Enzyme-labeled Antigen Method: Histochemical Detection of Antigen-specific Antibody-producing Cells in Tissue Sections of Rats Immunized With Horseradish Peroxidase, Ovalbumin, or Keyhole Limpet Hemocyanin

The enzyme-labeled antigen method is a histochemical technique that visualizes antigen-specific antibody-producing cells in tissue sections, originally documented in 1968. In this study, we attempted to reemerge this hidden but potentially useful method in rat models immunized with horseradish peroxidase (HRP), ovalbumin (OA), or keyhole limpet hemocyanin (KLH). After repeated immunization in footpads, popliteal, groin, and axillary lymph nodes and spleen were sampled. Paraformaldehyde-prefixed frozen sections were incubated with HRP, biotinylated OA, or biotinylated KLH. Proteinase K pretreatment and the secondary use of HPR-labeled streptavidin were applied in the latter two situations. Plasma cells producing antigen-specific antibodies were visualized. Proportions of antigen-specific antibody-producing cells in total plasma cells shown with the immunoperoxidase method for rat immunoglobulins were evaluated. The percentage of antigen-specific plasma cells reached ∼50% of total plasma cells in the regional lymph nodes. The specificity was confirmed by (a) negativity in non-immune rat tissue, (b) negativity with indifferent antigen probes, and (c) abolishment of the reactivity with the corresponding rat serum. In buffered formalin-fixed, paraffin-embedded tissues, fewer plasma cells were labeled for HRP and KLH antibody reactivity after strong proteolysis and prolonged incubation. Expectedly, this method allows us to observe antigen-specific antibody-producing cells under varied pathological conditions. (J Histochem Cytochem 57:101–111, 2009)     

Application of an enzyme‐labeled antigen method for visualizing plasma cells producing antibodies against Strep A,a carbohydrate antigen of Streptococcus pyogenes,in recurrent tonsillitis

Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme‐labeled antigen method is a novel histochemical technique that visualizes specific antibody‐producing cells in tissue sections by employing a biotin‐labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde‐fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme‐labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR‐detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti‐Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A‐reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders.     

Nippostrongylus brasiliensis: Indirect fluorescent antibody studies of immunity in mice

Antibodies against Nippostrongylus brasiliensis were demonstrated by the indirect fluorescent antibody technique in four pools of sera from mice 3 weeks after one or more exposures to this parasite. These antibodies combined with antigens in sections of N. brasiliensis and included IgG, IgM, and IgA classes of immunoglobulins. Antibodies of the first two classes produced intense fluorescence in many areas of the parasites, whereas IgA usually evoked only minimal reactions at most of these same sites. Results produced by all pools of antisera were similar, while pools of sera from nonparasitized mice were essentially negative.The principal site of fluorescence in sections treated with antisera was in the cellular cytoplasm and in the microvillar layer of the intestinal epithelium. The nuclei in these cells did not fluoresce. The cuticle, lateral canals, body wall musculature, and reproductive organs fluoresced to a lesser extent. In this group of structures, the most notable fluorescence occurred in the cytoplasm of developing ova, in shells of completed eggs, and in muscle cells of the vagina. Slight autofluorescence was observed in the cuticle, cytoplasm of cells of the lateral canals, substances in the lumina of the lateral canals, and in the extracellular substance of the excretory glands.     

Schistosoma japonicum: Immunological response to circulating polysaccharide antigens in rabbits with a light infection

To study the detectability of circulating polysaccharide antigens and the immunological response to such antigens in rabbits with a light Schistosoma japonicum infection, sera of five rabbits infected with 50 cercariae were studied up to 29 weeks post infection (p.i.). While one rabbit developed no worm burden, the other rabbits developed low worm burdens (4 to 16 worms). In the sera of these rabbits, the only polysaccharide antigen demonstrable with immunoelectrophoresis (IEF), was the circulating anodic antigen (CAA). With the enzyme-linked immunosorbent assay (ELISA), CAA was detectable from 5 to 6 weeks p.i. in the sera of the two rabbits with the highest number of worm couples. The lowest CAA level which was detectable in unconcentrated sera from which serum proteins had been removed was 125 ng CAA/ml, corresponding with a worm burden of 4.5 worm/kg body wt. During the entire infection, CAA-specific immune complexes were only demonstrable in very low concentrations. Antibodies against polysaccharide antigens were assessed with immunofluorescent antibody (IFA) on Rossman's fixed sections of adult worms, with the ELISA, and with IEF. Specific IgA, IgG, and IgM antibodies were detectable from 2 to 3 weeks p.i. with IFA and ELISA. These early antibodies were shown to be directed against gut-associated antigens, while antibodies against parenchyma-associated antigens were found later in the infection. With IEF, antibodies against two trichloroacetic acid (TCA)-soluble antigens were detectable, including the major, S. japonicum-specific antigen 2.     

Shared antigens between human malignant melanoma cells and Mycobacterium bovis (BCG).

This study was undertaken to investigate the antigenic relationships between human malignant melanoma cells and Mycobacterium bovis (BCG). Rabbits were immunized with sonicates of BCG or with malignant melanoma cells from different patients and the resulting antisera were tested for their capacity to bind radiolabeled soluble extracts prepared from BCG and melanoma cells. The binding of antibodies to radiolabeled antigens was studied by precipitation of radiolabeled antigen-antibody complexes by anti-rabbit immunoglobulin. Antibodies in sera from rabbits immunized with either BCG (anti-BCG) or melanoma cells (anti-melanoma) bound both the labeled BCG and melanoma antigens. Control antisera, from rabbits immunized with human acute or chronic lymphatic leukemia cells or with normal human spleen cells, did not bind significant amounts of radiolabeled BCG. Antibodies in sera from rabbits immunized with normal spleen cells bound small but significant amounts of radiolabeled melanoma antigens. Binding by anti-BCG and anti-melanoma to the radiolabeled antigens was studied before and after absorption of antisera with cells from human melanoma, leukemia, guinea pig hepatoma, and normal human spleen cells. Inhibition studies using unlabeled BCG extracts also were carried out. The absorption and inhibition studies confirmed that the binding reactions were specific and that antigens from five melanoma patients shared antigenic determinants with BCG.     

Immunohistochemical characterization of B cells and T cells in musk shrew (Suncus murinus) lymphoid tissues using monoclonal antibodies

We have developed and characterized three monoclonal antibodies (mAbs), which recognize the surface antigens of musk shrew B cells and T cells. About 30% of all lymph node cells reacted with the mAb ST1. Staining of frozen sections showed that ST1-positive cells were located in the primary lymphoid follicles of lymph nodes, and were absent in the thymus. mAbs ST4 and ST2 were also surface-reactive. The ST1-negative (about 60% of the total) lymph node cells reacted positively with ST4, and about 30% of these ST4-positive cells were recognized by ST2. The distribution of ST4-positive cells was shown by staining of lymph node sections to be identical to that of T cells reported in other species. Western blot analysis showed that the apparent molecular weights of the antigens recognized by ST1 and ST4 were 70000 and 74000, and 60000 and 64000, respectively. These findings suggest that the antigens detected by ST1, ST4, and ST2 are the musk shrew homologues of pan-B cells, pan-T cells, and T cell subsets, respectively. The three mABs may facilitate immunohistochemical analysis of the cellular immune response in this species.     

Antibody reaction of human anti-Toxoplasma gondii positive and negative sera with Neospora caninum antigens

Anti-Neospora caninum antibody was detected in anti-Toxoplasma gondii positive and negative human sera by ELISA, western blot and immunofluorescence assay (IFA). Twelve cases out of 172 (6.7%) Toxoplasma-positive sera cross-reacted with both T. gondii and N. caninum antigens, and one out of 110 Toxoplasma-negative sera reacted with N. caninum antigen by ELISA. By western blot, all 12 sera reacted with T. gondii antigens with various banding patterns but specifically at 30 kDa (SAG1) and 22 kDa (SAG2) bands. With N. caninum antigen, the number of reactive bands was reduced, however a 43 kDa band reacted in three cases in Toxoplasma-positive sera in addition to one in Toxoplasma-negative control sera. All sera of the Toxoplasma-positive group labeled surface membrane of T. gondii, but reacted differently with N. caninum. Fluorescence was detected in surface membrane, subcellular organelles, or both in N. caninum. And one case in the Toxoplasma-negative group also reacted with N. caninum strongly in subcellular organelles. This suggested that the antibody against N. caninum may be present in human sera although the positive rate was very low in this study. The possibility of human infection with N. caninum remains to be evaluated further.     

Concentration of antibodies against Porphyromonas gingivalis is increased before the onset of symptoms of rheumatoid arthritis

BackgroundThe periodontal pathogen Porphyromonas gingivalis is hypothesized to be important in rheumatoid arthritis (RA) aetiology by inducing production of anti-citrullinated protein antibodies (ACPA). We have shown that ACPA precede RA onset by years, and that anti-P. gingivalis antibody levels are elevated in RA patients. The aim of this study was to investigate whether anti-P. gingivalis antibodies pre-date symptom onset and ACPA production.MethodsA case–control study (251 cases, 198 controls) was performed within the Biobank of Northern Sweden. Cases had donated blood samples (n = 422) before the onset of RA symptoms by 5.2 (6.2) years (median (interquartile range)). Blood was also collected from 192 RA patients following diagnosis. Antibodies against P. gingivalis virulence factor arginine gingipainB (RgpB), and a citrullinated peptide (CPP3) derived from the P. gingivalis peptidylarginine deiminase enzyme, were analysed by ELISA.ResultsAnti-RgpB IgG levels were significantly increased in pre-symptomatic individuals (mean ± SEM; 152.7 ± 14.8 AU/ml) and in RA patients (114.4 ± 16.9 AU/ml), compared with controls (p < 0.001). Anti-CPP3 antibodies were detected in 5 % of pre-symptomatic individuals and in 8 % of RA patients, with elevated levels in both subsets (4.33 ± 0.59 and 9.29 ± 1.81 AU/ml, respectively) compared with controls (p < 0.001). Anti-CPP3 antibodies followed the ACPA response, with increasing concentrations over time, whilst anti-RgpB antibodies were elevated and stable in the pre-symptomatic individuals with a trend towards lower levels after RA diagnosis.ConclusionsAnti-P. gingivalis antibody concentrations were significantly increased in RA patients compared with controls, and were detectable years before onset of symptoms of RA, supporting an aetiological role for P. gingivalis in the development of RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-016-1100-4) contains supplementary material, which is available to authorized users.     

Serodiagnosis of environmental mycobacterial infections

To demonstrate the usefulness of enzyme-linked immunosorbent assay for serodiagnosis of mycobacterioses due to environmental mycobacteria we utilized a panel of glycolipid antigens selective for Mycobacterium avium-intracellulare, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium scrofulaceum and Mycobacterium gordonae. The levels of circulating antibodies were determined against the environmental mycobacteria, and Mycobacterium tuberculosis in human immunodeficiency virus-negative and -positive patient sera. The method used immunomagnetic separation of the antigens, with covalent immobilization of antibodies to superparamagnetic amine and carboxyl terminated particles in solutions of the specific antigens. Enzyme-linked immunosorbent assay was performed on 195 patient sera: 34 with infections due to environmental mycobacteria, 114 with tuberculosis, 47 with other respiratory diseases. There were 46 human immunodeficiency virus-1 infected individuals. Among the 34 infections due to environmental mycobacteria, 9 patients were singularly infected with an environmental mycobacterium, and 25 co-infected with both M. tuberculosis and an environmental mycobacterium. Sensitivity, specificity and false positivity ranges were determined for each of the volunteer groups: tuberculosis positive, human immunodeficiency virus negative; tuberculosis positive, human immunodeficiency virus positive; those with infections due to individual environmental mycobacteria (such as M. scrofulaceum and M. kansasii); and those with other respiratory diseases. We demonstrate that such multiple assays, can be useful for the early diagnosis of diverse environmental mycobacterial infections to allow the start of treatment earlier than henceforth.     

A Hospital-Based Serological Survey of Cryptosporidiosis in the Republic of Korea

The seroprevalence of cryptosporidiosis was examined using patients'' sera collected from hospitals located in 4 different areas of the Republic of Korea. ELISA was used to measure antibody titers against Cryptosporidium parvum antigens from a total of 2,394 serum samples, which were collected randomly from patients in local hospitals; 1) Chungbuk National University Hospital, 2) Konkuk University Hospital, 3) local hospitals in Chuncheon, Gangwon-do (province), 4) Jeonnam National University Hospital, from 2002 through 2003. Of the 2,394 samples assayed, 34%, 26%, and 56% were positive for C. parvum-specific IgG, IgM, and IgA antibodies, respectively. Positive IgG titers were most common in sera from Jeonnam National University Hospital, Gwangju, Jeollanam-do, and positive IgM titers were most common in sera from Chungbuk National University Hospital, Cheongju, Chuncheongbuk-do. The seropositivity was positively correlated with age for both the IgG and IgA antibodies but was negatively correlated with age for the IgM antibodies. Western blotting revealed that 92%, 83%, and 77% of sera positive for IgG, IgM, and IgA ELISA reacted with 27-kDa antigens, respectively. These results suggested that infection with Cryptosporidium in hospital patients occurs more commonly than previously reported in the Republic of Korea.     

Human follicular dendritic cells express CR1, CR2, and CR3 complement receptor antigens

The expression of complement receptors by human follicular dendritic cells (FDC) was investigated by immunohistochemical techniques by using polyclonal and monoclonal antibodies to antigenic determinants of CR1, CR2, and CR3. Upon optical immunohistochemical examination of frozen sections from human reactive lymph nodes and tonsils by a three-step immunoperoxidase technique, a strong staining of cell bodies and cytoplasmic extensions of FDC was observed in germinal centers with anti-CR1 and anti-CR2 antibodies. Staining for these antigens was also found on cytoplasmic extensions of FDC in the mantle zone and on the plasma membrane of B cells in the entire follicles. Staining of FDC with anti-CR2 antibody was more intense than that of B lymphocytes. Monoclonal antibodies directed against epitopes of the alpha-chain of CR3 weakly stained FDC in follicles in a similar pattern to that which was observed on adjacent sections with mouse monoclonal antibody KIM4 that only recognizes FDC in human lymph nodes. Immunoelectron-microscopy was performed on frozen sections of a lymph node involved with a centroblastic centrocytic B malignant lymphoma and a reactive tonsil with the use of rabbit F(ab')2 anti-CR1 antibodies and mouse monoclonal anti-CR2 antibody. All the plasma membrane of the cell body and cytoplasmic extensions of FDC in germinal centers and in the mantle zones homogeneously stained for CR1 and CR2 antigens. Fibroblastic reticulum cells were negative. The plasma membrane of tumoral B lymphocytes strongly stained with anti-CR1 and weakly stained with anti-CR2 antibodies. The presence of CR1, CR2, and CR3 on FDC is a unique surface characteristic of these cells that should optimally allow the cells to bind antigen/antibody complexes bearing any type of C3 fragment.     

Immunochemical evidence for the transmembrane orientation of glycophorin A. Localization of ferritin-antibody conjugates in intact cells.

Antibodies were raised in rabbits to a 51-amino acid cyanogen bromide-derived peptide of human erythrocyte glycophorin A which has been shown to represent the C-terminal end of the 131-residue polypeptide chain. Antibodies prepared by immunoadsorption were found to be directed against a chymotryptic-derived peptide (residues 102 to 118) of glycophorin A but were unreactive with either intact or proteolytically modified red blood cells. No cross-reactivity was observed with glycophorin B of human or sialoglycoproteins prepared from red blood cells of other mammalian species. Ferritin-antibody conjugates of such sera were applied to thin sections of intact red blood cells (frozen or protein embedded) and were found to localize exclusively to sites distributed uniformly along the inner surfaces of the membrane. No staining was seen on sections prepared from red blood cells from other species nor on sections of human red cells pretreated with unconjugated antisera. These results provide additional evidence in intact, fixed human erythrocytes that glycophorin A has a transmembrane orientation.     

Tumor-associated neural differentiation antigens detected on C 1300 neuroblastoma cells by hybridoma monoclonal autoantibodies

Immune isoantisera and hybridoma monoclonal autoantibodies against syngeneic C1300 neuroblastoma (NB) cells were produced from BALB/c mice. Isoantisera were obtained (i) from mice immunized with membrane preparations from cloned NB cells and (ii) from mice bearing NB tumors. After repetitive absorptions on several different syngeneic or allogeneic tumor cell lines and syngeneic normal kidney, liver, spleen, bone marrow, and brain mouse tissue powders, these sera still retained antibodies reacting with tissue-differentiation antigens present on both NB cells and normal nerve sympathetic cells on cryostat whole body sections of neonatal mice. Monoclonal autoantibodies against NB cells were the products of the fusion between plasmacytoma cells and spleen cells from mice bearing syngeneic NB tumors. These anti-NB monoclonal antibodies revealed a restricted spectrum of distinct alloantigenic specificities against syngeneic bone marrow, fetal and adult brain cells, and nerve sympathetic cells present on neonatal rather than adult mice. A mixture of four monoclonal antibodies, recognizing, respectively, an epitope of the Ia complex and three distinctive neuronal-restricted antigens, proved to be a powerful and specific probe for histological immunodiagnosis of neuroblastoma, on cryostat sections of NB tumors, metastases, and tumor-draining lymph nodes.     

Detection and Assay of Avian Tumor Virus Group-Specific Antigen and Antibody by the Paired Radioiodine-Labeled Antibody Technique

The paired radioiodine-labeled antibody technique (PRILAT) was applied to the detection and quantitation of avian tumor virus group-specific (gs) antigens and antibody. The technique proved to be specific, repeatable, and appreciably more sensitive than the microcomplement-fixation test for avian leukosis (COFAL). The PRILAT facilitated direct measurement of comparative antigen content of several types of transformed, neoplastic, or virus-infected cells and the magnitude of nonspecific antibody binding by appropriate control cells. The versatility of the technique was illustrated by application to the detection and quantitation of gs antibody content of chicken, turkey, pigeon, and hamster sera. Antibodies were detected in COFAL-negative sera from hamsters bearing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus. Sera from chickens bearing similar tumors were not positive for gs antibodies, although sera from turkeys and chickens immunized with avian leukosis virus did contain gs antibodies.     

LOCALIZATION OF ANTIBODIES BY ELECTRON MICROSCOPY IN DEVELOPING ANTIBODY-PRODUCING CELLS

The development of antibody-producing cells in the early stages of the secondary or hyperimmune response has been studied with the electron microscope in lymph nodes of adult chinchilla rabbits immunized with ferritin or apoferritin. The intracellular distribution of antiferritin antibodies was determined in the lymph node cells at 1 to 5 days after a booster injection, employing the labelling technique previously used by the authors (12) to demonstrate the localization of antibodies in mature plasma cells. Antibodies were first detected at 48 hours in blasts; i.e., cells which have a poorly developed endoplasmic reticulum and a cytoplasm filled with many ribosomes grouped in clusters. The label was subsequently found in forms intermediate between blasts and plasma cells (plasmoblasts, immature plasma cells), in which the endoplasmic reticulum appeared progressively more developed. Antiferritin antibodies were also found in cells in mitosis. In all the above cell types, antigen-antibody precipitates were consistently found in the perinuclear space and in the cisternae of the granular endoplasmic reticulum, from an early stage in the development of the latter. Evidence was also obtained for the presence of antibody in the Golgi area. The results are discussed in relation to the possible cellular sites of antibody synthesis.     

Profiling serum antibodies to Mycobacterium tuberculosis proteins in rhesus monkeys with nontuberculous Mycobacteria

Recent evidence indicates that the prevalence of diseases caused by nontuberculous mycobacteria (NTM) has been increasing in both human and animals. In this study, antibody profiles of NTM in rhesus monkeys (Macaca mulatta) were determined and compared with those of monkeys infected with Mycobacterium tuberculosis complex (MTBC). Antibodies against 10 M. tuberculosis proteins, purified protein derivative (PPD), and mammalian old tuberculin (MOT) were detected in 14 monkeys naturally infected with NTM by indirect ELISA. Sera from 10 monkeys infected with MTBC and 10 healthy monkeys were set as controls. All antigens showed high serological reactivities to MTBC infections and low reactivities in healthy monkeys. NTM infections showed strong antibody responses to MOT and PPD; moderate antibody responses to 16kDa, U1, MPT64L, 14kDa, and TB16.3; and low antibody responses to 38kDa, Ag85b, CFP10, ESAT-6, and CFP10-ESAT-6. According to the criteria of MTBC, only CFP10, ESAT-6, and CFP10-ESAT-6 showed negative antibody responses in all NTM infections. Taken together, these results suggest that positive results of a PPD/MOT-based ELISA in combination with results of antibodies to M. tuberculosis-specific antigens, such as CFP10 and ESAT-6, could discriminate NTM and MTBC infections. Two positive results indicate an MTBC infection, and a negative result for an M. tuberculosis-specific antigen may preliminarily predict an NTM infection.     

On the dynamics of population changes of plasma cells during antibody formation

The dynamics of changes of the types of all plasma cells and of the antibody-synthesizing cells against the given antigen were studied on regionary lymph nodes of rabbits immunized twice with diphtheria toxoid (staining of sections of the nodes with methyl-green-pyronine and the indirect method of Coons for antibodies). On the basis of comparison of the ratio of young plasma cells to mature plasma cells characteristics of the course of differentiation of plasma cells synthesizing and not synthesizing antibodies were evaluated. It was shown that differentiation of plasma cells does not proceed in the same way in the secondary and primary antibody formation. In the last case, differentiation of antibody-synthesizing cells proceeds differently from cells not synthesizing antibodies. Data obtained on differences in histogenesis of plasma cells are discussed.     

Effect of Porphyromonas gingivalis outer membrane vesicles on gingipain-mediated detachment of cultured oral epithelial cells and immune responses

Porphyromonas gingivalis is a major etiological agent of periodontal diseases and the outer membrane vesicles (OMVs) contain virulence factors such as LPS and gingipains. In this study, we investigated the potential role of the OMVs in host immune response and tissue destruction during P. gingivalis infection. Firstly, we found that sera from periodontitis patients had significantly stronger reactivity against an OMV-producing wild type strain than the isogenic OMV-depleted strain. OMVs were found to be highly antigenic, as absorption of patient sera with OMVs greatly reduced reactivity with whole cells of P. gingivalis. LC-MS/MS analysis of OMVs revealed multiple forms of gingipains and several gingipain-related proteins. Western blots of OMVs using patient sera revealed a conserved immunoreactive antigen profile resembling the profile of OMV antigens that were recognized by gingipain antiserum, suggesting a potential role of OMV-associated gingipains in triggering antibody-mediated immune responses to P. gingivalis infection. When OMVs were added to a monolayer of an oral squamous epithelial cell line, OMVs caused cell detachment, which was inhibited by preincubating OMVs with anti-gingipain antiserum. These data suggest that gingipain-laden OMVs may contribute to tissue destruction in periodontal diseases by serving as a vehicle for the antigens and active proteases.     

Embedding and Fixation Techniques for Immunohistochemical Staining with Anti-DNA Polymerase α and Ki-67 Monoclonal Antibodies to Analyze the Proliferative Potential of Tumors

Anti-DNA polymerase a and Ki-67 are monoclonal antibodies that recognize nuclear antigens expressed in proliferating cells. In this study, we evaluated various methods of embedding and fixing brain tumor specimens to optimize staining with these antibodies. In fresh frozen sections, postfixation with 4% paraformaldehyde, 100% methanol, 95% ethanol and 10% buffered formalin were tested; also tested were prefixation with 4% paraformaldehyde followed by freezing and fixation with 100% methanol, 95% ethanol, or 10% buffered formalin followed by embedding in paraffin. For both antibodies, postfixation of fresh frozen sections with 4% paraformaldehyde at 4 C gave the most intense staining and lowest background activity while preserving histological features. This technique can be used in routine clinical practice to predict the growth potential of tumors.     

Seroprevalence of Encephalitozoon cuniculi and Toxoplasma gondii in domestic rabbits (Oryctolagus cuniculus) in China

The breeding of domestic rabbits (Oryctolagus cuniculus) for human consumption has a long tradition in China. Infections that can affect the production of meat or even be transmitted from animals to humans are important to monitor, especially for public health reasons as well as for their impact on animal health. Thus, a total of 1,132 domestic rabbit sera from 4 regions in China were collected for serological screening for Encephalitozoon cuniculi and for Toxoplasma gondii by ELISA and modified agglutination test (MAT), respectively. Antibodies to E. cuniculi were detected in 248/1,132 (21.9%) sera tested while antibodies against T. gondii revealed a seroprevalence of 51/1,132 (4.5%). We believe that the present results are of epidemiological implications and public health importance due to the acknowledged susceptibility of humans to E. cuniculi and T. gondii infections. Therefore, routine screening tests of domestic rabbits are proposed considering the zoonotic potential of these parasites.