Abstract:
An improved method for the manufacture of a medicament, which method includes the use of tissues, larval forms or derivatives of insects that have been

Description:
FIELD OF THE INVENTION 
       [0001]    This invention relates to immunity generation. 
         [0002]    In British Patent Specification No. 2 368 016 there is described a method for the manufacture of a medicament, which method includes the use of tissues, larval forms or derivatives of insects that have been fed on a food containing pathogens. This method is hereinafter referred to as “a method as defined” and results in the expression of anti-microbial peptides which are hereinafter referred to as AMPs. 
         [0003]    It is an object of the present invention to provide improvements in the method as defined. 
       SUMMARY OF THE INVENTION 
       [0004]    According to a first aspect of the present invention there is provided a method as defined in which the expression of AMPs is modulated (maximized) by selectively altering the number or nature of the pathogens that are used. 
         [0005]    As applied to the feeding of larvae with bacteria, it has been found that different AMPs are stimulated maximally by different doses of bacteria. Thus, Diptericin expression is favoured by high doses, whereas Sapecin by low doses. 
         [0006]    The optimal bacterial/larval combination that has been found to date is  Lucilia serritica  stimulated by  Pseudomonas syringae , a  bacterium  which is not pathogenic to humans or other animals and is found in the environment. 
         [0007]    Thus, according to a second aspect of the present invention, there is provided a method as defined which includes the stimulation of  Lucilia serricata  by  Pseudomonas syringae.    
         [0008]    Exposure of 4-day-old larvae to this  bacterium  for twelve hours has been found to increase AMP expression up to 30-fold as compared to unstimulated larvae. The larvae can be stimulated with leave or dead bacteria without affecting larval feed consumption or final weight. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0009]      FIG. 1  shows the number of  Camphylobacter  in the caecum of birds fed diets supplemented with soya, unstimulated larvae (unstim) or stimulated larvae (stim). The bars indicate significant differences between groups, 
           [0010]      FIG. 2  shows the numbers of lactic acid bacteria in the caecum of birds fed diets supplemented with soya, unstimulated larvae (unstim) or stimulated larvae (stim). No significant differences were seen between groups, 
           [0011]      FIG. 3  shows the number of  enterobacteria  in the caecum of birds fed diets supplemented with soya, unstimulated larvae (unstim) or stimulated larvae (stim). No significant differences were seen between groups. 
           [0012]      FIG. 4  shows median levels of Gallicin 11 expression in the caecal tonsil in birds fed either control or unstimulated larval or stimulated larval diets. The asterisk indicates significant differences using Dunn&#39;s post hoc test. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0013]    Day-old Ross broiler chicks were housed in biosecure facilities and fed commercial chick crumb supplements with 10% of either soya meal, crushed unstimulated  Lucilia  larvae or crushed unstimulated  Lucilia  larvae. 
         [0014]    Insect larvae reared in a milk powder/sucrose/wheat germ/agar mixture for four days were removed from this medium and washed. They were then introduced into fresh growth medium without added bacteria (unstimulated) or containing approximately 5×10 7    Pseudomonas syringae  per gram (stimulated). After 24 hours on this medium, the larvae were removed, washed to remove all traces of growth medium and freeze-dried. Once dry, they were crushed using a pestle and mortar to obtain a coarse powder for addition to the chick crumb. 
         [0015]    At three days old, birds were infected orally with 10 5  cfu  Campylobacter jejuni  strain M1. 
         [0016]    At eight days old, birds were euthanased and the caeca removed and examined for  Campylobacter , lactic acid bacteria and  enterobacteria  by serial dilution and spread plating on mCCDA, MRS, and VRBG agars respectively. Differences in the level of bacteria found in the birds were analysed using a Kruskal-Wallis test with Dunn&#39;s post test with P&lt;0.05 being regarded as significant. 
         [0017]    The use of feed containing stimulated larvae resulted in a reduction of  campylobacter  in the caecum ( FIG. 1 ); no effect was seen on numbers of lactic bacteria or  enterobacteria  ( FIGS. 2 and 3 ). 
         [0018]    In terms of colonization, 13% of soya fed birds were negative for  campylobacter  in the caecum after eight days, 18% of birds fed unstimulated larvae and 41% of birds fed stimulated larvae. The feed containing stimulated larvae resulted in a significant reduction (P&lt;0.05) in the number of birds colonized with  campylobacter  in the caecum as compared to the soya-supplemented control. 
         [0019]    Larvae of  Lucilia serricata, Calliphora vacciniae  and  Musca domestica  were assessed for their expression of antimicrobial peptides (AMPs) following stimulation with three different species of bacteria. As bacterial simulation was found to increase AMP expression, experiments have been carried out to investigate the optimal parameters for stimulation of the larvae. 
         [0020]    The optimal bacterial/larval combination has been found to be  Lucilia serricata  stimulated by  Pseudomonas  syringe, a  bacterium  which is not pathogenic to animals or humans. Exposure of four-day old larvae to this  bacterium  for twelve hours has been found to increase AMP expression by up to thirty-fold over unstimulated larvae. 
         [0021]    Larvae can be stimulated with live or dead bacteria and this does not affect larval feed consumption or final weight. There is also evidence of an interaction between pathogenicity of the  bacterium , time and dose in affecting AMP expression. 
         [0022]    In vitro experiments have been carried out using powder prepared from  Lucilia serricata  larvae stimulated by  Pseudomonas syringae  testing its effectiveness as an antibacterial agent against  Campylobacter jejuni , with powder from unstimulated larvae used as a control. 
         [0023]    Over a three-hour time period,  Campylobacter  bacteria incubated with powder from unstimulated larvae were able to grow whereas the bacterial cultures exposed to 1% w/v powder from larvae stimulated with  Pseudomonas syringae  showed significant bacterial death (P=0.0015). 
         [0024]      Lucilia serricata  larvae challenged with  Pseudomonas syringae  were fed to broiler chicks. The chicks were then infected orally to test their immunity. 
         [0025]    Adult  Lucilia serricata  were allowed to lay eggs in a 1:1:1 mixture of wheat germ, yeast and milk powder that had been solidified with agar. This mixture containing eggs was split into portions and at hatch larvae were either challenged with  Pseudomonas syringae  or left unchallenged. After 4 days larvae were harvested, washed to remove feed and freeze dried to a moisture content of 15%. Larvae were then ground to a fine powder in a chilled pestle and mortar. 
         [0026]    Groups of 18 day-old Ross broiler chicks were housed in biosecure facilities and fed commercial chick crumb supplemented with 10% w/w of either soya meal (to balance protein levels with feed containing larvae), 10% w/w crushed unstimulated  Lucilia  larvae or 10% w/w crushed stimulated  Lucilia  larvae. 
         [0027]    At 3 days old birds were infected orally with 10 5  cfu  Campylobacter jejuni  strain M1. 
         [0028]    At 8 days old birds were euthanased and the caecal tonsil immediately placed into RNAlater. mRNA was extracted using a Qiagen kit and examined for Gallicin 11 expression by quantitative RT-PCR using the primers D11F cagaattgcagaaagccaca and D11R ttctacgtgtgcgtgtgtga with chicken beta actin as a housekeeping gene. Expression of Gallicin 11 was expressed relative to beta actin expression. 
         [0029]    Differences between groups of birds were examined for significance using a Kruskal-Wallis test with Dunn&#39;s post hoc test as data were not normally distributed. 
         [0030]    The results of the test are shown in  FIG. 4  of the accompanying drawings. Drawing A is a box and whiskers plot where each box represents the first to third quartile with the median as the line inside the box. The whiskers show the range of data. Drawing B is a histogram showing the median level of expression. 
         [0031]    Gallicin 11 was chosen as this has been shown to be expressed in the intestine by others (Hong at al. Poult. Sci. (2012) 1082). 
         [0032]    Diet had a significant effect on Gallicin 11 expression (P=0.0007). In birds fed a diet containing stimulated larvae relative levels of Gallicin 11 expression in the caecum were significantly higher than in birds fed a diet containing unstimulated larvae. This shows that feeding of stimulated larvae, which contain higher amounts of insect AMPs, stimulates host AMP expression. This would give an added stimulation of antibacterial activity in the intestine over and above the provision of fly AMPs in the feed itself.