An automated, multiplex-tandem PCR platform for the diagnosis of gastrointestinal nematode infections in cattle: An Australian-European validation study

Abstract

Detecting the genera and species of gastrointestinal (GI) nematode infections in faecal samples obtained from cattle requires the incubation of faeces ('larval culture') followed by identification of the third-stage larvae that are harvested after 10-14days. Substantial research in the development of PCR-based methods for the rapid and specific identification GI nematodes has been conducted for small ruminants, whilst only few such assays have been developed for cattle. In the present paper we describe the development of an automated, robotic PCR platform for the detection and genus and/or species-specific identification of GI nematodes from bovine faecal samples. This test was then validated using samples from different regions of three countries (Australia, Belgium and Scotland). The PCR platform was found to be highly sensitive and specific for the identification of the important GI nematodes in naturally infected cattle (both estimates >90%). The PCR platform can also estimate the percentage of genera or species present in a mixed-species infection, and was found superior to larval culture in terms of speed (1-2days versus 1-2 weeks for culture), sensitivity and specificity. The PCR was simple to use and the operator requires no knowledge or experience to identify the nematodes present, compared to larval culture where even experienced operators can make substantial errors due to considerable overlap in the published characteristics of key species.

Multiplexed-tandem PCR (MT-PCR) assay to detect and differentiate gastrointestinal nematodes of alpacas

“…We modified the two MT-PCR assays, originally developed for the identification of GINs of cattle [ 16 ], and sheep [ 14 , 15 ] (AusDiagnostics Pty. Ltd., Mascot, New South Wales, Australia) to accurately detect and differentiate the seven common GINs of alpacas, including C .…”

“…Conventionally, data on the detection of GINs using molecular methods are compared with those of LC [ 16 , 20 ]. However, we used the morphological identification of adult female and male worms from the same animals because such data are more accurate and specific, and were incidentally available to us from a previous study (Rashid et al, unpublished data).…”

“…This assay consists of two amplification steps where the primary amplification involves a ‘target enrichment’ using outer primer sets with a small number of PCR cycles, whereas the secondary amplification ‘quantification step’ utilises target-specific, nested or inner primers to target a region within the product from the primary amplification [ 11 ]. To date, MT-PCR has been applied for simultaneous detection of a number of pathogens of veterinary and medical significance, including fungi [ 12 ], enteric pathogens of humans [ 13 ], GINs of sheep [ 14 , 15 ] and cattle [ 16 ], and toxigenic cyanobacteria [ 17 ].…”

“…Furthermore, a high prevalence of a stomach worm, C. mentulatus , was found in alpacas (Rashid et al, unpublished data). Given that very little is known about the epidemiology and diagnosis of GINs of SACs and reliable keys for the identification of L3s of some nematodes (such as C. mentulatus ) that infect alpacas and llamas remain undescribed, we modified two existing semi-quantitative MT-PCR assays for the GINs of sheep [ 15 , 16 ] and cattle [ 16 ] to accurately detect and differentiate the common GINs, including C . mentulatus , Cooperia spp., Haemonchus spp., Oesophagostomum spp., O. ostertagi , T. circumcincta and Trichostrongylus spp.…”

Sheep Nemabiome Diversity and Its Response to Anthelmintic Treatment in Swedish Sheep Herds

“…These include external factors such as climate and managerial factors affecting the exposure to the free living stages [4], and not at least selection reinforced by use of anthelmintic compounds [5].On top of this parasites are in uenced by host immunity affected by previous exposure [6], presence of other animals that may act as reservoirs [7], Traditional parasitological diagnostic techniques based on microscopical examination of fecal eggs counts (FEC) and larval cultures can provide rough measures about the nematode species or genus composition in sheep [8]. However, a disadvantage with these diagnostic tools is that they rely on trained expertise that is rare to found today, but it is also getting clearer that they may have major constraints in terms of both sensitivity and speci city [9]. Thus, there is a need to utilize improved diagnostics for the investigation of complex nematode communities.…”