Suis DNA Research Articles

Морфофункциональные изменения СОЖ откормочных свиней при хеликобактериозе

Abstract. The purpose is to study the features of morphofunctional changes in the gastric mucosa of fattening pigs in gastric ulcer disease and to establish a possible correlation between pathological changes and colonization of the gastric mucosa of H. suis. Methods. The study is based on pathoanatomic, microscopic, molecular genetic and histological research methods. Results. In 86 % of cases, significant pathomorphological changes in the gastric mucosa of pigs were detected during a pathoanatomical autopsy. Spiral-shaped microorganisms in smears were found in 28 %, H. suis DNA on the gastric mucosa – in 76 %. In the examined samples in which H. DNA was found. suis in the gastric mucosa, 76.3 % revealed various pathologies in the esophagus, 92% revealed various pathologies on the gastric mucosa. In 24 %, when H. suis DNA was not detected, 33.3 % showed no visible damage and 66.7 % revealed various pathologies. The results of the correlation between morphological changes in the gastric mucosa and its infection with helicobacter reached the limits of statistical reliability. Histological methods revealed that active chronic gastritis was detected in 48.0 %, inactive chronic gastritis was detected in 34.0 % and the norm (almost the norm) was found in 8.0 %. Chronic gastritis during exacerbation was reliably diagnosed in 55.2 % of cases when H. suis DNA was detected on the gastric mucosa. Chronic gastritis during remission was diagnosed in 34.2 % of cases when H. suis DNA was detected on the gastric mucosa. The results presented in the work reflect the undoubted connection between the presence of H. suis and the development of gastritis in pigs.

Detection of DNA of bacteria of the genus <i>Helicobacter</i> in pig meat

Helicobacters different from Helicobacter pylori, colonizing various animals, are also capable of causing gastritis, stomach ulcers and MALT – lymphomas in humans. Helicobacter suis is most often isolated among similar bacteria from the mucous membrane of the human stomach. In spite of its name this microorganism can also infect cats and dogs. At the same time the spread of Helicobacter suis among the pig population in different countries varies between 10.8-90.0% but in most studies it is in average 60%. It is obvious that such extensive spread of Helicobacter suis and close contacts with infected animals can contribute to occupational infections of human beings. In addition, the consumption of infected and insufficiently heat-treated pork meat is a possible route of transmission of this microorganism. In our studies on the presence of Helicobacter suis DNA in various parts of pig carcasses immediately after slaughter determined that DNA samples in 26.7% of bacterial were found on the oral mucosa, 13.3% – along the neck lines section, 6.6% - thoracic section and 0% – hock section. According to the available literature the isolation of Helicobacter suis by the bacteriological method presents great difficulties and the detection of nucleic acids of the microorganism remains as one of the few available diagnostic methods. To detect the DNA of living Helicobacter suis in pork we used the protocol for processing pork samples with ethidium monoazide in combination with PCR to study pig meat (pork neck) from various manufacturers purchased on the shelves of Kazan markets. As a result, the DNA of living Helicobacter suis was found in 0.5% of the samples.

Detection of Streptococcus suis using the optimized real-time polymerase chain reaction protocol

The article presents the results of studies on the detection of Streptococcus suis by real-time polymerase chain reaction. Isolation and species identification of the studied isolates of streptococci was carried out according to morphological, cultural, biochemical and biological properties by conventional methods. The study of cultural characteristics of growth was carried out using conventional bacteriological methods on the brain heart infusion broth (BHI) and BHI agar with the addition of 5% sheep blood (blood BHI agar). To confirm biochemical properties as a confirmatory method, API 20 STREP test kit (bioMerieux, France) was used. In addition, to differentiate S. suis from the non-pathogenic species of streptococci, the hemolysis test was used. As a result of the studies, it was found that the use of the real-time PCR (polymerase chain reaction) method makes it possible to detect S. suis in an amount of 1 x 104 genome copies in the sample. All described validation parameters for the qualitative detection of S. suis DNA by real-time PCR meet international requirements, which guarantees accurate and reliable results. In Ukraine only a diagnostic test kit for convential PCR has been developed for the detection of swine streptococcosis. This approach is more time consuming and complex in comparison with the real-time PCR approach. We recommend that diagnostic laboratories implement this method in their practice. This will increase the number of effective diagnostic tools available to veterinarians on pig farms when they order laboratory tests. The high analytical sensitivity limit of a test is an essential parameter when screening is the focus, and obtaining false negative results causes a risk of the development of infection process among pig populations within infected herds. Our study showed that microbiological diagnostic methods to determine morphological and cultural properties can identify S. suis at the genus level. Determination of biochemical properties using the API 20 STREP test kit can be used to identify S. suis 1 and 2 serotypes. The conventional method and real-time PCR have 100% specificity and can be used to identify S. suis of different serotypes. Real-time PCR is a 2 to 4 times more sensitive limit than conventional PCR depending on the serotype being studied, and can be used to more accurately identify streptococcal DNA. It was found that the use of the real-time PCR method makes it possible to detect S. suis in an amount of 1 x 104 copies of the genome in the sample. Additionally, it was found that all the studied validation parameters of the qualitative method for determining S. suis DNA by real-time PCR meet international requirements, which guarantees accurate and reliable results.

Impacto econômico de lesões pleurais crônicas e consequente desqualificação de carcaças para exportação durante inspeção em frigoríficos de suínos

ABSTRACT: Chronic pleuritis is the main reason for sending pig carcasses to the Department of Final Inspection (DIF), condemnation and led to economic losses to industries and producers. Most pleura lesions detected after slaughter are sequelae from bacterial infections by agents that do not pose risks to pork consumers. The objective of the present study was to generate science-based information for decision making in the evaluation and destination of chronic pleuritis by the Federal Inspection Service (SIF). Therefore, 200 carcasses, with and without pleurisy, from a swine slaughterhouse with SIF were assessed following the visual classification of the inspection agent. The study was carried out in two stages. In stage 1, 50 carcasses with pneumonic lesions adjacent to chronic pleuritis and 50 carcasses with only chronic pleuritis lesions were evaluated, through macroscopy, histopathology, and bacterial culture. In stage 2, 50 swine carcasses with chronic pleuritis and 50 without this lesion were sampled in the parietal pleura region to bacterial culture and PCR. The economic impact of not exporting these carcasses with chronic pleuritis was also assessed. Considering the stages of evolution of the lesions, the macroscopic examination showed high correlation with the histological examination. There was no bacterial isolation through pleural swabs, regardless of the presence or not of adjacent pulmonary lesions. Isolation was restricted to the adjacent pulmonary lesions of 70% samples, with Pasteurella multocida type A found in 48% of them, followed by P. multocida type D and Streptococcus suis in 12%, and Actinobacillus pleuropneumoniae in 3%. Only Streptococcus suis DNA was detected in 5/100 samples, with no correspondence to the isolation of viable bacteria. The reliability demonstrated in the macroscopic evaluation carried out during inspection, the absence of viable bacteria in the chronic pleural lesions, and the negative economic impact suggest that carcasses with chronic pleuritis can be submitted to pleura removal, with no need of sending to DIF.

ОСОБЕННОСТИ ДИАГНОСТИКИ И ТЕРАПИИ СТРЕПТОКОККОЗА СВИНЕЙ, ОСЛОЖНЕННОГО РРСС НА ПЛЕМЕННОЙ ФЕРМЕ

Pathological phenomena began to appear in April in piglets after weaning from sows and in piglets weighing up to 30 kg. Clinical signs: cough, diarrhea, vomiting, tremor, lameness and extensive eczema-like skin lesions (spots of hyperemia on the skin of the breast and abdominal part of the body, redness of the eyelids), palpation - increased tactile sensitivity. The disease often proceeded in a septic form and was accompanied by fever. A significant part of the diseased pigs died. Mortality among piglets with streptococcosis varied from 66.6 to 100%. Bacteriological and molecular genetic (PCR) diagnostics of streptococcosis and reproductive and respiratory syndrome were carried out. As a result of PCR with porcine brain samples, Streptococcus suis DNA was isolated in 100% of cases. In the study of PCR swabs from the vagina of sows, Streptococcus suis DNA was isolated in 79% of cases. The use of bacteriological and molecular genetic (PCR) diagnostics of streptococcosis and PRRS virus made it possible to establish an etiological diagnosis - streptococcosis in association with PRRS. For treatment, an antibacterial drug of the third generation cephalosporin group Naxcel (Naxcel) was used, containing ceftiofur, Enroxil 10% injection solution, containing enrofloxacin as an active substance, as well as oral veterinary drugs Trisulfon and Pulmokit. Pregnant sows and piglets after weaning, without signs of infection with the reproductive and respiratory syndrome virus, were treated with antibacterial drugs (Naxel, Enroxil, Trisulfon and Pulmokit), containing ceftiofur and enrofloxacin, which allowed to inactivate Streptococcus suis in the body of piglets. The use of specific prophylaxis of the reproductive and respiratory syndrome on pregnant sows and piglets after weaning, without signs of disease, using the Unistrain PRRS vaccine against PRRS, contributed to the improvement of the epizootic situation for bronchopulmonary diseases on the breeding farm. Comprehensive recreational activities made it possible to sanitize the livestock from bacterial infections, including streptococcosis.

Gastric Helicobacter suis Infection Partially Protects against Neurotoxicity in A 6-OHDA Parkinson's Disease Mouse Model.

The exact etiology of Parkinson’s disease (PD) remains largely unknown, but more and more research suggests the involvement of the gut microbiota. Interestingly, idiopathic PD patients were shown to have at least a 10 times higher prevalence of Helicobacter suis (H. suis) DNA in gastric biopsies compared to control patients. H. suis is a zoonotic Helicobacter species that naturally colonizes the stomach of pigs and non-human primates but can be transmitted to humans. Here, we investigated the influence of a gastric H. suis infection on PD disease progression through a 6-hydroxydopamine (6-OHDA) mouse model. Therefore, mice with either a short- or long-term H. suis infection were stereotactically injected with 6-OHDA in the left striatum and sampled one week later. Remarkably, a reduced loss of dopaminergic neurons was seen in the H. suis/6-OHDA groups compared to the control/6-OHDA groups. Correspondingly, motor function of the H. suis-infected 6-OHDA mice was superior to that in the non-infected 6-OHDA mice. Interestingly, we also observed higher expression levels of antioxidant genes in brain tissue from H. suis-infected 6-OHDA mice, as a potential explanation for the reduced 6-OHDA-induced cell loss. Our data support an unexpected neuroprotective effect of gastric H. suis on PD pathology, mediated through changes in oxidative stress.

Clinical, haematological and pathomorphological findings in Mycoplasma suis infected pigs

BackgroundMycoplasma 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.ResultsDuring 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.ConclusionIn 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.

Presence of Helicobacter pylori and H. suis DNA in Free-Range Wild Boars.

Simple SummaryHelicobacter pylori and H. suis are associated with gastric pathologies in humans. To obtain better insights into the potential role of wild boars as reservoirs of these pathogens, gastric samples of 14 animals were tested for the presence of H. pylori and H. suis DNA. Two wild boars were found PCR-positive for H. pylori and one for H. suis. This indicates that these microorganisms may colonize the stomach of wild boars.Helicobacter pylori (H. pylori) is a Gram-negative bacterium that infects half of the human population worldwide, causing gastric disorders, such as chronic gastritis, gastric or duodenal ulcers, and gastric malignancies. Helicobacter suis (H. suis) is mainly associated with pigs, but can also colonize the stomach of humans, resulting in gastric pathologies. In pigs, H. suis can induce gastritis and seems to play a role in gastric ulcer disease, seriously affecting animal production and welfare. Since close interactions between domestic animals, wildlife, and humans can increase bacterial transmission risk between species, samples of gastric tissue of 14 free range wild boars (Sus scrofa) were evaluated for the presence of H. pylori and H. suis using PCR. Samples from the antral gastric mucosa from two animals were PCR-positive for H. pylori and another one for H. suis. These findings indicate that these microorganisms were able to colonize the stomach of wild boars and raise awareness for their putative intervention in Helicobacter spp. transmission cycle.

Peptide ELISA and FRET-qPCR Identified a Significantly Higher Prevalence of Chlamydia suis in Domestic Pigs Than in Feral Swine from the State of Alabama, USA.

Chlamydia suis is an important, highly prevalent, and diverse obligate intracellular pathogen infecting pigs. In order to investigate the prevalence and diversity of C. suis in the U.S., 276 whole blood samples from feral swine were collected as well as 109 fecal swabs and 60 whole blood samples from domestic pigs. C. suis-specific peptide ELISA identified anti-C. suis antibodies in 13.0% of the blood of feral swine (26/276) and 80.0% of the domestic pigs (48/60). FRET-qPCR and DNA sequencing found C. suis DNA in 99.1% of the fecal swabs (108/109) and 21.7% of the whole blood (13/60) of the domestic pigs, but not in any of the assayed blood samples (0/267) in feral swine. Phylogenetic comparison of partial C. suis ompA gene sequences and C. suis-specific multilocus sequencing typing (MLST) revealed significant genetic diversity of the C. suis identified in this study. Highly genetically diverse C. suis strains are prevalent in domestic pigs in the USA. As crowding strongly enhances the frequency and intensity of highly prevalent Chlamydia infections in animals, less population density in feral swine than in domestic pigs may explain the significantly lower C. suis prevalence in feral swine. A future study is warranted to obtain C. suis DNA from feral swine to perform genetic diversity of C. suis between commercial and feral pigs.

Seroprevalence and Molecular Identification of Brucella spp. in Camels in Egypt.

Brucellosis is one of the most important worldwide zoonoses of many countries including Egypt. Camel brucellosis has not gained much attention in Egypt yet. This study is focused on the three governorates with the highest camel populations and the largest camel markets in the country to determine the disease seroprevalence and identify the Brucella species in local camel holdings. In total, 381 serum samples were collected from male and female camels from Giza, Aswan, and Al-Bahr Al-Ahmar (the Red Sea) governorates. Samples were serologically examined using the Rose–Bengal plate test (RBPT), indirect ELISA (i-ELISA), competitive ELISA (c-ELISA) and complement fixation test (CFT). Brucella antibodies were detected in 59 (15.5%), 87 (22.8%), 77 (20.2%) and 118 (31.0%) of sera by RBPT, i-ELISA, c-ELISA and CFT, respectively. Using real-time PCR, Brucella DNA was amplified in 32 (8.4%) seropositive samples including Brucella abortus (25/32), Brucella suis (5/32) and Brucella melitensis (2/32), defining a complex epidemiological status. To the best of our knowledge, this is the first study reporting Brucella suis DNA in camel serum. The risk-associated factors including age, sex, breed and geographical distribution were statistically analyzed, showing non-significant association with seroprevalence. The results of this study will raise awareness for camel brucellosis and help develop effective control strategies.

First molecular detection of Mycoplasma suis in the pig louse Haematopinus suis (Phthiraptera: Anoplura) from Argentina

Porcine haemoplasmosis caused by Mycoplasma suis affects the global pig industry with significant economic losses. The main transmission route of M. suis is through the blood and some haematophagous arthropods, like flies and mosquitoes, could be the vectors to this pathogen. However, the presence of M. suis in pig haematophagous ectoparasites in natural conditions has not yet been studied. The most frequent ectoparasite in pigs is the blood-sucking louse Haematopinus suis, an obligate and permanent parasite. Therefore, this work aims to study the occurrence of M. suis in H. suis samples from both domestic and wild pig populations from Argentina; using the 16S rRNA gene. A total of 98 sucking lice, collected from domestic and wild pigs from Buenos Aires Province in central Argentina, were examined. We found M. suis DNA in 15 H. suis samples (15.30%). Positive lice were detected from all studied populations. This is the first report of M. suis presence in H. suis, being also the first detection in a pig ectoparasite species. We conclude that H. suis could serve as a mechanical vector for M. suis. This information not only extends the knowledge about the pathogen spectrum potentially transmitted by H. suis, but may be also useful in epidemiological studies about Mycoplasma.

Co-Occurrence of Chlamydia suis DNA and Chlamydia suis-Specific Antibodies in the Human Eye.

Chlamydia suis is a swine pathogen that causes economic losses due to reproductive failure. Recently, C. suis has been detected in human eyes. However, knowledge of the zoonotic potential is still limited. C. suis infections in swine could present a risk for public health because (1) tetracycline-resistant C. suis strains are emerging in the pork industry, (2) tetracycline resistance gene transfers in vitro from C. suis to the human pathogen Chlamydia trachomatis and as previously demonstrated, (3) C. suis and C. trachomatis can be both present in the human eye. Pig farmers were sampled during a seminar in West-Flanders. Conjunctival swabs for detection of C. suis and C. trachomatis and for the detection of mucosal antibodies against C. suis and C. trachomatis were collected. The farmers completed a questionnaire designed to assess information on the following: (1) the health status of their pigs, (2) administration of veterinary drugs, (3) their professional and nonprofessional activities, (4) general health status, (5) smoking habits, (6) use of medication, (7) allergies, and (8) clinical signs/history. Thirty-three on 40 (82.5%) farmers participated. None of the conjunctival swabs contained C. trachomatis DNA and mucosal antibodies against C. trachomatis were not detected. Six of 33 (18.2%) farmers had C. suis DNA in their eyes and 22 of 33 (67%) swabs contained C. suis-specific mucosal antibodies. The older the farmer, higher the chance of finding C. suis antibodies in the eye. There was a significant correlation between the presence of conjunctivitis in the pigs and the occurrence of C. suis DNA in the eye of their owner. This study shows that C. suis may transfer from pigs to the human eye as specific mucosal antibodies were detected in conjunctivae of pig farmers. Veterinarians, general practitioners, and occupational physicians should be aware of the zoonotic potential of C. suis.

Presence of Helicobacter suis on pork carcasses

Helicobacter (H.) suis is a world-wide spread pathogen which not only colonizes the stomach of pigs, but is also the most prevalent gastric non-H. pylori Helicobacter (NHPH) species in humans. H. suis infections are associated with gastric lesions both in pigs and in humans. Recently, the presence of viable H. suis bacteria has been demonstrated in minced pork, suggesting that manipulation or consumption of contaminated pig meat is a possible route of transmission of this zoonotic agent. The main goal of this study was to determine the extent of pork carcass contamination with H. suis at slaughter. In two consecutive studies, the occurrence of H. suis DNA was assessed in scalding water, head and mouth swabs, mesenteric lymph nodes, palatine tonsils and on the chest, shoulder and ham region of pork carcasses from three slaughterhouses using qPCR with ureA gene based H. suis-specific primers. H. suis DNA was detected on carcasses in all slaughterhouses, in 8.3% of all 1083 samples. It was found in all sampled matrices, except for the palatine tonsils and scalding water samples. Contamination levels of dressed pork samples did not exceed 184 genomic equivalents per 100cm2 (shoulder, ham) or 300cm2 (chest). All positive PCR products were subjected to sequence analysis of the ureA gene to confirm the identification of H. suis bacteria. Using multilocus sequence typing (MLST) on a selection of the positive samples, 5 unique sequence types (STs) could be assigned. Multiple H. suis strains were present on samples derived from one specific pig herd. Since H. suis DNA was detected in 11% (n: 90) of the mesenteric lymph nodes derived at the slaughterhouse, it was determined whether these organisms can colonize the mesenteric lymph nodes after experimental infection. Despite high-level colonization of the porcine stomachs with the H. suis strain, no H. suis DNA was detected in the mesenteric lymph nodes at four weeks after experimental infection. This might indicate that its presence in these tissues of slaughtered pigs is due to contamination during the slaughter process, but further studies are necessary to confirm this. In conclusion, we demonstrate a relatively high prevalence of H. suis on pork carcasses.

Quantitative PCR analysis of Mycoplasma suis shedding patterns during experimental infection

The uncultivable hemotrophic bacterium Mycoplasma suis causes infectious anemia in pigs worldwide. The mechanisms by which M. suis is transmitted from pig to pig are largely unknown. Thus, the present study aimed at investigating urine, feces, saliva, nasal and vaginal secrets as well as environmental samples for the presence of M. suis DNA to get insights into potential transmission routes. Seven pigs were experimentally infected with M. suis KI3806. Samples were taken for 8 days post infection (p.i.). A quantitative LightCycler msg1 PCR was used to detect and quantify M. suis. Shedding was found in saliva as well as nasal and vaginal secrets from day 6 p.i. on with a quantity of 3.4×102 to 2.7×105M. suis/swab. In urine M. suis DNA could be detected in 100.0% of the samples from day 6 p.i. on with a quantity of 4.7×102 to 6.3×105M. suis per mL. When shedding patterns were correlated to the median bacterial blood loads shedding was observed at loads of 2.0×109–7.0×1010M. suis per mL blood. No M. suis DNA could be amplified from feces. Dust and water samples of the pig drinking troughs were positive for M. suis on days 2 and 6 post infection, air samples were M. suis-negative throughout the experiment. Our results indicate that blood independent direct transmission as well as indirect transmission via environmental contamination could play a role in the epidemiology of M. suis infections.

Identification of Helicobacter suis in pig-producing regions of the United States

Objectives: To develop a non-culture-based method to determine levels of Helicobacter suis infection in porcine stomachs and to test the method in a sample of pigs from a variety of regions in the United States. Materials and methods: A polymerase chain reaction (PCR) assay was developed to quantitate total Helicobacter generic DNA and Helicobacter suis species-specific DNA in pig stomachs. Primers were derived from 16s ribosomal RNA (rRNA) gene sequences, selected on the basis of relative conservation and divergence of sequences across the various Helicobacter species. The assay was standardized using cloned 16s rRNA sequences and was initially tested with DNA isolated from cultured H suis. Gastric mucosal scrapings were collected from pigs in three geographic regions of the United States, including the North (Minnesota and Michigan), East Central (Iowa), and South (Oklahoma and North Carolina). Results: Of a total of 118 pigs tested, approximately half (55.1%; 95% CI, 46.1%-63.8%) were positive for H suis DNA. Helicobacter suis DNA was detected in pigs from all states tested. Implications: Helicobacter suis is present in US pigs and may be relevant to pig health and production. This quantitative PCR assay will facilitate further study of H suis in pigs, including potential therapeutic and prophylactic interventions.

Survival of Helicobacter suis bacteria in retail pig meat

Helicobacter (H.) suis colonizes the gastric mucosa of pigs world-wide and is the most prevalent non-Helicobacter pylori Helicobacter species in humans. This agent might be transmitted to humans by manipulation or consumption of contaminated pork. H. suis is a very fastidious micro-organism and is extremely difficult to isolate. Therefore, we developed a non-culture dependent, quantitative detection method allowing differentiation of viable from dead H. suis bacteria in pork. This was established by a combination of ethidium bromide monoazide (EMA) treatment and real-time (RT)-PCR. This EMA RT-PCR was applied to 50 retail pork samples. In two samples, viable H. suis bacteria were detected. Sequence analysis of the obtained PCR products confirmed the presence of H. suis DNA. Viable H. suis bacteria persisted for at least 48h in experimentally contaminated pork. In conclusion, consumption of contaminated pork may constitute a new route of transmission for H. suis infections in humans.

Identification of the virB operon genes encoding the type IV secretion system, in Colombian Brucella canis isolates

Canine brucellosis is a zoonotic disease caused by Brucella canis. The establishment of intracellular replicative niches of B. canis is mediated by proteins secreted by the type IV secretion system, which is encoded by the virB operon. The characterization of such genes has been conducted in other species of the genus, but not in B. canis.We report the design of a multiplex PCR test for the detection of the virB operon genes of B. canis. Primers for each of the 12 genes were designed and evaluated using bioinformatics tools. A multiplex PCR assay was standardized and applied to 36 isolates obtained from infected dogs of Aburrá Valley kennels, as well to the Brucella abortus, Brucella melitensis, Brucella suis and Brucella ovis DNA strains. As a result of the in silico design, a pair of primers for each gene was selected. All species and isolates evaluated showed evidence of the presence of the entire virB operon.

The surface-localised α-enolase of Mycoplasma suis is an adhesion protein

Mycoplasma suis belongs to the haemotrophic mycoplasmas which colonise red blood cells of a wide range of vertebrates. Adhesion to red blood cells is the crucial step in the unique lifecycle of M. suis. Due to the lack of a cultivation system, identification of adhesion structures has been difficult. So far, only one adhesion protein, i.e. MSG1 was identified. In order to determine further adhesion molecules of M. suis, we screened genomic M. suis libraries and performed Southern blot hybridisation analyses of genomic M. suis DNA. The α-enolase of M. suis was identified and analysed genetically and functionally. The encoding gene has 1623bp in size. The deduced amino acid sequence showed an overall identity of 59.6–65.1% to α-enolases of other pathogenic mycoplasmas. The 540aa M. suis α-enolase displays a size extension of about 90aa in comparison to α-enolases of other mycoplasmas. Recombinant α-enolase expressed in Escherichia coli demonstrated immunogenicity in experimentally infected pigs. Immunoblot, confocal laser scanning microscopy and immune electron microscopy analysis using antibodies against recombinant α-enolase, indicate the membrane and surface localisation of native α-enolase in M. suis, though no typical signal sequences exist. Furthermore, we showed that recombinant α-enolase binds to porcine erythrocyte lysate in a dose-dependent manner. E. coli transformants which express α-enolase on their surface acquire the ability to adhere to porcine red blood cells. In conclusion, our observations indicate that α-enolase could be involved in the adhesion of M. suis to porcine red blood cells.

Development of a real-time polymerase chain reaction assay for detection of Actinobacillus suis in porcine lung

In the current study, the development and validation of a real-time polymerase chain reaction (PCR) assay using a TaqMan-labeled probe for the detection of Actinobacillus suis from porcine lung samples is described. This real-time PCR amplified a 110-bp region of the 23S ribosomal RNA gene from A. suis but not from other bacteria. First, the assay was validated with 183 bacterial strains representing different species of bacteria. Subsequently, 85 porcine lung specimens that were declared A. suis-positive and -negative by bacterial culture and identification were tested to assess whether it can be performed directly on tissue specimens. The bacterial culture results and real-time PCR results agreed across all the samples tested assigning 100% positive and negative predictive values to the PCR. Further, the detection limit of the assay was 380 colony-forming units (CFU) per ml or approximately 1 CFU per reaction. In conclusion, the TaqMan real-time PCR assay described herein is a highly specific, sensitive, and reproducible test, which can be used to detect A. suis DNA in porcine lung specimens, thus providing a timely diagnosis.

Possible pathogenic interplay between Chlamydia suis, Chlamydophila abortus and PCV-2 on a pig production farm

A concurrent outbreak of chlamydial disease in boars, sows and gilts and postweaning multisystemic wasting syndrome (PMWS) in weaned piglets was investigated on a large pig production farm in Estonia....