Article — Artikel Microscopic morphology of Dichapetalum cymosum (Hook.) Engl. as an aid in the identification of leaf fragments from the digestive tract of poisoned animals D van der Merwea* and L du Plessisb samples were obtained from 2 plants that ABSTRACT differed morphologically regarding the Dichapetalum cymosum (Hook.) Engl. (Poison leaf; gifblaar) is a major cause of acute livestock density of trichomes (hairs) on the leaf plant poisoning in southern Africa. Microscopic identification of leaf fragments found in surface. Leaves of plant species with retic- the digestive tract of poisoned animals can assist in the diagnosis of poisoning when ulated leaf vein patterns, including Burkea D. cymosum poisoning is suspected. The microscopic morphology of D. cymosum leaves are africana Hook. f., Combretum zeyheri Sond., described using standard staining and microscopy methods for histopathology samples at Euclea crispa (Thunb.) Guerke subsp. many regional diagnostic laboratories. Morphological descriptions included structures in crispa and Lannea discolor (Sond.) Engl., the epidermis and mesophyll that were discernible using H & E staining. The microscopic were collected from the same habitat. morphology of D. cymosum was used to differentiate between leaf fragments of D. cymosum and other species from the same habitat with macroscopic features that resemble those of Tissue preparation D. cymosum, including Euclea crispa, Combretum zeyheri, Burkea afrikana and Lannea discolor. The method of plant tissue preparation Key words: Burkea africana, Combretum zeyheri, Dichapetalum cymosum, Euclea crispa, Lannea followed standard procedures for the discolor, plant poisoning, monofluoroacetate, rumen content microscopy. preparation and haematoxylin and eosin van der Merwe D, du Plessis L Microscopic morphology of Dichapetalum cymosum (H & E) staining of animal tissues for (Hook.) Engl. as an aid in the identification of leaf fragments from the digestive tract of histological examination in the Pathology poisoned animals. Journal of the South African Veterinary Association (2006) 77(4): 197–201 Section of the Department of Paraclinical (En.). Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Sciences, Faculty of Veterinary Science, Kansas State University, Manhattan, KS, USA. University of Pretoria. The procedures used are widely known and commonly available at regional veterinary diagnostic INTRODUCTION Owing to the rapidity of death in animals laboratories. The economic losses and social impact that have died of D. cymosum poisoning, Dichapetalum cymosum samples were of plant poisonings of livestock in southern leaf fragments of the plant that caused the also stained using McMannus’ Periodic Africa is considerable5. Dichapetalum poisoning can often be found in the diges- Acid Schiff’s (PAS) method to compare it cymosum (Hook.) Engl. (poison leaf; tive tract of poisoned animals. The presence withH&Estaining. PAS staining is com- gifblaar), which occurs in southern Angola, of D. cymosum leaf fragments in the diges- monly available at diagnostic laboratories north-eastern Namibia, Botswana, south- tive tract of an animal that has died due to for detecting fungi in histological tissue ern Zambia, western and southern suspected plant poisoning is therefore sections. It is an efficient stain for polysac- Zimbabwe, southern Mozambique and considered to be an important factor in charides found in cell walls. northern and eastern South Africa, is the diagnosis of D. cymosum poisoning4. highly toxic to livestock2. It causes around Microscopic examination of leaf frag- Microscopy 8 % of annual cattle losses due to plant ments found in the digestive tract of Microscopy was with a Leitz Laborlux D poisonings in South Africa5. poisoned animals can aid in the identifi- microscope fitted with ×10 eyepieces and The toxic principle of D. cymosum is cation of D. cymosum leaf fragments re- ×10, ×25, ×40, and ×100 (oil) objectives. monofluoroacetate7. Monofluoroacetate covered from rumen content. This study All diagnostic features could be discerned itself is not toxic, but it is converted in reports the characterisation of the micro- using ×10, ×25 and ×40 objectives. the animal to monofluorocitrate. Mono- scopic morphology of D. cymosum leaves fluorocitrate inhibits the enzyme aconi- discernible by light microscopy using RESULTS tase, thereby the tricarboxylic acid cycle tissue sections prepared by standard and cellular respiration6. It causes sudden histopathology procedures. Epidermis (outer cell layer) death in animals, 4 to 24 hours after they The epidermis has 1 adaxial (facing the have ingested lethal amounts of plant MATERIALS AND METHODS stem, usually the ‘top’ surface of the leaf material4. and 1 abaxial (facing away from the stem) Plant samples cell layer. The cuticle (waxy outer mem- aDepartment of Diagnostic Medicine/Pathobiology, Dichapetalum cymosum samples were brane) is well developed on both sides of College of Veterinary Medicine, Kansas State University, collected from the northern slopes of the leaf. Manhattan, KS, USA. bVetpath Veterinary Pathologists, Department of Pathol- the Magaliesberg in the vicinity of the Stomata (openings on leaf surface that ogy, Faculty of Veterinary Science, University of Pretoria, Florauna suburb of Pretoria in the allows for gas exchange) (Fig. 1) are rare Private Bag X04, Onderstepoort, 0110 South Africa. Gauteng Province of South Africa. Leaf or absent on the adaxial surface, but *Author for correspondence. E-mail: [email protected] samples were fixed in 10 % buffered relatively common on the abaxial surface Received: March 2006. Accepted: November 2006. formalin. Mature and immature leaf where they are randomly distributed. 0038-2809 Jl S.Afr.vet.Ass. (2006) 77(4): 197–201 197 Fig. 1: Stomatal structure in the abaxial epi- Fig. 2: Abaxial side of a Dichapetalum Fig. 4: Large vein of a Dichapetalum cymo- dermis of a Dichapetalum cymosum leaf. cymosum leaf showing the epidermis with sum leaf showing the bundle sheath, stomata, spongiform parenchyma and a bundle sheath extensions, inner sheath, Stomata consist of 2 guard cells (cells lining secondary vein. xylem and phloem. the stomata openings) and 2 subsidiary cells (cells that connect guard cells with vesicles with yellow to orange coloured the spongiform parenchyma of mature typical epidermal cells) and are paracytic, content onH&Estaining are scattered leaves, but may be small or absent in meaning that the longitudinal axes of the through all parenchymal cell types. immature leaves. guard cells and the subsidiary cells are Palisade parenchyma (columnar cells Larger veins (Fig. 4) are surrounded by parallel to the stomatal apertures. Each with longitudinal axes perpendicular to bundle sheaths, consisting of a single guard cell is supported by a single subsid- the epidermis) occurs only on the adaxial layer of tightly packed, thin-walled paren- iary cell. Guard cells do not span the side of the leaf (Fig 3). They occur in 1 to chyma cells, which are elongated in the entire thickness of the epidermis. The 3 layers with 2 layers being the most direction of the veins. Multi-layered guard cells are smaller than the surround- prevalent. The cell walls typically have a bundle sheath extensions, consisting of ing epidermal cells, with diameters of one beaded appearance due to multiple areas more or less elongated cells with unequally third to one half of subsidiary cells. The of cell wall thickening. thickened walls, connect the veins with guard cells are raised above the surface of Spongiform parenchyma (irregularly the epidermis and are present on both the epidermal layer. The cuticle extends shaped cells) occurs only on the abaxial sides of the leaf where large veins are onto the outside surface of the guard cells. side of the leaf (Fig. 2). The cell walls present. In these areas palisade and It does not extend over the guard cells typically have a beaded appearance spongiform layers are absent. into the stomatal lumen. Guard cell wall similar to that of the palisade cells. Sclerenchymal cells, a supporting tissue protuberances are present on the outer Large intercellular spaces are present in consisting of multilayered, elongated guard cell walls. These protuberances are cells with markedly thickened walls, form visible in dorsoventral sections as small, the inner sheath layer around larger veins horn-like ridges in the centre of the outer (Fig. 4). cell walls and they appear to be part of the The xylem (Fig. 4) consists of variably cuticle. In dorsoventral section, the guard shaped cells arranged in dorsoventral cells and subsidiary cells usually appear rows on the adaxial side of the vascular rounded, compared to cube or brick- bundles of large veins. The phloem con- shaped surrounding epidermal cells. sists of variably shaped cells in a random Large, intercellular substomatal cham- pattern on the abaxial side of the xylem of bers consistently occur in mature leaves large veins. The cells of the phloem stain (Fig. 2), but are absent or small in imma- more basophilic than the cells of the ture leaves. xylem and the bundle sheaths onH&E Trichomes (hairs) are unicellular and staining. non-flattened. They vary in density from very dense to very sparse or absent. Venation characteristics Venationis reticulate (Fig. 5). Side-veins Mesophyll (internal leaf tissues) branch in offset pairs and occasionally One hypodermal cell layer (layer/layers oppositely, from a central main vein, of cells beneath the epidermis that differ