Advances in laboratory diagnosis of parasitic infections of sheep

Veterinary Parasitology 189 (2012) 52–64

Contents lists available at SciVerse ScienceDirect

Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar

Advances in laboratory diagnosis of parasitic infections of sheep J. Demeler, E. Schein 1 , G. von Samson-Himmelstjerna ∗ Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Königsweg 67, 14163 Berlin, Germany

a r t i c l e

i n f o

a b s t r a c t

Keywords: Anthelmintic efficacy Biomarker ELISA FLOTAC Molecular diagnosis Serology

Parasitic infections constitute an important group of diseases in sheep concerning the health status, welfare and productivity. On a global scale, there are considerable differences concerning the epidemiological situation with respect of the various parasite species. However, there are also numerous species, which occur on all continents and, potentially, in every country. Accordingly, the present review aims to providing an overview about the recent developments in methods and technologies for the laboratory diagnosis of parasite infections in sheep. Following in principle a systematic order the review encompasses publications addressing the diagnosis of helminthes (i.e., trematodes, cestodes and nematodes) and arthropod species. New approaches using conventional (e.g., microscopic), immunological and molecular techniques are being considered. The diagnosis of anthelmintic resistance is highlighted separately, due to its significant importance. The review ends with an outlook into the future by discussing most recent technological advances, which might become of use for the diagnosis of parasite infections in sheep in the future. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

‘targeted selective treatment’ or targeted treatment. Furthermore, they already were shown to be of relevance during programmes aiming at tempero-spatial disease and risk mapping (Musella et al., 2011). Various host material matrices are being used to test for parasite infections, e.g., blood, serum, faeces, liquor, skin scrapings or saliva, depending on the parasite under detection and its localisation within the host. A large range of different techniques, including basic microscopy based-methods, molecular, immunological or recent nanotechnological tools, have been adopted to provide specific diagnostic solutions for the detection of parasite infections in sheep. The review attempts to provide an overview, which, due to the broad spectrum of organisms encompassed, cannot be complete, but rather exemplifies the most promising new tools, particularly with a focus on those offering new opportunities for testing in the field.

The diagnosis of parasitic infections in small ruminants has great importance due to the major threats this group of diseases pose to the welfare and productivity of ruminants and particularly of grazing sheep. A range of new methods and procedures aiming at the direct or indirect detection of parasite infections through laboratory examinations have been described in the last decade. Sensitive, reliable and cost-effective laboratory test systems are desirable for conducting epidemiological surveys, as well as a prerequisite for routine on farm parasite monitoring. The latter has become of increasing interest recently along with the advance of high throughput multiplex screening tools, such as LUMINEX or high throughput mass spectrometry testing. These approaches will potentially play an important role as indicators for anthelmintic treatment decisions in optimised helminth control strategies such as

∗ Corresponding author. E-mail address: [email protected] (G. von Samson-Himmelstjerna). 1 Deceased. 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.03.032

2. Advances in laboratory diagnosis In this text, major advances in the laboratory diagnosis of helminth infections and arthropod infestations in sheep

J. Demeler et al. / Veterinary Parasitology 189 (2012) 52–64

and published in general during the past decade are being reviewed following in principle a systematic order. Protozoan infections have recently been covered by De Waal (2012). 2.1. Trematodes 2.1.1. Dicrocoelium dendriticum To date the diagnosis of Dicrocoelium dendriticum infections in sheep is mainly based on the finding of adult stages in the liver or of eggs in the faeces (Otranto and Traversa, 2002). The latter requires flotation solutions with particularly high density, as, otherwise, the sensitivity of detection will be low (Rehbein et al., 1999). For the coproscopical analysis of D. dendriticum infections in sheep, the recently developed FLOTAC method was comparatively assessed using also a simple flotation and the standard McMaster method (Rinaldi et al., 2011). Using different flotation solutions including zinc-sulphate, both in terms of sensitivity as well as reproducibility, the FLOTAC method provided the best results and was considered to be as ‘gold standard’. For serological examination of D. dendriticum infections, several ELISA-based methods have been described in the past (Otranto and Traversa, 2002). In a more recent study using excretory/secretory antigen from adult D. dendriticum, seroconversion was detected until 30 days post infection in all four experimentally infected sheep and, thus, approximately 4 weeks before the first positive coproscopic diagnosis (Broglia et al., 2009). Based on sera from experimentally infected animals, a testsensitivity of 88% was calculated, while the specificity of 84% was assessed using serum samples from animals naturally infected with Fasciola hepatica and Paramphistomum spp., as well as those from experimental nematode infections. Similar findings were reported by Manga-González and González-Lanza (2005), who also described changes in several blood parameters, like increased aspartate aminotransferase and ␥-glutamyl transpeptidase concentrations and leucocyte numbers. First attempts were made to molecularly characterise specific D. dendriticum antigens suitable for immunodiagnostic (Revilla-Nuín et al., 2005). Partial N-terminal sequencing of a 130 kD sized antigen, which was derived from excretory/secretory fractions, as well as somatic extracts suggested that both sources contained the same putatively globin-like protein. Western blot and preliminary ELISA-data indicate that this antigen may be suitable for D. dendriticum immunodiagnostic. Molecular investigations of the ribosomal DNA can provide additional information on the specific distinction and phylogenetic relationship of different Dicrocoelium species (Otranto et al., 2007; Maurelli et al., 2007) and allowed the development of a specific and sensitive PCR-based protocol for the detection of D. dendriticum DNA (Capuano et al., 2007). 2.1.2. Fasciola hepatica For the direct detection of F. hepatica infections in sheep, the standard protocol remains the classical sedimentation method. However, recent data showing significantly higher sensitivity and reproducibility of the FLOTAC method

53

using zinc-sulphate as flotation medium (specific gravity 1.35) compared with the sedimentation methods, when performing coproscopical diagnosis in experimentally infected rats, have indicated that this procedure might represent a valuable diagnostic alternative also in sheep (Duthaler et al., 2010). Numerous recent publications describe the development and use of ELISA-based methods for the direct or indirect detection of F. hepatica infections in sheep. Most publications address the use of serum as a matrix; however, several approaches were also described for the detection of fluke antigen in the faeces and antibodies in milk or saliva (Table 1). In principle, the serological detection of infection allows a considerably earlier diagnosis than the coproscopical examination, with, in some assays, first positive test findings as soon as 2 weeks post-infection (Mezo et al., 2007). However, also when testing for coproantigens in faeces, detection of infection 4–7 weeks and 3–6 weeks prior to patency was recorded for F. hepatica and Fasciola gigantica, respectively (Valero et al., 2009). The molecular detection of fluke infections in sheep can be achieved through the PCR-based amplification of specific DNA sequences. The identification of suitable target sequences for species specific PCR primers, as well as the molecular differentiation of fluke isolates from varying geographic regions has been achieved through the genetic characterisation of ribosomal gene sequences: the internal transcribed spacer (ITS) (Nguyen et al., 2009; Capuano et al., 2007) regions, and mitochondrial gene sequences: the cytochrome C oxidase subunit I (COI) (Farjallah et al., 2009). Alternatively, instead of sequencing distinct genes for development of species specific PCR primers, McGarry et al. (2007) used sequences information from Randomly Amplified Polymorphic DNA (RAPD)-PCR generated fragments to obtain a method reliable for the molecular differentiation of F. hepatica and F. gigantica DNA. The application of sequence-related amplified polymorphism was shown to be suitable for detection of genetic variability in F. hepatica derived from different host species and geographic regions (Alasaad et al., 2008). In an earlier study, also employing RAPD-PCR, inter- and intra-species variations were demonstrated between different isolates of F. hepatica and F. gigantica obtained from cattle and sheep (Ramadan and Saber, 2004). Furthermore, by digestion through a suitable restriction enzyme PCR-amplified, ribosomal DNA was cut into fragments of characteristic length, allowing the unequivocal delineation of Fasciola isolates from different regions in China (Huang et al., 2004) or Iran (Rokni et al., 2010). In a similar approach, the 28 s ribosomal RNA gene sequence was amplified and restricted to develop a species specific assay for the differentiation of F. hepatica and F. gigantica (Marcilla et al., 2002). The use of mass spectrometry analysis allowed identification of F. hepatica infection-associated biomarkers in sheep serum and the characterisation of their expression profile during different phases of the infection (Rioux et al., 2008). It is hoped that this technological approach will contribute to an improved understanding of parasite-host interactions and diagnostics of fluke infections. Moreover, the sequencing and bioinformatic analysis of the liver fluke

54

J. Demeler et al. / Veterinary Parasitology 189 (2012) 52–64

Table 1 Recent publications describing the development of immunological tests for the detection of fluke infections in sheep. Antigen/antibody

Type

Matrix

ES Purified 28 kD antigen (F. gigantica) ES (F. gigantica)

Antibody-ELISA Indirect, sandwich ELISA (mAbs) Sandwich ELISA (polyclonal Abs) Indirect ELISA

Serum Saliva, serum

MM3 (F. hepatica, F. gigantica) ES (F. gigantica) MM3 MM3 Recombinant 2.9 kDa, ES ES (F. hepatica, F. gigantica) Cathepsin-L cysteine proteinase Somatic (F. gigantica)

Capture-ELISA

Serum, faeces

Antibody-ELISA Capture ELISA Capture ELISA Indirect ELISA

Serum Serum Serum, faeces Serum

Antibody-ELISA

ES (Commercial test from Institute Pourquier) ES, polyclonal anti IgG Abs ES (FPLC-fractionated) 28 kDa cysteine protease from ES (F. gigantica) Recombinant cathepsin L1 Cercarial antigen (F. hepatica) Recombinant cathepsin L-like protease

ES Crude GST (all F. gigantica)

Sensitivity

Specificity

Reference

Saliva: 65% Serum: 100% Serum: 92% Faeces: 97% ES: 95% Crude: 100% GST: 72% NA

Saliva: 100%

Novobilsky´ et al. (2007) Sabry and Taher (2008)

Serum: 89% Faeces: 93% ES: 97% Crude: 83% GST: 78% NA Nearly 100% 100% NA 2.9 kDa: 86% ES: 48% NA

El Ridi et al. (2007) Mezo et al. (2007) Mezo et al. (2004) Arias et al. (2007)

Serum

Nearly 100% 100% NA 2.9 kDa: 68% ES: 53% NA

Antibody-ELISA, dipstick ELISA Immunoblotting

Serum

NA

NA

Yadav et al. (2005)

Serum

NA

NA

Antibody-ELISA

Serum, milk

Serum: 97%

Serum: 99%

Yokannanth et al. (2005) Molloy et al. (2005)

Antibody ELISA, sanwich ELISA Antibody ELISA Antibody ELISA, dipstick-ELISA, Western blot Antibody-ELISA

Serum

NA

NA

Paz-Silva et al. (2003)

Serum Serum

100% Western blot: 100%

100% NA

Mezo et al. (2003) Dixit et al. (2002)

Serum

NA

NA

Carnevale et al. (2001)

Antibody-ELISA

Serum

100%

90%

Mousa (2001)

Antibody-ELISA

Serum

99%

97%

Cornelissen et al. (2001)

Serum, faeces Serum

El Amir et al. (2008) Awad et al. (2009)

Valero et al. (2009)

Phiri et al. (2006)

Antigen from F. hepatica if not otherwise stated, NA: not available.

transcriptome (Young et al., 2010) will certainly foster the development of future diagnostic methods for the detection of liver fluke infections in sheep and the assessment of the clinical, immunological and pathological effects. 2.1.3. Paramphistomum spp. The diagnosis of ruminal Paramphistomum infections in the routine diagnostics is based on faecal examination, usually by sedimentation technique; the flotation method is also feasible, providing even an opportunity for the indirect quantitative assessment of parasitic burden (Rieu et al., 2007). Since pathological changes are mainly caused during the long pre-patency period of the juvenile helminthes, it is of interest to establish diagnostic tools, which would allow the detection of pre-patent infections. In cattle, a 52 kDa P. cervi antigen derived from whole helminth preparations was found to provide high sensitivity and specificity when used in a serological test (Anuracpreeda et al., 2008). 2.1.4. Schistosoma spp. The sedimentation method currently remains the method of choice for the diagnosis of Schistosoma spp. infections in sheep (Schnieder, 2006). Alternative approaches have been developed in humans, like a dot-blot diagnostic test (Yisheng et al., 2005) or ELISA using saliva

(Santos et al., 2000). In sera from sheep infected with S. bovis, antibodies against distinct tegumental antigens like Lewis(x) and Lewis(Y) were detected (Ramajo-Hernández et al., 2007).

2.2. Cestodes 2.2.1. Moniezia spp. The coproscopical examination of faecal samples following processing by flotation remains the laboratory method of choice for the direct detection of Moniezia infections in sheep. By comparing the basic flotation, the McMaster and the FLOTAC methods Rinaldi et al. (2011) characterised the FLOTAC method as ‘gold standard’ for the detection of Moniezia eggs, due to highest sensitivity and lowest coefficient of variation. For differentiation between Moniezia expansa and Moniezia benedeni a Multilocus Enzyme Electrophoresis (MEE) approach has been used (Chilton et al., 2007). For 10 out of 19 genetic loci fixed differences between the two species were detected. Furthermore, it was encountered that there was a considerable within-species genetic difference, which exceeded the between-species difference, thus suggesting the presence of cryptic species.

J. Demeler et al. / Veterinary Parasitology 189 (2012) 52–64

2.2.2. Taenia spp. Sheep can be infected as intermediate hosts by a range of Taenia species, namely T. ovis, T. hydatigena and T. multiceps. Intra vitam diagnosis is challenging, as often clinical symptoms are not specific. The laboratory detection of infections can be based on the presence of antibodies or circulating antigen in serum samples. Earlier Western blot analyses of T. hydatigena cyst fluid revealed an immuno-reactive 36 kDa antigen (Kara et al., 2003); however, no data on sensitivity or specificity with respect to this antigen when being used in a serological test were provided. Direct evidence for Taenia infections in sheep can be provided by examination of biopsy samples with molecular tools, like PCR. Of importance for the control of Taenia infections in sheep flocks is also the diagnosis of the infection in the definitive host. In addition to coproscopy following flotation, molecular tools have been developed enabling the differentiation of eggs from different taeniid species (Mathis and Deplazes, 2006; Armua-Fernandez et al., 2011). Using NADH dehydrogenase I (ND1) and CO1 mitochondrial gene sequences, Varcasia et al. (2006) provided an assessment of genetic variability in T. multiceps isolates from mainly sheep and few dogs from different geographical regions. Through the recent identification and characterisation of mitochondrial genome sequences of several Taenia species additional options for the development of molecular diagnostic markers were provided (Jia et al., 2010; Lavikainen et al., 2010; Liu et al., 2010). 2.2.3. Echinococcus granulosus (E. hydatidosus) The laboratory diagnosis of Echinococcus granulosus infections in sheep is of relevance mainly within epidemiological studies in the context of public veterinary health issues, for example to assess the prevalence and/or the effectiveness of programmes to control this severe zoonotic parasite (Torgerson and Deplazes, 2009). To date immunodiagnostic is problematic, due to sometimes low sensitivity and/or specificity of serological tests (Gatti et al., 2007; Torgerson and Deplazes, 2009). Thus, while sensitivities of over 80% and specificities over 90% were achieved when using total hydatid liquid as antigen, it was recommended to confirm positive enzyme immunoassay results by secondary testing, e.g., by using Western blot analysis (Gatti et al., 2007). The comparative molecular analysis of mitochondrial DNA sequence information from E. granulosus isolates derived from different intermediate host species and geographical regions has contributed to a revision of the taxonomy of the genus Echinococcus with the suggestion to restrict the species name E. granulosus to the G1 and G2 genotypes, which use ruminants, pigs and camels as intermediate host (McManus and Thompson, 2003; Thompson, 2008; Thompson and McManus, 2002). The development of PCR primers specific for the newly suggested Echinococcus species has to overcome the problem of high sequence identity in target genes from the different species and genotypes (Boufana et al., 2008). Even highly sequence specific methods, like the single stranded conformational polymorphism analysis, did not provide a resolution suitable for the differentiation of all genotypes. Following amplification

55

of 12S rRNA, the analysis of G1 and G3 samples gave same band patterns (Simsek et al., 2011). Based on rDNA ITS1 PCR-RFLP the G1 and G6 (camel) genotypes were distinguished, which was confirmed by sequencing of CO1 and ND1 genes (Shahnazi et al., 2011). Similarly, through sequencing of CO1, ND1, ATPase subunit 6 and 12 rRNA gene sequences samples from G1, G3 and G7 were differentiated (Snábel et al., 2009), while Varcasia et al. (2007) used a semi-nested PCR approach and sequenced ND1 and CO1 genes to reveal the prevalence of G1, G3, G5 and G6/G7 genotypes in samples from sheep and goats. 2.3. Nematodes 2.3.1. Coproscopic and clinical methods The accurate diagnosis of gastrointestinal nematodes in sheep is essential for its effective control. This applies for routine monitoring, as well as for ensuring efficacy of an anthelmintic drug. In any investigation of infectious diseases, there is the need for sensitive and specific diagnostic procedures for the detection of the aetiological agent. Traditional parasitological diagnostic techniques (Bowman et al., 2009; Taylor, 2010) involving mainly microscopy have been complemented by a variety of new techniques and tools. To date, traditional methods are still routinely used despite the fact that they can be labour and time intense to perform. One of the most commonly used techniques for the diagnosis of parasites in the field is the examination of faecal samples. Faecal egg count techniques are generally considered to be straight forward and relatively easy to perform. A variety of different protocols, such as simple flotation methods, McMaster technique, Wisconsin flotation technique, FECPAK® and FLOTAC® , have been available for years. Generally, these differ in their sensitivity and, therefore, accuracy. Additionally, the reasons for the performance of a faecal egg count have broadened. If a treatment decision is required, simple methods, such as the McMaster, FECPAK® or Wisconsin technique, are satisfactory in terms of sensitivity and accuracy. If the faecal egg count is required for the verification of the efficacy of treatment and/or the detection of resistance to certain anthelmintics, sensitivity and accuracy are of greater importance. A relatively new coproscopical method, the FLOTAC® technique, has been reported to have significantly improved sensitivity (Rinaldi et al., 2011) for parasite egg counting in sheep. Another problem associated with gastrointestinal parasite infections is the fact that these usually occur as mixed infections. Different species vary significantly in pathogenicity and susceptibility to anthelmintics and, thus, the clinical consequences of infections (Leignel and Cabaret, 2001). Therefore, the identification of causative species is important. Nematodes with morphologically distinct egg shapes, e.g., Strongyloides or Trichuris, can be distinguished from strongyle eggs. However, aside from Nematodirus spp., there are only tiny differences between the eggs of trichostrongyle nematodes, making it difficult if not impossible to identify the eggs to species or even genus level (Mes et al., 2007). The use of lectin binding characteristics for the identification of different parasite species

56

J. Demeler et al. / Veterinary Parasitology 189 (2012) 52–64

has been evaluated by using flow cytometry (Colditz et al., 2002), revealing a species specific binding-reaction to different lectins. This was further developed into a more field-based method, where eggs were retrieved directly from faecal samples after sugar gradient centrifugation, stained and examined under a microscope (Jurasek et al., 2010). Another faecal based method, the use of a faecal occult blood test, was shown to have the potential to predict imminent Haemonchus infections in sheep (Colditz and Le Jambre, 2008). The identification of contributing species is also possible using the larval culture method, including microscopic examination of larvae under a microscope. Despite the report of a simplified guide for the differentiation of gastrointestinal larvae of parasitic nematodes in sheep (van Wyk et al., 2004), this method takes at least one week (larval culture), is laborious, can be inaccurate regarding different species, requires experienced personnel, but still has been reported to be unreliable (Dobson et al., 1992). Successful morphological identification and differentiation have been reported for other parasites, such as Thelazia (Naem, 2007) and Strongyloides species (Sato et al., 2008), by using scanning electron microscopy. 2.3.2. Immunological methods For serological and immunological detection of nematode infections several ELISA-based methods have been described for different species, mainly Haemonchus contortus and Teladorsagia circumcincta, in the past decade. Copro-antigen ELISAs were reported for the detection of T. circumcincta (Johnson et al., 2004). Various serum ELISAs were described for the detection of infections with H. contortus (Schallig et al., 1995; Kooyman et al., 1997; ˜ et al., 2000; Li et al., 2007; Prasad et al., Gómez-Munoz 2008), T. circumcincta (Huntley et al., 2001; Molina et al., 2009), Trichostrongylus (Shaw et al., 1998; Bendixsen et al., 2004) or Oesophagostomum (Jas et al., 2010). Further characterisation of proteins suitable for immunodiagnostic or vaccination is ongoing for a variety of different gastrointestinal parasites (Pernthaner et al., 2005; Redmond et al., 2006; Kiel et al., 2007). Generally, serological approaches have limitations regarding their ability to reliably distinguish between current and recent infections. Additionally, a commercially available saliva test for the detection of nematode infection in sheep has been reported recently. The test measures antibodies (IgA), which are considered to be directed against parasite larvae in the gut mucous of sheep. Animals with high antibody titres have been shown to have lower faecal egg counts and comparable better growth rates (CARLATM ). 2.3.3. Molecular methods PCR-based procedures have been proven to have greater sensitivity and specificity than ‘conventional’ diagnostic approaches reliant on microscopy and/or immunodetection. In addition, ongoing molecular epidemiological investigations on a variety of parasitic diseases are providing predictive data of immense value for control. The application of molecular biological methods to the diagnosis of parasites has increased rapidly in recent years. The value of such molecular tools is greatest, if they can be

applied directly to faecal or tissue specimens and if there is the potential to automate such procedures. In this respect, PCR-based techniques have provided veterinary parasitologists with very powerful tools. A number of PCR based assay for the identification/differentiation of strongylid eggs and larvae has been developed. In these assays, genetic markers in the first and second internal transcribed spacers (ITS-1, ITS-2), external transcribed spacers of nuclear ribosomal DNA and ribosomal DNA itself (18S, 28S) have been used. Bott et al. (2009) reported another approach for the diagnosis of strongylid infections in sheep, where microscopic and molecular methods were combined. Molecular assays for the detection of Bunostomum (Wang et al., 2012), Chabertia, Oesophagostomum (Bott et al., 2009; Roeber et al., 2011), Trichuris (Oliveros et al., 2000; Cutillas et al., 2004), Thelazia (Otranto et al., 2003; Otranto and Traversa, 2004), Strongyloides (Eberhardt et al., 2008), Nematodirus or Dictyocaulus have been reported recently (Wimmer et al., 2004). For the most important gastrointestinal nematodes of sheep, including the species Haemonchus, Trichostrongylus, Teladorsagia and Ostertagia, an enormous variety of molecular assays have been published. A detailed review of the advances in DNA-based diagnostic methods for nematode infections in livestock has been published by Gasser et al. (2008). If appropriate techniques for obtaining sufficient quantities of parasite material out of faecal samples could be developed and combined with molecular tools, such as PCR, then the major limiting factors for parasite detection and characterisation will be obviated. The limiting factors, which can impact negatively on the development of real time PCR-based techniques, such as faecal inhibitors (depending on environmental factors such as feed, forage, host condition), developmental stage of the eggs and problems due to sequence similarities, have been discussed in detail by Harmon et al. (2007). The remaining challenge of field applicability is likely to be overcome in the near future with advances in sampling protocols, storage of parasite isolates leading to the development of field-applicable and cost-effective assays. 2.4. Arthropods 2.4.1. Mites The verification of mites of small ruminants is usually achieved by examination of deep skin scrapings or skin biopsy, followed by microscopic differentiation. Species can be identified according to the region of lesions and based on morphological characteristics. The main causative agents are members of the following species: Psoroptes, Chorioptes, Sarcoptes and Demodex. Sarcoptes scabiei affects (sarcoptic mange) non-woolly skin and is usually found on the head, the face and, in very severe infestations, the abdominal wall and the scrotum. The characterisation of recombinant immunoreactive antigens of the mite S. scabiei has been discussed (Kuhn et al., 2008) and, recently, details of a serological test have been reported (Rodríguez-Cadenas et al., 2010). Chorioptes infestation (chorioptic mange) is not that often seen in sheep. Papules and crusts are particularly seen on the feet and legs. It has to be distinguished from

J. Demeler et al. / Veterinary Parasitology 189 (2012) 52–64

dermatitis (strawberry foot rot). There are currently no serological tests available for the diagnosis of this infestation. Psoroptes ovis is a widely prevalent and economically important mite infestation (psoroptic mange) of sheep in Northern Europe. Large, scaly, crusted lesions develop almost exclusively on wooly parts of the body. Due to biting and scratching, often intense pruritus manifests. Skin scrapings are taken from the superficial part of the lesions. Due to often latent infections, diagnosis of Psoroptes mites in skin scrapings is known not to be highly sensitive. A serological test has been reported, with a sensitivity and specificity over 90% and is at current recommended for the diagnosis of the disease (Ochs et al., 1999, 2001; Siegfried et al., 2004). Approaches towards vaccination against P. ovis have first been described by Smith et al. (2001, 2002) and further candidate vaccines were evaluated (Smith and Pettit, 2004; Nisbet and Huntley, 2006; Nisbet et al., 2008). Mange (ear mange) caused by Psoroptes cuniculi, usually affects the ears, but can spread to the head, neck, and body and cause severe irritation. Psorergates ovis is prevalent in Australia, New Zealand, South Africa and South America and possibly also present in Europe. Infestations with this mite are mainly inapparent and, therefore, often not recognised. No serological test is available for the diagnosis. The hair follicle mite Demodex has been reported in sheep, in which it causes typical lesions mainly in the anterior body region. 2.4.2. Ticks All ticks infesting sheep can generally be diagnosed directly by collecting the ticks from the animal, followed by differentiation using morphological characteristics. Depending on the environmental factors, a variety of different tick species can be found on sheep. In Europe, mainly hard ticks are infesting sheep, while soft ticks are extremely rarely seen. Often, only adult ticks are diagnosed, but larvae and nymphs are also common and, often, overlooked due to their small size. The most important tick species found in Europe are Ixodes ricinus, Haemaphysalis punctata, Dermacentor marginatus, Rhipicephalus bursa and Hyalomma spp. In para-Mediterranean, tropical and subtropical regions different Rhipicephalus, Hyalomma, Haemaphysalis, Amblyomma and Boophilus species are also present (Mohammed and Hassan, 2007; Pavlidou et al., 2008; Abunna et al., 2009; Nabian et al., 2009; Dehaghi et al., 2011; Omeragic, 2011); in the USA the emergence of Amblyomma americanum has been reported (Bowen et al., 2010). Besides skin lesions (particularly heavy infections) and reactions of the host to tick salivary toxins, pathogens such as Borrelia, Anaplasma, Rickettsia, Babesia species and various viruses are transmitted by ticks. The diagnosis of these pathogens is usually performed by the examination of the host blood and is described elsewhere. 2.4.3. Stationary hexapoda (sheep ked, lice & biting lice) Melophagus ovinus (sheep ked) are wingless, reddishbrown biting flies (dipterans) feeding on blood. They are stationary parasites, which spend the whole life (∼6 weeks) on the host. Direct damage results from bites causing blood

57

loss and irritations, emaciation and susceptibility to other diseases. They are found year-round and infest mainly areas around the neck, breast, shoulders, flank and rump in the greatest numbers, but tend not to feed on the back where the most debris collects in the wool. Their reddish excrements stain the wool in a typical manner. Close inspection of damaged and dirty wool by recommended parting procedures and additional examination of the skin reveals infestation. At current, no serological tests are available. Lice are wingless and flattened insects, living in the microenvironment provided by the skin and the wool/hair of sheep. They are largely host specific and primarily transmitted by contact. In temperate regions, such as most parts of Europe, lice are most abundant during the colder time of the year and often difficult to find in the warmer months. Sheep become infected with Damalinia (Bovicula) ovis (sheep biting louse or sheep body louse) or thee species of sucking lice, Linognathus pedalis (sheep foot louse), Linognathus ovillus (face and body louse) and Linognathus africanus (African blue louse). The sucking louse of goats, Linognathus stenopsis, can also infest sheep. Other chewing (biting) lice that may infect sheep, are Damalinia limbatus and Damalinia crassipes. Diagnosis of lice infestation is traditionally based on the presence of the lice. Depending on the different regions of infestation, the hair of the head, legs, feet, top line, face neck and tail region should be parted and the skin and coat examined with the help of light. Biting lice are active and can be seen moving through the hair, while sucking lice are slower and are usually found with their mouthparts embedded in the skin. However, traditional methods of detection, such as visual inspection of the animal by parting the fleece or the lamp test using parts of shorn wool are not sufficiently sensitive and therefore animals often are left untreated (Morcombe et al., 1996). The accuracy of visual inspection of wool lots as an indicator for lice infestation was evaluated by Ward and Armstrong (2000). They reported a sensitivity of 36% and suggested that 16 inspections of wool lots per flock of sheep could be used for an efficient monitoring. The sensitivity of a two-stage sampling method was evaluated by James et al. (2002). Additionally, the pruritic behaviour and fleece derangement can be used as indicators for lice infestation (James and Moon, 1998; James et al., 2007). Immunological approaches have been undertaken by Bany et al. (1995b). These authors showed that B. ovis antigen specifically induced proliferation of prescapular lymph node cells, indicating that an infestation sensitises T-lymphocytes and is detectable by a lymphocyte proliferation assay (Bany et al., 1995a). Recently, an abundant allergen from B. ovis was identified and partially characterised (Pfeffer et al., 2010), but more research is required to determine the biological function of this molecule. 2.4.4. Flies causing traumatic myiasis There are several species of myiasis producing flies, but myiasis in sheep is mainly caused by flies belonging to the species Lucilia (e.g., L. sericata, L. cuprina, L. illustris, L. caesar) and Wohlfahrtia (e.g., W. magnifica, W. vigil). Diagnosis of myiasis on an infested animal is relatively simple

58

J. Demeler et al. / Veterinary Parasitology 189 (2012) 52–64

and is performed by visual inspection of the wounds. To note that in Lucilia species, adults and larvae are parasitic, while only the larvae of Wohlfahrtia species are parasitic. The identification of adult flies is relatively difficult and mainly performed using morphological characteristics of the flies body and wings. A definitive diagnosis can be made only after extraction and identification of the larvae in the wounds. The caudal ends of several third-stage larvae infesting the wound should be sliced using a scalpel blade. When the sliced caudal ends are placed cut surface down on a glass slide, covered with a cover-glass and examined under a compound microscope, a dichotomous key can be used to identify the genera of flies involved in the infestation. The unique spiracular plates are distinct for a particular genus. Several specimens should be examined, because more than one genus may be present within the lesion. Molecular methods for identification of Wohlfahrtia species have also been established (Hall et al., 2009a, 2009b), by using a mitochondrial cytochrome b gene. An attempt to diagnose infested animals by the use of an electronic nose has been reported by Cramp et al. (2009), where under experimental conditions an electronic nose was able to correctly diagnose infested sheep as early as 24 h after infestation. Although immunisation against Lucilia has been evaluated in various studies, no serological diagnostic tool for the detection of infested animals is available at current. Presence of larvae in tissue does not produce a detectable immune response, making immunological diagnosis not possible for the detection of myiasis causative agents. 2.4.5. Oestrus ovis The sheep nose botfly, Oestrus ovis, is a cosmopolitan parasite with economic importance particularly in South Africa, Brazil and some para-Mediterranean countries. The larvae are normally found in the nostrils and paranasal sinuses of sheep and goats, but they have also been found in other hosts, such as dogs and humans. They rarely penetrate the ethmoid bone and reach the forebrain. However, it is possible that other factors facilitate entry of larvae into the brain. If the brain is injured, clinical signs, such as a high-stepping gait and incoordination, may mimic infection with Coenurus cerebralis. This condition is often referred to as false gid. Diagnosis of the larvae is difficult and restricted to visual inspection of the nostrils and interpretation of clinical signs, such as thickening of nasal mucosa, mucopurulent discharge, impaired respiration and sneezing. Once the larvae mature in the sinuses, they leave the nasal passage and drop out of the nostrils to the ground. Serological immune responses have been studied using intradermal tests (Ilchmann and Hiepe, 1985) and indirect haemagglutination (Bautista-Garfias et al., 1988). The use of an immunological detection method (ELISA) has first been reported by Yilma (1992) and further evaluated in different studies (Papadopoulos et al., 2001, 2006; Dorchies et al., 2003; Alcaide et al., 2005a). A dot ELISA was described by Duranton et al. (1995) and the first validated serodiagnostic ELISA was reported by Goddard et al. (1999). Tissue extracts from O. ovis larvae have been shown to induce specific antibody production in infected animals (Innocenti et al., 1995; Tabouret et al., 2001), whilst Alcaide

et al. (2005b) analysed different larval antigens for their use in an ELISA. These authors recommended an ELISA using excretory–secretory product (ESP) antigen from first stage larvae (L1) during winter and L2 ESP antigen in summer for diagnosis of ovine oestrosis. 3. Advances in the assessment of anthelmintic efficacy For the detection of anthelmintic resistance, the most important requirement is correct diagnosis of parasitic species in the host, particularly, if pre- and post-treatment samples would be compared. Problems and limitations of current diagnostic methods have been discussed in the previous section. For the assessment of anthelmintic efficacy in a range of helminths, several in vivo or in vitro methods are available, but only for very few parasitic species molecular tools have been developed. 3.1. In vivo and in vitro conventional methods One useful in vivo method is the controlled test, where infected animals are allocated to two groups (treatment and control). The reduction and/or elimination of parasites and, therefore, the efficacy of the treatment are calculated by counting remaining parasites in the host following necropsy. This method is very laborious, cost intense and, due to the necessity of post mortem examination, lacks the acceptance in farming communities. Another alternative is the faecal egg count reduction test (FECRT), where results of faecal egg counts before and after treatment are compared. The FECRT is simple, relatively easy to perform and currently the most commonly used method for the assessment of anthelmintic efficacy. Guidelines for the performance of a FECRT have been published (Coles et al., 1992) and reviewed (Coles et al., 2006) before. However, the data obtained by FECRT have been reported not to be highly reproducible (Miller et al., 2006) and a straight forward interpretation is hindered by a number of limiting factors associated with the FECRT. The accuracy of the FECRT is dependent on the sensitivity of the technique used for faecal egg counting. As outlined above, most currently available methods for faecal egg counting are still not highly accurate. Factors unrelated to treatment, such as non-uniform distribution of eggs in the faeces and inappropriate drug administration, can complicate the interpretation of FECRT data (Vidyashankar et al., 2007). Furthermore, the FECRT relies on the general assumption that faecal egg counts reflect adult parasitic burden, which is extremely different between parasitic species (Schnieder et al., 2006; Stear et al., 2006). Egg output by female parasites varies between species and may change under certain conditions (Gibbs, 1986; Anderson, 2000; Kumba et al., 2003; Jackson et al., 2006). Additionally, certain drugs, e.g., the macrocyclic lactones, may temporarily suppress egg laying (Le Jambre et al., 1995). Various attempts have been made to further optimise the procedure of the FECRT, including evaluation of egg counting methods (Cringoli et al., 2004; Le Jambre et al., 2007; Rinaldi et al., 2011), statistical approaches (Cabaret and Berrag, 2004; Torgerson et al., 2005; Dobson et al., 2009), determination of the best

J. Demeler et al. / Veterinary Parasitology 189 (2012) 52–64

sampling time (McKenna, 2010), comparison of available methods (McKenna, 2006, 2008) and handling of samples (Van Wyk and Van Wyk, 2002). In vitro tests are generally more cost effective than in vivo ones (Lacey et al., 1990). Generally, they involve incubation of free-living parasite stages in a range of drug concentrations followed by measurement of vitality in form of development, motility or migration. Data is used to generate dose–response-curves and EC50 or EC95 values are established. Currently, four main assays involving freeliving stages of a parasite are used, measuring: (i) hatching rates (e.g., the egg hatch assay), (ii) development rates (e.g., the larval development assay), (iii) motility/migration (e.g., larval motility assay, larval migration inhibition assay) and (iv) feeding (e.g., larval feeding assay). The egg hatch assay was first described by Le Jambre (1979). In the past, over 30 reports have been published using various modifications of the test for detection of resistance against benzimidazoles and/or levamisole. A standardised protocol for the performance of the test was published recently (von Samson-Himmelstjerna et al., 2009a). The usefulness of the test for detection of benzimidazole resistance has been evaluated in a number of field studies. The majority of studies revealed a good agreement between results obtained by FECRT and the egg hatch assay in sheep in the field (Maingi et al., 1998; Alvarez-Sánchez et al., 2006; Várady et al., 2006, 2007; Diez-Banos et al., 2008) indicating, that the test is an accurate, reliable and cost- effective alternative to the FECRT. The larval development assay was first reported in 1990 by Lacey et al. (1990) for the use of five different anthelmintics including benzimidazoles, levamisole and ivermectin. A variety of larval development assays have been published since (Taylor, 1990; Hubert and Kerboeuf, 1992; Gill et al., 1995; Gill and Lacey, 1998) and a commercial kit (DrenchRite® ) has been developed. A detailed description of a microagar-based larval development assay was published by Coles et al. (2006). The main advantage of the larval development assay compared to the egg hatch test is the possibility to screen for benzimidazole, levamisole and macrocyclic lactone resistance simultaneously on the same plate. Similar to the egg hatch test, it was shown that results obtained by the larval development assay were in good agreement with results obtained through FECRT in the field (Ancheta et al., 2004; Várady et al., 2006; Leathwick et al., 2006). Resistance against anthelmintics causing paralysis in somatic muscles can be assessed using larval motility or migration assays. Measurement of larval motility has been reported by observation, electronic detectors (e.g., micromotility metre) or migration through sieves. Adaptations for the use with different parasites species and drugs resulted in a wide range of similar assays being published (Taylor et al., 2002; Demeler et al., 2010). A standardisation of a larval migration inhibition assay has recently been published by Demeler et al. (2010), where highly reproducible results were obtained in a ring test. The field applicability of the assay has not been sufficiently studied and the potential problem of the assay to distinguish between species in naturally acquired mixed infections still needs to be solved (Kotze et al., 2006; Kleinschmidt, 2009).

59

Effects of anthelmintics on feeding have been evaluated using feeding assays with larval or adult stages of parasites (Geary et al., 1993; Jackson and Coop, 2000; Kotze and McClure, 2001; Sheriff et al., 2002; O’Grady and Kotze, 2004). Alvarez-Sanchez et al. (2005) reported a larval feeding inhibition assay suitable to distinguish ivermectin resistant and susceptible isolates of different nematodes. This assay has been evaluated for the assessment of anthelmintic efficacy (Diez-Banos et al., 2008) in the field. Resistance to macrocyclic lactones, as indicated by the FECRT, was confirmed on all farms using the larval feeding inhibition assay, indicating, that this in vitro assay has the potential to detect resistance to one of the most commonly used drug class. 3.2. Molecular methods To date methods for the molecular diagnosis of anthelmintic resistance in sheep nematodes have been developed and evaluated in the field only for the analysis of benzimidazole resistance. PCR based methods have been established for the assessment of presence/absence or the quantification of benzimidazole resistance-associated single nucleotide polymorphisms in the benzimidazole-target gene beta-tubulin isotype 1. In a recent paper, the mechanism of benzimidazole action, as well as conventional and real-time PCR protocols for the detection of SNPs at beta-tubulin codon 167 and 200 have been reviewed (von Samson-Himmelstjerna, 2006). For the determination of the resistance-associated TAC codon at position 200 robust PCR-restriction-fragmentlength-polymorphism techniques were developed for T. circumcincta (Shayan et al., 2007) and H. contortus (Tiwari et al., 2006). In addition to the codon 167 and 200 polymorphisms, which lead to an amino acid exchange from phenylalanine to tyrosine in resistant strains, a mutation within codon 198, encoding for alanine in resistant instead of glutamate, in susceptible individuals has been identified to confer benzimidazole resistance in some H. contortus isolates (Rufener et al., 2009a; Ghisi et al., 2007). The use of real-time PCR or pyrosequencing techniques allows the quantification of these alleles in DNA extracted from pools of nematode larvae and, thus the sensitive, reliable and cost-efficient assessment of benzimidazole resistance status in H. contortus field populations as part of routine diagnosis (von Samson-Himmelstjerna et al., 2009b; Höglund et al., 2009). The recent introduction of monepantel as the first commercialised member of the new nematocidal amino-acetonitrile derivatives drug class was already accompanied with the description of the drug target and even of a panel of mutations conferring reduced sensitivity to amino-acetonitrile derivatives in H. contortus (Kaminsky et al., 2008; Rufener et al., 2009b). The respective genetic changes in the monepantel-target, a nematode-specific nicotinic acetylcholine receptor of the DEG-3 subfamily, could become useful sites for molecular diagnosis of resistance. However, as the above-mentioned mutations were induced by experimental introduction or selection and while no field derived monepantel-resistant nematode isolates are available, it is unclear if they will be of practical relevance in this context.

60

J. Demeler et al. / Veterinary Parasitology 189 (2012) 52–64

Currently, there are no molecular tests available neither for the macrocyclic lactones, nor for the imidazothiazoles, nor for the tetrahydropyrimidines drug classes in any parasitic species. 4. Concluding remarks Current trends in diagnostics include the use of multiplex platforms (e.g., multi-plex PCR and in particular xMAP based LUMINEX systems). While multi-plex PCR systems have been widely applied to identify and specify viral (Vandenbussche et al., 2008; Abrahão et al., 2009) and bacterial (Pacheco et al., 2007; Moravkova et al., 2008; Berri et al., 2009; Hill et al., 2010) pathogens in sheep, the use of xMAP technology has, up to now, only been reported for exclusion of foot-and-mouth disease (Lenhoff et al., 2008). In general, xMAP technology has only rarely been used for simultaneous detection of parasites in clinical or environmental samples yet (McNamara et al., 2006; Bandyopadhyay et al., 2007; Li et al., 2010; Taniuchi et al., 2011). In addition to simple diagnosis of parasites, xMAP technology will probably also turn out to be a useful platform for discrimination of species, genospecies or strains by molecular techniques. Moreover, real-time highresolution-melt PCR and pyrosequencing might be used in the future for these purposes. A second innovative direction in diagnostics is the use of proteomic biomarkers to identify infected and/or diseased animals by rapid and relatively inexpensive analyses of serum samples using spectometry (in particular mass spectrometry, but also NMR spectometry) and multivariate analysis of spectra. For sheep, preliminary identification of such biomarkers has been described for scrapie (BatxelliMolina et al., 2010) and for fasciolosis. Despite its high costs regarding necessary technical equipment, the running costs of the method are fairly small, thus offering the opportunity to allow medium- to high-throughput screening of herds for multiple infectious agents with little hands-on time. Conflict of interest statement The authors declare that they are not aware of any conflict of interest concerning the writing and publication of this article. References Abrahão, J.S., Lima, L.S., Assis, F.L., Alves, P.A., Silva-Fernandes, A.T., Cota, M.M., Ferreira, V.M., Campos, R.K., Mazur, C., Lobato, Z.I., Trindade, G.S., Kroon, E.G., 2009. Nested-multiplex PCR detection of orthopoxvirus and parapoxvirus directly from exanthematic clinical samples. Virol. J. 6, 140. Abunna, F., Kasasa, D., Shelima, B., Megersa, B., Regassa, A., Amenu, K., 2009. Survey of tick infestation in small ruminants of Miesso district, West Harergie, Oromia Region, Ethiopia. Trop. Anim. Health Prod. 41, 969–972. ˜ Alasaad, S., Li, Q.Y., Lin, R.Q., Martín-Atance, P., Granados, J.E., Díez-Banos, P., Pérez, J.M., Zhu, X.Q., 2008. Genetic variability among Fasciola hepatica samples from different host species and geographical localities in Spain revealed by the novel SRAP marker. Parasitol. Res. 103, 181–186. Alcaide, M., Reina, D., Sánchez-López, J., Frontera, E., Navarrete, I., 2005a. Seroprevalence of Oestrus ovis (Diptera, Oestridae) infestations and

associated risk factors in ovine livestock from Southwestern Spain. J. Med. Entomol. 42, 327–331. Alcaide, M., Reina, D., Frontera, E., Navarrete, I., 2005b. Analysis of larval antigens of Oestrus ovis for the diagnosis of oestrosis by enzyme-linked immunosorbent assay. Med. Vet. Entomol. 19, 151–157. Alvarez-Sanchez, M.A., Perez Garcia, J., Bartley, D., Jackson, F., RojoVazquez, F.A., 2005. The larval feeding inhibition assay for the diagnosis of nematode anthelmintic resistance. Exp. Parasitol. 110, 56–61. Alvarez-Sánchez, M.A., Pérez-García, J., Cruz-Rojo, M.A., Rojo-Vázquez, F.A., 2006. Anthelmintic resistance in trichostrongylid nematodes of sheep farms in Northwest Spain. Parasitol. Res. 2006 (99), 78–83. Ancheta, P.B., Dumilon, R.A., Venturina, V.M., Cerbito, W.A., Dobson, R.J., LeJambre, L.F., Villar, E.C., Gray, G.D., 2004. Efficacy of benzimidazole anthelmintics in goats and sheep in the Philippines using a larval development assay. Vet. Parasitol. 120, 107–121. Anderson, R.C. (Ed.), 2000. Nematode Parasites of Vertebrates. Their Development and Transmission. , 2nd ed. CABI Publishing, Wallingford, UK, p. 672. Anuracpreeda, P., Wanichanon, C., Sobhon, P., 2008. Paramphistomum cervi: antigenic profile of adults as recognized by infected cattle sera. Exp. Parasitol. 118, 203–207. Arias, M., Morrondo, P., Hillyer, G.V., Sánchez-Andrade, R., Suárez, J.L., ˜ Lomba, C., Pedreira, J., Díaz, P., Díez-Banos, P., Paz-Silva, A., 2007. Immunodiagnosis of current fasciolosis in sheep naturally exposed to Fasciola hepatica by using a 2.9 kDa recombinant protein. Vet. Parasitol. 146, 46–49. Armua-Fernandez, M.T., Nonaka, N., Sakurai, T., Nakamura, S., Gottstein, B., Deplazes, P., Phiri, I.G., Katakura, K., Oku, Y., 2011. Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids. Parasitol. Int. 60, 84–89. Awad, W.S., Ibrahim, A.K., Salib, F.A., 2009. Using indirect ELISA to assess different antigens for the serodiagnosis of Fasciola gigantica infection in cattle, sheep and donkeys. Res. Vet. Sci. 86, 466–471. Bandyopadhyay, K., Kellar, K.L., Moura, I., Casaqui Carollo, M.C., Graczyk, T.K., Slemenda, S., Johnston, S.P., da Silva, A.J., 2007. Rapid microsphere assay for identification of Cryptosporidium hominis and Cryptosporidium parvum in stool and environmental samples. J. Clin. Microbiol. 45, 2835–2840. Bany, J., Pfeffer, A., Phegan, M.D., 1995a. Comparison of local and systemic responsiveness of lymphocytes in vitro to Bovicola ovis antigen and concanavalin A in B. ovis-infested and naive lambs. Int. J. Parasitol. 25, 1499–1504. Bany, J., Pfeffer, A., Phegan, M., Heath, A.C., 1995b. Proliferative responses of lymphocytes in Bovicola ovis-infested lambs. Int. J. Parasitol. 25, 765–768. Batxelli-Molina, I., Salvetat, N., Andréoletti, O., Guerrier, L., Vicat, G., Molina, F., Mourton-Gilles, C., 2010. Ovine serum biomarkers of early and late phase scrapie. BMC Vet. Res. 6, 49. Bautista-Garfias, C.R., Angulo-Contreras, R.M., Garay-Garzon, E., 1988. Serologic diagnosis of Oestrus ovis (Diptera:Oestridae) in naturally infested sheep. Med. Vet. Entomol. 2, 351–355. Bendixsen, T., Windon, R.G., Huntley, J.F., MacKellar, A., Davey, R.J., McClure, S.J., Emery, D.L., 2004. Development of a new monoclonal antibody to ovine chimeric IgE and its detection of systemic and local IgE antibody responses to the intestinal nematode Trichostrongylus colubriformis. Vet. Immunol. Immunopathol. 97, 11–24. Berri, M., Rekiki, A., Boumedine, K.S., Rodolakis, A., 2009. Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant’s clinical samples using multiplex PCR. BMC Microbiol. 9, 130. Bott, N.J., Campbell, B.E., Beveridge, I., Chilton, N.B., Rees, D., Hunt, P.W., Gasser, R.B., 2009. A combined microscopic-molecular method for the diagnosis of strongylid infections in sheep. Int. J. Parasitol. 39, 1277–1287. Boufana, B.S., Campos-Ponce, M., Naidich, A., Buishi, I., Lahmar, S., Zeyhle, E., Jenkins, D.J., Combes, B., Wen, H., Xiao, N., Nakao, M., Ito, A., Qiu, J., Craig, P.S., 2008. Evaluation of three PCR assays for the identification of the sheep strain (genotype 1) of Echinococcus granulosus in canid feces and parasite tissues. Am. J. Trop. Med. Hyg. 78, 777–783. Bowen, C.J., Jaworski, D.C., Wasala, N.B., Coons, L.B., 2010. Macrophage migration inhibitory factor expression and protein localization in Amblyomma americanum (Ixodidae). Exp. Appl. Acarol. 50, 343–352. Bowman, D.D., Lynn, R.C., Eberhard, M.L., 2009. Georgi’s Parasitology for Veterinarians, 9th ed. Saunders Elsevier, Edinburgh. Broglia, A., Heidrich, J., Lanfranchi, P., Nöckler, K., Schuster, R., 2009. Experimental ELISA for diagnosis of ovine dicrocoeliosis and application in a field survey. Parasitol. Res. 104, 949–953.

J. Demeler et al. / Veterinary Parasitology 189 (2012) 52–64 Cabaret, J., Berrag, B., 2004. Faecal egg count reduction test for assessing anthelmintic efficacy: average versus individually based estimations. Vet. Parasitol. 121, 105–113. Capuano, F., Rinaldi, L., Maurelli, M.P., Perugini, A.G., Musella, V., Cringoli, G., 2007. A comparison of five methods for DNA isolation from liver and rumen flukes to perform ITS-2+ amplification. Parassitologia 49, 27–31. Carnevale, S., Rodríguez, M.I., Guarnera, E.A., Carmona, C., Tanos, T., Angel, S.O., 2001. Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase. Diagn. Microbiol. Infect. Dis. 41, 43–49. Chilton, N.B., O’Callaghan, M.G., Beveridge, I., Andrews, R.H., 2007. Genetic markers to distinguish Moniezia expansa from M. benedeni (Cestoda: Anoplocephalidae) and evidence of the existence of cryptic species in Australia. Parasitol. Res. 100, 1187–1192. Colditz, I.G., Le Jambre, L.F., 2008. Development of a faecal occult blood test to determine the severity of Haemonchus contortus infections in sheep. Vet. Parasitol. 2008 (153), 93–99. Colditz, I.G., Le Jambre, L.F., Hosse, R., 2002. Use of lectin binding characteristics to identify gastrointestinal parasite eggs in faeces. Vet. Parasitol. 105, 219–227. Coles, G.C., Bauer, C., Borgsteede, F.H., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World Association for the Advancement of Veterinary Parasitology (WAAVP) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 44, 35–44. Coles, G.C., Jackson, F., Pomroy, W.E., Prichard, R.K., von SamsonHimmelstjerna, G., Silvestre, A., Taylor, M.A., Vercruysse, J., 2006. The detection of anthelmintic resistance in nematodes of veterinary importance. Vet. Parasitol. 136, 167–185. Cornelissen, J.B., Gaasenbeek, C.P., Borgsteede, F.H., Holland, W.G., Harmsen, M.M., Boersma, W.J., 2001. Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease. Int. J. Parasitol. 31, 728–737. Cramp, A.P., Sohn, J.H., James, P.J., 2009. Detection of cutaneous myiasis in sheep using an ‘electronic nose’. Vet. Parasitol. 166, 293–298. Cringoli, G., Rinaldi, L., Veneziano, V., Capelli, G., Scala, A., 2004. The influence of flotation solution, sample dilution and the choice of McMaster slide area (volume) on the reliability of the McMaster technique in estimating the faecal egg counts of gastrointestinal strongyles and Dicrocoelium dendriticum in sheep. Vet. Parasitol. 123, 121–131. Cutillas, C., Oliveros, R., de Rojas, M., Guevara, D.C., 2004. Determination of Trichuris skrjabini by sequencing of the ITS1-5.8S-ITS2 segment of the ribosomal DNA: comparative molecular study of different species of trichurids. J. Parasitol. 90, 648–652. De, Waal, 2012. Advances in diagnosis of protozoan diseases. Vet. Parasitol. 189, 65–74. Dehaghi, M.M., Fathi, S., Asl, E.N., Nezhad, H.A., 2011. Prevalence of ixodid ticks on cattle and sheep southeast of Iran. Trop. Anim. Health Prod. 43, 459–461. Demeler, J., Küttler, U., El-Abdellati, A., Stafford, K., Rydzik, A., Varady, M., Kenyon, F., Coles, G., Höglund, J., Jackson, F., Vercruysse, J., von Samson-Himmelstjerna, G., 2010. Standardization of the larval migration inhibition test for the detection of resistance to ivermectin in gastro intestinal nematodes of ruminants. Vet. Parasitol. 174, 58–64. Diez-Banos, P., Pedreira, J., Sanchez-Andrade, R., Francisco, I., Suarez, J.L., Diaz, P., Panadero, R., Arias, M., Painceira, A., Paz-Silva, A., Morrondo, P., 2008. Field evaluation for anthelmintic-resistant ovine gastrointestinal nematodes by in vitro and in vivo assays. J Parasitol. 94, 925–928. Dixit, A.K., Yadav, S.C., Sharma, R.L., 2002. 28 kDa Fasciola gigantica cysteine proteinase in the diagnosis of prepatent ovine fasciolosis. Vet. Parasitol. 109, 233–247. Dobson, R.J., Barnes, E.H., Birclijin, S.D., Gill, J.H., 1992. The survival of Ostertagia circumcincta and Trichostrongylus colubriformis in faecal culture as a source of bias in apportioning egg counts to worm species. Int. J. Parasitol. 22, 1005–1008. Dobson, R.J., Sangster, N.C., Besier, R.B., Woodgate, R.G., 2009. Geometric means provide a biased efficacy result when conducting a faecal egg count reduction test (FECRT). Vet. Parasitol. 161, 162–167. Dorchies, P., Wahetra, S., Lepetitcolin, E., Prevot, F., Grisez, C., Bergeaud, J.P., Hoste, H., Jacquiet, P., 2003. The relationship between nasal myiasis and the prevalence of enzootic nasal tumours and the effects of treatment of Oestrus ovis and milk production in dairy ewes of Roquefort cheese area. Vet. Parasitol. 113, 169–174. Duranton, C., Bergeaud, J.P., Dorchies, Ph., 1995. Dot enzyme linked immunosorbent assay for the rapid diagnosis of ovine oestrosis. Rev. Med. Vet. 146, 283–286. Duthaler, U., Rinaldi, L., Maurelli, M.P., Vargas, M., Utzinger, J., Cringoli, G., Keiser, J., 2010. Fasciola hepatica: comparison of the sedimentation

61

and FLOTAC techniques for the detection and quantification of faecal egg counts in rats. Exp. Parasitol. 126, 161–166. Eberhardt, A.G., Mayer, W.E., Bonfoh, B., Streit, A., 2008. The Strongyloides (Nematoda) of sheep and the predominant Strongyloides of cattle form at least two different, genetically isolated populations. Vet. Parasitol. 157, 89–99. El Amir, A., Rabee, I., Kamal, N., El Deeb, S., 2008. Evaluation of the diagnostic potential of different immunological techniques using polyclonal antibodies against Fasciola gigantica excretory/secretory antigens in sheep. Egypt. J. Immunol. 15, 65–74. El Ridi, R., Salah, M., Wagih, A., William, H., Tallima, H., El Shafie, M.H., Abdel Khalek, T., El Amir, A., Abo Ammou, F.F., Motawi, H., 2007. Fasciola gigantica excretory–secretory products for immunodiagnosis and prevention of sheep fasciolosis. Vet. Parasitol. 149, 219–228. Farjallah, S., Sanna, D., Amor, N., Ben Mehel, B., Piras, M.C., Merella, P., Casu, M., Curini-Galletti, M., Said, K., Garippa, G., 2009. Genetic characterization of Fasciola hepatica from Tunisia and Algeria based on mitochondrial and nuclear DNA sequences. Parasitol. Res. 105, 1617–1621. Gasser, R.B., Bott, N.J., Chilton, N.B., Hunt, P., Beveridge, I., 2008. Toward practical, DNA-based diagnostic methods for parasitic nematodes of livestock—bionomic and biotechnological implications. Biotechnol. Adv. 26, 325–334. Gatti, A., Alvarez, A.R., Araya, D., Mancini, S., Herrero, E., Santillan, G., Larrieu, E., 2007. Ovine echinococcosis. I: immunological diagnosis by enzyme immunoassay. Vet. Parasitol. 143, 112–121. Geary, T.G., Sims, S.M., Thomas, E.M., Vanover, L., Davis, J.P., Winterrowd, C.A., Klein, R.D., Ho, N.F., Thompson, D.P., 1993. Haemonchus contortus: ivermectin-induced paralysis of the pharynx. Exp. Parasitol. 77, 88–96. Ghisi, M., Kaminsky, R., Maser, P., 2007. Phenotyping and genotyping of Haemonchus contortus isolates reveals a new putative candidate mutation for benzimidazole resistance in nematodes. Vet. Parasitol. 144, 313–320. Gibbs, H.C., 1986. Hypobiosis in parasitic nematodes—an update. Adv. Parasitol. 25, 129–174. Gill, J.H., Lacey, E., 1998. Avermectin/milbemycin resistance in trichostrongyloid nematodes. Int. J. Parasitol. 28, 863–877. Gill, J.H., Redwin, J.M., Van Wyk, J.A., Lacey, E., 1995. Avermectin inhibition of larval development in Haemonchus contortus—effects of ivermectin resistance. Int. J. Parasitol. 25, 463–470. Goddard, P., Bates, P., Webster, K.A., 1999. Evaluation of a direct ELISA for the serodiagnosis of Oestrus ovis infections in sheep. Vet. Rec. 144, 497–501. ˜ Gómez-Munoz, M.T., Domínguez, I.A., Gómez-Iglesias, L.A., FernándezPérez, F.J., Méndez, S., de la Fuente, C., Alunda, J.M., Cuquerella, M., 2000. Serodiagnosis of haemonchosis with a somatic antigen (Hc26) in several breeds of sheep. J. Vet. Diagn. Invest. 12, 354–360. Hall, M.J., Adams, Z.J., Wyatt, N.P., Testa, J.M., Edge, W., Nikolausz, M., Farkas, R., Ready, P.D., 2009a. Morphological and mitochondrial DNA characters for identification and phylogenetic analysis of the myiasiscausing flesh fly Wohlfahrtia magnifica and its relatives, with a description of Wohlfahrtia monegrosensis sp. n. Wyatt & Hall. Med. Vet. Entomol. 23 (Suppl.), 59–71. Hall, M.J., Testa, J.M., Smith, L., Adams, Z.J., Khallaayoune, K., Sotiraki, S., Stefanakis, A., Farkas, R., Ready, P.D., 2009b. Molecular genetic analysis of populations of Wohlfahrt’s wound myiasis fly, Wohlfahrtia magnifica, in outbreak populations from Greece and Morocco. Med. Vet. Entomol. 23 (Suppl.), 72–79. Harmon, A.F., Williams, Z.B., Zarlenga, D.S., Hildreth, M.B., 2007. Real-time PCR for quantifying Haemonchus contortus eggs and potential limiting factors. Parasitol. Res. 101, 71–76. Hill, A.E., Dhungyel, O.P., Whittington, R.J., 2010. Diagnostic sampling strategies for virulent ovine footrot: simulating detection of Dichelobacter nodosus serogroups for bivalent vaccine formulation. Prev. Vet. Med. 95, 127–136. Höglund, J., Gustafsson, K., Ljungström, B.L., Engström, A., Donnan, A., Skuce, P., 2009. Anthelmintic resistance in Swedish sheep flocks based on a comparison of the results from the faecal egg count reduction test and resistant allele frequencies of the beta-tubulin gene. Vet. Parasitol. 161, 60–68. Huang, W.Y., He, B., Wang, C.R., Zhu, X.Q., 2004. Characterisation of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence. Vet. Parasitol. 120, 75–83. Hubert, J., Kerboeuf, D., 1992. A microlarval development assay for the detection of anthelmintic resistance in sheep nematodes. Vet. Rec. 130, 442–446. Huntley, J.F., Redmond, J., Welfare, W., Brennan, G., Jackson, F., Kooyman, F., Vervelde, L., 2001. Studies on the immunoglobulin E responses to

62

J. Demeler et al. / Veterinary Parasitology 189 (2012) 52–64

Teladorsagia circumcincta in sheep: purification of a major high molecular weight allergen. Parasite Immunol. 23, 227–235. Ilchmann, G., Hiepe, T., 1985. Immunologische Untersuchungen zur Intravitaldiagnostik der Östrose. Mh. Vet. Med. 40, 304–307. Innocenti, L., Masetti, M., Macchioni, G., Giorgi, F., 1995. Larval salivary gland proteins of the sheep nasal bot fly, (Oestrus ovis L.), are major immunogens in infested sheep. Vet. Parasitol. 60, 273–282. Jackson, F., Coop, R.L., 2000. The development of anthelmintic resistance in sheep nematodes. Parasitology 120 (Suppl.), 95–107. Jackson, R., Rhodes, A.P., Pomroy, W.E., Leathwick, D.M., West, D.M., Waghorn, T.S., Moffat, J.R., 2006. Anthelmintic resistance and management of parasites on beef cattle-rearing farms in the North Island of New Zealand. N. Z. Vet. J. 54, 289–296. James, P.J., Moon, R.D., 1998. Pruritus and dermal response to insect antigens in sheep infested with Bovicola ovis. Int. J. Parasitol. 28, 419–427. James, P.J., Garrett, J.A., Moon, R.D., 2002. Sensitivity of two-stage sampling to detect sheep biting lice (Bovicola ovis) in infested flocks. Vet. Parasitol. 103, 157–166. James, P.J., Bartholomaeus, F.W., Karlsson, L.J., 2007. Temporal relationship between infestation with lice (Bovicola ovis Schrank) and the development of pruritic behaviour and fleece derangement in sheep. Vet. Parasitol. 149, 251–257. Jas, R., Ghosh, J.D., Das, K., 2010. Polyclonal antibody based coproantigen detection immunoassay for diagnosis of Oesophagostomum columbianum infection in goats. Vet. Parasitol. 170, 262–267. Jia, W.Z., Yan, H.B., Guo, A.J., Zhu, X.Q., Wang, Y.C., Shi, W.G., Chen, H.T., Zhan, F., Zhang, S.H., Fu, B.Q., Littlewood, D.T., Cai, X.P., 2010. Complete mitochondrial genomes of Taenia multiceps, T. hydatigena and T. pisiformis: additional molecular markers for a tapeworm genus of human and animal health significance. BMC Genomics 11, 447. Johnson, D.A., Behnke, J.M., Coles, G.C., 2004. Copro-antigen capture ELISA for the detection of Teladorsagia (Ostertagia) circumcincta in sheep: improvement of specificity by heat treatment. Parasitology 129, 115–126. Jurasek, M.E., Bishop-Stewart, J.K., Storey, B.E., Kaplan, R.M., Kent, M.L., 2010. Modification and further evaluation of a fluorescein-labeled peanut agglutinin test for identification of Haemonchus contortus eggs. Vet. Parasitol. 169, 209–213. Kaminsky, R., Ducray, P., Jung, M., Clover, R., Rufener, L., Bouvier, J., Weber, S.S., Wenger, A., Wieland-Berghausen, S., Goebel, T., Gauvry, N., Pautrat, F., Skripsky, T., Froelich, O., Komoin-Oka, C., Westlund, B., Sluder, A., Mäser, P., 2008. A new class of anthelmintics effective against drugresistant nematodes. Nature 452, 176–180. Kara, M., Sarimehmetoglu, H.O., Gonenc, B., 2003. The determination of immune reactive proteins in Cysticercus tenuicollis cyst fluids by SDSPAGE and western blotting in sheep. Parasite 10, 141–145. Kiel, M., Josh, P., Jones, A., Windon, R., Hunt, P., Kongsuwan, K., 2007. Identification of immuno-reactive proteins from a sheep gastrointestinal nematode, Trichostrongylus colubriformis, using twodimensional electrophoresis and mass spectrometry. Int. J. Parasitol. 37, 1419–1429. Kleinschmidt, N., 2009. Untersuchung zum Vorkommen von Anthelminthikaresistenzen bei erstsömmrigen Rindern in norddeutschen Milchviehbetrieben. PhD thesis, University of Veterinary Medicine, Hannover, Germany. Kooyman, F.N., Van Kooten, P.J., Huntley, J.F., MacKellar, A., Cornelissen, A.W., Schallig, H.D., 1997. Production of a monoclonal antibody specific for ovine immunoglobulin E and its application to monitor serum IgE responses to Haemonchus contortus infection. Parasitol. 114, 395–406. Kotze, A.C., McClure, S.J., 2001. Haemonchus contortus utilises catalase in defence against exogenous hydrogen peroxide in vitro. Int. J. Parasitol. 31, 1563–1571. Kotze, A.C., Le Jambre, L.F., O’Grady, J., 2006. A modified larval migration assay for detection of resistance to macrocyclic lactones in Haemonchus contortus, and drug screening with Trichostrongylidae parasites. Vet. Parasitol. 137, 294–305. Kuhn, C., Lucius, R., Matthes, H.F., Meusel, G., Reich, B., Kalinna, B.H., 2008. Characterisation of recombinant immunoreactive antigens of the scab mite Sarcoptes scabiei. Vet. Parasitol. 153, 329–337. Kumba, F.F., Katjivena, H., Kauta, G., Lutaaya, E., 2003. Seasonal evolution of faecal egg output by gastrointestinal worms in goats oncommunal farms in eastern Namibia. Onderstepoort J. Vet. Res. 70, 265–271. Lacey, E., Redwin, J.M., Gil, L.J.H., Demargherit, i.V.M., Waller, P.J., 1990. A larval development assay for the simultaneous detection of broad spectrum anthelmintic resistance. In: Boray, J.C., Martin, P.J., Roush, R.T. (Eds.), Resistance of Parasites to Antiparasitic Drugs. MSD Agvet, New Jersey, pp. 177–184.

Lavikainen, A., Haukisalmi, V., Lehtinen, M.J., Laaksonen, S., Holmström, S., Isomursu, M., Oksanen, A., Meri, S., 2010. Mitochondrial DNA data reveal cryptic species within Taenia krabbei. Parasitol. Int. 59, 290–293. Le Jambre, L.F., 1979. Egg hatch as an in vitro assay of thiabendazole resistance in nematodes. Vet. Parasitol. 2, 385–391. Le Jambre, L.F., Gill, J.H., Lenane, I.J., Lacey, E., 1995. Characterisation of an avermectin resistant strain of Australian Haemonchus contortus. Int. J. Parasitol. 25, 691–698. Le Jambre, L.F., Dominik, S., Eady, S.J., Henshall, J.M., Colditz, I.G., 2007. Adjusting worm egg counts for faecal moisture in sheep. Vet. Parasitol. 145, 108–115. Leathwick, D.M., Miller, C.M., Atkinson, D.S., Haack, N.A., Alexander, R.A., Oliver, A.M., Waghorn, T.S., Potter, J.F., Sutherland, I.A., 2006. Drenching adult ewes: implications of anthelmintic treatments pre- and post-lambing on the development of anthelmintic resistance. N. Z. Vet. J. 54, 297–304. Leignel, V., Cabaret, J., 2001. Are Teladorsagia circumcincta (Nematoda) morphs equally able to survive under anthelmintic treatment in sheep on pastures? Parasitol. Res. 87, 687–692. Lenhoff, R.J., Naraghi-Arani, P., Thissen, J.B., Olivas, J., Carillo, A.C., Chinn, C., Rasmussen, M., Messenger, S.M., Suer, L.D., Smith, S.M., Tammero, L.F., Vitalis, E.A., Slezak, T.R., Hullinger, P.J., Hindson, B.J., Hietala, S.K., Crossley, B.M., McBride, M.T., 2008. Multiplexed molecular assay for rapid exclusion of foot-and-mouth disease. J. Virol. Methods 153, 61–69. Li, X., Du, A., Cai, W., Hou, Y., Pang, L., Gao, X., 2007. Evaluation of a recombinant excretory–secretory Haemonchus contortus protein for use in a diagnostic enzyme-linked immunosorbent assay. Exp. Parasitol. 115, 242–246. Li, W., Zhang, N., Gong, P., Cao, L., Li, J., Su, L., Li, S., Diao, Y., Wu, K., Li, H., Zhang, X., 2010. A novel multiplex PCR coupled with Luminex assay for the simultaneous detection of Cryptosporidium spp., Cryptosporidium parvum and Giardia duodenalis. Vet. Parasitol. 173, 11–18. Liu, G.H., Lin, R.Q., Li, M.W., Liu, W., Liu, Y., Yuan, Z.G., Song, H.Q., Zhao, G.H., Zhang, K.X., Zhu, X.Q., 2010. The complete mitochondrial genomes of three cestode species of Taenia infecting animals and humans. Mol. Biol Rep. 38, 2249–2256. Maingi, N., Bjørn, H., Dangolla, A., 1998. The relationship between faecal egg count reduction and the lethal dose 50% in the egg hatch assay and larval development assay. Vet. Parasitol. 77, 133–145. Manga-González, M.Y., González-Lanza, C., 2005. Field and experimental studies on Dicrocoelium dendriticum and dicrocoeliasis in northern Spain. J. Helminthol. 2005 (79), 291–302. Marcilla, A., Bargues, M.D., Mas-Coma, S., 2002. A PCR-RFLP assay for the distinction between Fasciola hepatica and Fasciola gigantica. Mol. Cell. Probes 16, 327–333. Mathis, A., Deplazes, P., 2006. Copro-DNA tests for diagnosis of animal taeniid cestodes. Parasitol. Int. 55 (Suppl.), 87–90. Maurelli, M.P., Rinaldi, L., Capuano, F., Perugini, A.G., Veneziano, V., Cringoli, G., 2007. Characterization of the 28S and the second internal transcribed spacer of ribosomal DNA of Dicrocoelium dendriticum and Dicrocoelium hospes. Parasitol. Res. 101, 1251–1255. McGarry, J.W., Ortiz, P.L., Hodgkinson, J.E., Goreish, I., Williams, D.J., 2007. PCR-based differentiation of Fasciola species (Trematoda: Fasciolidae), using primers based on RAPD-derived sequences. Ann. Trop. Med. Parasitol. 101, 415–421. McKenna, P.B., 2006. Further comparison of faecal egg count reduction test procedures: sensitivity and specificity. N. Z. Vet. J. 54, 365–366. McKenna, P.B., 2008. An examination of the relative reliability of laboratory case submissions in determining the prevalence of anthelmintic resistance in sheep nematodes in New Zealand, and the possible influence of test analysis methodology on such data. N. Z. Vet. J. 56, 55–59. McKenna, P.B., 2010. Has the optimum time for faecal nematode egg count reduction testing in sheep in New Zealand changed? N. Z. Vet. J. 58, 312–314. McManus, D.P., Thompson, R.C., 2003. Molecular epidemiology of cystic echinococcosis. Parasitology 127 (Suppl.), 37–51. McNamara, D.T., Kasehagen, L.J., Grimberg, B.T., Cole-Tobian, J., Collins, W.E., Zimmerman, P.A., 2006. Diagnosing infection levels of four human malaria parasite species by a polymerase chain reaction/ligase detection reaction fluorescent microsphere-based assay. Am. J. Trop. Med. Hyg. 74, 413–421. Mes, T.H., Eysker, M., Ploeger, H.W., 2007. A simple, robust and semiautomated parasite egg isolation protocol. Nat. Protoc. 2, 486–489. Mezo, M., González-Warleta, M., Ubeira, F.M., 2003. Optimized serodiagnosis of sheep fascioliasis by Fast-D protein liquid chromatography fractionation of Fasciola hepatica excretory–secretory antigens. J. Parasitol. 89, 843–849.

J. Demeler et al. / Veterinary Parasitology 189 (2012) 52–64 Mezo, M., González-Warleta, M., Carro, C., Ubeira, F.M., 2004. An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3). J. Parasitol. 90, 845–852. Mezo, M., González-Warleta, M., Ubeira, F.M., 2007. The use of MM3 monoclonal antibodies for the early immunodiagnosis of ovine fascioliasis. J. Parasitol. 93, 65–72. Miller, C.M., Waghorn, T.S., Leathwick, D.M., Gilmour, M.L., 2006. How repeatable is a faecal egg count reduction test? N. Z. Vet. J. 54, 323–328. Mohammed, M.S., Hassan, S.M., 2007. Distribution and population dynamics of ticks (Acari: Ixodidae) infesting sheep in Sennar State, Sudan. Onderstepoort J. Vet. Res. 74, 301–306. Molina, J.M., Hernández, Y., Ruiz, A., González, J.F., Argüello, A., Ferrer, O., Forbes, A.B., 2009. Preliminary study on the use of a Teladorsagia circumcincta bulk milk ELISA test in dairy goats under experimental conditions. Vet. Parasitol. 166, 228–234. Molloy, J.B., Anderson, G.R., Fletcher, T.I., Landmann, J., Knight, B.C., 2005. Evaluation of a commercially available enzyme-linked immunosorbent assay for detecting antibodies to Fasciola hepatica and Fasciola gigantica in cattle, sheep and buffaloes in Australia. Vet. Parasitol. 130, 207–212. Moravkova, M., Hlozek, P., Beran, V., Pavlik, I., Preziuso, S., Cuteri, V., Bartos, M., 2008. Strategy for the detection and differentiation of Mycobacterium avium species in isolates and heavily infected tissues. Res. Vet. Sci. 85, 257–264. Morcombe, P.W., Young, G.E., Ball, M.D., Dunlop, R.H., 1996. The detection of lice (Bovicola ovis) in mobs of sheep: a comparison of fleece parting, the lamp test and the table locks test. Aust. Vet. J. 73, 170–173. Mousa, W.M., 2001. Evaluation of cercarial antigen for the serodiagnosis of fasciolosis in experimentally and naturally infected sheep. Vet. Parasitol. 97, 47–54. Musella, V., Catelan, D., Rinaldi, L., Lagazio, C., Cringoli, G., Biggeri, A., 2011. Covariate selection in multivariate spatial analysis of ovine parasitic infection. Prev. Vet. Med. 99, 69–77. Nabian, S., Rahbari, S., Changizi, A., Shayan, P., 2009. The distribution of Hyalomma spp. ticks from domestic ruminants in Iran. Med. Vet. Entomol. 23, 281–283. Naem, S., 2007. Morphological differentiation among three Thelazia species (Nematoda: Thelaziidae) by scanning electron microscopy. Parasitol. Res. 101, 145–151. Nguyen, T.G., Van De, N., Vercruysse, J., Dorny, P., Le, T.H., 2009. Genotypic characterization and species identification of Fasciola spp. with implications regarding the isolates infecting goats in Vietnam. Exp. Parasitol. 123, 354–361. Nisbet, A.J., Huntley, J.F., 2006. Progress and opportunities in the development of vaccines against mites, fleas and myiasis-causing flies of veterinary importance. Parasite Immunol. 28, 165–172. Nisbet, A.J., Halliday, A.M., Parker, L., Smith, W.D., Kenyon, F., Knox, D.P., Huntley, J.F., 2008. Psoroptes ovis: identification of vaccine candidates by immunoscreening. Exp. Parasitol. 120, 194–199. ´ M., Mikes, L., Kovarcík, K., Koudela, B., 2007. ´ A., Kasny, Novobilsky, Humoral immune responses during experimental infection with Fascioloides magna and Fasciola hepatica in goats and comparison of their excretory/secretory products. Parasitol. Res. 101, 357–364. Ochs, H., Mathis, A., Deplazes, P., 1999. Single nucleotide variation in rDNA ITS-2 differentiates Psoroptes isolates from sheep and rabbits from the same geographical area. Parasitology 119, 419–424. Ochs, H., Lonneux, J., Losson, B.J., Deplazes, P., 2001. Diagnosis of psoroptic sheep scab with an improved enzyme-linked immunosorbent assay. Vet. Parasitol. 96, 233–242. O’Grady, J., Kotze, A.C., 2004. Haemonchus contortus: in vitro drug screening assays with the adult life stage. Exp. Parasitol. 106, 164–172. Oliveros, R., Cutillas, C., De Rojas, M., Arias, P., 2000. Characterization of four species of Trichuris (Nematoda: Enoplida) by their second internal transcribed spacer ribosomal DNA sequence. Parasitol. Res. 86, 1008–1013. Omeragic, J., 2011. Ixodid ticks in bosnia and herzegovina. Exp. Appl. Acarol. 53, 301–309. Otranto, D., Traversa, D., 2002. A review of dicrocoeliosis of ruminants including recent advances in the diagnosis and treatment. Vet. Parasitol. 107, 317–335. Otranto, D., Traversa, D., 2004. Molecular characterization of the first internal transcribed spacer of ribosomal DNA of the most common species of eyeworms (Thelazioidea: Thelazia). J. Parasitol. 90, 185–188. Otranto, D., Tarsitano, E., Traversa, D., De Luca, F., Giangaspero, A., 2003. Molecular epidemiological survey on the vectors of Thelazia gulosa, Thelazia rhodesi and Thelazia skrjabini (Spirurida: Thelaziidae). Parasitology 127, 365–373.

63

Otranto, D., Rehbein, S., Weigl, S., Cantacessi, C., Parisi, A., Lia, R.P., Olson, P.D., 2007. Morphological and molecular differentiation between Dicrocoelium dendriticum (Rudolphi, 1819) and Dicrocoelium chinensis (Sudarikov and Ryjikov, 1951) Tang and Tang, 1978 (Platyhelminthes: Digenea). Acta Trop. 104, 91–98. Pacheco, L.G., Pena, R.R., Castro, T.L., Dorella, F.A., Bahia, R.C., Carminati, R., Frota, M.N., Oliveira, S.C., Meyer, R., Alves, F.S., Miyoshi, A., Azevedo, V., 2007. Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samples. J. Med. Microbiol. 56, 480–486. Papadopoulos, E., Prevot, F., Jacquiet, P., Duranton, C., Bergeaud, J.P., Kalaitzakis, E., Dorchies, P., 2001. Seasonal variation of Oestrus ovisspecific antibodies in sheep and goats mixed flocks in Greece. Vet. Parasitol. 95, 73–77. Papadopoulos, E., Prevot, F., Diakou, A., Dorchies, P., 2006. Comparison of infection rates of Oestrus ovis between sheep and goats kept in mixed flocks. Vet. Parasitol. 138, 382–385. Pavlidou, V., Gerou, S., Kahrimanidou, M., Papa, A., 2008. Ticks infesting domestic animals in northern Greece. Exp. Appl. Acarol. 45, 195–198. Paz-Silva, A., Sanchez-Andrade, R., Suarez, J.L., Pedreira, J., Arias, M., Lopez, C., Panadero, R., Diaz, P., Diez-Banos, P., Morrondo, P., 2003. Prevalence of natural ovine fasciolosis shown by demonstrating the presence of serum circulating antigens. Parasitol. Res. 91, 328–331. Pernthaner, A., Shaw, R.J., McNeill, M.M., Morrison, L., Hein, W.R., 2005. Total and nematode-specific IgE responses in intestinal lymph of genetically resistant and susceptible sheep during infection with Trichostrongylus colubriformis. Vet. Immunol. Immunopathol. 104, 69–80. Pfeffer, A., Shoemaker, C.B., Shaw, R.J., Green, R.S., Shu, D., 2010. Identification of an abundant allergen from the sheep louse, Bovicola ovis. Int. J. Parasitol. 40, 911–919. Phiri, I.K., Phiri, A.M., Harrison, L.J., 2006. Serum antibody isotype responses of Fasciola-infected sheep and cattle to excretory and secretory products of Fasciola species. Vet. Parasitol. 141, 234–242. Prasad, A., Nasir, A., Singh, N., 2008. Detection of anti-Haemonchus contortus antibodies in sheep by dot-ELISA with immunoaffinity purified fraction of ES antigen during prepatency. Indian J. Exp. Biol. 46, 94–99. Ramadan, N.I., Saber, L.M., 2004. Detection of genetic variability in nonhuman isolates of Fasciola hepatica and Fasciola gigantica by the RAPD-PCR technique. J. Egypt. Soc. Parasitol. 34, 679–689. Ramajo-Hernández, A., Oleaga, A., Ramajo-Martín, V., Pérez-Sánchez, R., 2007. Carbohydrate profiling and protein identification of tegumental and excreted/secreted glycoproteins of adult Schistosoma bovis worms. Vet. Parasitol. 144, 45–60. Redmond, D.L., Smith, S.K., Halliday, A., Smith, W.D., Jackson, F., Knox, D.P., Matthews, J.B., 2006. An immunogenic cathepsin F secreted by the parasitic stages of Teladorsagia circumcincta. Int. J. Parasitol. 36, 277–286. Rehbein, S., Kokott, S., Lindner, T., 1999. Evaluation of techniques for the enumeration of Dicrocoelium eggs in sheep faeces. Zentralbl. Veterinarmed A 46, 133–139. ˜ B., González-Lanza, Revilla-Nuín, B., Manga-González, M.Y., Minambres, C., 2005. Partial characterization and isolation of 130 kDa antigenic protein of Dicrocoelium dendriticum adults. Vet. Parasitol. 134, 229–240. Rieu, E., Recca, A., Bénet, J.J., Saana, M., Dorchies, P., Guillot, J., 2007. Reliability of coprological diagnosis of Paramphistomum sp. infection in cows. Vet. Parasitol. 146, 249–253. Rinaldi, L., Coles, G.C., Maurelli, M.P., Musella, V., Cringoli, G., 2011. Calibration and diagnostic accuracy of simple flotation, McMaster and FLOTAC for parasite egg counts in sheep. Vet. Parasitol. 177, 345–352. Rioux, M.C., Carmona, C., Acosta, D., Ward, B., Ndao, M., Gibbs, B.F., Bennett, H.P., Spithill, T.W., 2008. Discovery and validation of serum biomarkers expressed over the first twelve weeks of Fasciola hepatica infection in sheep. Int. J. Parasitol. 38, 123–136. Rodríguez-Cadenas, F., Carbajal-González, M.T., Fregeneda-Grandes, J.M., Aller-Gancedo, J.M., Huntley, J.F., Rojo-Vázquez, F.A., 2010. Development and evaluation of an antibody ELISA for sarcoptic mange in sheep and a comparison with the skin-scraping method. Prev. Vet. Med. 96, 82–92. Roeber, F., Jex, A.R., Campbell, A.J., Campbell, B.E., Anderson, G.A., Gasser, R.B., 2011. Evaluation and application of a molecular method to assess the composition of strongylid nematode populations in sheep with naturally acquired infections. Infect. Genet. Evol. 5, 849–854. Rokni, M.B., Mirhendi, H., Mizani, A., Mohebali, M., Sharbatkhori, M., Kia, E.B., Abdoli, H., Izadi, S., 2010. Identification and differentiation of Fasciola hepatica and Fasciola gigantica using a simple PCR-restriction enzyme method. Exp. Parasitol. 124, 209–213.

64

J. Demeler et al. / Veterinary Parasitology 189 (2012) 52–64

Rufener, L., Kaminsky, R., Mäser, P., 2009a. In vitro selection of Haemonchus contortus for benzimidazole resistance reveals a mutation at amino acid 198 of beta-tubulin. Mol. Biochem. Parasitol. 168, 120–122. Rufener, L., Mäser, P., Roditi, I., Kaminsky, R., 2009b. Haemonchus contortus acetylcholine receptors of the DEG-3 subfamily and their role in sensitivity to monepantel. PLoS Pathog. 5, e1000380. Sabry, M.A., Taher, E.S., 2008. Saliva as an easy specimen for diagnosis of human and animal fascioliosis. Egypt J. Immunol. 15, 43–51. Santos, M.M., Garcia, T.C., Orsini, M., Disch, J., Katz, N., Rabello, A., 2000. Oral fluids for the immunodiagnosis of Schistosoma mansoni infection. Trans. R. Soc. Trop. Med. Hyg. 94, 289–292. Sato, H., Tanaka, S., Une, Y., Torii, H., Yokoyama, M., Suzuki, K., Amimoto, A., Hasegawa, H., 2008. The stomal morphology of parasitic females of Strongyloides spp. by scanning electron microscopy. Parasitol. Res. 102, 541–546. Schallig, H.D., Hornok, S., Cornelissen, J.B., 1995. Comparison of two enzyme immunoassays for the detection of Haemonchus contortus infections in sheep. Vet. Parasitol. 57, 329–338. Schnieder, T., 2006. Veterinärmedizinische Parasitologie, 6th ed. Parey Verlag, Stuttgart, pp. 180–188. Shahnazi, M., Hejazi, H., Salehi, M., Andalib, A.R., 2011. Molecular characterization of human and animal Echinococcus granulosus isolates in Isfahan, Iran. Acta Trop. 117, 47–50. Shaw, R.J., Gatehouse, T.K., McNeill, M.M., 1998. Serum IgE responses during primary and challenge infections of sheep with Trichostrongylus colubriformis. Int. J. Parasitol. 28, 293–302. Shayan, P., Eslami, A., Borji, H., 2007. Innovative restriction site created PCR-RFLP for detection of benzimidazole resistance in Teladorsagia circumcincta. Parasitol. Res. 100, 1063–1068. Sheriff, J.C., Kotze, A.C., Sangster, N.C., Martin, R.J., 2002. Effects of macrocyclic lactone anthelmintics on feeding and pharyngeal pumping in Trichostrongylus colubriformis in vitro. Parasitology 125, 477–484. Siegfried, E., Ochs, H., Deplazes, P., 2004. Clinical development and serological antibody responses in sheep and rabbits experimentally infested with Psoroptes ovis and Psoroptes cuniculi. Vet. Parasitol. 124, 109–124. Simsek, S., Balkaya, I., Ciftci, A.T., Utuk, A.E., 2011. Molecular discrimination of sheep and cattle isolates of Echinococcus granulosus by SSCP and conventional PCR in Turkey. Vet. Parasitol. 178, 367–369. Smith, W.D., Pettit, D.M., 2004. Immunization against sheep scab: preliminary identification of fractions of Psoroptes ovis which confer protective effects. Parasite Immunol. 26, 307–314. Smith, W.D., van den Broek, A., Huntley, J., Pettit, D., Machell, J., Miller, H.R., Bates, P., Taylor, M., 2001. Approaches to vaccines for Psoroptes ovis (sheep scab). Res. Vet. Sci. 70, 87–91. Smith, W.D., Bates, P., Pettit, D.M., Van Den Broek, A., Taylor, M.A., 2002. Attempts to immunize sheep against the scab mite, Psoroptes ovis. Parasite Immunol. 24, 303–310. Snábel, V., Altintas, N., D’Amelio, S., Nakao, M., Romig, T., Yolasigmaz, A., Gunes, K., Turk, M., Busi, M., Hüttner, M., Sevcová, D., Ito, A., Altintas, N., ´ P., 2009. Cystic echinococcosis in Turkey: genetic variability Dubinsky, and first record of the pig strain (G7) in the country. Parasitol. Res. 105, 145–154. Stear, M.J., Abuagob, O., Benothman, M., Bishop, S.C., Innocent, G., Kerr, A., Mitchell, S., 2006. Variation among faecal egg counts following natural nematode infection in Scottish Blackface lambs. Parasitology 132, 275–280. Tabouret, G., Prevot, F., Bergeaud, J.P., Dorchies, P., Jacquiet, P., 2001. Oestrus ovis (Diptera: Oestridae): sheep humoral immune response to purified excreted/secreted salivary gland 28 kDa antigen complex from second and third instar larvae. Vet. Parasitol. 101, 53–66. Taniuchi, M., Verweij, J.J., Noor, Z., Sobuz, S.U., Lieshout, L., Petri Jr., W.A., Haque, R., Houpt, E.R., 2011. High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites. Am. J. Trop. Med. Hyg. 84, 332–337. Taylor, M.A., 1990. A larval development test for the detection of anthelmintic resistance in nematodes of sheep. Res. Vet. Sci. 49, 198–202. Taylor, M.A., 2010. Parasitological examinations in shepe health management. Small Rumin. Res. 92, 120–125. Taylor, M.A., Hunt, K.R., Goodyear, K.L., 2002. Anthelmintic resistance detection methods. Vet. Parasitol. 103, 183–194. Thompson, R.C., 2008. The taxonomy, phylogeny and transmission of Echinococcus. Exp. Parasitol. 119, 439–446. Thompson, R.C., McManus, D.P., 2002. Towards a taxonomic revision of the genus Echinococcus. Trends. Parasitol. 18, 452–457.

Tiwari, J., Kumar, S., Kolte, A.P., Swarnkar, C.P., Singh, D., Pathak, K.M., 2006. Detection of benzimidazole resistance in Haemonchus contortus using RFLP-PCR technique. Vet. Parasitol. 138, 301–307. Torgerson, P.R., Deplazes, P., 2009. Echinococcosis: diagnosis and diagnostic interpretation in population studies. Trends. Parasitol. 25, 164–170. Torgerson, P.R., Schnyder, M., Hertzberg, H., 2005. Detection of anthelmintic resistance: a comparison of mathematical techniques. Vet. Parasitol. 128, 291–298. ˜ L., Mezo, M., Valero, M.A., Ubeira, F.M., Khoubbane, M., Artigas, P., Muino, Pérez-Crespo, I., Periago, M.V., Mas-Coma, S., 2009. MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica. Vet. Parasitol. 159, 77–81. van Wyk, J.A., Cabaret, J., Michael, L.M., 2004. Morphological identification of nematode larvae of small ruminants and cattle simplified. Vet. Parasitol. 119, 277–306. Van Wyk, J.A., Van Wyk, L., 2002. Freezing of sheep faeces invalidates Haemonchus contortus faecal egg counts by the McMaster technique. Onderstepoort J. Vet. Res. 69, 299–304. Vandenbussche, F., Vanbinst, T., Vandemeulebroucke, E., Goris, N., Sailleau, C., Zientara, S., De Clercq, K., 2008. Effect of pooling and multiplexing on the detection of bluetongue virus RNA by real-time RT-PCR. J. Virol. Methods 152, 13–17. Várady, M., Cernanská, D., Corba, J., 2006. Use of two in vitro methods for the detection of anthelmintic resistant nematode parasites on Slovak sheep farms. Vet. Parasitol. 2006 (135), 325–331. Várady, M., Cudeková, P., Corba, J., 2007. In vitro detection of benzimidazole resistance in Haemonchus contortus: egg hatch test versus larval development test. Vet. Parasitol. 2007 (149), 104–110. Varcasia, A., Lightowlers, M.W., Cattoli, G., Cancedda, G.M., Canu, S., Garippa, G., Scala, A., 2006. Genetic variation within Taenia multiceps in Sardinia, Western Mediterranean (Italy). Parasitol. Res. 99, 622–666. Varcasia, A., Canu, S., Kogkos, A., Pipia, A.P., Scala, A., Garippa, G., Seimenis, A., 2007. Molecular characterization of Echinococcus granulosus in sheep and goats of Peloponnesus, Greece. Parasitol. Res. 101, 1135–1139. Vidyashankar, A.N., Kaplan, R.M., Chan, S., 2007. Statistical approach to measure the efficacy of anthelmintic treatment on horse farms. Parasitology 134, 2027–2039. von Samson-Himmelstjerna, G., 2006. Molecular diagnosis of anthelmintic resistance. Vet. Parasitol. 136, 99–107. von Samson-Himmelstjerna, G., Coles, G.C., Jackson, F., Bauer, C., Borgsteede, F., Cirak, V.Y., Demeler, J., Donnan, A., Dorny, P., Epe, C., Harder, A., Hoglund, J., Kaminsky, R., Kerboeuf, D., Kuttler, U., Papadopoulos, E., Posedi, J., Small, J., Varady, M., Vercruysse, J., Wirtherle, N., 2009a. Standardization of the egg hatch test for the detection of benzimidazole resistance in parasitic nematodes. Parasitol. Res. 105, 825–834. von Samson-Himmelstjerna, G., Walsh, T.K., Donnan, A.A., Carrière, S., Jackson, F., Skuce, P.J., Rohn, K., Wolstenholme, A.J., 2009b. Molecular detection of benzimidazole resistance in Haemonchus contortus using real-time PCR and pyrosequencing. Parasitology 136, 349–358. Wang, C.R., Gao, J.F., Zhu, X.Q., Zhao, Q., 2012. Characterization of Bunostomum trigonocephalum and Bunostomum phlebotomum from sheep and cattle by internal transcribed spacers of nuclear ribosomal DNA. Res. Vet. Sci. 92, 99–102. Ward, M.P., Armstrong, R.T., 2000. Inspection of wool lots at sales as a diagnostic test for louse infestation. Vet. Parasitol. 90, 119–128. Wimmer, B., Craig, B.H., Pilkington, J.G., Pemberton, J.M., 2004. Noninvasive assessment of parasitic nematode species diversity in wild Soay sheep using molecular markers. Int. J. Parasitol. 34, 625–631. Yadav, S.C., Saini, M., Raina, O.K., Nambi, P.A., Jadav, K., Sriveny, D., 2005. Fasciola gigantica cathepsin-L cysteine proteinase in the detection of early experimental fasciolosis in ruminants. Parasitol. Res. 97, 527–534. Yilma, J.M., 1992. Contribution a l’etude de l’epidemiologie, du diagnostic immunologique et de la physiopathologie de l’oestrose ovine (Oestrus ovis Linne 1761). These. INP, Toulouse, France. Yisheng, L., Wenping, D., Kuiyang, Z., Linlin, F., Ming, C., 2005. Comparative study on rapid dot-immunogold staining and two immunogold silver staining assays for diagnosing schistosomiasis japonica. Southeast Asian J. Trop. Med. Public Health 36, 79–82. Yokannanth, S., Ghosh, S., Gupta, S.C., Suresh, M.G., Saravanan, D., 2005. Characterization of specific and cross-reacting antigens of Fasciola gigantica by immunoblotting. Parasitol. Res. 97, 41–48. Young, N.D., Hall, R.S., Jex, A.R., Cantacessi, C., Gasser, R.B., 2010. Elucidating the transcriptome of Fasciola hepatica – a key to fundamental and biotechnological discoveries for a neglected parasite. Biotechnol. Adv. 28, 222–231.