Acute phase protein response in Alpine ibex with sarcoptic mange

Md. Mizanur Rahman a,b, Cristina Lecchi a, Cristina Fraquelli a,Paola Sartorelli a,c, Fabrizio Ceciliani a,c,*a Department of Animal Pathology, Hygiene and Veterinary Public Health, Via Celoria 10, 20133 Milano, Italyb Department of Medicine & Surgery, Chittagong Veterinary and Animal Sciences University, Zakir Hossain Road, Chittangong 4202, Bangladeshc Interdepartmental Center for Studies on Mammary Gland (CISMA), Universita di Milano, Italy

Veterinary Parasitology 168 (2010) 293–298

A R T I C L E I N F O

Article history:

Received 22 May 2009

Received in revised form 26 November 2009

Accepted 3 December 2009

Keywords:

Acute phase proteins

Sarcoptes scabiei

Mange

Alpine ibex

Capra ibex

A B S T R A C T

The acute phase proteins (APP) are a group of serum proteins that change their

concentration in animals following external or internal challenges, such as infection,

inflammation or stress. The concentrations of four APPs, including serum amyloid A (SAA),

haptoglobin (Hp), a1-acid glycoprotein (AGP) and ceruloplasmin (Cp) were determined in

serum collected from healthy Alpine ibexes (Capra ibex) and ibexes with Sarcoptes scabiei

mange. Primary structures of all four APPs were determined by cDNA sequencing. The

concentrations of all four APPs were higher in serum of animals with clinical signs of

sarcoptic mange when compared to healthy animals. Two of the APPs, including SAA and

AGP, acted as major APPs, since their serum concentrations were increased more than 10-

folds when compared to healthy animals (P< 0.001). The other two APPs, including Hp and

Cp, acted as minor acute phase proteins, as their concentrations were increased from two

to five folds (P< 0.001).

These findings provide a remarkable potential as diagnostic markers for the early

detection of sarcoptic mange in free ranging animals.

� 2009 Elsevier B.V. All rights reserved.

Contents lists available at ScienceDirect

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1. Introduction

Sarcoptic mange is a severe contagious disease world-wide affecting humans and other mammalians caused bythe burrowing mite Sarcoptes scabiei (Arlian, 1989;Burgess, 1994; Walton et al., 2004). Scabies epizooticshave been reported in almost all European wild ruminants,including chamois (both subspecies Rupicapra pyrenaica

and Rupicapra rupicapra) (Leon-Vizcaıno, 1990; Rossi et al.,2007), ibex (both subspecies Capra pyrenaica and Capra

ibex) (Leon-Vizcaıno et al., 1999; Schaschl, 2003; Onder-sheka et al., 1968; Rossi et al., 1995), Barbary sheep(Ammotragus lervia) (Gonzalez-Candela et al., 2004), reddeer (Cervus elaphus) (Oleaga et al., 2008a), roe deer

* Corresponding author at: Department of Animal Pathology, Hygiene

and Veterinary Public Health, Via Celoria 10, 20133 Milano, Italy.

Tel.: +39 02 50318100; fax: +39 02 50318095.

E-mail address: fabrizio.ceciliani@unimi.it (F. Ceciliani).

0304-4017/$ – see front matter � 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2009.12.001

(Capreolus capreolus) (Oleaga et al., 2008b) and differentother wild animals around the world (Pence and Ueck-ermann, 2002).

Sarcoptic mange first induces a typical hypercheratosisand local inflammation, followed by a hyperplasia in thestratum granulosum. Eventually, affected areas featuresevere alopecia and scab formation (Arenas et al., 2002;Arlian et al., 1990). Prolonged infestations with scabies, orinfestations in already debilitated animals, have effects onorgans (Burgess, 1994) and even body weight and size(Serrano et al., 2007). The blood oxidant/antioxidantbalance is likely to be modified as well, as it was foundin sarcoptic dogs (Camkerten et al., 2009). Affected animalsoften succumb to the infestation (Pence and Ueckermann,2002). Immune response against mange infestation is notcompletely understood. Scabies extracts modulateimmune response of the host by down-regulating thekeratinocyte expression of IL-1 – receptor antagonist andby increasing the expression level of other cytokines,including IL-6, granulocyte-colony stimulating factor

M.M. Rahman et al. / Veterinary Parasitology 168 (2010) 293–298294

(G-CSF) and IL-10 (Arlian et al., 2003, 2006). Moreover,cutaneous lesions caused by sarcoptic mange are likely toserve as a route for secondary invaders, such as bacteria forexample. A systemic inflammatory status due to mangeinfestation probably occurs, but it has not been demon-strated, at least so far. This acute phase reaction issupposed to be caused by the invasion of bacteria fromcutaneous lesions or by the immunomodulatory activity ofmange products on pro-inflammatory cytokines, includingIL-6 and IL-1b which, in turn, up-regulate the expression ofacute phase proteins (Suffredini et al., 1999).

The acute phase proteins (APPs) are a group ofstructurally unrelated serum proteins that change inconcentration in animals subjected to external or internalchallenges, such as infection, inflammation, surgicaltrauma or stress. The circulating concentration of theAPP is species-specific and related to the severity of thedisorder and the extent of tissue damage in the affectedanimal. Since APP’s serum amount may increase (positiveAPPs) or decrease (negative APPs), it may also providediagnostic and prognostic information, thus representing apowerful means of monitoring animal health (Petersenet al., 2004).

To the best of knowledge of the authors, the few reportson haematological and biochemical intervals in ibex(Sartorelli et al., 1991; Perez et al., 1999, 2003; Degiorgiset al., 2000; Lastras et al., 2000) did not include APPs.Therefore, the dynamic of acute phase response in ibex isstill unknown.

The present study was carried out to gain insight intothe acute phase reaction during S. scabiei infestation inibex. In detail, our experiments aimed, in a first instance,to determine the primary structures of the APPs: SAA, Hpand AGP. These proteins were selected because they arethe most common APPs in ruminants (Petersen et al.,2004), but also because a very recent report demonstratedthat these three proteins increased after pro-inflamma-tory challenge in goats (Gonzalez et al., 2008). Cerulo-plasmin concentration was also studied, since severalreports demonstrated that this protein is an indicator ofinfection in ruminants (Murata et al., 2004), and itsamount can be measured in wild ruminants as well(Barboza and Blake, 2001). This information was con-sidered the requisite to reach the second objective, thatwas to compare the serum APP concentrations betweenhealthy ibexes and those affected by sarcoptic mange.Results are presented hereby.

2. Materials and methods

2.1. Identification of animals and diagnosis of mange

The study was carried out on a population of wildAlpine ibex that was located in Dolomite Alps, NorthernItaly (Universal Transverse Mercator coordinates: 32T712826 mE 5150746 mN) during a major outbreak of S.

scabiei epidemic that occurred between years 2004 and2005. Blood samples were collected from 43 males (meanage: 5.7 years� 2.45 s.d.) and 10 females (mean age: 6.9years� 4.53 s.d.) from years 2003 to 2005. Age of animalswas determined by horn segment counts (Habermehl, 1985).

APP concentration was assayed from 15 clinicallyhealthy ibexes (Group 1) (4 females and 11 males). Theseanimals were also sarcoptes mange negative, as deter-mined by ELISA search for specific antibodies (Rambozziet al., 2004). A second group (Group 2: 38 animals – 6females and 32 males) included animals with evidentclinical signs of sarcoptic mange, as determined by skinlesions associated with detection of the causal agent S.

scabiei from cutaneous biopsies.Blood samples were obtained by jugular veinpuncture

of ibexes captured for management/therapy purposesduring the summer season (from July to September). Bloodcontaining vials were refrigerated at 4 8C, left to clot andthen centrifuged at 2500� g for 15 min. Serum wasseparated and stored at �20 8C until analysed for Cp,SAA, AGP and Hp.

2.2. Experimental protocol

In a first series of experiments the determination of theprimary structure of the four APP included in this study, i.e.SAA, Hp, AGP and Cp, was carried out. Following this first,preliminary phase, the immunological methods availablefor the measurement of APP in cattle were tested for cross-reactivity in Alpine ibex by means of Western blottingusing, as primary antibodies, those included in thecommercial kit for APP determination.

Once the method is validated, the concentration of APPin serum obtained from Alpine ibex affected by sponta-neous mange infestation was measured.

2.3. Determination of primary structure of ceruloplasmin,

haptoglobin, a1-acid glycoprotein and serum amyloid A

Total RNA was extracted from euthanized ibex liverusing the RNeasy Mini Kit (Qiagen) according to themanufacturer’s protocol. The reverse transcription (RT)reaction was carried out on 1 mg RNA using iSCRIPT cDNASYNTESIS Kit (Bio-Rad, Segrate, Italy). The cDNA was usedas the template for the PCR (Eppendorf Mastercycler1)(Eppendorf, Milan, Italy).

PCR reactions were performed in 10 ml final volumeunder the following condition: 1� buffer Eppendorf,1.5 mM MgCl2, 0.2 mM of each dNTP, 1 mM of eachprimer and 0.5 unit of Taq Polymerase (Eppendorf,Milan, Italy).

The primers used to amplify the coding sequences ofibex AGP, SAA, Hp and Cp are listed in SupplementaryMaterial, Table 1, together with their thermal profiles.

PCR products were applied on a 1.5% agarose gelelectrophoresis and the segments of predicted molecularweight obtained were gel-purified using the QIAquick gelextraction kit (Qiagen, Milan, Italy) and then werequantified by NanoDrop1 ND-1000. The fragments weresequenced directly with ABI technology using an auto-mated DNA sequencer (ABI PRISM 310 Genetic Analyzer).The predicted amino acid sequence was obtained usingthe ExPASy proteomic server (www.expasy.ch). Inter-proscan and prosite (www.ebi.ac.uk) analysis was carriedout to detect post-translational modifications of the fourproteins.

M.M. Rahman et al. / Veterinary Parasitology 168 (2010) 293–298 295

2.4. Testing the cross-reactivity of the commercially available

assays in ibex

Serum SAA and AGP concentrations were determinedby using immunoenzymatic assays. The cross-reactivity ofthe antibodies included in the commercially available testswith ibex proteins had never been tested before in thisspecies. Therefore, a preliminary step to validate thepossible utilization of the commercially available assayswith ibex serum was carried out. These experiments wereperformed by testing 1 ml of ibex serum with SDS-PAGE ona 12% (for AGP detection) and 15% (for SAA detection)polyacrylamide gel, and Western blotting onto nitrocellu-lose. The membranes were then incubated with differentconcentrations of anti-boAGP and anti-SAA primaryantibodies (1:100, 1:200, 1:500, 1:1000, 1:2000, 1:5000,1:10,000, 1:20,000 and 1:40,000) using a multiscreenapparatus (Bio-Rad, Segrate, Italy) and incubated for 1, 2 or3 h at RT. Both antibodies were those included in thecommercial kits, and were made available by the courtesyof Dr Martin Gallagher, from Tridelta Company. Serum AGPpositive bands were visualized by immunostaining usingan anti-bovine AGP polyclonal antibody which was, afterpreliminary experiments, eventually utilized at a concen-tration of 1:40,000 for 45 min of incubation at roomtemperature. Detection was carried out by enhancedchemiluminescence (ECL) using a ImmobilonTM WesternChemiluminescence HRP Substrate (Millipore, Vimodrone,Italy). Bovine purified AGP (Ceciliani et al., 2007) was usedas positive control (50 ng each lane).

Serum SAA positive bands were visualized by immu-nostaining using anti-SAA biotinylated monoclonal anti-body (1:400 dilution from the original antibody included inthe kit, incubated overnight at 4 8C in order to reduce thebackground), followed by a streptavidin–biotin peroxi-dase-conjugated complex, using the Vectastain ABC kit(Vector Laboratories, Peterborough, UK) and developedusing ECL as previously described.

2.5. Determination of serum APP concentrations in ibex

Once the cross-reactivity of commercially availableassays in ibex is assessed, the concentration of each APPwas determined as follows.

The concentration of AGP was determined in ibexserum by a radial immunodiffusion assay commercial kit(Bovine a1 AG Plate, Tridelta Development Ltd., Kildare,Ireland). 5 ml of serum from both infected and non-infected animals was loaded onto multiwell plates andincubated at 37 8C for 48–72 h for ring formation. BovineAGP was utilized as standard for quantification of ibex AGP.AGP’s concentration was determined in mg/ml from themanufacturer’s guidelines.

Serum SAA concentrations were determined by meansof a commercially available sandwich ELISA assay (PhaseSAATM assay, Tridelta Development Ltd., Kildare, Ireland).The experiment was carried out according to the manu-facturer’s instructions, as previously described for bovineserum (Eckersall et al., 2006).

The concentration of Hp in serum samples wasdetermined using a Hp-haemoglobin binding assay (Tri-

delta Development Limited), the basis of which is thehaemoglobin binding method (Eckersall et al., 1999).

Ceruloplasmin concentration was determined by anindirect assay, which measures oxidase activity by amodified kinetic procedure originally developed for sheepplasma (Smith and Wright, 1974). To facilitate compar-isons with data from other laboratories, the oxidaseactivity has been converted to the concentration ofceruloplasmin (mg/l) on the assumption that the slopeof its relationship with TCA-soluble copper (mmol/l) has astoichiometrically determined value of 22 mg/mmol cop-per (Mckenzie et al., 1997). Each serum sample wasassayed once for TCA-soluble copper and oxidase activity.

2.6. Statistical analysis

Differences between groups were compared by meansof non-parametric Mann–Whitney test, because the valuesobserved for the different APP did not fit with normaldistribution, as assayed by Kolmogorov–Smirnov test. Thestatistical analysis of the data was carried out with theStatistica software (Stat Soft. Inc.). Statistical significancewas accepted at P< 0.05.

3. Results

3.1. Sequence analysis

The cDNA sequences of AGP, SAA, Hp and Cp obtainedfrom ibex liver have been deposited in NCBI GenBankunder the accession number of EU884571, EU884570,FJ194972 and FJ855480, respectively.

Their corresponding polypeptide backbones are shownin Fig. 1 Suppl., which also presents homology comparisonsbetween ibex and other ruminants sequenced so far, aswell as the description of post-translational modifications.

3.2. Assessment of the cross-reactivity of immunological

assays for acute phase proteins

The assays used to determine the concentration of SAAand AGP in ibex serum were based on immunologicalmethods, such as ELISA and Radial Immuno Diffusion(RID). As a prerequisite for their utilization in ibex, both ofthem had to be tested for the cross-reactivity of the anti-bovine SAA and AGP antibodies included in the kit withibex SAA and AGP, respectively.

Western blotting experiments were therefore carriedout to determine (a) whether the specific antibodyincluded in the kit cross-reacted with the respectiveprotein and (b) if the antibodies did not cross-react withserum proteins other than those targeted by this experi-ments.

Results are presented in Fig. 2 Suppl. The anti-bovineAGP antibody included in the kit cross-reacts with a bandwith a Mw of 42–45 kDa, with an electrophoretical pattern(molecular weight and presence of high molecular weightglycoforms) consistent with that of bovine AGP (Fig. 2ASuppl.).

The same experiment was carried out using anti-bovineSAA antibody. Results, presented in Fig. 2B Suppl., indicate

Table 1

Mean and standard deviation APP concentration in ibex.

Acute phase

proteins

Group I (healthy

animals)

Group II (infested

animals)

Mean s.e. Mean s.e.

SAA (mg/ml) 8.7 �0.13 130.7 �0.16

Hp (mg/l) 0.58 �0.09 3.72 �0.65

AGP (mg/ml) Under detection

limit

0.42 �0.36

Cp (mg/ml) 1.1 �0.13 2.31 �0.16

Acute phase protein concentrations in ibex: means and standard errors

(s.e.).

M.M. Rahman et al. / Veterinary Parasitology 168 (2010) 293–298296

that also the anti-bovine SAA is specific for ibex SAA, whichbands at a Mw of 19 kDa. Ibex serum does not apparentlypresents the low molecular protein (approximately14 kDa) which is on the contrary present in bovine serum.

In conclusion, this series of preliminary experimentsdemonstrated that the two kits that were utilized todetermine the concentration of AGP and SAA in ibex serumcan cross-react with their respective antigens in ibexserum.

3.3. Acute phase protein concentration measurement in ibex

serum

Serum concentration of the acute phase proteins SAA,Hp, AGP and Cp were determined on 15 healthy ibexes and37 animals with sarcoptic mange clinical signs. Results arepresented in Fig. 1 and Table 1.

Normal levels of SAA are significantly increased inanimals with clinical signs of mange (Z = 3.15; P = 0.0013).Non-pathological levels of Hp are statistically different(Z = 3.86; P = 0.0001) in animals with clinical mange,whose concentration of Hp is increased. It was not possibleto determine the concentration of AGP in healthy animalssince values were under the detection limit of the assay(except the value for one ibex, shown as a dot in Fig. 1C). Onthe contrary, the serum concentration of AGP is signifi-cantly increased (Z = 3.81; P = 0.00001) in ibexes withclinical signs of mange.

Finally, Fig. 1D presents the concentration of Cp inhealthy animals which also significantly increased duringmange (Z = 3.67; P = 0.0002). No statistically significantrelationship between sex/age and APP concentration wasfound.

4. Discussion

This study presents for the first time a wide survey ofthe acute phase proteins expression in serum of ibexesaffected or not affected by S. scabiei infestation. Resultsindicated that the concentrations of all the proteinsinvestigated, including SAA, AGP, Hp and Cp, significantlyincreased in infected ibexes.

Fig. 1. Serum concentration of ibex acute phase proteins. APP

measurement in ibex serum from non-pathologic (Group I) and

sarcoptic mange-affected (Group II). Small boxes (&) show the

median. Boxes indicate value from 25th to 75th percentiles, while

whiskers indicate the distribution of all the values. Significance was

declared for ***P< 0.05.

M.M. Rahman et al. / Veterinary Parasitology 168 (2010) 293–298 297

From a clinical perspective, only AGP and SAA may beconsidered as ‘‘major acute phase proteins’’ duringsarcoptic mange in ibex, since their concentrationincreased more than 10-fold during disease (Gabay andKushner, 1999). The increase of AGP concentration isremarkable, since this protein is not detectable in animalswithout clinical signs of sarcoptic mange. The increase ofCp and Hp concentration in affected animals, whilestatistically significant, is modest. These two proteinsmight therefore be classified as minor acute phase proteinsin ibex, since their concentration is increased from two tofive folds if compared to healthy animals.

The detection of the four APP, some of them at highconcentration, in the serum of mange-affected ibexesindicated that a systemic reaction has occurred, probablybecause the local inflammation is sufficiently intense toinduce a measurable acute phase reaction. The highnumber of macrophages, which has been reported to bethe histological hallmark of mite infestation in ibex (Leon-Vizcaıno et al., 1999) are likely to be the main source ofpro-inflammatory cytokines which eventually induce theacute phase reaction in clinically affected animals.

The pool of pro-inflammatory cytokines is probablyincreased by keratinocytes and fibroblasts. Both cell typeshave been already reported as possible source of pro-inflammatory cytokines, if adequately stimulated (Hein-rich et al., 1990). Scabies extracts may further contribute tocytokine pool since they have been shown to up-regulatethe secretion of IL-1b and down-regulate the expression ofIL-1 receptor antagonist, which is an anti-inflammatorycytokine, by keratinocytes, as well as IL-6 secretion byfibroblasts (Arlian et al., 2003). Finally, Streptococcus

pyogenes and Staphylococcus aureus pyoderma are frequentsequels of scabies, if mite infestation is left untreated(Brook, 1995). Therefore, the possible presence of highconcentrations of bacterial endotoxins in circulation mayfurther contribute to the up-regulation of APP geneexpression in liver (Gabay and Kushner, 1999). The factthat S. scabiei infestation can cause a systemic reaction toinflammation can be further confirmed by the increasedleukocytosis, in particular neutrophilia, which has beenreported in other species (Arlian et al., 1988).

In conclusion, while the experimental design of thepresent investigation shares the weak points of most of thestudies carried out on free ranging ruminant populations,this study fulfilled its main task that was to assess the APPprofile during sarcoptic mange after proper validation ofthe respective immunological assays.

This information provides a remarkable potential forthe use in free ranging animal clinical practice, since thesebiological markers may be very useful for early diagnosis ofsarcoptic mange. Furthermore, these results represent astarting point for investigation on other domestic animalsaffected by S. scabiei infestation. Of course, a more accuratediagnostic study in asymptomatic animals is recom-mended for further implementation of this finding, forexample in domestic animals, where no information aboutacute phase reaction during ectoparasitic disease is stillavailable.

The identification of early affected animal is ofparamount importance for managing this parasitic disease,

since it may reduce the possibility that infected animalsare introduced in unaffected populations. This wouldprobably contribute to reduce the high prevalence ofsarcoptic mange infestation in ibex population.

Acknowledgments

We acknowledge the precious support of Mr MartinGallagher, from Tridelta Ltd. Company, for the gift on anti-bovine AGP and anti-bovina SAA antibodies, which wereuseful to validate the utilization of SAA and AGP bovine kiton ibex. We also thank Prof Claudio Genchi, Prof PaoloLanfranchi and Dr Nicola Ferrari for the critical reading ofthe paper and their precious support.

This work was financed by Grant First 2006 funded toDr Ceciliani and PUR 2008 funded to Prof Sartorelli.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.vetpar.2009.

12.001.

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