Modulation of the spleen transcriptome in domestic turkey (Meleagris gallopavo) in response to aflatoxin B1 and probiotics.

Poultry are highly susceptible to the immunotoxic effects of the food-borne mycotoxin aflatoxin B1 (AFB1). Exposure impairs cell-mediated and humoral immunity, limits vaccine efficacy, and increases the incidence of costly secondary infections. We investigated the molecular mechanisms of AFB1 immunotoxicity and the ability of a Lactobacillus-based probiotic to protect against aflatoxicosis in the domestic turkey (Meleagris gallopavo). The spleen transcriptome was examined by RNA sequencing (RNA-seq) of 12 individuals representing four treatment groups. Sequences (6.9 Gb) were de novo assembled to produce over 270,000 predicted transcripts and transcript fragments. Differential expression analysis identified 982 transcripts with statistical significance in at least one comparison between treatment groups. Transcripts with known immune functions comprised 27.6 % of significant expression changes in the AFB1-exposed group. Short exposure to AFB1 suppressed innate immune transcripts, especially from antimicrobial genes, but increased the expression of transcripts from E3 ubiquitin-protein ligase CBL-B and multiple interleukin-2 response genes. Up-regulation of transcripts from lymphotactin, granzyme A, and perforin 1 could indicate either increased cytotoxic potential or activation-induced cell death in the spleen during aflatoxicosis. Supplementation with probiotics was found to ameliorate AFB1-induced expression changes for multiple transcripts from antimicrobial and IL-2-response genes. However, probiotics had an overall suppressive effect on immune-related transcripts.

Amanitin toxicosis in two cats with acute hepatic and renal failure.

Amanitin is a toxic cyclopeptide present in several species of poisonous mushrooms. Amanitin toxicosis was diagnosed in 2 cats from separate premises. Both cats initially had lethargy and vomiting, and they rapidly developed depression and neurological signs over 24-48 hours. Marked elevation of alanine aminotransferase was the primary finding, with subsequent serum chemistry values compatible with hepatic and renal failure. Histopathological findings consisted of submassive to massive acute hepatic necrosis, renal proximal tubular epithelial necrosis, and foci of necrosis and inflammation in the gastrointestinal tract. Amanitin exposure was confirmed postmortem by detection of α-amanitin in the kidney by liquid chromatography-mass spectrometry. A similar clinical course and pathological changes are reported in human and canine amanitin intoxication; however, gastrointestinal lesions are not typically described.

Early diagnosis of amanitin exposure (amatoxicosis) in a dog with a point-of-care diagnostic test.

OBJECTIVE: To describe the rapid diagnosis, treatment, and clinical course of a dog that ingested an amanitin-containing mushroom. CASE SUMMARY: A 2-month-old female intact Australian Shepherd presented with diarrhea and vomiting, along with a possible mushroom exposure. Upon presentation, the dog's urine was collected and tested positive by a point-of-care rapid diagnostic test specific for detecting amanitins, the causative agents of amatoxicosis. NEW OR UNIQUE INFORMATION PROVIDED: This is the first reported case of amatoxicosis that was diagnosed using a point-of-care test prior to starting treatment. An early diagnosis helps to guide early treatment decisions in this frequently fatal toxicosis.

Clinical recovery of 5 dogs from amatoxin mushroom poisoning using an adapted Santa Cruz protocol for people.

OBJECTIVE: To describe the clinical course, treatment, and outcome of 5 dogs following ingestion of toxic Amanita spp. mushrooms containing amatoxins using an adapted version of the Santa Cruz protocol developed for people. CASE SERIES SUMMARY: Five dogs were presented with clinical signs compatible with amanitin toxicity with witnessed ingestion noted in 3 of 5 dogs. Clinical findings included acute onset vomiting and diarrhea, lethargy, and hepatopathy including signs of fulminant hepatic failure (increased liver enzyme activities, hyperbilirubinemia, prolonged clotting times, and hypoglycemia were noted among these cases). Urine toxicological screening confirmed the presence of Amanita toxins in 4 cases with expert mycologist speciation in the fifth. Core interventions included percutaneous biliary drainage, use of octreotide, and early nil per os orders. All dogs survived to discharge with this treatment strategy. NEW OR UNIQUE INFORMATION PROVIDED: This case series describes the use of a modified version of the Santa Cruz protocol to address amatoxin-induced fulminant hepatic failure in dogs. The protocol was safe, well tolerated, and all patients made a full clinical recovery.

[Acute liver failure in a dog after mushroom intake, presumably of the genus Amanita]. / Akutes Leberversagen bei einem Hund nach Aufnahme eines Pilzes, vermutlich der Gattung Amanita.

A 4-year-old, neutered male Husky-mix dog weighing 29.4 kg that reportedly ingested a mushroom most likely of the genus Amanita one day prior to presentation exhibited signs of diarrhea, vomitus, inappetence and progressively worsening lethargy. Clinical chemistry revealed hypoglycemia, hyperbilirubinemia, decreased prothrombin and thromboplastin time, as well as increased liver enzyme activities. Despite hospitalization and supportive therapy over a period of 3 days the dog's general condition worsened leading to euthanasia. The pathomorphological findings were characterized by hemorrhage in several organs, hemorrhagic ingesta, icterus, and marked hepatic cellular necrosis.

Rapid, Sensitive, and Accurate Point-of-Care Detection of Lethal Amatoxins in Urine.

Globally, mushroom poisonings cause about 100 human deaths each year, with thousands of people requiring medical assistance. Dogs are also susceptible to mushroom poisonings and require medical assistance. Cyclopeptides, and more specifically amanitins (or amatoxins, here), are the mushroom poison that causes the majority of these deaths. Current methods (predominantly chromatographic, as well as antibody-based) of detecting amatoxins are time-consuming and require expensive equipment. In this work, we demonstrate the utility of the lateral flow immunoassay (LFIA) for the rapid detection of amatoxins in urine samples. The LFIA detects as little as 10 ng/mL of α-amanitin (α-AMA) or γ-AMA, and 100 ng/mL of ß-AMA in urine matrices. To demonstrate application of this LFIA for urine analysis, this study examined fortified human urine samples and urine collected from exposed dogs. Urine is sampled directly without the need for any pretreatment, detection from urine is completed in 10 min, and the results are read by eye, without the need for specialized equipment. Analysis of both fortified human urine samples and urine samples collected from intoxicated dogs using the LFIA correlated well with liquid chromatography-mass spectrometry (LC-MS) methods.

Failure of benzylpenicillin, N-acetylcysteine and silibinin to reduce alpha-amanitin hepatotoxicity.

BACKGROUND: Intoxications caused by amanitin-containing mushrooms represent an unresolved problem in clinical toxicology. The objective of this study was a comparative evaluation of benzylpenicillin (Bp), acetylcysteine (ACC) and silibinin (Sil) efficacy as antidotes in hepatocytes intoxicated with alpha-amanitin (alpha-AMA). MATERIALS AND METHODS: All experiments were performed on cultured canine hepatocytes. Cytotoxicity evaluation of cultured cells (MTT assay, extracellular lactate dehydrogenase activity) was performed at 12, 24 and 48 h of exposure to alpha-AMA and/or antidotes. RESULTS: Following 24 and 48 h exposure there was a significant decline of hepatocyte viability and an increase of lactate dehydrogenase activity in groups exposed to alpha-AMA and in groups exposed simultaneously to alpha-AMA and antidotes. Moreover, hepatocyte viability and lactate dehydrogenase activity in all these groups were similar. Administration of studied antidotes without alpha-AMA, was not associated with any adverse effects in hepatocytes. CONCLUSION: All antidotes tested in this study against alpha-AMA were not effective in canine hepatocyte cultures.

Fatal Amanita muscaria poisoning in a dog confirmed by PCR identification of mushrooms.

Diagnosing mushroom poisoning in dogs can be difficult and often includes identification of suspect mushrooms. Visual identification may be hindered by mastication, oral medications, or poor quality of environmental mushroom samples. Other analytical techniques may thus be necessary to aid in mushroom identification. A 5-y-old neutered male Labrador Retriever dog developed acute onset of vomiting, diarrhea, tremors, seizures, and somnolence. The dog was treated at a veterinary clinic and was briefly stabilized, but died during transport to an emergency clinic. On postmortem examination at the University of Kentucky Veterinary Diagnostic Laboratory, the dog's stomach was full of mushrooms covered with activated charcoal. Mushrooms were damaged, fragmented, and discolored, precluding accurate visual identification. Mushroom pieces were sent to the Department of Plant Pathology at the University of California-Davis for PCR identification; the neurotoxic mushroom Amanita muscaria was identified. A qualitative liquid chromatography-mass spectrometry (LC-MS) method was developed to detect ibotenic acid and muscimol, the toxic compounds present in A. muscaria. Mushrooms, stomach contents, and urine were analyzed by LC-MS; ibotenic acid and muscimol were detected in all samples. Because identification of ingested mushrooms is sometimes necessary to confirm mushroom poisoning, PCR can identify ingested mushrooms when visual identification is unreliable.

Mushroom Poisoning Cases in Dogs and Cats: Diagnosis and Treatment of Hepatotoxic, Neurotoxic, Gastroenterotoxic, Nephrotoxic, and Muscarinic Mushrooms.

Ingestion of poisonous mushrooms by small animals can lead to liver failure, neurotoxicity, or gastrointestinal irritation. Although amanita poisoning can be lethal, ingestion of other toxic mushrooms is generally self-limiting and not life threatening. Most cases are undiagnosed, as routine diagnostic tests only exist for amanitins and psilocin. Early detection of amanitin exposure can greatly aid in the therapeutic intervention by allowing veterinarians to make timely decisions regarding patient management. Treatment is generally supportive, but specific therapeutic measures exist for amanitin and psilocin exposures.

Diagnosis of Amanita toxicosis in a dog with acute hepatic necrosis.

Poisoning with amanitin-containing hepatotoxic mushrooms demands extensive efforts from clinicians, toxicologists, and pathologists. Presumptive diagnoses are established by positive identification of the suspect mushroom along with the occurrence of consistent clinical signs. If the animal dies, hepatic lesions may suggest exposure to amanitin-containing mushrooms; however, lesions are nonspecific. A 15-week-old female Dachshund was presented to the referring veterinarian for acute onset of lethargy that quickly progressed to sternal recumbency. Despite supportive care, the dog remained lethargic and died approximately 12 hours after initial presentation. A pale tan liver was noted at necropsy. Microscopically, the liver showed panlobular coagulative necrosis of hepatocytes. A presumptive diagnosis of amanitin poisoning was based on suspect history of exposure to mushrooms, clinical signs, and pathologic findings. Exposure to amanitin was confirmed through detection of alpha-amanitin in the liver by liquid chromatography/mass spectrometry. The objective of this case report is to illustrate the essential components to a successful diagnostic work-up of a suspect case of hepatotoxic mushroom poisoning. Although hepatotoxic mushroom poisoning has been documented in dogs before, confirmatory techniques for biologic specimens have not been used previously in diagnostic investigations.

Acute Inocybe mushroom toxicosis in dogs: 5 cases (2010-2014).

OBJECTIVE: To describe the clinical course, treatment, and outcome of 5 dogs following ingestion of mushrooms belonging to the Inocybe genus. CASE SERIES SUMMARY: Five dogs with witnessed Inocybe ingestions were presented with clinical signs compatible with poisoning. Vomiting, ptyalism, diarrhea, depression, and tachycardia were common clinical findings in the dogs in this case series. The prognosis with Inocybe toxicosis appears to be excellent as all dogs fully recovered following supportive care. NEW OR UNIQUE INFORMATION PROVIDED: This is the first reported case series of Inocybe mushroom ingestions in dogs where identification of the mushrooms were confirmed by an expert mycologist.

A novel orellanine containing mushroom Cortinarius armillatus.

Orellanine (3,3',4,4'-tetrahydroxy-2,2'-bipyridine-1,1'-dioxide) is a tetrahydroxylated di-N-oxidized bipyridine compound. The toxin, present in certain species of Cortinarius mushrooms, is structurally similar to herbicides Paraquat and Diquat. Cortinarius orellanus and Cortinarius rubellus are the major orellanine-containing mushrooms. Cortinarius mushrooms are widely reported in Europe where they have caused human poisoning and deaths through accidental ingestion of the poisonous species mistaken for the edible ones. In North America, Cortinarius orellanosus mushroom poisoning was recently reported to cause renal failure in a Michigan patient. Cortinarius mushroom poisoning is characterized by delayed acute renal failure, with some cases progressing to end-stage kidney disease. There is debate whether other Cortinarius mushroom contain orellanine or not, especially in North America. Currently, there are no veterinary diagnostic laboratories in North America with established test methods for detection and quantitation of orellanine. We have developed two diagnostic test methods based on HPLC and LC-MSMS for identification and quantitation of orellanine in mushrooms. Using these methods, we have identified Cortinarius armillatus as a novel orellanine-containing mushroom in North America. The mean toxin concentration of 145 ug/g was <1% of that of the more toxic C. rubellus. The HPLC method can detect orellanine at 17 µg g(-1) while the LC-MSMS method is almost 2000 times more sensitive and can detect orellanine at 30 ng g(-1). Both tests are quantitative, selective and are now available for veterinary diagnostic applications.

Suspected mycotoxicosis in laying hens.

A flock of 15,000 leghorn-type hens experienced a 10% drop in egg production. Enlarged, blue combs and diarrhea were also present. Clinical signs, postmortem and histopathological lesions, and response to therapy were compatible with a diagnosis of mycotoxin intoxication. However, all feed samples submitted were negative for mycotoxins, so the diagnosis could not be confirmed.

Prevention of aflatoxicosis by addition of hydrated sodium calcium aluminosilicate to the diets of growing barrows.

Hydrated sodium calcium aluminosilicate (HSCAS), an anticaking agent for mixed feed, was added to the diets of growing barrows and was evaluated for its potential to ameliorate the clinical signs of aflatoxicosis. The experimental design consisted of 6 treatments of 5 barrows each at concentrations of 0 g of HSCAS and 0 g of aflatoxin (AF)/kg of feed (control), 5 g of HSCAS/kg of feed (0.5%), 20 g of HSCAS/kg of feed (2.0%), 3 mg of AF/kg of feed, 5 g of HSCAS (0.5%) plus 3 mg of AF/kg of feed, or 20 g of HSCAS (2.0%) plus 3 mg of AF/kg of feed. Barrows were maintained in indoor concrete-floored pens, with feed and water available ad libitum for 28 days (from the age of 7 to 11 weeks). Barrows were observed twice daily and were weighed weekly, and blood samples were obtained weekly for hematologic and serum biochemical measurements. At the termination of the study, barrows were euthanatized and necropsied. Body weight gains were diminished significantly (P less than 0.05) by consumption of 3 mg of AF/kg of feed, whereas body weight gain in barrows consuming diets containing HSCAS or HSCAS plus AF did not differ from that in control barrows. Serum enzymatic activities of alkaline phosphatase and gamma-glutamyl transferase and prothrombin time were increased in barrows consuming 3 mg of AF/kg of feed, but not in those consuming HSCAS or HSCAS plus AF.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxicosis in cattle pastured in a field of sweet corn.

Aflatoxicosis was diagnosed in a small herd of cattle having access to moldy, unharvested sweet corn. Necropsy of 1 cow that died revealed anasarca and a pale tan liver. In this cow, microscopic examination revealed edema of all soft tissues and liver lesions consistent with aflatoxicosis. Samples of corn taken from the field contained 2,365 ng of aflatoxin/g of corn. Weather conditions were conducive to the formation of aflatoxins by Aspergillus flavus and A parasiticus.

Aflatoxicosis in Iowa swine: eight cases (1983-1985).

During 1983, 1984, and 1985, aflatoxicosis was diagnosed in 8 Iowa swine herds after the herds were fed corn from the 1983 corn crop. As a result of the diagnosis, the associated environmental conditions, clinical signs of aflatoxicosis, macroscopic and microscopic lesions, aflatoxin concentrations detected in feeds, and management of affected swine were reviewed. Concentrations of aflatoxin in shelled corn and complete feed were as high as 2,020 ng and 1,200 ng of aflatoxin (B1 and B2)/g of feed, respectively. Clinical signs of aflatoxicosis included decreased feed consumption and weight loss. Some pigs died acutely, but death often was preceded by a period of clinical disease. Greater morbidity and mortality were observed in swine herds that consumed greater concentrations of aflatoxin.

Toxic mushrooms.

Poisonous mushrooms contain toxins that are as diverse as the mushrooms themselves. Clinical syndromes often involve multiple organ systems, and progression of clinical signs is often directly related to the quantity eaten. Diagnostic detection of the toxins is rarely an option; rather, diagnosis is based on a history of possible exposure and identification of mushroom species in the stomach contents and environment. Treatments are usually based on clinical signs, as most mushroom toxins are without an antidote. There are exceptions, however, and prompt identification of mushroom species involved is vital whenever possible. Collection of the toxicologic minimum database and gastrointestinal decontamination are important in all cases where mushroom ingestion is suspected.

An experimental mycotoxicosis in sheep and goats caused by Drechslera campanulata, a fungal pathogen of green oats.

Field outbreaks of a syndrome of unknown aetiology associated with the grazing of green oats (Avena sativa) in the south-western Cape Province were characterized by diarrhoea, photosensitivity and death in goats and by diarrhoea and a reduction in milk production in cows. A phytopathogenic fungus, Drechslera campanulata, was isolated from conspicuous reddish-brown leaf spots on oat plants collected from both outbreaks. Pure cultures on autoclaved maize of D. campanulata isolates from oat leaves implicated in both field outbreaks, as well as a Canadian isolate, proved to be highly toxic to ducklings, goats and sheep. Characteristic clinical signs of the fatal mycotoxicosis caused by D. campanulata culture material in goats and sheep were anorexia, apathy, diarrhoea and ruminal stasis. Photosensitivity, however, was not induced. Necrosis of the forestomach mucosa was the most characteristic gross pathological change. Histopathological findings included mild focal erosions to severe, diffuse, coagulative necrosis of the mucosa in the rumen, reticulum and omasum and congestion and haemorrhages in the abomasum. These results provide circumstantial evidence that green oat leaves infected by D. campanulata may cause outbreaks of a mycotoxicosis in grazing animals.

Hepatogenous photosensitivity diseases in South Africa.

Various hepatogenous photosensitivity diseases of ruminants in South Africa, caused by plants, fungi and an alga, are described. Information is given on botanical, mycological, toxicological, clinical and pathological aspects of the diseases. The intoxications were grouped according to the primary site of involvement and type of lesions in the liver. The aetiology, pathogenesis, and diagnosis of these conditions received special attention and the most important features are illustrated in colour.

Granulocytopaenia and thrombocytopaenia in dairy cattle--a suspected mycotoxicosis.

During a 6-week period, 22 Dairy Shorthorn cows and heifers died with granulocytopaenia and thrombocytopaenia. Clinical signs observed in the affected animals included increased salivation, pyrexia, depression, rumenal stasis, bilateral epistaxis, melaena, increased bleeding after removal of retained foetal membranes and rapid weight loss. Despite intensive antibiotic and vitamin K therapy and blood transfusions, all affected animals died. The aetiological agent, thought to be a fungal toxin, could not be isolated from post mortem specimens or pasture samples.