Genetic diversity of Mycoplasma hyopneumoniae isolates of abattoir pigs

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, is present in swine herds worldwide. However, there is little information on strains infecting herds in Canada. A total of 160 swine lungs with lesions suggestive of enzootic pneumonia originating from 48 different farms were recovered from two slaughterhouses and submitted for gross pathology. The pneumonic lesion scores ranged from 2% to 84%.Eighty nine percent of the lungs (143/160) were positive for M. hyopneumoniae by real-time PCR whereas 10% (16/160) and 8.8% (14/160) were positive by PCR for M. hyorhinis and M. flocculare, respectively. By culture, only 6% of the samples were positive for M. hyopneumoniae (10/160). Among the selected M. hyopneumoniae-positive lungs (n = 25), 9 lungs were co-infected with M. hyorhinis, 9 lungs with PCV2, 2 lungs with PRRSV, 12 lungs with S. suis and 10 lungs with P. multocida. MLVA and PCR-RFLP clustering of M. hyopneumoniae revealed that analyzed strains were distributed among three and five clusters respectively, regardless of severity of lesions, indicating that no cluster is associated with virulence. However, strains missing a specific MLVA locus showed significantly less severe lesions and lower numbers of bacteria. MLVA and PCR-RFLP analyses also showed a high diversity among field isolates of M. hyopneumoniae with a greater homogeneity within the same herd. Almost half of the field isolates presented less than 55% homology with selected vaccine and reference strains.     

Occurrence and severity of lung lesions in slaughter pigs vaccinated against Mycoplasma hyopneumoniae with different strategies

Different vaccination strategies against Mycoplasma hyopneumoniae have been adopted worldwide. Reports from the field indicate varying levels of protection among currently available vaccines. The goal of the present study was to compare the efficacies of three widespread commercial vaccination strategies against M. hyopneumoniae under field conditions. 20 farms were included. 14 farms used different single dose vaccines (vaccine 1 [V1], 8 herds; vaccine 2 [V2], 6 herds); another 6 farms (V3) used a two dose vaccination strategy. Gross lesions of 854 lungs and histopathology from 140 lungs were quantified, and a quantitative PCR was applied to detect M. hyopneumoniae and porcine circovirus 2 (PCV2) DNA in lung tissue (n = 140). In addition, porcine reproductive and respiratory disease virus (PRRSV), swine influenza virus (SIV), Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida were tested by qualitative PCR. 53% of lungs were positive for M. hyopneumoniae. 55.9% of lungs showed macroscopic enzootic pneumonia (EP)-like lesions. Lung lesion scores (P < 0.001) and M. hyopneumoniae-loads (P < 0.008) differed significantly among the vaccination groups, with the most severe cases and highest amounts occurring in V1. Histological alterations differed (P < 0.001) between V1 and V3. Lung lesion scores and histopathological changes were significantly correlated, with prevalence and load of M. hyopneumoniae indicating that the applied diagnostic tools are valuable in confirming the prevalence and severity of M. hyopneumoniae infections. Comparing different vaccination strategies against M. hyopneumoniae indicates varying levels of protection. M. hyopneumoniae is still a major problem despite the widely applied vaccination.     

A comparative study of the complement fixation test for mycoplasma hyopneumoniae in SPF and non-SPF herds of swine in Illinois

Serum samples from 458 swine from eight specific pathogen-free (SPF) herds swine were compared to samples from 178 swine from eight non-SPF herds in Illinois using the complement fixation (CF) test for Mycoplasma hyopneumoniae antibodies. In SPF herds, 9.4% of the samples gave positive test results, compared to 51.7% of the samples from non-SPF herds. The range of reactor rates in SPF herds was 0–26.9% and in non-SPF herds 3.4–100%. Used on herd basis with some modification, the CF test should be useful in monitoring state and nationally accredited SPF swine herds.     

Adaptation of a latex agglutination tube test for diagnosis of Mycoplasma hyopneumoniae swine pneumonia

A tube latex agglutination test (LAT) for diagnosis of Mycoplasma hyopneumoniae swine pneumonia was developed. In pigs inoculated with M. hyopneumoniae, LAT antibodies were generally detected 2–3 weeks post-inoculation, which was 1 week or more before complement-fixing antibodies were first detected in the corresponding pigs. Correlation of LAT results and gross and microscopic lung lesions of corresponding pigs revealed that LAT titers persisted after the pneumonic lesions had resolved.Latex agglutination titers in pigs exposed by contact with M. hyopneumoniae inoculated pigs were demonstrated 4–12 weeks post-contact.Latex agglutination antibodies were detected up to 48 weeks post-inoculation in pigs inoculated with M. hyopneumoniae and this was similar to the duration of detectable indirect hemagglutination titers. Complement-fixation titers, however, were demonstrated no longer than 28 weeks post-inoculation in corresponding pigs.Evaluation of repeatability of LAT results revealed some variation of LAT titers of sera titered on four different occasions.Sera from pigs inoculated with other swine mycoplasmas, including M. hyosynoviae and M. hyorhinis, did not react in the M. hyopneumoniae LAT. In addition, no detectable titers were demonstrated in the M. hyopneumoniae LAT using sera from pigs infected with Metastrongylus spp., Ascaris suum, or in sera from pigs vaccinated with any of four commonly used swine vaccines.     

Herd specific risk factors for Mycoplasma hyopneumoniae infections in suckling pigs at the age of weaning

Background

Mycoplasma hyopneumoniae is the etiologic agent of enzootic pneumonia mainly occurring in fattening pigs. It is assumed that horizontal transmission of the pathogen during nursery and growing phase starts with few suckling pigs vertically infected by the sow. The aim of the present study was the exploration of the herd prevalence of M. hyopneumoniae infections in suckling pigs followed by an investigation of various herd specific factors for their potential of influencing the occurrence of this pathogen at the age of weaning.

Results

In this cross-sectional study, 125 breeding herds were examined by taking nasal swabs from 20 suckling pigs in each herd. In total, 3.9% (98/2500) of all nasal swabs were tested positive for M. hyopneumoniae by real-time PCR. Piglets tested positive originated from 46 different herds resulting in an overall herd prevalence of 36.8% (46/125) for M. hyopneumoniae infection in pigs at the age of weaning. While the herds were epidemiologically characterized, the risk for demonstration of M. hyopneumoniae was significantly increased, when the number of purchased gilts per year was more than 120 (OR: 5.8), and when the number of farrowing pens per compartment was higher than 16 (OR: 3.3). In herds with a planned and segregated production, where groups of sows entered previously emptied farrowing units, the risk for demonstration of M. hyopneumoniae in piglets was higher in herds with two or four weeks between batches than in herds with one or three weeks between batches (OR: 2.7).

Conclusions

In this cross-sectional study, several risk factors could be identified enhancing the probability of breeding herds to raise suckling pigs already infected with M. hyopneumoniae at the time of weaning. Interestingly, some factors (farrowing rhythm, gilt acclimatisation issues) were overlapping with those also influencing the seroprevalences among sows or the transmission of the pathogen between older age groups. Taking the multifactorial character of enzootic pneumonia into account, the results of this study substantiate that a comprehensive herd specific prevention programme is a prerequisite to reduce transmission of and disease caused by M. hyopneumoniae.     

Immunological characterization of peripheral blood leukocytes using vaccine for mycoplasmal pneumonia of swine (MPS) in swine line selected for resistance to MPS

This study was conducted to evaluate immunological changes in peripheral blood leukocytes in pigs that were genetically selected for their improved resistance to mycoplasmal pneumonia of swine (MPS), using MPS vaccine as an antigen. Twelve castrated MPS‐selected Landrace pigs were compared with the same number of pigs from a nonselected line by using a time‐course analysis at the hematological level. After the second sensitization with MPS vaccine, the percentages of B cells, CD4⁺ T cells, and natural killer (NK) cells in total leukocytes were lower in the selected line than in the nonselected line, whereas the percentage of granulocytes in total leukocytes increased in the MPS‐selected line. We also assessed the proliferative ability of peripheral blood mononuclear cells (PBMCs) stimulated with Mycoplasma hyopneumoniae, lipopolysaccharide or concanavalin A, and found that although the proliferative ability of the PBMC was not different between the two lines at a steady state, the nonselected line showed a significantly higher proliferative ability after sensitization with MPS vaccine than the selected line regardless of antigens used. These results thus indicate that the selection of pigs on the basis of MPS resistance changes their immunophenotype, and would give us beneficial information for the prevention of MPS infection.     

Natural history of influenza in swine in Hawaii: swine influenza virus (Hsw1N1) in herds not infected with lungworms

To obtain more information on mechanisms by which influenza virus is perpetuated in swine, retrospective and prospective seroepizootiologic observations were made in swine herds in Hawaii, beginning in 1974. An epizootic of swine influenza (Hsw1N1) virus was observed in November and December 1976, involving 31 of 41 herds. Features of the epizootic included (1) infection of all herds within one geographic location, during a short period; (2) no obvious introduction of virus from the outside in most herds; (3) epizootics mainly in herds with serologic history of infection; (4) no evidence that lungworms were involved; and (5) little clinical disease associated with infection. There was evidence of viral activity in some herds before the epizootic period and afterward in two of three herds monitored. Evidence of viral transmission by feral animals was not obtained. Data indicated that swine influenza virus persists through latently or chronically infected swine, with epizootics occurring when herd immunity reaches a critically low degree.     

Dynamics of Mycoplasma hyopneumoniae infection in 12 farms with different production systems

This study had 2 objectives: 1) to determine the involvement of Mycoplasma hyopneumoniae in respiratory outbreaks in herds of pigs, with the use of a nested polymerase chain reaction (nPCR) and an enzyme-linked immunosorbent assay (ELISA); and 2) to determine if the dynamics of M. hyopneumoniae infection differ between 3-site versus 1- or 2-site production systems (in which at least farrowing/gestation and nursery pigs are on the same site). Animals of different ages from 12 Spanish farms with respiratory problems were randomly sampled. Blood samples and nasal swabs were collected in a single farm visit, and ELISA and nPCR tests, respectively, were performed. All the farms demonstrated M. hyopneumoniae. According to the proportions of infected animals and the appearance of clinical signs in the different age groups, the farms were divided into 2 groups: farms in which M. hyopneumoniae probably played an important role in the observed respiratory outbreak and farms in which M. hyopneumoniae was not the main agent involved in the outbreak. Although seroconversion occurred in most herds in the finishing units, the number of seropositive pigs in the first group of farms was greater than the number in the second group. Statistically significant differences (P < 0.0001) between farms with a 1- or 2-site production system versus those with a 3-site production system were detected in nPCR results but not in rates of seroconversion. The farm effect also had a great influence on both controlled parameters: the pathogen’s DNA and antibody detection. Thus, although M. hyopneumoniae was present in all the studied farms, there were significant differences in the infection dynamics and clinical implications according to the type of production system, and M. hyopneumoniae colonization and seroconversion were greatly influenced by the effect of the individual farm.     

Seroprevalence and risk factors associated with seropositivity in sows from 67 herds in north-west Germany infected with Mycoplasma hyopneumoniae

Risk factors for the spread of Mycoplasma hyopneumoniae in sows have not been studied although vertical transmission from sows to their offspring is considered a significant risk factor in the development of enzootic pneumonia in growers and finishers. Seropositivity for M. hyopneumoniae in sows, as assessed by commercial ELISA, is a possible indicator of infection pressure among sows. The objective of this study was to estimate seroprevalence and associated risk factors of a sow being seropositive for M. hyopneumoniae. A cross-sectional study was carried out in 2578 sows from 67 herds in north-west Germany. Data concerning general herd characteristics, acclimatisation practices, indoor and outside contacts, as well as data describing the immediate local environment were collected during a herd visit via questionnaire. Blood samples were seropositive in 65% of the 2578 sows, and all herds had ≥14% seropositive sows. Data analysis was performed in two steps. First, univariate analysis of predictor variables for the risk of a sow being seropositive for M. hyopneumoniae was performed using chi-square test. Secondly, all variables associated with the risk of a sow being seropositive (P ≤ 0.25) were included in a multivariate model using a generalised linear model. The risk of a sow being seropositive for M. hyopneumoniae was increased in herds with two- or three-site production (OR 1.50), when piglets were not vaccinated against M. hyopneumoniae (OR 1.81), in herds with a 2-week farrowing intervals (OR 1.84) and in herds without all-in/all-out management of the farrowing units (OR 1.37). The lack of an acclimatisation period for replacement boars was also associated with the risk of a sow being seropositive (OR 2.10). The results indicate that M. hyopneumoniae seropositivity is common in sows in north-west Germany and is influenced by various management factors. It is recommended that evaluation of sow herd management should be included in any strategic health plan to control M. hyopneumoniae infection.     

Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce inflammation and apoptosis in porcine peripheral blood mononuclear cells in vitro

Mycoplasma hyopneumoniae is the causative agent of swine enzootic pneumonia (EP), a disease that causes considerable economic losss in swine industry. Lipid-associated membrane proteins (LAMPs) of mycoplasma play important roles in causing mycoplasma diseases. The present study explores the pathogenic mechanisms of M. hyopneumoniae LAMPs by elucidating their role in modulating the inflammation, apoptosis, and relevant signaling pathways of peripheral blood mononuclear cells (PBMCs) of pig. LAMP treatment inhibited the growth of PBMCs. Up-regulation of cytokines, such as IL-6 and IL-1β, as well as increased production of nitric oxide (NO) and superoxide anion were all detected in the supernatant of LAMPs-treated PBMCs. Furthermore, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMPs of M. hyopneumoniae induced a time-dependent apoptosis in lymphocyts and monocytes from PBMCs, which was blocked by NOS inhibitor or antioxidant. In addition, LAMPs induced the phosphorylation of p38, the ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2, activation of caspase-3 and caspase-8, and poly ADP-ribose polymerase (PARP) cleavage in PBMCs. These findings demonstrated that M. hyopneumoniae LAMPs induced the production of proinflammatory cytokines, NO and reactive oxygen species (ROS), and apoptosis of PBMCs in vitro through p38 MAPK and Bax/Bcl-2 signaling pathways, as well as caspase activation.     

Evidence of long distance airborne transport of porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae to be transported over long distances via the airborne route was evaluated. A source population of 300 grow-finish pigs was experimentally inoculated with PRRSV MN-184 and M. hyopneumoniae 232 and over a 50-day period, air samples were collected at designated distances from the source herd using a liquid cyclonic collector. Samples were tested for the presence of PRRSV RNA and M. hyopneumoniae DNA by PCR and if positive, further characterized. Of the 306 samples collected, 4 (1.3%) were positive for PRRSV RNA and 6 (1.9%) were positive for M. hyopneumoniae DNA. The PRRSV-positive samples were recovered 4.7 km to the northwest (NW) of the source population. Four of the M. hyopneumoniae-positive samples were obtained at the NW sampling point; 2 samples at approximately 2.3 km and the other 2 samples approximately 4.7 km from the source population. Of the remaining 2 samples, one sample was obtained at the southeast sampling point and the other at the southwest sampling point, with both locations being approximately 4.7 km from the source. The four PRRSV-positive samples contained infectious virus and were ≥ 98.8% homologous to the MN-184 isolate used to inoculate the source population. All 6 of the M. hyopneumoniae-positive samples were 99.9% homologous to M. hyopneumoniae 232. These results support the hypothesis that long distance airborne transport of these important swine pathogens can occur.     

Immunogenic properties of Landrace pigs selected for resistance to mycoplasma pneumonia of swine

Mycoplasma pneumonia of swine (MPS) lung lesions and immunogenic properties were compared between a Landrace line that was genetically selected for reduced incidence of pulmonary MPS lesions, and a non‐selected Landrace line. The MPS‐selected Landrace line showed significantly lower degrees of pulmonary MPS lesions compared with the non‐selected Landrace line. When changes in immunity before and after vaccination were compared, the percentage of B cells in the peripheral blood of the MPS‐selected Landrace line was significantly lower than that of the non‐selected line. Furthermore, the concentration of growth hormone and the mitogen activity of peripheral blood mononuclear cells in the MPS‐selected Landrace line showed significantly (P < 0.05) lower increases after vaccination than the non‐selected line. Conversely, the concentration of peripheral blood interferon (IFN)‐γ and salivary immunoglobulin A (IgA) after Mycoplasma hyopneumoniae vaccination was significantly higher in the MPS‐selected Landrace line than in the non‐selected line. Gene expression of toll‐like receptor (TLR)2 and TLR4 was significantly higher in the MPS‐selected Landrace line in immune tissues, with the exception of the hilar lymph nodes. The present results suggest that peripheral blood IFN‐γ, salivary IgA TLR2, and TLR4 are important immunological factors influencing the development of MPS lesions.     

Exposure of feral swine (Sus scrofa) in the United States to selected pathogens

Feral swine (Sus scrofa) are widely distributed in the United States. In 2011 and 2012, serum samples and tonsils were recovered from 162 and 37 feral swine, respectively, in the US to evaluate exposure to important swine endemic pathogens. Antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) were found in 2.5% and 25.3% of tested sera, respectively. Positive serological reactions against Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae have been detected in 19.7% and 69.7% of animals. More than 15% of animals presented antibodies against these 2 pathogens simultaneously. Most animals were also seropositive for Lawsonia intracellularis. Feral swine can also be involved in transmission of zoonotic agents. Almost 50% of animals possessed antibodies against Salmonella. In addition, 94.4% of animals were carriers of Streptococcus suis in their tonsils. In conclusion, feral swine may be considered as a potential reservoir for different endemic diseases in domestic pigs, as well as for important zoonotic agents.     

Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations

Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.     

Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to pig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, but current vaccines offer only partial control and there is a need for improved preventative strategies. A major barrier to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack of tools to genetically manipulate the organism. We describe the development and optimisation of the first successful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids contain the origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. With these plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generating tetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNA over serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. In addition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising the conditions necessary for successful transformation, we have used this system to determine the minimum functional oriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintained over multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we have generated a set of plasmids that will be valuable in studies of M. hyopneumoniae pathogenesis and provide a major step forward in the study of this important swine pathogen.     

Interaction between single-dose Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus vaccines on dually infected pigs

The objective of this study was to determine the effects of Mycoplasma hyopneumoniae and/or porcine reproductive and respiratory syndrome virus (PRRSV) vaccination on dually infected pigs. In total, 72 pigs were randomly divided into nine groups (eight pigs per group), as follows: five vaccinated and challenged groups, three non-vaccinated and challenged groups, and a negative control group. Single-dose vaccination against M. hyopneumoniae alone decreased the levels of PRRSV viremia and PRRSV-induced pulmonary lesions, whereas single-dose vaccination against PRRSV alone did not decrease nasal shedding of M. hyopneumoniae and mycoplasma-induced pulmonary lesions in the dually infected pigs. The M. hyopneumoniae challenge impaired the protective cell-mediated immunity induced by the PRRSV vaccine, whereas the PRRSV challenge did not impair the protective cell-mediated immunity induced by the M. hyopneumoniae vaccine. The present study provides swine practitioners and producers with efficient vaccination regimes; vaccination against M. hyopneumoniae is the first step in protecting pigs against co-infection with M. hyopneumoniae and PRRSV.     

Current perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection

Mycoplasma hyopneumoniae is the principal aetiological agent of enzootic pneumonia (EP), a chronic respiratory disease that affects mainly finishing pigs. Although major efforts to control M. hyopneumoniae infection and its detrimental effects have been made, significant economic losses in pig production worldwide due to EP continue. M. hyopneumoniae is typically introduced into pig herds by the purchase of subclinically infected animals or, less frequently, through airborne transmission over short distances. Once in the herd, M. hyopneumoniae may be transmitted by direct contact from infected sows to their offspring or between pen mates.The ‘gold standard’ technique used to diagnose M. hyopneumoniae infection, bacteriological culture, is laborious and is seldom used routinely. Enzyme-linked immunosorbent assay and polymerase chain reaction detection methods, in addition to post-mortem inspection in the form of abattoir surveillance or field necropsy, are the techniques most frequently used to investigate the potential involvement of M. hyopneumoniae in porcine respiratory disease. Such techniques have been used to monitor the incidence of M. hyopneumoniae infection in herds both clinically and subclinically affected by EP, in vaccinated and non-vaccinated herds and under different production and management conditions. Differences in the clinical course of EP at farm level and in the efficacy of M. hyopneumoniae vaccination suggest that the transmission and virulence characteristics of different field isolates of M. hyopneumoniae may vary. This paper reviews the current state of knowledge of the epidemiology of M. hyopneumoniae infection including its transmission, infection and seroconversion dynamics and also compares the various epidemiological tools used to monitor EP.     

Genetic selection for resistance to mycoplasmal pneumonia of swine (MPS) in the Landrace line influences the expression of soluble factors in blood after MPS vaccine sensitization

We recently developed a Landrace line that is resistant to mycoplasmal pneumonia of swine (MPS) infection by genetic selection for five generations, and we reported that the immunophenotype of this line is different from that of the non‐selected line in terms of changes in peripheral blood leukocyte population after MPS vaccination. This study followed up previous findings demonstrating changes in soluble factors in blood, namely, hormones, Mycoplasma hyopneumoniae‐specific immunoglobulin G (IgG), and cytokines. These two lines were injected with MPS vaccine on days ?7 and 0 after blood sampling on those days, and blood samples were collected on days ?14, ?7, 0, 2, 7 and 14. We found changes in the levels of many hormones and cytokines in both lines. However, we found that only growth hormone (GH) and interferon (IFN)‐γ levels were statistically different between these two lines. GH concentration was reduced (day 0) and IFN‐γ concentration was increased (day 14) in the MPS‐selected line compared with the non‐selected line, despite unchanged IFN‐γ messenger RNA expression in blood cells. Although detailed mechanisms underlying these phenotypes remain unsolved, these traits would be useful to improve MPS resistance in pig production and provide an insight into MPS infection.     

A comparative study of ELISA and other methods for the detection of Brucella antibodies in bovine sera

Enzyme-linked immunosorbent assay (ELISA), using β-galactosidase and a fluorigenic substrate, was used for the detection of antibodies to Brucella abortus in bovine sera.Among 677 animals from 9 brucellosis-free herds, none reacted in the ELISA. Among 785 animals from 23 brucellosis-infected herds, 336 were positive in ELISA, 229 in the slow agglutination test (SAT), 185 in the complement fixation test (CFT), and 165 in the Rose-Bengal test (RBT).Experimental infections were conducted with two B. abortus strains. At slaughter on day 101, after intraconjunctival infection of heifers with B. abortus strain 19 organisms, 3 animals were positive in the SAT, 3 in the CFT, 4 in the RBT and 11 in the ELISA, and Brucella organisms could be cultivated from 10 animals; among these, 2 scored positive in the SAT, 3 in the CFT, 3 in the RBT and 8 in the ELISA test. Seventeen heifers were infected with organisms of B. abortus strain 2308. On day 101, 11 heifers were found to be carriers, all of which yielded positive results in the CFT, RBT and ELISA tests, but not in the SAT.     

Genotype distribution of Mycoplasma hyopneumoniae in swine herds from different geographical regions

Genetic heterogeneity of Mycoplasma hyopneumoniae in pigs has been reported, however there has been limited reproducibility on the molecular methods employed so far. The aim of this study was to modify and standardize a high-resolution multiple locus variable number tandem repeat analysis (MLVA), to investigate the genetic variability of M. hyopneumoniae circulating in the United States of America (USA), Brazil, Mexico and Spain. The MLVA was standardized on the basis of the number of tandem repeats in two Mycoplasma adhesins, P97 and P146, which are proteins involved in the adherence of the pathogen to cilia. A total of 355 samples obtained from the four countries were analyzed. The Simpson's diversity index for the assay was D = 0.976 when samples from all countries were combined. A large number of MLVA types (n = 139) were identified, suggesting that multiple M. hyopneumoniae variants are circulating in swine. The locus P97 had 17 different types with 2–18 repeats. The P146 locus showed higher heterogeneity, with 34 different types, ranging from 7 to 48 repeats. MLVA types that presented more than 30 repeats in P146 were found in Spain and Brazil, while shorter repeats were observed in the USA and Mexico. This simplified MLVA method proved to be an efficient tool for typing M. hyopneumoniae with a high degree of stability, repeatability, and discriminatory power. In conclusion, M. hyopneumoniae showed a high variable number tandem repeat heterogeneity and this assay can be applied in molecular epidemiology investigations within farms and productions systems.