Clinico-Microbiological and Therapeutic Aspects of Glanders in Equines

Ghulam MUHAMMAD*, M. Zargham KHAN and Muhammad ATHAR Department of Clinical Medicine and Surgery, Faculty of Veterinary Science, University of Agriculture, Faisalabad 38040, Pakistan

The role of glanders therapy as an alternative to test and slaughter policy was investigated in J. Equine Sci. 13 draught equines (1 horse, 2 mares and 10 mules). The disease was diagnosed clinically and Vol. 9, No. 3 confirmed by isolation of Burkholderia mallei on blood agar and sterile potato slices. Mallein pp. 93–96, 1998 test was positive in 6 (46.15 %) of the 13 animals. All isolates from the 13 equines were sensitive to chloramphenicol, danofloxacin, norfloxacin and co-trimoxazole whereas penicillin and cephalexin were the least effective in vitro antibiotics. These animals were treated for 4 days with Ringer’s-lactate-dextrose 500 ml + dimetyl-sulfoxide 60–80 ml intravenously and Inj. Norfloxacin 5% 35–50 ml intramuscularly (1st round of treatment). Five animals were treated again similarly (2nd round) after two weeks of the first round. All treated animals showed a dramatic improvement in the severity of the disease condition which lasted for 2–3 weeks after which the signs rebounded. Even the second round of treatment was met with the same fate. The treatment was not associated with any side-effect except in two cases which suffered injection site swelling. The possibility of a cure in clinical glanders using a longer course of treatment is proposed. Key words: glanders, chemotherapy, equines, Burkholderia mallei

Glanders is one of the oldest known diseases free of this disease. The present study was, therefore, principally in equines, and at one time it was widespread designed to investigate the role of therapy of glanders as throughout Europe, Asia, Africa, and to a lesser extent, an alternative to animal destruction. the Western hemisphere. Through test and eradication methods, sanitation, veterinary surveillance and because of the decrease in density of the horse population, the Materials and Methods disease has been eliminated or controlled in most well developed countries. The disease, however, is still Animals: Over an 18-months period (September 1995 prevalent in developing countries such as India [10], to March, 1997), one horse (8 years old), 2 draught mares China [11], Turkey [2], Iraq [3], and Pakistan [4] as well (7 years old and 17 years old) and 10 mules (5–16 years as Eastern Europe and North Africa [13]. old) were brought to the outdoor clinics of the In Pakistan, the statutory requirements call for Department of Clinical Medicine and Surgery, University detection and destruction of the animals affected with of Agriculture, Faisalabad, with clinical signs compatible glanders [7]. However, the incredibly low indemnity with those of glanders [13]. All animals were kept for paid to the owners and lack of any rigorous draught purpose. implementation of the Glanders & Farcy Act (1899) do Diagnosis of the disease: The diagnosis of the disease not oblige the owners to let their animals be destroyed. was based on clinical signs; intradermo-palpebral mallein Because of international trade and movement of equines, test (using Mallein supplied by Veterinary Research the glanders may spread to countries which are currently Institute, Lahore, Pakistan); haematological examination (on blood samples collected before treatment) [5]; and This article was submitted July 24, 1998 and was accepted duration of the existence of the disease before seeking November 6, 1998. *corresponding author. professional advice. 94 G. MUHAMMAD, M.Z. KHAN, M. ATHAR

Isolation of Burkholderia mallei from the pus: The pus Table 1. Clinical signs and their frequencies noted among 13 was aspirated aseptically from the unopened skin equines suffering from glanders swellings and cultured on blood agar, MacConkey’s agar Clinical Signs Frequency and sterile potato slices placed in a jar. Gram-stained a) Fever: smears prepared from the pus aspirated from unopened - Mild (38.9–39.4°C) 7 soft swellings on the skin were examined microscopically - Moderate (39.4–40°C) 6 (100 ×). In addition, blood drawn aseptically was also b) Inappetance, depression, cough, dyspnoea, debility cultured on blood agar and MacConkey’s agar. and a quick loss of stamina when put to work: 13 c) Bilateral nasal discharge: Antibiotic sensitivity test: Antibiotic susceptibility of the - with blood 12 isolates to antibiotics/antibacterials was performed on - without blood 1 Mueller-Hinton medium. Staphylococcus aureus ATCC d) Nasal septum ulcers: 12 25923 (American-type Culture Collection. Rockville, e) Nodules and mound-like ulcers on: Maryland, USA) was used as a quality control. The - groin 4 isolates were categorized as sensitive, intermediate, and - stifle 4 - flank 5 resistant according to the size of the zone of inhibition of - perineal region 6 antibiotics/antibacterials [12]. - medial aspect of thigh (Fig. 1) 11 Treatment of the disease: The affected animals were - forelegs 5 treated for 4 days as follows: f) Cording of lymphatics: 6 a) Ringer’s-lactate-dextrose (5%) 500 ml + dimethyl- g) Slight enlargement of submaxillary lymph nodes sulfoxide (DMSO) (BDH Limited, Poole, UK) 60– without any tenderness or heat: 12 h) Haematuria: 1 80 ml (depending upon the body weight) slow IV; b) Inj. Norfloxacin 5% (Tarobina Corp., Pakistan) 35– 50 ml (depending upon the body weight) IM at 2 different sites. leukocytic count (4,900–14,800/mm3). The treatment was repeated at 24 hr intervals. The Microbiological Findings: The yellowish pus aspirated response to the treatment was monitored by examination aseptically from unopened matured subcutaneous on day 7, 14, 21, and 28 post-treatment. Five animals nodules when cultured on blood agar yielded numerous were treated again similarly on day-15 thru 18 post-first small non-haemolytic slimy, light greyish-yellow colonies round of treatment and examined weekly for the next 3 at 48 hr which turned granular and yellowish brown at weeks. 72 hr. In 5 cases, culture of blood on blood agar yielded skimpy growth at 72 hr. Growth on potato slices was characterized by the presence of light yellowish pigment Results resembling honey at 48 hr which turned deep reddish brown at 72 hr. A chocolate coloured fluid accumulated Important clinical signs and their frequencies among at the bottom of the jar. There was no growth on 13 equines are summarized in Table 1. MacConkey’s agar. Gram-stained smears of aspirated Mallein test was positive in 6 (46.15%) (Fig. 2) and pus as well as the growth on the blood agar and potato negative in 7 (53.85%) cases. As learned from owners, slices revealed irregularly staining Gram-negative the disease had existed for 15 days, 1 month, 1–2 months, microorganisms with beaded and filamentous and 2–4 months, respectively in 3,3,4, and 3 cases before appearance. On the basis of the microbiological features seeking our professional advice. In 6 cases, there was a together with characteristic clinical signs and results of history of co-dwelling with animals showing signs mallein test (in 6 cases positive) described in the suggestive of glanders. Three mules afflicted with this foregoing, a confirmatory diagnosis of implication of condition belonged to one owner. There was no history Burkholderia mallei, the etiological agent of glanders was of signs suggestive of glanders among the owners or the reached. All isolates were sensitive to chloramphenicol, attendants. danofloxacin, norfloxacin, and co-trimoxazole, whereas Haematological Findings: Haematological examination penicillin-G and cephalexin were least effective in vitro before commencing therapy revealed an accelerated ESR antibiotics (Table 2). (132–142 mm/first hour) increased fibrinogen (2393– Response to Treatment: All treated animals showed a 2789 mg/dl), decreased haemoglobin (4.8–6.8 g/dl) and dramatic improvement in the severity of the disease haematocrit (18–22%) values, and a varying total condition as reflected by improved feed intake, drying GLANDERS IN EQUINES 95

Fig. 1. Glander’s nodules on the caudo-medial aspect of thigh Fig. 2. Positive mallein reaction in a horse. of a mule.

in Pakistan at present) and the lack of stern enforcement Table 2. Antibiogram profiles of Burkholderia mallei isolates of glanders control laws as well as government’s (n=13) to 11 different antibiotics/antibacterials preoccupation with other more pressing socioeconomic No. of Isolates Antibiotic/Antibacterial issues do not create a milieu conducive for the adoption Sensitive Intermediate Resistant of test and slaughter glanders policy. This scenario would Cephalexin - 3 10 undoubtedly continue in the foreseeable future. It was Chloramphenicol 13 - - against this backdrop that we attempted to investigate Danofloxacin 13 - - the role of therapy as an alternative to animals’ Erythromycin 10 3 - destruction. Kanamycin 10 2 1 Norfloxacin was selected because recently another Lincomycin 8 2 3 Metronidazole 2 - 11 member of quinolone group viz ofloxacin proved to be Norfloxacin 13 - - the most effective antibiotic in the treatment of golden Penicillin G - 2 11 hamsters infected subcutaneously by glanders bacillus Streptomycin 11 2 - [9]. Norfloxacin was adjunct with DMSO because this Co-trimoxazole 13 - - chemical not only enhances penetration of co- administered drugs into the infection foci [6], but also antibiotic-resistant and antibiotic-sensitive of the ruptured cutaneous nodules, marked reduction in microorganisms have been shown to be more sensitive nasal discharge, and improvement in general demeanor. to antibiotics after exposure to DMSO [8, 18]. This improvement lasted for 2–3 weeks after which the Of the 13 cases of glanders which were confirmed signs rebounded. In five animals, even a second round bacteriologically, only 6 (46.15%) reacted positive on of treatment was met with the same response. No side- mallein testing. Poor sensitivity of mallein test in acute effects were noted except swelling at injection site in two and advanced form of glanders has already been animals. A temporary improvement prompted the documented [10, 14]. Because of poor sensitivity of owners to sell their animals as a salvage measure. mallein test, the use of other diagnostic techniques such as indirect fluorescent antibody technique [11], Dot ELISA [16], micro-complement fixation test [15], and Discussion micro-ELISA test [1] has been advocated. Haematuria observed in one of the subjects of the present study may Because of the risk to both man and animals, glanders be ascribed to metastatic spread to the kidneys [17]. is a notifiable disease in most countries of the world. Haematological alterations reflected anaemia and a Infected animals should be slaughtered at once and the chronic inflammatory response [5]. carcass disposed off properly. Treatment of equine Inside the body, the glanders bacilli have been shown glanders is forbidden in most countries [17]. to be contained in firm nodules of varying sizes showing Ridiculously low indemnity (equivalent of only $ 1.1 chronic inflammatory changes [3]. Because of the poor 96 G. MUHAMMAD, M.Z. KHAN, M. ATHAR penetration of antibiotics into these niches, even with an Savchenko, S.T. and Lozovaia, N.A. 1994. antibiotic effective against the organism under in vitro Effectiveness of treatment of experimental glanders conditions, it may not be possible to rid these sites of the after aerogenic infection. Antibiot. Khimioter 39: 9– infection. This may account for the recrudescence of 10. the disease after the cessation of antibiotic therapy. 10. Jana, A.M., Gupta, A.K., Pandya, G., Verma, R.D. 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