Epidemiology of epizootic lymphangitis of carthorses in northern Ethiopia using conventional diagnostic methods and nested polymerase chain reaction

Abstract

Background: Epizootic lymphangitis (EL), caused by Histoplasma capsulatum variety farciminosum (HCF) is a contagious, chronic disease of equines, characterized by development of nodular lesions in the lymph nodes, lymphatic vessels and skin. It is one of the most important diseases of equines in Ethiopia, causing significant economic loss, particularly in the livelihood of carthorse owners. To date there is neither effective diagnostic nor control measure implemented in the country. Furthermore, there is a shortage of data on the epidemiology of the disease in different regions of this country. The aim of this study was to investigate epidemiology of EL in northern Ethiopia, using the conventional methods as well as nested polymerase chain reaction (PCR).

Results: The presence of HCF genetic material was confirmed in 44% (84/191) of the carthorses. Subclinical infection was observed in 18.2% (22/121) of the apparently healthy carthorses. Considering the nested PCR as a gold standard, sensitivity and specificity of clinical examination were 74% and 92.5%, respectively, while the area under the ROC curve (AUR) was 0.83 (95% confidence interval, 0.77, 0.896). Moreover, a moderate (k = 0.675) agreement observed between the nested PCR and clinical examination.

Conclusions: This study demonstrated widespread occurrence of EL in northern Ethiopia, and the advantage of the nested PCR in detecting infection of HCF, even before the clinical symptoms became apparent.

Keywords: Epizootic lymphangitis; Gram stain; Histoplasma capsulatum var farciminosum; PCR.

Fig. 1

Clinical forms of epizootic lymphangitis. a. cutaneous form in fore limb, b. pulmonary form and c. cutaneous form in hind limb

Fig. 2

Gram stained smears of pus samples from Closed EL nodules. Green arrow=. Individual Histoplasma casulatum var farciminosum (HCF) yeast cell; black arrow= HCF yeast cells intracellularly within the macrophages

Fig. 3

Gel electrophoresis of nested PCR amplification products obtained from DNA extracted from horse buffy coat samples. Lane 1: negative control, lanes 2-5 buffy coat DNAs of apparently healthy horses that gave positive amplicon, M= marker, lanes 6-9 buffy coat DNAs of apparently healthy horses with no amplification, lanes 10-15 buffy coat DNAs of clinically suspected EL cases giving positive amplification except lane 12, and lane 16 positive control

Fig. 4

Map of study areas. Map produced by authors. The shape files for the country, study regions and districts were obtained from the Institute of Climate and Society of Mekelle University. Maps were produced using the applications of QGIS version 3.12.3 software