The development of an “in vivo assay technique” as a tool for measuring protective immune responses of vaccine against myiasis in sheep

An “in vivo assay technique” is urgently needed for measuring protective immune effects of a myiasis vaccine in sheep. Such a technique is being developed simultaneously with the development of a vaccine against myiasis caused by the screwworm fly Chrysomya bezziana under a collaborative project undertaken by Balitvet, ITB and CSIRO (Australia) and funded by ACIAR. Experiments were conducted in naive sheep. C. bezziana larvae were allowed to develop on abraded skin in aluminium rings which had been attached to the sheep by means of a glue (Aibon) on the day prior to infection. Rings were arranged on clipped areas close to the mid line of the sheep’s back, two rings on the right side and two rings on the left. Four trials were performed, involving studies on the effects of including wet sponges in the rings to maintain humidity (Trial 1); the effects of sponge and blended meat as counting and transferring media during infection (Trial 2); the effects of the repellants citronella, eucalyptus oil and neem extract in assisting the recovery of larvae (Trial 3); and the effects of the reducing the infective dose from 50 to 25 1st instar larvae/ring and using a fine brush for counting and transferring larvae instead of using a forceps as in the previous groups (Trial 4) on the larval recovery rates (LRR). The results indicated that the inclusion of wet sponges in the rings, the use of sponge and blended meat as counting and transferring media during infection, and the application of repellants all increased the LRR to some extent; however, variations among individual rings remained high. On the other hand, the reduction of infective dose of larvae from 50 to 25 1st instar larvae/ring and using a fine brush for counting and transferring larvae sharply increased the LRR while substantially decreasing the coefficient variations. Key words : Myiasis, Chrysomya bezziana, larval recovery rat

Orientation by gravid Australian sheep blowflies, Lucilia cuprina (Diptera: Calliphoridae), to fleece and synthetic chemical attractants in laboratory bioassays

Gravid females of Lucilia cuprina (Wiedeman) in a laboratory cage orientated positively to samples of fleece. Newly-wetted fleece was significantly more attractive than dry fleece, an effect resulting from the action of water on the fleece and not just addition of water vapour to the volatile fleece kairomones. Fleece contaminated with serous exudate, resulting from myiasis by L. cuprina, was much more attractive than wet, uncontaminated fleece from the same sheep. Kairomones from wetted fleece consistently augmented the attractive effects of 2-mercaptoethanol and indole in separate experiments, and of hydrogen sulphide (released from saturated aqueous sodium sulphide solution) in one trial out of three, but not overall. It is suggested that volatile fleece kairomones play a part in eliciting orientation to sheep by gravid L. cuprina. Fleece kairomones may augment the efficacy of kairomones released by putrefactive conditions in the fleece, which are known to predispose sheep to fly strike. They may also provide an input which helps to retain L. cuprina populations in the sheep's peridomestic precinct

Oviposition behaviour of Dacus tryoni: The effects of some sugars and salts

Gravid Queensland fruit flies (Dacus tryoni) are stimulated by the presence of β-D(-) fructose to lay significantly more eggs in an agar substrate. Fructose is only effective when accessible to the tarsal and/or labellar gustatory sensilla; it greatly increases oviposition through holes in an impenetrable membrane. Threshold for the fructose effect is 4 mM, maximal response being at 50 mM and above. Sucrose and glucose are not oviposition stimulants for D. tryoni. In the field situation D. tryoni probably uses fructose as a marker to locate breaks in the skin of ripe fruit, where insertion of the ovipositor is easier. The flies are deterred from ovipositing in fructose agar by the presence of molar calcium chloride, even when this is inaccessible to the tarsal and labellar gustatory sensilla. Molar sodium chloride is not inhibitory. Calcium ions apparently exert their inhibitory effect via gustatory sensilla located on the ovipositor

The origin of sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), attractants in media infested with larvae

Laboratory bioassays with gravid females of Lucilia cuprina (Wiedemann) were used to isolate the source(s) of olfactory attractants emanating from larvae-infested media. Adults were not attracted by odours from axenic (micro-organism-free) larvae, but volatiles from xenic larvae were highly attractive. The attractants proved to be kairomones not pheromones, as odours from other species of calliphorids and a sarcophagid species were also attractive. Axenic, proteinaceous media produced a low level of attractive volatiles, which was increased by the activities of axenic larvae growing on the media. A greater degree of attraction occurred to odours from xenic media, and this too was much increased by the actions of growing larvae. The order of attractiveness of such volatiles is therefore: xenic with larvae >> xenic without larvae > axenic with larvae > axenic without larvae. It is concluded that larvae-infested media owe their great attractiveness to the volatiles produced by the action of micro-organisms, not to specific larval volatiles. Larval activity accentuates the output of attractive volatiles from both xenic and axenic proteinaceous media, possibly due to the effects of digestive enzymes, pH changes, mechanical mixing, warming or aeration or a combination of some or all of these factors

Attractants for the gravid Queensland fruit fly Dacus tryoni

The vapours of certain pure chemicals, typical of ripe fruits, elicited characteristic components of ovipositional behaviour from gravid Dacus tryoni (Froggat) in an olfactometer: the flies walked and flew upwind to the source of the vapour and then probed with their ovipositors. A range of alcohols, acids, ketones and esters having 2-6 carbon atoms were effective (1 and 10% of iso-butyric acid, n-butyric acid, methyl butyrate, ethyl butyrate, 2-butanone, ethyl lactate and ethyl acetate; and 10% concentrations of ethanol and 2-propanone). The most effective were 4-6 carbon acids, esters and ketones. Behavioural threshold for n-butyric acid vapour at 26°C was obtained from a 5×10-3% dilution in paraffin oil; maximum fly response occurred at about 200 times this concentration. Low concentrations of the 15-carbon sesquiterpene, α-farnesene, were also very effective, despite its lower volatility. These results suggest that at least three different types of alfactory sensory neurones are involved in the identification of fruit attractants by gravid D. tryoni

The development of an “in vivo assay technique” as a tool for measuring protective immune responses of vaccine against myiasis in sheep

An “in vivo assay technique” is urgently needed for measuring protective immune effects of a myiasis vaccine in sheep. Such a technique is being developed simultaneously with the development of a vaccine against myiasis caused by the screwworm fly Chrysomya bezziana under a collaborative project undertaken by Balitvet, ITB and CSIRO (Australia) and funded by ACIAR. Experiments were conducted in naive sheep. C. bezziana larvae were allowed to develop on abraded skin in aluminium rings which had been attached to the sheep by means of a glue (Aibon) on the day prior to infection. Rings were arranged on clipped areas close to the mid line of the sheep’s back, two rings on the right side and two rings on the left. Four trials were performed, involving studies on the effects of including wet sponges in the rings to maintain humidity (Trial 1); the effects of sponge and blended meat as counting and transferring media during infection (Trial 2); the effects of the repellants citronella, eucalyptus oil and neem extract in assisting the recovery of larvae (Trial 3); and the effects of the reducing the infective dose from 50 to 25 1st instar larvae/ring and using a fine brush for counting and transferring larvae instead of using a forceps as in the previous groups (Trial 4) on the larval recovery rates (LRR). The results indicated that the inclusion of wet sponges in the rings, the use of sponge and blended meat as counting and transferring media during infection, and the application of repellants all increased the LRR to some extent; however, variations among individual rings remained high. On the other hand, the reduction of infective dose of larvae from 50 to 25 1st instar larvae/ring and using a fine brush for counting and transferring larvae sharply increased the LRR while substantially decreasing the coefficient variations

Development of myiasis vaccine: In vitro detection of immunoprotective responses of peritrophic membrane protein, first instar larva Ll supernatant and pellet antigen of fly Chrysomyia bezziana in sheep

Myiasis control by means of individual treatment of animals which are mainly rised extensively is time consumed and expensive. The alternative way to control this disease by vaccination is considered effective and economically accepted. However the expected vaccine is now still being developed under a collaborative project between CSIRO, Inter-University Centre on Biotechnology-ITB and Research Institute for Veterinary Science and funded by ACIAR. There are several antigens have been identified as vaccine candidates and an in vitro bioassay technique has been developed for assessing the immunoresponses of vaccine in sheep. Three antigens were used for vaccines in this study, these included protein peritrophic membrane (PM), soluble extract (SE) and pellet extract (PE) of 1st instar larvae of Chrysomya bezziana. Twenty four experimental sheep were divided into 4 groups of 6 animals, 3 groups of animals were injected with PM, SE and PE vaccines with the dose rate of 0.5 g PM/head, 0.8 g PE/head and 4.2 ml LE/head respectively, and the other one group was injected with 4 ml PBS/head as a control group. Vaccination with the same dose was repeated 4 weeks after the 1st vaccination as a booster, and 2 weeks after the booster the sheep were challenged with live larvae, 3 days after challenge animals were killed. Sera were collected at the day of vaccination, 4 weeks after vaccination, 2 weeks after booster, and 3 days after challenge. An in vitro bioassay technique was conducted by culturing 1st instar larvae on five media containing sera collected from each experimental animal. The effects of sera on cultivated larvae were assessed by means of larval weight and larval mortality rate. The results indicated that the growth rate and survival of cultivated larvae in media containing anti-PM sera were significantly lower (P<0.01) compared to the larvae cultivated on media with sera on the day of vaccination. The larval weight depression by anti- PM sera collected at 3 days after challenge was 65% of that larvae cultivated on media with sera collected on the day of vaccination. Anti-PM sera depressed the growth rate and survival of larvae significantly greater (P<0.05) than that of anti-PE or anti-LE sera. It is concluded that PM has the best immunoresponses and as the candidate of choice for myiasis vaccine

Development of myiasis vaccine: In vitro detection of immunoprotective responses of peritrophic membrane protein, first instar larva Ll supernatant and pellet antigen of fly Chrysomyia bezziana in sheep

Myiasis control by means of individual treatment of animals which are mainly rised extensively is time consumed and expensive. The alternative way to control this disease by vaccination is considered effective and economically accepted. However the expected vaccine is now still being developed under a collaborative project between CSIRO, Inter-University Centre on Biotechnology-ITB and Research Institute for Veterinary Science and funded by ACIAR. There are several antigens have been identified as vaccine candidates and an in vitro bioassay technique has been developed for assessing the immunoresponses of vaccine in sheep. Three antigens were used for vaccines in this study, these included protein peritrophic membrane (PM), soluble extract (SE) and pellet extract (PE) of 1st instar larvae of Chrysomya bezziana. Twenty four experimental sheep were divided into 4 groups of 6 animals, 3 groups of animals were injected with PM, SE and PE vaccines with the dose rate of 0.5 g PM/head, 0.8 g PE/head and 4.2 ml LE/head respectively, and the other one group was injected with 4 ml PBS/head as a control group. Vaccination with the same dose was repeated 4 weeks after the 1st vaccination as a booster, and 2 weeks after the booster the sheep were challenged with live larvae, 3 days after challenge animals were killed. Sera were collected at the day of vaccination, 4 weeks after vaccination, 2 weeks after booster, and 3 days after challenge. An in vitro bioassay technique was conducted by culturing 1st instar larvae on five media containing sera collected from each experimental animal. The effects of sera on cultivated larvae were assessed by means of larval weight and larval mortality rate. The results indicated that the growth rate and survival of cultivated larvae in media containing anti-PM sera were significantly lower (P0.01) compared to the larvae cultivated on media with sera on the day of vaccination. The larval weight depression by anti- PM sera collected at 3 days after challenge was 65% of that larvae cultivated on media with sera collected on the day of vaccination. Anti-PM sera depressed the growth rate and survival of larvae significantly greater (P0.05) than that of anti-PE or anti-LE sera. It is concluded that PM has the best immunoresponses and as the candidate of choice for myiasis vaccine.   Key words : In vitro bioassay, myiasis, immunoresponses, Chrysomya bezzian