Gentamicin-attenuated Leishmania infantum: cellular immunity production and protection of dogs against experimental canine leishmaniasis

An attenuated line of Leishmania infantum (L. infantum H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. Here, we show that L. infantum H-line induced significantly higher levels of IFN-γ and lower levels of IL-10 compared with those in dogs infected with L. infantum wild type (WT). Anti-Leishmania-specific total IgG, IgG1, and IgG2 antibodies were present in the serum of all infected dogs, with levels of IgG2 subclass highest in the sera of dogs inoculated with L. infantum H-line. Relatively high levels of IgG1 were found in the sera of dogs infected with L. infantum WT. Six of seven dogs immunized intradermally (i.d.) with the attenuated line later showed a positive skin test to leishmanin, whereas the dogs infected with L. infantum WT did not. No clinical abnormalities were observed, and no parasites found in the visceral organs of the dogs inoculated intravenously (i.v.) with L. infantum H-line over 24 months post-inoculation. Dogs which had been immunized with L. infantum H-line i.d. 12 months previously were protected against challenge with L. infantum WT. These data suggest that the L. infantum H-line was safe and induced a protection which is correlated with cellular immunity in dogs.     

IgG antibody responses in mice coinfected with Toxocara canis and other helminths or protozoan parasites

The immune response expressed by IgG antibodies in BALB/c mice experimentally infected with Toxocara canis, was studied with the aim of verifying the possible in vivo cross-reactivity between antigens of T. canis and other parasites (Ascaris suum, Taenia crassiceps, Schistosoma mansoni, Strongyloides venezuelensis and Toxoplasma gondii). Experiments included three groups of mice: one infected only by T. canis, another with one of the other species of parasites and a third concomitantly infected with T. canis and the other species in question. Animals were bled by orbital plexus at 23, 38 and 70 days post infection (p.i.). Sera were analyzed for anti-Toxocara antibodies by ELISA and Immunoblotting, using excretion-secretion antigens (ES), obtained from culture of third-stage larvae of T. canis. For all experiments a control group comprised by ten non-infected mice was used. Only in the case of A. suum infection, in these experimental conditions, the occurrence of cross-reactivity with T. canis was observed. However, in the case of co-infection of T. canis - S. mansoni, T. canis - S. venezuelensis and T. canis - T. crassiceps the production of anti-Toxocara antibodies was found at levels significantly lower than those found in mice infected with T. canis only. Co-infection with S. mansoni or S. venezuelensis showed lower mortality rates compared to what occurred in the animals with single infections. Results obtained in mice infected with T. canis and T. gondii showed significant differences between the mean levels of the optical densities of animals infected with T. canis and concomitantly infected with the protozoan only in the 23rd day p.i.     

Toxocara infestations in humans: symptomatic course of toxocarosis correlates significantly with levels of IgE/anti-IgE immune complexes

Infestations of humans with the parasitic nematode T. canis are common in both developing and industrialized countries. Most infestations induce a clinically inapparent course of infection, however, severe clinical manifestations, i.e. visceral larva migrans (VLM) or ocular larva migrans (OLM) syndromes are observed. To find an explanation for the different courses of toxocarosis we examined several serological parameters: the expression of (i) specific IgE (Immunoblot, IB), (ii) specific IgG subclasses (IgG1–4, ELISA) and the formation of (iii) IgE/anti-IgE immune complexes. Serum samples were obtained from persons with symptomatic (VLM, OLM) and asymptomatic course (AS) of the infestation. As antigen, T. canis excretory/secretory (TES) antigen from L3 larvae was used. Reactivity of IgE against SDS-PAGE separated TES antigens was marginally higher in toxocarosis patients (35%) than in asymptomatics (24%), but without statistical significance. TES-specific IgG (1–4), predominant subclass in all three groups was IgG1, followed by IgG2, IgG4 and IgG3. Subclasses IgG1, 2, 4 showed significant differences between patients with VLM associated symptoms and asymptomatic persons ( P  < 0.001) but not between patients with OLM associated symptoms and asymptomatics. Significantly elevated levels of IgE/anti-IgE immune complexes were detected in sera of patients with symptomatic course of the disease, both VLM and OLM ( P  < 0.001). Whereas specific IgG may act via antibody dependent cell-mediated cytotoxicity mechanisms, IgE/anti-IgE immune complexes might possibly participate in VLM and OLM by inducing type III hypersensitivity.     

Coccidiosis: characterization of antibody responses to infection with Eimeria nieschulzi

Summary The antibody responses of rats to infection with the intestinal intracellular protozoan parasite Eimeria nieschulzi were examined by a sensitive radio-immunoassay with a soluble preparation of sporulated oocysts as antigen. Specific antibodies of the IgM, IgGl, IgG2a and IgG2b isotypes were found in the blood circulation and IgA antibodies were detected in the bile and in intestinal washings. The IgM response was rapid, its peak was relatively brief and it was not recalled by the reinoculation of oocysts. There were some differences between the responses in the different subclasses of IgG but they all reached a peak between 20–30 days after the initiation of the primary infection and there was an anamnestic response to a challenge inoculation of oocysts. IgA antibodies to E. nieschulzi antigen in the bile and in intestinal washings increased and decreased after both primary and secondary inocula. Antibodies of all isotypes tested were virtually absent in the blood circulation of infected athymic rats. These findings are discussed with reference to antibody responses to other parasitic infections and to the role of antibodies in immunity to coccidiosis.     

Use of ELISA employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of canine visceral leishmaniasis

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.     

Seroprevalence and geographic distribution of Dirofilaria immitis and tick-borne infections (Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, and Ehrlichia canis) in dogs from Romania

Tick-borne diseases are of great concern worldwide. Despite this, in Romania there is only limited information regarding the prevalence of vector-borne pathogens in dogs. In all, 1146 serum samples were tested by SNAP(?) 4Dx(?) (IDEXX Laboratories, Inc., Westbrook, ME) for Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies, and for Dirofilaria immitis antigen. The correlation between positive cases and their geographic distribution, as well as potential risk factors (age, sex, breed, type of dog, habitat, and prophylactic treatments) were evaluated. Overall, 129 dogs (11.3%) were serologically-positive to one or more of the tested pathogens. The seroprevalence for the four infectious agents were: A. phagocytophilum 5.5% (63/1146), D. immitis 3.3% (38/1146), E. canis 2.1% (24/1146), and B. burgdorferi 0.5% (6/1146). Co-infection with E. canis and A. phagocytophilum was registered in 2 dogs (0.2%). The geographical distribution of the seropositive cases suggests clustered foci in southern regions and in the western part of the country for D. immitis, and in the southeastern region (Constan?a County) for E. canis. A. phagocytophilum and B. burgdorferi showed a homogenous distribution, with a tendency for Lyme-positive samples to concentrate in central Romania. For D. immitis, A. phagocytophilum, and E. canis, administering prophylactic treatments was a risk factor associated with infection. Another associated risk factor was the type of dog (stray dogs were at risk being positive for D. immitis, shelter dogs for E. canis, and hunting dogs for B. burgdorferi). The prevalence of D. immitis was significantly higher in males and in dogs older than 2 years. This survey represents the first data detailing A. phagocytophilum and E. canis seroprevalence in Romanian dogs, and the most comprehensive epidemiological study on vector-borne infections in dogs from this country.     

Leucine rich repeats are the main epitopes in Leishmania infantum PSA during canine and human visceral leishmaniasis

The PSA protein is one of the major antigens of the surface of the Leishmania infantum parasite membrane. We describe the immune humoral response against the PSA in dogs and human patients with visceral leishmaniasis caused by L. infantum. The immunodominant region of the PSA was determined by subcloning, expression and purification of three fragments covering the complete protein. The analysis revealed that the antibodies are mostly directed against the central region, which is formed exclusively by leucine rich repeats. This region is recognized by 100% of the sera from the infected dogs and 40% of the human sera. These percentages are significantly higher than those observed when the complete protein was used as antigen. The analysis of the isotype of the G immunoglobulins raised against the immunodominant determinants of the PSA indicates that both IgG1 and IgG2 classes are produced during natural infections but that the IgG2 predominates over that of the IgG1.     

Interferon-gamma and the induction of protective lgG2a antibodies in non-lethal Plasmodium berghei infections of mice

Mice treated with anti-IFN-gamma monoclonal antibodies were unable to recover from infection with an attenuated variant of P. berghei (Pb XAT) which causes non-lethal malaria in normal mice. On the other hand, treatment with anti-lL-4 monoclonal antibodies had no effect on the course of infection. IFN-gamma was produced by spleen cells in vitro during the early phase of the infection. Treatment with anti-IFN-gamma suppressed development of an anti-plasmodial IgG2a immunoglobulin isotype in the serum of infected mice whereas anti-IL-4 interfered with the IgGl response. An lgG2a fraction of immune serum collected from mice that had recovered from Pb XA T transferred immunity to naive mice but the IgGl fraction did not. When glutaraldehyde fixed parasitized erythrocytes were incubated with immune serum in suspension, specific IgG2a antibodies were detected by fluorescein staining on the membranes of cells infected with mature stages of parasites. These results indicate that IFN-gamma is a key to inducing B cells to produce the protective anti-plasmodial IgG2a immunoglobulin isotype. Antibody-dependent cell-mediated parasite killing seems to be involved in the mechanism of recovery from infection with Pb XAT.     

Seroepidemiological studies on human pulmonary dirofilariasis in Spain

Preliminary data are presented on a serological survey of the human population of an area with a high prevalence of Dirofilaria immitis infection in dogs. Five per cent of the sera were positive for IgG. Simultaneous radiological study revealed a pulmonary nodule in one of the positive patients, and in this case the evolution of IgG and IgM levels was followed over a period of three months. None of the positive sera reacted with Toxocara canis antigens.     

Specific antibody responses in dogs experimentally infected with Echinococcus granulosus

Six dogs reared helminth-free were divided into 2 groups. Four dogs were infected per os with 200,000 protoscoleces each of Echinococcus granulosus and 2 were kept as uninfected controls. All the dogs were kept together until 32 days after infection, when 1 infected dog was killed, its intestine removed and the contents examined to confirm that the infection with E. granulosus had been successful. The remaining 3 infected dogs were transferred to high security housing and their feces inspected daily to establish the time infections became patent. The infected and control dogs were bled every 5 days for 75 days from the time of infection and the sera were stored at -70 degrees C. Sera were tested by the enzyme-linked immunosorbent assay (ELISA) for antibodies to E. granulosus scolex excretory/secretory (ES) antigen, protoscolex antigen and oncosphere antigen. Antibodies to scolex ES antigen and protoscolex antigen were detected in the sera of infected dogs within 2 weeks of infection. Antibody titers rose rapidly and remained at a high level until the dogs were killed 75 days after infection. Antibodies in these sera did not cross react with antigens prepared from Taenia ovis, T. hydatigena, T. pisiformis, Ancylostoma caninum, Trichuris vulpis and Toxocara canis.     

Association between the IgG subclass response, inflammation and disease status in Helicobacter pylori infection

BACKGROUND: In many viral, bacterial and parasitic infections the Immunoglobulin G (IgG) subclass response has been shown to correlate with severity of inflammation and disease outcome. The aim of the present study was to investigate the association between the IgG subclass response to Helicobacter pylori infection and disease and inflammation. METHODS: Eighty-three symptomatic patients undergoing endoscopic examination were included in the study. Upon endoscopic examination, the presence of ulceration was noted and biopsy specimens were collected from the gastric antrum, body and transitional zone. Blood was also collected from each patient. Gastric biopsy sections were graded using the Sydney system. H. pylori specific IgG, IgG1, IgG2, IgG3 and IgG4 were measured by ELISA. The IgG subclass was also examined retrospectively in sera collected from 20 patients previously proven to have duodenal ulcer (DU). RESULTS: The results of histological examination and IgG serology showed 35 subjects to be H. pylori negative and 48 to be H. pylori positive. Of the 48 H. pylori positive subjects, 25 were diagnosed with functional dyspepsia (FD), 14 with current DU and 9 with evidence of past DU. Significantly higher levels of IgG2 antibodies were found in patients with DU as compared with patients with FD (P < 0.01). In addition, significantly higher IgG3 subclass antibody levels were associated with chronic inflammatory cells in the body (P < 0.05) and active inflammatory cells in the transitional zone (P < 0.01). A significantly increased level of IgG1 antibodies was associated with lower levels of colonization in the gastric antrum. CONCLUSION: The results of this study suggest that the IgG subclass response in subjects infected with H. pylori may be a marker of DU disease as well as increased levels of inflammation.     

Immunological and ultrastructural aspects of the cell-mediated killing of Dirofilaria immitis microfilariae

Summary Neutrophils in the presence of serum from dogs with occult Dirofilaria immitis infections were shown to be cytotoxic to D. immitis microfilariae recovered from the blood of microfilaraemic dogs. This cytotoxicity was correlated with the presence of IgM and IgG antibodies on the cuticular surface of microfilariae incubated in the sera from occult dogs. Such antibodies were not observed on the surface of microfilariae incubated in sera from microfilaraemic or normal dogs. The neutrophil attack was directed at the cuticular crypts, at which sites the worms appeared to be structurally most vulnerable because of the absence of the outer layer of the cuticle. The IgM antibodies were shown to be bound preferentially to these sites. Our data suggested that the neutrophil-mediated toxicity involved both hydrogen peroxide release and degranulation.     

Follow-up of Leishmania infantum naturally infected dogs treated with allopurinol: immunofluorescence antibody test,ELISA and Western blot

Fourteen dogs naturally infected with Leishmania infantum and treated with allopurinol were monitored clinically and serologically with immunofluorescent antibody test (IFAT, amastigotes and promastigotes), enzyme linked-immunosorbent-assay (ELISA, IgG1 and IgG2) and Western blotting (WB). In all dogs therapy lead to clinical improvement together with decreasing specific antibodies in IFAT, ELISA and WB, demonstrating the usefulness of serology for follow-up. Although IgG1 and IgG2 varied considerably between individual animals, IgG2 of all dogs was predominantly in both ELISA and WB. This suggests the value of monitoring the IgG2 response (especially against 29 and 67 kDa antigens) in the follow-up of treated dogs.     

Antibodies to the Plasmodium falciparum rhoptry protein RAP-2/RSP-2 in relation to anaemia in Cameroonian children

Previous studies have implicated reactive antibodies to the low molecular weight rhoptry‐associated proteins (RAP‐1, RAP‐2/RSP‐2 and RAP‐3) in erythroid cell destruction during Plasmodium falciparum infection. In this pilot study, the frequency, specificity and functional capacity of naturally acquired anti‐RAP‐2/RSP‐2 antibodies were investigated in the sera of anaemic and nonanaemic malaria‐infected Cameroonian children. All sera recognized RAP‐2/RSP‐2 by FACS, irrespective of the clinical status of the subjects. However, the anaemic children showed higher levels of IgG antibodies than the nonanaemic group, while both groups showed similar levels of IgM antibodies. Only few individuals had detectable levels of RAP‐2/RSP‐2‐specific IgG1 and IgG3 subclass antibodies, while no IgG2 and IgG4 subclass antibodies were detected in these subjects. By ELISA, the anaemic group tended to show higher levels of antibodies to RAP‐2/RSP‐2 regarding all antibody classes tested, except for IgG4 and IgE. Unexpectedly, sera from the nonanaemic group activated complement to a greater extent than those from the anaemic group. These results need to be confirmed in extended studies but indicate that the effector functions of the RAP‐2/RSP‐2‐reactive antibodies may be more important than their amounts. Such antibodies could play a role in both immunity and pathogenesis during P. falciparum infection.     

Diagnostic values of IgG4 in human gnathostomiasis

The diagnostic values of immunoglobulin G subclass antibodies from patients with gnathostomiasis were assessed by immunoblot technique. Antigen was prepared from crude extracts of Gnathostoma spinigerum advanced third-stage larvae obtained from naturally infected eels. The sera were obtained from 14 parasite-confirmed gnathostomiasis cases, 63 patients with other helminthic infections and 13 healthy controls. Nine prominent IgG4 reactive bands appeared with molecular weights of 94, 51, 47, 43, 38, 24, 21, 20 and 15 kDa. The diagnostic sensitivity of each of the nine reactive bands ranged from 100% to 64.3% in 14 parasite-confirmed gnathostomiasis cases. All (100%) confirmed cases recognized the 21 kDa antigenic band, but not other helminthic infections or parasite-free control. Recognition of 21 kDa antigen in G. spinigerum advanced third-stage larvae crude extracts is the most specific diagnostic marker for human gnathostomiasis, with 100% sensitivity and specificity. The 20 and 24 kDa protein bands were additional diagnostic bands for confirming diagnosis of infection where the 21 kDa band was faint. No specific binding of IgG1, IgG2, or IgG3 antibodies was observed in any sera from confirmed gnathostomiasis cases.     

Recognition of different Toxoplasma antigens by IgM and IgG antibodies in mothers and their congenitally infected newborns

The protein-blotting technique was used to determine the antigens of Toxoplasma gondii that were recognized by IgG and IgM antibodies in sera of congenitally infected newborns and their mothers. Patterns of IgG and IgM blots with sera from newborns revealed antigen-antibody reactions (bands) that were not present in the respective blots obtained with sera from their mothers. This was true for 50% of 24 congenitally infected newborns. In contrast, such a difference was noted in only one (5%) of 21 newborns who were not congenitally infected but whose mothers had serological evidence of acute infection with T. gondii acquired during gestation. Our results suggest that the protein-blotting method or an adaptation may be valuable for study of the immune response of the mother, fetus, and newborn to various antigens of infectious organisms and for diagnosis of congenital infections in the newborn.     

Seroprevalence and risk factors for canine toxocariasis by detection of specific IgG as a marker of infection in dogs from Salvador, Brazil

Toxocara canis is a highly prevalent worldwide canine nematode responsible for enzootic and zoonotic infections. It is considered to be one of the main agents of human visceral and ocular larva migrans. False negative diagnosis may occur because adult infected dogs with "dormant" larvae may have negative fecal test results since they usually do not shed parasite eggs in their stools. During pregnancy, the larvae become active and infect the offspring through the placenta. A serological test can distinguish infected animals, thus increasing the accuracy of the diagnosis for epidemiological studies and prophylactic purposes. In the present work a serological investigation was carried out to study the risk factors for the acquisition of this infection in 301 dogs inhabiting the city of Salvador, northeast Brazil. A validated questionnaire was applied to the donors and caretakers to assess animal management practices. All dogs were submitted to clinical evaluation and blood collection. Serum samples were analyzed for IgG antibodies against excretory-secretory products of T. canis larvae, used as antigens, by indirect ELISA. The overall seroprevalence of anti-T. canis IgG antibodies was 82.7%. Risk factors for T. canis infection included sex, area of origin within the city, homemade leftover food intake, failure to receive regular vaccination against infectious diseases and lack of preventive anti-helminthic treatment. Most of these risk factors suggest a lack of veterinary care and poverty. The high frequency of seropositivity found for toxocariasis in dogs suggests that results based on parasitological fecal examination could underestimate the actual prevalence of the infection.     

SERUM IgG SUBCLASSES IN PATIENTS WITH AN INCREASED SUSCEPTIBILITY TO RESPIRATORY TRACT INFECTIONS

Abstract Serum IgG subclass concentrations were assayed in 45 patients with chronic respiratory tract infections and 16 patients with recurrent acute respiratory tract infections. Eleven of these 61 patients, all but one with recurrent acute infections, were IgA-deficient but the remainder had normal or high serum immunoglobulin concentrations. Only 4% of patients with chronic infections were lgG2-deficient. The prevalence of lgG2 deficiency amongst patients with recurrent acute infections was greater (31%), but in most cases this appeared to be due to an association with IgA deficiency. Owing to the limits of assay sensitivity it was not possible to determine whether any patient was lgG4-deficient, but the number of sera with undetectable lgG4 was greater in patients with recurrent acute infections than in controls (37.5%vs 10%, p<0.01), although such patients were mainly those with IgA and lgG2 deficiency. None of the patients had IgGl or lgG3 deficiency; in fact lgG3 concentrations were higher than those of controls in both groups of patients (ρ<0.001) and IgGl concentrations were higher than those of controls in patients with recurrent infections (ρ<0.01). Thus, unequivocal IgG subclass deficiency is uncommon in non-lgA-deficient patients, but those with lgG2 deficiency may have an immune defect requiring further investigation.     

Detection of circulating parasite antigens in canine dirofilariasis by counterimmunoelectrophoresis

Circulating Dirofilaria immitis antigen was detected in sera from 24 of 24 infected dogs by counterimmunoelectrophoresis (CIE). Parasite antigen was not detected in sera from uninfected dogs or dogs with Dipetalonema reconditum infection. In experimentally infected dogs, the antigen was first detectable 6.5-8.5 months after infection. Preliminary evidence suggests that the antigen is present in male and female adult worms but not in microfilariae. Sera from dogs with microfilaremic and amicrofilaremic infections contained statistically equivalent amounts of D. immitis antigen. However, a significant correlation was observed between serum parasite antigen content and the number of adult worms present in individual dogs at necropsy. Previous studies from several laboratories have shown that microfilarial counts and serum antibody titers are not related to adult worm counts in canine dirofilariasis or other filarial infections. Thus, CIE detection of D. immitis antigenemia represents a significant improvement over previously available diagnostic techniques because it is more sensitive than microfilarial tests, more specific than antibody tests, and the only test that has been related to infection intensity.     

Serum IgG and IgA antibody responses to Campylobacter pylori in a group of healthy asymptomatic volunteers

Sera from 17 healthy asymptomatic volunteers were tested for presence of IgG and IgA antibodies against Campylobacter pylori and correlated with endoscopic biopsy findings. Three volunteers infected with C. pylori had the highest IgG antibody titers of the group. None of 14 C. pylori free subjects had significant IgG antibody levels. IgA antibody titers were negative in all subjects regardless of state of infection, in contrast to control sera from symptomatic C. pylori infected patients who manifested high IgA antibody levels.