Observed management practices in relation to the risk of infection with paratuberculosis and to the spread of Mycobacterium avium subsp. paratuberculosis in Swiss dairy and beef herds

Abstract BACKGROUND: Many studies have been conducted to define risk factors for the transmission of bovine paratuberculosis, mostly in countries with large herds. Little is known about the epidemiology in infected Swiss herds and risk factors important for transmission in smaller herds. Therefore, the presence of known factors which might favor the spread of paratuberculosis and could be related to the prevalence at animal level of fecal shedding of Mycobacterium avium subsp. paratuberculosis were assessed in 17 infected herds (10 dairy, 7 beef). Additionally, the level of knowledge of herd managers about the disease was assessed. In a case-control study with 4 matched negative control herds per infected herd, the association of potential risk factors with the infection status of the herd was investigated. RESULTS: Exposure of the young stock to feces of older animals was frequently observed in infected and in control herds. The farmers' knowledge about paratuberculosis was very limited, even in infected herds. An overall prevalence at animal level of fecal shedding of Mycobacterium avium subsp. paratuberculosis of 6.1% was found in infected herds, whereby shedders younger than 2 years of age were found in 46.2% of the herds where the young stock was available for testing. Several factors related to contamination of the heifer area with cows' feces and the management of the calving area were found to be significantly associated with the within-herd prevalence. Animal purchase was associated with a positive herd infection status (OR = 7.25, p = 0.004). CONCLUSIONS: Numerous risk factors favoring the spread of Mycobacterium avium subsp. paratuberculosis from adult animals to the young stock were observed in infected Swiss dairy and beef herds, which may be amenable to improvement in order to control the disease. Important factors were contamination of the heifer and the calving area, which were associated with higher within-herd prevalence of fecal shedding. The awareness of farmers of paratuberculosis was very low, even in infected herds. Animal purchase in a herd was significantly associated with the probability of a herd to be infected and is thus the most important factor for the control of the spread of disease between farms.

Mycobacterium avium Subsp. avium infection in four veal calves: differentiation from intestinal tuberculosis

Mycobacterium avium subsp. avium (Maa) is an intracellular pathogen belonging to the Mycobacterium avium-intracellulare complex (MAC). Reservoirs of MAC are the natural environment, wildlife and domestic animals. In adult bovine, MAC infections are typically caused by Mycobacterium avium subsp. paratuberculosis (Map). Maa infections in bovine are rarely reported but may cause clinical disease and pathological lesions similar to those observed in paratuberculosis or those induced by members of the Mycobacterium tuberculosis complex (MTBC). Therefore, differentiation of MAC from MTBC infection should be attempted, especially if unusual mycobacterial lesions are encountered. Four veal calves from a fattening farm dying with clinical signs of otitis media, fever, and weight loss were submitted for necropsy. Samples from affected organs were taken for histologic investigation, bacteriologic culture, and bacterial specification using PCR. Macroscopic thickening of the intestinal mucosa was induced by granulomatous enteritis and colitis. Intracytoplasmic acid-fast bacteria were detected by Ziehl-Neelsen stains and PCR revealed positive results for Mycobacterium avium subsp. avium. Clinical and pathological changes of Maa infection in veal calves had features of Mycobacterium avium subsp. paratuberculosis and the MTBC. Therefore, Mycobacterium tuberculosis complex infection should be considered in cases of granulomatous enteritis in calves.

Mycobacterium avium subspecies paratuberculosis and Crohn's disease: a systematic review and meta-analysis

This systematic review assesses the evidence for an association between Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn's disease. We analysed 28 case-control studies comparing MAP in patients with Crohn's disease with individuals free of inflammatory bowel disease (IBD) or patients with ulcerative colitis. Compared with individuals free of IBD, the pooled odds ratio (OR) from studies using PCR in tissue samples was 7.01 (95% CI 3.95-12.4) and was 1.72 (1.02-2.90) in studies using ELISA in serum. ORs were similar for comparisons with ulcerative colitis patients (PCR, 4.13 [1.57-10.9]; ELISA, 1.88 [1.26-2.81]). The association of MAP with Crohn's disease seems to be specific, but its role in the aetiology of Crohn's disease remains to be defined.

Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis

BACKGROUND: Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP. RESULTS: By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%). CONCLUSIONS: There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease

Partial overlap of anti-mycobacterial, and anti-Saccharomyces cerevisiae mannan antibodies in Crohn's disease

AIM: To test whether humoral immune reaction against mycobacteria may play a role in anti-Saccharomyces cerevisiae antibodies (ASCA) generation in Crohn's disease (CD) and/or whether it correlates with clinical subtypes. METHODS: The dominant ASCA epitope was detected by Galanthus nivalis lectin (GNL)-binding assay. ASCA and IgG against mycobacterial lysates (M avium, M smegmatis, M chelonae, M bovis BCG, M avium ssp. paratuberculosis (MAP)] or purified lipoarabinomannans (LAM) were detected by ELISA. ASCA and anti-mycobacterial antibodies were affinity purified to assess cross-reactivities. Anti-mycobacterial IgG were induced by BCG-infection of mice. RESULTS: GNL bound to different extents to mycobacterial lysates, abundantly to purified mannose-capped (Man) LAM from M tuberculosis, but not to uncapped LAM from M smegmatis. Fifteen to 45% of CD patients but only 0%-6% of controls were seropositive against different mycobacterial antigens. Anti-mycobacterial IgG correlated with ASCA (r = 0.37-0.64; P = 0.003-P < 0.001). ASCA-positivity and deficiency for mannan-binding lectin synergistically associated with anti-mycobacterial IgG. In some patients, anti-mycobacterial antibodies represent cross-reactive ASCA. Vice-versa, the predominant fraction of ASCA did not cross-react with mycobacteria. Finally, fistulizing disease associated with antibodies against M avium, M smegmatis and MAP (P = 0.024, 0.004 and 0.045, respectively). CONCLUSION: Similar to ASCA, seroreactivity against mycobacteria may define CD patients with complicated disease and a predisposition for immune responses against ubiquitous antigens. While in some patients anti-mycobacterial antibodies strongly cross-react with yeast mannan; these cross-reactive antibodies only represent a minor fraction of total ASCA. Thus, mycobacterial infection unlikely plays a role in ASCA induction.

Two novel antibiotic resistance genes, tet(44) and ant(6)-Ib, are located within a transferable pathogenicity island in Campylobacter fetus subsp. fetus

New tetracycline and streptomycin resistance genes, tet(44) and ant(6)-Ib, were identified in Campylobacter fetus subsp. fetus within a transferable pathogenicity island that is typically unique to Campylobacter fetus subsp. venerealis. The 640-amino-acid tetracycline resistance determinant, Tet 44, belongs to a class of proteins that confers resistance to tetracycline and minocycline by ribosomal protection. The 286-amino-acid streptomycin resistance determinant, ANT(6)-Ib, belongs to a family of aminoglycoside nucleotidyltransferases. The resistance phenotypes were demonstrated by gene inactivation and expression.

Comparative analysis of four lipoproteins from Mycoplasma mycoides subsp. mycoides Small Colony identifies LppA as a major T-cell antigen

Control of contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), remains an important goal in Africa. Subunit vaccines triggering B and T-cell responses could represent a promising approach. To this aim, the T-cell immunogenicity of four MmmSC lipoproteins (LppA, LppB, LppC and LppQ), present in African strains and able to elicit humoral response, was evaluated. In vitro assays revealed that only LppA was recognized by lymph node lymphocytes taken from three cattle, 3 weeks after MmmSC exposure. Maintenance of the LppA-specific response, relying on CD4 T-cells and IFN gamma production, was then demonstrated 1 year after infection. LppA is thus an important target for the CD4 T-cells generated early after MmmSC infection and persisting in the lymph nodes of recovered cattle. Its role as a protective antigen and ability to in vivo trigger both arms of the host immune response remain to be evaluated.

Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ(-) mutant vaccine strain used for DIVA-strategies

Contagious bovine pleuropneumonia (CBPP) is the most serious cattle disease in Africa, caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC). CBPP control strategies currently rely on vaccination with a vaccine based on live attenuated strains of the organism. Recently, an lppQ(-) mutant of the existing vaccine strain T1/44 has been developed (Janis et al., 2008). This T1lppQ(-) mutant strain is devoid of lipoprotein LppQ, a potential virulence attribute of M. mycoides subsp. mycoides SC. It is designated as a potential live DIVA (Differentiating Infected from Vaccinated Animals) vaccine strain allowing both serological and etiological differentiation. The present paper reports on the validation of a control strategy for CBPP in cattle, whereby a TaqMan real-time PCR based on the lppQ gene has been developed for the direct detection of M. mycoides subsp. mycoides SC in ex vivo bronchoalveolar lavage fluids of cows and for the discrimination of wild type strains from the lppQ(-) mutant vaccine strain.

A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony

During evolution, pathogenic bacteria have developed complex interactions with their hosts. This has frequently involved the acquisition of virulence factors on pathogenicity islands, plasmids, transposons, or prophages, allowing them to colonize, survive, and replicate within the host. In contrast, Mycoplasma species, the smallest self-replicating organisms, have regressively evolved from gram-positive bacteria by reduction of the genome to a minimal size, with the consequence that they have economized their genetic resources. Hence, pathogenic Mycoplasma species lack typical primary virulence factors such as toxins, cytolysins, and invasins. Consequently, little is known how pathogenic Mycoplasma species cause host cell damage, inflammation, and disease. Here we identify a novel primary virulence determinant in Mycoplasma mycoides subsp. mycoides Small Colony (SC), which causes host cell injury. This virulence factor, released in significant amounts in the presence of glycerol in the growth medium, consists of toxic by-products such as H2O2 formed by l-alpha-glycerophosphate oxidase (GlpO), a membrane-located enzyme that is involved in the metabolism of glycerol. When embryonic calf nasal epithelial cells are infected with M. mycoides subsp. mycoides SC in the presence of physiological amounts of glycerol, H2O2 is released inside the cells prior to cell death. This process can be inhibited with monospecific anti-GlpO antibodies.

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be grouped into a single subspecies

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be combined into one taxon on the basis of several contributions on both DNA sequence and protein analyses reported in the literature. Moreover, for the differentiation and identification of mycoplasmas of the "mycoides cluster", we investigated the rpoB gene, encoding the beta-subunit of the RNA polymerase. A segment of 527 bp of the rpoB gene was amplified from 31 strains of ruminant mycoplasmas by PCR. The nucleotide sequences were determined and aligned, and accurate genetic relationships were calculated. Cluster analysis of rpoB DNA allowed species differentiation within the "mycoides cluster" and confirmed that M. mycoides subsp. capri and M. mycoides subsp. mycoides LC cannot be distinguished from each other. "Mycoplasma mycoides subsp. capri" is proposed as a common name for both subspecies.

Genomic differences between type strain PG1 and field strains of Mycoplasma mycoides subsp. mycoides small-colony type

The recently accomplished complete genomic sequence analysis of the type strain PG1 of Mycoplasma mycoides subsp. mycoides small-colony type revealed four large repeated segments of 24, 13, 12, and 8 kb that are flanked by insertion sequence (IS) elements. Genetic analysis of type strain PG1 and African, European, and Australian field and vaccine strains revealed that the 24-kb genetic locus is repeated only in PG1 and not in other M. mycoides subsp. mycoides SC strains. In contrast, the 13-kb genetic locus was found duplicated in some strains originating from Africa and Australia but not in strains that were isolated from the European outbreaks. The 12- and 8-kb genetic loci were found in two and three copies, respectively, in all 28 strains analyzed. The flanking IS elements are assumed to lead to these tandem duplications, thus contributing to genomic plasticity. This aspect must be considered when designing novel diagnostic approaches and recombinant vaccines.

Molecular mechanisms of pathogenicity of Mycoplasma mycoides subsp. mycoides SC

Mycoplasma mycoides subsp. mycoides SC, the aetiological agent of contagious bovine pleuropneumonia (CBPP), is considered the most pathogenic of the Mycoplasma species. Its virulence is probably the result of a coordinated action of various components of an antigenically and functionally dynamic surface architecture. The different virulence attributes allow the pathogen to evade the host's immune defence, adhere tightly to the host cell surface, persist and disseminate in the host causing mycoplasmaemia, efficiently import energetically valuable nutrients present in the environment, and release and simultaneously translocate toxic metabolic pathway products to the host cell where they cause cytotoxic effects that are known to induce inflammatory processes and disease. This strategy enables the mycoplasma to exploit the minimal genetic information in its small genome, not only to fulfil the basic functions for its replication but also to damage host cells in intimate proximity thereby acquiring the necessary bio-molecules, such as amino acids and nucleic acid precursors, for its own biosynthesis and survival.

Beta-D-glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H2O2 production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-beta-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val204, from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala204. RESULTS: Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val204, but not strains with the Bgl isoform Ala204, do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., beta-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val204 show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala204. Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H2O2 production. Rather, the viability during addition of beta-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val204 than for those with the isoform Ala204. CONCLUSION: Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val204 with low hydrolysing activity are more prone to survive in environments that contain high levels of beta-D-glucosides, thus contributing in some extent to mycoplasmaemia.

Domain analysis of lipoprotein LppQ in Mycoplasma mycoides subsp. mycoides SC

The lipoprotein LppQ is the most prominent antigen of Mycoplasma mycoides subsp. mycoides small colony type (SC) during infection of cattle. This pathogen causes contagious bovine pleuropneumonia (CBPP), a devastating disease of considerable socio-economic importance in many countries worldwide. The dominant antigenicity and high specificity for M. mycoides subsp. mycoides SC of lipoprotein LppQ have been exploited for serological diagnosis and for epidemiological investigations of CBPP. Scanning electron microscopy and immunogold labelling were used to provide ultrastructural evidence that LppQ is located to the cell membrane at the outer surface of M. mycoides subsp. mycoides SC. The selectivity and specificity of this method were demonstrated through discriminating localization of extracellular (i.e., in the zone of contact with host cells) vs. integral membrane domains of LppQ. Thus, our findings support the suggestion that the accessible N-terminal domain of LppQ is surface exposed and such surface localization may be implicated in the pathogenesis of CBPP.

The AsaP1 peptidase of Aeromonas salmonicida subsp. achromogenes is a highly conserved deuterolysin metalloprotease (family M35) and a major virulence factor

Infections by the bacterium Aeromonas salmonicida subsp. achromogenes cause significant disease in a number of fish species. In this study, we showed that AsaP1, a toxic 19-kDa metallopeptidase produced by A. salmonicida subsp. achromogenes, belongs to the group of extracellular peptidases (Aeromonas type) (MEROPS ID M35.003) of the deuterolysin family of zinc-dependent aspzincin endopeptidases. The structural gene of AsaP1 was sequenced and found to be highly conserved among gram-negative bacteria. An isogenic Delta asaP1 A. salmonicida subsp. achromogenes strain was constructed, and its ability to infect fish was compared with that of the wild-type (wt) strain. The Delta asaP1 strain was found to infect Arctic charr, Atlantic salmon, and Atlantic cod, but its virulence was decreased relative to that of the wt strain. The 50% lethal dose of the AsaP1 mutant was 10-fold higher in charr and 5-fold higher in salmon than that of the wt strain. The pathology induced by the AsaP1-deficient strain was also different from that of the wt strain. Furthermore, the mutant established significant bacterial colonization in all observed organs without any signs of a host response in the infected tissue. AsaP1 is therefore the first member of the M35 family that has been shown to be a bacterial virulence factor.