Across Africa, wild giraffes suffer from a variety of skin disorders of mostly unknown etiology. With their populations already threatened from anthropogenic factors, it is important to understand infectious disease risks to giraffes. Here we describe filarid parasites and a portion of their genetic sequence associated with skin disease in Rothschild's giraffes (Giraffa camelopardalis rothschildi) in Uganda.
Populations of Rothschild's giraffes (Giraffa camelopardalis rothschildi) are distinct and threatened across their shrinking ranges in Kenya and Uganda. A 2016 survey in Murchison Falls National Park (MFNP) in Uganda revealed that about one third of giraffes at MFNP had rash-like lesions of unknown etiology, and the International Union for the Conservation of Nature lists diseases of unknown causes as one of their major threats (Fennessy et al. 2018).
Conditions referred to as giraffe skin disease have affected wild giraffes across Africa (Muneza et al. 2016). Although other known skin diseases in wild giraffes have been diagnosed by examination of clinical specimens (Muneza et al. 2016), investigations to date have yielded few diagnostic specimens or definitive diagnoses for affected giraffes. Evidence of bacterial infection and an unidentified spirurid nematode (order Spirurida) have been reported in Masai giraffe (Giraffa c. tippelskirchi) in Tanzania (Mpanduji et al. 2011; Muneza et al. 2016) and an association was found between affected giraffes and soil type, which might influence the ground-dwelling life stages of nematodes (Lee and Bond 2016). Filarial worms (superfamily Filaroidea) have been reported, but not fully described, from Rothschild's giraffes in Uganda (Kalema 1996). Similar lesions associated with filarial parasites of the genus Stephanofilaria have been seen in black rhinoceros (Diceros bicornis) and white rhinoceros (Ceratotherium simum) in Kenya (Stephanofilaria dinnicki), hippopotamus (Hippopotamus amphibious) in South Africa (Stephanofilaria thelazoides), and domestic pigs (Sus scrofa) in the Democratic Republic of Congo (Stephanofilaria Boomkeri; Kock and Kock 1990; Boomker et al. 1995; Bain et al. 1996; Mutinda et al. 2012).
In April of 2014, the Uganda Wildlife Authority immobilized and sampled seven adult Rothschild's giraffes from MFNP. Giraffes were immobilized with etorphine hydrochloride and azaperone at approximately 0.015 mg/kg and 0.033 mg/kg, respectively with 50,000 IU hyaluronidase added to enhance bioavailability. Whole blood, along with skin scrapings, swabs, and biopsies were collected from lesions on their necks and shoulders (Fig. 1). Skin biopsies were preserved in 10% formalin and processed for histopathology examination by light microscopy in the Pathology Department at National Zoological Park of the Smithsonian Institution (Washington, DC, USA). All seven biopsies had a severe, chronic, regionally extensive, eosinophilic, neutrophilic dermatitis with intralesional adult or larval microfilarid nematodes (Fig. 2). Adult nematodes were found in the deep dermis or follicular epidermis at the dermal-epidermal junction and microfilaria were in the dermis. Hyperplastic dermatitis with ulceration and superficial bacterial colonies was also noted along with presumptive superficial dermal lymphangiectasia. Adult nematodes were 120–150 µm in diameter with a thick, ridged cuticle, multicellular gastrointestinal tract, pseudocoelom, peripheral musculature and muscular pharynxes, and reproductive tracts with large numbers of microfilarid larvae (Fig. 2). No definitive diagnosis or identification of the nematodes beyond family Filariidae was possible from microscopic analysis.
Further identification was pursued using PCR at the Smithsonian Center for Conservation Genomics. We extracted DNA from formalin fixed and skin biopsies preserved in RNAlater tissue stabilization solution (Invitrogen, Carlsbad, California, USA), dried blood spots, serum, and skin scrapes preserved dry or in phosphate buffered saline or RNAlater solutions. Only dry skin scrapes (all four chosen for extraction) yielded DNA that generated filarial sequences. We extracted DNA with the Qiagen Blood and Tissue extraction kit (Qiagen, Valencia, California, USA) and used it for PCR following published protocols designed to differentiate filarial parasites (Nuchprayoon et al. 2005). In brief, primers FL1-F (5′-TTCCGTAGGT-GAACCTGC-3′) and Di5.8s 660-R (5′-AC-CCTCAACCAGACGTAC-3′) were used to amplify a 600 base pair fragment of the conserved internal transcribed spacer 1 region. PCR products of the appropriate size on gel electrophoresis were excised from the gel, cleaned (QIAquick, Qiagen) and DNA sequenced in both directions by an ABI PRISMS 3130 Genetic Analyzer (Applied Biosystems, Foster City, California, USA). End-read and other obvious errors were corrected with inspection of electropherograms, and the cleaned DNA sequences were used to detect similar sequences using BLAST (Altschul et al. 1990).
Skin scrapes from the four giraffe samples extracted all yielded filarial sequences of varying length and quality. The consensus sequence of 460 base pair length did not match any sequences in GenBank with more than 92% fidelity. The closest matches were to Onchocerca sp. (92%, accession no. DQ317639), Protospirura (92%, JF514771), Rumenfilaria andersoni (90% accession no. KT873731), Stegophorus macronectes (90%, HE793715), Gongylonema sp. (89% accession no. LC026029 and LC330994), and Cystidicola stigmatura (89%, AY161297). We found no published sequences in GenBank for any Stephanofilaria nor Parafilaria, which, with low percentage homology for Onchocerca sp., were the main differentials based on histopathology. Although we did not attempt it, skin scrapes can yield diagnostic results for stephanofilariasis by microscopy, potentially providing a simpler, field-based diagnostic for future cases (Hansen and Perry 1994).
Four affected giraffes were selected for treatment in November 2015. Giraffes were immobilized as before and treated with ivermectin (Noromectin, Norbrook Laboratories Limited, Newry, Northern Ireland) at an estimated dose of 0.2 mg/kg by subcutaneous injection. Animals showed visual resolution of lesions after 4 mo. Affected giraffes at MFNP showed no significant health effects other than skin lesions; however, individual giraffes are not easily or routinely monitored. Histopathological examination of the lesions demonstrated associated bacterial infection as well as disruption of the lymphoid system, so secondary or long-term, health-related complications in affected giraffes is possible.
We have been unable to find or generate a comparable DNA sequence from a known Stephanofilaria sp. specimen to genetically confirm our diagnosis. Mostly recognized in domestic cattle, different species of Stephanofilaria in various geographic regions are usually each associated with lesions on specific anatomic areas of the body (e.g., Stephanofilaria assamensis in India causing chronic head and neck lesions in cattle and domestic buffalo). In East Africa, stephanofilariasis is a recently documented disease of concern in both black and white rhinoceros but remains poorly understood in terms of disease ecology (Mutinda et al. 2012).
Wildlife habitat loss and associated increased human and domestic animal contact with wildlife species are widely recognized drivers of interspecies disease transmission to and from wild animals. Added to the threats that giraffes face from poaching and habitat loss, infectious diseases including those that might be new or recently emerging need to be understood to inform management and conservation planning. The conditions collectively referred to as giraffe skin disease are of increasing concern but have undergone little systematic diagnostic investigation. Detection and tentative identification of this treatable filarial parasite in affected giraffes at MFNP contribute to our understanding of giraffe skin diseases. These skin lesions are likely not a significant health threat to the remaining 1,400 individual Rothschild's giraffes.
We thank Robert Aruho for assistance collecting and shipping samples and Mark Eberhard for assistance with identification of parasites sections from histopathology images. In addition to in-kind support from all partners, partial funding was provided by Sea World Bush Gardens Conservation Fund.