VETERINARSKI ARHIV 68 (5), 155-162, 1998

ISSN 0372-5480
Printed in Croatia





Glanders in horses: clinical, biochemical and serological studies in Iraq

Falah K. Al-Ani1*, Odeh F. Al-Rawashdeh1, Ali H. Ali2, and Farook K. Hassan2

1Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid, Jordan

2Department of Veterinary Microbiology, College of Veterinary Medicine, Baghdad University, Baghdad, Iraq





* Contact address:
Prof. Dr. Falah K. Al-Ani,
Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid, P.O. Box: 620026 Southdistrict, Jordan,
Phone: 962 2 295 111, Fax: 962 2 295 123, E-mail: alan@just.edu.jo


AL-ANI, F. K., O. F. AL-RAWASHDEH, A. H. ALI, F. K. HASSAN: Glanders in horses: clinical, biochemical and serological studies in Iraq. Vet. arhiv 68, 155-162, 1998.

ABSTRACT

Epidemiological and diagnostic aspects of glanders were studied, using a total of 134 horses. Based on clinical examination and risk status the animals were divided into four different groups. Samples for bacteriological isolation and serological testing were collected and a pure culture of Burkholderia mallei was isolated from all horses, which developed the classical picture of glanders. The cutaneous form of the disease was characterised by the appearance of rounded, raised and firm nodules along the course of lymphatic vessels. The results of antibiotic sensitivity tests indicated that B. mallei is sensitive to baytril, gentamicin, erythromycin, oxytetracycline and ampicillin. Infected horses were shown to develop an antibody titer of 1:80 or more.

Key words: glanders, Burkholderia mallei, Pseudomonas mallei, horse, Iraq



Introduction

Glanders is an old-age respiratory disease of horses occurring in different parts of the world. The disease has been reported in Iraq (Al-Ani et al., 1987; Al-Kafawi et al., 1977) in Egypt and certain parts of Africa (Henning, 1956) as well as in Italy (Battelli et al., 1973), in India (Gangulee et al., 1966; Verma et al., 1994), in China (Zhang and Lu, 1983; Ma et al., 1986) and in Russia (Shumilov, 1974). On the other hand, glanders has been eradicated from certain parts of the world through the mass slaughter of affected and suspect animals. The disease has been completely eradicated from the United States, England and Australia (Henning, 1956). Glanders is of major concern in the Middle East where it is widely distributed, has a high incidence in endemic areas, and affects all breeds and ages of horses and mules. A vaccine for the disease is still undergoing trials, making the control and eradication of glanders difficult (Zhang and Lu, 1983). The aims of the present study were to describe the clinical features of glanders, biochemical properties of isolated B. mallei and the application of a passive haemagglutination test in the diagnosis of infected horses.

Materials and methods

Animals

The study was conducted on Arabian-breed horses intended for use as racehorses. They were kept in a large-scale racehorse maintenance facility of about 25 hectares. The horses were divided into four groups according to the following criteria:

Group 1: 12 horses confirmed as being infected with glanders by bacteriological isolation of B. mallei from cutaneous nodules.

Group 2: 49 racehorses, 3-7 years old, which were in direct contact with infected horses.

Group 3: 37 mares, 5-15 years old, kept for breeding purposes, which were in contact with infected horses.

Group 4: 36 Arabian-breed horses, 2-15 years old, brought from an area known to be free from glanders. No antibody titres for glanders were detected in their sera by complement fixation test, and the animals were kept under observation for one year prior to the study.

Clinical and laboratory examination

The horses were examined clinically every day. The skin and nasal passages were carefully examined for any gross lesions. All horses were routinely tested by the intradermo-palpebral mallein test.

Samples for bacteriological isolation were collected aseptically and streaked on potato-dextrose agar (DIFCO) containing 3% glycerin. B. mallei was identified morphologically and biochemically according to the methods described by Miller et al. (1948) and Rogul et al. (1970). Mller-Hinton agar was used for sensitivity testing of the isolates by the disc diffusion method, in accordance with the method of Quinn et al. (1994). The antimicrobial agents used were: ampicillin (10 g), penicillin G (10 I.U.), streptomycin (10 g), sulphamethoxasole/ trimethoprim (25 g), amoxycillin (25 g), erythromycin (25 g), baytril (5 g), chloramphenicol (30 g), tetracycline (10 g) and gentamicin (10 g).

Blood was obtained from the jugular veins of all horses and sera were separated and stored at -20 oC until used in the antibody detection assay. The indirect passive haemagglutination test was performed using the microtitre technique according to the method of Zhang and Lu (1983). A titre of 1:80 and over was considered positive.

Results

The early clinical signs of glanders were characterised by slight serous nasal discharge, gradual loss of weight and a sudden decrease in appetite. A few horses developed slight stiffness of the forelegs while others had oedema of one hind leg, or both hind legs. Some days to weeks later, the nasal discharge became mucopurulent and was associated with the enlargement of one or both submaxillary lymph nodes. The cutaneous nodules were characterised by the appearance of round, raised and firm nodules along the course of lymphatic vessels. These lesions would appear anywhere on the body but were mostly observed on the head, neck, back and legs (Fig. 1). After about 3-7 days, the lesions opened up and discharged a creamy pus. Two horses developed severe orchitis. The scrotal sac of both testicles became enlarged and painful. These lesions were probably an extension of an existing infection, although they could have resulted from direct contact with contaminated bedding. Careful examination of the visible portions of the nasal mucosa frequently revealed deep ulcers, particularly over the nasal septum. Healing of the ulcers resulted in characteristic star-shaped cicatrices. These lesions are pathognomonic for glanders. Auscultation of the lungs revealed vesicular murmur and rales. One mare, with advanced cutaneous lesions of glanders, aborted at 7 months gestation. Also, B. mallei was isolated from milk collected from a mare with mastitis. However, glanders bacilli is not a major cause of mastitis in mares.

Fig. 1.

Fig. 1. A horse with cutaneous nodules of glanders on the legs


Fig. 2.

Fig. 2. A horse with glanders and with positive mallein test

The chronic form of the disease developed as a sequel to either non- clinical infections or non-fatal acute cases, and was manifested by progressive loss of body weight, a "run-down appearance" and unthrifty hair coat. Such horses are unfit for racing purposes. These horses produced a positive mallein test and developed detectable circulating antibodies, which could be detected by serological tests (Fig. 2). Fresh pus samples streaked on potato-dextrose agar (DIFCO) containing 3% glycerol revealed the appearance of small, round, amorphous, translucent colonies. A number of biochemical tests were applied to the B. mallei isolates (Table 1). The biochemical tests most commonly used are: glucose, fructose, galactose and motility test. Furthermore, intraspecific variation in some biochemical reactions is often considerable, rendering biochemical test application of less value. Based on biochemical reactions the isolates of B. mallei were seen to fall into three groups (Table 1). Antibiotic sensitivity testing showed that erythromycin, oxytetracycline, gentamicin, sulphamethoxazole/trimethoprim, ampicillin and baytril provide large zones of inhibition.

Table 1. Biochemical properties of different isolates of Burkholderia mallei from horses tested in Iraq

Property

Type-1 isolates
(N=9)

Type-2 isolates
(N=4)

Type-3 isolates
(N=1)

Oxidase

+

+

+

Catalase

+

+

+

Indole

-

-

-

Nitrate reduction

-

-

slight

Methyl red and Voges-Proskauer

-

-

-

H2S production

slight

slight

slight

NH3 production

slight

slight

slight

Gelatin liquefaction

-

-

-

Motility test

-

-

-

Glucose

+

+

+

Fructose

+

+

delayed

Galactose

+

+

+

Mannose

+

+

delayed

Lactose

+

+

delayed

Sucrose

-

-

-

Maltose

+

-

-

Mannitol

+

+

+

Arginine dihydrolase

+

+

+

Serological testing showed that eleven of the twelve horses in group 1 had a titre between 1:80 and 1:640. The remaining animals and a mare in an advanced stage of glanders gave a reading of 1:20 titre. Five of the horses in group 2 had a titre of 1:80 and also gave a positive mallein test. Fifteen months following isolation, two of these horses developed the cutaneous form of glanders, from which B. mallei were isolated. Six mares from group 3 had a titre of 1:80 and over. None of the horses in group 4 had a titre exceeding 1:20. Therefore, the passive agglutination test can be routinely used for herd diagnosis and survey purposes. The presence of antibodies in horses with no history of lesions suggested that some infections may be unapparent or unobserved.

Discussion

The glanders bacillus is one of several species belonging to the genus Burkholderia (Yabuuchi et al., 1992) and its new name Burkholderia mallei differential of the various species in the genus is based on the 16s rRNA sequences, DNA-DNA homology values, cellular lipid and fatty acid composition, and phenotypic characteristics (Yabuuchi et al., 1992). The medium used routinely for the isolation and growth of B. mallei is Brain Heart infusion Agar (DIFCO) alone, or with 2-3% glycerol or Potato-dextrose agar (Rogul et al., 1970; Quinn et al., 1994). Variations in biochemical reactions of B. mallei isolates reported in this paper correspond to previous reports (Redfearn et al., 1966). Horses are believed to be the principal reservoir of B. mallei infection, although mules, donkeys, goats and swine can also be naturally infected (Radostits et al., 1994). The epidemiological significance of infection in these species is unknown. The literature contains voluminous descriptions of clinical signs and the many antibody prevalence studies which reflect the extent of natural infections, indicate that glanders is widely distributed in horse populations in many parts of the world (Battelli et al., 1973; Verma, 1981; Al-Ani et al., 1987). In Iraq, the case-fatality rate is considerably higher in breeding mares than in young horses (Al-Ani et al., 1987). This is probably due to the effect of age, exposure and other stress factors resulting from management practices. When one case occurs, other cases are usually also present in a group of horses reared together. Most infected animals survive with serious harm and are thus unfit for racing purposes (Al-Ani et al., 1987).

Asymptomatic carriers, as well as horses in the acute and convalescent stages of the disease, shed the organisms in their respiratory and cutaneous secretions (Radostits et al., 1994). Transmission occurs by direct contact through droplet spread, and indirect contact with many contaminating environmental sources, including water or feed. Contaminated bedding, fences, stanchions, surgical instruments, needles, halters, ropes and chains can presumably also serve as an intermediate source of infection (Radostits et al., 1994). The presence of asymptomatic carriers indicates generally a low susceptibility to clinical illness. Specific immunity following recovery from the disease is of uncertain importance in providing future protection. Mohler and Eichhorn (1914) reported the failure of immunisation of horses by means of dead bacilli. Inoculated animals develop agglutinins and complement fixation antibodies, but they remained susceptible to either natural or artificial infection. Such horses usually also give a positive mallein test.

The traditional, and still current, basis of glanders diagnosis is by mallein testing (Hagebock et al., 1993). However, there are several disadvantages of mallein testing, including negative results in horses in an advanced stage of the disease (Verma, 1981). False positive reactions are also encountered, particularly between B. mallei infection and Streptococcus equi infection (Al-Ani et al., 1993). Despite these detractions, the mallein test remains a valuable screening test. The existence of cross-reactions between glanders and strangles in the mallein test was not ascertained from this study. However, Misra and Arora (1990) found serological a cross-reaction between Pseudomonas mallei and some other bacteria.

The results of the study indicated that glanders could occur in horses of any age, although the prevalence of infection increases with age, and that contact with infected adult horses seems to be the major influencing factor (Zubaidy and Al-Ani, 1978). A serological diagnosis of glanders can be made by demonstrating a titre of specific antibody when haemagglutination, complement fixation and counter immuno-electrophoresis tests are employed (Gangulee et al., 1966; Sen et al., 1968; Verma, 1981; Jana et al., 1982; Zhang and Lu, 1983). Our results showed that a titre of 1:80 or over in the passive haemagglutination test can be regarded as a positive reaction. In India, an haemagglutination titre of 1:640 is considered to be positive (Gangulee et al., 1966).

References

Al-Ani, F. K., A. K. Al-Delaimi, A. H. Ali (1987): Glanders in horses: clinical and epidemiological studies in Iraq. Pakistan Vet. J. 7, 126-129.

Al-Ani, F. K., A. H. Omran, F. S. Al-Zubaidy (1993): A micro-enzyme-linked immunosorbent assay (ELISA) for detection of antibody to Pseudomonas mallei infection in horses. Pakistan Vet. J. 13, 70-73.

Al-Kafawi, A. A., F. K. Al-Ani, L. S. Al-Bassam, A. Y. Youkob (1977): Hematological changes in Arabian horses infected with glanders. Vet. Rec. 101, 427.

Battelli, C., T. Contente, G. Corsalini, P. Goffredo, P. Lazari, V. Puccini, L. Sobrero (1973): Glanders in a group of lions in captivity. Vet. Italica 24 , 87-112.

Gangulee, R. C, G. P. Sen, G. L. Sharma (1966): Serological diagnosis of glanders by haemagglutination test. Indian Vet. J. 43, 386.

Hagebock, J. M., L. K. Schlater, W. M. Frerichs, D. P. Olson (1993): Serologic responses to the mallein test for glanders in solipeds. J. Vet. Diagn. Investig. 49, 97-99.

Henning, M. W. (1956): Animal Diseases in South Africa, 3rd ed. Central News Agency Ltd. Kelvin.

Jana, A. M., A. K. Cupta, G. Pandy, R. D. Verma, K. M. Rao (1982): Rapid diagnosis of glanders in equine by counter immuno-electrophoresis. Indian Vet. J. 59, 5-9.

Ma, C. L., S. M. Fan, X. Wang, L. Jiang, S. Q. Fang (1986): Diagnosis of glanders in horses by the indirect fluorescent antibody technique. Chinese J. Vet. Sci. Technol. 9, 3-5.

Miller, W. R., L. Pannel, L. Cravitz, W. A. Tanner, M. S. Ingalls (1948). Studies on certain biological characteristics of Malleomyces mallei and Malleomyces pseudomallei. 1. Morphology, cultivation, viability and isolation from contaminated specimens. J. Bacteriol. 55, 115-126.

Misra, V. C., A. K. Arora (1990): Serological cross-reaction between Pseudomonas mallei and some other bacteria. Indian J. Comp. Micro. Imm. Infe. Dis. 11, 28-31.

Mohler, J., N. Eichhorn (1914). Immunization tests with glanders vaccine. J. Comp. Pathol. 27, 183-185.

Quinn, P. J., M. F. Carter, B. Markey, G. R. Carter (1994): Clinical Veterinary Microbiology. Wolfe Publishing Company. London.

Redfearn, M. S., N. J. Allerani, B. Y. Stainer (1966): A comparative study of Pseudomonas pseudomallei and Bacillus mallei. J. Gen. Microb. 43, 293-313.

Radostits, O. M., D. C. Blood, C. C. Gay (1994): Veterinary Medicine. Bailliere Tindall. London.

Rogul, M., J. J. Brendle, D. K. Haapala, A. D. Alexander (1970): Nucleic acid similarities among Pseudomonas pseudomallei, Pseudomonas multicerans and Actinobacillus mallei. J. Bacteriol. 101, 827-853.

Sen, G. P., G. Singh, T. P. Jeshi (1968): Comparative efficacy of serological tests in the diagnosis of glanders. Indian Vet. J. 45, 286-292.

Shumilov, K. V. (1974). Comparative study of methods of diagnosis of glanders in horses in Mangolia. Trudy. Veses. Nogo. Institut. Eksperimintalnaia Veterinaria 42, 272-282.

Verma, R. D. (1981). Glanders in India with special reference if incidence and epidemiology. Indian Vet. J. 58, 177-183.

Verma, R. D., K. S. Venkateswaran, J. K. Sharma, G. S. Agarwal (1994): Potency of partially purified malleo-proteins for mallein test in the diagnosis of glanders in equines. Vet. Microbiol. 41, 391-397.

Yabuuchi, E., Y. Kosako, H. Oyaizu (1992). Proposal of Burkholderia genus and transfer of seven species of the genus Pseudomonas homology. J. Microbiol. Immunol. 36, 1251-1275.

Zhang, W. D., Z. B. Lu (1983): Application of an indirect haemagglutination test for the diagnosis of glanders and melioidosis. Chinese J. Vet. Med. 9, 8-9.

Zubaidy, A. J., F. K. Al-Ani (1978): Pathology of glanders in horses in Iraq. Vet. Pathol. 15, 566-568.

Received: 12 August 1997
Accepted: 8 September 1998



AL-ANI, F. K., O. F. AL-RAWASHDEH, A. H. ALI, F. K. HASSAN: Sakagija u konja: klinicka, biokemijska i seroloska istrazivanja u Iraku. Vet. arhiv 68, 155-162, 1998.

SAZETAK

Na 134 konja istrazivane su epidemioloske i dijagnosticke osobitosti sakagije. Zivotinje su podijeljene u 4 skupine prema klinickim znakovima i stupnju rizika. Prikupljeni su uzorci za seroloske pretrage i za izolaciju bakterije, te su uzgojene ciste kulture Burkholderia mallei od svih konja koji su razvili klasicnu sliku sakagije. Osobitosti koznog oblika bolesti bili su okrugli, uzdignuti i tvrdi cvorovi uzduz limfnih zila. Rezultati odredivanja osjetljivosti bakterije prema antibioticima pokazali su da je B. mallei osjetljiva na baitril, gentamicin, eritromicin, oksitetraciklin i ampicilin. Utvrdeno je da su zarazeni konji razvili titar protutijela od 1:80 i vise.

Kljucne rijeci: sakagija, maleus, Burkholderia mallei, Pseudomonas mallei, konj, Irak


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