Kinetics of the inflammatory response during experimental Babesia rossi infection of Beagle dogs

Abstract

Background: Babesia rossi, the most virulent canine Babesia parasite, causes severe clinical disease and death in dogs in sub-Saharan Africa. Complications and multiple organ dysfunction seen with babesiosis are likely caused by the effects of an unfocused, imbalanced and excessive inflammatory response. The aim of this study was to investigate markers of inflammation and cytokine kinetics from the point of inoculation with B. rossi throughout the course of clinical disease and determine if infectious dose influenced rate and severity of disease progression. Hypothesis: Dogs infected with a higher inoculum dose would experience more severe clinical disease and systemic inflammation over a shorter disease course. Measurable differences would be found between the baseline, low dose and high dose groups for clinical, clinicopathological and cytokine variables. Materials and Methods: This experimental study was performed on 6 healthy sterilised male beagle dogs. One dog was splenectomised and used to raise a viable parasite inoculum. Three dogs were given a high infectious dose (HD group) and 2 dogs a low infectious dose (LD group) of parasite. Appetite, habitus, clinical examination, glucose, lactate and CBC was performed daily and EDTA plasma was stored at -80⁰C. C-reactive protein and albumin were determined every second day. Cytokines were quantified on the stored plasma using a canine specific cytokine magnetic bead panel (Milliplex©). Dogs were monitored and parasitaemia was determined daily until predetermined endpoints for treatment were reached. The dogs in the high dose group were treated at 96 hours and those in the low dose group were treated at 108 hours post infection. Results: The infection was allowed to run its course for 4 days prior to intervention. The HD group was treated at 96 hours and the LD group was treated 12 hours later, at 108 hours. No significant difference was noted for baseline data between the LD and HD groups for any variable. Post inoculation, initial parasitaemia occurred at 24 hours in the HD group and 72 hours in the LD group. The rate of increase in parasitaemia in the HD group was considerably faster than that seen in the LD group. The mean temperature peaked 36-hours earlier in the HD group. The pyrexia persisted for at least 24 hours after treatment in both groups. In addition to the difference seen in vital parameters between the two groups, the HD group also demonstrated a more pronounced decline in habitus and appetite during the course of the infection. The red cell count showed a significant decline from 96 hours in the HD group, worsening after treatment with the lowest count at 120 hours. The dogs in the HD group required multiple blood transfusions before the red cell count stabilised and started to improved. Although there was a drop in the red cell counts of the LD group after treatment, it resulted in tolerable clinical levels of anaemia which did not require transfusions. The C- reactive protein also peaked 36-hours earlier in the HD group. A neutropenia was seen in both groups, but the nadir was earlier and more dramatic in the HD group. Thirteen cytokines were evaluated in total and the results are divided into 4 groups by pattern of change. The categories for the kinetic patterns identified include: a. Cytokines that rose during the infection and fell after treatment: IFNγ and KC-like. Both these cytokines peaked earlier in the high dose group and declined rapidly after treatment. b. Cytokines that rose and remained high even after treatment: MCPI-1, IL-6, IL-8 and IL-10. Both MCP-1 and IL-6 gradually increased during infection in the HD group with minimal changes in the LD group. After treatment, these cytokines increased drastically in the HD group, with only 1 dog in the LD group showing an increase at 192 hours. IL-8 had a different kinetic pattern between HD and LD groups, not only a delay in the increase. The anti-inflammatory cytokine IL-10 increased progressively during parasite replication in both groups. c. Cytokines that rose dramatically after treatment: GM-CSF, TNFα, IL-2 and IL-7 were all markedly increased after treatment in the HD group with moderate increases seen in one dog the LD group. d. Cytokines that showed no distinct pattern of change: IL-15, IL-18 and IP-10. Conclusion: Our findings suggest that the initiation of inflammation occurs before the onset of clinical disease with a possible imbalance in the pro- and anti-inflammatory cytokine concentrations during parasite replication. Infectious dose influenced the course of inflammation as well as the course and severity of disease. Treatment of the infection did not result in the resolution of inflammation. In fact, many markers of inflammation and cytokines were significantly increased following treatment. This is in agreement with the hypothesis that severe inflammation and complications are associated with an unfocused, imbalanced and pronounced inflammatory response that may even be perpetuated by chemotherapeutic intervention.