- Email: [email protected]
Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil
TOXCON4629_proof ■ 14 July 2013 ■ 1/9 Toxicon xxx (2013) 1–9
Contents lists available at SciVerse ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
1 2 3 q 4 5 a b b a a 6 Q3 C.H.S. De Oliveira , J.D. Barbosa , C.M.C. Oliveira , E. Bastianetto , M.M. Melo , 7 c c a a, * M. Haraguchi , L.G.L. Freitas , M.S. Xavier , R.C. Leite 8 a Escola de Veterinária, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Minas Gerais, Av. Antônio Carlos 9 6627, CEP 30123-970 Belo Horizonte, MG, Brazil 10 b Central de Diagnóstico Veterinário, Faculdade de Veterinária, Universidade Federal do Pará, Brazil 11 c Laboratório de Farmacologia, Instituto Biológico de São Paul, Brazil 12 13 14 a r t i c l e i n f o a b s t r a c t 15 16 Article history: The aim of this paper is to report the study of hepatogenous photosensitization in buf17 Received 7 January 2013 faloes during two outbreaks provoked by ingestion of Brachiaria decumbens in Minas Received in revised form 26 June 2013 18 Gerais state, Brazil. Ten young buffaloes in outbreak 1 and seven buffaloes in outbreak 2 Accepted 2 July 2013 19 were intoxicated by B. decumbens. Nine clinically healthy buffaloes raised under the same Available online xxxx 20 conditions as the sick animals served as the control group. All animals were subjected to 21 clinical examination, and serum was collected to measure gamma-glutamyl transferase Keywords: (GGT), aspartate aminotransferase (AST), direct bilirubin (DB), indirect bilirubin (IB) and 22 Buffalo total bilirubin (TB) as indicators of liver function and urea and creatinine as indicators of 23 Photosensitization renal function. Histopathology of liver fragments from five different animals was carried Brachiaria decumbens 24 out. During the outbreaks and every two months for one year, samples of grass from Saponin 25 paddocks where the animals got sick were collected for quantitative evaluation of the Protodioscin 26 saponin protodioscin, combined with observations of pasture characteristics and daily 27 rainfall. Clinical signs included apathy, weight loss, restlessness, scar retraction of the ears 28 and intense itching at the skin lesions, mainly on the rump, the tail head, neck and hin29 dlimbs, similar to the signs observed in other ruminants. Only the GGT enzyme presented 30 significantly different (P < 0.01) serum levels between intoxicated animals (n ¼ 17) and 31 healthy animals (n ¼ 9), indicating liver damage in buffaloes bred in B. decumbens pastures. Microscopy of the liver showed foamy macrophages and lesions of liver disease associated 32 with the presence of crystals in the bile ducts, which have also been found in sheep and 33 cattle poisoned by grasses of the genus Brachiaria. During the outbreaks, protodioscin 34 levels were higher than 3%, and shortly after, these levels were reduced to less than 0.80%, 35 suggesting a hepatic injury etiology. The outbreaks took place at the beginning of the rainy 36 season, and there was a positive correlation between saponin and the amount of rainfall, as 37 well as between saponin and the amount of green leaves in the pasture. These findings 38 indicate that the grass was more toxic in this period. This is the first report of photosen39 sitization by B. decumbens in buffalo. 40 Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. 41 42 43 44 45 46 q This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, 47 and reproduction in any medium, provided the original author and source are credited. * Corresponding author. Tel.: þ55 31 34092127; fax: þ55 31 34092080. 48 E-mail addresses: [email protected], [email protected] (R.C. Leite). 49
Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil
0041-0101/$ – see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.07.001
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99
TOXCON4629_proof ■ 14 July 2013 ■ 2/9 2
100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160
C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
1. Introduction Hepatogenous photosensitization is a disease that occurs when hepatotoxic substances – poisonous plants, mycotoxins, drugs or infectious agents – injure the liver sufficiently to prevent the excretion of phylloerythrin, which is a metabolite produced in the gastrointestinal tract as a result of chlorophyll metabolism. Phylloerythrin is a photodynamic agent that it is eliminated by the liver. When the liver is injured, phylloerythrin accumulates in the blood stream, allowing sunlight to cause skin lesions (Scheie et al., 2002; Smith, 2000). Clinical signs of photosensitization in cattle are characterized by erythema, wrinkling and crust formation over large skin areas, especially when the skin is not pigmented and is exposed to a high amount of sunlight, such as the back, loin and neck. Additional clinical signs are flank and caudal fold edema, loss of appetite, excitability, itching, tearing, polyuria, edema and scar retraction of the ears, keratitis and blindness, jaundice, weakness, dehydration and death. Thus, hepatogenous photosensitization causes vast economic losses due to production deficiencies (Stannard, 1994). Considering animals raised on Brachiaria spp. pastures, the etiology of hepatic injury is a major issue. In New Zealand, the mycotoxin sporidesmin produced by the fungus Pithomyces chartarum, which grows mainly in senescent plant material under high temperature and humidity, is the causative agent (Lancashire and Keogh, 1968; Smith, 2000). In Brazil, isolates of P. chartarum involved in photosensitization outbreaks failed to produce the mycotoxin, and cases have occurred in pastures with low counts of fungal spores, so the presence of steroidal saponins in the grass has been identified as the causative agent of hepatic injury (Brum et al., 2007; Meagher et al., 1996). Animals intoxicated by Brachiaria spp. develop cholangiohepatopathy lesions in association with crystalloid material deposition in the biliary system (Meagher et al., 2001). For crystal formation, a source of steroid substance in the animal’s diet is needed (Flayoen et al., 1991), as steroidal saponins present in Brachiaria spp. Saponins are steroid or polycyclic terpene glycosides characterized by the presence of a lipophilic portion (steroid or triterpene) and a hydrophilic portion (sugar). They are also broadly distributed in plants and are produced in the plant secondary metabolism (Schenkel et al., 2007). This type of intoxication has well-known effects in cattle, sheep and horses, but no report on this disease was found in buffaloes. Therefore, the aim of this study is to report the occurrence of two hepatic photosensitization outbreaks in buffaloes caused by Brachiaria decumbens ingestion in Minas Gerais state, Brazil, and to describe the clinical and pathological characteristics, to evaluate the saponin level in the pasture, and to assess liver and kidney serum biochemistry in these buffaloes.
neighboring farms (19 290 41.7400 S, 44 300 29.7500 W) which are located in the central area of Minas Gerais state. Both farms had the same owner, and the buffaloes belonged to the Murrah breed. The outbreaks occurred during the months of October and November 2009. Ten animals of outbreak 1 were examined, two with clinical signs of photosensitization and eight without clinical signs, all crated in the same paddock (paddock A). Seven animals of outbreak 2 were examined, all with clinical signs of photosensitization, and they were crated in two paddocks (paddocks B and C). A third group (control group) consisted of nine buffaloes without clinical or laboratory changes indicating deficiency of liver function, which belonged to another farm (19 260 30.1200 S, 44 280 26.1400 W) where the disease did not occur (paddock D). All animals were raised on B. decumbens pastures. All animals were subjected to clinical examination in accordance with Dirksen et al. (1993). Epidemiological data were obtained through a questionnaire applied to the owner and to the employees of the properties as well as through onsite observation. Data were collected regarding the animals’ breed, sex and age, and also about management details, the time of year and places where the disease had occurred, the number of animals in the batch, the grass species and pasture characteristics, the presence of toxic plants in the pastures, and outbreaks that had occurred on other occasions and which animal species had been affected by them. 2.2. Biochemical tests Using jugular venipuncture and a vacuum collection system, blood was collected to measure the serum levels of gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), direct bilirubin (DB), indirect bilirubin (IB) and total bilirubin (TB) as indicators of liver function, and urea and creatinine to assess renal function. Bilirubin was measured with a Doles Kit (Doles Reagents and Laboratory Equipment, Goiania, Brazil). All of the other measurements were performed using commercial Cepa Kits (MBiolog Diagnostics, Minas Gerais, Brazil). These measurements were performed according to the manufacturers’ recommendations and in accordance with their processing through a BIOPLUS-2000 semi-automatic biochemical analyzer. 2.3. Pathology tests Necropsy and histopathological examination of liver fragments from one buffalo in the terminal stage of the disease were performed, from outbreak 1 (paddock A). We also performed histopathology on liver biopsy specimens from four sick buffaloes from outbreak 2 (paddocks B and C). The fragments were fixed in 10% formalin, processed according routine histopathological methods, embedded in paraffin, cut at 5 mm thickness, stained with hematoxylin and eosin (HE) and analyzed with an optical microscope (Luna, 1968).
2. Materials and methods 2.4. Saponin study 2.1. Outbreaks Two hepatogenous photosensitization outbreaks in buffaloes were examined. These events took place in two
During the outbreaks (October 21, 2009 and November 6, 2009) and every two months for one year (January 26, May 25, July 1 and September 23, 2010), samples of B.
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221
TOXCON4629_proof ■ 14 July 2013 ■ 3/9 C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282
decumbens from the paddocks where the animals got sick were collected to quantify the saponin protodioscin. These data were combined with the observation of pasture characteristics and daily rainfall. The grass samples were collected with the hands, grabbing and pulling it out of random points, simulating a grazing buffalo. The grass height and general conditions of each paddock were estimated semi-quantitatively by adopting a score of þ, þþ, and þþþ, respectively, for a low (up to 30%), moderate (30–70%) and high amount (above 70%) of green leaves of B. decumbens (GL) and senescent material (SM) at the time of each gathering. The average daily rainfall was calculated in millimeters from daily rainfall data retrieved from Sete Lagoas Meteorological Station throughout the experimental period, i.e., from September 2009 to September 2010. The station was located 42 km away from both farms considered in this study (INMET, 2010). Protodioscin was quantified by high-performance liquid chromatography (HPLC) using evaporative light-scattering detection (ELSD) in accordance with a modified method proposed by Ganzera et al. (2001) (Wysocki et al., 2009). 2.5. Statistical analysis Data for all variables were analyzed using descriptive statistics. An error of 5% was considered to calculate mean values, standard deviation and the mean confidence interval length. The Mann–Whitney test was used to compare means, considering a 5% margin of error (P < 0.05) to distinguish the groups for the studied variables (Sampaio, 2010). Spearman’s correlation test was used to compare the values of the variable saponin with average daily rainfall and with pasture characteristics such as height, number of green leaves and amount of senescent material (Sampaio, 2010), considering the value of P < 0.05 as significant. All analyses were performed using the software SAEG-D, version 9.1. 3. Results Two photosensitization outbreaks in herds of Murrah buffaloes from the same genetic group were identified in two neighboring farms belonging to the same landowner. Both farms were located in the city of Fortuna de Minas, in the central region of Minas Gerais, Brazil. In outbreak 1, occurring on October 21, 2009, the batch containing 62 Murrah buffaloes aged 3–5 months had two female buffaloes (4 months old) with skin lesions on the rump, neck and limbs, areas that were clinically consistent with photosensitization. These animals were given water and mineral supplementation ad libitum and grazed in B. decumbens pastures that presented height of 25 cm during the outbreak, many green leaves and a moderate amount of senescent material. We observed no presence of poisonous plants in the paddock (paddock A). The owner reported the occurrence of similar cases in previous years. After two weeks, one animal died and the other one recovered. Eight other animals from the same batch did not show any clinical signs but underwent biochemical evaluation of the liver and kidneys.
3
Outbreak 2 was diagnosed 16 days later on the neighboring property in a batch of 226 buffaloes from the same breed and the same genetics. These victims were of both sexes, aged 6–8 months, recently weaned and fed with a barley-based dietary supplement due to low availability of pasture. The animals were placed in B. decumbens paddocks (paddocks B and C). These paddocks had been left at rest for more than 30 days and presented pastures with large amounts of buds and green leaves reaching approximately 50 cm above the soil and an absence of toxic plants. In addition, the occurrence of this disease had never been recorded in these paddocks. According to the owner’s report, 25 days after introducing these animals into paddocks B and C, six animals showed apathy, and deaths began to be observed in both paddocks. Some animals presented skin lesions. On our technical visit (November 6, 2009), six animals had died already and seven others had skin lesions (Fig. 1), which we examined. The animals were aged 5–7 months, 3 were males and 4 were females, and all of them were Murrah buffaloes. During clinical examination of all nine buffaloes in both outbreaks (2 from outbreak 1 and 7 from outbreak 2), we identified extensive dry areas, which were mummified in appearance with cracks and ulcerations, in addition to shedding, mainly on the rump (Fig. 2). We also detected crusted lesions over the tail head, hindlimbs, ears and back of the animals. Other symptoms were restlessness, intense itching, apathy, weight loss, eye discharge, scar retraction of the ears and dehydration. The necropsy performed on the buffalo with terminal illness showed a slightly yellowish carcass, and the liver exhibited a hardened consistency, hypertrophy, and rounded edges with a nutmeg pattern on its surface. The gallbladder had become quite distended (Fig. 3), with thick and lumpy bile. Liver histopathology revealed mild biliary stasis, artery hypertrophy, and incipient necrosis of the hepatocytes in the central-lobular area that was characterized by nuclear chromatin condensation and by cytoplasm appearing to be more eosinophilic than normal, as well as rare necrosis or lysed hepatocytes (Fig. 4). Diffuse hepatocellular swelling – from moderate to severe – light megalocytosis with individual hepatocyte necrosis and with intracytoplasmic eosinophilic globules were observed in the hepatic biopsy fragments from four animals that presented clinical signs. We also verified the presence of foamy macrophages in 2 animals and crystals in the bile ducts of one animal (Fig. 5). There was no significant difference in liver or kidney biochemical parameters between the animals from outbreak 1 (n ¼ 10) and outbreak 2 (n ¼ 7). Irrespective of the presence of skin lesions associated with photosensitization, these animals were grouped into a group poisoned by B. decumbens (n ¼ 17) and this group was compared with control group (n ¼ 9). Table 1 presents the mean values of GGT, AST, DB, IB, TB, urea and creatinine as well as their respective confidence intervals for the two groups of animals. Serum GGT was significantly greater in the intoxicated animal group (P < 0.01). However, despite the slightly higher values of other liver function variables in the intoxicated animals, these differences were not statistically significant (P > 0.05) (Fig. 6).
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343
TOXCON4629_proof ■ 14 July 2013 ■ 4/9 4
344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404
C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
Fig. 1. Buffalo featuring skin lesions due to hepatic photosensitization.
Regarding the steroidal saponins, the protodioscin percentage found in B. decumbens pastures during the outbreaks of photosensitization in buffaloes was high. This percentage was 3.24% for paddock A in the first outbreak, 3.26% and 3.54% for paddocks B and C, respectively, in the
second outbreak and 1.60% for paddock D, adopted as a control paddock (Table 2). Average daily rainfall analysis for the study area revealed that in September 2009, there was almost no rainfall, with a daily average of 1.83 mm. Thus, a much
Fig. 2. Buffalo presenting skin lesions due to hepatic photosensitization, characterized by dry areas, mummified appearance, with cracks and ulcerations, and shedding mainly on the rump.
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465
TOXCON4629_proof ■ 14 July 2013 ■ 5/9 C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526
5
Fig. 3. Enlarged liver and gallbladder from a buffalo intoxicated by Brachiaria decumbens.
higher rainfall average was observed in the month of October, 5.69 mm, coinciding with the development of hepatic photosensitization outbreaks in buffaloes and high levels of protodioscin in the pastures (Fig. 7). Rainfall averages were high until March 2010, when the dry period began. During this period, levels of saponin in all three paddocks where the outbreaks took place remained low (less than 0.80%) and did not significantly increase during the period between April and September 2010 (Fig. 7). The Spearman correlation test detected a positive correlation
Fig. 4. Incipient necrosis of hepatocytes (arrow) in the central-lobular area and rare necrotic hepatocytes or lysed hepatocytes. HE, obj. 25.
between daily rainfall and saponin level in the pasture, with a coefficient of 0.5893 and significance of P < 0.05. Regarding pasture characteristics, no significant correlation between saponin content and pasture height was identified. A positive correlation was detected between saponin and quantity of green leaves, and a negative correlation was detected between saponin and quantity of senescent material (Table 3). 4. Discussion and conclusion B. decumbens, Brachiaria humidicola and Brachiaria brizantha poisoning are reported in Brazil and elsewhere in the world, affecting cattle, sheep, goats and horses (Driemeier et al., 2002; Graydon et al., 1991; Lemos et al., 1998; Meagher et al., 1996; Motta et al., 2000; Smith and Miles, 1993; Stegelmeier, 2002). This is the first report of hepatic photosensitization outbreaks in buffaloes, which involved a large number of animals and the death and disposal of sick animals. In the reported outbreaks, photosensitization by B. decumbens affected buffaloes, which were the only species present on the studied properties. The farm owner reported that seven years ago, when there were sheep and goats on his property, photosensitization outbreaks affected most animals, especially young ones. According to Lemos et al. (1996), poisoning mainly affects young animals (lambs and calves), it may also occur in infant animals and in animals under 30 days old, which is consistent with what was observed in this study: the age range of affected buffaloes was 3–8 months. The clinical signs observed in both outbreaks were similar to those observed in cattle (Motta et al., 2000), but
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587
TOXCON4629_proof ■ 14 July 2013 ■ 6/9 6
588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648
C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
Fig. 5. Histological section of liver biopsy from a buffalo intoxicated by Brachiaria decumbens. Presence of crystals in the bile duct (arrow), light megalocytosis (arrowhead), foamy macrophages (white arrow) and moderate hepatocellular swelling. HE, obj. 40.
jaundice, a sign quite frequent in photosensitization in cattle, sheep and goats, was not found in this study. The signs observed in buffaloes that previously developed hepatogenous photosensitization by copper poisoning (Minervino et al., 2010) were similar to those observed in the current buffaloes poisoned by B. decumbens, especially when considering the areas affected by lesions, which were mainly the hindlimbs, tail, neck, and dorsal part of the back. Edema in the ears, around the eyes, and in the mandible, as well as keratitis and blindness, were not observed in this study. These findings differ from those of Albernaz et al. (2010) concerning sheep intoxicated by B. brizantha in Pará state, Brazil. Increased levels of hepatic enzymes are detected in diseases that evolve with liver injury (Lemos et al., 1997). This fact is of great value for the diagnosis of liver damage in animals poisoned by Brachiaria spp. According Dirksen et al. (1993), GGT displays increased activity in the cholestatic hepatic diseases, as observed in cases of photosensitization by Brachiaria spp. Comparing the serum Table 1 Confidence interval, means and standard deviations of GGT, AST, DB, IB, BT urea and creatinine in animals poisoned by Brachiaria decumbens (Intoxicated) and the control group (Control). Parameters
Group
Mean
Standard deviation
Confidence interval
GGT, U/L
Intoxicated Control Intoxicated Control Intoxicated Control Intoxicated Control Intoxicated Control Intoxicated Control Intoxicated Control
102.78* 14.19* 229.76 213.78 0.66 0.41 0.55 0.43 1.21 0.85 40.26 43.19 1.18 1.25
84.52 3.31 107.85 53.49 1.07 0.23 0.62 0.22 1.62 0.43 21.79 10.14 0.55 0.32
59.33–146.24 11.64–16.73 174.32–285.21 172.66–254.90 0.11–1.21 0.24–0.59 0.23–0.87 0.27–0.60 0.38–2.04 0.51–1.18 29.05–51.46 35.39–50.39 0.90–1.47 1.00–1.49
AST, U/L DB, mg/dL IB, mg/dL BT, mg/dL Urea, mg/dL Creatinine, mg/dL
*Significant difference between groups by Mann–Whitney test (P < 0.01).
biochemistry from intoxicated animals and apparently healthy animals, only GGT was significantly different between the two groups, so we chose it to assess the risk of hepatogenous photosensitization development in B. decumbens pastures. GGT is connected to the cell membrane and is located mostly in canaliculi, bile ducts and, to a lesser extent, in the hepatocytes. In hepatic diseases that evolve with cholestasis, serum GGT is elevated due to the membrane solubilization caused by bile salts that accumulate in the liver and in the ducts (Kaneko et al., 2008). AST and bilirubin were similar between the intoxicated animals and the control group, although the results were higher for the first group. Nonetheless, when the results were compared to normal parameters for cattle, both groups presented non-standard results, which shows the necessity of establishing normal biochemical parameters for buffaloes raised in the Brazilian farming system. The statistical similarity of total bilirubin and its fractions between the studied groups could explain the absence of jaundice in buffaloes poisoned by B. decumbens, contrary to previous results in other ruminants (Stannard, 1994). But the necropsy performed on the buffalo with terminal illness showed a slightly yellowish carcass, indicating that the jaundice can appears later in fatally poisoned animals. Alterations in kidney biochemistry parameters have been observed in sheep and cattle intoxicated by Brachiaria spp. (Albernaz et al., 2010). No similar result was found in the buffaloes in this study. Histological lesions observed in the livers of buffaloes with hepatogenous photosensitization were similar to those observed in other ruminants, including the presence of biliary stasis and crystals in the bile ducts. This finding confirms that photosensitization in animals that ingest Brachiaria spp. is associated with crystalloid material deposition in the bile ducts and cholestasis (Cruz et al., 2000; Lemos et al., 1997; Meagher et al., 2001; Pires et al., 2002). Foamy macrophages and hepatocellular swelling have been observed in buffaloes raised in Brachiaria spp. pastures in Pará state, Brazil, but they showed no clinical
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709
TOXCON4629_proof ■ 14 July 2013 ■ 7/9 C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770
7
importance of this injury in the pathogenesis of hepatogenous photosensitization is not fully understood (Tokarnia et al., 2000, 2002). During the outbreaks, the levels of protodioscin in B. decumbens were high, ranging from 3.24% to 3.54%, while in other periods these levels were lower than 0.80%. These data corroborate what was discovered in outbreaks of hepatogenous photosensitization in sheep in pastures with B. decumbens in Mato Grosso do Sul, Brazil, where the protodioscin content was 2.36% (Brum et al., 2007). Therefore, the intake of steroidal saponins from brachiaria pastures by buffaloes may be one factor triggering photosensitization in these ruminants. Brum et al. (2009) analyzed the concentrations of steroidal saponins in B. decumbens and B. brizantha at different stages of plant development, finding that the contents ranged from 0.5 to 2.1% and were higher in more developed plants. These results suggest that the grass is more toxic during its maturation phase and that photosensitization outbreaks in the midwest region of Brazil occur in pastures left at rest for more than 30 days. These conditions are similar to those described for the pasture involved in outbreak 2, which was at rest for more than 30 days and had plants in an advanced stage of development. Daily rainfall data confirmed the owner’s observations, who claimed the disease occurred at the beginning of the rainy season, which coincided with increased levels of protodioscin in the pasture. The occurrence of outbreaks during the growth of pastures at the beginning of the rainy season has also been reported for other ruminants (RietCorrea et al., 2007). The content of saponins and sapogenins in plants may vary depending on the place of cultivation, environmental factors, age and the stage of plant development (Brum et al., 2009; Meagher et al., 1996). In this study, although we detected a correlation between the amount of rainfall, green leaves and senescent material, these variables are not sufficient to explain the increased levels of saponins in the pasture. A more detailed study, including the combination
Fig. 6. Graphical representation of the means of variables of liver and kidney function from buffaloes intoxicated or not by Brachiaria decumbens. * Significant difference between groups by Mann–Whitney test (P < 0.01).
signs of photosensitization (Riet-Correa et al., 2010). Foamy macrophages and hepatocellular swelling were also observed in buffaloes with clinical signs in this study. The presence of foamy macrophages in the liver, lymph nodes and spleen of buffaloes and cattle kept on Brachiaria spp. pastures has been observed in Brazil. However, the
Table 2 Protodioscin levels (%) and pasture characteristics (height in cm, green leaves – GL, senescent material– SM) according to sampling dates over one year (10/ 21/2009–09/23/2010), in Minas Gerais, Brazil. Paddock
10/21/2009
11/06/2009
Pasture
Protodioscin
Height
GL
SM
A B C D
9.8
þþþ
þþ
Paddock
05/25/2010
Protodioscin
Height
GL
SM
25 50 50 17
þþþ þþþ þþþ þþþ
þ þ þ þ
Protodioscin
Height
GL
SM
20 40 45
þþ þþ þþ
þþ þþ þþ
0.41 0.41 0.8 nd
Pasture
Protodioscin
Height
GL
SM
1.82 3.26b 3.54b 1.6
30 45 40
þþþ þþþ þþþ
þþ þ þ
Protodioscin
Pasture
07/01/2010
Pasture
A B C D
3.24a nd nd nd
01/26/2010
Pasture
0.74 0.67 0.39 nd
09/23/2010
Pasture Height
GL
SM
25 40 50
þ þ þþ
þþþ þþþ þþþ
0.31 0.41 0.5 nd
Protodioscin
Height
GL
SM
17 22 25
þþ þþ þþ
þþþ þþþ þþþ
0.13 0.17 0.15 nd
nd: not determined. a Period of outbreak 1. b Period of outbreak 2.
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831
TOXCON4629_proof ■ 14 July 2013 ■ 8/9 8
832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892
C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 Fig. 7. Graphical representation of the average daily rainfall and levels of protodioscin saponin in three paddocks of Brachiaria decumbens (A, B and C) between October 2009 and September 2010. 912 913 914 propose that older animals that have adapted to the presof other variables with assessments of plant physiology, 915 ence of saponins in grass and are more resistant to photowould be needed to understand the behavior of these 916 sensitization. The owner and employees of the two substances. 917 properties considered in this study claimed to observe the Saponins are products obtained from plant secondary 918 disease only in young animals. metabolism. They function in the plant defense processes, 919 Saponins can kill or at least damage ruminal protozoa by and they can be produced in larger quantities under 920 complex formation with sterol membranes. However, this stressful situations, such as fungal and bacterial attack, 921 effect is transient, so the number of protozoa in the rumen ensuring survival benefits for the ecosystem to which the 922 of sheep returns to normal after 14 days of Enterolobium plant belongs (Wina et al., 2005). The first rains that 923 cyclocarpum feeding, a plant that also contains steroidal occurred in this outbreak, associated with rising tempera924 saponins (Ivan et al., 2004, cited Wina et al., 2005). Q1 925 ture and insolation, could have inflicted some type of stress on the grass, leading to the high content of protodioscin, as Albernaz et al. (2010) found that lambs fed with ruminal 926 previously suggested (Barbosa et al., 2009). fluid of adult sheep before being placed on B. brizantha 927 On the other hand, a photosensitization outbreak in pastures had lower levels of hepatic-injury enzymes and a 928 sheep in Pará state, Brazil, occurred during the less rainy lower rate of photosensitization in comparison to a control 929 season, and the pasture (B. brizantha) had reduced and group. Animals’ mechanisms of adaptation to steroidal sa930 senescent residual mass (Albernaz et al., 2010). However, ponins are not yet fully understood and deserve further 931 the levels of saponins methylprotodioscin and protodioscin investigation. 932 found by those authors in the pasture (0.92% and 0.88% In the first outbreak we studied, the morbidity rate was 933 during the outbreaks) were similar to the protodioscin 6.45%, and the mortality rate was 1.61%. In the second 934 levels observed in B. decumbens in this study outside of the outbreak, the morbidity rate was 5.75%, and the mortality 935 rainy season. These data may indicate that buffaloes are rate was 2.65%. Motta et al. (2000) and Riet-Correa et al. 936 more resistant than sheep to intoxication by saponins. (2007) reported that morbidity rates in cattle outbreaks 937 Many authors describe the occurrence of photosensitimay vary from 0.11% to 64%, and mortality can reach 14%, 938 zation in young animals, and we also observed photosenwhich agrees with our findings. 939 sitization mainly in young buffaloes. This result leads us to The involvement of just a few animals in these two 940 outbreaks of photosensitization, despite being exposed to 941 the same epidemiological conditions and having ingested 942 Table 3 high concentrations of protodioscin, demonstrates an in943 Spearman’s correlation for saponins versus pasture characteristics and dividual variation in susceptibility among the animals, a 944 daily rainfall; P values and numbers of pairs (n). fact that deserves further attention. 945 Correlation Spearman’s P n In conclusion, Murrah buffaloes are sensitive to 946 coefficient poisoning by B. decumbens, and steroidal saponins may be 947 Saponin versus green leaves 0.6727 0.0031* 17 responsible for causing liver damage. This is the first report 948 Saponin versus 0.702 0.0017** 17 of this disease in buffaloes. In the studied region, the 949 senescent material beginning of the rainy season was the period of greatest 17 Saponin versus height 0.4515 0.0688ns 950 Saponin versus rainfall 0.5893 0.0128** 17 risk for outbreaks because of higher levels of protodioscin 951 in the pastures compared to other periods. Buffaloes appear 952 *Significant at P < 0.01. **Significant at P < 0.05. ns: not statistically to be more resistant to protodioscin poisoning, a possibility significant. 953 Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
TOXCON4629_proof ■ 14 July 2013 ■ 9/9 C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969Q2 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013
that should be investigated in other studies. The enzyme GGT should be used to evaluate liver function in buffaloes fed with B. decumbens. 5. Ethical statement The authors declare that the study entitled “Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil” was conducted according to ethical guidelines published in the ELSEVIER website. Uncited reference Robey et al., 2006. Acknowledgments We would like to thank Prof. Carlos Tokarnia and Prof. David Driemeier for histological analysis. CNPq, Capes, Fapemig and INCT-Pecuária provided financial support. Pró-Reitoria de Pesquisa da UFMG funded the English proofreading. Conflict of interest The authors declare that there are no conflicts of interest. References Albernaz, T.T., Silveira, J.A.S., Silva, N.S., Oliveira, C.H.S., Belo Reis, A.S., Oliveira, C.M.C., Duarte, M.D., Barbosa, J.D., 2010. Fotossensibilização em ovinos associada à ingestão de Brachiaria brizantha no estado do Pará. Pesq. Vet. Bras. 30 (9), 741–748. Barbosa, M.F., Brum, K.B., Fernandes, C.E., Martins, C.F., Monteiro, L.C., Vandeufrazio, S.C., Rezende, K.G., Riet-Correa, F., Haraguchi, M., Junior, H.L.W., Lemos, R.A.A., 2009. Variations of saponin level x maturation in Brachiaria brizantha leaves. In: 8th International Symposium on Poisonous Plants. João Pessoa, Paraíba, Brazil, p. 13. Brum, K.B., Haraguchi, M., Lemos, R.A.A., Riet-Correa, F., Fioravanti, M.C.S., 2007. Crystal-associated cholangiopathy in sheep grazing Brachiaria decumbens containing the saponin protodioscin. Pesq. Vet. Bras. 27, 39–42. Brum, K.B., Haraguchi, M., Garutti, M.B., Nóbrega, F.N., Rosa, B., Fioravanti, M.C.S., 2009. Steroidal saponin concentrations in Brachiaria decumbens and B. brizantha at different developmental stages. Ciência Rural 39 (1), 279–281. Cruz, C., Driemeier, D., Pires, V.S., Colodel, E.M., Taketa, A.T.C., Schenkel, E.P., 2000. Isolation of steroidal sapogenins implicated in experimentally induced cholangiopathy of sheep grazing Brachiaria decumbens in Brazil. Vet. Hum. Toxicol. 42 (3), 142–145. Dirksen, G., Gründer, H.D., Stöber, M., 1993. Exame Clínico dos Bovinos. Guanabara Koogan, Rio de Janeiro. Driemeier, D., Colodel, E.M., Seitz, A.L., Barros, S.S., Cruz, C.E.F., 2002. Study of experimentally induced lesions in sheep by grazing Brachiaria decumbens. Toxicon 40, 1027–1031. Flayoen, A., Hjorth Tønnesen, H., Grønstøl, H., Karlsen, J., 1991. Failure to induce toxicity in lambs by administering saponins from Narthecium ossifragum. Vet. Res. Commun. 15, 483–487. Ganzera, M., Bedir, E., Khan, I.A., 2001. Determination of steroidal saponins in Tribulus terrestris by reversed-phase high-performance liquid chromatography and evaporative light scattering detection. J. Pharm. Sci. 90, 1752–1758. Graydon, R.J., Hamid, H., Zaha, R.I.P., Gardiner, C., 1991. Photosensitization and crystal-associated cholangiohepatopathy in sheep grazing Brachiaria decumbens. Aust. Vet. J. 68 (7), 234–236.
9
INMET, 2010. Instituto Nacional de Meteorologia. Ministério da Agricultura Pecuária e Abastecimento. Available at: www.inmet.gov.br (accessed 12.02.10.). Kaneko, J.J., Harvey, J.W., Bruss, M.L., 2008. Clinical Biochemistry of Domestic Animals, 6a ed. Academic Press, Burlington. Lancashire, J.A., Keogh, R.G., 1968. Facial eczema and grazing management. Sheepfarming Annu., 29–35. Lemos, R.A.A., Ferreira, L.C.L., Silva, S.M., Nakazato, L., Salvador, S.C., 1996. Fotossensibilização e colangiopatia associada a cristais em ovinos em pastagem com Brachiaria decumbens. Ciência Rural 26, 109–113. Lemos, R.A.A., Salvador, S.C., Nakazato, L., 1997. Photosensitization and crystal associated cholangiohepatopathy in cattle grazing Brachiaria decumbens in Brazil. Vet. Hum. Toxicol. 39, 376–377. Lemos, R.A.A., Nakazato, L., Herrero Jr., G.O., Silveira, A.C., Porfírio, L.C., 1998. Fotossensibilização e colangiopatia associada a cristais em caprinos mantidos sob pastagens de Brachiaria decumbens no Mato Grosso do Sul. Ciência Rural 28, 507–510. Luna, L.G., 1968. Manual of Histological Staining Methods of the Armed Force Institute of Pathology, third ed. McGraw-Hill, New York. Meagher, L.P., Miles, C.O., Fagliari, J.J., 1996. Hepatogenous photosensitization of ruminants by Brachiaria decumbens and Panicum dichotomiflorum in the absence os sporidesmin: lithogenic saponins may be responsible. Vet. Hum. Toxicol. 38 (4), 271–274. Meagher, L.P., Smith, B.L., Wilkins, A.L., 2001. Metabolism of diosgeninderived sapoinins: implications for hepatogenous photosensitization diseases in ruminants. Anim. Feed Sci. Technol. 91, 157–170. Minervino, A.H.H., Barrêto Júnior, R.A., Rodrigues, F.A.M.L., Ferreira, R.N.F., Reis, L.F., Headley, S.A., Ortolani, E.L., 2010. Hepatogenous photosensitization associated with liver copper accumulation in buffalos. Res. Vet. Sci. 88, 519–522. Motta, A.C., Rivero, G.R., Schild, A.L., Riet-Correa, F., Méndez, M.C., Ferreira, J.L., 2000. Fotossensibilização hepatógena em bovinos no sul do Rio Grande do Sul. Ciência Rural 30 (1), 143–149. Pires, V.S., Taketa, A.T.C., Gosmann, G., Schenkel, E.P., 2002. Saponins and sapogenins from Brachiaria decumbens Stapf. J. Braz. Chem. Soc. 13 (2), 135–139. Riet-Correa, B., Riet-Correa, F., Oliveira Junior, C.A., Duarte, V.C., RietCorrea, G., 2010. Alterações histológicas em fígados e linfonodos de búfalos (Bubalus bubalis) mantidos em pastagens de Brachiaria spp. Pesq. Vet. Bras. 30 (9), 705–711. Riet-Correa, F., Schild, A.L., Lemos, R.A.A., Borges, J.R.J., 2007. Doenças de ruminantes e equídeos, third ed. Pallotti, Santa Maria. Robey, R.W., Fetsch, P.A., Polgar, O., Dean, M., Bates, S.E., 2006. The livestock photosensitizer, phytoporphyrin (phylloerythrin), is a substrate of the ATP-binding cassette transporter ABCG2. Res. Vet. Sci. 81 (3), 345–349. Sampaio, I.B.M., 2010. Estatística Aplicada à Experimentação Animal, third ed. Fundação de Estudo e Pesquisa em Medicina Veterinária e Zootecnia, Belo Horizonte. Scheie, E., Flaoyen, A., Moan, J., Berg, K., 2002. Phylloerythrin: mechanisms for cellular uptake and location, photosensitization and spectroscopic evaluation. N. Z. Vet. J. 50 (3), 104–110. Schenkel, E.P., Gosman, G., Athayde, M.L., 2007. Saponinas. In: Simões, C.M.O., Schenkel, E.P., Gosman, G., Mello, J.C.P., Mentz, L.A., Petrovick, P.R. (Eds.), Farmacognosia: da Planta ao Medicamento, sixth ed. UFRGS, Porto Alegre, pp. 711–740. Smith, B.L., 2000. Effects of low dose rates of sporidesmin given orally to sheep. N. Z. Vet. J. 48, 176–181. Smith, B.L., Miles, C.O., 1993. A letter to the editor: a role for Brachiaria decumbens in hepoatogenous photosensitization of ruminants? Vet. Hum. Toxicol. 35 (3), 256–257. Stannard, A.A., 1994. Moléstias da pele: dermatopatias. In: Smith, B.P. (Ed.), 1994. Tratado de Medicina Interna de Grandes Animais, vol. 2. Manole, São Paulo, pp. 1061–1117. Stegelmeier, B.L., 2002. Equine photosensitization. Clin. Tech. Equine Pract. 1 (2), 81–88. Tokarnia, C.H., Döbereiner, J., Peixoto, P.V., 2000. Plantas Tóxicas do Brasil. Helianthus, Rio de Janeiro. Tokarnia, C.H., Döbereiner, J., Peixoto, P.V., 2002. Poisonous plants affecting livestock in Brazil. Toxicon 40, 1635–1660. Wina, E., Muetzel, S., Becker, K., 2005. The impact of saponins or saponin containing plant materials on ruminant productions: a review. J. Agric. Food Chem. 53, 8093–8105. Wysocki Jr., H.L., Freitas, L.G.L., Araújo, A.L.L., Santos Jr., H.L., Castro, M.B., Haraguchi, M., 2009. Relação dos níveis de protodioscina em diferentes estágios de crescimento de Brachiaria decumbens com quadros de fotossensibilização em ovinos. Biológico 71, 34. São Paulo.
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073
Contents lists available at SciVerse ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
1 2 3 q 4 5 a b b a a 6 Q3 C.H.S. De Oliveira , J.D. Barbosa , C.M.C. Oliveira , E. Bastianetto , M.M. Melo , 7 c c a a, * M. Haraguchi , L.G.L. Freitas , M.S. Xavier , R.C. Leite 8 a Escola de Veterinária, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Minas Gerais, Av. Antônio Carlos 9 6627, CEP 30123-970 Belo Horizonte, MG, Brazil 10 b Central de Diagnóstico Veterinário, Faculdade de Veterinária, Universidade Federal do Pará, Brazil 11 c Laboratório de Farmacologia, Instituto Biológico de São Paul, Brazil 12 13 14 a r t i c l e i n f o a b s t r a c t 15 16 Article history: The aim of this paper is to report the study of hepatogenous photosensitization in buf17 Received 7 January 2013 faloes during two outbreaks provoked by ingestion of Brachiaria decumbens in Minas Received in revised form 26 June 2013 18 Gerais state, Brazil. Ten young buffaloes in outbreak 1 and seven buffaloes in outbreak 2 Accepted 2 July 2013 19 were intoxicated by B. decumbens. Nine clinically healthy buffaloes raised under the same Available online xxxx 20 conditions as the sick animals served as the control group. All animals were subjected to 21 clinical examination, and serum was collected to measure gamma-glutamyl transferase Keywords: (GGT), aspartate aminotransferase (AST), direct bilirubin (DB), indirect bilirubin (IB) and 22 Buffalo total bilirubin (TB) as indicators of liver function and urea and creatinine as indicators of 23 Photosensitization renal function. Histopathology of liver fragments from five different animals was carried Brachiaria decumbens 24 out. During the outbreaks and every two months for one year, samples of grass from Saponin 25 paddocks where the animals got sick were collected for quantitative evaluation of the Protodioscin 26 saponin protodioscin, combined with observations of pasture characteristics and daily 27 rainfall. Clinical signs included apathy, weight loss, restlessness, scar retraction of the ears 28 and intense itching at the skin lesions, mainly on the rump, the tail head, neck and hin29 dlimbs, similar to the signs observed in other ruminants. Only the GGT enzyme presented 30 significantly different (P < 0.01) serum levels between intoxicated animals (n ¼ 17) and 31 healthy animals (n ¼ 9), indicating liver damage in buffaloes bred in B. decumbens pastures. Microscopy of the liver showed foamy macrophages and lesions of liver disease associated 32 with the presence of crystals in the bile ducts, which have also been found in sheep and 33 cattle poisoned by grasses of the genus Brachiaria. During the outbreaks, protodioscin 34 levels were higher than 3%, and shortly after, these levels were reduced to less than 0.80%, 35 suggesting a hepatic injury etiology. The outbreaks took place at the beginning of the rainy 36 season, and there was a positive correlation between saponin and the amount of rainfall, as 37 well as between saponin and the amount of green leaves in the pasture. These findings 38 indicate that the grass was more toxic in this period. This is the first report of photosen39 sitization by B. decumbens in buffalo. 40 Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. 41 42 43 44 45 46 q This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, 47 and reproduction in any medium, provided the original author and source are credited. * Corresponding author. Tel.: þ55 31 34092127; fax: þ55 31 34092080. 48 E-mail addresses: [email protected], [email protected] (R.C. Leite). 49
Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil
0041-0101/$ – see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.07.001
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99
TOXCON4629_proof ■ 14 July 2013 ■ 2/9 2
100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160
C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
1. Introduction Hepatogenous photosensitization is a disease that occurs when hepatotoxic substances – poisonous plants, mycotoxins, drugs or infectious agents – injure the liver sufficiently to prevent the excretion of phylloerythrin, which is a metabolite produced in the gastrointestinal tract as a result of chlorophyll metabolism. Phylloerythrin is a photodynamic agent that it is eliminated by the liver. When the liver is injured, phylloerythrin accumulates in the blood stream, allowing sunlight to cause skin lesions (Scheie et al., 2002; Smith, 2000). Clinical signs of photosensitization in cattle are characterized by erythema, wrinkling and crust formation over large skin areas, especially when the skin is not pigmented and is exposed to a high amount of sunlight, such as the back, loin and neck. Additional clinical signs are flank and caudal fold edema, loss of appetite, excitability, itching, tearing, polyuria, edema and scar retraction of the ears, keratitis and blindness, jaundice, weakness, dehydration and death. Thus, hepatogenous photosensitization causes vast economic losses due to production deficiencies (Stannard, 1994). Considering animals raised on Brachiaria spp. pastures, the etiology of hepatic injury is a major issue. In New Zealand, the mycotoxin sporidesmin produced by the fungus Pithomyces chartarum, which grows mainly in senescent plant material under high temperature and humidity, is the causative agent (Lancashire and Keogh, 1968; Smith, 2000). In Brazil, isolates of P. chartarum involved in photosensitization outbreaks failed to produce the mycotoxin, and cases have occurred in pastures with low counts of fungal spores, so the presence of steroidal saponins in the grass has been identified as the causative agent of hepatic injury (Brum et al., 2007; Meagher et al., 1996). Animals intoxicated by Brachiaria spp. develop cholangiohepatopathy lesions in association with crystalloid material deposition in the biliary system (Meagher et al., 2001). For crystal formation, a source of steroid substance in the animal’s diet is needed (Flayoen et al., 1991), as steroidal saponins present in Brachiaria spp. Saponins are steroid or polycyclic terpene glycosides characterized by the presence of a lipophilic portion (steroid or triterpene) and a hydrophilic portion (sugar). They are also broadly distributed in plants and are produced in the plant secondary metabolism (Schenkel et al., 2007). This type of intoxication has well-known effects in cattle, sheep and horses, but no report on this disease was found in buffaloes. Therefore, the aim of this study is to report the occurrence of two hepatic photosensitization outbreaks in buffaloes caused by Brachiaria decumbens ingestion in Minas Gerais state, Brazil, and to describe the clinical and pathological characteristics, to evaluate the saponin level in the pasture, and to assess liver and kidney serum biochemistry in these buffaloes.
neighboring farms (19 290 41.7400 S, 44 300 29.7500 W) which are located in the central area of Minas Gerais state. Both farms had the same owner, and the buffaloes belonged to the Murrah breed. The outbreaks occurred during the months of October and November 2009. Ten animals of outbreak 1 were examined, two with clinical signs of photosensitization and eight without clinical signs, all crated in the same paddock (paddock A). Seven animals of outbreak 2 were examined, all with clinical signs of photosensitization, and they were crated in two paddocks (paddocks B and C). A third group (control group) consisted of nine buffaloes without clinical or laboratory changes indicating deficiency of liver function, which belonged to another farm (19 260 30.1200 S, 44 280 26.1400 W) where the disease did not occur (paddock D). All animals were raised on B. decumbens pastures. All animals were subjected to clinical examination in accordance with Dirksen et al. (1993). Epidemiological data were obtained through a questionnaire applied to the owner and to the employees of the properties as well as through onsite observation. Data were collected regarding the animals’ breed, sex and age, and also about management details, the time of year and places where the disease had occurred, the number of animals in the batch, the grass species and pasture characteristics, the presence of toxic plants in the pastures, and outbreaks that had occurred on other occasions and which animal species had been affected by them. 2.2. Biochemical tests Using jugular venipuncture and a vacuum collection system, blood was collected to measure the serum levels of gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), direct bilirubin (DB), indirect bilirubin (IB) and total bilirubin (TB) as indicators of liver function, and urea and creatinine to assess renal function. Bilirubin was measured with a Doles Kit (Doles Reagents and Laboratory Equipment, Goiania, Brazil). All of the other measurements were performed using commercial Cepa Kits (MBiolog Diagnostics, Minas Gerais, Brazil). These measurements were performed according to the manufacturers’ recommendations and in accordance with their processing through a BIOPLUS-2000 semi-automatic biochemical analyzer. 2.3. Pathology tests Necropsy and histopathological examination of liver fragments from one buffalo in the terminal stage of the disease were performed, from outbreak 1 (paddock A). We also performed histopathology on liver biopsy specimens from four sick buffaloes from outbreak 2 (paddocks B and C). The fragments were fixed in 10% formalin, processed according routine histopathological methods, embedded in paraffin, cut at 5 mm thickness, stained with hematoxylin and eosin (HE) and analyzed with an optical microscope (Luna, 1968).
2. Materials and methods 2.4. Saponin study 2.1. Outbreaks Two hepatogenous photosensitization outbreaks in buffaloes were examined. These events took place in two
During the outbreaks (October 21, 2009 and November 6, 2009) and every two months for one year (January 26, May 25, July 1 and September 23, 2010), samples of B.
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221
TOXCON4629_proof ■ 14 July 2013 ■ 3/9 C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282
decumbens from the paddocks where the animals got sick were collected to quantify the saponin protodioscin. These data were combined with the observation of pasture characteristics and daily rainfall. The grass samples were collected with the hands, grabbing and pulling it out of random points, simulating a grazing buffalo. The grass height and general conditions of each paddock were estimated semi-quantitatively by adopting a score of þ, þþ, and þþþ, respectively, for a low (up to 30%), moderate (30–70%) and high amount (above 70%) of green leaves of B. decumbens (GL) and senescent material (SM) at the time of each gathering. The average daily rainfall was calculated in millimeters from daily rainfall data retrieved from Sete Lagoas Meteorological Station throughout the experimental period, i.e., from September 2009 to September 2010. The station was located 42 km away from both farms considered in this study (INMET, 2010). Protodioscin was quantified by high-performance liquid chromatography (HPLC) using evaporative light-scattering detection (ELSD) in accordance with a modified method proposed by Ganzera et al. (2001) (Wysocki et al., 2009). 2.5. Statistical analysis Data for all variables were analyzed using descriptive statistics. An error of 5% was considered to calculate mean values, standard deviation and the mean confidence interval length. The Mann–Whitney test was used to compare means, considering a 5% margin of error (P < 0.05) to distinguish the groups for the studied variables (Sampaio, 2010). Spearman’s correlation test was used to compare the values of the variable saponin with average daily rainfall and with pasture characteristics such as height, number of green leaves and amount of senescent material (Sampaio, 2010), considering the value of P < 0.05 as significant. All analyses were performed using the software SAEG-D, version 9.1. 3. Results Two photosensitization outbreaks in herds of Murrah buffaloes from the same genetic group were identified in two neighboring farms belonging to the same landowner. Both farms were located in the city of Fortuna de Minas, in the central region of Minas Gerais, Brazil. In outbreak 1, occurring on October 21, 2009, the batch containing 62 Murrah buffaloes aged 3–5 months had two female buffaloes (4 months old) with skin lesions on the rump, neck and limbs, areas that were clinically consistent with photosensitization. These animals were given water and mineral supplementation ad libitum and grazed in B. decumbens pastures that presented height of 25 cm during the outbreak, many green leaves and a moderate amount of senescent material. We observed no presence of poisonous plants in the paddock (paddock A). The owner reported the occurrence of similar cases in previous years. After two weeks, one animal died and the other one recovered. Eight other animals from the same batch did not show any clinical signs but underwent biochemical evaluation of the liver and kidneys.
3
Outbreak 2 was diagnosed 16 days later on the neighboring property in a batch of 226 buffaloes from the same breed and the same genetics. These victims were of both sexes, aged 6–8 months, recently weaned and fed with a barley-based dietary supplement due to low availability of pasture. The animals were placed in B. decumbens paddocks (paddocks B and C). These paddocks had been left at rest for more than 30 days and presented pastures with large amounts of buds and green leaves reaching approximately 50 cm above the soil and an absence of toxic plants. In addition, the occurrence of this disease had never been recorded in these paddocks. According to the owner’s report, 25 days after introducing these animals into paddocks B and C, six animals showed apathy, and deaths began to be observed in both paddocks. Some animals presented skin lesions. On our technical visit (November 6, 2009), six animals had died already and seven others had skin lesions (Fig. 1), which we examined. The animals were aged 5–7 months, 3 were males and 4 were females, and all of them were Murrah buffaloes. During clinical examination of all nine buffaloes in both outbreaks (2 from outbreak 1 and 7 from outbreak 2), we identified extensive dry areas, which were mummified in appearance with cracks and ulcerations, in addition to shedding, mainly on the rump (Fig. 2). We also detected crusted lesions over the tail head, hindlimbs, ears and back of the animals. Other symptoms were restlessness, intense itching, apathy, weight loss, eye discharge, scar retraction of the ears and dehydration. The necropsy performed on the buffalo with terminal illness showed a slightly yellowish carcass, and the liver exhibited a hardened consistency, hypertrophy, and rounded edges with a nutmeg pattern on its surface. The gallbladder had become quite distended (Fig. 3), with thick and lumpy bile. Liver histopathology revealed mild biliary stasis, artery hypertrophy, and incipient necrosis of the hepatocytes in the central-lobular area that was characterized by nuclear chromatin condensation and by cytoplasm appearing to be more eosinophilic than normal, as well as rare necrosis or lysed hepatocytes (Fig. 4). Diffuse hepatocellular swelling – from moderate to severe – light megalocytosis with individual hepatocyte necrosis and with intracytoplasmic eosinophilic globules were observed in the hepatic biopsy fragments from four animals that presented clinical signs. We also verified the presence of foamy macrophages in 2 animals and crystals in the bile ducts of one animal (Fig. 5). There was no significant difference in liver or kidney biochemical parameters between the animals from outbreak 1 (n ¼ 10) and outbreak 2 (n ¼ 7). Irrespective of the presence of skin lesions associated with photosensitization, these animals were grouped into a group poisoned by B. decumbens (n ¼ 17) and this group was compared with control group (n ¼ 9). Table 1 presents the mean values of GGT, AST, DB, IB, TB, urea and creatinine as well as their respective confidence intervals for the two groups of animals. Serum GGT was significantly greater in the intoxicated animal group (P < 0.01). However, despite the slightly higher values of other liver function variables in the intoxicated animals, these differences were not statistically significant (P > 0.05) (Fig. 6).
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343
TOXCON4629_proof ■ 14 July 2013 ■ 4/9 4
344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404
C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
Fig. 1. Buffalo featuring skin lesions due to hepatic photosensitization.
Regarding the steroidal saponins, the protodioscin percentage found in B. decumbens pastures during the outbreaks of photosensitization in buffaloes was high. This percentage was 3.24% for paddock A in the first outbreak, 3.26% and 3.54% for paddocks B and C, respectively, in the
second outbreak and 1.60% for paddock D, adopted as a control paddock (Table 2). Average daily rainfall analysis for the study area revealed that in September 2009, there was almost no rainfall, with a daily average of 1.83 mm. Thus, a much
Fig. 2. Buffalo presenting skin lesions due to hepatic photosensitization, characterized by dry areas, mummified appearance, with cracks and ulcerations, and shedding mainly on the rump.
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465
TOXCON4629_proof ■ 14 July 2013 ■ 5/9 C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526
5
Fig. 3. Enlarged liver and gallbladder from a buffalo intoxicated by Brachiaria decumbens.
higher rainfall average was observed in the month of October, 5.69 mm, coinciding with the development of hepatic photosensitization outbreaks in buffaloes and high levels of protodioscin in the pastures (Fig. 7). Rainfall averages were high until March 2010, when the dry period began. During this period, levels of saponin in all three paddocks where the outbreaks took place remained low (less than 0.80%) and did not significantly increase during the period between April and September 2010 (Fig. 7). The Spearman correlation test detected a positive correlation
Fig. 4. Incipient necrosis of hepatocytes (arrow) in the central-lobular area and rare necrotic hepatocytes or lysed hepatocytes. HE, obj. 25.
between daily rainfall and saponin level in the pasture, with a coefficient of 0.5893 and significance of P < 0.05. Regarding pasture characteristics, no significant correlation between saponin content and pasture height was identified. A positive correlation was detected between saponin and quantity of green leaves, and a negative correlation was detected between saponin and quantity of senescent material (Table 3). 4. Discussion and conclusion B. decumbens, Brachiaria humidicola and Brachiaria brizantha poisoning are reported in Brazil and elsewhere in the world, affecting cattle, sheep, goats and horses (Driemeier et al., 2002; Graydon et al., 1991; Lemos et al., 1998; Meagher et al., 1996; Motta et al., 2000; Smith and Miles, 1993; Stegelmeier, 2002). This is the first report of hepatic photosensitization outbreaks in buffaloes, which involved a large number of animals and the death and disposal of sick animals. In the reported outbreaks, photosensitization by B. decumbens affected buffaloes, which were the only species present on the studied properties. The farm owner reported that seven years ago, when there were sheep and goats on his property, photosensitization outbreaks affected most animals, especially young ones. According to Lemos et al. (1996), poisoning mainly affects young animals (lambs and calves), it may also occur in infant animals and in animals under 30 days old, which is consistent with what was observed in this study: the age range of affected buffaloes was 3–8 months. The clinical signs observed in both outbreaks were similar to those observed in cattle (Motta et al., 2000), but
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587
TOXCON4629_proof ■ 14 July 2013 ■ 6/9 6
588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648
C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
Fig. 5. Histological section of liver biopsy from a buffalo intoxicated by Brachiaria decumbens. Presence of crystals in the bile duct (arrow), light megalocytosis (arrowhead), foamy macrophages (white arrow) and moderate hepatocellular swelling. HE, obj. 40.
jaundice, a sign quite frequent in photosensitization in cattle, sheep and goats, was not found in this study. The signs observed in buffaloes that previously developed hepatogenous photosensitization by copper poisoning (Minervino et al., 2010) were similar to those observed in the current buffaloes poisoned by B. decumbens, especially when considering the areas affected by lesions, which were mainly the hindlimbs, tail, neck, and dorsal part of the back. Edema in the ears, around the eyes, and in the mandible, as well as keratitis and blindness, were not observed in this study. These findings differ from those of Albernaz et al. (2010) concerning sheep intoxicated by B. brizantha in Pará state, Brazil. Increased levels of hepatic enzymes are detected in diseases that evolve with liver injury (Lemos et al., 1997). This fact is of great value for the diagnosis of liver damage in animals poisoned by Brachiaria spp. According Dirksen et al. (1993), GGT displays increased activity in the cholestatic hepatic diseases, as observed in cases of photosensitization by Brachiaria spp. Comparing the serum Table 1 Confidence interval, means and standard deviations of GGT, AST, DB, IB, BT urea and creatinine in animals poisoned by Brachiaria decumbens (Intoxicated) and the control group (Control). Parameters
Group
Mean
Standard deviation
Confidence interval
GGT, U/L
Intoxicated Control Intoxicated Control Intoxicated Control Intoxicated Control Intoxicated Control Intoxicated Control Intoxicated Control
102.78* 14.19* 229.76 213.78 0.66 0.41 0.55 0.43 1.21 0.85 40.26 43.19 1.18 1.25
84.52 3.31 107.85 53.49 1.07 0.23 0.62 0.22 1.62 0.43 21.79 10.14 0.55 0.32
59.33–146.24 11.64–16.73 174.32–285.21 172.66–254.90 0.11–1.21 0.24–0.59 0.23–0.87 0.27–0.60 0.38–2.04 0.51–1.18 29.05–51.46 35.39–50.39 0.90–1.47 1.00–1.49
AST, U/L DB, mg/dL IB, mg/dL BT, mg/dL Urea, mg/dL Creatinine, mg/dL
*Significant difference between groups by Mann–Whitney test (P < 0.01).
biochemistry from intoxicated animals and apparently healthy animals, only GGT was significantly different between the two groups, so we chose it to assess the risk of hepatogenous photosensitization development in B. decumbens pastures. GGT is connected to the cell membrane and is located mostly in canaliculi, bile ducts and, to a lesser extent, in the hepatocytes. In hepatic diseases that evolve with cholestasis, serum GGT is elevated due to the membrane solubilization caused by bile salts that accumulate in the liver and in the ducts (Kaneko et al., 2008). AST and bilirubin were similar between the intoxicated animals and the control group, although the results were higher for the first group. Nonetheless, when the results were compared to normal parameters for cattle, both groups presented non-standard results, which shows the necessity of establishing normal biochemical parameters for buffaloes raised in the Brazilian farming system. The statistical similarity of total bilirubin and its fractions between the studied groups could explain the absence of jaundice in buffaloes poisoned by B. decumbens, contrary to previous results in other ruminants (Stannard, 1994). But the necropsy performed on the buffalo with terminal illness showed a slightly yellowish carcass, indicating that the jaundice can appears later in fatally poisoned animals. Alterations in kidney biochemistry parameters have been observed in sheep and cattle intoxicated by Brachiaria spp. (Albernaz et al., 2010). No similar result was found in the buffaloes in this study. Histological lesions observed in the livers of buffaloes with hepatogenous photosensitization were similar to those observed in other ruminants, including the presence of biliary stasis and crystals in the bile ducts. This finding confirms that photosensitization in animals that ingest Brachiaria spp. is associated with crystalloid material deposition in the bile ducts and cholestasis (Cruz et al., 2000; Lemos et al., 1997; Meagher et al., 2001; Pires et al., 2002). Foamy macrophages and hepatocellular swelling have been observed in buffaloes raised in Brachiaria spp. pastures in Pará state, Brazil, but they showed no clinical
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709
TOXCON4629_proof ■ 14 July 2013 ■ 7/9 C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770
7
importance of this injury in the pathogenesis of hepatogenous photosensitization is not fully understood (Tokarnia et al., 2000, 2002). During the outbreaks, the levels of protodioscin in B. decumbens were high, ranging from 3.24% to 3.54%, while in other periods these levels were lower than 0.80%. These data corroborate what was discovered in outbreaks of hepatogenous photosensitization in sheep in pastures with B. decumbens in Mato Grosso do Sul, Brazil, where the protodioscin content was 2.36% (Brum et al., 2007). Therefore, the intake of steroidal saponins from brachiaria pastures by buffaloes may be one factor triggering photosensitization in these ruminants. Brum et al. (2009) analyzed the concentrations of steroidal saponins in B. decumbens and B. brizantha at different stages of plant development, finding that the contents ranged from 0.5 to 2.1% and were higher in more developed plants. These results suggest that the grass is more toxic during its maturation phase and that photosensitization outbreaks in the midwest region of Brazil occur in pastures left at rest for more than 30 days. These conditions are similar to those described for the pasture involved in outbreak 2, which was at rest for more than 30 days and had plants in an advanced stage of development. Daily rainfall data confirmed the owner’s observations, who claimed the disease occurred at the beginning of the rainy season, which coincided with increased levels of protodioscin in the pasture. The occurrence of outbreaks during the growth of pastures at the beginning of the rainy season has also been reported for other ruminants (RietCorrea et al., 2007). The content of saponins and sapogenins in plants may vary depending on the place of cultivation, environmental factors, age and the stage of plant development (Brum et al., 2009; Meagher et al., 1996). In this study, although we detected a correlation between the amount of rainfall, green leaves and senescent material, these variables are not sufficient to explain the increased levels of saponins in the pasture. A more detailed study, including the combination
Fig. 6. Graphical representation of the means of variables of liver and kidney function from buffaloes intoxicated or not by Brachiaria decumbens. * Significant difference between groups by Mann–Whitney test (P < 0.01).
signs of photosensitization (Riet-Correa et al., 2010). Foamy macrophages and hepatocellular swelling were also observed in buffaloes with clinical signs in this study. The presence of foamy macrophages in the liver, lymph nodes and spleen of buffaloes and cattle kept on Brachiaria spp. pastures has been observed in Brazil. However, the
Table 2 Protodioscin levels (%) and pasture characteristics (height in cm, green leaves – GL, senescent material– SM) according to sampling dates over one year (10/ 21/2009–09/23/2010), in Minas Gerais, Brazil. Paddock
10/21/2009
11/06/2009
Pasture
Protodioscin
Height
GL
SM
A B C D
9.8
þþþ
þþ
Paddock
05/25/2010
Protodioscin
Height
GL
SM
25 50 50 17
þþþ þþþ þþþ þþþ
þ þ þ þ
Protodioscin
Height
GL
SM
20 40 45
þþ þþ þþ
þþ þþ þþ
0.41 0.41 0.8 nd
Pasture
Protodioscin
Height
GL
SM
1.82 3.26b 3.54b 1.6
30 45 40
þþþ þþþ þþþ
þþ þ þ
Protodioscin
Pasture
07/01/2010
Pasture
A B C D
3.24a nd nd nd
01/26/2010
Pasture
0.74 0.67 0.39 nd
09/23/2010
Pasture Height
GL
SM
25 40 50
þ þ þþ
þþþ þþþ þþþ
0.31 0.41 0.5 nd
Protodioscin
Height
GL
SM
17 22 25
þþ þþ þþ
þþþ þþþ þþþ
0.13 0.17 0.15 nd
nd: not determined. a Period of outbreak 1. b Period of outbreak 2.
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831
TOXCON4629_proof ■ 14 July 2013 ■ 8/9 8
832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892
C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 Fig. 7. Graphical representation of the average daily rainfall and levels of protodioscin saponin in three paddocks of Brachiaria decumbens (A, B and C) between October 2009 and September 2010. 912 913 914 propose that older animals that have adapted to the presof other variables with assessments of plant physiology, 915 ence of saponins in grass and are more resistant to photowould be needed to understand the behavior of these 916 sensitization. The owner and employees of the two substances. 917 properties considered in this study claimed to observe the Saponins are products obtained from plant secondary 918 disease only in young animals. metabolism. They function in the plant defense processes, 919 Saponins can kill or at least damage ruminal protozoa by and they can be produced in larger quantities under 920 complex formation with sterol membranes. However, this stressful situations, such as fungal and bacterial attack, 921 effect is transient, so the number of protozoa in the rumen ensuring survival benefits for the ecosystem to which the 922 of sheep returns to normal after 14 days of Enterolobium plant belongs (Wina et al., 2005). The first rains that 923 cyclocarpum feeding, a plant that also contains steroidal occurred in this outbreak, associated with rising tempera924 saponins (Ivan et al., 2004, cited Wina et al., 2005). Q1 925 ture and insolation, could have inflicted some type of stress on the grass, leading to the high content of protodioscin, as Albernaz et al. (2010) found that lambs fed with ruminal 926 previously suggested (Barbosa et al., 2009). fluid of adult sheep before being placed on B. brizantha 927 On the other hand, a photosensitization outbreak in pastures had lower levels of hepatic-injury enzymes and a 928 sheep in Pará state, Brazil, occurred during the less rainy lower rate of photosensitization in comparison to a control 929 season, and the pasture (B. brizantha) had reduced and group. Animals’ mechanisms of adaptation to steroidal sa930 senescent residual mass (Albernaz et al., 2010). However, ponins are not yet fully understood and deserve further 931 the levels of saponins methylprotodioscin and protodioscin investigation. 932 found by those authors in the pasture (0.92% and 0.88% In the first outbreak we studied, the morbidity rate was 933 during the outbreaks) were similar to the protodioscin 6.45%, and the mortality rate was 1.61%. In the second 934 levels observed in B. decumbens in this study outside of the outbreak, the morbidity rate was 5.75%, and the mortality 935 rainy season. These data may indicate that buffaloes are rate was 2.65%. Motta et al. (2000) and Riet-Correa et al. 936 more resistant than sheep to intoxication by saponins. (2007) reported that morbidity rates in cattle outbreaks 937 Many authors describe the occurrence of photosensitimay vary from 0.11% to 64%, and mortality can reach 14%, 938 zation in young animals, and we also observed photosenwhich agrees with our findings. 939 sitization mainly in young buffaloes. This result leads us to The involvement of just a few animals in these two 940 outbreaks of photosensitization, despite being exposed to 941 the same epidemiological conditions and having ingested 942 Table 3 high concentrations of protodioscin, demonstrates an in943 Spearman’s correlation for saponins versus pasture characteristics and dividual variation in susceptibility among the animals, a 944 daily rainfall; P values and numbers of pairs (n). fact that deserves further attention. 945 Correlation Spearman’s P n In conclusion, Murrah buffaloes are sensitive to 946 coefficient poisoning by B. decumbens, and steroidal saponins may be 947 Saponin versus green leaves 0.6727 0.0031* 17 responsible for causing liver damage. This is the first report 948 Saponin versus 0.702 0.0017** 17 of this disease in buffaloes. In the studied region, the 949 senescent material beginning of the rainy season was the period of greatest 17 Saponin versus height 0.4515 0.0688ns 950 Saponin versus rainfall 0.5893 0.0128** 17 risk for outbreaks because of higher levels of protodioscin 951 in the pastures compared to other periods. Buffaloes appear 952 *Significant at P < 0.01. **Significant at P < 0.05. ns: not statistically to be more resistant to protodioscin poisoning, a possibility significant. 953 Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
TOXCON4629_proof ■ 14 July 2013 ■ 9/9 C.H.S. De Oliveira et al. / Toxicon xxx (2013) 1–9
954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969Q2 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013
that should be investigated in other studies. The enzyme GGT should be used to evaluate liver function in buffaloes fed with B. decumbens. 5. Ethical statement The authors declare that the study entitled “Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil” was conducted according to ethical guidelines published in the ELSEVIER website. Uncited reference Robey et al., 2006. Acknowledgments We would like to thank Prof. Carlos Tokarnia and Prof. David Driemeier for histological analysis. CNPq, Capes, Fapemig and INCT-Pecuária provided financial support. Pró-Reitoria de Pesquisa da UFMG funded the English proofreading. Conflict of interest The authors declare that there are no conflicts of interest. References Albernaz, T.T., Silveira, J.A.S., Silva, N.S., Oliveira, C.H.S., Belo Reis, A.S., Oliveira, C.M.C., Duarte, M.D., Barbosa, J.D., 2010. Fotossensibilização em ovinos associada à ingestão de Brachiaria brizantha no estado do Pará. Pesq. Vet. Bras. 30 (9), 741–748. Barbosa, M.F., Brum, K.B., Fernandes, C.E., Martins, C.F., Monteiro, L.C., Vandeufrazio, S.C., Rezende, K.G., Riet-Correa, F., Haraguchi, M., Junior, H.L.W., Lemos, R.A.A., 2009. Variations of saponin level x maturation in Brachiaria brizantha leaves. In: 8th International Symposium on Poisonous Plants. João Pessoa, Paraíba, Brazil, p. 13. Brum, K.B., Haraguchi, M., Lemos, R.A.A., Riet-Correa, F., Fioravanti, M.C.S., 2007. Crystal-associated cholangiopathy in sheep grazing Brachiaria decumbens containing the saponin protodioscin. Pesq. Vet. Bras. 27, 39–42. Brum, K.B., Haraguchi, M., Garutti, M.B., Nóbrega, F.N., Rosa, B., Fioravanti, M.C.S., 2009. Steroidal saponin concentrations in Brachiaria decumbens and B. brizantha at different developmental stages. Ciência Rural 39 (1), 279–281. Cruz, C., Driemeier, D., Pires, V.S., Colodel, E.M., Taketa, A.T.C., Schenkel, E.P., 2000. Isolation of steroidal sapogenins implicated in experimentally induced cholangiopathy of sheep grazing Brachiaria decumbens in Brazil. Vet. Hum. Toxicol. 42 (3), 142–145. Dirksen, G., Gründer, H.D., Stöber, M., 1993. Exame Clínico dos Bovinos. Guanabara Koogan, Rio de Janeiro. Driemeier, D., Colodel, E.M., Seitz, A.L., Barros, S.S., Cruz, C.E.F., 2002. Study of experimentally induced lesions in sheep by grazing Brachiaria decumbens. Toxicon 40, 1027–1031. Flayoen, A., Hjorth Tønnesen, H., Grønstøl, H., Karlsen, J., 1991. Failure to induce toxicity in lambs by administering saponins from Narthecium ossifragum. Vet. Res. Commun. 15, 483–487. Ganzera, M., Bedir, E., Khan, I.A., 2001. Determination of steroidal saponins in Tribulus terrestris by reversed-phase high-performance liquid chromatography and evaporative light scattering detection. J. Pharm. Sci. 90, 1752–1758. Graydon, R.J., Hamid, H., Zaha, R.I.P., Gardiner, C., 1991. Photosensitization and crystal-associated cholangiohepatopathy in sheep grazing Brachiaria decumbens. Aust. Vet. J. 68 (7), 234–236.
9
INMET, 2010. Instituto Nacional de Meteorologia. Ministério da Agricultura Pecuária e Abastecimento. Available at: www.inmet.gov.br (accessed 12.02.10.). Kaneko, J.J., Harvey, J.W., Bruss, M.L., 2008. Clinical Biochemistry of Domestic Animals, 6a ed. Academic Press, Burlington. Lancashire, J.A., Keogh, R.G., 1968. Facial eczema and grazing management. Sheepfarming Annu., 29–35. Lemos, R.A.A., Ferreira, L.C.L., Silva, S.M., Nakazato, L., Salvador, S.C., 1996. Fotossensibilização e colangiopatia associada a cristais em ovinos em pastagem com Brachiaria decumbens. Ciência Rural 26, 109–113. Lemos, R.A.A., Salvador, S.C., Nakazato, L., 1997. Photosensitization and crystal associated cholangiohepatopathy in cattle grazing Brachiaria decumbens in Brazil. Vet. Hum. Toxicol. 39, 376–377. Lemos, R.A.A., Nakazato, L., Herrero Jr., G.O., Silveira, A.C., Porfírio, L.C., 1998. Fotossensibilização e colangiopatia associada a cristais em caprinos mantidos sob pastagens de Brachiaria decumbens no Mato Grosso do Sul. Ciência Rural 28, 507–510. Luna, L.G., 1968. Manual of Histological Staining Methods of the Armed Force Institute of Pathology, third ed. McGraw-Hill, New York. Meagher, L.P., Miles, C.O., Fagliari, J.J., 1996. Hepatogenous photosensitization of ruminants by Brachiaria decumbens and Panicum dichotomiflorum in the absence os sporidesmin: lithogenic saponins may be responsible. Vet. Hum. Toxicol. 38 (4), 271–274. Meagher, L.P., Smith, B.L., Wilkins, A.L., 2001. Metabolism of diosgeninderived sapoinins: implications for hepatogenous photosensitization diseases in ruminants. Anim. Feed Sci. Technol. 91, 157–170. Minervino, A.H.H., Barrêto Júnior, R.A., Rodrigues, F.A.M.L., Ferreira, R.N.F., Reis, L.F., Headley, S.A., Ortolani, E.L., 2010. Hepatogenous photosensitization associated with liver copper accumulation in buffalos. Res. Vet. Sci. 88, 519–522. Motta, A.C., Rivero, G.R., Schild, A.L., Riet-Correa, F., Méndez, M.C., Ferreira, J.L., 2000. Fotossensibilização hepatógena em bovinos no sul do Rio Grande do Sul. Ciência Rural 30 (1), 143–149. Pires, V.S., Taketa, A.T.C., Gosmann, G., Schenkel, E.P., 2002. Saponins and sapogenins from Brachiaria decumbens Stapf. J. Braz. Chem. Soc. 13 (2), 135–139. Riet-Correa, B., Riet-Correa, F., Oliveira Junior, C.A., Duarte, V.C., RietCorrea, G., 2010. Alterações histológicas em fígados e linfonodos de búfalos (Bubalus bubalis) mantidos em pastagens de Brachiaria spp. Pesq. Vet. Bras. 30 (9), 705–711. Riet-Correa, F., Schild, A.L., Lemos, R.A.A., Borges, J.R.J., 2007. Doenças de ruminantes e equídeos, third ed. Pallotti, Santa Maria. Robey, R.W., Fetsch, P.A., Polgar, O., Dean, M., Bates, S.E., 2006. The livestock photosensitizer, phytoporphyrin (phylloerythrin), is a substrate of the ATP-binding cassette transporter ABCG2. Res. Vet. Sci. 81 (3), 345–349. Sampaio, I.B.M., 2010. Estatística Aplicada à Experimentação Animal, third ed. Fundação de Estudo e Pesquisa em Medicina Veterinária e Zootecnia, Belo Horizonte. Scheie, E., Flaoyen, A., Moan, J., Berg, K., 2002. Phylloerythrin: mechanisms for cellular uptake and location, photosensitization and spectroscopic evaluation. N. Z. Vet. J. 50 (3), 104–110. Schenkel, E.P., Gosman, G., Athayde, M.L., 2007. Saponinas. In: Simões, C.M.O., Schenkel, E.P., Gosman, G., Mello, J.C.P., Mentz, L.A., Petrovick, P.R. (Eds.), Farmacognosia: da Planta ao Medicamento, sixth ed. UFRGS, Porto Alegre, pp. 711–740. Smith, B.L., 2000. Effects of low dose rates of sporidesmin given orally to sheep. N. Z. Vet. J. 48, 176–181. Smith, B.L., Miles, C.O., 1993. A letter to the editor: a role for Brachiaria decumbens in hepoatogenous photosensitization of ruminants? Vet. Hum. Toxicol. 35 (3), 256–257. Stannard, A.A., 1994. Moléstias da pele: dermatopatias. In: Smith, B.P. (Ed.), 1994. Tratado de Medicina Interna de Grandes Animais, vol. 2. Manole, São Paulo, pp. 1061–1117. Stegelmeier, B.L., 2002. Equine photosensitization. Clin. Tech. Equine Pract. 1 (2), 81–88. Tokarnia, C.H., Döbereiner, J., Peixoto, P.V., 2000. Plantas Tóxicas do Brasil. Helianthus, Rio de Janeiro. Tokarnia, C.H., Döbereiner, J., Peixoto, P.V., 2002. Poisonous plants affecting livestock in Brazil. Toxicon 40, 1635–1660. Wina, E., Muetzel, S., Becker, K., 2005. The impact of saponins or saponin containing plant materials on ruminant productions: a review. J. Agric. Food Chem. 53, 8093–8105. Wysocki Jr., H.L., Freitas, L.G.L., Araújo, A.L.L., Santos Jr., H.L., Castro, M.B., Haraguchi, M., 2009. Relação dos níveis de protodioscina em diferentes estágios de crescimento de Brachiaria decumbens com quadros de fotossensibilização em ovinos. Biológico 71, 34. São Paulo.
Please cite this article in press as: De Oliveira, C.H.S., et al., Hepatic photosensitization in buffaloes intoxicated by Brachiaria decumbens in Minas Gerais state, Brazil, Toxicon (2013), http://dx.doi.org/10.1016/j.toxicon.2013.07.001
1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073