Tuberculosis In Animals Research Articles

Rapid detection of Mycobacterium tuberculosis complex in cattle and lechwe (Kobus leche kafuensis) at the slaughter house

The detection and diagnosis of tuberculosis (TB) in food-producing animals is critical to human health. In this study we applied the loop-mediated isothermal amplification (LAMP) system to detect Mycobacterium tuberculosis complex (MTC) directly in 57 cattle and six lechwe (Kobus leche kafuensis) carcasses exhibiting lesions characteristic of TB. The samples were first subjected to Ziehl-Neelsen microscopy, followed by culture and LAMP assay. In addition, multiplex-PCR was used to determine the species involved. Of the samples from the cattle, 84.2% (95% confidence interval: 71.6-92.1) were found positive with Ziehl-Neelsen microscopy, 93.0% (95% confidence interval: 82.2-97.7) with culture, and 94.7% (95% confidence interval: 84.5-98.6) with the LAMP system while the Kobus leche kafuensis samples were all positive for all techniques used. These results indicate that the LAMP system can be used to augment the detection and surveillance of TB in animals; hence can be a very useful tool in the veterinary field and in public health.

Determinantes do uso de medidas sanitárias de controle da brucelose e tuberculose bovinas

A pecuária bovina é um dos mais importantes setores do agronegócio brasileiro. Entretanto, a sanidade dos rebanhos brasileiros - peça fundamental para a atividade - ainda é uma questão pouco estudada em termos econômicos. Assim, este trabalho buscou identificar os principais determinantes do uso das medidas preventivas estabelecidas pelo Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Animal (PNCEBT). Para isso, utilizou-se um modelo Tobit em painel. Os principais resultados indicam que o início efetivo do programa, em 2004, estimulou significativamente o aumento das medidas. Também observou-se que a vacinação não é influenciada pelos preços pagos tanto para carne quanto para leite, mas que esses afetavam positivamente o número de exames de tuberculose. Variáveis relativas às transferências do governo federal por convênio aos estados e às exportações de carne ou leite não foram significativas. Concluiu-se que o PNCEBT vem apresentando bons resultados. Além disso, podem ser planejados incentivos econômicos aos produtores, de modo a estimular o aumento das práticas preventivas estudadas.

Molecular epidemiology of human and animal tuberculosis in Ibadan, Southwestern Nigeria

From 2005 to 2007, Mycobacterium tuberculosis complex (MTC) strains were isolated from cattle, goats and pigs samples collected at the Bodija abattoir and from human samples from tuberculosis patients and livestock traders at the Akinyele cattle market in Ibadan, Southwestern Nigeria. Seventy four isolates obtained from humans (24) and livestock (50) were identified as MTC strains. Thirty two isolates were spoligotyped. Nineteen of these 32 isolates were identified as M. tuberculosis whilst 13 were identified as Mycobacterium bovis. M. bovis was isolated from two humans, whereas M. tuberculosis was isolated from a bovine, a pig and a goat. All the M. bovis isolates identified in this study belonged to the Africa 1 clonal complex. Multiple locus VNTR [variable number of tandem repeats] analysis (MLVA) was carried out on the 74 isolates. Three major clusters were defined. Group A consisted of 24 M. tuberculosis isolates (MLVA genotypes 1–18). One strain was isolated from a bovine and one from a pig. Group B consisted of 49 M. bovis strains (MLVA genotypes 19–48), mainly of cattle origin but also included four goat, nine pig and two human isolates. Group C consisted of a single M. tuberculosis isolate (MLVA genotype 49) obtained from a goat. Spoligotyping and MLVA confirmed it as clustering with the East Africa Indian clade found in humans in Sudan and the Republic of Djibouti. The isolation of three M. tuberculosis strains from livestock raises the question of their epidemiological importance as a source of infection for humans.

Rapid detection of Mycobacterium tuberculosis complex in cattle and lechwe (Kobus leche kafuensis) at the slaughter house

The detection and diagnosis of tuberculosis (TB) in food-producing animals is critical to human health. In this study we applied the loop-mediated isothermal amplification (LAMP) system to detect Mycobacterium tuberculosis complex (MTC) directly in 57 cattle and six lechwe (Kobus leche kafuensis) carcasses exhibiting lesions characteristic of TB. The samples were first subjected to Ziehl-Neelsen microscopy, followed by culture and LAMP assay. In addition, multiplex-PCR was used to determine the species involved. Of the samples from the cattle, 84.2% (95% confidence interval: 71.6-92.1) were found positive with Ziehl-Neelsen microscopy, 93.0% (95% confidence interval: 82.2-97.7) with culture, and 94.7% (95% confidence interval: 84.5-98.6) with the LAMP system while the Kobus leche kafuensis samples were all positive for all techniques used. These results indicate that the LAMP system can be used to augment the detection and surveillance of TB in animals; hence can be a very useful tool in the veterinary field and in public health.

Tuberculosis in Birds: Insights into the Mycobacterium avium Infections.

Tuberculosis, a List B disease of World Organization for Animal Health, caused by M. avium or M. genavense predominantly affects poultry and pet or captive birds. Clinical manifestations in birds include emaciation, depression and diarrhea along with marked atrophy of breast muscle. Unlike tuberculosis in animals and man, lesions in lungs are rare. Tubercular nodules can be seen in liver, spleen, intestine and bone marrow. Granulomatous lesion without calcification is a prominent feature. The disease is a rarity in organized poultry sector due to improved farm practices, but occurs in zoo aviaries. Molecular techniques like polymerase chain reaction combined with restriction fragment length polymorphism and gene probes aid in rapid identification and characterization of mycobacteria subspecies, and overcome disadvantages of conventional methods which are slow, labour intensive and may at times fail to produce precise results. M. avium subsp. avium with genotype IS901+ and IS1245+ causes infections in animals and human beings too. The bacterium causes sensitivity in cattle to the tuberculin test. The paper discusses in brief the M. avium infection in birds, its importance in a zoonotic perspective, and outlines conventional and novel strategies for its diagnosis, prevention and eradication in domestic/pet birds and humans alike.

A Review of Bovine Tuberculosis in the Kafue Basin Ecosystem

The Kafue basin ecosystem is the only remaining natural habitat for the endangered Kafue lechwe antelope (Kobus leche Kafuensis). However, hydroelectricity power production, large-scale sugar plantations, commercial fishing and increasing livestock production are threatening its natural existence and sustainability. Further, increasing human settlements within and around the Kafue basin have resulted in decreased grazing grounds for the Kafue lechwe antelopes despite a corresponding increase in cattle population sharing the same pasture. Baseline epidemiological data have persistently reported findings of bovine tuberculosis (BTB) in both wild and domestic animals, although these have been deficient in terms of describing direct evidence in the role of either lechwe antelopes or cattle in the reported observations. Despite the current literature being deficient in establishing the casual role and transmission patterns of BTB, a bimodal route of infection at the livestock/wildlife interface has been postulated. Likewise, it is not known how much of (BTB) has the potential of causing disease in humans. This paper, seeks to underline those aspects that need further research and update available data on BTB in the Kafue basin with regards to the prevalence, distribution, risk factors, threats on wildlife conservation, livestock production, public health implications, and possible mitigatory measures.

Mycobacterium indicus pranii as stand-alone or adjunct immunotherapeutic in treatment of experimental animal tuberculosis.

Background & objectives:Mycobacterium w (M.w) is a saprophytic cultivable mycobacterium and shares several antigens with M. tuberculosis. It has shown good immunomodulation in leprosy patients. Hence in the present study, the efficacy of M.w immunotherapy, alone or in combination with multi drug chemotherapeutic regimens was investigated against drug sensitive M. tuberculosis H37Rv and three clinical isolates with variable degree of drug resistance in mice.Methods:BALB/c mice were infected with M. tuberculosis H37Rv (susceptible to all first and second line drugs) and three clinical isolates taken from the epository of the Institute. The dose of 200 bacilli was used for infection via respiratory route in an aerosol chamber. Chemotherapy (5 days/wk) was given one month after infection and the vaccinated group was given a dose of 1×107 bacilli by subcutaneous route. Bacterial load was measured at 4 and 6 wk after initiation of chemotherapy.Results:M.w when given along with chemotherapy (4 and 6 wk) led to a greater reduction in the bacterial load in lungs and other organs of TB infected animals compared to. However, the reduction was significantly (P<0.05) more in terms of colony forming units (cfu) in both organs (lungs and spleen).Conclusion:M.w (as immunomodulator) has beneficial therapeutic effect as an adjunct to chemotherapy.

Multihost Tuberculosis: Insights from the Portuguese Control Program

This paper describes the current situation of animal tuberculosis in Portugal, reviewing the accomplishments and constraints of the 2001–2009 period. Notwithstanding the substantial progress achieved with the implementation of a comprehensive test and cull scheme, notification, postmortem inspection and surveillance at slaughterhouses, herd and animal prevalence have unexpectedly increased in 2009. In parallel, the recent awareness of tuberculosis in local free-ranging wildlife species causes concern regarding the final steps towards eradication, demanding new approaches to the existing disease control policies.

Inhalable microparticles modify cytokine secretion by lung macrophages of infected mice

Inhalable microparticles containing a large payload of isoniazid (INH) and rifabutin (RFB) in equal proportions show extremely high efficacy against experimental animal tuberculosis (TB). It was investigated whether inhaled microparticles affect the cytokine environment in the lung lumen, and cytokine secretion by airway and lung macrophages recovered from mice infected with Mycobacterium tuberculosis (Mtb). We attempted to determine whether the cytokine environment of the mouse lung receives significant contribution by lung macrophages, and whether these macrophages maintain a profile of cytokine secretion that is consistent with the cytokine environment of the lung. Groups of mice were infected intravenously with Mtb H37Ra and treated with (a) 5 mg/Kg each of INH and RFB administered by oral gavage; or, (b) 2.5 mg/Kg of the same and an additional ∼2.5 mg/Kg in the form of inhaled microparticles; or, (c) ∼2.5 mg/Kg by inhalation alone. Bronchioalveolar lavage (BAL) was carried out and recovered macrophages cultured. BAL Fluid and culture supernatants were assayed for tumor necrosis factor (TNF-α), interferon (IFN-γ), interleukin (IL)-12 and IL-10 by ELISA and amounts compared with both infected and uninfected, untreated controls. Inhaled microparticles enhanced secretion of TNF-α and supported IFN-γ secretion despite upregulated IL-10. Oral chemotherapy with the same drugs enhanced IL-12 and downregulated TNF-α. Differences in cytokine profiles suggest distinct effects of drug delivery modalities on innate immune strategies mobilized during host response. These differences might account, in part, for the extraordinary efficacy of the microparticles.

Gold nanoprobe assay for the identification of mycobacteria of the Mycobacterium tuberculosis complex

Members of the Mycobacterium tuberculosis complex (MTC) are causative agents of human and animal tuberculosis. This complex encompasses several phylogenetically related species, including M. tuberculosis, the main aetiological agent of human tuberculosis, and Mycobacterium bovis, the causative agent of bovine tuberculosis, a relevant worldwide zoonosis. Clear epidemiological evaluation of appropriate and effective treatment requires unambiguous differentiation between MTC members. Routine diagnosis has been increasingly relying on the molecular identification of MTC members. In the present study, we report the use of a gold nanoparticle-based approach for the sensitive, specific and fast identification of MTC and for the differentiation of M. bovis and M. tuberculosis using the gyrB locus as target. This gold nanoprobe strategy relies on the colorimetric differentiation of specific DNA sequences based on differential aggregation profiles in the presence or absence of specific target hybridization. Three nanoprobes were designed and successfully used for the specific identification of members of MTC, M. bovis and M. tuberculosis.

At Least in Some Settings, Tuberculosis Acts as a Zoonosis

The distribution of Mycobacterium bovis and Mycobacterium tuberculosis, the agents of tuberculosis in animals and humans, is far more jumbled among host species than many people realize, according to participants of the symposium, “Transmission of Tuberculosis: Is TB more of a Zoonosis than We Thought?” convened as part of the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), held during September 2009 in San Francisco, Calif. Based on tuberculosis (TB) disease patterns on farms, zoos, and other settings, a substantial and growing number of species is susceptible to this disease, with both those pathogens crossing species barriers, albeit at a frequency that proves difficult to estimate.

Investigations on Deer to Deer and Deer to Cattle Transmission of the Vaccine Mycobacterium bovis Bacillus Calmette-Guérin (BCG)

Mycobacterium bovis is the causative agent of tuberculosis in animals and can cause tuberculosis in humans clinically indistinguishable from that caused by Mycobacterium tuberculosis. Efforts to eradicate bovine tuberculosis have significantly decreased prevalence in developed countries. However, some countries have found it impossible to eradicate bovine tuberculosis due to the presence of a wildlife reservoir of M. bovis. In Michigan, USA there exists a reservoir of M. bovis in free-ranging white-tailed deer. Vaccination with M. bovis BCG is one approach to controlling tuberculosis in wildlife. Nevertheless, use of a live vaccine generates concerns about exposure risk to non-target species, including domestic livestock. Unintentional exposure of cattle to BCG may result in increased numbers of false positive tuberculin skin test reactions. Twenty-nine white-tailed deer received 1 SC dose of 107 colony-forming units of M. bovis BCG Danish 1331 (n=19) or no vaccination (n=10). Vaccinated and non-vaccinated deer were comingled with opportunity for direct and indirect contact. Twelve unvaccinated Holstein calves were housed in a separate paddock with no means of direct contact with deer; however, indirect contact through the sharing of feed and water was permitted. After 180 days, 11 out of 15 vaccinated deer and 4 out of 8 non-vaccinated deer were classified as reactors using the tuberculin skin test. All 12 calves were categorized as non-reactors, by both the tuberculin skin test and BOVIGAM™ assay. Vaccination of free-ranging white-tailed deer with BCG Danish is unlikely to have a deleterious effect on tuberculosis surveillance measures in cattle.

Gold nanoprobes assay for identification of mycobacteria from theMycobacterium tuberculosiscomplex

Members of the Mycobacterium tuberculosis complex (MTC) are causative agents of human and animal tuberculosis. This complex encompasses several phylogenetically related species, including M. tuberculosis, the main aetiological agent of human tuberculosis, and Mycobacterium bovis, the causative agent of bovine tuberculosis, a relevant worldwide zoonosis. Clear epidemiological evaluation of appropriate and effective treatment requires unambiguous differentiation between MTC members. Routine diagnosis has been increasingly relying on the molecular identification of MTC members. In the present study, we report the use of a gold nanoparticle-based approach for the sensitive, specific and fast identification of MTC and for the differentiation of M. bovis and M. tuberculosis using the gyrB locus as target. This gold nanoprobe strategy relies on the colorimetric differentiation of specific DNA sequences based on differential aggregation profiles in the presence or absence of specific target hybridization. Three nanoprobes were designed and successfully used for the specific identification of members of MTC, M. bovis and M. tuberculosis.

Identification of Novel Mycobacterium bovis Antigens by Dissection of Crude Protein Fractions

Culture filtrate and cell extracts from Mycobacterium bovis cultures contain molecules which could promote protective immunity to tuberculosis in animals. Different protein fractions of M. bovis cultures were obtained by elution electrophoresis and were tested in experimentally infected cattle. The fractions that elicited gamma interferon (IFN-gamma) responses were resolved by two-dimensional gel electrophoresis, and individual proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The open reading frames were cloned, expressed as their recombinant forms, and retested with naturally and experimentally infected animals. Eleven protein fractions were highly reactive, from which the Rv1636, HspX, Rv0138, Rv2524, EsxI, and Rv3740 recombinant proteins were obtained. EsxI and HspX were the antigens most recognized by the IFN-gamma release assay. In summary, a proteomic approach allowed the identification of novel antigens useful for the diagnosis of bovine tuberculosis.

Bacterial Thymidine Kinase as a Non-Invasive Imaging Reporter for Mycobacterium tuberculosis in Live Animals

BackgroundBacteria can be selectively imaged in experimentally-infected animals using exogenously administered 1-(2′deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-[125I]-iodouracil ([125I]-FIAU), a nucleoside analog substrate for bacterial thymidine kinase (TK). Our goal was to use this reporter and develop non-invasive methods to detect and localize Mycobacterium tuberculosis.Methodology/Principal Findings We engineered a M. tuberculosis strain with chromosomally integrated bacterial TK under the control of hsp60 - a strong constitutive mycobacterial promoter. [125I]FIAU uptake, antimicrobial susceptibilities and in vivo growth characteristics were evaluated for this strain. Using single photon emission computed tomography (SPECT), M. tuberculosis Phsp60 TK strain was evaluated in experimentally-infected BALB/c and C3HeB/FeJ mice using the thigh inoculation or low-dose aerosol infection models. M. tuberculosis Phsp60 TK strain actively accumulated [125I]FIAU in vitro. Growth characteristics of the TK strain and susceptibility to common anti-tuberculous drugs were similar to the wild-type parent strain. M. tuberculosis Phsp60 TK strain was stable in vivo and SPECT imaging could detect and localize this strain in both animal models tested.ConclusionWe have developed a novel tool for non-invasive assessment of M. tuberculosis in live experimentally-infected animals. This tool will allow real-time pathogenesis studies in animal models of TB and has the potential to simplify preclinical studies and accelerate TB research.

Tissue-specific changes in mRNA expression of Abc and Slc transporters in murine pulmonary tuberculosis

A pulmonary tuberculosis mouse model was used to assess the pharmacodynamic and pharmacokinetic characteristics of tuberculosis therapeutics. While membrane transporters play important roles in drug disposition and physiological homeostasis, their expressional changes and contribution have never been analysed in a tuberculosis animal model.The mRNA expression level of 20 Abc family transporters and 32 Slc family transporters in tuberculosis-infected mice were compared with those in naïve uninfected mice using real-time polymerase chain reaction (PCR). Mycobacterium tuberculosis infection induced many dramatic expression changes of families of both Abc transporters and Slc transporters at 4 and 8 weeks, as observed in the livers, kidneys, and intestines of test mice — and in a different mode, in the lungs and spleens as well. These changes were dependent on the tuberculosis progression with the tissue-specific manner, that is, in the lungs, the number of transporters of which the expression level changed due to M. tuberculosis infection had increased, and the magnitude of change also greater at 8 weeks, while in the spleen, the transcription of most transporters except Mrps had not changed or had recovered back to the same level of naïve transcription at 8 weeks.Understanding the expression changes of transporters will assist in setting up rational preclinical dosing plans through the ability to predict the pharmacokinetics of new anti-tuberculosis chemotherapeutics and, furthermore, will assist in the design of safer and more efficient drug regimens.

Tuberculosis peritoneal por Mycobacterium bovis en paciente cirrótico

Isolated peritoneal tuberculosis is an uncommon extrapulmonary form of presentation of tuberculosis in industrialized countries. In most cases, this disease is the result of reactivation and secondary hematogenous spread of a latent infection. Although the suspected diagnosis is given by clinical manifestations and analysis of ascitic fluid (lymphocytic predominance, albumin gradient between serum and ascitic fluid ⩽1 g/dl and adenosine deaminase concentration ⩾39 U/L), microbiologic assessment is required for the definitive diagnosis. Mycobacterium bovis causes tuberculosis in animals. Transmission to humans is rare in developed areas, given that it usually occurs through ingestion of unpasteurized contaminated milk. We present a patient with cirrhosis who developed ascites caused by an exceptional infection in our setting.

Immunological Parameters to Define Infection Progression and Therapy Response in a Well-Defined Tuberculosis Model in Mice

To evaluate novel approaches for tuberculosis (TB) diagnostics and treatment, well-validated animal TB models are needed. Especially the emergence and spread of drug resistant TB requires innovative therapy and accurate parameters for monitoring success or failure of therapy. We developed a TB model in BALB/c mice, in which Mycobacterium tuberculosis (Mtb) infection was induced through the natural respiratory route, mimicking human TB infection. The lung showed a mild inflammatory infiltrate consisting of granulomas in the first phase of infection, followed by progressive increase of pneumonic lesions resulting in extensive lung consolidation in the chronic phase. Dissemination to the extra-pulmonary sites was observed. The model was validated in terms of therapeutic outcome. The 26-week standard therapy administered in human pharmacokinetic-equivalent doses, resulted in complete elimination of Mtb in all infected organs, without relapse of infection in the post-treatment period. However, a 13-week therapy, simulating patient non-adherence resulted in relapse of infection. In our quest to find biomarkers for monitoring success or failure of therapy, the concentrations of various cytokines in serum and lung, determined by cytometric bead array (CBA), were evaluated in relation to the in situ cytokine expression in the lung, assessed by immunohistochemistry. The level of IFN-gamma concentration in serum increased with infection progression, and decreased during effective therapy, and as such appeared to be an appropriate immunological parameter for success or failure of therapy. Relapse of infection, after inappropriate therapy, manifested as an increase in the serum IFN-gamma concentration.

EPIZOOTIOLOGICAL CHARACTERISTICS OF BOVINE TUBERCULOSIS IN SOUTH BAČKA REGION

Bovine tuberculosis caused by Mycobacterium bovis is a chronicle, infective disease associated with formation of specific inflammatory granulomas. Infection usually remains subclinical for a long period, but even when the clinical are present, they are not pathognomic. Bovine tuberculosis can spread to humans through inhalation of infectious droplets and by ingestion of raw milk, and on the rare occasion through consumption of meat products. Since the disease remains a great economic concern for cattle production operations and due to its zoonotic nature, most of the countries initiated a program for the control and eradication of tuberculosis in domestic animals. In South Baåka region (SBD) three tuberculous foci were detected on the territory of Žabalj, Novi Sad and Titel municipality. The first 11 reactors were detected in the year 2004 during the diagnostic examination that was conducted according to the Program of Measures for infectious disease survaillance. The number of animals with a positive reaction to the intradermal tuberculin test was 113, 54 and 142, in 2006, 2007 and 2008, respectively. The diagnosis was further confirmed by gama-interferon test, gross pathology and histopathological examination, and definitively confirmed by M. bovis isolation from the lymph node and lung lesions. The number of estimated tuberculin reactors was 320, located in six settlements and 37 husbandries. The percent of infected animals in some herds ranged from 11.10 to 59.18%. The objective of this investigation was to determine the routes of infection and to identify risk factors that contributed to the tuberculosis breakdown in the tuberculous hot spots. An epizootiological evaluation revealed that the high herd prevalence and high animal incidence of bovine TBC in South Baåka region was associated with the following factors: lack of TBC diagnosis in pastured animals in swampy areas for a long period, grazing areas are often flooded resulting in bad quality fodder and cattle malnutrition, presence of other domestic animals on the pastures, co-mingling of animals from different herds is frequent, common water through, presence of different wild animal species on pasture (as a potential reservoir of the disease), uncontrolled movement and illegal trade of infected animals, natural breeding as well as overpopulation in the sheds during winter housing.

Acute and Persistent Mycobacterium tuberculosis Infections Depend on the Thiol Peroxidase TPX

The macrophage is the natural niche of Mycobacterium tuberculosis infection. In order to combat oxidative and nitrosative stresses and persist in macrophages successfully, M. tuberculosis is endowed with a very efficient antioxidant complex. Amongst these antioxidant enzymes, TpX is the only one in M. tuberculosis with sequence homology to thiol peroxidase. Previous reports have demonstrated that the M. tuberculosis TpX protein functions as a peroxidase in vitro. It is the dominant antioxidant which protects M. tuberculosis against oxidative and nitrosative stresses. The level of the protein increases in oxidative stress. To determine the roles of tpx gene in M. tuberculosis survival and virulence in vivo, we constructed an M. tuberculosis strain lacking the gene. The characteristics of the mutant were examined in an in vitro stationary phase model, in response to stresses; in murine bone marrow derived macrophages and in an acute and an immune resistant model of murine tuberculosis. The tpx mutant became sensitive to H2O2 and NO compared to the wild type strain. Enzymatic analysis using bacterial extracts from the WT and the tpx mutant demonstrated that the mutant contains reduced peroxidase activity. As a result of this, the mutant failed to grow and survive in macrophages. The growth deficiency in macrophages became more pronounced after interferon-γ activation. In contrast, its growth was significantly restored in the macrophages of inducible nitric oxide synthase (iNOS or NOS2) knockout mice. Moreover, the tpx mutant was impaired in its ability to initiate an acute infection and to maintain a persistent infection. Its virulence was attenuated. Our results demonstrated that tpx is required for M. tuberculosis to deal with oxidative and nitrosative stresses, to survive in macrophages and to establish acute and persistent infections in animal tuberculosis models.