DNA barcoding as new diagnostic tool to lethal plant poisoning in herbivorous mammals.

Reliable identification of plant species in the digestive tract of a deceased animal often represents the major key to diagnose a lethal intoxication with poisonous plants in veterinary pathology. In many cases, identification of the species is challenging or even impossible because the diagnostic morphological features have been degraded, and because the interpretation of such features requires a considerable expertise in plant anatomy and biodiversity. The use of DNA barcoding markers can support or even replace classical morphological assessment. While these markers have been widely used for plant taxonomy, their forensic application to clarify causes of animal poisoning is novel. In addition, we use specific single-nucleotide polymorphisms as fingerprints. This allows for a clear decision even in cases, where the conventionally used statistical e-values remain ambiguous. In the current work, we explore the feasibility of this strategy in a couple of exemplary cases, either in concert with anatomical diagnostics, or in cases where visual species identification is not possible, or where chemical toxin detection methods are not well established, complex, time consuming and expensive.

Identification of lamb flocks susceptible and resistant against Brachiaria poisoning / Identificação de rebanhos de cordeiros suscetíveis e resistentes a intoxicação por braquiária

This study was designed to assess the influence of genetic resistance against brachiaria poisoning in sheep. Two groups of sheep, one identified as susceptible (formed by two ewes and one ram) and the other as resistant against brachiaria poisoning (formed by three ewes and one ram) were selected. Sheep considered susceptible were those that presented clinical signs of brachiaria poisoning at any time of their life; resistant sheep were those that even raised on Brachiaria spp. pastures, did not developed any sign of the poisoning during their life. The offspring of the two flocks (15 lambs from the sensitive flock and 9 lambs from the resistant flock) were placed into brachiaria pasture (initially Brachiaria decumbens and B. brizantha,and only B. decumbens after weaning) and followed up during two years (2013-2014). The determination of protodioscin levels in B. decumbens pasture was performed only in 2014 and revealed significant amounts of the toxic principle. Eleven lambs of the susceptible group were affected to some degree of brachiaria poisoning and six died; no lamb of the resistant group was affected. Clinical signs consisted of varying degrees of subcutaneous edema of the face and, erythema and loss of hair of the ears, crusts on the skin of ears, around the eyes and on planum nasale, scar deformation of the ears, and bilateral ocular discharge; affected lambs also sought for shadowy shelters and they were poor doers. Several sheep recovered from the condition and then relapsed. Necropsy findings in six lambs included pale mucous membranes, emaciation, dermatitis, scar deformation of the ears, large yellow livers with marked lobular pattern, and moderate infestation by Haemonchus contortus...

Este estudo avaliou a resistência genética na ocorrência de intoxicação por braquiária em ovinos. Foram selecionados dois grupos de ovinos, um identificado como suscetível (formado por duas ovelhas e um carneiro) e o outro como resistente (formado por três ovelhas e um carneiro). Foram considerados suscetíveis ovinos que apresentaram sinais de intoxicação por Brachiaria spp. em algum ponto de suas vidas e resistentes aqueles ovinos que, mesmo criados em pastagem de braquiária, nunca desenvolveram qualquer sinal da intoxicação. A progênie desses dois grupos (15 cordeiros do grupo suscetível e 9 no grupo resistente) foi colocada numa pastagem de braquiária (inicialmente Brachiaria decumbens e B. brizantha e, após o desmame, apenas B. decumbens) e acompanhada durante dois anos (2013-2014). A determinação dos níveis de protodioscina em B. decumbens foi realizada apenas em 2014 e foram encontradas quantidades significativas do princípio tóxico. Onze cordeiros do grupo suscetível foram afetados por algum grau de intoxicação por braquiária; nenhum cordeiro do grupo resistente foi afetado. Os sinais clínicos consistiam de graus variáveis de edema subcutâneo da face e eritema e alopecia da pele das orelhas, crostas na pele das orelhas e ao redor dos olhos e no plano nasal, retração cicatricial das orelhas, fotofobia e corrimento ocular bilateral. Três cordeiros apresentaram desenvolvimento retardado. Vários cordeiros se recuperaram da condição, mas posteriormente quando foram colocados na pastagem apresentaram recidivas. Achados de necropsia em seis cordeiros incluíam mucosas pálidas, pobre condição corporal, dermatite, deformação cicatricial das orelhas, fígado aumentado de volume, amarelo e com padrão lobular evidenciado e graus moderados de infestação por Haemonchus contortus...

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.

Sleep time following anesthesia in mouse lines selected for resistance or susceptibility to fescue toxicosis.

In previous work, a mouse line selected for resistance (R) to fescue toxicosis had higher activities of two hepatic Phase II detoxification enzymes than a mouse line selected for fescue toxicosis susceptibility (S). The primary objective of the present study was to determine whether those same lines also differed in hepatic Phase I enzyme activity, estimated from sleep time (ST) following sodium pentobarbital anesthesia. Additional objectives were to determine whether ST differences between lines were modulated by endophyte-infected fescue in the diet (with or without an enzyme inducer) and whether ST of individual mice was correlated with the effect of a toxin-containing diet on the postweaning growth of those mice. In Exp. I, 24 males from each line were randomly assigned to each of five diets: control (commercial rodent food meal); E+ (50% endophyte-infected fescue seed, 50% control); E+P (the E+ diet supplemented with 1,000 ppm phenobarbital); E- (50% endophyte-free fescue seed, 50% control); and E-P (the E- diet supplemented with 1,000 ppm phenobarbital). After 4 wk on these diets, ST was measured on all the mice. A second ST was recorded on each mouse by randomly sampling one-fourth of the population after 1, 2, 3, or 4 wk on a pelleted rodent food diet. Regardless of diet, R mice had shorter first and second ST than S mice (P < 0.01), suggesting higher hepatic Phase I microsomal enzyme activity. Mice on both phenobarbital-supplemented diets had shorter first ST than mice whose diets did not include that microsomal enzyme inducer (P < 0.01). In Exp. II, ST was measured on male and female R and S mice (n = 280) after they had been fed the E- diet for 2 wk, then the E+ diet for 2 wk, and then a pelleted rodent food diet for 2 wk. Growth response to the E+ diet was the percentage of reduction in gain on the E+ diet compared to gain on the E- diet the previous 2 wk. As in Exp. I, S mice slept longer than R mice (P < 0.01). The residual correlation between ST and gain reduction associated with the E+ diet equaled 0.04. Thus, an animal's apparent Phase I enzyme activity did not predict its growth rate depression on the toxin-containing diet. Based on these and previous studies, divergent selection for toxicosis response in mice was successful partially by causing divergence in activities of hepatic Phase I and II detoxification enzymes.

Inbreeding and reproduction in mice divergently selected for response to a dietary toxin.

This study was conducted to determine whether inbreeding coefficients of selected parents or of progeny differed between lines of mice selected for increased or decreased responsiveness to a nutritional toxicosis. A second objective was to determine whether the influence of inbreeding of parents and/or progeny on reproductive traits differed between those lines. Mice were selected divergently for 8 generations for the effect on post-weaning growth of endophyte-infected fescue seed in their diet. Forty pairs (or in Generation 7, 20 pairs) were selected and mated per generation in each line. Inbreeding increased 0.5 to 0.6% per generation in both lines, a rate close to that predicted from genetic theory. Inbreeding coefficients of selected parents were not higher in the susceptible than in the resistant line. A difference would have been expected if the inbreeding coefficient had been correlated with susceptibility to toxicosis. The magnitudes of inbreeding depression for reproductive traits did not differ significantly between lines. The average inbreeding coefficient of the potential litter tended to be higher in nonfertile than fertile matings (P = 0.10), but inbreeding coefficients of sires and dams did not differ between successful and unsuccessful matings. Inbred litters tended to be born earlier than noninbred litters (P = 0.10). Inbred dams produced smaller litters than noninbred dams (main effect P < 0.05) but only when the litter also was inbred (interaction P < 0.01). Sex ratio was not influenced by inbreeding of sire, dam or litter, but there was a higher proportion of male progeny in the susceptible than in the resistant line (P = 0.01). To avoid reduced reproductive fitness, laboratory animal populations should be managed to minimize inbreeding of progeny and dam.

Increased IL-5 and IL-10 transcription in bronchial cells after Sauropus androgynus ingestion.

An outbreak of rapidly progressive obstructive lung disease in Taiwan was noted in some patients regularly ingesting the vegetable Sauropus androgynus because of its perceived weight reducing properties. Bronchoalveolar lavage (BAL) prior to administration of medication was performed in two women who had regularly ingested this vegetable and had developed obstructive ventilatory defects. Both patients showed a significant increase in neutrophils and, to a lesser extent, of eosinophils in the lavage fluid as compared to two unrelated controls. Interleukin (IL)-1 beta, IL-2, IL-5, IL-10, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha in cells recovered from BAL fluid were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Augmented expression of the IL-10 gene was detected in only two patients who had regularly ingested S. androgynus. Our results suggest that altered cytokine expression and infiltration of eosinophils and neutrophils may be involved in the pathology of obstructive lung disease caused by regular ingestion of S. androgynus.

Variation among inbred and linecross mice in response to fescue toxicosis.

Variation in response to fescue toxicosis was examined in inbred and linecross mice. In Exp. 1, exposure to a 50% endophyte-infected tall fescue diet (E+) reduced ADG of males from six inbred lines, but ADG of males from one line was modestly higher on E+. Lines differed (P < .01) for reproductive organ weight, but the diet x line interaction was not significant. In Exp. 2, an apparently susceptible (C57) and an apparently resistant line (FVB) were mated to produce inbred and linecross offspring. The reduction in weight gain caused by the E+ diet did not differ significantly among the genetic groups. In Exp. 3, C57 and C57 backcrosses had smaller reductions in ADG during E+ vs control feeding periods than FVB and FVB backcrosses (P < .10). In Exp. 4, the E+ diet reduced litter size of mates of C57 males by one pup, whereas litter size of mates of FVB males was four pups larger (interaction P = .07). Neither diet, line, nor their interaction affected male reproductive organ weights or tissue proportions in testis cross-sections. In Exp. 5, the E+ diet did not affect weight gain of C57 or FVB males, but effects of the E+ diet on litter size of mates were similar to those in Exp. 4. Percentage of abnormal sperm was increased in C57 males on the E+ diet but decreased in FVB males (Exp. 5). Differences among inbred lines in susceptibility to fescue toxicosis may depend on severity of the challenge and life cycle stage when the challenge is imposed.

Differences between progeny of beef sires in susceptibility to fescue toxicosis.

Fifteen progeny of two Polled Hereford sires were fed endophyte-infected tall fescue seed (E+) to investigate variability in susceptibility to fescue toxicosis. One sire, bred in Missouri, was reputed to produce calves that were resistant to fescue toxicosis. The Control sire, from Virginia, had unknown merit for susceptibility. There were two phases in which E+ was included in the diet (2 and 4) and three in which it was not (1, 3, and 5). Voluntary feed intake, serum prolactin, cholesterol, and alkaline phosphatase concentrations; ability to maintain homeothermy (afternoon minus morning rectal temperature); and heat-transfer inefficiency (afternoon rectal minus afternoon surface temperatures) were monitored. Intake and prolactin concentration were decreased by E+, but sire groups did not differ in responses across phases. Cholesterol levels were lower during E+ phases, and the rate of increase in cholesterol concentration after withdrawal of E+ differed between groups. Alkaline phosphatase concentrations were lower during E+ phases; calves from the Control sire showed greater reduction than calves sired by the Missouri sire. Ability to maintain homeothermy was reduced by E+, but the reduction did not differ between sire groups. Heat-transfer inefficiences were not indicative of fescue toxicosis in this study. Evidence was not compelling to support the existence of differences between the sire groups for susceptibility to toxicosis. However, progeny of the Missouri bull had higher feed intake and lower rectal and body surface temperatures through nearly all phases of the trial. This may account for the reputation of the Missouri bull to sire calves with relatively high resistance to fescue toxicosis.