The Interplay of Animal African Trypanosomiasis Prevalence, Iron Homeostasis, and Transferrin Receptor Variability in Livestock of the Greater Accra Region, Ghana

Abstract

Background African trypanosomiasis is a parasitic disease that can infect both humans and animals. In humans, the disease is known as human African trypanosomiasis (HAT), while the animal form is called animal African trypanosomiasis (AAT). Although the burden of HAT has significantly decreased over the years, AAT remains a significant constraint to livestock production in several sub-Saharan African countries, including Ghana. Several species of African trypanosomes can infect humans, domestic and/or wild animals. As majority of parasite, trypanosomes rely on host-derived iron for survival and disease establishment. Consequently, the parasites express a divergent transferrin receptor (TfR) that facilitates host transferrin uptake to satisfy their iron needs. Previous studies reported that TfR diversification enables trypanosomes to expand their mammalian host reservoirs, but these studies were primarily performed using in-vitro systems. It remains unknown whether this phenomenon occurs during natural infections. Additionally, TfR is co-transcribed from the same polycistronic transcription unit as Variant Surface Glycoproteins (VSGs), which are critical for immune evasion. There are ~10 million densely packed VSG molecules per trypanosome cell that shield invariant surface proteins from host-derived antibodies. VSGs can exist either as homodimers or trimers, but whether VSG oligomerisation influences the uptake of essential ligands such as transferrin remains unknown. Numerous studies have identified iron deficiency as the primary cause of anaemia during AAT, yet only a limited number have described changes in iron status in naturally-infected livestock. In light of these knowledge gaps, this study sought to examine the prevalence of AAT in cattle, sheep, goats, and pigs in the Greater Accra Region, Ghana, assess changes in the iron status of livestock during trypanosome infection, determine whether TfR variability facilitates trypanosome colonisation of livestock during natural infections, and finally, examine whether VSG oligomerisation influences the uptake of host ligands. Methods Blood samples were collected from 456 animals (155 cattle, 138 sheep, 132 goats, and 31 pigs) from six farms in the Greater Accra region. DNA was extracted from the blood collected, and nested diagnostic polymerase chain reactions (PCRs) were performed using Trypanosoma species specific primers amplifying tubulin gene. Subsequently, PCR amplicons were subjected to Sanger sequencing for trypanosome species validation. ELISA and colourimetric assays were performed using the serum to determine ferritin concentration, total serum iron (TSI), transferrin saturation (TS), and total iron-binding capacity (TIBC) as indicators of iron status during infections. Additionally, the haematocrit (HCT) levels were compared between infected and non-infected livestock. To determine the variable region of TfR, RNA was extracted from the blood of livestock infected with T. b. brucei. Following this, cDNA was synthesised and used to perform conventional PCR. Finally, cell lines expressing different VSGs belonging to the main classes (dimers and trimers) were used to measure transferrin uptake. Results Of the 456 animals screened, 73 (16%) were infected, of which 50 (11%) were infected with T. brucei, 16 (3.5%) with T. theileri, and 5 (1.1%) with T. congolense. Mixed infections with T. congolense and T. theileri were observed only in two animals at two study sites. Overall, sheep were the most infected, with an infection of rate 29.7% (41/138), compared to 16.8% (26/155) in cattle, 3.8% (5/132) in goats, and 3.2% (1/31) in pigs. An increase in ferritin concentration was observed in infected cattle (Mean = 394 ng/mL) compared to non-infected controls (Mean = 283 ng/mL); the difference observed was statistically significant (p-value = 0.047). However, the ferritin concentration in infected and non-infected sheep was low (Mean = 39 ng/mL). TIBC was very high in infected cattle and sheep (p-value = 0.0001) compared to non-infected animals, while TSI and TS showed a decrease in all the infected livestock compared to non-infected controls. Moreover, a significant reduction in HCT levels in goats was observed compared to cattle and sheep (p-value = 0.0001); however, the difference between infected and non-infected animals was not significant. TfR sequences from sheep, cattle, and goats were compared, a huge diversity was observed between and within the different livestock species in the variable region of ESAG7 that was analysed. Finally, a significant reduction in transferrin uptake was observed in trimeric VSG expressing cell lines compared to dimeric VSG cell lines (p-value = 0.001). Conclusion The prevalence of AAT in this study was low; the predominant circulating trypanosome species was T. brucei, and sheep were the most infected animals. This is the first study to report a high prevalence of AAT in sheep in the Greater Accra Region. Therefore, as seen in cattle, equal attention should be given to other livestock when implementing control measures for AAT elimination. Furthermore, the data showed that during AAT, there are significant changes in the iron status of infected livestock. Also, for the first time, this study reported diversity in TfR sequences in trypanosomes from naturally infected livestock. Therefore, this suggests that parasites express a unique TfR in different mammal hosts. Finally, this study provided evidence that the VSG structure may influence nutrient uptake.