Mapping of the distribution of Mycobacterium bovis strains involved in bovine tuberculosis in Mozambique

Machado, Adelina da Conceicao (2015-12)

Thesis (PhD)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: Bovine tuberculosis (BTB), caused by bacteria of the Mycobacterium tuberculosis complex is reported to cause economic and public health negative impact in countries where it is prevalent. The control of the disease has been a difficult task worldwide. The main object of this thesis was to use molecular tools to generate useful information to contribute to the design of appropriate BTB control measures in Mozambique. To do so we considered a deep knowledge of the BTB history in Mozambique to be essential. The search was largely based on the reports produced annually by the Veterinary Services and other available information. We found reports of BTB in Mozambique as early as 1940. These cases were mainly identified as a result of post-mortem meat inspection. The higher numbers of cases reported were from 8 locations, namely Maputo, Magude, Vilanculos, Beira, Chimoio, Tete, Quelimane and Nampula, and served as a basis to decide the locations to perform prevalence and molecular epidemiologic studies. Prevalence studies were done in 10 districts selected based on the history of a high number of BTB case reports (intentionally biased towards locations presumably with higher prevalence), a high cattle density, but also to represent districts from the south, centre and north of Mozambique. A representative sample was defined, based on all livestock areas or villages in Massingir and Govuro Districts or by randomly selecting small-scale and commercial herds in 8 districts, specifically Manhiça, Chibuto, Buzi, Gondola, Mutarara, Mogovolas, Angoche and Mecanhelas. Results were obtained from 6983 cattle tested using tuberculin testing. Apparent prevalence varied from 0.98% in Massingir to 39.6% in the Govuro, with prevalence as high as 71.4% in some livestock areas/herds. The analysis of risk factors showed no noteworthy difference with respect to the sex of the animal. Younger age had significantly lower odds of infection compared to the older age class. There was a tendency of cattle from small-scale herds to have lower prevalence when compared to the commercial herds. From the prevalence studies, 187 tissue and 41 milk samples from BTB reactors were collected. Additionally 220 tissue samples were obtained from the Central Veterinary Laboratory routine diagnostic work. Samples were subject to bacteriological culture and a collection of 170 M. bovis isolates were obtained. Eight additional isolates were supplied from another study. All isolates were subjected to molecular typing using spoligotyping, and a sub-sample using MIRU-VNTR and regions of difference (RD) analysis. Fifteen different spoligotype patterns were identified of which 8 were not previously registered in the Mbovis.org database. The pattern SB0961 accounted for 61% of the isolates and was found in all areas of the country investigated. We hypothesize that this was one of the first clones to be introduced in Mozambique. Twenty-nine isolates had the pattern SB0140, which is specific for the European 1 (Eu1) clonal complex. Eleven isolates with this spoligotype were subjected to RD analysis, and all isolates had the Eu1 specific deletion. These were all isolated from cattle from the south of Mozambique and the majority from commercial farms that imported cattle, mainly from South Africa, where the Eu1 clonal complex is common. There were no isolates of the African 1 (Af1) or African 2 (Af2) clonal complexes that are frequent in Central-West Africa and East Africa, respectively. The clones identified from different farms and districts, strongly suggest routes of transmission and/or common source of infection. In conclusion, our results show a potential increase in the prevalence of BTB in Mozambique even taking into consideration i) that the selection of locations in our study was biased towards locations with a history of higher BTB prevalence and ii) the use of a more sensitive technique i.e. the testing in the middle neck region as opposed to the testing in the caudal fold as used in previous studies. Even if no cattle to human transmission was found in studies done in Mozambique so far, the evidence of M. bovis shedding through milk and the lack of correct practices to prevent animal to human transmission (consumption of raw milk), strongly suggests that there is zoonotic risk; a subject that needs to be investigated. The results presented in this work also strengthen the need to reinforce the current regulations that require a negative BTB test result before cattle importation. The same should be enforced for the internal movements, as the frequency of shared genotypes (Spoligotype and MIRU) from cattle originating from different parts of the country strongly suggest intra-contry transmission of BTB.

AFRIKAANSE OPSOMMING: Beestering (BTB), wat veroorsaak word deur bakterieë van die Mycobacterium tuberculosis kompleks, het ‘n negatiewe impak op die ekonomiese en publike gesondheid in lande waar dit voorkom. Die beheer van die siekte is ‘n moeilike taak wêreldwyd. Die hoofdoel van hierdie tesis was om molekulêre toetse te gebruik om nuttige inligting te genereer wat sal bydra tot die ontwikkeling van toepaslike BTB beheermaatrëels in Mosambiek. Om dit te kon doen, was dit noodsaaklik om ‘n indiepte kennies te hê van BTB geskiedenis in Mosambiek. Die soektog was gebaseer op jaarlikse verslae van Veearts Dienste en ander beskikbare inligting. Ons het verslae gevind van BTB in Mosambiek so vroeg as 1940. Hierdie gevalle is hoofsaaklik geïdentifiseer as gevolg van roetine na-doodse inspeksie van vleis. Hoër getalle van sulke gevalle is geïdentifiseer in 8 distrikte, naamlik Maputo, Magude, Vilanculos, Beira, Chimoio, Tete, Quelimane en Nampula; en het gedien as ‘n basis vir die seleksie van studieareas vir die voorkoms studies. Voorkoms studies is uitgevoer in 10 distrikte gekies op grond van die geskiedenis van 'n hoër aantal BTB gevalle in hierdie areas (doelbewus bevooroordeeld teenoor plekke vermoedelik met 'n hoër voorkoms), asook‘n hoë digtheid beeste, maar ook om distrikte in die suide, middel en noorde van Mosambiek te verteenwoordig. ‘n Verteenwoordigende steekproef is geïdentifiseer gebaseer op al die vee-gebiede of dorpe in Massingir and Govuro distrikte óf deur kleinskaalse en kommersiële kuddes lukraak te kies in 8 distrikte, spesifiek Manhica, Chibuto, Busi, Gondola, Mutarara, Mogovolas, Angoche en Mecanhelas. Resultate is verkry deur 6983 beeste te toets met behulp van die tuberkulien vel toets. Skynbare voorkoms het gewissel van 0,98 % in Massingir tot 39,6 % in Govuro, met voorkoms so hoog as 71,4 % in sommige vee gebiede/ kuddes. Die ontleding van risiko faktore het geen noemenswaardige verskil met betrekking tot die geslag van die dier gewys nie. Jonger ouderdom diere het ‘n aansienlike laer kans van infeksie gehad in vergelyking met die ouer ouderdom klas. Daar was 'n neiging van beeste van kleinskaalse kuddes om ‘n laer voorkoms te hê in vergelyking met die kommersiële kuddes. Van die voorkoms studies, is 187 weefsel- en 41 melkmonsters van BTB reaktors ingesamel. ‘n Addisionele 220 weefselmonsters is verkry vanaf die Sentrale Veterinêre Laboratorium se roetine diagnostiese werk. Monsters was onderhewig aan bakteriologiese kweking en 'n versameling van 170 M. bovis isolate is verkry. Agt bykomende isolate is voorsien deur 'n ander studie. Alle isolate was onderhewig aan molekulêre-tipering met behulp van spoligotipering en ‘n subgroep met behulp van MIRU-VNTR en analise van genomies diverse areas. Vyftien verskillende spoligotipering patrone is geïdentifiseer, waarvan 8 nie voorheen in die Mbovis.org databasis geregistreer is nie. Die SB0961 patroon is geïdentifiseer vir 61% van die isolate en gevind in alle dele van die land wat ondersoek was. Ons hipotese is dat hierdie een van die eerste klone was wat voorgestel is in Mosambiek. Nege en twintig isolate het die SB0140 patroon gehad wat spesifiek is aan die Europese 1 (EU1) klonale kompleks. Elf isolate met hierdie spoligotipering patroon is verder geanaliseer om genomies diverse areas te identifiseer, waarvan almal die Eu1 spesifieke delesie getoon het. Hierdie isolate is almal geïsoleer uit beeste van die suide van Mosambiek, asook beeste gevind op kommersiele plase wat hoofsaaklik vanuit Suid Afrika invoer- waar die EU1 klonale kompleks algemeen is. Daar is geen isolate van die Afrikaans 1 (AF1) of Afrikaans 2 (AF2) klonale komplekse nie, dikwels gevind in onderskeidelik Sentraal-Wes-Afrika en Oos- Afrika. Isolate wat in verskillende plase en distrikte geïdentifiser is dui roetes van transmissie en/ of a gemeenskaplike bron van infeksie aan. Ten slotte, ons resultate dui op 'n moontlike toename in die voorkoms van BTB in Mosambiek, selfs met inagneming dat i) die keuse van areas in ons studie is bevooroordeeld teenoor areas met 'n geskiedenis van hoër BTB voorkoms en ii) die gebruik van 'n meer sensitiewe tegniek d.w.s. toetsing in die middel nekgebied i.p.v. toetsing in die stert vou soos gebruik in vorige studies. Selfs al is geen bees-na-mens-oordrag gevind nie, is die bewys van M. bovis oordrag deur melk en die gebrek aan korrekte prosedures om dier-na-mens-oordrag te voorkom (verbruik van nie-gepasturiseerde melk), ‘n sterk bewys van die soönotiese risiko; ‘n onderwerp wat ondersoek moet word. Die resultate van hierdie ondersoek beklemtoon die behoefte om die huidige regulasies wat ‘n negatiewe BTB toetsuitslag vereis voor beeste ingevoer word, te versterk. Dieselfde maatreëls moet ingestel word vir interne beweging van beeste, omdat die frekwensie van gedeelde genotipes (Spoligotipering en MIRU) tussen beeste met oorsprong uit verskillende dele van die land aandui dat interne oordrag van BTB plaasvind.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98114
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