African trypanosomiasis (AT) is a debilitating parasitic disease endemic in sub-Saharan Africa. AT is caused by species of the Trypanosoma genus and affects both humans and animals. The etiological agents of human African trypanosomiasis (HAT) are Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense while animal African trypanosomiasis (AAT) is caused by Trypanosoma brucei brucei, Trypanosoma congolense, Trypanosoma evansi, Trypanosoma equiperdum, and Trypanosoma vivax. Although HAT has not been completely eliminated, it is no longer considered a public health problem in many endemic countries due to sustained control efforts and a significant decline in incidence. AAT however, continues to exert significant burden on livestock production, leading to economic losses and exacerbated poverty in affected regions, as extensive areas of arable land remain uncultivated due to the disease (Tora and Dana, 2024; Franco et al., 2024).

The prominent species, T. brucei brucei, has a complex life cycle that involves both the tsetse fly vector and mammalian hosts. This parasite utilizes procyclin, and immune modulatory mechanisms such as variant surface glycoprotein-mediated antigenic variation to sustain infection within the immunologically hostile vector mid-gut and mammalian blood respectively (Bockstal et al., 2011; Frenkel et al., 2016; Radwanska et al., 2018).

However, the co-evolution of trypanosomes and their hosts has led to the development of physiological, genetic and immune-based resistant mechanisms in certain species (Mulla and Rickman, 1988; Capewell et al., 2015). Notably, anti-Trypanosoma apolipoprotein L1 (Apo L1) mediates resistance in humans and some primates (Pays et al., 2006; Vanhollebeke and Pays, 2010). Similarly, xanthine oxidase, metalloproteinase, glycoprotein and a 115-kD protein are anti-Trypanosoma factors characterized in Cape buffalo, African saw-scale vipers, mosquitoes, and African hedgehogs, respectively (Reduth et al., 1994; Nok et al., 2002; Abdullahi et al., 2020; Ilu et al., 2023; Muranjan et al., 1997; Wang et al., 2002). Trypanotolerance of Ndama and West African short-horn cattle is attributed to genetic process, while neutrophils and macrophages mediate resistance against Trypanosoma carasi in zebrafish (Jacobs et al., 2021; Murray and Trail, 1984).

On the other hand, the complement system of chicken serum mediate in vitro lysis of Trypanosoma cruzi and confers resistance against Chagas disease (Kierszenbaum et al., 1976, 1981; Minter-Goedbloed and Croon, 1981). Intriguingly, indigenous African chickens (Gallus gallus domesticus) exhibit unique resistance to African trypanosomiasis (Minter-Goedbloed, 1981; Joshua et al., 1978a, Joshua et al., 1978b; Dina and Arowolo, 1988). Pathogenic human and animal African trypanosomes rarely cause disease in chickens, as they maintain covert infections detectable only through xenodiagnoses; a characteristic similar to pigeons and Guinea fowls (Corson, 1935; Mandal et al., 2008; Hussain et al., 2021). Several mechanisms including splenic immunity, xanthine oxidase activity, elevated leukocyte counts and inherently high body temperature are thought to mediate resistance to trypanosome infection in indigenous chickens (Joshua, 1983; Felicite et al., 2020; Dina and Arowolo, 1988; Oyewale, 1992). However, these speculations lack experimental validations.

Despite the apparent resistance of indigenous chickens to AT, natural trypanosome infection, and experimental transmission between chickens and tsetse flies are documented (Corson, 1935; Duke, 1935; Zillmann and Mehlitz, 1979). Similarly, surveillance studies reveal that domestic chickens harbor avian specific trypanosomes (Sehgal et al., 2006; Boonchuay et al., 2022). These observations underscore the potential of indigenous chickens to become active drivers in the epidemiology of AT, due to their proximity to humans and livestock especially in settings where livestock and poultry farming are jointly practiced.

Considering the substantial burden of AAT and the WHO's 2030 goal for HAT elimination, identifying and characterizing potential animal reservoirs—such as chickens—is critical for understanding the persistence and possible reemergence of African trypanosomiasis. Given the tendency of surveillance programs to overlook avian species, characterizing cryptic trypanosome infections in chickens and related birds could significantly expand the current understanding of AT epidemiology.

While previous reports have noted low-grade or latent infections in chickens and proposed humoral mechanisms of resistance, the immunological basis for this resistance and the presence or absence of intrinsic trypanolytic activity in chicken blood remain unclear. Similarly, these experiments utilized molecularly uncharacterized parasites species or involved isolated natural infection case.

Therefore, this study investigated the cryptic infection mechanism of T. brucei brucei in both immunocompetent and immunocompromised chicken models. We also explored the potential transmissibility of the parasite from chickens to mammalian hosts using xenodiagnosis and further evaluated whether humoral immune responses or intrinsic blood components might contribute to the observed suppression of parasitemia.