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Glanders is a highly infectious and zoonotic disease of solipeds caused by Burkholderia mallei. Progressive loss of efficiency and fatal outcome resulted in massive economic losses which forced veterinary authorities throughout the world to implement disease control measures; these included mass testing using the complement fixation test and/or malleinisation, and the culling of positives. This led to the eradication of glanders from Western Europe and North America in the 1950s. However, in the last decade, the number of outbreaks in Asia and South America increased steadily and glanders regained the status of a re-emerging transboundary disease. Pakistan has been an endemic country for the past 120 years but concise data on the presence of disease are not available.
A total of 533 serum samples were collected from draught equines, a suspected risk group for glanders, from various districts of the Pakistani Punjab. The complement fixation test (CFT) and the highly sensitive Western blot technique were used for serodiagnosis. No positive animal (horse, mule, and donkey) was found. Glanders seems to be restricted to remote, sporadic pockets of endemicity and may cause outbreaks after being introduced into naive populations by (asymptomatic) shedders.
Various serological tests were used for the diagnosis of glanders in the past but still complement fixation test (CFT) is the internationally prescribed test for trading equines. A new immunoblot (IB) technique has recently been introduced to overcome the well known shortcomings of CFT i. e. a considerable number of false positive and negative results and anticomplementary effect of sera. The objective of this study was the comparative evaluation of two glanders CFT antigens commercially available at Central Veterinary Institute of Wageningen UR, Lelystad, The Netherland (CIDC) and at c.c.pro GmbH, Oberdorla, Germany (c.c.pro) in an glanders endemic area regarding specificity and sensitivity. A total of 1,678 serum samples from the endemic region (Province Punjab, Pakistan) and a non-endemic area (Germany) were analysed. All sera tested positive or suspicious with CFT were analysed by the confirmatory IB to exclude CFT false positive results. Both CFT antigens showed 100% sensitivity. The use of CIDC or c.c.pro antigen resulted in specificities of 77.45% or 75.71% for sera from endemic area and 93.75% or 94.79% for sera from non-endemic areas, respectively. The results demonstrate the different performances of identical tests in different epidemiologically settings. Based on these results, the combined use of CFT and IB is highly suggestive for the serodiagnosis of glanders. Good agreement was calculated between CFT (using either c.c.pro or CIDC antigen) and IB.
In response to third objective of comparatively evaluation of three commercially available antigens: (c. c. pro, CIDC and USDA) using sera from glanders free (Germany) and glanderous/immunised animals, the sensitivity and specificity of three commercially available complement fixation test (CFT) antigens from c.c.pro (c.c.pro), Central Veterinary Institute of Wageningen UR (CIDC) and the United States Department of Agriculture (USDA) were comparatively evaluated by testing 410 sera collected from glanders-endemic and non-endemic areas (200 true negative randomly collected sera and 210 sera collected from experimentally immunised animals (12 rabbits, 19 horses), clinical-positive (135) and culture-positive (44) horses, donkeys and mules). Immunoblotting (IB) was used as gold standard test. Highest sensitivity was shown for the CIDC antigen (100%) followed by the c.c.pro antigen (99.39%). However, the USDA antigen showed substantially less (P<0.05) sensitivity (62.19%). Highest specificity was found for the USDA antigen (100%) followed by the CIDC (97.5%) and c.c.pro antigen (96.5%). Positive and negative predictive values for each antigen were calculated to be: 95.88 and 99.48 (c.c.pro), 97.04 and 100 (CIDC), 100 and 76.33% (USDA). Almost perfect agreement (0.96) was found between CFT using either c.c.pro or CIDC and IB. Due to almost perfect agreement (0.96), CFT using c.c.pro or CIDC antigen can be combined with IB to increase the detection rate of glanders among infected animals.