Kinetics of antibody response in BALB/c and C57BL/6 mice bitten by Phlebotomus papatasi
Authors: Jitka Hostomska
Lucia Pohankova
Petr Volf
Lenka Zidkova
Michaela Vlkova
Jesus G Valenzuela
Jan Drahota
Iva Rohousova
Journal: PLoS Neglected Tropical Diseases (2012)
Discipline: IMMUNOLOGY
Keywords: Phlebotomine sand flies; Leishmania parasites; anti-saliva antibodies; BALB/c mice; C57BL/6 mice
Affiliations: Department of Parasitology, Faculty of Science, Charles University in Prague, Prague, Czech Republic. 

Abstract:  Background: Phlebotomine sand flies are blood-sucking insects transmitting Leishmania parasites. In bitten hosts, sand fly saliva elicits specific immune response and the humoral immunity was shown to reflect the intensity of sand fly exposure. Thus, anti-saliva antibodies were suggested as the potential risk marker of Leishmania transmission. In this study, we examined the long-term kinetics and persistence of anti-Phlebotomus papatasi saliva antibody response in BALB/c and C57BL/6 mice. We also tested the reactivity of mice sera with P. papatasi salivary antigens and with the recombinant proteins. Methodology/principal findings: Sera of BALB/c and C57BL/6 mice experimentally bitten by Phlebotomus papatasi were tested by ELISA for the presence of anti-saliva IgE, IgG and its subclasses. We detected a significant increase of specific IgG and IgG1 in both mice strains and IgG2b in BALB/c mice that positively correlated with the number of blood-fed P. papatasi females. Using western blot and mass spectrometry we identified the major P. papatasi antigens as Yellow-related proteins, D7-related proteins, antigen 5-related proteins and SP-15-like proteins. We therefore tested the reactivity of mice sera with four P. papatasi recombinant proteins coding for most of these potential antigens (PpSP44, PpSP42, PpSP30, and PpSP28). Each mouse serum reacted with at least one of the recombinant protein tested, although none of the recombinant proteins were recognized by all sera. Conclusions: Our data confirmed the concept of using anti-sand fly saliva antibodies as a marker of sand fly exposure in Phlebotomus papatasi-mice model. As screening of specific antibodies is limited by the availability of salivary gland homogenate, utilization of recombinant proteins in such studies would be beneficial. Our present work demonstrates the feasibility of this implementation. A combination of recombinant salivary proteins is recommended for evaluation of intensity of sand fly exposure in endemic areas and for estimation of risk of Leishmania transmission. 

Retracted: No

PubPeer comments:
 Figure 3. The negative control blot strips from the BALB c and C57Bl 6 mice appear to look very similar. ![file] This issue was reported to the journal in October 2015, to which the journal responded with the standard  Thank you, we will look into this reply. I sent a friendly reminder in November 2016, but the journal never replied to that email. Maybe the authors could look into this? , 
 There is an additional question I have about Figure 4 (not reported to the journal in 2015). Could the authors please check the figure? * Two panels in Figure 4A appear to look unexpectedly similar. Shown with green boxes. * One panel appears to show a repetitive background. Shown with dark blue boxes. ![file] , 
 There are differences that can be observed with higher contrast and magnification. Some of the markings on the edge of the strips are pencil marks to cut strips correctly.![file] Petr Volf , 
 #2 Two strips in Fig. 4 marked in green were cut from the same membrane (same antigen separated by SDS PAGE) but they were incubated with different sera. Strip with two blue boxes is incubated with negative serum. Repetitive background in its part might be due to reflection of light (probably the strip was not optimally flat when the picture was taken). Please note that the background is different in the rest of strip. Again, there are pencil marks to cut strips correctly. Petr Volf ,