Today, serodiagnostic tests for Mycoplasmasuis 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 postin- oculation 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 immu- noglobulins 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.
Pigs are popular animal models in biomedical research. RNA-Seq is becoming the predominant tool to investigate transcriptional changes of the pig’s response to infection. The high sensitivity of this tool requires a strict control of the study design beginning with the selection of healthy animals to provide accurate interpretation of research data. Pigs chronically infected with Mycoplasmasuis often show no obvious clinical signs, however the infection may affect the validity of animal research. The goal of this study was to investigate whether or not this silent infection is also silent at the host transcriptional level. Therefore, immunocompetent pigs were experimentally infected with M. suis and transcriptional profiles of whole blood, generated by RNA-Seq, were analyzed and compared to non-infected ani- mals. RNA-Seq showed 55 differentially expressed (DE) genes in the M. suis infected pigs. Down-regulation of genes related to innate immunity (tlr8, chemokines, chemokines receptors) and genes containing IFN gamma-activated sequence (gbp1, gbp2, il15, cxcl10, casp1, cd274) suggests a general suppression of the immune response in the infected animals. Sixteen (29.09%) of the DE genes were involved in two protein interaction networks: one involving chemokines, chemokine receptors and interleukin-15 and another involving the complement cascade. Genes related to vascular permeability, blood coagulation, and endothelium integrity were also DE in infected pigs. These findings suggest that M. suis subclinical infection causes significant alterations in blood mRNA levels, which could impact data interpretation of research using pigs. Screening of pigs for M. suis infection before initiating animal studies is strongly recommended.
In all cases in which blood samples were PCR positive, direct DNA sequencing was performed to identify patho- gen species down to the level of strain when possible. Ref- erence sequences for this study included the following GenBank accession numbers: AY055469 (Anaplasma phagocytophilum); AF399916 (Anaplasma platys); KU710803 (Babesia vogeli); AY618928 (large unnamed Babesia sp. “coco”); AF271081 (Babesia gibsoni); NC_005956.1 (Bartonella henselae Houston I); AF369529 (B. henselae SA2); DQ059763 (Bartonella vinsonii berkhof- fii genotype II); DQ059764 (B. vinsonii berkhoffii genotype III); AF312490 (Bartonella koehlerae); AB896695 (Barto- nella clarridgeiae); NC_007354 (Ehrlichia canis); NR_044747 (Ehrlichia ewingii); AY529641 (Mycoplasma haemocanis); GQ129113 (Mycoplasma haematoparvum); NR_103930 and AB610849 (Mycoplasmasuis); and CP000848 (Rickettsia rickettsii). Sequences were compared to the GenBank database using the Basic Local Alignment Search Tool and homology data was generated using AlignX (Vector NTI suite 11.5.1, Invitrogen Inc., USA) software.
Die porcine Eperythrozoonose, verursacht durch Mycoplasmasuis, ist eine weltweit verbreite- te Infektionskrankheit und verläuft meist als latente Infektion. Zum Ausbruch der Erkrankung kommt es nach Belastungen wie Geburt, Umstallen, Absetzen, schlechten Haltungs- und Füt- terungsbedingungen oder weiteren Infektionskrankheiten. Ebenso zählen zootechnische Ein- griffe und Rangkämpfe zu den auslösenden Ursachen. Das Krankheitsbild wird bestimmt von einer hämolytischen Anämie, Ikterus und je nach Schweregrad Apathie, Anorexie, Dyspnoe und Fieber bis 42°C. Die erkrankten Tiere zeigen zyanotische Veränderungen an den Akren, bevorzugt an den Rändern der Ohrmuscheln. Durch die lebenslange Persistenz des Erregers im Körper kann es zu wiederholten Infektionsschüben kommen. Aufgrund dessen treten ver- zögertes Wachstum und somit verlängerte Aufzucht- und Mastperioden sowie erhöhte Anfäl- ligkeit gegenüber anderen Krankheiten auf. Die Tiere zeigen erhöhte Empfänglichkeit für Durchfall- und Respirationskrankheiten.
Porcine eperythrozoonosis is a disease with worldwide distribution caused by the unculturable hemotrophic bacterium Mycoplasmasuis. Current serological testing utilizes crude M. suis antigens purified from the blood of experimentally infected pigs. These antigens show high variability and are restricted to specialized labora- tories. We evaluated a novel serological assay based on two recombinant M. suis antigens (rMSG1 and rHspA1). Antigen specificity was proven by means of sera raised against nonhemotrophic mycoplasma and other relevant bacteria. Using experimental and convalescent-phase sera, rMSG1 and rHspA1 enzyme-linked immunosorbent assays (ELISAs) demonstrated sensitivities, specificities, and predictive values (94.0 to 100.0%) equal to or higher than those of the M. suis whole-cell ELISA. Field samples from 120 weaning piglets grouped by quantitative PCR results were used to evaluate the diagnostic capability of the new ELISA systems in comparison to that of the whole-cell ELISA. Assuming a 100.0% specificity of the PCR, the whole-cell ELISA, rHspA1 ELISA, and rMSG1 ELISA showed specificities of 84.8%, 83.8%, and 90.6% and sensitivities of 61.5%, 74.0% and 58.1%, respectively. Cohen’s kappa coefficients comparing the recombinant ELISAs to the whole-cell ELISA indicate moderate to substantial agreement. The detection of anti-MSG1 and/or anti-HspA1 antibodies in pigs was significantly correlated with decreased hematocrit, erythrocyte numbers, and hemoglobin concen- trations, indicating that a single seropositive result is connected with clinical and etiological significance. In conclusion, rMSG1 and rHspA1 are sensitive and specific serological and infection markers which are for the first time used independently of animal experiments. They are especially fit to be used in routine diagnosis, pathogenesis studies, and large-scale epidemiological investigations.
selectin and P-selectin are up-regulated by inflammation and mediate leukocyte capture and rolling on the endo- thelium . The up-regulation of E-selectin is asso- ciated with organ dysfunction and septic shock , both of which are seen in acute IAP. The time point of a maximum upregulation of activation markers by LPS and TNF-alpha might not represent the time point of a maximum upregulation by M. suis. This could explain the relatively smaller but significant difference between M. suis and negative control in case of E-selectin and PECAM-1. ICAM-1 is involved in leukocyte rolling and arrest on endothelial cells , as well as the movement of neutrophils and monocytes on the endothelium . Recently, ICAM-1 was identified as the receptor for rhinovirus . PECAM-1 is involved in the removal of apoptotic neutrophils from the body and makes up a large proportion of the endothelial cell intercellular junction [61,62], where it mediates transendothelial mi- gration via homotypic binding to PECAM-1 on leuko- cytes . In summary, these in vitro findings, including the up-regulation of endothelial adhesion receptors, to- gether with the observed structural changes of the endo- thelial layer in vivo, i.e. microvilli- and gap-formation, demonstrate that M. suis infection results in activation of EC.
Bisherige Untersuchungen zur intrauterinen Übertragung von M. suis kamen zu unterschiedlichen Ergebnissen, sodass keine definitive Aussage getroffen werden konnte. Erstmals wurde von Smith an der Universität Illinois über die Möglichkeit einer vertikalen Übertragung des Erregers berichtet, welcher mittels Wright- Giemsa gefärbten Ausstrichen in den Blutproben aller Ferkel eines mit M. suis infizierten Mutterschweines den hämotrophen Erreger nachweisen konnte (SMITH und RAHN, 1975). Um andere Infektionswege auszuschließen wurde in dieser Versuchsanordnung statt des natürlichen Geburtsweges ein Kaiserschnitt durchgeführt (SMITH und RAHN, 1975). Auch Henderson et al. beschrieben eine vertikale Transmission von M. suis. Da der Untersuchungszeitpunkt jedoch erst nach Kolostrumaufnahme und engem Kontakt zwischen Muttertier und Ferkel stattfand, kann eine Infektion post partum nicht sicher ausgeschlossen werden (HENDERSON et al., 1997). In der vorliegenden Arbeit wurde aufgrund der Versuchsanordnung, mit einer Beprobung der Ferkel unmittelbar nach Geburt und vor Aufnahme von Kolostrum sowie vor Kontakt zu anderen Tieren, die Wahrscheinlichkeit einer Infektion post partum minimiert. Eine Infektion der Ferkel auf dem Geburtsweg ist ebenso wenig wahrscheinlich, da laut den Untersuchungen von STADLER et al. (2014) M. suis bei nicht splenektomierten Schweinen frühestens 2 Tage nach experimenteller Infektion in der PCR detektierbar ist.
relative ease of use, ethical acceptability, and cost. Here we describe the development and characterization of mouse mod- els of infection with aerosolized B. melitensis and B. suis bac- teria and demonstrate the utility of these models in evaluating vaccines and therapeutics for inhalational brucellosis using the model live attenuated B. melitensis Rev.1 vaccine. A mouse model of intranasal (i.n.) infection with B. melitensis has pre- viously been described (12), and since we started this work, two other laboratories have described studies of infection of mice with aerosolized Brucella bacteria. Kahl-McDonagh et al. de- scribed aerosol infection of BALB/c mice with B. abortus 2308 and B. melitensis 16 M and the use of these models for the evaluation of protective efficacy of deletion mutants (10). Ol- sen et al. similarly described the infection of BALB/c mice with aerosolized strain 16 M or 2308 and demonstrated that vacci- nation with the live attenuated animal vaccine B. abortus RB51 provided protection against intraperitoneal but not aerosolized B. abortus challenge (13). Our findings both support and ex- tend the data generated in these studies.
is thought to share many of the virulence factors of Actinoba- cillus pleuropneumoniae and other gram-negative organisms such as capsule (slime) and lipopolysaccharide (23, 36). There are several lines of evidence that A. suis also carries RTX (repeats in toxin) toxins that are related to the Apx toxins of A. pleuropneumoniae (7, 8, 16). RTX toxins are pore-forming protein exotoxins with hemolytic and/or cytotoxic activity, so- named because of the presence of characteristic glycine-rich repeats (25, 39). Most RTX operons have an A gene that encodes the toxin (which typically has hemolytic and/or cyto- toxic activity), a C gene that encodes a protein required for the modification and activation of the structural A protein, and B and D genes that encode the proteins required for signal se- quence-independent transport. Using the leukotoxin (lktA) de- terminant from Pasteurella haemolytica, Burrows and Lo (7) reported the cloning and sequencing of genes with a high degree of sequence homology to the apxIIC and apxIIA genes of A. pleuropneumoniae. They designated these genes ashC and ashA, respectively, and noted that there were no linked trans- port (BD) gene homologs. Two immunological studies also supported the notion that A. suis isolates could produce an Apx-like toxin. In 1989, Devenish et al. (8) showed that con- valescent-phase serum from an A. suis-infected pig could rec- ognize a 104-kDa hemolysin from A. pleuropneumoniae and, conversely, that antisera to the A. pleuropneumoniae hemolysin cross-reacted with a high-molecular-weight protein from the supernatant of A. suis cultures. Later, Kamp et al. (16) re- ported that culture filtrates of A. suis were strongly cytotoxic and hemolytic and that monoclonal antibodies made to the ApxI and ApxII toxins from A. pleuropneumoniae reacted strongly with these filtrates in dot blot experiments.
The response of these mycoplasma-contaminated HeLa cells to EMCV markedly differed from that of mycoplasma-free cultures, as documented by a very strong TUNEL signal in the overwhelming majority of cells (Fig. 1B) and very intense DNA degradation to heterogeneous fragments, in particular, oligo- nucleosome-sized ones (Fig. 2B), the both features being typ- ical of apoptosis. The cells, however, did not display such apoptosis-associated signs as plasma membrane blebbing (Fig. 1B, inset) or nuclear fragmentation, although deformation of nuclei and moderate chromatin condensation could readily be observed upon Hoechst-33342 staining (Fig. 1B).These nuclear alterations, typical of CPE associated with EMCV reproduc- tion, did not appear to differ significantly from those observed in mycoplasma-free EMCV-infected cells (Fig. 1A). Remark- ably, the presence of mycoplasma did not appreciably change HeLa cell response to poliovirus infection: in line with previ- ous results (3, 80), there was no (or nearly no) signs of apop- tosis upon productive infection with this virus, as judged by the TUNEL assay (Fig. 1B) or DNA electrophoresis (see Fig. 4B). The mycoplasma-contaminated HeLa cells responded to known viral and nonviral inducers of apoptosis, such as abor- tive poliovirus infection (Fig. 1) and CHI (Fig. 1 and 2) qual- itatively in the same way as did mycoplasma-free cells, i.e., by development of TUNEL-positive staining and by DNA frag- mentation detectable electrophoretically. The presence of my- coplasma did not markedly alter rapid apoptotic response of HeLa cells to another apoptosis inducer, ActD (not shown).
Rates of urogenital mycoplasmosis associated with Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urea- lyticum, and Ureaplasma parvum have been reported numerous times, and frequently show a wide range of findings. Differing diagnostic techniques, population targeting, temporal and spatial data collection, and coincident infections make the conclusions from these analyses difficult to compare. We generated a single data set including the infection rate, geographic location, year, study population, diagnostic method, and clinical signs for these organisms by perform- ing literature searches with the species names and compiling the findings. Studies focusing on basic research or report- ing clinical surveys where these criteria were not reported were excluded. A statistical analysis of the dataset parameters found that: diagnostic method does not significantly correlate with positive specimen rate but does correlate with the year of publication, and the number of publications correlated significantly with year, indicating that this topic is of growing interest. Further analysis indicated that Ureaplasma species infection rate is significantly higher in pregnant women across all studies. Associations with distinct clinical presentation could not be made on datasets assembled across studies due to the number of confounding variables presented in each. The generated data set represents a large amount of temporal, geographic, and clinical data that can be utilized in future communications.
Mycoplasma pneumoniae (MP) was first isolat- ed from cultures of sputum of a patient with primary atypical pneumonia . What was referred to as “Eaton’s agent” was shown to be a Mycoplasma species. MP is a very small bac- terium in the class Mollicutes. It is a human pathogen that causes MP, a form of atypical bacterial pneumonia related to cold agglutinin disease. MP is characterized by the absence of a peptidoglycan cell wall and resulting resis- tance to many antibacterial agents. The persis- tence of MP infections even after treatment is associated with its ability to mimic the compo- sition of the cell surfaces of hosts.
It is not yet clear how exactly H. suis might influence ulcer development in the Pars œsophagea, but altera- tions in gastric acid secretion might be involved. No clear effects on the markers for gastric acid secretion or num- ber of parietal cells, D-cells and G-cells, and no lesions in the Pars œsophagea were detected in the H. suis- infected 2–3 months old pigs (acute phase of infection). In a later phase of infection (6–8 months H. suis infected pigs), markers for gastric acid secretion were downregu- lated, genes encoding somatostatin were upregulated and the number of G-cells was decreased, indicating inhibi- tion of gastric acid secretion. In this age group, lesions in the Pars œsophagea were present in several animals. The prevalence of severe lesions was extremely high in H. suis infected adult sows (chronic phase of infection). Markers for gastric acid secretion were upregulated and the number of G-cells was increased in this age group, indicating increased gastric acid secretion. We hypoth- esize that decreased gastric acid secretion in the glandu- lar part of the stomach may affect the composition of the Pars œsophageal microbiota which may affect develop- ment of lesions in this non-glandular part of the stomach. Indeed, higher numbers of a recently described Fusobac- terium species, designated F. gastrosuis, were detected in the Pars œsophagea of H. suis-infected 6–8 months old pigs than in non-infected pigs of the same age group . Increased production of gastric acid during the chronic phase of infection might further aggravate severity of lesions in this stomach region, which is not protected by mucus. Further studies in which for instance the gastric microbiota and pH are determined in H. suis-infected and non-infected pigs, are necessary to confirm or reject this hypothesis.
It has been previously reported that the number of overlapping genes increases with the genome size and the number of ORFs 5 . In contrast to this notion, in the current study it is observed that M. penetrans with a comparatively larger genome size and higher number of genes is found to have lesser number of overlapping gene pairs than that of M. hominis (Table 1). It has been reported earlier that Mycoplasma genitalium with the smallest genome has the largest proportion of overlapping gene pairs
The eradication of avian metapneumovirus is based on the serologic elimination of infectious carriers, antibiotic programmes, and vaccination. Currently, vaccines such as Gallivac (live attenu- ated vaccine), Poulvac and Talovac (inactivated vac- cine) are registered and used in the Czech Republic. Mycoplasma gallisepticum and Mycoplasma syno- viae are still detected on turkey and chicken farms. Mycoplasma infections are distributed worldwide. The prevalence has increased in Holland, especially of M. synoviae, and in herds of commercial lay- ers it has risen by 73% and in meat turkey by 16% (Feberwee et al. 2008). The prevalence of M. syno- viae was monitored using the PCR method in herds of laying hens (75%) in Germany, however, PCR analysis failed to detect M. gallisepticum in any of the collected samples. (Kohn et al. 2009).
the bacterial chromosome as a whole, the ICE have core and accessory components to their gene complement. The conjugative machinery appears to be relatively well conserved between the ICE, with all of the streptococcal Tn5252-like elements sharing a large recombination protein, as well as VirB4-type and VirD4- type proteins. Furthermore, a genus-wide comparison of streptococcal ICE reveals a surprising level of similarity in their complement of cargo genes. The pezAT addiction toxin system, first identified in the chromosome of S. pneumoniae, is found on elements present in S. suis, S. pneumoniae and S. agalactiae, presumably aiding fixation of the transposon following integration. Bacteriocins, either alone or as part of a gene cluster encoding the accompanying processing machinery, are present on all the sequenced S. suis and S. pneumoniae  elements. Many of the other cargo coding sequences appear to be involved in increasing the stress tolerance of the host: abi genes are common, and the ICE of S. dysgalactiae subsp. equisimilis and S. agalactiae 2603V/R encode multiple heavy metal resistance genes. Of greater clinical importance, sequenced streptococcal ICE can carry genes for resistance to tetracycline, erythromycin, chloramphenicol, trimethoprim, aminoglycosides and streptomycin.
Mycoplasma genitalium (MG) can cause nongonococcal urethritis and is potentially associated with urethri- tis, endometritis, and cervicitis. Several assays have been developed to detect MG. Molecular amplification assays for organism detection can be problematic due to the potential for false-positive and false-negative results. Confirmatory testing is often required in these situations, requiring additional time and resources. Use of multigene targets could integrate both detection and verification at lower cost. Utilizing two targets, the MgPa adhesion gene and the 16S rRNA gene, a multitarget real-time (MTRT) PCR for the detection of MG was developed. Samples from patients attending sexually transmitted disease clinics were collected in dupli- cate. Urine samples from males (n ⴝ 286) and self-collected vaginal swabs from females (n ⴝ 321) were analyzed by MTRT PCR for MG and the Gen-Probe transmission-mediated amplification (TMA) assay, which targets MG rRNA for detection (TMA-MG research use only). Utilizing the criteria of any two targets being positively amplified, the MTRT PCR had a sensitivity and specificity of 91.8% (101 positive samples/110 samples tested) and 99.5% (495/497), respectively, with a positive predictive value (PPV) of 98.1% (101/103) and a negative predictive value (NPV) of 98.2% (495/504). The Gen-Probe TMA-MG assay had a sensitivity, specificity, PPV, and NPV of 98.1% (108/110), 98.1% (488/497), 92.3% (108/117), and 99.5% (488/490), respec- tively. Comparison between the MTRT PCR and TMA-MG assay by kappa statistic analysis indicated that an overall kappa value was 0.941 (95% confidence interval, 0.907 and 0.976). Both assays demonstrated accuracy in the detection of MG from urine samples from male patients and self-collected vaginal swabs from female patients.
In the present study, histological analysis of the stom- achs of H. suis-infected pigs at slaughter age, revealed that H. suis bacteria are often observed in close vicin- ity of parietal cells and they even can be observed inside the canaliculi of parietal cells, which reveals a direct interaction of H. suis and parietal cells in situ. Upon co-incubation of isolated parietal cells with live H. suis, a considerable number of H. suis bacteria were found near or potentially in the canaliculi of the isolated pari- etal cells, which further confirmed the direct interplay between this bacterium and parietal cells in vivo and in vitro. Longer times of incubation of H. suis with iso- lated parietal cells showed similar results, and the most plausible explanation for this may be that a longer incu- bation time decreases the bacterial viability due to the improper medium and gas environment for this fastidi- ous bacterium, requiring vigorous culture conditions. Future experiments should attempt to identify the possi- ble mechanisms of adhesion.
natural immunity against disease. Serological analyses could have clarified this issue, however, employees did not give their consent for blood sampling. Contact sur- faces at both locations were equally positive by both PCR and culture. Thus, Chlamydia suis could have ended up in the eyes through direct contact of hands with ‘contami- nated’ contact surfaces. Besides, bioaerosol monitoring demonstrated high amounts (score 4) of viable C. suis in the air of the slaughtering and bleeding area. On the other hand, the air of the intestine washing room was C. suis negative, which could indicate that the air is not the main C. suis transmission route. However, further studies on larger risk populations should be conducted to get more insights into transmission routes and clinical conse- quences of C. suis in humans.