Abstract Trypanosomatid parasites are notorious for the human diseases they cause throughout Africa and South America. However, non-pathogenic trypanosomatids are also found worldwide, infecting a wide range of hosts. One example is Trypanosoma (Megatrypanum) theileri, a ubiquitous protozoan commensal of bovids, which is distributed globally. Exploiting knowledge of pathogenic trypanosomatids, we have developed Trypanosoma theileri as a novel vehicle to deliver vaccine antigens and other proteins to cattle. Conditions for the growth and transfection of T. theileri have been optimised and expressed heterologous proteins targeted for secretion or specific localisation at the cell interior or surface using trafficking signals from Trypanosoma brucei. In cattle, the engineered vehicle could establish in the context of a pre-existing natural T. theileri population, was maintained long-term and generated specific immune responses to an expressed Babesia antigen at protective levels. Building on several decades of basic research into trypanosomatid pathogens, Trypanosoma theileri offers significant potential to target multiple infections, including major cattle-borne zoonoses such as Escherichia coli, Salmonella spp., Brucella abortus and Mycobacterium spp. It also has the potential to deliver therapeutics to cattle, including the lytic factor that protects humans from cattle trypanosomiasis. This could alleviate poverty by protecting indigenous African cattle from African trypanosomiasis.

Author Summary Single-celled parasites of the order Kinetoplastida are responsible for devastating diseases of humans and animals, including African trypanosomiasis, Chagas' disease and leishmaniasis. However, there are also many species of trypanosomatids that do not cause disease and are distributed globally. One example is Trypanosoma (Megatrypanum) theileri, which is restricted to bovids and ubiquitous in cattle herds worldwide. This organism is maintained extracellularly in the blood and tissues long-term without any observed ill effects on host health or productivity. Using knowledge of gene expression and protein trafficking in pathogenic trypanosomatids, we have successfully developed, from first principles, Trypanosoma theileri as a delivery system for vaccine antigens and therapeutics. Procedures for the growth, transfection and heterologous gene expression of T. theileri have been developed, and the delivery of a vaccine antigen derived from Babesia divergens evaluated in vivo. Our results demonstrate the ability of T. theileri to be used as a flexible and easily manipulated protein delivery system suitable for the control of cattle pathogens and cattle-borne zoonoses. In one notable application, we propose that the system could allow the expression of serum trypanolytic factors in cattle, with the potential to alleviate poverty in Africa through the killing of pathogenic trypanosomatids in livestock.

Citation: Mott GA, Wilson R, Fernando A, Robinson A, MacGregor P, Kennedy D, et al. (2011) Targeting Cattle-Borne Zoonoses and Cattle Pathogens Using a Novel Trypanosomatid-Based Delivery System. PLoS Pathog 7(10): e1002340. https://doi.org/10.1371/journal.ppat.1002340 Editor: Elisabetta Ullu, Yale University, United States of America Received: June 20, 2011; Accepted: September 14, 2011; Published: October 27, 2011 Copyright: © 2011 Mott et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work in this manuscript received contributory (10%) funding from Intervet Schering Plough as part of a contribution to a UK BBSRC Industrial Partnership Award. The information contained within the manuscript is protected by a patent to the University of Edinburgh. The work was funded through a Wellcome Trust grant (067592) and BBSRC Industrial Partnership award (BB/F00057X/1). The Industrial Partnership award received 10% funding from Intervet Schering Plough. Scientists at Intervet Schering Plough contributed reagents and data analysis advice but had no influence over the interpretations of the work. The manuscript was reviewed and approved for submission by Intervet Schering Plough but these partners exerted no influence on the material contained within the submission excepting to ensure that there was no inadvertent disclosure of IP protected information. Work in Keith Matthews' laboratory is supported by a Programme grant from the Wellcome Trust (088293) and by a Wellcome Trust Strategic Award for the Centre for Immunity, Infection and Evolution (095831). Jacqui Matthews and David Kennedy are funded by the Moredun Group and the Scottish Government's Rural and Environment Science and Analytical Services Division. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: Work in this manuscript received contributory (10%) funding from Intervet Schering Plough as part of a contribution to a UK BBSRC Industrial Partnership Award. The information contained within the manuscript is protected by a patent to the University of Edinburgh.

Introduction Human health is intimately linked to animal health through the impact of infectious agents on livestock productivity and their potential for zoonosis [1]. Indeed, animal borne disease represents the major source of both emergent and resurgent pathogens in humans, this affecting communities in both the developed and developing world. One major source of such zoonotic infections is cattle, which threaten human health in the developed world through their capacity to transmit bacterial infections including Escherichia coli, Salmonella spp., Campylobacter spp., Brucella spp. and mycobacteria. In the developing world, livestock are also a reservoir for Human African Trypanosomiasis (HAT) caused by Trypanosoma brucei rhodesiense. This parasite, and Trypanosoma brucei gambiense, is closely related to Trypanosoma brucei brucei, which cannot infect humans. The basis of this host restriction is that human serum contains a trypanolytic component of high-density lipoprotein, ApoLI, that kills T. brucei brucei [2], but to which the human infective parasites have evolved resistance [3], [4]. Although trypanosomes are important causes of human and animal disease, many species are non-pathogenic. One of these is Trypanosoma (Megatrypanum) theileri, a cosmopolitan parasite of bovids that infects most cattle worldwide [5]–[11]. Although one report describes reduced milk yield in three infected cows [12], the ubiquity of infection with this organism in cattle herds suggests that it has no significant impact on health or productivity in healthy animals, while cases of disease in immuno-compromised animals are sufficiently rare as to merit individual case reports [13]–[15]. The parasite is transmitted in the contaminated faeces of tabanid flies and gains entry into the host through breaks in the skin or via contamination of the oral mucosa [16]. Thereafter, it lives extracellularly, being sustained at a low level (∼100 organisms/ml) for the life of the host. Being non-pathogenic, systemic, related to the genetically well-characterised T. brucei, and sustained long-term, we conceived that T. theileri would make a suitable protein delivery system in cattle, able to generate immunity to expressed antigens. As a naturally non-pathogenic kinetoplastid, T. theileri offers considerable advantages over alternative vaccine delivery systems that comprise engineered, attenuated pathogens such as Salmonella [17], [18], Mycobacterium [19], [20], E. coli [21], Vibreo cholera, Listeria or Shigella [22]. Furthermore, being maintained over long periods at low level, T. theileri offers the potential to generate sustained immune responses of greater efficacy than conventional vaccination approaches and to deliver therapeutic proteins of benefit to bovine health or to limit the zoonotic potential of cattle borne diseases.

Discussion Our work describes a naturally non-pathogenic eukaryotic organism, T. theileri, engineered to deliver antigens and therapeutic proteins to cattle. Importantly, T. theileri is a flexible and adaptable system for the targeted expression of individual, or cocktails of multiple, heterologous proteins simultaneously. This allows for the targeting of different pathogens, or multiple defined antigens of a specific pathogen, providing an effective approach to limiting the transmission of disease from livestock to man, as well as maintaining bovine health and maximising bovine productivity. This system has many advantages over traditional vaccine delivery technologies, including the possibility of oral delivery, a natural route of T. theileri infection, and the potential for sustained immune stimulation through prolonged infection without adverse consequence on productivity or health. It also provides an explicit example of an unanticipated applied benefit derived from the extensive research investment in the basic biology of kinetoplastid pathogens. A number of kinetoplastid parasites have been successfully transfected to drive ectopic expression of endogenous genes or heterologous expression of reporter genes[35] . In general, these have been used for the analysis of gene function in the transfected organisms or for protein expression in vitro. In particular, the kinetoplastid protozoa T. brucei, Crithidia fasciculata, Leishmania amazonensis and Leishmania tarentolae have each been engineered as eukaryotic vehicles for the expression of proteins which are appropriately post-translationally modified, that retain biological activity, or which are suitable for meaningful inhibition studies for drugs targeted against kinetoplastids [36]–[40]. In each case, the basic organisation of the expression vectors employed was similar. However, there is no predictable cross functionality of RNA processing signals . For example, in one study, transfection of a range of kinetoplastid protozoa with a common expression construct developed in Leishmania major resulted in reporter gene expression in other Leishmania spp., but not in T. brucei or T. cruzi [41]. The specificity of RNA processing signals among kinetoplastids necessitated that we isolated intergenic sequences from T. theileri. To our knowledge, no protein coding gene sequence have been characterised in these organisms to date. Despite this, we were able to make use of the tandem arrangement of several conserved housekeeping genes in trypanosomatids to isolate intergenic sequences between alpha and beta tubulin genes, beta and alpha tubulin genes, actin genes and the PFR genes. Conservation in these genes allowed successful amplification between genes using degenerate oligonucleotides. In each case, the predicted protein sequences were highly similar to those in other kinetoplastids over the sequenced region, this being reinforced by the reactivity of monoclonal antibodies specific for alpha tubulin, PFRA and BiP in T. brucei with T. theileri (Figure 1A, Figure 2 and data not shown). Extensive protein coding similarity is also evident from a whole genome analysis of T. theileri that we have recently completed (our unpublished observations). Despite this similarity in coding regions, there was no recognisable similarity in the intergenic regions of each gene between species. This emphasised the requirement to use endogenous intergenic sequences in the developed expression constructs. Indeed, transient transfection of T. theileri with a reporter construct generated for use in T. brucei did not result in detectable heterologous gene expression (unpublished observations). Cloning and sequencing of 5′RACE products derived from the actin gene transcript identified the SL sequence common to all trypanosomatid mRNAs. Interestingly, the sequence of the leader RNA did not match the SL sequence of a previous T. theileri isolate (K127), which was reported to exhibit a 1 nucleotide distinction from the sequence identified on the majority of trypanosomatid parasites . Instead, the leader sequence identified in the isolate of T. theileri used in our study matched exactly the trypanosomatid consensus and shared with T. theileri D30 . This suggests variation in this sequence amongst different T. theileri isolates. Previous approaches have used kinetoplastid parasites as vehicles for protein expression in vitro, or for potential medicinal use via expression of biomolecules from attenuated pathogens in vivo. In these, and other cases of pathogen-based vaccine vectors (for example, using attenuated Salmonella , Mycobacterium , E. coli , V. cholera, Listeria or Shigella), the vehicles have a potential to revert to pathogenicity raising issues of long-term efficacy and safety. Moreover, recombination with non-attenuated pathogens can generate vaccine escape mutants, as observed after pneumococcal vaccination in the USA [42]. By exploiting an already non-pathogenic organism (T. theileri) for protein expression in its natural host, the possibility of reversion to, or unanticipated, pathogenicity is greatly reduced. Supporting this, the T. theileri isolate used in our studies has been maintained in tissue culture for over 6 years and yet, when inoculated into cattle, generated no ill effects and was sustained at low abundance throughout a 12-week trial. The transgenic parasites could also establish and be sustained in the context of a pre-existing natural infection. This is an important consideration since T. theileri is almost ubiquitous in cattle herds worldwide with, for example, 44%–80% incidence levels reported in New York State and up to 93% in Louisiana [43]. A survey of local adult cattle in Edinburgh revealed that all animals tested (16/16) were positive for T. theileri infection by the nested PCR assay for tubulin intergenic sequence (data not shown). T. theileri has clear potential as a flexible vaccine vehicle, able to target a wide range of pathogens, including viruses (e.g. Foot and Mouth Disease Virus, Bovine Viral Diarrhea Virus), bacteria (e.g. E. coli, Salmonella spp., Campylobacter spp., Brucella spp. and mycobateria), ectoparasites (e.g. ticks, mites, lice) as well as pathogenic eukaryotic parasites (Babesia spp., Neospora caninum, Coccidia spp., Trichomonas spp. and helminths). However, T. theileri also has potential to deliver therapeutic proteins such as antimicrobials systemically in cattle, able to limit the impact of bacterial infections, for example. Furthermore, we propose that it may be possible to avoid the need to engineer transgenic cattle to express ApoLI or mutant variants [44], [45], allowing indigenous and disease-resistant African livestock to combat pathogenic trypanosome infection via T. theileri directed ApoL1 expression. Such an approach would require that the ApoL1 is expressed in a form that is non-immunogenic and active in cattle. Nonetheless, this could provide a simple and cost effective route to limiting the effects of trypanosome infection on the productivity or zoonotic potential of indigenous African cattle. This has the potential to increase their productivity and eliminate an important reservoir for human disease, alleviating poverty and disease in afflicted regions.

Materials and Methods Ethics statement Animal trials in this manuscript were reviewed and approved through the ethical review committee at Moredun Research Institute (Experiment approval no. E28/10). Experiments were carried out under a UK Home Office Licence (PPL 60/4044) in accordance with the UK Animals (Scientific Procedures) Act, 1986. Cell culture Bovine conditioned media was produced by growing Madin-Darby Bovine Kidney (MDBK) cells in Eagle's Minimum Essential Medium with Earle's Balanced Salt Solution and sodium bicarbonate (Sigma, M2279) supplemented with 1% MEM non-essential amino acids (Invitrogen, 11140), 1% L-Glutamine solution (from 200 mM solution, Sigma, G7513), and 10% FCS until confluence (2–3 days). The MDBK-conditioned media was then harvested and filtered prior to use. T. theileri were cultured in 50% HMI-9 medium [46] supplemented with 20% FCS and 10% Serum+ and 50% MDBK-conditioned media as described above. Creation of recombinant cell lines The plasmid backbone used for construction of all expression vectors was derived from the pGemT Easy plasmid (Promega). All elements of the vector were prepared through PCR of the noted sequence, followed by cloning into the vector backbone, and construction was accomplished through the insertion of appropriate restriction sites at the 5′ and 3′ ends of each segment. The segments used for the construction of the expression cassettes and the corresponding primers used in this study include: the 5′ fragment of the T. theileri SSU rRNA gene (SSU5-ApaI-For and SSU5-AvrII-Rev), the 3′ fragment of the T. theileri SSU rRNA gene (SSU3-PacI-For and 3SSU-Rev-XmaI), the trans-splicing addition site from the actin IR sequence (splice-AvrII-For and splice-FseI-Rev), the Bd37 coding sequence (Bd37-Core-F-FseI or Bd37-Core-F-AvrII and Bd37-Core-F-AvrII and Bd37-Core-R-HindIII), the CAT coding sequence (CAT For FseI or CAT For XhoI and CAT Rev XbaI or CAT Rev AscI), the blasticidin resistance cassette (BSDKpn-F and BSDBgl-R), the beta-alpha tubulin IR sequence (ba-tub-AscI-For or ba-tub-BglII and ba-tub-KpnI-Rev or ba-tub-PacI), the T. brucei BiP N-terminal fragment sequence (BiP-For-FseI or BiP-For-FseI-SpeI and BiP-Rev-XhoI), and the GPI addition signal (GPI-For-HindIII and GPI-Rev-AscI). In fusion protein cassettes, care was taken to preserve the reading frame and appropriate start and stop codons were provided. The plasmids containing the expression cassettes were then digested with appropriate restriction enzymes to liberate the plasmid backbone from the linear expression cassette, which was isolated via gel electrophoresis and gel purification. The linear cassette was then purified by ethanol precipitation and resuspended in 5 ml of TE buffer (1 mM Tris-HCl (pH 8) and 0.1 mM EDTA). From a culture of logarithmically growing T. theileri parasites, 10 ml of culture at ∼5×105 cells/ml were used for each transfection. Cells were centrifuged at 1000 × g, for 10 minutes at room temperature and resuspended in 1 ml of sterile PBS to wash the cells. Cells were then re-centrifuged and resuspended in 100 µl Ingenio transfection buffer (Mirus Bio). The cells were added to the prepared linear DNA and transferred to a cuvette for the Nucleofector II electroporation device (Lonza). Transfection was done with Nucleofector program X-001 (recommended for mouse CD8+ T cells) and cells transferred to a culture flask containing 10 ml of pre-warmed media and incubated for 24 hrs at 37°C, in 5 % CO 2 . Cells were then transferred to media containing the selective drug concentration (10 µg/ml of blasticidin) and plated using a variety of dilutions into a 24-well cell culture plate. After selection clones were recovered under normal cell culture conditions. Immunofluorescence assay The localisation of the BiP-Bd37 fusion protein was determined using 2% paraformaldehyde fixed cells permeabilised, or not, with TBS: 0.1% Triton X-100 for 2 minutes. Cells were quenched for 30 minutes with TBS:0.1% glycine and then blocked for 1 hr with TBS: 1%BSA. Cells were then reacted with anti-BiP antibody (a gift of Jay Bangs, University of Wisconsin) diluted 1∶200 in TBS:1%BSA, washed three times with TBS and then incubated with anti-rabbit FITC conjugated antibody (Sigma) diluted 1∶100 inTBS:1%BSA. Prior to mounting in MOWIOL, cells were stained with 4′, 6-diamidino-2-phenylindole for 5 minutes to visualize cellular DNA. Experimental animals, sampling and DNA extraction Calves (6-weeks old) were injected intravenously with 1×105 T. theileri parasites. Blood samples were taken weekly into EDTA-containing tubes (Becton-Dickinson) and DNA extracted. DNA extraction from whole blood samples was performed as follows: 1 ml of blood was mixed thoroughly with 0.5 ml of RBC lysis buffer (0.32 M sucrose, 10 mM Tris-HCl pH 7.5, 5 mM MgCl 2 , 0.75% Triton X-100) in a microfuge tube. The samples were then centrifuged at 14, 000 rpm for 1 minute to pellet all cells and the supernatant removed. The pellets were repeatedly resuspended and recovered from 0.5 ml aliquots of RBC lysis buffer until no red blood cells were present. The resulting pellets were resuspended in 100 µl of lysis buffer (50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl 2 , 0.1 mg/ml gelatin, 0.45% NP40, 0.45% Tween-20, 60 µg/ml proteinase K) and kept at 55°C for 60 minutes. The samples were then incubated at 95°C for 10 minutes prior to storage at −20°C until use. Nested PCR Nested PCR reactions were designed to amplify either the T. theileri β-α tubulin intergenic sequence (to identify any T. theileri population) or the Bd37 coding sequence (specific for the vaccine vehicle). The primers used were as follows: Tub Diagnostic F1: 5′-AGTAGCAACGACAGCAGCAGT-3′ Tub Diagnostic R1: 5′-GTAAAGTGTTTGAAGAAGAGCTCG-3′ Tub Diagnostic F2: 5′-CGATTCTCTTCGCCTGTTTGT-3′ Tub Diagnostic R2: 5′-ACTAACCGCGACCAAAGAAGT-3′ Bd37 Diagnostic F1: 5′-GCTCACAGGAGCCAGCAGCGG-3′ Bd37 Diagnostic R1: 5′-CCAGAGCTTTGAGATTAGCTGGTA-3′ Bd37 Diagnostic F2: 5′-ACGCAGCAAGGTGGTGCGAA-3′ Bd37 Diagnostic R2: 5′-AGCAAGGCCTCACCGCCCTTGGC-3′ Each 25 µl reaction contained the following components: 5 µl of template, 1X PCR buffer, 0.2 mM of each dNTP, 1.25 mM MgCl 2 , 0.4 µM of each primer and 0.25 U GoTaq Flexi DNA Polymerase (Promega). The first stage PCR reactions were heated to 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 45 seconds and elongation at 72°C for 45 seconds. Following the final cycle, the reactions were elongated for a further 4 minutes. The second stage nested PCR reaction was conducted using the same conditions with 5 µl of the first reaction as template. The resulting products were electrophoresed on a 1% Tris-acetic acid-EDTA agarose gel, stained with ethidium bromide and visualized. Serum preparation and ELISA assay Serum samples were prepared from 10 ml of fresh blood and stored at −20°C until use. Recombinant (E. coli) expressed His-tagged Bd37 antigen was diluted to 5 µg per ml in coating buffer (0.01 M sodium carbonate pH 9.6), and 100 µl were added to each well of microtiter plates and incubated overnight at 37°C. The coating buffer was removed and 200 µl blocking buffer (10% horse serum in 10 mM PBS) were added, and incubated at 37°C for 60 minutes. The plates were washed 3 times with 200 µl washing buffer (10 mM PBS, pH 9.6 with 1% Tween-20). Bovine serum samples were diluted in blocking buffer as appropriate, and 100 µl were incubated in the coated and incubated at 37°C for 60 minutes. For standard ELISA plates were washed and incubated with HRP-conjugated anti-bovine IgG antibody diluted 1∶1000 in blocking buffer and incubated at 37°C for 60 minutes. For competitive ELISA assays, 100 µl of mouse monoclonal anti-Bd37 antibody (1 mg/ml diluted 1∶1000 in blocking buffer) were added to each well and incubated at 37°C for 60 minutes. Plates were washed and incubated with HRP-conjugated anti-mouse IgG antibody diluted 1∶1000 in blocking buffer and incubated at 37°C for 60 minutes. Plates were washed, treated with TMB supersensitive substrate (Sigma), stopped with 2M sulphuric acid and the OD was measured at 450 nm in an ELISA plate reader. Statistics To analyse serum antibody titres, a Kruskal-Wallis one way ANOVA was used.

Acknowledgments We thank Jay Bangs for plasmids containing the N-BiP sequence and for the gift of antibody to T. brucei BiP. We thank Keith Gull for the gift of antibody to T. brucei alpha tubulin. We also thank members of the Matthews lab for helpful suggestions and encouragement throughout the course of this work.

Author Contributions Conceived and designed the experiments: KRM GAM RW. Performed the experiments: GAM RW AF AR PM DK. Analyzed the data: KRM GAM RW DS JBM. Contributed reagents/materials/analysis tools: DS. Wrote the paper: KRM GAM.