Toxoplasma gondii is an important foodborne opportunistic pathogen that causes a severe disease in immunocompromised patients. The pathology and immune responses associated with the ensuing disease have not been well described in strains from different parts of the world. The aim of the present study is to determine the IFN-γ and IL-10 variations and organ pathology in immunocompetent and immunocompromised mice infected with T. gondii isolated from a Kenyan chicken. Two groups of BALB/c mice were infected with T. gondii cysts and administered with dexamethasone (DXM) in drinking water. Other two groups: infected untreated and uninfected mice were kept as controls. The mice were euthanized at various time points: blood collected for serum and assayed for IFN-γ and IL-10 variations. After infection, significant (p<0.05) elevated levels of IFN-γ and IL-10 were observed. A significant decline in IFN-γ and IL-10 levels (p<0.05) was observed after dexamethasone treatment. Histological sections in the liver, heart, and spleen of the mice administered with DXM revealed various degrees of inflammation characterized by infiltration of inflammatory cells. The dexamethasone-treated mice presented with progressively increased (p<0.001) inflammatory responses is compared with the infected untreated mice.
- Toxoplasma gondii
- organ pathology
Toxoplasmasmosis, cause by
Cases of toxoplasmosis have been reported in Kenya with the earliest study documented in 1968 . Since then,
Infection of immunologically competent persons with
In a study carried out in Kenya, a neurological murine model of chronic toxoplasmosis in BALB/c was developed in BALB/c mice using
Despite the fact that there is a high burden of toxoplasmosis and transmission in Africa , there are no studies which have evaluated the immunopathology of Toxoplasma isolates from these countries. Further, there is little information available regarding the immune responses inherent to reactivated toxoplasmosis. Acute and chronic infections in the neurological model described above  was associated with increase in both IgM and IgG levels but following dexamethasone treatment, IgM levels declined but IgG levels continued on rising. The current study therefore sought to determine the profile of IFN-γ and IL-10, and organ pathology in immunocompetent and immunosuppressed mice infected with
2. Materials and methods
2.1. Laboratory animals and ethical clearance
Prior to commencement of the study, all protocols and procedures used were reviewed and approved by the Institute of Primate Research, Institutional Animal Care and Use committee (Approval number: IRC/21/11). A total of 84 female BALB/c white mice were obtained from the rodent breeding facility Institute of Primate Research, Nairobi, Kenya. The mice were 6–8 weeks old and weighed 20–30 g. The mice were housed under standard laboratory conditions, in plastic cages (medium size cages; length 16.9 inches, width 10.5 inches, and height 5 inches) with wood shaving bedding and nesting material. Food (Mice Pellets®, Unga Feeds Ltd., Kenya), and drinking water were provided ad libitum.
T. gondii isolate and expansion
Prior to commencement of experimental work, the presence of
2.3. Experimental design
The BALB/c mice were intraperitoneally infected with 15
At 42 dpi, 48 BALB/c mice previously infected with 15 cysts each, were divided into three groups of 16 mice each. The mice were treated with Dexamethasone (Decadron DexPak PHARMA Links, India) at dosages of 2.66 mg/kg (Group 1) and 5.32 mg/kg (Group 2) daily in drinking water over a period of 6 weeks [24, 29, 30]. Sixteen infected nontreated mice were used as controls (Group 3). Another 16 uninfected control mice were given untreated water (Group 4). The mice were monitored daily over 6 weeks for survival analysis and any clinical signs and mortalities were recorded. After every 2 weeks, four mice from each group were serially euthanized using concentrated carbon dioxide and sampling done as previously described above. Mice that showed any severe clinical signs of toxoplasmosis were anesthetized immediately using concentrated carbon dioxide and sampling of blood, done. The liver, heart and spleen were collected and preserved in 10% formalin.
2.4. IFN-γ and IL-10 levels
Serum for cytokine activities was prepared as previously described by Parasuraman
2.5. Histological analysis
Liver, spleen and heart were processed for paraffin embedding and sectioning. To determine the histological changes, tissue sections were stained with hematoxylin and eosin and observed under light microscope. The inflammation was assessed and scored histologically. The severity of the histopathological lesions in the heart was evaluated by grading the lesions using a modified random scale as previously described .
In the liver, the inflammatory lesions were quantified based on the degree of lymphocyte infiltration and hepatocyte necrosis as previously described . Segments of spleen were scored for the enlargement of lymphocyte infiltrated areas and for the increased numbers of macrophages and necrotic cells previously described .
In these organs, the inflammatory changes were examined in two noncontinuous sections (40 μ distance between them) from each mouse in 25 microscopic fields using a 40× objective. The total inflammation score was determined from the summed scores of each mouse from each group or sampling time point and used for data analysis.
2.6. Data analysis
The results were entered into MS Excel program (Microsoft, USA) before being exported to GraphPad prism version 5.0 (GraphPad Software, USA) for statistical analysis. Statistical differences between the mice groups were determined by ANOVA; groups were considered statistically different if P ≤ 0.05.
3.1. IFN-γ Levels
The mean of IFN-γ cytokine levels in the infected mice are as shown in Figure 1. There was a progressively significant (p < 0.001) increase in IFN-γ from 3.5 pg/ml (95%; CI: 2.93–4.07 at day 0 reaching 10.59 pg/ml, (95% CI: 9.03–12.15) at 35 dpi. The noninfected control group did not display any significant increase in IFN-γ cytokine levels and remained decreased at all time points compared to the infected group.
After treatment with dexamethasone, IFN-γ productions levels progressively declined at time points between 42 and 84 dpi. The decline in the 2.66 mg/kg/day of dexamethasone treated mice (Group 1) was from 17.84 pg/ml (95% CI: 1.60–34.08) at 42 dpi to 10.02 pg/ml (95% CI: 2.98–17.07) at 84 dpi (Figure 2).
The corresponding decline in the 2.66 mg/kg/day of dexamethasone treated mice (Group 2) was from 15.51 pg/ml (95% CI:−0.64–31.66) at 42 dpi to 7.89 pg/ml (95%; CI: 3.02–12.73.50) at 84 dpi. The decrease in IFN-γ levels was associated with increased dose, although the difference between the 2 doses were not significant (P > 0.05). The IFN-γ levels in the infected nontreated mice (Group 3) increased from 21.48 pg/ml (95%CI: 10.59–32.38) at 42 dpi to 26.38 pg/ml (95% CI: 20.01–32.75) at 56 dpi and thereafter, a progressive decline in IFN-γ levels reaching 13.53 pg/ml (95% CI: 0.42–26.64) and 11.03 pg/ml (95% CI: 5.43–16.64) at 70 and 84 dpi, respectively. Mice in the infected nontreated group (Group 3) maintained significantly (P < 0.001) increased levels of IFN-γ compared to the infected treated mice (Figure 2).
3.2. IL-10 levels
The levels of IL10 also increased following
Following dexamethasone treatment, the levels of IL-10 maintained a downward trend (Figure 4). In the mice treated with 2.66 mg/kg/day of DXM, the levels ranged between 135.66 pg/ml (95% CI: 82.79–188.54) at 42 dpi and dropped to 71.73 pg/ml (95% CI: 45.67–97.79) at 84 dpi. In the group treated with 5.32 mg/kg/day, the IL-10 level was 116.92 pg/ml (95% CI: 89.69–144.15) at 42 dpi and dropped to 55.59 pg/ml (95% CI: 40.77–70.43) at 84 dpi. The infected group (group 3) recorded a decreased IL-10 concentration ranging between 141.97 pg/ml (95% CI: 134.26–149.68) at 42 dpi and 99.71 pg/ml (95% CI: 77.16–122.27) at 84 dpi. Mice in the infected group recorded significantly (P < 0.01) elevated IL-10 levels compared to the treated groups at all time points.
3.3. Histological changes in the peripheral organs of BALB/c mice infected with
In general, the histopathological changes in the liver, heart and spleen of infected mice consisted of mild-to-moderate congestion and detectable multifocal or focal inflammatory infiltrate. Between 3 and 14 dpi, the liver showed increased pathology characterized by hepatic necrosis, infiltration of lymphocytes and macrophages scattered in portal triad areas (Figure 5). The inflammatory scores increased from 1.2 (±0.49) at 3 dpi to 2.0 (±0.316) at 7 dpi. The highest inflammatory score was recorded at 14 dpi (2.8 ± 0.2) and thereafter, a progressive significant decline in inflammatory score (P < 0.001) at 42 dpi (1.4 ± 0.4) was observed.
Following dexamethasone treatment, the mice treated with 2.66 mg/kg/day (Group 1) and 5.32 mg/kg/day (Group 2) of dexamethasone showed varied degrees of inflammatory responses. For the mice treated with 2.66 mg/kg/day of dexamethasone, an inflammatory score of 1.4 (±0.245) and 2.0 (±0.00) was observed between 56 and 84 dpi, respectively, while the mice treated with 5.32 mg/kg/day (Group 2) of dexamethasone recorded an inflammatory score of 1.6 (±0.245) and 2.6 (±0.25) at 56 and 84 dpi, respectively. On the other hand, the infected nontreated mice presented an inflammatory score of 0.6 (±0.245) at 42 dpi but did not significantly (P > 0.05 change with the progression of the infection maintaining at 0.8 (±0.2) at 56, 70 and 84 dpi. Although the treated mice presented with progressively increased inflammatory scores there was no significant difference (P > 0.05) in the liver inflammatory response between the same groups.
In the heart of infected mice, the histopathological lesions were relatively fewer compared to those in liver and were characterized by inflammatory infiltrates (Figure 6). The inflammatory score at 7 dpi was 1.75 (±0.25) and this was followed by a significant (P < 0.001) decrease reaching the lowest inflammatory score of 1.25 (±0.25) at 35 dpi. However, treatment with dexamethasone markedly increased the severity and number of myocardial lesions in these infected animals. The toxoplasma infected group (Group 3) presented with higher inflammatory lesions at the time of treatment (day 42 dpi; P < 0.01). However, at 56, 70 and 84 dpi, an increasing inflammatory score was noted although there was no significant difference (P > 0.05). All the heart tissues of mice from group 1 recorded an inflammatory score of 1.25 (±0.25) at 56, 70 and 84 dpi (P > 0.05) while group 2 recorded a significant (p < 0.01) inflammatory score of 1.25 (±0.25); 1.5 (±0.289) and 2.5 (±0.289) at 56, 70 and 84 dpi, respectively. The uninfected control group (Groups 4) did not show any myocardial lesions at all time points.
The spleen was also affected by
In the present study, BALB/c mice infected with
In the current study, the IFN-γ levels were significantly depressed in the dexamethasone-treated
In the current study, the IL-10 levels were also elevated during the acute and chronic infection and there was also a decline in immunosuppressed mice (42–84 dpi). This anti-inflammatory cytokine, has the ability to antagonize T helper 1 (Th1) responses . IL-10 is considered to be an inhibitor of Th1 and Th2 immune responses [47, 48, 49]. Therefore, the role of IL-10 cytokines secreted by macrophages, monocytes, B cells, and CD4+ and CD8+ T cells during both the acute and the chronic phases of infection in both immunocompetent and immunosuppressed mice is to acts broadly on accessory cells and adaptive cells responses to downregulate or limit the consequences of an exaggerated inflammatory response and major histocompatibility complex and costimulatory molecule expression [20, 47, 50, 51]. This cytokine also prevents tissue immune destruction through immunomodulation  and has been identified as a factor induced by
The results of the present study showed that chronically infected nontreated mice had an increase in mononuclear cells organ infiltrations upon infection. The recruitment of inflammatory cells as was the case in these organs, is one of the most important immune mechanisms induced by IFN-γ and is geared towards control of parasite multiplication. These cells could also be responsible for the higher levels of cytokines observed in the initial stage of
The results of this study indicates that immunological and pathological features of
This work was funded by Jomo Kenyatta University of Agriculture and Technology (JKUAT)-Research production and Extension. The authors are grateful to the technical assistance provided by IPR staff including Samson Mutura, Tom Adino, Esther Kagasi and Caroline Jerono.
The authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this book chapter.
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