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Today, serodiagnostic tests for Mycoplasma suis infections in pigs have low accuracies. The development of novel serodiagnostic strategies requires a detailed analysis of the humoral immune response elicited by M. suis and, in particular, the identification of antigenic proteins of the agent. For this study, indirect enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses were performed using pre- and sequential postinoculation sera from M. suis-infected and mock-infected control pigs. M. suis purified from porcine blood served as the antigen. Eight M. suis-specific antigens (p33, p40, p45, p57, p61, p70, p73, and p83) were identified as targets of the immunoglobulin G (IgG) antibody response during experimental infection, with p40, p45, and p70 being the preferentially recognized M. suis antigens. Besides the M. suis-specific antigens, porcine immunoglobulins were identified in blood-derived M. suis preparations. By immunoglobulin depletion, the specificity of the M. suis antigen for use in indirect ELISA was significantly improved. M. suis-specific Western blot and ELISA reactions were observed in all infected pigs by 14 days postinfection at the latest and until week 14, the end of the experiments. During acute clinical attacks of eperythrozoonosis, a derailment of the antibody response, determined by decreases in both the M. suis net ELISA values and the numbers of M. suis-specific immunoblot bands, was accompanied by peaking levels of autoreactive IgG antibodies. In conclusion, the M. suis-specific antigens found to stimulate specific IgG antibodies are potentially useful for the development of novel serodiagnostic tests.

Background

In autoimmune haemolytic anaemia (AIHA), autoreactive antibodies directed against red blood cells are up-regulated, leading to erythrocyte death. Mycoplasma suis infections in pigs induce AIHA of both the warm and cold types. The aim of this study was to identify the target autoantigens of warm autoreactive IgG antibodies. Sera from experimentally M. suis-infected pigs were screened for autoreactivity.

Results

Actin-reactive antibodies were found in the sera of 95% of all animals tested. The reactivity was species-specific, i.e. reactivity with porcine actin was significantly higher than with rabbit actin. Sera of animals previously immunised with the M. suis adhesion protein MSG1 showed reactivity with actin prior to infection with M. suis indicating that molecular mimicry is involved in the specific autoreactive mechanism. A potentially cross-reactive epitope was detected.

Conclusions

This is the first report of autoreactive anti-actin antibodies involved in the pathogenesis of autoimmune haemolytic anaemia.

The use of recombinant fragments of the major surface glycoprotein (Msg) of Pneumocystis jirovecii has proven useful for studying serological immune responses of blood donors and human immunodeficiency virus (HIV)-positive (HIV+) patients. Here, we have used ELISA to measure antibody titres to Msg fragments (MsgA, MsgB, MsgC1, MsgC3, MsgC8 and MsgC9) in sera isolated in the USA and Spain, to determine whether geographical location affects serological responses to these antigens. Blood donors from Seville exhibited a significantly greater antibody titre to MsgC8, and significantly lower responses to MsgC3 and MsgC9, than did Cincinnati (USA) donors. Spanish blood donors also exhibited elevated responses to MsgC1, MsgC8 and MsgC9 as compared with Spanish HIV+ patients . HIV+ patients who had Pneumocystis pneumonia (PcP+) exhibited a higher response to MsgC8 than did HIV+ PcP- patients. These data show that geographical location plays a role in responsiveness to Msg fragments. Additionally, these fragments have utility in differentiating between patient populations.

Streptococcus suis is an important porcine pathogen causing meningitis and other invasive diseases in piglets of different ages. Application of S. suis serotype 2 bacterins to specific-pathogen-free (SPF) weaning piglets has been demonstrated to protect against the homologous serotype. However, autogenous S. suis bacterins are also applied to sows and suckling piglets in the field. Therefore, comparative evaluation of different bacterin immunization regimes, including sow vaccination, was performed in this study. The main objectives were to determine the immunogenicity of an S. suis bacterin in sows prepartum and its influence on active immunization of piglets. Experimental infection of 6- and 8-week-old weaning piglets was performed to elucidate protective efficacies. Humoral immune responses were investigated by an enzyme-linked immunosorbent assay (ELISA) measuring muramidase-released protein (MRP)-specific IgG titers and by opsonophagocytosis assays. Bacterin application elicited high MRP-specific IgG titers in the serum and colostrum of sows, as well as opsonizing antibodies. Piglets from vaccinated sows had significantly higher MRP-specific titers than respective piglets from nonvaccinated sows until 6 weeks postpartum. Vaccination of suckling piglets did not result in high MRP-specific titers nor in induction of opsonizing antibodies. Furthermore, neither vaccination of suckling nor of weaning piglets from immunized sows was associated with a prominent active immune response and protection at 8 weeks postpartum. However, protection was observed in respective 6-week-old weaning piglets, most likely because of protective maternal immunity. In conclusion, this study provides the first results suggesting protective passive maternal immunity for S. suis serotype 2 after bacterin vaccination of sows and a strong inhibitory effect on active immunization of suckling and weaning piglets, leading to highly susceptible growers.

Helicobacter (H.) suis is a porcine and human gastric pathogen. Previous studies in mice showed that an H. suis infection does not result in protective immunity, whereas immunization with H. suis whole-cell lysate (lysate) protects against a subsequent experimental infection. Therefore, two-dimensional gel electrophoresis of H. suis proteins was performed followed by immunoblotting with pooled sera from H. suis- infected mice or mice immunized with lysate. Weak reactivity against H. suis proteins was observed in post-infection sera. Sera from lysate-immunized mice, however, showed immunoreactivity against a total of 19 protein spots which were identified using LC-MS/MS. The H. suis urease subunit B (UreB) showed most pronounced reactivity against sera from lysate-immunized mice and was not detected with sera from infected mice. None of the pooled sera detected H. suis neutrophil-activating protein A (NapA). The protective efficacy of intranasal vaccination of BALB/c mice with H. suis UreB and NapA, both recombinantly expressed in Escherichia coli (rUreB and rNapA, respectively), was compared with that of H. suis lysate. All vaccines contained choleratoxin as adjuvant. Immunization of mice with rUreB and lysate induced a significant reduction of H. suis colonization compared to non-vaccinated H. suis-infected controls, whereas rNapA had no significant protective effect. Probably, a combination of local Th1 and Th17 responses, complemented by antibody responses play a role in the protective immunity against H. suis infections.

Cameroon lacks the capacity for routine Pneumocystis pneumonia (PcP) diagnosis thus, the prevalence of Cameroonian exposure to this microbe is unknown. It is known that Pneumocystis infecting different mammalian host species represent diverse phylogenetic backgrounds and are now designated as separate species. The highly sensitive nature of ELISA and the specificity afforded by using human-derived P. jirovecii Msg peptides has been shown to be useful for serological analysis of human sera. Thus, sera from patients in Yaoundé, the capital city of Cameroon, were analyzed for anti-P. jirovecii antibodies by enzyme-linked immunosorbent assay (ELISA) using three recombinant major surface glycoprotein (Msg) peptide fragments, MsgA1, MsgB, and MsgC1. Based on serum recognition of one or more of the three fragments, 82% of the total samples analyzed was positive for antibodies to P. jirovecii Msg, indicating high prevalence of P. jirovecii infection or colonization among Cameroonians. Different Msg fragments appear to be recognized more frequently by sera from different geographic regions of the globe. Antibodies in the Cameroonian serum samples recognized MsgA>MsgC>MsgB, suggesting that different P. jirovecii strains exist in different parts of the world and/or human populations differ in their response to P. jirovecii. Also, HIV+ patients diagnosed with respiratory infections (such as TB and pneumonia) and maintained on trimethoprim/sulfamethoxazol prophylaxis had relatively lower anti-Msg titers. Whether PcP prophylaxis has significant effects on the quality of life among HIV+ patients in Cameroon warrants further investigation.

Background

Mycoplasma suis belongs to a group of highly specialized hemotrophic bacteria that attach to the surface of host erythrocytes. Hemotrophic mycoplasmas are uncultivable and the genomes are not sequenced so far. Therefore, there is a need for the clarification of essential metabolic pathways which could be crucial barriers for the establishment of an in vitro cultivation system for these veterinary significant bacteria.

Inorganic pyrophosphatases (PPase) are important enzymes that catalyze the hydrolysis of inorganic pyrophosphate PPi to inorganic phosphate Pi. PPases are essential and ubiquitous metal-dependent enzymes providing a thermodynamic pull for many biosynthetic reactions. Here, we describe the identification, recombinant production and characterization of the soluble (s)PPase of Mycoplasma suis.

Results

Screening of genomic M. suis libraries was used to identify a gene encoding the M. suis inorganic pyrophosphatase (sPPase). The M. suis sPPase consists of 164 amino acids with a molecular mass of 20 kDa. The highest identity of 63.7% was found to the M. penetrans sPPase. The typical 13 active site residues as well as the cation binding signature could be also identified in the M. suis sPPase. The activity of the M. suis enzyme was strongly dependent on Mg2+ and significantly lower in the presence of Mn2+ and Zn2+. Addition of Ca2+ and EDTA inhibited the M. suis sPPase activity. These characteristics confirmed the affiliation of the M. suis PPase to family I soluble PPases. The highest activity was determined at pH 9.0. In M. suis the sPPase builds tetramers of 80 kDa which were detected by convalescent sera from experimentally M. suis infected pigs.

Conclusion

The identification and characterization of the sPPase of M. suis is an additional step towards the clarification of the metabolism of hemotrophic mycoplasmas and, thus, important for the establishment of an in vitro cultivation system. As an antigenic and conserved protein the M. suis sPPase could in future be further analyzed as a diagnostic antigen.

A recombinant antigen cocktail enzyme-linked immunosorbent assay (ELISA) for diagnosis of contagious bovine pleuropneumonia (CBPP) was developed after careful selection of antigens among one-third of the surface proteome proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony (M. mycoides SC). First, a miniaturized and parallelized assay system employing antigen suspension bead array technology was used to screen 97 bovine sera for humoral immune responses toward 61 recombinant surface proteins from M. mycoides SC. Statistical analysis of the data resulted in selection of eight proteins that showed strong serologic responses in CBPP-affected sera and minimal reactivity in negative control sera, with P values of <10−6. Only minor cross-reactivity to hyperimmune sera against other mycoplasmas was observed. When applied in an ELISA, the cocktail of eight recombinant antigens allowed a fivefold signal separation between 24 CBPP-affected and 23 CBPP-free sera from different geographical origins. No false-positive results and only two false-negative results were obtained. In conclusion, the selected recombinant mycoplasma antigens qualified as highly specific markers for CBPP and could be employed in both a suspension bead array platform and a cocktail ELISA setting. This set of proteins and technologies therefore offers a powerful combination to drive and further improve serological assays toward reliable, simple, and cost-effective diagnosis of CBPP.

The reactivity of monoclonal antibody (MAb) 12G12 was analyzed in regard to the main biovars of Brucella species and some members of the families Enterobacteriaceae and Vibrionaceae which present serological cross-reactions with the smooth lipopolysaccharide (S-LPS) of Brucella species. This MAb was strictly directed against the common specific epitope of the Brucella S-LPS. It recognized all of the smooth Brucella strains and biovars except B. suis biovar 2. In order to improve the specificity of the serological diagnosis of brucellosis, a competitive enzyme-linked immunosorbent assay (cELISA) was developed with the horseradish peroxidase-conjugated MAbs 12G12 and S-LPS of B. melitensis Rev1. The specificity of the cELISA was analyzed with 936 serum samples from healthy cattle. The assay was evaluated with sera from heifers (n = 18) experimentally infected with B. abortus 544. After infection, the performance of the cELISA was in agreement with those of the complement fixation test and the rose Bengal plate test. Finally, the specificity of the assay was also evaluated in regard to false-positive serological reactions by using sera from heifers experimentally infected with Yersinia enterocolitica 0:9 (n = 4) and with field sera presenting false-positive reactions (n = 74). The specificity of the cELISA was greater than the specificities of the complement fixation test and the rose Bengal plate test. Indeed, the new assay detected only 31 of the 101 false-positive serum samples detected by at least one serological test.

Mycoplasma suis, a member of the hemotrophic mycoplasma (HM) group, parasitize erythrocytes of pigs. Increasing evidence suggests that M. suis is also a zoonotic agent. Highly pathogenic strains of M. suis (e.g., M. suis KI3806) have been demonstrated to invade erythrocytes. This complete sequenced and manually annotated genome of M. suis KI3806 is the first available from this species and from the HM group. The DNA was isolated from blood samples of experimentally infected pigs due to the lack of an in vitro cultivation system. The small circular chromosome of 709,270 bp, encoding an unexpectedly high number of hypothetical proteins and limited transport and metabolic capacities, could reflect the unique lifestyle of HM on the surface of erythrocytes.

An indirect enzyme-linked immunosorbent assay (IELISA), a competitive ELISA (CELISA), and a fluorescence polarization assay (FPA) for the presumptive serological diagnosis of swine brucellosis were evaluated using two populations of swine sera: sera from brucellosis-free Canadian herds and sera from Argentina selected based on positive reactions in the buffered antigen plate agglutination test (BPAT) and the 2-mercaptoethanol (2-ME) test. In addition, sera from adult swine from which Brucella suis was isolated at least once for each farm of origin were evaluated. The IELISA, CELISA, and FPA specificity values were 99.9, 99.5, and 98.3%, respectively, and the IELISA, CELISA, and FPA sensitivity values relative to the BPAT and the 2-ME test were 98.9, 96.6, and 93.8%, respectively. Actual sensitivity was assessed by using 37 sera from individual pigs from which B. suis was cultured, and the values obtained were as follows: BPAT, 86.5%; 2-ME test, 81.1%; IELISA, 86.5%; CELISA, 78.5%; and FPA, 80.0%.

Capsular polysaccharide antigens of serotypes 1, 2, 5 and 7 of Actinobacillus pleuropneumoniae were used in enzyme-linked immunosorbent assays (ELISAs) to test sera from experimentally infected and field pigs. Specific reactions were found in sera of experimental pigs with antigens of serotypes 1, 5 and 7 whereas the serotype 2 antigen was cross-reactive. A 1:200 serum dilution was used for testing of 300 sera from 21 swine herds in southern Ontario. Cases of pleuropneumonia had occurred in 11 of these herds, but not in the others. The negative cut-off value was the mean optical density at 405 nm (OD405) + three standard deviations (SD) for 16 negative reference sera. Sera from four pigs naturally infected with Actinobacillus suis were tested and found to react to varying degrees with each of the antigens. Therefore a second cut-off value was determined as the mean OD405 + 2 SD for the A. suis sera. Sera which, in the ELISA produced OD readings above the latter cut-off were considered positive for antibodies to A. pleuropneumoniae; those which were lower than the former cut-off were considered negative. Readings between the two cut-off values may have been due to low positive titers or cross-reactivity, possibly with A. suis, and could not be used to predict pleuropneumonia. Of the pleuropneumonia-free herds, none had positive reactors to serotypes 5 or 7, whereas one and two herds had positive reactors to serotypes 1 and 2, respectively. Of the pleuropneumonia positive herds, six had positive reactors to serotype 1, one to serotype 2, four to serotype 5, and eight to serotype 7.

An enzyme-linked immunosorbent assay was developed using a heat-killed Brucella suis antigen for detecting antibodies in the sera of swine from which B. suis was isolated. Optimal enzyme-linked immunosorbent assay reactions were obtained using heat-killed B. suis antigen at a concentration comparable to McFarland Standard No. 1. Statistically significant differences were observed in the enzyme-linked immunosorbent assay results of 40 animals from which B. suis was isolated and the results for 48 noninfected swine at serum dilutions of 1:25 and 1:50 (P < 0.0001). The enzyme-linked immunosorbent assay is a rapid reproducible test which can be readily automated that appears to have practical value for screening large numbers of breeding and slaughter swine for brucellosis.

An autogenous vaccine was developed, using sonicated bacteria, with a strain of Streptococcus suis capsular type 1/2. The objectives of this study were to evaluate the antibody response following vaccination and to assess the changes in antibody levels in pigs from a herd showing clinical signs of S. suis capsular type 1/2 infection in 6- to 8-week-old pigs. An enzyme-linked immunosorbent assay using the vaccine antigen was standardized. Results from a preliminary study involving 2 control and 4 vaccinated 4-week-old pigs indicated that all vaccinated pigs produced antibodies against 2 proteins of 34 and 43 kDa, respectively, and, in 3 out of 4 vaccinated pigs, against the 117-kDa muramidase-released protein. For the serologic profile, groups of 30 pigs from the infected herd were blood sampled at 2, 4, 6, 8, and 10 weeks of age. The lowest antibody level was observed between weeks 6 and 8, presumably corresponding to a decrease in maternal immunity. A marked increase was seen at 10 weeks of age, shortly after the onset of clinical signs in the herd. For the vaccination field trial, newly weaned, one-week-old piglets were divided into 2 groups of 200 piglets each (control and vaccinated); blood samples were collected from 36 piglets in each group at 2-week intervals for 12 weeks. A significant increase (P 0.05) in antibody response was observed 4 weeks following vaccination and the level of antibodies stayed high until the end of the experiment. In the control group, the increase was only observed at 13 weeks of age, probably in response to a natural infection. The response to the vaccine varied considerably among pigs and was attributed, in part, to the levels of maternal antibodies at the time of vaccination. No outbreak of S. suis was observed in the control or vaccinated groups, so the protection conferred by the vaccine could not be evaluated.

Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactivity with patient sera and the most antigenic was further assessed for its serodiagnostic potential by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA based on the recombinant protein was equivalent in sensitivity to the commercial test (Serodia Myco II; Fujirebio Inc.) to which it was compared. Southern and Western blotting data suggested that the recombinant protein derived from the 116-kDa protein of M. pneumoniae could provide a species-specific diagnostic tool, although further assessment is required.

Summary

Objectives

To characterize the seroepidemiological features of Pneumocystis jirovecii infection in healthy Chilean children using overlapping fragments (A, B, C) of the P. jirovecii major surface glycoprotein (Msg).

Methods

Serum antibodies to MsgA, MsgB, and MsgC were measured every 2 months by enzyme-linked immunosorbent assay (ELISA) in 45 Chilean infants from about age 2 months to 2 years.

Results

Peak antibody levels (usually reached at age 6 months) and the force (or rate) of infection were somewhat greater for MsgC than for MsgA. Significant seasonal variation in antibody levels was only found with MsgA. Respiratory infections occurred in most children, but nasopharyngeal aspirates were of limited value in detecting the organism. In contrast, serological responses commonly occurred, and higher levels only to MsgC were significantly related to the number of infections.

Conclusions

Serological responses to recombinant Msg fragments provide new insights into the epidemiological and clinical features of P. jirovecii infection of early childhood. MsgA, the amino terminus fragment, is more sensitive in detecting seasonal influences on antibody levels, whereas MsgC is better able to detect changes in antibody levels in response to clinical infection.

Twenty Hereford heifers approximately 9 months of age were vaccinated with saline (control) or 2 × 1010 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 107 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.

A rabbit homologous polyclonal antiserum to the 104-kilodalton hemolysin of Actinobacillus pleuropneumoniae serotype 1 strain CM-5 was specifically produced and used in an antigen capture enzyme-linked immunosorbent assay (ELISA) to detect swine serum antibodies to this potentially important virulence factor. Sera from pigs experimentally infected with the most common disease-producing serotypes (serotypes 1, 2, 5, and 7) of A. pleuropneumoniae produced positive results in this ELISA. Of 144 serum samples collected from 10 herds free of pleuropneumonia and 155 serum samples from 11 herds with a history of the disease, 68 (47%) and 148 (95%), respectively, were found positive by the ELISA. In addition, pigs naturally infected with Actinobacillus suis produced antibodies which seroreacted in this ELISA. The results indicated that a high proportion of swine have antibodies seroreactive with the 104-kilodalton hemolysin produced by A. pleuropneumoniae.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM antibodies against Mycoplasma pneumoniae, performed with commercial antigen and reagents, is compared with the complement fixation test (CF) in a serological study of 209 human sera. Concordant results were usually obtained by CF test and by IgG ELISA in sera from patients with recent M pneumoniae infection. In contrast, when used for an immunological survey of a general population, approximately 27% of the sera negative in the CF test were positive for IgG by the ELISA, and sera with low CF titres were found to have a broad range of IgG titre by the ELISA. This may be due to the greater sensitivity of the ELISA technique and/or to different types of antibody measured by both tests. IgM was detected by ELISA in sera from all patients with recent M pneumoniae infection diagnosed on the basis of clinical findings and by CF assay. Occasionally false-positive IgM antibodies were due to rheumatoid factor (RF); this potential interference necessitates routine testing of IgM antibody positive sera for RF.

Background

The immune responses to Pneumocystis jirovecii major surface glycoprotein (Msg) in individuals with human immunodeficiency virus (HIV) infection are poorly understood.

Methods

We examined the sequential serologic responses to recombinant Msg carboxyl terminus fragments (MsgC1, MsgC3, MsgC8, and MsgC9) by enzyme-linked immunosorbent assay in a cohort of individuals with HIV infection for the 5.5 years before death and autopsy. Analyses included mean antibody levels by status at death (Pneumocystis pneumonia, P. jirovecii colonization, or neither), factors associated with high antibody levels, and antibody responses before and after active Pneumocystis pneumonia.

Results

Patients who died from Pneumocystis pneumonia had higher levels of antibody to MsgC8 than did patients who died from other causes. Previous episode of Pneumocystis pneumonia, geographic location, and age were independent predictors of high levels of anitbodies to most or all Msgs. Failure to take Pneumocystis pneumonia prophylaxis was associated with high levels of antibody to MsgC1. Patients who developed and recovered from active Pneumocystis pneumonia during the study exhibited an increase in serum antibody levels that persisted for months after the infection, whereas patients who developed another acquired immunodeficiency syndrome–defining illness did not.

Conclusions

Serum antibodies to Msgs are important markers of P. jirovecii infection in patients with HIV infection and are influenced by host and environmental factors in complex ways.

The enzyme-linked immunosorbent assay (ELISA) indicated significant cross-reactivity between the antigens of Mycoplasma hyopneumoniae ( HyoP ) and M. flocculare (Floc), another porcine mycoplasma of wide distribution but uncertain pathogenic significance, when porcine antisera of each specificity were tested against HyoP antigen. The titers of the anti-Floc sera ranged from threefold to 13-fold less than the titer of the anti- HyoP reference serum at different times after immunization. These values ranged from onefold less than to fourfold greater than the minimal positive titer of 80. The antisera to the other porcine mycoplasmal antigens [i.e. M. hyorhinis ( HyoR ) and M. hyosynoviae ( HyoS )] reacted less strongly to HyoP antigen but titers only slightly less than to slightly greater than the minimal positive titer were noted for some sera. Cross-reactivity was also detected by the complement fixation test, although the titers for this test were generally lower than for the ELISA, presumably reflecting lower sensitivity of the complement fixation test. Positive indirect hemagglutination titers to HyoP antigen were also observed for both anti-Floc sera obtained at one or more times during the immune response. With two exceptions (one anti- HyoR serum with a complement fixation titer of 16 and one anti- HyoR serum with an indirect hemagglutination titer of 10), none of the anti- HyoR or anti- HyoS sera had detectable indirect hemagglutination or complement fixation titers to HyoP antigen at any time after immunization. The levels of cross-reactivity detected by the complement fixation test and indirect hemagglutination and, especially, the ELISA would be of significance for the development of any practical sero-diagnostic test for mycoplasmal pneumonia of swine.

Mycoplasma suis belongs to the hemotrophic mycoplasma group and causes infectious anemia in pigs. According to the present state of knowledge, this organism adheres to the surface of erythrocytes but does not invade them. We found a novel M. suis isolate that caused severe anemia in pigs with a fatal disease course. Interestingly, only marginal numbers of the bacteria were visible on and between the erythrocytes in acridine orange-stained blood smears for acutely diseased pigs, whereas very high loads of M. suis were detected in the same blood samples by quantitative PCR. These findings indicated that M. suis is capable of invading erythrocytes. By use of fluorescent labeling of M. suis and examination by confocal laser scanning microscopy, as well as scanning and transmission electron microscopy, we proved that the localization of M. suis was intracellular. This organism invades erythrocytes in an endocytosis-like process and is initially surrounded by two membranes, and it was also found floating freely in the cytoplasm. In conclusion, we were able to prove for the first time that a member of the hemotrophic mycoplasma group is able to invade the erythrocytes of its host. Such colonization should protect the bacterial cells from the host's immune response and hamper antibiotic treatment. In addition, an intracellular life cycle may explain the chronic nature of hemotrophic mycoplasma infections and should serve as the foundation for novel strategies in hemotrophic mycoplasma research (e.g., treatment or prophylaxis).

Sera from six outbreaks of legionellosis and four outbreaks of pneumonia of other etiologies were tested with the indirect immunofluorescence assay (IFA) as currently performed. The current IFA is at least as sensitive as the original test in detecting cases of Legionnaires disease (78 to 91%). By using Center for Disease Control criteria for a positive (fourfold increase in titer during convalescence to greater than or equal to 128) or presumptive (single titer greater than or equal to 256) serological test, the specificity exceeded 99%. No cross-reactions against Legionella pneumophila antigens were observed among sera from epidemic cases of Q fever, tularemia, and psittacosis; the only positive L. pneumophila IFA titer among the epidemic Mycoplasma pneumonia sera was reduced to a negative titer with an immunosorbent extracted from Escherichia coli strain O13:K92:H4. The slight increase in specificity (to 100%), however, was offset by a slight decrease in sensitivity. The sensitivity of the IFA was maximal when a conjugate that detected immunoglobulins G, M, and A was used. IFA titers were not significantly altered by replacing the monovalent serogroup 1 antigen with a polyvalent antigen (serogroups 1 through 4) nor by the presence of rheumatoid factor or heat-labile serum factors.

Images

Hemotrophic mycoplasmas, bacteria without cell walls whose niche is the erythrocytes of their hosts, have never been cultivated in vitro. Therefore, knowledge of their pathogenesis is fundamental. Mycoplasma suis infects pigs, causing either acute fatal hemolytic anemia or chronic low-grade anemia, growth retardation, and immune suppression. Recently, the complete genomes of two hemotrophic mycoplasma species, M. suis and M. haemofelis, were sequenced, offering new strategies for the analysis of their pathogenesis. In this study we implemented a proteomic approach to identify M. suis proteins during acute infection by using tandem mass spectrometry. Twenty-two percent of the predicted proteins encoded in M. suis strain KI_3806 were identified. These included nearly all encoded proteins of glycolysis and nucleotide metabolism. The proteins for lipid metabolism, however, were underrepresented. A high proportion of the detected proteins are involved in information storage and processing (72.6%). In addition, several proteins of different functionalities, i.e., posttranslational modification, membrane genesis, signal transduction, intracellular trafficking, inorganic ion transport, and defense mechanisms, were identified. In its reduced genome, M. suis harbors 65.3% (strain Illinois) and 65.9% (strain KI_3806) of the genes encode hypothetical proteins. Of these, only 6.3% were identified at the proteome level. All proteins identified in this study are present in both M. suis strains and are encoded in more highly conserved regions of the genome sequence. In conclusion, our proteome approach is a further step toward the elucidation of the pathogenesis and life cycle of M. suis as well as the establishment of an in vitro cultivation system.

Background

Little is known about the serologic responses to Pneumocystis jirovecii major surface glycoprotein (Msg) antigen in African cohorts, or the IgM responses to Msg in HIV-positive and HIV-negative persons with respiratory symptoms.

Methods

We conducted a prospective study of 550 patients, both HIV-positive (n = 467) and HIV-negative (n = 83), hospitalized with cough ≥2 weeks in Kampala, Uganda, to evaluate the association between HIV status, CD4 cell count, and other clinical predictors and antibody responses to P. jirovecii. We utilized ELISA to measure the IgM and IgG serologic responses to three overlapping recombinant fragments that span the P. jirovecii major surface glycoprotein: MsgA (amino terminus), MsgB (middle portion) and MsgC1 (carboxyl terminus), and to three variations of MsgC1 (MsgC3, MsgC8 and MsgC9).

Results

HIV-positive patients demonstrated significantly lower IgM antibody responses to MsgC1, MsgC3, MsgC8 and MsgC9 compared to HIV-negative patients. We found the same pattern of low IgM antibody responses to MsgC1, MsgC3, MsgC8 and MsgC9 among HIV-positive patients with a CD4 cell count <200 cells/µl compared to those with a CD4 cell count ≥200 cells/µl. HIV-positive patients on PCP prophylaxis had significantly lower IgM responses to MsgC3 and MsgC9, and lower IgG responses to MsgA, MsgC1, MsgC3, and MsgC8. In contrast, cigarette smoking was associated with increased IgM antibody responses to MsgC1 and MsgC3 but was not associated with IgG responses. We evaluated IgM and IgG as predictors of mortality. Lower IgM responses to MsgC3 and MsgC8 were both associated with increased in-hospital mortality.

Conclusions

HIV infection and degree of immunosuppression are associated with reduced IgM responses to Msg. In addition, low IgM responses to MsgC3 and MsgC8 are associated with increased mortality.