Age, sex and breed effect on laboratory parameters in natural Babesia canis infection.

We tested the hypothesis that age, breed, and sex are related to hematology, biochemistry, acute phase proteins (APPs), seroreactivity and level of parasitemia in dogs with an acute phase response (APR) due to Babesia canis infection. The study enrolled 61 privately owned dogs that naturally acquired B. canis infection. Groups were formed according to the age: young dogs less than one year, and adult dogs more than one year old. Moreover, the group of males was compared to females and purebred to mixed breed dogs. Seroreactivity was tested with immunofluorescence antibody test, level of parasitemia with real-time polymerase chain reaction (real-time PCR), hematology, and biochemistry with automatic analyzers, serum amyloid A with enzyme-linked immunosorbent assay, fibrinogen with heat precipitation and ceruloplasmin and paraoxonase-1 with manual spectrophotometric methods. For protein separation agarose gel electrophoresis was used. The main changes in the whole population of B. canis-infected dogs were fever, pancytopenia, and change in APPs level. One-third of young, and 96% of adult dogs were seropositive (P < 0.001). The level of parasitemia was higher in the young dogs (P < 0.001). Erythroid lineage parameters (P < 0.01), and leukocytes (P < 0.05) were lower in the young, when compared to the adult dogs. Young dogs had lower total globulins (P < 0.001), ß- and γ-globulins (P < 0.001), and higher α-globulins (P = 0.022) than adult dogs. Young dogs had higher concentrations of phosphate (P = 0.003) and cholesterol (P < 0.001) and lower amylase (P = 0.014) and lipase activity (P = 0.020) than adult ones. Male dogs had lower neutrophil count than females (P = 0.035), and purebred dogs had more band neutrophils than mixed breed dogs (P = 0.004). In conclusion, dogs with natural Babesia canis infection at a young age have more severe anemia and APR including leukopenia than adults. Male and purebred dogs might also have more severe APR than females and mix-breeds, as they have more pronounced changes related to the myeloid lineage.

Seasonal differences in the intensity of acute phase response in dogs infected with Babesia canis.

The highest number of acute Babesia canis cases in dogs is recorded over the February-May (Feb-May) period, which also represents the optimal climate conditions for tick activity in Belgrade, Serbia. A possibility that the acute phase response is more intense in dogs developing the disease in the Feb-May period compared with the response in other time periods of the year was tested. A total of 63 client-owned dogs with acute B. canis infection were enrolled and the routine hematology and biochemistry parameters-serum amyloid A (SAA), IgG against B. canis, level of parasitemia, ceruloplasmin (CER), paraoxonase-1 (PON-1), and fibrinogen-were measured. Acute phase indexes (API) were calculated as (SAA×CER)/(Iron×PON-1) and (SAA×CER)/(Albumin×Iron). Statistics included Kruskal-Wallis test and logistic regression analysis. The results showed that in the Feb-May period, the following parameters were lower: creatinine, albumin, iron, and level of parasitemia. Furthermore, increased API values were more probable in the Feb-May than in the other periods. Together, higher acute phase response intensity and presumptive hemodilution in the Feb-May period indicate a more acute course of B. canis infection than in other time periods of the year.

Low serum levels of promatrix metalloproteinase-2 and -9 occur during acute Babesia canis infection in dogs.

Inflammation is a hallmark of the acute Babesia canis infection. Promatrix metalloproteinase (proMMP)-2 and -9 are involved in inflammation, but their levels have not been analyzed in canine babesiosis. We hypothesized that in dogs infected with B. canis, serum proMMP-2 and -9 levels change between presentation and recovery. Degree of the change differs if dogs develop systemic inflammatory response syndrome (SIRS). This study included 24 dogs with an acute B. canis infection, at presentation and after two weeks. We used routine hematology and biochemistry methods, spectrophotometry for the acute-phase proteins, microscopy for parasitemia and zymography for (pro)MMPs. In vitro endothelial cells and leukocyte short-term cultures, and platelet lysates were used to detect specific MMP activity. Statistical analyses included Wilcoxon test for paired samples, Mann-Whitney U test and Spearman's rank correlation. Our results showed that endothelial cells, leukocytes and platelets are the source of proMMP-2 and proMMP-9. Furthermore, both proMMPs were lower at presentation than after recovery (p < 0.001). At presentation, proMMP-9 levels correlated with parasitemia (rho = -0.616, p = 0.009), total leukocyte (rho = 0.704, p < 0.001) and neutrophil counts (rho = 0.741, p < 0.001). Extent of alterations in proMMP-2 levels between presentation and recovery was lower (p = 0.038) in dogs with SIRS than in non-SIRS dogs, while levels of proMMP-9 were comparable between these groups. Our conclusion is that during the acute B. canis infection, low serum levels of proMMP-2 and proMMP-9 at presentation reflect thrombocytopenia and leukopenia. Decreased proMMP-2 level could be associated with SIRS.

Systemic inflammatory response syndrome in dogs naturally infected with Babesia canis: Association with the parasite load and host factors.

The common signs of canine babesiosis caused by an infection with Babesia canis are fever, anorexia, lethargy, pulse alterations, anemia, and occasionally mild icterus. Dogs with these clinical signs can be divided into two groups: those with acute-phase reaction and those with systemic inflammatory response syndrome (SIRS). Factors associated with the occurrence of SIRS in canine babesiosis have not been thoroughly researched. This article outlines a cross-sectional study of 54 client-owned dogs with an acute B. canis infection, and evaluates the differences in age, gender, laboratory findings, parasite load, and seroreactivity against B. canis between the SIRS and the SIRS-free dogs. We have analyzed a complete blood count, serum biochemistry, serum amyloid A, ceruloplasmin, paraoxonase-1, serology, and PCR testing using standard methodologies. The frequency of SIRS among the investigated dogs reached 0.59. Male dogs and those seronegative against B. canis, were more frequent in the SIRS group, whilst age and parasite load could not be associated with the presence of SIRS. Dogs with SIRS had a lower count of total leukocytes, neutrophils, lymphocytes, and monocytes, and a lower concentration of iron and bilirubin compared with SIRS-free dogs. No significant differences in the concentration of acute-phase proteins have been noticed to exist between the groups of dogs. Further, the seronegative dogs had a lower count of lymphocytes and monocytes and a higher parasite load than the seroreactive dogs. Multivariate logistic regression analysis has identified leukopenia (<6 × 109/L) and monocytopenia (<0.2 × 109/L) as independent associates of SIRS in the investigated dogs, thus implying that these routine tests could be used as reliable markers for SIRS.

A short-term and long-term relationship between occurrence of acute canine babesiosis and meteorological parameters in Belgrade, Serbia.

Belgrade, the capital of the Republic of Serbia, is an endemic location for canine babesiosis caused by Babesia canis. This tick-borne disease occurs seasonally in regions with moderate continental climate. However, recent clinical data show that: 1) some cases of canine babesiosis have been recorded during the winter, and 2) canine babesiosis is spreading to the northern parts of Europe with a colder climate, which is a region previously free of this disease. Our study investigates the occurrence of canine babesiosis in different seasons over 2013-2016, and explores a short-term link between meteorological parameters and number of diagnosed cases of canine babesiosis in Belgrade. It also looks into possible long-term correlations that span one year before the onset of the disease. Based on 872 recorded cases over four years, our results show a bimodal seasonal distribution of canine babesiosis, with a pronounced peak in the spring, and a less conspicuous one in the autumn. Throughout the year, even over the coldest and warmest periods, there is a broad range of temperatures and relative humidities when the disease is recorded. Over one year prior to the spring and autumn onset of the disease, we found a noticeable impact of temperature and relative humidity, and to a lesser extent, of atmospheric pressure and cloud cover, on the number of diagnosed cases. These findings imply short-term and long-term relationships between occurrence of acute canine babesiosis and certain meteorological parameters, and they open further questions that need to be investigated in order to understand the epidemiology of this disease.