Role of nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis

Nitric oxide (NO) is a crucial mediator in host defense and is one of the major killing mechanisms within macrophages. Its induction is highly affected by the types of cytokines and the infectious agents present. In the current study, NO production was evaluated after in vitro infection of unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8 h, 3 and 6 days of culture for cows in different stages of disease. In addition, the effects of in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor β (TGF-β) as well as the pro-inflammatory cytokine IFN-γ were correlated with the level of NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation of NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to the addition of MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was further boosted with the combination of both in vitro MAP infection and IFN-γ stimulation. Alternatively, nonspecific stimulation with LPS from Escherichia coli O111:B4-W resulted in an upregulation of NO production in all infected groups at 3 and 6 days after in vitro infection. Finally, the in vitro exposure to inhibitory cytokines such as IL-10 and TGF-β prior to MAP infection or LPS stimulation resulted in the downregulation of this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level of NO production was associated with cows in the subclinical stage of MAP infection. As well, the results demonstrated an increase in NO production upon infection with MAP and in the presence of exogenous IFN-γ. Finally, the results suggest an important role of IL-10 and TGF-β on the profile of NO production which may explain the low NO production in MAP clinically infected cows.     

Distribution of IS900 restriction fragment length polymorphism types among animal Mycobacterium avium subsp. paratuberculosis isolates from Argentina and Europe

Sixty-one Mycobacterium avium subsp. paratuberculosis isolates from cattle and deer from the Buenos Aires province, an important livestock region in Argentina, were typed by restriction fragment length polymorphisms (RFLP) analysis based on IS900. Four different RFLP patterns (designated ‘A’, ‘B’, ‘C’ and ‘E’) were identified in BstEII digests of genomic DNA. The most frequently observed type, pattern ‘A’, was found in 46 isolates (75%). The second, pattern ‘E’, included 8 isolates (13%), while the third, pattern ‘B’, included 6 isolates (10%). Pattern ‘C’ was found for only one isolate. All of the deer isolates were classified as pattern ‘A’, while cattle isolates represented all four RFLP patterns. Twenty-one isolates representing the four different BstEII-RFLP patterns were digested with PstI. Twenty isolates showed identical PstI-RFLP pattern. BstEII-RFLP patterns from Argentine cattle and deer were compared with patterns found in cattle, goat, deer, rabbit, and human isolates from Europe. The most common pattern in Argentina, pattern ‘A’, was identical to a less frequently occurring pattern R9 (C17) from Europe. The other Argentine patterns ‘B’, ‘C’ and ‘E’, were not found in the Europe. These results indicate that the distribution of M. avium subsp. paratuberculosis genotypes in the Buenos Aires province of Argentina is different from that found in Europe.     

Identification of differentially expressed genes in ileum, intestinal lymph node and peripheral blood mononuclear cells of sheep infected with Mycobacterium avium subsp. paratuberculosis using differential display polymerase chain reaction

Discovery of differentially expressed genes aids in understanding molecular mechanisms underpinning normal and pathological states. When studying animals such as sheep where the entire genome has not been characterized, techniques that do not require knowledge of gene sequences are particularly advantageous. We used one such technique, differential display polymerase chain reaction (DD-PCR), to identify genes that had different degrees of expression in response to Mycobacterium avium subsp. paratuberculosis (M. ptb), the organism that causes Johne's disease in ruminants. Differentially expressed genes were validated by quantitative PCR using especially selected reference genes established in this study. Sheep (n = 47) were classified according to history of exposure to M. ptb and infection status by histology and faecal and tissue culture. Differences in levels of gene expression were analyzed using restricted maximum likelihood (REML) in a linear mixed model. Five genes from the ileum and 17 genes from lymph node were differentially expressed in ovine Johne's disease. Expression of seven of these genes was also significantly different in peripheral blood mononuclear cells. Genes identified in association with M. ptb infection had a wide range of functions in pathways including: antigen presentation, signal transduction and cell differentiation, TLR signaling, immune cell activation and chemokine functions, granulomatous inflammation, Th1 suppression and apoptosis.     

Unique genotypes of Mycobacterium avium subsp. paratuberculosis strains of Type III

Mycobacterium avium subsp. paratuberculosis (MAP) strains with two new IS900 restriction fragment length polymorphism (RFLP) BstEII types intermediate suspected to belong to the MAP Type III group were isolated from migrating sheep in Germany. Such strains have only been sporadically identified in a few studies. For a better understanding of the genomic diversity of MAP with regard to specific host associations, geographic origin, and the discussed classification into Type I, Type II and Type III, these isolates were further characterized.Using IS900-RFLP, the isolates showed unique fingerprint patterns after BstEII-, PstI-, PvuII- and BamHI-digestion which had not been published before. Additionally, using gyrB-PCR-restriction endonuclease analysis (PCR/REA) and mycobacterial interspersed repetitive unit (MIRU)-PCR, the two strains showed differences to known patterns of the Type I as well as the Type II group. Unique genotypes were also obtained with multilocus short sequence repeat (MLSSR) sequencing and MIRU-variable-number tandem-repeat (VNTR) typing.As expected, genomic profiles identical to the Type I and different from the Type II group were detected by IS1311-PCR/REA, IS1311 sequencing as well as by Large Sequence Polymorphism analysis (LSP^A 8, 17, 20, 4-II, and 18).In addition to distinct growth characteristics, the unique genotypes of the studied sheep strains support their affiliation to the assumed third group within the MAP subspecies and suggest the existence of different genotypes within this Type III group. The results could serve as further evidence that Type I and Type III groups are more closely related to each other than to the bovine Type II group.     

Mycobacterium porcinum strains isolated from bovine bulk milk: implications for Mycobacterium avium subsp. paratuberculosis detection by PCR and culture

In this study, the isolation of 52 mycobactin-independent fast growing mycobacteria from 631 bulk milk samples (8.2%), is reported. These strains, isolated during a bulk milk survey for Mycobacterium avium subsp. paratuberculosis (Map), strongly affected Map detection both by PCR and by culture, as they gave a positive IS900 PCR signal and resulted to totally inhibit the growth of Map when spotted on HEYM slants already inoculated with 200 microl of 10-fold dilutions containing from 5 x 10 to 5 x 10(3)Map cells/ml. 16S rRNA gene sequencing, using the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems), was performed on a subset of six strains, identifying Mycobacterium porcinum with 100% homology in all six cases. The 52 strains were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene, which confirmed the identification of M. porcinum for all the isolates. Using specific primers designed on the Map-IS900 sequence and on the M. porcinum sequence determined in this study, a 1385bp sequence from the M. porcinum genome was characterized. This IS900-like sequence showed 82% homology with Map IS900. From our findings the following results emerged: (a) any culture showing one or more M. porcinum colonies represents a potential "false negative" result and should therefore be considered as contaminated; (b) IS900-like elements could be more widespread than was previously thought; (c) IS900 PCR positive results should be interpreted cautiously, as confirmed by the evidence that the primer pair used in this study resulted not to be specific.     

Quantitative real-time PCR and culture examination of Mycobacterium avium subsp. paratuberculosis at farm level

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in ruminants and may contribute to Crohn's disease in humans. The aim of this study was to determine the occurrence and quantity of MAP in cattle feces and milk in the Iranian context. In addition, we evaluated the effect of cattle age as well as farming system as risk factors contributing to MAP load. For this, a total sample of 373 consisting of 150 cattle feces (CF), 150 individual cow's milk (ICM), as well as 73 bulk-tank milk (BTM) was collected randomly and regardless of the cattle health status. The samples were assayed using F57 quantitative real-time PCR (qPCR) and culture method. According to the results of qPCR which was found ∼10 times more sensitive than culture assay, MAP was detected in 68.66% (103/150) of the CF, 12% (18/150) of the ICM and 52.05% (38/73) of the BTM samples. In contrast to the previous reports, the quantity of MAP in the BTM (2.03–5.97 log cfu/50 ml) was statistically (p < 0.01) higher than the ICM (0.90–1.97 log cfu/50 ml). Data suggested a direct relation (p < 0.01) between the cattle age and the quantity of MAP in the CF samples, while the relation was not statistically significant (p > 0.05) for the ICM. In addition, MAP load in the BTM samples obtained from traditional farms was significantly (p < 0.01) higher than that of the industrial ones, while the differences in CF and ICM was not significant (p > 0.05).     

Characterization of pathogen-specific expression of host immune response genes in Anaplasma and Mycobacterium species infected ruminants

Anaplasma and Mycobacterium species are among the most prevalent bacterial pathogens in European red deer (Cervus elaphus) in south-central Spain and are known to modify gene expression in ruminants. In this study, we used microarray hybridization and real-time RT-PCR analyses to characterize global gene expression profiles in red deer in response to Anaplasma ovis and A. ovis/Mycobacterium bovis/Mycobacterium avium sub. paratuberculosis (MAP) infections, compare the expression of immune response genes between red deer infected with A. ovis, M. bovis and A. ovis/M. bovis/MAP, and characterize the differential expression of immune response genes identified in red deer in cattle infected with M. bovis and Anaplasma marginale. Global gene differential expression in A. ovis- and A. ovis/M. bovis/MAP-infected deer resulted in the modification of common and pathogen-specific cellular biological processes. The differential expression of host immune response genes showed pathogen and host-specific signatures and the effect of infection with multiple pathogens on deer immune response. These results suggested that intracellular bacteria from Anaplasma and Mycobacterium genera produce similar genes expression patterns in infected ruminants. However, pathogen and host-specific differences could contribute to disease diagnosis and treatment in ruminants.     

Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded “echA12_2” in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2–7 months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.     

Tissue localisation of Mycobacterium avium subspecies paratuberculosis following artificially induced intracellular and naked bacteraemia

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease or paratuberculosis, a chronic enteritis of ruminants. While Johne's disease is primarily expressed in the gastrointestinal tract, isolation of MAP from extra-intestinal tissues indicates that microbial dissemination via the haematogenous route may occur during the infection. This study examined the movement of peripheral blood mononuclear cells (PBMCs) infected with MAP and the dissemination of MAP following mycobacteraemia induced by IV inoculation over a time frame of 3 days.     

The contribution of molecular biology to Mycobacteriumavium subspecies Paratuberculosis research

Molecular biology has contributed to our knowledge and understanding of the structure of Mycobacteriumavium subspecies paratuberculosis and has been particularly useful in determining those components that elicit immune responses in the host or discriminate M. avium paratuberculosis from other closely related environmental mycobacteria. As such, it has made a significant impact in the field of diagnosis, and has been instrumental in the development of specific and sensitive diagnostic tests. The next decade will see exciting new developments in paratuberculosis research as a consequence of substantial advances made in the construction of gene transfer systems in mycobacteria. These will provide opportunities for applying new strategies to determine the genetic basis for pathogenesis and the mechanisms of drug resistance and will offer new prospects for the rational design of efficient vaccines.     

Occurrence of Mycobacterium avium subspecies paratuberculosis and Neospora caninum in Alberta cow-calf operations

Mycobacterium avium subsp. paratuberculosis (MAP) and Neospora caninum (NC) are two pathogens causing important production limiting diseases in the cattle industry. Significant impacts of MAP and NC have been reported on dairy cattle herds, but little is known about the importance, risk factors and transmission patterns in western Canadian cow-calf herds. In this cross-sectional study, the prevalence of MAP and NC infection in southwest Alberta cow-calf herds was estimated, risk factors for NC were identified, and the reproductive impacts of the two pathogens were assessed.     

Tactics of Mycobacterium avium subsp. paratuberculosis for intracellular survival in mononuclear phagocytes

Johne''s disease is a condition that refers to chronic granulomatous enteritis in ruminants. It is believed that survival and replication of Mycobacterium (M.) paratuberculosis in mononuclear phagocytes plays an important role in the pathogenesis of Johne''s disease. However, it is not clear how M. paratuberculosis survives for long time periods in mononuclear phagocytes, nor is it clear which factors trigger multiplication of these bacilli and result in the development of Johne''s disease. Investigating the intracellular fate of M. paratuberculosis is challenging because of its very slow growth (more than two months to form visible colonies on media). Existing animal models also have limitations. Despite those obstacles, there has been progress in understanding the intracellular survival tactics of M. paratuberculosis and the host response against them. In this review, we compare known aspects of the intracellular survival tactics of M. paratuberculosis with those of other mycobacterial species, and consider possible mycobactericidal mechanisms of mononuclear phagocytes.     

First in vitro isolation of Besnoitia besnoiti from chronically infected cattle in Germany

Besnoitia besnoiti was in vitro isolated during the first recorded outbreak of bovine besnoitiosis in Germany. Molecular characterization of the new isolate, named Bb-GER1, revealed almost 100% identity with other B. besnoiti isolates obtained in Portugal, Spain, Israel or South Africa, when partial sequences of the 18S ribosomal RNA gene, of the internal transcribed spacer 1 and of the 5.8S RNA gene were compared. Cystozoites obtained from skin tissue of one bull were infectious for γ-interferon knockout (GKO) mice by intraperitoneal (ip) inoculation. Tachyzoites were detected in the peritoneal cavity, spleen, liver and lung of the mice 5 days post-infection. The parasite could be maintained in GKO mice by ip inoculation for at least 5 passages. Peritoneal washings containing tachyzoites were obtained from infected mice and used to infect five cell lines (Vero, MARC-145, NA42/13, BHK₂₁, KH-R). The best growth of tachyzoites was observed in BHK₂₁ cells, but replication occurred to a smaller extent also in MARC-145, NA42/13 and KH-R cells. Subsequent comparative analyses revealed that after direct infection of these cell lines with cystozoites derived from bovine skin, the growth was best in NA42/13 cells. Considerable replication was also observed in the BHK₂₁ and KH-R cell lines. Our observations on the growth characteristics of Bb-GER1 partially contrast those for other isolates. The preferential growth in particular cell lines may be characteristic for particular B. besnoiti isolates. A potential association between growth properties and differences in virulence remains to be established. This is the first in vitro isolation of B. besnoiti from cattle in Germany.     

Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strain

In this study we have characterized M. bovis isolates from a herd of cattle in Uvalde, Texas in which 52 of the 193 animals selected at random in 1994 from a herd of 331 were caudal fold skin-test positive. Thirty-two of 52 skin-test positive cattle had gross lesions at slaughter, and isolations of M. bovis were made from 29 animals. The herd was comprised of Red Devon cattle purchased between 1978 and 1980 (n = 26) and breeding bulls (n = 3) introduced at later times, and all were tuberculosis test negative at the time of purchase. Other animals were natural additions (offspring) of these cattle. One additional animal, a Holstein present on the ranch at the time of purchase in 1976, was retained to nurse orphaned and weak calves. Using several molecular fingerprinting techniques we have verified a clonal relationship among the M. bovis isolates consistent with infection originating with a single strain. The molecular fingerprint patterns demonstrate the stability of the profiles despite persistence and spread of the organism within the herd for two decades and confirms their use in epidemiological tracing.     

Molecular identification and characterization of Mycobacterium avium subspecies paratuberculosis in free living non-human primate (Rhesus macaques) from North India

In recent years, Mycobacterium avium subspecies paratuberculosis (MAP) has emerged as major animal pathogen with significant zoonotic concerns, worldwide. MAP infection is endemic in domestic and wild ruminant population in India. However, information on MAP infection in free ranging animal species and non human primates is limited. Present study aimed to estimate the status of MAP infection in free living Rhesus macaques suffering with multiple clinical conditions (coughing and loose stool). A total of 25 stool samples were collected from six colonies of Rhesus macaques from Mathura region (North India) and screened for the presence of MAP, using microscopic examination and IS900 PCR, directly from stool samples. PCR positive DNA samples were further genotyped using IS1311 PCR-restriction enzyme analysis. Of the 25 stool samples, 10 (40.0%) and 2 (8.0%) were positive for MAP using microscopic examination and direct IS900 PCR, respectively. IS900 PCR positive DNA samples were genotyped as ‘Indian Bison type’, which is a major MAP genotype infecting domestic and wild ruminant species and human beings in India. Prevalence of MAP in Rhesus macaques (Indian monkeys) was moderately high and confirmed interspecies sharing of MAP between domestic livestock and non-human primates. Presence of MAP in non-human primates, support the etiological role of MAP in inflammatory bowel disease patients. Indian monkeys may serve as model for understanding the role of non-human primates in sustenance, transmission and pathogenesis of MAP infection.     

Growth pattern and partial proteome of Mycobacterium avium subsp. paratuberculosis during the stress response to hypoxia and nutrient starvation

Mycobacterium avium subsp. paratuberculosis is an important pathogen that causes Johne's disease in animals and has been implicated in Crohn's disease in man yet few data exist on its physiological adaptation in either the host or the environment. In this study, the proteomic responses of the two distinct strains of M. a. paratuberculosis, cattle (C) and sheep (S), to hypoxia and starvation were studied in vitro. Nutrient starvation inhibited growth of both strains and was lethal for S strain after 12 weeks. Hypoxia induced a state of very low metabolic activity but rapid resuscitation occurred upon restoration of an aerobic atmosphere, consistent with the dormancy response of other mycobacteria. A total of 55 protein spots differentially expressed in response to starvation and/or hypoxic stress in one or both strains were identified from 2D gels and classified based on biological function. Antioxidant enzymes, oxidoreducatse enzymes and proteins involved in amino acid metabolism, fatty acid metabolism, ATP and purine biosynthesis, proteolysis, cell wall synthesis, protein synthesis, signal recognition and hypothetical proteins with putative functions including dormancy response regulators and universal stress proteins were identified. These proteins are potential screening targets for future diagnosis, prevention and control of M. a. paratuberculosis infection and their identification will assist understanding the pathogenesis of diseases caused by this organism.     

Candidate gene and genome-wide association studies of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep: a review

Paratuberculosis (Johne's disease), caused by Mycobacterium avium subspecies paratuberculosis, is responsible for significant economic losses in livestock industries worldwide. This organism is also of public health concern due to an unconfirmed link to Crohn's disease. Susceptibility to paratuberculosis has been suggested to have a genetic component. In livestock, a number of candidate genes have been studied, selected on their association to susceptibility in other mycobacterial diseases, their known role in disease pathogenesis or links to susceptibility of humans to Crohn's disease. These genes include solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1), toll-like receptors, caspase associated recruitment domain 15 (CARD15, formerly NOD2), major histocompatibility complex (MHC) and cytokines (interleukin-10 and interferon-gamma) and their receptors. Genome wide association studies have attempted to confirm associations found and identify new genes involved in pathogenesis and susceptibility. There are a number of limitations and difficulties in these approaches, some peculiar to paratuberculosis but others generally applicable to identification of genetic associations for complex traits. The technical approaches and available information for paratuberculosis have expanded rapidly, particularly relating to sheep and cattle. Here we review the current published evidence for a genetic association with paratuberculosis susceptibility, technological advances that have progressed the field and potential avenues for future research.