Trichomonas foetus is the most prevalent protozoan parasite causing Bovine Trichomonosis in cattle. Invasive Species Compendium-2023 reported a worldwide prevalence of Bovine Trichomoniasis. Trichomonas foetus (T,foetus) the causative organism of Bovine Trichomonosis was found to be more prevalent in North America, South America, Southern parts of Asia, including INDIA. The infection in the herd is characterized by frequent abortions, presence of large number of unbred cows (Collántes et al.2018). Cows usually get infected through sexual transmission from infected bulls, artificial insemination with the infected semen. Except for few chemical methods of surface sterilization (Martin et al., 2023), till date there are no legal options proposed for elimination of parasite. Periodic screening, culling of infected animals, and strictly limiting the entry of infected animals were the stringent control strategies used certain parts of Wyoming, USA, that resulted in the eradication of Bovine Trichomonosis (Yao.2021). As the bulls were found to be the lifetime carrriers, and limited screening screening facilities seem to be the predisposing factors for several bulls found to be remain non-evaluated in herds and pose to be major risk factor for transmission of the disease (Oyherant 2019).
Apart from widespread transmission among cattle, several spill over incidents of T.foetus causing zoonotic disease in humans were also reported. T.foetus infection was found to be associated with clinical symptoms like Pneumocystis pneumonia (Duboucher et al., 2006), Meningo-encephalitis (Yao et al., 2012), Peritonitis (Zalonis et al., 2011) and Cholecystitis (Suzuki et al., 2016) in immune-compromised individuals. As a few most recent case controlled studies have reported a higher prevalence rates of 17.23% (Viola et al., 2023), 16.5% (Calvani et al., 2023), its quite alarming scenario to address the major challenges involved in surveillance and herd management practices. In view of the above prevailing circumstances, regular screening and early diagnosis are most important strategies to prevent further transmission in cows and isolating the infected cattle from mating would further prevent spreading in the herd.
As per the recommendations of World Organization for Animal Health (OIE, 2019) several advancements in developments of pathogens detection methods. Advanced culture methods like, InPouch TF-Bovine kit is the easy-to-use proprietary medium for the specific culture of Trichomonas foetus (12-011-001, 1Biomed Diagnostics, USA, 2023), Invitro culture methods using Bovine Cervico-vaginal mucus(Paradiso et al., 2023) as well as Expansion microscopy for immunofluorescence based specific detection of T.foetus proteins (Bandiera et al., 2023) are the most popular methods developed for specific detection of T.foetus from clinical specimens. RT-PCR based assays were developed for T.foetus DNA detection with improved sensitivity and specificity of the pathogen detection (Chroeder et al., 2023, Polo et al., 2023) However, these techniques are not very suitable for onsite screening by the farmer as well relatively not suitable for large scale screening.
Due to the cost associated with bulk culturing of T.foetus, long incubation periods and the zoonotic potential (human Transmission) associated with T.foetus cells, the earlier assays exhibit limited scope for scaling up and commercialization. Hence, in this current study we have designed a most suitable protein for development of recombinant protein based serological assays.
In our earlier studies on “ Comparative Omics based approach to identify immunogenic proteins of Trichomonas foetus” we have proposed several antigenic proteins suitable for developing serological testing kits (Karli et al.,2020, Abdala et al 2023). Though several highly expressed outer membrane proteins were listed as possible antigenic markers of Trichomonas foetus, the Cysteine proteases gain lot of significance due to their extracellular secretion into the cervical and uterine tissue. As reported by Tolbert et al. (2016) T.foetus was found to use a variety of strategies such as destruction of Complement, Leucocyte Phagocytosis, Apoptosis and suppression of Pro-inflammatory cytokine expression to avoid detection by the host immune system. Recent reports on existence of pseudocyst form of T.foetus further raises the concern on other possible modes of transmission in herds, without being detected in normal screening protocols (Martinez et al., 2023). In this context, choosing a membrane protein for detection of T.foetus specific antibodies in the serum was found to be quite inappropriate.
Earlier studies have also demonstrated that T.foetus was found to secrete several proteins that were found to be directly involved in the lysis of genital mucosal cells of Cattle. Singh et al. (2005) highlighted the role of CPs in the destruction of endometrial cells leading to early abortions and reproductive failure in cattle. Huang et al. (2013) suggested that in the transcriptomics and Proteomics studies Cysteine Proteases were found to be the predominant proteins that get secreted extracellularly. CP8 was found to be the most predominantly expressed among about 12 CPs involved in virulence. Morin et al. (2014) in the Comparative transcriptomics studies have also clearly suggested CP8 were the most abundantly expressed transcripts among all other CPs. Similarly, Stroud et al. (2016) also showed that CP8-induced host cell damage to the most predominant mechanism of T. foetus induced pathogenesis in cattle. Based on above knowledge, CP8 was found to be a highly expressed extracellular protein among about 20 CPs of T.foetus. Cysteine Protease8 (CP8)-mediated tissue damage was found to be the most notable mechanism of T. foetus-induced pathogenesis in Cattle. However, Huang et al. (2013) demonstrated that Cysteine protease8 was the highly expressed protein followed by CP13 and CP16. Hence, in this current study, we hypothesized to engineer a chimera protein from all above three CPs using Immunoinformatics and Bioinformatics tools.