Mallein Research Articles

Serological and Bacteriological Surveillance of Glanders Among Horses in Central Region of Iran.

Glanders is the oldest and very contagious disease among horses caused by Burkholderia mallei. The disease occurs as a chronic form in horses. Hence, because of the prolonged shedding, numerous horses can potentially get infected by one horse with glanders. Glanders is endemic in Iran and this causes occasional occurrence in horse population of the country. The aim of this study was to determine the incidence of B.mallei infection in horses in two central provinces of Iran. A total of 517 serum samples were collected from stable horses in Tehran and Alborz provinces. Among the studied horses, seven presented fever, anorexia, dyspnea, subcutaneous abscesses, nasal and cutaneous discharges, emaciation, and lymphadenopathy. Nasal and ocular discharges and subcutaneous abscesses were sampled for bacterial culture and PCR. The sera were examined by means of complement fixation test (CFT) and indirect enzyme-linked immunosorbent assay (iELISA). Seropositive cases were further examined by Mallein test. The results derived from the present study indicated that only 1.35% of the studied horses were positive in CFT, iELISA and Mallein test, of which only in 42.85% B.mallei was successfully cultured on blood agar and glycerinated nutrient media and confirmed by PCR. Periodic serological tests along with quarantine can benefit reduction of the occurrence of the disease in horses in Iran.

Designing of Western Blot Technique for Glanders Diagnosing in Iran

Burkholderia mallei is the etiologic agent of glanders. It is difficult to diagnose this zoonotic disease in its early stages. Some methods such as the complement fixation test (CFT) cause some problems for veterinary authorities and financial losses to animal owners due to false-positive results. The mallein test requires appropriate laboratory equipment and skilled personnel. To quickly and accurately diagnose the disease, especially in areas where animals cannot be kept, new methods (such as the Western blot test [WBT]) should be used to identify the disease. This study designed and optimized the Western blot (immunoblot) test using sera from 84 glanderous equids, and the sensitivity and specificity of ELISA and CFT were compared with the WBT. ELISA tests are based on B. mallei antigens whereas a purified lipopolysaccharide-containing B. mallei antigen is used in the WBT. The sensitivity and specificity of the tests were estimated using the cut-off values recommended by the test developers. The WBT and ELISA were significantly more specific than the CFT. The ELISA based on B. mallei antigens was significantly less sensitive than the CFT. Given their comparable sensitivities and specificities, the CFT (95.7%, 98.5%), the WBT (95%, 100%) and the ELISA (85%, 100%) should be further developed. The CFT is still the prescribed technique for serological investigation of equids for trade purposes to certify individual animals without glanders. Therefore, more efforts should be made to further improve and optimize the WBT and ELISA tests.

Development of Rose Bengal test against mallein test for rapid diagnosis of equine glanders.

Burkholderia mallei, the etiologic agent of the disease known as glanders. Clinical and bacteriological diagnosis of glanders is difficult in the early stages of the disease. Currently, mallein (allergic hypersensitivity test) is used for the diagnosis of glanders. The mallein test requires an experienced laboratory person and lasts 48h. Therefore, in order to quickly diagnose the disease, especially in areas (such as the borders of the country) that cannot be kept animals, new methods should be used to identify the disease. The Rose Bengal is a serological diagnostic test and has been recommended by the World Organization for Animal Health (OIE). In this study, the Rose Bengal test (RBT) was evaluated for the diagnosis of equine glanders, and its diagnostic was compared with mallein test. Sera from 70 naturally infected culture-positive horses, 3 equines that were sensitized by injecting antigen and 110 healthy equines were tested. Specificity and sensitivity of RBT and mallein test when testing culture-positive equines were calculated. Diagnosis of glanders with both methods yield the same results, but Rose Bengal test is much faster than mallein test for diagnosis of equine glanders. By comparative RBT with mallein test, it can be considered, RBT test has been used for rapid detection of glanders with features such as, ease of use and can be applicable without specialized equipment and trained personnel. Because the RBT is simpler and rapid to perform, the inclusion of the test as a supplementary test for the diagnosis of glanders in field conditions is recommended.

Generation of Monoclonal Antibodies Against Recombinant Capsular Antigen of Burkholderia mallei and Its Potential Application in Glanders Disease Diagnosis

Glanders is primarily a disease of solipeds, most mammals have a certain degree of susceptibility and infection has been reported in goats, sheep, camels, cats, dogs and various other carnivores, human cases of glanders have rarely been reported. Glanders was eradicated in many countries through the use of the mallein test and also with the application of countermeasures like intensive blood testing, rigorous culling of positive animals and strict trading restrictions but sporadic cases still occur in Asia, Africa, the Middle East, and South America. The similarities that exist between Burkholderia mallei causative agent of Glanders and its closely related bacterial species Burkholderia pseudomallei, the causative agent of melioidosis are well documented. Due to its high homology and sharing of many antigens, serological and immunological tests developed for these agents are cross-reactive. In the present study, efforts were made for the development of specific monoclonal antibodies specific to a 14 kDa recombinant capsular protein of B. mallei that does not cross-react to B. pseudomallei. For this purpose genes of 14 kDa recombinant capsular protein of B.mallei was cloned and expressed in pQE expression system. After fusion 114 hybrids were screened for reactivity and 15 were found reactive and out of which 8 were stabilized and preserved. The specificity of the antibodies was checked with different bacterial strains and the isotypes of all the clones were determined. One clone was specifically reactive to B. mallei and 2 were reactive to B. mallei and B. pseudomallei and 5 clones were crossreactive. These specific monoclonal antibodies will be helpful in the standardization of antigen detection system for Glanders.

Comparative Evaluation of Rose Bengal Plate Agglutination Test, Mallein Test, and Some Conventional Serological Tests for Diagnosis of Equine Glanders

The Rose Bengal plate agglutination test (RBT) was evaluated for the diagnosis of equine glanders, and its diagnostic efficiency was compared with that of mallein and other serological tests, including indirect hemagglutination test (IHAT), complement fixation test (CFT), and modified counter immunoelectrophoresis test (mCIET). Sera from 70 naturally infected culture-positive, 96 potentially exposed cohorts, and 110 healthy equines were tested. All tests but mCIET showed 100% specificity when testing the sera from glanders-negative equines. The calculated sensitivities of RBT, IHAT, CFT, mCIET, and mallein test when testing culture-positive equines were 90.0, 97.1, 91.4, 81.4, and 75.7%, respectively. The RBT was significantly (P < 0.05) more sensitive than the mallein test and mCIET. The positive and negative predictive values of each test (RBT, IHAT, CFT, mallein test, and mCIET) were as follows: 100 and 94, 100 and 98.2, 100 and 96.7, 100 and 86.6, and 90.5 and 88.6, respectively. On comparing glandered and nonglandered animals, the highest agreement (0.987) was found between RBT and CFT followed by RBT and IHAT (0.940), RBT and mallein test (0.871), and RBT and mCIET (0.852). Because the RBT is simpler and rapid to perform, the inclusion of the test as a supplementary test for the diagnosis of glanders in field conditions is recommended.

Equine glanders in Turkey

In the course of an epidemiological study of glanders on a number of Turkish islands in the Sea of Marmara, 1128 horses were examined by using the intracutaneous mallein test....

Clinico-Microbiological and Therapeutic Aspects of Glanders in Equines.

The role of glanders therapy as an alternative to test and slaughter policy was investigated in 13 draught equines (1 horse, 2 mares and 10 mules). The disease was diagnosed clinically and confirmed by isolation of Burkholderia mallei on blood agar and sterile potato slices. Mallein 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.

Potency of partially purified malleo-proteins for mallein test in the diagnosis of glanders in equines

Malleo-proteins from synthetic broth mallein of six strains of Pseudomonas mallei were separated by trichloroacetic acid precipitation, amonium sulfate precipitation and Ultrogel AcA 34 gel filtration chromatography. When tested comparatively with Dutch PPD mallein as standard on P. mallei-sensitized and normal horses all the strains were found to be malleinogenic, trichloroactive acid precipitated proteins were comparable to Dutch PPD mallein in potency and innocuity whereas ammonium sulfate-precipitated proteins elicited non-specific reactions. Ultrogel AcA 34 chromatographed high molecular weight proteins (MW range > 350 000) were having equal or higher potency and without adverse reactions and low molecular weight proteins (MW range 120 000–170 000) nearly inactive. Ultrogel AcA 34 column fractionation studeis revealed that mallein activity was associated with high molecular weight proteins and represented a purer sensitin than PPD mallein for the purpose of mallein test.

Serologic Responses to the Mallein Test for Glanders in Solipeds

Serologic Responses to the Mallein Test for Glanders in Solipeds

The Old and the New of Veterinary Science and Practice

The paper recalls how matters veterinary were regarded forty-six years ago, what has been achieved since, and future progress is reflected.THE PAPER IS DIVIDED INTO PARTS RELATING RESPECTIVELY TO: (a) medicine; (b) surgery (c) teaching and research; (d) administration.Formerly, glanders and farcy, and rabies, though acknowledged as contagious and specific, were also believed to be of spontaneous origin. Experiences with regard to these two diseases, and the mallein test for glanders, are related. The discovery of the Bacillus anthracis led to the development of veterinary research, but for some time confusion existed. Tuberculosis was believed to be endogenous and the result of the absorption of caseous products of a previous inflammation. Treatment of "milk fever" in cows by udder inflation and biochemistry in relation to that disease are considered.The advance in veterinary surgery stands out most prominently; in canine practice, operations are now attempted which were never thought possible in the early days. Allusion is made to the recent formidable operations for the cure of "windsucking" in horses, and for traumatic pericarditis in bovines. The powers of observance of the old practitioners in diagnosing lameness, and some of the old methods of treatment for lameness, are supported.The great progress in veterinary research is referred to, also its advantages from an imperial point of view. The causative agents of those diseases which are at present ultravisible, particularly foot-and-mouth disease, will probably be found, and better methods of prevention result.In training, thorough instruction in animal physiology, animal nutrition and biochemistry is advocated, also affiliation of veterinary colleges to universities, the individuality of such colleges, and the one-portal system of qualification being maintained.

Melioidosis and its Relation to Glanders

(1) It is impossible to distinguish the lesions of melioidosis from those of glanders by means of inspection with the naked eye or even with the microscope.(2) The symptoms and course of the two diseases are similar. Melioidosis generally runs a more acute course.(3) The mallein test was applied in a case of chronic human melioidosis and gave a positive reaction. The mallein was obtained from Java.(4) The blood of this chronic case agglutinated the Muktesar and Java strains of B. mallei to full titre and the Minett strain to 10 per cent, of this amount. One of these strains (Muktesar) absorbed the agglutinins for the homologous bacillus.(5) There are very few horses in the Federated Malay States. Melioidosis is primarily a disease of rodents. All attempts to infect horses have been unsuccessful.(6) Melioidosis broke out spontaneously among the rabbits, guinea-pigs and rats at the Institute for Medical Research in 1913. The animals became infected through eating contaminated food.(7) Cases of infection in wild rats and in a domestic cat have occurred far away from this laboratory and independently of it.(8) Rabbits, guinea-pigs and rats have been infected experimentally, by feeding, by subcutaneous inoculation, by scarification and by the application of infective material to the nasal mucosa.(9) B. whitmori, whose growth in cultures occur in two forms, a commoner corrugated form and a mucoid form which gives origin to the corrugated type.(10) B. whitmori differs from B. mallei in the following particulars. It is actively motile. It forms a corrugated growth on glycerine agar, a white opaque growth on ordinary agar and a pellicle on the surface of broth. It grows more rapidly than B. mallei, and it liquefies gelatine in a few days.(11) B. whitmori resembles B. mallei in the following particulars. The morphology of the organisms is similar. Young cultures of the mucoid form are indistinguishable from B. mallei, by inspection. The growth of the mucoid form on potato is similar. The action on milk and carbohydrates differs in degree only. Both organisms produce Strauss's reaction in guinea-pigs.(12) Five strains of B. mallei were compared with fourteen strains of B. whitmori. The cultures of B. mallei comprised three from the National Collection at the Lister Institute, one from Muktesar and one from Java. The cultures of B. whitmori had been isolated, some from human cases of melioidosis and some from animals which had acquired the disease naturally.(13) The serological reactions of these organisms, namely agglutination, absorption and complement-fixation tests, showed that the cultures of B. whitmori were a homogeneous group, but the five strains of B. mallei were sharply divided into two sub-groups by their serological reactions. One sub-group includes the Muktesar and Java strains; the other comprises the three strains from the National Collection (Minett, Egypt and A.).(14) The first sub-group of B. mallei is very closely related to B. whitmori. The serological reactions of the strain from Muktesar are almost identical with those of B. whitmori.(15) The three strains of B. mallei from the National Collection, which form the second sub-group, are only distantly related to the Muktesar sub group and to B. whitmori.

A Typical Reaction to Mallein

A Typical Reaction to Mallein

The Value of the Intra-Palpebral Mallein Test in the Diagnosis of Glanders

Summary There are certain factors that should be taken into consideration in interpreting these serological results. The fact that the sera of all the suspicious horses and controls were heated at 56°C. for one-half hour would tend to cut down the agglutinating titer of those sera. However, as the same technic was carried out in both test and control animals the results have a true relative significance. This fact may account for the absence of agglutinins in such a large percentage of the normal control horses. The results definitely indicate that in horses giving a suspicious intra-palpebral mallein reaction of this type the complement fixation test is of more value than the agglutination reaction. In the former 75.4 per cent were positive while in the latter only 57.4 per cent were positive, even when all the borderline cases were included. These observations agree fairly well with published statistics. In Povitzky's recent article (this Journal, 1918, 3, 463) she records work done in the laboratories of the New York City Department of Health on 123 horses which were proven at autopsy to have glanders. The complement fixation reaction was positive in 75.6 per cent of these cases, while the agglutination reaction was positive in 64.2 per cent. In her work she used horse sera which had not been heated to 56°C. for one-half hour, but again this would be partly neutralized by her using a titer of 1:1000 as necessary for a positive result. The consensus of opinion is that the agglutination reaction gives higher positive results in acute cases, while in chronic or subacute cases the complement fixation is the more reliable.

A New Method of Diagnosing Glanders: Preliminary Note on the Intrapalpebral Test with Mallein

A New Method of Diagnosing Glanders: Preliminary Note on the Intrapalpebral Test with Mallein

Mallein and Tuberculin.

Mallein and Tuberculin.

The Value of the Agglutination Reaction and of Bacteriological Methods in the Diagnosis of Glanders

The readiness with which the agglutination test can be carried out when compared with the mallein test for glanders has made it the subject of much investigation by those interested in comparative medicine. In attempting to establish the agglutination test as a routine method of diagnosis we were confronted by many questionable cases that proved puzzling to the diagnostician, and although the delicacy and specificity of the reaction seemed to be very well established by many observers it was deemed advisable to undertake a thorough study of the method with the hope of coming to some practical conclusion as to the disposition of these anomolous cases. Therefore while very little that is new is here presented our experience may prove of some interest and value in the practical application of the test. In confirming the results of the test by bacteriological findings some new facts were developed that have not heretofore been mentioned by any of the observers along these lines. During the course of this work all clinical observations and autopsies have been made and blood specimens furnished by Dr. H. D. Gill, veterinarian for the State Department of Health, New York, Dr. K. S. Silkman, veterinarian for the Department of Health, New York City, and Dr. R. H. Kingston, New York City. Dr. E. P. Miller and Dr. R. E. Pick have also rendered valuable assistance

The Mallein Reaction in Non-Glandered Horses

The Mallein Reaction in Non-Glandered Horses

The Agglutination Method of Diagnosis in the Control of Glanders

In the control of glanders the first and most important requirement is an accurate diagnosis. The inoculation method of Strauss gave much assistance in cases of apparent lesions, from which inoculating material could be obtained. This, however, did not afford assistance in cases of horses which had been exposed to the disease, but which did not exhibit symptoms or lesions of the malady. Mallein was welcomed as a means of detecting the disease in cases of this kind and in obscure cases generally, but its application has not been altogether satisfactory. Its limitations have not been so clearly defined as those of tuberculin, and the obstacles and complications in its use render many of the results difficult of interpretation. For these reasons a method that can be applied by a trained man, eliminating nearly if not all of the troublesome features of the mallein test, is worthy of careful consideration. The agglutination method of diagnosis, which was first applied to glanders by M'Fadyean, was recommended in the work of control of the disease by Schiitz and Miessner, and by Schniirer, and more recently studied in this laboratory, offers a possible solution for several of the difficult features in the diagnosis of glanders. The method as formulated by Schiitz and Miessner has been officially adopted for the diagnosis of glanders generally in Prussia. When there are cases of suspected glanders, the veterinarian or official in charge applies for sterile bottles, which are forwarded from the laboratory to receive the blood. The specimen of blood and the horse from which it was taken are given a number for identification, and the samples are sent to the laboratory. The test is made and the results reported, with directions for the disposition of the animal. In Austria, Schniirer makes the diagnosis of glanders in suspected cases for the Austrian army. His method of procedure is practically the same as that of Schiitz and Miessner, and he reports it as more

Transmission of Glanders by a Horse Which Had Reacted to Mallein but Externally Appeared Healthy, and Re-Infection of Two Horses Which Had Ceased to React to Mallein

Transmission of Glanders by a Horse Which Had Reacted to Mallein but Externally Appeared Healthy, and Re-Infection of Two Horses Which Had Ceased to React to Mallein

Rules for Judging the Reaction to Mallein

Rules for Judging the Reaction to Mallein