Anaemia in advanced chronic fasciolosis

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Acta Tropica 108 (2008) 35–43

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Anaemia in advanced chronic fasciolosis

M.A. Valeroa,∗, N. Gironèsb, M.A. García-Bodelóna, M.V. Periagoa, I. Chico-Calerob,M. Khoubbanea, M. Fresnob, S. Mas-Comaa

a Departamento de Parasitología, Facultad de Farmacia, Universidad de Valencia, Av. Vicente Andrés Estellés s/n, 46100 Burjassot, Valencia, Spainb Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, 28049 Madrid, Spain

a r t i c l e i n f o

Article history:Received 20 September 2007Received in revised form 23 August 2008Accepted 26 August 2008Available online 3 September 2008

Keywords:FasciolosisHaematologyImmunologyWistar modelAdvanced chronic phase

a b s t r a c t

The association between fasciolosis-induced anaemia and related factors has been quantified in a rodentmodel. Haematological parameters were analysed in Wistar rats at 20 and 60 weeks post-infection (p.i.).Pigment stones and bile specimens were collected. Serum IgG1, IgG2a and IgE were determined in ratserum samples. Cytokine levels have been correlated with haematological parameters. The screening forgastrointestinal bleeding was carried out. Bacteriological bile cultures revealed viable bacteria in 53.8% ofspecimens at 60 weeks p.i. The results show that the type of anaemia in fasciolosis might be considereda biomarker of the chronicity period of the disease, changing from normocytic to macrocytic in the earlychronic period (20 weeks p.i.) and to microcytic in the advanced chronic period (60 weeks p.i.). Likewise,changing from normochromic in the early chronic period to hypochromic in the advanced chronic period.Multivariate analysis suggested an association between anaemia and the following factors: fluke burden,eggs per gram of faeces, body area of parasite, presence of blood in faeces, IgG1 and eosinophil levels, and %of splenic weight. Of all variables analysed, the fluke burden is the one which presents the highest anaemiarisk, even exceeding the variable presence of blood in faeces. The development of anaemia appears tobe complex and may involve multiple mechanisms. However, to the mechanisms that until now explainFascioliosis-related anaemia (compensatory increase in erythrocyte production and a continuous drain oniron stores resulting from the parasites’ blood-sucking activities) the following causes ought to be added:haemolysis of red blood cells, the general effects of inflammation on erythropoiesis, concomitant parasiticand bacterial infections and pre-morbid nutritional abnormalities. Extrapolation to human fasciolosis isdiscussed. The results of the rodent model lead to the assumption that a high risk of anaemia in subjectswith a heavy parasitic burden in human hyperendemic areas of fasciolosis is to be expected.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Fasciolosis is considered an important human disease caused bytwo liver fluke species: Fasciola hepatica and Fasciola gigantica (Fas-ciolidae). Several geographic areas have been described as endemicfor the disease in humans, including hypoendemic, mesoendemicand hyperendemic areas, with prevalences and intensities rang-ing from low to very high (Mas-Coma, 2004, 2005; Mas-Coma etal., 1999a, 2003, 2005; Curtale et al., 2007; Parkinson et al., 2007).The last stage of the disease in humans encompasses an obstruc-tive or chronic phase which may develop after months to yearsof infection (Mas-Coma et al., 1999b), including mild to moder-ate anaemia, especially in heavy infections. It has been argued thatblood loss into the bile is most probably an important factor, if notthe only one, contributing to severe anaemia (Dawes and Hughes,

∗ Corresponding author. Tel.: +34 96 3544536; fax: +34 96 3544769.E-mail address: [email protected] (M.A. Valero).

1970). The relationship between liver fluke infection and anaemia inhuman fasciolosis endemic areas has not been demonstrated, giventhat numerous nutritional deficiencies and infectious diseases co-exist in liver fluke infected subjects and that both are related to anincreased risk of anaemia when present (Salem et al., 1993; Curtaleet al., 1998, 2003). Consequently, it is necessary to quantify the asso-ciation between fasciolosis infection and anaemia, and to assess therelated factors.

A useful approach for pathological research in the advancedchronic period appears to be the laboratory Wistar rat modelbecause the rat’s resistance level, susceptibility and pathologymimic chronic disease in humans (Valero et al., 2000, 2003,2006a,b; Gironès et al., 2007). Therefore, a parasite isolate and lym-naeid snail vectors from a human fasciolosis hyperendemic areawere used for experimental procedures in the Wistar rat model. Notonly the presence of blood in faeces, but also the cytokine and anti-body response as well as the relative splenic weight were assessed.The correlation between fasciolosis, anaemia and immune responsehas never been studied in a rat model at such an advanced chronic

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36 M.A. Valero et al. / Acta Tropica 108 (2008) 35–43

phase and during such a long time-span of 60 weeks post-infection(p.i.) as described in the present work.

2. Materials and methods

2.1. Animals and experimental design

The study was approved by the institutional committee on ani-mal care of University of Valencia (Spain). Wistar rats (80–100 gbody weight; Iffa Credo, Barcelona, Spain) were housed in micro-isolator boxes and maintained in a pathogen-free room, electricallyheated with a 12 h/12 h light/darkness cycle (i.e. conditions in com-pliance with the European Agreement of Strasbourg, 18 March1986). A balanced commercial rodent diet (Panlab Chow A04,Barcelona, Spain) and water were provided ad libitum. Parasiteand lymnaeid vectors used were strains from human endemicareas. Metacercariae of F. hepatica were obtained from experi-mentally infected Galba truncatula snails (Mas-Coma et al., 2001).Metacercariae were inoculated with an orogastric syringe with20 metacercariae per rat. Necropsy was carried out at the endof the study using Fluothane® (Zéneca Farma, S.A., Pontevedra,Spain) anaesthesia followed by exsanguination. Before dissec-tion, the rats were weighed and their body size was measuredin mm, i.e. the distance between the nose and the begin-ning of the tail. In the rat model, fasciolosis is considered anadvanced chronic disease from 100 days post-infection onwards(Valero et al., 2003). Dissection was carried out at 20 weeksp.i. (experiment 1: 6 infected rats and 8 control rats) and 60weeks p.i. (experiment 2: 22 infected rats and 46 control rats),respectively. F. hepatica worms, bile samples and stones were col-lected under a dissecting microscope in sterile conditions. Theliver stones were classified as brown pigment stones by energy-dispersive X-ray microanalysis (Valero et al., 2000). The spleenswere removed from the groups of infected and control animals atthe end of the two experiments and weighed. The relative splenicweight was calculated as percentage of body weight (% splenicweight).

2.2. Morphometric measurement techniques

The adult fluke body area (BA) (mm2) was measured by acomputer image analysis system (CIAS) with specialized software(ImagePro® Plus, version 4.5 for Windows, Media Cybernetics Inc.,Silver Spring, USA) (Valero et al., 1998, 2001, 2005). Thus, for eachinfected rat, the liver fluke total body area was calculated addingthe adult body area of each parasite present.

2.3. Haematological samples

Blood samples were collected at 20 weeks p.i. (experiment 1)and 60 weeks p.i. (experiment 2) in vials containing ethylene-diamine-tetracetic acid (EDTA) as anticoagulant. Total red bloodcell (RBC) count (M/�l), haemoglobin (HGB) (g/dl), haematocrit(HCT) (%), the mean corpuscular volume (MCV) (fl), the meancorpuscular haemoglobin concentration (CHC) (pg) were calcu-lated using an automatic blood analyser (Sysmex© XE-2100).The percentage of eosinophils (EOS) was calculated throughthin blood films stained with Giemsa. At least 100 cells werecounted on each film. The percentage of reticulocytes (RET)was additionally calculated in experiment 1. Serum iron levels(Mcg/dl) were automatically calculated by means of standard-ised enzymatic methods (BM/Hitachi 917-rack) (experiments 1 and2).

2.4. Microbiological analyses

Bile samples were simultaneously collected from the commonbile duct (CBD) in experiments 1 and 2. The bile specimens werecultured aerobically using standard methods. Bile specimens werecollected in thioglycolate with resazurin medium (bioMerieux®,Marcy l’Etoile, France), and cultured by inoculation onto an EMBplate (Eosin-methylene blue agar), TSS (Trypcasa Soy Agar + 5%sheep blood) and SGC (Sabouraud Gentamicin ChloramphenicolAgar) (bioMerieux®, Marcy l’Etoile, France), and incubated for 24and 48 h at 37 ◦C (Valero et al., 2006a).

2.5. Cytokine assays

The data on the serum cytokine assay used in this work waspublished by Gironès et al. (2007). Serum cytokine assays wereperformed in rat serum samples in experiment 1 following themanufacturers’ guidelines. Rat IL-4 Eli-pair, Rat IFN-� Eli-pair andRat TNF-� Eli-pair were from Diaclone Research. Rat IL-10 ELISASet was from BD Biosciences. Rat IL-1� ELISA Set was from R&DSystems. In experiment 2 cytokines in infected rat serum were notquantified because previous studies had already demonstrated thatthey show basal levels from 20 weeks p.i. on (Gironès et al., 2007).

2.6. Serum IgG1, IgG2a and IgE measurements

In experiments 1 and 2, serum IgG1, IgG2a and IgE were deter-mined in rat serum samples using detergent soluble F. hepaticaextract in ELISA. Mature flukes were removed from the bile ductsof bovine livers. For the preparation of a detergent soluble extract,adult flukes were killed by freezing at −70 ◦C, washed in phos-phate buffered saline (PBS) and drained. One gram of wet weight ofminced tissue was incubated in 10 ml of 50 mM Tris pH 7.4, 150 mMNaCl, 1% DOC and 1 �g/ml of protease inhibitors (leupeptin, pep-statin, aprotinin) and 1 mM PMSF. The extract homogenate wasobtained by disruption with a polytron for 3 min on ice, followedby centrifugation at 14,000 rpm in a minifuge for 15 min. The quan-tification of protein in the resulting supernatant was carried outby the BCA method. A total of 100 �g of protein extract per well ofa 96 well plate was used. Coating of the wells was carried out inPBS overnight at 4 ◦C. Blocking was done for 1 h at room tempera-ture with PBS containing 0.2% Tween-20 and 3% low fat dry milk.Wells were washed 3 times with PBS containing 0.05% Tween-20and 1% low fat dry milk. Sera were diluted 1:50 in PBS and incu-bated for 1 h at room temperature. Wells were washed 3 times withPBS containing 0.05% Tween-20 and 1% low fat dry milk. Antibod-ies against IgG1, IgG2a and IgE coupled to horseradish peroxidase(Serotec) were used at 1:10,000 dilutions and incubated for 1 h atroom temperature. Wells were washed 5 times with PBS containing0.05% Tween-20 and 1% low fat dry milk, and developed with phe-nilendyamine (OPD) for 30 min at room temperature. Absorbanceat 450 nm was measured in an EL 340 Biokinetics microplate reader(Biotek Instruments) (Gironès et al., 2007).

2.7. Erythrocyte agglutination assay

The direct antiglobulin test (DAT), also known as the directCoombs test, demonstrates the presence of antibodies on thesurface of red blood cells and is the hallmark of autoimmunehaemolysis. The patient’s red blood cells are mixed with rabbit ormouse antibodies against human IgG. Agglutination of the patient’santibody-coated red blood cells by anti-IgG serum constitutes apositive test. The basis of this test was adapted to rat host species,and DAT was therefore used in this study. A flow cytometry assaywas used to detect anti-RBC antibodies in rat serum samples in

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the two experiments. Erythrocytes were purified from normal ratblood by gradient centrifugation with NycoPrep 1.077A from Axis-Shield. Purified erythrocytes were blocked with staining buffer (PBSsupplemented with 1% foetal bovine serum and 1% bovine serumalbumin) for 20 min at 4 ◦C, and incubated with chronically infectedrat serum, negative and positive control sera for 20 min at 4 ◦C. Ery-throcytes were washed with staining buffer and incubated withgoat anti-rat IgG antibody coupled to Alexa 488. After washing threetimes in staining buffer, erythrocytes were analysed in a FacScal-ibur cytometer using the Cell Quest software (Becton Dickinson).Negative control sera were used to set erythrocyte negative popula-tions. Samples above the negative values were considered positive.A mouse anti-rat endothelium antibody that recognizes a 90 kDaerythrocyte antigen (Serotec) was used as positive control.

2.8. Detection of fluke eggs in faeces

In both experiments the number of eggs per gram of faeces(epg) shed by each rat was calculated using the Kato-Katz technique(Helm-Test®, Belo Horizonte, Brazil). Faecal pellets were collecteddirectly from the cages of each animal and were kept in a closedPetri dish before examination to avoid desiccation (Valero et al.,2002). The samples were taken at the following time points: at 7,10 and 14 weeks p.i. in experiment 1, and at 41, 46 and 51 weeksp.i. in experiment 2. The average epg obtained in each of the threesamples was calculated for each rat.

2.9. Detection of blood in faeces

The screening for gastrointestinal bleeding was carried out inall rats using the guaiac test procedure, Tri-slide® stool bloodtest (Cenogenics Corporation, Morganville, USE). The samples weretaken at the following time points: at 7, 10 and 14 weeks p.i. inexperiment 1, and at 41, 46 and 51 weeks p.i. in experiment 2.

2.10. Statistical analyses

Haematological data (RBC, HGB, HCT, MCV, CHC, EOS, Fe, RET),weight, body size, % splenic weight and IgG1, IgG2a and IgE levels ofthe rats were compared by non-parametric Mann–Whitney test. Fora proper description, some transformations were necessary: log eof IgG1 [ln(IgG1)], log e of IgG2a [ln(IgG2a)], log e of IgE [ln(IgE)].

Liver fluke total body size measurements and egg counts wereconsidered after converting individual values to logarithms (log e)with the (n + 1) transformation [ln(BA) and ln(epg), respectively](Stephenson, 1987). Bivariant correlations (Pearson’s correlation)were calculated for haematological data, cytokine levels (TFN-�,IFN-�, IL-10, IL-4 and IL-1�), immunoglobulin subclasses [ln(IgG1),Ig(G2a), Ig(E)], fluke burden, ln(epg) and ln(BA). Relative risks (RR)were estimated using a multivariate logistic regression [in the con-text of risk factors, the resulting Exp(B) are estimates of RR] usingSPSS 14.0. Six multiple logistic regression models were performed.The following were used as independent variables: presence of par-asites in the CBD, lithiasis, rat body weight, ln(IgG1) and EOS inmodel 1; fluke burden, lithiasis, rat body weight, ln(IgG1) and EOSin model 2; ln(epg), lithiasis, rat body weight, ln(IgG1) and EOSin model 3; ln(BA), lithiasis, rat body weight, ln(IgG1) and EOS inmodel 4; lithiasis, ln(IgG1), EOS, presence of blood in faeces andpresence of anti-RBC antibodies in model 5; and ln(IgG1) and %splenic weight in model 6. Results were considered statisticallysignificant when P < 0.05.

3. Results

3.1. Experimental infection

Wistar rats were infected as described in Section 2 and differentparameters were followed. Two different experiments were set toanalyse haematological parameters at 20 weeks p.i. (experiment 1)and 60 weeks p.i. (experiment 2). The value of HGB average − S.D.in non-infected control rats was used as cut-off to define anaemia(13.6 g/l in both experiments). Table 1 summarizes the results of theexperimental infections. At 20 weeks p.i., anaemia was detected in66.6% of the infected rats. Total values of RBC, HGB and HCT weresignificantly higher in the non-infected controls when compared tothe parasitized rats. Reticulocyte values were significantly higher inthe parasitized rats when compared to the non-infected controls.No statistically significant differences were found in the values ofMCV, CHC and Fe between infected and control rats, although 2out of 6 infected rats showed high MCV values in relation to theaverage + S.D. values obtained in controls. Bacterobilia associatedwith infection was not detected at 20 weeks p.i. Brown pigmentstones were detected simultaneously with the liver fluke in thecommon bile duct in 3 out of 6 infected rats. Statistically significant

Table 1Average ± S.D. in control and infected rats for: total red blood cell (RBC) count (M/�l), haemoglobin (HGB) (g/dl), haematocrit (HCT %), mean corpuscular volume (MCV) (fl),mean corpuscular haemoglobin concentration (CHC) (pg), percentage of reticulocytes (RET %), percentage of eosinophils (EOS %), iron serum level (Fe) (Mcg/dl), percentageof rats with RBC antibodies (anti-RBC Ab%), rat body weight (g), rat body size (mm), number of liver flukes detected in each rat (fluke burden), log e of the average number ofeggs per gram of faeces [ln(epg)], log e of liver flukes total body size (BA) [ln(BA)] and relative splenic weight calculated as a percentage of rat body weight (% splenic weight)

Experiment 1 Experiment 2

Infected, 20 wpi Control, 20 wpi Infected, 60 wpi Control, 60 wpi

RBC 6.75 ± 0.77* 8.28 ± 1.01 7.67 ± 1.42* 8.34 ± 1.52HGB 10.40 ± 2.61* 14.84 ± 1.27 12.76 ± 2.86* 15.05 ± 1.42HCT % 41.22 ± 6.51* 48.57 ± 6.09 47.59 ± 13.01* 55.42 ± 5.81MCV 68.20 ± 6.34 66.44 ± 2.08 62.19 ± 7.60* 66.42 ± 4.10CHC 17.09 ± 1.87 17.77 ± 0.85 16.39 ± 2.20* 17.89 ± 0.95RET % 5.57 ± 1.22* 2.64 ± 0.31 – –EOS % 3.83 ± 1.33a 2.89 ± 1.36 4.75 ± 1.98 3.28 ± 1.10Fe 150.00 ± 28.53 192.00 ± 31.37 169.48 ± 73.19* 214.68 ± 48.27Anti-RBC Ab % 0 0 13.64 0Rat body weight 412.50 ± 54.13 447.63 ± 24.16 482.82 ± 51.11* 516.28 ± 55.17Rat body size 193.67 ± 9.13 193.38 ± 7.95 194.09 ± 8.91 198.09 ± 16.22Fluke burden 3.33 ± 1.86 0 2.05 ± 1.70 0ln(BA) 5.98 ± 0.54 0 5.41 ± 0.73 0ln(epg) 7.55 ± 0.38 0 7.01 ± 0.61 0% Splenic weight 0.15 ± 0.02 0.13 ± 0.02 0.16 ± 0.07* 0.13 ± 0.06

* P < 0.05.a Values published by Gironès et al. (2007).

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Fig. 1. Liver fluke infection at 20 weeks post-infection (p.i.): serum cytokine levels [tumour necrosis factor (TNF-�), interferon (IFN-�), interleukin (IL), IL-10, IL–4, and IL-1�](pg/ml) and serum iron levels (Fe) (Mcg/dl) in infected rats with iron deficiency (n = 4) vs rats without iron deficiency (infected and control rats, n = 6). The bars denote S.E.s.

differences in weight and body size of the rats were not detectedbetween infected and control groups. In experiment 2, anaemiawas detected in 12 out of 22 infected rats (54.5%). Total values ofRBC, HGB, HCT, MCV, HCM and Fe were significantly higher in thenon-infected controls when compared to the parasitized rats. Allmicrobiological analyses of the bile samples from the control ratswere negative, but bacterobilia associated with liver-fluke infec-tion was detected in 7 out of 13 infected rats (Escherichia coli in 3rats and Enterococcus faecalis in 4 rats). Brown pigment stones weredetected simultaneously with liver flukes in the common bile ductsin 12 out of 22 infected rats. The weight of the non-infected controlswas significantly higher when compared to the parasitized rats.

3.2. Haematological parameters and cytokine production

In a previous study (Gironès et al., 2007) at 20 weeks p.i., we didnot detect IFN-� above basal values, but in some rats TNF-�, IL-10,IL-4 and IL-1� were above basal values. For the first time, thesecytokine levels were correlated with haematological parameters.The comparison of TNF-�, IL-10 e IL-4 levels in the specimens withanaemia (n = 4) vs the specimens without anaemia (n = 2) showsthat TNF-� increased only in the two rats without anaemia, but IL-4, IL-10 and IL-1� levels increased in the rats with anaemia. Thecytokine values of rats with serum iron deficiency and rats withnormal serum iron levels are shown in Fig. 1. Increased TNF-�, IL-10,and IL-1� levels were detected in rats with serum iron deficiency.

The bivariant correlation between haematological (RBC, HGB, HCT,MCV and CHC) and cytokine (TNF-�, IL-10, IL-4 and IL-1�) levels ofeach rat was carried out. A significant positive correlation betweenIL-4 and MCV (r = 0.84) was detected.

3.3. Eosinophilia

The bivariant correlation between RBC, HGB, HCT, CHC, MCV, Feand EOS was calculated in both experiments. Significant negativecorrelations were obtained between EOS and RBC (r = −0.42), HGB(r = −0.46), HCT (r = −0.55), HCM (r = −0.36) and MCV (r = −0.36).

3.4. Antibody production

Specific IgG1, IgG2a and IgE responses were induced in infectedrats respect to non-infected control rats (Figs. 2 and 3). A pre-dominance of the IgG1 vs the IgG2a subclass was observed inserum samples at 20 (Fig. 2) and 60 (Fig. 3) weeks p.i. No sta-tistically significant differences in values of IgG1, IgG2a and IgEwere detected between rats with anaemia and without anaemiain either experiment, but at 60 weeks p.i., significant negativecorrelations were obtained between IgG1 vs RBC (r = −0.31) andHGB (r = −0.46), IgG2a vs RBC (r = −0.41), HGB (r = −0.51) andHCT (r = −0. 43) and between IgE vs HGB (r = −0.31), IgG1 vsRBC (r = −0.31) and HGB (r = −0.46), and between IgE vs HGB(r = −0.31).

Fig. 2. Liver fluke infection at 20 weeks post-infection (p.i.): immunoglobulin (Ig) IgG1, IgG2a and IgE levels in infected rats with anaemia (n = 4), infected rats withoutanaemia (n = 2) vs control rats (n = 4). The bars denote S.E.s. OD, optical density.

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Fig. 3. Liver fluke infection at 60 weeks post-infection (p.i.): immunoglobulin (Ig) IgG1, IgG2a and IgE levels in infected rats with anaemia (n = 12), infected rats withoutanaemia (n = 10) vs control rats (n = 12). The bars denote S.E.s. OD, optical density.

3.5. Egg production, parasite size and fluke burden

Bivariant correlations between RBC, HGB, HCT, CHC, MCV, Fe,ln(epg), ln(BA) and fluke burden were calculated in the two exper-iments (Fig. 4). Significant negative correlations were obtainedbetween ln(epg) vs RBC (r = −0.37), HGB (r = −0.56), HCT (r = −0.43),HCM (r = −0.45), MCV (r = −0.34) and Fe (r = −0.38); ln(BA) vs RBC(r = −0.40), HGB (r = −0.60), HCT (r = −0.47), HCM (r = −0.50), MCV(r = −0.32) and Fe (r = −0.39); and between fluke burden vs RBC(r = −0.47), HGB (r = −0.70), HCT (r = −0.54), HCM (r = −0.63), MCV(r = −0.39) and Fe (r = −0.38).

3.6. Study of splenomegaly

The % splenic weight obtained in infected and control rats inboth experiments is shown in Table 1. The % splenic weight wassignificantly higher in the parasitized rats when compared to thenon-infected controls. The bivariant correlation between RBC, HGB,HCT, CHC, MCV, Fe and % splenic weight was calculated. Significantnegative correlations were obtained between % splenic weight vsHGB (r = −0.44), HCT (r = −0.48) and MCV (r = −0.39).

3.7. Study of blood in faeces

Occult blood loss was not detected in any control rat, while bloodpresence in faeces was detected in infected rats independent ofthe intensity of infection. In the 3 samples analysed from each rat,blood emission was detected in all samples in some rats, while itwas intermittent in others. In experiment 1, in the 3 samples anal-ysed from each rat, 33.3% were positive in one sample, 33.3% in twosamples and 33.3% in all 3 samples. In relation to time p.i., in exper-iment 1 the stool blood test resulted positive in 33.3% at 7 weeksp.i., 66.6% at 10 weeks p.i. and 100% at 14 weeks p.i.

In experiment 2, in the 3 samples analysed from each rat, 9%were negative in any of the samples taken, 22.8% were positive inone sample, 13.6% in two samples and 54.5% in all 3 samples. Inrelation to time p.i., the stool blood test resulted positive in 59.1%at 41 weeks p.i., 68.2% at 46 weeks p.i. and 81.8% at 51 weeks p.i.Blood detection was not always associated with anaemia, i.e. inexperiment 1 the maximum percentage of infected rats with occultblood loss was 100%, but anaemia was only present in 66.6%. Inexperiment 2 the maximum percentage of infected rats with occultblood loss was 81.8%, and anaemia was detected in 54.5%, while 4.5%

of the rats without detection of blood in faeces presented anaemia.These results indicate the intervention of another mechanism otherthan extracorporeal loss of iron.

3.8. Autoimmune haemolysis

Anti-RBC antibodies in rat serum samples were not detectedin experiment 1 (0.0%). In experiment 2, anti-RBC antibodies weredetected in serum in three rats (13.6%) and they also presentedlower HGB levels.

3.9. Risk factors for anaemia

Six multiple logistic regression models were used. The RR ofanaemia associated with the presence of parasites in model 1(Table 2) showed a RR of 83.82. The results of this multiple logisticregression analysis support the impression that anaemia is associ-ated with advanced chronic fasciolosis. The results of these multiplelogistic regression analyses (models 2, 3, 4) show that anaemia isnegatively associated with burden, activity and development of theparasite, which is reflected in the relative risk values of anaemiaassociated with fluke burden (RR: 139.42), ln(eggs per gram of fae-ces) (RR: 2.92) and ln(body area) (RR: 6.42).

The RR of anaemia associated with presence of blood in fae-ces in model 5 (Table 2) showed a remarkable RR increase (116.40)when taking immunological response levels (ln(IgG1) and EOS) intoaccount as covariates. The RR of anaemia associated with ln(IgG1)and % splenic weight was analysed through logistic regression inmodel 6. The inclusion of % splenic weight as a covariate increasesthe anaemia risk in the dependent variable ln(IgG1) to 5.07, indicat-ing that splenomegaly partly explains this process. Of all variablesanalysed, the fluke burden is the one which presents the high-est anaemia risk, even exceeding the variable presence of bloodin faeces.

4. Discussion

Anaemia is a global health problem in low- and middle-incomecountries due to its negative effects on physical development, intel-lectual performance, work capacity and pregnancy (De Maeyer etal., 1989).

Anaemia due to fasciolosis was traditionally thought to berelated to a direct loss of whole blood caused either through inges-

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Fig. 4. Fasciolosis and anaemia: (A) Eggs per gram of faeces (epg) calculated through ln e of epg [ln(epg)] vs haemoglobin levels (HGB). (B) Liver fluke total body size (BA)calculated through ln e of BA [ln(BA)] vs haemoglobin levels (HGB). (C) Fluke burden vs haemoglobin levels (HGB).

tion of blood by the flukes or haemorrhage induce by them (Toddand Ross, 1966; Chen and Mott, 1990; Behm and Sangster, 1999).However, there is disagreement on whether haematophagia occurs,and if it does, whether it is sufficient to produce the anaemic symp-toms observed. It has been estimated that blood is lost at the rateof 0.2–0.5 ml per day per fluke. Despite the fact that protein com-ponents of plasma and blood cells are reabsorbed after digestion inthe intestine, considerable amounts of iron are lost, which are notreabsorbed. Under these circumstances, the rate of erythropoiesisis increased but is limited in the later stages of the infection by theavailability of dietary iron and protein, which depends on the qual-ity of the diet and intensity of anorexia (Behm and Sangster, 1999).The results of our study on the presence of blood in faeces show thatblood loss occurs at 20 and 60 weeks p.i. Although this phenomenonis not present in all anaemic-parasitized rats, it does not appearto be the only cause of liver-fluke related anaemia. The samples

taken at 20 weeks p.i. show the presence of blood in faeces caus-ing the loss of iron. This blood loss is chronic and remains until 60weeks p.i., possibly resulting in iron deficiency anaemia. Erythro-cyte production requires iron. Ferropenic erythropoiesis leads tothe development of microcytosis and hypochromia, a consequenceof the decrease of the synthesis of haemoglobin through the lack ofiron (Sans Sabrafen et al., 2006).

Different types of anaemia have been described as macrocyticand normochromic in various liver fluke-infected animal species(Sinclair, 1964, 1965; Ross et al., 1967; Haroun and Hussein, 1975;Ganga et al., 2006). Human fasciolosis has been associated withnormocytic hypochromic anaemia and to a lesser extent withmacrocytic and microcytic hypochromic anaemia (Bassiouny et al.,1991), with significant increases of reticulocyte counts (El-Shazlyet al., 2005). The results obtained in this study indicate the occur-rence of different types of anaemia related to the various fasciolosis

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Table 2Results of multivariate logistic regression analysis of different factors obtained in the course of experimental fascioliasis in Wistar rats and regression coefficients[Exp(B) = RR = relative risk of presence of anaemia] with significance in different models

Model 1 Model 2 Model 3 Model 4 Model 5 Model 6

RR P RR P RR P RR P RR P RR P

Presence of parasites 83.83 0.044Fluke burden 139.42 0.040ln(epg) 2.92 0.021ln(BA) 6.42 0.023Lithiasis – NS – NS – NS – NS – NSRat body weight – NS – NS – NS – NSln(IgG1) – NS – NS – NS – NS – NS 5.08 0.042EOS – NS – NS – NS – NS – NSBlood in faeces 116.41 0.017RBC Ab – NS% Splenic weight – NS

Presence of parasites = presence/absence of liver flukes; fluke burden = number of liver flukes/rat; ln(epg) = log e of eggs per gram of faeces/rat; ln(BA) = log e of the liverfluke total body size (BA) calculated through the addition of the adult body area of each of the parasites present in the common bile duct; lithiasis = presence/absence oflithiasis in the common bile duct; rat body weight in g; ln(IgG1) = log e of IgG1 serum levels; EOS = % of eosinophils; Blood in faeces = presence/absence of blood in faeces;RBC Ab = presence/absence of red blood cell antibodies; % splenic weight = relative splenic weight calculated as a percentage of rat body weight; NS = not significant. 6 models(model 1, 2, 3, 4, 5 and 6) were used in the multivariate logistic regression analysis including presence/absence of anaemia as dependent variable: Model 1 including presenceof parasites in the CBD, lithiasis, rat body weight, ln(IgG1) and EOS as independent variables. Model 2 including fluke burden, lithiasis, rat body weight, ln(IgG1) and EOS asindependent variables. Model 3 including ln(epg), lithiasis, rat body weight, ln(IgG1) and EOS as independent variables. Model 4 including ln(BA), lithiasis, rat body weight,ln(IgG1) and EOS as independent variables. Model 5 including lithiasis, ln(IgG1), EOS, presence of blood in faeces and presence of RBC antibodies as independent variables.Model 6 including ln(IgG1) and % splenic weight as independent variables.

infection periods: mainly normocytic (in 4 out of 6 infected ratsstudied), secondly macrocytic (in 2 out of 6 infected rats studied) at20 weeks p.i.; mainly microcytic (in 13 out of 22 infected rats stud-ied) and secondly macrocytic (in 5 out of 22 infected rats studied) at60 weeks p.i. The extrapolation of our results to human fasciolosismight explain the different types of anaemia previously described.The type of anaemia in fasciolosis could be considered a biomarkerof the chronicity period of the disease, changing from normocyticto macrocytic in the early chronic period and to microcytic in theadvanced chronic period. Likewise, changing from normochromicin the early chronic period to hypochromic in the advanced chronicperiod.

The negative correlation between the haematological param-eters vs fluke burden, eggs per gram of faeces and total parasitebody size has been demonstrated. In human populations, HGB lev-els appear related to epg levels of this parasite (Curtale et al., 1998,2003, 2005). In hyperendemic zones, the majority of adult humansubjects should be in the chronic phase and may frequently haveaccumulated fluke burdens due to the high contamination risk(Mas-Coma et al., 1999b). Consequently, a high risk of anaemia maybe expected in human subjects inhabiting areas where fasciolosisis highly endemic and where high egg outputs detected in humanssuggest that liver fluke burdens may also be very high.

Our results suggest that the host’s immunological response,mainly increased IgG1, IgE and EOS levels, may also contribute tothe pathophysiology of fasciolosis-related anaemia. In human fas-ciolosis, IgG, IgM and IgE are usually elevated and total and specificIgE levels are positively correlated with egg burden, patient age andclinical features (Mas-Coma et al., 2000; Salem et al., 1987).

To date, no study has investigated the contribution of autoim-mune haemolysis to fasciolosis related-anaemia. Our data showthe presence of anti-RBC antibodies in a low percentage of F. hep-atica infected rats at 60 wpi and might lead to the considerationof autoimmune haemolysis. In fasciolosis, as in schistosomiasis,autoimmune haemolysis might be produced when the erythrocytesurface either adsorbs molecules of antigens produced by the par-asite or when the erythrocyte surface is damaged in some way,thus providing binding sites on its surface (Bruninga, 1971). Nev-ertheless, it must be pointed out that the production of theseantibodies is not enough to attribute autoimmune haemolysisto infection. Consequently, the question between DAT positive

anaemia and the presence of autoimmune haemolysis in rats withfasciolosis remains unsolved. In humans, the application of Coombsdirect antiglobulin test has never been recommended in fasciolosispatients, and consequently information on this aspect is lacking.Nevertheless, in one Peruvian child with mild anaemia and fasci-olosis to whom DAT was applied, the result was positive (Marcos etal., 2005).

Another recognized cause of anaemia is the accelerated destruc-tion of the reticulocytes in peripheral blood (Todd and Ross, 1966).Our results indicate for the first time that splenic hypertrophypartly explains the anaemia process. Anaemia and splenomegalyare commonly observed in human fasciolosis (Mas-Coma et al.,2000; Hassan et al., 2000; Torres et al., 2004).

Anaemia of inflammation, also known as anaemia of chronic dis-ease, is associated with infectious agents, cancer, and autoimmunedisorders (Weiss and Goodnough, 2005). Inflammatory cytokinesadversely affect erythropoiesis as well as RBC life span. Specifically,TNF-�, IFN-�, and IL-1� inhibit differentiation and proliferation oferythroid progenitor cells (Weiss and Goodnough, 2005). The Th-1 cytokine, IFN-�, is involved in the pathogenesis of anaemia ofinflammation (Means, 1995; Weiss et al., 1995). In contrast to therelationship of the Th-1 response to anaemia, certain lines of evi-dence suggest that a dominant Th-2 response may be protectiveagainst anaemia. Th-2 cytokines (such as IL-4 and IL-10) down-regulate the production of Th-1 cytokines by feedback inhibition. Inmice, Th-2 cytokines stimulate bone marrow formation and protectfrom severe anaemia (Kühn et al., 1993; Van Vlasselaer et al., 1995).Interestingly, IL-10, which, in general, plays an anti-inflammatoryrole, has also been associated with anaemia of inflammation (Tilget al., 2002). The role of cytokines in the pathogenesis of anaemiain parasitic infections has also been demonstrated, e.g. in malariaand schistosomiasis (Menendez et al., 2000; Friedman et al., 2005).Development of severe malarial anaemia may be inversely cor-related with serum IL-4 levels (Biemba et al., 2000). IL-10 andthe dangers of immune polarization: excessive type 1 and type 2cytokine responses induce distinct forms of lethal immunopathol-ogy in murine schistosomiasis. Certain cytokines are involved inboth immune activation and iron metabolism. However, the inter-action between cytokine production and iron status is not clear, e.g.the interactions between malaria, iron deficiency and immunity arecomplex. The balance between pro-inflammatory cytokines (such

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42 M.A. Valero et al. / Acta Tropica 108 (2008) 35–43

as TNF-�, IFN-�) and anti-inflammatory cytokines (such as IL-4 andIL-10) is important not only in malaria disease presentation but alsoin iron homeostasis. Inflammatory cytokines have been associatedwith both the pathogenesis of severe anaemia in Plasmodium falci-parum (Othoro et al., 1999) and the regulation of iron metabolism(Weiss et al., 1997). Maintenance of cellular iron homeostasis isnot only a general prerequisite for the growth and proliferation ofall cells but is also of central importance for the regulation of theimmune function. Cytokine secretion is supposed to be modulatedby the iron status (Helyar and Sherman, 1987; Munoz et al., 1994).

In the rodent model the immune reaction to fasciolosis appearsto be characterized by a predominant Th-2 response in a chronicinfection, but the presence of Th-1 cytokines has also been demon-strated (Gironès et al., 2007). At 20 weeks p.i., a positive relationshipbetween IL-4 and MCV was detected in our study. IL-4 appears toplay an important role in modulating the host protective immuneresponse to certain helminths, such as Trichuris muris in mice (Elseet al., 1994). Since the levels of IL-4 vary as the infection progresses,our results point to the fact that the type of anaemia may be directlyrelated to the time of infection and the clinical periods of the dis-ease. Furthermore, our observations might imply that increasedIL-4 levels are detected in rats with normal iron serum levels, whileincreased TNF-�, IL-1� and IL-10 levels are found in rats with adeficiency of iron serum levels. However, due to the low number ofinfected rats in experiment 1, the possible effect of cytokine levels inanaemia-related fasciolosis could not be assessed. More studies willbe required to corroborate our findings and elucidate the effect ofthe iron status on various cytokine profiles during fasciolosis infec-tion. The coincidence of several factors associated with anaemia ofinflammation as well as fasciolosis highlights the need for morefocused research. For example, in addition to directly affectingerythropoiesis, IL-1� or TNF-� also decrease appetite which maylead to malnutrition and its attendant consequences, such as iron-deficiency anaemia (Tracey and Cerami, 1990).

As fasciolosis is a potent inducer of Th2 responses, the abilityto mount any effective Th1 responses against bacteria and otherpathogens is impaired (O’Neill et al., 2000). The predominance of aTh2 response during early chronic infection decreases in advancedchronic infection, which is characterized by a persistent immunesuppression (Gironès et al., 2007). A high bacterobilia risk inadvanced experimental chronic fasciolosis has been described in arat model and supports this fact (Valero et al., 2006a). Co-infectionsin liver-fluke-infected subjects are common in human fasciolosisendemic areas (Esteban et al., 1997a,b, 1999, 2002, 2003). Mod-erate or severe anaemia in cases of Fasciola co-infection has beendescribed in children (Marcos et al., 2005).

In conclusion, a correlation between fasciolosis, anaemia andimmune response has been demonstrated. The development ofanaemia appears to be complex and may involve multiple mech-anisms. However, to the mechanisms that until now explainFascioliosis-related anaemia (compensatory increase in erythro-cyte production and a continuous drain on iron stores resultingfrom the parasites’ blood-sucking activities) the following causesought to be added: haemolysis of red blood cells, the general effectsof inflammation on erythropoiesis, concomitant parasitic and bac-terial infections and pre-morbid nutritional abnormalities.

Conflicts of interest

All authors: no conflicts.

Acknowledgments

We would like to thank Dr. M.D. Bargues (Valencia, Spain)for providing the liver fluke metacercariae used. We gratefully

acknowledge the Servicio Central de Soporte a la InvestigaciónExperimental (Universidad de Valencia) for the access to theirinstrumental facilities, and the Servicio de Análisis Clínicos del Hos-pital General de la Defensa de Valencia (Spain) for the biochemical,haematological and microbiological determinations.

Financial support: Haematological and parasitological researchfunded by Projects No. BOS2002-01978 and No. SAF2006-09278 ofthe Spanish Ministry of Science and Technology, Madrid, Spain; theRed de Investigación de Centros de Enfermedades Tropicales–RICET(Projects No. C03/04, No. PI030545 and No. RD06/0021/0017 of theProgramme of Redes Temáticas de Investigación Cooperativa) of theFondo de Investigación Sanitaria (FIS), Spanish Ministry of Health,Madrid, and Projects No. 03/113, GV04B-125 and GVCOMP2006-106 of Conselleria de Empresa, Universidad y Ciencia, Valencia,Spain. Immunological work funded by Spanish Ministry of Educa-tion, Health and Sport, Ministry of Science and Technology, andPlan Nacional de Salud (Project No. SAF2002-03356 and SAF2004-0519) and CAM (08.3/0023.1/2001), the Red Temática Cooperativade Investigación en SIDA (RIS G03/173) and the Red Temática Recava(C03/ 01).

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