Experimental mycotoxic nephropathy in pigs provoked by a mouldy diet containing ochratoxin A and...

This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

http://www.elsevier.com/copyright

Author's personal copy

Experimental and Toxicologic Pathology 64 (2012) 733–741

Contents lists available at ScienceDirect

Experimental and Toxicologic Pathology

journa l homepage: www.e lsev ier .de /e tp

Experimental mycotoxic nephropathy in pigs provoked by a mouldy dietcontaining ochratoxin A and fumonisin B1

Stoycho D. Stoeva,∗, Dimitrina Gundashevaa, Ivan Zarkovb, Teodora Mirchevac,Dimitrina Zapryanovac, Stefan Denevd, Yuri Miteve, Hristo Daskalov f,Mike Duttong, Mulunda Mwanzag, Yves-Jacques Schneiderh

a Department of General and Clinical Pathology, Faculty of Veterinary Medicine, Trakia University, Students Campus, 6000 Stara Zagora, Bulgariab Department of Epizootology, Faculty of Veterinary Medicine, Trakia University, Students Campus, 6000 Stara Zagora, Bulgariac Department of Biochemistry, Faculty of Veterinary Medicine, Trakia University, Students Campus, 6000 Stara Zagora, Bulgariad Department of Microbiology, Faculty of Agriculture, Trakia University, Students Campus, 6000 Stara Zagora, Bulgariae Department of Ecology and Zoochygiene, Faculty of Agriculture, Trakia University, Students Campus, 6000 Stara Zagora, Bulgariaf National Reference Laboratory, National Reference Centre of Food Safety, National Diagnostic and Research Veterinary Institute, 15 Pencho Slaveykov, 1606 Sofia, Bulgariag Food, Environment and Health Research Group, Faculty of Health Science, University of Johannesburg, Doornfontein 2028, Gauteng, PO Box 17011, South Africah Institut des Sciences de la Vie, Universite Catholique de Louvain, Croix du Sud 5, B 1348 Louvain-la-Neuve, Belgium

a r t i c l e i n f o

Article history:Received 16 October 2010Accepted 13 January 2011

Keywords:Ochratoxin AFumonisin B1MycotoxinsMycotoxic porcine nephropathyPathologyImmunosuppression

a b s t r a c t

Mycotoxic nephropathy was induced in eighteen young pigs by mouldy diets containing 0.5 ppm ochra-toxin A (OTA) and/or 10 ppm fumonisin B1 (FB1) for three months. While the most obvious damagesprovoked by OTA were seen in the kidneys as expressed by the strong degenerative changes in proximaltubules and fibrosis in kidneys, FB1 was found to induce an increase in permeability of vessels mainly inlung, brain, cerebellum or kidneys and slight to moderate degenerative changes in kidneys. Pathomor-phological damages in pigs exposed to both mycotoxins simultaneously present a combination of themain lesions provoked by each mycotoxin alone being stronger in their expression. Biochemical inves-tigations as expressed by the increase of serum creatinine, urea and enzyme activity of ASAT/ALAT andby the decrease of serum cholesterol, total protein, albumin and glucose were strongest in pigs exposedto both mycotoxins simultaneously as can be anticipated form the strongest lesions in the kidneys. Bothmycotoxins and their combination were found to disturb powerfully humoral immune response in allexperimental pigs as expressed by the strong decrease in antibody titer against Morbus Aujesky at days21 and 35 after vaccination. Having in mind that the feed levels of the both mycotoxins as well as theexposure time and the pathological findings corresponded to those in some spontaneous cases of porcinenephropathy in Bulgaria and South Africa, it can be concluded that the same mycotoxins are involved inthe observed field cases of that nephropathy.

© 2011 Elsevier GmbH. All rights reserved.

1. Introduction

The mycotoxic nephropathy (MN) is a renal disorder causedby alimentary ingestion of secondary fungal metabolites possess-ing nephrotoxic properties and encountered in feeds/foods mademainly from cereals or fibrous plants, and kept in storehouse con-

Abbreviations: BEN, balkan endemic nephropathy; CIT, citrinin; FB1, fumonisinB1; MVN, microvirusneutralization test; MN, mycotoxic nephropathy; MPN, myco-toxic porcine nephropathy; MCN, mycotoxic chicken nephropathy; OTA, ochratoxinA; PA, penicillic acid.

∗ Corresponding author at: Department of General and Clinical Pathology, Fac-ulty of Veterinary Medicine, Trakia University, Students Campus, 6000 Stara Zagora,Bulgaria. Tel.: +359 42 670540.

E-mail address: [email protected] (S.D. Stoev).

ditions and increased humidity. There are however some variancesin the manifestation of the disease, especially in the clinicomor-phological picture, which in many cases is influenced by variousother mycotoxins in addition to ochratoxin A (OTA) (Stoev et al.,2010a,b) as well as by secondary intestinal bacterial diseases asa result of the pronounced immunosuppression in the affectedanimals (Stoev et al., 2000a,b; Stoev, 2008). Although many nephro-toxic fungal compounds such as fumonisins are known, only OTAand partly citrinin (CIT) are usually associated with spontaneouscases of MN. Recently, however, the spontaneous nephropathy inBulgaria (Stoev et al., 2010a), which is observed frequently dur-ing the meat inspection and which differs morphologically fromthe classical description of mycotoxic porcine/chicken nephropa-thy (MPN/MCN) in Denmark was found to have multi-mycotoxicetiology involving several mycotoxins such as OTA, penicillic acid

0940-2993/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.doi:10.1016/j.etp.2011.01.008


734 S.D. Stoev et al. / Experimental and Toxicologic Pathology 64 (2012) 733–741

(PA) and fumonisin B1 (FB1). Moreover, spontaneous MN in SouthAfrica was also found to have the same multimycotoxic etiology(Stoev et al., 2010b).

Recently, a potent synergistic effect was found between OTAand PA, mycotoxins produced by the same ochratoxinogenic fungi,when the same mycotoxins were given simultaneously to pigs orchicks (Micco et al., 1991; Stoev et al., 2000a, 2001, 2004). The pro-duction of multiple toxins by one or several fungi, as is sometimesthe natural situation, presents a problem that has not been suf-ficiently investigated. Such mixtures of toxins may have additiveor synergistic effects in farm animals, which could be responsiblefor the different pathology observed (Stoev et al., 2001, 2010a,b).Recently, it was suggested that combined administration of targetmycotoxins as OTA, FB1 and PA may be responsible for the MPN inBulgaria (Stoev et al., 2010a), associated with relatively low meancontamination by OTA in feeds (207.10 ± 65.14 ppb in 1993 and114.06 ± 35.79 ppb in 1994) (Stoev et al., 1998a,b).

In this regard, the pathomorphological picture of some spon-taneous MPN or MCN were found to differ from the classicaldescription of MPN/MCN as made in Scandinavia by the exten-sive vascular changes, which may result largely from the effectsof nephrotoxic metabolites other than OTA (Stoev et al., 1998a,2002a) or may be attributed to synergistic effects between OTA andother mycotoxins, and the increase of OTA toxicity (Stoev, 2008).The low contamination levels of OTA in fed forages (200–400 ppb)and the high incidence of MPN (Stoev et al., 1998a,b) and MCN(Stoev et al., 2002a) in Bulgaria suppose a possible synergistic oradditive interaction between OTA and other mycotoxins enhanc-ing the nephrotoxicity of OTA and/or having additional nephrotoxiceffect, which needs to be proved. Such an interaction betweenOTA and other mycotoxins needs to be further investigated. Thedifferent macroscopic and microscopic lesions found in variousnephropathies in different countries should be related to theexposure time to the mouldy feed as well as to the different con-centrations or combinations of nephrotoxic mycotoxins ingestedby affected pigs or chicks (Stoev, 2008).

It has been reported that FB1 and OTA co-occurred in maize inCroatia (Jurjevic et al., 1999; Domijan et al., 2005). In addition, FB1and OTA were found to contaminate the feeds from farms withknown nephropathy problems in Bulgaria (with mean levels of FB13000–5000 ppb and OTA of 200–400 ppb) and South Africa (withmean levels of FB1 above 5000 ppb and OTA of 60–70 ppb). How-ever, only scarce data are available about a combined exposure toOTA and FB1 or PA, which might spontaneously occur under fieldconditions (Stoev et al., 2010a,b).

In the literature, there are only a few in vitro experiments,reporting some synergistic interactions between OTA and FB1(Klaric et al., 2007; Creppy et al., 2004; Stoev et al., 2009). It is impor-tant to recognize that in vitro experiments can mainly demonstratedirect synergistic effects of mycotoxins, but only in vivo studiescan clearly show the real interaction between mycotoxins in addi-tion to their absorption, distribution, bioavailability, metabolismand excretion. Therefore, it is already crucial to determine whetherfeeding such mycotoxin contaminated mouldy feed to pigs/chickscould reproduce the same functional and morphological changesin kidneys observed in spontaneous porcine/chicken nephropathyat contamination levels and periods corresponding to the levelsand periods of exposure to feed naturally contaminated with thesame combination of mycotoxins. In addition, it will be helpful toestablish, whether co-administration of OTA and FB1 has a realsynergistic or additive effects as co-administration of PA and OTA(Stoev et al., 2001, 2004).

Having in mind the circumstance, that there are not any use-ful data in regard to the combined toxic effects of OTA and FB1 onpigs, which might spontaneously occur under field conditions, theobjective of this study was to carry out some experimental tox-

icological, biochemical, immunological and pathomorphologicalinvestigations in order to assess the responsibility of both myco-toxins for the different pathology, characteristic for spontaneousMPN, largely encountered in some regions in Bulgaria. The feedlevels of both mycotoxins, used in the present experiment, corre-sponded to some high levels of OTA and FB1 found in the farmswith MPN, but were a little higher than the mean feed values of thesame mycotoxins (Stoev et al., 2010a,b).

2. Materials and methods

2.1. Ochratoxin A production

Aspergillus ochraceus (isolate D2306, as used by Stoev et al.,2000a,b, 2002b) was grown on sterilised shredded wheat (40 g) in500 ml conical flasks, moistened by a 40% (v/w) addition of ster-ile water and incubated on a rotary shaker at 27 ◦C for 2 weeks.The brown granular product, which bore no obvious sign of fungalgrowth or sporulation, was sterilised at 80 ◦C for 1 h (yield 2 kg)and stored at −20 ◦C. A sample was analysed by HPLC with diodearray detection for OTA and found to contain ∼2 mg/g. No othermycotoxins (e.g. PA) were present, although there was typically asmall additional proportion of the biologically inactive deschloro-analogue ochratoxin B. The ochratoxin-rich moulded shreddedwheat was then homogenised into pig ration (diluted more than2000-fold) to give the required concentration of OTA in diet.

2.2. Fumonisin B1 production

Fusarium verticillioides (isolate MRC 826) was grown on moist-ened ground corn kernels (50 g ground maize kernels in 1000 mlconical flasks were moistened by addition of 70 ml sterile waterand then autoclaved 30 min at 121 ◦C and 120 kPa) as describedby Alberts et al. (1993). The moistened ground corn kernels weredirectly inoculated with 1 ml of a spore suspension (lyophilizedconidia of F. verticillioides MRC 826 in 100 ml of sterile distilledwater) and incubated on a rotary shaker at 25 ◦C in the dark for2 weeks. The medium was then freeze-dried. A sample was ana-lysed by HPLC using the fluorescence detector for FB1 and foundto contain ∼8–10 mg/g. The FB1-rich moulded ground maize ker-nels were then homogenised into pig ration to give the requiredconcentration of FB1 in diet.

The used fungus is an endophytic plant pathogen that causesear rot of maize. The particular isolate was isolated from maizein the former Transkei region of South Africa. It initially producedabout 17 g/kg of fumonisin B1 in maize culture material. Currentlyit still produces around 8–9 g/kg FB1 in maize cultures. This strainwas used under material transfer agreement with Medical ResearchCouncil of PROMEC Unit, Tygerberg, South Africa.

2.3. HPLC analysis

FB1 extraction and clean-up was performed according to Hinojoet al. (2006) using the SAX column. OTA extraction and purificationwas performed as recently described (Stoev et al., 2010a,b). HPLCquantification of OTA or FB1 in the experimental materials wasperformed using the system Shimadzu (Kyoto, Japan), consistingof liquid chromatograph LC 20A fitted to degasser DGU 20A3, autosampler (injection) SIL 20A, communications bus module CBM 20A,column oven CTO 20A, photodiode array detector SPD M20A andfluorescence detector RF 10AXL all connected to gigabyte computerwith Intel Core DUO with Microsoft XP.

For HPLC analysis of OTA photodiode array (PDA) detector SPDM20A with column Waters Symetry C18 (250 mm long, 4.6 mminternal diameter) were used accordingly, with appropriate mobilephase, flow rate and running time according to Kokkonen et al.


S.D. Stoev et al. / Experimental and Toxicologic Pathology 64 (2012) 733–741 735

(2005) and Abdulkadar et al. (2004) with little modifications.Reagents and HPLC conditions for OTA were: mobile phase – ace-tonitrile/water (50/50); flow rate – 0.8 ml/min; injection volume– 10 �l per sample; running time – 30 min; column temperature –30 ◦C.

For FB1, RF-10AXL fluorescence detector (FD) was used with thesame column and the following settings: wavelength of 420 nmexcitation and 500 nm emission. Reagents and HPLC conditions forFB1 were: mobile phase – acetonitrile/water in the ratio 60/40; flowrate – 1 ml/min; injection volume – 10 �l per sample; running time– 30 min; column temperature – 30 ◦C. Derivatization reagent usedfor FB1 analysis was naphthalene dicarboxyaldehyde (NDA) andHPLC was performed according to Bennett and Richard (1994) –residues were dissolved with 1 ml methanol; into 100 �l of sampleor mycotoxin standard dissolved with methanol in HPLC vial, thefollowing reagents were added in sequence and mixed after eachaddition: 100 �l 0.05 M sodium borate buffer (pH 9.5), 50 �l sodiumcyanide and 50 �l NDA. This was followed by incubation in a waterbath set at 50 ◦C for 20 min, after which the samples were cooledand diluted with 700 �l 0.05 M phosphate buffer (pH 7)-acetonitrile(40/60).

2.4. Experimental design

Twenty-four young pigs (Landrace × Bulgarian white from acommercial stock source in Bulgaria) were purchased at about eightweeks of age and approximately equal body weight (12–14 kg).After that, pigs were arranged in four groups of six (3 male and3 female) and fed ad libitum either a good quality commercialpig starter ration (controls) or the same ration artificially con-taminated with the fermented OTA-rich shredded wheat or/andFB1-rich ground maize kernels to give the desired contaminationlevels of both mycotoxins in the diet as mentioned in Table 1. Freshdrinking water and feeds were available ad libitum during the fol-lowing three months.

To protect against developing secondary bacterial enteric dis-ease, to which pigs given OTA or FB1 may be susceptible (Stoev et al.,2000b), pigs were given Dor-novTM (Dorvet Ltd., Nes-Ziona, Israel)at a prophylactic dose in drinking water for the first experimen-tal week, and were then similarly given RidzolTM (Merck, Sharp &Dohme, Holland) up to day 14. This 2-week prophylactic treatmentwas repeated again after 1 month.

Blood (from v. cava cranialis) for clinical biochemistry andimmunological investigations was taken after premedication with“Combistress” (Acepromasini maleus-KELA-Laboratoria B-2320,Hoogstraten, Belgium) at the end of the 21st and 35th day aftervaccination or 35th and 49th day from the beginning of the exper-iment. Clinical biochemistry parameters as well as antibody titerswere measured in samples of both blood collection dates.

The Trakia University Animal Care Ethic Committee approvedthe study protocol and the pigs were housed, maintained andslaughtered in accordance with the Bulgarian Welfare Regulations.

2.5. Immunization

On day 14 of the experiment all pigs were immunised against theMorbus Aujeszky by intramuscular injection (2 ml). Pseudorabies(virus of Aujeszky), vaccine strain MK-35, was used after adaptationin cell line “Calf trachea”. After 3 passages virus was titrated anddemonstrated titer 105.5 TKID50/0.1 ml.

2.6. Measurement of humoral immune response viamicrovirusneutralization (MVN) test

Subsequent development of humoral immune response wasmeasured by demonstration of virusneutralization antibodies in

pigs on 21st and 35th day after inoculation of the vaccine. Vac-cinal strain MK-35 was used for MVN test. MVN was accomplishedin 96-well PVC plates of Limbro (Flow LaboratoriesTM) followingthe method prescribed by World Organisation for Animal Health(OIE) in the Manual of Diagnostic Tests and Vaccines for TerrestrialAnimals (Part 2, Section 2.2, Chapter 2.2.2 – Aujeszky’s Disease).Cell line of calf trachea was cultured in medium MEM enrichedwith lactate albumin hydrolysate and foetal calf serum. The ther-molabile virus neutralizing inhibitors were separated by heatingsera at 56 ◦C for 30 min. The same medium was used to prepareseries of double dilutions of sera from 1:4 up to 1:1024. Aliquotsof 100 TKID50/ml from a viral strain (MK-35) adapted to a cellline of calf trachea were added to each dilution. After 1 h of incu-bation at 37 ◦C cell suspension adjusted to 2.5 × 105 cells/ml wasadded. All ingredients took part in the reaction in 0.05 ml quan-tities. The plates were incubated in a thermostat at 37 ◦C for 7days before reading of the results. As final was accepted the titer,that at the highest dilution protected 50% of the cells (cell linefrom calf trachea) from cytopathic effect (CPE) in the monolayer inseparate well.

2.7. Pathomorphology

At the end of the 3 month experimental period, all pigs wereslaughtered and examined macroscopically and materials for his-tological examination were taken from kidney, liver, spleen, lung,heart, brain, cerebellum, intestine and mesenteric or kidney’slymph nodes, and fixed in 10% neutral buffered formalin. Fixed tis-sues were embedded in paraffin wax, sectioned at 6 �m and stainedwith hematoxylin–eosin. Periodic acid – Schiff stain was also usedfor proving of lipoproteid, glycoproteid or mucoproteid substancesin various tissues and cell components, and especially to establish apossible thickening of basement tubular membranes (with lipopro-tein structure). Some materials were stained according to Weigertiron hematoxylin for proving the presence or absence of fibrin inlymphatic cysts or edematous areas.

2.8. Clinical biochemistry

Serum was analysed within 1–2 h of collection. Serum creati-nine was measured via measuring the absorbance of its orange-redcoloured complex with picric acid in alkaline solution accordingto modified method of Bartels and Bohmer (1971). Serum albu-min was measured via measuring the absorbance of its colouredcomplex with bromocresol green in citrate buffer as describedby Rodkey (1964). Serum total protein was measured via mea-suring the absorbance of its purple complex with cupric ions inalkaline solution (Weichselbaum, 1946). Serum urea was mea-sured via enzymatic method as described by Berthelot (1859),whereas serum glucose was determined after enzymatic oxida-tion in the presence of glucose oxidase according to Barham andTrinder (1972). Serum cholesterol was determined after enzy-matic hydrolysis and oxidation as described by Schettler andNussel (1975). Photometric and/or enzymatic colorimetric testswere purchased from Human Gesellschaft fur Biochemica undDiagnostica mbH (D-65205 Wiesbaden, Germany). The activitiesof aspartate aminotransferase (ASAT) (Bergmeyer et al., 1976) andalanine aminotransferase (ALAT) (Bergmeyer and Horder, 1980)were measured by kinetic methods according to recommenda-tions of the Expert Panel of International Federation of ClinicalChemistry (IFCC) using liquiUV Test of Human Gesellschaft fur Bio-chemica und Diagnostica mbH (D-65205 Wiesbaden, Germany).The same tests were performed using Automatic Biochemistry Ana-lyzer SINNOWA BS-3000P (Sinnowa Med Sci & Technol, Co LtdNajing, China).



Table 1Mean clinical biochemistry values and antibody titers in the blood of pigs (6 pigs in each group) fed on diet containing 0.5 ppm OTA and/or 10 ppm FB1 at the end of the 21stand 35th day after immunization against the Morbus Aujeszky or 35th and 49th day from the beginning of the experiment.

Mycotoxin (feed levels) OTA + FB1 (0.5 ppm + 10 ppm) OTA (0.5 ppm) FB1 (10 ppm) Control

35th day of the experiment (21st day after immunization)Creatinine (�mol/l) 2023 ± 5.6 1913 ± 3.9 1722c ± 1.2 153c ± 2.7Urea (mmol/l) 9.163 ± 0.27 7.163c ± 0.32 3.82c ± 0.14 3.18c ± 0.29Glucose (mmol/l) 4.682 ± 0.14 5.04 ± 0.20 5.44a ± 0.15 5.74b ± 0.26Cholesterol (mmol/l) 2.261 ± 0.09 2.28 ± 0.05 2.50 ± 0.14 2.75a ± 0.18Total protein (g/l) 61.21 ± 1.17 63.9 ± 1.38 67.7a ± 1.26 66.8a ± 1.49Albumin (g/l) 31.02 ± 1.06 31.42 ± 1.95 40.1a ± 2.98 41.5b ± 1.48ASAT (U/L) 52.12 ± 3.1 45.6 ± 1.83 41.5a ± 1.57 38.3b ± 2.25ALAT (U/L) 71.23 ± 2.05 66.33 ± 2.08 52.9c ± 0.48 49.8c ± 1.37Antibody titer 203 ± 5.3 693 ± 38.0 413 ± 19.7 384c ± 80.9

49th day of the experiment (35th day after immunization)Creatinine (�mol/l) 1943 ± 2.3 1713c ± 3.4 1462c ± 2.5 132c ± 1.2Urea (mmol/l) 10.173 ± 0.64 6.743c ± 0.37 3.47c ± 0.14 2.59c ± 0.16Glucose (mmol/l) 4.78 ± 0.27 4.98 ± 0.22 5.35 ± 0.28 5.51 ± 0.15Cholesterol (mmol/l) 2.282 ± 0.13 2.521 ± 0.16 3.03 ± 0.10 3.34b ± 0.33Total protein (g/l) 60.63 ± 1.05 64.02 ± 1.99 70.1b ± 1.44 72.2c ± 1.55Albumin (g/l) 29.03 ± 1.27 32.63 ± 0.82 39.6c ± 1.18 42.3c ± 0.76ASAT (U/L) 57.93 ± 3.53 49.91 ± 1.64 41.1c ± 1.69 39.1c ± 1.28ALAT (U/L) 73.42 ± 4.79 65.91 ± 7.06 51.6a ± 1.77 47.0b ± 2.24Antibody titer 173 ± 3.2 623 ± 38.8 323 ± 7.1 426c ± 53.9

±SEM (standard error of the mean). 1 – Significant difference (p < 0.05); 2 – very significant difference (p < 0.01); 3 – extremely significant difference (p < 0.001) comparedto controls. a – Significant difference (p < 0.05); b – very significant difference (p < 0.01); c – extremely significant difference (p < 0.001) compared to pigs given OTA and FB1simultaneously.

2.9. Statistical methods

Student’s t-test, in addition to non-parametric Mann–Whitney-U-test was used to estimate significance of differences between themean values of measured parameters of the clinical biochemistry.

3. Results

3.1. Clinical biochemistry and immunology

Biochemical investigations revealed that the concentrations ofserum creatinine and urea were increased in different degrees inthe pigs of all experimental groups. These increases were more pro-nounced in the groups treated with OTA being highest in the grouptreated with both mycotoxins simultaneously (Table 1).

Such increases were also seen for serum enzyme activity of ASATand ALAT, but only in the pigs of groups treated with OTA beinghighest in the group exposed to both mycotoxins (Table 1).

The serum levels of cholesterol were decreased only in the pigstreated with both mycotoxins as well as in the pigs exposed to OTAalone, but only at day 49 of the experiment (Table 1).

Similar trends were seen in the serum levels of albumin and totalproteins, which were significantly decreased only in the pigs of bothgroups treated with OTA, but such decreases, were not observed inthe pigs exposed to FB1 alone (Table 1).

A temporary decrease was also seen in the serum level of glu-cose, but only in the pigs exposed to both mycotoxins at day 35 ofthe experiment (Table 1).

A strong disturbance was found in humoral immune responseof all experimental pigs as expressed by antibody titer against theMorbus Aujesky. The same was well pronounced in all experimen-tal groups at day 21 and day 35 after immunization (Table 1).

3.2. Clinical signs

There were only scarce clinical signs confined mainly to polyuriaand polydipsia seen in the pigs of both groups exposed to OTA.Transient cases of diarrhoea were only seen in a few pigs from thegroup exposed to both mycotoxins OTA and FB1 simultaneously.

3.3. Macroscopic and microscopic pathology

3.3.1. OTA-treated groupGenerally, the most obvious pathomorphological damages in

the pigs of the group exposed to OTA alone were seen in the kidneys.In the kidneys, the only macroscopic lesions were a few faint

grey-white foci on the surface. On the cut surface, the grey-whitefoci were confined mainly to the ventral part of the cortex andresembled pale grey strips orientated radially to the renal pelvis.The main renal microscopical changes were seen in the epitheliumof proximal tubules and consisted of cloudy swelling, granular orvacuolar degeneration and desquamation. Karyolysis, karyopykno-sis and rarely karyomegaly were seen in the nuclei of epithelialcells (Fig. 1). Some proximal tubules were occluded by the swollenepithelial cells, whereas others were atrophied and surroundedby connective tissue. Some dilated tubules were lined by squa-mous epithelium or contained proteinaceous and necrotic debrisin the lumen. Cellular casts were sometimes observed in convo-luted or collecting tubules (Fig. 2). Telangiectasis, minute lymphaticcysts or slight enlargement of lymphatic spaces containing serousfluid and lined by endothelial cells were sometimes encountered

Fig. 1. Granular degeneration and karyopyknosis or karyolysis in epithelial cells ofproximal tubules. Kidney of pig fed on diet containing 0.5 ppm OTA for 3 months.HE × 260.



Fig. 2. Granular degeneration in epithelial cells of proximal tubules (a). Prolifera-tion of fibroblastic connective tissue and mononuclear cells in the interstitium (b).Granular casts in the lumen of some tubules (c). Kidney of pig fed on diet containing0.5 ppm OTA for 3 months. HE × 200.

in the cortex. Capillary endothelial proliferation was seen in therenal interstitium. A limited proliferation of fibroblastic connectivetissue and a focal infiltration of mononuclear inflammatory cellswere observed in some regions of renal interstitium in the ventralcortex (Fig. 2). Focal interstitial fibrosis and glomerular sclerosiswere rarely seen in the cortex. Large retention cysts or pronouncedinterstitial fibrosis were not seen in any of the kidneys. Only slightcystiform dilatation was observed in some tubules of the kidneys.

Slight degenerative changes were also seen in the duodenal orjejunal mucosa. The mesenteric lymph nodes were slightly edema-tous or congested. The mucosal lesions consisted of a few little fociof necrosis and loss of surface epithelium. Small foci of mononu-clear inflammatory cells and neutrophils were seen in the laminapropria. A slight hyperpalasia of Goblet cell, slight submucosaledema or congestion of vessels were also seen.

Slight degenerative changes or depletion of cells were some-times observed in the lymphoid tissue of lymph follicles of thekidney’s lymph nodes or mesenteric lymph nodes.

In the liver, only the irregular staining or fatty changes, char-acterized by a slight cytoplasmic vacuolization in the hepatocytes,were observed.

Pathological changes were not observed in the brain, heart andlung of pigs from this group.

3.3.2. FB1-treated groupGenerally, the most obvious damage in the pigs of this group

was seen in the permeability of vessels, which was responsible forperivascular and especially pericapillary edema in the lung, brain,cerebellum and kidneys.

In the kidneys, the persistent pathomorphological changes werecharacterized by slight to moderate vacuolar or granular degen-eration in the epithelium of proximal tubules, hyperaemia ofvessels and peritubular capillaries, slight activation of capillaryendothelium, slight mononuclear proliferation in the interstitium,perivascular or pericapillary edema and enlargement of lymphaticspaces (Fig. 3). Proteinaceous debris was seen in the lumen of sometubules.

In the brain, pathomorphological picture was dominated by vac-uolization or slight colliquative/lytic changes in the cortex of thebrain, especially under the meninges as well as by lytic changes insome of the neurons and glia cells (Fig. 4). Slight perivascular andpericellular edema was observed. Edema and hyperaemia in themeninges were also seen (Fig. 5).

Fig. 3. Degenerative changes in tubular epithelium (a). Hyperaemia of peritubu-lar capillaries (b). Kidney of pig fed on diet containing 10 ppm FB1 for 3 months.HE × 300.

Fig. 4. Perivascular and pericellular edema (a). Lytic changes in some of the neu-rons and glia cells (b). Brain of pig fed on diet containing 10 ppm FB1 for 3 months.HE × 260.

Fig. 5. Hyperaemia of vessels and edema in the meninges (a). Vacuolization andlytic changes under the meninges (b). Brain of pig fed on diet containing 10 ppmFB1 for 3 months. HE × 200.



Fig. 6. Lymphatic cysts or enlargement of lymphatic spaces containing serousfluid in the interstitium (a). Proliferation of fibroblastic connective tissue, capillaryendothelium and mononuclear cells in the interstitium (b). Kidney of pig fed on dietcontaining 0.5 ppm OTA and 10 ppm FB1 for 3 months. HE × 200.

In the cerebellum, the most obvious pathomorphological dam-ages were the edema and slight degenerative changes (mainly lyticchanges) in the region of the Purkinje’s cells as well as the lytic orcolliquative changes in the corpus medullare or laminae medullaresof white matter and rarely in molecular or granular layers. Perivas-cular edema in molecular and granular layers was seen.

In the lung, pathomorphological picture was characterized byedematous changes, accumulation of serous or sero-fibrinous exu-date in interlobular tissue or alveolar lumina and thickening ofinteralveolar septa, because of epithelial hyperplasia and/or accu-mulation of fibrin in the septa. Slight proliferation of bronchial andbronchiolar epithelium was also observed. Slight mononuclear andfibroblastic proliferation in the interstitium was seen.

In the spleen, there was only a slight edema in the trabeculae.The slight depletion of cells in the white pulp, especially under thecapsule, was not persistent.

In the liver, only irregular staining (eosinophilia or cloudyswelling) or slight granular degeneration in some hepatocytes andslight to moderate hyperaemia of vessels were the most obviousdamages.

Pathological changes were not observed in the heart of pigs fromthis group.

3.3.3. OTA + FB1-treated groupGenerally, the main pathomorphological damages in the pigs of

this group can be considered as a combination of the main lesionsobserved in the above two groups and therefore were seen in thekidneys and permeability of vessels.

The surface of kidneys bore small grey-white foci confinedmainly in the ventral part of the cortex, which look like as pale greystrips with radial orientation. Histological examination revealedstrong damages in the epithelium of proximal tubules, where aci-dophilia, cloudy swelling and granular or vacuolar degenerationwere evident. Some of the tubules contained proteinaceous ornecrotic debris in the lumen. Cellular or hyaline casts were alsoseen in convoluted or collecting tubules. Hyperaemia of vesselsand peritubular capillaries, lymphatic cysts or enlargement of lym-phatic spaces containing serous fluid were often encountered inthe cortex (Fig. 6). Vascular damages consisted mainly of perivas-cular or pericapillary edema and capillary endothelial proliferationin the renal interstitium. A proliferation of connective tissue andfocal infiltration of mononuclear inflammatory cells was seen inthe interstitium. Focal interstitial fibrosis, atrophy of tubules andglomerular sclerosis were seen in some of the ventral regions of thecortex. A few retention cysts formations were also observed in thekidneys.

Fig. 7. Edema in interlobular and interalveolar tissue. Lung of pig fed on diet con-taining 0.5 ppm OTA and 10 ppm FB1 for 3 months. HE × 200.

In the brain, vacuolization or slight colliquative changes inthe cortex were seen. The lytic changes in some neurons andglia cells, the perivascular or pericellular edema, especially underthe meninges, were also obvious. Edema and hyperaemia in themeninges were found.

The main lesions in the cerebellum consisted of edema and lyticor slight colliquative changes in the region of Purkinje’s and corpusmedullare or laminae medullares of the white matter, and rarelyin molecular or granular layers. A perivascular edema was alsoobserved.

The lung was edematous with accumulation of serous or sero-fibrinous exudate in interlobular tissue or alveolar lumina andthickening of interalveolar septa due to epithelial hyperplasiaand/or accumulation of fibrin (Fig. 7). Bronchial and/or bronchio-lar epithelium was slightly proliferated in some regions and slightmononuclear or fibroblastic proliferation was seen in the intersti-tium.

Duodenal or jejunal mucosa revealed slight degenerativechanges. The mesenteric lymph nodes were slightly prominent,edematous or congested. A few little foci of necrosis and loss ofsurface epithelium in the mucosa were seen. A slight mononuclearinflammatory cells proliferation was sometimes seen in the lam-ina propria. Goblet cell hyperplasia, slight submucosal edema andcongestion of vessels were also found.

Slight degenerative changes or depletion of cells were observedin the lymphoid tissue of lymph follicles of the kidney’s lymphnodes (Fig. 8) or mesenteric lymph nodes and spleen. A slight edemain the trabeculae was seen in the spleen and lymph nodes. A slightdepletion of cells in the white pulp, mainly under the capsule, wassometimes present in the spleen.

In the liver, only irregular staining, slight granular degenera-tion or fatty changes in some hepatocytes and slight to moderatehyperaemia of vessels were the most obvious damages.

Pathological changes were not observed in the heart of pigs fromthis group.

Macroscopical and histological examination of the internalorgans from the control pigs did not reveal any pathologicalchanges.

4. Discussion

In the experiment performed, stronger nephrotoxic damages,supported by the respective biochemical indices, were found in thepigs exposed to both mycotoxins simultaneously compared to pigsexposed to any single mycotoxin.



Fig. 8. Degenerative changes or depletion of cells in the lymphoid tissue of regionallymph nodes of kidneys (a). Focal edema in the trabeculae (b). Lymph nodes of kidneyof pig fed on diet containing 0.5 ppm OTA and 10 ppm FB1 for 3 months. HE × 100.

The macroscopic and especially microscopic changes in thepresent nephropathy, especially vascular damages and edematouschanges in the kidneys of pigs exposed to both mycotoxins simulta-neously were found to be very similar to those seen in spontaneouscases of MPN in Bulgaria (Stoev et al., 1998a). The interstitial cystswere considered to be dilated lymphatics or telangiectasis. Thissuggests for a lymph stasis in the kidneys, and could thereforeexplain the more pronounced fibrosis in the ventral part of the cor-tex, possibly due to gravitational forces (Stoev et al., 2001). Thevascular changes observed in the present experiment also contrastwith the pathology in chronic Danish MPN (Krogh, 1976; Elling,1977), but were similar to spontaneous Bulgarian cases of MPN(Stoev et al., 1998a). The strong vascular lesions and edematouschanges in most of the internal organs in pigs exposed to bothmycotoxins are obviously due to the synergistic or additive effectsbetween OTA and FB1 as the same were not observed in nephropa-thy provoked by pure OTA (Elling et al., 1985) or in spontaneousDanish MPN (Elling, 1977). However, such changes often accom-pany spontaneous MPN in Bulgaria.

It is known that OTA inhibits the mitochondrial transport ofphosphate and the respiration of whole mitochondria by acting as acompetitive inhibitor of carrier proteins located on the inner mito-chondrial membrane (Meisner and Chan, 1974). It can be expectedthat the lost integrity of the membrane of cell organelles (Ellinget al., 1985; Stoev et al., 2001), due to specific inhibition of proteinsynthesis by OTA (Creppy et al., 1983), implies leakage of materialsfrom the organelles into the cytosol. In such a way, the reduc-tion of structural integrity of membranes could be associated withan increase of various lysosomal and peroxisomal enzymes in thesoluble cell fractions and subsequent degenerative changes in theepithelium (Elling et al., 1985).

The most obvious indicators for significant kidney damageappeared to be the concentrations of serum creatinine and urea,which were clearly increased in all experimental groups and espe-cially in those exposed to OTA, being significantly higher in pigsof the group exposed to both mycotoxins simultaneously. Theobserved decrease in serum protein, albumin or glucose could beresulted from impaired proximal tubular function of kidneys andconsequent disturbance in their reabsorption or from impairedglomerular filtration, which appeared to be significant only in thepigs given both mycotoxins. On the other hand, it has been reportedthat OTA can impair protein synthesis (Creppy et al., 1983), whichmight result to lower levels of total protein in the blood (Stoev et al.,2000b, 2001). It has been shown that FB1 could also impair proteinsynthesis in cells (Abado-Becognee et al., 1998). In the same study,it has been suggested that the observed synergism in cytotoxicity

of FB1 and OTA could be related to the ability of both toxins toimpair protein synthesis and increase lipid peroxidation producingreactive oxygen species (Creppy et al., 1984; Rahimtula et al., 1988;Abado-Becognee et al., 1998). Due to the fact that these toxins areincreasing reactive oxygen species production, the feeding animalswith diet containing antioxidants may provide a good preventionmeans (Creppy et al., 2004).

A decrease of sterol carrier protein as a result of decreased pro-tein synthesis could also affect cholesterol synthesis in the liver ofOTA treated pigs. It is known that OTA competitively inhibits mito-chondrial transport carrier proteins in liver mitochondria (Meisnerand Chan, 1974), which may result in decreased energy for choles-terol synthesis in more advanced stages of MN (Stoev et al., 2000b).The found increase in the serum enzymes ASAT and ALAT may bedue to the pronounced degenerative changes in kidneys or liver inOTA treated pigs (Stoev et al., 2000b). The liver lesions might haveresulted from enterohepatic recirculation of OTA (Fuchs, 1988) orto FB1 action (Voss et al., 2001; Diaz et al., 2001).

Recently, it was shown that immunosuppression is the firstexpressed toxic effect of OTA that may become evident clinicallybefore nephropathy and its associated biochemical changes (Stoevet al., 2000b). OTA suppression of humoral and cellular immunity,defined in principle (NNT, 1991), has been demonstrated in prac-tice by the present experiments via significantly lower antibodytiter on days 21 and 35 after immunization, as might be expectedfrom impaired, though not totally inhibited, protein synthesis. Hav-ing in mind the circumstance that the amount of OTA used in thepresent experiment is around the average values for natural con-tamination in Bulgaria (200–400 ppb) (Stoev et al., 1998a, 2010a),lower values may still be immunosuppressive, especially over along period. In natural conditions OTA is only part of a complexarray of mould metabolites, such as FB1, which may be synergisticwith it in immunosuppression.

It is important to mention that FB1 may also contribute to theimmunosuppressive effect of OTA and the increase in secondarybacterial infections observed in pigs with spontaneous MPN (Stoevet al., 2000b), having in mind the observed increase of intestinal col-onization by pathogenic Escherichia coli found in FB1-treated pigs(Oswald et al., 2003).

Mycotoxins have also been suspected as a possible etiologicalagent in Balkan endemic nephropathy (BEN) in humans (Krogh,1972; Stoev, 1998). It has to be emphasized that pathologicalchanges in kidneys in spontaneous MPN in Bulgaria, includingfibrotic changes and contraction of kidneys in later stages of MPN,resemble much more to those in BEN in humans, than in Dan-ish MPN (Stoev, 1998). Moreover, there are many other strikingsimilarities between BEN in humans and Bulgarian MPN as thelow food/feed concentrations of OTA, various kinds of tumoursin kidneys (pigs) or urinary tract (humans), retention tubularcyst formations, vascular damages, electron dense formations ormyelin-like figures in mitochondria or nuclei of epithelial cells,etc. The same damages have not been found in classical MPNas described in Denmark or elsewhere. All these discrepanciesbetween Bulgarian MPN and classical MPN, in addition to the sim-ilarities between Bulgarian MPN and BEN, could be due to theinteractions between OTA and other mycotoxins, which need tobe studied further. Our arguments can be also supported by somerecent experiments, in which we found electron-dense formationsin nuclei and myelin-like figures in mitochondria of kidneys in pigsexposed to very low contamination levels of OTA together withPA (Stoev et al., 2001). The same damages resemble much moreto those in spontaneous MPN and are unusual for classical DanishMPN (Elling, 1977; Elling et al., 1985).

OTA and FB1 were reported to induce in vitro and in vivo degen-erative and apoptotic changes in rat kidney (Dragan et al., 2001;Petrik et al., 2003). A synergistic effect between OTA and FB1, which



was proved in vitro (Klaric et al., 2007; Creppy et al., 2004) is in linewith some in vivo data from turkey poults experiment (Kubenaet al., 1997). The DNA damage provoked by the combined treat-ment with OTA and FB1, measured either by the standard cometassay or Fpg-modified comet assay, showed a synergistic increasein kidney cells in vivo as indicated by the tail length, tail intensityand OTM (olive tail moment), even at doses that correspond to thedaily human exposure in Europe (Domijan et al., 2006). In the sameexperiment, the tail intensity and OTM of the kidney cells of ratsreceiving combined treatment of OTA and FB1 was much higherthan would be a simple sum of values caused by the respectivedoses of either mycotoxin alone.

High contamination levels of OTA and FB1 (up to 40 mg/kg) havebeen also found in some pig feeds (Diaz et al., 2001) and werereported to provoke the death of the same pigs as pathologicalpicture revealed pathological signs of both toxins, e.g. pulmonaryedema, liver and kidney lesions.

On the other hand, FB1 (Gelderblom et al., 1992; Howard et al.,2001) was found to be carcinogenic mycotoxin and may interact inthis dimension with OTA, which is also proven carcinogen. More-over, FB1 was found to have a pronounced nephrotoxic effect onanimal kidneys (Voss et al., 2001; Bucci et al., 1998), which can beadditive to nephrotoxic effect of OTA.

Our recent investigations on the cytotoxic effect of differentcombinations of OTA, PA, CIT and FB1 on human peripheral bloodmononuclear cells measured by MTT assay revealed additive or syn-ergistic effects of the following mycotoxins: OTA, CIT and FB1 ascompared to any single mycotoxin (Stoev et al., 2009).

Because of the reported synergistic/additive effects betweenOTA and FB1 (Klaric et al., 2007; Creppy et al., 2004) as has been alsoconfirmed in the present study, simultaneous exposure to thosemycotoxins might be of significant importance and could be cru-cial for development of chronic renal failure observed in MPN orBEN, especially after long-term ingestion of the same mycotoxins.The interaction between OTA, FB1 and possibly some other co-contaminants such as PA of CIT in commercial chicken/pig rations orhuman food would be very important and could explain the signifi-cance of the relatively lower doses of OTA that commercial chickens(Stoev et al., 2000a), pigs (Stoev et al., 1998a,b) or humans (Stoev,1998) may ingest via the feed/food. The present findings shouldstimulate broader consideration of mould metabolites in chronicrenal disease.

The present study is complementary to that of Stoev et al.(2001) and suggests that combined exposure to various mycotox-ins usually has synergistic or additive nephrotoxic effect on farmanimals greater than could be expected from the action of anysingle mycotoxin. In the mentioned above study, we managed toinduce macroscopic kidney damage using a mouldy diet containingsuch low levels of OTA as 180 ppb in combination with PA (Stoevet al., 2001). The observed changes were similar to those causedby much higher concentration of pure OTA in feed (Krogh et al.,1974).

Conflict of interest statement

None of the authors of this paper has a financial or personalrelationship with other people or organizations that could inap-propriately influence or bias the content of the paper.

Acknowledgements

This research has been financially supported in part by MarieCurie Outgoing International Fellowship within 6th EuropeanCommunity Framework Programme, Department of Science andTechnology in South Africa, UK Royal Society Joint Project with Cen-

tral and Eastern Europe and Foundation of Ministry of Science andEducation of Bulgaria.

References

Abado-Becognee K, Mobio TK, Ennamany R, Fleurat-Lessard F, Shier WT, Badria F,et al. Cytotoxicity of fumonisin B1: implication of lipid peroxidation and inhibi-tion of protein and DNA syntheses. Arch Toxicol 1998;72:233–6.

Abdulkadar AHW, Al-Ali AA, Al-Kildi M, Al-Jedah JH. Mycotoxins in food productsavailable in Qatar. Food Control 2004;15:543–8.

Alberts JF, Gelderblom WCA, Vleggaar R, Marasas WFO, Rheeder JP. Production of[14C]Fumonisin B1 by Fusarium moniliforme MRC 826 in corn cultures. ApplEnviron Microb 1993;59(8):2673–7.

Barham D, Trinder P. An improved color reagent for the determination of bloodglucose by the oxidase system. Analyst 1972;97:142–5.

Bartels H, Bohmer M. Eine mikrometode zur kreatininibestimmung. Clin Chim Acta1971;32:81–5.

Bennett GA, Richard JL. Liquid chromatographic method for analysis of the naph-thalene dicarboxaldehyde derivative of fumonisins. J Assoc Anal Chem Int1994;77(2):501–6.

Bergmeyer HV, Horder M. IFCC methods for measurement of catalytic concentrationsof enzymes. Clin Chim Acta 1980;105:147–72.

Bergmeyer HU, Bowers GN, Hörder M, Moss DW. Provisional recommendations onIFCC methods for the measurement of catalytic concentrations of enzymes. Part2. IFCC method for aspartate aminotransferase. Clin Chim Acta 1976;70:19–42.

Berthelot M. Rep Chem Appl 1859;1:284.Bucci TJ, Howard PC, Tolleson WH, Laborde JB, Hansen DK. Renal effects of fumonisin

mycotoxins in animals. Toxicol Pathol 1998;26:190–4.Creppy EE, Stormer FC, Kern D, Roschenthaler R, Dirheimer G. Effects of ochratoxin A

metabolites on yeast phenylalanyl-tRNA synthetase and on the growth and “invivo” protein synthesis of hepatoma cells. Chem Biol Interact 1983;47:239–47.

Creppy EE, Röschenthaler R, Dirheimer G. Inhibition of protein synthesis in miceby ochratoxin A and its prevention by phenylalanine. Food Chem Toxicol1984;22:883–6.

Creppy EE, Chiarappa P, Baudrimont I, Borracci P, Moukha S, Carratù M. Synergis-tic effects of fumonisin B1 and ochratoxin A: are “in vitro” cytotoxicity datapredictive of “in vivo” acute toxicity. Toxicology 2004;201:115–23.

Diaz CT, Sogbe E, Ascanio E, Hernandez M. Ochratoxin A and fumonisin B1 naturalinteraction in pigs. Clinical and pathological studies. Revista Cientifica Facultadde Ciencias Veterinarias 2001;7:314–21.

Domijan A, Peraica M, Jurjevic Z, Ivic D, Cvjetkovic B. Fumonisin B1, fumonisin B2,zearalenone and ochratoxin A contamination of maize in Croatia. Food AdditContam 2005;22(7):677–80.

Domijan A, Zeljezic D, Kopjar N, Peraica M. Standard and Fpg-modifed cometassay in kidney cells of ochratoxin A-and fumonisin B1-treated rats. Toxicology2006;222:53–9.

Dragan YP, Bidlack WR, Cohen SM, Goldsworthy TL, Hard GC, Howard PC, et al. Impli-cations of apoptosis for toxicity, carcinogenicity, and risk assessment: fumonisinB1 as an example. Toxicol Sci 2001;61:6–17.

Elling F. Morphological aspects of mycotoxic nephropathy. In: Rodricks J, Hessel-tine C, Mehlman M, editors. Mycotoxins in human and animal health. Illinois:Pathotox. Publishers, Inc.; 1977. p. 499–506.

Elling F, Nielsen JP, Lillehoj EB, Thomassen MS, Stormer FC. Ochratoxin A – inducedporcine nephropathy: enzyme and ultrastructural changes after short-termexposure. Toxicon 1985;23(2):247–54.

Fuchs R. Distribution and fate of ochratoxin A in experimental animals. DoctoralThesis. University of Uppsala 1988; pp. 1–43.

Gelderblom WCA, Marasas WFO, Farber E. The cancer initiating potential of thefumonisin B mycotoxins. Carcinogenesis 1992;13:433–7.

Hinojo MJ, Medina A, Valle-Algarra FM, Gimeno-Adelantado JV, Jimenez M, Mateo R.Fumonisin production in rice cultures of Fusarium verticillioides under differentincubation conditions using an optimized analytical method. Food Microbiol2006;23:119–27.

Howard PC, Warbritton A, Voss KA, Lorenzen RJ, Thurman JD, Kovach RM, et al.Compensatory regeneration as a mechanism for renal tubule carcinogenesis offumonisin B1 in F344/N/Nctr BR rat. Environ Health Persp 2001;109:309–14.

Jurjevic Z, Solfrizzo M, Cvjetkovic B, Avantaggiato G, Visconti A. Ochratoxin A andfumonisins (B1 and B2) in maize from Balkan nephropathy endemic and nonendemic areas of Croatia. Mycotox Res 1999;15:67–80.

Klaric M, Rumora L, Ljubanovic D, Pepeljnjak S. Cytotoxicity and apoptosis inducedby fumonisin B1, beauvericin and ochratoxin A in porcine kidney PK15 cells:effects of individual and combined treatment. Arch Toxicol 2007;82:247–55.

Kokkonen M, Jestoi M, Rizzo A. The effect of substrate on mycotoxin production ofselected Penicillium strains. Int J Food Microbiol 2005;99:207–14.

Krogh P. Mycotoxic porcine nephropathy: a possible model for Balkan endemicnephropathy. In: Puchlev A, editor. Proceeding of the Second InternationalSymposium on Endemic Nephropathy. Sofia: Publishing Houses of BulgarianAcademy of Science; 1972. p. 266–70.

Krogh P, Axelsen N, Elling F, Gyrd-Hansen N, Hald B, Hyldgaard-Jensen J, et al.Experimental porcine nephropathy: changes of renal function and structureinduced by ochratoxin A-contaminated feed. Acta Pathol Microbiol Scand A1974;246(Suppl.):1–21.

Krogh P. Mycotoxic nephropathy. Advances in veterinary science and comparativemedicine, vol. 20. New York, NY: Academic Press; 1976. pp. 147–170.



Kubena LF, Edrington TS, Harvey RB, Phillips TD, Sarr AB, Rottinghaus GE. Indi-vidual and combined effects of fumonisin B1 present in Fusarium moniliformeculture material and diacetoxyscirpenol or ochratoxin A in turkey poults. PoultSci 1997;76:256–64.

Meisner N, Chan S. Ochratoxin A, an inhibitor of mitochondrial transport system.Biochemistry New York 1974;13:2795–800.

Micco C, Miraglia M, Onori R, Libanori A, Brera C, Mantovani A, et al. Effect ofcombined exposure to ochratoxin A and penicillic acid on residues and toxi-city in broilers. La Ravista della Societa Italiana di Scienza dell’Allimentazione1991;20(3):101–8.

The Nordic Working Group on Food Toxicology and Risk Evaluation (NNT). NordiskeSeminar-og Arbejdsrapporter (1991: 545). Health Evaluation of ochratoxin A inFood Products 1991; pp. 5–26.

Oswald IP, Desautels C, Laffitte J, Fournout S, Peres SY, Odin M, et al. Mycotoxinfumonisin B-1 increases intestinal colonization by pathogenic Escherichia coli inpigs. Appl Environ Microbiol 2003;69(10):5870–4.

Petrik J, Zanic-Grubisic T, Barisic K, Pepeljnjak S, Radic B, Ferenicic Z, et al. Apo-ptosis and oxidative stress induced by ochratoxin A in rat kidney. Arch Toxicol2003;77:685–93.

Rahimtula AD, Bereziat JC, Bussachini-Griot V, Bartsch H. Lipid peroxidation as pos-sible cause of Ochratoxin A toxicity. Biochem Pharmacol 1988;37:4469–77.

Rodkey FL. Estimation of albumin. Clin Chem 1964;10:606–10.Schettler G, Nussel E. Method for triglycerides. Arb Med Soz Med Prav Med

1975;10:25–9.Stoev SD. The role of ochratoxin A as a possible cause of Balkan endemic nephropathy

and its risk evaluation. Vet Hum Toxicol 1998;40(6):352–60.Stoev SD, Hald B, Mantle P. Porcine nephropathy in Bulgaria: a progressive syndrome

of complex of uncertain (mycotoxin) etiology. Vet Rec 1998a;142:190–4.Stoev SD, Stoeva J, Anguelov G, Hald B, Creppy EE, Radic B. Haematological, bio-

chemical and toxicological investigations in spontaneous cases with differentfrequency of porcine nephropathy in Bulgaria. J Vet Med A 1998b;45:229–36.

Stoev SD, Anguelov G, Ivanov I, Pavlov D. Influence of ochratoxin A and an extractof artichoke on the vaccinal immunity and health in broiler chicks. Exp ToxicolPathol 2000a;52:43–55.

Stoev SD, Goundasheva D, Mirtcheva T, Mantle PG. Susceptibility to secondary bacte-rial infections in growing pigs as an early response in ochratoxicosis. Exp ToxicolPathol 2000b;52:287–96.

Stoev SD, Vitanov S, Anguelov G, Petkova-Bocharova T, Creppy EE. Experimentalmycotoxic nephropathy in pigs provoked by a mouldy diet containing ochra-toxin A and penicillic acid. Vet Res Commun 2001;25(3):205–23.

Stoev SD, Daskalov H, Radic B, Domijan A, Peraica M. Spontaneous mycotoxicnephropathy in Bulgarian chickens with unclarified mycotoxin aetiology. VetRes 2002a;33(1):83–94.

Stoev SD, Paskalev M, MacDonald S, Mantle PG. Experimental one year ochratoxinA toxicosis in pigs. Exp Toxicol Pathol 2002b;53:481–7.

Stoev SD, Stefanov M, Denev S, Radic B, Domijan A, Peraica M. Experimen-tal mycotoxicosis in chickens induced by ochratoxin A and penicillic acidand intervention by natural plant extracts. Vet Res Commun 2004;28(8):727–46.

Stoev SD. Complex etiology prophylaxis and hygiene control in mycotoxicnephropathies in farm animals and humans. Int J Mol Sci 2008;9:578–605, Spe-cial Issue “Mycotoxins: Mechanisms of Toxicological Activity – Treatment andPrevention”, Section “Molecular Pathology”.

Stoev SD, Denev S, Dutton M, Nkosi B. Cytotoxic effect of some mycotoxins and theircombinations on human peripheral blood mononuclear cells as measured byMTT assay. Open Toxinol J 2009;2:1–8.

Stoev SD, Dutton M, Njobeh P, Mosonik J, Steenkamp P. Mycotoxic nephropathyin Bulgarian pigs and chickens: complex aetiology and similarity to BalkanEndemic Nephropathy. Food Addit Contam A 2010a;27(1):72–88.

Stoev SD, Denev S, Dutton M, Njobeh P, Mosonik J, Steenkamp P, et al. Complex eti-ology and pathology of mycotoxic nephropathy in South African pigs. MycotoxRes 2010b;26(1):31–46.

Voss KA, Riley RT, Norred WP, Bacon CW, Meredith FI, Howard PC. An overview ofrodent toxicities: liver and kidney effects of fumonisins and Fusarium monili-forme. Environ Health Persp 2001;109:259–66.

Weichselbaum TE. Estimation of serum total protein by Biruet method. Am J ClinPathol 1946;16:40–8.

Experimental mycotoxic nephropathy in pigs provoked by a mouldy diet containing ochratoxin A and...

Documents

Transcript of Experimental mycotoxic nephropathy in pigs provoked by a mouldy diet containing ochratoxin A and...