Prevalence, molecular characterization, transmission dynamics and cost analysis of bovine tuberculosis in Morocco

Bovine tuberculosis is a chronic disease, caused by M. bovis, mycobacteria which belongs to the mycobacterium tuberculosis complex; it is notable for having one of the broadest spectrum of hosts. The preferred host of M. bovis is cattle, but it has the ability to infect humans and a wide range of domestic animals. In Morocco, cattle production is one of the most important components of the agricultural economy; a sector which contributes heavily to the development of the Moroccan economy. The development of this sector is faced by many problems, like poor infrastructure, lack of services and climate change, in addition to infectious diseases like bovine tuberculosis Bovine tuberculosis is a zoonosis which affects the livestock industry, the public health sector and wildlife reservoirs. BTB has also effects like international trade restrictions for countries where BTB is endemic. Tourism and other areas of public and private interest could also be affected indirectly by BTB infection. The respiratory route is considered to be the primary mode of infection between cattle. In addition, M. bovis is largely transmitted to humans through consumption of unpasteurized milk, but there is also the possibility of inhalation of aerosols due to contact with cattle. Bovine tuberculosis in endemic in Morocco, the prevalence in Moroccan cattle is estimated at 18% (95% CI: 16.5%-20.3%), and 33% (95% CI: 31%-35%) at the individual and the herd level respectively, but the human burden needs further clarification. A prevalence study have been conducted in Sidi Kacem province in Morocco in 2012, 1201 cattle were screened using single comparative intradermal tuberculin skin test, the apparent prevalence was 20.4% and 57.7% in the individual and herd level respectively. The individual prevalence found in the present study is in line with the last national survey conducted in 2004 in collaboration with the FAO in Morocco. Consequently, Morocco is in an endemic stable state, similarly to other African countries. The livestock production sector in Morocco is continuously growing, due to the ambitious “plan Maroc Vert” launched in 2008, and also to the increasing demand of animal protein in Morocco. Consequently, livestock production system in Morocco is moving to intensified and irrigation rearing systems. Those factors in Sidi Kacem have been shown to be associated with higher risk of BTB compared to the extensive livestock system. In order to investigate BTB molecular epidemiology in Morocco. Bovine tuberculosis samples were collected from two slaughterhouses in Morocco, Rabat and El Jadida, 8658 animals were examined, 3.7% of them showed gross visible lesions suggesting bovine tuberculosis. However this prevalence reflects the prevalence in young bulls and old cows rather than the prevalence in the whole cattle population. Molecular characterization of the samples collected from the previously reported slaughterhouses has shown grown cultures in 225 isolates, 63.6% (n=143) have been confirmed to be M. bovis (absence of the RD4). From 134 samples analyzed using spoligotyping, 43 different spoligotypes were found; ten of them were new patterns (23%), they were submitted to the M.bovis database and they were given new reference numbers. The most prevalent spoligotypes were SB0121, SB0265, and SB0120, which were already reported in many other countries, mainly in Algeria, Spain, Tunisia, and also in the United States and Argentina. Spoligotypes of African 1 and African 2 clonal complexes were not reported among the characterized isolates. Considering the localization of Af 1 and Af 2 in West Central Africa and East Africa respectively, we could consider Sahara as a potential efficient barrier preventing the introduction of BTB to Morocco from West Central and East Africa. More molecular characterization is needed to investigate the strains circulating in the south and the north of Morocco. In order to investigate more deeply transmission dynamics of BTB in Africa, an overall study using whole genome sequencing and including several African countries is needed. The present thesis presents the first cattle to cattle and cattle to human compartmental deterministic mathematic model. Bovine tuberculosis reproductive number was consequently calculated, it was found to be equal to 1.375, in the range of both low and high risk areas. The sensitivity analysis of the model showed that the birth rate and the sensitivity of the single comparative intradermal tuberculin skin test are the most sensitive parameters of the model for the total cost and the time to elimination respectively. High birth rate values lead to an increased cattle population yielding higher costs for elimination. In the other hand, low test sensitivity cases low detection of infected animals and therefore less culling which leads to a longer time to elimination. Simulation of test and slaughter interventions led to a decline of BTB prevalence depending on the proportion of testing (p). Using a severe cut off (2mm) for the SICTT, the time of freedom from BTB ranged from 75 years for p=20% to 25 years for p=50%. The cumulated cost was largely stable ranging from 1.47*10^9 (p=100%, time to disease freedom of 12 years) to 1.87*10^9 (p=20%, time to disease freedom of 12 years). Deterministic and matrix models were used to develop a demographic model of Moroccan cattle population based on real data. The cost of bovine tuberculosis was consequently calculated using the established model. The productivity losses triggered by BTB (5%) were estimated for 18 years, applying Leslie matrix with and without BTB. Cattle Moroccan population was compared with and without the disease, and the loss in term of animal numbers was then calculated. Considering the productivity loss, the asset value of the living animals lost due to BTB in year 18 is 98 Million Euro. The present thesis informs Moroccan stakeholders involved in bovine tuberculosis regarding the updated prevalence in Sidi Kacem Area, molecular epidemiology of BTB among slaughtered cattle, the time frame, and range of cost and levels of intervention, in addition to the cost of BTB considering productivity losses. Further research is needed in Morocco, in one hand, investigations of the molecular epidemiology of BTB in the north and the south of the country will give more insight about the dynamics of BTB in Morocco, a broader investigation using whole genome sequencing including several African countries could be even more efficient. In the other hand a herd based transmission model will provide a more realistic cost estimation of BTB intervention in Morocco. Elimination of bovine tuberculosis is a costly and long process, the achievement of BTB control of Morocco will need the commitment of the different stakeholders involved. In addition, public-private collaborations could be helpful in order to achieve a sustainable control intervention of BTB in Morocco

Prevalence of Bovine Tuberculosis Gross Lesions in Doukkala slaughterhouses, Morocco

In Morocco, tuberculosis (TB) is a major zoonotic disease with very serious hygienic and economic impacts. Our study aimed to investigate bovine tuberculosis in two Moroccan slaughterhouses in Doukkala area based on the observation of macroscopic gross lesions. 4198 cattle were examined for tuberculosis lesions during the period of end of May 2014 to July 2015.The results showed that 64.4% of cattle with TB lesions were male with a dominant age of less than or equal to 2 years (67.2%). Females represented only 35.6% with a dominant age between 4 and 8 years (62.3%). The dominant breed was the cross breed of imported and local breeds with 95.4%. Tuberculosis-like lesions were observed in 194 animals (4.6%) including 10 (5.15 %) of generalized and 184 (94.85 %) of localized lesions, mainly encountered in the lungs (44.83%), the head lymph nodes (42.32%) the liver and its associated lymph nodes (9.41%). An estimated overall daily prevalence of 4.6% and a daily average of 5.4% (95% CI: 4.7 - 6.1%) were noted. Compared data obtained from the slaughter database 1.97 % (95% CI: 1.8 – 2.4), this prevalence is more than twice of estimated average (RR=2.34). This assumes that routine inspection leaves more than 50% of animals with suspicious lesions. On the other hand, 390 gross lesions were collected for culturing. An isolation frequency of Mycobacterium of 85.6% was obtained and the LPJ and Herrold seem to be the best culture media

The prevalence of brucellosis and bovine tuberculosis in ruminants in Sidi Kacem Province, 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 ruminantowning 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 (R) 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

Molecular characterization of bovine tuberculosis strains in two slaughterhouses in Morocco

Bovine tuberculosis (BTB) is caused by Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. Mycobacterium bovis have been described to be responsible of most cases of bovine tuberculosis. Although M. tuberculosis, M. africanum and non-complex mycobacteria were isolated from cattle. In Morocco, so far, no molecular studies were conducted to characterize the strains responsible of BTB. The present study aims to characterize M. bovis in Morocco. The present study was conducted in slaughterhouses in Rabat and El Jadida. Samples were collected from 327 slaughtered animals with visible lesions suggesting BTB.; A total of 225 isolates yielded cultures, 95% (n = 215) of them were acid-fast (AF). Sixty eight per cent of the AF positive samples were confirmed as tuberculous mycobacteria (n = 147), 99% of these (n = 146) having RD9 and among the latter, 98% (n = 143) positive while 2% (n = 3) negative for RD4 A total of 134 samples were analyzed by spoligotyping of which 14 were in cluster and with 41 different spoligotypes, ten of them were new patterns (23%). The most prevalent spoligotypes were SB0121, SB0265, and SB0120, and were already identified in many other countries, such as Algeria, Spain, Tunisia, the United States and Argentina.; The shared borders between Algeria and Morocco, in addition to the previous importation of cattle from Europe and the US could explain the similarities found in M. bovis spoligotypes. On the other hand, the desert of Morocco could be considered as an efficient barrier preventing the introduction of BTB to Morocco from West Central and East Africa. Our findings suggest a low level endemic transmission of BTB similar to other African countries. However, more research is needed for further knowledge about the transmission patterns of BTB in Morocco

CtpB Facilitates Mycobacterium tuberculosis Growth in Copper-Limited Niches

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

Partial rank correlation coefficients (PRCC) sensitivity analysis of time to elimination (left) and total cost (right) on parameter values.

<p>Partial rank correlation coefficients (PRCC) sensitivity analysis of time to elimination (left) and total cost (right) on parameter values.</p

Prevalence of tuberculin positive cattle depending on the proportion of test and slaughter between 0 and 1 (in steps of 0.1) with sensitivity and specificity of the 4mm cutoff test (left) and the 2mm cuttoff test (right).

<p>Prevalence of tuberculin positive cattle depending on the proportion of test and slaughter between 0 and 1 (in steps of 0.1) with sensitivity and specificity of the 4mm cutoff test (left) and the 2mm cuttoff test (right).</p

Parameters of the BTB cattle-human transmission model assuming a stable prevalence (endemic stability).

<p>Parameters of the BTB cattle-human transmission model assuming a stable prevalence (endemic stability).</p

Initial values for the BTB cattle-human transmission model (S3 Supporting information).

<p>Initial values for the BTB cattle-human transmission model (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005214#pntd.0005214.s003" target="_blank">S3 Supporting information</a>).</p