Canine Antibodies against Salivary Recombinant Proteins of Phlebotomus perniciosus: A Longitudinal Study in an Endemic Focus of Canine Leishmaniasis

Abstract

BackgroundPhlebotomine sand flies are vectors of Leishmania parasites. During blood feeding, sand flies deposit into the host skin immunogenic salivary proteins which elicit specific antibody responses. These anti-saliva antibodies enable an estimate of the host exposure to sand flies and, in leishmaniasis endemic areas, also the risk for Leishmania infections. However, the use of whole salivary gland homogenates as antigen has several limitations, and therefore, recombinant salivary proteins have been tested to replace them in antibody detection assays. In this study, we have used for the first time sand fly salivary recombinant proteins in a longitudinal field study on dogs.Methodology/Principal FindingsSera from dogs naturally exposed to P. perniciosus bites over two consecutive transmission seasons in a site endemic for canine leishmaniasis (CanL) were tested at different time points by ELISA for the antibodies recognizing whole saliva, single salivary 43 kDa yellow-related recombinant protein (rSP03B), and a combination of two salivary recombinant proteins, 43 kDa yellow-related protein and 35.5 kDa apyrase (rSP01). Dogs were also tested for Leishmania infantum positivity by serology, culture, and PCR and the infection status was evaluated prospectively. We found a significant association between active CanL infection and the amount of anti-P. perniciosus saliva antibodies. Importantly, we detected a high correlation between IgG antibodies recognizing rSP03B protein and the whole salivary antigen. The kinetics of antibody response showed for both a whole saliva and rSP03B a similar pattern that was clearly related to the seasonal abundance of P. perniciosus.ConclusionsThese results suggest that P. perniciosus rSP03B protein is a valid alternative to whole saliva and could be used in large-scale serological studies. This novel method could be a practical and economically-sound tool to detect the host exposure to sand fly bites in CanL endemic areas.

Longitudinal monitoring of anti‐saliva antibodies as markers of repellent efficacy against Phlebotomus perniciosus and Phlebotomus papatasi in dogs

“…The same unexplained variability in dogs was also reported in Kostalova et al . (). Sandflies have been shown to have biting preferences for certain dogs (Bongiorno et al ., ).…”

“…Salivary glands were dissected from 4–6‐day‐old adult female P. perniciosus and P. papatasi sandflies from laboratory colonies kept in standard conditions (Volf & Volfova, ). Salivary glands were pooled in groups of 20, mixed with 20 µL of Tris‐NaCl buffer (20 m m Tris, 150 m m NaCl; pH 7.4) and submitted to three consecutive freeze–thaw cycles to obtain SGH that were then stored at − 20 °C until use (Kostalova et al ., ).…”

“…Positive control sera for P. perniciosus and NC sera for both sandfly species originated from the Italian study mentioned previously (Kostalova et al ., ). Positive control sera for P. papatasi were sourced from this study and were selected from among those with the highest ODs in a preliminary evaluation of 40 samples from the second season when ODs were expected to be highest.…”

“…Moreover, immunoglobulin G (IgG) and subclass Ig2 elevation decreased shortly after the sandfly challenge was removed, thus allowing the investigation of recent sandfly exposure. In addition, longitudinal studies in naturally exposed dogs showed that anti‐sandfly saliva antibodies follow a seasonal pattern that correlates with sandfly abundance (Kostalova et al ., ; Quinnell et al ., ).…”

Monitoring Leishmania infection and exposure to Phlebotomus perniciosus using minimal and non-invasive canine samples

“…Phlebotomus perniciosusspecific salivary recombinant proteins have been produced, to overcome difficulties in obtaining appropriate amounts of whole salivary gland homogenates (SGH), especially in large scale epidemiological studies [13]. The evaluation of different recombinant antigens of P. perniciosus revealed yellow-related protein (rSP03B) and/ or apyrase (rSP01) as the most promising candidates to replace SGH in the detection of P. perniciosus exposure in dogs [14][15][16] and other mammalian hosts [17].…”