Development of quantitative PCR and digital PCR for the quantification of Leishmania infantum in dogs.

Leishmaniasis is a zoonotic disease with worldwide distribution. In the Americas, the causative agent of the visceral form is the protozoa Leishmania (Leishmania) infantum. Transmission to the host or vertebrate reservoir occurs through the bite of infected arthropod females like Lutzomyia longipalpis. The epidemiological connection between the infection in dogs and humans generate constant studies about the relationship between the parasite and the canine host, including the development of methods and tests for the detection and quantification ofLeishmania (L.) infantum. Both conventional PCR (cPCR) and quantitative PCR (qPCR) can be used in the diagnosis of the parasite. Dropet Digital PCR (ddPCR) is another useful tool. Knowing the parasite load and its relationship with the clinical signs of naturally infected dogs is useful in research development and for establishing treatments that reduce the transmission of the disease. In this study, thirty-nine clinical samples of spleen from dogs naturaly infected by L. infantum were collected after necropsy. Two molecular tools were used to quantify the parasite load (qPCR and ddPCR) and there was 100% agreement in the results of the them. The tools developed in this work are important for the detection of L. infantum in dogs and humans. Droplet Digital PCR does not require a standard curve and is easy to standardize. In such manner, this new tool can generate more in-depth information in the broad debate about parasitic loads and the pathogenesis of leishmaniasis.

A1 noradrenergic neurons lesions reduce natriuresis and hypertensive responses to hypernatremia in rats.

Noradrenergic neurons in the caudal ventrolateral medulla (CVLM; A1 group) contribute to cardiovascular regulation. The present study assessed whether specific lesions in the A1 group altered the cardiovascular responses that were evoked by hypertonic saline (HS) infusion in non-anesthetized rats. Male Wistar rats (280-340 g) received nanoinjections of antidopamine-ß-hydroxylase-saporin (A1 lesion, 0.105 ng.nL(-1)) or free saporin (sham, 0.021 ng.nL(-1)) into their CVLMs. Two weeks later, the rats were anesthetized (2% halothane in O2) and their femoral artery and vein were catheterized and led to exit subcutaneously between the scapulae. On the following day, the animals were submitted to HS infusion (3 M NaCl, 1.8 ml • kg(-1), b.wt., for longer than 1 min). In the sham-group (n = 8), HS induced a sustained pressor response (ΔMAP: 35±3.6 and 11±1.8 mmHg, for 10 and 90 min after HS infusion, respectively; P<0.05 vs. baseline). Ten min after HS infusion, the pressor responses of the anti-DßH-saporin-treated rats (n = 11)were significantly smaller(ΔMAP: 18±1.4 mmHg; P<0.05 vs. baseline and vs. sham group), and at 90 min, their blood pressures reached baseline values (2±1.6 mmHg). Compared to the sham group, the natriuresis that was induced by HS was reduced in the lesioned group 60 min after the challenge (196±5.5 mM vs. 262±7.6 mM, respectively; P<0.05). In addition, A1-lesioned rats excreted only 47% of their sodium 90 min after HS infusion, while sham animals excreted 80% of their sodium. Immunohistochemical analysis confirmed a substantial destruction of the A1 cell group in the CVLM of rats that had been nanoinjected withanti-DßH-saporin. These results suggest that medullary noradrenergic A1 neurons are involved in the excitatory neural pathway that regulates hypertensive and natriuretic responses to acute changes in the composition of body fluid.