Evidence of Homeostatic Regulation in Mycobacterium avium Subspecies paratuberculosis as an Adaptive Response to Copper Stress.

BACKGROUND: Bacteria are capable of responding to various stressors, something which has been essential for their adaptation, evolution, and colonization of a wide range of environments. Of the many stressors affecting bacteria, we can highlight heavy metals, and amongst these, copper stands out for its great antibacterial capacity. Using Mycobacterium tuberculosis (Mtb) as a model, the action of proteins involved in copper homeostasis has been put forward as an explanation for the tolerance or adaptive response of this mycobacteria to the toxic action of copper. Therefore, the aim of this study was to confirm the presence and evaluate the expression of genes involved in copper homeostasis at the transcriptional level after challenging Mycobacterium avium subsp. paratuberculoisis (MAP) with copper ions. METHODOLOGY: Buffer inoculated with MAP was treated with two stressors, the presence of copper homeostasis genes was confirmed by bioinformatics and genomic analysis, and the response of these genes to the stressors was evaluated by gene expression analysis, using qPCR and the comparative ΔΔCt method. RESULTS: Through bioinformatics and genomic analysis, we found that copper homeostasis genes were present in the MAP genome and were overexpressed when treated with copper ions, which was not the case with H2O2 treatment. CONCLUSION: These results suggest that genes in MAP that code for proteins involved in copper homeostasis trigger an adaptive response to copper ions.

Experimental evidence of the anti-bacterial activity pathway of copper ion treatment on Mycobacterium avium subsp. paratuberculosis.

Copper causes significant damage to the integrity of many bacteria, mainly at the DNA level, through its redox states, as well as its reactive oxygen species (ROS) generating capacity at the cellular level. But whether these mechanisms also apply to Mycobacterium avium subsp. paratuberculosis (MAP) is unknown. In the present study, we have evaluated whether copper ions produce damage at the DNA level of MAP, either through their redox states or through ROS production. MAP-spiked PBS was first supplemented with different copper chelators (2) and ROS antioxidants (3), followed by treatment with copper ions at 942 ppm. MAP DNA integrity (qPCR, magnetic phage separation) was then evaluated. We found that bathocuproine (BCS), as a chelator, and D-mannitol, as an antioxidant of hydroxyl radicals, had a significant protective effect (P < 0.05) on DNA molecules, and that EDTA, as a chelator, and D-mannitol, as an antioxidant had a significant positive effect (P < 0.05) on the viability of this pathogen in contrast to the control and other chelators and anti-oxidants used. In light of the reported findings, it may be concluded that copper ions within MAP cells are directly related to MAP DNA damage.

Understanding the antibacterial mechanisms of copper ion treatment on Mycobacterium avium subsp. paratuberculosis.

Copper and its alloys are natural and very well-proven antimicrobial materials. The mechanisms of action through which copper is highly effective have been described at the molecular and cellular level. However, both the design of the studies carried out and the nature of the microorganisms studied have meant that this research has been of limited scope. In the present study, we examined the action mechanisms of a copper ion treatment on the integrity of Mycobacterium avium subsp. paratuberculosis (MAP), a highly resistant animal pathogen. The copper ion treatment applied to MAP cells, resulted in nucleic acid degradation and disintegration, increased ROS production and protein alteration. However, the observed susceptibility of MAP to copper-based treatment was dose-dependent. Finally, it had no effect on the integrity of the MAP cell wall. This new evidence about the observed tolerance in the MAP cell wall against the copper ions, may help us to understand how we can improve the proposed copper-based treatment, and finally achieve a totally effective alternative to control MAP in calf´s milk.

More Insights about the Efficacy of Copper Ion Treatment on Mycobacterium avium subsp. paratuberculosis (MAP): A Clue for the Observed Tolerance.

BACKGROUND: Scientific evidence is scarce for the antimicrobial effect of copper on bacteria characterized as more resistant. Using Mycobacterium avium subsp. paratuberculosis (MAP), a highly resistant microorganism, as a pathogen model, copper ion treatment has shown a significant bactericidal effect; however, the sustainability of MAP against copper toxicity was also reported in several studies. Accordingly, the present study aimed to evaluate the impacts of copper on MAP. METHODOLOGY: This study considered physicochemical properties and copper concentration in a buffer since it could modulate MAP response during the application of copper treatment. RESULTS: Despite the efficacy of copper ions in significantly reducing the MAP load in Phosphate Buffered Saline, some MAP cells were able to survive. The copper concentration generated by the copper ion treatment device increased significantly with increasing exposure times. MAP bacterial load decreased significantly when treated with copper ions as the exposure times increased. An increase in pH decreased oxygen consumption, and an increase in conductivity was reported after treatment application. CONCLUSIONS: Even with higher concentrations of copper, the efficacy of MAP control was not complete. The concentration of copper must be a key element in achieving control of highly resistant microorganisms.

Attempted Control of Paratuberculosis in Dairy Calves by Only Changing the Quality of Milk Fed to Calves.

One of the important routes of Mycobacterium avium subsp. paratuberculosis (MAP) transmission in dairy calves is milk. The aim of the present study was to assess the efficacy of milk treatments to prevent MAP infection transmission to calves. A one-year longitudinal study was carried out. Newborn calves were assigned to one of four experimental groups: 5 calves received naturally MAP-contaminated milk, 5 calves received copper treated milk, 4 calves were fed calf milk replacer, and 3 were fed UHT pasteurized milk. MAP load in milk was estimated. Infection progression was monitored monthly. After one year, calves were euthanized, and tissue samples were cultured and visually examined. MAP was undetectable in milk replacer and UHT milk. Copper ion treatment significantly reduced the number of viable MAP in naturally contaminated milk. Fecal shedding of MAP was observed in all study groups but began earlier in calves fed naturally contaminated milk. Paratuberculosis control programs must place multiple hurdles between the infection source, MAP-infected adult cows, and the most susceptible animals on the farm, young calves. As our study shows, strict dependence on a single intervention to block infection transmission, no matter how important, fails to control this insidious infection on dairy farms.

Bayesian latent class estimation of sensitivity and specificity parameters of the PMS-PCR test for the diagnosis of cattle sub-clinically infected with Mycobacterium avium subsp. paratuberculosis.

The objective of this study was to estimate the performance of the peptide magnetic separation PCR test (PMS-PCR) for the diagnosis of Mycobacterium avium subsp. paratuberculosis (MAP) in sub-clinically infected dairy cattle. Twenty-one herds were randomly selected from a source population of 131 commercial dairy herds with a known history of MAP infection, located in the De Los Rios and De Los Lagos regions, in southern Chile. In the selected herds, all milking cows with ≥2 parities and without any clinical signs were sampled, collecting feces and blood-serum samples. The PMS-PCR test was used to analyze the fecal samples, while serum samples were analyzed using a commercial ELISA kit. A Bayesian latent class model was used to estimate the sensitivity (Se) and specificity (Sp) of the diagnostic tests. A total of 1381 animals were sampled in the 21 selected dairy herds, with an average sample size of 65 animals per herd (range 10-721). The PMS-PCR test had a greater Se than the ELISA test, with a median of 85.5 % (posterior probability interval (PPI) 95 %: 79.3-91.0%), while the ELISA test presented a median of 21.7 % (95 % PPI: 18.3-25.4%). On the other hand, the ELISA test had a better Sp than the PMS-PCR test, with a median of 97.7 % (95 % PPI: 96.6-98.5%), whereas PMS-PCR presented a median of 90.8 % (95 % PPI: 88.3-93.9%). Model results showed that PMS-PCR has a better Se than all available tests for MAP diagnosis in subclinical animals. However, this test should be used with care in herds with high infection rates, where a high MAP environmental load is expected, potentially increasing the frequency of false positive cases due to the pass-through phenomenon.

Is the transmission of Mycobacterium avium subspecies paratuberculosis (MAP) infection through milk intended to feed calves an overlooked item in paratuberculosis control programs?

As in many parts of the world, Chile shows a high Mycobacterium avium subsp. paratuberculosis (MAP) infection rate. Evidently, the control recommendations have been inefficient. In the author's opinion, the potential risk of MAP transmission to susceptible calves through milk consumption is largely overlooked. Two observational studies were performed, one to confirm MAP shedding in young stock in a high MAP-infected dairy herd and MAP load in milk intended to feed these calves was estimated. In parallel, in a second study, we estimated the relationship between MAP herd seroprevalence and the cow MAP shedding level as well as the presence of this pathogen in milk used to feed dairy calves. In the first study, 53.7% of cows and 22.5% of calves showed positive culture results. Among all cows tested, 9 (2.19%) animals had a positive serum ELISA, and MAP load in milk reached 106 bacteria/mL. In the second study, three seroprevalence categories were established as follows: high (≥ 9%), medium (> 5% and ≤ 9%), and low (≤ 5%). Statistical significant differences among these categories were observed. Animals from the high seroprevalence category shed significantly more MAP than the others. However, in the low category, heavy shedder animals were also observed. Finally, in all study herds, MAP presence in milk intended to feed calves was reported, even from herds without ELISA-positive animals.

Risk factors for Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium bovis coinfection at individual animal level in southern Chile cattle populations.

Mycobacterium avium subsp. paratuberculosis (MAP) causes a chronic intestinal infection mainly in domestic and wild ruminants and is transmitted primarily by the fecal-oral route. Mycobacterium bovis (M. bovis) produces a chronic infection principally of the respiratory system. It affects most domestic mammals, wild species, and humans and is spread via the respiratory or oral route. It is important to note that M. bovis is considered a major zoonotic agent. The term coinfection refers to the coexistence of two or more infectious agents in the same host. The goal of the present study was to assess management factors that may favor coinfection with MAP and M. bovis in cattle at an individual level. A cross-sectional study was conducted including 366 cattle from 11 herds. Diagnostic information for both pathogens and individual characteristics of the animals and management practices applied on them was collected from each herd. The results indicated a set of variables being more frequent in the coinfected group of animals and mainly related with biosecurity measures. This study provided regionally based data that may be used to design future control plans for both cattle infections in southern Chile.