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Ability of a proteinase inhibitor mixture to kill poultry red mite, Dermanyssus gallinae in an in vitro feeding system
Veterinary Parasitology 141 (2006) 380–385 www.elsevier.com/locate/vetpar
Short communication
Ability of a proteinase inhibitor mixture to kill poultry red mite, Dermanyssus gallinae in an in vitro feeding system R. McDevitt a,*, A.J. Nisbet b, J.F. Huntley b a
b
Avian Science Research Centre, SAC, West Mains Road, Scotland, Edinburgh EH9 3JG, UK Moredun Research Institute, Pentlands Science Park, Bush Loan, Scotland, Penicuik EH26 0PZ, UK Received 15 March 2006; received in revised form 28 April 2006; accepted 15 May 2006
Abstract The development of a reliable in vitro feeding system has enabled the rapid testing of presumptive anti-mite factors, a mixture of protease inhibitors (PI), by incorporating these into the blood feed. Mites were collected from a caged-hen facility and underwent a regime of starvation under varying conditions of temperature and darkness to determine the optimum conditions that encouraged mites to feed in the in vitro system. The efficacy of two anti-coagulants, heparin (HS) and acid citrate glucose (ACD), on mite feeding rates and mortality was evaluated. The ability of a mixture of PI to kill mites was also evaluated. The rate of feeding was greater in mites that were starved and cooled for between 7 and 30 days compared with mites that were not starved or cooled. The percentage of mites that fed when presented with HS treated blood (70%) was greater when compared with ACD treated blood (48%). The mortality of mites fed blood treated with HS + PI increased to 45% compared with a mortality level of 5% in mites fed on blood treated with HS alone. A reliable in vitro method for feeding D. gallinae which has the potential to be used to rapidly screen blood-borne products for their efficacy in reducing mite numbers has been developed. # 2006 Elsevier B.V. All rights reserved. Keywords: Dermanyssus gallinae; Poultry red mite; In vitro feeding; Proteinase inhibitors; Mite mortality
1. Introduction The poultry red mite, Dermanyssus gallinae, is an important ectoparasite of many poultry species, and it can cause significant health and welfare issues for laying hens (Chauve, 1998). Low-level infestations affect egg production by decreasing egg numbers and also increasing the number of eggs rejected on the grounds of their blood-stained appearance (Chauve, 1998).
* Corresponding author at: Avian Science Research Centre, SAC, Scotland, Ayr KA6 5HW, UK. Tel.: +44 1292 525113; fax: +44 1292 525098. E-mail address: [email protected] (R. McDevitt). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.05.013
Severe infestations can cause a serious reduction in bird performance (Pilarczyc et al., 2004) and can result in the development of anaemia (Kirkwood, 1967; Kilpinen et al., 2005; Fiddes et al., 2005), which can be fatal (Abrahamsson et al., 1998; Pilarczyc et al., 2004; Kilpinen et al., 2005). Surveys of the incidence of laying hen farms affected by poultry red mite vary from 60 to 70% in Scandinavia (Hoglund et al., 1995; Ruff, 1999) and 60% in the UK (Fiddes et al., 2005). Infestations by the poultry red mite are more prevalent in alternative poultry systems, such as barn or aviary systems (Hoglund et al., 1995) and in free range flocks (Fiddes et al., 2005), compared with conventional cage systems. In the EU in particular, there is an increased consumer-driven movement away from conventional production systems to
R. McDevitt et al. / Veterinary Parasitology 141 (2006) 380–385
extensive alternative systems, and therefore the control of poultry red mite will become increasingly important in the future (Chauve, 1998). The main methods of control of the poultry red mite are to use a combination of house sanitisation followed by spraying with a variety of acaricides and the use of desiccant dusts, such as diatomous earth or silica (Chauve, 1998; Fiddes et al., 2005). However, the control of poultry red mite is difficult to manage due to a combination of physical and chemical factors (Hoglund et al., 1995; Nordenfors and Hoglund, 2000). The laying hen is typically housed in cage or aviary type systems which contain a considerable amount of ‘‘furniture’’, such as perches, feeding troughs and nest boxes, which are often constructed from wood or metal with unsealed edges. Poultry red mites feed on hens during the night and retreat to these harbourages during the day from which they are difficult to dislodge. In addition, the mites can survive without a blood-meal for up to 9 months (Nordenfors et al., 1999) and can re-appear in a house that was thought to have been de-contaminated. The lifecycle of the mite can be completed in a week (Nordenfors et al., 1999; Nordenfors and Hoglund, 2000) especially in warm conditions, which means that the numbers of mites in a poultry house increases substantially in the summer (Nordenfors and Hoglund, 2000). Although there are a number of chemical pesticides available for mite control (Chauve, 1998) there are two aspects of chemical control that cause concern, namely resistance to pesticides and chemical residues in poultry products. There is evidence that the poultry red mite has developed heritable resistance to acaricides, such as pyrethroids, in a number of EU countries (Beugnet et al., 1997; Nordenfors et al., 2001; Fiddes et al., 2005). In addition, residues of acaricides, such as propoxur, in eggs from laying hens have been detected at more than 6 times the permitted levels in the EU (Hamscher et al., 2003). Residue levels are also greater in eggs from hens in conventional cage systems compared with eggs from alternative production systems (Hamscher and Nau, 2003; Hamscher et al., 2003). There is therefore, an increasing need to develop non-chemical methods of control for poultry red mite (Fiddes et al., 2005). The development of resistance to chemical treatments, coupled with a movement towards extensive systems that are more susceptible to mite infestation, means that alternative methods of protecting birds from poultry red mite infestation such as vaccination, are required. Vaccines based on antibodies that target intestinal surface antigens ingested in the blood feed have been shown to be effective in controlling the cattle tick, Boophilus microplus (Willadsen et al., 1995), and a
381
similar approach may be effective for the red mite. Intestinal proteinase activity is essential for adequate nutrient absorption in mites, therefore, the inhibition of these and other proteinases is likely to be lethal. The incorporation of enzyme inhibitors in the blood meal of the mites could provide an assessment of the efficacy and measure of the assay’s usefulness to investigate anti-mite antibodies directed at gut membrane antigens. Although populations of red mites in the laboratory have been successfully fed, this has generally relied on the use of live hens (Reynaud et al., 1997; Kilpinen, 2005) and in order to assist vaccine development, a reliable rapid in vitro method of feeding D. gallinae is required. An artificial feeding system for poultry mites was first developed by Kirkwood (1971), and more recently some success in feeding mites was reported by Bruneau et al. (1999) in a system for maintaining the life cycle of D. gallinae. The present paper describes a modification of the latter in vitro feeding system for maintaining D. gallinae in culture. A number of studies were conducted to ascertain; the optimum maintenance conditions for holding the mites prior to feeding, the most appropriate anticoagulant to use and the ability of a mixture of proteinase inhibitors in the blood to kill mites. 2. Materials and methods Mites were collected from a typical cage laying hen facility and placed in large colonies (n 500) in vented plastic tissue culture flasks. The mites were maintained initially at room temperature (20 2 8C) for between 7 and 21 days in the dark, and were then transferred to a refrigerator (LecTM, L955WS) and kept at a lower temperature (5 1 8C) for between 7 and 23 days. During the maintenance period the mites had no access to blood and were in a starved condition. Freshly collected mites (no pre-conditioning) and mites conditioned for between 7 and 30 days were used in the in vitro feeding chambers. Blood was collected from chickens at 42 days of age following electrical stunning and exsanguination. The blood was collected into tubes and admixed with one of two anticoagulants; 20 mM heparin (HS) or acid citrate glucose (ACD). The ACD was prepared according to the method described by Kirkwood (1971) where 2 g of disodium citrate and 3 g of glucose were dissolved in 120 ml of distilled water. Blood collection tubes were treated with either 250 ml of HS in a 5 ml volume or 5 ml of ACD in a 30 ml volume. The treated blood was used for feeding experiments on the day of collection. The feeding chambers were constructed from glass Pasteur pipettes with the dispensing end sealed closed
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Fig. 1. Diagram of the in vitro feeding mechanism.
with a blow-torch and the open end covered with a plastic blood reservoir which contained the treated blood (Fig. 1). The blood reservoir was constructed from an inverted pipette tip covered with a 1 cm2 strip of chicken skin, so that the external surface of the skin was exposed to the mites present in the chamber. The internal surface of the skin was in direct contact with the treated blood. The chicken skin was harvested from freshly killed day-old chicks, plucked and placed in a freezer ( 18 2 8C) for between 1 and 14 days before use. Mites were removed from the colonies and placed in the feeding chambers in groups of 10–20, along with a strip of Whatmann filter paper (10 mm 50 mm). Between 200 and 400 ml of freshly collected blood, treated with either HS or ACD, was placed in the reservoir and the chambers were placed in a temperature controlled incubator (Sanyo Gallenkamp) at temperatures between 30 and 40 8C, with a relative humidity of 65–75%. For each experiment, between six and eight chambers of each treatment were used and the experiments were repeated on three separate occasions. In order to facilitate enumeration, each chamber held between 10 and 20 adult mites. The feeding chambers
containing the mites were placed in the incubator at T0 of an experiment and were observed 4, 16, 24, 40 and 48 h later, using a stereomicroscope (SwiftTM, Stereo Eighty) at ten-fold magnification, for evidence of mite feeding and mortality. Mites were determined to have fed if they were engorged and/or had changed in colour from pale grey or brown to bright red. Mites were deemed to be dead if they were immobile and unresponsive to the stimulus of a long needle introduced into the feeding chamber. The optimum time for determining mite feeding rates was found to be 16 h after chambers were placed in the incubator. A mixture of water-soluble proteinase inhibitors (PI) was used to determine whether a blood borne substance could successfully kill mites in the in vitro feeding system. The mixture had broad specificity for the inhibition of serine, cysteine, aspartic, and metallo proteinases and contained; 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), E-64, bestatin, leupeptin, aprotinin, and sodium EDTA (Sigma–Aldrich, P2714). The proteinase inhibitor mixture, supplied as a lyophilized powder, was added to 100 ml of distilled water and divided into 1 ml aliquots. A single 1 ml aliquot of PI mixture was added to the standard volume of HS used to treat the blood meal. This volume contained proteinase inhibition capacity sufficient to inhibit 1 mg of pancreatin. Mites, which had been preconditioned by starvation for 7 days at 20 2 8C and 7 days at 5 1 8C were then allowed to feed on blood containing HS plus proteinase inhibitor treated (HS + PI) or HS-treated blood only. Mite feeding rates and mortality levels were determined, as described above, in 12 chambers for each treatment after 16 h access to the bloodmeal in darkness at 40 0.5 8C and 70% RH. Two repetitions of the experiment were performed. Data were analysed by one-way ANOVA or by general linear model (GLM) using Minitab (Release 13). The effect of anticoagulant type and conditioning of mites on feeding success was tested by GLM where anticoagulant type, maintenance conditions and the interaction between these two main effects were included in the model. The reproducibility of the in vitro feeding system was tested using a GLM where the effect on feeding success of the main effects of anticoagulant type, repeat experiment number and the interaction between these two main terms was determined. The comparison between the effects of HS and HS + PI treated blood on the feeding and mortality levels in mites were made using one-way ANOVA. Data are presented as mean S.E.M. unless otherwise stated.
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3. Results Several factors affected the success rate of the in vitro feeding system in getting mites to feed, including duration of starvation and the temperature mites were kept at during starvation. The number of mites that successfully fed in the in vitro system increased with the duration of the period of starvation (P 0.001), pooled for maintenance temperature. Approximately 25% of mites fed within 1 day of collection, i.e., freshly collected. This rose to 50% when the mites had been starved for 7 days at room temperature, and increased further to a successful feeding rate of 76% when mites had been starved and cooled for 30 days (Fig. 2). Mites that had been kept chilled (5 1 8C) were significantly (P 0.001) more likely to feed (65% compared with 24%), compared with mites that had been kept at room temperature (20 3 8C). The in vitro feeding model developed in the present study was successful in encouraging mites to feed on blood kept patent by either of two anticoagulants tested. Around 70% of mites presented with blood treated with HS fed compared with 48% of mites given ACD treated blood (P 0.001). There was a significant interaction between anticoagulant type and mite maintenance conditions, as a greater (P 0.05) percentage of mites that were presented with HS treated blood successfully fed when starved and cooled for 30 days compared with mites presented with ACD treated blood (Fig. 3). There was no effect of conditioning, i.e. starvation and cooling, on the feeding success rate of mites presented with ACD treated blood. The reproducibility of the in vitro feeding system was good with the percentage of mites feeding on HS treated blood ranging from 68 to 72% on three
Fig. 2. Percentage of mites that fed on HS treated blood in the in vitro feeding system, following starvation and cooling for varying periods. Where; mites starved for 24 h at room temperature (24-h), mites starved for 7 d at room temperature (within 7 d) and mites starved for 30 d at 5 1 8C (conditioned). Data are mean S.E.M.
Fig. 3. Percentage of mites that fed on heparin (HS) or acid citric glucose (ACD) treated blood in the in vitro feeding system, following starvation at 20 3 8C for 7 d (7 d) or starvation and cooling at 5 1 8C for 30 d (30 d). Data are mean S.E.M.
Fig. 4. The percentage of mites that fed on heparin (HS) or acid citric glucose (ACD) treated blood in the in vitro feeding system, on three consecutive experiments. Data are mean S.E.M.
consecutive experiments using mites conditioned for 30 days (Fig. 4). In contrast, the percentage of mites feeding on ACD treated blood in the same experiments ranged from 38 to 55%, though percentage feeding rate did not differ significantly within treatment (HS or ACD) between consecutive experiments.
Fig. 5. The percentage of mites that fed and that died following presentation of HS treated blood, with (HS + PI) or without (HS) the addition of a proteinase inhibitor Data are mean S.E.M.
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The addition of the proteinase mixture to HS treated blood (HS + PI) did not affect the percentage of mites observed to feed in the in vitro feeding system (Fig. 5). However, the addition of a proteinase inhibitor mix to HS treated blood resulted in a significant increase (P 0.01) in mite mortality from 5 to 45% (Fig. 5). When mite mortality was corrected for the number of mites that fed, the mortality of mites presented with HS + PI treated blood increased (P 0.05) from 13 to 64%. 4. Discussion Poultry red mites are very sensitive to temperature and humidity (Kirkwood, 1971; Zeman, 1988; Nordenfors et al., 1999). The success of the in vitro feeding system used in the present study depended on the environmental conditions in which the feeding system was housed and the type of preconditioning that the mites underwent prior to being placed in the feeding system. Early pilot studies (not presented) confirmed that a temperature of 40 8C and a humidity >70%, as well as being maintained in the dark, was required in order to encourage mites to feed freely on the blood meal provided (see also Kirkwood, 1971; Bruneau et al., 1999). Poultry red mites also feed best when a period of starvation and cooling is observed (Kirkwood, 1971; Bruneau et al., 1999; this study). Previous studies have found that using either ACD (Kirkwood, 1971) or HS (Bruneau et al., 1999) resulted in mite feeding levels of 70% and 45%, respectively. Although the in vitro feeding system used in the present study is a modified version of that developed by Bruneau et al. (1999), the feeding rates obtained in the present study were higher when HS was used as the anticoagulant in the blood meal and reproducibility was consistent, 70% of mites presented with HS treated blood feeding in each experiment. In addition to some minor physical differences in the in vitro feeding apparatus between studies, the subcutaneous fat on the chicken skin used in the present study was not removed nor was the skin washed with any solution. The presence of subcutaneous fat on the skin may have served to attract mites and encourage feeding (Zeman, 1988). Although there are a number of methods of control for poultry red mite, both chemical and physical, there is an increasing number of problems with these control methods. There is therefore a pressing need to develop alternative methods of control for poultry red mite, preferably non-chemical, so that the twin concerns of resistance (Beugnet et al., 1997; Chauve, 1998; Nordenfors et al., 2001) or residues occurring in poultry products (Hamscher and Nau, 2003; Hamscher
et al., 2003), cease to be a constraint. The development of an appropriate vaccine against poultry red mite would be a potential alternative to the use of chemical acaracides (Shyrock, 2004; Nisbet and Huntley, 2006) and a vaccine has already been successfully developed against another ectoparasite, the tropical cattle tick, Boophilus microplus (Willadsen et al., 1995). The primary purpose in refining the in vitro feeding system for poultry red mite in the present study was to develop a tool that would allow the screening of anti-mite substances, such as antibodies, contained in chicken blood for efficacy in killing mites. A mixture of proteinase inhibitors dissolved in the blood meal did not reduce the number of mites feeding on the blood, but increased considerably the mortality of the mites feeding on this blood meal. This finding supports the potential use of the in vitro feeding system as a tool for rapidly screening chicken anti-mite antibodies for efficacy in killing mites and therefore as potential vaccine candidates. In conclusion, we have developed a reliable in vitro feeding system for the poultry red mite and in so doing have detailed the conditions for the maintenance of mites in the laboratory that encourages optimum feeding rates. The inclusion of a mixture of proteinase inhibitors in the blood meal of the mites increased mite mortality nine-fold and supports the validity of the use of the model for screening potential blood borne antimite products, such as antibodies. The in vitro model can be used to rapidly screen potential anti-mite antibodies for use in vaccine development against poultry red mite. Acknowledgements The authors are grateful to Genomia for financial support. SAC and MRI receive funding from the Scottish Executive Environment and Rural Affairs Department. References Abrahamsson, P., Fossum, O., Tauson, R., 1998. Health of laying hens in an Aviary system over five batches of birds. Acta Vet. Scand. 39, 367–379. Beugnet, F., Chauvre, C., Gauthey, M., Beert, L., 1997. Resistance of the red poultry mite to pyrethroids in France. Vet. Rec. 140, 577– 579. Bruneau, A., Dernberg, A., Chauve, C., Zenner, L., 1999. First in vitro cycle of the chicken mite, Dermanyssus gallinae (DeGeer 1778), utilizing an artificial feeding device. Parasitology 123, 583–589. Chauve, C., 1998. The poultry red mite, Dermanyssus gallinae (De Greer, 1778): current situation and future prospects for control. Vet. Parasitol. 79, 239–245.
R. McDevitt et al. / Veterinary Parasitology 141 (2006) 380–385 Fiddes, M.D., Le Gresley, S., Parsons, D.G., Epe, C., Coles, G.C., Stafford, K.A., 2005. Prevalence of the poultry red mite (Dermanyssus gallinae) in England. Vet. Rec. 157, 233–235. Hamscher, G., Nau, H., 2003. Effects of animal housing facilities on residues and contaminants in food. Dtsch. Tierarztl. Wochenschr. 110, 316–319. Hamscher, G., Priess, B., Hartung, J., Nogossek, M.I., Glunder, G., Nau, H., 2003. Determination of propoxur residues in eggs by liquid chromatography-diode array detection after treatments of stocked housing facilities for the poultry red mite (Dermanyssus gallinae). Ana. Chim. Acta 2, 19–26. Hoglund, J., Nordenfors, H., Uggla, A., 1995. Prevalence of the poultry red mite, Dermanyssus gallinae, in different types of production systems for egg layers in Sweden. Poult. Sci. 74, 1793–1798. Kirkwood, A.C, 1967. Anaemia in poultry infested with the red mite Dermanyssus gallinae. Vet. Rec. 80, 514–516. Kirkwood, A.C., 1971. In vitro feeding of Dermanyssus gallinae. Exp. Parasitol. 29, 1–6. Kilpinen, O., 2005. How to obtain a bloodmeal without being eaten by a host: the case of the poultry red mite, Dermanyssus gallinae. Physiol. Entomol. 30, 232–240. Kilpinen, O., Roepstoeff, A., Permin, A., Norgaard-Nielsen, G., Lawson, L.G., Simonsen, H.B., 2005. Influence of Dermanyssus gallinae and Ascaridia galli infections on behaviour and health of laying hens (Gallus gallus domesticus). Brit. Poult. Sci 46, 26–34. Nisbet, A.J., Huntley, J.F., 2006. Progress and opportunities in the development of vaccines against mites, fleas and myiasis-causing flies of veterinary importance. Parasite Immunol. 28, 165–172.
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Nordenfors, H., Hoglund, J., 2000. Long term dynamics of Dermanyssus gallinae in relation to mite control measures in aviary systems for layers. Brit. Poult. Sci. 41, 533–540. Nordenfors, H., Hoglund, J., Uggla, A., 1999. Effects of temperature and humidity on oviposition, molting and longevity of Dermanyssus gallinae (Acari: Dermanyssidae). J. Med. Entomol. 36, 68– 72. Nordenfors, H., Hoglund, J., Tauson, R., Chirico, J., 2001. Effect of permetherin impregnated plastic strips on Dermanyssus gallinae in loose housing systems for laying hens. Vet. Parasitol. 102, 121– 131. Pilarczyc, B., Balicka-Ramisz, A., Ramisz, A., Pajak, B., 2004. Influence of Dermanyssus gallinae on health and production in layers. Med. Wet. 60, 874–876. Reynaud, M.C., Chauve, C., Beugnet, F., 1997. Dermanyssus gallinae (DeGeer 1778): reproduction experimentale du cycle et essai de traitement par la moxidectine et l’ivermectine. Rev. Med. Vet. 148, 433–438. Ruff, M.D., 1999. Important parasites in poultry production systems. Vet. Parasitol. 84, 337–347. Shyrock, T.R., 2004. The future of anti-infective products in animals health. Nat. Rev. Microbiol. 2, 425–430. Willadsen, P., Bird, P., Cobon, G.S., Hungerford, J., 1995. Commercialisation of a recombinant vaccine against Boophilus microplus. Parasitology 110, 43–50. Zeman, P., 1988. Surface skin lipids of birds – a proper host karimone and feeding inducer in the poultry Red Mite, Dermanyssus gallinae. Exp. Appl. Acarol. 5, 163–173.
Short communication
Ability of a proteinase inhibitor mixture to kill poultry red mite, Dermanyssus gallinae in an in vitro feeding system R. McDevitt a,*, A.J. Nisbet b, J.F. Huntley b a
b
Avian Science Research Centre, SAC, West Mains Road, Scotland, Edinburgh EH9 3JG, UK Moredun Research Institute, Pentlands Science Park, Bush Loan, Scotland, Penicuik EH26 0PZ, UK Received 15 March 2006; received in revised form 28 April 2006; accepted 15 May 2006
Abstract The development of a reliable in vitro feeding system has enabled the rapid testing of presumptive anti-mite factors, a mixture of protease inhibitors (PI), by incorporating these into the blood feed. Mites were collected from a caged-hen facility and underwent a regime of starvation under varying conditions of temperature and darkness to determine the optimum conditions that encouraged mites to feed in the in vitro system. The efficacy of two anti-coagulants, heparin (HS) and acid citrate glucose (ACD), on mite feeding rates and mortality was evaluated. The ability of a mixture of PI to kill mites was also evaluated. The rate of feeding was greater in mites that were starved and cooled for between 7 and 30 days compared with mites that were not starved or cooled. The percentage of mites that fed when presented with HS treated blood (70%) was greater when compared with ACD treated blood (48%). The mortality of mites fed blood treated with HS + PI increased to 45% compared with a mortality level of 5% in mites fed on blood treated with HS alone. A reliable in vitro method for feeding D. gallinae which has the potential to be used to rapidly screen blood-borne products for their efficacy in reducing mite numbers has been developed. # 2006 Elsevier B.V. All rights reserved. Keywords: Dermanyssus gallinae; Poultry red mite; In vitro feeding; Proteinase inhibitors; Mite mortality
1. Introduction The poultry red mite, Dermanyssus gallinae, is an important ectoparasite of many poultry species, and it can cause significant health and welfare issues for laying hens (Chauve, 1998). Low-level infestations affect egg production by decreasing egg numbers and also increasing the number of eggs rejected on the grounds of their blood-stained appearance (Chauve, 1998).
* Corresponding author at: Avian Science Research Centre, SAC, Scotland, Ayr KA6 5HW, UK. Tel.: +44 1292 525113; fax: +44 1292 525098. E-mail address: [email protected] (R. McDevitt). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.05.013
Severe infestations can cause a serious reduction in bird performance (Pilarczyc et al., 2004) and can result in the development of anaemia (Kirkwood, 1967; Kilpinen et al., 2005; Fiddes et al., 2005), which can be fatal (Abrahamsson et al., 1998; Pilarczyc et al., 2004; Kilpinen et al., 2005). Surveys of the incidence of laying hen farms affected by poultry red mite vary from 60 to 70% in Scandinavia (Hoglund et al., 1995; Ruff, 1999) and 60% in the UK (Fiddes et al., 2005). Infestations by the poultry red mite are more prevalent in alternative poultry systems, such as barn or aviary systems (Hoglund et al., 1995) and in free range flocks (Fiddes et al., 2005), compared with conventional cage systems. In the EU in particular, there is an increased consumer-driven movement away from conventional production systems to
R. McDevitt et al. / Veterinary Parasitology 141 (2006) 380–385
extensive alternative systems, and therefore the control of poultry red mite will become increasingly important in the future (Chauve, 1998). The main methods of control of the poultry red mite are to use a combination of house sanitisation followed by spraying with a variety of acaricides and the use of desiccant dusts, such as diatomous earth or silica (Chauve, 1998; Fiddes et al., 2005). However, the control of poultry red mite is difficult to manage due to a combination of physical and chemical factors (Hoglund et al., 1995; Nordenfors and Hoglund, 2000). The laying hen is typically housed in cage or aviary type systems which contain a considerable amount of ‘‘furniture’’, such as perches, feeding troughs and nest boxes, which are often constructed from wood or metal with unsealed edges. Poultry red mites feed on hens during the night and retreat to these harbourages during the day from which they are difficult to dislodge. In addition, the mites can survive without a blood-meal for up to 9 months (Nordenfors et al., 1999) and can re-appear in a house that was thought to have been de-contaminated. The lifecycle of the mite can be completed in a week (Nordenfors et al., 1999; Nordenfors and Hoglund, 2000) especially in warm conditions, which means that the numbers of mites in a poultry house increases substantially in the summer (Nordenfors and Hoglund, 2000). Although there are a number of chemical pesticides available for mite control (Chauve, 1998) there are two aspects of chemical control that cause concern, namely resistance to pesticides and chemical residues in poultry products. There is evidence that the poultry red mite has developed heritable resistance to acaricides, such as pyrethroids, in a number of EU countries (Beugnet et al., 1997; Nordenfors et al., 2001; Fiddes et al., 2005). In addition, residues of acaricides, such as propoxur, in eggs from laying hens have been detected at more than 6 times the permitted levels in the EU (Hamscher et al., 2003). Residue levels are also greater in eggs from hens in conventional cage systems compared with eggs from alternative production systems (Hamscher and Nau, 2003; Hamscher et al., 2003). There is therefore, an increasing need to develop non-chemical methods of control for poultry red mite (Fiddes et al., 2005). The development of resistance to chemical treatments, coupled with a movement towards extensive systems that are more susceptible to mite infestation, means that alternative methods of protecting birds from poultry red mite infestation such as vaccination, are required. Vaccines based on antibodies that target intestinal surface antigens ingested in the blood feed have been shown to be effective in controlling the cattle tick, Boophilus microplus (Willadsen et al., 1995), and a
381
similar approach may be effective for the red mite. Intestinal proteinase activity is essential for adequate nutrient absorption in mites, therefore, the inhibition of these and other proteinases is likely to be lethal. The incorporation of enzyme inhibitors in the blood meal of the mites could provide an assessment of the efficacy and measure of the assay’s usefulness to investigate anti-mite antibodies directed at gut membrane antigens. Although populations of red mites in the laboratory have been successfully fed, this has generally relied on the use of live hens (Reynaud et al., 1997; Kilpinen, 2005) and in order to assist vaccine development, a reliable rapid in vitro method of feeding D. gallinae is required. An artificial feeding system for poultry mites was first developed by Kirkwood (1971), and more recently some success in feeding mites was reported by Bruneau et al. (1999) in a system for maintaining the life cycle of D. gallinae. The present paper describes a modification of the latter in vitro feeding system for maintaining D. gallinae in culture. A number of studies were conducted to ascertain; the optimum maintenance conditions for holding the mites prior to feeding, the most appropriate anticoagulant to use and the ability of a mixture of proteinase inhibitors in the blood to kill mites. 2. Materials and methods Mites were collected from a typical cage laying hen facility and placed in large colonies (n 500) in vented plastic tissue culture flasks. The mites were maintained initially at room temperature (20 2 8C) for between 7 and 21 days in the dark, and were then transferred to a refrigerator (LecTM, L955WS) and kept at a lower temperature (5 1 8C) for between 7 and 23 days. During the maintenance period the mites had no access to blood and were in a starved condition. Freshly collected mites (no pre-conditioning) and mites conditioned for between 7 and 30 days were used in the in vitro feeding chambers. Blood was collected from chickens at 42 days of age following electrical stunning and exsanguination. The blood was collected into tubes and admixed with one of two anticoagulants; 20 mM heparin (HS) or acid citrate glucose (ACD). The ACD was prepared according to the method described by Kirkwood (1971) where 2 g of disodium citrate and 3 g of glucose were dissolved in 120 ml of distilled water. Blood collection tubes were treated with either 250 ml of HS in a 5 ml volume or 5 ml of ACD in a 30 ml volume. The treated blood was used for feeding experiments on the day of collection. The feeding chambers were constructed from glass Pasteur pipettes with the dispensing end sealed closed
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Fig. 1. Diagram of the in vitro feeding mechanism.
with a blow-torch and the open end covered with a plastic blood reservoir which contained the treated blood (Fig. 1). The blood reservoir was constructed from an inverted pipette tip covered with a 1 cm2 strip of chicken skin, so that the external surface of the skin was exposed to the mites present in the chamber. The internal surface of the skin was in direct contact with the treated blood. The chicken skin was harvested from freshly killed day-old chicks, plucked and placed in a freezer ( 18 2 8C) for between 1 and 14 days before use. Mites were removed from the colonies and placed in the feeding chambers in groups of 10–20, along with a strip of Whatmann filter paper (10 mm 50 mm). Between 200 and 400 ml of freshly collected blood, treated with either HS or ACD, was placed in the reservoir and the chambers were placed in a temperature controlled incubator (Sanyo Gallenkamp) at temperatures between 30 and 40 8C, with a relative humidity of 65–75%. For each experiment, between six and eight chambers of each treatment were used and the experiments were repeated on three separate occasions. In order to facilitate enumeration, each chamber held between 10 and 20 adult mites. The feeding chambers
containing the mites were placed in the incubator at T0 of an experiment and were observed 4, 16, 24, 40 and 48 h later, using a stereomicroscope (SwiftTM, Stereo Eighty) at ten-fold magnification, for evidence of mite feeding and mortality. Mites were determined to have fed if they were engorged and/or had changed in colour from pale grey or brown to bright red. Mites were deemed to be dead if they were immobile and unresponsive to the stimulus of a long needle introduced into the feeding chamber. The optimum time for determining mite feeding rates was found to be 16 h after chambers were placed in the incubator. A mixture of water-soluble proteinase inhibitors (PI) was used to determine whether a blood borne substance could successfully kill mites in the in vitro feeding system. The mixture had broad specificity for the inhibition of serine, cysteine, aspartic, and metallo proteinases and contained; 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), E-64, bestatin, leupeptin, aprotinin, and sodium EDTA (Sigma–Aldrich, P2714). The proteinase inhibitor mixture, supplied as a lyophilized powder, was added to 100 ml of distilled water and divided into 1 ml aliquots. A single 1 ml aliquot of PI mixture was added to the standard volume of HS used to treat the blood meal. This volume contained proteinase inhibition capacity sufficient to inhibit 1 mg of pancreatin. Mites, which had been preconditioned by starvation for 7 days at 20 2 8C and 7 days at 5 1 8C were then allowed to feed on blood containing HS plus proteinase inhibitor treated (HS + PI) or HS-treated blood only. Mite feeding rates and mortality levels were determined, as described above, in 12 chambers for each treatment after 16 h access to the bloodmeal in darkness at 40 0.5 8C and 70% RH. Two repetitions of the experiment were performed. Data were analysed by one-way ANOVA or by general linear model (GLM) using Minitab (Release 13). The effect of anticoagulant type and conditioning of mites on feeding success was tested by GLM where anticoagulant type, maintenance conditions and the interaction between these two main effects were included in the model. The reproducibility of the in vitro feeding system was tested using a GLM where the effect on feeding success of the main effects of anticoagulant type, repeat experiment number and the interaction between these two main terms was determined. The comparison between the effects of HS and HS + PI treated blood on the feeding and mortality levels in mites were made using one-way ANOVA. Data are presented as mean S.E.M. unless otherwise stated.
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3. Results Several factors affected the success rate of the in vitro feeding system in getting mites to feed, including duration of starvation and the temperature mites were kept at during starvation. The number of mites that successfully fed in the in vitro system increased with the duration of the period of starvation (P 0.001), pooled for maintenance temperature. Approximately 25% of mites fed within 1 day of collection, i.e., freshly collected. This rose to 50% when the mites had been starved for 7 days at room temperature, and increased further to a successful feeding rate of 76% when mites had been starved and cooled for 30 days (Fig. 2). Mites that had been kept chilled (5 1 8C) were significantly (P 0.001) more likely to feed (65% compared with 24%), compared with mites that had been kept at room temperature (20 3 8C). The in vitro feeding model developed in the present study was successful in encouraging mites to feed on blood kept patent by either of two anticoagulants tested. Around 70% of mites presented with blood treated with HS fed compared with 48% of mites given ACD treated blood (P 0.001). There was a significant interaction between anticoagulant type and mite maintenance conditions, as a greater (P 0.05) percentage of mites that were presented with HS treated blood successfully fed when starved and cooled for 30 days compared with mites presented with ACD treated blood (Fig. 3). There was no effect of conditioning, i.e. starvation and cooling, on the feeding success rate of mites presented with ACD treated blood. The reproducibility of the in vitro feeding system was good with the percentage of mites feeding on HS treated blood ranging from 68 to 72% on three
Fig. 2. Percentage of mites that fed on HS treated blood in the in vitro feeding system, following starvation and cooling for varying periods. Where; mites starved for 24 h at room temperature (24-h), mites starved for 7 d at room temperature (within 7 d) and mites starved for 30 d at 5 1 8C (conditioned). Data are mean S.E.M.
Fig. 3. Percentage of mites that fed on heparin (HS) or acid citric glucose (ACD) treated blood in the in vitro feeding system, following starvation at 20 3 8C for 7 d (7 d) or starvation and cooling at 5 1 8C for 30 d (30 d). Data are mean S.E.M.
Fig. 4. The percentage of mites that fed on heparin (HS) or acid citric glucose (ACD) treated blood in the in vitro feeding system, on three consecutive experiments. Data are mean S.E.M.
consecutive experiments using mites conditioned for 30 days (Fig. 4). In contrast, the percentage of mites feeding on ACD treated blood in the same experiments ranged from 38 to 55%, though percentage feeding rate did not differ significantly within treatment (HS or ACD) between consecutive experiments.
Fig. 5. The percentage of mites that fed and that died following presentation of HS treated blood, with (HS + PI) or without (HS) the addition of a proteinase inhibitor Data are mean S.E.M.
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The addition of the proteinase mixture to HS treated blood (HS + PI) did not affect the percentage of mites observed to feed in the in vitro feeding system (Fig. 5). However, the addition of a proteinase inhibitor mix to HS treated blood resulted in a significant increase (P 0.01) in mite mortality from 5 to 45% (Fig. 5). When mite mortality was corrected for the number of mites that fed, the mortality of mites presented with HS + PI treated blood increased (P 0.05) from 13 to 64%. 4. Discussion Poultry red mites are very sensitive to temperature and humidity (Kirkwood, 1971; Zeman, 1988; Nordenfors et al., 1999). The success of the in vitro feeding system used in the present study depended on the environmental conditions in which the feeding system was housed and the type of preconditioning that the mites underwent prior to being placed in the feeding system. Early pilot studies (not presented) confirmed that a temperature of 40 8C and a humidity >70%, as well as being maintained in the dark, was required in order to encourage mites to feed freely on the blood meal provided (see also Kirkwood, 1971; Bruneau et al., 1999). Poultry red mites also feed best when a period of starvation and cooling is observed (Kirkwood, 1971; Bruneau et al., 1999; this study). Previous studies have found that using either ACD (Kirkwood, 1971) or HS (Bruneau et al., 1999) resulted in mite feeding levels of 70% and 45%, respectively. Although the in vitro feeding system used in the present study is a modified version of that developed by Bruneau et al. (1999), the feeding rates obtained in the present study were higher when HS was used as the anticoagulant in the blood meal and reproducibility was consistent, 70% of mites presented with HS treated blood feeding in each experiment. In addition to some minor physical differences in the in vitro feeding apparatus between studies, the subcutaneous fat on the chicken skin used in the present study was not removed nor was the skin washed with any solution. The presence of subcutaneous fat on the skin may have served to attract mites and encourage feeding (Zeman, 1988). Although there are a number of methods of control for poultry red mite, both chemical and physical, there is an increasing number of problems with these control methods. There is therefore a pressing need to develop alternative methods of control for poultry red mite, preferably non-chemical, so that the twin concerns of resistance (Beugnet et al., 1997; Chauve, 1998; Nordenfors et al., 2001) or residues occurring in poultry products (Hamscher and Nau, 2003; Hamscher
et al., 2003), cease to be a constraint. The development of an appropriate vaccine against poultry red mite would be a potential alternative to the use of chemical acaracides (Shyrock, 2004; Nisbet and Huntley, 2006) and a vaccine has already been successfully developed against another ectoparasite, the tropical cattle tick, Boophilus microplus (Willadsen et al., 1995). The primary purpose in refining the in vitro feeding system for poultry red mite in the present study was to develop a tool that would allow the screening of anti-mite substances, such as antibodies, contained in chicken blood for efficacy in killing mites. A mixture of proteinase inhibitors dissolved in the blood meal did not reduce the number of mites feeding on the blood, but increased considerably the mortality of the mites feeding on this blood meal. This finding supports the potential use of the in vitro feeding system as a tool for rapidly screening chicken anti-mite antibodies for efficacy in killing mites and therefore as potential vaccine candidates. In conclusion, we have developed a reliable in vitro feeding system for the poultry red mite and in so doing have detailed the conditions for the maintenance of mites in the laboratory that encourages optimum feeding rates. The inclusion of a mixture of proteinase inhibitors in the blood meal of the mites increased mite mortality nine-fold and supports the validity of the use of the model for screening potential blood borne antimite products, such as antibodies. The in vitro model can be used to rapidly screen potential anti-mite antibodies for use in vaccine development against poultry red mite. Acknowledgements The authors are grateful to Genomia for financial support. SAC and MRI receive funding from the Scottish Executive Environment and Rural Affairs Department. References Abrahamsson, P., Fossum, O., Tauson, R., 1998. Health of laying hens in an Aviary system over five batches of birds. Acta Vet. Scand. 39, 367–379. Beugnet, F., Chauvre, C., Gauthey, M., Beert, L., 1997. Resistance of the red poultry mite to pyrethroids in France. Vet. Rec. 140, 577– 579. Bruneau, A., Dernberg, A., Chauve, C., Zenner, L., 1999. First in vitro cycle of the chicken mite, Dermanyssus gallinae (DeGeer 1778), utilizing an artificial feeding device. Parasitology 123, 583–589. Chauve, C., 1998. The poultry red mite, Dermanyssus gallinae (De Greer, 1778): current situation and future prospects for control. Vet. Parasitol. 79, 239–245.
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