Paralogs vs. genotypes? Variability of Babesia canis assessed by 18S rDNA and two mitochondrial markers.

Canine babesiosis caused by Babesia canis sensu stricto became an emerging disease of dogs across Europe calling for attention also in countries where it was an only rare imported disease. An easy accessibility of molecular methods and the growing amount of sequencing data led to the description of intraspecific variability in 18S rDNA sequences designated as "genotypes". Using material from a homogenous cohort of dogs with microscopically confirmed canine babesiosis caused by B. canis, we evaluated Babesia intraspecific variability and amplification sensitivity of three different genes (18S rDNA, COI, Cytb) to assess their potential as diagnostic or phylogenetic markers. In raw sequencing data obtained, we observed at least 3 ambiguous positions in up to 86% of chromatograms within the ∼560 bp fragment of 18S rDNA suggesting the existence of several, not identical copies of this gene. Our COI haplotype analysis resulted in a star-like pattern indicating a recent origin of most haplotypes, but not supporting the existence of two dominant haplotypes. Similarly, the Cytb sequences obtained from samples with all variants of 18S rDNA were identical. We corroborate previous observations from three other European countries and bring the evidence of the existence of 18S rDNA paralogs in B. canis genome replacing currently used "genotype" theory.

Dermacentor reticulatus (Fabricius, 1794) and Babesia canis (Piana et Galli-Valerio, 1895) as the parasites of companion animals (dogs and cats) in the Wroclaw area, south-western Poland.

UNLABELLED: Tests performed in 2013 and 2014 revealed the occurrence of three tick species parasitizing pet cats and dogs in the Wroclaw Agglomeration. In total, 1,455 tick specimens were removed from 931 hosts (760 dogs and 171 cats) in 18 veterinary clinics. The dominant tick species was Ixodes ricinus (n=1272; 87.4%), followed by I. hexagonus (n=137; 9.4%) and Dermacentor reticulatus (n=46; 3.2%). Females were the most often collected development stage among I. ricinus and D. reticulatus, and nymphs among I. hexagonus. Additionally, D. reticulatus ticks (n=337) were then collected from vegetation in the Wroclaw area to detect Babesia canis; however, none was found positive. Only 9.0% of dog blood samples sent to VETLAB were positive for Babesia spp. Negative results for B. canis from ticks may result from the short period of the occurrence of D. reticulatus in the Wroclaw area and therefore the vectorpathogen cycle may not have been fully established at the time of the study. Nevertheless, D. reticulatus is expanding its range, and the size of its population in the Wroclaw Agglomeration is increasing. The presence of the pathogenic Babesia spp. combined with the occurrence of its main vector¸ D. reticulatus, suggests that the epizootiological situation in the area can change and may pose a new veterinary problem in the future. KEY WORDS: Dermacentor reticulatus, Babesia canis, pets, Wroclaw, Poland.

Proteomic evaluation of inflammatory proteins in rat spleen interstitial fluid and lymph during LPS-induced systemic inflammation reveals increased levels of ADAMST1.

The spleen is a part of the immune system and is involved in the response to a systemic inflammation induced by blood borne pathogens that may induce sepsis. Knowledge about the protein composition of the spleen microenvironment in a control situation and during systemic inflammation may contribute to our understanding of the pathophysiology of sepsis. To our knowledge, the proteome of the fluid phase of the spleen microenvironment has not previously been investigated. In order to access the proximal fluid surrounding the splenic cells, we collected postnodal efferent spleen lymph from rats by cannulation, and spleen interstitial fluid (IF) by centrifugation. The origin of the isolated spleen IF was assessed by the extracellular tracer (51)Cr-EDTA and the plasma tracer (125)I-HSA. Spleen lymph, IF, and plasma samples were collected during lipopolysaccharide (LPS) induced systemic inflammation and analyzed using a cytokine multiplex assay and, for the first time, using label-free mass spectrometry based proteomics. The concentrations of TNF-α, IL-1ß, IL-6, and IL-10 increased severalfold in all fluids after LPS exposure. In total, 281, 201, and 236 proteins were identified in lymph, IF, and plasma, respectively, and several of these were detected after LPS only. A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) was detected by proteomics (the pro- region) in lymph only after LPS. ADAMTS1 was assessed by ELISA (the metalloproteinase domain), and the concentration was significantly higher in IF and lymph than in plasma in a control situation, showing local production in the spleen. A dramatic increase in ADAMTS1 was detected in lymph, IF, and plasma after LPS exposure. In conclusion, the procedures we used to isolate IF and lymph from the spleen during LPS enabled detection of locally produced proteins. Furthermore, we have demonstrated that the inflammatory proteome is different in the spleen microenvironment when compared to that in plasma.