**2. Advances in animal disease diagnostics**

#### **2.1 Synthesis of Noble metal nanoparticles**

Throughout the emergence of nanotechnology, there have been many techniques developed on how to synthesize nanoparticles which include physical, chemical, and biological approaches. Among the three, synthesis of nanoparticles from physical and chemical methods are considered the best methods for they can provide more uniform-sized nanoparticles with long-term stability. Biological approach on the other hand is also used to lessen the production of toxic by-products from physical and mostly from chemical approaches [21].

#### *2.1.1 The Turkevich method*

The most common method of synthesis of nanoparticles is probably through the Turkevich method used to make spherical gold and silver nanoparticles [22]. This method is a chemical approach which makes use of a single phase water-based reduction where gold or silver salt undergo reduction by citrate (sodium tri-citrate) at boiling temperature (100°C). The citrate ions, which serves as both reducing and non-aggregation agent, stabilize the nanoparticles by providing negatively charged ions which gets absorbed onto the surface of each particle (see **Figure 1**). Individual particles which are now stabilized and surrounded by negative charges will tend to repel each other causing a more stable nanoparticle dispersion and preventing them from aggregation [23].

Furthermore, the concentration of the citrate ions used in the solution determines the average size of the nanoparticles. Higher concentration of citrate ions (citrate to gold ratio) produces smaller nanoparticle size (average of 10 nm) due to higher stabilization and particle repulsion. On the other hand, reducing the concentration of sodium tri-citrate limits the number of citrate ions that will stabilize the particles. This causes aggregation and forms bigger particles (>15 nm) [24].

**Figure 1.** *Synthesis of AuNP using Turkevich method [23].*

### *2.1.2 Physical methods of synthesizing nanoparticles*

Several ways of producing nanoparticles using physical methods are already reported [25, 26]. Generally, some of these methods are using Plasma, Chemical Vapor Deposition, Microwave Irradiation, Pulsed Laser, Sonochemical Reduction and Gamma Radiation.

#### *2.1.3 Green synthesis of nanoparticles*

Green synthesis or biological synthesis are alternative pathways to produce nanoparticles in an eco-friendly way. This approach (in comparison with the physical and chemical methods) has lower energy consumption, lower cost, and less harmful to the environment. This pathway utilizes the use of microorganisms or plants (phytosynthesis) as source of reducing agents [26]. The main limitation of this approach is how to control the size and shape of the product. Different phyto-chemical compositions from organic sources have different influences on the particles' size and shape which can be attributed to purity of the extract used as reducing agents [27].

#### **2.2 Characterization of synthesized gold nanoparticles**

Synthesis of nanoparticles are verified mainly through their size and shape using Scanning Electron or Transmission Electron Microscopes (SEM/TEM). Additional characterization methods include spectroscopic analysis (UV–Vis Spectroscopy), dynamic light scattering (DLS), Zeta Potential, Inductively-coupled Plasma Mass Spectroscopy (ICP-MS), dark field microscopy, and more [28, 29]. Aside from their size and shapes, nanoparticles can have other unique properties based on their method of synthesis and precursor metals. These characteristics affect how they react with light due to surface plasmon resonance [30]. A good example on how to demonstrate the effect of size of nanoparticle on how they interact with light can be seen in **Figure 2** [31].

### **2.3 Advances in animal disease diagnostics using Noble metals**

Serological (e.g. Enzyme-linked immunosorbent assay – ELISA) and molecular detection (e.g. PCR) of different animal pathogens has been one of the routine diagnostic techniques is most animal disease diagnostics. However, this requires well-trained laboratory technicians and expensive, sophisticated equipment [3]. Thus, the LAMP method developed by Japanese researchers that is claimed to be cost-effective without sacrificing sensitivity and specificity became a promising point-of-care molecular method [4]. However, this technique still has drawbacks such as less versatility compared to PCR, cannot be used in cloning purposes, limitation in multiplexing and difficulty in primer designing.

Colorimetric-based nanoparticle DNA detection is an eye-catching method due to its rapidity and cost-effectiveness compared to current generation of DNA detection or amplification. This method enables a direct or visual detection of amplified DNA even without expensive, sophisticated equipment. The incorporation of nanoparticles in platforms such as LAMP addresses the issue with regards to false positive results due to the addition of intercalating dyes as amplification indicators. Furthermore, hybridizations of nanoparticles with complementary DNA make this method more specific and overcoming the weaknesses of test format such as LAMP. Thus, LAMP and other

*Application of Noble Metals in the Advances in Animal Disease Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.99162*

**Figure 2.**

*Variations in color of gold nanoparticle suspension as particle size increases. Synthesized gold nanoparticles tend to have wine-red color (average size of 10–15 nm) and turns blue to purple as particles aggregate and get bigger.*

point-of-care diagnostic tools coupled with nanoparticle has become a promising, sensitive, specific, cost-effective and rapid animal disease diagnosis techniques.

### *2.3.1 Point-of-care animal disease diagnosis using gold nanoparticles (AuNPs)*

AuNPs are the most studied nanoparticle and has a fascinating property for biosensing. Furthermore, AuNPs can be synthesized to gold nanoprobes (AuPr) for detection of colorimetric detection of different animal diseases.

#### *2.3.1.1 Bacterial diseases*

Paratuberculosis or Johne's Disease, caused by *Mycobacterium avium subsp. paratuberculosis* (MAP), is a chronic gastro-enteric disease of ruminants marked with diarrhea and irreversible wasting leading to death [32, 33]. The problem with paratuberculosis is that it can exist in the herd for years and remain undetectable. However, recent reports reveal that an estimated 200–250 million USD is lost in US Cattle industry due to paratuberculosis [11]. Furthermore, sub-clinically infected cattle produce 15–16% less milk that amounts to 1300–1500 pound's loss in every lactation. In addition, there is no approved treatment for paratuberculosis and no effective vaccine available. Thus, screening and removal of the infected animal from the herd is the most effective way of controlling and preventing spread of paratuberculosis [33]. Ganareal *et al.* [11] developed a gold-nanoparticle based probe for the colorimetric detection of MAP DNA. The developed nano-probe was specific to detect MAP with a detection limit of 103 ng of MAP PCR product per reaction. Furthermore, UV–Vis and SEM showed dispersion and aggregation of the AuNPs for the positive and negative results with no observed particle growth.

#### *2.3.1.2 Viral diseases*

Foot-and-Mouth Disease (FMD) is one of the most devastating and highly contagious disease of cloven-hoofed animals (e.g. ruminants and swine) that may threaten food security [34]. The causative agent, Foot-and-Mouth Disease virus (FMDV) has multiple modes of transmission, fast replication rate and viral excretion that makes FMD one of the acute and highly contagious diseases of cloven-hoofed animals [34, 35]. Southeast and East Asian countries such as Cambodia, Laos, Thailand, Vietnam and China show varying FMD prevalence [34]. Eradication and control strategy for FMD is mainly controlled by vaccination. However, discrimination between naturally infected versus immunized animals against FMD is a must especially in the event of mass importation of cloven-hoofed animals. Furthermore, the rapidity of development of antibody against FMD and the differentiation of naturally infected vs. immunized animals are important in the disease control and prevention strategies. Conventionally, serological methods such as FMD structural proteins (SPs)-based virus neutralization test (VNT), liquid phase blocking enzyme-linked immunosorbent assay (LPB-ELISA) and solid-phase competition ELISA (SPCE) can evaluate the antibody level and non-structural proteins (NSPs)-based ELISA can discriminate naturally infected from immunized animals. However, with the advent and success of immunochromatographic strip (ICS) in the field due to its high specificity, sensitivity, rapidity, low cost and portability for field detection and high sample throughput, Yang *et al.* [34] developed an immunochromatographic gold nanoparticle strip that can differentiate FMD type O-naturally infected from immunized animals using serum. Both epitopes of FMDVs SPs (T1) and NSPs (T2) were dispensed in the nitrocellulose membranes to be the two test lines and as for control line a goat anti-pig antibody IgG was used. The result of their experiment shows 95.17% and 100% specificities for T1 and T2, respectively with the sensitivity comparable to the commercial ELISA kits. Furthermore, the coincidence rate of the develop AuNP strip is 95.5% and 93.13% for 3ABC monoclonal antibody (Mab)-ELISA and LPB-ELISA, respectively. Thus, the developed AuNP strip can provide a point-of-care differentiation test between naturally-infected and immunized FMD animals that is easy-to-use, economical, faster without sacrificing sensitivity and specificity of the test.

Nam *et al.* [36] developed a bio-barcode amplification (BCA) and used to measure respective protein and nucleic acid targets of different living organisms. In 2011, Ding *et al.* [35] developed an AuNP improved immuno-PCR for the detection of FMDV. The target particles were captured using a polyclonal antibody on a microplate followed by the addition of primers with AuNP and FMDV Mab 1D11 to form the sandwich complex. Then, immuno-complex will be formed and the signal DNA will be released by heating and then characterized by PCR and real time PCR. The developed FMDV BCA has a detection limit of 10 fg/ml purified FMDV particles and can detect clinical samples of FMDV with high sensitivity as compared to the traditional ELISA techniques with 100 ng/ml. Thus, FMDV AuNP BCA provided a detection test for FMDV with high sensitivity.

Bluetongue disease (BTD) is an arthropod-borne viral disease that affects ruminants worldwide. Bluetongue can cause massive socio-economic effects and is one of OIE listed diseases [36, 37]. Diagnosis of BTD includes viral isolation, serology and molecular diagnostics. In 2011, Yin *et al.* [37] developed a BCA BTD VP7 test. However, traditional BCA is time-consuming and complex. Thus, Yin *et al.* [38] improved their previous BCA BTD VP7 [36] test by incorporating gold nanoparticle probe to make the test easy and more sensitive to detect BTD VP7. Their platform

### *Application of Noble Metals in the Advances in Animal Disease Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.99162*

captures the protein VP7 using AuPr coated with the anti-VP7 polyclonal antibodies and single-stranded signal DNA. Then, magnetic microplate (MMP) probes coated with the anti-VP7 monoclonal antibodies were added to form the sandwich immunocomplex. Using PCR and real-time fluorescence PCR using Taqman probe, the singlestranded signal DNA in the immuno-complex can be detected. This technique has a detection limit of 10−2 fg/ml which is 8 orders of magnitude (100,000,000x) greater than conventional antigen capture ELISAs and 1 order (10x) than conventional BCA. The developed AuNP BCA test is a highly sensitive and an easier detection test for VP7 protein of bluetongue. Furthermore, this technique can be modified to measure the presence of other proteins.

Caprine arthritis encephalitis virus (CAEv) is one of the economically important diseases of goats that causes mostly polyarthritis in adults and progressive paresis (leukoencephalomyelitis) in kids. However, other clinical manifestations include interstitial pneumonia, mastitis and chronic wasting diseases that lead to eventual death of the animal. Detection of CAEv infection is mostly done through serological testing such as Agar Gel Immunodiffusion (AGID) and Enzyme-linked immunosorbent assay (ELISA) [39, 40]. However, application of polymerase chain reaction to detect CAEv became a routine assay due to its rapidity and ability to detect CAEv in early stage of the disease [41]. Furthermore, Huang *et al.* [42] and Balbin *et al.* [41] optimized loopmediated isothermal amplification (LAMP) to detect CAEv. Moreover, Balbin *et al.* [43] developed a LAMP test coupled with AuPr that can provide a specific colorometric detection CAEv. The specificity of this test was evaluated by subjecting other economically important small ruminant pathogens such as *Leptospira* spp., Bovine Leukemia Virus (BLV), *Trypanosoma evansi*, *Babesia* spp., *Anaplasma marginale,* and *Theileria* spp. The AuPr was not able to hybridize with the DNA amplification products of these pathogens, thus the designed oligonucleotide in the AuPr is only CAEv-specific. Furthermore, the result of AuPr colorimetric detection corroborated with the result of SYBR green and gel electrophoresis result of CAEv LAMP amplification.

Acute hepatopancreatic necrosis disease (AHPND) is one viral disease that causes devastating economic effects due to 100% mortality that occurs at 35 days after stocking of shrimp post-larvae in ponds [44–46]. De Guia *et al.* [12] developed a AuPr-based detection *pir*Avp toxin gene that causes AHPND without PCR amplification. The sensitivity of the developed test was as low as 20 fg/μl of extracted genomic DNA and positive samples had decreased absorbance value of 0.048 from 0.210 as compared to the negative controls with 0.137 absorbance value. Thus, most of the AuNPs aggregated due to the presence of *pir*Avp toxin in the samples. Furthermore, the sensitivity of this technique was tested with AHPND uninfected shrimp samples and non-vibrio DNA extracts of *Staphylococcus haemolyticus* isolate 1, *Staphylococcus haemolyticus* isolate 2, *Plesiomonas shigelloides, Staphylococcus arlettae, Edwardshiella tarda, Bacillus cereus* and *Citrobacter freundii.* The specificity and sensitivity of the test was conducted in 5 replications to assure the reliability of the test results. The positive result of the test will reveal a colorimetric change from pink red to purple, while negative will retain the pink red color.

## *2.3.1.3 Fungal diseases*

Epizootic ulcerative syndrome (EUS) also known as mycotic granulomatosis, red spot disease or ulcerative mycosis is an economically important disease of wild and cultured fresh-water and estuarine finfish species [45]. This disease is caused by a fungus, *Aphanomyces invadans*. The conventional detection method for the

disease includes culturing of causative agent, gross observation of clinical signs and symptoms and histopathology [45]. Molecular techniques such as PCR and fluorescent *in-situ* hybridization (FISH) have also been used for diagnosis of EUS [47, 48]. Furthermore, electrochemical DNA biosensors have been used to detect diseases for their relatively lower cost, higher sensitivity and specificity, portability, greater analyte discrimination, fast result and easy-to-use [49]. Thus, application of noble metal nanoparticle on these electrochemical DNA biosensors have been used to further improve disease diagnosis [47, 48]. Kuan *et al.* [49] developed an EUS electrochemical genosensors for the detection of *18S rRNA* and the internal transcribed spacer (IRS) of *A. invadans.* Kuan *et al.* research group described their platform as novel application for the detection of PCR product from real sample of *A. invadans* using a premix of sandwich hybridization assay. This assay was easier to use and more specific and sensitive compared to conventional techniques. The limit of detection of the EUSgenosensor was 0.5 fM (4.99 zmol) of linear DNA target and 1 fM (10 zmol) of PCR product. The developed EUS-genosensor will be highly suitable for surveillance and diagnostics of EUS in the aquaculture industry worldwide.

### *2.3.1.4 Parasitic diseases*

Visceral Leishmaniasis, caused by *Leishmania infantum* that is transmitted by sandflies, is a fatal zoonotic diseases of domesticated dogs, wild canids and humans [50, 51]. Canine Leishmaniasis (CanL) can be diagnosed through the use of parasitological [52, 53], serological [50] and molecular testing approaches [54–58]. However, the limitations of using this test to diagnose CanL are reported to be the need to skilled workers/laboratory staff, expensive and the need to send samples to reference laboratories [50]. Furthermore, a lateral flow assay (LFA) test for the detection of CanL, however, the detection limit is the drawback as it cannot detect low level of CanL antibody in the blood [59–61]. Molecular tool, PCR, have proven effective as it is considered as the confirmatory and gold standard test. However, molecular diagnostic tool has its drawbacks like the need of expensive and sophisticated equipment for the precise and repeated heating required for amplification [50]. Thus, de la Escosura-Muñiz *et al.* [50] developed a point-of-care test kit for the detection of CanL using primers labeled by AuNPs and magnetic beads (MBs) using isothermally amplified DNA products. This test kit successfully discriminated CanL infected blood from healthy dog's blood. Further qualitative studies revealed that less than 1 *Leishamania* parasite can be detected per microliter of blood (8 x 10−3 parasites per isothermal amplification reaction). The result of study of de la Escosura-Muñiz *et al.* [50] provided a pioneering approach to advance diagnostic testing in animals using noble metals as it makes diagnostics faster, economical and easy-to-use.
