Application of Molecular and Serological Diagnostics in Veterinary Parasitology
Dr Shraddha Sinha
Department of Veterinary Medicine,
College of Veterinary Science and Animal Husbandry Anjora, Durg
Collection and Submission of Samples Diagnosis of parasitic infections depends on several factors, such as collection of the sample, transport of the sample to the laboratory, and method of laboratory evaluation. Diagnostic stages of most parasites can be detected in faeces, blood, sputum, or skin scrapings. However, infections of immature parasites and latent and occult infections present a diagnostic challenge.
Important factors to be considered in the diagnosis of parasitism and the interpretation of results are:
- Age of the host,
- Previous exposure to parasites (resistance),
- Time of the year (spring rise),
- Physiological relationship (peri parturient rise),
- Geographical location,
- Previous use of anthelmintics,
- History of clinical disease,
- and other considerations.
Proper collection and submission of samples to the laboratory increase the accurate diagnosis of parasitic infection.
Faecal Samples-
Faeces must be fresh for accurate results.
As faeces age, a diagnosis is complicated because many parasite eggs develop and hatch into larvae. Contaminants such as free-living soil nematodes, fly larvae, mites, and other arthropods often invade faeces, and complicate a diagnosis.
At least 10 gm of fresh faeces should he collected. If samples are more than two hours old, samples should be stored at 4°C until examined. Many parasite stages can be stored at 4°C for at least two months with minimal development. For routine shipment to the laboratory, samples can he cooled to 4°C then packed with ice or other coolant (blue ice) for shipment via any of the 24- to 48-hour transport services. Faecal samples are best stored and sent whirl-pack hags, plastic, sandwich bags, plastic containers, disposal laboratory gloves turned inside out, or rectal palpation gloves turned inside out. All samples should be clearly labelled with black indelible marker with the number of the animal, date, and the person responsible for the sample.
If coolants are not available, samples can be stored indefinitely in 1O% formalin (one part faeces, nine parts 10% formalin), but limitations must be noted. Approximately 50% of the ruminant strongyle eggs were detected in faeces stored in 10% formalin for 200 days. Storage by freezing is very inefficient, and storage in 70% ethyl alcohol or 100% methyl alcohol is unacceptable.
When Giardia sp. is suspected, faeces can he placed in polyvinyl alcohol fixative at a ratio of one part faeces to two parts polyvinyl alcohol or in 5% formalin for fixation and transport. Also, faecal smears on slides can be stained with Gormori’s trichrome, iron-hematoxylin, clorzol black, or Giemsa stains and submitted to the laboratory in standard slide mailers.
NUCLEIC ACID BASED ASSAYS
The routine laboratory diagnosis for veterinary parasites involves conventional methods, such as optical microscopy for morphological identification of parasites. However, the conventional methods are not much reliable and have reduced sensitivity and specificity. Moreover, these methods are sometimes not applicable to micro parasites. Therefore, with the advancements in molecular biology techniques, the molecular methods are now more indicated. Several molecular based laboratory techniques such as polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), loop-mediated isothermal amplification (LAMP), Luminex x MAP-based technology and real-time PCR are used for identification of several parasitic species.
Polymerase Chain Reaction (PCR)
PCR utilizes the power of DNA polymerase enzyme to amplify a small number of targeted DNA using specific primers. It can selectively amplify the targeted sequence from a mixture of genomes. PCR acts as a major tool for identification of parasites because it is generally difficult to obtain sufficient amount of parasitic material for conventional assays.
PCR can utilize all biological samples such as faeces, blood, skin scrapings, urine and meat for parasitic identification. The detection limit of PCR is higher than conventional method (light microscopy). Therefore, it is useful for detecting low number of parasites in faecal samples. It can also be used for detection of non-intestinal parasites such as Leishmania and Plasmodium.
In a recent study, Toxoplasma gondii parasite has been genetically characterized using PCR based molecular techniques. The seven different species of Eimeria (E. acervulina, E. brunetti, E. tenella, E. maxima, E. necatrix, E. mitis and E. Praecox) have been detected using PCR from Egyptian baladi chickens. PCR using cytochrome c oxidase subunit 1 (cox1) gene- based identification and subsequently intra-specific variation study was done for Habronema muscae in horses.
PCR can also be combined with other molecular methods such as RFLP (restriction fragment length polymorphism) or nested PCR to genotype the organisms.
In a recent study PCR-RFLP assay was successfully used for identification of Toxoplasma gondii and differentiation of Fasciola hepatica from Fasciola gigantica. PCR can be modified to multiplex-PCR to detect several pathogens in a single reaction.
Although PCR is much sensitive and specific than conventional methods, it has limitation of being time consuming. It also needs equipment (thermal cycler) and gives qualitative results. Moreover, this technique is still expensive in several countries. In veterinary parasitology, this technique is more used in research purpose.
Real-Time Polymerase Chain Reaction (Real time PCR or qPCR)
This technique enables the monitoring of PCR amplification in real time along with specificity and sensitivity of original PCR assay.
The Real-time PCR assay provides quantification of the sample using several fluorescent agents such as TaqMan probes, SYBR Green dye and Scorpion primers.
The parasitic nucleic acids from various biological and environmental samples can be quantified to provide information about the intensity of infection.
The SYBR Green dye based Real-time PCR assays have been validated for several parasitic species such as Cryptosporidium, Trypanosoma, Leishmania and Mecistocirrus digitatus.
The gastrointestinal infection of nematode in small ruminants was successfully detected using Real-time PCR. A Real-time PCR assay has been developed for Toxoplasma gondii quantification.
Real-time PCR assay can easily detect differential gene expression due to different strains of parasites in tissues
The Real-Time PCR is rapid, specific, sensitive and quantitative technique to detect parasite in clinical samples.
Different studies of leishmaniosis such as animal models, vectorial capacity, diagnosis, drug efficacy have been investigated using Real-time PCR
A new version of real-time PCR i.e. multiplex real-time PCR was used for identification of Sarcocystis spp. in cattle in a single reaction
The Real-time PCR amplification protocols, DNA extraction, choice of primer sets may cause diversification in results and lead to difficult in standardization of assay
Loop-Mediated Isothermal Amplification (LAMP)
LAMP is an extremely sensitive and specific method of nucleic acid amplification which can discriminate single nucleotide differences. In LAMP assay, six different sequences of target genes are identified by a set of four primers.
The DNA polymerase used (Bst DNA polymerase) in LAMP assay can work under isothermal condition with high specificity and low sensitivity to inhibitors in sample.
It amplifies the DNA only when all primers bind to targeted DNA.
The results can be seen directly by adding SYBR green, HNB or Calcein dye. Therefore, electrophoresis is not required which also reduces the time for interpretation of result.
This technique can synthesize 20 μg of DNA from 25 μL of reaction volume within one hour of time. Moreover, a novel OmniAmp DNA polymerase has been validated for LAMP assay of DNA and RNA both.
Earlier, this technique was used for diagnosis of many humans and animal viral and bacterial pathogens. However, now this method is also validated for many parasites such as Plasmodium spp., Cryptosporidium spp., Taenia spp., Trypanosoma spp., Fasciola hepatica, Schistosoma spp., Toxoplasma gondii, Theileria spp., Echinococcus spp and Babesia spp.
It can also be used for identification of parasites in their vectors.
Plasmodium spp. and Dirofilaria immitis have been detected in mosquitos using this technique.
Also, it could detect the parasites in intermediate host of Schistosoma i.e., miracidium can be detected after first day of snails’ exposure.
In a recent study, LAMP assay was used for diagnosis of seven chicken Eimeria species. The LAMP assay is ten times more sensitive than conventional PCR in identification of Fasciola spp. in snails and stool samples.
The multiplex LAMP (mLAMP) assay was standardized for simultaneous identification of bovine protozoan parasites i.e., Babesia bovis and Babesia bigemina.
Although LAMP is simple and low cost DNA amplification technique, it has some limitations. The LAMP assay product is not easily degraded hence, chance of carry over contamination may exist.
It is also affected by the amplification time limit. The LAMP products usually take 60 minute to 120 minute for amplification and negative control may get amplified at 180 minute.
LAMP is also less versatile than PCR because it is not useful for cloning reactions.
Luminex
It is a bead-based xMAP (multianalyte profiling) technology which combines fluorescent microspheres (beads), flow cytometry, lasers and digital signal processing technology.
It has the capability of simultaneous analysis of 100 different analytes in a single tube.
The 100 distinct sets of Luminex tiny beads (microspheres) coated with specific reagent of particular bioassay allow the detection of specific analytes in a sample.
In Luminex compact analyser, lasers excite the internal dyes and identify each microsphere beads.
Random Amplified Polymorphic DNA (RAPD)
It is a PCR reaction which amplifies random segments of DNA.
It uses several arbitrary, short primers of 8-12 nucleotides in length and a large template of DNA for PCR amplification. By resolving the resulting amplification patterns on agarose gel electrophoresis, a semi unique profile can be visualized from a RAPD reaction.
Several studies on nematodes of human and plant origin have showed the high efficiency of RAPD marker in identification and differentiation of parasites at species level
RAPD is also useful for study of genetic differences in population of microorganism because it reveals polymorphism in non-coding region of genome.
RAPD is increasingly used in parasitology due to its fast, simple and inexpensive nature since it does not require prior knowledge of the DNA sequence or DNA hybridization
Amplified Fragment Length Polymorphism (AFLP)
It is a technique which allows detection of DNA polymorphism without the knowledge of actual DNA sequence. This technique utilizes the PCR to selectively amplify the restriction fragments of digested genomic DNA.
The entire analysis completes in four steps: restriction endonuclease digestion of DNA, ligation, amplification and polyacrylamide gel analysis.
The presence or not of DNA fragments in gel reveals the polymorphisms.
This method is highly advantageous because of its reproducibility, its ability to search entire genome for polymorphisms and possibility of being used against parasites without any prior genetic information.
However, AFLP has some of the limitations such as it is difficult to develop locus specific markers from individual genome fragments of a species.
Probe Hybridization
The hybridization probe is a 100-1000 bases long fragment of DNA or RNA used for detection of presence of nucleotide sequences complementary to probe sequence.
The probe hybridizes to single-stranded nucleic acid sequence. The nucleotide sequence of probe allows probe-target base pairing due to nucleotide base complementarity between the target and probe. The labelled probe is hybridized to the target RNA (Northern blotting) or ssDNA (Southern blotting) immobilized on a membrane or hybridised in situ.
To detect hybridization, probe is tagged with a molecular marker of either radioactive (P32, I125 etc.) molecules or non-radioactive fluorescent molecules (Digoxigenin).
The probe hybridization-based assays have been used for diagnosis of animal parasites.
The probe hybridization assay is relatively easy to perform. However, it has creation limitations such as it is difficult to differentiate between species when using 16S rRNA sequences due to similarity.
Moreover, nucleic acid sequence data base is constantly becoming larger thus the possibility of a random hybridization between a specifically designed probe and an unknown target organism cannot be ruled out.
Restriction Fragment Length Polymorphism (RFLP)
This technique utilizes the specific restriction enzyme to digest the genomic DNA sequence in small fragments followed by separation on gel electrophoresis.
The gel electrophoresis showed different patterns of nucleic acid fragment which help in identification of parasite species or genotype. It allows the detection of multiple genotypes in a single reaction. The RFLP is a widely used technique for Toxoplasma gondii genotyping.
Despite its many benefits, the RFLP analysis is slow and tedious process than the newer DNA analysis techniques. Moreover, it requires relatively larger sample size than other forms of nucleic acid analysis techniques.
Microarray
The nucleic acid microarray is a collection of microscopic nucleic acid spots attached to a solid glass surface (microscope slide).
Each spot consists of picomoles of specific nucleic acid sequence called probes (Bumgarner, 2013). The probes are allowed to hybridize labelled target nucleic acid i.e., cDNA or cRNA (anti-sense RNA). Probe-target hybridization can be detected and quantified using silver, fluorophore or chemiluminescence-labelled targets.
This technique is used to measure expression levels of several genes of same or different species simultaneously. Microarray has also been used for diagnosis of animal parasites.
GENOME SEQUENCE STUDY OF ANIMAL PARASITES
The veterinary parasitology has utilized the traditional immunological and empirical drug screening methods for development of vaccines, diagnostic tools and drugs. However, these conventional approaches have some limitations such as inadequate efficacy of vaccines, cross reactive of parasitic diagnosis methods and drug resistance of parasites. The drug resistance against many well-known antiparasitic drugs threatened the livestock production in several parts of the world.
In the last few years, the gene sequencing and complete genome sequencing is becoming popular in identifying the new pathogens and its potential target genes.
In complete genome sequencing the whole genomic DNA is cut in smaller fragments, sequence the fragments individually and then assemble the fragmented sequence to get whole genome sequences.
The complete genome sequence study of animal parasites is a need for modern nucleic acid based diagnostic assays. However, complete genome sequence study of only few animal parasites have been studied so far due to its costly nature and technical expertise required.
PROTEIN BASED ASSAYS
These assays are categorised into antibody and antigen detection assays. There are several protein-based assays such as enzyme-linked immunosorbent assay (ELISA), dot-ELISA, Falcon Assay Screening Test ELISA (FAST-ELISA), indirect or direct immunofluorescent antibody (IFA or DFA) tests, immunoblotting, complement fixation test (CFT) are used for parasitic disease diagnosis. These tests are more sensitive and specific than microscopy and allow post-therapeutic parasitic clearance.
Enzyme-linked immunosorbent assay (ELISA)
ELISA test uses components of the immune system to detect immune responses against pathogens. The ELISA test involves antigen, antibody and an enzyme to detect specific immune response. The antigens are attached to polystyrene microtiter plate surface and a specific antibody is applied over the surface to bind the antigen. The antibody is conjugated with an enzyme (HRPO- Horseradish peroxidase). Finally, a substrate is added to produce a detectable colour change in reaction mixture. Based on use or not of a secondary antibody, the ELISA test may be either direct or indirect. This test is successfully used for diagnosis of several parasitic diseases in both humans and animals.
There are several types of ELISA test such as dot-ELISA, FAST-ELISA etc, which are increasingly in use in veterinary parasitology.
Dot-ELISA
In dot-ELISA, a nitrocellulose membrane is used for attachment of small amount of antigen. The dotted membrane is incubated in antigen specific antibody followed by enzyme conjugated anti-antibody. Finally, a chromogenic substrate is added which causes precipitation of a visible coloured dot on the membrane. This technique is fast cost-effective and easily interpretive.
Falcon Assay Screening Test-ELISA (FASTELISA) FAST-
ELISA uses synthetic and recombinant peptides to detect antibody responses to antigens. This method was used for diagnosis of fasciolosis, schistosomiasis and taeniasis.
Luciferase Immunoprecipitation System (LIPS)
It is a modified ELISA-based assay. The specific antibodies in serum are identified by light production. The antigen of choice is fused with an enzyme reporter Renilla luciferase (Ruc) which is expressed as a Ruc-fusion in mammalian cells. The Ruc-fusion protein extract is incubated with protein A/G beads and test serum. The Ruc-antigen fusion along with antibody complex immobilizes on the A/G beads. The antibody can be quantitated by measurement of light production after addition of coelenterazine substrate
This method is rapid and accurate in detection of pathogen. It produces low backgrounds as compared to ELISA which enhances the accuracy of separation between positive and negative samples (increases sensitivity and specificity). The LIPS assay has been successfully used for diagnosis of Strongyloides stercoralis using recombinant antigen from infected sera sample.
Radioimmuno- Assay (RIA)
RIA is used for detection and quantitation of antigen in a sample using specific antibodies. It is very sensitive and specific. It can detect as low as few picograms of antigen using antibodies of high affinity. This assay is based on competitive binding, where a radioactive antigen competes with a non-radioactive antigen for a fixed number binding sites on antibody.
Fluorescent Antibody Test (FAT)
In FAT, antibody is tagged by fluorescent dye which is used for visualization of antigen in clinical specimens. The immunoglobulin conjugated with fluorescent dye in antigen-antibody complex produce visible glow when examined under a fluorescent microscope. The fluorescent dye can be tagged directly with primary antibody (direct fluorescent antibody test) or with a secondary anti-antibody (Indirect Fluorescent Antibody Test). The FAT is used in diagnosis of several parasitic diseases.
Complement Fixation Test (CFT)
CFT is an immunological test used for detection of presence of either antigen or antibody in serum sample. It was used mostly for microorganisms which are not easily cultured in laboratory. Although, the critical study revealed its poor sensitivity and specificity in detection of parasites, it is still used for parasitic disease diagnosis. The CFT was found reproducible and reliable assay for Trypanosoma equiperdum sera sample
This test is used to confirm the clinical evidence and to detect latent infection of Trypanosoma equiperdum.
Therefore, it is used as recommended test by OIE (OIE, 2013). However, the specificity of this test was found less in Trypanosoma evansi diagnosis. The IFAT was also found more sensitive than CFT for diagnosis of equine piroplasmosis caused by Babesia caballi and Theileria equi. The CFT has some of the critical limitations such as time consuming, labour intensive and often non-specific (cross-reactivity) in nature.
