Molecular epidemiology of chlamydiae at shorebird-human interface in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Manawatū, New Zealand

Abstract

Chlamydiae (plural form of the bacteria, Chlamydia) can infect a broad range of avian hosts. The well-recognised Chlamydia, Chlamydia psittaci can cause avian chlamydiosis; a respiratory, ocular, and enteric disease. C. psittaci is also a zoonotic agent causing psittacosis in humans. Worldwide, migratory shorebirds are one of the principal hosts of C. psittaci. New Zealand has two Ramsar sites (internationally important wetlands) that serve as significant stopovers for migratory shorebirds using the East Asian-Australasian Flyway. However, there has been no prior surveillance conducted for chlamydiae in migratory shorebirds in New Zealand. The aim of this study was to perform a chlamydiae survey at four estuarine sites in New Zealand, in both shorebirds and the people that handle them.

Molecular methods are currently the most sensitive and rapid tests to detect C. psittaci in birds. Most laboratories have designed an in-house molecular assay or applied published protocols, sometimes with slight changes to the original procedure, for their studies with different purposes. Systematic review of 120 articles revealed that the sensitivity and specificity of a molecular test is dependent on the target genes, primer sequences, type of molecular test (quantitative PCR, conventional PCR), DNA extraction methods, and sampling methods. Of the currently available ten genomic targets to detect C. psittaci in birds, the ompA gene was the most widely used target gene. A testing strategy using a hierarchical approach that includes molecular tests of genus- and species-specific targets is recommended to facilitate detecting the well-recognised C. psittaci as well as other avian chlamydial species.

In this study, DNA extracted from choanal and cloacal swabs of 320 live shorebirds of 7 species from New Zealand was screened for chlamydiae by High-Resolution DNA Melt quantitative PCR (qPCR-HRM) analysis. Shedding of chlamydiae was detected in 71 out of 320 (22%; 95%CI 18-27%) shorebirds sampled. Due to low pathogen load, molecular characterisation of avian chlamydial at the species level was possible in only 14 out of 73 positive samples by applying a culture-independent sample preparation method – multiple displacement amplification (MDA). ompA (outer membrane protein A gene) sequencing of the MDA products revealed 100% identity with C. psittaci in one pied stilt, and 99.9% identity with the avian C. abortus strain in five bar-tailed godwits, four pied stilts, and four South Island pied oystercatchers.

DNA extracted from the nasal swabs of 27 people handling the shorebirds was screened using the same methodologies as above. qPCR-HRM results of the shorebird handlers showed the detection of chlamydiae in 14 out of 27 (52%; 95%CI 33-71%) participants. And the ompA sequencing results of human samples revealed 100% identity with C. psittaci in one sample, and 99.9% identity with the avian C. abortus strain in ten samples. To our knowledge, this is the first report of avian C. abortus strains in both shorebirds and people in New Zealand. The outcome from this study suggests potential transmission of chlamydiae among shorebirds populations, and between shorebirds and people. With this, we can raise awareness and inform disease prevention protocol for targeted people, in addition to conservation management strategies for shorebirds and public health plans.