Evaluation of Chlamydia LAMP Assays

C. psittaci LAMP assay was evaluated using: (1) 12 DNA samples extracted from previously characterised C. psittaci isolates (10 human, two parrot and one equine) (Table S1); (2) DNA extracted from 21 placental, foetal, nasal, lung and rectal swabs, and 1 each placental and foetal tissue sample taken from 20 equine hosts; and (3) three pigeon liver DNA extracts (Table S2). All samples were collected and submitted as part of routine diagnostic testing by field or district veterinarians to the State Veterinary Diagnostic Laboratory (SVDL), Elizabeth Macarthur Agricultural Institute (EMAI), Menangle, NSW, Australia, and as such do not require special animal ethics approval. DNA extracts from these samples were kindly provided by Dr. Cheryl Jenkins, and Dr. James Branley. The use of these swabs was considered by the University of The Sunshine Coast (USC) Animal Ethics Committee and the need for further ethics consideration was waived under exemption AN/E/17/19.
C. pecorum LAMP was evaluated using a: (1) 18 DNA samples extracted from previously characterised koala (n = 7), sheep (n = 4), cattle (n = 4) and pig C. pecorum (n = 3) cultures (Table S1); (2) 16 sheep and 13 cattle ocular, rectal, and tissue swab DNA samples; and (3) 34 ocular and urogenital (UGT) koala swab DNA samples (Table S3), all available in our collection. The use of these swabs, also collected by qualified veterinarians as a part of routine diagnostic testing, was considered and approved for exemption by the University of The Sunshine Coast (USC) Animal Ethics Committee (AN/E/14/01 and AN/E/14/31).
We also evaluated the specificity of the assays against DNA samples extracted from previously characterised (i) chlamydial isolates (koala C. pneumoniae LPColN, C. abortus S26/3, C. suis S45, C. trachomatis serovar D, C. murridarum Nigg, C. caviae GPIC) and uncultured Chlamydiales (Fritschea spp.); (ii) Gram negative Escherichia coli and Prevotella bivia; Gram positive Fusobacterium nucleatum, Staphylococcus epidermidis, S. aureus, Streptococcus spp., and Enterococcus faecalis; and (iii) commercially available human gDNA (Promega, Alexandria, NSW 2015), all available in our laboratory (Table S1).
In order to evaluate rapid swab processing, 18 ocular, cloacal and UGT (14 dry and four RNA-Later) clinical swabs taken from 14 koalas with presumptive chlamydiosis were used for testing without DNA extraction. Briefly, RNA-Later and dry swabs with added 500 µL TE buffer were vortexed vigorously for 5 min. 300 µL aliquots were then heated to 98 °C for 15 min to lyse DNA, following LAMP testing. The use of these swabs, collected as a part of routine diagnostic testing, is also under Animal Ethics approval exemption (AN/E/14/01). An aliquot of 50 µL of the swab suspension was used for LAMP and qPCR assays, while from the remaining volume of the swab suspension was used for DNA extraction, in order to compare swab suspension and its paired extracted DNA as a template in the assays.