Are coinfections a threat to drug control programmes for livestock trypanosomes?

非洲锥虫给撒哈拉以南非洲的畜牧业生产造成了巨大的经济成本，加剧了受影响地区的贫困。布鲁氏锥虫、刚果锥虫和间日锥虫三种致病物种共同传播，通过化学预防和药物治疗来控制感染。然而，物种之间的混合感染是常见的，一些文献报道和我们自己的数据表明，与单一物种或菌株的单一感染相比，锥虫物种和菌株之间的混合感染可以改善疾病的病理。这在暴露于次优剂量的共感染锥虫之间存在不同药物敏感性的环境中产生风险，或者在一些循环寄生虫种群中存在耐药性的情况下产生风险。具体地说，通过将混合感染恢复为只包含更具耐药性的菌株或物种的单一感染，药物干预可能会导致病理增强。因此，药物应用可能会导致疾病增加的反常结果。在这项建议中，我们将在小鼠模型和与疾病相关的牲畜宿主中探索合并感染、药物敏感性和病理学之间的相互作用。具体来说，我们将：1.利用已知的分子耐药/敏感机制，分别设计耐药和敏感的布鲁氏锥虫和刚果锥虫，这是治疗家畜锥虫最常用的药物。具体地说，核苷转运子TbAT1将在布氏锥虫中缺失以产生抗药性寄生虫，而贡高松将被改造成异源表达布氏锥虫AT1基因，从而产生一个Dinazene超敏感系。这将允许在我们的研究中精确地去除实验中与布鲁氏毛滴虫混合感染的弓形虫，避免不同野生型实验室和野外菌株可能表现出的不同的减氮烯敏感性的复杂性。这些工程化品系将被用来评估混合感染或合并感染后的小米那烯诱导的单一感染对小鼠体内锥虫种群的比例和分布及其病理的影响。这将通过IVIS成像对布鲁氏毛滴虫每个物种和宿主的寄生虫数量进行量化来实现。小鼠的病理评分将按照既定的标准进行。利用罗斯林研究所独特和专用的大型动物控制设施，这些工程化的品系将被用来测试混合感染，或在小氮烯诱导的单一感染之后，如何影响与疾病相关的牛宿主中的寄生虫流行和病理。当通过治疗干预将联合感染重定向到单一感染时，研究将确定对寄生虫流行和宿主病理的影响。这可能出人意料地对牲畜健康有害，因为在混合感染情况下存在不同的抵抗力。我们的实验可以优先对这种以前被忽视的威胁进行流行病学研究，并促进优化剂量的策略，或治疗患病动物和维持治疗效果的策略。这还将加快有针对性地采用可获得的替代类锥虫杀菌剂。撒哈拉以南非洲的农民和政策制定者没有预料到在混合感染环境中进行治疗干预可能产生不利影响，可能被忽视或当作轶事而不屑一顾。我们将确保通过我们计划的推广活动和以家畜锥虫为重点的合作，将我们的发现传播给科学界、政策制定者和农民。其中包括计划中的会议，例如莫里森将于2023年在坦桑尼亚组织的一次会议，与比尔和梅林达·盖茨基金会的合作，与肯尼亚国际牲畜研究所的联系，以及正在进行的非洲实地工作。

African trypanosomes cause substantial economic cost to livestock production in sub-Saharan Africa exacerbating poverty in afflicted regions. Three pathogenic species co-circulate, Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax, with infections being managed by chemoprophylaxis and drug therapy. However, coinfections between the species are common and several literature reports and our own data indicate that coinfection between trypanosome species and strains can ameliorate disease pathology with respect to monoinfections with a single species or strain. This generates a risk in settings where there is differential drug sensitivity among coinfecting trypanosomes exposed to suboptimal dosing, or where drug resistance is present in some circulating parasite populations. Specifically, by reverting coinfections to monoinfections comprising only the more resistant strain or species, drug intervention may lead to enhanced pathology. Consequently, drug application may generate a perverse outcome of increased disease.In this proposal we will explore the interaction between coinfection, drug sensitivity and pathology in both a mouse model and disease-relevant livestock host. Specifically, we will:1. Engineer drug resistant and sensitive Trypanosoma brucei and Trypanosoma congolense, respectively, using a known molecular resistance/sensitivity mechanism for diminazene, the most commonly used therapy for livestock trypanosomes. Specifically, the diminazene resistance determinant TbAT1, a nucleoside transporter, will be deleted in T. brucei to generate resistant parasites and T. congolense will be engineered to heterologously express the T. brucei AT1 gene, generating a diminazene super-sensitive line. This will allow the precision removal of T. congolense in experimental coinfections with T. brucei for our studies, avoiding the complexity of variable diminazene sensitivities different wild type laboratory and field strains may exhibit.2. The engineered lines will be used to evaluate the impact of coinfection, or coinfection followed by diminazene induced monoinfections, on the proportion and distribution of the trypanosome populations and their pathology in mice. This will be achieved by quantitating parasite numbers for each species and reservoirs of T. brucei by IVIS imaging. Murine pathology will be scored by established criteria.3. The engineered lines will be used to test how coinfection, or coinfection followed by diminazene-induced monoinfections, affects parasite prevalence and pathology in the disease-relevant bovine host using unique and dedicated large animal containment facilities at Roslin Institute.The studies will determine the consequences for parasite prevalence and host pathology when a coinfection is redirected to a monoinfection through therapeutic intervention. This could be unexpectedly harmful for livestock health where differential resistance exists in mixed infection scenarios. Our experiments could prioritise epidemiological studies of this previously overlooked threat, and promote strategies to optimise dosing, or to treat diseased animals and sustain therapeutic efficacy. This would also accelerate the targeted adoption of alternative trypanocides as they become available.The potential for adverse impact of therapeutic intervention in coinfection settings is unanticipated among farmers and policymakers in sub-Saharan Africa and may have been overlooked or dismissed as anecdote. We will ensure our findings are disseminated to the scientific community, policymakers and famers through our planned outreach activities and collaborations focused on livestock trypanosomes. These include planned meetings, for example a meeting Morrison is organising in Tanzania in 2023, and collaborative work with the Bill and Melinda Gates Foundation, links with the International livestock research Institute in Kenya and ongoing field work in Africa.