Evaluation of functional activity of leukocytes in dogs naturally infected with protozoon Babesia canis

Babezioza pasa je vektorski-prenosivo oboljenje izazvano protozoama roda Babesia. U Srbiji, najčešći uzročnik babezioze pasa je Babesia canis. Bolest može proteći asimptomatski ili klinički manifestno kao nekomplikovana ili komplikovana babezioza. U zavisnosti od toga da li su zadovoljeni klinički i laboratorijski kriterijumi za sindrom sistemskog inflamatornog odgovora (systemic inflammatory response syndrome, SIRS), psi sa nekomplikovanom babeziozom mogu biti SIRS pozitivni ili negativni, pri čemu ne dolazi do poremećaja funkcije organa. Komplikovana babezioza podrazumeva prisustvo SIRS praćeno poremećajem funkcije organa. Intenzitet odgovora akutne faze na infekciju B. canis zavisi od različitih faktora poput imunskog odgovora psa, koinfekcije ili neinfektivnih komorbiditeta, broja infektivnih krpelja i količine sporozoita oslobođenih iz pljuvačne železde krpelja, kao i virulencije soja B. canis. Tokom odbrane od patogena, leukociti prepoznaju specifične domene na njegovoj površini i aktiviraju mehanizme urođenog i stečenog imuniteta. Očuvanost funkcionalne aktivnosti leukocita ogleda se kroz sposobnost neutrofilnih granulocita i monocita/makrofaga da fagocituju uzročnika i iniciraju sekreciju matriks metaloproteinaza (MMP) i stvaranje reaktivnih kiseonikovih radikala, odnosno kroz lučenje citokina koji će aktivirati T pomagačke limfocite i dalje omogućiti odgovor B limfocita, kao i bolju aktivaciju makrofaga. Pokazano je da odgovor akutne faze tokom infekcije pasa B. canis karakteriše poremećaj redoks homeostaze i značajno povišenje nivoa IL-8, MCP-1 i IFN-γ in vivo, međutim, u literaturi nisu dostupni podaci u vezi sa funkcionalnom aktivnošću leukocita pasa kod ovog oboljenja.U prvi deo studije je bilo uključeno 24 psa, kojima je krv uzorkovana na prezentaciji (akutna infekcija B. canis) i nakon dve nedelje (klinički oporavak). U drugi deo studije je bilo uključeno sedam akutno obolelih pasa i sedam zdravih pasa, koji su služili kao kontrole. Na osnovu kliničkih pokazatelja i rezultata rutinskih laboratorijskih testova, psi su klasifikovani u SIRS pozitivne i SIRS negativne pacijente, bez poremećaja funkcije organa i organskih sistema. Funkcionalna aktivnost neutrofilnih granulocita je ispitana u uzorcima krvi testovima fagocitoze i oksidativnog praska, odnosno određivanjem aktivnosti MMP-9 u plazmi metodom zimografije na gelu poliakrilamida. Procena funkcionalne aktivnosti ukupnih leukocita i T limfocita zasnivala se na određivanju koncentracije IL-8, MCP-1 i IFN-γ u plazmi pomoću komercijalnih ELISA testova, nakon inkubacije (24h, 37°C, 5% CO2) pune krvi uz dodatak različitih koncentracija solubilnih antigena B. canis, koji se koriste za vakcinaciju pasa protiv babezioze. Pokazatelj funkcije B limfocita/plazmocita predstavljala je koncentracija γ globulina u serumu, dobijena elektroforetskim razdvajanjem proteina na gelu agaroze. Proteini akutne faze (nivo ceruloplazmina, haptoglobina i paraoksonaze 1), parametri oksidativnog stresa (koncentracija malondialdehida i slobodnih tiolnih grupa) i antioksidativne zaštite (aktivnost katalaze i glutation peroksidaze) određeni su spektrofotometrijski u uzorcima eritrocita i seruma pasa na prezentaciji i oporavku.Dobijeni rezultati su pokazali prisustvo anemije i trombocitopenije kod svih, odnosno leukopeniju i SIRS kod polovine pasa na prezentaciji. Tokom dvonedeljnog oporavka, utvrđeno je da su se testirani parametri vratili u okvire referentnih vrednosti, čime je isključena mogućnost da je tokom infekcije došlo do oštećenja organa. Analiza funkcionalne aktivnosti leukocita sprovedena nakon inkubacije (24h, 37°C, 5% CO2) periferne krvi bez i uz stimulaciju solubilnim antigenima B. canis pokazala je da je fagocitna aktivnost neutrofilnih granulocita u bazalnim uslovima i nakon stimulacije izraženija kod akutno obolelih pasa. Analogne razlike u intenzitetu oksidativnog praska su bile uočene nakon stimulacije. Prisustvo SIRS nije bilo povezano sa opisanim razlikama...Babesiosis is a vector-borne disease caused by protozoa Babesia spp. In Serbia, Babesia canis is considered to be the main causative agent of canine babesiosis. The infection can be asymptomatic, but it is usually followed by clinical manifestation, through uncomplicated or complicated form. Based upon clinical and laboratory criteria for systemic inflammatory response syndrome (SIRS), dogs with uncomplicated babesiosis can be classified as SIRS positive or SIRS negative, both without organ dysfunction. Unlike uncomplicated, complicated babesiosis includes SIRS with organ dysfunction. The level of the acute phase response depends on the dogs’ immune response, coinfection/comorbidity, the number of infectious ticks and sporozoites in ticks’ saliva, as well as the virulence of the B. canis variant. When fighting the pathogen, leukocytes recognize specific patterns on its surface, which leads to the activation of innate and adaptive immune response mechanisms. Functional activity of leukocytes reflects through the ability of neutrophils and monocytes/macrophages to perform phagocytosis and initiate the release of matrix metalloproteinases (MMP) and reactive oxygen species, and through the secretion of cytokines that will activate T helper and consecutively B lymphocytes, as well as macrophages. It has been previously shown that in canine babesiosis disturbed redox homeostasis and in vivo increased levels of IL-8, MCP-1 and IFN-γ occur during the acute phase response, but there is no evidence regarding functional activity of leukocytes in dogs infected with B. canis.For the first part of the study, residual blood was collected from 24 dogs at presentation (acute B. canis infection) and after two weeks (recovery). The second part of the study included seven dogs with acute B. canis infection and seven healthy dogs. Dogs with uncomplicated babesiosis were classified as SIRS positive and SIRS negative based on clinical examination and laboratory results. Functional activity of neutrophils was evaluated after performing phagocytosis and oxidative burst tests in vitro, as well as polyacrylamide gel zymography for MMP-9 detection. Using commercial ELISA kits, functional activity of monocytes and T lymphocytes was evaluated by measuring the concentration of IL-8, MCP-1 and IFN-γ after incubation (24h, 37°C, 5% CO2) of blood samples stimulated with different concentrations of soluble antigens of B. canis that are used to vaccinate dogs against babesiosis. Sera levels of γ globulins, that were detected using agarose electrophoresis, reflected the functional activity of B lymphocytes/plasmocytes. Levels of the acute phase proteins (ceruloplasmin, haptoglobin and paraoxonase 1) as well as markers of oxidative stress (concentration of malondialdehyde and free thiol groups) and antioxidant protection (activity of catalase and glutathione peroxidase) were evaluated in erythrocyte and sera samples, using spectrophotometry.The results showed that all of the dogs had anemia and thrombocytopenia, while half of them had leukopenia and SIRS at presentation. After clinical recovery, almost all of the tested parameters returned to reference intervals, excluding the possibility of organ disfunction as a consequence of B. canis infection. After incubation (24h, 37°C, 5% CO2) under basal conditions and with addition of soluble antigens of B. canis, the number of neutrophils that performed phagocytosis was higher in acutely infected dogs. Under stimulated conditions, similar results were shown for oxidative burst in neutrophils of acutely infected dogs. These results were not different between SIRS positive and negative dogs. T lymphocytes of acutely infected dogs released higher levels of IFN-γ in both basal and stimulated conditions, and these concentrations were highest in the SIRS positive group. Leukocytes released higher levels of MCP-1 in both basal and stimulated conditions, but they were not different between SIRS positive and negative dogs. Concentration of IL-8 did not differ between treatments nor groups..