Occurrence of 'Candidatus Mycoplasma haemosuis' in fattening pigs, sows and piglets in Germany using a novel gap-based quantitative real-time PCR assay.

BACKGROUND: The appearance of the novel porcine haemotrophic mycoplasma (HM) species 'Candidatus Mycoplasma haemosuis' was reported in apparently healthy but also in clinically sick animals in China, Korea and in a case report from Germany. Outside of Asia, however, nothing further is known about the frequency of 'Ca. M. haemosuis' in pigs to date. To investigate the distribution of this novel HM species in Germany, fattening pigs, sows and pre-suckling piglets were examined using a herein developed quantitative real-time PCR assay (qPCR). Because the piglets were sampled before the first colostrum uptake, additional information on a possible vertical transmission from dams to their offspring was obtained. RESULTS: Our novel qPCR assay successfully detected 'Ca. M. haemosuis' in all blood samples from the 'Ca. M. haemosuis'-infected pigs. No cross-reactivity was detected when DNA from non-target Mycoplasma spp. and other bacterial species representing 105 bacteria/reaction were used as a template. The lower limit of detection of the qPCR was thus 10 gap gene copies per reaction and 2.5 x 103 genome equivalents (GE) per mL blood. 'Candidatus M. haemosuis' was detected by this qPCR in blood samples from a total out of 6.25% sows (13/208), 4.50% pre-suckling piglets (28/622) and 17.50% fattening pigs (35/200). On farm level, 3 out of 21 piglet producing farms (14.28%) and 9 out of 20 fattening farms (45.00%) were positive for 'Ca. M. haemosuis'. Co-infections with M. suis were evident in all age groups. CONCLUSION: 'Candidatus M. haemosuis' infection is present in German pig farms and the detection of the novel porcine HM species in piglets immediately after birth before colostrum intake indicates vertical transmission. The novel qPCR assay specific for 'Ca. M. haemosuis' described herein will be a prerequisite for future studies on the prevalence, epidemiology as well as the clinical and economic impact of 'Ca. M. haemosuis' infections.

Clinical, haematological and pathomorphological findings in Mycoplasma suis infected pigs.

BACKGROUND: Mycoplasma suis (M. suis) belongs to the group of haemotrophic mycoplasmas and is known as the causative agent of infectious anaemia in pigs. In the last few years valuable insights into the mechanism of adhesion and invasion, shedding patterns and cell tropism of M. suis were gained by the use of new molecular techniques. However, details on M. suis induced lesions as well as the distribution of M. suis in different organs are still lacking. Therefore, seven splenectomised pigs were experimentally infected and clinical and laboratory investigations as well as a detailed histopathological examination were performed. Detection and quantification of M. suis DNA in blood and various tissue samples was done using a quantitative real-time PCR. RESULTS: During the course of experimental infection, periodically occurring signs of infectious anaemia of pigs including severe icteroanaemia, fever, apathy and anorexia were observed. In addition, dermatological manifestations such as haemorrhagic diathesis presenting as petechiae occurred. The most important haematological alterations were normochromic, normocytic anaemia, hypoglycaemia as well as increased bilirubin and urea concentrations. Necropsy revealed predominant evidence of haemolysis with consecutive anaemia, as well as disseminated intravascular coagulation. M. suis was found in all investigated tissues with the highest copy numbers found in the kidneys. In Giemsa stained sections M. suis was only detected red blood cell (RBC)-associated. CONCLUSION: In the present study, no RBC independent sequestration of M. suis was detected in organs of experimentally infected pigs. Pathological findings are most likely resulting from haemolysis, consecutive anaemia as well as from disseminated intravascular coagulation and subsequent organ impairments.

Detection of Mycoplasma suis in pre-suckling piglets indicates a vertical transmission.

BACKGROUND: Transmission of Mycoplasma (M.) suis mainly occurs via iatrogenic or zootechnical manipulations or due to ranking fights. Other transmission routes including ingestion of secretes/excretes; blood-sucking arthropods and intra-uterine transmission have thought to play an epidemiological role without being experimentally proven. To investigate a vertical transmission of M. suis under field conditions blood samples from pre-suckling piglets and their corresponding dam were examined for M. suis by quantitative polymerase chain reaction (qPCR) in 21 farms in Southern Germany. RESULTS: A total of 14.35% of the 474 blood samples from pre-suckling piglets reacted qPCR positive. Additionally, M. suis was detected in 65 (31.25%) of the 208 sows at farrowing. On farm level, 16 (76.2%) of the 21 farms had at least one M. suis positive animal. M. suis positive farms had an average of 0.41 more stillborn piglets per litter than M. suis negative farms (p = 0.007). CONCLUSION: The present study provides further insights into M. suis infection dynamics as it is the first detection of M. suis in piglets immediately after birth prior to colostrum intake and the first large scale investigation of M. suis in sows at farrowing.

Updating the proteome of the uncultivable hemotrophic Mycoplasma suis in experimentally infected pigs.

Mycoplasma suis belongs to the hemotrophic mycoplasmas that are associated with acute and chronic anemia in a wide range of livestock and wild animals. The inability to culture M. suis in vitro has hindered its characterization at the molecular level. Since the publication of M. suis genome sequences in 2011 only one proteome study has been published. Aim of the presented study was to significantly extend the proteome coverage of M. suis strain KI_3806 during acute infection by applying three different protein extraction methods followed by 1D SDS-PAGE and LC-MS/MS. A total of 404 of 795 M. suis KI_3806 proteins (50.8%) were identified. Data analysis revealed the expression of 83.7% of the predicted ORFs with assigned functions but also highlights the expression of 179 of 523 (34.2%) hypothetical proteins with unknown functions. Computational analyses identified expressed membrane-associated hypothetical proteins that might be involved in adhesion or host-pathogen interaction. Furthermore, analyses of the expressed proteins indicated the existence of a hexose-6-phosphate-transporter and an ECF transporter. In conclusion, our proteome study provides a further step toward the elucidation of the unique life cycle of M. suis and the establishment of an in vitro culture. All MS data have been deposited in the ProteomeXchange with identifier PXD002294 (http://proteomecentral.proteomexchange.org/dataset/PXD002294).

Mycoplasma suis infection results endothelial cell damage and activation: new insight into the cell tropism and pathogenicity of hemotrophic mycoplasma.

Hemotrophic mycoplasmas (HM) are highly specialized red blood cell parasites that cause infectious anemia in a variety of mammals, including humans. To date, no in vitro cultivation systems for HM have been available, resulting in relatively little information about the pathogenesis of HM infection. In pigs, Mycoplasma suis-induced infectious anemia is associated with hemorrhagic diathesis, and coagulation dysfunction. However, intravasal coagulation and subsequent consumption coagulopathy can only partly explain the sequence of events leading to hemorrhagic diathesis manifesting as cyanosis, petechial bleeding, and ecchymosis, and to disseminated coagulation. The involvement of endothelial activation and damage in M. suis-associated pathogenesis was investigated using light and electron microscopy, immunohistochemistry, and cell sorting. M. suis interacted directly with endothelial cells in vitro and in vivo. Endothelial activation, widespread endothelial damage, and adherence of red blood cells to the endothelium were evident in M. suis-infected pigs. These alterations of the endothelium were accompanied by hemorrhage, intravascular coagulation, vascular occlusion, and massive morphological changes within the parenchyma. M. suis biofilm-like microcolonies formed on the surface of endothelial cells, and may represent a putative persistence mechanism of M. suis. In vitro analysis demonstrated that M. suis interacted with the endothelial cytoskeletal protein actin, and induced actin condensation and activation of endothelial cells, as determined by the up-regulation of ICAM, PECAM, E-selectin, and P-selectin. These findings demonstrate an additional cell tropism of HM for endothelial cells and suggest that M. suis interferes with the protective function of the endothelium, resulting in hemorrhagic diathesis.

Insights into the gene expression profile of uncultivable hemotrophic Mycoplasma suis during acute infection, obtained using proteome analysis.

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.

Occurrence of Mycoplasma parvum in German Pigs of Different Age Groups Using a Novel Quantitative Real-Time PCR Assay

Mycoplasma (M.) parvum is a hemotrophic bacterium circulating in the blood of pigs but is not considered a primary pathogen. Only a handful of studies dealing with this agent have been published since its first description in 1951, and many issues, including epidemiology and the impact of subclinical infections, are yet to be elucidated. This study aimed to establish a M. parvum specific real-time PCR for its detection and quantification in porcine blood and the application of this assay to obtain insights into the occurrence of M. parvum in German pigs. Furthermore, 16S rDNA amplicons of M. parvum positive blood samples were phylogenetically analyzed using MEGA 11 software. The established qPCR targeting the M. parvum glyceraldehyde-3-phosphate dehydrogenase encoding gene (gap) showed a lower detection limit of 10 gene copies per reaction and no cross-reactivity within the specificity test. A total of 36.0% (n = 72) of the sampled fattening pigs, 25.0% (n = 15) of the sows, and 4.37% (n = 8) of the boars tested M. parvum positive. The dendrogram showed the typical allocation of the M. parvum isolates into the "haemominutum group" subgroup within the hemotrophic Mycoplasma species. Both the novel established qPCR and the obtained epidemiological data can serve as an important basis for future studies dealing with M. parvum.

Complete genome sequence of the hemotrophic Mycoplasma suis strain KI3806.

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.

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.

Update on shedding and transmission routes of porcine haemotrophic mycoplasmas in naturally and experimentally infected pigs.

Horizontal transmission of Mycoplasma suis via parenteral exposure during standard practices or through bites during fightings have been identified as key epidemiological routes. However, as knowledge gaps on other potential shedding and transmission routes exist, the present study combines both laboratory experiments and field surveys to gain new insights into the epidemiology of porcine haemotrophic mycoplasmas. Splenectomised pigs were orally inoculated with a M. suis field strain and investigated for clinical signs related to infectious anaemia of pigs (IAP) and the presence of M. suis in blood, urine and saliva samples by qPCR. All blood samples were negative for M. suis and animals did not show obvious clinical signs of IAP throughout the entire study period. Additionally, urine, nasal and saliva samples from sows of conventional piglet producing farms and semen samples from a boar stud revealed no detection of M. suis and 'Candidatus Mycoplasma haemosuis' by qPCR. Thus, the results indicate that blood-independent transmission routes might be of minor relevance under field conditions.

Inorganic pyrophosphatase in uncultivable hemotrophic mycoplasmas: identification and properties of the enzyme from Mycoplasma suis.

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.

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.

Detection of a novel haemoplasma species in fattening pigs with skin alterations, fever and anaemia.

BACKGROUND: In a fattening farm in Southern Germany, skin alterations (urticaria, haemorrhagic diathesis) and high fever were observed in 30% of the pigs 2 weeks after arrival. Feed intake was severely compromised in affected pigs. METHODS: After detailed clinical observation, blood samples from affected pigs were collected for haematological, PCR and serological investigations. In addition, pathological investigations were performed on one pig. RESULTS AND CONCLUSION: Analysis of blood parameters revealed a normocytic, normochromic anaemia. A novel porcine haemoplasma species was detected in blood samples of affected pigs and spleen sample of the necropsied pig by PCR. Phylogenetic analyses based on the 16S rDNA showed 99% identity to a novel porcine haemoplasma ('Candidatus (Ca.) M. haemosuis') species which has recently been described in China. Interestingly, this is the first report of 'Ca. M. haemosuis' in pigs with clinical signs resembling those of Mycoplasma (M) suis and the first description of this novel haemoplasma species outside Asia. On-farm affected pigs were treated with oxytetracycline and non-steroidal anti-inflammatory drugs. Clinical signs improved after implementation of treatment and optimisation of management procedures. This case might indicate that other porcine haemoplasma species than M suis can induce fever and skin alterations and may have an economic impact on affected farms.

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.

Quantitative analysis of Mycoplasma wenyonii and 'Candidatus Mycoplasma haemobos" infections in cattle using novel gapN-based realtime PCR assays.

Hemotrophic mycoplasmas (HMs) are associated with anemia and other disease complexes in a wide range of livestock and wild animals. Two bovine HM species have been identified to date, i.e. Mycoplasma wenyonii and 'Candidatus Mycoplasma haemobos'. The study aim was to develop quantitative real-time PCR assays (qPCRs) to detect and quantify M. wenyonii and 'C. M. haemobos' and to apply these assays to DNA samples extracted from bovine blood collected in Germany (n = 220) from 22 herds. The qPCR assays specific for M. wenyonii and 'C. M. haemobos' were designed using the gapN of the respective hemoplasma species as gene target which encodes the NADP-dependent glyceraldehyde 3-phosphate dehydrogenases (GAPN). The sensitivity of both assays was 10 genome equivalents per reaction, corresponding to 2500 genome equivalents per ml blood. No cross-reactivity with non-target bovine HMs. and other bovine pathogens was observed. Bovine HM DNA was detected in 137 samples (62.27%) with 118 samples (53.64%) being positive for 'C.M. haemobos' and 19 samples (8.64%) being positive for M. wenyonii. Thereof, 11 animals (5.00%) were co-infected with both bovine HM species. The found herd prevalence for `C. M. haemobos` was 100.00%, and for M. wenyonii 36.36% with mean bacterial loads of 3.7 × 107 `C. M. haemobos`/mL blood and of 4.29 × 105M. wenyonii/mL blood respectively. Clinical and economic relevance of bovine HM species should be goal of future studies for which the novel gapN qPCR assays can serve as a valuable diagnostic tool.

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.

Spatial Variation of the Gut Microbiota in Broiler Chickens as Affected by Dietary Available Phosphorus and Assessed by T-RFLP Analysis and 454 Pyrosequencing.

Molecular fingerprinting and sequencing based techniques have been widely used to characterize microbial communities. Terminal restriction fragment length polymorphism (T-RFLP) and 454-pyrosequencing were used to determine the microorganisms present in the different sections of the chicken gastrointestinal tract (GIT) (crop, jejunum, ileum and caeca). Broilers fed with diets differing in phosphorous (P) and calcium (Ca) as well as in phytase levels were used to study the microbiota of the upper and lower part of the GIT. A database with terminal restriction fragments (T-RF) of the most important organism present in the different gastrointestinal sections was constructed. The analysis revealed a distinct microbial assemblage on each section. Regardless of the diet, crop, jejunum and ileum were mainly colonized by Lactobacillaceae, and caeca were the most diverse site. The correlation between Lactobacillus crispatus and L. reuteri was positive in the crop, but negative in the jejunum. In crop samples, higher P and Ca levels led to a shift in the abundance of L. reuteri and L. crispatus to L. salivarius and L. taiwanensis whereas in the ileum supplementation of phytase favored L. salivarius and L. taiwanensis but resulted in decreased abundance of L. crispatus. Both methods were correlating significantly, being T-RFLP a reliable fingerprinting method to rapidly analyze large numbers of samples in a cost-effective and rapid manner. Results are easy to interpret with no need of deep bioinformatics knowledge and can be integrated with taxonomic information.

Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera.

Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.