Clinical, clinicopathologic, and parasitologic observations of trypanosomiasis in dogs infected with North American Trypanosoma cruzi isolates

Nineteen purebred Beagles of various ages (4, 5, 13, and 47 weeks) were inoculated with North American Trypanosoma cruzi isolates obtained from an opossum (Tc-O), an armadillo (Tc-A), or a dog (Tc-D). Dogs were grouped on the basis of clinical outcome of infection. During the acute stage of disease, dogs of group 1 (n = 7 inoculated with Tc-O or Tc-A) died or were euthanatized because of the severity of disease. Dogs of group 2 (n = 5 inoculated with Tc-O or Tc-A) developed acute disease, but survived to develop chronic disease. Dogs of group 3 (n = 7 Tc-D-inoculated dogs) developed neither acute nor chronic disease. Dogs of group 4 (n = 4--2 dogs 13 weeks old and 2 dogs 47 weeks old) served as noninoculated controls. Clinical signs associated with severe acute myocarditis developed in dogs of groups 1 and 2 between postinoculation day (PID) 15 and 28. Generalized lymphadenopathy and lymphocytosis were observed in all dogs of groups 1, 2, and 3 between PID 14 and 17. Serum alanine transaminase and aspartate transaminase activities and urea nitrogen concentration were high, and glucose concentration was low prior to death of dogs in group 1. Serum activities of isoenzymes of creatine kinase were significantly (P less than 0.05) high in only 1 dog (group 1), whereas serum lactate dehydrogenase isoenzyme activities were not significantly high in any dog.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immune response and antigen recognition in non-pregnant ewes experimentally infected with Neospora caninum tachyzoites

The cellular and humoral responses as well as the antigen recognition during the acute stage of a Neospora caninum (NC) infection were investigated in non-pregnant ewes. The experimentally infected ewes developed specific lymphoproliferative and humoral responses within 2 weeks post-infection (PI). The magnitude of the cellular response showed large variations between animals. A significant decrease in the proliferative response to Con A mitogen and N. caninum, Toxoplasma gondii (TG) antigens was recorded on day 21 post-infection (PI). The humoral response and the pattern of antigen recognition were similar among infected ewes. Proteins of 44, 42, 40, 39 and 28 kDa were intensively recognized by the infected animals during the experiment. The 42 and 28 kDa antigens should be considered as useful for the diagnostic of N. caninum infection, as the intensity of recognition infection of the other antigens had decreased markedly 8 weeks post-infection. For some antigens a sequential recognition was recorded. The 59, 54 and 38-37 kDa proteins were frequently recognized by infected sera during the first weeks of the infection, but recognition of these antigens was absent or rare at the end of the experiment. These antigens could be related to the acute stage of the infection.     

Comparison of virologic and serologic responses of lambs and calves infected with bluetongue virus serotype 10

Four lambs and 3 calves, seronegative to bluetongue virus (BTV), were inoculated intravenously with a highly plaque-purified strain of BTV Serotype 10. A single calf and lamb served as controls and were inoculated with uninfected cell culture lysate. All BTV-inoculated lambs exhibited mild clinical manifestations of bluetongue, whereas infected calves were asymptomatic. Viremia persisted in BTV-infected lambs for 35-42 days, and for 42-56 days in BTV-infected calves. Neutralizing antibodies were first detected in sera collected at Day 14 post-inoculation (PI) from 2 BTV-infected calves and all 4 infected lambs, and at Day 28 PI in the remaining calf. The appearance of neutralizing antibody in serum did not coincide with clearance of virus from blood; BTV and specific neutralizing antibody coexisted in peripheral blood of infected lambs and calves for as long as 28 days. The sequential development, specificity and intensity of virus protein-specific humoral immune responses of lambs and calves were evaluated by immunoprecipitation of [35S]-labelled proteins in BTV-infected cell lysates by sera collected from inoculated animals at bi-weekly intervals PI. Sera from infected lambs and calves reacted most consistently with BTV structural proteins VP2 and VP7, and nonstructural protein NS2, and less consistently with structural protein VP5, and nonstructural protein NS1. Lambs developed humoral immune responses to individual BTV proteins more rapidly than calves, and one calf had especially weak virus protein-specific humoral immune responses; viremia persisted longer in this calf than any other animal in the study. The clearance of virus from the peripheral blood of BTV-infected lambs and calves is not caused simply by the production of virus-specific neutralizing antibody, however the intensity of humoral immune responses to individual BTV proteins might influence the duration of viremia in different animals.     

Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta,Argentina

Some Leishmania species affect humans in two principal forms: visceral and cutaneous leishmaniosis (CL). Several studies have identified dogs as the main reservoirs of the visceral leishmaniosis (VL) caused by Leishmania infantum. The purpose of this work was to carry out a survey of the canine population associated with human cases of American tegumentary leishmaniosis (ATL), in order to establish the clinical, parasitological, serological and immunological characteristics of the canine disease, in an endemic region for both ATL and Chagas' disease in the province of Salta, in northwestern Argentina. Two hundred and eight dogs from the endemic area were examined and 41 (19.7%) of them presented lesions compatible with leishmaniosis. In order to investigate the presence of antibodies against Leishmania spp. and Trypanosoma cruzi, sera were screened by ELISA using two complex antigens from these parasites and, because of cross-reactions between them, a specific antigen for diagnosis of T. cruzi infection. Sixty-two (29.8%) of 208 dogs were positive for the complex antigen F45 from Leishmania and 50 (24%) were positive for the complex antigen F105 from T. cruzi. Nine dogs (4.3%) were positive for the specific Ag163B6-cruzipain suggesting that these dogs were truly infected with T. cruzi. Furthermore, three of these nine dogs presented Leishmania sp. in their skin lesions and therefore were considered as infected by both, T. cruzi and Leishmania parasites. The prevalence of Leishmania infection detected by lesions and/or positive serology was 27.4% (57/208). On the basis of previous observations regarding the clustered appearance of human ATL, the dog population was divided into two groups: zone A, dogs living within a 100 m radius from houses with human cases, and zone B, dogs living beyond this limit. The prevalence of ATL in dogs was significantly higher in zone A (34.6%) than in zone B (7.3%), suggesting a strong correlation between canine and human cases. The average time required for a parasitological diagnosis by microscopy was six times longer for dog samples than human ones, and the average number of parasites per 100 microscopic fields was 14-fold lower in canine samples. The high prevalence of Leishmania infection and the close association with human cases, demonstrated that dogs are a very susceptible host for Leishmania infection, but the scarcity of parasites in their lesions suggests that they may not be the main reservoir of the parasite in this endemic area.     

IgG isotype profile is correlated with cardiomegaly in Beagle dogs infected with distinct Trypanosoma cruzi strains

A systematic study following infection by various strains of the protozoan parasite, Trypanosoma cruzi, and the simultaneous monitoring of the humoral immune response together with the elicited cellular response, could add greatly to our understanding of differences between strains of this important human pathogen. In that sense, acute and chronic infections with distinct T. cruzi strains (Y, Berenice-78 and ABC) in Beagle dogs were studied through a longitudinal evaluation of immunoglobulin G (IgG), IgG1 and IgG2 isotypes (by ELISA and flow cytometry (FC)), as well as measurements of peripheral blood mononuclear cell (PBMC) proliferation over a 100-week period, and their correlation with cardiomegaly. Our results show that infected animals presenting cardiomegaly showed lower or absent levels of IgG1 during the chronic phase of the infection, when compared to those that did not show an increase in heart weight. In that manner, our results suggest that IgG1 could be used as a marker for cardiac pathogenicity in Chagas disease.     

Prevalence of American trypanosomiasis (Chagas disease) among dogs in Oklahoma

OBJECTIVE: To determine the prevalence of Trypanosoma cruzi infection among dogs in Oklahoma. DESIGN: Cross-sectional study. ANIMALS: 301 owned or impounded dogs related by ownership or general geographic location to 3 dogs determined to have trypanosomiasis. PROCEDURES: Blood samples were obtained from dogs between November 1996 and September 1997. Infection status was determined by use of a radioimmunoprecipitation assay. Second blood samples were obtained from some of the seropositive dogs for study by hemoculture and polymerase chain reaction (PCR) assay. Sites where infected dogs were found were inspected for triatomine insects, and light traps were used for vector trapping. RESULTS: 11(3.6%) dogs were seropositive for T. cruzi infection. Ten of the 11 were owned rural hunting dogs. Protozoal organisms isolated from the blood of 1 seropositive dog were identified as T. cruzi by PCR testing. Only 1 adult Triatoma sanguisuga was captured in a light trap at a site near infected dogs; this insect was not infected. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings suggest that T. cruzi is enzootic in eastern Oklahoma. Measures that would reduce the risk of dogs acquiring T. cruzi infection are unlikely to be acceptable to their owners, and no effective drugs are available for treatment. The presence of T. cruzi-infected dogs poses a threat of transmission to persons at risk of exposure to contaminated blood Veterinarians who practice in the southern United States should be cognizant of this blood borne zoonosis and educate all personnel about appropriate precautions.     

The effect of immunosuppression on protective immunity of turkey poults against infection with turkey coronavirus

The objective of the present study was to evaluate the protective effect of humoral and cellular immunities on turkeys infected with turkey coronavirus (TCV). Two trials were conducted with two separate hatches of turkey poults. Turkey's were experimentally immunosuppressed with cyclosporin A (CsA) or cyclophosphamide (CY) and infected with TCV. Prior to infection, treatment with CsA selectively suppressed T cell activity as revealed by 2-3 fold decreased (p < 0.1) lymphocyte proliferation responses to a T cell mitogen, concanavalin A (Con A). Treatment with CY mainly induced B cell deficiency as indicated by significant reductions (p < 0.05) in antibody responses to sheep erythrocytes 7 days after injection. Body weight gain of turkeys treated with CY was significantly lower (p < 0.05) than that of untreated turkeys at 9 days post-infection (PI). Turkeys treated with CY had 1-2 fold higher immunofluorescent antibody assay (IFA) scores for TCV antigens (p < 0.05) in the intestine than untreated turkeys at 9 or 14 days PI. These results suggested that humoral immunity against TCV infection may be important in turkeys.     

Serological responses of dogs to cell wall and internal antigens of Brucella canis (B. canis)

The serological responses of dogs to cell wall and internal antigens of B. canis were studied in experimentally infected specific-pathogen-free (SPF) Beagles. Sera from infected and false positive field dogs also were examined. Cell wall antigens were extracted from B. canis by two procedures that employed either hot phosphate buffered saline (PBS) or sodium desoxycholate (SDC). Agar gel immunodiffusion (AGID) tests employing sera from experimentally infected SPF dogs were used to evaluate antigenic extracts. Extraction with PBS yielded two antigens; SDC extracted an antigen complex and sonication of PBS extracted cells liberated four internal antigens.Sera from field dogs that were negative for B. canis infection in repeated tests often had heterospecific antibodies. Such cross-reactive sere commonly gave “spur” (partial fusion) reactions with a positive reference serum when tested against the SDC cell wall antigen. In addition, false positive dogs did not have antibody to one of the cell wall antigens or to the internal antigens. In contrast, sera from infected field dogs commonly gave “identity” (fusion) reactions in the AGID test with two antigens in the SDC extract, and produced precipitin lines to one to four internal antigens.Examination of a library of sera obtained from experimentally infected SPF dogs over a period spanning 4$\text{1}\text{2}$ years revealed that none of the serodiagnostic tests employed (tube agglutination, slide agglutination, AGID) was accurate during the inital 12 weeks of infection; hemocultures were the most sensitive during this period. Tube and slide agglutination tests were initially sensitive, but they showed a lack of sensitivity and specificity after the bacteremic period ceased, as well as in their failure to exclude false positive reactions in field animals. Immunodiffusion tests that employed SDC or PBS extracts of B. canis cell walls were sensitive and accurate in identifying most infected dogs. After the bacteremia had ceased, however, AGID tests that employed cell wall antigens gave equivocal results. Immunodiffusion tests that employed sonicated (internal) antigens were sensitive shortly after the onset of bacteremia, and they had the advantage of detecting infected animals for at least 6 months following the cessation of bacteremia, a time when other serological tests gave equivocal results.     

Specific antibody responses to Taenia hydatigena, Taenia pisiformis and Echinococcus granulosus infection in dogs

Groups of dogs reared free of both nematodes and cestodes were infected with Taenia hydatigena, Taenia pisiformis or Echinococcus granulosus. After infections with the Taenia spp became patent, dogs were purged to remove the worms. They were later reinfected and the second infections again removed by purging after patency. A group of 3 uninfected worm free dogs was kept as age-matched controls. The dogs were bled at intervals of 5 days and their serums tested for antibodies using the enzyme-linked immunosorbent assay (ELISA) with excretory/secretory (ES) antigens collected during in vitro incubation of evaginated scoleces (scolex ES antigen) and oncosphere antigens. Antibodies to scolex ES antigen were detected by 3 weeks after infection with each cestode species whereas antibodies to oncosphere antigen were not detected until about one week after eggs were found in the faeces of the infected dogs. Antibody responses to both oncosphere and scolex ES antigens decreased rapidly following removal of the worms by purging. Uninfected control dogs were invariably negative to both oncospheral and scolex ES antigens. There were cross-reactions between the serums from dogs infected with T. pisiformis and T. hydatigena when tested with scolex ES antigens, but oncospheral antigens showed a high degree of species specificity. Scolex ES antigens from E. granulosus were compared with those prepared from T. hydatigena and T. pisiformis for their ability to discriminate between antibodies in serums collected from dogs 31 and 32 days after infection with 100,000 protoscoleces of E. granulosus or dogs infected with Taenia spp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Frequency of IFNγ-producing T cells correlates with seroreactivity and activated T cells during canine Trypanosoma cruzi infection

Vaccines to prevent Trypanosoma cruzi infection in humans or animals are not available, and in many settings, dogs are an important source of domestic infection for the insect vector. Identification of infected canines is crucial for evaluating peridomestic transmission dynamics and parasite control strategies. As immune control of T. cruzi infection is dependent on humoral and cell-mediated immune responses, we aimed to define a serodiagnostic assay and T cell phenotypic markers for identifying infected dogs and studying the canine T. cruzi-specific immune response. Plasma samples and peripheral blood mononuclear cells (PBMCs) were obtained from forty-two dogs living in a T. cruzi-endemic region. Twenty dogs were known to be seropositive and nine seronegative by conventional serologic tests two years prior to our study. To determine canine seroreactivity, we tested sera or plasma samples in a multiplex bead array against eleven recombinant T. cruzi proteins. Ninety-four percent (17/18) of dogs positive by multiplex serology were initially positive by conventional serology. The frequency of IFNγ-producing cells in PBMCs responding to T. cruzi correlated to serological status, identifying 95% of multiplex seropositive dogs. Intracellular staining identified CD4⁺ and CD8⁺ T cell populations as the sources of T. cruzi lysate-induced IFNγ. Low expression of CCR7 and CD62L on CD4⁺ and CD8⁺ T cells suggested a predominance of effector/effector memory T cells in seropositive canines. These results are the first, to our knowledge, to correlate T. cruzi-specific antibody responses with T cell responses in naturally infected dogs and validate these methods for identifying dogs exposed to T. cruzi.     

Electrocardiographic and echocardiographic features of trypanosomiasis in dogs inoculated with North American Trypanosoma cruzi isolates.

Purebred Beagles were inoculated with Trypanosoma cruzi isolates from a North American opossum or armadillo (Tc-W), and dog (Tc-D). Although Tc-D established infection in dogs, the dogs did not develop cardiac abnormalities. Dogs inoculated with Tc-W developed acute myocarditis associated with increases in P-R interval, atrioventricular block, depression of R wave amplitude and shifts in mean electrical axis. Echocardiograms were normal during this stage. Three Tc-W-inoculated dogs died during the acute stage. Following the acute stage, 5 of 8 Tc-W-inoculated dogs entered an indeterminate stage in which ECG changes were minor and echocardiograms were normal. Progression to the chronic stage in 5 of the 8 Tc-W-inoculated dogs was indicated by development of ventricular-based arrhythmias, mainly ventricular premature contractions, between postinoculation days 60 and 170. In some dogs, ventricular premature contractions were multifocal. Electrocardiographic abnormalities progressively degenerated to various forms of ventricular tachycardia. Worsening ECG coincided with loss of left ventricular function as measured by echocardiography. Mean percent ejection fraction and percentage of fractional shortening decreased to 63% and 52% of control values, respectively. The left ventricular free wall (LVFW) thickness decreased and % septal: % LVFW thickening ratio increased, indicating a relative preservation of septal wall motion and LVFW hypokinesis.     

Dehydroepiandrosterone increases resistance to experimental infection by Trypanosoma cruzi

Dehydroepiandrosterone (DHEA) enhances immune responses against a wide range of viral, bacterial, and parasitic pathogens. In a previous study, we reported that administration of DHEA significantly decreased the numbers of blood parasites in Trypanosoma cruzi experimental infection. The present study was undertaken to determine the effectiveness of DHEA in reducing the severity of acute phase T. cruzi infection of male and female Wistar rats. Animals were treated subcutaneously with 40 mg/kg body weight/day of DHEA. The concentration of nitric oxide (NO) was determined in spleen peritoneal cavity. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) were determined in the sera of uninfected and infected animals. DHEA treatment augments NO production for both sexes after in vitro LPS treatment for uninfected animals. Infection triggered enhanced NO levels although not significant. IL-2 and IFN-gamma were detectable in higher concentrations in treated and infected rats of both genders when compared to untreated controls. These data suggest that DHEA may have a potent immunoregulatory function that can affect the course of T. cruzi infection.     

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

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

Lymphocyte subsets and specific IgG antibody levels in clindamycin-treated and untreated dogs experimentally infected with Babesia gibsoni

This study was carried out to clarify the role of lymphocyte subpopulations and Babesia-specific antibody on the treatment of clindamycin in dogs infected with B. gibsoni. Ten beagle dogs were divided into two groups: an untreated group (5 dogs) and a clindamycin-treated group (5 dogs), which was administered clindamycin at 25 mg/ kg body weight, per os, q 12 hr from 7 days to 21 days post-infection (PI). On the acute stage of infection, clindamycin treatment resolved anaemia and other clinical findings. There were no significant differences between treated and untreated dogs either in parasitemia levels or Babesial IgG antibody levels. However, morphological changes that indicated degeneration in the majority of parasites were observed. The numbers of CD4(+) showed a significant increase in treated dogs, especially after treatment. On the chronic stage, CD4(+) cells maintained high level both of the treated and untreated dogs. Although parasitemia maintained low level, their relapses were occurred on the 49th day PI in treated dogs and on the 42nd and 63rd PI in untreated dogs. A rapid humoral antibody response was observed in treated dogs, however, lower humoral antibody responses in untreated dogs after relapses. The antibody levels of treated dogs were significantly higher than those of untreated dogs. These results suggested that clindamycin might not eliminate rapidly parasites from peripheral blood, but damage parasites, which might stimulate efficiently humoral and cellular immunity against Babesia infection, and result in an improvement of clinical conditions.     

SPF雏鸡感染REV后免疫器官免疫功能的动态变化

试验通过对 1日龄 SPF雏鸡人工感染网状内皮组织增殖病病毒 ( REV) ,观察了免疫器官胸腺、法氏囊和脾脏 T、B淋巴细胞增殖反应的动态变化。结果表明 ,雏鸡感染 REV后胸腺 T淋巴细胞的增殖反应于7、2 1、4 2和 4 9日龄明显降低 ( P<0 .0 5) ,于感染后 1 4、2 8和 3 5日龄极显著减弱 ( P<0 .0 1 ) ;脾脏 T淋巴细胞的增殖反应分别于感染后 1 4～ 2 8d和 3 5～ 4 9d较对照组雏鸡明显或极明显降低 ( P<0 .0 5,P<0 .0 1 )。法氏囊和脾脏 B淋巴细胞增殖反应分别在感染后 7～ 4 9d和 1 4～ 4 9d极显著减弱 ( P<0 .0 1 )。免疫器官胸腺重量和脾脏重量与体重比值及体重分别于感染后 7～ 4 9、7～ 3 5、7～ 4 9d明显降低 ( P<0 .0 5,P<0 .0 1 )。说明SPF雏鸡感染 REV后 ,免疫器官 (胸腺、法氏囊和脾脏 )淋巴细胞发生变性坏死、数量减少和功能降低 ,机体的细胞免疫和体液免疫功能均发生明显降低或抑制     

The immunologic response of dogs to Bartonella vinsonii subspecies berkhoffii antigens: as assessed by Western immunoblot analysis.

Bartonella vinsonii subspecies berkhoffii is a recently recognized zoonotic pathogen that causes endocarditis, granulomatous rhinitis, and granulomatous lymphadenitis in dogs. Isolation of B. vinsonii (berkhoffii) from blood or tissue samples is frequently unsuccessful; therefore, diagnosis is primarily dependent on serologic or molecular testing modalities. Because previous canine serologic studies have used an indirect immunofluorescence assay (IFA), without Western immunoblot (WI) confirmation, the overall objective of this study was to examine the diagnostic use of WI for confirmation of B. vinsonii (berkhoffii) infection in dogs. To confirm that agar-grown and cell culture-grown organisms yielded similar patterns of WI antigenic protein recognition, the 2 preparations were compared using IFA-reactive sera obtained from dogs experimentally infected with B. vinsonii (berkhoffii). Temporal changes in the pattern of antigenic protein recognition were characterized using sera obtained from dogs at various time points after experimental B. vinsonii (berkhoffii) infection. The specificity of B. vinsonii (berkhoffii) WI was examined by testing canine sera that were reactive to B. henselae, B. clarridgeiae, Ehrlichia canis, Rickettsia rickettsii, Babesia canis, Anaplasma phagocytophilum (previously E. equi), or Brucella canis antigens. Clinical accessions including serum samples obtained from B. vinsonii (berkhoffii) culture-positive dogs and B. vinsonii (berkhoffii) culture-negative dogs that were IFA seroreactive to B. vinsonii (berkhoffii) antigens were examined by WI. The results of this study indicate that WI using agar-grown or cell culture-grown B. vinsonii (berkhoffii) antigens produce identical patterns of antigenic protein recognition. After experimental infection, there is a progressive increase in the number of antigenic proteins that are recognized by WI, with the 33-kD antigen representing the first and the most persistent antigen recognized by B. vinsonii (berkhoffii)-infected dogs. Regarding specificity, sera from dogs that were reactive to various heterologous antigens did not recognize B. vinsonii (berkhoffii) antigens by IFA or WI, and sera from dogs experimentally infected with B. henselae did not recognize B. vinsonii (berkhoffii) antigens by WI. Regarding clinical accessions, there was good agreement between B. vinsonii (berkhoffii) IFA test results and WI analysis. Western immunoblot analysis can be used to detect or confirm exposure to B. vinsonii (berkhoffii) in dogs.     

A preliminary study to evaluate the immune responses induced by immunization of dogs with inactivated Ehrlichia canis organisms

Ehrlichia canis is an intracellular pathogen that causes canine monocytic ehrlichiosis. Although the role of antibody responses cannot be discounted, control of this intracellular pathogen is expected to be by cell mediated immune responses. The immune responses in dogs immunized with inactivated E. canis organisms in combination with Quil A were evaluated. Immunization provoked strong humoral and cellular immune responses, which were demonstrable by Western blotting and lymphocyte proliferation assays. By Western blotting antibodies to several immunodominant E. canis proteins were detected in serum from immunized dogs and antibody titres increased after each immunization. The complement of immunogenic proteins recognized by the antisera were similar to those recognized in serum from infected dogs. Upon challenge with live E. canis, rapid anamnestic humoral responses were detected in the serum of immunized dogs and primary antibody responses were detected in the serum from control dogs. Following immunization, a lymphocyte proliferative response (cellular immunity) was detected in peripheral blood mononuclear cells (PBMNs) of immunized dogs upon stimulation with E. canis antigens. These responses were absent from non-immunized control dogs until after infection with live E. canis, when antigen specific-lymphocyte proliferation responses were also detected in the PBMNs of the control dogs. It can be thus concluded that immunization against canine monocytic ehrlichiosis may be feasible. However, the immunization regimen needs to be optimized and a detailed investigation needs to be done to determine if this regimen can prevent development of acute and chronic disease.     

IgG antibodies from dourine infected horses identify a distinctive Trypanosoma equiperdum antigenic pattern of low molecular weight molecules

Diagnosis and control of dourine is strongly based on serological evidence, but knowledge of the humoral response of horses during infection is limited. In this study we developed a chemiluminescent immunoblotting (cIB) assay to characterise the Trypanosoma equiperdum antigen pattern recognised by IgGs from naturally or experimentally dourine-infected horses and analyse the kinetics of IgG humoral response following the infection. One compounding factor is that sera from uninfected animals often cross-react with T. equiperdum antigens. Development of the cIB assay was based on the hypothesis that serum IgGs from healthy and infected animals recognise different T. equiperdum antigen patterns. We used sera from 8 naturally infected horses which had recovered from Italian outbreaks and 2 experimentally infected mares. In addition, sera from 10 healthy control animals, eight of which were CFT positive but IFA negative for dourine, were collected from disease free regions. Sera were compared by the complement fixation test (CFT), indirect immune fluorescence (IFA) and the cIB assay.cIB analysis revealed that IgGs from infected horses, in contrast to IgGs from healhty horses, specifically recognise a T. equiperdum antigenic profile with low molecular weight bands ranging between 16 and 35 kDa. A time course experiment indicated that IgGs specific for the 16–35 kDa parasite protein fraction appear 17 days post-infection. The cIB assay confirmed all ten infected animals as positive and all controls as negative. This study demonstrated that analysis of IgGs by cIB can provide clear confirmation of trypanosome infection in horses, suggesting that this technique can be applied as a confirmatory serological test for dourine infection.     

Proliferative responses to canine thyroglobulin of peripheral blood mononuclear cells from hypothyroid dogs

The immune responses of hypothyroid dogs to canine thyroglobulin (cTg) were evaluated for the proliferative ability of peripheral blood mononuclear cells (PBMC). PBMC from three hypothyroid dogs with high titers of thyroglobulin autoantibody (TgAA) and 3 clinically normal dogs were cultured with 5, 10, or 20 microg/ml of cTg for 72 hr. The proliferative responses of the cells were determined by the level of incorporated BrdU. The numbers of cells expressing Thy-1, CD4, CD8 and IgG in the PBMC were counted by the immunofluorescence method. Proliferative responses to cTg were observed in the cells from hypothyroid dogs. The number of cells expressing IgG and CD8 in the hypothyroid dogs tended to be high compared with the clinically normal dogs. The CD4+ cells in cultures from hypothyroid dogs increased depending upon the amount of cTg. There was a significant (P<0.05) positive correlation between the number of CD4+ cells and the concentration of cTg in the cultures from hypothyroid dogs. These findings suggest a possible relationship between canine hypothyroidism and cellular immunity. Loss of self tolerance to thyroid antigens in CD4+ T cells may play an important role in the development of canine hypothyroidism.     

Use of two polysaccharide antigens in ELISA for the detection of antibodies to Echinococcus granulosus in sheep sera

Polysaccharide antigens were obtained from either the secretions produced during in vitro cultivation of Echinococcus granulosus protoscoleces or from mouse hydatid cyst membranes by phenol extraction. When either of these antigens was used in an enzyme-linked immunosorbent assay antibody activities were detected in sera from sheep infected 27 or more weeks earlier with at least 100 E granulosus eggs. These antibody responses were significantly higher (P less than 0.05) than those of sheep infected with Taenia hydatigena or T ovis and tested with the E granulosus antigens. Very high cross-reacting antibody responses in sera from sheep recently infected with T hydatigena were only detected with the protoscoleces secretions antigen. Neither antigen was sufficiently sensitive or specific for serodiagnostic use. However, when sera were first tested with one antigen and then with the other, and only sera that were positive in both tests were regarded as positive, the overall sensitivity and specificity of this two antigen method increased to about 80 per cent.