Experimental ochratoxicosis in broiler chickens

Abstract

This study describes the toxicity signs that developed when the diet of male broiler chickens was artificially contaminated with different levels of the mycotoxin ochratoxin A (OTA). Chicks were assigned randomly to three groups of 80 chicks that were fed a diet containing 0 oarts per billion (ppb) (control, group 1), 400 ppb (group 2) or 800 ppb (group 3) OTA from day 1 to 5 weeks of age. Signs of ochratoxicosis were assessed on the basis of changes in the following criteria: body weight, relative weights of two representative internal organs (gizzard and thymus), feed consumption, feed conversion ratio, mortality, thyroid activity, blood profile, humoral and cell mediated immunity. Feeding OTA at levels of 400 and 800 ppb (groups 2 and 3) significantly decreased the body weight, thymus weight, feed consumption, feed conversion ratio and thyroxine concentration (P<0.05). The OTA groups developed anaemia manifested by a significant decrease in the red blood cell count, packed cell volume percentage and haemoglobin concentration (P<0.05). By the end of the experiment both groups that received OTA showed a 37% reduction in red blood cell count compared with the control group. Furthermore, a significant decrease in the white blood cell count, humoral immune response and cell-mediated immunity was found in both groups fed ochratoxin compared with the control group (P<0.05). The reduction in the above parameters was more noticeable with time and was proportional to the level of OTA exposure. A significant increase in relative gizzard weight, cumulative mortality and triiodothyronine concentration was found in OTA-fed chicks (P<0.05). These data provide a description of ochratoxicosis in broilers that should be useful in diagnosis and in improved understanding of the practical implications on broiler performance and health, a problem that can threaten the poultry industry.

Ochratoxicose expérimentale chez le poulet de chair

Cette étude décrit les symptômes de toxicité qui se sont développés quand l'alimentation de poulets de chair mâle a été artificiellement contaminée avec différents niveaux de mycotoxine ochratoxine A (OTA). Les poussins ont été répartis de façon aléatoire en trois groupes de 80 poussins qui ont été nourris, de l'âge d'un jour à 5 semaines, avec des aliments contenant 0 ppb d'OTA (G1, témoin), 400 ppb (G2), ou 800 ppb (G3). Les symptômes d'ochratoxicose ont été évalués sur la base des changements des critères suivants : le poids du corps (BW) les poids relatifs de deux organes internes représentatifs (gésier et thymus), la consommation d'aliment, indice de conversion alimentaire (FCR), la mortalité, l'activité de la thyroïde, le profil sanguin, immunité humorale et l'immunité à médiation cellulaire. Les aliments contenant l'OTA à des taux de 400 et 800 ppb (G2 & G3) ont entraîné une diminution significative du BW, du poids du thymus, de la consommation alimentaire, du FCR et de la concentration de la thyroxine (T4) (P < 0,05). Les groupes OTA ont développé une anémie qui s'est manifestée par une diminution significative du nombre de globules rouges (RBC), de l'hématocrite (PCV) et de la concentration en hémoglobine (P < 0,05). A la fin de l'expérimentation les deux groupes qui ont reçu de l'OTA ont montré une réduction de 37% du nombre de des RBC comparés au groupe témoin. De plus, une diminution significative du nombre des globules blancs (WBC), de la réponse immunitaire humorale et de la réponse immunitaire à médiation cellulaire a été observée dans les deux groupes ayant reçu de l'aliment avec de l'ochratoxine comparés au témoins (P < 0,05). La diminution observée au niveau des paramètres précités a été plus importante dans le temps et a été proportionnelle à la concentration d'OTA. Une augmentation significative du poids relatif du gésier, de la mortalité cumulée et de la concentration en triiodothyronine (T3) a été notée chez les poulets ayant reçu un aliment avec de l'OTA (P < 0,05). Ces données fournissent une description de l'Ochratoxicose chez les poulets de chair qui devrait être utile en diagnostic et améliorer la compréhension des implications pratiques sur la santé et les performances des poulets de chair, un problème qui peut être une menace pour l'industrie de la volaille.

Experimentelle Ochratoxikose bei Broilerküken

Dieser Artikel beschreibt die Toxizitätsanzeichen, die nach Gabe von absichtlich mit verschiedenen Mengen des Mykotoxins Ochratoxin A (OTA) kontaminierten Futters an männliche Broilerküken entstanden. Die Küken wurden zufällig auf drei Gruppen von je 80 Broilerküken verteilt, die vom 1. Lebenstag bis zum Alter von 5 Wochen Futter mit 0 (Kontolle, G1), 400 (G2) oder 800 (G3) ppb OTA erhielten. Anzeichen einer Ochratoxikose wurden auf der Basis von Veränderungen der folgenden Parameter ermittelt: Körpergewicht, relatives Gewicht von zwei repräsentativen inneren Organen (Muskelmagen und Thymus), Futterverbrauch, Futterverwertung (FCR), Mortalitätsrate, Schilddrüsenaktivität, Blutwerte, humorale und zellvermittelte Immunität. Die Verfütterung von OTA in Mengen von 400 und 800 ppb (G2 & G3) reduzierte Körper- und Thymusgewicht, Futterverbrauch, FCR und die Thyroxin (T4)-Konzentration signifikant (p < 0,05). Die OTA-Gruppen entwickelten eine Anämie, die sich in einem signifikanten Rückgang (p < 0,05) der Anzahl der roten Blutkörperchen (RBC), des Hämatokritwerts und der Hämoglobulinkonzentration manifestierte. Am Versuchsende wiesen die beiden Gruppen, die OTA erhielten, im Vergleich zur Kontrollgruppe eine Reduktion der RBC-Zahl um 37 % auf. Darüber hinaus wurde in beiden mit Ochratoxin gefütterten Gruppen verglichen mit der Kontrollgruppe eine signifikante Verringerung der Anzahl der weißen Blutzellen, der humoralen Immunantwort und der zellvermittelten Immunität gefunden (p < 0,05). Die Reduktion der oben genannten Parameter wurde mit zunehmender Zeitdauer deutlicher und war proportional zur Menge des verabreichten OTAs. Außerdem wurde bei den mit OTA gefütterten Küken ein relativer Anstieg des Drüsenmagengewichts, der kumulativen Mortalität und der Triiodthyronin (T3)-Konzentration festgestellt (p < 0,05). Diese Beschreibung der Befunde bei einer Ochratoxikose in Broilern, die ein ernsthaftes Problem für die Geflügelindustrie sein kann, kann hilfreich sein für die Diagnostik sowie eine verbesserte Einsicht in die praktischen Auswirkungen einer Ochratoxikose auf die Leistung und Gesundheit von Broilern.

Ocratoxicosis experimental en pollos de engorde

Este estudio describe los signos de toxicidad que se desarrollaron cuando las dietas de pollos de engorde machos se contaminaron artificialmente con distintos niveles de la micotoxina ocratoxina A (OTA). Los pollos se distribuyeron aleatoriamente en tres grupos de 80 pollos que se alimentaron con una dieta que contenía 0 (control, G1), 400 (G2) o 800 (G3) ppb de OTA desde el primer día hasta las cinco semanas de vida. Los signos de ocratoxicosis se valoraron en base a cambios en los siguientes parámetros: peso corporal (BW), pesos relativos de dos órganos internos representativos (molleja y timo), consumo de pienso, índice de conversión del pienso (FCR), mortalidad, actividad tiroidea, perfil sanguíneo e inmunidad celular y humoral. El consumo de OTA a niveles de 400 y 800 ppb (G2 & G3) redujo de manera significativa el BW, el peso del timo, el consumo de alimento, FCR y la concentración de tiroxina (T4) (P < 0.05). Los grupos OTA desarrollaron anemia, evidente a través de una reducción significativa del recuento de eritrocitos (RBC), del porcentaje de volumen celular aglomerado (PCV) y de la concentración de hemoglobina (P < 0.05). Al final de la prueba experimental los dos grupos que recibieron OTA mostraron una reducción del 37% en el recuento de RBC en comparación con el grupo control. Además, también se observó una reducción significativa de la células blancas sanguíneas (WBC), en la respuesta inmune humoral y en la inmunidad mediada por células en ambos grupos alimentados con ocratoxina en comparación con el control (P < 0.05). La reducción de los parámetros mencionados anteriormente fue más evidente en el tiempo y fue proporcional al nivel de exposición a las ocratoxinas. Se halló un incremento significativo en el peso relativo de la molleja, en la mortalidad acumulada y en la concentración de triyodotironina (T3) en los pollos alimentados con OTA (P < 0.05). Estos resultados proporcionan una descripción de la ocratoxicosis en pollos de engorde que debería ser útil para el diagnóstico y para una mayor comprensión de las implicaciones prácticas que ésta tiene en el rendimiento y estatus sanitario de los pollos de engorde, un problema que puede amenazar a la industria avícola.

Introduction

The family of ochratoxins consists of three members known as ochratoxin A, ochratoxin B and ochratoxin C. They are the second major group of mycotoxins to be characterized after the discovery of aflatoxins. Structurally, the three toxins differ only very slightly from each other; however, these differences have marked effects on their respective toxic potentials, with ochratoxin A (OTA) being the most toxic of that family based on the median lethal dose and minimal growth inhibition in birds (Peckham et al., 1971; Chang et al., 1979). Considerable species and sex differences in sensitivity towards OTA acute toxicity and half-life have been demonstrated (O'Brien & Dietrich, 2005). Oral median lethal dose values have been shown to range from approximately 5.9 and 16.5 mg/kg body weight (BW) in turkeys and Japanese quail, respectively, to 0.5 and 2 to 4 mg/kg BW in ducks and chickens (Peckham et al., 1971; Huff et al., 1974). Variations in kinetic parameters may play a major role in explaining these differences, as current data suggest some species-specific, sex-specific, and age-specific variations in the expression levels of proteins capable of active OTA transport (Dietrich et al., 2005). OTA is produced by fungi from two genera, Aspergillus and Penicillium. The Aspergillus OTA producers include strains of seven species in section Circumdati (Aspergillus ochraceus, Aspergillus melleus, Aspergillus auricomus, Aspergillus ostianus, Aspergillus petrakii, Aspergillus sclerotiorum, and Aspergillus sulphureus), two species in section Flavi (Aspergillus alliaceus, and Aspergillus albertensis), two species in section Nigri (Aspergillus niger and Aspergillus carbonarius), and one species in section Aspergillus (Aspergillus glaucus) (Bayman et al., 2002). Two Penicillium species, Penicillium verrucosum and Penicillium nordicum, share the ability to produce OTA (Larsen et al., 2001). The natural occurrence of OTA in food and feedstuffs of plant and animal origin is common. Due to its long half-life OTA accumulates in the food chain, and threatens human and animal health because of its extreme toxicity, widespread occurrence and the variety of commodities that it can contaminate (Scott, 1978). OTA has been implicated in a diverse range of toxicological effects, including renal toxicity, mutagenicity, teratogenicity, neurotoxicity and immunotoxicity in both animals and man (O'Brien & Dietrich, 2005).

OTA causes significant losses to the poultry industry due to its effects on performance and health. It causes a reduction in growth rate and feed consumption, poorer feed conversion and increased mortality (Peckham et al., 1971; Huff et al., 1974; Verma et al., 2004). Furthermore it produces a reduction in total blood proteins (Huff et al., 1988; Stoev et al., 2000), suppression of immune function (Chang et al., 1979; Dwivedi & Burns, 1984a and b; Stoev et al., 2000, 2002; Santin et al., 2002; Politis et al., 2005) and impairment of blood coagulation (Raju & Devegowda, 2000). OTA induces degenerative changes and an increase in the weight of the kidney and liver, as well as a decrease in the weights of the lymphoid organs (Stoev et al., 2000 2002).

The objective of the present study was to describe the collective changes associated with an induced experimental ochratoxicosis in male broiler chicks by feeding them for 5 weeks on diets containing known concentrations of OTA at levels similar to the exposure that might spontaneously occur in the field.

Materials and Methods

Two hundred and forty 1-day-old male Ross broiler chicks were randomly assigned equally after weighing to three groups of 80 chicks. OTA was added to their diet as follows:

•	Group 1 (G1) served as a control with 0 µg/kg OTA (0 parts per billion (ppb)).
•	Group 2 (G2) diet was supplemented with 400 µg/kg OTA (400 ppb).
•	Group 3 (G3) diet was supplemented with 800 µg/kg OTA (800 ppb).

The groups were kept under similar and standard hygienic and environmental conditions, and were supplied with feed and water ad libitum throughout the experimental period of 5 weeks. They were fed a commercial starter ration (23.8% crude protein and 3045 kcal ME/kg) from day 1 to 18 days, then a grower ration (22.8% crude protein and 3160 kcal metabolizable energy (ME)/kg) from day 19 to 5 weeks. These commercial broiler starter and grower diets (to which OTA was added), were formulated to meet or exceed the nutritional requirements of broilers recommended by the National Research Council (1994) and were free from any medication. The diet was tested for two other mycotoxins, aflatoxin and zeralenone, by immunoaffinity, which showed them to be below the detection limits of 1 and 10 µg/kg, respectively.

OTA was supplied by A. ochraceus NRRL 3174 culture. Production and extraction of OTA were performed by the method of Davis et al. (1969) and purification and clean-up of the toxin were performed according to Nesheim (1969). Quantitative determination of OTA was conducted using a basic methanolic extraction, monoclonal antibody immunoaffinity column clean-up and a fluorometric detection method with a detection limit of 0.7 µg/kg according to Scott & Kanhere (1995). Pure chloroform-extracted dry OTA was dissolved in 95% ethanol and mixed with a small portion of feed, the ethanol was allowed to evaporate and then it was mixed with the remainder of the feed in a horizontal mixer to provide 400 or 800 µg OTA/kg finished feed. During the preparation of the control diet, an equal amount of ethanol was added and allowed to evaporate in a fashion similar to that of the OTA-treated feed.

Measurements and statistical analysis

At the end of each week, from weeks 1 to 5, broiler chicks were weighed individually to the nearest gram to determine the average weekly body weight. Feed consumption was recorded daily and the mean was calculated weekly up to the end of the experiment. Feed conversion ratios (FCRs) were calculated for each group at the end of the experiment as grams of feed/grams of gain. The mortality rate was calculated weekly for each group through the experimental period as a percentage of the total number of birds. Eight birds of each group were selected randomly, starved overnight and then humanely killed to extract the gizzard and thymus. These were blotted dry, weighed individually and expressed as a percentage of the respective body weight.

Blood samples were collected weekly from the eight sacrificed birds of each group in a heparinized tube to determine the haemoglobin (Hb) concentration by a spectrophotometric method. Packed cell volume (PCV) percentages were determined by microhaematocrit, and red blood cell (RBC) and white blood cell (WBC) counts using a haemocytometer, all as described by Dacie & Lewis (1991). A second blood sample from each bird was collected in a sterile, dry centrifuge tube without anticoagulant and allowed to clot, and serum was separated by centrifugation and stored at −20°C until the determination of thyroxine (T4) and triiodothyronine (T3) by a radioimmunoassay technique based on the method described by Chopra (1972). The humoral immune response was determined by haemagglutination test against injection of sheep red blood cells (SRBCs) according to the method described by Wegman & Smithies (1966). Five representative 19-day-old chicks from each group were injected with SRBCs. Blood samples were collected from the brachial vein of each chick 3, 7 and 10 days thereafter and the serum was separated to determine antibodies to SRBCs. The cell-mediated immune response was determined by a cutaneous delayed hypersensitivity test at 37 days of age on 10 chicks from each group using phytohaemagglutinin-L (PHA-L) according to McCorkle et al. (1980).

The data obtained in this study were analysed statistically by the general linear model procedure with Statistical Analysis System software (SAS Institute, 1992). One-way and two-way analysis of variance models were fitted to the data as appropriate.

Results

Effect of OTA on productive performance

The results in Table 1 show that body weight was significantly decreased in both OTA-treated groups G2 and G3 throughout the duration of the experiment. It was more noticeable in birds in G3 consuming the higher level of OTA (800 ppb) than those in G2 consuming the lower level (400 ppb) when both were compared with control birds in G1. The extent of body weight depression was not only proportional to the exposure level of OTA, but it was also proportional to the exposure period, because the body weight reduction became more severe after a longer period of exposure to the higher level of OTA. Table 1 shows that OTA treatment affected the amount of feed consumed per chicks per week. There was a trend for reduced feed consumption throughout the experimental period in both groups of birds fed the OTA-containing diet. This reduction in feed consumption was more noticeable with time and with the higher level of OTA (G3). Feed conversion ratio results (Table 1) show that the quantity of feed required per unit of weight increased in both OTA-fed groups. The feed conversion ratios were 1.85 and 1.99 for the G2 (400 ppb) and G3 (800 ppb) levels of OTA, respectively, compared with 1.65 for G1. Results in the same table also demonstrate that the effect of OTA on cumulative feed conversion ratio was dose dependent.

Table 1.  Effect of dietary OTA levels on body weight, feed consumption and cumulative FCR of male broiler chicks

Group (OTA level, ppb)	1 week old	2 weeks old	3 weeks old	4 weeks old	5 weeks old	FCR (g feed/g body weight)
Body weight (g)
G1 (0)	184.20^A±2.19	448.58^A±4.99	870.95^A±8.29	1340.50^A±12.12	1744.60^A±16.45
G2 (400)	135.45^B±1.89	315.63^B±7.25	512.63^B±9.64	726.76^B±17.90	895.13^B±29.69
G3 (800)	105.91^C±1.58	234.75^C±6.29	368.67^C±8.21	486.09^C±11.76	585.04^C±22.93
Feed consumption (g)
G1 (0)	150.84	338.70	590.72	806.94	1003.80	1.657
G2 (400)	103.75	234.15	338.90	424.10	556.90	1.852
G3 (800)	92.81	146.18	229.73	311.87	387.85	1.997
Values presented as mean±standard error. Means in a column with different uppercase superscripts are significantly different (P < 0.05).

Mortality was increased in G2 and G3, with the total number of deaths being three and seven respectively, compared with one death in the control group. This gave a cumulative mortality percentage of 1.25, 5.23, and 12.98% for groups G1, G2, and G3, respectively, and indicated that a direct relationship existed between OTA level in the diet and the mortality percentage.

Effect of OTA on gizzard and thymus weights

Table 2 presents the changes in the relative weights of two representative internal organs chosen because they are sensitive in their response to OTA dietary contamination. The relative gizzard weight increased significantly in broiler chicks that were subjected to OTA in their diets when compared with the control birds. This increase was seen in the first week of age and was more pronounced in G3 with the presence of high level of OTA, which confirms that the effect of OTA was dose dependent. Table 3 demonstrates that there was a significant decrease in the relative thymus weight of the OTA-treated birds compared with that of the control birds. This decrease became more severe with time and with the higher level of OTA (800 ppb). The differences between G2 and G3 were significant only at the first and fourth weeks of age.

Table 2.  Effect of dietary OTA levels on the relative gizzard and thymus weights of male broiler chicks

Group (OTA level, ppb)	1 week old	2 weeks old	3 weeks old	4 weeks old	5 weeks old
Relative gizzard weight (g/100 g)
G1 (0)	6.25^B±0.15	4.34^C±0.12	3.49^C±0.11	3.53^C±0.14	2.49^B±0.05
G2 (400)	6.41^B±0.18	5.29^B±0.13	4.65^B±0.14	3.92^B±0.11	4.04^A±0.12
G3 (800)	7.76^A±0.15	5.98^A±0.11	5.19^A±0.14	4.97^A±0.09	4.24^A±0.06
Relative thymus weight (g/100 g)
G1 (0)	0.2308^A±0.010	0.2765^A±0.007	0.3367^A±0.009	0.3844^A±0.008	0.3763^A±0.009
G2 (400)	0.2423^A±0.110	0.1980^C±0.004	0.1553^B±0.006	0.1520^B±0.009	0.1371^BC±0.006
G3 (800)	0.1936^B±0.007	0.2099^BC±0.009	0.1460^B±0.007	0.1036^C±0.004	0.1814^C±0.007
Values presented as mean±standard error. Means in a column with different uppercase superscripts are significantly different (P < 0.05).

Table 3.  Effect of dietary OTA levels on thyroid hormones (T3 and T4) activity of male broiler chicks

Group (OTA level, ppb)	1 week old	2 weeks old	3 weeks old	4 weeks old	5 weeks old
T3 (nmol/l)
G1 (0)	7.788^A±0. 58	6.175^AB±0.61	5.371^B±0.73	6.171^B±0.37	7.211^B±0.22
G2 (400)	9.746^A±0.27	6.692^AB±0.52	7.482^AB±0.31	8.181^AB±1.56	9.569^AB±0.93
G3 (800)	9.952^A±0.96	7.326^A±0.62	8.004^A±0.66	10.071^A±1.11	9.712^A±0.86
T4 (ng/ml)
G1 (0)	9.728^A±0.53	11.577^A±1.14	13.209^A±1.15	13.208^A±0.42	14.996^A±1.28
G2 (400)	9.246^A±0.89	9.168^AB±1.06	10.489^AB±0.94	11.810^AB±0.96	13.287^A±1.29
G3 (800)	5.159^B±0.65	6.309^B±1.43	8.143^B±1.03	9.324^B±1.21	9.402^B±0.49
Values presented as mean±standard error. Means in a column with different uppercase superscripts are significantly different (P < 0.05).

Effect of OTA on the thyroid hormones

Results in Table 3 shows that the presence of OTA in the broiler diets at 400 and 800 ppb caused a significant increase in serum T3 concentration when compared with the control birds after 2 weeks of age. The higher dietary level of OTA led to a greater T3 concentration in G3 than in G2, but differences were only significant at the fourth week of age. The data show that T3 values decreased during the second and third weeks, and then increased thereafter.

An inverse relationship was shown between the dietary OTA level and the serum T4 concentration. Both OTA levels caused a decrease in T4 concentration but reductions were only significant in G3, receiving the higher OTA level, from the first week of the experiment and throughout.

Effect of ochratoxin A on haematological parameters

RBC count. The RBC counts were significantly decreased in the broilers in G2 and G3 compared with the control G1 (Table 4) at each time point. The effect of OTA on RBC counts appeared dose dependent up to week 4.

Table 4.  Effect of dietary OTA levels on haematological indices of male broiler chicks

Group (OTA level, ppb)	1 week old	2 weeks old	3 weeks old	4 weeks old	5 weeks old
RBC count (10^6×mm^3)
G1 (0)	3.924^A±0.15^a	4.903^A±0.79	5.588^A± 0.31	5.654^A±0.35	5.341^A±0.26
G2 (400)	2.454^B±0.15	3.561^B±0.18	3.997^B±0.28	4.116^B±0.25	3.366^BC±0.34
G3 (800)	1.707^C±0.17	2.768^C±0. 22	3.154^C±0.28	2.684^C±0.15	3.363^C±0.26
Hb concentration (g/dl)
G1 (0)	8.634^A±0.42	9.057^A±0.43	10.463^A±0.35	11.568^A±0.34	13.220^A±0.32
G2 (400)	8.261^A±0.37	9.034^AB±0.46	8.822^B±0.46	9.914^BC±0.29	12.426^A±0.37
G3 (800)	6.437^B±0.45	7.953^B±0.41	9.149^B±0.27	9.248^C±0.35	10.661^B±0.45
PCV (%)
G1 (0)	30.6^A±0.81	34.0^A±0.71	36.0^A±0.89	37.4^A±0.93	38.2^A±1.11
G2 (400)	29.0^A±1.14	33.4^AB±1.03	33.6^AB±1.17	36.2^AB ±0.80	35.4^AB ±1.03
G3 (800)	30.0^A±1.41	31.0^B±0.63	33.2^B±0.86	32.0^B ±1.05	34.0^B±1.05
WBC count (10^3×mm^3)
G1 (0)	14.25^A±0.52	13.25^A±0.45	11.00^A±0.46	14.75^A±1.30	15.13^A ±1.36
G2 (400)	11.13^B±0.69	6.00^BC±0.59	6.25^BC±0.53	8.38^B±1.79	4.50^B±0.46
G3 (800)	5.13^C±0.81	4.00^C±0.46	3.38^C±0.53	5.00^C±1.35	4.50^B±0.68
Values presented as mean±standard error. Means in a column with different uppercase superscripts are significantly different (P < 0.05).

Haemoglobin concentration and PCV

Table 4 shows that both the Hb concentration and PCV were directly affected by OTA as a significant reduction occurred in both parameters in G2 and G3. Hb concentration values of the control group were significantly higher than those of G3. The values of G3 were lower than the values of G2, the differences between them being significant only at the first and the fifth weeks. Regardless of the treatment, Hb concentration values and PCV had a tendency to increase gradually with time.

Effect of OTA on WBC

Table 4 shows that feeding OTA at 400 or 800 ppb caused a significant reduction in WBC counts throughout the experiment as compared with the control group. Birds receiving OTA at 800 ppb in their diet (G3) had lower WBC count values than those receiving 400 ppb (G2), the differences between them being significant only at the fourth week of age.

Effect of ochratoxin A on immunological parameters

Humoral immune response. G2 and G3 showed significant sharp reductions in antibody titre determined by the haemagglutination test against SRBCs at all test periods (3, 7 and 10 days post immunization) when compared with the titre produced in the control birds, G1 (Table 5). The decrease in titre was significantly more pronounced at the higher level of OTA (800 ppb) than the lower level (400 ppb). Regardless of the dietary OTA level used, the antibody titre against SRBCs increased to reach a peak at 7 days post immunization, and then decreased thereafter at 10 days post immunization.

Table 5.  Effect of dietary OTA levels on the immune system function of male broilers

Group (OTA level, ppb)	Humoral immune response			Wattle thickness before injection (mm)		Wattle thickness 24 h post injection^a (mm)
	3 d.p.i.	7 d.p.i.	10 d.p.i.	Right wattle	Left wattle	Right wattle	Left wattle	Mean wattle index^b (mm)	Mean relative response^c
HA test^d
G1 (0)	5.60^A±0.67	8.00^A±0.32	4.40^A±0.50
G2 (400)	2.20^B±0.66	4.80^BC±0.49	1.60^B±0.51
G3 (800)	1.40^B±0.75	3.00^C±1.05	1.20^B±0.58
Cell-mediated immune response
G1 (0)				1.50±0.03	1.50±0.03	2.30±0.04	1.50±0.03	0.79^A±0.033	1.54^A±0.027
G2 (400)				1.47±0.03	1.47±0.03	1.96±0.04	1.47±0.03	0.49^B±0.037	1.34^B±0.030
G3 (800)				1.42±0.05	1.42±0.05	1.83±0.05	1.42±0.05	0.41^B±0.023	1.29^B±0.017
Values presented as mean±standard error. Means in a column with different uppercase superscripts are significantly different (P < 0.05). d.p.i., days post immunization. ^aRight wattle was injected with PHA; left wattle was injected with sterile physiological saline and served as control. ^bMean wattle index = wattle thickness post injection – wattle thickness before injection (for right wattle). ^cRelative response = thickness of wattle after injection/thickness of wattle before injection (for right wattle). ^dHaemagglutination test to determine the antibody titre to SRBCs.

Cell-mediated immune response

Table 5 shows that dietary OTA supplementation to broiler chickens at both 400 and 800 ppb significantly inhibited the wattle response to injection with PHA antigen. Both the wattle index and relative response decreased significantly in groups of birds G2 and G3 compared with the control birds, reflecting the detrimental effects of OTA on cell-mediated immunity in broilers.

Discussion

The results of the current study confirm that OTA is an important mycotoxin in broilers because of the magnitude of detrimental effects induced. Feeding OTA to broiler chicks significantly decreased the body weight, feed consumption, FCR, T4, RBCs, WBCs, Hb concentration, PCV, relative thymus weight, and humoral and cell-mediated immune responses. On the other hand, OTA significantly increased the mortality rate, relative gizzard weight and T3. The OTA responses presented in this study were dose and time dependent.

The decrease in broiler body weight due to ochratoxicosis was in agreement with several previous reports using dietary OTA inclusion rates of 567 ppb (Garcia et al., 2003), 0.5 to 2 parts/10⁶ (Prior et al., 1980; Campbell et al., 1983; Kubena et al., 1988; Raju & Devegowda, 2000; Kumar et al., 2003), 1 to 4 parts/10⁶ (Gibson et al., 1989; Verma et al., 2004), 5 parts/10⁶ (Stoev et al., 2002) and up to 8 parts/10⁶ (Huff et al., 1974, 1980, 1988).

Decreased feed consumption, FCR and body weight in G2 and G3 may well have resulted from the decreased serum T4 and the increased serum T3 reported in this study. Body weights and T4 concentrations in the control group G1 were significantly higher than in the OTA-treated groups. Wentworth & Ringer (1986) reported that T3 and T4 had a significant effect on growth and feed efficiency of broiler chickens and that treatment with T3 lowered feed consumption. On the other hand, Prior et al. (1980) observed that the loss in body weight during ochratoxicosis was not due to a direct effect of OTA, but rather to the reduced feed intake that led to a decreased total serum proteins or hypoproteinaemia. The present study showed that feed intake was reduced in broilers fed OTA-contaminated diets, which confirms the above findings. Furthermore, the FCR was altered in a manner consistent with dietary OTA level and agreed with the findings of several others (Gibson et al., 1989; Elkady, 1993; Raju & Devegowda, 2000; Garcia et al., 2003; Verma et al., 2004).

The increase in percentage mortality of broilers fed an OTA-supplemented diet reached 12.98% for G3 and was in accordance with the findings of many investigators. In this respect Gibson et al. (1989), feeding OTA contaminated diets at 2 to 4 parts/10⁶, reported mortality that reached 21.9% in OTA-treated broiler chicks versus 2.5% for the controls. Kumar et al. (2003) reported a mortality of 14.3% in chicks infected with Escherichia coli but fed no OTA, and an increase to 35.7% mortality in E. coli-infected chicks fed 2 parts/10⁶ OTA. An increase in mortality as a result of the OTA in the diet was also reported in other species of poultry. For example, Chang et al. (1981) found that a concentration of 8 µg/g OTA in feed for turkey poults caused a significant increase in the cumulative mortality reaching 40% at 3 weeks from hatch.

According to O'Brien & Dietrich (2005), OTA has been suggested by various researchers to mediate its toxic effects via induction of apoptosis, disruption of mitochondrial respiration and/or the cytoskeleton, or via the generation of DNA adducts.

The significant increase in the gizzard relative weight due to OTA exceeded the percentage increase that was found by other workers exposing birds to dietary OTA at 1 to 8 parts/10⁶ (Huff et al., 1974; Gibson et al., 1989; Elkady, 1993; Stoev et al., 2002). In the present study, the gizzard weight almost doubled in G2 and G3 in comparison with G1. Raju & Devegowda (2000) found that exposing broiler chicks to OTA-contaminated ration at 2 parts/10⁶ from 1 to 35 days of age resulted in a 14.6% increase in gizzard weight. They suggested that toxin affinity for the gastrointestinal tract affected several physiological systems that led to dose-related weight alterations during ochratoxicosis. Moreover, Huff et al. (1988) attributed the gizzard sensitivity to OTA to the toxin irritative properties when in direct contact. They ranked the relative sensitivity of the organs to OTA from the most sensitive to the least sensitive as follows: gizzard > kidney > spleen > liver, based upon the time at which the significant changes occurred on the relative organ weight. In contrast to its effect on the gizzard, OTA caused a significant reduction in the relative weight of the thymus in the broiler chicks. Similar results were reported when OTA was administered to specific pathogen-free Plymouth Rock chicks at 1 or 5 parts/10⁶ (Stoev et al., 2002) or to broiler chicks at 2 and/or 4 parts/10⁶ (Dwivedi & Burns, 1984a; Elkady, 1993; Kumar et al., 2004) for periods ranging from 21 to 70 days. Results from all previous reports agreed that the greater the dose used, the greater the decrease in thymus weight, and this was found to be true also when lower doses (130, 305, and 790 ppb) were used by Stoev et al. (2000). All three doses significantly decreased the relative thymus weight and depleted it of lymphocytes, causing histological changes in chicks exposed to this toxin for up to 70 days. Because the thymus is the primary determinant of cell-mediated immunity, its regression at both examined levels of OTA imply that cellular immunity in broilers is impaired during ochratoxicosis.

The broiler chicks in G2 and G3 suffered a significant reduction in RBC count, Hb concentration and PCV in comparison with G1. These results are in agreement with Mohiuddin et al. (1993) who added OTA at concentrations of 0.75, 1.5 or 3.0 mg/kg diet of broiler chicks for 4 weeks and found a significant decrease in RBC count in all treated groups, while Stoev et al. (2000) showed only a significant decrease of RBC count in response to 5 parts/10⁶ and not 1 parts/10⁶ OTA. Over a range of OTA exposure rates starting from 0.5 parts/10⁶ (Agawane & Lonkar, 2004) and up to 4.0 parts/10⁶ (Huff et al. 1988), an anaemia characterized by a significant decrease in PCV and Hb concentration levels was reported and attributed to iron deficiency or as a consequence of a disturbance in the haemopoietic system.

The significant decrease in WBC count of broilers in G2 and G3 reached 30% of the control value at the end of the test period. Chang et al. (1979), and Mohiuddin et al. (1993) reported similar results when OTA was supplemented into broiler diets at levels of 0.5 to 8.0 µg/g feed from 1 day to 3 weeks of age, and 0.75 to 3 µg/g feed from 4 to 8 weeks of age, respectively. Leucocytopaenia was induced at even the lowest dose and reached 46% of the control value for the highest dose (Chang et al., 1979). The decrease in number of leucocytes was reported to be a reflection of a decrease primarily of lymphocytes, and to a lesser extent monocytes (Chang et al., 1979) or heterophils (Chang et al., 1981; Mohiuddin et al., 1993). Such a lymphocytopaenia may be a sensitive and useful indicator of ochratoxicosis that possibly may occur due to a direct effect on germinal centres of lymphoid tissues and implies alteration of the immune function. The detrimental effects of OTA on WBC counts were also found in male turkey poults fed diets contaminated with OTA at 4 and 8 µg/g feed from hatching to 3 weeks of age (Chang et al., 1981), and in Japanese quail administered with OTA by oesophageal intubation at 50 µg/bird per day for 60 days (Farshid & Rajan, 1996).

The significant decrease in antibody produc tion against SRBCs and mean wattle thickness in response to PHA injection indicated a reduced humoral and cellular immune response in the OTA-treated groups. This agrees with previous reports showing that OTA fed to chickens at concentrations of 130 to 790 ppb, 5 parts/10⁶ (Stoev et al., 2000, 2002); 2 parts/10⁶ (Singh et al., 1990; Santin et al., 2002) or 4 parts/10⁶ (Elkady, 1993) reduced either humoral or cellular immune response, or both. Furthermore, OTA fed to broilers alone (4 parts/10⁶) decreased humoral immunity, and in combination with aflatoxin (2 parts/10⁶) decreased humoral and cellular immunity (Verma et al., 2004). OTA was detrimental also to quail (Farshid & Rajan, 1996) and turkey immunity (Dwivedi & Burns, 1985). Suppression of humoral and cellular immunity by OTA reduces the immune response of chickens to vaccination against Newcastle disease virus strain B1 (Stoev et al., 2000, 2002) or LaSota strain (Santin et al., 2002), and allows development of secondary bacterial infections (Kumar et al., 2003, 2004)

Two findings that were reported in this study, a reduced leucocyte count and thymus weight, implied that the functioning of the immune system is altered. Regression of other lymphoid organs, the spleen and bursa of Fabricius, were reported during ochratoxicosis (Dwivedi & Burns, 1984a; Singh et al., 1990; Elkady, 1993; Stoev et al., 2000, 2002; Kumar et al., 2004; Verma et al., 2004) and was found to be accompanied by a decrease in the number of lymphoid cells present (Peckam et al., 1971; Stoev et al., 2000, 2002; Kumar et al., 2004) and in the immunoglobulin-containing cells (Dwivedi & Burns, 1984b). Closer histopathological examination revealed degenerative changes in the germinal centres of the thymus and spleen, and hence a possible explanation for the dietary OTA-induced lymphocytopaenia (Stoev et al., 2000, 2002). Therefore, O'Brien & Dietrich (2005) attributed the OTA-impaired immunity to a reduction in the proliferating, activation and differentiation of lymphocytes. That, in addition to the decrease in monocytes (Chang et al., 1979) and heterophils (Chang et al., 1981; Mohiuddin et al., 1993), leads to the reported leucocytopaenia. Moreover, OTA not only decreases differential WBC counts, but it also significantly reduces the phagocytic activity of splenic macrophages and impairs the T-lymphocyte function (Singh et al., 1990). When the antigen enters the circulation, it induces T cells to produce lymphokines that attract basophils or marrow-derived monocytes; thus, T-cell inhibition by OTA intoxication renders this chemotactic ability of leucocytes inefficient and explains the reported decrease in CMI. Recently, Politis et al. (2005) showed that several functional properties of macrophages and heterophils were depressed in chicks fed OTA at 0.5 parts/10⁶, thus confirming that the immune system is a primary target of OTA detrimental effects.

Results of the present study reinforce the negative influence of ochratoxins on broiler chickens’ performance and internal organ function. These detrimental effects observed when OTA at the concentration of >400 ppb was fed to broiler chicks definitely pose a potential risk of a significant health and economic impact on poultry industry.

Acknowledgments

The Department of Biological Applications, NRC-EAEA, is thanked for making research funds available.