Article — Artikel

Microscopic morphology of cymosum (Hook.) Engl. as an aid in the identification of fragments from the digestive tract of poisoned animals

D van der Merwea* and L du Plessisb

samples were obtained from 2 that ABSTRACT differed morphologically regarding the (Hook.) Engl. (Poison leaf; gifblaar) is a major cause of acute livestock density of trichomes (hairs) on the leaf poisoning in . Microscopic identification of leaf fragments found in surface. of plant species with retic- the digestive tract of poisoned animals can assist in the diagnosis of poisoning when ulated leaf vein patterns, including 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: , 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 , of D. cymosum leaf fragments in the diges- monly available at diagnostic laboratories north-eastern , , south- tive tract of an animal that has died due to for detecting fungi in histological tissue ern , western and southern suspected plant poisoning is therefore sections. It is an efficient stain for polysac- , southern and considered to be an important factor in charides found in cell walls. northern and eastern , is the diagnosis of D. cymosum poisoning4. highly toxic to livestock2. It causes around Microscopic examination of leaf frag- Microscopy 8 % of annual 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. 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 which runs from the leaf stem through structurally from the tissues beneath the length of the leaf blade. Large side- them) is present under the adaxial epider- veins anastomose with neighbouring mis (Fig. 3). No hypodermis occurs be- Fig. 3: Adaxial side of a Dichapetalum large side-veins about three quarters of neath the abaxial epidermis (Fig. 2). cymosum leaf showing the epidermis and the way to the leaf margin to form a closed Parenchyma cells that contain large palisade parenchyma. system. The central vein and large side

198 0038-2809 Tydskr.S.Afr.vet.Ver. (2006) 77(4): 197–201 . Central vein with alternatelying branch- side-veins Large side-veins anastomosethree about quarters of themargin way to the leaf Only the centralridges vein only on form the abaxial palpable andleaf adaxial surfaces Stomata mostly found inepidermis the abaxial Guard cells raised above leaf surface Guard cell diameter isthat a of third subsidiary cells to half Cuticle extends intolumen the stomatal Cuticle forms a thin,each curved guard ridge cell on Terminal vein endingsfrom are areoles absent or(1–2) are low in number On adaxial and abaxial sides 1–3 cell layers on adaxial side; 1on layer abaxial side The epidermis consists of a single cell layer The cuticle is thickerside on the adaxial Central Substomatal chambers present Trichomes only onsurface the abaxial leaf ns Absent only from very small veins Central vein with alternatelying branch- side-veins Large side-veins anastomosehalfway about between the central veinthe and leaf margin Central vein and large side-veins form faintly palpable ridgesadaxial leaf only surface while onvein the only the central forms a prominentthe abaxial ridge leaf on surface epidermis Guard cells not raised above leafface sur- Guard cell diameter isthat a of third subsidiary cells to half Cuticle extends intolumen the stomatal Cuticle ridges on guardor cells absent are low Areoles contain 1–3endings terminal vein Only on adaxial side of leaf 1–2 cell layers The epidermis consists of a single cell layer The cuticle is welladaxial developed and abaxial on side the Only on abaxial side of leaf Substomatal chambers present No trichomes Multi-cellular, stellate trichomes Stomata mostly found in the abaxial branching alternately Central vein with side-veins Large side-veins anastomoseto close the leaf margin Central vein and large side-veins form palpable ridges onlyleaf on surface the abaxial Areoles are rich in terminalings vein (up end- to 10) Only on adaxial side of leaf 1–3 cell layers The epidermis consists of a single cell layer The cuticle is welladaxial developed and abaxial on side the Only on abaxial side of leaf Substomatal chambers present seriate, non-flattened hairs and multi- cellular, sessile, flattened,hairs peltate Trichomes common on both leaffaces sur- Stomata mostly found inepidermis the abaxial Guard cells not raised above leafface sur- Guard cell diameter is about a quarter that of subsidiary cells Cuticle extends intolumen the stomatal Cuticle forms aeach guard horn-like cell ridge on

Dichapetalum cymosum, Burkea africana, Combretum zeyheri, Euclea crispa and Lannea discolor Central vein with alternately tositely oppo- branching side-veins Large side-veins anastomoseto close the leaf margin Central vein and large side-veins form palpable ridges onsurface the while on adaxial theonly leaf abaxial the surface central veinonly is towards palpable the base and of the leaf endings (up to 5) No hypodermal cell layersOnly on adaxial side of leaf 1–3 cell layers No hypodermal cell layers No hypodermal cell layers No hypodermal cell layers The epidermis consists of a single cell layer The cuticle is markedly thickeradaxial on side the Only on abaxial side of leaf Substomatal chambers present No trichomes Two trichome types: unicellular, uni- Stomata mostly found inepidermis the abaxial Guard cells not raised above leafface sur- Guard cell diameter is aboutof half subsidiary that cells Cuticle extends intolumen the stomatal Cuticle forms 2 thin, curvedeach ridges on guard cell hypodermal cell layer is present Central vein with alternately tobranching oppositely side-veins Large side-veins anastomose about three quarters of the way to the leaf margin Central vein andpalpable ridges large on the adaxial side-veins and abaxial leaf form surfaces Areoles contain 1–3 terminal vein endings Areoles usually contain terminal vein under the adaxial epidermis 1–3 cell layers layer The cuticle isadaxial and well abaxial side developed on the Substomatal chambers present hairs Trichomes vary insparse density to very fromfaces dense and very are on not both always present leaf sur- Dichapetalum cymosum Burkea africana Combretum zeyheri Euclea crispa Lannea discolor dermis Guard cells raised above leaf surface Guard cell diameter is a third tosubsidiary half cells that of Cuticle does not extend intolumen the stomatal Cuticle forms aguard horn-like cell ridge on each Anatomical features of leaves used to differentiate between Venation Reticulate Reticulate Reticulate Reticulate Reticulate Hypodermal cell layerPalisade parenchyma One Only on adaxial side of leaf Epidermal cell layer The epidermis consists of a single cell Spongiform parenchyma Only on abaxial side of leaf Trichomes Trichomes are unicellular, non-flattened Bundle sheath extensions Present only on large veins Absent only from very small veins Absent only from very small veins Absent only from very small vei Stomata Stomata mostly found in the abaxial epi- Table 1:

0038-2809 Jl S.Afr.vet.Ass. (2006) 77(4): 197–201 199 Table 2: Summary of Dichapetalum cymosum morphological structures that are useful for the microscopic identification of leaf fragments.

Structure(s) Description

Venation Reticulate Central vein with alternately to oppositely branching side-veins Large side-veins anastomose about three quarters of the way to the leaf margin Central vein and large side-veins form palpable ridges on the adaxial and abaxial leaf surfaces Areoles usually contain 1–3 terminal vein endings Epidermal cell layer The epidermis consists of a single cell layer The cuticle is well developed on the adaxial and abaxial side Trichomes Trichomes are unicellular, non-flattened hairs Trichomes vary in density from very sparse to very dense on both leaf surfaces and are not always present Stomata Guard cells raised above leaf surface Guard cell diameter is a third to half that of subsidiary cells Cuticle does not extend into the stomatal lumen Cuticle forms a horn-like ridge on each guard cell Hypodermal cell layer One hypodermal cell layer is present under the adaxial epidermis Palisade parenchyma Only on adaxial side of leaf 1–3 cell layers Spongiform parenchyma Only on abaxial side of leaf Substomatal chambers present Bundle sheath extensions Present only on large veins veins form palpable ridges on both the Comparison with other reticulately- (gousiektebossie), P. thamnus Robyns adaxial and abaxial leaf surfaces. Smaller veined plant species (smooth gousiektebossie), veins anastomose to form networks Samples of other reticulately-veined zeyheri (Sond.) Robyns (goorappel), P. covering the whole leaf blade. Areoles plant species that share D. cymosum habitat chamaedendrum (Kuntze) Robyns (small contain 1–3 terminal vein endings that in the Magaliesberg region of Gauteng, goorappel) and the shrublet-form of Och- end blindly in the mesophyll. Except including Burkea africana, Combretum na pulchra Hook. (lekkerbreek) include for areoles, the venation characteristics zeyheri, Euclea crispa and Lannea discolor, the leaf attachment pattern, the presence/ described above can be seen macroscopi- were compared with D. cymosum. Note absence of stipules, the relative colour of cally on whole leaves and large leaf frag- that this does not represent a complete list the adaxial and abaxial leaf surfaces, the ments. of plants with leaf fragments that may leaf venation pattern and fruit and flower resemble D. cymosum macroscopically, characteristics4. Identification of plant but only those that were found in associa- fragments in rumen content, however, tion with D. cymosum at a single locality. requires a different approach because All plants with similar growth forms to D. most of these characteristics can be lost cymosum, such as capensis Harv., after an animal chews and ingests leaves. were not included if their leaf venation Growth form is irrelevant when only leaf patterns were not reticulated and leaf fragments are available for examination. fragments could easily be differentiated The venation pattern is often the only from that of D. cymosum macroscopically. recognisable macroscopic feature dis- The results are summarised in Table 1. cernible in leaf fragments found in the gastrointestinal tract. The reticulate DISCUSSION venation of D. cymosum leaves is not Post mortem examinations of animals unique and is found in a number of other that have died from D. cymosum poisoning species that share the same habitat. Leaf usually do not reveal any lesions of diag- fragment identification based on reticu- nostic significance4. A high-performance late venation alone is therefore not a com- liquid chromatographic technique has pletely reliable proof that a poisoned been developed for the estimation of animal had ingested D. cymosum leaves. monofluoroacetate concentrations in Microscopic examination of plant rumen contents8, but the technique is not samples offers a means of identifying performed routinely and is not readily typical histological features3,9. It provides available. The presence of undigested access to multiple identification features D. cymosum leaf fragments in conjunction on relatively small leaf fragments. Histo- with circumstantial evidence is often the logical sections of leaf fragments can be most practical method used to confirm a prepared using standardised, routine diagnosis of poisoning4. techniques that are available at many The macroscopic characteristics typically regional laboratories. H & E staining was used to differentiate between D. cymosum found to be adequate for the visualisation Fig.5:Schematic representation of the retic- and other species with similar growth of diagnostic features using light micros- ulate venation of Dichapetalum cymosum forms, such as (grysappel), copy. In this study, PAS staining com- leaves. Pachystigma pygmaeum (Schltr.) Robyns pared withH&Estaining resulted in

200 0038-2809 Tydskr.S.Afr.vet.Ver. (2006) 77(4): 197–201 more vivid staining of cell walls, and content is examined shortly after ingestion 2. De Sousa Correia R I, Van Rensburg L 2000 both methods resulted in satisfactory of plant material. However, extended Geographical distribution and local occur- rence of Dichapetalum cymosum (Hook) visualisation of diagnostic features. PAS exposure to the digestive tract secretions Engl. (gifblaar) in Namibia and surround- staining may be useful if increased and flora may degrade or destroy diag- ing areas. In Vermeulen C (ed.) Agricola contrast is desired. nostic morphological features. It should 2000 Nr II. Ministry of Agriculture, Water Direct comparison between unknown also be noted that the presence of poten- and Rural Development, Windhoek plant material and known standards, tially toxic plant fragments in the diges- 3. Fahn A 1982 Plant anatomy (3rd edn). Perga- mon Press, Oxford using the same instrumentation, magnifi- tive tract does not prove the absorption of 4. Kellerman T S, Coetzer J A W, Naudé T W, cation and settings, is a practical an effective lethal doses of toxin, and should be inter- Botha C J 2005 Plant poisonings and myco- 1 approach to plant fragment identification . preted with caution in the absence of sup- toxicoses of livestock in southern Africa (2nd It would be of practical value to those who porting circumstantial and symptomatic edn). Oxford University Press, Cape Town might use this diagnostic approach to pre- evidence. 5. Kellerman T S, Naudé T W, Fourie N 1996 pare reference slides containing stained The distribution, diagnoses and estimated ACKNOWLEDGEMENTS economic impact of plant poisonings and and permanently mounted cross-sections mycotoxicoses in South Africa. Onderste- of D. cymosum leaves. Reference slides We thank Dr J V van Greuning (Botany poort Journal of Veterinary Research 63: 65–90 should represent the morphological vari- Department, University of Pretoria) for 6. Liebecq C, Peters R A 1989 The toxicity of ability of the species in the area of interest, advice on plant morphology and diag- fluoroacetate and the tricarboxylic acid such as hairy/smooth leaves and mature/ nostic features. We thank Beatrix Gehring cycle. 1949. Biochimica et Biophysica Acta 1000: 254–269 for the line drawing showing the vena- immature plants. Table 2 represents a 7. Marais 1944 Monofluoroacetic acid, the summary of morphological characteristics tion pattern of a typical D. cymosum leaf. toxic principle of ‘gifblaar’ Dichapetalum of D. cymosum that are commonly discern- We thank the staff of the Pathology Sec- cymosum (Hook) Engl. Onderstepoort Journal ible in leaf fragments and that are likely to tion of the Department of Clinical Sci- of Veterinary Science and Animal Industry. 20: be useful in the identification of leaf frag- ences, Faculty of Veterinary Science, 67–73 University of Pretoria, for processing and 8. Minnaar P P,Swan G E, McCrindle R I, De ments. It would be imprudent to depend BeerWHJ,Naudé T W 2000 A high- on a single characteristic for identifica- mounting plant tissue samples. performance liquid chromatographic tion. Combining a number of characteris- method for the determination of mono- tic histological features significantly REFERENCES fluoroacetate. Journal of Chromatographic reduces the risk of misidentification. 1. Bates L S, Barefield L, Landgraf W, Sample Science 38(1): 16–20 R, Bax B, Jaconis S 1992 Manual of microscopic 9. Storr G M 1961 Microscopic analysis of Use of microscopy to identify plant frag- analysis of feedstuffs (3rd edn).The American faeces, a technique for ascertaining the diet ments can help to confirm the diagnosis Association of Feed Microscopists, Cham- of herbivorous mammals. Australian Journal of plant poisoning if the gastrointestinal paign, USA of Biological Science 14: 157–164

0038-2809 Jl S.Afr.vet.Ass. (2006) 77(4): 197–201 201