Parasitic infections of small ruminants are one of the major concerns and cause significant losses to the livestock industry in India. Accurate diagnosis of the parasitic diseases are essential to study the epidemiology, parasite control strategies, which assists in the improving the productivity of sheep in world wide. However, current routine diagnostic techniques have limitations, in sensitivity and/or specificity, which hamper efforts to investigate the epidemiology of key species. Development of the drug resistance in parasites of sheep, is a major need for advanced methods of diagnosis. Diagnosis of parasitic diseases is regularly done by conventional methods, but DNA based approaches of diagnosis can overcome the disadvantages of previous methods. To assess the molecular and genetic host parasitic interaction different advanced molecular tools are required and it is useful in the diagnosis of parasitic diseases. Various molecular diagnostic techniques that have been developed for diagnosis of parasites include conventional PCR, RAPD-PCR, RFLP-PCR, multiplex-PCR, real-time PCR, reverse transcriptase PCR, Nested PCR, In Situ PCR, SSCP-PCR PCR-ELISA, RLB, micro-arrays, LAMP and microsatellite etc.
Diagnosis is the critical component in successful control of animal diseases. Diagnostic methods for the different parasitic diseases evolved from traditional detection of parasite by microscopy, to detect parasite antigens/antibodies by immunological methods and parasite DNA detection by molecular methods (Sivajothi et al., 2012; 2014). These developments have led to availability of highly specific, sensitive, simple and cost-effective tests for detection of parasites in infected animals, carriers and vectors. Validated tests however need to be made available in future for constructing more reliable epidemiological pictures of parasitic diseases of small ruminants for implementing control programmes more effectively (Comes et al., 1995; Sivajothi and Reddy, 2014). Haemoprotozoans were diagnosed by the based on the clinical symptoms, demonstrating the causative agent by wet blood examination, serological techniques (Sivajothi et al., 2013). Although, microscopy is still considered as a gold standard in the diagnosis of many parasitic diseases, it cannot be applied to all situations particularly where the diagnostic requirements demand defining the carrier status (Shahnawaz et al., 2012). Although, the use of various serological methods provide definite clues about the parasitic infection in general, but these tests have some limitations (Weiss, 1995; Sivajothi and Reddy, 2017). Several decades back, different types of DNA hybridization probes were developed which can be suites for development of the polymerase chain reaction diagnostic modalities. The ability of PCR to detect very small quantities of a target material and the absence of the need to use radioactive elements are two of the advantages of PCR compared with hybridization techniques (Ala and Wayne, 2005). However, more accurate identification of a PCR product may require the use of specific nucleic acid probes. But, it is not evident, with exception of RLB which is now being commercially produced, that the use of the technique will spread as a routine diagnostic tool in the laboratories. The use of molecular biology tools based on nucleic acid for tick-borne diseases will therefore, for sometime continue to be used in research activities rather than for day-to-day diagnosis in the laboratories (Dey and Singh, 2009). However, recombinant antigens based ELISAs may be available for routine diagnosis in the field.
Different Advanced Diagnostic Methods
First approach in the diagnosis of the parasitic diseases is by the detection of parasitic eggs in the feces and isolation of the parasite DNA. PCR and the sequencing is the main molecular approaches used for detection of genetic variation, identification of nematodes of livestock. Gene specific PCR primer pairs have been used to detect polymorphisms in the haemoprotozoans (Wimmer et al., 2004). Nuclear and mitochondrial genomes are important sources of DNA markers that have been used for nematode identification of species or strains. Mitochondrial DNA (mtDNA) evolves to determine the closely related nematodes. It has been successfully practiced for the identification of the diseases in interbreeding populations and cryptic species. DNA sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) or external transcribed spacer (ETS) of nuclear ribosomal DNA (rDNA) have been used extensively for nematodes identification (Amarante et al., 2014). The magnitude of sequence variation in both rDNA regions within a species is considerably less than the levels of sequence differences among species. It can be useful in determination of the nematode parasites including Haemonchus, Teladorsagia, Ostertagia, Trichostrongylus, Cooperia, Nematodirus, Bunostomum, Oesophagostomum and Chabertia (Gasser et al., 2008). The development of the multiplex PCR gave the opportunity to diagnose in a single reaction different nematode genera and/or species (Zarlenga et al., 2001). Real-time PCR assess the quantification of the DNA which indicates the proportion of each genus or species in given sample (Bott et al., 2009).
Tanaka et al. (1993) utilized a probe derived from a gene encoding a 32 kDa intra-erythrocytic piroplasm surface protein of Theileria sergenti (Theileria orientalis). It was sensitive to detect four parasites per microlitre of blood with a 10 µl sample. Sensitivity of the identification of the strains improved by using random amplified polymorphic deoxyribonucleic acid ‘DNA’ (RAPD). Moreover, several real-time PCR assay has been developed for diagnosis and quantitation of many tick bone parasites in recent years (Dong et al., 2013; Schotthoefer et al., 2013; Bloch et al., 2013). The sensitivity and specificity of molecular methods is very high and over the years a number of different approaches have been developed to detect Babesia spp. in the hosts and vectors. Deoxyribonucleic acid (DNA) probing was the first developed method, which was used to detect babesial DNA from parasitized blood (Buening et al., 1990). The level of sensitivity was high as the PCR product was detected in blood samples containing approximately 20 µl of packed cell with a parasitemia of 0.000001%.
PCR based on msp4 gene for A. marginale and Anaplasma ovis (de la Fuente et al., 2003) have also been developed. Sequencing of gltA and ompA genes, identification of Rickettsia species based on the sizes of highly variable intergenic spacers, namely, dksA-xerC, mppA-purC, and rpmE-tRNAfMet was carried out. Application of multiplex PCR for simultaneous amplification of 3 spacers combined with capillary electrophoresis separation technique is simple, accurate and high-throughput fragment sizing with considerable time and cost savings (Nakaoa et al., 2013). PCR base tests including PCR ELISA and duplex PCR have been developed and applied successfully with high sensitivity and specificity to differentiate tick borne haematozoan diseases (Torina et al., 2008; Ashuma et al., 2013; Sharma et al., 2013).
Random amplification of Polymorphic DNA-PCR also known as AP-PCR (arbitrary primed PCR), in this primers of arbitrary sequences obtained by end product, utilized further to amplify fragments of the genome. It is a very simple technique; routinely used to differentiate the species polymorphisms of Plasmodium, Trypanosoma and other haemo protozoans (Hajjaran et al., 2004).
PCR – RFLP
PCR – Restriction Fragment Length Polymorphism is used for diagnosis of species and genotypes of different parasites. In this, digestion of the PCR products obtained from parasitic gene amplification, by restriction enzymes. These enzymes cut DNA into fragments of certain sizes, whose analysis on agarose or polyacrylamide gel results in different patterns of fragment sizes, enabling the identification. Zaeemi et al. (2011) were able to differentiate among Theileria lestoquardi, T. ovis, and T. annulata in case of sheep. Recently, semi nested PCR-RFLP was used for detection of persistent anaplasmosis (Jaswal et al., 2014). The RFLP technique is currently one of the most commonly used molecular methods for diagnosis of species and genotypes of parasites such as Toxoplasma gondii (Quan et al., 2008). This technique was first used to detect variations at the DNA level (Carpentieri et al., 2008). It is recommended for the detection of the multiple genotypes in the given single sample.
It is to study the two or more target loci from the organisms are amplified using mixture of locus-specific primer pairs in a single reaction. It is recommended for the detection of deletions or duplications in a large gene of particular organisms (Markoulatos et al., 2002). Most commonly Multiplex PCR had been employed in detection of concurrent infections of economically important haemoprotozoans (Kaur et al., 2012).
Real-time PCR is one of the very simple and fast amplification system with less cross contamination. In this, no need to perform gel electrophoresis to visualize the PCR products (Mackay, 2004). This technique involves the analysis of genome using fluorogenic probes that release fluorescent signals during amplification. Real-time PCR has engendered wider acceptance of PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carryover contamination Jeong et al. (2003) applied real-time PCR for diagnosis and quantification of T. sergenti using specific primer for 33 kDa gene. A pan-Theileria FRET-qPCR can detect all recognized Theileria spp. of ruminants in a single reaction has also been developed (Yang et al., 2014).
In n-PCR, two separate amplifications are done. The first uses a set of primers that yields a large product, which is then used as a template for the second amplification. The second set of primers anneal to sequences within the initial product producing a second small product. The sensitivity and severity of amplifications are improved by n-PCR, because this eliminates non-specific amplification products (Chaisi et al., 2013).
In situ PCR
PCR reagents are placed on top of fixed and permeabilized tissue specimens or cells attached to the glass slide and PCR carried out in specialised thermal cycler block. This method combines the sensitivity of PCR with histological localization. This method also reduces the sample contamination. Following amplification, the labelled amplicon is detected with the cells by standard immune-cytochemical staining (Edwards and Gibbs, 1994).
Single Standard Conformation Polymorphism PCR (SSCP-PCR)
This is a simple and efficient method used to detect any small alteration in PCR-amplified product and is based on electrophoretic detection of conformational changes in single standard DNA molecules resulting from point mutations or other for rapid analysis of mutations. The PCR based SSCP can be employed for identifying parasites to species or strains where morphology is unreliable and has been used in identification of hookworms, Strongyloides, Schistosoma and Echinococcus species (Gasser, 2006).
In this, both the sensitivity of ELISA and the specificity of PCR are combined together for detection of parasitic genome. The PCR products are hybridized to an immobilized capture probe. It is used alternate to the real time PCR and useful for detecting and differentiating between multiple targets. This technique has been used in detection and quantification of Trypanosoma evansi in animals and vectors. The sensitivity limit of PCR-ELISA was 0.01 pg, which corresponded to one parasite/ml of blood. No cross reactivity of the assay was observed against Babesia spp., Theileria spp. and host DNA (Chansiri et al., 2002).
Two integrated approaches were developed to detect several Theileria or Babesia spp. in one assay (Allsop et al., 1993). Using these approaches, multiple species can be detected in one assay without performing independent PCR reactions for each parasite. One of such techniques, reverse line blot (RLB) hybridization, combines a genus specific PCR with hybridization to membrane bound type/species- specific oligonucleotide for differential detection. This technique can differentiate all known Theileria and Babesia spp. of importance in cattle in the sub-tropics on the basis of their differences in 18S subunit rRNA gene sequences (Gubbels et al., 1999). The specificity of the techniques result from the fact that amplified conserved domains of the 18 srRNA genes of the parasites are hybridized to species specific oligonucleotide immobilized on a solid membrane.
It is loop mediated isothermal amplification (LAMP) and it is a rapid, simple and sensitive technique (Notomi et al., 2000). This is a novel strategy for gene amplification which relies on the auto-cycling strand displacement synthesis of target deoxyribonucleic acid (DNA) by Bst DNA polymerase under isothermal conditions. Further improvement of the technique has been achieved by the use of additional loop primers, which increased its efficiency and rapidity (Nagamine et al., 2002). The LAMP technique allows visual detection of amplified products through the addition of fluorescent dyes such as SYBR Green (Poon et al., 2006) and measurement of turbidity. Unlike PCR, LAMP is carried out at a temperature range of 60 to 65°C eliminating the need of a thermal cycler. In addition, the reaction can be carried out without the need of DNA extraction. The method has been successfully developed for the detection of several TBDs (Salih et al., 2012). Recently, parasitologists have adapted the LAMP technique to detect several parasitic diseases, including the human parasites Cryptosporidium, Entamoeba histolytica, Plasmodium, Trypanosoma, Taenia, Schistosoma, Fasciola hepatica and Fasciola gigantica, and Toxoplasma gondii, and animal parasites such as Theileria and Babesia. Also, this technique could detect the miracidium after the first day of exposure in snails, the intermediate hosts of Schistosoma (Kumagai et al., 2010). Nkouawa et al. (2010) compared LAMP with multiplex PCR in the differential detection of Taenia in stool samples from patients with taeniasis. LAMP, with no false positives, showed greater sensitivity (88.4%) than multiplex PCR (37.2%), demonstrating a high value in detecting molecular taeniasis. Ai et al. (2010) performed DNA analysis of Fasciola sp. in mollusks that are intermediate hosts and in stool samples. The results indicated that the LAMP assay is approximately ten times more sensitive than conventional specific methods such as PCR. These findings suggest that the LAMP method for specific species may have a potential clinical application for detection and differentiation of Fasciola species, especially in endemic countries.
It is one of the costly procedures commonly known as gene chip, DNA chip, or biochip and it was developed for mapping of genes to detect a wide variety of pathogens through multi-gene detection. It consists of glass slide or silicon chip or nylon membrane, onto which the nucleic acid sequences from thousands of different genes are attached at fixed locations (Seitzer et al., 2007). It combines the DNA amplification with subsequent hybridization to oligonucleotide probes specific for multiple target sequences. It allows analysis of a larger number of genetic features in a single trial. It has been used in detection and genotyping of Plasmodium, Toxoplasma, and Trypanosoma (Duncan, 2004).
Microsatellites are the short DNA sequences which consist of tandem repeats of one to six nucleotides, with approximately one hundred repeats.
Table 1: The advances of the methodology over time in diagnosis of parasitic diseases
|Methodology||Source of parasitic DNA for diagnosis||Species diagnosed||Comments||Reference|
|PCR||eggs or larvae||Ost, Coop, Nema, Haem, Tricho||Single eggs or larvae could be differentiated to genus level without previous DNA extraction||Schnieder et al. (1999)|
|PCR-based assay using species-specific primer pairs||eggs and cultured larvae||Bt, Co, Df, Nb, Nf, Ta, Tc, Tv, To||Qualitative||Wimmer et al. (2004)|
|Multiplex PCR test||adult worm||Cc||Qualitative||Amarante et al. (2014)|
|Real-time PCR (RT PCR)||Eggs||Oo, Hp, Or, Coo, Tc||Qualitative||Zarlenga et al. (2001)
|RT PCR and meltingcurve analysis||first stage larvae derived from overnight cultures||Hc, Ol, Tcol, Cc||Quantitative assays based on genus-specific primer and probe combinations||Samson-Himmelstjerna et al. (2002)|
|Semi-automated, multiplexed-tandem PCR platform||Eggs
|Hc, Tc, Tricho spp., Coo, Oc, Ov, Co
Hc, Tc, Tricho spp., Co, Ov
|Semi-quantitation of parasite DNA in faeces
Estimate the proportion of eggs of the different species/genera in a sample
|Roeber et al. (2012)|
|RT PCR||Eggs from crude faecal egg preparations||Trichostrongylids||For identification of species or resistance alleles, using different post PCR methods||Blouin et al. (1998)|
|Loop-mediated isothermal amplification||Amplification from relatively crude samples||Hc||Allows detection of Haemonchus in a faecal samples containing two eggs per gram||Marra et al. (2010)|
|A closed-tube RT PCR||L3 larvae||Hc, Tc, Tcol, Ns, Ov, Ta, Tv, Cc, Co||Identification of individual strongylid nematode larvae||Marra et al. (2010)|
Bt- Bunostumum trigonocephalum; Cc- Cooperia curticei; Co- Chabertia ovina; Coo- C. oncophora;
Df- Dictyocaulus filiaria; Hc- Haemonchus contortus; Hp- H. placei; Nb- Nematodirus battus;
Nf- Nemtodirus filicollis; Ns- Nematodirus spathiger; Oo- O. ostertagi; Oc- Oesophagostomum columbianum; Or- O. radiatum; Ov- Oesophagostomum venulosum; Ol- Ostertagia leptospicularis; Ta- Trichostrongylus axei; Tc- Teladorsagia circumcincta; Tcol- Trichostrongylus colubriformis; Tv- Trichostrongylus vitrinus; To- Trichuris ovis.
Microsatellites are abundant in eukaryotic genomes and can mutate rapidly by loss or gain of repeat units. These are utilized because of the frequent polymorphism, codominant inheritance, high reproducibility, high resolution of the genes. Due to presence of the technical difficulties by PCR, it was utilised for the very few parasites. Recently its use on diagnosis of Trichostrongyloid parasites had been reported (Temperley et al., 2009).
The ultimate in diagnosis has been the development of robotic platforms that make possible separation of eggs from feces, obtaining good quality DNA from eggs for amplification, and finally, produce a result indicating the degree of the infection by the different parasite species that commonly cause mix infection.