Suis Antigens Research Articles

Development of a Multiplex Bead Assay to Detect Serological Responses to Brucella Species in Domestic Pigs and Wild Boar with the Potential to Overcome Cross-Reactivity with Yersinia enterocolitica O:9.

The aim of this study was to develop a multiplex bead assay using a Brucella rLPS antigen, a Brucella suis smooth antigen, and a Yersinia enterocolitica O:9 antigen that not only discriminates Brucella-infected from Brucella-uninfected pigs and wild boar, but also overcomes the cross reactivity with Y. enterocolitica O:9. Sera from 126 domestic pigs were tested: 29 pigs were Brucella infected, 80 were non-infected and 17 were confirmed to be false positive serological reactors (FPSR). Sera from 49 wild boar were tested: 18 were positive and 31 were negative. Using the rLPS antigen, 26/29 Brucella-infected domestic pigs and 15/18 seropositive wild boar were positive, while 75/80 non-Brucella infected domestic pigs, all FPSR, and all seronegative wild boar were negative. Using the smooth B. suis 1330 antigen, all Brucella-infected domestic pigs, 9/17 FPSR and all seropositive wild boar were positive, while all non-infected pigs and 30/31 seronegative wild boar were negative. The ratio of the readouts from the smooth B. suis antigen and Y. enterocolitica O:9 antigen enabled discriminating all Brucella infected individuals from the FPSR domestic pigs. These results demonstrate the potential of this assay for use in the surveillance of brucellosis, overcoming the cross-reactivity with Y. enterocolitica.

Carrier status of Streptococcus suis in the palatine tonsils of apparently healthy slaughtered pigs of India

ABSTRACT Streptococcus suis is an emerging bacterial pathogen of huge economic impact to the swine industry worldwide. The information regarding the carrier status of S. suis in the slaughtered pigs along with its genetic characterization is not available in Indian pig population, which needs to be addressed for the therapeutic and preventive measures. In the present study, 563 palatine tonsils of apparently healthy slaughtered pigs were probed for the prevalence, and genetic characterization of S. suis and prevalence were found to be 15.45% and 32.68% by bacteriological and molecular methods, respectively. In 87 isolates recovered, 6 cps-types were detected showing the predominance of serotype 7 (24.13%) and 5 (18.39%), whereas 11 cps-types were detected in tonsillar DNA involving cps-types 9 (28.26%) and 7 (14.13%) as the major serotypes with arcA+/sly+/epf+/mrp− being the prevalent genotype. The histopathological changes with the immunodetection of S. suis antigen confirmed its persistence in asymptomatic carriers. Of 87 bacterial isolates, 7 isolates (serotypes 7 & 2) were pathogenic to Swiss albino mice showing the classical lesions of meningitis and septicemia. The presence of virulent serotypes of S. suis in healthy slaughtered pigs suggests a great health risk to the people engaged in piggery operations and in-contact pigs.

Engineering Antigens to Assemble into Polymer Particle Vaccines for Prevention of Streptococcus suis Infection.

Streptococcus suis is a zoonotic pathogen affecting pigs and humans. This bacterium causes severe economic losses in the swine industry and poses a serious threat to public health and food safety. There is no effective commercial vaccine available for pigs or humans. In this study, we applied the biopolymer particle (BP) vaccine technology to incorporate seven conserved S. suis antigens (38 kDa protein (38), enolase (Enol), SSU1915, SSU1355, SSU0185, SSU1215, and SSU1773 (SSU1 and SSU2)). Two combinations of these antigens (38 and Enol; all SSU antigens designated as SSU1 and SSU2) were engineered to mediate production of BPs coated with either antigens 38 and Enol or SSU1 and SSU2 inside recombinant Escherichia coli. The isolated and purified empty BPs, 38-BP-Enol and SSU1-BP-SSU2, showed size ranges of 312–428 nm and 292–344 nm with and without the QuilA® adjuvant, respectively, and all showed a negative surface charge. Further characterization of purified BPs confirmed the presence of the expected antigen-comprising fusion proteins as assessed by tryptic peptide fingerprinting analysis using quadrupole time-of-flight mass spectrometry and immunoblotting. Vaccination with 38-BP-Enol and SSU1-BP-SSU2 formulated with and without QuilA® adjuvant induced significant antigen-specific humoral immune responses in mice. Antigen-coated BPs induced significant and specific Ig (IgM + IgG) and IgG immune responses (1.0 × 106–1.0 × 107) when compared with mice vaccinated with empty BPs. Functionality of the immune response was confirmed in challenge experiments using an acute murine S. suis infection model, which showed 100% survival of the 38-BP-Enol and SSU1-BP-SSU2 vaccinated mice compared to 70% survival when vaccinated with empty BPs. Overall, our data suggest that S. suis antigen-coated BPs could be developed into particulate vaccines that induce protective immunity against S. suis infections.

Effects of the dietary fibre inulin and Trichuris suis products on inflammatory responses in lipopolysaccharide-stimulated macrophages

Consumption of fermentable dietary fibres, such as inulin, or administration of helminth products (e.g. Trichuris suis ova) have independently been shown to alleviate inflammation in vivo. We recently found that dietary inulin and T. suis infection in pigs co-operatively suppressed type-1 inflammatory responses in the gut, suggesting the potential of dietary components to augment anti-inflammatory responses induced by certain helminths. Here, we explored whether T. suis antigens and inulin could directly suppress inflammatory responses in vitro in a cooperative manner. T. suis soluble products (TsSP) strongly suppressed lipopolysaccharide (LPS)-induced IL-6 and TNF-α secretion from murine macrophages and induced an anti-inflammatory phenotype as evidenced by transcriptomic and gene pathway analyses. Inulin regulated the expression of a small number of genes and transcriptional pathways in macrophages after exposure to LPS, but did not enhance the suppressive activity of TsSP, either directly or in co-culture experiments with intestinal epithelial cells. Culture of macrophages with short-chain fatty acids, the products of microbial fermentation of inulin, did however appear to enhance TsSP-mediated inhibition of TNF-α production. Our results confirm a direct role for helminth products in suppressing inflammatory responses in macrophages. In contrast, inulin had little capacity to directly modulate LPS-induced responses. Our results suggest distinct mode-of-actions of T. suis and inulin in regulating inflammatory responses, and that the role of inulin in modulating the response to helminth infection may be dependent on other factors such as production of metabolites by the gut microbiota.

Meningoencephalitis and septicemia in a 4-week-old piglet due to dual streptococcal infections

A 4-week-old male crossbred piglet was presented for necropsy examination with history of neurological signs progressing to death. Grossly, the meninges covering the brain were thickened, opaque containing purulent exudates. Besides, the other visceral organs like lungs, heart, spleen, lymph nodes, kidneys and intestine were severely congested. Histologically, suppurative meningoencephalitis was observed in addition to suppurative bronchopneumonia, hepatitis, nephritis and splenitis indicating the septicemia in pig. Immunolabelling of S. suis antigen was observed in meninges, brain, lungs, spleen, tonsil and lymph nodes. Both S. suis and S. dysgalactiae subspecies equisimilis were isolated from the brain, lungs and spleen. The biochemical characterization by API 20 strep kit and genomic detection test by PCR further confirmed both the isolates. This is the first report of dual infection of S. suis and S. dysgalactiae subspecies equisimilis infection in a pig with severe disseminated suppurative meningoencephalomyelitis.

An Infectious Pig Model of Crohn's Disease.

Over the last several years, we have demonstrated that intestinal lymphangitis and lymphatic obstruction are fundamental lesions in Crohn's disease, for which no therapy is currently available. There is an infectious enteritis of young pigs that offers an opportunity to understand how the lymphangitis of CD may have been initiated. The pathology of chlamydial enteritis was described earlier, from 1987 to 2009. Tissue blocks and hematoxylin and eosin-stained slides from Chlamydia suis-inoculated young pigs were provided by D. Rogers and F. Guscetti. Experimental animals, gnotobiotic and conventional, had been autopsied 4, 7, and 10 days postinoculation. Serial sections of intestine were immunostained with a Chlamydia trachomatis antibody, which cross-reacted with C. suis antigen, and compared with hematoxylin and eosin preparations. Immunohistochemistry revealed antigen in villous epithelial cells of jejunum and ileum and in the endothelium of lacteals and lymphatics by day 4. This was accompanied by lymphatic endothelial necrosis, lymphangitis, and inflammatory lymphatic obstruction, through several layers of the affected intestinal segments, days 4 through 10. Although the original authors documented lesions to define the porcine disease, here the author characterizes the lymphangitis as a model for understanding Crohn's disease and suggests a chlamydial origin for the latter.

Colloidal gold-based immunochromatographic strip test compromising optimised combinations of anti-S. suis capsular polysaccharide polyclonal antibodies for detection of Streptococcus suis

A rapid diagnosis kit that detects Streptococcus suis (S. suis) antigens from urine with an immunochromatographic stripe (ICS) test was developed in this study. The ICS test was produced using colloidal gold coated with polyclonal antibodies (pAbs) against S. suis. The pAbs were developed from rabbits immunised with S. suis serotype 2 capsular polysaccharides (CPS). Development of the pAbs was investigated to establish their binding to CPS and to determine the maximum sensitivity of two combination antibodies for the ICS test. The results of the ICS optimisation revealed that the combinations of pAb C-N1 and pAb C-N2 had the highest sensitivity to CPS. The minimum limitation of ICS sensitivity indicated 1.0×104 colony forming units (CFU) and a CPS concentration of 0.05µg. The assay time for detection of S. suis antigens is less than 15min, which is suitable for rapid detection. A cross-reactive test was also conducted, and it detected no other bacteria (Streptococcus pneumoniae, Streptococcus agalactiae, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae). The cross-reactivity of other serotypes in S. suis was also investigated, and tests for serotypes of 1, 1/2, 3, 4, 5, 6, 7, 8, 9, 14, and 16 were positive. This study presents the first report of a development of an ICS that enables the quantitative detection of streptococcal antigens. The S. suis ICS provides several advantages over other methods, including the speed and simplicity of use.

Identification of Mycoplasma suis antigens and development of a multiplex microbead immunoassay

The aims of the current study were to identify Mycoplasma suis antigens and develop a multiplex microbead immunoassay (MIA). A M. suis-expression library was screened for immunogens using sera from infected pigs. Based on bioinformatics, putative antigens were identified within positive inserts; gene fragments were expressed and purified as polyhistidine fusion proteins, and immunoreactivity was confirmed by Western blot. Selected antigens were used to develop a MIA. Sera from noninfected and infected pigs were used to set the median fluorescent intensity (MFI) cutoffs and as positive controls, respectively. Assay specificity was tested using sera from pigs seropositive for other pathogens (2 different pigs seropositive for each pathogen). Samples from 51 field pigs and 2 pigs during the course of acute (pig 1) and chronic (pig 2) infections were tested using MIA, indirect hemagglutination assay (IHA), and quantitative polymerase chain reaction (qPCR). Sixteen reactive plaques (52 genes) were detected. A heat-shock protein (GrpE), a nicotinamide adenine dinucleotide-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPN), and 4 proteins from paralogous gene families (PGFs) were identified as antigens by Western blot. While GrpE, GAPN, and 1 PGF protein were strong antigens, the others were not suitable as MIA targets. A MIA using GrpE, GAPN, and the strongly reactive PGF protein was developed. Cross-reactivity with sera from pigs infected with Mycoplasma hyopneumoniae, Porcine circovirus-2, Porcine parvovirus, Porcine reproductive and respiratory syndrome virus, and Porcine respiratory coronavirus with this MIA was not observed. Pig 2 was consistently positive by MIA and qPCR, whereas pig 1, initially negative, seroconverted before becoming qPCR positive. Only 2 samples (from pig 1) were IHA positive. Five (9.8%) field samples were qPCR positive and 40 (78.43%) were positive for all 3 MIA antigens; however, all were IHA negative. In summary, the MIA is specific and more sensitive than qPCR and IHA, providing simultaneous evaluation of antibody response to M. suis antigens.

Brucellosis of the European Brown Hare ( Lepus europaeus)

The European brown hare (Lepus europaeus) is an important reservoir of Brucella suis biovar 2 and also of the life-threatening zoonotic agent Francisella tularensis. Since both bacteria can produce similar gross pathological lesions in this species, laboratory tests are necessary for the final diagnosis. The aim of the present study was to develop an immunohistochemical method for the detection of B. suis infection and to describe the pathological and histological lesions caused by B. suis in European brown hares. Hyperimmune serum for immunohistochemistry (IHC) was produced by subcutaneous infection of mice with 2 × 10(9) colony forming units of live B. suis biovar 2, injected four times at 1-week intervals. The antiserum did not react with F. tularensis or Yersinia pseudotuberculosis in IHC and displayed only weak cross-reaction with B. canis. Numerous, yellow-white necrotic foci (0.1-0.5 cm diameter) were found in the spleen of five B. suis-infected female European brown hares and also in the lung, uterus, kidney or liver of four of these cases. Microscopically, the foci comprised single or coalescing granulomas with a central necrotic area. Both bacterial isolation and IHC gave positive results for B. suis infection in these animals. B. suis antigens were found as granular or amorphous extracellular material in the necrotic centre of several granulomas. IHC appears to be a suitable complementary diagnostic method for the detection of B. suis infection in the European brown hare.

Immune Responses and Protection against ExperimentalBrucella suisBiovar 1 Challenge in Nonvaccinated orB. abortusStrain RB51-Vaccinated Cattle

Twenty Hereford heifers approximately 9 months of age were vaccinated with saline (control) or 2 × 10(10) CFU of the Brucella abortus strain RB51 (RB51) vaccine. Immunologic responses after inoculation demonstrated significantly greater (P < 0.05) antibody and proliferative responses to RB51 antigens in cattle vaccinated with RB51 than in the controls. Pregnant cattle received a conjunctival challenge at approximately 6 months of gestation with 10(7) CFU of B. suis bv. 1 strains isolated from naturally infected cattle. The fluorescence polarization assay and the buffered acid plate agglutination test had the highest sensitivities in detecting B. suis-infected cattle between 2 and 12 weeks after experimental infection. Serologic responses and lymphocyte proliferative responses to B. suis antigens did not differ between control and RB51 vaccinees after experimental infection. No abortions occurred in cattle in either treatment group after challenge, although there appeared to be an increased incidence of retained placenta after parturition in both the control and the RB51 vaccination treatment groups. Our data suggest that the mammary gland is a preferred site for B. suis localization in cattle. Vaccination with RB51 did not reduce B. suis infection rates in maternal or fetal tissues. In conclusion, although B. suis is unlikely to cause abortions and fetal losses in cattle, our data suggest that RB51 vaccination will not protect cattle against B. suis infection after exposure.

Intranasal immunization with a live Streptococcus suis isogenic ofs mutant elicited suilysin-neutralization titers but failed to induce opsonizing antibodies and protection

In Europe, Streptococcus suis serotypes 2 and 9 are very important causative agents of meningitis, arthritis and septicemia in piglets. So far, no vaccine has been described which elicits protection against both serotypes. The working hypothesis of this study was that an isogenic serotype 2 mutant attenuated in virulence but not in colonization of the respiratory tract might induce protective immunity against both serotypes. Piglets were immunized with the attenuated S. suis serotype 2 strain 10Δofs2/12, which is deficient in expression of the serum opacity factor OFS. Three weeks after intranasal application of 10Δofs2/12 piglets were challenged intravenously with two strains representing the most important S. suis pathotypes in Europe, the homologous MRP+ EF+ SLY+ serotype 2 strain and a heterologous MRP* SLY+ serotype 9 strain. Application of the live vaccine elicited significant serum IgG responses against muramidase-released protein (MRP), extracellular factor (EF) and, most prominently, suilysin. Seroconversion against suilysin was accompanied with an increase of suilysin-neutralization titers in the vaccinated group. Though mortality was lower in the vaccinated groups, significant protection was not observed, neither against the homologous nor against the heterologous challenge. This was in agreement with the finding that the vaccinated piglets had low opsonizing antibody titers insufficient to mediate elimination of the two challenge strains by porcine neutrophils. In conclusion, a single intranasal application of the S. suis serotype 2 strain 10Δofs2/12 elicited humoral immune responses against different S. suis antigens but failed to induce sufficient opsonizing antibody titers and protective immunity against systemic serotypes 2 and 9 challenges.

Establishment of an Efficient Enzyme-Linked Immunosorbent Assay for the Detection of Eperythrozoon sius Antibody in Swine

The Eperythrozoon suis (E. suis) antigen was purified using a Sephadex G-200 chromatograph, and thereby, a high-affinity, specific E. suis antigen was collected and confirmed with Western blotting. Using this antigen, an enzyme-linked immunosorbent assay (ELISA) system to detect the antibody against E. suis in swine was established. There was no cross-reaction with swine sera, which were affected with Mycoplasmal pneumonia, swine fever, swine colibacillosis, or toxoplasmosis. A comparison of this ELISA system with an indirect hemagglutination (IHA) test using 78 swine samples revealed that the ELISA system significantly improved the sensitivity, specificity, and stability for the serodiagnosis of swine E. suis.

Experimental Infection of Conventional Pigs with Streptococcus suis serotype 2 by Aerosolic Exposure

Experimental Infection of Conventional Pigs with Streptococcus suis serotype 2 by Aerosolic Exposure

Eperythrozoon infection in young bulls with scrotal and hindlimb edema, a herd outbreak

Young (9 to 10 mo) Aberdeen-Angus bulls (n = 5) in excellent body condition and pastured with approximately 35 other bulls of similar age on a farm in southeastern Alabama had fever, harsh pulmonary sounds, increased respiratory rate and variable amounts of hindlimb and scrotal edema. Bulls had mild microcytic, normochromic anemia. Numerous eperythrozoon organisms were identified on blood smears. Indirect hemagglutination inhibition test results for Eperythrozoon suis antibodies were negative either because E. suis antigens do not cross react with cattle eperythrozoon organism antibodies or blood was collected before there was sufficient time for seroconversion. Bulls had swelling of the scrotal wall, soft testes, and poor semen quality, characterized by low progressive motility and a high percentage of spermatozoa with primary and secondary abnormalities. Some of these abnormalities may be attributed to the age of the bulls. However, loss of scrotal thermoregulation was a major cause of testicular degeneration leading to poor semen guality. Other bulls in the herd had fever, increased respiratory rate, and swollen hindlimbs or scrotum. Subsequent to administration of oxytetracycline, parasitemia resolved rapidly, and clinical signs gradually abated. Four of five bulls successfully passed breeding soundness examinations six months after initial clinical illness. The remaining bull failed twice due to the presence of testicular inflammation.

Evaluation of an enzyme-linked immunosorbent assay for detection of Eperythrozoon suis antibodies in swine

Summary An elisa was developed and tested to detect antibodies to Eperythrozoon suis in swine. Results were compared with those of the indirect hemagglutination (iha) test. Antigen isolated from swine heavily infected with E suis was used for both tests. Comparison of the elisa with the iha test revealed a significant (P &lt; 0.001) correlation between results. Of 114 samples obtained from 9 swine infected with E suis, 87.7% were seropositive (titer ≥ 200) via the elisa, and 80.7% were seropositive (titer ≥ 20) via the iha test. The sensitivity of the elisa was greater than that of the iha test. All blood samples obtained from specific-pathogen-free swine tested negative for E suis antibody. Cross-reactions were not observed between E suis antigen and antisera against various swine and cattle disease agents using elisa. We concluded that the elisa may be used for rapid and effective diagnosis of infection with E suis in swine.

Organ-specificity and diagnostic value of cell-mediated immunity against a liver-specific membrane protein: studies in hepatic and non-hepatic diseases.

In chronic active hepatitis (CAH, n=58) 70% of the HBsAg negative and 48% of the HBsAg positive cases showed a CMI against human liver specific proteins (HLPI). Using HBsAg as antigen only 12% of the HBsAg negative and 24% of the HBsAg positive cases gave a CMI response. On the basis of HBsAg and autoantibodies in the serum CAH patients could be divided into 4 subgroups. A close correlation between CMI against HLPI, sex, ANA and HL-A-8 could be detected. In a follow-up study of patients with acute virus B hepatitis (n=62) CMI against HBsAg was detected in 60% of the cases in the acute phase of the disease but in 15% only 3-6 months after the onset of the illness (n=40). In patients who developed a chronic HBsAg carrier status 3 of 5 cases remained persistently positive with HLPI as antigen in the migration inhibition test. - In non-hepatic diseases in which immunological abnormalities may be present (malignant diseases n=46, diabetes mellitus n=27, active tuberculosis, n=18 and untreated systemic lupus erythematodes, n=5) only 26% of patients with malignant diseases showed a migration inhibition with HLPI. - Using different antigens such as human liver specific proteins (HLP), rabbit liver specific proteins (RLP), brucella suis antigen and tuberculin it was possible to demonstrate the validity of the two-step migration inhibition test to detect CMI. The results with different antigens in hepatic and non-hepatic diseases demonstrated that cell-mediated immunity of HLPI is an organ specific immune reaction which is associated with acute and chronic active liver diseases as a time limited or long-lasting phenomenon. Positive reactions in some tumor patients suggest that different mechanisms may elicit an autoimmune reaction against liver antigens.

Standardization of Agglutinating Antigens for the Diagnosis of Brucellosis

SUMMARY Agglutinating antigens prepared from strains of Brucella abortus, B. melitensis and B. suis were standardized to equal sensitivity (1) by use of the International Standard anti-B. abortus serum, and (2) against a collection of 20 bovine (anti-abortus) sera according to the method recommended by the United States Department of Agriculture. In subsequent tests against sera obtained from a variety of animal species infected with different biotypes of B. abortus, B. suis and B. melitensis, it was evident that the sensitivity of the abortus and suis antigens remained the same but that the melitensis antigen differed from the other 2 in sensitivity except in tests with the sera of cattle infected with B. abortus. Two methods for standardizing B. melitensis antigens are proposed for investigation.

Antibody formation by cells isolated from donors of different ages after transfer to young rabbits and to x-irradiated adult rabbits.

To study the first phase of antibody formation by isolated cells, a method was developed for transferring isolated lymphoid cells stiinulated by antigen in vitro. Antibody formation by spleen cells isolated from adult, nonimmunized donors and mixed with Brucella suis antigen was compared after transfer to young rabbits and to adult recipients irradiated with 450 r. It was found that this dose did not produce complete inhibition and that the recipients still responded actively to the antigen. Therefore, only young animals are a suitable experimental model in which to study antibody formation by transferred isolated cells. Development of antibody in young rabbits was studied by transferring cells isolated from the spleen of animals aged 8-30 days and mixed with antigen in vitro. Antibodies were formed after the transfer of these cells to x-irradiated recipients, but by the active response of the latter to the antigen. Antibody formation by cells of donors of different ages can be determined after transfer to young animals. The youngest donors whose cells formed antibodies after transfer were 3-week-old rabbits, but demonstration of antibodies required the transfer of large numbers of cells. It is clear from these results that cells from adult donors which have contactedmore » antigen in vitro form antibodies when transferred to homologous newborn recipients. However, newborn rabbits are functionally too active to be altogether ideal and suitable recipients. The newborn destroy transferred cells within a few days, thus abolish their immunological activity. The discrepancy between the finding that antibodies can be obtained in young animals immunized soon after birth and failure of their isolated cells to perform the same function may be due to several factors. It is suggested that direct immunization experiments, using different antigens, may provide a more accurate answer. (H.H.D.)« less

A study of the formation of antibodies in new-born animals following transfer of nucleoprotein fractions

(1) Ribonucleoprotein and deoxyribonucleoprotein fractions were isolated from the spleen 24 and 48 hr. following the injection of a single dose ofBrucella suis antigen to an adult rabbit. Nucleoproteins transferred to 3–5-day-old new-born rabbits did not result in the appearance of detectable antibodies againstBrucella suis in the sera of the recipients. (2) Sera of new-born rabbits were also found to contain no antibodies againstBrucella suis, as long as whole spleen cells isolated from an immunized adult rabbit 24 hr. after the injection of theBrucella suis antigen were transferred. After transfer of cells isolated from the spleen 48 hr. after the injection of antigen there was in some cases an observable titre of antibodies amounting to 1∶8 or 1∶16. Significant titres (1∶32 to 1∶128) were found only after transfer of cells isolated 72 hr. after antigen injection, antibodies being then detectable in the donor serum. (3) A possible explanation of the differences in results obtained after the transfer of nucleoproteins when different antigens were used is discussed.

Study of antibody formation by the use of metabolic inhibitors

(1) In adult individuals the response to bacterial antigen usually has the character of a secondary reaction and is not influenced even by large doses of 6-mercaptopurine. The effect of 6-mercapto-purine on antibody response was therefore studied in young rabbits, in which a primary reaction is produced. It was found that doses of 0.3 and 0.5 mg./ 100 g. body weight led to complete inhibition of the antibody reaction after the injection ofSalmonella paratyphi B andBrucella suis antigen. (2) The phase of antibody formation influenced by 6-mercaptopurine was determined by transferring isolated spleen cells mixed with antigenin vitro to young rabbits. In all the experiments, 6-mercaptopurine was administered to the recipients subcutaneously in doses of 0.5 mg./ 100 g. for five days. If the administration of 6-mercaptopurine was initiated immediately after the transfer of normal cells mixed with antigenin vitro, antibody formation was completely inhibited. If the transferred cells were isolated from the donor during the phase of antibody production, however, the same doses of 6-mercaptopurine failed to inhibit the antibody reaction. Antibody formation was likewise not inhibited when the administration of 6-mercaptopurine was not begun until 72 hours after the transfer of normal cells mixed with antigenin vitro. When 6-mercaptopurine was administered 24 and 48 hours after transfer of the cells the antibody response was completely inhibited. This part of the experiments shows that 6-mercaptopurine is effective during the inductive phase of antibody formation only. (3) When 6-mercaptopurine was administered to one-month-old rabbits over a period of 14 days, during which 60 ml. blood and 500×106 spleen cells from an adult donor were administered, tolerance to a skin homograft from the corresponding donor was not induced.