title: Development and Evaluation of a Molecular Diagnostic Method for Rapid Detection of Histoplasma capsulatum var. farciminosum, the Causative Agent of Epizootic Lymphangitis, in Equine Clinical Samples.
abstract: Histoplasma capsulatum var. farciminosum, the causative agent of epizootic lymphangitis (EZL), is endemic in parts of Africa. Diagnosis based on clinical signs and microscopy lacks specificity and is a barrier to further understanding this neglected disease. Here, a nested PCR method targeting the internal transcribed spacer (ITS) region of the rRNA operon was validated for application to equine clinical samples. Twenty-nine horses with signs of EZL from different climatic regions of Ethiopia were clinically examined. Blood samples and aspirates of pus from cutaneous nodules were taken, along with blood from a further 20 horses with no cutaneous EZL lesions. Among the 29 horses with suspected cases of EZL, H. capsulatum var. farciminosum was confirmed by extraction of DNA from pus and blood samples from 25 and 17 horses, respectively. Positive PCR results were also obtained with heat-inactivated pus (24 horses) and blood (23 horses) spotted onto Whatman FTA cards. Two positive results were obtained among blood samples from 20 horses that did not exhibit clinical signs of EZL. These are the first reports of the direct detection of H. capsulatum var. farciminosum in equine blood and at high frequency among horses exhibiting cutaneous lesions. The nested PCR outperformed conventional microscopic diagnosis, as characteristic yeast cells could be observed only in 14 pus samples. The presence of H. capsulatum var. farciminosum DNA was confirmed by sequencing the cloned PCR products, and while alignment of the ITS amplicons showed very little sequence variation, there was preliminary single nucleotide polymorphism-based evidence for the existence of two subgroups of H. capsulatum var. farciminosum This molecular diagnostic method now permits investigation of the epidemiology of EZL.
authors.affiliation: Department of Epidemiology and Population Health, Institute of Infection and Global Health, University of Liverpool, Neston, Wirral, United Kingdom claire.scantlebury@liverpool.ac.uk aj55m@liverpool.ac.uk.|Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
authors.forename: C E
authors.initials: CE
authors.lastname: Scantlebury
authors.affiliation: Department of Epidemiology and Population Health, Institute of Infection and Global Health, University of Liverpool, Neston, Wirral, United Kingdom.
authors.forename: G L
authors.initials: GL
authors.lastname: Pinchbeck
authors.affiliation: Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
authors.forename: P
authors.initials: P
authors.lastname: Loughnane
authors.affiliation: SPANA Ethiopia, College of Veterinary Medicine and Agriculture, Addis Ababa University, Addis Ababa, Ethiopia.
authors.forename: N
authors.initials: N
authors.lastname: Aklilu
authors.affiliation: SPANA Ethiopia, College of Veterinary Medicine and Agriculture, Addis Ababa University, Addis Ababa, Ethiopia.
authors.forename: T
authors.initials: T
authors.lastname: Ashine
authors.affiliation: SPANA UK, London, United Kingdom.
authors.forename: A P
authors.initials: AP
authors.lastname: Stringer
authors.affiliation: Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
authors.forename: L
authors.initials: L
authors.lastname: Gordon
authors.affiliation: Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Marshall
authors.affiliation: Department of Epidemiology and Population Health, Institute of Infection and Global Health, University of Liverpool, Neston, Wirral, United Kingdom.|NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool, United Kingdom.
authors.forename: R M
authors.initials: RM
authors.lastname: Christley
authors.affiliation: Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom claire.scantlebury@liverpool.ac.uk aj55m@liverpool.ac.uk.
authors.forename: A J
authors.initials: AJ
authors.lastname: McCarthy
mesh_terms: D000818:Animals; D001769:Blood / Q000382:microbiology; D016925:Corynebacterium pseudotuberculosis / Q000302:isolation & purification; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D003937:Diagnosis, Differential; D005002:Ethiopia; D006658:Histoplasma / Q000145:classification / Q000302:isolation & purification*; D006660:Histoplasmosis / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D006734:Horse Diseases / Q000175:diagnosis* / Q000382:microbiology; D006736:Horses; D008205:Lymphangitis / Q000175:diagnosis* / Q000382:microbiology / Q000662:veterinary*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013492:Suppuration / Q000382:microbiology
chemical_list: D021903:DNA, Ribosomal Spacer