Acute phase response in dogs with babesiosis during different seasons and the relationship between the occurrence of acute disease and meteorological parameters

Babezioza pasa, uzrokovana infekcijom protozoom Babesia canis, sezonska je bolest na području Beograda, glavnog grada Republike Srbije. Sezone pojave oboljenja se poklapaju sa aktivnošću vektora krpelja Dermacentor reticulatus. Period inkubacije varira od četiri do 21 dan, a kliničku sliku karakteriše sistemska akutna inflamacija ili u težim slučajevima sindrom sistemskog inflamatornog odgovora (eng. Systemic Inflammatory Response Syndrome – SIRS). Povezanost klimatskih i epidemioloških faktora koja bi precizno pokazala i okarakterisala sezone u kojima se ovo oboljenje javlja na teritoriji Beograda, do sada nije ispitana. Sa kliničko-epidemiološkog aspekta, nameće se pitanje, da li je pojava SIRS-a vezana za pojedine sezone, odnosno da li postoje razlike u intezitetu odgovora akutne faze kod pasa obolelih u različitim sezonama. Hipoteza ove studije bila je da je tokom prolećne sezone, u kojoj je učestalost pojave pasa akutno inficiranih B. canis najveća, odgovor akutne faze najintenzivniji, odnosno da će reaktanti akutne faze pokazati najveće promene, a da će broj pasa sa SIRS-om, biti najzastupljeniji.Prvi deo studije obuhvatio je ispitivanje učestalosti pojave oboljenja na teritoriji Beograda tokom četiri godine (2013–2016), u različitim sezonama. Ispitan je kratkoročni uticaj (dve nedelje) i dugoročni uticaj meteoroloških parametara (godinu dana) koji su prethodili pojavi 872 zabeležena slučaja babezioze pasa. Drugi deo studije uključio je 66 pasa, sa akutnom infekcijom B. canis tokom 2017–2019. godine, od kojih je prikupljan višak pune krvi i seruma koji su preostali posle dijagnostike oboljenja. Rutinskim laboratorijskim testovima su izvedene hematološke i biohemijske analize, dok je elektroforeza proteina izvedena na agaroznom gelu. Parazitemija je određena komercijalnom metodom lančane reakcije polimeraze, dok je prisustvo IgG antitela protiv B. canis ispitano komercijalnim serološkim testom. Reaktanti/proteini akutne faze određeni su sledećim testovima: koncentracija serum amiloida A komercijalnom ELISA-om (eng. Enzyme Linked Immunosorbent Assay), koncentracija ceruloplazmina i aktivnost paraoksonaze 1 spektrofotometrijski, dok je za koncentraciju fibrinogena primenjen metod zagrevanja uz precipitaciju proteina. Klasifikacija pasa na SIRS pozitivne i negativne izvedena je prema odgovarajućim kliničkim i laboratorijskim promenama, dok su indeksi reaktanata akutne faze (IRAF) dobijeni računskim putem. Razlike između varijabli ispitane su pomoću Kruskal-Wallis, Mann-Whitney U testa i Chi-square testa. Veze između varijabli kvantifikovane su Spirmanovim koeficijentima korelacije odnosno logističkom regresionom analizom.Rezultati analize distribucije pojave babezioze pasa omogućavaju podelu kalendarske godine na četiri sezone: januar–februar, februar–maj, jun–septembar i oktobar–decembar. Broj dijagnostikovanih slučajeva pokazuje bimodalnu distribuciju sa najvećim brojem tokom kasne zime i proleća i nešto manje tokom jeseni. Pokazano je da su u sezoni sa najvećim brojem slučajeva (februar–maj) kratkoročni „optimalni“ uslovi za pojavu babezioze pasa temperatura (3–16) ˚C pri vlažnosti vazduha (53–87) %...Canine babesiosis, caused by infection with a protozoan Babesia canis, is a seasonal disease in the area of Belgrade, the capital of the Republic of Serbia. The seasons of the disease coincide with the activity of the vector Dermacentor reticulatus. The disease incubation period varies between 4 and 21 days, and the clinical picture is characterized by systemic acute inflammation or, in severe cases, Systemic Inflammatory Response Syndrome (SIRS). A connection between meteorological parameters and epidemiological factors that could help characterize the seasons in which this disease occurs in the region of Belgrade, has not been investigated previously. From the clinical-epidemiological aspect, there is a question whether the occurrence of SIRS is restricted to some seasons, or, in other words, whether there are differences in the intensity of the acute phase response in dogs infected in different seasons. This study hypothesized that during the spring season, when the frequency of dogs acutely infected with B. canis is the highest, the acute phase response is also the most intense, with acute-phase reactants showing the greatest changes, and the fraction of dogs with SIRS reaching its maximum.The first part of the study investigated the disease frequency in the Belgrade region in different seasons during four years (2013–2016) when a total of 872 were recorded. A short-term influence (two weeks prior) and long-term influence of meteorological parameters (up to one year prior) were analyzed.The second part of the study included 66 dogs with acute B. canis infection diagnosed between 2017 and 2019. Laboratory analyses were conducted using the excess of the whole blood and serum that remained after the diagnosis of the disease. Hematological and biochemical analyses were performed by routine laboratory tests, while agarose gel was used for electrophoretic analysis of proteins. Parasitemia was measured by a polymerase chain reaction method in a commercial laboratory. The presence of IgG antibodies against B. canis was tested by a commercial serological test. The following techniques were used for analyses of acute-phase proteins: commercial Enzyme Linked Immunosorbent Assay for serum amyloid A concentration, spectrophotometry for ceruloplasmin concentration and paraoxonase 1 activity, and heat denaturation method for fibrinogen. The dogs were classified as SIRS positive or negative according to a predefined set of clinical and laboratory changes. The indexes of acute phase reactants (IRAF) were calculated. The significance of the differences between the variables was tested using the Kruskal-Wallis, Mann-Whitney U, and Chi-square tests. The relationships between variables were quantified by Spearman correlation coefficients and logistic regression analyses.The number of diagnosed canine babesiosis cases showed a bimodal distribution wherein four seasons of the year can be distinguished: January–February, February–May, June–September and October–December. The highest number of the cases was recorded during late winter and spring, i.e., in the February–May season, and the other, less prominent maximum, occurred during autumn. In the season with the highest number of cases, from February to May, short-term (two weeks prior) "optimal" conditions for the occurrence of dog babesiosis were temperatures (3–16) ˚C and relative humidity (53–87) %..