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REVISTA BIO CIENCIAS

ISSN: 2007-3380

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Chronic exposure to aflatoxigenic fungi related to liver damage in pelt chinchillas (Chinchilla lanigera)

Exposición crónica a hongos productores de aflatoxinas relacionada a daños hepáticos en chinchillas (Chinchilla lanigera) destinadas a la producción de piel

Landa MF1, 2, González Pereyra ML1, 2, Pena G1, 2, Bagnis G1, 3 ,
Cavaglieri LR1, 2, Da Rocha Rosa CA4, 5, Dalcero AM1, 2*.

1Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas. Físico-Químicas y Naturales,
Universidad Nacional de Río Cuarto. Ruta N 36 Km 601. (5800) Río Cuarto, Córdoba, Argentina.
2Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).
3Facultad de Agronomía y Veterinaria, Universidad Nacional de Río Cuarto. Ruta N 36 Km 601. (5800)
Río Cuarto, Córdoba, Argentina.
4Departamento de Microbiologia e Imunología Veterinária. Universidade Federal Rural do Rio de Janeiro.
Instituto de Veterinária. Rodovia BR 465 Km 7. Seropédica 23890-000, Rio de Janeiro, Brazil.
5Conselho Nacional de Pesquisas Científicas (CNPq), Brazil.

Autor corresponsal:
Cavaglieri LR, Departamento de Microbiología e Inmunología, Universidad Nacional de Río Cuarto, Ruta Nº 36 Km 601. (5800) Río Cuarto,
Córdoba, Argentina. Telefono/Fax: 54-358-4676231. Correo Electrónico: lcavaglieri@arnet.com.ar ,  lcavaglieri@exa.unrc.edu.ar


Información del Artículo


Revista Bio Ciencias 2(2): 35-47

Recibido: 18 de julio de 2012
Aceptado: 07 de octubre de 2012


Palabras Claves / Key Words


Aflatoxins, Aspergillus spp., Chinchilla lanigera, feed, histopathology, toxicology / Aflatoxinas, Aspergillus spp., Chinchilla lanígera, histopatología, toxicología


Resumen


La piel de chinchilla es una de las más exóticas y apreciadas en el mercado internacional. La cría de estos animales es una actividad muy rentable. En Argentina, se han detectado casos de aflatoxicosis aguda en criaderos. Los objetivos de este trabajo fueron: i) estudiar la micobiota y los hongos productores de aflatoxina B1 (AFB1) presentes en alimento para chinchillas. ii) analizar la contaminación natural con AFB1 de estos alimentos iii) buscar lesiones histopatológicas en hígados de chinchillas de los criaderos. Se recolectaron muestras de diferentes alimentos (A: chinchilla piel, B: chinchilla madre, C: cubos de alfalfa) en una fábrica y un criadero localizados en la ciudad de Rio Cuarto, en la región central de Argentina. Los hígados de las chinchillas sacrificadas en el criadero fueron analizados macroscópica y microscópicamente. Los recuentos fúngicos totales fueron mayores a 1x104 UFC g-1. Aspergillus, Fusarium y Penicillium fueron los géneros prevalentes, mientras que A. flavus, A. fumigatus, F. verticillioides y F. proliferatum fueron las especies aisladas con mayor frecuencia. 50 % de las cepas de A. flavus aisladas de la fábrica y 69.7 % de las aisladas del criadero produjeron 2.78 a 8.64 µg g-1 y 0.66 a 58.8 µg g-1 de AFB1, respectivamente. Se detectó AFB1 sólo en las muestras del criadero, con mayor incidencia en el alimento C. Los niveles de toxina variaron entre 1.90 y 97.34 µg kg-1. Los niveles promedios en A y C fueron superiores a 20 µg kg-1. El análisis macroscópico de los hígados reveló apariencia, tamaño y color normal. El análisis microscópico indicó que 63.3 % de los hígados presentaron degeneración lipídica leve a moderada con vacuolización difusa del citoplasma, 9 % presentaron vacuolización lipídica intensa y 27.3 % degeneración hidrópica y vacuolización nuclear en los hepatocitos. El monitoreo periódico de La calidad de los alimentos e ingredientes usados en la alimentación de chinchillas puede evitar intoxicaciones agudas y pérdidas económicas causadas por la exposición crónica a aflatoxinas.


Abstract


Chinchilla pelt is a rare and expensive fur. Therefore, breeding these animals is a profitable activity. Confirmed acute cases of aflatoxin intoxication have been reported in Argentinean farms. The aims of this study were i) to evaluate mycobiota and AFB1-producing species in chinchilla feeds ii) to investigate their natural AFB1 contamination and iii) to analyze histopathological lesions in chinchilla livers. Feed samples (A: fur chinchillas, B: mother chinchillas, C: lucerne cubes) were collected from a factory and a farm. Livers of sacrificed chinchilla from the farm were macroscopically and microscopically examined. Total fungal counts of feed C exceeded 1x104 CFU g-1. Aspergillus, Fusarium and Penicillium were the prevalent genera, while A. flavus, A. fumigatus, F. verticillioides and F. proliferatum were the prevalent species. 50 % of A. flavus strains from factory samples and 69.7 % from farm samples produced 2.78 to 8. 64 µg g-1 and 0.66 to 58.8 µg g-1 AFB1, respectively. Aflatoxin B1 was detected only in feeds from the farm, finding the highest incidence in feed C. Toxin levels varied between 1.90 and 97.34 µg kg-1 AFB1. Mean levels in feed A and C exceeded 20 µg kg-1. Macroscopic examination of livers revealed normal appearance, size and color. However, histopathological examination indicated 63.3 % showed slight to moderate lipid degeneration with diffuse cytoplasm vacuolation, 9 % intense lipid cytoplasm vacuolation and 27.3 % hydropic degeneration and nuclear vacuolation in hepatocytes. A periodic monitoring of aflatoxins in feeds and their ingredients can prevent acute outbreaks and economic losses caused by chronic exposure.


Introducción


Fungal contamination of harvest products and animal feeds with fungi and mycotoxins is a worldwide concern. Mycotoxins have been detected in several commodities, ingredients and final products destined to animal feeding throughout the world (Whitlow and Hagler, 2002; Monbaliu et al., 2010).

Aflatoxins (AFs) are secondary metabolites produced by toxigenic strains of A. flavus and A. parasiticus, mainly. Chemically, AFs belong to the group of bifuran coumarins, being aflatoxins B1 (AFB1), the most toxic and hazardous one. Aflatoxin B1 is hepatotoxic, highly mutagenic, carcinogenic and probably teratogenic to animals (Smith and Moss, 1985). Aflatoxin B1  has been classified as a class 1 human carcinogen by the International Agency for Research on Cancer (IARC, 2002).

In all species, the liver is the primary target organ of acute injury caused by aflatoxins (AFs). The first step in the biotransformation of AFB1 takes place in the hepatocyte, with nonreversible detoxification via the formation of hydroxylated metabolites (AFM1, AFQ1, AFP1, AFB2a), followed either by reversible detoxification through aflatoxicol formation, or by activation through the generation of AFB1-8,9-epoxide (Neal, 1998). Acute aflatoxicosis is frequently associated with the ingestion of large doses of AFs, which cause typical hepatic changes, such as liver enlargement, color change, fat accumulation, and lipid vacuolation, which are confirmed by necropsy and histopathology (CAST, 2003; Newman et al., 2007).

Chinchillas (Chinchilla lanigera) are rabbit-sized crepuscular rodents native of the Andes Mountains in South America. At the present time, this species practically no longer exists in its natural habitat. Chinchillas are raised in farm for fur and pets.  Currently, chinchilla pelt along with Russian sable, are the most prestigious, rare and expensive furs.  The growing international demand for chinchilla fur makes the breeding of these animals a highly profitable activity in Argentina (González Pereyra et al., 2008a).

Chinchillas are known to be very sensitive to mycotoxins, and a large number of animals often die when acute aflatoxicosis occurs. Clinical signs that may indicate mycotoxicosis include low feed intake, diarrhea, weight loss, poor condition of the skin, fur discoloration, sudden death, and a predisposition to secondary infections (Labala, 2008). Confirmed cases of acute aflatoxin intoxication have been reported in Argentina (González Pereyra et al., 2008a). However, the mycobiota of chinchilla feeds and the identification of AFs producer species have not been reported yet. Since chinchillas are rare, expensive and very delicate animals, the mycotoxin content of their feed should be reduced to the minimum to avoid death, immunosuppression and fur loss. The use of quality feeds and feed ingredients is a key to minimize economic losses.

The aims of the present study were i) to evaluate the mycobiota and the presence of  AFB1 producer species in chinchilla feeds ii) to investigate the incidence of natural AFB1 contamination in these feeds and iii) to analyze histopathological lesions in chinchilla livers in search for typical changes associated to aflatoxin exposure.


Materials and Methods

Source of samples

A total of 77 chinchilla feed samples (5 kg each) were collected during 12 months (June 2009 to June 2010) from two different sources: a chinchilla farm and a feed factory both located in Rio Cuarto city, Córdoba Province, Argentina. Three samples of different chinchilla feeds were collected monthly: A) fur chinchillas, B) mother chinchillas and C) lucerne cubes. The latter was sampled only in the chinchilla farm, since it was not produced in the factory. The samples were homogenized and quartered to obtain a 1 kg laboratory sample. Water activity (aW) of the samples was measured using an AQUALAB CX2 (Decagon, Devices, Inc. USA) appliance. A 20 g aliquot from each was randomly selected for the analysis of the mycobiota and the rest was stored at 4 ºC until mycotoxin analysis.

Mycological survey

Total fungal counts were performed on two different culture media: dichloran rose bengal chloranphenicol agar (DRBC) for estimating total mycobiota (Abarca et al., 1994; ISO 21527-1) and dichloran 18 % glycerol agar (Pitt and Hocking, 1997; DG18) to favor xerophilic fungi development (ISO 21527-2). Quantitative enumeration was done using the plate count method. Twenty grams of each sample were homogenized in 180 mL 0.1 % peptone water solution for 30 min. Serial dilutions (10-2 to 10-4) were made and 0.1 mL aliquots were inoculated in triplicates on the solid media. After 7 days of incubation at 25 °C, plates containing 10–100 CFU were used for counting and the results were expressed as CFU per gram of sample (CFU g-1). On the last day of incubation, individual CFU g-1 counts for each colony type considered to be different were recorded. Colonies representative of each type were transferred to plates with malt extract agar (MEA). Fungal colonies were selected for identification, according to Pitt and Hocking (1997), Klich (2002) and Nelson et al., (1983), depending on the genus. The results were expressed as isolation frequency (percentage of samples in which each genus/species was present) and relative density (percentage of isolates of the same species among the total number of isolates of a certain genus).

Ability of Aspergillus section flavi to produce AFB1 in vitro

Ability of 20 Aspergillus section flavi strains to produce AFB1 in vitro was tested by thin layer chromatography (TLC) (Geisen, 1996). Strains were inoculated on MEA and incubated for 7 days at  25 ºC. Mycelium was transferred to previously weighted Eppendorff tubes. Mycelium weight was calculated by weight difference. Fifty µL of chloroform were added and the mixture was shaken for 20 min at 400 rpm. The mycelial mass was removed and the chloroform extract was evaporated under N2 flow. Five µL of each sample extract were spotted on silica gel TLC plates (Merk, Germany), 2 cm from the bottom edge. Different volumes of AFB1 standard solution were spotted in each plate along with the extracts. Plates were developed in chloroform:acetone (9:1 v/v) at room temperature. When the solvent front was 15 cm from the spot line, the plates were removed and dried to room temperature. Plates were examined under 365 nm UV light for the quantification of AFB1 through visual comparison with the standard solution of known concentration. Limit of detection (LOD) of this method was 5 µg kg-1.

Aflatoxin analysis of feed samples

Aflatoxin B1 analysis was performed by HPLC according to Trucksess et al., (1994). For each sample, 25 g of chinchilla feed were extracted with 100 mL acetonitrile:water (84:16, v/v). The mixture was shaken for 30 min in an orbital shaker and filtered through Whatman Nº4 filter paper (Whatman, Inc., Clifton, New Jersey, USA). An 8 mL aliquot was taken and placed into a 10 mL culture tube. An AflaPat Mycosep®228 clean-up column (Romer Labs Inc., Union, MO, USA) multifunctional column was used to obtain a purified extract (4 mL) that was collected and evaporated under N2  flow. Extracts were resuspended in 400 µL water:methanol:acetonitrile (4:1:1, v/v). Aliquots of 200 µL were derivatized with 700 µL trifluoroacetic acid:acetic acid:water (20:10:70, v/v). The derivatized extracts were analyzed by using an HPLC system. Chromatographic separations were performed on a C18 RP Phenomenex Luna (150 x 4.60 mm, 5 µ) column (Phenomenex, USA). A water: methanol: acetonitrile (4:1:1, v/v) solution was used as mobile phase at 1 mL min-1 flow rate. Fluorescence of aflatoxin derivatives was recorded at 360 nm excitation 460 nm emission wavelengths. Standard curves were constructed using AFB1 standard solutions of different concentration. The toxin was quantified by correlating peak heights of sample extracts with those of standard solutions in a calibration curve. The LOD of this method was 1 ng g-1.

Histopathological examination

          Fifteen chinchilla livers were obtained from the farm. All animals had been sacrificed for fur. Macroscopic characteristics of the organs were evaluated. These included general size (lateral width), weight, and color. Liver tissue for histological analysis was fixed in 10 % neutral buffered formalin and trimmed. They were processed routinely, embedded in paraffin, sectioned at 5-mm thickness, and stained with hematoxylin and eosin (H/E). An histopathological analysis was performed to evaluate hepatocellular characteristics and lesions such as cytoplasmic vacuolation, nodular hyperplasia, and bile-duct proliferation (CAST, 2003; Allameh et al., 2005; Miazzo et al., 2005).

Statistical analyses

Analysis of CFU g-1 was performed by Mixed and General Lineal Model. Fisher’s LSD test was done to compare means of treatments (Quinn and Keough, 2002). The analysis was conducted using PROC GLM in SAS (SAS Institute, Cary, NC, USA).


Results

Water activity values of feeds samples ranged from 0.402 to 0.613. Total fungal counts (CFU g-1) of chinchilla feeds varied from 1x102 to 1.4x106 (Table 1). Total fungal counts of lucerne cubes exceeded the limit established for good quality feeds and feed ingredients by the Good Manufacturing Practices (1x104 CFU g-1) (GMP, 2008). There was no significant difference between counts on DRBC and DG18 media, or between the different sources of samples (farm and factory). However, there was significant difference between the different kinds of feed (A, B and C) (Table 2 and 3).
The occurrence of Aspergillus, Fusarium and Penicillium genera was evaluated calculating isolation frequency. Aspergillus was the prevalent genera in feed A and feed B from the farm, while Fusarium was predominant in feed C. In feeds from the factory, the most frequent genera found was Fusarium followed by Penicillium and Aspergillus (Figure 1).

 

Table 1.
Fungal counts in chinchilla feeds.

Total fungal counts (UFC g-1)

Source of Samples

Kind of sample

DRBC

Contaminated samples exceeding recomended limits (%)*

DG18

Farm

feed A

1.0 x102- 1 x104

0

2.0 x102- 1.0 x104

feed B

1.0 x102- 3.6x103

0

1.0 x102-7.0 x103

feed C

1.0 x102- 1.4x106

77.7

1.0 x102- 6.0 x105

Factory

feed A

2.0 x102- 6.0x102

0

7.0 x102- 7.0 x103

feed B

2.0 x102- 3.0x103

0

2.0 x102- 3.0 x103

 

 

 

 

 

Total fungal counts (UFC g-1) obtained from three different kinds of chinchilla feeds (A: fur chinchillas, B: mother chinchillas and C: lucerne cubes) on DRBC and DG18 media.
* Recommended limits established by Good Manufacturing Practices, GMP (2008).

Table 2.
Statistical analysis of fungal counts in chinchilla feeds

Región

Estaciones Climáticas

Tprom

Tmax

Tmin

Hr

Prec
mm3

Costa Norte

Acaponeta

26.83

28.41

25.61

73.86

0.00

Rosamorada

21.41

28.03

17.19

87.39

0.00

Promedio

24.12

28.22

21.40

80.62

 

 

 

 

 

 

 

 

Costa Sur

Compostela-Las Varas

24.78

28.48

22.34

87.54

288.60

Bahía de Banderas-Valle de Banderas

25.02

29.14

22.75

86.51

81.00

 Promedio

24.90

28.81

22.54

87.03

 

 

 

 

 

 

 

 

Centro

Tepic

21.71

29.13

16.91

71.71

30.00

 

 

 

 

 

 

 

Sierra

Huajicori

20.34

28.88

15.56

73.60

0.00

El Nayar-Mesa del Nayar

20.43

28.71

13.67

23.63

0.00

 Promedio

20.38

28.80

14.61

48.61

 

Analysis of CFU g-1 by Mixed and General Lineal Model. Software: PROC GLM in SAS (SAS Institute, Cary, NC, USA).

 

Table 3.
Statistical analysis of fungal counts in chinchilla feeds
.

Kind of feed

Mean

SE*

A

1.67 b

0.13

B

2.01 b

0.16

C

4.82 a

0.29

Fisher’s LSD test tp compare mean values. Different letters indicate statistically significant difference.
* Standard error.

Figure 1. Mycobiota of chinchilla feeds. Isolation frequency (%) of fungal genera identified in different kinds of chinchilla feeds (A: fur chinchillas, B: mother chinchillas and C: Lucerne cubes) from different sources (farm and factory).

 

Table 4.
Aspergillus and Fusarium species in chinchilla feeds.

Fungal species relative density (%)

Farm

Factory

feed A

feed B

feed C

feed A

feed B

A. flavus                 83.3
A. fumigatus           16.6
F. verticillioides      78.9
F. proliferatum        15.8
F. oxysporum           5.3

A. flavus                 55.5
A. fumigatus          16.6
A. versicolor           22.2
A. alliaceus            15.8
F. verticillioides         50
F. proliferatum       37.5
F. oxysporum         12.5

A. flavus                100
F. verticillioides    66.7
F. oxysporum       33.4

A. flavus                 100
F. verticillioides      71.4
F. proliferatum       28.6

A. flavus                 100
F. verticillioides        75
F. proliferatum          25

Distribution (%) of Aspergillus and Fusarium species in different kinds of chinchilla feeds (A: fur chinchillas, B: mother chinchillas and C: Lucerne cubes) from different sources (farm and factory).

 

Table 5.
Aflatoxins in chinchilla feeds.

Kind of feed

AFB1 levels (µg kg-1)

Contaminated samples (%)

Samples exceeding permitted limit*

Range

Mean

Feed A

41.2

28.6

3.84- 97.34

24.76

Feed B

33.3

0

1.90- 9.74

5.66

Feed C

55.5

60

1.93- 40.4

23.64

Aflatoxin B1 levels (µg kg-1) and contamination frequency of different feeds (A: fur chinchillas, B: mother chinchillas and C: Lucerne cubes) sampled in a chinchilla farm. * 20 µg kg-1< according to Good Manufacturing Practices (GMP, 2008)


Aspergillus and Fusarium species were identified. Aspergillus flavus, A. fumigatus, F. verticillioides and F. proliferatum were the most frequently isolated species in all different chinchilla feeds (Table 4). 

All Aspergillus spp. strains isolated from the factory samples were identified as A. flavus and 50 % of them were able to produce AFB1 in values ranging from 2.8 to 8.64 µg g-1, while 72 % of Aspergillus spp. strains isolated from farm samples were identified as A. flavus, and 69, 7% of them were able to produce 0.66 to 58.8 µg g-1  AFB1.

 

Figure 2. Histopathology of chinchilla livers. Histopathology of liver sections from slaughtered chinchillas from the farm stained with Hematoxylin and Eosin (H/E). A) Slightly diffuse cytoplasmic vacuolation of hepatocytes present in a section of a normal chinchilla liver. B) Moderated diffuse cytoplasmic vacuolation and necrosis localized in the periportal region.

Aflatoxin B1 was detected in the three types of chinchilla feed from the farm. Lucerne cubes (feed C) showed the highest percentage of contaminated samples (55.5 %), with levels between 1.97 and 40.40 µg kg-1 AFB1. Contamination percentage of pelletized feeds A and B were 33.3 and 41.2 %, respectively. Aflatoxin B1 levels detected ranged from 3.84 to 97.34 µg kg-1 in feed A and 1.9 to 9.74 µg kg-1 in feed B. The mean AFB1 levels in feed A and C exceeded the limit established by the Good Manufacturing Practices, which is 20 µg kg-1 (GMP, 2008)(Table 5). Aflatoxin B1 was not detected in any of the samples from the factory.

The macroscopic examination of livers revealed they had a normal appearance, normal size, sharp borders and a reddish-brown color. Histopathology the livers indicated 63.3 % of the organs showed slight to moderate lipid degeneration, with diffuse cytoplasm vacuolation, 9 % showed intense lipid cytoplasm vacuolation, while 27.3 % showed hydropic degeneration and even nuclear vacuolation of hepatocytes from H/E stained tissue sections (Figure 2).


 Discussion

The mycobiota, water activity, natural occurrence of AFB1 in chinchilla feed samples and the aflatoxigenic capacity of A. flavus isolates obtained from these were evaluated.  The macroscopic and microscopic characteristics and lesions in the livers of animals exposed to AFB1 natural contamination levels were also studied.

In general, total fungal counts (CFU g-1) on the three types of feed were moderate. Pelletized feeds did not exceed the feed hygienic quality limit (1x104 CFU g-1) while lucerne cubes did slightly surpassed this limit (GMP, 2008). These results differ from studies that informed counts highly exceeding 1x104 CFU g-1 in different feeds intended for poultry, swine and cattle (Magnoli et al., 2002; Accensi et al., 2004; Rosa et al., 2006; Cavaglieri et al., 2009; González Pereyra et al., 2008a; González Pereyra et al., 2008b; González Pereyra et al., 2008c; González Pereyra et al., 2009; González Pereyra et al., 2011) and agree with others (Fraga et al., 2007; Oliveira et al., 2006) that reported lower counts in poultry feed samples. In our study, Aspergillus and Fusarium species showed the highest isolation frequencies, followed by Penicillium spp. Many studies have encountered species of these three genera as the dominant mycobiota in many animal feedstuffs (Magnoli et al., 2002; González Pereyra et al., 2008b; González Pereyra et al., 2008c; González Pereyra et al., 2009; González Pereyra et al., 2011;  Bragulat et al., 1995; Richard et al., 2007; Ghiasian and Maghsood, 2011). In our study, A. flavus was the prevalent species. This result concurs with many studies carried out by other authors who encountered this species in the highest frequency in cereals and different feeds and feed ingredients (Pitt and Hocking, 1997; Accensi et al., 2004; Adebajo et al., 1994; Dalcero et al., 1997; Sanchis et al., 1993; Pitt et al., 1994; Saleemi et al., 2010). Aspergillus species belong to the fungal flora that typically appears during storage at low aw in substrates such as grains and mixed feeds. The most frequently isolated species in this study have been described as moderately xerophilic (A. flavus) and slightly xerophilic fungi (A. fumigatus) (Lacey and Magan, 1991). All samples showed Fusarium spp. contamination. As it has been informed for other animal feeds, F. verticilliodes was the prevalent species (Oliveira et al., 2006; Campos et al., 2008).

A high percentage of the A. flavus strains assayed were AFB1 producers. Several authors have reported production of aflatoxins B and G by A. flavus isolated from maize, feeds and other substrates (Saleemi et al, 2010; El-Kady et al., 1994; Jan et al., 1995; Horn and Dorner, 1999; Gatti et al., 2003).

The GMP (GMP, 2008) regulations on product standards in the animal feed sector established that the current maximum permitted level for AFB1 for poultry feeds is 20 µg kg-1. The mean AFB1 levels of feeds A and C collected from the farm slightly exceeded this limit, while feed B and the feeds from the factory did not. In some samples from the farm, AFB1 levels were as high as 97 and 40 µg kg-1 for feeds A and C, respectively. Even though amounts of toxin detected on our chinchilla feeds were not enough to cause dramatically adverse effects in animals, such as an acute mycotoxicosis, it is a sign that the feed used in the farm was not of the best quality, and the levels of toxin detected could affect young animals (Jones et al., 1982). Furthermore, sublethal doses of mycotoxins produce a chronic toxicity that can result in liver cancer. Consumption of low doses of AFs for an extended period of time can cause reduction of the feed intake and feed conversion, weight loss and weak fur. The liver is the primary target organ of acute injury from AFs ingestion in all species. The diagnosis of mycotoxicoses includes the analysis of the feed as well as the histopathology since clinical signs can be nonspecific and confusing. In a recent research, livers from 9 chinchillas that died naturally during the disease outbreak in a farm and livers from healthy chinchillas slaughtered for commercial pelt recovery were analyzed for their macroscopic and microscopic characteristics through necropsy and histopathology (González Pereyra et al., 2008a). Histopathologic analysis revealed hepatocellular changes typical of AFs intoxication such as cytoplasmic vacuolation, nodular hyperplasia, and bile-duct proliferation. Moreover, aflatoxins B1, B2, G1and G2  levels high enough for causing an acute outbreak were detected in the feed consumed by the animals. In the present study, macroscopic inspection of the livers did not reveal the typical characteristics of acute toxicity such as general enlargement, yellowish coloration, hypertrophy, rounded hepatic borders or increased friability. Only slight histological changes that indicated hepatic toxicity (lipid vacuolation of hepatocites) were observed in the microscopic analysis of H/E stained liver sections.

Analyses of the pelletized feed for AFB1 by HPLC revealed that the feed samples were contaminated. The presence of AFB1 producer species and the detection of this toxin (even in low levels) indicated that contamination in these kinds of feeds exists and constitutes a hazard for the animals, farmers and feed factory workers.

The current study revealed that toxigenic fungal species can contaminate feed intended for chinchillas in breeding farms. Toxigenic strains of A. flavus able to produce AFB1 as well as the toxin itself were detected in feeds inducing moderate changes in the animals liver histopathology. This fact suggests that periodic monitoring of the feeds and their ingredients would be required in order to prevent acute outbreaks and economic losses caused by chronic AFs exposure.


Acknowledgements

We thank Mr. Sergio Alaminos, Ms. Fabiana and Mr. Ignacio Corteggiano, for their assistance in providing the samples and the livers for the present study. This study was carried out with grants from CAPES/SPU project Nº 048/10 period 2010-2013 and PID- 105/08-Agencia Córdoba Ciencia Project (Ministerio de Ciencia y Tecnología) period 2009-2011.


Literatura citada


Abarca ML, Bragulat MR, Castella G, Cabañes FJ. Mycoflora and aflatoxin-producing strains in animal mixed feeds. Journal of Food Protection 1994; 57: 256-258.

Accensi F, Abarca ML, Cabañes FJ. Occurrence of Aspergillus species in mixed feeds and component raw materials and their ability to produce ochratoxin A. Food Microbiology 2004; 21: 623–627.

Adebajo LO, Idowu AA, Adesanya OO. Mycobiota and mycotoxins production in Nigeria corn and corn-based snacks. Mycopathologia 1994; 126: 183–192.

Allameh A, Safamehr A, Mirhadi SA, Shivazadd M, Abyanehe MR, Naderia AA. Evaluation of biochemical and production parameters of broiler chicks fed ammonia treated aflatoxin contaminated maize grains. Animal Feed Science and Technology 2005; 122: 289–301.

Bragulat MR, Abarca ML, Castella O, Cabañes J. Mycological survey on mixed poultry feeds and mixed rabbit feeds. Journal of the Science of Food and Agriculture 1995; 67: 215–220.

Campos SG, Cavaglieri LR, Fernández JMG, Dalcero AM, Kruger LA, Keller M, et al. Mycobiota and aflatoxins in raw materials and pet foods in Brazil. Journal of Animal Physiology and Animal Nutrition 2008; 92: 377-383.

Cavaglieri LR, Keller KM, Pereyra CM, González Pereyra ML, Alonso VA, Rojo FG, et al. Fungi and natural incidence of selected mycotoxins in barley rootlets. Journal of Stored Products Research 2009; 45 suppl 3: 147-150.

Council for Agricultural Science and Technology (CAST). Mycotoxins: risks in plants animal and human systems. Task Force Report No. 139. Ames, IA, USA, 2003.

Dalcero A, Magnoli C, Chiacchiera S, Palacios G, Reynoso M. Mycoflora and incidence of aflatoxins B1, zearalenone and deoxinyvalenol in poultry feeds in Argentina. Mycopathologia 1997; 137: 179-184.

El-Kady I, El-Maraghy S, Zohri AN. Mycotoxin producing potential of some isolates of Aspergillus flavus and Eurotium groups from meat products. Microbiology Research 1994; 149: 297–307.

Fraga ME, Curvello F, Gatti MJ, Cavaglieri LR, Dalcero AM, Rosa CAR. Potential Aflatoxin and Ochratoxin A Production by Aspergillus Species in Poultry Feed Processing. Veterinary Research Communications 2007; 31 suppl 3: 343-353.

Gatti MJ, Fraga ME, Magnoli C, Dalcero AM, Rosa CAR. Mycological survey for potential aflatoxin and ochratoxin producers and their toxicological properties in harvested Brazilian black pepper. Food Addititives and Contaminants 2003; 20 suppl 12: 1120–1126.

Geisen R. Multiplex polymerase chain reaction for the detection of potential aflatoxin and sterigmatocystin producing fungi. Systems of Applied Microbiology 1996; 19: 388-392.

Ghiasian SA, Maghsood AH. Occurrence of aflatoxigenic fungi in cow feeds during the summer and winter season in Hamadan, Iran. African Journal of Microbiology Research 2011; 5 suppl 5: 516-521.

González Pereyra ML, Alonso VA, Sager R, Morlaco MB, Magnoli CE, Astoreca AL, et al. Fungi and selected mycotoxins from pre- and post- fermented corn silage. Journal of Applied Microbiology 2008b; 104 suppl 4: 1034-1041.

González Pereyra ML, Carvalho CQ, Tissera JL, Keller KM, Magnoli CE, Rosa CAR, et al. An outbreak of acute aflatoxicosis on a chinchilla (Chinchilla lanigera) farm in Argentina. Journal of Veterinary Diagnostic Investigation 2008a; 20: 853–856.

González Pereyra ML, Chiacchiera SM, Rosa CAR, Sager RL, Dalcero AM, Cavaglieri LR. Comparative analysis of the mycobiota and mycotoxins present in trench type corn silos and silo bags. Journal of the Science of Food and Agriculture 2011; 91 suppl 8: 1474-1481.

González Pereyra ML, Keller KM, Keller LAM, Cavaglieri LR, Queiroz BD, Tissera JL, et al. Mycobiota and mycotoxins of equine feedstuffs in the central region of Argentina. Revista Brasileira de Medicina Veterinâria 2009; 31 suppl 1: 24-29.

González Pereyra ML, Pereyra CM, Ramirez ML, Rosa CAR, Dalcero AM, Cavaglieri LR. Determination of mycobiota and mycotoxins in pig feed in central Argentina. Letters of Applied Microbiology 2008c; 46 suppl 5: 555-561.

Good Manufacture Practices (GMP). Certification Scheme Animal Feed Sector 2006, Including Residue Standards. Version, March 28th, 2008.

Horn BN, Dorner JW. Regional differences in production of aflatoxin B1 and cyclopiazonic acid by soil isolates of Aspergillus flavus along a transect within the United States. Applied and Environmental Microbiology 1999; 65 suppl 4: 1444–1449.

International Agency for Research on Cancer (IARC). Aflatoxins (group 1). In IARC monographs on the evaluation of carcinogenic risks to humans 2002; 82: 9–13.

ISO 21527-1: Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of yeasts and moulds. Part 1: Colony count technique in products with water activity greater than 0. 95. Uganda National Bureau of Standards (UNBS-Ed.)

ISO 21527-2: Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of yeasts and moulds. Part 2: Colony count technique in products with water activity less than or equal to 0. 95. Uganda National Bureau of Standards (UNBS-Ed.)

Jan SK, Singh PP, Amarjt S. Observations of occurrence of poultry diseases associated with mycotoxins in feeds. Indian Journal of Animal Science 1995; 65: 1063–1067.

Jones FT, Hagler WM, Hamilton PB. Association of low levels of aflatoxin in feed with productivity losses in commercial broiler operation. Poultry Science 1982; 61: 861–868.

Klich MA. Identification of Common Aspergillus Species. Utrecht, Netherlands: CBS, 2002.

Labala, J. Micotoxinas de chinchillas. Accessed Jan. 16th, 2008. Text in Spanish. http://www.cuencarural.com/granja/chinchillas/micotoxinas-de-chinchillas.

Lacey J, Magan N. Fungi in cereal grains, their occurrence and water and temperature relationships. In: Cereal Grain, Mycotoxins, Fungi and Quality in Drying and Storage. Edited by Chelkowski J. Amsterdam: Elsevier 1991, 77–118.

Magnoli C, Chiacchiera SM, Miazzo R, Palacio G, Angeletti A, Hallak C, et al. The mycoflora and toxicity of feedstuffs from a production plant in Cordoba, Argentina. Mycotoxin Research 2002; 18: 7–22.

Miazzo R, Peralta MF, Magnoli C, Salvano M, Ferrero S, Chiacchiera SM, et al. Efficacy of Sodium Bentonite as a Detoxifier of Broiler Feed Contaminated with Aflatoxin and Fumonisin. Poultry Science 2005; 84: 1–8.

Monbaliu S, Van Poucke C, Detavernier C, Dumoulin F, Van De Velde M, Schoeters E, et al. Occurrence of mycotoxins in feed as analyzed by a multi-mycotoxin LC-MS/MS method. Journal of Agricultural and Food Chemistry 2010; 58: 66–71.

Neal GE. Participation of animal biotransformation in mycotoxin toxicity. Revue de Medicine Vetétérinaire 1998; 149: 555–560.

Nelson PE, Toussoun TA, Marasas WFO. (Eds). Fusarium Species. An Illustrated Manual for Identification. University Park, PA/London, UK: The Pennsylvania State University Press, 1983.

Newman SJ, Smith JR, Stenske KA, Newman LB, Dunlap JR, Imerman PM, et al. Aflatoxicosis in nine dogs after exposure to contaminated commercial dog food. Journal of Veterinary Diagnostic Investigation 2007; 19: 168–175.

Oliveira GR, Ribeiro JM, Fraga ME, Cavaglieri LR, Direito GM, Keller KM, et al. Mycobiota in poultry feeds and natural occurrence of aflatoxins, fumonisins and zearalenone in the Rio de Janeiro State, Brazil. Mycopathologia 2006; 162: 355-362.

Pitt JI, Hocking AD, Bhudhasamai K, Miscamble BF, Wheeler KA, Tanboon EK. The normal mycoflora of commodities from Thailand. 2. Beans, rice, small grains and other commodities. International Journal of Food Microbiology 1994; 23: 35–53.

Pitt JI & Hocking AD (Eds.). Fungi and Food Spoilage. London, UK: Chapman & Hall, 1997.
Quinn GP & Keough MJ (Eds.). Experimental Design Data analysis for biologists. Cambridge, United Kingdom: Cambridge University Press, 2002.

Richard E, Heutte N, Sage L, Pottier D, Bouchart V, Lebailly P, et al. Toxigenic fungi and mycotoxins in mature corn silage. Food and Chemycal Toxicology 2007; 45: 2420–2425.

Rosa CAR, Ribeiro JMM, Fraga ME, Gatti MJ, Cavaglieri LR, Magnoli CE, et al. Mycobiota of poultry feeds and ochratoxin-producing ability of isolated Aspergillus and Penicillium species. Veterinary Microbiology 2006; 13: 89–96.

Saleemi MK, Khan MZ, Khan A, Javed I. Mycoflora of poultry feeds and mycotoxins producing potential of Aspergillus species. Pakistan Journal of Botany 2010; 42 suppl 1: 427-434.

Sanchis V, Sanclemente A, Usall J, Viñas I. Incidence of mycotoxigenic Alternaria alternata and Aspergillus flavus in barley. Journal of Food Protection 1993; 56: 246–248.

Smith JE, Moss MO. Mycotoxins: Formation, Analysis and Significance. John Wiley & Sons, 1985. Chichester, UK, 240.

Trucksess MW, Stack ME, Nesheim S, Albert RH, Romer TR. Multifunctional column coupled with liquid chromatography for determination of aflatoxins B1, B2, G1, G2 in corn, almonds, Brazil nuts, peanuts and pistachio nuts: collaborative study. Journal of the AOAC International 1994; 6: 1512–1521.

Whitlow LW, Hagler WM. Mycotoxins in feeds. Feedstuffs 2002; 74: 1–10.

 

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