Glanders has been eliminated from the western world by means of a rigorous test and culling policy, frequently accompanied by sensible compensation schemes
[18, 19]. However, in countries with low or no compensation, albeit strict legislation, implementation of these regulations is extremely difficult, and as a consequence regions of endemicity may be formed or continue to persist
. These regions not only pose a local public health threat but may also have a major impact on international horse transportation, especially with regard to equestrian sport and breeding. Highly valuable animals are regularly transported to and from endemic regions and thus pose a risk of reintroduction of disease to so far disease free areas. Availability of effective treatment schemes and post-exposure prophylaxis could help to reduce spread of disease. In recent years, evidence has accumulated from in vitro antibiotic sensitivity testing and in vivo treatment studies that certain antibiotics such as enrofloxacin, erythromycin, doxycycline, and sulphonamides might be effective against B. mallei[1, 21, 22]. The present study therefore, aimed at assessing the effectiveness of a treatment protocol of 12 week duration based on the parenteral administration of enrofloxacin and trimethoprim + sulfadiazine, followed by the oral administration of doxycycline during a confined glanders outbreak in horses in Pakistan. Enrofloxacin is a fluoroquinolone antibiotic with an antibacterial activity against a broad range of Gram- positive and Gram-negative bacteria. Its mode of action is not fully known, but it is believed to interfere with bacterial DNA synthesis by inhibiting bacterial DNA gyrase
. Trimethoprim reversibly inhibits the bacterial synthesis of tetrahydrofolic acid, which is essential for the de novo synthesis of Thymidine monophosphate, a precursor of the DNA metabolite Thymidine triphosphate
. Sulfadiazine, like other sulphonamides, interferes with the bacterial synthesis of dihydrofolic acid, ultimately leading to bacterial death by starvation
. Doxycycline is a semi-synthetic tetracycline with an antibacterial activity against a broad range of Gram-positive and Gram-negative bacteria. Tetracyclines inhibit protein synthesis by binding to the 30S subunit of susceptible organisms
Several rather anecdotal reports describing the successful treatment of horses with glanders exist. Early attempts towards the end of the 19th century and in the very beginning of the 20th century to “vaccinate” or even cure horses using mallein or other heat or chemically inactivated B. mallei preparations, i. e. “anamorve”, and “farase” did not lead to satisfactory results and were thus aborted
[24, 25]. However, a few decades later, new approaches based on the application of different sulphonamides combined with mallein or “anamorve”, i.e. heat-inactivated, and formolized B. mallei showed a more promising outcome. Back in 1951, Deyhimi and Katai succeeded in treating 141 glanderous horses and 18 mules by means of using various sulphonamides (sulphathiazole, sulphanilamide, sulphadiazine, sulphamezathine) for thirty consecutive days in combination with “anamorve” or mallein
. Subsequent post mortem studies done 12 and 30 months after end of treatment on three horses revealed non specific findings and no cultures could be obtained. Moreover, inoculation of hamsters with samples obtained from these horses did not induce any kind of reaction. A similar treatment protocol was used by Fathi et al.
, who managed to cure 384/400 glanderous horses and mules by the combined application of sulphonamides given for 30 consecutive days with a formolized preparation of B. mallei or mallein. Hu et al. used sulphamezathine (sulfadimidin) and mallein to treat artificially infected acutely diseased horses and asses and apparently cured 41/48 of the former and 17/23 of the latter
. In a more recent study, Muhammad et al. managed to alleviate clinical symptoms in 13 draught equines (horse, mare, and mule) by applying dimethylsulfoxide, a free radical scavenger with anti-inflammatory effects and norfloxacin, a fluoroquinolone antibiotic, for four consecutive days
. All animals showed a temporary improvement which generally lasted for 2–3 weeks. In all studies mentioned including our own successful outcome was dependant on duration of therapy. The significance of treatment duration on disease outcome has also been shown for small laboratory animals such as mouse and hamster, and man
[1, 29, 30]. Infected mice treated for ten days with ceftazidime, a third generation cephalosporin and levofloxacin, a fluoroquinolone, survived for 34 days, however treatment did not result in complete clearance of infection, and bacteria were still found in lungs and spleens
. Likewise, onset of antibiotic treatment after infection with B. mallei and efficacy of the antibiotic used influenced disease outcome in hamsters significantly
. Prolonged treatment duration possibly prevents the establishment of chronic disease. Chronic glanders as a sequel to non-apparent or non-fatal infection
[24, 31] poses a particular challenge to the practitioner as it can go unnoticed for a long period of time. It is still not clear what causes the “dormant state” and what triggers off disease. Histopathological studies in horses assessing samples obtained from glanderous lesions of the nasal septum have detected bacteria in the vicinity of extensive accumulations of disintegrating neutrophilic granulocytes and macrophages, indicative of intracellular invasion and survival
. However, recent data obtained from a respiratory cell model suggest that B. mallei actually induces apoptotic cell death
. This might offer an explanation for the protraction or recurrence of disease. Depending on the amount of released bacteria and the individual immune status of the affected animal, the disease can continue as a non-apparent infection or exacerbate.
The results of the present study suggest that long term treatment, i.e. a minimum of 12 weeks, of glanderous horses with a combination of various antibiotics could apparently eliminate the pathogenic agent from the infected animal host. Induction of immunosupression in six randomly selected horses did not lead to recrudescence of disease. Moreover, when the treated animals were returned to the polo club and housed with other horses, no further cases were reported. Cultivation is considered a method with low sensitivity, especially in chronic cases. Under ideal circumstances, one viable bacterium is hypothetically sufficient for cultivation. Based on the assumption that immunosupression will activate infection and the number of viable bacteria is high, one can assume an increase in sensitivity. In order to determine the success of treatment is not sufficient to rely on cultivation results alone; all techniques applied, i.e. the clinical picture, pathology/necropsy results, microbiology, and health status of the sentinel animals must be evaluated in their entirety.
Of the treated horses, seven are still active at the polo club and two are kept for breeding purposes and are still healthy to date (September 2012; data not shown). The remaining eight were sold and their whereabouts are unknown to the authors. A further shortcoming of this study is that no animal was submitted to immunosuppressive therapy after the one year observation treatment to induce recrudescence. However, if the horses were chronically ill, constant stress due to training and pregnancy ought to trigger off acute disease. Ever since this outbreak, horses at the polo club have been monitored regularly with the mallein test and none of the treated animals has been tested positive so far (data not shown). It must be stressed that any animal (treated or untreated) with a positive complement fixation test (CFT) or positive indirect haemagglutination test (IHAT) is subject to national and international restrictions and regulations concerning movement control, re-testing, quarantine and culling. Of note are the findings that on day 0, prior to malleinisation, 22/23 horses where positive in the IHAT, and titres increased further during the treatment period. One explanation for this rise in titres is the malleinisation, which is in accordance with previous findings
. A further influencing factor might be the antibiotic-induced release of B. mallei antigen. The gradual but continuous decline of the titres could be an indication of effective and successful therapy.
Although the presented study has shown very promising results, they must be treated with some caution. B. mallei strains from the Punjab represent only a small part of the genetically diverse pathogen
. Therefore, more studies, possibly from small confined outbreaks in different endemic regions are needed to test if this treatment regime is effective on other B. mallei strains.