Reactivity of purified and axenic amastigotes as a source of antigens to be used in serodiagnosis of canine visceral leishmaniasis

Although there is a great diversity of techniques and antigens used in the serodiagnosis of canine visceral leishmaniasis (CVL), total sensitivity and specificity have not yet been found. Since the use of amastigote forms in the indirect immunofluorescence assay has shown an improvement in the specificity of the test for the diagnosis of CVL, the performance of amastigotes forms of L. (L.) infantum chagasi as antigen source were evaluated in automatized ELISA test using crude antigen of axenic amastigote and purified amastigote from spleen of hamster chronically infected comparing with ELISA using total antigen produced with promastigote forms of L. (L.) infantum chagasi. One hundred and fifteen sera from dogs with positive parasitological diagnosis by PCR were used. The animals were classified into 2 groups: symptomatic (n = 67) and asymptomatic (n = 48) animals, in accordance with the clinical signs and laboratory tests were. As control, ninety-four sera from dogs with negative parasitological diagnosis were included. No significant difference was found in sensitivity, specificity, predictive values and accuracy between ELISA using whole antigens produced with both axenic and purified amastigotes in comparison with promastigotes forms. Correlation and concordance between the three total antigens tested in ELISA was observed. According to the similar performance among antigens, data pointed out to use antigen from promastigote forms for diagnosing canine leishmaniasis, especially due the easily in the production, lower cost and the abundance of correlative literature.     

Gentamicin-Attenuated Leishmania infantum Vaccine: Protection of Dogs against Canine Visceral Leishmaniosis in Endemic Area of Southeast of Iran

An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. A vaccine trial was conducted using 103 naive dogs from a leishmaniosis non-endemic area (55 vaccinated and 48 unvaccinated) brought into an endemic area of southeast Iran. No local and/or general indications of disease were observed in the vaccinated dogs immediately after vaccination. The efficacy of the vaccine was evaluated after 24 months (4 sandfly transmission seasons) by serological, parasitological analyses and clinical examination. In western blot analysis of antibodies to L. infantum antigens, sera from 10 out of 31 (32.2%) unvaccinated dogs, but none of the sera from vaccinated dogs which were seropositive at >100, recognized the 21 kDa antigen of L. infantum wild-type (WT). Nine out of 31 (29%) unvaccinated dogs, but none of vaccinated dogs, were positive for the presence of Leishmania DNA. One out of 46 (2.2%) vaccinated dogs and 9 out of 31 (29%) unvaccinated dogs developed clinical signs of disease. These results suggest that gentamicin-attenuated L. infantum induced a significant and strong protective effect against canine visceral leishmaniosis in the endemic area.     

Uncinaria stenocephala: assessment of antigens for the immunodiagnosis of canine uncinariosis

Although Uncinaria stenocephala is the most frequent hookworm in the intestine of dogs from Northern, Central and Southern Europe, little is known about its host-parasite relationship. Three groups of sera from dogs (Group 1: dogs naturally infected only by U. stenocephala; Group 2: helminth-free dogs at necropsy, and Group 3: dogs parasitized by other helminths) were analyzed by ELISA using U. stenocephala antigens from adult worms (somatic and excretory-secretory antigens) and from L3 larvae (somatic antigens). All three sources of antigens were found to be suitable for immunodiagnosis of canine uncinariosis with up to 90% efficacy. However, an analysis to assess the diagnostic value of the different antigens demonstrated that the adult excretory-secretory antigens had a higher diagnostic efficacy (96.7%), indicating that this is the best antigen source for the diagnosis of Uncinaria infection.     

Characterization of the immune response to Leishmania infantum recombinant antigens

Leishmaniases have a high prevalence in tropical countries. In order to improve existing diagnostic systems based on total Leishmania proteins, and to identify antigen candidates for vaccine development, an intensive search for the identification of antigens was performed using molecular biology techniques. In this study, the immune response to three L. infantum recombinant antigens was evaluated. Upon stimulation with KMP11, mononuclear cells from leishmaniasis patients produced high levels of IL-10, while a predominant IFN-gamma production could be observed in cultures stimulated with H2A and soluble Leishmania antigen. All the recombinant antigens induced very little IL-5. KMP11 decreased IFN-gamma production by 48% in cultures of peripheral blood mononuclear cells from cutaneous leishmaniasis patients who had been stimulated with soluble Leishmania antigen. Furthermore, antibodies to KMP11 were detected in the sera from all patients with visceral leishmaniasis and in the majority of the sera from patients with cutaneous leishmaniasis or individuals with asymptomatic L. chagasi infection. Thus, KMP11 is recognized by cells and sera of patients with different clinical forms of leishmaniasis, and KMP11, through IL-10 production, proved to be a potent antigen in modulating type 1 immune response.     

Isolation of Leishmania infantum, zymodeme MON-1 from canine and human visceral leishmaniasis on Margarita Island, Venezuela.

An increase in the incidence of human visceral leishmaniasis (HVL) has been detected in recent years on Margarita Island, located off the NE coast of Venezuela. Recent studies have revealed reactivity to rK39 antigen (Leishmania chagasi) in 20% of 541 sera from domestic dogs in endemic communities; PCR reactions were positive using primers for the L. donovani complex. Here we report that isolates from human and canine infection, identified by isoenzyme analysis, correspond to L. infantum, zymodeme MON-1. This appears to be the first isolation and identification of an isolate from HVL on Margarita Island and demonstrates the presence of this zymodeme in the canine population.     

T cell response of asymptomatic Leishmania chagasi infected subjects to recombinant leishmania antigens.

In areas of Leishmania chagasi transmission the ability to control leishmania infection is associated with IFN-gamma production. In visceral leishmaniasis down-regulation of T cell responses is mediated by interleukin-10 (IL-10). In this study we evaluated the lymphoproliferative response, IFN-gamma and IL-10 production on lymphocyte cultures stimulated with recombinant leishmania antigens in subjects with asymptomatic L. chagasi infection. There was a statistically significant difference in the lymphoproliferative response of the subjects with asymptomatic infection as compared to patients with visceral leishmaniasis and healthy subjects with respect to crude antigens (p<0.01), gp-63 (p<0.05) and hsp-70 (p<0. 01), as well as between asymptomatic L. chagasi infected subjects and patients with visceral leishmaniasis with respect to the response to all antigens tested. The IFN-gamma production observed in the group with asymptomatic infection with all the three recombinant antigens tested was higher (p<0.01) than that observed in patients with visceral leishmaniasis and in healthy subjects. Furthermore, in individuals with asymptomatic infection, IL-10 levels in cultures stimulated with recombinant antigens were very low. This study shows that lymphocytes from individuals with asymptomatic L. chagasi infection are able to recognize recombinant leishmania antigens with production of a cytokine that is associated with leishmania killing.     

Brugia malayi: antibody responses to larval antigens in infected and immunized jirds.

Vaccination with irradiated third stage Brugia malayi larvae (L3) has been reported to induce partial protective immunity to L3 challenge in jirds. The purpose of this study was to identify antigens that may be targets of protective immunity in this model. Jirds were immunized by s.c. injection of irradiated L3 and challenged either s.c. or i.p. Necropsy was performed 11 wk after challenge. Partial protection was achieved in s.c. challenged animals; worm recovery was only 41% of that observed in unvaccinated controls, and worms recovered from immunized animals were stunted. Worm recoveries in immunized animals that were challenged i.p. did not differ from those of unimmunized controls. Group differences in parasite antigen levels in sera collected 2-11 wk after larval challenge were consistent with parasitological findings obtained at necropsy. Antibody studies compared prechallenge sera from immunized animals to sera from infected (unimmunized) controls. Antibody responses to L3 surface antigens (assessed by IFA) were much stronger after immunization than after infection. Immunoblot studies showed preferential recognition of several L3 antigens (97, 54, 48, and 40 kDa) by antibodies in sera from immunized animals. Additional studies are needed to determine whether immunization with such preferentially recognized antigens can induce protection to larval challenge comparable to or better than that observed with live vaccines.     

BCG expressing LCR1 of Leishmania chagasi induces protective immunity in susceptible mice

Cellular immune responses are required for protective immunity against Leishmania chagasi. Immunization strategies using live intracellular bacteria (e.g., bacille-Calmette Guerin strain of Mycobacterium bovis) expressing recombinant antigens can induce cellular immune responses to these antigens. Previous studies demonstrated that the L. chagasi antigen LCR1 stimulates IFN-gamma production from T cells of infected BALB/c mice, and immunization with recombinant LCR1 partially protects against L. chagasi infection. To determine whether live bacteria could enhance the immunization potential of LCR1, we engineered BCG expressing LCR1 (BCG-LCR1). Subcutaneous immunization with BCG-LCR1, but not with BCG containing plasmid only (BCG-pMV261), elicited better protective immunity against L. chagasi infection than LCR1 protein alone. BCG-LCR1 administered intraperitoneally did not protect. Splenocytes from mice immunized s.c. with either BCG-LCR1 or BCG-pMV261 and then infected with L. chagasi promastigotes had increased antigen-induced IFN-gamma and reduced IL-10 production compared to splenocytes of control mice. We propose that BCG-LCR1 promotes a Th1-type protective immune response, and it may be a useful component of a Leishmania vaccine.     

Monoclonal antibodies to parasite antigens found in the serum of Dirofilaria immitis-infected dogs

We recently reported that parasite antigens are detectable in the serum of Dirofilaria immitis-infected dogs by counterimmunoelectrophoresis (CIE). Hybridoma cell lines that produce monoclonal antibodies specific for these antigens were obtained by immunizing mice with a partially purified antigen preparation, fusing spleen cells with SP-2 myeloma cells, and screening cell culture supernatants for antibody by ELISA and CIE inhibition. Antibodies specific for two epitopes shared by the two major circulating parasite antigens were identified. Immunoperoxidase studies showed that the epitopes recognized by the monoclonals were widely distributed in D. immitis, but the female uterus and eggs were particularly strongly labeled. A monoclonal antibody-based ELISA was developed to measure parasite antigens in dog sera. Parasite antigens were detected in 45 of 46 sera from infected dogs but were absent in sera from uninfected dogs and sera from dogs infected with Dipetalonema reconditum. Serum antigen content was significantly correlated with the number of female worms recovered from infected dogs (r = 0.82, p less than 0.001). Antigenemia was first detected 6 mo after infection, and antigen levels remained fairly stable between 9 and 21 mo after infection. Parasite antigen detection with this monoclonal antibody-based ELISA appears to be superior to previously described diagnostic methods for canine dirofilariasis in terms of sensitivity, specificity, and relation to infection intensity.     

Immunity of dogs against Babesia canis, its vector tick Dermacentor reticulatus, and Ixodes ricinus in endemic area

Previous epidemiological studies allowed us to accurately define endemic areas of canine babesiosis and tick distribution in southeastern France (Martinod, 1983). Using a micro-ELISA test 100 dogs sera were tested with 3 antigens: Babesia canis, Dermacentor reticulatus and Ixodes ricinus. Antibodies against B. canis and its vector D. reticulatus were detected in an endemic area, sometimes with high levels (optical density 1.38 and 0.80 respectively). A correlation factor and regression lines were found between ELISA activity of B. canis and vector tick antigens, even for dogs which never showed any babesiosis symptoms. These results were compared with those of an area without any babesiosis. Furthermore I. ricinus antigens detected ELISA activity in sera of dogs; some cross reactions were observed between I. ricinus and D. reticulatus antigen.     

Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs

The sensitivities of spleen and lymph node cultures for the diagnosis of canine visceral leishmaniasis were compared in 64 anti-Leishmania antibody positive dogs from an endemic area in Brazil. The sensitivity of spleen cultures for Leishmania detection was 97.9%; in lymph node cultures it was 25%. Positive spleen culture was more frequent (p = 0.048, Fisher's exact probability test) in symptomatic (28 out of 33 animals) than in asymptomatic animals (19 out of 31 animals). These results support the use of spleen instead of lymph node aspiration as the choice method for the parasitological diagnosis of the infection.     

The increased presence of repetitive motifs in the KDDR-plus recombinant protein,a kinesin-derived antigen from Leishmania infantum,improves the diagnostic performance of serological tests for human and canine visceral leishmaniasis

Visceral leishmaniasis (VL) is caused by protozoa belonging to the Leishmania donovani complex and is considered the most serious and fatal form among the different types of leishmaniasis, if not early diagnosed and treated. Among the measures of disease control stand out the management of infected dogs and the early diagnosis and appropriate treatment of human cases. Several antigens have been characterized for use in the VL diagnosis, among them are the recombinant kinesin-derived antigens from L. infantum, as rK39 and rKDDR. The main difference between these antigens is the size of the non-repetitive kinesin region and the number of repetitions of the 39 amino acid degenerate motif (6.5 and 8.5 repeats in rK39 and rKDDR, respectively). This repetitive region has a high antigenicity score. To evaluate the effect of increasing the number of repeats on diagnostic performance, we designed the rKDDR-plus antigen, containing 15.3 repeats of the 39 amino acid degenerate motif, besides the absence of the non-repetitive portion from L. infantum kinesin. Its performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and rapid immunochromatographic test (ICT), and compared with the kinesin-derived antigens (rKDDR and rK39). In ELISA with human sera, all recombinant antigens had a sensitivity of 98%, whereas the specificity for rKDDR-plus, rKDDR and rK39 was 100%, 96% and 71%, respectively. When evaluated canine sera, the ELISA sensitivity was 97% for all antigens, and the specificity for rKDDR-plus, rKDDR and rK39 was 98%, 91% and 83%, respectively. Evaluation of the ICT/rKDDR-plus, using human sera, showed greater diagnostic sensitivity (90%) and specificity (100%), when compared to the IT LEISH (79% and 98%, respectively), which is based on the rK39 antigen. These results suggest that the increased presence of repetitive motifs in the rKDDR-plus protein improves the diagnostic performance of serological tests by increasing the specificity and accuracy of the diagnosis.     

Effect of vaccination with a recombinant fusion protein encoding an astacinlike metalloprotease (MTP-1) secreted by host-stimulated Ancylostoma caninum third-stage infective larvae

Laboratory dogs were vaccinated intramuscularly with a recombinant fusion protein (expressed and isolated from Escherichia coli) formulated with the Glaxo SmithKline Adjuvant System 02 (AS02). The fusion protein encoded Ac-MTP-1, a developmentally regulated astacinlike metalloprotease secreted by host-stimulated Ancylostoma caninum third-stage larvae (L3). Control dogs were injected intramuscularly with an equivalent amount of AS02 adjuvant alone. The vaccinated and control dogs were then challenged by s.c. injection of 500 L3 of the canine hookworm A. caninum. The vaccinated dogs developed prechallenge immunoglobulin G2 (IgG2) antibody responses specific to anti-Ac-MTP-1-fusion protein with titers ranging between 1:40,000 and 1:364,000, whereas they developed antigen-specific immunoglobulin E antibody responses with titers ranging between 1:500 and 1:1,500. By immunoblotting, canine sera obtained from the vaccinated dogs recognized a protein of the estimated apparent molecular weight of Ac-MTP-1 in activated L3 secretory products. Spearman rank order correlations between the canine intestinal adult hookworm burden and quantitative egg counts at necropsy and anti-Ac-MTP-1 IgG2 antibody titers revealed a statistically significant inverse association (r = -0.89; P = 0.04), suggesting that this molecule offers promise as a recombinant vaccine.     

Use of recombinant antigens of Borrelia burgdorferi and Anaplasma phagocytophilum in enzyme-linked immunosorbent assays to detect antibodies in white-tailed deer

Serum samples obtained from white-tailed deer (Odocoileus virginianus) in Connecticut (n=218) and South Carolina (n=20) (USA) during the period 1992-2002 were analyzed for antibodies to whole-cell or recombinant antigens (i.e., fusion proteins) of Borrelia burgdorferi sensu stricto and Anaplasma phagocytophilum, etiologic agents of Lyme borreliosis and granulocytic ehrlichiosis, respectively. In enzyme-linked immunosorbent assays (ELISAs) with whole-cell B. burgdorferi, the overall seropositivity rate for Connecticut (53%) exceeded that for South Carolina (30%). In separate tests of seven recombinant antigens of B. burgdorferi by an ELISA, seroprevalence for the VlsE antigen was highest (48%) in Connecticut followed by outer surface protein (OspF) (21%), whereas serum reactivities to the protein (p) 41-G antigen (55%) and VlsE (25%) were most frequent for South Carolina sera. In analyses for antibodies to the recombinant protein (p) 44 antigen of A. phagocytophilum, seroprevalences of 52% and 25% were recorded for Connecticut and South Carolina samples, respectively. These findings paralleled those determined by indirect fluorescent antibody staining methods with whole cells (43% and 30%). Moreover, there was good agreement (74%) in results of Western blot analyses and an ELISA when a subset of 39 sera was screened with whole-cell or recombinant p44 antigens of A. phagocytophilum. An ELISA with highly specific recombinant VlsE or p44 antigens can be used in conjunction with other antibody tests to determine whether deer living in different regions of eastern United States were exposed to B. burgdorferi or A. phagocytophilum.     

Serological diagnosis of brucellosis caused by Brucella canis

Blood serum samples from 2,328 dogs were tested to detect antibodies against Brucella canis with the agar gel immunodiffusion (AGID) and 2-mercaptoethanol slide agglutination test (ME-SAT) using Brucella ovis as the antigen. All blood serum samples were also evaluated for antibodies against Brucella abortus and Brucella melitensis using the Rose Bengal test. Twentyfive (1.07%) of the sera evaluated were considered positive with AGID test. Only 4 (16%) of these blood serum samples were positive when evaluated with ME-SAT. The 25 AGID positive samples and 25 AGID negative serum samples were also examined by: the complement fixation test (CFT) using B. ovis hot saline extract (HSE) as the antigen, indirect enzyme linked immunosorbent assay (ELISA) and immunoblotting (IB) using B. canis and B. ovis HSE antigens. Two positive canine sera from culture positive dogs and the serum of an experimentally RM6/66 B. canis-infected rabbit were employed as positive controls and one serum from a known uninfected dog as a negative control. ELISA with B. canis antigen gave 9 (18%) positive results (6 AGID-positive and 3 AGID-negative sera). ELISA performed with B. ovis antigen detected 15 (30%) positive samples (10 AGID-positive, 5 AGID-negative and 8 B. canis ELISA positive sera). IB analysis of known positive controls sera employing B. canis antigen detected bands with molecular weights of 94-80, 64-50, 35, 32-30, 28, 23, 20-18, 15-12 kDa. The same sera tested with B. ovis antigen revealed bands of 35, 32-30, 25, 23, 20-18, 15-12 kDa. No bands were observed with the negative control serum and the 50 canine tested sera.     

Leishmania infection in humans, dogs and sandflies in a visceral leishmaniasis endemic area in Maranhão, Brazil

Leishmania infection in humans, dogs and sandflies was examined in the endemic visceral leishmaniasis (VL) municipality of Raposa, state of Maranh?o, Brazil. In this study, we examined Leishmania chagasi infection in the blood serum of both humans and Canis familiaris and the natural Leishmania sp. infection rate in the sandfly vector, Lutzomyia longipalpis. Enzyme-linked immunosorbent assay, indirect immunofluorescence reaction and polymerase chain reaction were performed to detect Leishmania infections in humans, dogs and sandflies, respectively. Overall, 186 out of 986 studied human beings were infected with L. chagasi parasites, representing an infection prevalence of 18.9%. An even higher infection rate was detected in dogs, where 66 (47.8%) out of 138 were infected. Among all Lu. longipalpis captured (n = 1,881), only 26.7% were females. The Leishmania infection frequency for the vector Lu. longipalpis was 1.56%. Remarkably, all infected sandflies were found in the peridomiciliary area. Furthermore, a high incidence of asymptomatic forms of VL in the human and canine populations was observed. The results of this study suggest autochthonous transmission of L. chagasi in this endemic area for visceral leishmaniasis because infection by Leishmania sp. was identified in all important elements of the transmission chain.     

A Comparison of Two Serological Methods for Detecting the Immune Response after Rabies Vaccination in Dogs and Cats being Exported to Rabies-free Areas

Levels of rabies virus neutralizing antibody in sera from dogs and cats were titrated to endpoint by the Rapid Fluorescent Focus Inhibition Test (RFFIT) and retested by the RFFIT and the Fluorescent Antibody Virus Neutralization test (FAVN). The two tests were compared for their ability to detect the 0.5 international units/ml (I.U.) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. No difference was observed in sensitivity or specificity for either method in tests of 168 sera from unvaccinated animals or 70 sera from vaccinated animals with high levels of neutralizing antibody (an initial RFFIT titre of > or = 1.0 I.U.). Test to test variation occurred for results obtained by both RFFIT and FAVN for 95 sera from vaccinated animals with low to moderate levels of neutralizing antibody (RFFIT titre < 1.0 I.U.). No significant differences were detected for the 95 sera in the frequency for one methodology more often than the other to have a positive response (> or = 0.5 I.U.), nor were significant differences detected for the symmetry (P = 0.43) or the marginal homogeneity (P = 0.39) of results obtained by the two methods. Both methods can adequately identity unvaccinated animals, but false positive and false negative results are possible for either method when a single test is used to measure the antibody response of low-responding vaccinated animals. Nucleotide sequence analysis identified several amino acid differences in stocks of the challenge rabies virus from different laboratories. The small differences in neutralizing antibody titre that may result from mutations in the challenge virus are not important for evaluating immunity induced by vaccines which are themselves prepared from a variety of different rabies virus strains, but differences in the challenge virus, rather than differences in methodology, may account for at least some of the discrepant results reported in inter-laboratory surveys. Comparative studies of serological methods for measuring rabies antibodies should use well-characterized unpassaged virus stocks obtained from a single reference laboratory.     

Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96.8%), whereas 85 sera were positive with DAT (sensitivity=91.4%) and 89 sera were positive with whole cell ELISA (sensitivity=95.7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89.2%) and whole cell ELISA was negative in 114 cases (specificity=87.7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.     

A method for studies of an El Tor-associated antigen of Vibrio cholerae O1

A method for studying the biotype El Tor associated mannose-sensitive haemagglutinin (MSHA) of V. cholerae O1 has been developed. By using crude MSHA adsorbed to chicken erythrocytes as solid phase antigen in an enzyme-linked immunosorbent assay (ELISA), antisera against V. cholerae of the El Tor biotype reacted in high titre with the MSHA-coated cells, whereas antisera against vibrios of the classical biotype did not bind significantly, i.e. in higher titre than pre-immune sera. The binding of anti-MSHA serum, or a monoclonal antibody against MSHA, to the MSHA-coated erythrocytes could be efficiently inhibited by crude MSHA as well as by El Tor vibrios whereas neither V. cholerae lipopolysaccharide nor different strains of classical vibrios had any inhibitory effect. These results support the existence of an El Tor-associated immunogen. They also suggest a possibility of determining antibodies against different haemagglutinins in ELISA without having access to purified antigens.