Does tail docking prevent Cochliomyia hominivorax myiasis in sheep? A six-year retrospective cohort study.

Tail docking is a husbandry practice widely incorporated in sheep farms around the world. It is an irreversible mutilation that impairs animal welfare, both immediately and in the longer term. The defence of tail docking as a practice is centred around the perception that doing so contributes to the promotion of local hygiene, allowing the use of the wool, facilitating reproductive management and reducing the chances of myiasis, a disease caused by the invasion of blowfly larvae in the tissues of warm-blooded animals. However, current understanding of farm animal welfare questions the need to maintain practices such as tail docking. Thus, the aim of this study was to evaluate the effect of tail docking on the incidence of Cochliomyia hominivorax myiasis in sheep in an experimental flock in Brazil during a six-year retrospective cohort study. Relative risk, odds ratio and incidence rate ratio were the association measures adopted. A total of 4,318 data-points were collected and supplied the analytical model. Tail docking did not decrease the risk and, on the contrary, was found to increase the chances of sheep being affected by myiasis. The results support the hypothesis that tail docking is not a protective factor against the occurrence of myiasis and further fuel calls for a rethink of tail docking being deployed as a blanket measure in the prevention of myiasis in sheep.

Complete Mitochondrial Genome for Lucilia cuprina dorsalis (Diptera: Calliphoridae) from the Northern Territory, Australia.

The Australian sheep blowfly, Lucilia cuprina dorsalis, is a major sheep ectoparasite causing subcutaneous myiasis (flystrike), which can lead to reduced livestock productivity and, in severe instances, death of the affected animals. It is also a primary colonizer of carrion, an efficient pollinator, and used in maggot debridement therapy and forensic investigations. In this study, we report the complete mitochondrial (mt) genome of L. c. dorsalis from the Northern Territory (NT), Australia, where sheep are prohibited animals, unlike the rest of Australia. The mt genome is 15,943 bp in length, comprising 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a non-coding control region. The gene order of the current mt genome is consistent with the previously published L. cuprina mt genomes. Nucleotide composition revealed an AT bias, accounting for 77.5% of total mt genome nucleotides. Phylogenetic analyses of 56 species/taxa of dipterans indicated that L. c. dorsalis and L. sericata are the closest among all sibling species of the genus Lucilia, which helps to explain species evolution within the family Luciliinae. This study provides the first complete mt genome sequence for L. c. dorsalis derived from the NT, Australia to facilitate species identification and the examination of the evolutionary history of these blowflies.

Using weather data to predict the presence of Lucilia spp. on sheep farms in New Zealand.

Flystrike remains an important animal health issue on New Zealand sheep farms. To date no useful predictive tool to assist farmers to develop control options has been available. The aim of this study was to use National Institute of Water and Atmospheric Research (NIWA) virtual climate station data in New Zealand to develop a weather-based model to accurately predict the presence of Lucilia spp. on sheep farms throughout New Zealand. Three LuciTrap® baited fly traps were positioned on each of eight sheep farms throughout New Zealand (5 in the North Island and 3 in the South Island). The traps were put out for both the 2018-2019 and 2019-2020 seasons. They were emptied each week and the flies morphologically identified; with the counts of Lucilia cuprina and L. sericata combined as Lucilia spp. The count data for Lucilia spp. for each week of trapping was transformed into a binary outcome and a generalised linear mixed effects models fitted to the data, with farm as a random effect. The dependent variable was Lucilia spp. flies caught, yes or no, and the independent variables were mean weekly climate variables from the nearest NIWA virtual climate station to that farm. The model was trained on the 2018-2019 catch data and tested on the 2019-2020 catch data. A cut point was identified which maximised the model's ability to correctly predict whether Lucilia spp. were present or not for the 2019-2020 catch data, and the sensitivity, specificity, accuracy, and area under the curve (AUC) of the model calculated. The final model included just 3 significant variables, mean weekly 10 cm soil temperature, mean weekly soil moisture index, and mean weekly wind speed at 10 m. Mean weekly 10 cm soil temperature accounted for 64.7% of the variance explained by the model, mean weekly soil moisture index 34.7% and mean weekly wind speed at 10 m only 0.6%. The results showed that the predictive model had a sensitivity of 0.93 (95% CI = 0.80-0.98) and a specificity of 0.75 (95% CI = 0.62-0.85), using a cut point for the probability of Lucilia spp. being present on farm = 0.383. This model provides New Zealand farmers with a tool which will allow them to know when Lucilia spp. flies will likely be present and thus more accurately plan their interventions to prevent flystrike.

Resistance to dicyclanil and imidacloprid in the sheep blowfly, Lucilia cuprina, in Australia.

BACKGROUND: The sheep blowfly, Lucila cuprina, is a myiasis-causing parasite responsible for significant production losses and welfare issues for the Australian sheep industry. Control relies largely on the use of insecticides. The pyrimidine compound, dicyclanil, is the predominant control chemical, although other insecticides also are used, including imidacloprid, ivermectin, cyromazine and spinosad. We investigated in vitro resistance patterns and mechanisms in field-collected blowfly strains. RESULTS: The Walgett 2019 strain showed significant levels of resistance to both dicyclanil and imidacloprid, with resistance factors at the IC50 of 26- and 17-fold, respectively, in in vitro bioassays. Co-treatment with the cytochrome P450 inhibitor, aminobenzotriazole, resulted in significant levels of synergism for dicyclanil and imidacloprid (synergism ratios of 7.2- and 6.1-fold, respectively), implicating cytochrome P450 in resistance to both insecticides. Cyp12d1 transcription levels were increased up to 40-fold throughout the larval life stages in the resistant strain compared to a reference susceptible strain, whereas transcription levels of some other cyp genes (6g1, 4d1, 28d1) did not differ between the strains. Similar resistance levels also were observed in flies collected from the same property in two subsequent years. CONCLUSION: This study indicates that in vitro resistance to both dicyclanil and imidacloprid in this field-collected blowfly strain is likely mediated by cytochrome P450, with Cyp12d1 implicated as the enzyme responsible; however, it remains possible that another P450 also may be involved. A common resistance mechanism for the two drugs has important implications for drug rotation strategies designed to prolong the useful life of flystrike control chemicals. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Control of sheep flystrike: what's been tried in the past and where to from here.

Flystrike remains a serious financial and animal welfare issue for the sheep industry in Australia despite many years of research into control methods. The present paper provides an extensive review of past research on flystrike, and highlights areas that hold promise for providing long-term control options. We describe areas where the application of modern scientific advances may provide increased impetus to some novel, as well as some previously explored, control methods. We provide recommendations for research activities: insecticide resistance management, novel delivery methods for therapeutics, improved breeding indices for flystrike-related traits, mechanism of nematode-induced scouring in mature animals. We also identify areas where advances can be made in flystrike control through the greater adoption of well-recognised existing management approaches: optimal insecticide-use patterns, increased use of flystrike-related Australian Sheep Breeding Values, and management practices to prevent scouring in young sheep. We indicate that breeding efforts should be primarily focussed on the adoption and improvement of currently available breeding tools and towards the future integration of genomic selection methods. We describe factors that will impact on the ongoing availability of insecticides for flystrike control and on the feasibility of vaccination. We also describe areas where the blowfly genome may be useful in providing impetus to some flystrike control strategies, such as area-wide approaches that seek to directly suppress or eradicate sheep blowfly populations. However, we also highlight the fact that commercial and feasibility considerations will act to temper the potential for the genome to act as the basis for providing some control options.

Ovine Toll-like Receptor 9 (TLR9) Gene Variation and Its Association with Flystrike Susceptibility.

Toll-like receptors (TLRs) are a family of proteins that play a role in innate immune responses by recognising pathogen-associated molecular patterns derived from various microbes. Of these receptors, TLR9 recognises bacterial and viral DNA containing unmethylated cytosine-phosphate-guanine (CpG) motifs, and variation in TLR9 has been associated with resistance to various infectious diseases. Flystrike is a problem affecting the sheep industry globally and the immune response of the sheep has been suggested as one factor that influences the response to the disease. In this study, variation in ovine TLR9 from 178 sheep with flystrike and 134 sheep without flystrike was investigated using a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) approach. These sheep were collected from both commercial and stud farms throughout New Zealand and they were of 13 different breeds, cross-breds and composites. Four alleles of TLR9 were detected, including three previously identified alleles (*01, *02 and *03) and a new allele (*04). In total six single nucleotide polymorphisms (SNPs) were found. Of the three common alleles in the sheep studied, the presence of *03 was found to be associated with a reduced likelihood of flystrike being present (OR = 0.499, p = 0.024). This suggests that variation in ovine TLR9 may affect a sheep's response to flystrike, and thus the gene may have value as a genetic marker for improving resistance to the disease.

Ovine FABP4 Variation and Its Association With Flystrike Susceptibility.

Flystrike is a major cost and a welfare issue for the New Zealand sheep industry. There are several factors that can predispose sheep to flystrike, such as having fleecerot, a urine-stained breech, and "dags" (an accumulation of fecal matter in the wool of the breech). The FABP4 gene (FABP4) has been associated with variation in ovine fleecerot resistance, with a strong genetic correlation existing between fleecerot and flystrike occurrence. In this study, blood samples were collected from sheep with and without flystrike for DNA typing. PCR-SSCP analyses were used to genotype two regions of ovine FABP4. Sheep with the A 1 variant of FABP4 were found to be less likely (odds ratio 0.689, P = 0.014) to have flystrike than those without A 1. The likelihood of flystrike occurrence decreased as copy number of A 1 increased (odds ratio 0.695, P = 0.006). This suggests that FABP4 might be a candidate gene for flystrike resilience in sheep, although further research is required to verify this association.

Dicyclanil resistance in the Australian sheep blowfly, Lucilia cuprina, substantially reduces flystrike protection by dicyclanil and cyromazine based products.

Late in 2017, field samples of the Australian sheep blowfly, Lucilia cuprina, were submitted by sheep producers from three states of Australia (South Australia, Victoria and New South Wales). Some were collected by submitters concerned about shortened periods of flystrike protection from dicyclanil based products. Neonate larval offspring from the NSW field samples survived and successfully completed their life cycles following exposure to dicyclanil and cyromazine at susceptible discriminating concentrations in vitro. The in vivo study reported here used dicyclanil resistant neonate larvae to assess the flystrike protection provided by a cyromazine jetting fluid and a number of dicyclanil based spray-on products, when applied to sheep six weeks after shearing. The two dicyclanil resistant blowfly strains used in this study showed in vitro resistance ratios, at the LC50, of approximately 13- and 25-fold relative to a dicyclanil and cyromazine susceptible strain. Compared to the levels of resistance that L. cuprina has developed to other insecticides these are relatively low, however, three dicyclanil based spray-on products (active ingredient 12.5 g/L, 50 g/L and 65 g/L) had protection periods reduced by 73%, 78% and 69% respectively when compared to the maximum protection periods claimed by the manufacturer. A 50% and a 33% reduction in protection period was also observed to a cyromazine and an ivermectin based jetting fluid respectively. In contrast, protection periods were attained or exceeded regardless of the treatment used against field derived dicyclanil susceptible neonate larvae. For the first time we confirm that dicyclanil resistance enables the completion of the L. cuprina life cycle following flystrike initiation on dicyclanil or cyromazine treated sheep when insecticide levels are considered high and protective. This study also provides in vivo information on the effect of dicyclanil resistance on the protection provided by a product with an active ingredient belonging to an unrelated insecticide group. Dicyclanil resistance is of major concern to the Australian sheep industry.

Remote Identification of Sheep with Flystrike Using Behavioural Observations.

Flystrike is a major problem affecting sheep in Australia. Identification of 'flystruck' individuals is crucial for treatment; but requires labour-intensive physical examination. As the industry moves toward more low-input systems; there is a need for remote methods to identify flystruck individuals. The aim of this study was to investigate the behaviour of sheep with breech flystrike within a paddock setting. Video footage of sixteen Merino sheep; eight later confirmed with flystrike and eight without; was collected as they moved freely within the paddock with conspecifics. Quantitative behavioural measurements and a qualitative behavioural assessment (QBA) were conducted and compared to their breech conditions (i.e., faecal/urine staining; flystrike severity). Both qualitative and quantitative assessments indicated behavioural differences between flystruck and non-flystruck animals. Flystruck sheep had a behavioural profile characterised by restless behaviour; abnormal postures and reduced grazing time (p < 0.05). Furthermore; flystruck sheep were scored to have a more 'exhausted/irritated' demeanour using QBA (p < 0.05). The behavioural responses also corresponded to the flystrike severity scores and condition of the breech area. We conclude that remotely assessed behaviour of flystruck sheep diverges markedly from non-flystruck sheep; and thus could be a low-input method for identifying and treating affected animals.

The Effects of Diets and Long-term Laboratory Rearing on Reproduction, Behavior, and Morphology of Lucilia cuprina (Diptera: Calliphoridae).

The Australian sheep blow fly, Lucilia cuprina (Wiedemann), is commonly reared in the laboratory for many sequential generations on simple, fixed diets, so it can be used in veterinary, medical, and forensic studies. To investigate the effect of diet and long-term laboratory rearing on L. cuprina, flies were fed with two different diets (sugar and milk-sugar) over a year and F1, F6, and F11 generations were used for comparisons based on the number of eggs, attraction to wool and liver, and wing size. The results showed that the number of eggs of gravid flies, and the attractiveness of wool and liver did not differ significantly between diets and generations, but gravid flies were more attracted to wool and liver than non-gravid flies (P < 0.05). Moreover, in the F1 generation, thorax length and wing aspect ratio were significantly longer than in the F6 and F11 generations (P < 0.05), and the wing length was significantly longer than in the F11 generation (P < 0.05). It was concluded that neither diet nor long-term laboratory rearing affect potential fecundity or the behavioral responses of L. cuprina, but the gravidity of flies affects their behavioral response, and long-term laboratory rearing significantly affects fly morphology, apparently explaining a loss in flight performance.

Isolation of two insecticidal toxins from venom of the Australian theraphosid spider Coremiocnemis tropix.

Sheep flystrike is caused by parasitic flies laying eggs on soiled wool or open wounds, after which the hatched maggots feed on the sheep flesh and often cause large lesions. It is a significant economic problem for the livestock industry as infestations are difficult to control due to ongoing cycles of larval development into flies followed by further egg laying. We therefore screened venom fractions from the Australian theraphosid spider Coremiocnemis tropix to identify toxins active against the sheep blowfly Lucilia cuprina, which is the primary cause of flystrike in Australia. This screen led to isolation of two insecticidal peptides, Ct1a and Ct1b, that are lethal to blowflies within 24 h of injection. The primary structure of these peptides was determined using a combination of Edman degradation and sequencing of a C. tropix venom-gland transcriptome. Ct1a and Ct1b contain 39 and 38 amino acid residues, respectively, including six cysteine residues that form three disulfide bonds. Recombinant production in bacteria (Escherichia coli) resulted in low yields of Ct1a whereas solid-phase peptide synthesis using native chemical ligation produced sufficient quantities of Ct1a for functional analyses. Synthetic Ct1a had no effect on voltage-gated sodium channels from the American cockroach Periplanata americana or the German cockroach Blattella germanica, but it was lethal to sheep blowflies with an LD50 of 1687 pmol/g.

Estimates of genetic parameters for breech strike and potential indirect indicators in sheep.

AIM: To determine flystrike incidence, and estimate genetic parameters and potential indicator traits to reduce breech strike susceptibility in Romney sheep in New Zealand. METHODS: Seventeen Romney-based flocks, on 11 farms from throughout New Zealand, were enrolled in a case-control study in 2009/10 and 2010/11. Farmers observed lambs with flystrike and recorded dag score, breech bareness score and flystrike location for each lamb. Flystrike location was classed as breech, body, belly, shoulders, head or feet. Paternity of lambs was determined from tissue samples collected from lambs and all potential sires on farms. Control lambs without flystrike were selected by matching birth year, flock and sex. Due to the majority of strike occurring in the breech, genetic parameters for breech strike were explored. Dag score and breech bareness were investigated as indirect indicators of breech strike. Heritabilities and genetic and phenotypic correlations were estimated for breech strike, dag score and breech bareness using an animal model. RESULTS: For the 2009/10 season, 484 cases of flystrike were recorded with mean incidence rate per farm of 1.76 (min 0.47, max 2.95)%. For the 2010/11 season, 352 cases were recorded with a mean incidence rate per farm of 2.54 (min 0.43, max 8.18)%. Over both years 694/792 (88%) cases of flystrike occurred on the breech. Heritability on the observed scale for breech strike was 0.32 (SE 0.10). Heritabilities for dag score and breech bareness were 0.23 (SE 0.09) and 0.35 (SE 0.11), respectively. Breech strike had a high positive genetic correlation with dag score (0.71) and a low negative genetic correlation with breech bareness (-0.17). Breech strike had a high phenotypic correlation with dag score (0.62) and negative phenotypic correlation with breech bareness (-0.06). CONCLUSIONS: The high genetic and phenotypic correlations between breech strike and dag score makes dag score a viable option for indirect selection for breech strike resistance in Romney sheep in New Zealand. The heritability, genetic and phenotypic correlations require validation in other dual-purpose breeds, before breeding values for breech strike can be implemented, for use throughout the New Zealand sheep industry.

Survival advantage of cyromazine-resistant sheep blowfly larvae on dicyclanil- and cyromazine-treated Merinos.

OBJECTIVE: To determine whether a cyromazine-resistant strain of Lucilia cuprina was able to establish strikes sooner than a susceptible strain on cyromazine- or dicyclanil-treated sheep. METHODS: Groups of 7 sheep were treated with cyromazine or dicyclanil in accordance with label directions. Beginning 5 weeks after treatment, 5 sheep from each group were challenged by implantation of neonate larvae belonging to the cyromazine-resistant strain 'Nimmitabel-selected' and the susceptible blowfly strain 'Field 2011' according to standard larval implant technique. The implant sites were alternated between the shoulder, mid-back and rump within the treatment zone. Similarly, implants of the resistant and susceptible strain larvae were alternated at each challenge such that the strain implanted on the left side of the sheep at one challenge was implanted on the right side at the next. Challenges were conducted at 3-weekly intervals until the susceptible larvae formed strikes on at least 2 of the 5 sheep in a treatment group or until 29 weeks after treatment. RESULTS: Sheep treated with cyromazine or dicyclanil were protected from flystrike by the cyromazine-susceptible strain for periods consistent with, or longer than, the registered product label claims. The cyromazine-resistant strain created strikes several weeks sooner after treatment than did the susceptible strain. Accordingly, the protection periods provided by cyromazine and dicyclanil against the resistant strain were reduced from 14 and 18-24 weeks to <8 weeks and <11 weeks, respectively. CONCLUSION: Resistance, even in the pure-breeding resistant strain, was not so severe as to cause treatment failure with cyromazine or dicyclanil, but was sufficient to reduce the protection period provided. It is recommended that producers adopt management practices that minimise the development of resistance to these and other compounds.

Breech-strike on mulesed, clipped and unmulesed Merino ewes and hoggets in south-eastern Australia.

OBJECTIVE: Compare breech-strike on Merino ewes and hoggets that were mulesed, had breech and tail clips applied ('clipped') or left unmulesed and treated strategically with long-acting insecticide. DESIGN: A cohort study on two farms in southern Victoria. METHODS: Three treatment groups were established at lamb marking in 2008 and 2009 on each farm. The unmulesed group was treated with a long-acting insecticide in early spring. The prevalence of breech-strike and key risk factors, including presence of dag, urine stain and breech wrinkle, were compared between groups. RESULTS: Breech-strike was detected from October to December on 1.9% and 7.2% of mulesed, and on 14.8% and 12.5% of clipped ewes, respectively, on each farm. Thus, clipped ewes had a relative risk of breech-strike 7.8- and 1.7-fold that of mulesed ewes. Unmulesed ewes treated with insecticide had similar or less breech-strike compared with mulesed ewes (3.4% and 1.4%), but significantly more dag, stain and breech wrinkle. From January, breech-strike on unprotected unmulesed ewes was 8.5% and 2.8%, compared with 3.5% and 0% on mulesed ewes. CONCLUSION: Early-season treatment of unmulesed sheep prevented most breech-strikes during spring and early summer on both hoggets and breeding ewes, confirming it as a short- to medium-term option for the control of breech-strike. Some benefits were associated with the use of clips but, to control breech-strike, clipped sheep should be treated the same as unmulesed sheep. Genetic selection to reduce the prevalence of dag will be required for future strategies to control breech-strike on Merino sheep in south-eastern Australia.

Effective control of a suspected cyromazine-resistant strain of Lucilia cuprina using commercial spray-on formulations of cyromazine or dicyclanil.

OBJECTIVE: To demonstrate the protection of Merino sheep from flystrike by Lucilia cuprina with cyromazine or dicyclanil in an implant study and in the field. METHODS: In the implant study, sheep were treated with cyromazine or dicyclanil and implanted with 1st-stage larvae from a newly isolated field strain of L. cuprina (CYR-LS) or a reference strain (DZR50), then assessed over 3 days and compared with the implants on untreated control sheep. In the field study, weaner lambs were treated with cyromazine or dicyclanil and monitored weekly for flystrike over 18 weeks of grazing on the same farm from which the L. cuprina were isolated. RESULTS: Implant study: cyromazine (6%) provided effective protection against CYR-LS and DZR50 L. cuprina for a minimum of 13 and 10 weeks, respectively. Dicyclanil (5%) provided at least 18 weeks' protection against both strains. Field study: only 1 of 386 lambs in the cyromazine-treated group was struck in the first 14 weeks of the trial. No strikes occurred in the 198 sheep treated with dicyclanil (5%). Rainfall, temperature and flytrap data indicated consistent fly pressure during the study. CONCLUSIONS: Based on the results of these studies, there was no evidence of reduced susceptibility to cyromazine or dicyclanil and the periods of protection of sheep against L. cuprina were unaffected and consistent with the registered label claims.

Transgenic sexing system for genetic control of the Australian sheep blow fly Lucilia cuprina.

The New World screwworm and the Australian sheep blowfly Lucilia cuprina are devastating pests of livestock. The larvae of these species feed on the tissue of the living animal and can cause death if untreated. The sterile insect technique or SIT was used to eradicate screwworm from North and Central America. This inspired efforts to develop strains containing complex chromosomal rearrangements for genetic control of L. cuprina in Australia. Although one field trial was promising, the approach was abandoned due to costs and difficulties in mass rearing the strain. As the efficiency of SIT can be significantly increased if only sterile males are released, we have developed transgenic strains of L. cuprina that carry a dominant tetracycline repressible female lethal genetic system. Lethality is due to overexpression of an auto-regulated tetracycline repressible transactivator (tTA) gene and occurs mostly at the pupal stage. Dominant female lethality was achieved by replacing the Drosophila hsp70 core promoter with a Lucilia hsp70 core promoter-5'UTR for tTA overexpression. The strains carry a dominant strongly expressed marker that will facilitate identification in the field. Interestingly, the sexes could be reliably sorted by fluorescence or color from the early first instar larval stage as females that overexpress tTA also overexpress the linked marker gene. Male-only strains of L. cuprina developed in this study could form the basis for a future genetic control program. Moreover, the system developed for L. cuprina should be readily transferrable to other major calliphorid livestock pests including the New and Old World screwworm.