gamma-globulins, with a substantial decrease of the albumin/globulin ratio.     

A model to simulate the effect of vaccination against Boophilus ticks on cattle

In this study, serum antibodies to Sarcoptes scabiei var. canis (SS), Dermatophagoides farinae (DF), and D. pteronyssinus (DP) were determined in 19 healthy, random-source dogs prior to infestation with scabies then again during a primary infestation, cure and challenge infestation with scabies. Prior to scabies infestation, serum of 11 dogs contained faintly detectable amounts of IgE and/or IgG to proteins in SS extract, probably resulting from sensitization to dust mites that share cross-reactive antigenic epitopes with SS. After becoming infested with scabies, the response to SS antigens became stronger with antibodies appearing to more antigens as the scabies infestation progressed. Three of the newly recognized proteins were 170, 155 and 142/133 kD and could be used in a diagnostic test since antibodies to them appeared during the primary infestation.

In addition, during the primary infestation, 14 of 15 dogs developed IgE to 1–11 new SS proteins in addition to an increase in IgE binding to those proteins recognized prior to infestation. Overall, the strongest antibody responses (IgE and IgG) were exhibited during cure of the first infestation, when dead mites were still present in the stratum corneum. As expected, the antibody response was strong and rapid during challenge when the infestation self-cured. The immunogenic SS proteins identified by serum antibody binding during challenge, when the hosts self-cured, are candidates for inclusion in a vaccine. These candidate proteins are 200, 185, 170, 155, 142/133, 112, 97, 74, 57, 45/42, 32 and 22 kD.

Some of the proteins in SS that exhibited new or increased antibody binding during the experiment also had IgE and IgG binding to proteins with similar molecular weights in DF and DP extracts. These results illustrate the difficulties involved in understanding and interpreting serum antibody for developing a serological test for the diagnosis of scabies, isolating relevant SS antigens that could be included in a vaccine for prevention of scabies, and for understanding the immune response mechanism to scabies.     

Detection of antiplatelet immunoglobulin in thrombocytopenic dogs

Indirect enzyme-linked immunosorbent assays (ELISA) were used to detect platelet-associated immunoglobulin in sera from dogs with immune-mediated thrombocytopenia (IMT) and/or other autoimmune syndromes. One ELISA, utilizing whole platelets as the antigen substrate, readily detected antibody associated with platelets, either as specific, antiplatelet antibody or as immune complexes. This assay apparently lacked specificity because of the position reactions with sera from dogs with miscellaneous autoimmune disorders and no concurrent thrombocytopenia. Although the second ELISA, utilizing immunoaffinity purified platelet antigens was not influenced as much by immune complexes, absorbance values apparently were slightly increased. However, a small number of dogs with non-thrombocytopenic autoimmune disease tested positive. Immunoadsorption and Western immuno-blotting techniques demonstrated a complex pattern of specificities for antiplatelet antibodies. Clinical significance of these findings is discussed.     

Serum antibody to Sarcoptes scabiei and house dust mite prior to and during infestation with S. scabiei

In this study, serum antibodies to Sarcoptes scabiei var. canis (SS), Dermatophagoides farinae (DF), and D. pteronyssinus (DP) were determined in 19 healthy, random-source dogs prior to infestation with scabies then again during a primary infestation, cure and challenge infestation with scabies. Prior to scabies infestation, serum of 11 dogs contained faintly detectable amounts of IgE and/or IgG to proteins in SS extract, probably resulting from sensitization to dust mites that share cross-reactive antigenic epitopes with SS. After becoming infested with scabies, the response to SS antigens became stronger with antibodies appearing to more antigens as the scabies infestation progressed. Three of the newly recognized proteins were 170, 155 and 142/133 kD and could be used in a diagnostic test since antibodies to them appeared during the primary infestation.In addition, during the primary infestation, 14 of 15 dogs developed IgE to 1–11 new SS proteins in addition to an increase in IgE binding to those proteins recognized prior to infestation. Overall, the strongest antibody responses (IgE and IgG) were exhibited during cure of the first infestation, when dead mites were still present in the stratum corneum. As expected, the antibody response was strong and rapid during challenge when the infestation self-cured. The immunogenic SS proteins identified by serum antibody binding during challenge, when the hosts self-cured, are candidates for inclusion in a vaccine. These candidate proteins are 200, 185, 170, 155, 142/133, 112, 97, 74, 57, 45/42, 32 and 22 kD.Some of the proteins in SS that exhibited new or increased antibody binding during the experiment also had IgE and IgG binding to proteins with similar molecular weights in DF and DP extracts. These results illustrate the difficulties involved in understanding and interpreting serum antibody for developing a serological test for the diagnosis of scabies, isolating relevant SS antigens that could be included in a vaccine for prevention of scabies, and for understanding the immune response mechanism to scabies.     

Identification of two highly performing Leishmania infantum antigens for serodiagnosis of canine leishmaniosis

In the aim of improving serodiagnosis of canine leishmaniosis, we analysed the humoral immune response of dog against Leishmania infantum parasite. The antigenic reaction of L. infantum polypeptides with sera from 31 dogs with parasitologically confirmed leishmaniosis was studied by using the immunoblot technique. Electrophoretic profile of the parasite extract showed more than 50 polypeptides, with molecular weights ranging from 12 to 170 kDa. Among these polypeptides, 37 antigen components, ranging from 14 to 91 kDa, were recognised by antibodies of L. infantum infected dogs. Three polypeptides (14, 16 and 76 kDa) reacted with all of the 31 serum samples. The other most frequently recognised antigens were those of 29.5, 32, 46, 59 and 66 kDa with a sensitivity of 87.1%, 93.6%, 96.8%, 87.1% and 80.6%, respectively. The 14 and 16 kDa bands were the most intense and remained detectable until a serum dilution of 1:6400. No reaction of these two major antigens was observed with sera collected from 50 Leishmania-free dogs, living in the leishmaniosis-free region of Rabat in Morocco, whereas the crude antigen used in IFAT or ELISA lead to three false positive results. Four antigen components of 29, 41, 55, and 70 kDa were recognised by some sera samples from negative controls. These results demonstrated the potential interest of the fractions of 14 and 16 kDa in immunodiagnosis of canine leishmaniosis.     

Use of Echinococcus granulosus worm antigens for immunodiagnosis of E. granulosus infection in dogs.

Echinococcus granulosus worm excretory/secretory antigens (WES) were used in ELISA for diagnosis of E. granulosus infection in dogs and compared with protoscolex somatic antigens (PSM). Sera from 224 dogs were tested. There was no correlation between ELISA absorbance values and E. granulosus worm burdens using either antigen. There was a significant linear relationship between absorbance values of sera tested in the ELISA using WES (W-ELISA) and the ELISA using PSM (P-ELISA). However, there was a small but significant difference between the absorbance values of the sera tested against the two antigens. Western blot analysis of WES using sera from E. granulosus-infected and uninfected dogs revealed antigenic components of relative molecular mass (Mr) larger than 94,000, Mr 94,000-68,000 and Mr 43,000-39,000 in worms, and these were specific for E. granulosus and not identified in PSM; these antigenic differences may be responsible for differences in reactivity in ELISA. The sensitivities of W-ELISA and P-ELISA were 80.8% and 75.6%, respectively. The specificities of W-ELISA and P-ELISA were 93.7% and 97.9%, respectively. The reduced specificity in W-ELISA was mainly attributable to increased background reactivity of sera from Taenia hydatigena-infected dogs. Despite the reduction in specificity, both ELISAs are valuable epidemiological tools to determine the prevalence of antibody to E. granulosus in dog populations and to monitor the success of hydatid control campaigns.     

Differential extraction of antigens of Anaplasma marginale

Strain-, group-, and genus-specific antigens of the Florida isolate of Anaplasma marginale had different solubilities in zwitterionic, nonionic, and anionic detergents. On the basis of their solubility in nonionic detergent, antigens were grouped into 3 classes: (i) a 108-kilodalton (kD) group-specific antigen and a series of poorly defined antigens (70 to 100 kD) were completely soluble in nonionic detergent; (ii) 96 kD and 75 kD group-specific antigens, and a 91-kD strain-specific antigen were completely insoluble in nonionic detergent; and (iii) a 47-kD group-specific antigen and a 41-kD genus-specific antigen were partially soluble in nonionic detergent. A mercaptoethanol-sensitive antigen of 200 kD also was present in the insoluble fraction. Differences in solubility of taxonomic antigens of Anaplasma may reflect differences in function. Antigens totally insoluble in nonionic detergent were possibly integral membrane proteins, and those completely soluble in nonionic detergent were more likely peripheral membrane proteins that were less tightly bound to hydrophobic components of the membrane. Seemingly, all antigens were associated with membranes. Bovine preimmune and immune sera used did not react with any proteins of noninfected RBC.     

Development of ELISAs based on recombinant antigens for the detection of Toxoplasma gondii-specific antibodies in sheep and cats.

ELISAs using recombinant parasite polypeptides as antigens were developed to measure Toxoplasma gondii-specific antibodies in the sera of sheep and cats. Compared with an ELISA based on traditional parasite antigen, the ELISA for sheep sera had a sensitivity of 79% and a negative predictive value of 80%, and the ELISA for cat sera had a sensitivity of 100% and a negative predictive value of 100%. Both ELISAs had specificities of 100% and positive predictive values of 100%. These ELISAs appear to be a useful cost-effective alternative to ELISAs based on traditional parasite antigen for the measurement of T. gondii-specific antibodies in the sera of sheep and cats.     

Hematological findings and antibody responses in Syrian hamster (Mesocricetus auratus) infected with Babesia microti

Hematological findings during the course of infection and the antibody response in Syrian hamsters infected with Babesia microti were examined. A macrocytic hypochromic anemia with an increase of the reticulocyte count was detected as a rise in the parasitized erythrocyte rate. White blood cell counts also remarkedly increased with the increases of both neutrophils and active-shaped monocytes, and thus they particularly play an important role in eliminating the parasite. In Western blotting with the sera from the hamsters infected with B. microti, a 38 kDa protozoan antigen reacted to the early-term sera, and additionally 28, 32, and 34 kDa antigens also reacted to the medium- and latter-term, and convalescent sera. These antigens were immunodominant and the antibodies against these antigens had also important roles for inhibition of this parasite.     

Antibody response against endogenous stages of an attenuated strain of Eimeria tenella

The application of attenuated vaccines for the prevention of chicken coccidiosis has increased exponentially in recent years. In Eimeria infections, protective immunity is thought to rely on a strong cell mediated response with antibodies supposedly playing a minor role. However, under certain conditions antibodies seem to be significant in protection. Furthermore, antibodies could be useful for monitoring natural exposure of flocks to Eimeria spp. and for monitoring the infectivity of live vaccines. Our objective was to investigate the chicken antibody response to the different parasite life cycle stages following infection with an attenuated strain of Eimeria tenella. Western blotting analysis of parasite antigens prepared from the lining of caeca infected with the attenuated strain of E. tenella revealed two dominant antigens of 32 and 34 kDa, apparently associated with trophozoites and merozoites that were present at high concentrations between 84 and 132 h post-infection. When cryosections of caeca infected with E. tenella were probed with IgY purified from immune birds the most intense reaction was observed with the asexual stages. Western blotting analysis of proteins of purified sporozoites and third generation merozoites and absorption of stage-specific antibodies from sera suggested that a large proportion of antigens is shared by the two stages. The time-courses of the antibody response to sporozoite and merozoite antigens were similar but varied depending on the inoculation regime and the degree of oocyst recirculation.     

A comparison of serologic tests for the detection of serum antibodies to whole-cell and recombinant Borrelia burgdorferi antigens in cattle

Serum samples from healthy dairy and beef cattle, living in tick-infested areas of Connecticut, USA, were analyzed by polyvalent enzyme-linked immunosorbent assays (ELISA), indirect fluorescent antibody (IFA) staining methods, or Western blot procedures to detect antibodies to tick-borne agents. Of the 80 sera tested by ELISA with whole-cell or 10 separate recombinant antigens (fusion proteins) of Borrelia burgdorferi sensu stricto, 57 (71%) were positive to 1 or more antigens, while 36 (45%) reacted to whole-cell antigens by IFA staining methods. Three (4%) of 80 samples had antibodies to Anaplasma phagocytophilum. There were antibodies to outer surface protein (Osp) A, OspB, OspC, OspE, OspF, protein (p) 41-G, p35, p37, and VlsE antigens of B. burgdorferi, but there was no reactivity to the p39 antigen by ELISA. Western immunoblots of a subset of 9 sera verified antibody presence in all samples and showed distinct reactivities to multiple proteins having molecular masses of about 31 kilodaltons (kDa), 34 kDa, 35 kDa, 41 kDa, and 83/93 kDa. High specificity (97%) was noted when 16 cattle sera containing antibodies to Leptospira interrogans serovars, Brucella sp., Anaplasma marginale, or A. phagocytophilum were tested by ELISA with separate whole-cell or recombinant B. burgdorferi antigens. An ELISA and Western blot analyses can be used to confirm the exposure of cattle to B. burgdorferi.     

Local and systemic immune responses to Echinococcus granulosus in experimentally infected dogs

Local and systemic immune responses were studied in six dogs experimentally infected with the dog/sheep tapeworm Echinococcus granulosus. All dogs developed similar IgG antibody response to parasite antigens. In contrast, IgE and IgA responses differed widely. No relationship between IgA responses and parasite burden at the end of the infection were observed. Further, clear differences in the anti-parasite IgA response in serum as compared with specific IgA forming cells in mesenteric lymph nodes were observed within the same dog. An inverse association of anti-parasite IgE and parasite load seemed to be present, with the strongest IgE response in the one dog that had no worms in the intestine at the end of the experiment. No differences were observed in the numbers of intestinal mast cells and goblet cells among all infected dogs. However, the dog with no detectable parasite load had a marked reduction of detected mast cells in the submuscular and muscular layer of the mucosa. Our data give new insight into the immune response of dogs during E. granulosus infection and provide information that may be useful for the rational design of vaccines for the control of hydatid disease.     

Detection of anti-Babesia gibsoni heat shock protein 70 antibody and anti-canine heat shock protein 70 antibody in sera from Babesia gibsoni-infected dogs

Antibodies that recognized either Babesia gibsoni or canine red blood cell (RBC) 70-kilodalton (kDa) protein were detected in serum from acutely and chronically B. gibsoni-infected. In those sera, antibodies that reacted with recombinant B. gibsoni and canine heat shock protein 70 (rBgHsp70 and rcHsp70) were detected; therefore, B. gibsoni and canine RBC 70-kDa proteins seemed to be BgHsp70 and cHsp70, respectively. In infected dogs, the amounts of these antibodies increased after infection. Interestingly, polyclonal antibody raised against rBgHsp70 in two rabbits reacted not only with rBgHsp70 but also with rcHsp70 and native cHsp70 from canine RBCs. Because BgHsp70 showed high homology with cHsp70 (70.8%), anti-rBgHsp70 antibody might cross-react with cHsp70. Additionally, the localizations of both BgHsp70 and cHsp70 were observed by indirect fluorescence assay. As a result, cHsp70 was not found on the membrane surface of erythrocytes, suggesting that erythrocytes would not be targets of anti-cHsp70 antibody. Meanwhile, only exoerythrocytic parasites were stained by anti-rBgHsp70 antibody. This result showed that BgHsp70 would be expressed on the surface of parasites during the exoerythrocytic stage. These results indicated that BgHsp70 was a highly immunogenic protein in canine B. gibsoni infection, and that exoerythrocytic parasites might be targets of anti-BgHsp70 antibody.     

The immune response and PBMC subsets in canine visceral leishmaniasis before, and after, chemotherapy

Peripheral blood mononuclear cell subsets, in vitro lymphoproliferative response to leishmanial antigen, and Leishmania-specific serum antibody levels were examined in 11 dogs, naturally infected with L. infantum, and 9 healthy control dogs. A decrease in the percentage of CD4+ T-cells and an increase in the proportion of gammadelta T-cells and sIgG+ B-cells were observed during canine visceral leishmaniasis (CVL). These changes may be responsible for the marked humoral response and the absence of in vitro lymphoproliferation to mitogen and specific parasite antigens. This possibility was supported by the analysis of these subsets after treatment with amphotericin B. One month after therapy, a significant increase in the percentage of CD4+ T-cells and a decrease of gammadelta T-cells and sIgG+ B-cells were observed. At the same time, the lymphocyte blastogenesis assay with leishmanial antigen was positive and the levels of specific antibodies to Leishmania were significantly lower than before the treatment. Five months after therapy, lymphocyte proliferative response to LSA disappeared, antibody and lymphocyte subsets levels returned to those observed during CVL. Therapeutic failure in CVL is associated with the inability of antileishmanial drugs to completely revert the profound immunodepression induced by the infection and prevent relapse.     

Serological evaluation of experimentally infected dogs by LicTXNPx-ELISA and amastigote-flow cytometry

Canine visceral leishmaniasis (CVL) is characterized by a high incidence of asymptomatic infections. Because of the high prevalence of asymptomatic dogs in the endemic areas of visceral leishmaniasis (VL), a sensitive test is required for an accurate diagnosis. In this study, we evaluated the detection of symptomatic and asymptomatic Leishmania infantum infection in dogs using the secreted LicTXNPx antigen (Leishmania infantum cytosolic tryparedoxin peroxidase) in an ELISA format and compared it to soluble Leishmania antigens from promastigote or amastigote forms (SPLA and SALA) and two other unrelated secreted Leishmania proteins (LiTXN1 and TDR1). Moreover, we evaluated the diagnostic potential using the promastigote or amastigote-flow cytometric methodologies. The assays utilized sera collected from a cohort of L. infantum experimentally infected dogs, in which the intravenous or intradermal parasite injection mimics a symptomatic or asymptomatic pattern of infection, respectively. Our study indicated that anti-LicTXNPx antibodies were present in both symptomatic and asymptomatic experimental infections. Among the different Leishmania recombinant proteins tested, LicTXNPx showed a good predictive correlation with total soluble promastigote or amastigote Leishmania antigens, suggesting this antigen as a good candidate for a marker in either symptomatic or asymptomatic infection. The use of flow cytometry using both forms of live parasites was also tested with the same group of dogs. Amastigotes were shown to have more advantages than promastigotes for the serological diagnostic in both symptomatic and asymptomatic dogs, since higher continuous levels of anti-amastigote antibodies were detected during the course of experimental infection. Moreover, additional studies were done using sera from non-infected dogs and clinically asymptomatic and symptomatic dogs with confirmed naturally occurring L. infantum infections. The sensitivities of amastigote and promastigote flow cytometry were 96% vs. 89%, respectively, while the specificity for both was 93.2%. Therefore, our findings showed for the first time the potential of amastigote-flow cytometry regarding their applicability to detect both symptomatic and asymptomatic VL canine infections.     

Detection of antibody to Toxocara vitulorum perieneteric fluid antigens (Pe) in the colostrum and serum of buffalo calves and cows by Western blotting

Toxocara vitulorum, a nematode parasite in the small intestine of cattle and water buffaloes, causes high morbidity and mortality of 1-3 months old buffalo calves. This research evaluated the specific perieneteric antigens (Pe) reactivity of anti-T. vitulorum-Pe antibody (Tv-Pe-Ab) in both immune sera and colostrum from buffalo cows immediately post-partum from buffalo cows. The presence of Tv-Pe-Ab in sera of buffalo newborn calves was also examined at 1 day before and after suckling the colostrum as well as in sera from naturally infected calves at the beginning and peak of the maximum infection and then again during the period of rejection and post-rejection of the parasite. Pe antigens were characterized for Tv-Pe-Ab by SDS-PAGE and Western blot (WB). The SDS-PAGE showed that Pe contained nine protein bands (11, 14, 31, 38, 58, 76, 88, 112 and 165 kDa). All Pe bands were recognized by Tv-Pe-Ab in sera and colostrum of buffalo cows. Only the serum antibodies of buffalo calves at 1 day of age after suckling the colostrum and during the beginning of T. vitulorum infection recognized Pe antigen's nine bands. In contrast, serum antibodies from 1-day-old buffalo calves, taken before suckling colostrum, did not react with any protein band. In suckling calves, which reached peak egg output, rejection and post-rejection stages of the infection, serum Tv-Pe-Ab reactivity with lower molecular weight protein bands (11-76 kDa) was lost and only reactivity with the Pe protein bands of higher molecular weight (88, 112 and 165 kDa) remained.