| dcterms.abstract | Wolbachia species are bacteria which are endosymbionts of arthropods, as well as of
the parasites from the Onchocercidae family. They belong to alpha proteobacteria and
they are closely related to Ehrlichia and Anaplasma species. Filarial-related
Wolbachia species are classified into C, D, and J supergroup, while the F supergroup
is common to filarial and arthropod-related Wolbachia species. In the filariae, these
species are found in the reproductive tract of females as well as in their hypodermis.
They are dominantly transmitted vertically, while the horizontal transfer mechanism
and direction are not entirely clear. It is believed that Wolbachia species are important
for the biosynthesis of heme, riboflavin and nucleotides, and thus affect the fertility
and vitality of the filariae. Due to this mutualistic relationship, antibiotics from the
group of tetracyclines are introduced into the therapeutic protocol for the treatment of
heartworm disease in dogs. Wolbachia species are mostly proven by PCR assays
based on different genes (wsp, 16S rRNA, ftsZ, groEL, CoxA, fbpA, and dnaA). The
aim of this study was to demonstrate the presence and to carry out the molecular
characterization of Wolbachia species in dogs positive to microfilariae and/or antigen
Dirofilaria immitis.
In this study in total 89 dogs, from the area of Novi Sad, Serbia were included. The
first group included 39 dogs which were brought to the veterinary clinics in order to
be examined to the infection caused by D. immitis while the second group included
50 dogs from dogs shelter. In all dogs, a modified Knot test was performed to
demonstrate microfilariae, a rapid immunoenzyme test (SNAP HTWM, IDEXX,
USA) to demonstrate the antigen of gravid adult D. immitis female, as well as
conventional PCR for 12S rDNA, 5.8S-ITS2-28S and COI gene to demonstrate the
presence and confirmation of the identity of the microfilariae. Afterwards, in the
microfilaria and/or antigen D. immitis positive dogs, a conventional PCR was
performed for wsp and 16S rRNA gene of Wolbachia species, whereas the PCR
products positive for wsp gene were sequenced.In 4 dogs which were brought to the veterinary clinics for examination of the infection
caused by D. immitis, the presence of D. immitis (3 dogs had co-infection with D.
repens), as well as the presence of wsp and 16S rRNA genes of Wolbachia species
were confirmed, while other 6 dogs from this group, in which the presence of D.
immitis was also confirmed, were positive only on the 16S rRNA gene of Wolbachia
species. An analysis of the wsp gene sequences pointed out the similarity of 95.62%
with Wolbachia endosymbiont of Paratrechina longicornis isolate 9 (BLAST access
number KU527461.1). In this group of dogs, the prevalence of Wolbachia species was
25.64%. In 6 dogs from dog shelter, which were positive to D. immitis, there was no
evidence of the presence of the Wolbachia species. Also, in the case of 3 dogs from
dog shelter where the presence of the microfilariae gene was confirmed, but without
the confirmed identity of D. immitis and D. repens, the presence of the genus
Wolbachia species was not demonstrated.
In the further course of the study, identity confirmation of microfilariae in dogs from
shelter should be done by 5,8S-ITS2-28S gene sequencing, and determination of the
affinity of the Wolbachia to supergroup by sequencing of 16S rRNA gene, as well as
phylogenetic analysis. | En |
