Characterisation of Giardia duodenalis assemblages from veterinary clinical cases in Ireland

Giardia duodenalis is a flagellated intestinal protozoan and a common cause of diarrhoea in humans and animals worldwide. Infection typically occurs through waterborne transmission or the faecal-oral route. Genetic studies recognise eight assemblages (A-H) with varying host specificity and zoonotic potential, making accurate assemblage discrimination essential within a One-Health framework. This study aimed to identify assemblages present in clinical cases received at a veterinary diagnostic laboratory in Ireland using a multilocus genotyping (MLG) approach and critically evaluate the performance and limitations of commonly used molecular markers. A total of 140 faecal samples from dogs, cats, cattle, lemurs, seals and alpacas were analysed. Samples were submitted to the UCDVH diagnostic laboratory from a range of sources. Following detection of G. duodenalis cysts by faecal flotation or lateral flow test, assemblages were characterised using Sanger sequencing of fragments of the β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes. Analysis of the bg locus identified assemblages A-F. Assemblages C and D were detected in canines as expected; however, assemblage E, typically associated with ruminants, was also detected across most host species except lemurs. Agreement between bg and gdh was moderate to substantial for assemblages B (κ=0.59) and F (κ=0.79), but slight for assemblage E for canine samples (κ=0.04), which was predominantly reassigned to C and D at the gdh locus. The tpi locus showed substantial to perfect agreement with gdh for B and E, though 42.9% of samples failed to amplify. Only 18/140 (12.9%) samples showed concordant assemblages across all three loci. The observed inter-locus discordance highlights the complexity of assemblage assignment in natural infections and underscores limitations of MLG, including potential mixed infections. These findings emphasise the need for critical interpretation of discordant MLG results, particularly in multi-host settings where epidemiological inferences may be misleading.