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Hosts counteract infections using two distinct defence strategies, resistance (reduction in pathogen fitness) and tolerance (limitation of infection damage). These strategies have been minimally investigated in multi-host systems, where they may vary across host species, entailing consequences both for hosts (virulence) and parasites (transmission). Comprehending the interplay among resistance, tolerance, virulence and parasite success is highly relevant for our understanding of the ecology and evolution of infectious and parasitic diseases. Our work investigated the interaction between an insect parasite and its most common bird host species, focusing on two relevant questions: (i) are defence strategies different between main and alternative hosts and, (ii) what are the consequences (virulence and parasite success) of different defence strategies? We conducted a matched field experiment and longitudinal studies at the host and the parasite levels under natural conditions, using a system comprising Philornis torquans flies and three bird hosts - the main host and two of the most frequently used alternative hosts. We found that main and alternative hosts have contrasting defence strategies, which gave rise in turn to contrasting virulence and parasite success. In the main bird host, minor loss of fitness, no detectable immune response, and high parasite success suggest a strategy of high tolerance and negligible resistance. Alternative hosts, on the contrary, resisted by mounting inflammatory responses, although with very different efficiency, which resulted in highly dissimilar parasite success and virulence. These results show clearly distinct defence strategies between main and alternative hosts in a natural multi-host system. They also highlight the importance of defence strategies in determining virulence and infection dynamics, and hint that defence efficiency is a crucial intervening element in these processes., (Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The last 50 years of research into infections in Australia and New Zealand caused by larvae of the sheep blowfly, Lucilia cuprina, have significantly advanced our understanding of this blowfly and its primary host, the sheep. However, apart from some highly effective drugs it could be argued that no new control methodologies have resulted. This review addresses the major areas of sheep blowfly research over this period describing the significant outcomes and analyses, and what is still required to produce new commercial control technologies. The use of drugs against this fly species has been very successful but resistance has developed to almost all current compounds. Integrated pest management is becoming basic to control, especially in the absence of mulesing, and has clearly benefited from computer-aided technologies. Biological control has more challenges but natural and perhaps transformed biopesticides offer possibilities for the future. Experimental vaccines have been developed but require further analysis of antigens and formulations to boost protection. Genetic technologies may provide potential for long-term control through more rapid indirect selection of sheep less prone to flystrike. Finally in the future, genetic analysis of the fly may allow suppression and perhaps eradication of blowfly populations or identification of new and more viable targets for drug and vaccine intervention. Clearly all these areas of research offer potential new controls but commercial development is perhaps inhibited by the success of current chemical insecticides and certainly requires a significant additional injection of resources., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

Understanding the spatial scale and temporal pattern of disease incidence is a fundamental prerequisite for the development of appropriate management and intervention strategies. It is particularly critical, given the need to understand the elevated risks linked to climate change, to allow the most likely changes in the distribution of parasites and disease vectors to be predicted under a range of climate change scenarios. Using statistical models, the spatial distribution and climatic correlates of a range of parasites and diseases have been mapped previously, but their development into dynamic, predictive tools is less common. The aim of the work described here, was to use a species distribution model to characterise the environmental determinants of the monthly occurrence of ovine cutaneous myiasis (blowfly strike) by Lucilia sericata, the most frequent primary agent of northern European myiasis, and to then use this model to describe the potential spatial changes that might be expected in response to predicted climate change in Great Britain. The model predicts that the range of elevated temperatures predicted by current climate change scenarios will result in an increase in the risk of strike and an elongated blowfly season. However, even for the most rapid warming scenario predictions over the next 70 years, strike is not predicted to occur throughout the winter. Nevertheless, in this latter case, parts of central and southern England are likely to become too hot and dry for strike by L. sericata, to persist in mid-summer. Under these conditions, it is possible that other, more pathogenic Mediterranean agents of myiasis, such as Wolfhartia magnifica, could potentially replace L. sericata. Where the phenology of strike is altered by climate change, as predicted here, significant changes to the timing and frequency of parasite treatments and husbandry practices, such as shearing, will be required to manage the problem. The results suggest that the modelling approach adopted here could be usefully applied to a range of disease systems., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Among arthropod diseases affecting animals, larval infections - myiases - of domestic and wild animals have been considered important since ancient times. Besides the significant economic losses to livestock worldwide, myiasis-causing larvae have attracted the attention of scientists because some parasitise humans and are of interest in forensic entomology. In the past two decades, the biology, epidemiology, immunology, immunodiagnosis and control methods of myiasis-causing larvae have been focused on and more recently the number of molecular studies have also begun to increase. The 'new technologies' (i.e. molecular biology) are being used to study taxonomy, phylogenesis, molecular identification, diagnosis (recombinant antigens) and vaccination strategies. In particular, more in depth molecular studies have now been performed on Sarcophagidae, Calliphoridae and flies of the Oestridae sister group. This review discusses the most topical issues and recent studies on myiasis-causing larvae using molecular approaches. In the first part, PCR-based techniques and the genes that have already been analysed, or are potentially useful for the molecular phylogenesis and identification of myiasis-causing larvae, are described. The second section deals with the more recent advances concerning taxonomy, phylogenetics, population studies, molecular identification, diagnosis and vaccination.

The larvae of the fly Lucilia cuprina cause a cutaneous myiasis in mammalian hosts, particularly sheep. The glycoprotein, peritrophin-95, isolated from Lucilia cuprina larval peritrophic matrix, is a candidate vaccine antigen. This protein induced an immune response in vaccinated sheep that inhibited larval growth. Recombinant forms of peritrophin-95 were produced in bacteria and baculovirus-infected insect cells. The bacterial protein was not glycosylated and incorrectly folded whereas the insect cell-expressed protein was glycosylated and probably correctly folded. Sheep immunised with purified native peritrophin-95 generated strong larval growth inhibitory activity in their sera, whereas sheep immunised with either recombinant form of peritrophin-95 generated only relatively weak inhibitory activity. Ingested ovine antibodies to native peritrophin-95 mediated the anti-larval growth activity and this was independent of the presence of ovine complement. The activity was associated with IgG(1) and IgG(2) but not IgM. There were strong antibody responses to both the correctly folded native peritrophin-95 polypeptide and the oligosaccharides present on this glycoprotein. Immuno-affinity isolation of antibody to the peritrophin-95 polypeptide and antibody to peritrophin-95 oligosaccharides demonstrated that the larval growth inhibitory activity resided with both antibodies. Lectin blots and ELISA data showed substantial differences between the oligosaccharides attached to native peritrophin-95 and insect cell-expressed recombinant peritrophin-95. It was concluded that the oligosaccharides attached to native peritrophin-95 and its unique polypeptide structure are essential for the induction of larval growth inhibitory activity in the sera of sheep vaccinated with this antigen.

A series of experiments were carried out to investigate the role of proteinase enzymes in the growth of larvae of the sheep blowfly, Lucilia cuprina. First, instar larvae were incubated on an artificial growth media in the presence of various concentrations of inhibitors of all the major proteinase classes. Inhibitors of serine proteinases and aminopeptidases were found to cause significant growth inhibition and in some cases death of the larvae within 24 h, suggesting that these enzymes were the major classes involved in protein digestion in the gut of the insect. A second group of experiments analysed the effects of two inhibitors from the same or different proteinase classes in the growth media. Synergistic inhibition of larval growth was observed with the incorporation of inhibitors of serine proteinases and aminopeptidases. The results suggest that these classes of proteinases are both central to protein digestion in this insect, probably in the gut, and that the inhibition of both types of activity leads to an almost complete blockade of digestion. Testing in vivo gave similar results with infections on sheep skin inhibited by either serine proteinase or aminopeptidase enzyme inhibitors and the combination of both stopped the infection process. The role of aminopeptidases in larval metabolism and as potential targets for blowfly control agents is examined.

Whole first-instar Lucilia cuprina larvae were homogenised and sequentially extracted with a series of buffers of progressively more severe solubilising power. The final extract, using a buffer containing 6 M-urea, was fractionated by preparative isoelectric focussing. At each step in this process, protein fractions were tested in sheep vaccination trials for their ability to induce immune responses affecting the growth of L. cuprina larvae which fed on the sera from vaccinated sheep. One isoelectric focussing fraction (pH 5.9-6.7) containing a number of larval proteins induced an immune response which inhibited the growth of larvae by a mean of 84 +/- 7% in an in vitro feeding bioassay. The recovery of larvae after feeding on sera from sheep vaccinated with this fraction was significantly reduced by 35 +/- 13%. This antilarval effect was shown to be mediated by ingested ovine antibodies. Immunofluorescence and immunogold localisations showed that the immune response was directed at proteins from the larval peritrophic membrane, larval cuticle and, to lesser extent, basement membranes and microvilli of digestive epithelial cells. Electron microscopic examination of larvae feeding on sera from sheep vaccinated with this fraction showed that the normally semi-permeable peritrophic membrane was blocked on the luminal side by an electron-lucent layer of undefined composition. It is postulated that this layer prevents nutrients from moving from the gut to the underlying digestive epithelial cells, thereby starving the larvae. The sera from sheep vaccinated with another isoelectric focussing fraction (pH 3.4-5.5) reduced the mean larval weight by 56 +/- 13% without significant effects on larval survival.

The nasal bot fly, Oestrus ovis, was investigated to establish which specific cuticular component is most immunogenic to infested sheep and how larval cuticle attains a protective role, if any, against the host immune system. To accomplish these goals, larval cuticle was extracted by a variety of agents and tested against immune sera from infested sheep and experimentally immunized rabbits. The cuticle substructure remaining after extraction was examined to localize various immunogenic components. O. ovis larval integument comprises an inner cellular layer, the epidermis, and an overlying cuticle layer. In 3rd instar larvae, the cuticle comprises 2 additional layers: the procuticle with numerous pore canals and the epicuticle which includes the wax canals. Three additional layers, altogether comprising the cuticulin layer, are present external to the epicuticle. The epicuticle is completed by apposition of an amorphous electrondense material extending for up to 1 micron in thickness. When fixed with ruthenium red, cuticle becomes heavily stained all along the epicuticular surface in larvae of all developmental stages. However, in 3rd instar larvae, ruthenium red deposits are restricted to the cuticulin layer alone. By gel electrophoresis, 3rd instar larval cuticle is shown to contain a number of polypeptides ranging in molecular weight from 180 to 4.5 kDa. The number and relative concentration of low molecular weight polypeptides was shown to vary in relation to the extraction media employed. Cuticular fragments examined after extraction exhibit an altered ultrastructure. When tested by immunoblotting, the cuticular polypeptides most reactive against sheep antisera are in the range of 180-56 kDa. A similar reaction was also detected with sera from rabbits infested experimentally with O. ovis larvae. Results are interpreted in relation to differential polypeptide distribution within the larval cuticle and to accessibility of the host immune system.

Blowfly strike is a cutaneous myiasis in sheep caused by infestations of larvae principally from the family Calliphoridae, particularly the species Lucilia cuprina and Lucilia sericata. These larval infestations cause considerable economic losses to the wool industry. Established control methods have served the industry well in the past, but there are growing deficiencies with these methods. In particular, there is widespread resistance to organophosphorus insecticides and potential difficulties associated with the presence of chemical residues derived from insecticides in wool and waste products which must be disposed of by the industry. There is also growing opposition to the radical surgical procedures used to decrease the susceptibility of sheep to blowfly strike. Consequently, there is a need for the development of alternative control measures. This review examines critically the present control methods and discusses the range of options available for the development of new control strategies. Many of the latter involve novel approaches which will strongly complement current control measures.

To consider the evolutionary origin of the ectoparasitic habit in the blowfly genus Lucilia (Diptera:Calliphoridae), phylogenetic analyses of mitochondrial DNA sequence data were performed for 10 species, including all the common Lucilia agents of myiasis, collected from Africa, Australasia, North America and Europe. Complementary genetic distance and parsimony analyses are used to consider inter and intraspecific relationships within the genus with reference to previous morphological work. The results support the hypothesis of independent multiple evolution of the ectoparasitic habit in Lucilia sericata, Lucilia cuprina and the Lucilia caesar/Lucilia illustris group and suggest that it has coevolved in relatively recent history along with the domestication and husbandry of sheep. The geographic differences in pathogenic importance of various species of Lucilia also suggest that there is a strong climatic influence determining which species has dominated. Lucilia cuprina has become the predominant pathogenic species in sub-tropical and warm temperate habitats (e.g., Australia and South Africa), L. sericata in cool temperate habitats (e.g., Europe and New Zealand) and L. caesar and L. illustris become more common in sheep myiasis in more northerly Palaearctic regions.

Intensive lymphocytic infiltration of the underlying dermis occurs during cutaneous myiasis caused by larvae of the blow fly, Lucilia cuprina. To determine the effect of this infiltrate on larval growth, monoclonal antibodies (mAb) to CD4, CD8 or WC1 lymphocyte subset determinants were injected intravenously before and during experimental infection of sheep with larvae. The effect of intravenous injection of mAb to ovine interferon (IFN) gamma was also examined. The experiments were performed in 18-month-old maiden ewes with genetic resistance or susceptibility to the disease complex, bacterial dermatitis/cutaneous myiasis. mAbs induced profound depletion of CD8+ and WC1+ subpopulations from blood and skin at sites of larval growth. mAb to CD4+ gave only a moderate reduction in lymphocytes from blood or skin. mAb treatments did not modify larval growth or survival at 20 or 50 h after infection. Larval growth rates did not differ between resistant and susceptible genotypes. No evidence was found for a role of T lymphocyte subpopulations or the cytokine IFN, in modifying larval growth during the first 50 h of infection. It seems unlikely that T lymphocyte-dependent immunological effector mechanisms contribute to the lower prevalence of fly strike seen in the resistant genotype in the field.

Investigation of restriction fragment length polymorphisms (RFLPs) associated with immunoglobulin E (IgE) and cytokine genes in the sheep genome revealed polymorphisms in the IgE constant heavy chain, interferon gamma and interleukin 4 genes. No polymorphisms were found in interleukin 1 beta or tumour necrosis factor alpha. PstI and BamHI RFLPs in the IgE gene showed differences in frequency between animals selected for resistance or susceptibility to fleece rot and blowfly strike.

A new monoclonal raised against sheep IgE was used to examine sera and wound exudates from sheep which had been struck by Lucilia cuprina in the field. The antibody was also used to detect the presence of IgE in sera and skin sections from sheep which had been artificially infected with fly larvae 3 times. Neither total, nor L. cuprina specific circulating IgE could be detected in serum or wound exudates from struck sheep. Cell bound IgE was, however, identified by the monoclonal in skin sections from struck sheep and from a control sheep which had not been struck. No difference in the number of IgE positive cells was observed between the control and 2 of the 3 artificially infected sheep, and none of the latter showed an increase in IgE positive cells even after 3 infections. One sheep showed twice as many IgE positive cells as the other treated sheep and the third larval infection was difficult to establish and limited in size and severity. This suggests a relationship between innate resistance to strike and the number of IgE positive cells present in skin.

Sheep were immunised with ovalbumin and then infected with the sheep blowfly, Lucilia cuprina in order to study immunoglobulin and specific antibody degradation at the wound site. Serum and wound exudates were collected over the infection period and the dry weight and protein content of the exudates were determined. Exudates were analysed by SDS-PAGE and immunoblotting for IgG degradation. Levels of IgG and specific anti-ovalbumin antibodies in the exudates were measured by ELISA. The total weight of exudates increased over the whole period of the infection, while protein content increased in the first 24 h and then remained relatively constant. Immunoglobulin was present 6 h after infection and levels increased with protein content. However, the levels of IgG measured were quite different depending on the secondary antibody used in the ELISA. A monoclonal antibody measured mainly intact IgG while a polyclonal anti-IgG measured intact and degraded IgG. This allowed an estimation that approximately 60% of the IgG in exudates was degraded from 6 h after infection. Assays in vitro showed that L. cuprina larval enzymes degraded sheep antibody. However, measurement of specific anti-ovalbumin levels in exudates suggested that although high levels of antibody were degraded this did not necessarily decrease the level of antigen binding. As a result, IgG degradation may assist and not hinder vaccine development by allowing antibody fragments to penetrate the peritrophic membrane and access gut cell antigens.

Sheep were vaccinated with two purified serine proteases, LCT25a and LCT25b, isolated from the secretory and excretory material from first instar larvae of Lucilia cuprina. The immunization produced a strong antibody response to LCT25b and a weaker response to LCT25a as measured by ELISA. However, neither protease induced an ovine immune response which affected the development of first instar larvae growing on sera derived from these sheep. Further, direct in vivo bioassays of larval growth on the backs of vaccinated sheep also indicated a lack of induction of an immune response which prevented establishment of the larvae. Sera from unvaccinated sheep which had previous experience of blowfly strike, in general, strongly recognised the serine protease LCT25b. It was concluded from all of these results that serine proteases from the secretory and excretory material of L. cuprina are unlikely to be effective antigens in a vaccine designed to protect sheep from blowfly strike.

A culture system has been established to produce gram amounts of peritrophic membrane from larvae of the sheep blowfly, Lucilia cuprina. Peritrophic membrane obtained from this culture has been used to immunize sheep. The immunization produced an immune response which resulted in the average weight of larvae on immunized sheep being only 50% of that of larvae grown on control sheep (P < 0.05). Fractionation of the components of the peritrophic membrane followed by immunization trials showed that the protective antigen fraction comprised material that could only be solubilized by harsh agents such as 4 M-urea. Even after solubilization by 4 M-urea, the protective antigens were able to produce a protective immune response which reduced growth of larvae on immunized sheep to 55% of larvae grown on control sheep (P < 0.05). This immune response which reduced growth of the larvae did not cause gross morphological damage to the larvae.

The specific serum antibody responses of sheep exposed to 10 consecutive infections of L. cuprina have been analysed by enzyme-linked immuno-sorbent assay and immunoblotting using monoclonal antibodies specific for sheep immunoglobulin isotypes. Recognition of a number of larval excretory-secretory products by IgM antibodies appeared to be non-specific. IgG1 was the major antibody class stimulated by the infection protocol and marked increases in antibody to specific excretory-secretory antigens were observed. Three molecules of 35, 30 and 25 kDa were particularly recognized although the extent of recognition of these molecules varied considerably between individual sheep serum. A pooled serum composed of sera collected after five to seven infections significantly inhibited larval growth in in vitro cultures when compared to a sera pool consisting of sera collected both prior to infection and after infections 1 and 2. The degree of inhibition was greater when serum with high specific antibody titre was used.

Sheep repeatedly infected with L. cuprina at 2- but not 4-week intervals developed partial resistance to infection after five infections, as measured by larval recovery. However, resistance did not persist for more than three infections. Skin weal responses were measured after injection of larval products simultaneously with each infection. The only correlation between weal size and larval recoveries occurred at infection 1 and indicated a relationship between skin sensitivity and innate rather than acquired resistance. The results suggest that resistance to L. cuprina can develop after repeated infections but that it is short lived and requires frequent larval exposure. A role for hypersensitivity responses was not confirmed by the weal responses but was suggested by the size of wound developed per larva recovered.

Research into vaccination against flystrike is aimed at either controlling the predisposing condition, fleece rot, or direct control of the fly maggots. A vaccine against the major bacterial species found in fleece rot lesions, Pseudomonas aeruginosa, is undergoing field trials and results suggest that this vaccine may reduce fleece rot incidence. Problems to be investigated include the existence of variants of P. aeruginosa in the field and the involvement of other species of bacteria in fleece rot. Strategies for direct vaccination include immunization with larval products involved in wound formation and larval nutrition and immunization against novel antigens usually from the gut of first instar larvae. Both methods have resulted in significant inhibition of larval growth. Analysis of larval products has revealed a number of active proteases which degrade skin proteins such as collagen. Inhibition of these enzymes with plasma enzyme inhibitors also affects larval growth in vitro. Antibodies raised against these enzymes are being tested for inhibitory effects against larvae and used to isolate cDNA clones from Lucilia cuprina libraries. Antigens from the gut are able to induce antibodies inhibitory to larval growth both in vitro and in vivo. Isolation of these antigens is proceeding in a number of laboratories. Problems still to be analysed include whether growth inhibition produces effective protection in the field and whether sufficient antibody will have early access to the larvae to significantly affect them.

The effect on subsequent larval survival of infesting sheep repeatedly with larvae of Lucilia cuprina was assayed in vivo and in vitro. One in vivo assay technique, in which implanted larvae were grown to third instar, indicated a significant reduction in larval survival; another in vivo technique, in which larvae were allowed to develop to second instar in small aluminium rings attached to the sheep, indicated no reduction in larval growth or survival. Larvae of Lucilia cuprina grown in vitro on media containing sera from previously infested sheep were significantly retarded in growth after 20 h compared with controls; no difference was detected when larvae were allowed to develop to pupation on two changes of the same media. No significant differences in survival of larvae either to 20 h or to pupation were obtained between the two treatments. ELISA antibody levels against crude soluble larval material were significantly higher for sera from infested sheep than for control sera, and the regression of antibody level on mean larval weight obtained after 20 h growth in vitro was significant. The immunoglobulin fraction isolated from sera of infested sheep significantly retarded larval growth when incorporated with normal serum in growth media. These results are consistent with an effect of specific anti-larval antibody produced by sheep in response to infestation.