Mycotoxic porcine nephropathy and spontaneous occurrence of ochratoxin A residues in kidneys of slaughtered swine

Zbornik Matice srpske za prirodne nauke / Proc. Nat. Sci, Matica Srpska Novi Sad,¥ 116, 81—90, 2009

UDC 637.5'64:615.919DOI:10.2298/ZMSPN0916081M

D r a g a n R. M i l i ã e v i ã1*, V e r i c a B. J u r i ã2 ,A l e k s a n d r a D a k o v i ã3 , M i l j a n J o v a n o v i ã4 ,S r ð a n M. S t e f a n o v i ã1 , Z o r a n I. P e t r o v i ã1

1 Institute of Meat Hygiene and Technology, Kaãanskog 13,11000 Belgrade, Serbia, E-mail: [email protected]

2 Department for Animal Sciences, Faculty of Agriculture,University of Novi Sad, Trg Dositeja Obradoviãa 10,21000 Novi Sad, Serbia, E-mail: [email protected]

3 Institute for Technology of Nuclear and other Mineral Raw Materials,Franche d'Epere 86, 11000 Belgrade, Serbia, E-mail: [email protected]

4 Department of Pathomorphology, Faculty of Veterinary Medicine,University of Belgrade, Bulevar Osloboðenja 18, 11000 Belgrade,Serbia, E-mail: [email protected]

MYCOTOXIC PORCINE NEPHROPATHYAND SPONTANEOUS OCCURRENCE OF OCHRATOXINA RESIDUES IN KIDNEYS OF SLAUGHTERED SWINE

ABSTRACT: In order to find information on the occurrence of mycotoxic porcinenephropathy in Serbia, during a six month period (2006/2007) samples of kidney from indi-vidual healthy slaughtered pigs were collected (n=90) and analyzed by HPLC for ochratoxinA. In addition, histological examinations were carried out. The incidence of OTA in kidneywas 33,3% and varied between 0.17—52.5 ng/g. Histopathological examination of kidneysconfirmed tubulopathies with oedema and cell vacuolization. In addition, hemorrhages andnecrosis of proximal kidney tubulesÿ cells were found. These findings indicate that it islikely that most of the kidney injury is related to ochratoxin A and other nephrotoxic com-pounds which enhance the toxicity of OTA.

KEY WORDS: Ochratoxin A, pig, nephropathy

INTRODUCTION

Ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by severalspecies in the Aspergillus and Penicillium genera. It is detected widely as acontaminant of agricultural commodities, especially cereals (20). As cereals arewidely used in animal feed, and because OTA is relatively stable in vivo,whence it is further transmitted to animal edible tissues (8), especially inkidney and liver. Major target for the toxicity of ochratoxin A in mammalianspecies is kidney, where the toxin primarily affects the proximal convoluted

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tubules (2, 14, 17, 18). OTA has been causally associated with nephropathy inpigs and poultry. Among farmed animals, pigs are particularly sensitive toOTA. This mycotoxin plays a special role in the genesis of swine mycotoxicnephropathy, a common disease in Scandinavia (10, 16, 23). It exhibits alsoimmunosuppressive, teratogenic, nephrotoxic and genotoxic properties in se-veral animal species (12).

Occurrence of OTA has been recognized as a potential human health ha-zard and OTA contamination is of public health significance since it is asso-ciated with Balkan nephropathy, a kidney disease in humans (4) described inseveral rural regions of Bulgaria, Romania, Serbia, Croatia, and Bosnia, andassociated with an increased incidence of tumors of the upper urinary tract.However, causality has not yet been established. Special attention has beenpaid to OTA since 1993, when the International Agency for Research on Can-cer classified this toxin as a possible human carcinogen (group 2B) (24). Hu-man exposure to OTA can occur directly by consumption of food containingthe toxin, or indirectly by consumption of animal tissue exposed to contami-nated materials (8).

Up to now, there has been little published information on the occurrenceof porcine nephropathy and content of ochratoxin A in kidneys of Serbianslaughtered pigs. Therefore, the aim of this paper was to evaluate the naturaloccurrence of porcine nephropathy and OTA content in kidneys from healthyslaughtered pigs originating from different regions of Serbia. Also, the purposeof this paper is to briefly review risk assessments of OTA in order to highlightthe critical issues that these assessments have identified.

MATERIALS AND METHODS

Reagents

OTA were purchased from Sigma-Aldrich Chemie GmbH. A workingstandard OTA for HPLC containing 25 ng/mL methanol was prepared dailyjust before starting the injection of a series of samples. Other reagents wereHPLC grade. All other chemicals were reagent grade or chemically pure.

Sample collection

During six month period (September 2006/Februar 2007), sample of kid-ney per animal was collected from healthy slaughtered pigs (n = 90) origina-ting from three different regions of Serbia. Slaughtered pigs were randomlysampled in the slaughterhouse during meat inspection, and whole kidneys weresampled from each pig. The samples were homogenized and stored at –18°Cbefore analysis. No preservatives were added.

Microscopical examination: Kidney samples were fixed in 10% neutralbuffered formalin and absolute alcohol for 5 to 7 days, processed by routine

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methods, sectioned at 5—8 µm, and stained with hematoxylin and eosin (HE)for light microscopy.

Extraction and clean-up for ochratoxin analyses

Kidney Analyses were performed by the method of M a t r e l l a et. al(2006) (21), which briefly includes double extraction with acidic ethyl acetate.The organic phase was removed and extracted with 0.5M NaHCO3 pH 8.4.The organic phase was evaporated to dryness under N2 steam, reconstituted in150 µl mobile phase and a 20 �l aliquot was injected. The detection limit forOTA in organs was 0.01 ng/g with a 61% mean recovery from artificially con-taminated samples at 3 ng/g.

Chromatographic conditions (HPLC)

An aliquot of 50 �l for kidneys samples were injected into WatersSymmetry Shield RP 18, high pressure liquid chromatography column (lengthand inner diameter 150x4,6 mm, particle size 5 �m, of the HPLC system(Waters Alliance). The column was eluted with 4% acetic acid and acetonitrile(32:68 v/v) at 25°C and a flow rate of 1 mL/min. Measurements were per-formed by fluorescence detection at wavelengths of 334 nm (excitation) and460 nm (emission) gain 10.

Statistical analysis

Descriptive statistics of the data set were performed with a standard pro-grammed and included arithmetic mean, standard deviation, coefficient of va-riation, minimum and maximum. Statistical differences in the mean levels ofOTA contamination across the three groups of positive samples were deter-mined by one-way ANOVA (p < 0.05).

RESULTS AND DISCUSSION

The incidences and mean values of ochratoxin A in swine kidney and theresults of pathomorphology examination are summarized in Table 1 and Figure1 and 2.

Ochratoxin A in Kidney

The incidence of OTA in kidneys was 33.3% in the range 0.17—52.5with the mean level 1.26 ng/g. The majority of samples (16.6%) containedOTA at low levels (0.01—1.0 ng/g). The concentrations in ten samples (11.1%)ranged between 1—5 ng/g, while ochratoxin A in five (5.5%) samples was

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greater than 5 ng/g. In regard to regional distribution of OTA, the occurrenceof OTA among the three regions whence samples were collected are almost si-milar and varied between 26.6% (region Vladimirci) and 36.6% (region Sentaand Bogatiã) (Table 1), but the mean level of contamination is different. Thehighest OTA level (52.5 ng/g, mean 2.20 ng/g), with the highest coefficient ofvariation (4.33) was found in the samples originating from region Bogatiã. In2.2% samples of kidneys, OTA levels were considerably higher and greatlyexceeded the permissible levels for this toxin established in Serbia, includingthose proposed by the SCF (1998) (27), and JECFA (2001) (10 ng/g) (13).

Tab. 1 — Incidence of Ochratoxin A in kidney of slaughtered pigs

Region N n (%)ng/g

X±Sd C.V. % Range

Vladimirci 30 8 (26.6) 0.42±1.2 2.96 0.18—6.5Senta 30 11 (36.6) 1.14±3.3 2.89 0.17—17.0

Bogatiã 30 11 (36.6) 2.2±9.54 4.33 0.26—52.5

Total 90 30 (33.3) 1.26±5.85 4.64 0.17—52.5

N — total number of analyzed samples, n — number of positive samples, X — arithmetic mean(conc. below LOD are regarded as zero), C.V. — coeff. of variation.

Comparison of data obtained in this trial, along with theother recently pu-blished data for the occurrence of OTA in pig edible tissues, shows that thefound levels are comparable with levels in other European countries or Canada(5, 7, 9, 23). The incidence of OTA in kidneys was 33.3% and almost compa-rable with the incidence reported by Koller (1991) in Austria (41.9%) or byGareis and Scheuer (1999) in Germany (41.9%), and in Serbia (41.6%) (22).The mean level (1.26 � 5.85 ng/g) was lower than those reported in Austria(2.5 ng/g) (15) and recently published data in Serbia (3.12 ng/g) (22), buthigher then the mean level found in Germany (0.43 ng/g) (8).

Patomorphology Examination

Gross pathology

All 90 pigs were slaughtered during the study period. Kidneys submittedto the laboratory were pale, swollen and enlarged and changed in colour fromnormal mahogany to tan, as follows: 43 (47.7%) had “mottled or pale kid-neys", 27 (30%) had enlarged kidneys and 11 (12.2%) were smaller than nor-mal. The only macroscopic lesions observed in few cases were small grey--white foci on the kidney surface. No obvious difference was observed be-tween the right and left kidney. No significant changes were seen in other or-gans.

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Histological examination

Histological findings of renal tissues and incidence of ochratoxin A inkidneys from slaughtered pigs are summarized in Figure 1 and 2. Histologicalexamination showed two types of changes: degenerative — affecting epithelialcells in some proximal tubules of pigs, and proliferative changes in the inter-stitium. The major renal histopathological changes were in the epithelium ofproximal tubules (Fig. 2). Dystrophy, (moderate to obvious degenerative chan-ges, Fig. 1A), swelling, vacuolization and lipophilic degeneration, were themain changes in the tubular epithelial cells. The majority of glomeruli exhi-bited mild or moderate exudates in the Bowman's capsular spaces as well ashypercellularity of vascular loops. In addition vascular changes expressed as ahyperaemia of blood vessels, moderate to marked hemorrhages of some renalcortical regions occurred occasionally (Fig. 1D). In the interstitium of the renalcortical regions, there was limited proliferation of connective tissue (Fig. 1B)and focal infiltration of mononuclear inflammatory cells which was sometimes

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Fig. 1 — Dystrophy and vacuolar degeneration in the epithelium of proximaltubules' cells (A), Focal interstitial fibrosis (B), Necrosis of proximal tubules'

cells (C) and Hemorrhages in cortex (D)

accompanied by small granulomas. OTA analysis of the kidney samples withdegenerative changes of moderate to marked cloudy swelling revealed the inci-dence of OTA in 32.2% samples at concentration levels up to 52.5 ng/g (Fig.2). Additionally, vascular changes, as well as fatty changes were observed insix kidneys of pigs in which ochratoxin A was detected up to 6.5 ng/g, whilefocal interstitial fibrosis and necrosis of proximal tubules' cells were only seenin one pig kidney in which ochratoxin A was detected up to 52.5 ng/g. The le-sions produced by higher OTA levels were more severe and widespread, inclu-ding degeneration, atrophy, glomerular swelling and sclerosis and interstitialnephritis (Fig. 2).

The macroscopic and microscopic changes observed in our study weremore similar to those reported by other papers (28, 29, 30). These findingsconfirm that high affinity of OTA towards serum proteins, allows its accumu-lation in the organs of animals (11). Kidney is the main target of OTA. Thishigh susceptibility of kidney is, at least partly, the result of OTA-toxicokine-tics. Renal blood flow per tissue weight is extremely high, which results in thedelivery of relativly large amounts of OTA in comparison to other organs.Furthermore, free OTA is secreted in the proximal tubule and subsequently re-absorbed, mainly in the proximal straight tubule, the thick ascending limb ofthe loop of Henle and the collecting duct (1, 6, 25, 27). The inhibition of pro-tein synthesis and damaged energy production in the mitochondria could beconsidered as the most important factors for degenerative changes in the epi-thelial cells of proximal tubules where ochratoxin A was detected. While agree-ing that the most important toxicological target of OTA in a pig is kidney, theprincipal descriptions of the pathology of MPN vary considerably with respect

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Fig. 2 — Summary of histological findings (PH) of renal tissues and incidence of OTAin kidney from slaughtered pigs (n=90). Necrosis of proximal tubules' cells (1),

hypercellularity of vascular loop (2), vascular changes (3), exudates in Bowman's space (4),focal interstitial nephritis (5), dystrophy of proximal tubules' cells (6), swelling of

proximal tubules' cells (7), renal hemorrhages (8), fatty changes of proximal tubules' cells

to some other details, and according to the dosing regime and the duration ofOTA exposure. Enlarged kidneys are indicative of renal inflammation and pro-liferative lesions following chronic exposure to OTA (28, 30). Therefore, itseems that MPN observed in Serbia may have a multitoxic aetiology becauseit cannot be explained by the concentration of OTA alone. The lack of astrong correlation among histopathological changes and incidence of OTA inkidney (33.3% kidney samples were positive, at levels ranging up to 52.5ng/g) found in our trial might thus explain the result of OTA-toxicokinetics, aswell as possible synergism between OTA and other nephrotoxic mycotoxins orcompounds which enhance the toxicity of OTA. Such synergism betweenOTA and other mycotoxins under field conditions may be responsible for theMPN in Serbia, which is associated with relatively low mean contamination byOTA in feed. The production of multiple toxins by one or several fungi, whichis sometimes completely normal, presents a problem that has not been suffi-ciently investigated.

However, it should be remembered that when comparing data factorssuch as climate conditions during harvest, practices for grain/feed storage etchave influence on the ochratoxin A level in edible tissues. The data obtainedin this trial show that there should be more concern for the livestock industry.

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26. S c h w e r d t, G., G e k l e, M., F r e u d i n g e r, R., M i l d e n b e r g e r, S., S i l -b e r n a g l, S. (1997): Apical-to-basolateral transepithelial transport of ochratoxinA by two subtypes of Madin-Darby canine kidney cells, Biochim. Biophys. Acta1324: 191—199.

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29. S t o e v, S. D., V i t a n o v, S., A n g u e l o v, G., T. K., P e t k o v a - B o c h a -r o v a and C r e p p y, E. E. (2001): Experimental mycotoxic nephropathy in pigsprovoked by a diet containing ochratoxin A and penicillin acid, Vet. Res. Com-mun., 25 (3): 205—23.

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MIKOTOKSIÅNA NEFROPATIJA SVIWA I ZASTUPQENOSTREZIDUA OHRATOKSINA A U BUBREZIMA ZAKLANIH SVIWA

Dragan R. Miliãeviã1*, Verica B. Juriã2, Aleksandra Dakoviã3,Miqan Jovanoviã4, Srðan Stefanoviã1, Zoran I. Petroviã1

1 Institut za higijenu i tehnologiju mesa,Kaãanskog 13, 11000 Beograd, Srbija

2 Department za stoåarstvo, Poqoprivredni fakultet,Univerziteta u Novom Sadu, Trg Dositeja Obradoviãa 10,

21000 Novi Sad, Srbija3 Institut za tehnologiju nuklearnih i drugih mineralnih

sirovina, Franše d'Eperea 86, 11000 Beograd, Srbija4 Department za patomorfologiju, Fakultet veterinarske medicine,

Univerziteta u Beogradu, Bulevar Osloboðewa 18, 11000 Beograd, Srbija

Rezime

U ciqu sticawa saznawa o zastupqenosti mikotoksiåne nefropatije svi-wa i uticaja OTA na vrstu i lokalizaciju patomorfoloških promena na bubre-zima, na liniji klawa tokom veterinarsko-sanitarnog pregleda redovno zakla-nih sviwa poreklom sa farmi iz Vojvodine i Srbije, uzimani su uzorci bubre-ga za analizu. Tokom šestomeseånih ispitivawa ukupno je analizirano 90 uzo-

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raka bubrega zaklanih sviwa. Prisustvo rezidua OTA u uzorcima bubrega pore-klom iz ispitivanih regiona utvrðeno je kod 33,3% ispitanih uzoraka, u koli-åini od 0,17 do 52,5 ng/g. Zastupqenost rezidua OTA bila je najveãa u uzorcimabubrega poreklom sa lokaliteta Senta i Bogatiã (36,6%), dok je najveãi prose-åan sadrÿaj rezidua OTA (–2,2 �g/kg) utvrðen u uzorcima bubrega poreklom salokaliteta Bogatiã. Patohistološkim pregledom bubrega najåešãe su utvrðenetubulopatije sa edemom i vakuolizacijom ãelija. Takoðe, utvrðene su hemoragijei nekroza ãelija proksimalnih bubreÿnih tubula.

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