Oral exposure to culture material extract containing fumonisins predisposes swine to the development of pneumonitis caused by Pasteurella multocida

Toxicology 213 (2005) 34–44

Oral exposure to culture material extract containing fumonisins predisposes swine to the development of pneumonitis caused by Pasteurella multocida David. J. Halloy a,1 , Pascal G. Gustin a,1 , Sandrine Bouhet b , Isabelle P. Oswald b,∗ a

Department of Functional Sciences, Unit of Pharmacology, Pharmacotherapy and Toxicology, Faculty of Veterinary Medicine, University of Li`ege, Liege, Belgium b INRA, UR66, Laboratory of Pharmacology and Toxicology, 180 Chemin de Tournefeuille, BP3, 31931 Toulouse, France Received 26 March 2005; received in revised form 8 May 2005; accepted 11 May 2005 Available online 23 June 2005

Abstract Fumonisin B1 (FB1 ) is a mycotoxin produced by Fusarium verticillioides and F. proliferatum that commonly occurs in maize. In swine, consumption of contaminated feed induces liver damage and pulmonary edema. Pasteurella multocida is a secondary pathogen, which can generate a respiratory disorder in predisposed pigs. In this study, we examined the effect of oral exposure to fumonisin-containing culture material on lung inflammation caused by P. multocida. Piglets received by gavage a crude extract of fumonisin, 0.5 mg FB1 /kg body weight/day, for 7 days. One day later, the animals were instilled intratracheally with a non toxin producing type A strain of P. multocida and followed up for 13 additional days. Pig weight and cough frequency were measured throughout the experiment. Lung lesions, bronchoalveolar lavage fluid (BALF) cell composition and the expression of inflammatory cytokines were evaluated at the autopsy. Ingestion of fumonisin culture material or infection with P. multocida did not affect weight gain, induced no clinical sign or lung lesion, and only had minimal effect on BALF cell composition. Ingestion of mycotoxin extract increased the expression of IL-8, IL-18 and IFN-␥ mRNA compared with P. multocida infection that increased the expression of TNF-␣. The combined treatment with fumonisin culture material and P. multocida delayed growth, induced cough, and increased BALF total cells, macrophages and lymphocytes. Lung lesions were significantly enhanced in these animals and consisted of subacute interstitial pneumonia. TNF-␣, IFN-␥ and IL-18 mRNA expression was also increased. Taken together, our data showed that fumonisin culture material is a predisposing factor to lung inflammation. These results may have implications for humans and animals consuming FB1 contaminated food or feed. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Mycotoxin; Swine; Fumonisin; Susceptibility; Infection; Immune response

∗ 1

Corresponding author. Tel.: +33 5 6128 54 80; fax: +33 5 61 28 53 10. E-mail address: [email protected] (I.P. Oswald). These authors equally contributed to this work.

0300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2005.05.012

D.J. Halloy et al. / Toxicology 213 (2005) 34–44

1. Introduction Mycotoxins are secondary metabolites of fungi that may contaminate animal and human feeds at all stages of the food chain. Since they contaminate approximately 25% of the World cereal crop production, they are representing a major risk factor affecting human and animal health (Finks-Gremmels, 1999; Oswald et al., 2005). Fumonisin B1 (FB1 ) belongs to the fumonisin family of toxins which are produced by Fusarium verticillioides and Fusarium proliferatum, fungi that commonly contaminate maize (Bezuidenhout et al., 1988). Recent surveys on fumonisins in food and feed throughout the World, including the United States and most European countries, have raised concerns about the extent of FB1 contamination of maize and its implications for food safety (Shephard et al., 1996; Scudamore et al., 1998). FB1 was found in 50% of maize samples collected between 1988 and 1991 from the midwestern United States (Murphy et al., 1993). Up to 10% of these samples had toxin concentrations between 10 and 50 ppm. Similarly, another survey of fumonisins in maize gluten and other maize products in the United Kingdom found these mycotoxins in almost every sample at concentrations of up to 32 ppm (Scudamore et al., 1998). Fumonisin B1 causes a variety of species-specific toxicological effects in domestic and laboratory animals. It induces leukoencephalomalacia in horses, pulmonary edema in pigs, nephrotoxicity in rats, rabbits and lambs as well as hepatotoxicity in all species examined (reviewed in Bolger et al., 2001). This toxin has also been reported to be a carcinogen in rodents and a contributing factor in human esophageal cancers (Howard et al., 2001; Rheeder et al., 1992). The inhibition of ceramide synthase was shown to be the primary biochemical effect of fumonisin. As a result of this inhibition, sphingoid bases and sphingoid base metabolites accumulate leading to the depletion of more complex sphingolipids (Merrill et al., 1996). In pigs, Pasteurella multocida type A is the most frequent secondary pathogen, which can generate a respiratory disorder, called swine pneumonic pasteurellosis (Chung et al., 1994). Unless some predisposing damage has occurred, P. multocida is considered incapable of invading the lung (Ciprian et al., 1994).

35

Primary infections with bacteria such as Mycoplasma hyopneumoniae, Bordetella bronchiseptica, or viruses (pseudorabies virus, porcine reproductive and respiratory syndrome virus) have been shown to predispose pigs to P. multocida pneumonia (Amass et al., 1994; Carvalho et al., 1997; Brockmeier et al., 2001). Cytotoxin from Actinobacillus pleuropneumoniae, lipopolysaccharide from gram negative bacteria or ammonia are also known to promote P. multocidainduced pneumonia (Chung et al., 1994; Hamilton et al., 1999; Halloy et al., 2004a). Ingestion of high doses of FB1 induces pulmonary edema in pigs (reviewed in Haschek et al., 2001). However, only few data are available on the effect of ingestion of low doses of this toxin on the development of pulmonary inflammatory processes (Smith et al., 1996; Zomborszky-Kovacs et al., 2002). The objective of the present paper was to determine if ingestion of fumonisin culture material could predispose conventional piglets to the development of pneumonia induced by P. multocida type A.

2. Materials and methods 2.1. Animals Twenty conventional piglets (9.6 ± 2.1 kg) were used in this study. They were acquired locally from a herd not infected by mycoplasma and vaccinated against PRRS virus. Animals acclimatized for seven days in a room of the animal facility of the University Liege with minimal air pollution. They were weighted at days 0, 7, 13 and 20, the last day of the experiment. Animals were fed with pellets and had access to water ad libitum. The experimental protocol was approved by the Ethics Committee of the University of Liege. 2.2. Toxin and bacterial isolate Fumonisin was administrated as a soluble crude extract obtained after in vitro culture of the Fusarium verticilloides strain NRRL 34281 as already described (Oswald et al., 2003; Tran et al., 2003). Briefly, sterilized maize inoculated with the fungal strain was incubated for 4 weeks at 25 ◦ C. The culture was extracted with acetonitrile-water, filtered, and concentrated. The

36

D.J. Halloy et al. / Toxicology 213 (2005) 34–44

purity of the crude extract was of 54% FB1 , 8% FB2 and 9% FB3 (Tran et al., 2003). We verified that it did not produce detectable amounts of fusariotoxins: zearalenone, deoxynivalenol, fusarochromanone, and trichothecenes. A field isolate of non-toxigenic P. multocida type A from a pig with bronchopneumonia was used (Halloy et al., 2004a). 2.3. Experimental design Piglets were randomly assigned to four groups. From day 0 to 6, toxin-treated pigs were given by gavage 0.5 mg/kg (body weight)/day of FB1 containing culture extracts under a volume of approximately 4 mL. Based on average feed consumption from piglets of this age, the dose used corresponds to feed contaminated with 5–8 ppm of FB1 . Control piglets received 4 mL of sterile water. One day after the end of the toxin treatment (day 7), piglets were anaesthetized with a combination of xylazine (Rompun® Bayer, Bruxelles, Belgium, 2 mg/kg im), ketamin (Imalgene® 1000, M´erial, Brussels, Belgium, 10 mg/kg im), and thiopental (Pentothal® , Abott, Louvain-LaNeuve, Belgium, 10 mg/kg iv). Within each group, half of the pigs were intra-tracheally injected with 5 mL of an overnight culture of P. multocida (>2.109 colony forming unit/mL) through a needle introduced in the middle extra-thoracic segment of the trachea. Control non-infected animals received 5 mL of sterile growth medium. Body temperatures were recorded daily throughout the experiment. Every day, the cough frequency was individually counted between 08:30 and 09:00 h am by one trained person observing the animals identified by a number on their back. The cumulative cough count (CCC) was calculated by adding the daily cough counts from the beginning of the experiment up to the end of the protocol with the aim of quantifying a clinical parameter integrating the evolution of the pathological process during the course of protocol (Halloy et al., 2004a). On the last day of the experiment (day 20), pigs were euthanized with an intravenous overdose of thiopental. Following exsanguination, a complete necropsy was performed, and lung samples were collected in order to measure the volume of the lung lesions and to perform bacteriology, histopathology and cytokine analyses.

2.4. Pathology The extent of macroscopic lesions was determined as already described (Halloy et al., 2004a). Briefly, the lung volume was determined by water displacement and the proportion of injured lung tissue was assessed visually on lungs transversely cut into slices. After examining macroscopic lung lesions, specimens were collected and fixed in 4% formaldehyde. The fixed samples were embedded in paraffin, sectioned, stained with hematoxylin and eosin and observed under a light microscope. 2.5. Bronchoalveolar lavage (BAL) and lung cell analysis At the end of the experiment, BAL were performed on anesthetized animals by bronchoscopy. Twenty mL of sterile phosphate-buffered saline were injected in the right principal bronchi and the first 5 mL of BAL fluid (BALF) recovered were analyzed for total cell number and differential cell count as already described (Halloy et al., 2004a). 2.6. Quantification of cytokine by RT-PCR Lung samples of the right cardiac lobe, collected after euthanasia, were maintained in Trizol (Life Technologies, Eragny, France) at −80 ◦ C before being homogenized using a Cat homogenizer. Total RNA was extracted, resuspended in EDTA-DEPC ultrapure water and quantified spectrophotometrically. A reverse transcriptase PCR (RT-PCR) procedure was performed to determine relative quantities of mRNA encoding for cytokines and cyclophilin as already described (Dozois et al., 1997; Fournout et al., 2000; Darwich et al., 2003). Briefly, 1.5 ␮g of RNA was reverse transcribed (transcriptase Point Mutant; Promega, Charbonni`eres, France) and then amplified (Invitrogene, Cergy Pontoise, France). Primer sequences for IFN␥, TNF-␣, IL-8, IL-18, and cyclophilin have already been described (Darwich et al., 2003; Dozois et al., 1997; Fournout et al., 2000). PCR products were analyzed by electrophoresis. Agarose gels were stained with ethidium bromide and the intensity of each PCR band was quantified by densitometry using the Quantity One program (Bio-Rad, Hercules, USA). Band intensities for cytokine mRNA were

D.J. Halloy et al. / Toxicology 213 (2005) 34–44

normalized to constitutively express “house-keeping” gene, cyclophilin. 2.7. P. multocida isolation and identification At necropsy, samples of pneumonic lungs were collected from caudal and cranial lobes, homogenized in sterile saline and plated as already described (Halloy et al., 2004a). Colonies were characterized by using standard laboratory procedures and were identified using API 20 NE kit (Bio-Merieux, Marcy-l’Etoile, France). Same bacterial analyzes were performed on BALF samples.

37

tribution, non parametric tests were used to analyse these results. For paired CCC data, Kruskal–Wallis analysis of variance and Mann–Whitney U-test were performed. The non paired CCC data were analysed with Friedman analysis of variance and compared with the Wilcoxon test. All data were expressed as mean ± standard deviation (S.D.) while the p-value defining significance was set at 0.05.

3. Results 3.1. Effect of FB1 and/or P. multocida on clinical signs and performance

2.8. Statistical analysis Data within and between groups were compared by one-way or two-way analysis of variance (ANOVA). When the F-test was significant (p < 0.05), further differences between means were determined by the least square difference (LSD) Fisher procedure. As data for cumulative cough count (CCC) had a non-normal dis-

We first examined the effects of fumonisin culture material and/or P. multocida on daily weight gain and clinical signs. The daily weight gain was not different in control piglets and in animals receiving either mycotoxin extract or P. multocida. By contrast, piglets fed with fumonisin culture material and inoculated with P. multocida presented a lower weight gain

Fig. 1. Change in mean daily weight gain measured during the course of the experiment. Values are expressed as mean ± S.D. (n = 5). Means without a common letter differ (p < 0.05).

38

D.J. Halloy et al. / Toxicology 213 (2005) 34–44

compared to the other groups, and their daily weight gain remained constant throughout the experiment (Fig. 1). No significant hyperthermia was recorded in any group. The rectal temperature of the piglets ranged from 39.4 ± 0.18 to 39.9 ± 0.09 ◦ C in control animals; from 39.5 ± 0.07 to 39.8 ± 0.07 ◦ C in FB1 treated piglets; from 39.1 ± 0.18 to 39.5 ± 0.31 ◦ C in P. multocida treated animals and from 39.2 ± 0.14

to 39.7 ± 0.19 ◦ C in animal receiving the mycotoxin extract and the bacterial infection. Control piglets, as well as animals receiving only fumonisin culture material or only instilled with P. multocida, did not present any clinical sign throughout the study. By contrast, piglets treated with mycotoxin extract and inoculated with P. multocida were afflicted with coughing. As illustrated in Fig. 2, a significant difference between cumulative cough count

Fig. 2. Change in mean cumulative cough count (CCC) measured during the course of the experiment. Values are expressed as mean ± S.D. (n = 5). Values measured from day 9 to 20 in piglets treated with fumonisin culture material and inoculated with P. multocida are significantly (p < 0.05) different from that measured at day 0 in the same group and from those measured at the same moment in the other animal group.

D.J. Halloy et al. / Toxicology 213 (2005) 34–44

(CCC) measured from day 9 to 20 was observed in animals receiving both mycotoxin extract and P. multocida when compared to the values obtained in the other groups. 3.2. Effect of FB1 and/or P. multocida on the lung At autopsy, we first determined the extent of macroscopic lesions such as congestion and red or grey hepatisation in the different groups of animals. As classically observed in conventional piglets, low volume macroscopic lung lesions were detected in all

39

groups and were preferentially located in caudal lung lobes (Hall et al., 1990; Maes et al., 2001). The size of these lesions varied from 10.0 ± 8.5 cm3 in control piglets to 15.8 ± 4.8 cm3 in animals treated with FB1 and 11.4 ± 6.2 cm3 in those infected with P. multocida. Lung damages were significantly more extended in animals treated with both mycotoxin extract and P. multocida. The mean size of lesion measured in four animals was 23.6 ± 2.2 cm3 while the last animal of this group showed larger (195 cm3 ) and more severe lesions. Histology was also performed on lung samples fixed in formaldehyde. In control animals, classical

Fig. 3. Lung parenchyma of a control animal (A), an animal treated with fumonisin culture material (B), an animal inoculated with P. multocida (C), an animal treated with Fumonisin B1 and inoculated with P. multocida (D). (A) Free of inflammatory cells in alveolar wall and lumina. Respiratory bronchiole with smooth muscle lined by flattened epithelial cells and a normal arteriole. (B) Thickening of the alveolar septa by a moderate macrophage infiltrate and a slight lymphocytic inflammatory infiltrate. (C) Minimal inflammatory infiltrate consisting of macrophages and lymphocytes observed in the alveolar wall. (D) An exudate consisting of macrophages and lymphocytes was present in the alveoli. Alveolar wall congestion and inflammatory infiltrate were also seen (×248).

40

D.J. Halloy et al. / Toxicology 213 (2005) 34–44

Table 1 BAL cell composition and cytokine mRNA levels in the lung of piglets Animal treatment Control

Fumonisin culture material

Broncho-alveolar lavage cell compositiona Total cellsb 1.50 ± 0.23 a Macrophages 1.27 ± 0.24 a Neutrophils 0.16 ± 0.04 ab Lymphocytes 0.08 ± 0.03 a Cytokinesc TNF-␣b IFN-␥ IL-8 IL-18

94.5 82.4 87.7 89.6

± ± ± ±

9.8 a 11.0 a 6.6 a 5.5 a

P. multocida

1.29 0.99 0.21 0.03

± ± ± ±

0.52 a 0.38 a 0.11 b 0.03 a

0.86 0.78 0.06 0.09

± ± ± ±

0.44 a 0.37 a 0.05 a 0.03 a

84.8 210.7 241.6 235.1

± ± ± ±

5.7 a 35.3 b 49.9 b 53.4 b

161.3 191.5 161.4 151.0

± ± ± ±

24.0 b 49.7 ab 18.0 ab 16.1 ab

Fumonisin culture material + P. multocida 3.49 2.77 0.14 0.58

± ± ± ±

1.31 b 1.05 b 0.06 b 0.23 b

149. 3 235.7 177.4 196.6

± ± ± ±

9.62 b 29.1 b 31.9 ab 25.1 b

a

Results are expressed as 106 cells/mL of BALF. Data represent the mean ± S.D. from a group of 5 piglets, means in the same line without a common letter differ (p < 0.05). c Total RNA was isolated from lung tissues and assayed for expression of cytokine and cyclophilin genes by RT-PCR. Cytokine mRNA is normalized to the housekeeping gene. b

chronic lesions were observed. The alveolar spaces were well expanded and the bronchiolar lumina were free of inflammatory cells (Fig. 3A). Sometimes, the alveolar walls were thickened by the presence of a slight lymphocytic and a moderate macrophage infiltrate. Lungs from mycotoxin-treated piglets showed a minimal enlargement of the alveolar septa due to an increase in the macrophage and lymphocyte number. There was no inflammation of the bronchial wall (Fig. 3B). Similar histological profiles were seen in P. multocida-infected animals although a subtle lymphocytic infiltrate was also seen in the bronchiolar and bronchial walls (Fig. 3C). In animals treated with both mycotoxin extract and P. multocida, the lesions were located in the cranial as well as in the caudal lobes. An exudate consisting of macrophages and lymphocytes was also present in the alveoli. The alveolar walls and bronchial parenchyma were thickened by the presence of a moderate inflammatory infiltrate (Fig. 3D). At the autopsy, important changes were also recorded in the broncho-alveolar lavage of piglets fed with FB1 and inoculated with P. multocida. A significant increase in the number of total cells as well as a specific increase in macrophages and lymphocytes was observed in this group of animal (Table 1). Nevertheless, piglets receiving only fumonisin containing culture extracts presented a slight but significant neutrophilia compared to the piglets from the other groups.

Lung tissues sampled at the autopsy were also analyzed for cytokine mRNA expression using semiquantitive RT-PCR. Indeed FB1 has been shown to induce the expression of various inflammatory cytokines (Bhandari et al., 2002). As shown in Table 1, exposure to culture material extract containing fumonisin significantly increased the expression of IL-8, IL18 and IFN-␥ mRNA. Infection with P. multocida significantly increased only the expression of TNF␣. When piglets were fed with fumonisin extract and infected with P. multocida, they presented an increased expression of TNF-␣, IFN-␥ and IL-18 mRNA. However, these increases were not higher than that observed in animals treated with either mycotoxin culture material or bacteria alone. Bacteriological analyses were performed on homogenized lung samples and BALF collected in all groups. All were negative for P. multocida and no other living pathogen was isolated in any group.

4. Discussion Several bacteria such as Mycoplasma hyopneumoniae, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae or viruses (pseudorabies virus, porcine reproductive and respiratory syndrome virus) have been shown to predispose pigs to P. multocida, induced

D.J. Halloy et al. / Toxicology 213 (2005) 34–44

pneumonia (Dugal et al., 1992; Chung et al., 1994; Ciprian et al., 1994; Brockmeier et al., 2001). Similarly, aerial pollutants such as gaseous ammonia could be involved in the development of P. multocida, induced atrophic rhinitis in swine (Hamilton et al., 1996). In the present study, conventional piglets bred in field conditions were used to investigate the impact of fumonisin, a feed contaminant. We demonstrated that this mycotoxin is also a predisposing agent to the development of lung pneumonia caused by P. multocida. In swine, fumonisin toxicosis is characterized by injury to the pulmonary, hepatic, cardiovascular, and immune systems (Haschek et al., 2001). An early and widespread alteration of the sphingolipid metabolism has also been demonstrated with consequences on growth rate and carcass composition. Pigs receiving FB1 –contaminated feed at concentrations above 92 ppm or the toxin at concentration above 16 mg/kg body weight develop lethal pulmonary edema within 4–7 days (reviewed in Haschek et al., 2001). Pulmonary oedema were also observed by pathologic examination and/or computer tomography in weaning pigs receiving 10–40 ppm of FB1 for an exposure of 4 weeks (Zomborszky-Kovacs et al., 2002; Dilkin et al., 2003). Exposition of the pigs to concentrations of toxin below 10 ppm did not induce clinical signs and significant performance impairment (Zomborszky-Kovacs et al., 2002). Lower concentrations of FB1 than the ones that induce pathology may be present in animal feed (Murphy et al., 1993; Shephard et al., 1996; Scudamore et al., 1998). In the present study, piglets were fed for 7 days, with 0.5 mg of FB1 containing culture extracts/kg of body weight/day corresponding to a feed contaminated with 5–8 ppm of toxin in the feed. At this concentration, we did not observe any inflammation of the bronchial wall and only noticed a minimal enlargement of the alveolar septa due to the higher presence of lymphocytes and macrophages (Fig. 3). At the autopsy, i.e. 13 days after the end of the mycotoxin exposure, the piglets also showed an increase in the number of neutrophils in BALF and an elevated expression of mRNA encoding for three pro-inflammatory cytokines (IL-8, IL-18 and IFN-␥). The neutrophilia observed in BALF is consistent with an increased expression of IL8 mRNA, as this cytokine is known to be a a chemotactic factor that recruits neutrophils to the inflammatory site (Baggiolini et al., 1994; Lin et al., 1994).

41

An increased inflammatory cytokine expression has been noticed in the lung of FB1 -treated piglets has already been observed in the liver and the lung of mice subcutaneously treated with FB1 (Bhandari et al., 2002; He et al., 2002). Similarly, mice and pig feed with high doses of FB1 demonstrated an elevated production of NO by splenic macrophages and TNF-like activity in their serum, suggestive of an inflammatory process (Guzman et al., 1997; Dresden et al., 2002). A protective role of this cytokine in the FB1 - induced toxicity has been described in mice carrying the human TNF-␣ transgene (Sharma et al., 2000). However in mice lacking either one or both receptor for TNF-␣ an increased heptotoxicity was observed in FB1 treated mice (Sharma et al., 2002) suggesting a more complex implication of this cytokine in FB1 induced pathology. Results obtained in vitro, also indicate an apoptosis of the targeted cells upon FB1 treatment (DombrinkKurtzman, 2003). Likewise, an apototic effect of FB1 has been described in porcine alveolar macrophages treated in vitro with the toxin (Liu et al., 2002). One of the main findings of the present study is that ingestion of fumonisin culture material predisposes the piglets to the pathology induced by P. multocida. Indeed, the sequential treatment of the animals with the toxin extract and the bacteria induced several changes that were not observed in control animals or in the ones receiving only fumonisin containing culture extracts or P. multocida. It reduced the growth rate of the animals (Fig. 2), induced coughing (Fig. 2) as well as extending lesions of sub-acute interstitial pneumonia (Fig. 3). Treatment with the combination of fumonisin culture extract and P. multocida also significantly increased the total number of cells present in the BALF as well as the number of macrophages and lymphocytes (Table 1). The clinical signs observed in the animals treated with mycotoxin and then infected with P. multocida were characteristic of a developing pneumonia (Hall et al., 1990). The mechanism by which FB1 predisposes pigs to lung pathology induced by P. multocida is not known; however it may be related with the ability of FB1 to inhibit pulmonary intravascular macrophages from removing particulate matter and bacteria from the circulation (Smith et al., 1996). Alternatively, the unique susceptibility of porcine pulmonary capillary endothelium to the toxin (Gumprecht et al., 2001) may lead to pulmonary damages and initiate a lung inflammatory process that will be increased

42

D.J. Halloy et al. / Toxicology 213 (2005) 34–44

by the bacterial infection. Finally, the alteration of sphingolipid metabolism by FB1 (Merrill et al., 1996) which leads to an increased concentration of sphinganine 1-phosphate may promote the inflammatory process as already suggested in asthma (Jolly et al., 2002; Cinque et al., 2003). In pigs submitted to the combined effect of fumonisin culture material and P. multocida, no bacterial colonization of the lung was observed. This is not surprising since, as already mentioned, respiratory epithelial alterations are needed to allow sustained P. multocida colonization and to induce a clinical pneumonic pasteurellosis (Dugal et al., 1992; Chung et al., 1994; Ciprian et al., 1994; Brockmeier et al., 2001). However, it has been previously demonstrated that a transient colonization by P. multocida, not detected at the end of the trial, was sufficient to induce a marked pulmonary inflammation in piglets sensitized with a low dose of E. coli endotoxins (Halloy et al., 2004a, b). Since conventional piglets were used, intervention of other pathogens can not be excluded. However, no other bacteria was detected in the lungs at the end of the trial including common pathogens known to predispose to P. multocida (Amass et al., 1994; Carvalho et al., 1997; Brockmeier et al., 2001). Indeed, the serological test performed indicated that M. hyopneumoniae was not involved in the inflammatory response and bacterial cultures allowed us to exclude other common pathogens such as B. bronchiseptica and A. pleuropneumoniae. Finally, viral implication was minimized by using piglets vaccinated against PRRSV. Several studies have indicated that mycotoxins could increase the susceptibility of animals to infectious diseases and decrease vaccinal efficacy (Tai and Pestka, 1988; Taranu et al., 2005; Marin et al., 2002; Bouhet et al., 2004). As far as FB1 is concerned, ingestion of this toxin increases bacterial colonization of the intestinal tract by a pathogenic strain of Escherichia coli (Oswald et al., 2003) and decreases clearance of Pseudomonas aeruginosa after intravenous infections (Smith et al., 1996). However, a recent paper (Dresden et al., 2002) also indicates that diets contaminated with 50 or 150 ppm of FB1 enhanced the resistance of mice to parasitic infection. In conclusion, we found that a low oral dose of fumonisin containing culture material may predispose piglets to the development of lung pneumonia induced by P. multocida. Animals treated with fumon-

isin extract and P. multocida, showed delay of growth, cough and a lung inflammatory process. Considering that high concentrations of FB1 may sometime be present in animal feeds and human food preparations (Murphy et al., 1993; Shephard et al., 1996; Scudamore et al., 1998), further studies are needed to identify the mechanism(s) by which this mycotoxin acts on the respiratory tract to synergize with the effects of bacteria.

Acknowledgements The technical assistance of F. Delvaux, D. Beerens and A. Bru is gratefully appreciated. Thanks are also due to Dr. Neil Ledger for his help with the English text. This work was supported by the Ministry of Agriculture (DG6), Brussels, Belgium, part by the Fonds de la Recherche (University of Li`ege), the R´egion Midi-Pyr´en´ees (DAER-Rech/99008345) France, and by the Transversalit´e INRA (Mycotoxines-P00263) Paris, France. Sandrine Bouhet was supported by a Fel´ lowship from the Minist`ere de l’Education Nationale, de la Recherche et de la Technologie, Paris, France.

References Amass, S.F., Clark, L.K., van Alstine, W.G., Bowersock, T.L., Murphy, D.A., Knox, K.E., Albregts, S.R., 1994. Interaction of Mycoplasma hyopneumoniae and Pasteurella multocida infections in swine. J. Am. Vet. Med. Assoc. 204, 102–107. Baggiolini, M., Dewald, B., Moser, B., 1994. Interleukin-8 and related chemotactic cytokines-CXC and CC chemokines. Adv. Immunol. 55, 97–179. Bezuidenhout, S.C., Gelderblom, W.C.A., Gorst-Alleman, C.P., Horak, R.M., Marasas, W.F.O., Spiteller, G., Vleggaar, R., 1988. Structure elucidation of the fumonisin, mycotoxins from Fusarium moniliforme. J. Chem. Soc. Chem. Commun., 743–745. Bhandari, N., Brown, C.C., Sharma, R.P., 2002. Fumonisin B1induced localized activation of cytokine network in mouse liver. Food Chem. Toxicol. 40, 1483–1491. Bolger, M., Coker, R.D., Dinovi, M., Gaylor, D., Gelderblom, M.O., Paster, N., Riley, R.T., Shephard, G., Speijers, J.A., 2001. Safety evaluation of certain mycotoxins in food. In: Food and Agriculture Organization of the United Nations, paper 74. World Health Organization Food Additives, pp. 103–279. Bouhet, S., Hourcade, E., Loiseau, N., Fikri, A., Martinez, S., Roselli, M., Galtier, P., Mengheri, E., Oswald, I.P., 2004. The mycotoxin, fumonisin B1 alters the proliferation and the barrier function of porcine intestinal epithelial cells. Toxicol. Sci. 77, 165–171. Brockmeier, S.L., Palmer, M.V., Bolin, S.R., Rimler, R.B., 2001. Effects of intranasal inoculation with Bordetella bronchisep-

D.J. Halloy et al. / Toxicology 213 (2005) 34–44 tica, porcine reproductive and respiratory syndrome virus, or a combination of both organisms on subsequent infection with Pasteurella multocida in pigs. Am. J. Vet. Res. 62, 521–525. Carvalho, L.F., Segales, J., Pijoan, C., 1997. Effect of porcine reproductive and respiratory syndrome virus on subsequent Pasteurella multocida challenge in pigs. Vet. Microbiol. 55, 241–246. Chung, W.B., Backstrom, L.R., Collins, M.T., 1994. Experimental model of swine pneumonic pasteurellosis using crude Actinobacillus pleuropneumoniae cytotoxin and Pasteurella multocida given endobronchially. Can J. Vet. Res. 58, 25–30. Cinque, B., Di Marzio, L., Centi, C., Di Rocco, C., Riccardi, C., Grazia, Cifone, M., 2003. Sphingolipids and the immune system. Pharmacol. Res. 47, 421–437. Ciprian, A., de la Cruz, T.A., Garza, M., 1994. Mycoplasma hyopneumoniae: interaction with other agents in pigs, and evaluation of immunogens. Arch. Med. Res. 25, 235–239. Darwich, L., Pie, S., Rovira, A., Segales, J., Domingo, M., Oswald, I.P., Mateu, E., 2003. Cytokine mRNA expression profiles in lymphoid tissues of pigs naturally affected by postweaning multisystemic wasting syndrome. J. Gen. Virol. 84, 2117–2125. Dilkin, P., Zorzete, P., Mallmann, C.A., Gomes, J.D., Utiyama, C.E., Oetting, L.L., Correa, B., 2003. Toxicological effects of chronic low doses of aflatoxin B1 and fumonisin B1-containing Fusarium moniliforme culture material in weaned piglets. Food Chem. Toxicol. 41, 1345–1353. Dombrink-Kurtzman, M.A., 2003. Fumonisin and beauvericin induce apoptosis in turkey peripheral blood lymphocytes. Mycopathologia 156, 357–364. Dozois, C.M., Oswald, E., Gautier, N., Serthelon, J.P., Fairbrother, J.M., Oswald, I.P., 1997. A reverse transcription-polymerase chain reaction method to analyze porcine cytokine gene expression. Vet. Immunol. Immunopathol. 58, 287–300. Dresden, O.C., Pittman, Noblet, G., Enongene, E.N., Bacon, C.W., Riley, R.T., Voss, K.A., 2002. Host resistance to Trypanosoma cruzi infection is enhanced in mice fed Fusarium verticillioides (=F. moniliforme) culture material containing fumonisins. Food Chem. Toxicol. 40, 1789–1798. Dugal, F., Belanger, M., Jacques, M., 1992. Enhanced adherence of Pasteurella multocida to porcine tracheal rings preinfected with Bordetella bronchiseptica. Can. J. Vet. Res. 56, 260–264. Finks-Gremmels, J., 1999. Mycotoxins: their implications for human and animal health. Vet. Q. 21, 115–120. Fournout, S., Dozois, C.M., Odin, M., Desautels, C., Peres, S., Herault, F., Daigle, F., Segafredo, C., Laffitte, J., Oswald, E., Fairbrother, J.M., Oswald, I.P., 2000. Lack of a role of cytotoxic necrotizing factor 1 toxin from Escherichia coli in bacterial pathogenicity and host cytokine response in infected germfree piglets. Infect. Immun. 68, 839–847. Gumprecht, L.A., Smith, G.W., Constable, P.C., Haschek, W.M., 2001. Species and organ specificity of fumonisin-induced endothelial alterations: potential role in porcine pulmonary edema. Toxicology 160, 71–79. Guzman, R.E., Bailey, K., Casteel, S.W., Turk, J., Rottinghaus, G., 1997. Dietary Fusarium moniliforme culture material induces in vitro tumor necrosis factor-alpha like activity in the sera of swine. Immunopharmacol. Immunotoxicol. 19, 279–289.

43

Hall, W.F., Bane, D.P., Kilroy, C.R., Essex-Sorlie, D.L., 1990. A model for the induction of Pasteurella multocida type-A pneumonia in pigs. Can. J. Vet. Res. 54, 238–243. Halloy, D.J., Bouhet, S., Oswald, I.P., Goret-Nicaise, M., Kobisch, M., Mainil, J., Gustin, P.G., 2004a. Pathophysiological changes occurring during Escherichia coli endotoxin and Pasteurella multocida challenge in piglets: relationship with cough and temperature and predictive value for intensity of lesions. Vet. Res. 35, 309–324. Halloy, D.J., Kirschvink, N.A., Vincke, G.L., Hamoir, J.N., Delvaux, F.H., Gustin, P.G., 2004b. Whole body barometric plethysmography: a screening method to investigate airway reactivity and acute lung injuries in freely moving pigs. Vet. J. 168, 276– 284. Hamilton, T.D., Roe, J.M., Hayes, C.M., Jones, P., Pearson, G.R., Webster, A.J., 1999. Contributory and exacerbating roles of gaseous ammonia and organic dust in the etiology of atrophic rhinitis. Clin. Diagn. Lab. Immunol. 6, 199–203. Hamilton, T.D., Roe, J.M., Webster, A.J., 1996. Synergistic role of gaseous ammonia in etiology of Pasteurella multocida-induced atrophic rhinitis in swine. J. Clin. Microbiol. 34, 2185–2190. Haschek, W.M., Gumprecht, L.A., Smith, G., Tumbleson, M.E., Constable, P.D., 2001. Fumonisin toxicosis in swine: an overview of porcine pulmonary edema and current perspectives. Environ. Health Perspect. 109 (Suppl. 2), 251–257. He, Q., Bhandari, N., Sharma, R.P., 2002. Fumonisin B1 alters sphingolipid metabolism and tumor necrosis factor alpha expression in heart and lung of mice. Life Sci. 71, 2015–2023. Howard, P.C., Eppley, R.M., Stack, M.E., Warbritton, A., Voss, K.A., Lorentzen, R.J., Kovach, R.M., Bucci, T.J., 2001. Fumonisin B1 carcinogenicity in a two-year feeding study using F344 rats and B6C3F1 mice. Environ. Health Perspect. 109 (Suppl. 2), 277–282. Jolly, P.S., Rosenfeldt, H.M., Milstien, S., Spiegel, S., 2002. The roles of sphingosine-1-phosphate in asthma. Mol. Immunol. 38, 1239–1245. Lin, G., Pearson, A.E., Scamurra, R.W., Zhou, Y., Baarsch, M.J., Weiss, D.J., Murtaugh, M.P., 1994. Regulation of interleukin8 expression in porcine alveolar macrophages by bacterial lipopolysaccharide. J. Biol. Chem. 269, 77–85. Liu, B.H., Yu, F.Y., Chan, M.H., Yang, Y.L., 2002. The effects of mycotoxins, fumonisin B1 and aflatoxin B1, on primary swine alveolar macrophages. Toxicol. Appl. Pharmacol. 180, 197– 204. Maes, D.G., Deluyker, H., Verdonck, M., Castryck, F., Miry, C., Vrijens, B., Ducatelle, R., De Kruif, A., 2001. Non-infections factors associated with macroscopic and microscopic lung lesions in slaughter pigs from farrow-to-finish herds. Vet. Rec. 148, 41–46. Marin, D.E., Taranu, I., Bunaciu, R.P., Pascale, F., Tudor, D.S., Avram, N., Sarca, M., Cureu, I., Criste, R.D., Suta, V., Oswald, I.P., 2002. Changes in performance, blood parameters, humoral and cellular immune responses in weanling piglets exposed to low doses of aflatoxin. J. Anim. Sci. 80, 1250–1257. Merrill, A.H., Wang, E., Vales, T.R., Smith, E.R., Schroeder, J.J., Menaldino, D.S., Alexander, C., Crane, H.M., Xia, J., Liotta, D.C., Meredith, F.I., Riley, R.T., 1996. Fumonisin toxicity and sphingolipid biosynthesis. Adv. Exp. Med. Biol. 392, 297–306.

44

D.J. Halloy et al. / Toxicology 213 (2005) 34–44

Murphy, P.A., Rice, L.G., Ross, P.F., 1993. Fumonisin B1, B2 and B3 content of Iowa, Wisconsin and Illinois corn and corn screening. J. Agric. Food Chem. 41, 263–266. Oswald, I.P., Desautels, C., Laffitte, J., Fournout, S., Peres, S.Y., Odin, M., Le Bars, P., Le Bars, J., Fairbrother, J.M., 2003. Mycotoxin fumonisin B1 increases intestinal colonization by pathogenic Escherichia coli in pigs. Appl. Environ. Microbiol. 69, 5870–5874. Oswald, I.P., Marin, D.E., Bouhet, S., Pinton, P., Taranu, I., Accensi, F., 2005. Immunotoxicological risk of mycotoxin for domestic animals in Europe. Food Add. Cont. 22, 354– 360. Rheeder, J.P., Marasas, W.F.O., Thiel, P.G., Sydenham, E.W., Shepard, G.S., van, Schalkwyk, D.J., 1992. Fusarium moniliforme and fumonisins in corn in relation to human esophagal cancer in Transkei. Phytopathology 82, 353–357. Scudamore, K.A., Nawaz, S., Hetmanski, M.T., 1998. Mycotoxins in ingredients of animal feeding stuffs. II. Determination of mycotoxins in maize and maize products. Food Addit. Contam. 15, 30–55. Sharma, R.P., Bhandari, N., Tsunoda, M., Riley, R.T., Voss, K.A., 2000. Fumonisin hepathotoxicity is reduced in mice carrying the human tumor necrosis factor a transgene. Arch. Toxicol. 74, 238–248.

Sharma, R.P., He, Q., Meridith, F.I., Riley, R.T., Voss, K.A., 2002. Paradoxicoal role of tumor necrosis factor a in fumonisin induced hepathotoxicity in mice. Toxicology 180, 221–232. Shephard, G.S., Thiel, P.G., Stockenstrom, S., Sydenham, E.W., 1996. Worldwide survey of fumonisin contamination of corn and corn-based products. J. AOAC Int. 79, 671–687. Smith, G.W., Constable, P.D., Smith, A.R., Bacon, C.W., Meredith, F.I., Wollenberg, G.K., Haschek, W.M., 1996. Effects of fumonisin-containing culture material on pulmonary clearance in swine. Am. J. Vet. Res. 57, 1233–1248. Tai, J.H., Pestka, J.J., 1988. Impaired murine resistance to Salmonella typhimurium following oral exposure to the trichothecene T-2 toxin. Food Chem. Toxicol. 26, 691–698. Taranu, I., Marin, D.E., Bouhet, S., Pascale, F., Bailly, J.D., Miller, J.D., Pinton, P., Oswald, I.P., 2005. Mycotoxin, Fumonisin B1, alters the cytokine profile and decreases the vaccinal antibody titer in pigs. Toxicol. Sci. 84, 301–307. Tran, S.T., Bailly, J.D., Tardieu, D., Durand, S., Benard, G., Guerre, P., 2003. Sphinganine to sphingosine ratio and predictive biochemical markers of fumonisin B1 exposure in ducks. Chem Biol Interact. 146, 61–72. Zomborszky-Kovacs, M., Vetesi, F., Horn, P., Repa, I., Kovacs, F., 2002. Effects of prolonged exposure to low-dose fumonisin B1 in pigs. J. Vet. Med. B Infect. Dis. Vet. Public Health 49, 197–201.