VETERINARSKI ARHIV 69 (4), 179-189, 1999

ISSN 1331-8055 Published in Croatia

Some antidotes and paraclinical investigations in experimental intoxication with ochratoxin A
and penicillic acid in chicks

Stoytcho Stoev¹*, Geno Anguelov², Dimitar Pavlov³,
and Ljubomir Pirovski⁴

¹Department of Pathomorphology, Faculty of Veterinary Medicine,
Trachian University, Stara Zagora, Bulgaria

²Department of Pathophysiology, Faculty of Veterinary Medicine,
Trachian University, Stara Zagora, Bulgaria

³Department of Plant Science, Faculty of Zootechnics,
Trachian University, Stara Zagora, Bulgaria

⁴Agrobiochem, Stara Zagora, Bulgaria

* Contact address:
Prof. Dr. Stoytcho Dimitrov Stoev,
Department of Pathomorphology, Faculty of Veterinary Medicine, Trachian University, Students campus, 6000 Stara Zagora,
Fax: 359 42 45101, E-mail: s-stoev@hotmail.com

STOEV, S., G. ANGUELOV, D. PAVLOV, LJ. PIROVSKI: Some antidotes and paraclinical investigations in experimental intoxication with ochratoxin A and penicillic acid in chicks. Vet. arhiv 69, 179-189, 1999.

ABSTRACT

The objects of this study were the main haematological and biochemical changes in experimental ochratoxicosis in 85 broiler chicks fed on a diet containing: 130, 300 and 800 ppb ochratoxin A (OTA) and 1000-2000 ppb penicillic acid (PA), corresponding to those in the field. Biochemical and haematological changes were characteristic in increased serum levels of uric acid and glucose, decreased serum levels of cholesterol and total protein, and slight normocyte anaemia. These changes were induced by several times lower contamination levels of OTA in feed than the levels described in literature of pure OTA, due to the probable synergism between OTA and PA. A positive protective effect of 5% total water extract of Artichoke and Rosallsat against the toxic effect of OTA was established.

Key words: mycotoxic nephropathy, ochratoxicosis, ochratoxin A, penicillic acid, chicks

Introduction

The toxic effect of nephrotoxic mycotoxin ochratoxin A (OTA) on broiler chicks is well known, but so far there are only scarce data about combined exposure to OTA and penicillic acid (PA) (MICCO et al., 1991), which could occur spontaneously in the field. These mycotoxins are metabolite products of the fungi Aspergillus and Penicillium, encountered mainly in forages stored for a long time in high humidity. Information about the antidote effects of various supplements against toxicity of OTA is also scarce (HAAZELE et al., 1993). Consequently, the aim of the present study was to elucidate the combined toxic effect of OTA and PA on some paraclinical parameters in experimentally provoked mycotoxic nephropathy in chicks, as well as to elucidate the supposed protection of Rosallsat and Artichoke-extracts against that toxicity.

The artichoke has been recommended as a diuretic agent (renal stimulant) in cardiac and renal insufficiency (GAHNIAN and ASENOV, 1986). It is known that the water extract of artichoke leaves (prepared as infusum by steam) contains various biologically active substances such as cynarine and flavonoids, as well high levels of vitamin C and calcium. Cynarine stimulates the metabolism of cholesterol and decreases serum urea and lipids, and also increases biliary secretion (GAHNIAN and ASENOV, 1986). Moreover, cynarine and flavonoids have a potent protective effect against hepatotoxic damage. The high content of calcium and vitamin C in such water extracts (prepared by steam, but not by boiling) probably also have a potent detoxicative effect against OTA (HAAZELE et al., 1993). Rosallsat was a further plant extract (a polyextract of bulbus Alii sativi and seminum Rosai canina) used in addition to artichoke-extract to elucidate the supposed protection against the toxicity of OTA. This extract was found to inhibit lipid peroxidation (unpublished data), which was usually enhanced by OTA (RAHIMTULA et al., 1988).

Materials and methods

Contamination of forages with OTA was performed using strains of Aspergillus ochraceus (obtained from P. Golinski): K101 producing 320 ppm OTA and 750 ppm PA per kg solid medium, and K103 producing 225 ppm OTA and 1000 ppm PA per kg. Contamination of barley flakes with ochratoxin A was performed by the following procedure: 40 ml sterilized salt solution (1 g NaCl, 2 mg ZnCl₂, 100 ml tap water) was added to 100 g barley flakes autoclaved at 120 °C for 30 minutes. Strains A. ochraceus grown out on maltager plate (1×1 cm) for one week were transferred to the sterile barley flakes and were incubated at 30 °C for two weeks in the dark, as well as being shaken every day. After the incubation period the barley flakes were finally dried. The concentration of OTA and PA in moulded barley flakes was determined by HPLC (high performance liquid chromatography) technique (PHILLIPS et al., 1981; HALD et al., 1993). Next, ochratoxin A-contaminated barley flakes were mixed with the feeds for chicks in order to obtain the following contamination levels of OTA in the feed: 130 ppb, 300 ppb and 800 ppb. Only the concentration of OTA was maintained at the same levels, whereas the concentration of PA in the fed forages ranged from 1000 to 2000 ppb.

Two-week-old broiler chicks (four line broiler hybrid Cornish × Plymouth Rock) were purchased. Commercially prepared broiler-complete feed containing the mentioned above feed levels of OTA, and drinking water, were available ad libitum. Broiler chicks were grouped in 4 experimental groups and 1 control group (20 birds in each) as well as 1 experimental group of 5 chicks (group V), and fed respectively: control group-OTA free standard diet; group I-130 ppb OTA in diet; group II-300 ppb OTA; group III-800 ppb OTA; group IV-800 ppb OTA and 5% total water extract of artichoke (Cynara scolymus L) added daily to the feed in a concentration of 5 ml/kg mean body mass. Extract of artichoke was prepared as infusum by steam from dried artichoke leaves and was found to contain the following biological active compounds: cynarine and flavonoids, as well high levels of calcium and ascorbic acid; group V-800 ppb OTA and 0.6 ml Rosallsat per kg body mass daily, given per os (Rosallsat is a new, natural, galenic phytosubstance of balanced polyvitamin, phytoncide and steroid-saponin composition, described in detail in patent No 98915 BG/1994, given as a supplement to the feed and which contains the biological active compound, allicin).

Blood samples were taken and examined for various haematological and biochemical parameters and acid-base balance after 6 weeks, as well as after 10 weeks, from the beginning of the experiment. Blood sugar and serum cholesterol were determined by the Boehringer Mannheim (Germany) test; total serum protein by the Bio-La-Test of Lachema Diagnostica, Czech Republic; uric acid by the EnzUric-FT-test of Labordiagnostica-Gopecke, Germany; serum urea by Enymatic (Germany) colorimetric test; serum enzyme levels of ASAT (asparate-aminotransferase), ALAT (alanine-aminotransferase) and LDH (lactate dehydrogenase) by the Cormay (Poland) test; leucocytes and erythrocytes were determined using the Bürker chamber; differential white blood cell counts were made on blood smears stained with Giemsa's and May Grünwald's solutions; acid-base balance and haemoglobin were determined using an apparatus for blood gaseous analysis (ABL-3, of Radiometer, Denmark).

Student's t-test was used to estimate significant differences between the mean values of various parameters in different groups of chicks.

Results

The main clinical signs were observed predominantly in chicks in group III and sometimes in group II: depression, weakness and dullness, growth depression, as well as nervous symptoms: torticollis, emprosthotonus, lurching, tremors, sitting posture. These clinical signs were especially well expressed after 6 weeks from the beginning of the experiment.

Biochemical investigations at the 42^nd and 70^th days revealed a significant increase in serum values of uric acid in groups I and II, and especially in group III (543.8 and 534.1), compared with the control group (174.4 and 162.6). No significant increase was observed in artichoke-treated group IV and Rosallsat-treated group V, compared with the control group. Values of uric acid were significantly lower in chicks of groups IV and V, compared with group III. A significantly lesser increase in that parameter was also established in groups I and II (Table 1).

Table 1. Mean serum levels (±SEM) of glucose, total protein, cholesterol and uric acid in experimental intoxication with ochratoxin A and penicillic acid in broiler chicks (42^nd and 70^th days)

¹significant difference towards control group (P<0.05); ²very significant difference towards control group (P<0.01); ³extremely significant difference towards control group (P<0.001); ^asignificant difference towards III group (P<0.05); ^bvery significant difference towards III group (P<0.01); ^cextremely significant difference towards III group (P<0.001)

Serum levels of total protein were significantly decreased in all experimental groups compared with the control group. A significantly lesser decrease in that parameter was established in groups I, II, IV and V. Moreover, a significant difference was observed between the mean values of that parameter in groups IV and III (Table 1).

Serum levels of glucose were significantly increased in all ochratoxin A-treated groups compared with the control group. A significantly lesser increase of serum glucose was observed in groups I and II. No significant difference was observed between the mean values of that parameter in groups III, IV and V (Table 1).

Serum levels of cholesterol were significantly decreased in groups I, II, III and IV in an OTA-dose-dependent manner compared with the control group. No significant decrease was established in serum levels of that parameter in chicks of the Rosallsat-treated group V, compared with the control group. A statistically significant increase of serum values of cholesterol was established in groups IV and V (antidote-treated groups) compared with group III at the 70^th day (Table 1).

Biochemical investigations of serum enzyme levels revealed no significant difference between experimental and control groups. There was only a slight (not positive) increase of serum levels of LDH and ASAT in chicks of group III compared with the control parameters (Table 2).

Table 2. Mean serum levels (±SEM) of LDH, ASAT, ALAT and urea in experimental intoxication with ochratoxin A and penicillic acid in broiler chicks (42^nd day)

Haematological investigations revealed a statistically significant decrease of red blood cell count (Er) in groups II, III, IV and V compared with the control group at the 70^th day, but no significant decrease was observed in low-dosed group I. No significant difference in white blood cell count (Le) and thrombocytes (Thr), compared with control parameters, was observed in any of the experimental groups. (Table 3).

Table 3. Mean serum values (±SEM) of red blood cell count (Er), white blood cell count (Le) and thrombocytes (Thr) in experimental intoxication with ochratoxin A and penicillic acid in broiler chicks (70^th day)

²very significant difference towards control group (P<0.01); ³extremely significant difference towards control group (P<0.001); ^asignificant difference towards III group (P<0.05); ^cextremely significant difference towards III group (P<0.001)

Some changes were observed in differential white blood cell count. A significant eosinopenia was observed in groups I, II and III compared with the control group at the 70^th day, but no significant changes were observed in that parameter in artichoke-treated group IV and Rosallsat-treated group V, compared with the control parameter (Table 4).

Table 4. Mean serum values (±SEM) of lymphocytes (Ly), monocytes (Mo), neutrophils (Ne), eosinophils (Eo) and basophils (Ba) in experimental intoxication with ochratoxin A and penicillic acid in broiler chicks (70^th day)

¹significant difference towards control group (P<0.05); ²very significant difference towards control group (P<0.01); ^asignificant difference towards III group (P<0.05); ^bvery significant difference towards III group (P<0.01)

A statistically significant decrease of haemoglobin values was found in all ochratoxin A-treated groups at the 42^nd day, but such a decrease was not observed in low-dosed group I or in artichoke-treated group IV and Rosallsat-treated group V at the 70^th day, compared with the control parameter (Table 5, Table 6).

Table 5. Mean serum values (±SEM) of blood pH, Hb, pCO₂, pO₂, HCO₃, TCO₂, ABE, SBE, SBC, SAT and O₂(ct) in blood in experimental ochratoxicosis in broiler chicks in experimental intoxication with ochratoxin A and penicillic acid in broiler chicks (42^nd day)

¹significant difference towards control group (P<0.05); ²very significant difference towards control group (P<0.01); ^asignificant difference towards III group (P<0.05)

Table 6. Mean serum values (±SEM) of blood pH, Hb, pCO₂, pO₂, HCO₃, TCO₂, ABE, SBE, SBC, SAT and O₂(ct) in blood in experimental ochratoxicosis in broiler chicks in experimental intoxication with ochratoxin A and penicillic acid in broiler chicks (70^th day)

¹significant difference towards control group (P<0.05); ²very significant difference towards control group (P<0.01); ³extremely significant difference towards control group (P<0.001); ^asignificant difference towards III group (P<0.05); ^bvery significant difference towards III group (P<0.01); ^cextremely significant difference towards III group (P<0.001)

Investigation of the acid-base balance at the 42^nd and 70^th days of the experiment showed no essential changes (Table 5, Table 6).

Discussion

Analysis of the obtained results demonstrates that the intensity of changes in various haematological and biochemical parameters depended on the quantity of OTA in feed. In chicks of artichoke-treated group IV and Rosallsat-treated group V, the changes in various haematological and biochemical parameters were more similar to those observed in group I, which was exposed to approximately 6 times lower contamination levels of OTA. This contributed significantly to clarification of the protective effect of these antidotes against the toxic effect of OTA.

Serum levels of uric acid were most significantly influenced by the quantity of OTA in fed forages. That parameter usually provides an exact assessment of the impairment of kidney function in chicks (MANNING and WYATT, 1984). The significant increase in serum levels of uric acid in group II, and especially in group III, suggests that the kidney function in chicks of these groups was severely impaired. The absence of a positive increase in that parameter in antidote-treated groups suggests a slight impairment in kidney function in these groups.

The increased concentration of serum glucose in all experimental groups is probably due to compensative hyperglycaemia (compensatory reaction of macro-organism) as a consequence of glucosuria and impaired resorption of glucose in the proximal tubules.

The decreased levels of serum total protein are in complete accord with previously observed impairment of protein synthesis by OTA (CREPPY et al., 1983). Impaired kidney function may also contribute to that decrease.

The low levels of serum cholesterol in OTA-treated groups are most probably due to the inhibition of cholesterol biosynthesis in the liver. It has been shown that OTA competitively inhibits mitochondrial transport carrier proteins in liver mitochondria of rats (MEISNER and CHAN, 1974), which could be responsible for the decrease in production of the energy necessary for cholesterol synthesis. Decreased sterol carrier, as a result of decreased protein synthesis, could also affect cholesterol synthesis. The protective effect of artichoke-extract and Rosallsat on the liver function may contribute to the higher cholesterol levels in antidote-treated groups.

The absence of significant changes in serum levels of the studied enzymes could reflect the slight degenerative changes in liver (unpublished data).

Analysis of serum values of Hb and Er shows that a slightly pronounced anaemia was evident in high dosed groups. This could be considered a result of an impairment of haemopoesis in bone marrow (unpublished data). The present results also suggest that the haemopoesis was favourably influenced by artichoke-extract and Rosallsat at the 70^th day of experiment.

Analysis of the values of acid-base balance shows no essential changes. The higher values of pH, pCO₂, HCO₃, TCO₂ and ABE at the 70^th day compared with the same parameters at the 42^nd day could be due to age-related changes.

Changes in various haematological and biochemical parameters similar to those described have also been observed by some other authors, although in higher contamination levels of pure OTA in feed (HARVEY et al., 1992; MOHIUDIN et al., 1993). The pronounced toxic effect of OTA in the present study could be due to a synergistic toxic interaction between OTA and PA, which has been seen in mice (SANSING et al., 1976; SHEPHERD et al., 1981) and poultry (MICCO et al., 1991). The increased toxicity of OTA (in combination with PA) is probably due to an impaired mechanism of detoxication of OTA by PA. It has been found that PA inhibits carboxypeptidase activity "in vivo" and "in vitro" (PARKER et al., 1982), which could be responsible for the impaired detoxication of OTA in the gut and the enhanced toxicity of OTA.

On the other hand, it has been found that some metabolites provide protection against the toxic effect of OTA. Such metabolites are phenylalanine and ascorbic acid (HAAZELE et al., 1993). In our study, a similar protection of artichoke-extract and Rosallast against the toxicity of OTA was established. This could be due to the high levels of ascorbic acid, calcium and flavonoids (in artichoke extract) as well as other biologically active compounds, such as allicin or high levels of vitamins A, F and E (in Rosallsat) contained in these antidotes. Moreover, it is known that cynarine content in artichoke extract increases biliary secretion (GAHNIAN and ASENOV, 1986), which probably augments the importance of the hepatobiliary route of excretion of OTA. The importance of the urinary route of excretion of OTA could be also increased by improvement of diuresis and cardiac activity in artichoke-treated animals, predominantly due to the cynarine compound of the extract. The stimulation of cholesterol metabolism, provoked by the same compound, is also of great importance in the observed protection against toxicity of OTA.

Conclusions

The main haematological and biochemical changes in the studied intoxication in broiler chicks are: an increase of serum levels of uric acid and glucose, a decrease of serum levels of total protein and cholesterol, haemoglobinopenia and eosinopenia. These changes were observed in chicks exposed to very low levels of OTA, encountered frequently in the field. A synergistic effect was observed between ochratoxin A and penicillic acid in experimental ochratoxicosis in broiler chicks, fed on a mouldy diet contaminated by Aspergillus ochraceus strain.

The toxic effect of both toxins was stronger than would be anticipated from the action of single OTA.

The 5% water-extract of artichoke and Rosallsat have a protective effect against the toxicity of ochratoxin A, expressed by a statistically significant protection against the OTA-induced changes in serum levels of uric acid, cholesterol and total protein, as well as against the changes in blood haemoglobin and eosinophils.

Acknowledgements
We thank Prof. Piotr Golinski (Department of Chemistry, Academy of Agriculture, Poznan, Poland) for the supply of reliable producers of ochratoxin A. The authors thank Prof. Benedicte Hald (Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, Frederiksberg, Denmark) for ochratoxin A analysis of fed forages. This study was financially supported by the Ministry of Science and Education Foundation, Bulgaria.

References

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HARVEY, R. B., M. H. ELISSALDE, L. F. KUBENA, E. A. WEAVER, D. E. CORRIER, B. A. CLEMENT (1992): Immunotoxicity of ochratoxin A to growing gilts. Am. J. Vet. Res. 53, 1966-1970.

MANNING, R. O., D. WYATT (1984): Toxicity of Aspergillus ochraceus contaminated wheat and different chemical forms of ochratoxin A in broiler chicks. Poultry Sci. 63, 458-465.

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Received: 12 June 1998
Accepted: 18 October 1999

STOEV, S., G. ANGUELOV, D. PAVLOV, LJ. PIROVSKI: Istrazivanje nekih antidota i laboratorijskih pokazatelja pri pokusnom otrovanju pilica ohratoksinom A i penicilinskom kiselinom. Vet. arhiv 69, 179-189, 1999.

SAZETAK

Istrazivane su glavne hematoloske i biokemijske promjene u pokusnoj ohratoksikozi u 85 pilica koji su u hrani primali 130, 300 i 800 ppb ohratoksina A (OTA) i 1000-2000 ppb penicilinske kiseline (PA) te su usporedeni s nalazima u 20 pilica koji nisu primali OTA i PA. Primijenjeni otrovi su izazvali povisenje razine mokracne kiseline i krvnog secera, te smanjenje razine kolesterola i ukupnih bjelancevina u krvnom serumu kao i laganu normocitnu anemiju. Ove promjene su bile izazvane s nekoliko puta manjim koncentracijama OTA u hrani, nego sto su opisane u literaturi s cistim OTA, sto je vjerojatno posljedica sinergistickog djelovanja OTA i PA. Utvrdeno je pozitivno zastitno djelovanje 5% vodenog iscrpka articoke i Rosallsata protiv otrovnog djelovanja ohratoksina A.

Kljucne rijeci: mikotoksicka nefropatija, ohratoksikoza, ohratoksin A, penicilinska kiselina, pilici