Aflatoxicosis in a canine associated with consumption of contaminated food - case report

[Aflatoxicose em canino associada ao consumo de ração contaminada - relato de caso]

ABSTRACT

Aflatoxins are mycotoxins produced by fungi that cause severe poisoning, mainly affecting the liver. Aflatoxicosis is rarely reported in dogs, but this species is extremely sensitive. The objective of this study was to report anatomopathological aspects of a case of aflatoxicosis in a male mongrel dog associated with ingestion of contaminated feed. The animal was treated at UNICRUZ Veterinary Hospital with a history of jaundice and apathy, which led to death within three days. At necropsy, marked jaundice of mucous membranes, subcutaneous tissue and intima of arteries, and a friable liver with accentuated lobular pattern were observed. Histopathological evaluation revealed intense vacuolization of hepatocytes, megalocytosis, fibrosis, proliferation of bile ducts and bilestasis. Samples of feed ingested by the animal were sent to Mycotoxicological Analysis Laboratory of UFSM, confirming high levels of aflatoxin. These poisonings occur due to ingestion of contaminated ingredients used in manufacture of dog food. The production of mycotoxins results from storage failures and poor quality of ingredients, which are often undetectable. The necropsy and histopathological findings, associated with clinical condition, were compatible with aflatoxicosis. The diagnosis was confirmed by assessing the aflatoxin levels in commercial food ingested by the animal.

Keywords:
hepatotoxicity; liver failure; mycotoxin; Aspergillus spp

RESUMO

Aflatoxinas são micotoxinas produzidas por fungos responsáveis por causar intoxicações graves, afetando principalmente o fígado. A aflatoxicose é pouco relatada em cães, contudo essa espécie é extremamente sensível. O objetivo deste trabalho é relatar os aspectos anatomopatológicos de um caso de aflatoxicose em um canino, macho, sem raça definida, associado à ingestão de ração contaminada. O animal foi atendido no Hospital Veterinário da Unicruz, com histórico de icterícia e apatia, com evolução para óbito em três dias. Na necropsia, observou-se marcada icterícia das mucosas, subcutânea e íntima das artérias, fígado friável com acentuação do padrão lobular. A avaliação histopatológica revelou vacuolização intensa dos hepatócitos, megalocitose, fibrose, proliferação de ductos biliares e bilestase. Amostras da ração ingerida pelo animal foram encaminhadas ao Laboratório de Análises Micotoxicológicas da UFSM, confirmando altos níveis de aflatoxina. Essas intoxicações ocorrem pela ingestão de ingredientes contaminados usados na fabricação de rações para cães. A produção de micotoxinas resulta de falhas no armazenamento e da baixa qualidade dos ingredientes, muitas vezes imperceptíveis. Os achados de necropsia e histopatológicos, associados ao quadro clínico, foram compatíveis com aflatoxicose. O diagnóstico foi confirmado por meio da avaliação dos níveis de aflatoxina na ração comercial ingerida pelo animal.

Palavras-chave:
hepatotoxicidade; insuficiência hepática; micotoxina; Aspergillus spp

INTRODUCTION

Mycotoxins are odorless, tasteless and invisible substances produced by mycotoxigenic fungi (Mallmann et al., 2009), which can cause poisoning in animals and humans when contaminated grains or by-products are consumed (Wouters et al., 2013). Some of these substances have carcinogenic, teratogenic and immunosuppressive effects, and can cause hepatotoxicosis, lesions in the digestive tract and decreased reproductive rates (Mallmann et al., 2009). The presence of aflatoxins in foods intended for pet feeding has been reported, along with environmental factors that contribute to fungal proliferation and mycotoxin production (Gomes et al., 2014). The production of mycotoxins in Brazil is favored by the climatic conditions of tropical and subtropical regions, mainly in low-quality grains stored in inadequate places with high humidity (Wouters et al., 2013).

Aflatoxin is one of the main mycotoxins related to food contamination and outbreaks of poisoning in animals, being produced by fungi of the genus Aspergillus spp. and having hepatotoxic potential (Gomes et al., 2014 and Wang et al., 2023). Although there are different subtypes of aflatoxins, AFB1 (aflatoxin B1) is the most pathogenic among them (Dereszynski et al., 2008 and Wang et al., 2023). The canine species is highly sensitive to poisoning due to the low concentration of the enzyme glutathione S-transferase, which leads to the occurrence of acute, subacute or chronic poisoning (Yang et al., 2023). Poisoned animals commonly present jaundice. At necropsy, the liver is yellowish and with an accentuated lobular pattern (McGavin and Zachary, 2013).

Microscopically, there is hepatic degeneration and necrosis with proliferation of ducts, megalocytosis and bilestasis (McGavin and Zachary, 2013). In chronic cases, fibrosis between the hepatocyte cords can be observed (Gomes et al., 2014, Guterres et al., 2017). Brazilian legislation defines the maximum tolerated limits of aflatoxins in commercial dog food as 50µg/kg (Brazil, 1988), however, outbreaks of poisoning with high mortality rates have been recorded in this species (Dereszynski et al., 2008; Bruchim et al., 2012 and Guterres et al., 2017).

Cases of aflatoxicosis in dogs are possibly underestimated (Dereszynski et al., 2008), as they are often overlooked and/or confused with other diseases due to the similarity of clinical signs associated with liver damage (Guterres et al., 2017). From this perspective, the objective of this work was: (i) to report histopathological findings of a case of aflatoxicosis in a canine, (ii) to analyze the levels of aflatoxins present in the food consumed by the animal, and (iii) to associate the histopathological findings with the mycological analysis of the food to determine the conclusive diagnosis.

CASUISTRY

A four-year-old male canine of mixed breed with a history of jaundice and apathy, with no history of previous contact with dogs at house, was treated at the Veterinary Hospital of the University of Cruz Alta (Unicruz) in August 2022. The body was sent to the Veterinary Pathology Laboratory of Unicruz for necropsy, performed according to the technique described by Bonel et al. (2020). During the necropsy, samples from all organs were collected and stored in 10% buffered formalin. After 48 hours, the samples were cleaved, routinely processed, cut into 5µm thick sections, and stained using hematoxylin and eosin (HE) and Masson's trichrome techniques. Subsequently, the histological slides were evaluated using a ZEISS Axio Scope A1 optical microscope and photomicrographed using a digital image capture system using an Axiocam 105 color camera (ZEISS®, Germany).

Samples of the feed consumed by the animal during a period of approximately 25 days were sent to the Mycotoxicological Analysis Laboratory (LAMIC) of the Federal University of Santa Maria (UFSM) for quantification of aflatoxins. The analysis was performed in duplicate using the liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) technique, performed according to the LAMIC standard operating procedure - POP 40, according to the methodology described by Sulyok et al., (2006). The limits of quantification (LQ) and recovery coefficients (CR) were adopted for: aflatoxin B1 (AFB1) of 1μg/kg and 94.5%; for aflatoxin B2 (AFB2) of 1μg/kg and 80.0%; for aflatoxin G1 (AFG1) of 1μg/kg and 88.5% and for aflatoxin G2 (AFG2) of 1μg/kg and 88.1%. The uncertainty estimates regarding the quantification of aflatoxins in food were: 1.0μg/kg (0.20μg/kg) for AFB1; 1.0μg/kg (0.21μg/kg) for AFB2; 1.0μg/kg (0.20μg/kg) for AFG1 and 1μg/kg (0.20μg/kg) for AFG2. To interpret the tolerable levels of aflatoxins present in food, the values established by the Ministry of Agriculture, Livestock and Food Supply Ordinance No. 7 (Brazil, 1988) were used, which determines the maximum tolerated limits (MTL) of contaminants in food or any raw material used directly or indirectly in the manufacture of feed for animal feed (Brazil, 2011).

At necropsy, marked jaundice was observed in the mucous membranes, subcutaneous tissue and intima of the arteries (figures 1A, 1B). There were also hemorrhagic areas in the subcutaneous tissue. The liver was friable, with an accentuated lobular pattern and yellowish areas interspersed with reddish areas (figure 1C). Histopathological evaluation of the liver revealed intense cytoplasmic vacuolization of hepatocytes, megalocytosis, areas of random fibrosis, proliferation of bile ducts, and marked bile stasis and loss of normal organ architecture (Figure 2). Areas of tubular degeneration and congestion were observed in the kidneys. Marked perineural and perivascular edema was observed in the central nervous system. Toxicological analysis of the feed revealed aflatoxin levels of 107.0μg/kg, 9.0μg/kg, and 2.5μg/kg (ppb), respectively, for AFB1, AFB2, and AFG1.

DISCUSSION

The clinical picture, macroscopic and microscopic lesions observed in this case, in addition to the toxicological analysis of the food provided to the dog are consistent with aflatoxin poisoning as described by Bruchim et al. (2012); Mehrzad et al. (2020); Bates (2021).

Observations by Bruchim et al. (2012) found that, in an outbreak of aflatoxicosis that affected 50 dogs, 78% of the animals presented lethargy, 76% vomiting, 74% anorexia, 66% depression, 60% melena and 36% hematuria and diarrhea, and all animals presented marked jaundice. In the animal analyzed in this study, generalized jaundice and apathy were observed, suggesting a rapid progression of the clinical picture to death, without manifestation of other clinical signs.

Figure 1
Macroscopic lesions associated with aflatoxicosis in a dog. A. Marked jaundice in the intima of the aorta. B. Icteric oral cavity mucosa. C. Liver with accentuated lobular pattern and yellowish areas interspersed with reddish areas.

Figure 2
Microscopic lesions associated with aflatoxicosis in a canine, visualized in the liver parenchyma. A. Bilestasis, stained with H&E, 40X magnification with 40µm scale bar. B. Megalocytosis (arrows) and cytoplasmic vacuolization, stained with H&E, 40X magnification with 40µm scale bar. C. General view of the liver parenchyma showing cytoplasmic vacuolization and loss of normal organ architecture, stained with H&E, 10X magnification with 100µm scale bar. D. General view of the liver parenchyma showing areas of fibrosis and loss of normal organ architecture, stained with Masson’s trichrome, 10X magnification with 100µm scale bar.

Despite the rapid clinical progression, it is worth noting that, according to the observations made, the histopathological findings were compatible with a case of aflatoxicosis. Hepatotoxicosis caused by aflatoxins presents, in the initial stages, histological alterations, such as central lobular necrosis and intense cytoplasmic vacuolization. In chronic cases, however, there is marked megalocytosis with considerable replacement of liver tissue by connective tissue and proliferation of bile ducts (Mehrzad et al., 2020 and Bates, 2021), similar to that observed in this case.

The marked jaundice observed in the present case is due to the accumulation of bilirubin in the tissues due to obstruction of bile canaliculi and bile stasis, frequently observed in liver lesions associated with severe cases of portal fibrosis (Bates, 2021). Furthermore, fat mobilization, in cases of inadequate diet, causes hepatic steatosis, interfering with apoprotein synthesis and causing weight loss in the animal (McGavin and Zachary, 2013), which was also observed in this case.

The presence of edema and marked cytoplasmic vacuolization observed in the brain corroborates the findings of hepatic encephalopathy (Bruchim et al., 2012 and Yu et al., 2019). The occurrence of marked vacuolization between the white and gray matter of the brain, and perineural and perivascular edema are the main lesions observed in these conditions, being reported in severe cases of aflatoxicosis (Yu et al., 2019). Furthermore, in the kidneys, the congestion and tubular degeneration observed were similar to the observations of Hennemann et al. (1996) analyzing the renal function of dogs experimentally challenged with aflatoxins.

The feed consumed by the animal was within the expiration date and was produced by a registered company and was composed of ingredients based on corn and rice - two cereals with a high potential for the presence and proliferation of mycotoxins, which are probably the source of the contamination, as observed by Martínez et al. (2021). Furthermore, the feed sold in sealed packaging suggests that the contamination came from the inadequate raw material used for manufacturing. This reinforces the lack of changes in the visual appearance of the feed which, according to Ekici and Yipel (2022), may be related to the use of inadequate raw material that favored fungal proliferation. Even though aflatoxin B1 level was 107µg/kg, which is within the margin that induces chronic cases, the consumption period related is our case is not enough to characterized chronic aflatoxicosis. The latter is developed under prolonged consumption of diets containing moderate levels of aflatoxin, with lesions being observed in animals fed for a period of six to eight weeks with levels between 50 and 300 µg/kg of aflatoxin B1 (Yang et al., 2023 and Wang et al., 2023). The aflatoxin levels in the feed consumed by the dog were above those established by Brazilian legislation, with the high value of aflatoxin B1 being the main toxic component of the total aflatoxins, given that it is the most hepatotoxic of the aflatoxins (Wang et al., 2023). In the canine analyzed in this study, the histopathological findings indicated the occurrence of aflatoxicosis, and confirmation of the diagnosis was based on the mycotoxicological analysis of the food ingested by the animal. Thus, the interaction between the analyses enabled confirmation of aflatoxin poisoning, making it possible to track the toxic effects on the animal based on the levels of mycotoxin found in the feed.

CONCLUSION

Based on the macroscopic and histopathological lesions observed, together with the presence of aflatoxins at levels higher than the limits established by Brazilian legislation in the commercial feed consumed by the animal, it was possible to establish the definitive diagnosis of aflatoxicosis. This case highlights the importance of performing necropsies on animals with a similar clinical history, considering individual susceptibility. It is also recommended to mycologically evaluate the feed consumed, even if it is commercial and within the expiration date, to quantify the levels of aflatoxins, in order to obtain a conclusive diagnosis.

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Publication Dates

• Publication in this collection
  14 July 2025
• Date of issue
  Jul-Aug 2025

History

• Received
  12 Aug 2024
• Accepted
  29 Jan 2025