A comparison of coprological, serological and molecular methods for the diagnosis of horse infection with Anoplocephala perfoliata (Cestoda, Cyclophyllidea)

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Veterinary Parasitology 152 (2008) 271–277 www.elsevier.com/locate/vetpar

A comparison of coprological, serological and molecular methods for the diagnosis of horse infection with Anoplocephala perfoliata (Cestoda, Cyclophyllidea) Donato Traversa a, Gianluca Fichi b, Michela Campigli b, Anna Rondolotti c, Raffaella Iorio a, Christopher J. Proudman d, Duccio Pellegrini e, Stefania Perrucci b,* b

a Department of Comparative Biomedical Sciences, Faculty of Veterinary Medicine, University of Teramo, Italy Department of Animal Pathology, Food Prophylaxis and Hygiene, Faculty of Veterinary Medicine, University of Pisa, Italy c Virbac s.r.l., Milan, Italy d Equine Division, Faculty of Veterinary Science, University of Liverpool, Liverpool, UK e Veterinary Pratictioner, Pisa, Italy

Received 31 October 2007; received in revised form 19 December 2007; accepted 21 December 2007

Abstract Anoplocephala perfoliata (Cestoda, Cyclophyllidea), the commonest intestinal tapeworm of horses, can cause colic, intussusceptions, ileal impactions and intestinal perforations. Common diagnostic techniques for A. perfoliata infection, i.e. coprology and serology, show inherent limitations in terms of sensitivity and specificity and new approaches are thus required. Hence, the present study compared the reliability of coprological, serological (i.e. ELISA) and molecular (i.e. nested PCR) methods in detecting A. perfoliata infection in naturally infected horses and in horses treated with a combination of ivermectin and praziquantel. Of 42 horses subjected to coprological examination, 16 and 26 resulted negative and positive, respectively for the presence of A. perfoliata eggs at the coprological examination. The 26 coprologically positive animals were also positive by nested PCR. Fifteen out of the 16 horses coprologically negative were negative at the molecular assay, while one yielded a PCR product detectable on an agarose gel. Eighteen out of 26 positive horses were treated with a combination of ivermectin 18.7 mg/g and praziquantel 140.3 mg/g and resulted subsequently negative by coprology and nested PCR performed 2 weeks after treatment. All infected and untreated animals had a high ELISA test optical density indicating high infection intensity and associated risk of colic. However, high optical density values were also obtained in four horses post-treatment and in three horses that were negative on molecular and coprological analysis. The results of the present work indicate that the nested PCR assay represents a valid method for the specific molecular detection of A. perfoliata in faecal samples collected from naturally infected horses and may have advantages over coprological and serological approaches for diagnosing A. perfoliata infection. # 2008 Elsevier B.V. All rights reserved. Keywords: Anoplocephala perfoliata; Horse; Coprology; ELISA; Nested PCR; Diagnosis; Treatment

1. Introduction * Corresponding author at: Dipartimento di Patologia Animale, Profilassi ed Igiene degli Alimenti, Facolta` di Medicina Veterinaria, Universita` degli Studi di Pisa, Viale delle Piagge 2, 56124 Pisa, Italy. Tel.: +39 050 2216949; fax: +39 050 2216941. E-mail address: [email protected] (S. Perrucci). 0304-4017/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.12.032

Anoplocephala perfoliata (Cestoda, Cyclophyllidea) represents the commonest intestinal tapeworm of horses (Matthews et al., 2004; Gasser et al., 2005). Although considered harmless for a long time, recent reports have identified a strong association between A. perfoliata

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infection and several intestinal diseases, e.g. colics, intussusceptions, ileal impactions and intestinal perforations (Proudman et al., 1998; Matthews et al., 2004; Gasser et al., 2005). Infection by A. perfoliata is globally distributed in different parts of the world with prevalence rates of about 13–82%, depending on the different countries and the diagnostic method used; higher prevalence values were found at necropsy (Gasser et al., 2005; Slocombe et al., 2007). The diagnosis of A. perfoliata infection in live animals commonly relies on egg detection by coprological methods based on the use of flotation and sedimentation techniques (Meana et al., 1998; Proudman and Trees, 1999). Among these coprological methods, the diagnostic test reported by Proudman and Edwards (1992) showed the highest diagnostic sensitivity (i.e. up to 61%). Although this sensitivity seems to be lower than values required for diagnostic tests, the technique is particularly useful to detect horses with heavy parasitic burdens (Matthews et al., 2004; Gasser et al., 2005). Serodiagnostic assays (ELISAs) based on somatic or excretory/secretory antigens of A. perfoliata proved capable of detecting circulating IgG antibody levels which correlated with number of parasites and, thus, the risk of severe pathological lesions (Hoglund et al., 1995; Proudman and Trees, 1996a). The sensitivity and specificity of such approach resulted of 70% and 95%, respectively (Proudman and Trees, 1996b; Matthews et al., 2004). Indeed, variability in antibody response to parasite infection could affect the sensitivity of this serological method and, in some cases, it cannot be possible to discriminate between false and true positives (Hoglund et al., 1995; Proudman and Trees, 1996a). Recently, a polymerase chain reaction (PCR)-based assay has been proposed for the diagnosis of horse tapeworm infection (Drogemuller et al., 2004). In particular the Internal Transcribed Spacer 2 (ITS2) of the ribosomal DNA (rDNA) was shown to contain genetic markers for the specific PCR-based detection of A. perfoliata DNA in horse faecal samples spiked with parasitic tissue (Drogemuller et al., 2004). Thus, the aims of this study were to compare the reliability of such a PCR assay with that of traditional coprological and serological methods to diagnose A. perfoliata infection using faecal samples from naturally infected horses and to evaluate its ability in detecting the absence of tapeworm DNA in stool samples collected from horses treated with a combination of ivermectin (18.7 mg/g) and praziquantel (140.3 mg/g) (Equimax1, Virbac).

2. Materials and methods 2.1. Study design Forty-two horses bred in farms, including an organic one, located in Tuscany region (central Italy) were randomly selected for the trial. A faecal sample of about 50 g was collected from the rectum of each horse and divided into a 40 g aliquot for coprological examination for A. perfoliata eggs and a 10 g aliquot for molecular analysis, stored at 20 8C until used. With the exception of eight horses bred in the organic farm, all horses scored positive (i.e. 18 out of the 26 horses, see Section 3) at the coprological examination for the presence of A. perfoliata eggs were treated with a combination of ivermectin (18.7 mg/g) and praziquantel (140.3 mg/g) (Equimax1, Virbac) at the dosage of 1.07 g/100 kg of body weight and subjected again to coprological analysis and molecular analysis 15 days post-treatment. Also, blood samples were collected from eight and nine horses positive and negative for A. perfoliata at the first coprological examination, respectively, and from eight horses 12 weeks after treatment with Equimax1. Blood samples were centrifuged at 2500 rpm for 10 min and sera were frozen at 20 8C pending examination with an anti-12/13 kDa IgG(T) ELISA test for A. perfoliata (Proudman and Trees, 1996b). 2.1.1. Coprological technique The method reported by Proudman and Edwards (1992) was used to isolate tapeworm eggs from individual horse faecal samples. Briefly, the faeces were placed in a beaker and mixed vigorously with 5– 10 ml of tap water to obtain a pasty consistency. The resulting mixture was then strained through a coarse sieve and the liquid collected in two 10-ml centrifuge tubes. Both tubes were then centrifuged at 2700 rpm for 10 min, the supernatant was discarded and the faecal plugs were re-suspended in tap water. Both tubes were spun again for 10 min at 2700 rpm and the supernatant from both tubes was discarded. The faecal plugs were re-suspended in saturated sucrose solution (prepared by dissolving 450 g of granulated sugar in 350 ml of warm water) and spun at 2700 rpm for 10 min. On removal from the centrifuge, the tubes were filled to the brim with saturated sucrose solution and a coverslip applied to the top. After the tubes had been allowed to stand for 2 h, the coverslips were placed on a microscope slide and microscopically examined at 10 and 20 magnification for the presence of tapeworm eggs.

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2.1.2. Serological test Twenty-five sera, obtained from eight positive, nine negative and eight treated animals (see Section 2.1), were analysed for the presence of antibodies against A. perfoliata with the method reported by Proudman and Trees (1996b). Immunology plates were incubated at 4 8C for 16 h with the capture antigen at concentration of 2 mg protein per well (0.1 ml volume) in a carbonate/ bicarbonate coating buffer (0.1 M, pH 9.6). The plates were washed three times with 2% SMP/PBS–0.3% Tween and then blocked for 2 h at room temperature with 0.1 ml per well of test serum, diluted 1/800 in 2% SMP/PBS and incubated for 1 h at room temperature. Plates were washed three times, and 0.1 ml of goat antihorse IgG conjugated to horseradish peroxidase (1/1000 in 2% SMP/PBS) was added to each well and incubated for 1 h at room temperature. Two washes with 2% SMP/ PBS–Tween and a final wash PBS–Tween were carried out prior to incubation of 100 ml azino-bis(3-ethylbenzthiazoline-6-sulphonic) acid at a concentration of 0.5 mg/ml in a citrate buffer (pH 4.2) in each well. Plates were incubated for 20 min in the dark at room temperature before optical density (OD) was read at 405 nm on a plate reader. Interpretation of the ELISA OD results relies upon the correlation between OD and infection intensity (Proudman and Trees, 1996b). Values less than 0.200 indicate zero or low infection intensity; values between 0.200 and 0.600 offer some evidence of tapeworm infection but at a level that is unlikely to be clinically significant; values greater than 0.600 are consistent with high infection intensity and a high risk of tapewormassociated disease. 2.1.3. Molecular procedures Forty-two faecal samples collected pre-treatment and 18 faecal samples collected 2 weeks post-treatment from animals that were coprologically positive (see Section 2.1) were subjected to molecular analysis. Each 10 g sample was slowly de-frosted at room temperature and then concentrated as described by Proudman and Edwards (1992). Two hundred microliter of the concentrated suspension of each faecal sample were subjected to three freeze/thaw cycles (liquid nitrogen 50 , 95 8C 50 ) and centrifuged at 13,000 rpm for 2 min. Genomic DNA was extracted from the resulting pellet with a commercial kit (QIAamp DNA Mini Stool Kit (Qiagen S.p.A., Milan, Italy)) and stored at 20 8C pending molecular analysis. Regions of A. perfoliata ITSs were amplified from all the DNA extracts (42 pre-treatment and 18 posttreatment) based on the nested PCR protocol reported

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by Drogemuller et al. (2004). PCR mixtures (25 ml) in both steps consisted of 12.5 ml ReadyMix RedTaq (Sigma, St. Louis, MO), 50 pmol of each forward and reverse primers, 0.5 mg bovine serum albumin, 2.5 ml of template (DNA extracted in the first step, first-round amplicon in the second step), and rinsed with distilled PCR water (Sigma, St. Louis, MO). Primer set S18 (50 -TAACAGGTCTGTGATGCC-30 ) and L3T (50 -CAACTTTCCCTCACGGTACTTG-30 ) (conserved among tapeworms) was used in the first round of PCR, followed by a second round using the A. perfoliata-specific primer set AP-ITS-2-3F (50 AATTGTGGGGGCTTCTCTTA-30 ) and AP-ITS-2-2R (50 -ACCTCGTGCCTTTCTTTAT-30 ) (Drogemuller et al., 2004). Cycling of both rounds was performed as follows: initial step at 94 8C for 7 min, followed by 40 cycles of 94 8C for 30 s (denaturation), 50 8C for 30 s (annealing) and 72 8C for 2 min (extension), and a final extension at 72 8C for 12 min. Appropriate control samples, i.e. samples without genomic DNA and a known positive control (DNA extracted from A. perfoliata specimen) were included in each amplification run. The specificity of this PCR assay when used on faeces collected from horses with multiple parasitic infections was also verified. In particular, 40 DNA extracts obtained from faeces collected in a previous study (Traversa et al., 2004) from horses infected with pinworms and/or cyathostomins and/or stomachworms and/or botfly (but not with tapeworms) were also tested in the nested PCR assay. All generated PCR products were subjected to electrophoresis in 2%-TBE agarose gels, stained with ethidium bromide, detected upon ultraviolet transillumination and photographed with the BioRad GelDoc documentation system. All second-round amplicons produced using primer set AP-ITS-2-3F/AP-ITS-2-2R and detected on agarose gels were purified over mini-columns (UltrafreeDA, Millipore, Bedford, MA) and subjected to automated sequencing (MWG Biotech/M-Medical, Milan, Italy). Sequences were determined in both orientations (using the same primers), aligned each other using the ClustalX program (Thompson et al., 1997), and verified by eye to maximize overall similarity. Also, sequences were compared with those of tapeworms available in the GenBankTM database with the Nucleotide–Nucleotide BLAST software (Altschul et al., 1997). The absence of PCR inhibition by faecal contaminants in the DNA samples was verified as described by Traversa et al. (2004).

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3. Results Sixteen and 26 out of the 42 horses included in the trial tested negative and positive, respectively for the presence of A. perfoliata eggs at the coprological examination pre-treatment. Fifteen out of the 16 horses coprologically negative were also negative at the molecular assay, while one sample yielded an amplicon detectable on an agarose gel (Fig. 1). The 26 coprologically positive animals were all positive by molecular diagnostic assay (Table 1 and Fig. 2). Eighteen of the 26 infected animals treated with the ivermectin/praziquantel combination tested negative at the post-treatment coprological analysis and did not produce any PCR product detectable on an agarose gel (Table 1). All the PCR amplicons produced at the second PCR round were of the expected size, i.e. 250 bp (Drogemuller et al., 2004) and no variation in size appeared on the agarose gels. Using the nested PCR, no amplicons were produced for any of the 40 control samples from horses infected with other parasites (Traversa et al., 2004) and there was no evidence of PCR inhibition in any of the faecal samples which were negative by coprological analysis. All the amplicons obtained were sequenced and demonstrated to be the appropriate ITSs region of A. perfoliata with a 99.5% mean value similar to the

Fig. 2. Example of an agarose gel showing amplicons amplified by the second round of the nested PCR from faecal samples collected from coprologically positive horse. Lane M: size marker; lane 1: sample no. 3; lane 2: sample no. 7; lane 3: positive control (Anoplocephala perfoliata DNA); lane 4: negative control.

respective sequences deposited in the GenBankTM by Drogemuller et al. (2004). Serologically, all infected and untreated animals gave high ELISA OD results indicating high infection intensity with increased risk of associated colic (Table 1). However, similar high values were obtained in four horses examined post-treatment and in three horses which were negative by molecular and coprological analysis (Table 1).

4. Discussion

Fig. 1. Example of an agarose gel showing a nested PCR product amplified from a faecal sample collected from a coprologically negative horse. Lane M: size marker; lanes 1–3: positive control (Anoplocephala perfoliata DNA); lanes 2–4: sample no. 17; lanes 5 and 6: negative control. Lanes 1, 2 and 5: first PCR round and lanes 3, 4 and 6: second PCR round.

The present work compared classical (i.e. coprological and serological) and innovative (i.e. DNA in vitro amplification) methods to detect infection by A. perfoliata in naturally infected horses. All methods undoubtedly have strength and weaknesses that should be taken into account in the diagnosis of this parasitosis. Coprological techniques for the detection of tapeworm eggs in the faeces of infected horses are easy to perform, inexpensive and require no sophisticated equipments or reagents; however, as previously reported (Proudman and Edwards, 1992; Matthews et al., 2004), the herein employed coprological approach showed to be messy

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Table 1 Untreated and treated horses (H) resulted positive (+) and negative () at the coprological (C), molecular (PCR) and serological (ELISA) analysis for Anoplocephala perfoliata Untreated horses H

C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

ELISAa

PCR

+ + + + + + + +                

+ + + + + + + +         +       

OD

I

1.858 2.275 2.443 2.158 2.248 2.082 2.198 2.267 2.482 1.339 0.117 1.373 0.528 0.254 0.045 0.398 0.401 n.p. n.p. n.p. n.p. n.p. n.p. n.p.

High High High High High High High High High High Low High Moderate Moderate Low Moderate Moderate n.p. n.p. n.p. n.p. n.p. n.p. n.p.

Treated horses Post-treatment c

Pre-treatment H

C

PCR

ELISAa, OD

C

PCR

ELISAb, OD

I

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

+ + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + +

n.p. n.p. 2.275 1.858 n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p.

                 

                 

0.223 2.423 1.084 0.659 0.574 0.301 0.600 0.302 n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p.

Moderate High High High Moderate Moderate High Moderate n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p. n.p.

OD: ELISA optical density; I: interpretation of the ELISA results; n.p.: not performed. a Sera collected at the same time of faecal collection. b Sera collected 12 weeks after the anthelmintic treatment. c Faecal samples collected 15 days after the treatment with the association of ivermectin 18.7 mg/g and praziquantel 140.3 mg/g (Equimax1, Virbac).

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and time consuming and, more importantly, lacked sensitivity. ELISA is commonly being used for clinical purposes to establish the relationship between tapeworm infection and symptomatic disease, with the aim to identify those horses that would benefit of an anti-cestode treatment. The serological approach has also proved useful to define the extent of equine tapeworm infection in different regions of the world (Matthews et al., 2004), even though its sensitivity can be impaired by individual variability in the antibody response to A. perfoliata infection, previous anthelmintic treatments and low OD range (see below); also, in some cases, the ELISAs are not able to discriminate between false and true positives (Hoglund et al., 1995; Proudman and Trees, 1996a). Hence, despite its importance in equine clinical medicine, A. perfoliata infection is likely to have been underestimated to date due to the inherent limitations of coprological and serological diagnostic approaches. PCRs specific for diagnostic markers within the rDNA showed to be powerful in overcoming the constraints of the classical diagnostic approaches for a range of parasites of veterinary and human importance. As far as horse endoparasites are concerned, molecular approaches recently allowed to achieve the diagnosis in vivo of gastric habronemosis (Traversa et al., 2004) and to conduct ante mortem molecular epidemiological surveys on this parasitosis whose classical diagnosis is unreliable (Traversa et al., 2006). Furthermore, the characterization of genetic markers within sequences of ribosomal DNA has allowed the investigation of interand intra-specific variation of different cestode groups (Bowles and McManus, 1993; Bowles et al., 1995; Van Herwerden et al., 2000). Thus, it is noteworthy that the present results indicate that the nested PCR previously developed (Drogemuller et al., 2004) to detect and amplify A. perfoliata DNA in horses faecal samples spiked with parasite material represents a valid method for the specific molecular diagnosis of A. perfoliata infection also from faecal samples collected in the field. In particular, the specificity of the primer set AP-ITS-23F/AP-ITS-2-2R, previously evaluated by Drogemuller et al. (2004) in the diagnostic amplification only of A. perfoliata DNA, has been herein confirmed against a panel of different DNA samples from pinworms, cyathostomins, stomachworms and botflies (cf. Section 2.1.3). Since the internal primer set has not been evaluated against Anoplocephala magna DNA (Drogemuller et al., 2004), non-specific positive PCR results cannot be completely ruled out. However, the high stringency condition of the molecular protocols and the nucleotidic distance features of rDNA of tapeworms

(Drogemuller et al., 2004), can lead us to consider the nested PCR with the primer sets S18/L3T and AP-ITS2-3F/AP-ITS-2-2R the most powerful species-specific approach for diagnosing the A. perfoliata infection in horses. The finding of a PCR-positive sample that was negative on coprological examination suggests lower sensitivity of classical copromicrospic approaches in comparison with more sensitive molecular methods. Even though the present molecular approach should be compared with the animal parasitological status at the necropsy (as previously elucidated for gastric habronemosis by Traversa et al. (2004)), the results confirms the advantages of (semi)-nested PCR approaches over the coprology, as previously indicated for other helminth-caused infections. For example, it has been demonstrated that molecular approaches achieve positive results using faecal samples that are negative at the coprologic methods, being able to specifically amplify as little as 0.14 fg of parasitic DNA present in the sample (Traversa et al., 2004). Also, the study carried out on Habronema stomachworms showed that the diagnostic sensitivity of PCR-based semi-nested approaches is 97%, thus with a very low probability of false negative results. The negative results obtained by coprological and molecular analysis of faecal samples from animals 15 days after treatment with Equimax1 indicates the efficacy of the ivermectin (18.7 mg/g) and praziquantel (140.3 mg/g) combination in the treatment of A. perfoliata-infected horses. It is also proposed that the nested PCR may be a useful tool for monitoring response to anti-cestode treatment in horses, other than for epidemiological purposes, such as to investigate distribution and prevalence of A. perfoliata infection in equids in several geographical areas. Nevertheless, the qualitative PCR-based approach herein used does not allow an estimation of the parasitic burden, thus impairing the clinical interpretation of the parasitological status of the infected animal/s (i.e. relationship between number of harboured tapeworms and risk of colic) that at the moment is possible only with the serological methods, even though partially. In this view it could be of importance to further modify this nested PCR protocol, e.g. by developing a (semi)quantitative real-time PCR as recently validated for Strongylus vulgaris (Nielsen et al., 2008). To evaluate animals at the risk of colic and for a comparison with the other diagnostic methods used in the present study, sera of eight positive, nine negative and eight treated animals, were analysed for the presence of antibodies against A. perfoliata with an

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ELISA method (Proudman and Trees, 1996b). Seven (three negative and four treated) horses that were negative by molecular and coprological analysis gave serologically positive results suggesting high infection intensity (Table 1). These results highlight the limitations of the anti-12/13 kDa ELISA test, particularly its lack of specificity and sensitivity to recent changes in parasite status, e.g. following anthelmintic treatment. Several factors can impair such a serological test, e.g. variability in antibody response to parasite infection, reexposure to infection after treatment, rate of decay of antibody levels in horses post-treatment and parasite modification of the host’s immune system (Hoglund et al., 1995; Proudman and Trees, 1996b). However, until now serodiagnostic assays (ELISA) were the only available diagnostic tool to detect horses with high tapeworm infection intensity (Hoglund et al., 1995; Proudman and Trees, 1996b) and consequently at risk of tapeworm-associated intestinal disease. In conclusion, the results obtained in the present study, although preliminary, strongly indicate that the nested PCR approach for the specific detection of A. perfoliata DNA could provide a useful clinic, diagnostic and research tool, even though further studies are needed for its definitive validation, e.g. by increasing the number of surveyed animals, autopsy confirmation of the molecular results and evaluation of quantitative PCR-based protocols. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Bowles, J., McManus, D.P., 1993. Rapid discrimination of Echinococcus species and strains using a polymerase chain reaction-based RFLP method. Mol. Biochem. Parasitol. 57, 231–239. Bowles, J., Blair, D., McManus, D.P., 1995. A molecular phylogeny of the genus Echinococcus. Parasitology 110, 317–328. Drogemuller, M., Beeliz, P., Pfister, K., Schnieder, T., Samson-Himmelstjerna, G., 2004. Amplification of ribosomal DNA of Anoplocephalidae: Anoplocephala perfoliata diagnosis by PCR as a

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