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Molecular Diagnosis of Parasitic Diseases in Sheep – A Review

Sirigireddy Sivajothi Bhavanam Sudhakara Reddy
Vol 8(2), 14-24
DOI- http://dx.doi.org/10.5455/ijlr.20170922043628

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.


Keywords : Advanced Molecular Diagnosis Sheep Parasitic Diseases

Introduction

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

PCR

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).

RAPD-PCR 

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.

Multiplex PCR

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

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).

Nested PCR  

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).

PCR-ELISA

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).

RLB

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.

 

LAMP

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.

DNA Microarrays

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

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

Undeveloped 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).

Conclusion

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.

References

  1. Ai L, Li C, Elsheikha HM, Hong SJ, Chen JX, Chen SH (2010) Rapid identification and differentiation of Fasciola hepatica and Fasciola gigantica by a loopmediated isothermal amplification (LAMP) assay. Vet Parasitol 174(3-4): 228-33.
  2. Ala L, Wayne J (2005) Molecular approaches to detect and study the organisms causing bovine tick borne diseases: babesiosis and anaplasmosis. Afr. J. Biotechnol 4: 292-302.
  3. Allsop BA, Baylis HA, Allsop MT, Cavaher-smith T, Bishop RP, Camngton DM, Sohanpal B, Spooner P (1993) Discrimination between six species of Theileria using oligonucleotide probes which detected small subunit ribosomal RNA sequences. Parasitology 107:157-165.
  4. Amarante MRV, Bassetto CC, Neves JH, Amarante AFT (2014) Species-specific PCR for the identification of Cooperia curticei (Nematoda: Trichostrongylidae) in sheep. Journal of Helminthology 88 (4): 447-452.
  5. Ashuma, Sharma A, Singla LD, Kaur P, Bal MS, Batth BK, Juyal PD (2013) Prevalence and haemato-biochemical profile of Anaplasma marginale infection in dairy animals of Punjab (India). Asian Pac. J. Med. 6:139-144.
  6. Bloch EM, Lee TH, Krause PJ, Telford SR, Montalvo L, Chafets D, Usmani-Brown S, Lepore TJ, Busch MP (2013) Development of a real-time polymerase chain reaction assay for sensitive detection and quantitation of Babesia microti Transfusion 53: 2299-2306.
  7. Blouin MS, YowelL CA, Courtney CH, Dame JB (1998) Substitution bias, rapid saturation, and the use of mtDNA for nematode systematic. Molecular Biology and Evolution 15 (12): 1719-1727
  8. Bott NJ, Campbell BE, Beveridge I, Chilton NB, Rees D, Hunt PW, Gasser RB (2009) Acombined microscopic-molecular method for the diagnosis of strongylid infections in sheep. International Journal for Parasitology 39(11): 1277-1287
  9. Buening G M, Barbet A, Myler P, Mahan S, Nene V, McGuire TC (1990) Characterisation of a repetitive DNA probe for Babesia bigemina. Vet. 36:11-20.
  10. Carpentieri-Pipolo V, Gallo-Meagher M, Dickson DW, Gorbet DW, Mendes ML, de Souza SGH (2008) Comparacao entre metodos de marcacao da sonda de RFLP R2430E utilizada na selecao de cultivares de amendoim resistente a Meloidogyne arenaria. Cienc Agrar 29(4):783-8.
  11. Chaisi ME, Janssens ME, Vermeiren L, Oosthuizen MC, Collins NE, Geysen D (2013) Evaluation of a real-time PCR Test for the detection and discrimination of Theileria Species in the African Buffalo (Syncerus caffer). PLoS One 17:e75827.
  12. Chansiri K, Khuchareontaworn S and Sarataphan, 2002. PCR-ELISA for diagnosis of Trypanosoma evansi in animals and vector. Mol Cell Probes 16: 173-177.
  13. Comes AM, Humbert JF, Carbaret J, Elard L (1995) Using molecular tools for diagnosis in veterinary parasitology. Vet. Parasitol. 27:333342.
  14. de la Fuente  J,  Van  Den  Bussche RA,  Prado T,  Kocan KM  (2003) Anaplasma marginale major surface protein 1a genotypes evolved under positive selection pressure but are not markers for geographic strains. J. Clin. Microbiol. 41:1609-1616.
  15. Dey A, Singh S (2009) Progress of science from microscopy to microarrays (Part 1): Diagnosis of parasitic diseases. J. Lab. Phys. 1: 2-6.
  16. Dong T, Qu Z, Zhang L (2013) Detection of phagocytophilum and E. chaffeensis in patient and mouse blood and ticks by a duplex realtime PCR assay. PLoS One 8: e74796.
  17. Duncan R (2004) DNA microarray analysis of protozoan parasite gene expression: outcomes correlate with mechanisms of regulation. Trends Parasitol. 20:211-216.
  18. Edwards MC, Gibbs RA (1994) Multiplex PCR: advantages, development, and applications. PCR Methods Appl. 3:S65-S75.
  19. Gasser RB. (2006) Molecular tools – advances, opportunities and prospects. Vet. Parasitol. 136(2):69-89.
  20. Gasser RB, Bott, NJ, Chilton NB, Hunt P, Beveridge I, Toward practical DNA-based diagnostic methods for parasitic nematodes of livestock — Bionomic and biotechnological implications. Biotechnology Advances, 26 (4), 325-334.
  21. Gubbels JM, de Vos AP, van der Weide M, Viseras J, Schouls LM, de Vries E, Jongejan F (1999) Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridization. J. Clin. Microbiol. 37:1782-1789.
  22. Hajjaran H, Mohebali M, Razavi MR, Rezaei S, Kazemi B, Edrissian GH (2004) Identification of Leishmania species isolated from human cutaneous leishmaniasis using Random Amplified Polymorphic DNA (RAPD-PCR). Iranian J. Public Health 33:8-15.
  23. Jaswal H, Bal MS, Singla LD, Amrita, Kaur P, Mukhopadhyay, Juyal PD (2014) Application of msp1β PCR and 16S rRNA semi nested PCRRFLP for detection of persistent anaplasmosis in tick infested cattle. Int. J. Adv. Res. 2:188-196.
  24. Jeong W, Hweon CH, Kang SW, Paik SG (2003) Diagnosis and quantification of Theileria sergenti using TaqMan PCR. Vet. Parasitol. 111:287-295.
  25. Johnson PC, Webster LM, Adam A, Buckland R, Dawson DA, Keller LF (2006)Abundant variation in microsatellites of the parasitic nematode Trichostrongylus tenuis and linkage to a tandem repeat. Mol Biochem Parasitol 148(2): 210-8.
  26. Kaur P, Sharma A, Singla LD, Juyal PD (2012) Molecular detection of anaplasmosis and babesiosis by duplex PCR in cattle. Crop Improv. 1395-1396.
  27. Kumagai T, Furushima-Shimogawara R, Ohmae H, Wang TP, Lu S, Chen R (2010) Detection of early and single infections of Schistosoma japonicum in the intermediate host snail, Oncomelania hupensis, by PCR and loop-mediated isothermal amplification (LAMP) assay. Am J Trop Med Hyg 83(3):542-8.
  28. Mackay IM (2004) Real time PCR in the microbiology laboratory. Clin. Microbiol. Infect. Dis. 10:190-212.
  29. Markoulatos P, Siafakas N, Moncany M (2002) Multiplex polymerase chain reaction: a practical approach. J. Clin. Anim. 16:47-51.
  30. Marra NM, Chiuso-MinicuccI F, Machado GC, Zorzella-pezavento SFG, França TGD, Ishikawa LLW, Amarante AFT, Sartori A, Amarante MRV (2010) Faecal examination and PCR to detect Strongyloides venezuelensis in experimentally infected Lewis rats. Memórias do Instituto Oswaldo Cruz, 105 (1): 57-61
  31. Nagamine K, Hase T, Notomi T (2002) Accelerated reaction by loop mediated isothermal amplification using loop primers. Mol. Cell. Probes 16:223-229
  32. Nakaoa R, Qiu Y, Igarashi M, Magona JW, Zhou L, Ito K, Sugimoto C (2013) High prevalence of spotted fever group rickettsiae in Amblyomma variegatum from Uganda and their identification using sizes of intergenic spacers. Ticks Tick-borne Dis. 4:506-512.
  33. Nkouawa A, Sako Y, Li T, Chen X, Wandra T, Swastika IK, Nakao M (2010) Evaluation of a loopmediated isothermal amplification method using fecal specimens for differential detection of Taenia species from humans. J Clin Microbiol 48(9):3350-2.
  34. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28:E63.
  35. Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W, Tangpukdee N, Yuen KY, Guan Y, Looareesuwan S, Peiris JS (2006) Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heattreated blood by loop-mediated isothermal amplification. Clin. Chem. 52:303-306.
  36. Quan JH, Kim TY, Choi IU, Lee YH (2008) Genotyping of a Korean isolate of Toxoplasma gondii by multilocus PCR-RFLP and microsatellite analysis. Korean J Parasitol. 46(2):105-8.
  37. Roeber F, Jex AR, Campbell AJD, Nielsen R, Anderson GA, Stanley KK, Gasser RB (2012) Establishment of a robotic, high-throughput platform for the specific diagnosis of gastrointestinal nematode infections in sheep. International Journal for Parasitology, 42(13-14): 1151-1158.
  38. Salih DA, Ali, AM, Liu Z, Bakheit MA, Taha KM, EL Imam AH, Kullmann B, El Hussein AM, Ahmed JS, Seitzer U (2012) Development of a loop-mediated isothermal amplification method for detection of Theileria lestoquardi. Parasitol. Res. 110: 533-538.
  39. Samson-Himmelstjerna GA, von Harder A, Schnieder T (2002) Quantitative analysis of ITS2 sequences in trichostrongyle parasites. International Journal for Parasitology, 32 (12) : 1529-1535.
  40. Schnieder T, Heise M, Epe C (1999) Genus-specific PCR for the differentiation of eggs or larvae from gastrointestinal nematodes of ruminants. Parasitology Research, 85 (11):  895-898.
  41. Schotthoefer AM, Meece JK, Ivacic LC, Bertz PD, Zhang K, Weiler T, Uphoff TS, Fritsche TR (2013) Comparison of a real-time PCR method with serology and blood smear analysis for diagnosis of human anaplasmosis: importance of infection time course for optimal test utilization. J. Clin. Microbiol. 51:2147-2153.
  42. Seitzer U, Bakheit MA, Salih DA, Ali A, Haller D, Yin H, Schnittger L, Ahmed JS (2007) From molecule to diagnostic tool: Theileria annulata surface protein TaSP. Parasitol. Res. 101:S217–S223.
  43. Shahnawaz M, Shahardar RA, Wani ZA, Bhat SA, Shah SN (2012) Prevalence and Seasonal Variation of Ovine Platyhelminth Parasitism in Ganderbal Area of Kashmir Valley, International Journal of Livestock Research 2(1): 184-192.
  44. Sharma A, Singla LD, Tuli A, Kaur P, Batth BK, Javed M, Juyal PD (2013) Molecular prevalence of Babesia bigemina and Trypanosoma evansi in dairy animals from Punjab, India by duplex PCR: A step forward to detection and management of concurrent latent infections. Biomed Res. Int. Article ID 893862:8 p
  45. Sivajothi S, Rayulu VC, Malakondaiah P, Sreenivasulu D (2013) Colloidal dye immunobinding assay for detection of Trypanosoma evansi antibodies in animals. Intern J Livest Res 3(3):48–56.
  46. Sivajothi S, Rayulu VC, Malakondaiah P, Sreenivasulu D, Reddy BS (2014) Detection of antibodies against Trypanosoma evansi in sheep by indirect ELISA in Rayalaseema region of Andhra Pradesh. J Adv Vet Res 4: 88-92.
  47. Sivajothi S, Rayulu VC, Reddy BS (2012) Development of slide enzyme linked immunosorbent assay (SELISA) for detection of Trypanosoma evansi infection in bovines. J Adv Vet Res 2:15–17.
  48. Sivajothi S, Reddy BS (2014) Immature paramphistomosis in a sheep herd. Int J Biol Res 2: 140-142.
  49. Sivajothi S, Sudhakara Reddy B (2017) Therapeutic Efficacy of Closantel Against Different Gastrointestinal Parasites in Sheep. Arch Parasitol 1: 111.
  50. Tanaka M, Onoe S, Matsuba T, Katayama S, Yamanaka M, Yonemichi H, Hiramatsu K, Baeck B, Sugimoto C, Onuma M (1993) Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA. J. Clin. 31:25652569.
  51. Temperley ND, Webster LM, Adam A, Keller LF, Johnson PC (2009) Cross-species utility of microsatellite markers in Trichostrongyloid nematodes. J Parasitol. 95(2): 487-9.
  52. Torina A, Alongi A, Naranjo V, Estrada-Pena A, Vicente J, Scimeca S (2008) Prevalence and genotypes of Anaplasma species and habitat suitability for ticks in a Mediterranean ecosystem. Appl. Environ. Microbiol.74:7578-7584.
  53. Weiss JB (1995) DNA probes and PCR for diagnosis of parasitic infections. Clin. Microbiol. Rev. 8:113-130.
  54. Wimmer B, Craig BH, Pilkington JG, Pemberton JM (2004) Non-invasive assessment of parasitic nematode species diversity in wild Soay sheep using molecular markers. International Journal for Parasitology 34 (5): 625-631.
  55. Yang Y, Mao Y, Kelly P, Yang Z, Luan L, Zhang J, Li J, El-Mahallawy HS, Wang C (2014) A pan-Theileria FRET-qPCR survey for Theileria in ruminants from nine provinces of China. Parasit. Vectors 7:413.
  56. Zaeemi M, Haddadzadeh H, Khazraiinia P, Kazemi B, Bandehpour M (2011) Identification of different Theileria species (Theileria lestoquardi, Theileria ovis, and Theileria annulata) in naturally infected sheep using nested PCR-RFLP. Parasitol. Res. 108:837843.
  57. Zarlenga DS, Chute MB, Gasbarr ELC, Boyd PC (2001) A multiplex PCR assay for differentiating economically important gastrointestinal nematodes of cattle. Veterinary Parasitology, 97 (3): 201-211.
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