Angus J.D. Campbell

Differences in a ribosomal DNA sequence of Strongylus species allows identification of single eggs

In the current study, molecular techniques were evaluated for the species identification of individual strongyle eggs. Adult worms of Strongylus edentatus, S. equinus and S. vulgaris were collected at necropsy from horses from Australia and the U.S.A. Genomic DNA was isolated and a ribosomal transcribed spacer (ITS-2) amplified and sequenced using polymerase chain reaction (PCR) techniques. The length of the ITS-2 sequence of S. edentatus, S. equinus and S. vulgaris ranged between 217 and 235 nucleotides. Extensive sequence analysis demonstrated a low degree (0-0.9%) of intraspecific variation in the ITS-2 for the Strongylus species examined, whereas the levels of interspecific differences (13-29%) were significantly greater. Interspecific differences in the ITS-2 sequences allowed unequivocal species identification of single worms and eggs using PCR-linked restriction fragment length polymorphism. These results demonstrate the potential of the ribosomal spacers as genetic markers for species identification of single strongyle eggs from horse faeces.

Improving the nutrition of Merino ewes during pregnancy and lactation increases weaning weight and survival of progeny but does not affect their mature size

Lamb growth to weaning, and during the period immediately following weaning, influences post-weaning mortality and may affect mature size. The hypothesis tested in the experiments reported in this paper was that changes in the maternal liveweight of Merino ewes during pregnancy and lactation could predict the weight at weaning, post-weaning survival and mature size of their progeny. At two sites (Vic. and WA) in each of two years, a wide range in the liveweight profiles of ewes was generated during pregnancy and lactation by varying the amount of supplements fed and feed on offer grazed. Across the four experiments this resulted in progeny weights ranging from 13.8 to 28.3 kg just before weaning. Lamb growth was primarily related to the amount of feed on offer during lactation, but was also related to the liveweight change of the ewe during pregnancy. These relationships were consistent in both experiments at each site. Weaning weight was strongly associated with post-weaning survival at the Vic. site. Survival rates decreased significantly when weaning weights were below 20 kg. These results indicate that management of ewe and lamb nutrition to maximise growth of lambs before weaning and growing weaners at 30 g/day or more after weaning are important for optimal post-weaning survival. The findings also suggest that the mature size of offspring is unlikely to be adversely affected by pre-weaning nutrition within the range of nutritional scenarios during pregnancy and lactation that are likely to be experienced within the Australian sheep industry.

Longitudinal prevalence, oocyst shedding and molecular characterisation of Cryptosporidium species in sheep across four states in Australia.

The prevalence of Cryptosporidium in sheep in the eastern states of Australia has not been well described, therefore a study of the prevalence, oocyst concentration, species and subtypes of Cryptosporidium were assessed from lamb faecal samples at three sampling periods (weaning, post-weaning and pre-slaughter) from eight farms across South Australia, New South Wales, Victoria and Western Australia. A total of 3412 faecal samples were collected from approximately 1182 lambs across the four states and screened for the presence of Cryptosporidium using a quantitative PCR (qPCR) at the actin locus. Positives were typed at the 18S locus and at a second locus using C. parvum and C. hominis specific qPCR primers. The overall prevalence was 16.9% (95% CI: 15.6-18.1%) and of the 576 positives, 500 were successfully genotyped. In general, the prevalence of Cryptosporidium was higher in WA than the eastern states. Cryptosporidium prevalence peaked at 43.9% and 37.1% at Pingelly (WA2) and Arthur River (WA1), respectively during weaning and at Pingelly (WA2) during pre-slaughter (36.4%). The range of oocyst shedding at weaning overall across all states was 63-7.9×10(6) and the median was 3.2 × 10(4) oocysts g(-1). The following species were identified; C. xiaoi (69%-345/500), C. ubiquitum (17.6%-88/500), C. parvum (9.8%-49/500), C. scrofarum (0.8%-4/500), mixed C. parvum and C. xiaoi (2.4%-12/500), C. andersoni (0.2%-1/500) and sheep genotype 1 (0.2%-1/500). Subtyping of C. parvum and C. ubiquitum isolates identified IIa and IId subtype families within C. parvum (with IId as the dominant subtype) and XIIa within C. ubiquitum. This is the first published description of C. parvum subtypes detected in lambs in Australia.

A Molecular Diagnostic Tool to Replace Larval Culture in Conventional Faecal Egg Count Reduction Testing in Sheep

The accurate diagnosis of parasitic nematode infections in livestock (including sheep and goats) is central to their effective control and the detection of the anthelmintic resistance. Traditionally, the faecal egg count reduction test (FECRT), combined with the technique of larval culture (LC), has been used widely to assess drug-susceptibility/resistance in strongylid nematodes. However, this approach suffers from a lack of specificity, sensitivity and reliability, and is time-consuming and costly to conduct. Here, we critically assessed a specific PCR assay to support FECRT, in a well-controlled experiment on sheep with naturally acquired strongylid infections known to be resistant to benzimidazoles. We showed that the PCR results were in close agreement with those of total worm count (TWC), but not of LC. Importantly, albendazole resistance detected by PCR-coupled FECRT was unequivocally linked to Teladorsagia circumcincta and, to lesser extent, Trichostrongylus colubriformis, a result that was not achievable by LC. The key findings from this study demonstrate that our PCR-coupled FECRT approach has major merit for supporting anthelmintic resistance in nematode populations. The findings also show clearly that our PCR assay can be used as an alternative to LC, and is more time-efficient and less laborious, which has important practical implications for the effective management and control strongylid nematodes of sheep.

Analysis of nucleotide variation within the triose-phosphate isomerase gene of Giardia duodenalis from sheep and its zoonotic implications

This study explored the genetic composition of Giardia in fecal samples from 284 individual lambs on pasture‐based sheep farms in three regions of Victoria, Australia. An approach, combining targeted sequencing, phylogenetic analysis and PCR‐coupled restriction endonuclease fingerprinting, was used to identify and genetically categorize Giardia present in 43 (15.1%) of the 284 samples and to infer their zoonotic potential. The specific identity and genetic classification were based on the phylogenetic analysis of sequence data for a portion of the triose‐phosphate isomerase gene. Fourteen different sequence variants (including seven sequences that contained between one and five polymorphic sites) representing two distinct assemblages of Giardia (recognized in the current literature) were defined, of which 13 were new records. One dominant sequence type (with accession no. GQ444447, representing a genotype within assemblage A) has been detected previously in humans and is thus considered to be of zoonotic potential. (Nucleotide sequences reported in this article are available in the GenBank database under accession nos. GQ444447–GQ444451 and GQ444454–GQ444462).

Establishment of a robotic, high-throughput platform for the specific diagnosis of gastrointestinal nematode infections in sheep.

The accurate diagnosis of strongylid nematode infections is central to investigating their epidemiology and for parasite control. To overcome major limitations in sensitivity or specificity of traditional methods, including faecal egg count (FEC) and/or larval culture (LC), we evaluated and established a semi-automated, high throughput multiplexed-tandem PCR (MT-PCR) platform for the diagnosis of gastrointestinal strongylid nematode infections in sheep, and established its diagnostic sensitivity (100%) and specificity (87.5%) based on the testing of 100 faecal DNA samples from helminth-free sheep and 30 samples from sheep with infections confirmed by necropsy. Subsequently, the platform was employed to test 219 faecal samples from sheep with naturally acquired infections from various geographical localities within Australia and the results compared with those from conventional LC using 139 of the 219 samples. The results obtained using both MT-PCR and LC correlated significantly for most nematodes examined, but revealed that Oesophagostomum venulosum and Chabertia ovina (parasites of the large intestine) were significantly under-represented in the LC results. The results showed that Trichostrongylus spp. (87%), Teladorsagia circumcincta (80%) and Haemonchus contortus (67%) had the highest prevalences, followed by O. venulosum (51%) and C. ovina (12%). The molecular-diagnostic platform established can be used for species- or genus-specific diagnosis of patent nematode infections within 24h (compared with 7-10 days for LC), and is a sensitive and cost effective tool for routine application in research and service laboratories.

Development of a quantitative PCR (qPCR) for Giardia and analysis of the prevalence, cyst shedding and genotypes of Giardia present in sheep across four states in Australia

A novel quantitative PCR (qPCR) for Giardia at the glutamate dehydrogenase (gdh) locus was developed and validated. The qPCR was used to screen a total of 3412 lamb faecal samples collected from approximately 1189 lambs at three sampling periods (weaning, post-weaning and pre-slaughter) from eight farms across South Australia (SA), New South Wales (NSW), Victoria (Vic) and Western Australia (WA). The overall prevalence was 20.2% (95% CI 18.9-21.6) and of the 690 positives, 473 were successfully typed. In general, the prevalence of Giardia varied widely across the different farms with the highest prevalence in one WA farm (42.1%) at pre-slaughter sampling and the lowest prevalence in one Victorian farm (7.2%) at weaning. The range of cyst shedding at weaning, post-weaning and pre-slaughter overall across all states was 63-1.3×10(9) cysts g(-1) (median=1.7×10(4)), 63-1.1×10(9) cysts g(-1) (median=9.6×10(3)), 63-4.7×10(9) cysts g(-1) (median=8.1×10(4)) respectively. Assemblage specific primers at the triose phosphate isomerase (tpi) locus identified assemblage A in 22.4% (106/473) of positive samples typed, assemblage E in 75.9% (359/473) and mixed A and E assemblages in 1.7% (8/473) of samples. A subset of representative samples from the 8 farms (n=32) were typed at both the gdh and beta-giardin loci and confirmed these results and identified sub-assemblage AII in 16 representative assemblage A isolates across the 8 farms. This demonstrates a prevalence of Giardia previously not recognised in Australian sheep, highlighting a need for further research to quantify the production impacts of this protozoan parasite.

Longitudinal prevalence and faecal shedding of Chlamydia pecorum in sheep

The prevalence and faecal shedding of Chlamydia spp. in sheep in Australia has not been well described. Two species-specific quantitative PCRs (qPCRs) targeting the chlamydial outer membrane protein cell surface antigen gene (ompA) were validated and used to determine the prevalence and faecal shedding of C. abortus and C. pecorum from faecal samples of lambs at three sampling times (weaning, post-weaning and pre-slaughter) from eight farms in South Australia, New South Wales, Victoria and Western Australia. A total of 3412 faecal samples were collected and screened from approximately 1189 lambs across the four states. C. abortus was not detected in any of the samples screened. The overall prevalence of C. pecorum was 1027/3412 (30.1%) and median bacterial concentrations at weaning, post-weaning and pre-slaughter were 1.8 × 10(7), 1.2 × 10(7) and 9.6 × 10(5)/g faeces, respectively. A subset of C. pecorum positive samples from each farm, (n = 48) was sequenced to confirm their identity. The present study demonstrates that C. pecorum is prevalent in Australian sheep, highlighting a need for further research on the impact of this bacterium on production.

Musculoskeletal injury rates in Thoroughbred racehorses following local corticosteroid injection

A retrospective cohort study was performed to compare the rates of musculoskeletal injury (MSI) in horses receiving local corticosteroid injection (LCI) with those that were untreated and those prior to treatment. Of the 1911 study horses, 392 had been treated. A LCI was defined as any injection of corticosteroid into or adjacent to a synovial structure, muscle, or tendon/ligament. A MSI was defined as any limb injury identified by a veterinarian, following which the horse did not race for at least 6 months, or was retired. Hazard ratios (HR) comparing hazard of injury following injection to that in non-injected horses and prior to injection were calculated using Cox proportional hazards models. At least one LCI was administered to 392 horses (20.5%; median 2, range 1-16). Most LCIs were performed bilaterally (70.9%) and intra-articularly into the carpal (49.7%) or fore fetlock (29.3%) joints. There were 219 MSIs of which carpal injuries (47%), fore fetlock (22%) and forelimb tendon injuries (16%) were the most common. The incidence rate of MSI in untreated horses and those prior to injection was 1.22 (95% CI 1.04-1.44) injuries/100 horse-months, and following LCI the hazard of MSI was greater (HR 4.83, 3.54-6.61, P<0.001). The hazard ratio returned to levels indistinguishable from before treatment after 49 days. The hazard of MSI in horses following second and subsequent LCIs in the data collection period was greater than in horses following their first LCI (HR 2.10, 1.31-3.36, P=0.002). There was a positive association between LCI and subsequent musculoskeletal injury rates which was most likely due to progression of the musculoskeletal condition which prompted treatment. Assuming horses that received LCI were at increased risk of MSI subsequently, any beneficial effects of the LCI were insufficient to counter this increased risk for at least 49 days after the injection.

Greater intensity and frequency of Cryptosporidium and Giardia oocyst shedding beyond the neonatal period is associated with reductions in growth, carcase weight and dressing efficiency in sheep

Associations between intensity and frequency of Cryptosporidium and Giardia shedding with growth, carcase weight and dressing% were investigated using a longitudinal study of 1182 lambs on eight Australian farms. Live weight was recorded and faecal samples were collected on three sampling occasions; weaning (approximately 12 weeks of age), post-weaning (approximately 19 weeks) and pre-slaughter (approximately 29 weeks). Hot standard carcase weight (HSCW) and dressing% were measured at slaughter. Faecal samples were screened for presence and concentration of Cryptosporidium, Giardia and Haemonchus oocysts using a quantitative PCR. Trichostrongylid eggs were quantified with modified McMaster faecal worm egg count (WEC). Protozoan shedding intensity was categorised as high (above median oocyst concentration in positive sheep), low (below median oocyst concentration in positive sheep) or not detected. Shedding was also categorised for shedding type (no shedding, single Giardia infection, single Cryptosporidium infection, concurrent Giardia and Cryptosporidium infection) and lambs were categorised for frequency of shedding (shedding identified on 0, 1, 2 or 3 occasions). Associations of parasite shedding intensity category, shedding type, shedding frequency, WEC and Haemonchus status (positive or negative) with lamb production were assessed using general linear models (HSCW and dressing%) and linear mixed effects models (live weight). High Cryptosporidium parvum shedding was associated with lower live weight, ranging 2.31-4.52kg over the 3 sampling occasions. Cryptosporidium parvum shedding was associated with less HSCW in high (3.22kg less) and low (3.22kg less) shedding lambs post-weaning, and high (2.21kg less) and low (2.60kg less) shedding lambs pre-slaughter as well as lower dressing% (2.7% lower in high shedding lambs post-weaning). Cryptosporidium (all species) shedding pre-slaughter was associated with reduced dressing% in both high (1.25% lower) and low (1.21% lower) shedding lambs. Giardia shedding pre-slaughter was associated with 0.59kg less HSCW in high shedding lambs. Increased frequency of C. parvum and Giardia shedding in a specific animal (repeated detection) were associated with reduced HSCW and dressing%. Concurrent Giardia and Cryptosporidium shedding pre-slaughter was associated with reduced dressing%. No statistically significant main effects for either WEC (P>0.05) or Haemonchus status (P>0.05) were identified for any of the sheep meat productivity measures (live weight, HSCW and dressing%). The findings suggest naturally acquired Cryptosporidium and Giardia infections in grazing sheep are associated with depressed growth, carcase weight and dressing efficiency beyond the neonatal period in sheep representing a range of genetic backgrounds and different sheep production environments.