Las competencias

DNA extraction and qPCR of Haemonchus, Teladorsagia and Trichostrongylus

are described in detail in Chapter 2, sections 2.6 and 2.7.5. Briefly, faecal samples were screened and quantified for the presence of the Trichostrongylid nematodes using quantitative PCR (qPCR) as previously described by Harmon et al. (2007) and Ryan (2016) (see section 2.7.5).

7.2.5. Statistical analyses

Prevalences with 95% intervals were calculated as proportion of goats that were Trichostrongylid nematode-positive on at least one occasion. Quantitative PCR total WEC was calculated by the sum total of egg counts calculated for Haemonchus,

Trichostrongylid detection category as detected (McMaster WEC ≥50 epg or qPCR total WEC ≥1 epg) or not detected (WEC less than 50 epg and qPCR 0) each for McMaster and total qPCR.

Analyses were performed using IBM SPSS statistics (version 24, IBM Corporation). Nematode species prevalences were compared using Chi-square test with Fishers exact test for significance. The WEC for each time point were compared using a general linear model with time point included as a fixed factor and post-hoc test to compare differences. Associations between McMaster WEC and total qPCR were determined using bivariate correlations with Pearson co-efficient. Agreement between methods for detection were evaluated using kappa statistic.

7.3. Results

The prevalence of Trichostrongylid nematodes is shown in Table 7.1.

Teladorsagia was not detected in any of the samples. Haemonchus and Trichostrongylus

Table 7.1. Prevalence of Trichostrongylid nematodes as determined by qPCR, in 125 rangeland goats over 4 monthly sampling occasions (S1-S4).

Sampling occasion

S1 S2 S3 S4

H. contortus 24 (17.2, 32.0) a _{0 (0, 2)} b _{0 (0, 2)} b _{0 (0, 2)} b

T. circumcincta 0 (0, 2) 0 (0, 2) 0 (0, 2) 0 (0, 2)

Trichostrongylus spp. 26.4 (19.3, 34.6) a _{0 (0, 2)} b _{0 (0, 2)} b _{0 (0, 2)} b

ab _{Point prevalence values in rows with different superscripts are significantly different (P<0.05).}

McMaster WEC and qPCR WEC are shown in Table 7.2. The McMaster WEC were highest at S1. The McMaster WEC were below levels where clinical haemonchosis was likely. The qPCR WEC were very high, and the levels observed were not biologically plausible for WEC in otherwise healthy goats, especially considering the McMaster WEC (Table 7.2). For example, 39/52 positive samples had qPCR WEC >10 000 epg, and 29/52 were >100 000 epg.

Table 7.2. Worm egg counts (WEC) determined by MacMaster and qPCR for 125 rangeland goats over 4 sampling occasions (S1-S4) approximately one month apart.

S1 S2 S3 S4

McMaster WEC (eggs per gram)

Mean  SE 450 ± 59 a 250± 37 b 150± 29 bc 100±27 c

Range 0–1850 0–1350 0–1100 0–1600

qPCR WEC (eggs per gram)*

Mean  SE 1.47x109 ± 9.0x108 0 0 0

No correlation (Pearson correlation co-efficient -0.077; P=0.393) between McMaster WEC and qPCR WEC were identified at S1, the only sampling occasions at which qPCR-positive samples were identified.

Agreement between qPCR and McMaster for detection of Trichostrongylid are shown in Table 7.3. Agreement between the methods for detection was very poor (kappa 0.056; P=0.160). Overall, 7.2% of samples were qPCR-positive/McMaster-negative and 19.8% of samples were McMaster-positive/qPCR negative.

Table 7.3. Agreement between qPCR and McMaster for detection of Trichostrongylid nematodes in goat faecal samples (n=500).

7.4. Discussion

Haemonchus contortus and Trichostrongylus spp. were identified in captured rangeland goats using qPCR. Both species have been previously reported in rangeland goats in arid regions of Australia (Beveridge et al., 1987), and are considered important nematodes in livestock (Cole, 1986). It was not surprising that Tel. circumcincta was not identified in faecal samples. The goats were sourced from remote arid and semi-arid regions where conditions are generally not conducive to survival of this parasite.

Quantitation for WEC with qPCR was not achieved in this study and the accuracy of qPCR for qualitative identification of Trichostronglid nematode species could not be validated because larval cultures were not performed to confirm species present. By the

qPCR

Not detected Detected Total

McMaster < 50 (not detected) 349 36 385

> 50 (detected) 99 16 115

time poor agreement between the methods was recognised, samples had been frozen and were not suitable for larval cultures. As such, only McMaster WEC are included as co- variates for analyses in Chapter 8 (conducted to determine relationships for pathogen detection with weight, growth, faecal consistency and scouring in goats), and analyses for associations between nematode species identification and production parameters should be interpreted with caution.

There was no correlation between WEC determined by McMaster and qPCR, and similarly the level of agreement for detection was very poor. Other studies have reported challenges in achieving precise quantitation with qPCR (Harmon et al., 2007). In the present study, WEC quantitation with qPCR greatly overestimated WEC relative to McMaster method at S1, and the values observed at this time point were so high as to not be plausible. The reason for this was not determined, but it is possible that eggs in faecal samples had embryonated in the period between sample collection and DNA extraction. Larval development would increase the amount of parasite DNA present relative to unembryonated Trichostrongylid eggs. It was also possible that Trichostrongylid genera not included in qPCR assay were present, and contributed to McMaster WEC. For example, Cooperia, Chabertia, Oesphagostamum have been reported in Australian goats (Edgar, 1936; Beveridge et al., 1987). The presence of other genera would also explain the observation that 20% of samples were McMaster-positive/qPCR negative. Larval cultures that would have confirmed the presence of other genera were not performed.

The observation that 7.2% of samples were qPCR-positive/McMaster-negative could be explained by a number of factors. It is possible that the qPCR assay was detecting nematode DNA in small numbers of eggs in faeces (below normal detection limit for McMaster WEC). The assay was performed using faecal extraction, not purified eggs, and it is possible faecal material contained nematode DNA not derived from eggs (for

Mean McMaster WEC were 100 epg at S2, S3 and S4, despite moxidectin treatment immediately after S1 and S2. It was not clear whether this represents reinfection, treatment failure or anthelmintic resistance. Treatment of goats with moxidectin is an off-label usage in Australia. The moxidectin dose (0.4 mg/kg) was consistent with recommendations by Kaplan (2014). The pre-patent period for H. contortus and Trichostrongylus spp. are 18–21 and 15-23 days respectively. Goats did not have access to pasture, so substantial reinfection was unlikely (Besier et al., 2016), but it is possible that some faecal contamination of feed and straw bedding had occurred. Moxidectin does have label claim in sheep for 91 days protection for H. contortus and 49 days Trichostrongylus spp. (Vibrac Australia, 2011). Assuming this applies to goats, a fully effective dose at S1 and S2 would have been expected to have reduced re-infection. Moxidectin resistance has not been reported in Trichostrongylus spp. for goats, but H. contortus resistance has been widely reported in Australia for sheep (Besier and Love, 2003; Le Jambre et al., 2005; Woodgate and Besier, 2010). However, these goats were wild prior to capture, and the goat parasite population is unlikely to have been exposed to moxidectin treatment, making resistance less likely to be present at meaningful levels. The results suggest that any off-label use of moxidectin without WEC monitoring to determine efficacy should be discouraged in goat depots. Furthermore, as rangeland goats are wild and not habituated to close handling, oral dosing may render both goats and operator at risk of injury (Lyndal-Murphy et al., 2007). The use of non-chemical alternative management methods that include grazing rotations, nutritional supplements and bioactive tanniferous forages to control nematodes in Australian goats have been suggested.

7.5. Conclusion

The present study identified Haemonchus and Trichostrongylus spp. druing the first sampling following capture and transport of rangeland goats with no detection of

Teladorsagia using qPCR assays. Conventional McMaster technique was also carried out. However, as no larval culture were conducted, quantitation for WEC with qPCR was not achieved. Therefore, further qPCR analyses based on larval culture are required to obtain more comparable data to WEC. Further studies are also required to determine the extent of production loss associated with strongyle faecal carriages in rangeland goats.

CHAPTER 8.

ASSOCIATIONS

FOR

ENTERIC