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Bovine tuberculosis (BTB) is an endemic zoonosis in Morocco caused by Mycobacterium bovis, which infects many domestic animals and is transmitted to humans through consumption of raw milk or from contact with infected animals. The prevalence of BTB in Moroccan cattle is estimated at 18%, and 33% at the individual and the herd level respectively, but the human M. bovis burden needs further clarification. The current control strategy based on test and slaughter should be improved through local context adaptation taking into account a suitable compensation in order to reduce BTB prevalence in Morocco and decrease the disease burden in humans and animals. We established a simple compartmental deterministic mathematical model for BTB transmission in cattle and humans to provide a general understanding of BTB, in particular regarding transmission to humans. Differential equations were used to model the different pathways between the compartments for cattle and humans. Scenarios of test and slaughter were simulated to determine the effects of varying the proportion of tested animals (p) on the time to elimination of BTB (individual animal prevalence of less than one in a thousand) in cattle and humans. The time to freedom from disease ranged from 75 years for p = 20% to 12 years for p = 100%. For p > 60% the time to elimination was less than 20 years. The cumulated cost was largely stable: for p values higher than 40%, cost ranged from 1.47 to 1.60 billion euros with a time frame of 12 to 32 years to reach freedom from disease. The model simulations also suggest that using a 2mm cut off instead of a 4mm cut off in the Single Intradermal Comparative Cervical Tuberculin skin test (SICCT) would result in cheaper and quicker elimination programs. This analysis informs Moroccan bovine tuberculosis control policy regarding time frame, range of cost and levels of intervention. However, further research is needed to clarify the national human-bovine tuberculosis ratio in Morocco.

Bovine tuberculosis (BTB) and brucellosis are major endemic zoonoses in ruminants in Morocco that impact on both animal and human health. This study presents an assessment of the epidemiological and socioeconomic burden of bacterial zoonoses in Sidi Kacem Province in Northern Morocco from a cross-sectional survey of 125 cattle and/or small ruminant-owning households. In total, 1082 sheep and goats were examined from 81 households. The single intradermal comparative cervical test to screen for bovine tuberculosis was undertaken on 1194 cattle from 123 households and all cattle were blood sampled. Cattle and small ruminant sera were tested for brucellosis using the standard Rose Bengal Test (sRBT) and the modified Rose Bengal Test (mRBT). Bacteriology was performed on 21 milk samples obtained from cattle that were seropositive for brucellosis for isolation and phenotyping of circulating Brucella strains. Individual and herd prevalence for BTB in cattle of 20.4% (95% CI 18%-23%) and 57.7% (95% CI 48%-66%), respectively, were observed in this study. The prevalence of brucellosis in cattle at individual and herd level was 1.9% (95% CI 1.2%-2.8%) and 9% (95% CI 4.5%-1.5%), respectively. Brucella pathogens were isolated from three cattle milk samples and were identified as B. abortus using Bruceladder® multiplex PCR and B. abortus biovar 1 by classical phenotyping. All small ruminants were seronegative to sRBT, two were positive to mRBT. A higher risk of BTB and brucellosis was observed in cattle in intensive livestock systems, in imported and crossed breeds and in animals from larger herds (>15). The three risk factors were usually present in the same herds, leading to higher transmission risk and persistence of both zoonoses. These results highlight the importance of implementing control strategies for both BTB and brucellosis to reduce productivity losses and the risk of transmission to humans. Prioritising control for BTB and brucellosis in intensive livestock production systems is essential for human and animal health.

Copper is required for aerobic respiration by Mycobacterium tuberculosis and its human host, but this essential element is toxic in abundance. Copper nutritional immunity refers to host processes that modulate levels of free copper to alternately starve and intoxicate invading microbes. Bacteria engulfed by macrophages are initially contained within copper-limited phagosomes, which fuse with ATP7A vesicles that pump in toxic levels of copper. In this report, we examine how CtpB, a P-type ATPase in M. tuberculosis, aids in response to nutritional immunity. In vitro, the induced expression of ctpB in copper-replete medium inhibited mycobacterial growth, while deletion of the gene impaired growth only in copper-starved medium and within copper-limited host cells, suggesting a role for CtpB in copper acquisition or export to the copper-dependent respiration supercomplex. Unexpectedly, the absence of ctpB resulted in hypervirulence in the DBA/2 mouse infection model. As ctpB null strains exhibit diminished growth only in copper-starved conditions, reduced copper transport may have enabled the mutant to acquire a “Goldilocks” amount of the metal during transit through copper-intoxicating environments within this model system. This work reveals CtpB as a component of the M. tuberculosis toolkit to counter host nutritional immunity and underscores the importance of elucidating copper-uptake mechanisms in pathogenic mycobacteria.

Although interactions with alveolar macrophages have been well characterized for Mycobacterium tuberculosis, the roles epithelial cells play during infection and disease development have been less studied. We have previously shown that deletion of gene rv3351c reduces M. tuberculosis replication in and necrosis of A549 human type II pneumocyte cells. In the present study, we report that rv3351c is required for lipid raft aggregation on A549 cell plasma membranes during M. tuberculosis infection. Lipid raft aggregation was also induced directly by recombinant Rv3351c protein. A Δrv3351c deletion mutant was less effective than wild type M. tuberculosis at circumventing phagolysosome fusion in A549 cells as evidenced by increased co-localization with lysosomal markers LAMP-2 and cathepsin-L by the mutant bacilli. These observations indicate a role for Rv3351c in modification of the plasma membrane to facilitate trafficking and survival of M. tuberculosis bacilli through alveolar epithelial cells, and support the hypothesis that M. tuberculosis has mechanisms to target the alveolar epithelium. Preliminary data also demonstrate that like the type II pneumocyte-targeting M. tuberculosis secreted protein heparin-binding filamentous hemagglutinin (HBHA), Rv3351c is detected by the host cellular and humoral immune responses during infection, and may play an important role in mycobacterial dissemination from the lungs.Author summaryMycobacterium tuberculosis is the leading causes of death due to a single infectious agent and many facets regarding the pathogenesis of this organism remain unknown. This facultative intracellular bacterial pathogen often establishes infection through inhalation of the bacilli into the alveoli of the lungs. Interactions with alveolar macrophages have been well characterized and it had been assumed that these interactions with phagocytic cells primarily determine the fate of the disease. However, alveolar epithelial cells, such as type II pneumocytes, play important roles in disease progression of other bacterial and viral respiratory pathogens, which provided the impetus to more-closely examine pneumocyte-M. tuberculosis interactions. We describe in this study the role of the M. tuberculosis rv3351c gene product in the internalization and survival of this pathogen in human type II pneumocytes. We previously showed that a Δrv3351c mutant replicates less efficiently and generates less necrosis than the parental M. tuberculosis strain in this cell type. We demonstrate herein that Rv3351c protein induces lipid raft aggregation on the membranes of alveolar epithelial cells and that M. tuberculosis Δrv3351c traffics through LAMP-2-labeled endosomes 30% more frequently than the parent strain. This trafficking toward phagolysosomes may underlie the reduced replication and cytotoxicity of the mutant. The role of Rv3351c in trafficking and survival of M. tuberculosis bacilli through epithelial cells ultimately resulting in dissemination from the lungs may begin with modifications to the plasma membrane prior to attachment. Such a mechanism of activity suggests Rv3351c as a potential vaccine target to train the host immune system to bind and eliminate the protein before it modulates the alveolar epithelium.

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