Associations among Haemophilus parasuis, Mycoplasma hyorhinis, and porcine reproductive and respiratory syndrome virus infections in pigs with polyserositis.

The objective of this study was to determine the associations among Haemophilus parasuis, Mycoplasma hyorhinis, and porcine reproductive and respiratory syndrome (PRRS) virus (EU-field strain) infections in 95 pigs with polyserositis. A significant association between H. parasuis and M. hyorhinis was identified. H. parasuis and M. hyorhinis were significantly more often detected in PRRS virus positive pigs.

Association entre infections avecHaemophilus parasuis, Mycoplasma hyorhinis,et le virus du syndrome dysgénésique et respiratoire du porc chez les porcs atteints de polysérosite. L'objectif de l'étude était d'étudier l'association de Haemophilus parasuis, Mycoplasma hyorhinis, et du SDRP (souche européenne-sauvage) chez 95 porcs atteints de polysérosite. Une association significative a été mise en évidence entre H. parasuis et M. hyorhinis. H. parasuis et M. hyorhinis ont été significativement plus fréquemment détectés chez SDRP positif porcs.(Traduit par les auteurs).

Hemotrophic mycoplasmas induce programmed cell death in red blood cells.

Hemotrophic mycoplasmas (HM) are uncultivable bacteria found on and in the red blood cells (RBCs). The main clinical sign of HM infections is the hemolytic anemia. However, anemia-inducing pathogenesis has not been totally clarified. In this work we used the splenectomized pig as animal model and Mycoplasma suis as a representative for hemotrophic mycoplasmas to study anemia pathogenesis. Eryptosis, i.e. programmed cell death of RBCs, is characterized by cell shrinkage, microvesiculation and phosphatidylserine (PS) exposure on the outer membrane. The eryptosis occurrence and its influence on anemia pathogenesis was observed over the time-course of M. suis infections in pigs using 3 M. suis isolates of differing virulence. All 3 isolates induced eryptosis, but with different characteristics. The occurrence of eryptosis could as well be confirmed in vitro: serum and plasma of an acutely ill pig induced PS exposure on erythrocytes drawn from healthy pigs. Since M. suis is able to induce eryptotic processes it is concluded that eryptosis is one anemia-inducing factor during M. suis infections and, therefore, plays a significant role in the pathogenesis of infectious anemia due to HM infection.

Antibodies to actin in autoimmune haemolytic anaemia.

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.

Prevalence of Mycoplasma suis in slaughter pigs, with correlation of PCR results to hematological findings.

Porcine infectious anemia is a well-known disease that occurs worldwide and is caused by the unculturable hemotrophic bacterium Mycoplasma suis. The actual prevalence and impact of M. suis infections, however, remain fairly unknown. This study examined the prevalence of M. suis in post-weaning pigs by employing a quantitative real-time LightCycler PCR. M. suis infections were detected in 164 out of 1176 feeder pigs (20-30 kg; 13.9%) as well as on 79 out of 196 pig farms (40.3%). The comparison of PCR results with microscopic investigation of acridine-orange-stained blood smears revealed a considerably lower sensitivity of the microscopic method: only 35 out of 1176 blood smears were microscopically positive. The microscopic detection of M. suis was shown to be closely linked to the bacterial load in the blood. M. suis infection is associated with significantly decreased hematocrit, erythrocyte counts and hemoglobin concentrations as well as significantly higher bilirubin concentrations. Furthermore, M. suis blood loads were significantly associated with erythrocyte count, hematocrit, hemoglobin, glucose and iron concentrations indicating that high M. suis loads are connected with clinical anemia. In conclusion, this study has shown, that M. suis infections are often under-diagnosed in pig husbandry and can therefore lead to considerable economic profit losses in pig husbandry. Furthermore, our study has shown that the LightCycler PCR could be an appropriate tool for a sufficiently coherent identification of M. suis in latent carrier animals in view of introducing effective treatment and disease control measures.

[Examination of the compatibility of a Salmonella Typhimurium-live vaccine Salmoporc for three day old suckling piglets]. / Uberprüfung der Verträglichkeit des Salmonella Typhimurium-Lebendimpfstoffes Salmoporc für drei Tage alte Saugferkel.

The present study intended to investigate the compatibility of the orally applicated Salmonella Typhimurium live vaccine Salmoporc on day 3 and 21, respectively. Piglets which only received saline solution orally were used as negative control. During eight hours following vaccination, fecal consistency, body temperature as well as body condition were evaluated. Furthermore, in addition to the daily measures of body temperature and body condition, weekly weight controls as well as bacteriological examination referring to the duration of excretion of the vaccine strain, were carried out until the end of the study. Additionally, distribution and persistence of the pathogen in different tissues were examined. Using serological determination of salmonella antibodies, immune response was scrutinised. Oral vaccination resulted in a significant rise of the body temperature.The vaccine strain could be isolated from fecal samples until the 28th day (seven days after the second vaccination). The vaccination strain persisted until six weeks after the second vaccination in organs of the piglets, whereas the last detection was from samples of small and large intestine. Field strains could neither be isolated from fecal nor from organ samples of vaccinated and control group. Until the end of the study, control animals were negative by bacteriological examination of fecal and organic samples. Seroconversion was observed from day seven after the second vaccination. Mean concentration of antibodies was significantly higher in vaccinated than in control animals three weeks after vaccination.

MSG1, a surface-localised protein of Mycoplasma suis is involved in the adhesion to erythrocytes.

Mycoplasma suis is a member of the group of uncultivable haemoplasmas which colonise erythrocytes of a wide range of vertebrates. Adhesion to erythrocytes is the crucial step in the unique haemoplasma life cycle. Due to the lack of a cultivation system, no adhesion structures have been identified so far. In order to determine potential adhesion molecules of M. suis, we screened genomic M. suis libraries. The protein MSG1 with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) similarity was identified. The encoding gene msg1 is 1011bp in size. The overall homology of the deduced amino acid sequence to GAPDHs of other pathogenic mycoplasmas ranged from 52.6% to 54.5%. Recombinant MSG1 expressed in Escherichia coli exhibited GAPDH activity. Immunoblot and immunoelectron microscopy analyses using antibodies against rMSG1 verified the membrane and surface localisation of native MSG1 in M. suis. Furthermore, we showed that rMSG1 binds to erythrocyte lysate in a dose-dependent manner. E. coli transformants which express MSG1 on their surface acquire the ability to adhere to porcine erythrocytes. This adhesion could be specifically and significantly inhibited by rMSG1 and antibodies to MSG1. In conclusion, our studies indicate that the membrane-associated MSG1 represents the first putative adhesion protein identified in the group of haemoplasmas.

First identification and functional characterization of an immunogenic protein in unculturable haemotrophic Mycoplasmas (Mycoplasma suis HspA1).

The antigenic structures of the haemotrophic Mycoplasma suis, an epicellular parasite of porcine erythrocytes, are largely unknown due to its unculturability. In this study, serological proteome and mass spectrometry analyses allowed the characterization of M. suis proteins targeted by the porcine antibody response: two proteins with characteristics of heat shock proteins, two proteins with characteristics of glycolytic enzymes, a RNA helicase- and an actin-like protein. The DnaK-like protein of M. suis (HspA1) was further analysed genetically and functionally. Its encoding gene (M. suis a1 gene) is 1.830 bp in size and corresponds to a 67 kDa protein. Immunoelectron microscopy verified the surface accessibility of HspA1 in M. suis. Recombinant HspA1 expressed in Escherichia coli demonstrated ATPase activity and antigenicity in experimentally infected pigs. In conclusion, this first identification and recombinant expression of an antigenic protein of M. suis provides the basis for the development of vaccines and new in vitro diagnostic assays.

First LightCycler real-time PCR assay for the quantitative detection of Mycoplasma suis in clinical samples.

Mycoplasma suis cannot be cultivated in vitro. Therefore, PCR-based methods are irreplaceable for the diagnosis of M. suis infections especially when clinical symptoms are not evident. Currently, no easy and reliable method allowing the quantitative detection of M. suis is available. This report describes the development of a quantitative LightCycler PCR assay based on the msg1 gene of M. suis (LC MSG1 PCR). No PCR signals were obtained with closely related haemotrophic and non-haemotrophic mycoplasmas, with other bacteria, and with M. suis-free blood and tissue arguing for a high analytical specificity. Test sensitivity was found to be 100%, and test specificity 96.7%. To test the diagnostic suitability of the LC MSG1 PCR, 25 pigs with clinical porcine eperythrozoonosis and 25 healthy pigs were investigated. All ill pigs revealed a positive real-time PCR result whereas only one healthy pig was detected to be M. suis-infected. M. suis was quantitatively detected in 19 blood specimens of 100 sows from Switzerland and in 17 of 160 post-weaning piglets from Germany. In conclusion, this new LC MSG1 PCR assay represents a powerful tool for the improvement of the current M. suis diagnosis and for prevalence and pathogenesis studies.

Development of a diagnostic PCR assay based on novel DNA sequences for the detection of Mycoplasma suis (Eperythrozoon suis) in porcine blood.

An efficient method of control of porcine eperythrozoonosis (PE) caused by Mycoplasma suis is eradication of infection by detection and removal of infected carrier animals. At present, only a few tests are available for the diagnosis of these latent M. suis infections in pigs. The objective of this study was to develop a PCR assay based on novel DNA sequences for the identification of M. suis-infected pigs. A 1.8 kb EcoRI DNA fragment of the M. suis genome was isolated from the blood of pigs experimentally infected with M. suis. Specificity of the DNA fragment was confirmed by DNA sequence analysis and PCR using primers directed against sequences contained in the 1.8 kb fragment. PCR products of 782 bp in size were amplified only from M. suis particles prepared from the blood of experimentally infected pigs but not from any controls, comprising blood from gnotobiotic piglets and a panel of bacteria including other porcine mycoplasmas. PCR results were confirmed by dot blot hybridisation. The applicability of the PCR assay to diagnose M. suis infections in pigs was evaluated by investigating blood samples from 10 symptomatic pigs with clinical signs typical of porcine eperythrozoonosis and blood samples from 10 healthy pigs. The M. suis-specific PCR product was amplified from all samples taken at episodes of acute disease as well as from samples taken during the latent stage of infection, thus demonstrating the suitability of the PCR assay for detecting latent infected carrier animals.

Vaccination of piglets at 1 week of age with an inactivated Mycoplasma hyopneumoniae vaccine reduces lung lesions and improves average daily gain in body weight.

The field efficacy and safety of a single-dose inactivated Mycoplasma hyopneumoniae vaccine, Suvaxyn MH-One, was evaluated in 4-5-day-old piglets on a commercial farm with a history of Mycoplasma disease in Southern Germany. The piglets were injected intramuscularly with the vaccine or saline (control group) and raised under commercial conditions to slaughter weight. The efficacy of the vaccine was determined by comparing the lung lesions associated with infection by M. hyopneumoniae in control and vaccinated pigs post mortem. In this analysis the vaccinated pigs had the lower mean percentage lung lesion at 5% compared to 9% in controls. Of the vaccinated pigs 52.3% were shown to have low levels of lung lesions between 0% and 5% and no more than 5.4% were shown to have levels above 20%. In contrast, the pigs administered saline showed 36.5% in the lower category (0-5%), while 18.3% showed lesions greater than 20%. There were significant differences in the mean body weight of pigs at the final two weight measurements at approximately 21 weeks and 26 weeks of age, with those receiving Suvaxyn MH-One being on average 5 kg heavier at each time point. There was also a significant increase in average daily gain in the vaccinated animals compared to the control group, particularly in the period from vaccination to the final two body weight measurements on day 138 and 166, from weaning at day 28 to the final two body measurements and from mid-way during finishing at day 84 to the final two body weight measurements. Vaccination had no adverse impact on appetite, although small numbers of vaccinated and control pigs did show mild signs of coughing, sneezing, respiratory distress or depression. There was no adverse impact on rectal temperatures and no signs of injection site reactions during the course of the study. We can conclude that vaccination with Suvaxyn MH-One to pigs at less than 1 week of age is effective in reducing lung lesions resulting from M. hyopneumoniae and also aids growth performance by reducing weight losses and improving average daily gain.

Evaluation of carbon dioxide anaesthesia for the castration of male suckling piglets by stress hormone concentrations, behaviour and clinical factors.

The objective of this study was to examine the effect of an anaesthesia using 70% carbon dioxide and 30% oxygen on endocrine stress reaction, behaviour and clinical parameters of male suckling piglets during castration. One hundred and seventy one male piglets, three to five days of age, were allocated to two experiments. They were assigned either to the procedures control handling, control castration, handling under anaesthesia or castration under anaesthesia in each experiment. In Experiment 1, adrenaline and noradrenaline plasma concentrations were measured in blood samples taken before (-15 min) and after (immediately, 2 min) handling/castration. In Experiment 2, behavioural observations and clinical parameters such as heart and respiratory rate, oxygen saturation, reflexes and recovery time were assessed at several sampling times. Measurement of adrenaline and noradrenaline concentrations revealed an increase in all groups after handling/castration (p < 0.0167), but higher concentrations were seen in the anaesthetized groups (25 to 93 times) than in control groups (two to four times). The excessive endocrine reaction suggests that carbon dioxide inhalation causes a more stressful situation in piglets compared to castration without anaesthesia. Behavioural abnormalities, significant decreases in the heart rate, the respiratory rate and the oxygen saturation (p < or = 0.001) including a cardiac arrhythmia (extrasystoles) underline the impression that CO2 inhalation anaesthesia negatively affects animal welfare. Based on the results of this study, this anaesthetic method is unsuitable to reduce stress induced by castration. Further research on alternatives is necessary to ensure the well-being of the piglets during castration.

[Investigation about the use of analgesics for the reduction of castration-induced pain in suckling piglets]. / Untersuchungen uber den Einsatz von Schmerzmitteln zur Reduktion kastrationsbedingter Schmerzen beim Saugferkel.

The pre-emptive use of analgesics for the reduction of pain induced by the castration of suckling piglets was investigated by measuring cortisol before as well as 30 min (minutes), 1 h (hour), 4 h and 24 h after castration/handling and by post surgical behaviour (0-60 min and 180-240 min after castration/handling). 245 male, 4 to 6 days old piglets with a good general condition were divided into twelve groups. The drugs meloxicam, flunixin, metamizole or carprofen, respectively, or saline solution in control piglets were administered 15 to 30 min before manipulation. All tested non-opioid analgesics reduced the rise of the cortisol concentration after castration. Piglets receiving meloxicam and flunixin had significantly lower values 30 min, 1 h and 4 h after castration than the control group, and already after 1 h they did not differ significantly from the corresponding handling groups. The frequency of occurrence of tail wagging, drooping the tail and changing the position was explicitly reduced when meloxicam and flunixin were injected before castration. The effect of flunixin was most clear. Results indicate that non-opioid analgesics, especially efficient anti-inflammatory drugs like meloxicam and flunixin, are capable of reducing castration-induced pain in piglets.

Vaccination with the Mycoplasma suis recombinant adhesion protein MSG1 elicits a strong immune response but fails to induce protection in pigs.

Mycoplasma suis is the unculturable pathogen of porcine infectious anemia. The study was aimed to determine the immunogenicity and protective efficacy of MSG1, an immunodominant adhesin of M. suis as the first vaccine candidate against M. suis. The results demonstrated that recombinant MSG1 and Escherichia coli transformants expressing MSG1 (E. coli_MSG1) induced a strong humoral and cellular immunity against M. suis. The induced antibodies were found to be functionally active as confirmed by an in vitro adhesion inhibition assay. Both, IgG1 and IgG2 antibodies were induced, but E. coli_MSG1 immune response was characterized by a significantly higher IgG1 antibody production. Both vaccine candidates failed to protect against M. suis challenge. However, E. coli_MSG1 vaccination has a considerable effect on the severity of the disease as shown by higher post-challenge hemoglobin and hematocrit values in comparison to control groups. This indicated that a high IgG1 antibody titer is negatively connected with severity of M. suis-induced anemia. Furthermore, the induction of monospecific anti-MSG1 antibodies by both vaccine candidates clearly allows for the differentiation between infected and vaccinated animals (DIVA principle). Overall, the importance of MSG1 as potential vaccine candidate remains to be established. Future studies will evaluate the conditions (i.e. adjuvant, vaccination scheme, and application route) to optimize the effects of E. coli_MSG1 vaccines.

[Effect of isoflurane inhalation anesthesia on postoperative pain due to castration of piglets]. / Auswirkung einer Isofluran-inhalations- narkose auf den postoperativen Kastrationsschmerz von Ferkeln.

Since April 2006 piglets in Germany can only be castrated without anesthesia in the first 7 days of life. However, a castration is a painful experience even for an animal of this young age. Whether the castration under isoflurane-anesthesia is a reasonable alternative to castration without anesthesia was tested in the following investigation at 206 4 to 6 day old piglets. The serum-cortisol-concentration was chosen as the parameter for the pain caused by castration. A part of the animals was castrated without anesthesia (group II, n = 42) or with anesthesia (group IV, n = 41). Additionally Meloxicam, a non-steroidal anti-inflammatory drug, was applicated to piglets castrated with anesthesia (group V, n = 41). For control another part of the animals were only handled without (group I, n = 41) or with anesthesia (group III, n = 41), but they were not castrated. Cortisol-concentration prior to castration was compared to the concentration 0.5, 1,4 and 24 hours after castration. In addition cortisol was compared between groups at all points of time. Cortisol did rise significantly in castrated animals with animals with or without anesthesia than in animals of the non-castrated control groups. Cortisol after castration was significantly lower in piglets with an application of Meloxicam prior to castration. The pain caused by castration is an explanation for the differences in cortisol-concentration between castrated and not-castrated animals. Regarding those results, we assume that castration with isoflurane-anesthesia does not fulfil the demand for reducing pain after castration compared to castrating piglets without anesthesia.

[Frequency of occurrence of pneumonia associated agents and their correlation with clinical and pathological-anatomical findings in pigs]. / Häufigkeit des Vorkommens pneumonie-assoziierter Erreger und deren Korrelation mit klinischen und pathologisch-anatomischen Befunden bei Schweinen.

The aim of this study was to investigate the incidence and influence of different causative organisms involved in the development of pneumonia and bronchopneumonia in pigs. Bronchoalveolar lavage fluid (BALF) from 339 pigs was examined molecular-biologically and culturally. The evaluation considered the following pathogens: Mycoplasma (M.) hyopneumoniae, Mycoplasma (M.) hyorhinis, PRRSV (US-Type), PRRSV (EU-Type), PCV-2, Influenzavirus Type A, alpha-haemolytic Streptococci, beta-haemolytic Streptococci, Pasteurella (P.) multocida, Bordetella (B.) bronchiseptica, Haemophilus (H.) parasuis and Actinobacillus (A.) pleuropneumoniae. This was followed by a pathological-anatomical examination in 168 pigs. Pathological-anatomical examination was evaluated for possible interstitial pneumonia, catarrhal-purulent bronchopneumonia and pleuritis. alpha-haemolytic Streptococci, PCV-2, H. parasuis, M. hyorhinis, M. hyopneumoniae, B. bronchiseptica, PRRSV (US-Type), P. multocida, PRRSV (EU-Type) and Influenzavirus Type A were evident in descending incidence in the BALF. A. pleuropneumoniae were only isolated culturally in two cases in the BALF. The frequency and semiquantitative detection rate in the bacteriological culture of alpha-haemolytic Steptococci, M. hyopneumoniae, P. multocida and B. bronchiseptica correlated significantly with the ascertained clinical evaluation score and the pathological-anatomical examination score. M. hyorhinis and Influenzavirus Type A only correlated with the severity degree of clinical appearance, while PCV-2 and PRRSV (US-Type) correlated with the frequency of pathological-anatomical changes. The higher the clinical score, the higher was the number of animals ascertained with five or more different pathogens. The more different causal agents were isolated in the BALF of one animal, the higher was the average pathological-anatomical examination score. For the diagnosis of pneumonia, especially when analysing facultative pathogens or secondary pathogens, a useful interpretation of analysis results is only possible in connection with a clinical and pathological evaluation.

Use of recombinant antigens to detect antibodies against Mycoplasma suis, with correlation of serological results to hematological findings.

Porcine eperythrozoonosis is a disease with worldwide distribution caused by the unculturable hemotrophic bacterium Mycoplasma suis. 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 laboratories. 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 concentrations, 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.

[Effect of analgesics on the castration of male piglets]. / Einfluss von Schmerzmitteln bei der Kastration männlicher Ferkel.

According to the current German animal welfare law, male piglets may be surgical castrated without anaesthesia up to four weeks of life. This surgical procedure is painful during and also after the operation, for newborn animals as well as for adults. This study was aimed to investigate the impact of preoperative application of analgesics (Meloxicam) on the postoperative castration - pain of four to six days old male piglets. In this investigation all animals were randomly distributed in three groups:the first one was only immobilized but had no surgery, the second one was castrated without analgesics, and the third group was castrated after application of Meloxicam. Blood samples were taken immediately before immobilization, castration or application of the analgesic as well as one, four and 28 hours afterwards to determine Cortisol-concentration in the blood serum and, via this stress-marker, to indirectly evaluate the postoperative und possible intraoperative castration-pain. As a result all piglets castrated without preoperative application of Meloxicam showed significantly increased Cortisol-concentration one and four hours after castration. In contrast, piglets castrated with analgesics resulted in no significant increase during the entire experiment.