Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 57 issue 5 (may 2023) : 535-546

SELEX- Aptamer Technology to Combat Diseases of Animals : Recent Advancement and Future Application: A Review

Ayan Mukherjee1,*, Arpana Verma3, Jagan Mohanarao Gali4, Indrajit Kar2, Dipak Dey5, Aditya Pratap Acharya1
1Department of Animal Biotechnology, West Bengal University of Animal and Fishery Sciences, Mohanpur, Kolkata-700 037, West Bengal, India.
2Department of Avian Science, West Bengal University of Animal and Fishery Sciences, Mohanpur, Kolkata-700 037, West Bengal, India.
3Division of Bio-informatics, Bose Institute, Kolkata-700 054, West Bengal, India.
4Department of Veterinary Physiology and Biochemistry, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih-796 014, Aizawl, Mizoram, India.
5Animal Resources Development Department, Government of West Bengal, Kolkata-700 106, West Bengal, India.
Cite article:- Mukherjee Ayan, Verma Arpana, Gali Mohanarao Jagan, Kar Indrajit, Dey Dipak, Acharya Pratap Aditya (2023). SELEX- Aptamer Technology to Combat Diseases of Animals : Recent Advancement and Future Application: A Review . Indian Journal of Animal Research. 57(5): 535-546. doi: 10.18805/IJAR.B-4304.
Aptamers are ‘nucleic acid antibodies’ that bind to target molecules with high affinity and specificity. The process by which aptamers are selected is called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). A plethora of aptamers have been selected against various targets, such as proteins, cells, microorganisms, chemical compounds, etc. Technical progress in the aspects of high throughput sequencing (HTS) technologies, bioinformatics, microfluidics and nanodevices have widened the scope of aptamers to contribute significantly in human as well as veterinary medicine. We have searched the Pubmed database and Google Scholar with the search term ‘aptamer’, ‘SELEX’, ‘animal diseases’. The search results have been categorized into bacterial, viral and protozoal diseases of veterinary importance. Here we have systematically described the strategy used to design aptamers for each disease and also shed light on the future research scope in this area. This article presents a comprehensive and up-to-date review of SELEX-aptamer technology, its modifications, aptamers selected against various animal pathogens, their potential to be applied in the future for diagnostic and therapeutic purposes.
The livestock sector plays a vital role in achieving food security by contributing to the bowl of the hunger-affected and malnourished human population in the form of meat, milk, eggs and other animal products. Demand for these animal products is increasing sharply as these are enriched with high-quality, balanced and highly bioavailable protein and numerous critical micronutrients, including iron, zinc and vitamin B12. Farm animals are critical to a sustainable agricultural system and especially for poor livestock keepers, traders and labourers in the developing world. Diseases of domestic livestock species, therefore, may have dire consequences on animal productivity and production, trade of animal products and human health and resulting adverse impact on overall economic development. Also, diseases of animals are major parameters that indicate the status of animal welfare in developed and developing countries (Kumar et al., 2017). Moreover, horizontal transmission of pathogens from animals to humans also poses serious public health threats worldwide (Hughes et al., 2010).

The significant practical approaches of mitigating animal diseases are disease forecasting, availability of swift, barn-side diagnostic tools that can detect the causative pathogen timely and accurately and the use of therapeutics. The development of simple, portable diagnostic devices is now considered a priority for animal diseases (Howson et al., 2017). The conventional clinical diagnosis method of the disease includes propagation and isolation from a culture which requires a definite the period and labour. Comparatively modern molecular diagnostic tools like polymerase chain reaction (PCR), real-time PCR (RT PCR) offer specificity, sensitivity and rapidity to the pathogen detection methods but they require isolated genetic materials, careful handling and sophisticated instruments. So, monitoring animal disease with these tools demands a dedicated workstation rather than an on-site execution. Therefore, to develop a rapid, sensitive and specific, field-level diagnostic assay for animal diseases has become a daunting task before the researchers across the globe.

Aptamers, a class of synthetic single-stranded DNAs or RNAs, which bind to target molecules with high affinity in three-dimensional shapes, have attracted increased attention in the last decades for diagnostic or therapeutic purposes. The term ‘aptamer’ was derived from the Latin word Aptus, meaning fit and the Greek word meros, meaning part or region. Aptamers have specificity and affinity like monoclonal antibodies; however, due to the several advantages like thermal and chemical stability, lower cost and less batch-to-batch variation aptamer-based approaches are promising alternative to immunological methods. Aptamers are selected and isolated by a specific strategy termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The method was ûrst developed by two independent groups 30 years ago in 1990 (Ellington and Szostak, 1990; Tuerk and Gold, 1990). This review focuses on SELEX procedure of aptamer development, modification of SELEX method, aptamers selected so far against pathogens causing diseases in animal, the future direction of aptamer-based research for the development of diagnostics and therapeutics against livestock  diseases.
 
Selex strategy
Principle of traditional SELEX methods
 
SELEX is an iterative process that starts with a vast library of 1014 to 1015 randomized nucleic acid sequences with the ability to bind to a target protein. The combinatorial library contains terminal fixed region at both the ends and internal variable region of 20-60 nt. About 1015 oligonucleotides (1-2 nmol) are incubated with target proteins. Oligonucleotides that bind to the target proteins are retrieved and unbound oligonucleotides are removed. The selected oligonucleotides are amplified by PCR for DNA-SELEX and reverse-transcription PCR for RNA-SELEX. Additional rounds of selection are performed with subpool of PCR amplified product until >90% aptamers bind to the target protein (enrichment). After that consensus motif between enriched aptamers are identified by cloning and sequencing of the selected oligonucleotides. Thus, with an iterative selection cycles comprising of binding, partitioning, recovery and re-amplification steps an enriched and dominant population of aptamer library species is developed (Fig 1). Traditional SELEX method has been evolved and several upgraded versions of the method have been introduced so far. Table 1 shows a various modified version of SELEX techniques widely applied to generate aptamers.

Fig 1: Schematic diagram of iterative SELEX process from DNA/RNA library. Single-stranded DNA or RNA library composed of 1014 to 1015 random oligonucleotides of 20-100 nucleotides length is mixed with the target of interest (incubation). Subsequently, the bound oligonucleotides are separated from unbound sequences (Partitioning) and amplified by PCR (Recovery). The resulting enriched DNA pool is used for the next round of selection. Aptamers are obtained after 10-20 iterative rounds.



Table 1: Different modifications of SELEX-Aptamer method.



Chemical modification of Aptamer
 
Despite possessing many of the advantages several inherent physicochemical features of aptamer like susceptibility to nuclease degradation and rapid renal filtration excretion restrict its use in in vivo condition. To safeguard the aptamers from nuclease digestion modifications are done at (a) ends of nucleic acid chain, (b) sugar ring of nucleoside and (c) phosphodiester linkage. Due to small mass (6-30 kDa) and short diameter (<5 nm) aptamers are easily excreted through renal filtration, even when using stabilizing backbone modifications. So bulky moieties like polyethylene glycol (PEG), cholesterol, organic or inorganic nanomaterials are attached at 5' end of aptamer to overcome renal filtration and extend circulation time.
 
Aptamers Against Major Pathogens Causing Diseases In Animal
 
Table 2 enlists handful number of aptamers selected so far against diseases in animals causing massive economic loss or having significance from public health point of view.

Table 2: The details of aptamers generated against important animal pathogens.


 
Aptamers against viral diseases
Foot and mouth disease
 
Foot-and-mouth disease (FMD) is a highly contagious vesicular disease that affects domestic and wild cloven-hoofed animal species. The causative agent of FMD is a small, non-enveloped, positive-sense, single stranded RNA (8.4 kb in length) virus belonging to the genus Aphthovirus of the family Picornaviridae called foot-and-mouth disease virus (FMDV). It spreads rapidly and is estimated to result in a 25% loss in productivity if not controlled (Brooksby, 1982). Not only across the world but in India also the disease has colossal economic impact estimated between US$6.5 and 21 billion a year, due to high morbidity of the disease and the huge numbers of animals affected (Sinha et al., 2017). Tackling the spread of the disease is major challenges due to high infectivity and transmissibility of the virus and ability of the virus to develop an asymptomatic carrier state. So, development of a prompt on-site diagnostic assay is requires immediate attention to check the disease in animal population. As far as FMDV diagnostic assays are concerned several rapid immunoassays (Bronsvoort  et al., 2004) and reverse transcriptase polymerase chain reactions (RT-PCR) or other nucleic acid amplification-based tests (Callahan et al., 2002; Collins et al., 2002; Shaw et al., 2007) with broad FMD serotype specificity are available. But there is no portable field tests for all FMD serotypes and strains and it needs at least 2 hours for results to confirm from the existing tests. Bruno et al., (2008) attempted to develop a competitive fluorescence resonance energy transfer (FRET)-aptamer-based strategy for detection of foot-and-mouth (FMD) disease within minutes. In a novel approach a 14-amino-acid peptide from the VP1 structural protein of FMDV was labeled with Black Hole Quencher-2 (BHQ-2) dye and allowed to bind to Polyclonal FMD DNA aptamers labelled with Alexa Fluor 546-14-dUTP. The study revealed a detectable, highly sensitive response within 10 minutes at the level of 25-250 ng/mL of FMD peptide.

Another approach to curb the transmission of FMDV is to stop the viral replication. An RNA dependent RNA polymerase (3D pol) is responsible for replication of viral genome via negative strand intermediates. The pioneering work to develop an aptamer with the ability to bind 3Dpol and inhibit FMDV replication was carried out by Ellingham et al., (2006). The group selected and characterized 3 aptamers (F38, F47 and F52) that can selectively bind to 3D pol and inhibit the replication machinery of FMDV. In a separate experiment a truncated version of F47 (F47tr) was found to inhibit replication elongation process and RNA-protein fibril formation in an in vitro elongation assay (Bentham et al., 2012). However, these aptamers were susceptible to degradation in serum as well as in cellular milieu as they were not chemically modified. Recently, modified versions of F-47 and F-52 (22 F-Cy3-47tr and 22 F-Cy3-52tr) were developed by same laboratory in the University of Leeds, UK. These modified aptamer species were found to be more stable and inhibit FMDV subgenomic replicon efficiently in in vitro in BHK-21 cells (Forrest et al., 2014). Fig 2 depicts the FMDV replication machinery within cellular environment of infected host and how aptamers precisely inhbit the transmission of virus from one affected host cell to another.

Fig 2: Aptamer targets the replication machinery of foot and mouth disease virus (FMDV) within host cell. Aptamer binds to VP1, a structural protein and inhibits capsid formation. Another aptamer binds viral 3D Polymerase thus inhibits viral RNA replication.


 
Rabies
 
Rabies is a viral disease that affects a wide range warm-blooded animals and causes about 60000 death annually all over the world (Yousaf et al., 2012). An epidemiologial survey in Chennai, India shows that Rabies is endemic in India (Bharathy and Gunaseelan, 2016). Rabies virion, is a bullet shaped, enveloped infectious particle (180 nm x 75 nm in size), having 12 Kb negative sense single-stranded RNA genome, belonging to the Lyssavirus genus of the Rhabdoviridae family and Mononegavirale order. Rabies virus (RABV) is transmitted by a rabid animal’s bite to healthy individual through saliva. Presently no treatment of rabies is available once symptoms are evident and death occurs within a week. First attempted a study to isolate DNA aptamer against RABV. In their initial approach aptamers were isolated through 35 iterative rounds of selection (Liang et al., 2012). The aptamer species were incubated further with baby hamster kidney (BHK)-21 cells infected with CVS11 strain of RABV. Uninfected BHK-21 cells were counter-selected and five aptamers were pinpointed to inhibit replication of RABV (Liang et al., 2012). However, inhibitory capacity of the selected aptamers was not satisfactory and they did not cross-react with other rabies strains. So in their next study the group used the same cell-SELEX approach to select a new batch of aptamers. Preliminarily 16 DNA aptamers were isolated, but only the aptamer, FO21, inhibited specifically the replication of RABV (Liang et al., 2013). Further the aptamer was modified with polyethylene glycol (PEG). Same study also revealed efficacy of the modified aptamer in in vivo condition. When mice were treated with FO21 aptamers for a day prior to challenge with RABV 87.5% of mice survived the infection (Liang et al., 2013). Subsequently an in vivo study by the same group revealed that aptamers FO24 and FO21, which target RABV-infected cells, can significantly protect mice from a lethal dose of the street rabies virus FJ strain (Liang et al., 2014). So, both the aptamers have the potential to be used to develop preventative antiviral therapy againstthis lethal disease.
 
Avian Influenza
 
The avian influenza or bird flu which is panzootic in poultry poses a serious threat to animal and human health (FAO 2006 and WHO 2007). The viruses cause a massive damage on the poultry industry in many developing countries due to high mortality in the flock and this directly or indirectly impacts both economic and social well-being. Major attention has been given towards diagnosis of Avian influenza virus because of substantial risk of infection in human and high frequency of mutations, resulting in appearance of new viral strains with epidemic or even pandemic danger. Influenza A viruses are enveloped RNA viruses with an eight-segmented, single-stranded, negative-sense genome belonging to the family Orthomyxoviridae. Hemagglutinin (HA), a glycoprotein expressed in high amounts on the viral surface, is a suitable target for aptamer-based antiviral therapy. It is responsible for fusion of virus with the host cell. At least 18 varieties of HA antigens are present on virus surface, therefore, it could serve as candidate protein for diagnosing and identifying influenza virus types and subtypes. Development of aptamer-based antiviral therapy against H5N1 virus was evident in 2011 when (Park et al., 2014) reported selection of an aptamer HAS15-5, which acts by blocking and inhibiting the receptor-binding domain of viral hemagglutinin (Park et al., 2011). Choi  et al., 2011 generated specific DNA aptamer (C7-35M) with high affinity to H9 peptide of H9N2, a low pathogenic avian influenza (LPAI) strain (Choi et al., 2011). 

Efforts are being made to immobilize aptamers on the biosensor chip so that full benefit of the technology can be obtained during field-level, rapid detection of the pathogen. In a novel aptasensor approach biotinylated specific DNA-aptamer, immobilized it on the sensor gold surface coated with streptavidin in a portable Surface Plasmon Resonance (SPR) biosensor for rapid detection of AIV H5N1 in poultry swab samples. The immobilized aptamers captured AIV H5N1 in a sample solution, which caused an increase in the refraction index (Bai et al., 2012).

Wang and Li generated an ssDNA crosslinked polymeric hydrogel construct that was able to detect avian influenza virus rapidly (Wang and Li 2013). Briefly, the aptamer hydrogel was immobilized on the gold surface of QCM sensor. In absence of H5N1 virus in the sample there occurs crosslinking between the aptamer and ssDNA in the polymer network and the hydrogel remained in the state of shrink. In case of H5N1 positive samples, the aptamer binds to virus and the linkage between the aptamer and ssDNA becomes disrupted resulting in the abrupt swelling of the hydrogel. The swollen hydrogel was monitored by the QCM sensor in terms of decreased frequency. Diba et al., (2015) developed a highly sensitive (detection limit 100 fMol) sandwich assay platform involving a surface formed aptamer-protein-antibody complex to obtain the highly selective and sensitive amperometric detection of H5N1 viral proteins using a gold nanoparticle (NP) modified electrode. Recently a specific DNA aptamer was immobilized onto the gold interdigitated microelectrode surface embedded within a microfluidics biochip. This microfluidic based aptasensor can detect as small as 0.0128 hemagglutinin units of H5N1 Avian Influenza Virus within 30 min in field condition (Lum et al., 2015).
 
Bovine viral diarrhoea
 
Bovine viral diarrhea is a significant infectious disease of livestock worldwide due to its endemic nature, clinical manifestations (Greiser-Wilke et al., 2003; Moennig et al., 2005). Persistently infected (PI) animals are particularly important in transmitting the pathogen as PI animals shed large amounts of virus throughout their lives (Burgstaller et al., 2016). A recent study has estimated BVD causes direct monetary loss that varies from 0.50 to 687.80 US dollars (USD) per animal (Richter et al., 2017). BVD virus, the causative agent of BVD, is a pestivirus in the Flaviviridae family. Once the virus enters a herd, transmission occurs rapidly and within weeks total herd is infected. Therefore, quick detection and identification of BVD virus is crucial to protect the herd from BVD outbreaks. Aptamer-based biosensors are promising sensory diagnostic tool over the contemporary laboratory based tests like RT-PCR, ELISA due to its short analysis times, affordability, miniaturized platforms, with low sample consumption and the possibility for measurements in complex samples (Daniels and Pourmand 2007). To this end, Park and coworker in 2014 applied an advanced graphene oxide (GO)-based immobilization-free SELEX method for obtaining ssDNA aptamers that can bind to a whole BVDV type 1 specifically. In this method two different aptamers specifically binding to bovine viral diarrhea virus type 1 (BVDV type 1) with high affinity were successfully screened. One aptamer (capturing aptamer) first binds with the whole BVDV Type 1. Another aptamer (reporting aptamer), conjugated with gold nanoparticle (AuNP), can also bind to BVDV Type 1 making a sandwich type sensing format-capturing aptamer-Whole BVDV Type1- AuNP labeled reporting aptamer. Significant signal enhancement occurs when BVDV is captured and it can sense as little as 800 copies/ml of virus (Park et al., 2014).
 
Infectious Bovine Rhinotracheitis
 
IBR is a highly contagious and infectious viral disease that affects younger and older cattle. The disease is clinically characterised by dyspnea, pyrexia, nasitis and conjunctivitis. The causative organisms Bovine herpes virus 1 (BoHV 1) also causes infectious pustular vulvovaginitis in the female and infectious balanoposthitis in the male and can cause abortions and foetal deformities. The world organization for animal health has placed BoHV-1 into category B for infectious diseases because of its worldwide prevalence and its significant impact on cattle industries (Muylkens et al., 2007). A sero prevalence study across different regions of India highlighted natural circulation of virus in the population (Patil et al., 2015). After causing an acute infection BoHV-1 remains in latent stage throughout the life in the animal. Following reactivation of BoHV-1 in carrier animal large amount of virus is shed and the disease is rapidly transmitted throughout the herd. Therefore, it is much essential to identify the carrier animal with suitable diagnostic tools and isolate them from the herd to break the cycle of BoHV-1 transmission (Workman et al., 2012). Considering the potential of aptamers as good candidates for diagnostic tools and antiviral agents (Gopinath et al., 2012; Lee et al., 2013) a study was carried out to develop aptamers that can inhibit entry of BoHV-1 into cells (Xu et al., 2017). Preliminarily nine aptamers were selected after eight iterative rounds of SELEX against purified immobilized BoHV-1. Out of nine candidate aptamers IBRV-A4 exhibited the greatest binding affinity and specificity for BoHV-1 with a Kd value of 3.519 ±0.4801nM. Further validation of its antiviral activity in Madin-darby Bovine Kidney (MDBK) cells verified that IBRV-A4 may be a novel tool for diagnosis and treatment of BoHV-1 infection in cattle.
 
Red-neck disease of turtle
 
The Chinese soft-shelled turtle (Trionyx sinensis) is cultured commercially in Asian countries, such as China, Japan, Vietnam and Korea due to its high nutritional and economic values (Chen et al., 1999). Also, T. sinensis is used as a model organism for developmental and biological research (Nagashima et al., 2009). However, ‘red-neck disease’ of this species of turtle caused by soft-shelled turtle iridovirus (STIV) causes great economic loss in aquaculture. With the aim to develop antiviral candidates for the control of STIV infections (Li et al., 2015) generated eight aptamers by whole-virus SELEX. All the selected aptamers inhibited STIV infection in vitro and in vivo, with aptamer QA-36 showing the greatest protective effect against STIV making it an appropriate candidate for antiviral therapy (Li et al., 2015).
 
Aptamers against bacterial diseases
Salmonellosis
 
Salmonellosis is manifested clinically as a syndrome of septicemia, typhocolitis, acute or chronic enteritis and abortion in cattle, sheep, pig and horse. Salmonella enterica subspecies enterica serotype Dublin (S. dublin) and Salmonella enterica subspecies enterica serotype Typhimurium (S. typhimurium) most commonly affect bovine. In poultry also S. typhimurium is major pathogen causing fowl paratyphoid. Human salmonellosis is generally foodborne and is contracted through consumption of contaminated food of animal origin such as meat, milk, poultry and eggs (Kemal, 2014). S. typhimurium is the leading cause of human salmonellosis cause a wide range of human health problems, including diarrhea, bacterial fever (typhoid), food poisoning, stomach flu and infectious diseases of the urinary tract, lung and kidney (Oh et al., 2017). Apart from public health issues related to S. typhimurium the fact that several isolates from S. typhimurium have developed resistance to the traditional antimicrobials and resistance to ciprofloxacin or cephalosporins in developed countries (Weill et al., 2006; Whichard et al., 2007) is matter of grave concern. So novel strategy like aptamer based therapeutic is need of the hour to prevent the spread the infection of this pathogenic organisms. To select DNA aptamers against S. enterica serovar Typhimurium, a mixture of outer membrane proteins (OMP) was used as the positive selection target (Joshi et al., 2009). The obtained aptamers, 33 and 45, were able to bind not only the target proteins but also the outer membrane proteins isolated from seven other serovars as well as the whole bacteria. These aptamers were subsequently immobilized on magnetic beads and applied to capture the pathogen from biological samples with subsequent PCR detection. Several modified 2’-F-RNA aptamers against the outer membrane protein, OmpC, of S. typhimurium were described (Han and Lee, 2013). One aptamer, I-2, possessed high affinity to the target protein (Kd = 20.27nM) and, in contrast to the other obtained aptamers, was able to bind intact S. typhimurium cells. To miniaturize the technology recently a QCM-based aptasensor was developed with an aptamer (B5, Kd = 58.5 nM) to detect S. typhimurium. This aptasensor is highly sensitive and able to detect 103CFU/mL of S. typhimurium with less than 1h (Wang et al., 2017).
 
Mycoplasma bovis mastitis
 
Mycoplasma bovis (M. bovis) mastitis is highly contagious and results in a severe milk production drop in an affected cow. A competitive enzyme linked aptamer assay using the biotin labeled aptamer of P48 protein was applied in an indirect diagnostic test to detect the disease. P48 protein is an ideal biomarker for M. bovis since it is consistently localized on the membrane surface of M. bovis. A single stranded DNA aptamer showing high affinity and specificity against P48 protein of M. bovis has been used in a competitive enzyme linked aptamer assay for the detection of M. bovis in sera (Fu et al., 2014).
 
Aptamers against hemoprotozoan parasite
Trypanosomosis
 
Trypanosomosis or ‘surra’ is a predominant hemoprotozoan diseases affecting human and animal population in the tropics (Kurup and Tewari, 2012) especially in the South East Asia (Bossard et al., 2010). Conventional diagnostic methods for most of the Trypanosoma infections rely on microscopical demonstration of infective stages in blood or tissue fluids which is not sensitive enough (100000 organism/ml of blood) to reduce false negative cases. Drugs currently used for treating parasite infections are not always effective and some of them have severe side effects. Aptamers are attractive tools for the development of alternative diagnostics and treatment methods. The aptamers against this fatal disease were generated against Variant Surface Globular (VSG) and other surface proteins of T.  brucei causing sleeping sickness in human. But these aptamers have potential to be used against T. evansi causing infection in animal as analysis of T. evansi variant surface glycoprotein (VSG) sequences shows extensive conservation of N-terminal sub-types, with extensive phylogenetic similarity with T.  brucei (Carnes et al., 2015). The group of Göringer used the cell-SELEX strategy to select RNA aptamers with high affinity for a 42 kDa protein located in the flagellar pocket of the infective blood life cycle stage of the T. brucei (Homann and Göringer, 1999). Further characterization of one aptamer molecule 2-16 revealed that target-bound aptamer is engulfed by receptor-mediated endocytosis and transported to the lysosome. Another investigation by same group revealed that aptamer can be used as a ‘piggy-back‘ delivery trypanocidal molecule to target the lysosomal compartment of trypanosome, so that aptamer 2-16 may represent a promising tool to facilitate drug delivery to lysosome (Homann and Göringer, 2001). VSG, that allows trypanosomes to escape the immune response of the infected host by antigenic variation, is an another target protein for aptamer development. Lorger et al., (2003) obtained RNA aptamers that bind the surface of live African trypanosomes using the structurally conserved C-end of VSG molecules (Lorger et al., 2003).
 
Aptamer: Future Application In Animal Disease Diagnosis And Therapeutics
 
Prompt and accurate detection of infectious animal pathogens is of major importance to minimize the economic consequences and emerging public health threats (Sinha et al., 2018). With the advent of newer technologies several improvements in development of animal disease diagnostic assays have been observed. All these assays require isolated genetic materials, careful handling, sophisticated instruments, skilled personnel and dedicated laboratory. Various reports of major outbreaks of deadly animal diseases across the globe have echoed the need of development of rapid, pointof-care testing (POCT) diagnostics for veterinary diseases. Therefore, innovative approaches based on combination of aptamer, microfluidics, nanotechnology is extremely required for developing portable, lab-on-chip diagnostic assays. A simplified view of future diagnostic approach based on aptamer has been described in Fig 3.

Fig 3: Simplified figure showing how aptamer-based biosensor (aptasensor) may be used to develop field-based diagnostics for animal diseases. After collections of biological samples from populations, samples will be passed through the lateral-flow channels on aptasensor where pathogen-specific aptamers are immobilized on surface. Binding with specific pathogen will produce signals that can be visualized.



Several intracellular pathogens like Brucella abortus, Listeria monocytogenes,Mycobacterium tuberculosis, Salmonella enterica cause severe pathogenic infection in animals. Some of the infectious organisms such as Listeria, Mycobacterium can resist and replicate even within phagocytic cells, which is the first-line of defense against invading organisms (Chono et al., 2008). The hydrophilicity of several antibiotics limit their ability to penetrate the cells and accumulation of antibiotics within lysosomes decreases their bioactivity. This results in limited intracellular activity against sensitive bacteria (Seral et al., 2003). Nanoparticle-based drug delivery system can release high concentrations of antimicrobial drugs at the site of infection, while keeping the total dose of drug administered low. Liposome is an efficient nanomaterial which can produce rapid distribution and enhance circulating time of encapsulated active drugs and thus, recognized as first-generation nanomedicines for clinical applications. Xing et al., (2013) developed AS1411 aptamer-targeted liposome NPs for breast cancer specific doxorubicin therapy (Xing et al., 2013). With the same AS1411 aptamer, Wu et al., (2013) developed a therapeutic aptamer-human serum albumin conjugate to deliver paclitaxel for breast cancer therapy (Wu et al., 2013). As far as animal diseases are concerned liposomal formulations have been used in several bacteria including Staphylococcus aureus, Salmonella species, Brucella species and Mycobacterium species (Swenson et al., 1998). MacLeod and Prescott demonstrated that liposomally entrapped gentamycin can kill Staphylococcus aureus mastitis in bovine (MacLeod and Prescott, 1988). Umpteen number of nanomaterials are developed that can specifically target medicine in infected animal cells. Coating of nanomaterial surfaces with aptamers furnishes the resultant nanomedicines with novel characteristics, such as: (i) reduced non-specific aggregation and improved stability; (ii) enhanced biocompatibility in vivo and (iii) acquisition of the “active targeting” function (Sun and Zu, 2015). Due to straightforwardness of programmable nucleic acid engineering, aptamers have been widely explored to be conjugated with small interfering RNA (siRNA), small hairpin RNA (shRNA) and microRNA (miRNA) for targeted delivery of these therapeutics for RNAi-based gene therapy of human diseases. Although this area is unexplored in animal disease management further attempts can be made with a vast array of small noncoding RNAs (sncRNAs) which modulate gene expression in wide spectrum of important animal pathogens. A short-hairpin RNA (RNAi-VP4) targeting viral VP4 gene has been observed to prevent FMDV infection in primary epithelium cells of transgenic bovine fetus (Wang et al., 2012). Several siRNAs against VP1 protin (Lv et al., 2009), viral Integrin-b6 receptors (Hu et al., 2017) have been observed to act as potent inhbitors of FMDV replication in different cell lines. Future research may be directed to search for cell-type specific aptamer with cell-SELEX approach and target the affected cell with highly efficient siRNAs, shRNAs or therapeutic agents (Fig 4).

Fig 4: Aptamer-conjugates are future therapeutics against animal diseases. Specific aptamers may be selected by cell-SELEX against animal cells where pathogens localize. Selected host cell-specific aptamers may be subsequently conjugated with siRNAs or miRNAs or shRNAs that can modulate gene expression of pathogen. Alternatively aptamerrs may be attached to nanoparticles (NP) like liposomes pre-loaded with therapeutic agents against pathogens. Aptamers will guide these small RNAs or NPs to specific cells and deliver them within the cell to show therapeutic effect.

This review shows promising data on application of SELEX-aptamer technique for rapid, cost-effective, ‘on-site’ detection and therapeutic development for animal diseases. The animal diseases discussed here are causing severe economic losses due to decreased animal productivity, increased treatment cost in present day scenario. Additionally, some of the diseases due to their zoonotic potential pose serious threat to public health. The time has become ripe to develop transportable, miniaturized, sensitive and specific diagnostic tools that can be used easily by the field-veterinarians, epidemiologists or competent authorities to control animal disease. Conventional diagnostic methods to diagnose them require time, trained professional and sophisticated instruments. Plethora of problems such as side effects of therapeutic agents and pathogen drug resistance constrain the development of new therapeutics in this area.

Since its inception in 1990 conventional SELEX technology has been modified continuously and an array of target-specific aptamers has been produced against wide variety of pathogens and diseases. However, development of clinically relevant aptamers with diagnostic and therapeutic significance is far from the expectation. So far, the U.S. Food and Drug Administration (FDA) has approved one RNA aptamer (Macugen®/pegaptanib) and ten aptamers are under clinical trials for the treatment of macular degeneration, coagulation, oncology and inflammation (Zhou and Rossi 2017). In spite of limited commercial success in last three decades after discovery aptamers are emerging as promising tools for pathogen detection, disease surveillance and disease treatment due to its judicious amalgamation with nanotechnology, microfluidics and next generation sequencing technologies. A market research report by Markets and Markets (Pune, India) has predicted that the global aptamers market is expected to reach $244.93 Million by 2020 from $107.56 Million in 2015, at a compound annual growth rate of 17.89% (https://www.marketsandmarkets.com/Market-Reports/aptamers-technology-market-1167.html).

Optimization of aptamer-based diagnostic and therapeutic tools for alleviating animal ailments and preventing economic loss require strategies like familiarization in the field condition, better stability of aptamer after immobilization. We predict that near future will witness an exponential growth of aptamer technology in the development of new efficacious aptamer-based theranostic agents towards animal diseases. This will in turn, reduce the incidences of animal disease outbreak and bolster food security across this planet.
Authors declare no conflict of interest.

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