J. Appl. Poult. Res.
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J APPL POULT RES 2008. 17:151-156. doi:10.3382/japr.2007-00058
© 2008 Poultry Science Association
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Field Reports

Ochratoxin A in Laying Hens: High-Performance Liquid Chromatography Detection and Cytological and Histological Analysis of Target Tissues

G. Bozzo, E. Ceci1, E. Bonerba, S. Desantis and G. Tantillo

Dipartimento di Sanità e Benessere degli Animali, Facoltà di Medicina Veterinaria, Università degli Studi di Bari, Strada Provinciale per Casamassima, km 3, 70010 (BA), Italy

1 Corresponding author: e.ceci{at}veterinaria.uniba.it


   SUMMARY
TOP
 SUMMARY
DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Ochratoxins are fungal secondary metabolites that contaminate grains, legumes, coffee, dried fruits, beer, wine, and meat. Ochratoxins are considered powerful nephrotoxins, carcinogens, teratogens, and immunotoxins in rats and likely in humans. In 2006 during a pilot program of Hazard Analysis Critical Control Point in the Apulian region, Southern Italy, ochratoxin A (OA) was detected in commercial feed for laying hens in 2 farms. The OA-contaminated feed had been administered to animals for at least 2 mo. Analysis by HPLC with fluorometric detection of the tissues of 4 layer hens that displayed gross and microscopical lesions identified OA in the kidney (8.7 to 16.9 µg/ kg, average 13.65 ± 3.58 µg/kg) and liver (3.7 to 5.1 µg/kg, average 4.43 ± 0.64 µg/kg) but not in the other tissues.

Key Words: ochratoxin A • high-performance liquid chromatography • kidney • liver


   DESCRIPTION OF PROBLEM
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Ochratoxins are fungal secondary metabolites that contaminate grains, legumes, coffee, dried fruits, beer, wine, and meat. Ochratoxins may be produced by several species of Aspergillus and Penicillium, including several members of the Aspergillus ochraceus group and Penicillium verrucosum type I and II. The most important of these toxins is ochratoxin A (OA), which is the most toxic and more common than ochratoxin B or ochratoxin C. Ochratoxins are considered powerful nephrotoxins, carcinogens, teratogens, and immunotoxins in rats and likely in humans [1, 2].

The role of OA as a major causative factor in mycotoxic porcine nephropathy in many European countries was first described by Krogh in 1976 [3]. Subsequently, OA has been suspected to be involved in the etiology of Balkan endemic nephropathy, a human disease characterized by progressive renal fibrosis and by tumors of the urinary tract, such as carcinoma of the renal pelvis, ureters, and bladder [3–6].

Naturally occurring outbreaks of ochratoxicosis in poultry in the United States were first documented by Hamilton et al. [7], with 5 independent episodes of ochratoxicosis in turkeys, 2 episodes in laying hens, and 2 episodes in broiler chickens being described. In 2006 during a program of Hazard Analysis Critical Control Point in the Apulian region, Southern Italy, OA was detected in commercial feed for laying hens in 2 farms. The OA-contaminated feed had been administered to animals for at least 2 mo. The laying hens in the flocks presented debility and reduced egg production, although there were no known infectious diseases or errors in management.

The aim of this study was to investigate the presence of OA throughout the chain production of the feed of the laying hen. The purpose was to detect OA in the feed and in the tissues of the animals and to describe OA-related gross, histological, and cytological lesions.


   MATERIALS AND METHODS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Samples
Ten samples of feed for laying hens were obtained from 2 farms (5 per farm) in the Apulia region, Southern Italy, and were screened for OA. Ten 11-mo-old ISA Brown layer hens from 2 farms (5 per farm) were subjected to postmortem inspection, and in 4 such animals, the OA levels were determined. The content of OA was estimated in the liver, kidney, and muscles by a HPLC with fluorimetric detection (HPLC-FL) method.

Analytical Reagents
Reagent preparations are described in the references and notes section [8–10].

Apparatus and Chromatographic Conditions
For the liquid chromatography analysis, an Agilent 1100 Series [11] equipped with pumps, a Rheodyne model 7125 injector (100-µL loop) [12], and a fluorescence detector were used. A LC column Restek C18 (5 µm; 250 x 4.6 mm i.d.) [13] was used with a mobile phase consisting of a mixture of water:acetonitrile:acetic acid (49.5:49.5:1 by volume), degassed at a flow rate of 0.9 mL/min. Detection of OA was carried out using 333 and 477 nm as wavelengths for excitation and emission, respectively [14].

Immunoaffinity Cleanup
To measure ochratoxin levels, samples are prepared by mixing with an extraction solution, followed by blending and filtering. The extract is then applied to the OchraTest WB column [15], which contains specific antibodies for OA. Using monoclonal affinity chromatography, OchraTest is the only ochratoxin test that produces precise numerical results. At this stage, the ochratoxin binds to the antibody on the column. The column is then washed to rid the immunoaffinity column of impurities. By passing methanol through the column, the ochratoxin is removed from the antibody. The methanol can then be injected into an HPLC system.

Sample Extraction and Cleanup of Animal Feeds
A 50-g aliquot of animal feed was extracted with 100 mL of acetonitrile:water (60:40) for 5 min. A 10-mL aliquot of the homogenate was filtered and diluted with 40 mL of PBS buffer. The diluted extract was loaded onto a Ochra Test WB column. After washing with 10 mL of PBS buffer and 10 mL of water, the mycotoxin was eluted with 1.5 mL of methanol. We added 1.5 mL of water to all samples before injecting onto the HPLC to make the solvent for the standards and samples similar to the mobile phase.

Sample Extraction and Cleanup of Tissues and Organs
A 20-g aliquot of layer hen tissues (samples of kidneys, livers, and muscles) was homogenized with 6 mL of 1 M phosphoric acid in an UltraTurrax T25 homogenizer for 5 min. A 2.5-g aliquot of the homogenate was transferred into a Pyrex centrifuge tube, extracted twice with 5 mL of ethyl acetate, and centrifuged for 5 min at about 350 g. The organic phases were combined, reduced to approximately 3 mL, and back-extracted with 3 mL of 0.5 M NaHCO3 (pH 8.4). The aqueous extract was loaded onto an OchraTest WB column. After washing with 10 mL of PBS buffer and 10 mL of water, the mycotoxin was eluted with 1.5 mL of methanol. We added 1.5 mL of water to all samples before injecting onto the HPLC to make the solvent for the standards and samples similar to the mobile phase [16, 17].

Samples for Cytological and Histological Test
At postmortem inspection of the layer hens, careful macroscopic examination was made on the organs usually targeted by OA-induced pathology. Based on necropsy evidence, 4 animals (2 per farm) were selected and used for cytological and histological examinations. Aliquots of thymus, bursa of Fabricius, spleen, Peyer’s patch, cecal tonsil, Harderian gland, liver, kidney, and muscles were sampled. For cytological examination, the samples were collected by needle aspiration. The slides for cytological analysis were prepared directly at the farm, fixed in methanol for 15 min, and stained by the May-Grunwald-Giemsa method. For histological examination, pieces of tissue were collected and fixed in 10% neutral buffered formalin. The samples were embedded in paraffin wax, sectioned at 4 µm, and stained by hematoxylin-eosin.


   RESULTS AND DISCUSSION
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
The limits of detection and quantitation of OA by HPLC-FL in the feed and tissue samples were 0.10 and 0.30 µg/kg, respectively. Recovery was 87 ± 13% as evaluated on spiked samples at the 10 µg/kg level, and day-to-day relative SD was 10%.

The OA was detected in all feed samples, and the values ranged between 160 and 332 µg/kg of dry weight with averages of 255 and 285 µg/kg of dry weight for the first and second farms, respectively. These levels of OA were higher than allowed by the Italian Ministerial Decree [18] (i.e., 100 µg/kg).

By HPLC-FL analysis of the tissues, OA was detected only in the kidneys and liver. The highest levels of OA were found in the kidneys (8.7 to 16.9 µg/kg, average 13.65 ± 3.58 µg/ kg), whereas OA concentration in the liver was 3.7 to 5.1 µg/kg (average 4.43 ± 0.64 µg/kg). In the 4 animals subjected to HPLC-FL, gross and microscopical lesions were evidenced exclusively in the kidneys and in the liver. Kidney gross lesions included congestion, hemorrhage, and marked increase in size. Cortical hyperemia and red streaks arranging radially were present, suggesting cortex infiltration (Figure 1Go).


Figure 1
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  		Figure 1. Kidney. Nephritis: congestion, hemorrhage, and cortical hyperemia. (Figure printed in color online.)

 
By cytological and histological analysis, mitotic figures and pyknotic nuclei of the proximal tubular cells were visible. The nuclei of the proximal tubular cells appeared enlarged heterogeneously (Figure 2Go), and there was degeneration of the epithelial cells in the whole cross-section of the proximal tubules, a pattern that is suggestive of tubulonephrosis. Smears for cytological analysis of kidney (Figure 3Go) are unusually rich in cells and contain primarily tubular epithelial cells. These cells have abundant cytoplasm and a round-oval, slightly eccentric nucleus. Tubulonephrosis has been associated with OA consumption. Tubular dilatation and hypertrophy, swelling of tubular epithelial cells, and desquamation of the tubular basament membrane has been described in young broiler chickens, with the changes usually affecting the proximal convoluted tubules but not being evident in the distal convoluted tubules [19].


Figure 2
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  		Figure 2. Kidney. Distension, enlargement, and degeneration of epithelial cells of the proximal tubules. Presence of pyknotic nuclei. Hematoxylin-eosin x 40. (Figure printed in color online.)

 

Figure 3
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  		Figure 3. Kidney. Cells with abundant cytoplasm and a round-oval, slightly eccentric nucleus. May-Grunwald-Giemsa x 100. (Figure printed in color online.)

 
The liver gross lesions included diffuse yellowish color of the surface that is suggestive of fatty infiltration of hepatocytes. Livers appeared enlarged, firm, and covered with scattered necrotic foci. In laying hens, such liver alteration is fairly common, and exogenous poisoning is the most frequent cause of this. Congestion and hemorrhage were also evident (Figure 4Go). By cytological analysis, a prominent feature was the presence of groups of vacuolated and heavily stained hepatocytes scattered among the pale-stained hepatic cells (Figure 5Go). In the histological sections, numerous hepatocytes with fatty infiltration were observable (Figure 6Go). In addition, groups of vacuolated hepatocytes and necrotic cells were found scattered throughout the liver parenchyma (Figure 6Go). Nodular inflammatory cells, arranged usually around the bile ducts, were also present (Figure 7Go), along with moderate focal fibrosis in the parenchyma.


Figure 4
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  		Figure 4. Liver. Enlargement, diffuse yellow color of the surface, and necrotic foci of the surface. (Figure printed in color online.)

 

Figure 5
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  		Figure 5. Liver. Numerous hepatocytes with fatty infiltration and vacuolated hepatocytes. May-Grunwald-Giemsa x 100. (Figure printed in color online.)

 

Figure 6
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  		Figure 6. Liver. Group of vacuolated and heavily stained hepatocytes. Hematoxylin-eosin x 20. (Figure printed in color online.)

 

Figure 7
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  		Figure 7. Liver. Nodular inflammatory cells around the bile ducts and focal fibrosis in the parenchyma. Hematoxylin-eosin x 40. (Figure printed in color online.)

 
The liver gross, histological, and cytological lesions observed in the layer hens mirror the OA-associated lesions described in previous studies [20, 21]. Our results showed alterations in liver histology localized in the biliary region. Similar findings have been described by Lee et al. [20] in mice, with OA-associated lesions developing mainly in the lumen of the biliary ducts. Discrete focal fibrosis in chicken liver parenchyma around the bile ducts and modest epithelial proliferation of biliary ducts have been observed after experimental consumption of OA [21]. In addition, enlargement and vacuolization have been described in liver parenchymal cells [22].


   CONCLUSIONS AND APPLICATIONS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES
 

  1. There is a possible association between consumption of OA-contaminated feed and development of macroscopical lesions in the liver and kidneys of layer hens. However, other factors may contribute to fatty infiltration in layer hens that are usually under strong physiological pressure to achieve optimal levels of production.
  2. More importantly, our study strengthens the notion that monitoring the quality of animal feed is a priority to guarantee animal health.
  3. Our findings warrant studies to investigate the possible accumulation of OA in the eggs to evaluate the risks of human exposure.


   ACKNOWLEDGMENTS
 
We thank V. Martella (Università degli Studi di Bari) for his editorial suggestions and comments.


   REFERENCES AND NOTES
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 

  1. Frisvad, J. C. 1995. Mycotoxins and mycotoxigenic fungi in storage. Pages 251–288 in Stored Grain Ecosystems. D. S. Jayas, N. D. White, and W. E. Muir, ed. Marcel Dekker, New York, NY.
  2. Romani, S., G. Sacchetti, C. Chaves López, G. Pinnavaia, and M. Dalla Rosa. 2000. Screening on the occurrence of ochratoxin A in green coffee beans of different origins and types. J. Agric. Food Chem. 48:3616–3619.[CrossRef][Web of Science][Medline]
  3. Krogh, P. 1976. Mycotoxic nephropathy. Pages 147–170 in Advances in Veterinary Science and Comparative Medicine. Vol. 20. Acad. Press. New York, NY.
  4. Krogh, P. 1980. Ochratoxins: Occurrence, biological effects and casual role in disease. Pages 673–680 in Natural Toxins. E. Eaker and T. Wadstrom, ed. Pergamon Press, Oxford, UK.
  5. IARC. 1993. Ochratoxin A. In Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines, and Mycotoxins. IARC Monographs on the Evolution of Carcinogenic Risks to Humans 56:489–521. Int. Agency Res. Cancer, Geneva, Switzerland.
  6. Marquardt, R. R., and A. A. Frohlich. 1992. A review of recent advances in understanding ochratoxicosis. J. Anim. Sci. 70:3968–3988.[Abstract]
  7. Hamilton, P. B., W. E. Huff, J. R. Harris, and R. D. Wyatt. 1982. Natural occurrences of ochratoxicosis in poultry. Poult. Sci. 61:1832–1841.[Medline]
  8. Supelco ochratoxin (product no. 46912) [9], packaged in sealed ampoules at a concentration of about 50 ng/ µL in benzene:acetic acid (99:1), was used as standard. This standard was prepared according to the AOAC official methods [10]. The standards for the OchraTest by HPLC were prepared as follows: a) ochratoxin-working solution 1: dilution 1:50 (in methanol) of Supelco ochratoxin A standard (50 ng/µL) and b) ochratoxin-working solution 2: diluting ochratoxin-working solution 1 (1 ng/µL) 10 times with methanol. The calibration curve was performed diluting ochratoxin-working solution 2 with methanol in order to obtain the following concentrations: 1.0, 10.0, 20.0, and 50.0 (ng/g). We added 1.5 mL of water to all our standards and samples before injecting onto the HPLC to make the solvent for the standards and samples similar to the mobile phase.
  9. Supelco, Bellefonte, PA.
  10. AOAC. 2001. Method 2001.01. J. AOAC Int. 84:1818–1827.[Medline]
  11. Agilent Technologies, Waldbronn, Germany.
  12. Rheodyne LLC, Rohnert Park, CA.
  13. Restek Corporation, Bellefonte, PA.
  14. Monaci, L., G. Tantillo, and F. Palmisano. 2004. Determination of ochratoxin A in pig tissue by liquid-liquid extraction/partition and high performance liquid chromatography. Anal. Bioanal. Chem. 378:1777–1782.[CrossRef][Medline]
  15. Vicam, Watertown, MA.
  16. Losito, I., L. Monaci, F. Palmisano, and G. Tantillo. 2004. Determination of ochratoxin A in meat products by high-performance liquid chromatography coupled to electrospray ionisation sequential mass spectrometry. Rapid Commun. Mass Spectrom. 18:1–7.[CrossRef][Medline]
  17. Matrella, R., L. Monaci, M. A. Milillo, F. Palmisano, and G. Tantillo. 2006. Ochratoxin A determination in paired kidneys and muscles samples from swine slaughtered in southern Italy. Food Control 17:114–117.[CrossRef]
  18. Ministerial decree (Italy). May 15, 2006. Ochratoxin A limit in feeds for animals.
  19. Dwivedi, P., and R. B. Burns. 1984. Pathology of ochratoxicosis in young broiler chicks. Res. Vet. Sci. 36:92–103.[Web of Science][Medline]
  20. Lee, S., J. T. Beery, and F. S. Chu. 1984. Immuno-histochemical fate of ochratoxin A in mice. Toxicol. Appl. Pharmacol. 72:218–227.[CrossRef][Medline]
  21. Birò, K., L. Solti, I. Barna-Vetrò, G. Bagò, R. Glavits, E. Szabò, and J. Fink-Gremmels. 2002. Tissue distribution of ochratoxin A as determined by HPLC and ELISA and histopathological effects in chickens. Avian Pathol. 31:141–148.[CrossRef][Medline]
  22. Kozaczynski, W. 1994. Experimental ochratoxicosis A in chickens. Histopathological and histochemical study. Arch. Vet. Pol. 34:205–219.[Medline]




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