Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System

Although genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with the susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection, only a few functional mutations for bovine paratuberculosis (PTB) have been characterized. Expression quantitative trait loci (eQTLs) are genetic variants typically located in gene regulatory regions that alter gene expression in an allele-specific manner. eQTLs can be considered as functional links between genomic variants, gene expression, and ultimately phenotype. In the current study, peripheral blood (PB) and ileocecal valve (ICV) gene expression was quantified by RNA-Seq from fourteen Holstein cattle with no lesions and with PTB-associated histopathological lesions in gut tissues. Genotypes were generated from the Illumina LD EuroG10K BeadChip. The associations between gene expression levels (normalized read counts) and genetic variants were analyzed by a linear regression analysis using R Matrix eQTL 2.2. This approach allowed the identification of 192 and 48 cis-eQTLs associated with the expression of 145 and 43 genes in the PB and ICV samples, respectively. To investigate potential relationships between these cis-eQTLs and MAP infection, a case–control study was performed using the genotypes for all the identified cis-eQTLs and phenotypical data (histopathology, ELISA for MAP-antibodies detection, tissue PCR, and bacteriological culture) of 986 culled cows. Our results suggested that the heterozygous genotype in the cis-eQTL-rs43744169 (T/C) was associated with the up-regulation of the MDS1 and EVI1 complex (MECOM) expression, with positive ELISA, PCR, and bacteriological culture results, and with increased risk of progression to clinical PTB. As supporting evidence, the presence of the minor allele was associated with higher MECOM levels in plasma samples from infected cows and with increased MAP survival in an ex-vivo macrophage killing assay. Moreover, the presence of the two minor alleles in the cis-eQTL-rs110345285 (C/C) was associated with the dysregulation of the eukaryotic elongation factor 1-α2 (eEF1A2) expression and with increased ELISA (OD) values. Finally, the presence of the minor allele in the cis-eQTL rs109859270 (C/T) was associated with the up-regulation of the U1 spliceosomal RNA expression and with an increased risk of progression to clinical PTB. The introduction of these novel functional variants into marker-assisted breeding programs is expected to have a relevant effect on PTB control.

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“…i.) was estimated as previously described 35 . The genotype T/T showed higher MAP uptake at 2 h p.i.…”

Section: Resultsmentioning

confidence: 99%

Identification of loci associated with susceptibility to bovine paratuberculosis and with the dysregulation of the MECOM, eEF1A2, and U1 spliceosomal RNA expression

Canive

Fernández-Jiménez

Casáis

et al. 2021

Sci Rep

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“…However, it is uncertain if differences exist between cattle and sheep strains of M. avium subsp. paratuberculosis, especially since it has been reported that growth curves between S-type (sheep and goat) strains were dissimilar from those of C-type stains (cattle and bison) in liquid culture (28). Furthermore, the growth of sheep strains of M. avium subsp.…”

Section: Discussionmentioning

confidence: 99%

Chemical Decontamination with N -Acetyl- l -Cysteine–Sodium Hydroxide Improves Recovery of Viable Mycobacterium avium subsp. paratuberculosis Organisms from Cultured Milk

et al. 2013

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8 CFU/ml of live M. avium subsp. paratuberculosis organisms. The results indicate that an increased length of exposure to NALC-NaOH from 5 to 30 min and an increased concentration of NaOH from 0.5 to 2.0% did not affect the viability of M. avium subsp. paratuberculosis. Additional treatment of milk samples with the antibiotics following NALC-NaOH treatment decreased the recovery of viable M. avium subsp. paratuberculosis cells more than treatment with NALC-NaOH alone. The Bactec 12B medium was the superior medium of the three evaluated for the isolation of M. avium subsp. paratuberculosis from milk, as it achieved the lowest threshold of detection. The optimal conditions for NALC-NaOH decontamination were determined to be exposure to 1.50% NaOH for 15 min followed by culture in Bactec 12B medium. This study demonstrates that chemical decontamination with NALC-NaOH resulted in a greater recovery of viable M. avium subsp. paratuberculosis cells from milk than from samples treated with hexadecylpyridinium chloride (HPC). Therefore, it is important to optimize milk decontamination protocols to ensure that low concentrations of M. avium subsp. paratuberculosis can be detected. M ycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease (JD) in ruminants, is transmitted primarily through the ingestion of contaminated feces, but it can also be transmitted through milk and in utero (1, 2). As the infection progresses, M. avium subsp. paratuberculosis can disseminate throughout the body, and it has been isolated from milk, supramammary lymph nodes, and lymph fluid samples from the udder (3-5). Contaminated colostrum or milk may be consumed by highly susceptible neonates and young stock, leading to possible infection with M. avium subsp. paratuberculosis and the persistence of JD within a herd. Studies enumerating the amount of M. avium subsp. paratuberculosis cells shed into milk have reported concentrations from Ͻ1 to 560 CFU/ml (3, 6). A meta-analysis of M. avium subsp. paratuberculosis in milk found that the prevalence rates of M. avium subsp. paratuberculosis in bulk tank milk samples collected from known infected herds and herds of unknown status were 6% and 1%, respectively, as detected by culture, and 68% and 22%, respectively, as detected by IS900 PCR (7). The huge discrepancies in the rates of prevalence between culture and PCR detection methods are because PCR detects both live and dead bacteria, whereas culture recovers only viable microorganisms.Because current PCR assays have not been developed to differentiate between viable and nonviable M. avium subsp. paratuberculosis organisms, culture methods must be used to enumerate the M. avium subsp. paratuberculosis organisms present in milk. Unfortunately, the culture of M. avium subsp. paratuberculosis from milk presents many difficulties because of the organism's low growth rate and fastidious nature compared with other organisms present in milk. Therefore, a decontamination and culturing protocol must be developed that will thwart the ...

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“…This assay was validated and optimized with a specific strain of M. avium subsp. paratuberculosis and would need to be validated for use with other strains or sample types, as growth dynamics in culture may vary (35,36). Furthermore, macrophages may not kill bacteria but may cause growth suppression/ inhibition, which may not be distinguishable in the assay (28).…”

Section: Discussionmentioning

confidence: 99%

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

Pooley

Silva

Purdie

et al. 2016

Appl Environ Microbiol

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Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCERapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method developed, reliable results can be obtained at 2 weeks. This method will be important for vaccine and antimicrobial screening work, as it will allow a greater number of candidates to be screened in the same amount of time, which will increase the likelihood that a favorable candidate will be found to be subjected to further testing. Determining the number of live and dead bacteria in a sample is a key requirement of most in vitro cellular infection/phagocytosis assays. Quantification of live and dead bacteria is generally done by assessing growth in culture (1). However, for fastidious slow-growing bacteria, such as Mycobacterium avium subspecies paratuberculosis, it can take months to obtain culture results (2-4). This becomes a significant obstacle for in vitro cellular infection assays, which precludes their use in large studies.In vitro cellular infection assays could be a rapid and costeffective alternative to animal trials for assessing M. avium subsp. paratuberculosis interactions with host immune cells. Through examination of interaction outcomes, thes...

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Quantification of Mycobacterium avium subsp. paratuberculosis Strains Representing Distinct Genotypes and Isolated from Domestic and Wildlife Animal Species by Use of an Automatic Liquid Culture System

Cited by 14 publications

References 43 publications

Identification of loci associated with susceptibility to bovine paratuberculosis and with the dysregulation of the MECOM, eEF1A2, and U1 spliceosomal RNA expression

Identification of loci associated with susceptibility to bovine paratuberculosis and with the dysregulation of the MECOM, eEF1A2, and U1 spliceosomal RNA expression

Chemical Decontamination with N -Acetyl- l -Cysteine–Sodium Hydroxide Improves Recovery of Viable Mycobacterium avium subsp. paratuberculosis Organisms from Cultured Milk

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

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