Understanding the strengths and weaknesses of DrenchRite and how it relates to faecal egg count reduction tests (FECRT) will lead to clearer interpretation of DrenchRite results and a better perspective on both tests. In previous studies efficacy was estimated for worms resistant to BZ, LEV and BZILEV combinations by FECRT, DrenchRite and post mortem worm counts. Correlations of in vivo efficacy with FECRT determined by them were: Trichostrongylus colubriformis 88%, Ostertagia circumcincta 56% and Haemonchus contortus 69%. The low correlation for O. circumcincta was associated with the variability in estimating species composition when O. circumcincta was present at low levels. Correlations of DrenchRite with in vivo efficacy were: O. circumcincta 87%, T. colubriformis 82% and H. contortus 80%. The correlation between resistance determined by Larval Development Assay (LDA) and FECRT was not high at 61% (averaged over drugs and species). Similar data for Macrocyclic Lactones (ML) resistant worms are not yet available.



Previous studies have shown for some LDA systems that eggs are more likely to be inhibited in development to L3s early (2-3 weeks post infection (PI)) and late (beyond 10 weeks PI) after infection and are most resilient to the effect of drugs at about 7 weeks PI. No systematic changes in DrenchRite results were observed over age of worm infection when tested weekly for 2 to 14 week old worms. 



Large bowel worms of sheep have not generally been examined under LDA conditions so a study was undertaken to determine the LD50s of susceptible Oesophagostomum columbianum (nodule worm) and Chabertia ovina (large mouthed bowel worm) on DrenchRite plates. LD50s for BZ and LEV were approximately 0.04 and 0.8 uM respectively for both worms. For the ML O. columbianum had a much higher LD50 (45 nM) than C. ovina (8 nM). In relation to the other common sheep worms, development characteristics of C. ovina in LDA is like H. contortus while O. columbianum is more like O. circumcincta. Both large bowel worms were very sensitive to BZILEV combinations, with LD50s 4 to 8 fold lower than those observed for H. contortus and O. circumcincta

Craven, J.H.

Dobson, R.J.

Stein, P.A.

English

Research Repository

HOW TO IMPROVE DRENCHRlTE® RJ Dobson  1, PA Stein  1, JH Craven2 lCSIRO,  Division  of Animal  Production,  McMaster  Laboratory,  Locked  Bag  1,  Blacktown  2148, Australia, 2Horizon Technology, PO Box 598, Roseville NSW 2064, Australia. Abstract Understanding  the  strengths  and  weaknesses  of DrenchRite  and  how  it relates  to  faecal  egg  count reduction tests (FECRT) will lead to clearer interpretation of  DrenchRite results and a better perspective on both tests.  In previous studies efficacy was estimated for worms resistant to BZ, LEV and BZILEV combinations by FECRT, DrenchRite and post mortem worm counts. Correlations of  in vivo efficacy with FECRT determined by them were: Trichostrongylus colubriformis 88%, Ostertagia circumcincta 56% and Haemonchus contortus 69%. The low correlation for 0. circumcincta was associated with the variability in  estimating  species  composition  when  O.  circumcincta  was present  at low  levels.  Correlations  of DrenchRite with in vivo efficacy were: O.  circumcincta 87%, T.  colubriformis 82% and H.  contortus 80%. The correlation between resistance determined by Larval Development Assay (LDA) and FECRT was not high at 61%  (averaged over drugs and species).  Similar data for Macrocyclic Lactones (ML)  resistant worms are not yet available. Previous  studies  have  shown  for  some  LDA  systems  that  eggs  are  more  likely  to  be  inhibited  in development to L3s early (2-3 weeks post infection (PI)) and late (beyond 10 weeks PI) after infection and are most resilient to the effect of drugs at about 7 weeks PI. No systematic changes in DrenchRite results were observed over age of  worm infection when tested weekly for 2 to 14 week old worms. Large bowel worms of sheep have not generally been examined under LDA conditions so  a study was undertaken to  determine the LD50s of susceptible Oesophagostomum columbianum (nodule worm) and Chabertia  ovina  (large  mouthed  bowel  worm)  on  DrenchRite plates.  LD50s  for  BZ and  LEV  were approximately 0.04 and 0.8 uM respectively for both worms. For the ML O.  columbiclnum had a much higher LD50 (45 nM) than C.  ovina (8 nM).  In relation to the other common sheep worms, development characteristics  of C.  ovina  in  LDA  is  like  H.  contortus  while  O.  columbianum  is  more  like  O. circumcincta.  Both large bowel worms were very sensitive to BZILEV combinations, with LD50s 4 to 8 fold lower than those observed for H. contortus and O. circumcincta. Introduction In  this  discussion  paper  we  examme  the relationship between post mortem worm  counts, FECRT and DrenchRite results (1).  The stability of  DrenchRite results in relation to worm age will be  reviewed  together  with  some  recent  results from LDA studies.  And the sensitivity of some large bowel parasites which are commonly found in worm egg counts is also described. FECRT, LDA vs. Post Mortem Worm Counts Sheep infected with mixtures of drug resistant and susceptible  strains  of  Haemonchus  contortus, Trichostrongylus  colubriformis  and  Ostertagia circumcincta were  treated with a benzimidazole (BZ),  levamisole  (LEV)  or  a  BZILEV combination to determine drug efficacy. Efficacy 16 was estimated by FECRT, LDA and post mortem worm counts (1). Correlation  of  in  vivo  efficacy  with  FECR (averaged  over  drugs,  and  expressed  as  the percentage  of  the  total  variance  that  was explained by the regression analysis) was best for T.  colubriformis  (88%)  and  worst  for  O. circumcincta  (56%)  with  H.  contortus intermediate at 69%.  The low correlation for  0. circumcincta was  associated with the  variability in  estimating  species  composition  when  0. circumcincta  was  present  at  low  levels.  For example,  if the  true  mean  contribution  of one species  is  9%  then  anything  between  3%  and 15% can be expected from a random sample, and the range for a mean of  4% is 0%-8%. Thiskind t of variability in % species composition can yield widely differing  results for FECR when the total egg count is high (ie. when H.  contortus and/or T. colubriformis are present in large numbers).  The current caution applied to FECRT is that the mean FEC  needs  to  be  greater  than  200  eggs/gram before  trusting  a  FECRT result.  An  additional restriction:  that  a  species  represent  more  than 15% of the egg count should be applied before a valid FECRT can be assumed for that species. Correlations  of  resistance  ratio,  measured  by LDA, with in vivo efficacy were more consistent. 0. circumcincta was best (87%), T.  colubriformis and  H.  contortus  were  similar  (82%  and  80% respectively). The correlation between resistance determine<! by LDA and FECRT was not high at 61% (averaged over drugs  and species).  This indicates that the two tests are measuring slightly different aspects of  anthelmintic resistance. The DrenchRite  assay,  used  to  estimate  drench efficacy,  is  based  on  the  correlation  of LDA resistance  ratio  and  in  vivo  efficacy  from  the above study (1). The logical approach is to check the validity of a new test against the established one, in this case the FECRT. It is valid to use the FECRT  as  a  guide  to  the  accuracy  of  the DrenchRite estimate of drug efficacy only for T. colubriformis  where  there  is  a  reasonable correlation (77%).  For O.  circumcincta  and H. contortus, the LDA gives a better estimate of in vivo  efficacy  than  the  FECRT  and  for  these species  the  two  tests  (LDAIFECRT)  correlate poorly (53%). A  similar  study  (to  1)  for  the  Macrocyclic Lactones (ML) has not been conducted' because of the  lack  of a  suitable  range  of ML  resistant species  and the  high cost of undertaking such a study.  LDA  has  been  shown  to  differentiate between  known  ML  resistant  and  susceptible strains of O.  circumcincta  (2)  and H.  contortus (3).  Unpublished  data  of  tlte  authors  using DrenchRite has demonstrated that ML susceptible and  resistant  (4)  T.  colubriformis  can  be distinguished  by LDA.  With  T.  colubrifonnis, known  susceptible  strains  are  divided  into  two populations by the  ML used  in  the DrenchRite plate (Table 1). Well 1 below is the control well, 17 the  and  the ML concentration doubles each well to the maximum concentration in well 12. This data shows between well 5 and 6 about 87% of the susceptible  T.  colubriformis are prevented from developing to L3s, while the remaining 13% of  the  susceptible  T.  colubriformis  are  not affected  till  well  10  (ie.  at  a  16  fold  higher concentration).  In  a  resistant  T.  colubriformis strain (4) no eggs are prevented from developing till well 11  (where about 40% are affected).  This division  of susceptible  T.  colubrifonnis  into  2 populations is not optimum for field detection of resistance particularly when  other worm species are present.  Changing the ML drug used in the assay may  overcome this  problem  but  this  will require continning research. Table 1 Percentage of eggs/well inhibited from development  to  Infective  T.  colubriformis Larvae (% Kill) Well #  1 %Kill  0 Well #  7 %Kill  90 2 o 8 84 3 o 9 87 4 o 10 100 5  6 o  86 11  12 100100 Effect of  Worm Age on LDSO  determined by LDA Systematic changes in the concentration of drug required  to  inhibit  development  of  50%  of larvae/eggs present ("LD50") were  observed  (2) for  BZ,  LEV and MLs  over time  after  a single infection  (ie.  with  worm  age).  In their  study Amarante et al (2) used a liquid medium for the LDA,  as  described by Hubert  and  Kerboeuf(5). They demonstrated that eggs are more likely to be inhibited in  development to  L3s  very early  and late  post  infection  (PI)  (ie.  2-3  weeks  PI  and beyond 10 weeks PI) and are most resistant to the effect of  drugs at about 7 weeks PI. The  authors  conducted  a  similar  study  with  a single batch of DrenchRite plates prepared prior to the study and used over a period of 16 weeks. This  assay  relies  on  a  drug/agar  matrix  as  the development  medium  as  opposed  to  a  liquid medium(2 and 5).  One sheep was infected with McMaster suscepti1?le  strain of T.  colubriformis. Two sheep were infected with another susceptible strain  of T.  colubriformis recently isolated from Armidale NSW.  When infections were patent (22 days post infection) DrenchRite assays were performed on each sheep and then weekly for 12 weeks  except  for  the  McMaster  sheep  which stopped shedding eggs after the 8th assay. Table 2 DrenchRite Well Number of  the LDSO for Susceptible T.  colubriformis strains in Rows G and H which contain different analogues of  a ML Assay # 1  2  3  4  5  6  7  8  9  10  11  12 RowG McM  6.5  6  6  4.5  3.5  5.5  5.5  5 Ar#1  5.5  4.5  5.5  4.5  5.5  5.5  5.5  5.S  6  4.5  5.5  5.5 Ar#2  4.5  4.5  5.5  4.5  5.5  5.5  5.5  5.5  6.5  5  6  5.5 RowH McM  6.5  6.5  6  5.5  4.5  5  6  6.5 Ar#1  5.5  5.5  5.5  5.5  5  5.5  6.5  6  5.5  5.5  7  6.5 Ar#2  5.5  5.5  6.5  6.5  5  6.5  6.5  9.5  8.5  5.5  6  7 Note: assay #1 was conducted 22 days post infection and the following assays weekly thereafter. For  BZ  virtually  no  variation  LD50S  was observed,  for  35  of the  36  assays  the  LD50 occurred between well 4 and 5 (ie.4.5),  and was 3.5 for the McMaster isolate on the 5th assay.  For LEV, one of  the sheep infected with the Armidale strain (Ar #1)  consistently had an  LD50  at well 4.5, the departures from this were at assays 6,  8, 10 and 12 when the LD50 was 5.5, 5.5, 5.5  and 4 respectively.  The other sheep infected with the Armidale  strain (Ar #2)  consistently showed  an LD50  for  LEV  at  well  5.5  departures  being  at assays 4, 5, 10 and 12 with LD50s at wells 4.5, 5, 5  and  4.5,  respectively.  The  McMaster strain(McM)  fluctuated  between  5.5  and 4.5  for LEV  in  no  consistent  way.  For  BZlLEy combinations the LD50 was found at well 5.5  for a little over 2/3  of the assays,  the remaining  10 assays  ranged  between  well  6  and  4.5  in  no consistent way.  Table 2 shows well numbers for ML  LD50s  for  both  worm  strains  and  both analogues  of ML used  in  the DrenchRite plate, and again no consistent trend emerges. A similar study to the above was conducted with BZ and ML resistant strains of H.  contortus but included additional ML only LDA plates  so the two  ML compounds  (rows  G  and  H)  could be replicated.  The  susceptible  McMaster  H. contortus  (McM  Hc)  strain  had  a  longer  pre-patent  period  (about  4  weeks)  than  the  other strains tested (2-3  weeks),  and at 5 weeks McM Hc  still had a low egg count.  The BZ resistant (DAP  Hc)  strain  was  isolated  at  McMaster Laboratory  from  local  sheep,  this  strain  is  also partially resistant to ML.  The other strain used (only  in  ML  LDA  assays)  was  ML  resistant (CA  VRS Hc) and was isolated at Armidale NSW (6).  Figure  1 shows  the  mean well  number  at each sampling time where the BZ-LD50 occurred. Where the  data is missing no DrenchRite  plate was  set up  for that strain at that sampling time. Data points are means of 2 or 4 replicates.  It is clear that little fluctuation occurred in BZ LD50s for either strain over the course of  the infection. l.~~  ... Figure 1: DrenchRite well number in which BZ LDSO occurs for McM Hc (susceptible. ) and DAP Hc (resistant +) strains over 14 weeks of infection. BZLD50s 7  ..  • •  .........  .....  • • • ~ 6 c 5 T  .. Q) E 4 +  ~.-<C  c 31  I  I  11=  ~  ~  ~  ~  ....I 4  5  6  7  8  9  10  11  12  13  14  15 Weeks post infection McM Hc and DAP Hc strains were susceptible to LEV and BZlLEV combinations (data not shown) and only random fluctuations  in LD50 for these drugs were observed over time.  Figure 2 shows the well number in which the ML LD50s (row H) occurred for  ~A  VRS Hc, DAP Hc and McM Hc (data points are  means  of 6,  2,  and  4 replicates respectively). Like  T.  colubriformis the  3 H  contortus strains display no consistent systematic change in LD50 over  time,  although  slight  divergence  in  ML LD50  lines by week 7 may be evident (see Figure 2).  No similar study has been undertaken using DrenchRite plates for Ostertagia spp. Figure 2 DrenchRite well number in which ML LDSO  occurs  for  McM Hc (susceptible  •  ), DAP Hc (tolerant +) and CA  VRS Hc (resistant .11.) strains over 13 weeks ofinfection. .. ~ c ~ <C 9 ML LD50s --4  5  6  7  8  9  10  11  12  13  14 Weeks post infection Large Bowel Worms Despite  rumours  to  the  contrary,  large  bowel worms, Oesophagostomum columbianum (nodule worm) and Chabertia ovina (large mouthed bowel worm) in particular, are still alive and well in the eastern states of Australia, frequently constituting up  to  30%  of the  worm  eggs  found  in  sheep faeces.  Because the behaviour of these parasites under LDA conditions has  not been studied,  we examined LD50s over time in DrenchRite plates. Susceptible strains of both species were  assayed weekly  for  5  weeks,  each  assay  was  replicated and  additional  ML  assays  were  conducted  each sampling  time.  O.  columbianum  and  C.  ovina have  pre-patent  periods  of  6  and  10  weeks , respectively.  When the  assays  were commenced the  O.  columbianum and  C.  ovina infections (in separate  sheep)  were  9  and  10  weeks  old respectively. 19 Table 3 DrenchRite well number in which the WSO for Large Bowel Worms occurred DRUG  BZ  LEV  BZlLEV  ML SPECIES O. columbianum 3.5 C. ovina  3.5 4.0 4.0 2.5 2.5 6.5 4.0 Development in control wells was normal for  0. columbianum (ie around 90%+ of eggs dispensed became L3s), however, for  C.  ovina this did not occur till week 12 despite a high egg count (about 500epg)  at  10  and  11  weeks  PI.  The  poor development  of  C.  ovina  immediately  after patency could mean that a high proportion of eggs were not fertile.  Apart from their relatively high sensitivity  to  the  BZlLEV  combination  (0. circumcincta,  T.  colubriformis  and  H  contortus have LD50s around well 5) O.  columbianum and C.  ovina  show  similar  sensitivities  to  O. circumcincta and H  contortus respectively. Conclusion The above data on changes in LD50 over period of infection  suggest  worm  age  is  unlikely  to influence  DrenchRite  results.  Knowing  the sensitivity of the  common large bowel  parasites will assist the users of  DrenchRite to determine if the broad spectrum anthelmintics are effective for these  parasites.  However,  the  difficulty encountered  when  developing  C.  ovina  to  L3s immediately  after  patency  does  imply  if some young infections have reduced egg viability this potentially  may  decrease  the  sensitivity  of a DrenchRite assay. Acknowledgements  - We  are  grateful  for  the financial support of  Horizon Technology. References 1  Lacey  E,  Craven  JH,  Gill  JH  and  Baker  PJ (1993)  The  Larval  Development  Assay  as  an Alternative  to  Faecal  Egg  Reduction  Test  for Field Diagnosis of Broad Spectrum Anthelmintic Resistance.  The  Australian  Society  for ParaSitology:  Program and Abstracts of Papers Annual  scientific  meeting  held at  Heron  Island September 28 - October 1, 1993 2 Amarante AFT, Pomroy WE, Charleston WAG, Leathwick  DM  and  Tornero  MTT  (1997) Evaluation of  a Larval Development Assay for the Detection  of  Anthelmintic  Resistance  in Ostertagia circumcincta Int J Parasitol 27:  305-311 3 Gill JH, Redwin JM, Van Wyk JA and Lacey E (1995)  Avermectin  Inhibition  of  Larval Development in Haemonchus contortus - Effects of Ivermectin Resistance Int J Parasitol 25:  463-470 4  Shoop  WL  (1993)  Ivermectin  resistance Parasitol Today 9:  154-159 5 Hubert J and KerboeufD (1992)  A microlarval development  test  assay  for  the  detection  of anthelmintic resistance in sheep nematodes  Vet Rec 130: 442-446 6 Le Jambre LF, Gill JH, Lenane IJ and  Lacey E (1995)  Characterisation  of  an  avermectin resistant  strain  of  Australian  Haemonchus contortus. Int J Parasitol25: 691-698. , 20 

A microlarval development test assay for the detection of anthelmintic resistance in sheep nematodes Vet Rec

Avermectin Inhibition of Larval Development in Haemonchus contortus -

Characterisation of an avermectin resistant strain of Australian Haemonchus contortus.

Evaluation of a Larval Development Assay for the Detection of Anthelmintic Resistance in Ostertagia circumcincta

How to improve Drenchrite

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How to improve Drenchrite

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