Reciprocal Titers Research Articles

Toxoplasma gondii and Chlamydophila abortus in Caprine Abortions in Tobago: a Sero‐Epidemiological Study

A sero-epidemiological study was conducted on a goat farm that experienced an abortion epidemic in the 2005 breeding season in Tobago. Serum samples of goats (aborting and non-aborting) and cats were collected, in addition to the use of stored sera from the farm sampled in 2003 and 2004. Farm records on the reproductive and mortality rates for year 2003, 2004 and 2005 were also reviewed. The sera were screened for Toxoplasma gondii antibodies using the latex agglutination test (LAT), Chlamydophila abortus with an enzyme-linked immunosorbent assay (ELISA) and Brucella abortus using the buffered plate agglutination test (BPAT). Farm records revealed that for the period 2003-2005, the average kid per doe rate decreased from 2.1 to 1.5, the mortality rate increased from 6.3% in 2002 to 19.4% in 2004 and the fertility rate decreased from 98-99% (2002-2004) to 89% (2005). There was a dramatic increase in the abortion rate from <1% (2002, 2003 and 2004) to 29.2% (2005). Of a total of 161 sera tested comprising 12 from 2003, 89 from 2004 and 70 from 2005, 0 (0.0%), 21 (23.6%) and 45 (64.3%) were positive for T. gondii agglutinins (i.e. titres > or =1 : 64) and the differences were statistically significant (P < 0.05; chi(2)). Of all serum samples tested, only 1 (1.1%) of 89 from 2004 was positive for C. abortus while all the sera tested were negative for B. abortus. Amongst the 24 does which aborted in 2005 and were available for testing in mid-2005, 15 (62.5%) had reciprocal titres of > or =1 : 2048, three (12.5%) each had titres of 1 : 1024, 1 : 256 and < or =1 : 16 i.e. negative. The seroprevalence and titres of does that aborted, 20 (87.0%) of 23, all with titres > or =1 : 256 suggesting current infection, were statistically significantly (P < 0.05; chi(2)) higher than was detected amongst does that delivered normal kids, 25 (53.25) of 47 with 22 (48.8%) having titres of > or =1 : 256. One (50.0%) of two cats caught and tested was seropositive with a reciprocal titre of 128. This is considered the first documentation of T. gondii agglutinins in caprine abortion as well the detection of C. abortus antibodies from livestock in Trinidad. It is concluded that of the three zoonotic abortifacient pathogens tested for, T. gondii appeared to have played some aetiological role in the abortion epidemic investigated.

Sero‐epidemiology of Canine Leptospirosis in Trinidad: Serovars, Implications for Vaccination and Public Health

A sero-epidemiological study on canine leptospirosis was conducted in house, stray, farm and hunting dogs, as well as in suspect cases of clinical canine leptospirosis. Serum samples were collected from apparently healthy (vaccinated and non-vaccinated), house dogs. A questionnaire was administered to the owners to elicit information on risk factors for leptospirosis. The microscopic agglutination test was used to screen for leptospirosis using 17 international serovars. Reciprocal titres of between 100 and <800 were considered as evidence of past exposure while reciprocal titres of 800 or greater were classified as suggestive of acute/current infection. Of a total of 419 serum samples tested, 61 (14.6%) were seropositive for Leptospira agglutinins, 23 (5.5%) had mixed infections and 16 (3.8%) had current infection. Amongst 50 suspected cases of clinical leptospirosis, 24 (48.0%) were seropositive and only 13 (26.0%) had current infection compared with 10 (6.3%) and three (1.9%) of 160 apparently healthy house dogs respectively. The difference was statistically significant (P < 0.05; chi2). Twelve (25.5%) of 47 hunting dogs, 10 (20.4%) of 49 farm dogs and five (4.4%) of 113 stray dogs were seropositive (P < 0.05; chi2). Overall, a total of nine serovars were detected with serovars mankarso, icterohaemorrhagiae RGA, autumnalis and copenhageni being involved in 29 (47.5%), 20 (32.8%), 25 (41.0%) and 10 (16.4%) respectively in 61 seropositive dogs (P < 0.05; chi2). Serovar mankarso was most predominant in seropositive apparently healthy dogs, 37.8% (14/37), suspected clinical cases of leptospirosis, 62.5% (15/24) compared with serovar icterohaemorrhagiae with a frequency of 21.6% (8/37) and 50.0% (12/24), the difference being statistically significant (P < 0.05; chi2). Although all vaccines used for prevention of canine leptospirosis in the country contain serovars canicola and icterohaemorrhagiae, serovar mankarso was mostly associated with infection and disease and may be a good candidate for inclusion in the vaccine used locally. The public health risk posed to owners of dogs infected with Leptospira cannot be over-emphasized considering the zoonotic nature of the disease.

Comparison of an indirect immunofluorescence assay, western blot analysis, and a commercially available ELISA for detection of Ehrlichia canis antibodies in canine sera

To examine the correlation between results for an indirect immunofluorescence assay (IFA) that uses Ehrlichia canis antigen as a substrate (ie, E canis-IFA), 2 western blot (WB) analyses, and a commercially available ELISA in the detection of E canis antibody in dog sera. 54 canine serum samples that were reactive on E canis-IFA and 16 canine serum samples that were E canis-IFA nonreactive. Serum samples were evaluated by use of 2 WB analyses and a commercially available ELISA. Correlation between results of the 3 testing modalities (ie, IFA, WB analyses, and the ELISA) was examined by use of nonreactive (E canis-IFA reciprocal titer, < 20), low-titer (reciprocal titer, 80 to 160), medium-titer (reciprocal titer, 320 to 2,560), and high-titer (reciprocal titer, 5,120 to > 20,480) serum samples. For all serum samples in the nonreactive (n = 16), medium-titer (17), and high-titer (18) groups, correlation of results among IFA, WB analyses, and the commercially available ELISA was excellent. A poor correlation was found between IFA results and those of WB analyses and the ELISA for serum samples in the low-titer group (19), with only 4 of the 19 serum samples having positive results on both WB analyses and the commercially available ELISA. The discrepancy between E canis-IFA, WB analyses, and the commercially available ELISA results for the low-titer serum samples may be related to a high IFA sensitivity or, more likely, a lack of specificity associated with cross-reactivity among Ehrlichia spp.

Diagnosis of systemic cryptococcosis by fecal cytology in a dog

A 3-year-old Boxer was presented with progressive diarrhea, vomiting, and lethargy of 5-months duration. The dog had watery black feces, a mature neutrophilia, and microcytic anemia. Cytologic evaluation of a direct fecal smear stained with Wright's-Giemsa revealed numerous encapsulated, narrow-based, budding organisms consistent with Cryptococcus sp. Pyogranulomatous inflammation and Cryptococcus organisms also were observed in ultrasound-guided fine-needle aspirates of the small intestine and mesenteric lymph nodes, and in histologic sections of colonic biopsies obtained by endoscopy. Multifocal chorioretinitis by fundic examination was consistent with systemic mycosis, and the reciprocal antigen titer (1600) on a cryptococcal antigen latex agglutination test for Cryptococcus neoformans was markedly increased. Using immunohistochemistry, the organism was identified further as C neoformans var. grubii (C neoformans var. neoformans serotype A). After 3 weeks of antifungal treatment, ultrasound examination revealed urinary bladder wall thickening, and Cryptococcus organisms were found in a urine sediment preparation. After 4 months of treatment, the dog was clinically normal and had no abnormal findings on CBC, serum biochemistry, urinalysis, or fecal cytology; however, the antigen titer remained unchanged, mesenteric lymphadenomegaly and jejunal wall thickening were still evident, and cytologic evaluation of fine-needles aspirates of the jejunal wall revealed budding Cryptococcus organisms. Intestinal involvement in dogs with cryptococcosis is rare, and diagnosis by fecal cytology has not been documented previously.

Evaluation of clinical characteristics, diagnostic test results, and outcome in horses with internal infection caused by Corynebacterium pseudotuberculosis: 30 cases (1995–2003)

To determine clinical signs, results of diagnostic testing, and outcome in horses with internal Corynebacterium pseudotuberculosis infection. Retrospective study. 30 horses. Information pertaining to clinical data, results of diagnostic tests, and costs of hospitalization and treatment was extracted from medical records of affected horses. Internal C. pseudotuberculosis infection was diagnosed on the basis of clinical signs, diagnostic imaging, and clinicopathologic data, including results of serologic tests and bacterial culture. The most common clinical signs were concurrent external abscesses, anorexia, fever, lethargy, weight loss, and signs of respiratory tract disease or abdominal pain. Clinicopathologic abnormalities included a geometric mean reciprocal serum synergistic hemolysin inhibition titer > or = 512, leukocytosis with neutrophilia, hyperglobulinemia, hyperfibrinogenemia, and anemia. Specific organ involvement was diagnosed in 27 of 30 horses. Affected organs included the liver (18 horses), lungs (12), kidneys (7), and spleen (3); multiple organs were affected in 10 horses. Treatment with antimicrobials for a median of 36 days (range, 7 to 97 days) was usually successful, yielding an overall survival rate of 71%. Early diagnosis and long-term antimicrobial treatment were important for a successful outcome in horses with internal C. pseudotuberculosis infection. Ultrasonographic imaging was an important technique for identifying specific organs affected, aiding in obtaining samples for a definitive diagnosis, and monitoring response to treatment. Pregnant mares with internal infections are at risk for fetal loss. Preexisting chronic organ disease may be associated with a poor prognosis.

Experimental Infection of White-Tailed Deer with Anaplasma phagocytophilum , Etiologic Agent of Human Granulocytic Anaplasmosis

Serologic and molecular evidence of Anaplasma phagocytophilum has been demonstrated in white-tailed deer (WTD; Odocoileus virginianus), and deer are an important host for the tick vector Ixodes scapularis. In this study, we describe experimental infection of WTD with A. phagocytophilum. We inoculated four WTD with a human isolate of A. phagocytophilum propagated in tick cells. Two additional deer served as negative controls. All inoculated deer developed antibodies (titers, > or =64) to A. phagocytophilum, as determined by an indirect fluorescent antibody test, between 14 and 24 days postinfection [p.i.]), and two deer maintained reciprocal titers of > or =64 through the end of the 66-day study. Although morulae were not observed in granulocytes and A. phagocytophilum was not reisolated via tick cell culture of blood, 16S reverse transcriptase nested PCR (RT-nPCR) results indicated that A. phagocytophilum circulated in peripheral blood of three deer through at least 17 days p.i. and was present in two deer at 38 days p.i. Femoral bone marrow from one deer was RT-nPCR positive for A. phagocytophilum at 66 days p.i. There was no indication of clinical disease. These data confirm that WTD are susceptible to infection with a human isolate of A. phagocytophilum and verify that WTD produce detectable antibodies upon exposure to the organism. Because adults are the predominant life stage of I. scapularis found on deer and because adult I. scapularis ticks do not transmit A. phagocytophilum transovarially, it is unlikely that WTD are a significant source of A. phagocytophilum for immature ticks even though deer have a high probability of natural infection. However, the susceptibility and immunologic response of WTD to A. phagocytophilum render them suitable candidates as natural sentinels for this zoonotic tick-borne organism.

Evaluation of the recombinant LipL32 in enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis

The recombinant leptospiral protein LipL32 was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLipL32 IgG ELISA). The microscopic agglutination test (MAT) of 150 serum samples from cattle suspected of leptospirosis showed that 125 (83.3%) samples had positive reciprocal agglutination titres, which ranged from 100 to 1600. The highest titres were observed for the serovars Hardjoprajitno and Bratislava. In the rLipL32 IgG ELISA, 83.3% of the samples were positive. The sensitivity of IgG ELISA for 125 bovine sera, which had MAT titres of greater than or equal to 100, was 100%. ELISA showed a specificity of 100% with 58 bovine sera, which were negative at a 1:50 dilution in the MAT for Leptospira interrogans serovars. When analytical specificity of the IgG ELISA was evaluted using 60 bovine serum samples from animals showing serum antibodies to other pathogens that cause abortion in cattle, such as Babesia sp., Anaplasma sp. and Brucella sp. and no cross-reaction was observed. The recombinant LipL32 IgG ELISA can be an alternative to the MAT for diagnosis of leptospiral infection in cattle.

Seroprevalence of antibodies against Coccidioides immitis in healthy horses

To determine the seroprevalence of antibodies against Coccidioides immitis in healthy horses residing in an area in which the organism is endemic. Prospective study. 197 healthy horses (in which coccidioidomycosis had not been previously diagnosed) that resided in an area of Arizona in which coccidioidomycosis is endemic. Of the horses evaluated at the Arizona Equine Medical and Surgical Center during a 6-month period, 197 with no clinical signs of coccidioidomycosis were randomly selected for inclusion in the study; sera were evaluated for IgM and IgG antibodies against C immitis via an immunodiffusion assay (IgG-positive samples were assessed quantitatively). Within 6 months, recheck titer evaluations were attempted for all seropositive horses. Serum antibodies against C immitis were detected in 8 of 197 horses (seroprevalence, 4.06%). Results of serologic assays were positive for IgG antibodies and negative for IgM antibodies in 7 horses and positive for both IgG and IgM antibodies in 1 horse; reciprocal serum IgG antibody titers were low (none > 8). Follow-up serologic data were obtained from 5 horses; compared with initial findings, horses had become seronegative or titers were unchanged or decreased. Duration of residence in the area was significantly shorter for seropositive horses than for seronegative horses. Serum antibodies against C immitis may rarely be detected in healthy horses residing in an area in which the disease is endemic; any horse with a detectable serum antibody titer should be reevaluated after an interval of at least 3 weeks.

67. Protective Immunity Against Yersinia pestis in Mice Immunized with an Ad-Based Vaccine Vector Expressing the Y. pestis F1 and V Antigens

The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, an extremely virulent and rapidly fatal disease that develops within days post-infection. In the context of a bioterror threat, an effective anti-plague vaccine must elicit rapid, robust and long acting protective immunity after a single dose. At present, no plague vaccines are available for use in the USA. Two subunits of Y. pestis, fraction 1 (F1) and V antigen have been identified as vaccine candidates based on: (1) F1 antigen is a temperature-regulated capsular protein of Y. pestis with anti-phagocytic activity in the host; (2) V antigen has local anti-inflammatory effects and influences the function of important virulence factors such as the Yersinia outer membrane proteins (Yops). It is known that protective immunity against Y. pestis challenge can be induced by multiple immunizations with recombinant F1 and V proteins and immediate protection can be achieved by passive transfer of immune sera against these antigens. Based on the knowledge that adenovirus (Ad) gene transfer vectors act as adjuvants in eliciting host immunity against the transgene they carry, replication-defective Ad gene transfer vectors encoding either the Y. pestis V antigen or the F1 protein (AdsecV and AdsecF1, respectively) were constructed. The F1 and V antigen genes were synthesized by an overlap PCR strategy using mammalian preferred codons, and fused to the human Ig signal sequence for extracellular secretion. We tested the hypothesis that AdsecV and AdsecF1 can stimulate strong protective immune responses without a requirement for repeat administration. To test the efficacy of the vaccines, BALB/c mice were immunized with a single intramuscular administration of either vector at a dose of 108 to 1011 particles units (pu). Four wk after immunization, mice were challenged in a BSL3 experimental animal facility (n=10/group) by the intranasal route with 3103 cfu of the fully virulent Yersinia pestis strain, CO92. At challenge time, anti-V antigen IgG reciprocal titers from AdsecV–immunized mice (n=10/group) were 666137 (108 pu), 3045735 (109 pu), 97032193 (1010 pu), and 7611215914 (1011 pu). In contrast, anti-F1 antigen IgG reciprocal titers from AdsecF1-immunized mice (n=10/group) were much lower with 122 (108 pu), 9135 (109 pu), 1154138 (1010 pu), and 939157 (1011 pu). All (10/10) of the animals survived the Y. pestis challenge in the group vaccinated with 1011 pu AdsecV, and 40% (4/10) survived in the 1010 pu AdsecV dose, while in all the other experimental groups immunized with AdsecV and in the control groups the mice died within 2 to 5 days after challenge. No mice vaccinated with AdsecF1 survived the Y. pestis challenge. These observations suggest that an Ad-based vaccine encoding for the V antigen of Y. pestis (AdsecV) is an excellent candidate as a effective single dose vaccine against the plague.

International collaborative study to evaluate a candidate reference preparation to define an appropriate specified limit of anti-D in intravenous immunoglobulin products

The aim of the study was to evaluate a lyophilized intravenous immunoglobulin (IVIG) preparation containing anti-D (02/228; nominal reciprocal titre of 8) for its suitability to define the maximum limit of anti-D in IVIG products when used in a proposed reference method of direct haemagglutination of papain-treated erythrocytes, in an international collaborative study. Twenty laboratories tested 02/228 along with a negative control IVIG preparation and four IVIG samples containing different levels of anti-D. Nineteen laboratories performed direct haemagglutination methodology using papain-treated erythrocytes; five of these laboratories and one additional laboratory performed their in-house haemagglutination methodology (all indirect antiglobulin tests). The mode titre of 02/228, obtained by using the proposed reference method, was 8 (62.5% of tests). However, there was wide variation in haemagglutination titres between laboratories for three of the four samples. Correcting the titres of the samples relative to those of the proposed reference preparation reduced the interlaboratory variability and increased the frequency of the mode titres in three out of four samples. The indirect antiglobulin tests also showed wide interlaboratory variability and were less sensitive than the direct method in four laboratories. Eleven of the 14 laboratories that expressed an opinion considered that the level of anti-D in 02/228 was appropriate to define a specified limit. Our results demonstrate the necessity of using a reference preparation to define the maximum level of anti-D in IVIG products and ensure sufficient sensitivity in haemagglutination testing methodology. On the basis of these results, members of the European Pharmacopoeia Expert Group 6B recommended revision of the appropriate monograph to include this new specification and test. The Food and Drug Administration in the USA intends to adopt the same maximal specification defined by the reference preparation and to recommend the same test for the safety of IVIG products. Preparations 02/228 and 02/226 were also established by the World Health Organization as International Reference Reagents to standardize haemagglutination testing for anti-D in normal IVIG products.

Prevalence of antibodies to Toxoplasmagondii and Neosporacaninum in red foxes ( Vulpesvulpes) from around the UK

The purpose of this study was to establish the prevalence of antibodies to Toxoplasma gondii and Neospora caninum in a random sample of red foxes from around the UK. Lung fluid from over 500 foxes was examined using an indirect fluorescent antibody test. Reciprocal titres of specific antibodies to T. gondii or N. caninum ranged from <1:16 to 1:1024. A titre of 1:128 or greater was deemed indicative of exposure to the parasite. One hundred and eleven (20%) of the 549 foxes tested were seropositive to T. gondii, and only five (0.9%) were seropositive to N. caninum. No correlation could be made between positive samples and geographical distribution, as sample numbers varied greatly between regions. The results of this study indicate that red foxes of the UK have more exposure to T. gondii than to N. caninum in their environment.

Immune response to Neospora caninum in naturally infected heifers and heifers vaccinated with inactivated antigen during the second trimester of gestation

The objective of this study was to compare the immune response to Neospora caninum in naturally infected heifers and heifers inoculated with a killed whole N. caninum tachyzoite preparation during the second trimester of gestation. Nine Holstein heifers were used in this study; three naturally infected heifers were born from seropositive dams, and six seronegative heifers were born from seronegative dams. Four seronegative heifers were subcutaneously vaccinated with a killed whole N. caninum tachyzoite preparation at weeks 13, 15 and 17 of gestation. A killed whole N. caninum tachyzoite preparation containing 45 mg of protein/5 ml dose was formulated with 70% of mineral oil adjuvant (13% consisting of Arlacel C, 85% Marcol 52 and 2% Tween-80). Similarly, two seronegative heifers (negative controls) were inoculated with mock-infected bovine monocytes in oil adjuvant. Humoral immune responses were tested by using an indirect fluorescent antibody test (IFAT) and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting isotype specific antibodies. Cellular immune responses were assessed by lymphocyte proliferation test (LPT) and IFN-γ production. N. caninum-specific antibody responses increased in immunized cattle by week 15 of gestation (mean reciprocal antibody titers 450 ± 252), peaked at week 23 (mean 16,000 ± 6400). Maximum antibody response in naturally infected heifers was observed at week 19 of gestation (mean: 3467 ± 2810). Mean serum IFAT titers were significantly higher in immunized heifers compared with those in naturally infected heifers from weeks 17 to 25 ( P < 0.05). Analysis of isotype specific antibodies in naturally infected heifers revealed a predominant IgG1 response in one heifer and a predominant IgG2 response in the other two. Similar titers of IgG1 and IgG2 occurred in immunized heifers. Control heifers remained seronegative throughout the study by IFAT and ELISA. Significant antigen-specific proliferation responses were only detected in naturally infected heifers in week 19 of gestation. Peripheral mononuclear blood cells (PMBC) from immunized animals produced IFN-γ in similar concentrations to those of infected animals ( P > 0.05). No abortion was seen in any experimental group; however, one calf from a vaccinated heifer died due to dystocia. All calves from vaccinated and control heifers were seronegative by IFAT at 6 months of age; in contrast, calves born from naturally infected heifers remained seropositive with titers ≥200. Killed vaccine induced similar immune responses to those found in chronically, naturally infected cattle which did not abort; however, different immune pathways may be followed in vaccinated and natural infected heifers with differences in degree of protective immunity.

Development and Validation of an Enzyme-Linked Immunosorbent Assay for Human Metapneumovirus Serology Based on a Recombinant Viral Protein

The human metapneumovirus (hMPV) is a newly reported respiratory virus belonging to the Paramyxoviridae family that has been associated with bronchiolitis and pneumonia in young children. We developed a simple enzyme-linked immunosorbent assay (ELISA) for hMPV serological testing using the nucleoprotein (N) from group A or B (N-A or N-B) as the antigen, and we evaluated it in both children and adults. The N proteins were first used in a Western immunoblot assay to identify hMPV-negative sera, which were then used to determine the cutoff value of the ELISA test. Subsequent evaluation of the ELISA-N test revealed that the mean reciprocal antibody titer of 20 randomly selected seropositive children was 143, compared to 69 for 20 seropositive adults. In a prospective evaluation of 71 adults with acute exacerbations of chronic obstructive pulmonary disease, 58 (81.6%) had prior hMPV antibodies and 3 (4.2%) had evidence of recent hMPV infection. In testing paired sera from adults (n = 4) with recent hMPV group A infection confirmed by reverse transcriptase PCR (RT-PCR), ELISAs using the N-A or N-B proteins were able to detect hMPV seroconversion. Moreover, testing of paired sera from three adults with a recent infection by the human respiratory syncytial virus confirmed by RT-PCR and serology did not reveal any increase in hMPV antibodies over time. The ELISA-N is a simple, objective, and specific serological test useful for detecting anti-hMPV antibodies following group A or B viral infections, which should permit a better understanding of the epidemiology of this virus.

Serologic Correlates of Protection against EnterotoxigenicEscherichia coliDiarrhea

We conducted a nested case-control study in 397 rural Egyptian children <36 months of age to assess the correlation between serum levels of antibodies against toxin and colonization factors (CFs) and the risk of homologous enterotoxigenic Escherichia coli (ETEC) diarrhea. Active case detection was performed via semiweekly home visits, and blood was obtained at 3-month intervals. After each serosurvey, case subjects were selected from children experiencing a CF antigen (CFA)/I-, CFA/II-, CFA/IV-, or heat-labile enterotoxin (LT)-ETEC diarrheal episode during the subsequent 3 months. Up to 5 control subjects per case subject were selected from children who did not experience an ETEC diarrheal episode during the corresponding interval. Serum titers of immunoglobulin G antibodies against CFA/I, coli surface antigen (CS) 3, CS6, and LT were measured by enzyme-linked immunosorbant assay. The distribution of serum titers of LT, CS3, and CS6 antibodies did not differ between the case and control subjects. For children <18 months of age, serum titers of CFA/I antibody were inversely related to the risk of CFA/I-ETEC diarrhea; reciprocal serum titers of CFA/I antibody > or =76 were associated with a 77% reduction in the odds of CFA/I-ETEC diarrhea. Induction of reciprocal serum titers of antibodies against CFA/I within or above the 76-186 range should be further evaluated as a predictor for assessment of the ability of candidate vaccines to protect against CFA/I-ETEC diarrhea.

Anti-Rhinosporidial Antibody Levels in Patients with Rhinosporidiosis and in Asymptomatic Persons, in Sri Lanka

The only report hitherto, from India in 1982, on anti-rhinosporidial antibody levels in patients with rhinosporidiosis recorded that antibody was not detected in Indian patients. The present report describes the use of the dot-ELISA assay of serum anti-rhinosporidial IgG, IgM and IgA and salivary sIgA in patients with diverse clinical presentations, in rural asymptomatic persons who had bathed in ground waters that probably harboured the causative pathogen, Rhinosporidium seeberi, and in laboratory persons who were exposed to R. seeberi. Ultrasonic extracts of purified endospores and sporangia of R. seeberi were used as antigen. The geometric mean (reciprocal) titres of serum antibody detected in patients were IgM 142.1, IgG 178.5, IgA 84.6, with ranges of 0-640, 30-960 and 0-160 respectively, salivary sIgA titres ranged from 0 to 18 with a mean of 4.6. The levels of antibody had no correlation with the site, the number of sporangia, duration and recurrence of the disease. Asymptomatic persons from the same endemic area as patients showed mean titres of IgM 89.6, IgG 69.1, IgA 95.5, with salivary sIgA titres of 3.1. Asymptomatic personnel who had been working in a laboratory where rhiniosporidial work was being done, showed mean titres of 169.6 IgM, 62.8 IgG, and 6.5 salivary sIgA. These results indicate that an anti-rhinosporidial antibody response occurs in rhinosporidial patients, as well as in asymptomatic persons who were exposed to R. seeberi in the environment. Anti-R. seeberi antibody does not appear to be protective in rhinosporidiosis since appreciable titres were present in patients with recurrent, single, multiple or disseminated lesions of long duration.

Serovar-specific prevalence and risk factors for leptospirosis among dogs: 90 cases (1997-2002).

To estimate serovar-specific prevalence of leptospirosis by use of veterinary teaching hospital and laboratory submission data; describe annual and seasonal patterns of leptospirosis; and identify risk factors for age, sex, and breed. Retrospective study. 90 dogs with leptospirosis. Hospital records of dogs examined at Purdue University Veterinary Teaching Hospital with a diagnosis of leptospirosis and laboratory records of dogs from which sera were tested for antibodies against Leptospira spp at Purdue University Animal Disease Diagnostic Laboratory from 1997 through 2002 were reviewed. The likely infecting Leptospira serovar was identified. Seasonal and annual prevalences were calculated by use of hospital population at risk (hospital cases) or serologic testing submissions (diagnostic laboratory cases). Age-, sex-, and breed-specific risk factors for hospital cases were estimated by odds ratios. Of the 39 hospitalized dogs identified, 34 had been serologically tested, and 22 of those were infected with Leptospira kirschneri serovar grippotyphosa. Of the 51 diagnostic laboratory cases, 59% had a reciprocal titer > or = 800 against serovar grippotyphosa. Diagnostic laboratory cases were more common in summer, whereas hospital cases of leptospirosis were more common in fall. Male dogs were at significantly greater risk of leptospirosis than female dogs; and dogs 4 to 6.9 years old were at significantly greater risk than dogs < 1 year old. L kirschneri serovar grippotyphosa infection was associated with most cases of leptospirosis in dogs. Use of an effective vaccine that includes this serovar is advisable for dogs at risk of leptospirosis.

569. Increased Antigen-Specific Humoral Response by Adenoviral Vector Prime and Adeno-Associated Viral Vector Boost

Top of pageAbstract Dissemination of anthrax spores in a bioweapons event could result in inhalational anthrax, a disease that resulted in 45% mortality in the recent US anthrax attacks. The current licensed vaccine and the experimental vaccine currently being assessed by the US Army elicit protective immune responses against protective antigen (PA), a component of lethal toxin. We have recently demonstrated (Tan et al, Human Gene Therapy 2003; 14:1673-1682) that a recombinant serotype 5 adenovirus vector expressing a synthetic gene for PA (Ad5sechPA) offers more robust protection against anthrax lethal toxin challenge than that achieved with purified recombinant PA protein. In this study we have tested the hypothesis that the efficacy of Ad5sechPA vaccination can be enhanced in prime-boost vaccination regimens. BALB/c mice were given intramuscular injections of the Ad5sechPA vaccine at a dose 107, 108, 109 particle units (pu) either once at wk 0, or twice at wk 0 and 4. Anti-PA IgG titer (by ELISA) and neutralizing antibody titer (NAb; reciprocal titer required for 50% inhibition of cytotoxicity by macrophage protection assay) were assessed and lethal toxin challenge was given at wk 6. In all dose groups, boost led to a higher reciprocal anti-PA ELISA titer in pooled sera rising from 200 to 670 in the 107 pu group; from 1,730 to 32,800 in the 108 group and from 2,550 to 40,200 in the 109 group (5 mice/group). Additionally, in all dose groups, boost led to a higher reciprocal neutralizing titer rising from undetectable to 35 in the 107 pu group; from 120 to 810 in the 108 group and from 120 to 1,400 in the 109 group (pooled sera from 5 mice/group). In response to lethal toxin challenge, boost immunization significantly increased protection: 100% of mice in the 108 pu prime-boost group survived the challenge compared to only 20% survival in the 108 pu single immunization group. In the 107 group there was no survival with or without boost and in the 109 group there was 100% survival regardless of boost. We conclude that an Ad5-based anthrax vaccine can be effectively used in prime-boost immunization to achieve more robust protective immunity than single immunization, at least when given via intramuscular route. These results have implications for applying Ad5-based vaccines to human populations.

1015. Induction of Protective Immunity to Anthrax Lethal Toxin with a Chimpanzee Adenovirus-Based Vaccine Carrier in the Presence of Pre-Existing Anti-Human Adenovirus Immunity

An intentional release of anthrax spores could result in widespread inhalational anthrax and a socioeconomic disaster, and thus development of a effective, rapidly acting vaccine is essential to protect susceptible individuals. The current vaccine licensed for use in the US has limited availability and requires multiple administrations for full protection. We have recently demonstrated (Tan et al, Hum Gene Ther. 2003, 14:1673) that a single intramuscular administration of a recombinant serotype 5 adenovirus (Ad) vector expressing protective antigen (PA), a component of anthrax lethal toxin, provides rapid protection against lethal toxin challenge in BALB/c mice. However, approximately 35 to 50% of humans have pre-existing neutralizing antibodies against Ad5 and this may limit the utility of the Ad5-based vaccine. Based on the concept that “seroswitch” of an Ad vector could circumvent pre-existing anti-Ad5 immunity, this study assesses the hypothesis that a recombinant adenoviral vector based on the chimpanzee-derived serotype AdC7 expressing the secreted form of PA (AdC7secPA) will protect against anthrax lethal toxin in the presence of pre-existing anti-Ad5 immunity. BALB/c mice (n=5/group) were immunized intramuscularly with 109 particle units (pu) AdC7secPA, Ad5secPA (a human serotype Ad5-based vector expressing a secreted form of PA) or AdNull (an Ad5 vector with no transgene). At 4 wk post-administration, anti-PA reciprocal titers were assessed by ELISA. Serum anti-PA antibody titers for Ad5secPA-immunized mice were 8800 ± 1600 (mean ± standard error). Serum antibody titers in response to AdC7secPA immunization were 130 ± 62 at a dose of 109 pu, 3000 ± 1300 at 1010 pu, and 14,000 ± 3400 at 1011 pu. No anti-PA titers were detectable for the AdNull control group. To assess the effect of pre-existing anti-Ad5 immunity on anti-PA antibody titers, mice were injected intramuscularly with 1010 pu of a control Ad5 vector to induce anti-Ad immunity. At 4 wk post-administration, with anti-Ad5 neutralizing titers of 84 ± 13, the pre-immunized mice received 1010 pu AdC7secPA. Four wk later, the reciprocal anti-PA antibody titers in Ad5 pre-immunized mice were 1300 ± 490, demonstrating the efficacy of AdC7secPA at stimulating anti-PA titers in the presence of anti-Ad5 immunity. To assess protection in vivo, AdC7secPA- and Ad5secPA-immunized mice were challenged with a lethal intravenous dose of B. anthracis lethal toxin. After 6 wk, 100% of Ad5sechPA (109 pu)-immunized mice survived, whereas 100% of naive control mice died. A dose of 109 pu AdC7secPA protected 60% of immunized mice and a dose of 1010 pu resulted in 100% survival. In Ad5 pre-immunized, AdC7secPA-vaccinated mice, survival after lethal toxin challenge was 100%. These data demonstrate that in the presence of pre-existing anti-Ad5 immunity, the chimpanzee based vector AdC7secPA is an effective vaccine for the development of protective immunity against PA.

558. Anti-SARS Humoral and Cellular Immunity Evoked by an Adenovirus Vector Expressing Spike Glycoprotein from SARS Coronavirus

Top of pageAbstract Severe acute respiratory syndrome (SARS) is a newly described disease caused by SARS-CoV, a novel coronavirus. Due to the limited treatment options and concern over consequences of SARS re-emergence, we have focused on using adenovirus (Ad)-based gene transfer vectors encoding SARS-CoV specific antigens to develop an effective vaccine. We tested the hypothesis that expression of the SARS-CoV spike glycoprotein (S), or its component parts S1 and S2, in E1-E3- Ad serotype 5 vectors can elicit cellular and humoral immune responses against S in immunized mice. A synthetic spike gene was made by an overlapping PCR strategy with codon optimization for mammalian cells. An Ad vector expressing S driven by a CMV promoter was constructed and protein expression was confirmed by Western analysis. To evaluate the immune responses stimulated by this vaccine, 1010 particle units (pu) of the vector was administered by intravenous, intramuscular or subcutaneous routes to C57Bl/6 mice and serum neutralizing antibody titers were measured in an assay assessing the neutralization of SARS-CoV in vitro. Subcutaneous immunization induced anti-SARS-CoV neutralizing antibodies at 2 wk (reciprocal neutralizing titer 32 ± 15) and at 5 wk (690 ± 470) post-administration. Mice immunized by either intramuscular or intravenous routes also developed neutralizing titers (380 ± 96 and 450 ± 78 respectively) by 5 wk post-administration. Immunization by all routes resulted in statistically similar titers (p>0.3 all pairwise comparisons). At a dose of 1011 pu, anti-SARS-CoV neutralizing titers were also measurable (700 ± 160, intravenous; 130 ± 20 intravenous; 450 ± 210 subcutaneous). In contrast, naive mice and mice immunized with AdNull (an Ad with no transgene) had no detectable neutralizing antibody titers at any time point. Cellular immunity stimulated by immunization was evaluated in BALB/c mice injected intravenously with AdnS or AdNull at a dose of 1011 pu. After 2 wk, splenic CD8+ T cells were isolated and exposed to syngeneic target cells stably expressing S. Accumulation of intracellular IFNγ was observed in 8% of CD8+ cells as opposed to 2.5% of cells exposed to syngeneic cells not expressing S. Similar results were observed in C57Bl/6 mice, with 11.5% of CD8+ cells accumulating intracellular IFNγ in response to syngeneic cells expressing S, as opposed to 3.3% of CD8+ cells exposed to syngeneic cell lines not expressing S. The AdNull vaccinated controls did not stimulate antigen-specific IFNγ accumulation in the splenic CD8+ cells. To determine the location of the major cellular epitopes in the spike glycoprotein, Ad vectors and target syngeneic cell lines expressing the N-terminal domain (S1) or the C-terminal domain (S2) of spike were constructed. The specific accumulation of IFNγ in immunized BALB/c mice at 2 wk post-administration was more dependent on the S2 domain than the S1 domain (19% specific intracellular IFNγ accumulation using the S2 domain vs 6.3% with S1). We conclude that immunization with an Ad vaccine vector expressing the SARS-CoV S elicits high titers of SARS-CoV-neutralizing antibodies and that the S2 domain of spike contains the immunodominant CD8+ T-cell epitopes.

372. Passive Immunotherapy for Anthrax Mediated by an Adenovirus Expressing Anti-Protective Antigen Single Chain Antibody

Bacillus anthracis was responsible for 45% of deaths in the 2001 US bioterrorism attacks, despite optimal medical therapy. Any delay in treatment of anthrax significantly affects the chances of survival, therefore, rapid therapy is required. Several vaccines have been developed against B. anthracis, but all require multiple administrations to elicit protective immunity. An anthrax vaccine composed of an adenovirus vector expressing the protective antigen (PA) component of anthrax lethal toxin only requires a single administration to be effective in protecting mice against the anthrax lethal toxin, but it takes >2 wk for anti-anthrax immunity to develop. To address the need for rapid protection, this study describes an in vivo gene transfer-based therapy with a human adenovirus (Ad)-based vector (AdαPAscAb) encoding a single chain antibody that neutralizes protective antigen (PA), a critical component of B. anthracis lethal toxin. To examine the expression and secretion of the anti-PA single chain antibody by AdαPAscAb, A549 cells were infected with AdαPAscAb and, as controls, cells were infected with AdαCTXscAb (an Ad vector encoding an irrelevant single chain antibody) or AdNull (an Ad vector with no transgene). Supernatants from cells infected with AdαPAscAb and AdαCTXscAb both contained antibody reactive with the common human Igκ light chain region. Western analysis of purified protective-antigen or lethal factor proteins with supernatants from AdαPAscAb-infected cells showed that the anti-PA single chain antibody specifically recognized PA. The in vivo expression profile of AdαPAscAb was assessed in serum 0 to 14 days after intravenous administration to BALB/c mice by quantifying anti-PA single chain antibody levels by ELISA and anti-PA neutralizing antibody levels. When measured with a PA-specific ELISA, the anti-PA serum antibody titer in mice receiving AdαPAscAb reached significant levels over a 2 wk period (peak reciprocal titer at day 5 = 12,400 ± 8800). Serum from AdαPAscAb-immunized mice, but not from AdαCTXscAb- or AdNull-immunized mice, had neutralizing activity that paralleled the ELISA titers (peak 50% neutralizing titer at day 5 = 260). Mice that had been injected with AdαPAscAb or AdNull were challenged with lethal toxin at 72 hr post-administration, using naive mice as an additional control. Over a seven day observation period, mice that received AdαPAscAb exhibited a significant survival advantage (100%) relative to naive mice (0%) or mice that received AdαCTXscAb (0%) or AdNull (0%). To determine the timing of protection by AdαPAscAb, mice were given a single intravenous administration with AdαPAscAb, AdαCTXscAb or AdNull at -14 to -1 day before, at the time of, or 0.5 days after challenge with lethal toxin. Although mice immunized at the time of challenge or post toxin administration did not survive, mice that were immunized as early as up to 14 days pre-challenge had a survival advantage relative to control animals (90% protection at day 1, 100% protection at day 2 to day 5, 10% protection at day 14). Passive immunotherapy with AdαPAscAb is a rapid, convenient approach to protect against anthrax or to use in conjunction with antibiotic treatment.