Ocurrence enzootic hematuria in buffaloes in Brazil: epidemiological, clinical, and pathological aspects / Ocorrência de hematúria enzoótica em búfalos no Brasil: aspectos epidemiológicos, clínicos e patológicos

It is reported the occurrence of enzootic hematuria (EH) in buffaloes in Brazil after performing an epidemiological survey and clinicopathological analises. To date, EH caused by ingestion of Pteridium esculentum subsp. arachnoideum, a radiomimetic plant popularly known as "bracken fern", has not been described in this species in Brazil. Bovine EH is responsible for high economic losses in Brazil's Southeast Region not only because of the deaths it causes, but also owing to its negative effect on productivity. In São José do Barreiro County, São Paulo, some farmers in areas with a high incidence of bovine EH have been replacing cattle with buffaloes, based on the premise that the latter would be more resistant to poisoning by ingestion of Pteridium spp. However, even though initial observations indicated that buffaloes are indeed less sensitive than cattle to the toxic principle of Pteridium spp., cases of hematuria in this species have been reported. According to preliminary date, EH only occurs in buffaloes over six years of age. Macroscopic examination revealed a thickened urinary vesicle mucosa, along with multiple foci of ulcerated, exophytic, verrucous, and pedunculated lesions. In one of the buffaloes studied, the bladder wall was ruptured and exhibited marked secondary inflammation. Histologically, neoplastic and non-neoplastic changes similar to those described in cattle poisoned by Pteridium spp. were observed. The neoplasms found included papilloma, carcinoma in situ, urothelial carcinoma (low and high grade), inverted, microcystic, and trabecular variants, urothelial carcinoma with divergent differentiation (squamous and glandular), squamous cell carcinoma, lymphangioma, hemangioma, and hemangiosarcoma. There was also coexistence of epithelial and mesenchymal neoplasms. Bovine papillomavirus particles were not detected by polymerase chain reaction in the bladder samples analyzed.

Descreve-se, através de levantamento epidemiológico e avaliação clínico-patológica, a ocorrência de hematúria enzoótica (HE) em búfalos no Brasil. Essa condição, causada pela ingestão da planta radiomimética Pteridium esculentum subsp. arachnoideum, conhecida popularmente como "samambaia" ou "samambaia do campo", até então não havia sido descrita nessa espécie no Brasil. Na Região Sudeste, a HE bovina é responsável por elevadas perdas econômicas, devidas não apenas aos óbitos, mas também em função da queda de produtividade. No município de São José do Barreiro/SP, alguns produtores de áreas com alta incidência de HE bovina, vêm substituindo os bovinos por búfalos, com base na premissa de que estes seriam mais resistentes à intoxicação. Embora, de acordo com observações iniciais, os búfalos realmente sejam menos sensíveis que os bovinos ao princípio tóxico de Pteridium spp., ainda assim, tem-se verificado a ocorrência de casos de hematúria nessa espécie. De acordo com o levantamento inicial, a HE só ocorre em búfalos com idade a partir de seis anos. Ao exame macroscópico, verificou-se a mucosa da bexiga espessa, com múltiplos focos de lesões ulceradas, exofíticas, papiliformes, verrucosas, pedunculadas. Histologicamente, foram observadas alterações neoplásicas e não neoplásicas semelhantes às descritas nos bovinos com HE. Entre as neoplasias foram encontrados papiloma, carcinoma in situ, carcinoma urotelial (baixo e alto grau), variantes invertida, microcística e trabecular, carcinoma urotelial com diferenciação divergente (escamosa e glandular), carcinoma de células escamosas, linfangioma, hemangioma e hemangiossarcoma. Ocorreu também coexistência entre neoplasias epiteliais e mesenquimais. Não foram detectadas partículas de papilomavírus bovino pelo teste PCR nas amostras de bexiga analisadas.

Fiddlehead Fern Poisoning: A Case Report.

Outdoor enthusiasts are at a high risk of poisonous side effects after ingestion of wild and raw edible fiddlehead ferns, such as the ostrich fern (Matteuccia struthiopteris) and bracken (Pteridium genus) species, in the United States and Canada. The acute onset of nonlethal side effects manifests with gastrointestinal signs and symptoms and can last from 24 h up to 3 d. This case report is the first to outline the presentation of ingestion of a wild fiddlehead plant in the Carrabassett Valley in Maine, as well as the supportive management for this concerning and self-limiting illness.

Bovine upper alimentary squamous cell carcinoma associated with bracken fern poisoning: Clinical-pathological aspects and etiopathogenesis of 100 cases.

Upper digestive tract (UDT) cancer is rare in cattle, however in Southern Brazil, the UDT squamous cell carcinomas (SCCs) are relatively common and have been associated with bracken fern consumption and the presence of papillomas. Although a theory of pathogenesis considers bovine papillomavirus type 4 (BPV-4) as a cofactor in the development of these SCCs, some aspects of the etiopathogenesis of this disease need to be more investigated. In fact, detection of BPV-4 in UDT papillomas is scarce in other regions of the world and has not been performed in Brazil. Therefore, this study had two aims: 1) to analyze the epidemiological, clinical and pathological aspects of 100 natural cases of SCCs in the UDT of cattle grazing on bracken fern (Pteridium arachnoideum) highly contaminated areas, investigating the associations between these parameters; and 2) to investigate the presence of papillomavirus DNA by polymerase chain reaction (PCR) in the UDT papillomas (n = 47) from 30 cattle that also had UDT SCCs. There were statistically significant associations between clinical signs and tumor localization in the UDT; between histological grade of differentiation and tumor localization; and a trend towards significant association between histological grade of differentiation and presence of metastases. The average age of cattle with oropharyngeal SCCs was 7.39 years, with statistically significant difference comparing to cattle with esophageal SCCs (8.6 years). No statistical association was observed among other clinical-pathological parameters (growth pattern and primary site of the tumor) analyzed. No BPV DNA was detected in papillomas by PCR. Therefore, these results suggest the possibility that papillomas of the UDT are not necessarily associated with BPV infection.

Multiple effects of bracken fern under in vivo and in vitro conditions.

Several toxic substances have been detected in plants which are responsible for animal and human diseases. Bracken fern (Pteridium aquilinum) is one example, widely distributed in many parts of the world. It is known to cause cancer in humans and other animals. In fact, man can be directly or indirectly exposed to the danger by consuming fern, contaminated water, milk, meat, and spore inhalation. Experimental studies have shown an association between bracken exposure and gastric cancer, and research has shown genotoxic and cytotoxic effects in vitro. This paper describes and reviews toxic, carcinogenic, genotoxic/cytotoxic, and immunomodulatory effects of bracken and included possible toxic agents. The chemistry of Ptaquiloside (PT) reactions is emphasized, along with bracken problems in livestock, possible pathways of exposure in man, and control for human health.

Intoxicação aguda por Pteridium arachnoideum e Pteridium caudatum em bovinos e distribuição das plantas em Mato Grosso / Acute poisoning by Pteridium arachnoideum and Pteridium caudatum in cattle and distribution of the plants in Mato Grosso

São descritos os aspectos epidemiológicos, clínicos e patológicos de um surto de intoxicação por Pteridium arachnoideum e Pteridium caudatum em bovinos no Estado de Mato Grosso. A distribuição dessas plantas no Estado, a intensidade de invasão de pastagens e alguns fatores associados à invasão das pastagens por Pteridium spp. são também descritos. Pteridium spp. foi observada em 83 propriedades de nove municípios de Mato Grosso e deste total, amostras de 22 propriedades foram coletadas para identificação taxonômica. Em 22 propriedades identifica-se P. arachnoideum e em duas dessas havia também P. caudatum. O desmatamento e a realização de queimadas parecem estar relacionados com a invasão de Pteridium spp. nas pastagens. Na propriedade em que ocorreu a doença, 306 bovinos foram introduzidos em uma pastagem formada por Brachiaria brizantha intensamente invadida por P. arachnoideum e P. caudatum e desses 22 bovinos adoeceram e morreram. Os principais sinais clínicos foram febre alta, apatia, fadiga, fraqueza e decúbito. Na necropsia havia graus variáveis de hemorragias em vários órgãos e cavidades e infartos ocasionais. Microscopicamente, a alteração mais importante consistiu em aplasia severa da medula óssea vermelha. Os achados epidemiológicos, clínicos e patológicos associados à identoificação taxonômica da planta fundamentam o diagnóstico de intoxicação aguda por P. arachnoideum e P. caudatum em bovinos. O crescente desmatamento e a frequente utilização de queimadas em Mato Grosso podem contribuir para que esta doença venha a ser uma importante fonte de prejuízos para a pecuária do Estado.

Epidemiological features, clinical signs and pathological findings of a spontaneous acute poisoning by Pteridium arachnoideum and Pteridium caudatum in cattle in the State of Mato Grosso, Brazil, are described. The plant distribution, magnitude and some aspects about the pasture invasion by P. arachnoideum and P. caudatum in the State are also described. Pteridium spp. were found in 83 farms from nine counties and 22 of them were submitted to taxonomic identification. P. arachnoideum was found in 22 farms and in two farms also P. caudatum. Deforestation and burning appear to be associated with pasture invasion by Pteridium spp. On the farm where the poisoning occurred 306 cattle were introduced into a pasture of Brachiaria brizantha intensely invaded by P. arachnoideum and P. caudatum. Twenty two cattle were poisoned by the plants and died. The main clinical signs were hemorrhages, high fever, apathy, fatigue, weakness and recumbency. Gross findings included variable degrees of hemorrhages in several organs and cavities with occasional infarcts. The most consistent histological finding was marked bone marrow aplasia. Epidemiological, clinical and pathological findings associated with the taxonomic identofication of the plant support the diagnosis of acute poisoning by P. arachnoideum and P. caudatum in cattle. The increasing rate of deforestation and frequent burning in Mato Grosso can contribute to this disease which becomes a major cause of economic losses to livestock production in the State.

The environmental and human effects of ptaquiloside-induced enzootic bovine hematuria: a tumorous disease of cattle.

In this review, we address the major aspects of enzootic bovine hematuria and have placed special emphasis on describing the etiology, human health implications, and advanced molecular diagnosis of the disease.Enzootic bovine hematuria (EBH) is a bovine disease characterized by the intermittent presence of blood in the urine and is caused by malignant lesions in the urinary bladder. This incurable disease is a serious malady in several countries across many continents. Accurate early-stage diagnosis of the disease is possible by applying advanced molecular techniques, e.g., detection of genetic mutations in the urine of cows from endemic areas. Use of such diagnostic approaches may help create an effective therapy against the disease.There is a consensus that EBH is caused primarily by animals consuming bracken fern (P. aquilinum) as they graze. The putative carcinogen in bracken is ptaquiloside(PT), a glycoside. However, other bracken constituents like quercetin, isoquercetin,ptesculentoside, caudatoside, astragalin, and tannins may also be carcinogenic.Studies are needed to identify the role of other metabolites in inducing urinary bladder carcinogenesis.The bovine papilloma virus is also thought to be an associated etiology in causing EBH in cattle. There is growing alarm that these fern toxins and their metabolites reach and contaminate the soil and water environment and that the carcinogen (PT)is transmitted via cow's milk to the human food chain, where it may now pose a threat to human health. An increased incidence of gastric and esophageal cancer has been recorded in humans consuming bracken ferns, and among those living for long periods in areas infested with bracken ferns.Although preliminary therapeutic vaccine trials with inactivated BPV-2 against EBH have been performed, further work is needed to standardize and validate vaccine doses for animals.

Expression of gap junction protein connexin 43 in bovine urinary bladder tumours.

The aetiopathogenesis of urinary bladder tumours in cattle involves prolonged ingestion of bracken fern and infection by bovine papillomavirus types 1 or 2 (BPV-1/2). The oncogenic activity of BPV is largely associated with the major oncoprotein E5. Gap junctions are the only communicating junctions found in animal tissues and are composed of proteins known as connexins. Alterations in connexin expression have been associated with oncogenesis. The present study investigated biochemically and immunohistochemically the expression of connexin 43 in samples of normal (n=2), dysplastic (n=3) and neoplastic (n=23) bovine urothelium. The tumours included 10 carcinomas in situ, five papillary urothelial carcinomas and eight invasive urothelial carcinomas. Normal and dysplastic urothelium had membrane expression of connexin 43, but this was reduced in samples of carcinoma in situ. Papillary urothelial carcinomas showed moderate cytoplasmic and membrane labelling, while invasive carcinoma showed loss of connexin 43 expression.

Morphological factors as indicators of malignancy of squamous cell carcinomas in cattle exposed naturally to bracken fern (Pteridium aquilinum).

Squamous cell carcinomas (SCCs) of the upper digestive tract (UDT) of cattle have been associated with chronic bracken fern (Pteridium aquilinum) toxicity and infection with bovine papillomavirus type-4. These tumours share some morphological similarities with human head and neck SCCs. In this study, morphological changes were correlated with the biological behaviour of 40 alimentary SCCs in cattle grazing on pastures with high bracken content. The majority of SCCs were localized to the cranial and caudal regions of the UDT (almost 45% each). More than 60% of the tumours were well differentiated and were found mostly in the cranial region. Metastasis occurred in 58% of the cases, mostly to regional lymph nodes. All poorly differentiated SCCs had evidence of metastasis. Morphological patterns characterized by islands and ribbons of neoplastic keratinocytes were more prominent in well differentiated SCCs. These patterns varied greatly in moderately differentiated SCCs. Poorly differentiated tumours were characterized by the presence of cellular aggregates and individual cells and these tumours had more marked desmoplasia. A significant positive association was established between lymphoplasmacytic inflammatory infiltration and tumour-associated tissue eosinophilia. Evaluation of argyrophylic nucleolar organizer regions (AgNORs) revealed higher proliferation indices in poorly differentiated tumours than in moderately or well differentiated lesions. There was significant correlation between the AgNOR index and histological grading. The morphological factors analyzed were all related to histological grading, which is the major factor predicting the biological behaviour of SCCs in cattle naturally exposed to bracken fern.

Intoxicação experimental aguda por samambaia (Pteridium aquilinum) em bovinos / Experimental acute poisoning by bracken fern (Pteridium aquilinum) in cattle

Este experimento foi delineado para investigar os seguintes pontos em relação à intoxicação aguda por samambaia (Pteridium aquilinum) em bovinos: 1) a intensidade da trombocitopenia em diferentes momentos da intoxicação e sua relação com possíveis déficits na hemostasia secundária, 2) a relação da neutropenia com as manifestações morfológicas de septicemia ocasionalmente observadas na necropsia, e 3) o mecanismo da anemia e sua relação com a perda de sangue, a vida média eritróide e a evolução da doença. As hastes superiores mais verdes de P. aquilinum foram administradas a quatro bovinos sem raça definida, com idade média de 1,5 ano e pesos entre 190-215 kg. Um bovino de idade e peso semelhantes foi usado como controle e, exceto por não ter recebido P. aquilinum, foi mantido nas mesmas condições que os outros quatro. Os quatro bovinos que receberam a planta morreram com quadro característico da intoxicação aguda por samambaia após receberem durante 53-58 dias, doses diárias de 8,0, 8,6, 10,2 e 10,6g/kg de peso corporal, que totalizaram, ao final do experimento, respectivamente, 112,7, 107,6, 85,7, 90,15 kg da planta, o que corresponde, respectivamente, a 59,3 por cento, 63,3 por cento, 47,4 por cento, 47,5 por cento da planta em relação ao peso dos bovinos. A doença caracterizou-se por febre de até 42,5 graus C e diversos graus de hemorragias observadas clinicamente, na necropsia e na histopatologia. A morte ocorria 6-7 dias após o início do quadro febril. As alterações hematológicas revelaram trombocitopenia e neutropenia acentuadas. Em dois dos quatro bovinos havia anemia leve. Não houve variações significativas nos tempos de coagulação dos bovinos intoxicados, quando avaliados os fatores de coagulação (secundária), excluindo-se assim a possibilidade da participação de distúrbios da hemostasia secundária na patogênese das hemorragias nessa intoxicação. A determinação dos produtos da degradação da fibrina no soro revelou dados conflitantes...

This experiment was design to address the following points in relation to the acute poisoning by bracken fern (Pteridium aquilinum) in cattle: 1) the severity of the thrombocytopenia in different stages of the intoxication and its relationship to possible deficits in the secondary hemostasis, 2) the relationship between neutropenia and the morphological signs of septicemia occasionally found at necropsy, and 3) the mechanism of anemia and its relationship with blood loss, medium life of erythrocytes and the progress of the disease. The fresh green upper parts of P. aquilinum were fed to four mixed breed calves with average age of 18 months and weights ranging from 190 to 215 kg. A calf of similar age and weight was kept together with the other four under the same conditions, except for the ingestion of P. aquilinum. The four fern-fed calves died with typical features of acute bracken fern poisoning after being fed with the plant for 53-58 days daily doses of 8.0, 8.6, 10.2, and 10.6g/kg body weight totaling at the end of the experiment, respectively, 59.3 percent, 63.3 percent, 47.4 percent, and 47.5 percent of bracken fern in relation to their body weight. The disease was characterized by fever up to 42.5 grades C and varying degrees of hemorrhages observed clinically, at necropsy and on histological examination. Death occurred 6-7 days after the onset of fever. The hematological changes consisted mainly of marked thrombocytopenia and neutropenia. Two of the four affected calves had mild anemia. The deficits in secondary hemostasis were mild in each case. There were no significant changes thus permitting to rule out the role of secondary hemostasis in the pathogenesis of the hemorrhages in the bracken fern poisoning. The measurement of fibrin degradation products in the serum showed conflicting results and did no allow for a solid conclusion regarding the role of disseminated intravascular coagulation in the pathogenesis of the hemorrhages...

A biological hazard of our age: bracken fern [Pteridium aquilinum (L.) Kuhn]--a review.

Bracken fern (Pteridium aquilinum) is the fifth most distributed common weed species of the world. Its ecological distribution is very wide, and the plant can grow and spread successfully on many types of soil. The cover of P. aquilinum is--in some cases--remarkable (e.g., in the United Kingdom). Bracken fern contains different poisonous agents: some cyanogen glycosides, factors (agents) of antithiamine character (thermolabile thiaminase and thermostable other compounds) and factors of carcinogenic activity (first of all ptaquiloside). This paper summarises and reviews different toxicological problems and poisonings caused by bracken fern in ruminants (cattle, sheep) and in non-ruminant animals (horses, pigs, rats, mice, etc.). The carcinogenic properties of the norsesquiterpene-type ptaquiloside make bracken fern a potent, living hazard. Recent investigations have shown that ptaquiloside pollution of different soil layers is a distinct possibility. Ptaquiloside may leach from the soil into the drinking water base. This ecotoxicological aspect seems to be the most hazardous phenomenon in relation to P. aquilinum and ptaquiloside. The carcinogenic effect of ptaquiloside is based on its hydrolysis, which leads to the formation of a dienon intermediate. It can produce DNA adducts, which are responsible for inducing carcinoma.

Investigation of a syndrome characterised by passage of red urine in smallholder dairy cattle in East Usambara Mountains, Tanzania.

A case-control study was carried out to investigate a syndrome in smallholder dairy cattle in East Usambara Mountains characterised by urination of clotted blood. Smallholder dairy farms with the problem (cases) were matched with nearest farms without the problem (controls). In total, 30 farmers from Mbomole (19), Shebomeza (9) and Mlesa (2) villages in Amani division participated in the study. Using a structured questionnaire, information on risk factors associated with conditions characterised by passage of red urine in cattle was collected. In addition, serum samples from 80 smallholder dairy animals were collected and submitted for serodiagnosis of leptospirosis and babesiosis by microscopic agglutination test (MAT) and an indirect enzyme-linked immunosorbent assay (ELISA), respectively. Laboratory analysis showed that the seroprevalence of leptospirosis and babesiosis was 21.3% and 46.3%, respectively and there was no significant difference between'case' and 'control' farms (P > 0.05), hence the occurrence of urination of clotted blood syndrome in Amani was not explained. However, bracken fern (Pteridium aquilinum) was found to be ubiquitous in the area, and also found to be widespread in all areas used as sources of animal fodder Given the presence and distribution of bracken ferns and clinical signs and post-mortem lesions described by informants, chronic bracken-fern poisoning is more likely to be associated with the syndrome affecting animals in the study area. However, further investigation is required to confirm this observation so that appropriate control strategies can be devised.

Molecular analysis of the fragile histidine triad (FHIT) tumor suppressor gene in vesical tumors of cattle with chronic enzootic hematuria (CEH).

The FHIT (fragile histidine triad) gene is a tumor suppressor gene known to be inactivated in many tumors including bladder tumors and is spanning FRA3B, a very active common fragile site in the human genome. We have recently isolated the bovine gene, and the aim of this study was to test whether FHIT presents altered expression patterns in vesical tumors of cattle with CEH (chronic enzootic hematuria). CEH is a common syndrome affecting Mediterranean cattle: clastogenic, mutagenic and cancerogenic substances released by the bracken fern (Pteridium spp) grazed by animals induce the formation of neoplastic lesions, among which bladder tumors have a high incidence. We analysed FHIT in 23 bladder tumors of CEH cattle looking at: 1) the methylation status of the CpG island comprising the promoter and part of exon 1; 2) the presence of altered FHIT transcripts; 3) the mRNA expression levels measured with a quantitative real time PCR (QRT-PCR) approach. Our results suggest that unlike in human tumors, FHIT in vesical tumors of CEH cattle is largely unmethylated. Furthermore, the same mRNA isoforms of FHIT were detected in tumors and in healthy tissues, including a novel isoform that was found in this study. Finally, QRT-PCR data did not reveal significantly altered expression profiles of FHIT transcripts. Further studies and larger sets of cases will be useful to confirm this finding, but the data seem to suggest that epigenetic modifications of FHIT and altered expression profiles are not a hallmark of bovine vesical tumors like they are in human tumors.

Bracken fern (Pteridium aquilinum) poisoning in cattle in southern Brazil.

Epidemiological data on Pteridium aquilinum intoxication in cattle the state of Santa Catarina were obtained by review of diagnostic records from 1987 to 2001. Of 3,407 necropsied cattle, 244 (7.16%) were diagnosed as intoxicated by Paquilinum; 122 of those were of the hemorrhagic form, 103 had tumors in the upper digestive tract, 19 were cases of chronic hematuria. Annual losses due to this intoxication in this State are estimated at 10,657 cattle. The highest incidence of the hemorrhagic form occurs in 1-3-y-old cattle between March and July, and most cases of tumors of the upper digestive tract affect cattle older than Sy. The highest incidence of digestive tract tumors is at the base of the tongue and pharynx, and the lowest frequency is in the rumen and esophagus. The large economic losses caused PaQuilinum in Santa Catarina call for improved control measures for the disease.