Molecular identification of naturally acquired strongylid infections in lambs - An investigation into how lamb age influences diagnostic sensitivity

Faecal samples (n= 1155) were collected from n= 111 (Farm A) and n= 124 (Farm B) 2-6 week old female lambs on two farms in southern Western Australia across five sampling occasions (spanning 8 months). Genomic DNA was extracted directly from faecal samples and screened by PCR for ITS-2 nuclear ribosomal DNA to detect patent strongylid infections, specifically Teladorsagia circumcincta, Trichostrongylus spp., Haemonchus contortus, Oesophagostomum spp. and Chabertia ovina. The minimum amount of extracted genomic DNA necessary for successful PCR amplification was 2.0-5.0. pg.During the five sampling occasions for the two farms, the sensitivities for WEC and PCR identification of strongylid infections varied, with levels of agreement between the two sets of diagnostic results ranging from 85 to 100%. Strongylid species prevalences were high (90.3-97.3%), with T. circumcincta and Trichostrongylus spp. the most prevalent species and together they were the most common mixed strongylid infection; H. contortus was not identified in either flock. T. circumcincta was the only species associated with an increased risk of non-pelleted faeces on Farm B, where T. circumcincta-positive lambs were 2.3 and 2.6 times more likely to have non-pelleted faeces than negative lambs at the second and final samplings, respectively. The highest strongylid prevalence, mixed strongylid prevalence and mean number of strongylid species detected per lamb coincided with the highest average flock faecal worm egg counts (WECs) on both farms. There was a positive correlation between the number of strongyle species detected per lamb and both WEC and adjusted WEC (P<0.01; r 2 0.026-0.591).These results indicate that strongylid eggs were likely to be the main source of strongylid DNA in the faecal DNA extracts. Despite the progress made by the molecular approach utilised in this study, it is incapable of distinguishing between patent and non-patent sources of strongylid DNA. However there is potential for further investigation into the development of a similar molecular procedure which could be used for early larvae detection on pastures

Prevalence and on-farm risk factors for diarrhoea in meat lamb flocks in Western Australia

Diarrhoea is a widespread problem for sheep enterprises worldwide. A cross-sectional epidemiological study was conducted using a questionnaire to determine the prevalence of diarrhoea and associated risk factors where there was evidence of recent diarrhoea (active diarrhoea or fresh faecal soiling of breech fleece) for meat lambs on farms in southern Western Australia during 2010. The response rate was 41.4% (139/336). Evidence of recent diarrhoea was reported on 64.8% of farms, with a mean of 6.9% lambs affected per farm. Location of a farm and a higher annual rainfall were associated with an increased diarrhoea prevalence. Binary logistic regression analysis suggested that the drinking water source was associated with the incidence of diarrhoea, since lamb flocks supplied with dam water were 117 times (95% CI: 18.2, 754.8) more likely to have observed diarrhoea or fresh breech fleece faecal soiling than lamb flocks supplied with other sources of water. Faecal worm egg counts were used by 65% of respondents to determine whether an anthelmintic treatment was warranted and 74% of respondents administered a treatment to their meat lambs. In response to a range of diarrhoea scenarios presented to respondents (5%, 25% and 50% of the flock with evidence of recent diarrhoea), 15.1% would have elected to administer an anthelmintic treatment regardless of differences in prevalence

Cryptosporidium and Giardia associated with reduced lamb carcase productivity

On two extensive sheep farms in southern Western Australia, 111 (Farm A) and 124 (Farm B) female crossbred lambs (2-6 weeks old) were randomly selected and individually identified using ear tags (a numbered tag and radio-frequency tag) at marking. On five separate occasions, faecal samples were collected and live weight, body condition score (BCS), faecal consistency score (FCS), breech fleece faecal soiling score and faecal dry matter percentage (DM%) were recorded. Lamb hot carcase weight (HCW) and dressing percentage were measured at slaughter. Faecal samples were screened by PCR for Cryptosporidium (18S rRNA, actin and 60 kDa glycoprotein [gp60] loci), Giardia duodenalis (glutamate dehydrogenase [gdh] and triosephosphate isomerise [tpi]) and Campylobacter jejuni (16S rRNA). Observation of Eimeria oocysts and faecal worm egg counts (WECs) were performed using a modified McMaster technique. The WECs were adjusted for FCS for analyses. Faecal samples were screened for patent strongylid infections using PCR (specifically ITS-2 nuclear ribosomal DNA for Teladorsagia circumcincta, Trichostrongylus spp. and Haemonchus contortus). Lambs positive for Cryptosporidium at least once had lighter HCWs by 1.25 kg (6.6%) (P = 0.029) and 1.46 kg (9.7%) (P < 0.001) compared to lambs never positive for Cryptosporidium for Farms A and B respectively. Similarly, dressing percentages were 1.7% (P = 0.022) and 1.9% (P < 0.001) lower in Cryptosporidium-positive lambs on Farms A and B respectively. Lambs positive for Giardia at least once had 0.69 kg (P < 0.001) lighter HCWs and 1.7% (P < 0.001) lower dressing percentages compared to lambs never positive for Giardia on Farm B only. Cryptosporidium-positive lambs at the second sampling were 4.72 (P = 0.010) and 3.84 (P = 0.002) times more likely to have non-pelleted faeces compared to Cryptosporidium-negative lambs for Farms A and B respectively. Breech fleece faecal soiling scores of Cryptosporidium-positive lambs were 3.36 (P = 0.026) and 2.96 (P = 0.047) times more likely to be moderate to severe (scores 3-5), compared to negative lambs at the second sampling for Farms A and B respectively. Live weight, growth rate and BCS were inconsistently associated with protozoa detection across different samplings and farms. Adjusted WEC was correlated positively with FCS and negatively with faecal DM%, differing between sampling occasions and farms. Campylobacter jejuni prevalence was very low (<1%). Adjusted WEC were not correlated with carcase attributes, growth rates or live weights. This study is the first to quantify productivity consequences of naturally acquired protozoa infections in lambs managed under extensive farming conditions

Comparison of molecular and McMaster microscopy techniques to confirm the presence of naturally acquired strongylid nematode infections in sheep

Patent strongylid nematode infections were identified using McMaster worm egg counts (WEC) and PCR assays (ITS-2 nuclear ribosomal DNA) to screen genomic DNA extracted directly from lamb faecal samples. Lambs from four different farms in southern Western Australia were sampled rectally on two separate occasions, with McMaster WECs and PCRs conducted on a total of 858 samples. Negative controls (n = 96) (WEC < 50 eggs per gram [epg]) and positive controls (n = 96) (faecal samples spiked with a 100 μL suspension of third-stage larvae (L3) containing approximately equal proportions of Teladorsagia circumcincta, Trichostrongylus colubriformis, Haemonchus contortus, Oesophagostomum spp. and Chabertia ovina) were generated. All control samples amplified in accordance with positive controls. High levels of agreement (Kappa values ≥ 0.93) were identified between the two diagnostic tests. PCRs detected an additional 2.0% of samples as strongylid-positive but there was no significant difference in the number of strongylid-positive samples identified using PCR or McMaster WEC

Impacts of naturally acquired protozoa and strongylid nematode infections on growth and faecal attributes in lambs

On two separate sampling occasions, faecal samples were collected from lambs (2-5 months of age) grazing pasture on two separate sheep farms in southern Western Australia. Live weight, body condition score (BCS), faecal consistency score (FCS) and faecal dry matter percentage (DM%) were measured. Faecal samples were screened by PCR for Cryptosporidium (18S rRNA, actin and 60kDa glycoprotein [gp60] loci), Giardia duodenalis (glutamate dehydrogenase [gdh] and β-giardin) and patent strongylid nematode infections (ITS-2 nuclear ribosomal DNA for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus spp. Chabertia ovina and Oesophagostomum spp.). Faecal worm egg counts (WECs) were performed using a modified McMaster WEC technique. The WECs were adjusted for FCS and transformed using log 10(adjusted WEC+25) prior to statistical analyses.Cryptosporidium, Giardia and Trichostrongylus spp. detected by PCR were associated with an increased risk of non-pelleted faeces (FCS ≥ 3.0) for both flocks. Cryptosporidium-positive lambs were 2.8-11.6 times more likely to have non-pelleted faeces and Giardia-positive lambs were 2.4-14.0 times more likely to have non-pelleted faeces compared to lambs negative for each respective parasite. Lambs positive for both Cryptosporidium and Giardia were 2.9-11.8 times more likely to have non-pelleted faeces than lambs positive for only one or neither of these parasites. Mixed internal parasite infections were found to have greater impacts on FCS and BCS than single infections. A higher number of internal parasites detected per lamb was associated with lower BCS and more loose faeces. The relationship between parasite detection and live weight or growth rate were inconsistent for both flocks. Adjusted WEC was correlated with FCS and faecal DM% for one flock only, although little or no correlation was found with live weight and growth rate for both flocks. Cryptosporidium ubiquitum and Cryptosporidium parvum were the most prevalent Cryptosporidium species isolated in the two flocks. Giardia assemblage E was the most commonly isolated genotype assemblage from both flocks, while assemblage A was isolated almost as frequently as assemblage E in the one flock. One flock was a potential source of zoonotic Cryptosporidium and the other flock was a potential source of zoonotic Giardia

Development of a modified molecular diagnostic procedure for the identification and quantification of naturally occurring strongylid larvae on pastures

A molecular procedure was developed to detect and quantify larvae of different strongylid parasite species recovered from pasture samples. Two lamb flocks (L and S) grazed separate paddocks with different natural larvae challenges (one low [Paddock L] and one high [Paddock S] challenge) on a commercial farm in Western Australia. Pasture samples were collected and analysed for larvae on 9 separate occasions from each paddock. Pregnant Merino ewes were sampled on 3 separate occasions (2 pre-partum and 1 post-partum). Following lambing, 203 female crossbred lambs were identified, from which faecal samples were collected across five separate samplings. Lamb production and faecal attributes were recorded. Genomic DNA was extracted directly from lamb faeces, in addition to the genomic DNA extracts from strongylid larval species recovered from pastures. Faecal worm egg counts (FWECs) were undertaken. Species-specific qPCRs and conventional PCRs (ITS-2 nuclear ribosomal DNA) were used to screen samples for strongylid species (Teladorsagia circumcincta, Trichostrongylus spp., Haemonchus contortus, Chabertia ovina and Oesophagostomum venulosum). Negative correlations (r 2>0.91) were found between qPCR C q values and log-transformed pasture larval counts for Trichostrongylus spp. and T. circumcincta. Moderate levels of agreement between pasture larval counts and qPCR results were observed (67%).A clear difference in pasture larval challenge levels was observed between the two flocks using both qPCR and conventional pasture larval counts. It is difficult to draw conclusions on the production performances of lambs from the two experimental flocks, as no further replicates were able to be conducted following this experiment. Flock L had higher dressing percentages than Flock S (P= 0.038), along with significantly higher faecal consistency and breech fleece faecal soiling scores at successive samplings. The molecular procedures utilised in this study have the potential to be beneficial for livestock grazing management strategies and parasite surveillance, however further investigation is necessary before they can become part of routine diagnostics

Associations between trichostrongylid worm egg count and productivity measures in Dorper lambs

Two flocks of pure bred Dorper lambs were managed separately according to sex (283 ewe lambs and 212 ram lambs) in southern Western Australia. Faecal sample collection, weighing and body condition assessments were performed for each lamb on 2 occasions, specifically pre-weaning (approximately 14 weeks of age) and post-weaning (approximately 9 months of age). Body condition score (BCS) was assessed using a scale of 1 (very thin, emaciated) to 5 (excessively fat). Faecal worm egg counts (WECs) were measured using a modified McMaster technique and larval cultures were performed to identify trichostrongylid nematode genera present. Eye muscle and c-site fat depths were measured using ultrasound at post-weaning. Lambs received an abamectin anthelmintic treatment at weaning (18 weeks of age). Worm egg count data was assessed for normality of data distribution and homogeneity of variance. This data was transformed using log10(WEC + 25) to stabilise variances between groups prior to statistical analyses and general linear models were used to assess relationships between WEC and productivity measures. Mean WECs were 564 eggs per gram of faeces (EPG) and 514 EPG at pre- and post-weaning in the ewe flock and 552 EPG and 480 EPG at pre- and post-weaning in the ram flock. Teladorsagia (Ostertagia) circumcincta, Trichostrongylus spp. and Oesophagostomum spp. larvae were identified. No lambs with WEC 1000 EPG post-weaning. Ewe and ram lambs with WEC > 1000 EPG at pre-weaning were 42 (12-145 95% CI) and 37 (9-153) times more likely to have WEC > 1000 EPG at post-weaning than lambs with WEC 501-1000 EPG at pre-weaning. There were no significant relationships between WEC and live weight in the ram flock, while relationships between WEC and live weight were inconsistent in the ewe lamb flock. There was no relationship between WEC and eye muscle or c-site fat depth. Significant negative relationships between WEC and BCS were identified at pre- and post-weaning for both flocks. Lambs with WEC 1000 EPG at pre- and post-weaning. In conclusion, high WEC was associated with lower body condition in Dorper lambs, however the relationship between WEC and live weight was inconsistent and there was no effect on eye muscle depth

Longitudinal investigation of protozoan parasites in meat lamb farms in southern Western Australia

In this study, 96 faecal samples were collected from pregnant Merino ewes, at two broad-acre, commercial sheep farms in southern Western Australia, on two separate occasions (16 and 2 weeks prior to lambing). Following lambing, 111 (Farm A) and 124 (Farm B) female crossbred lambs (2-6 weeks old), were individually identified using ear tags (a numbered tag and a radio-frequency tag). A total of 1155 faecal samples were collected only from these individually identified lambs on five separate sampling occasions. All samples were screened using PCR to detect Cryptosporidium (18S rRNA and actin loci) and Giardia duodenalis (glutamate dehydrogenase and triosephosphate isomerise loci). The overall prevalences (lambs positive for a parasite on at least one of the five samplings) at Farm A and B were 81.3% and 71.4%, respectively for Cryptosporidium and similarly 67.3% and 60.5% for Giardia, respectively. Cryptosporidium and Giardia prevalences at individual samplings ranged between 18.5 and 42.6% in lambs and were <10% in the ewes. Cryptosporidium xiaoi was the most prevalent species detected at all five samplings and was also isolated from lamb dam water on Farm B. Cryptosporidium ubiquitum was most commonly detected in younger lambs and Cryptosporidium parvum was detected in lambs at all five samplings, typically in older lambs and as part of a mixed species infection with C. xiaoi. A novel, possibly new genotype (sheep genotype I), was identified in six Cryptosporidium isolates from Farm B. Giardia duodenalis assemblage E was the most common genotype detected at all five samplings, with greater proportions of assemblage A and mixed assemblage A and E infections identified in older lambs. This longitudinal study identified high overall prevalences of Cryptosporidium and Giardia in lambs grazed extensively on pastures, while reinforcing that sampling a random selection of animals from a flock/herd on one occasion (point prevalence), underestimates the overall prevalence of these parasites in the flock/herd across an extended time period. Based on these findings, grazing lambs were identified as a low risk source of zoonotic Cryptosporidium and Giardia species/genotypes, with these protozoa detected at all five samplings in some lambs, indicating that these individuals were either unable to clear the naturally acquired protozoan infections or were repeatedly re-infected from their environment or other flock members

Dietary vitamin E and aspirin supplementation influence the performance and incidence of post-weaning colibacillosis in pigs experimentally infected with an enterotoxigenic strain of Escherichia coli

Increased biosynthesis of prostaglandin E2 (PGE2) from arachidonic acid, caused by immune system activation, negatively affects the performance and health of pigs (Wright et al., 2000). Xu et al. (1990) reported that supplementation of 125 ppm aspirin, an anti-inflammatory agent, improved the daily gain and feed conversion ratio of weaner pigs. Furthermore, in chickens, a combined supplementation of aspirin and vitamin E (Vit E) synergistically depressed PGE2 biosynthesis and reduced mortalities after an E. coli infection (Likoff et al., 1981). The current experiment tested the hypothesis that Vit E and aspirin supplementation would have a synergistic effect on reducing post-weaning colibacillosis (PWC) and improving performance in pigs experimentally infected with an enterotoxigenic strain of E. coli (ETEC)