Fall panicum (Panicum dichotomiflorum) toxicosis in three juvenile goats

Abstract

Consumption of certain grasses belonging to the genus Panicum has been found to cause hepatogenous photosensitization and crystal-associated cholangiohepatopathy in small ruminants, and liver disease in horses, in many areas of the world. We describe herein the clinical findings, microscopic lesions, and steroidal saponin analysis of Panicum dichotomiflorum associated with fatal toxicosis in 3 juvenile goats in Nebraska. The disease presentation in our case was fulminant, with anorexia, marked icterus, and death for all affected animals in less than a week. Photosensitization was not observed. The microscopic lesions consisted of severe crystal-associated cholangiohepatopathy and nephropathy, with aggregates of clear or refractile and birefringent, acicular crystals present within bile ducts, macrophages, hepatocytes, and renal tubules. High-performance liquid chromatography–mass spectrometry of the grass samples demonstrated that dichotomin was the major steroidal saponin present (0.89 µg/mg); protodioscin was also present (0.059 µg/mg). The findings were consistent with ingestion of steroidal saponins, and P. dichotomiflorum was identified as the predominant forage available.

The plant genus Panicum is large, containing 370–600 species of cool- and warm-season grasses that are distributed throughout the world in tropical and temperate regions.³ Several species are found in the grasslands of North America, where they may serve as an important source of forage for livestock. A few of these have been sporadically associated with hepatogenous photosensitization in grazing animals, particularly P. virgatum (switchgrass) and P. coloratum (klinegrass).⁵ Steroidal saponins have been determined to be the likely toxic principle, and can be detected in plant samples associated with outbreaks of toxicosis manifesting in photosensitization and liver disease, including crystal-associated cholangiohepatopathy, icterus, and elevation of serum hepatic enzymes.^2,9,11 It is thought that the bile duct crystals characteristic of poisoning are formed by glucuronic acid–conjugated sapogenin metabolites combining with Ca²⁺ to form an insoluble salt.^8,14 Many aspects of Panicum toxicosis are incompletely understood, particularly the conditions surrounding its sporadic occurrence and potential mechanisms contributing to hepatic damage. Herein, we describe a case of P. dichotomiflorum toxicosis in 3 goats.

Tissue and fecal samples obtained during the autopsy of a 3-mo-old Boer goat were received at the University of Nebraska–Lincoln Veterinary Diagnostic Center (Lincoln, NE) on September 2, 2015. The animal was 1 of 6 goats on a farm in Polk County, Nebraska. The owner had acquired the animals 1 mo prior and placed them in a small vacant livestock lot that was overgrown with grass and weeds. The goats did not receive supplemental feedstuffs because they were intended to forage the lot. The water source was a private well. During the week prior to case submission, a group of three, 3-mo-old, Boer goats developed anorexia and marked icterus. They were examined by a local veterinarian, who reported blood packed cell volumes of 0.42 L/L and 0.19 L/L, with profound leukocytosis estimated from the blood smears. The affected goats were treated with injectable oxytetracycline and oral albendazole (dose and brand not specified). The veterinarian who performed the autopsy on 1 goat that died 4 d after examination reported icterus and mild hepatomegaly. The rumen was filled with green forage and fluid, and there was minimal intestinal content. The 2 remaining affected goats died the following week but could not be evaluated postmortem. A Nubian nanny with 2 unweaned kids appeared unaffected.

Laboratory testing was performed using standard methods to evaluate for parasitic or infectious causes. No ova or parasites were observed on fecal floatation. No bacterial pathogens were detected in the liver with aerobic or Salmonella enrichment culture. Prevotella sp. and Porphyromonas sp. were isolated with anaerobic culture of the liver, and were considered to be contaminants. Leptospira was not detected with PCR on fresh kidney and liver samples. Serum biochemical analysis of 2 animals revealed marked azotemia (urea 55.0 and 38.2 mmol/L, reference interval [RI]: 2.1–8.6 mmol/L; creatinine 358 and 329 µmol/L, RI: 8–61 µmol/L), hyperbilirubinemia (381 and 265 µmol/L, RI: 2–12 µmol/L), hyperphosphatemia (3.59 and 4.59 mmol/L, RI: 0.97–1.94 mmol/L), elevated lactate dehydrogenase (17.4 and 48.2 µkat/L, RI: <8.5 µkat/L), and elevated aspartate aminotransferase (12.4 and 29.5 µkat/L, RI: <0.8 µkat/L). Alanine aminotransferase was mildly elevated in one animal (0.94 µkat/L, RI: <0.67 µkat/L). Alkaline phosphatase was within normal limits (<7.51 µkat/L). Hypoalbuminemia (27 and 33 g/L, RI: 37–50 g/L) and hypochloridemia (86 and 91 mmol/L, RI: 96–109 mmol/L) were also noted.

Formalin-fixed lung, lymph node, small and large intestine, liver, and kidney were available for examination. Microscopically, in a section of liver, bile ducts of all sizes were variably distended, containing mucus admixed with low numbers of necrotic epithelial cells and neutrophils, bile pigment, karyorrhectic debris, and aggregates of clear or refractile and birefringent, needle-like (acicular) crystals, 8–30 µm long (Fig. 1). Portal regions were frequently expanded by mild fibrosis and a pleocellular inflammatory cell infiltrate comprised of low numbers of neutrophils, lymphocytes, plasma cells, and macrophages. Occasionally, these inflammatory foci were comprised predominantly of clustered epithelioid macrophages and multinucleate giant cells that effaced small bile ducts and were centered on large, acicular crystals. Multifocal, mild biliary hyperplasia was noted. There was patchy, mild, vacuolar hepatopathy with intracytoplasmic lipid, and regions with mild attenuation of hepatic cords. Hepatocytes often contained small amounts of bile pigment and/or a few clear acicular crystals 4–6 µm long (Fig. 2). A few bile plugs were scattered in bile canaliculi. In a section of kidney, affecting 50% of the parenchyma and primarily in the cortex, there were scattered, poorly circumscribed regions of tubular degeneration and necrosis associated with mild inflammation and fibrosis. In the affected areas, the tubules were moderately dilated and lined by attenuated epithelium, containing hyaline eosinophilic or red-brown granular casts or cellular casts featuring numerous, variably sized, clear or refractile and birefringent, acicular crystals (Fig. 3). The inflammatory cells infiltrating the interstitium and tubules were primarily macrophages mixed with fewer lymphocytes and neutrophils. The majority of the renal tubular cells remaining throughout the cortex were heavily vacuolated, containing numerous small, clear, indistinct vacuoles, and occasionally containing small amounts of green-brown pigment (presumably bile). In sections of lymph node, the medulla was markedly expanded by edema and sheets of siderophages mixed with fewer eosinophils, neutrophils, and plasma cells. There were no significant findings in sections of lung and intestine. The acicular crystals within the liver and kidney did not stain for calcium with a Von Kossa stain. Copper assay on formalin-fixed tissues demonstrated 10 ppm in kidney and 23 ppm in liver (RI: 25–150 ppm).

Figures 1–4.

Fall panicum (Panicum dichotomiflorum) toxicosis in 3 juvenile goats.

Figure 1. A large bile duct in the liver of a goat is filled with neutrophils and inspissated bile containing aggregated, clear, acicular clefts. H&E. Original objective 40×. Figure 2. Scattered hepatocytes in the liver of a goat contain low numbers of acicular crystals (arrow) and/or lipid vacuoles. H&E. Original objective 40×. Figure 3. The cortical tubules in the kidney of a goat are markedly dilated and contain hyaline or cellular casts that are occasionally mixed with numerous acicular crystals (arrows). H&E. Original objective 40×. Figure 4. Lush growth of P. dichotomiflorum in the livestock lot grazed by the goats.

Based on these findings, ingestion of plants containing steroidal saponins was suspected. Many known causes of crystal-associated cholangiohepatopathy, such as Agave lecheguilla, Brachiaria decumbens, Narthecium ossifragum, and Tribulus terrestris, were eliminated based on available regional forages, and Panicum sp. was highly suspected.¹⁵ The referring veterinarian indicated that ~90% of the forage available was a distinct grass that grew in large clumps with tufted seed heads that were “sparkler-like” and stems that bent at prominent nodes (Fig.4). A grass sample from the lot was evaluated by the University of Nebraska–Lincoln Plant and Pest Diagnostic Clinic and identified as fall panicum (P. dichotomiflorum).

A sample of the grass was submitted to the USDA-ARS-Poisonous Plant Research Laboratory (Logan, UT) for steroidal saponin analysis by high-performance liquid chromatography–mass spectrometry (HPLC-MS), as reported previously.¹¹ Modifications of the reported method included a mobile phase of 78:22 (0.1% formic acid:acetonitrile), a total run time of 15 min, and the use of an Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) as a detector. A 5-point protodioscin (ChromaDex, Irvine, CA) standard curve in 0.1% formic acid:acetonitrile (90:10) over the range of 0.049–0.78 μg/mL was used to quantify protodioscin and dichotomin in the grass sample. Under these conditions, protodioscin and dichotomin eluted at 10.1 and 10.9 min, respectively. Peak areas of protodioscin and dichotomin were determined from reconstructed ion chromatograms of the respective MH⁺- H₂O ions (m/z = 1,031 and 1,177). Dichotomin was the major steroidal saponin present in the grass sample, with a concentration of 0.89 µg/mg; the concentration of protodioscin was 0.059 µg/mg.

P. dichotomiflorum is a native grass distributed across the continental United States, and introduced into areas of Canada and other parts of the world. It grows readily in disturbed, moist soils, and is a common weed in cultivated sites (USDA NRCS. Fall panicgrass Panicum dichotomiflorum Michx. In: PLANTS Database. Available from: https://plants.usda.gov/core/profile?symbol=padi). It has been reported that sheep, dairy cattle, and goats in New Zealand became photosensitized with crystalline debris in the bile ducts when grazing P. dichotomiflorum, but the associated lesions were not described further.^8,13,14 In one report of an outbreak of P. dichotomiflorum toxicosis in Brazil, nursing lambs and sheep <1 y of age were the primary animals photosensitized; donkeys, goats, and cattle in the same pasture were not affected.¹⁶ Histologic findings reported were scattered necrotic hepatocytes and foci of myocardial necrosis. In Virginia, hepatic disease in horses was associated with P. dichotomiflorum–containing hay.⁹ The affected horses were icteric and anorectic, with serum hepatic enzyme elevation. Photosensitization was not observed, although indoor housing and season were factors lowering solar exposure. Histologic changes in the liver included patchy, individual hepatocyte necrosis, hepatocyte vacuolation and swelling, and minimal-to-moderate fibrosis and bile duct proliferation. Feeding trials using the hay reproduced the disease in horses and sheep. In the latter 2 reports, clinical signs of hepatotoxicosis were evident 2–3 wk after exposure; acicular crystals were not detected in organs examined.^9,16

The findings in our case demonstrated severe disease and death in goats ~3 wk following the start of P. dichotomiflorum consumption. Distinct crystal-associated cholangiohepatopathy and nephropathy were observed, the latter of which has not been reported with P. dichotomiflorum toxicosis, to our knowledge. Other steroidal saponin-containing plants have occasionally been associated with renal lesions in addition to hepatopathy. Renal tubular dilation, and cell swelling and degeneration with some hyaline casts, were seen in sheep with experimental intoxication with P. miliaceum, with 2 of 5 sheep displaying crystalline material within renal tubular lumens.¹ Similar renal changes, including low numbers of intratubular crystals, were reported in an outbreak of photosensitizing hepatic disease of sheep grazing B. decumbens.⁷ Also in sheep, renal tubular necrosis and regeneration were reported with natural klinegrass (P. coloratum L.) toxicosis, and renal tubular cell swelling and degeneration were observed with experimental induction of “geeldikkop” with T. terrestris ingestion.^2,10 Moderate-to-severe, acute renal tubular necrosis was described in an outbreak of photosensitization of cattle grazing Phytolacca octandra.⁴ In these examples, intratubular acicular crystals seem to appear in the minority of cases. The cause of renal tubular epithelial degeneration and necrosis sometimes seen following ingestion of saponin-containing plants is not known. In our case, the red casts in renal tubules were suspected to contain hemoglobin, particularly with the history of anemia in one goat. Saponins have been known to lyse erythrocytes, but hemolysis could not be confirmed as the cause of casts with the tests performed.⁶ Hemoglobinuric nephrosis caused by copper toxicity was reasonably ruled out with copper analysis of tissues in conjunction with clinicopathologic data.

Groups of animals were differentially affected, with weaned goat kids all severely affected, but a nanny and 2 unweaned kids appeared healthy for a few months following until lost to follow-up. These differences could be the result of age and weaning status, as has been implicated with sheep intoxication with P. dichotomiflorum.¹⁶ It is unknown if there could be a breed difference in susceptibility to toxicosis, given that the Boer goats were affected whereas Nubian goats were not. It is notable that the animals were presented because of icterus and anorexia, rather than photosensitization, which was not reported. The case occurred in late summer with the animals in heavy sun exposure, thus photosensitization should have been obvious. However, no samples of skin were available for histologic examination to rule out early changes of photosensitization.

The documentation of steroidal saponin levels under these testing conditions may provide a basis for comparison to other cases of Panicum toxicosis analyzed similarly. However, it is unknown how the saponin levels may have varied over the period of exposure in our case and how this may be related to the presentation of disease. Similar mass spectrometry technique with quantification used for switchgrass (P. virgatum), which has known hepatotoxic potential for sheep and horses, demonstrated levels of dichotomin 1.11–2.96 µg/mg, compared to 0.89 µg/mg in P. dichotomiflorum found in our case.¹²

Authors of published works on steroidal saponin plant toxicity have speculated that bile crystals may not be the only toxic factor causing hepatic damage and photosensitization, and synergy with mycotoxins is a possibility.⁹ Animals exposed to the facial eczema toxin, sporidesmin, also experience biliary obstruction and photosensitization, and P. dichotomiflorum photosensitization is enhanced when concurrent with facial eczema.¹⁴ Facial eczema is not known to occur in Nebraska, nor has there been evidence that other photosensitizing mycotoxins occur frequently in regional forages. Therefore, our case furthers speculation that the toxic effects of steroidal saponins, here resulting in obvious crystal-associated cholangiohepatopathy and nephropathy, may be separate from factors leading to more general hepatocellular damage and photosensitization.