Acute hemorrhagic syndrome by bracken poisoning in cattle in Belgium

In August 2007, two Belgian blue cows which had been on pasture for three months, showed high fever (41.4°C), epistaxis, melena, cutaneous bleeding, a stiff gait and red lesions on the udder. Blood examination revealed severe pancytopenia, and bluetongue virus serotype 8 could be demonstrated by PCR. Despite blood transfusion and supportive treatment, both animals died within 6 days after the initial symptoms. At necropsy, an explicit case of a blood coagulation disorder was observed. Inspection at pasture, one week later, showed the presence of numerous regenerated young fronds of Pteridium aquilinum. Whereas the stiff gait and the red lesions on the udder were likely bluetongue virus associated, other symptoms were consistent with acute bracken poisoning (acute hemorrhagic syndrome). The present report illustrates that also in Belgium, where the density of bracken fern is relatively low, pastures should be carefully screened for the presence of young fronds.


INTRODUCTION
Bracken fern (Pteridium aquilinum) is one of the most commonly distributed weed species in the world.In both ruminants and non-ruminants, the deleterious effects of the ingestion of fresh or dried plants have been known since a long time.In cattle, both acute poisoning, presenting as a hemorrhagic syndrome due to bone marrow depression and chronic poisoning, presenting as tumor development in the bladder (bovine enzootic hematuria) and the gastrointestinal tract have been frequently described in different parts of the world (Naftalin and Cushnie, 1951;Evans and Mason, 1965;Evans, 1968;Jarrett et al., 1978;Hopkins, 1986;Smith et al., 1988;Bertone, 1990;Xu, 1992;Marrero et al., 2001;Gava et al., 2002;Karimuribo et al., 2008;Roperto et al., 2010).In Belgium on the other hand, bracken fern is less ubiquitous, except on woodland soils.This might explain why the plant was not included in a review on animal poisoning in Belgium (Vandenbroucke et al., 2010).Moreover, to the authors' knowledge, this is the fi rst article describing a case of acute bracken poisoning in cattle in Belgium.

Case history
At the end of April 2007, three three-year-old Belgian blue cows were pastured in a forested area in the province of Limburg in Belgium.Three months later (at the beginning of August 2007), one of the cows showed epistaxis, high fever, melena and a stiff gait, upon which the three animals were stabled immediately.After an initial on-site treatment with trimethoprim-sulfadoxin (Borgal ® , Virbac), fl unixin meglumine (Finadyne ® , Intervet) and doramectin (Dectomax ® , Eli Lilly), the affected cow was referred to the Faculty of Veterinary Medicine of Ghent University for further examination.Five days later, a second cow developed identical symptoms, while the third cow remained clinically healthy.At that time, bluetongue virus serotype 8 (BTV8) was circulating in the region (Wilson et al., 2007).

Clinical examination
The cow was anorectic, depressed, in sternal recumbency, and showed signs of dehydration (sunken eyes).Epistaxis from both nostrils (Figure 1), streaks of blood in the neck and fl ank regions (insect bites), scleral ecchymosis and petechial bleeding on the conjunctivae and gingival mucosae were obvious.On the udder and teats, red lesions were observed.The animal was severely pyretic (41.4°C), and had an elevated pulse and breathing rate.Regarding the gastro-intestinal tract, ruminal contractions were absent, and melena was observed.

Blood examination
Jugular plasma (ethylenediaminetetraacetic acid (EDTA) and citrate) and serum samples were collected for standard hematology, coagulation and biochemistry tests as well as for the detection of BTV8 (PCR) and bovine viral diarrhea virus (BVDV) (ELISA) antigen.

Treatment and further evolution
Upon arrival, an indwelling catheter was placed in the jugular vein, and a blood transfusion (eight liters) was performed.Further treatment consisted of perfusion with isotonic saline, cefquinome (1 mg/kg IV, Cobactan ® , Intervet) and fl unixin meglumine (2 mg/kg IV, Finadyne ® , Intervet).After this treatment, an initial improvement of the clinical condition and the packed cell volume was observed.However, four days later, a second blood transfusion was required, which was followed by an intravenous administration of dexamethasone (Rapidexon ® , Eurovet) in order to control vasculitis.Despite this therapy, the cow died during the following night (six days after the onset of the symptoms).
The second cow received a blood transfusion and antimicrobial and anti-infl ammatory drugs on the farm, but also died four days after the initial symptoms.The third animal did not develop any symptoms.

Post mortem examination
Necropsy demonstrated an explicit case of a blood coagulation disorder, with hemorrhagic content in the  abomasum as well as in the small and large intestines.Furthermore, numerous petechial hemorrhages were observed on the serosae of the abdominal organs.The rumen contained no plants that could be identifi ed with confi dence.After necropsy, no further histological analyses were performed.

Inspection of the pasture
Based on the clinical examination and hematology, bracken fern poisoning was suspected.Upon request, it was reported by the farmer that bracken fern was indeed present next to the pasture but not on the pasture.However, the inspection one week after the animals were stabled, revealed numerous young fronds within the enclosure of the pasture (Figure 2).Outside the fence, adult plants were ubiquitous, suggesting that the two affected cows had been ingesting the young fronds systematically.

DISCUSSION
Bracken fern (Pteridium aquilinum) belongs to the family of the Dennstaedtiaceae, and is an ancient and large (height: 0.5-4.0m) plant (Figure 3).The name refers to the double-headed eagle that can be recognized at cross-section of the stem (Figure 4).Bracken is distributed worldwide, except in very cold or dry regions.In Belgium, its distribution is mainly localized in forested areas, where plants can concentrate on the borders of pastures (Van Genderen et al., 1996).Bracken is a perennial fern that produces spores by the end of the third or fourth growing season.If spores have been shed under favorable conditions, new young plants will appear 6-7 weeks later.All parts of the plant, including the fronds and the rhizomes (or rootstocks), are toxic, and drying does not remove the toxicity (Osweiler, 1996).Rapid regeneration of the plant is observed in response to disturbances, such as cutting and fi re (Nicholls, 2001), suggesting a similar expansion after grazing.
The toxic substances are cyanogenic glycosides (mainly prunasin), a vitamin B 1 -decomposing enzyme (thiaminase) and factors with carcinogenic activity (particularly ptaquiloside) (Fenwick, 1988;Vetter, 2009).The cyanogenic glycosides have only very seldom led to cyanide poisoning in animals (Fenwick, 1988), while thiaminase induces a vitamin B 1 -deficiency in non-ruminants.Horses are particularly susceptible (Evans et al., 1951;Kelleway and Geovjian, 1978), while reports of acute bracken intoxication in pigs are less common, except in young animals after long term exposure (Harwood et al., 2007).Affected animals initially show anorexia and ataxia, followed by convulsions and death.Ruminants on the other hand are generally resistant, due to the synthesis of vitamin B1 by the ruminal microbiological fl ora.However, polioencephalomalacia associated with vitamin B 1 -defi ency has been experimentally induced in sheep using bracken fern rhizomes (Bakker et al., 1980).In cattle, the chronic uptake of the carcinogenic sesquiterpene glycoside ptaquiloside is responsible for the development of bladder tumors (referred to as bovine enzootic hematuria) and intestinal tumors (van der Hoeven et al., 1983;Smith et al., 1988;Potter 5).Its carcinogenicity is realized by the alkylation of DNA, resulting in ptaquilosin-DNA adducts (Prakash et al., 1996).Moreover, the association with the presence of bovine papillomaviruses (type 2 and type 4) has been suggested (Jarrett et al., 1978;Campo et al., 1992;Borzacchiello et al., 2003).The etiological agent responsible for acute bracken poisoning in cattle, causing acute hemorrhagic syndrome, has not been identifi ed yet, and is therefore described as aplastic anemia factor (Blowey and Weaver, 2003).However, ptaquiloside has been suggested by Hirono et al. (1984) and Radostitis et al. (2007), hence applying the term osteomyelotoxic ptaquiloside poisoning.Furthermore, it has been described that ptaquiloside concentrations in bracken vary among geographic locations and that the concentrations are related to the occurrence of both acute and chronic bracken poisoning in cattle (Smith and Seawright, 1995;Rasmussen et al., 2008).Also in sheep, long-term exposure to ptaquiloside induces tumors in the bladder, as well as bright-blindness, caused by progressive retinal degeneration (Hirono et al., 1993;Potter and Baird, 2000).Goats on the other hand are the only livestock that appear unsusceptible.Besides the toxic effects in animals, bracken has been reported to be harmful to human health.Primarily, the inhalation and subsequent ingestion of spores can be hazardous, as it has been shown that spore extracts can damage DNA and in this way exert carcinogenic effects (Povey et al., 1996;Simán et al., 2000).Secondly, the passage of ptaquiloside into milk from bracken fed cows has been demonstrated (Alsonso-Amelot et al., 1996).However, this risk should be reduced by dilutions during dairy production (Wilson et al., 1998).In geographically remote rural areas on the other hand, where locally produced milk is consumed, a larger incidence of gastric and oesophageal cancers has been reported (Villalobos-Salazar, 1989).
Cattle generally only graze bracken in the absence of more suitable feed, although it has been observed that individual animals can develop a taste for the plant (Anonymous, 2005).Young shoots and fronds are particularly preferred, while these parts accumulate some of the toxic substances (Fenwick, 1988;Gil da Costa et al., 2012).Furthermore, Hopkins (1990) noted considerably greater numbers of bracken intoxication in years with prolonged drought.Likewise, the month in which the three animals were placed on the pasture (April 2007) has been confi rmed to be the driest month in Belgium since the commencement of the observations of the Royal Meteorological Institute.These data suggest poor grass availability in this period.
The toxic effects of bracken fern appear to be cumulative and symptoms of acute bracken poisoning are observed one to three months after the transfer of the herd to a bracken-infested pasture, and even up to two weeks after the animals are removed from this pasture (Evans, 1968;Xu, 1992;Anonymous, 2005;Vetter, 2009).Data on the amount of ingested bracken during this period vary from 50 to around 100% of the body weight of the animal, indicating that the uptake of small amounts are negligible (Seifert, 1996;Radostits et al., 2007;Anjos et al., 2009;Vetter, 2009).The development of bovine enzootic hematuria and bladder tumors on the other hand requires a long-term bracken uptake, ranging from 225 to 550 days, depending on the provided amount of bracken (0.5-2 kg per day) (Fenwick, 1988;Vetter, 2009).
The symptoms of acute hemorrhagic syndrome include weakness, anorexia, high fever (40.5 to 42.5°C), breathing diffi culties and multiple hemorrhages.Furthermore, sudden death is occasionally observed (Blowey and Weaver, 2003).A bracken-induced depression of the bone marrow sequentially leads to severe leukopenia (particularly granulocytopenia) and thrombocytopenia, explaining the hemorrhages and the subsequent anemia (Naftalin and Cushnie, 1951;Fenwick, 1988;Xu, 1992;Anjos et al., 2009).Besides an acute bracken fern intoxication, blood coagulation disorders and pancytopenia can be caused by BVDV type II, auto-or allo-immune bone marrow destruction or any acute septicemic process (Braun et al., 1996;Ridpath et al., 2006;Pardon et al., 2010;Pardon et al., 2011).Based on the negative outcome of the BVDVantigen-test, the case history, the clinical signs, the blood results, the necropsy fi ndings and the inspection of the pasture, the most probable diagnosis for the two animals in this case was bracken fern intoxication.All symptoms described in the literature are consistent with the fi ndings in the two affected cows, except for the stiff gait and the red lesions on the udder and teats.These symptoms are most likely due to the concurrent BTV8 infection (Thiry et al., 2006;Elbers et al., 2008;Brenner et al., 2011).BTV8 induces fever as well, although the high fever observed in this case (41.4°C) was rather the consequence of the severe leukopenia, which resulted in a higher suscep- tibility to infections (Fenwick, 1988;Anonymous, 2005;Gil da Costa et al., 2012).The elevated liver enzymes can refl ect bracken-induced liver degeneration (Xu, 1992), while the increase in muscle enzymes can be explained by the prolonged decubitus.The observed hyperbilirubinemia and uremia on the other hand can be clarifi ed by the dehydrated condition of the animal, whereas hypokalemia could be the consequence of anorexia.
The prognosis of acute hemorrhagic syndrome is generally poor, and most animals (> 90%) die within 1-10 days after the onset of the symptoms (Anonymous, 2005;Radostitis et al., 2007;Anjos, 2009).Treatment with the frequently reported antidote for bovine bracken fern toxicosis, DL-batyl alcohol, is of limited value to stimulate the bone marrow, especially in clinically advanced cases, and is of course prohibited in food producing animals (Osweiler, 1996).Blood or platelet transfusion from healthy animals can be useful, yet large volumes are required (minimum of 2-4 l blood).Antimicrobial agents can be administered to prevent secondary infections, and the animals should be removed from the infested pasture (Plumlee, 2004).
Prevention is primarily based on the exclusion of animals from bracken-infested pastures or at least to limit the duration of grazing (alternated grazing on bracken-contaminated and non-contaminated pastures at three-week intervals) (Pinto et al., 2004;Plumlee, 2004).The control of bracken is diffi cult for a variety of reasons, including the extensive rhizome system (Stewart et al., 2008).Several techniques are available, such as herbicide application, cutting, rolling and burning (Stewart et al., 2007).Stewart et al. (2008) confi rmed that cutting twice a year (in June and in August) is generally the most successful treatment in the UK, although the use of the herbicide asulam (Asulox ® , United Phosphorus Limited) has been suggested to be effective as well.Besides the application of asulam by hand-operated sprayers, it can be used by ground-based vehicles and helicopters, due to its high specifi city to bracken.However, multiple treatments with asulam are necessary, and 100% control is only rarely achieved (Stewart et al., 2007;UPL, 2008).For this reason, UPL (2008) advises to retreat surviving plants as soon as they are fully expanded either in the year following the initial application or, more likely, in the second year.Besides the different possibilities to control bracken, it is important to inform the animal owners regarding its toxicity and to provide animals with suitable feed, particularly during dry periods.
In conclusion, the present report illustrates that despite the low density of bracken fern in Belgian pastures, lethal bracken poisoning can occur when the available forage is reduced by drought.As systematic grazing masks the presence of bracken, profound inspection of the pasture is advisable prior to the transfer of the herd.

Figure 1 .
Figure 1.Epistaxis in a three-year-old Belgian blue cow suffering from coagulation disorders due to acute bracken fern poisoning.

Figure 2 .
Figure 2. Adult plants of Pteridium aquilinum outside the pasture enclosure and regrowth of fronds within the enclosure observed one week after stabling of the animals.

Figure 4 .
Figure 4. Cross-section of the stem of bracken fern: a manner to identify Pteridium aquilinum (Prof.Dr. Verbeken).

Figure 5 .
Figure 5.The structure of ptaquiloside, one of the toxic substances of bracken fern (Pteridium aquilinum).