The hapten of deltamethrin: cyano (4-phenoxyphenyl) methyl 3-(2,2-dibromovinyl)-2,2- dimethylcycloprpoane carboxylate (Sigma-Aldrich, USA) was conjugated to BSA (Sigma-Aldrich, USA) according to the method described by Lee et al. (2002). Specifically, 0.05mmol hapten was dissolved in 4ml of ethanol and treated with 0.6ml of 1.0N hydrochloric acid. The resulting solution was stirred in an ice bath while 0.4ml of 0.2M sodium nitrite was added. Subsequently, 0.4ml of dimethylformammide (DMF) was added drop-wise to the solution to ensure thorough mixing.

In a separate step, 45mg of BSA was dissolved in 5.0ml mixture of 0.2M borate buffer (76.2g ml^-1 borax and 12.37g L^-1 at pH 8.8) and 1.2ml DMF. The activated hapten solution was added drop-wise to the stirred BSA solution. The resulting mixture was stirred in an ice bath for 45 minutes and then dialyzed against phosphate buffered saline (PBS) for 72 hours at 4°C. The purified conjugates were suspended in sterile distilled water and stored in aliquots at -20°C. The concentration of hapten-BSA was 2.8µg ml^-1 after measurement by UV-absorbance at 280nm. The product of conjugation contained large amounts of precipitate. The precipitate was included in the vaccination mixture to elicit an immune response in lactating sheep.

Two Merino adult non-pregnant lactating ewes of approximately 2 years old (Animal Care Unit, Australia) were used for the immunization experiment. They were numbered and housed in a communal area with their lambs at Large Animal Facility at University of Western Australia. Lambs were kept with their mothers for the welfare of animals and to maintain milk flow and production. They were provided with feed and water ad libitum and were maintained on a 12 hour light cycle.

Thirty nine adult female Balb/c mice, with approximate 30g body weight were used for the study (Animal Resources Centre, Australia). They were separated into five cages for the poisoning experiment. The mice were housed at Preclinical Animal Facility at University of Western Australia. They had free access to feed and water over the course of these experiments and were maintained on a 12 hour light cycle. All experimental procedures were performed after an initial one week adaptation period, using methods that were approved by Animal Ethics Committee at University of Western Australia (AEC permit number RA/3/100/811).

The inoculation formula used for sheep was 0.5ml of deltamethrin hapten-BSA conjugates per ml PBS (pH 7.0-8.0) emulsified in 0.5ml CFA. The booster formula was 0.5ml of deltamethrin hapten-BSA conjugates per ml PBS (pH 7.0-8.0) emulsified in 0.5ml incomplete Freund’s adjuvant (IFA).

The two ewes (No. 1 and 2) received an intramuscular injection in the left hind quadriceps with the inoculation formula at Day zero. Then, they received two boosters on Day 15 and 29 of the immunization period.

Milk samples (50ml each) were taken on Day 0, 8, 22 and 36. The samples were collected by manual milking. After collection, the samples were centrifuged at 10,000 for 10 minutes and stored in aliquots at -20°C. Samples from Day 0 were collected before the inoculation and used as the control for later milk samples.

Indirect Enzyme-Linked Immunosorbent Assay (ELISA) was used to measure the levels of anti-deltamethrin antibodies in the milk samples collected. A 96-well microtiter plate (Bio-Rad, NSW) was coated overnight at 4°C with 100µl/well of 20µg of deltamethrin hapten-BSA conjugate per ml of 0.1M carbonate coating buffer (0.03g Na₂CO₃ and 6.0g NaHCO₃ in 1,000ml distilled water at pH 9.6). The plate was washed twice with washing solution (0.05% Tween 20 in 10% phosphate buffered saline). The remaining protein-binding sites in the coated wells were blocked by adding 200µl carbonate blocking buffer (5.3g Na₂CO₃, 4.2g NaHCO₃ and bovine serum albumin in 1000ml distilled water at pH 9.6) then incubated overnight at 4°C prior to the assay. Next, the wells were washed twice to remove the blocking agent.

Serial dilutions of sheep’s immunoglobulin (BioCore, NSW) were prepared in the blocking buffer at the following concentrations: 0.1, 0.2, 0.3, 0.4 and 0.5µg/ml. Samples at dilutions 1:10, 1:100 and 1:1000 were added to wells and incubated 2 hours at room temperature. After incubation, the plates were washed and anti-sheep-peroxidase antibodies produced in donkey (Sigma-Aldrich, USA) were added. These antibodies were diluted to 1:5000 in blocking buffer to decrease non-specific binding. The plate was incubated for 1 hour at room temperature. To develop colour, 3,3’,5,5’-tetramethylbenzidine (Sigma, USA) was added to wells and incubated for 30 minutes in a dark place at room temperature. Finally, the reaction was stopped by adding 100µl stop solution (3.0M HCl) to each well. The absorbance readings were measured at 450nm using Plate Reader 200/2.0 (Bio-Rad, NSW). The concentration of anti-deltamethrin polyclonal antibodies was calculated by extrapolating the absorbance values using a constructed standard curve.

Thirty nine adult female Balb/c mice were randomly separated into five groups (two study, one control and two placebo). Each mouse had a unique number assigned. The two study and control groups were given a dose of deltamethrin LD₅₀ (20mg of deltamethrin per kg of body weight) ^{6, 7}. Group 1 received 0.2ml of deltamethrin LD₅₀ followed by 0.2ml of sheep’s milk with pAb. Group 2 received 0.1ml of deltamethrin LD₅₀ followed by 0.2ml of sheep’s milk with pAb. Group 3 received 0.1ml of deltamethrin LD₅₀ followed by 0.2ml of sheep’s milk without pAb as a control for the study. Further details are on Table 1. Special precautions were adopted during handling chemicals, especially, deltamethrin. Handling was according to precautions and conditions specified on Material Safety Data Sheet (Sigma-Aldrich, USA).

Figure 1

Table 1 The design of testing milk immunotherapy as a treatment of deltamethrin overdose. The number, type and description of the five groups of Balb/c mice used for all experiments

Assessment of toxicity was based on continuous visual observation^{7, 10, 22, 24}of the mice through all experiments. The mice were observed for an early toxicity sign displayed after the oral dose of deltamethrin LD₅₀. The early sign was somnolence which is a general indicator of depressed activity. The control group was designed to investigate whether the therapeutic effect was due to pAb or due to other milk components. To examine whether milk with or without pAb had any negative effect on the mice; two placebo groups were given saline orally instead of deltamethrin followed by milk with pAb for Group 4 and without pAb for Group 5.

Normal behavior of mice included general grooming, normal appetite, normal standing position, rounded abdominal lateral sides, inquisitive activity and widely open shiny eyes. The early symptoms of deltamethrin poisoning were somnolence (general depressed behavior), flat posture and eye squinting, while the later symptoms were twitching, tremors and convulsions (at a very advanced stage). Each mouse was given milk once she displayed somnolence. This was to avoid the severe pain of convulsions and to give the suggested therapy enough time to act.

The design of all experiments was based on previous studies of acute toxicity due to deltamethrin^{7, 10, 22, 24}. The test subjects became distinctly depressed and generally, there was less social activity among the animals. The mice retreat to a corner of their pen and show signs of distress. In addition, somnolence was the target stage of toxicity; an early sign of deltamethrin poisoning. The experiments were conducted by a veterinarian, Dr Shreen Nusair. The experiments were monitored by Dr Veronica Anderson, the resident veterinarian at the University of Western Australia and a member of the Animal Ethics Committee at UWA. The two independent observations made were filmed for validation.

The time each mouse succumbed to poisoning was recorded as well as the recovery time after receiving the treatment regime. The statistical package SPSS (SPSS Inc, USA) was used to analyze the data and to compare the study groups with each other and with the control. Mean ± Standard Deviation (M ± SD) were calculated. For all of the statistical comparisons, the value p < 0.05 was considered significant. All statistical analysis was conducted at the Statistics Clinic at University of Western Australia.

The physiochemical characters of the milk were yellowish white in color with pH of 6.8 and a freezing point of -0.56^oC. The composition of the milk was not measured in this specific study. The average fat content in the milk of Merino sheep is 6.1% (w/v); the average protein content is 6.5% (w/v) and the lactose content is 4.09% (w/v) ⁴.

The relative amount of anti-deltamethrin pAb in milk was measured following the primary (Day 0) and the two booster immunizations (Day 15 and 29). The milk samples collected at Day zero (before immunization) were used as controls (i.e. no pAb) for later samples (i.e. with pAb). Figure 2 shows that following primary immunization there was a 2.3-fold increase in anti-deltamethrin pAb in the milk of sheep No. 2 (0.43 µg/ml relative to 0.19 µg/ml) at 1:1000 dilution and a 4.3-fold increase in the milk of sheep No. 1 (0.39 µg/ml relative to 0.09 µg/ml) at the same dilution.

Figure 2

Figure 1 The concentration of anti-deltamethrin polyclonal antibodies (pAb) in the milk of sheep No. 1 and 2 measured by ELISA at a 1:1000 dilution during the immunization experiment. The measurements were taken at Day 0, 8, 22 and 36

The mice were visually monitored on continuous basis through all the experiments. The time for two events were recorded (Table 2), time elapsed before the mice responded to deltamethrin toxicity and time elapsed before the mice responded to immunotherapy (milk with pAb) and milk therapy (milk without pAb). The first elapsed time was recorded for each normal mouse when it displayed somnolence (general depressed activity). The second elapsed time was recorded for each poisoned mouse when it started to show recovery symptoms and return to normal behavior. An exception was in the control (Group 3); the mice displayed advanced symptoms after milk therapy without pAb. These symptoms were twitching and tremors. These animals failed to show normal behavior despite treatment with standard milk samples (i.e. without specific anti-deltamethrin antibodies).

The average time for Group 1 to respond to delatmethrin LD₅₀ dose and to display toxicity symptoms was 6.06 minutes, while that for Group 2 was 14.68 minutes. Group 2 and control took almost double the average time needed by Group 1 to respond to the oral dose of deltamethrin and to show somnolence. Group 1 took the shortest time for a group to develope toxicity symptoms in this study. Group 1 was compared to Group 2 to show the dose effect of deltamethrin toxicity and its role in the effeciency of the suggested immunotherapy.

Figure 3

Table 2 The time elapsed to respond to toxicity and the time elapsed to respond to immunotherapy was recorded for every mouse in Group 1 and 2. Then, results were compared to those of animals that received milk alone in Group 3.

Table 2 shows that the average time for Group 1 to respond to immunotherapy was 61.14 minutes, while that for Group 2 was 19.55 minutes; almost one third of Group 1. The control (Group 3) had the longest average time to respond to milk therapy (if at all); it was more than 93.57 minutes. The control mice displayed advanced symptoms of toxicity, twitching and tremors.

Table 3 shows the total response time during all experiments for the study, control and placebo groups. The average response time was the sum of the average time to develop toxicity symptoms and return to normal behavior. The average total elapsed time for Group 1 was 67.20 minutes. This was almost double that for Group 2 which was 34.23 minutes. The longest time was for the control group (>109.31 minutes). This group failed to show normal behavior when the experiment was terminated. The animals developed twitching and tremors instead. To test if the displayed symptoms were due to deltamethrin or milk itself, Group 4 and 5 were given saline as a placebo. Then, Group 4 was given milk with pAb and Group 5 was given milk without pAb. Only Group 4 displayed signs of discomfort due to milk ingestion with pAb. Data for Group 5 was not shown. The signs were flat back, inward sucktion of their abdomenal lateral side walls and seized inquzitive behavior for less than five minutes (Table 3).

For further illustration, Figure 2 illustrates the responce time to toxicity and the total elapsed time of the experiments for the study groups compared to control. In general, the Mean ± Standard Deviation (M ± SD) for the three groups was remarkably diffrent when compared to each other and the p-value was significant (p < 0.05) by a two way ANOVA.

Figure 4

Figure 2 The response time to toxicity and the total elapsed time for Group 1, 2 and Control (Group 3).

Figure 5

Table 3 The average time to develop symptoms compared to the average time to return to normal behavior. The average response time for the study, control and placebo groups following poisoning by deltamethrin then treatment by milk dose with or without antibodies.