Salmonella typhi is a facultative intracellular pathogen that causes typhoid fever in humans. In the present study, the effect of Salmonella typhi infection on hematological indices and spleen histology in Wistar rats was investigated and was followed by an evaluation of the ameliorative potential of the methanol leaf extract of Chromolaena odorata (MLECO) compared with ciprofloxacin treatment. The animals were divided into six groups: group 1 was normal control, group 2 was infected with Salmonella typhi without treatment (negative control), groups 3, 4 and 5 were Salmonella typhi infected and treated with 100 mg/kg, 200 mg/kg and 400 mg/kg of the extract respectively and group 6 was also Salmonella typhi infected and treated with 500 mg/70kg of ciprofloxacin. The animals were inoculated with a single infectious dose of Salmonella typhi bacteria and thereafter, treated with the extract and ciprofloxacin for a period of seventeen days, after the animals were confirmed infected. The rats were humanely sacrificed and blood samples taken for haematological investigations, and the spleen harvested and processed for histological examinations. Chromolaena odorata administration reversed the adverse hematotoxicity and histopathological changes in the spleen induced by Salmonella typhi infection.
Blood is one of the major homeostatic systems of the body and therefore supports normal integrity, viability and adaptive responses [
Leaves of Chromolaena odorata were fetched from Omuoko-Aluu community and cleaned of soil and dust by washing with tap water. The leaves were identified at the Department of Plant Science and Biotechnology, University of Pot Harcourt.
After collection, the leaves of the plant were shade-dried at room temperature (32˚C - 35˚C) to constant weight over a period of seven (7) days. The method employed a little modification of the cold maceration extraction method as described by [
All media used in this study were prepared following the manufacturer’s instruction. Salmonella typhi was isolated from typhoid patients in “University of Port Harcourt, Teaching Hospital. Bile salt broth (broth culture) [
Eighty five (85) animals were divided into 6 groups of 1 - 6. Group 1 (normal) had five (5) animals, Group 2 (negative control) had twenty (20) animals, while groups 3 - 6 each had fifteen (15) animals. Group 1 animals were not treated throughout the experiment but were given free access to normal animal feed and water ad labitum. Group 2 contained Salmonella typhi-infected rats not treated after disease induction. Group 3 contained Salmonella typhi-infected rats treated with 100 mg/kg (low dose) of MLECO. Group 4 contained Salmonella typhi-infected rats treated with 200 mg/kg (medium dose) of methanol leaf extract of Chromolena odorata. Group 5 contained Salmonella typhi-infected rats treated with 400 mg/kg (high dose) of methanol leaf extract of Chromolena odorata. Group 6 contained Salmonella typhi-infected rats treated with 500 mg/70kg of a standard antibiotic drug (Ciprofloxacin). On day 0, at five day intervals (i.e. 6, 11, and 16), 5 animals from each group were humanely sacrificed using diethyl ether as anesthesia, and blood was collected and the spleen removed for assessment of the hematological parameters and histopathological examination, respectively.
Eighty (80) animals (groups 2 - 6) were orogastrically challenged with an infective dose (2.0 × 108 cfu/ml) of Salmonella typhi. After infection had set in (through observation of signs like weakness, anorexia, non-productive cough, watery stool, standing of the hairs as in cold condition and isolation of the organism from the stool of the infected animals) (day 0), five animals were sacrificed and blood samples and spleen tissues collected for preliminary screening while the other 75 animals were treated with the methanol leaf extract of Chromolena odorata according to the different doses specified for each sub-group and the standard antibiotic (Ciprofloxacin), once daily, for seventeen days.
Stock solutions for the extract were prepared by dissolving 500 mg in 1 ml of dimethylsulfoxide (DMSO). An antibiotic control was made by dissolving 500 mg of ciprofloxacin in sterile distilled water. DMSO was also used as vehicle control in the study.
Blood was collected from each animal by cardiac puncture method after the animals were anaesthetized with diethyl ether in a desiccator. The blood was immediately transferred into appropriately labelled sample bottles containing anticoagulant and the spleen was removed aseptically and was weighed and a portion was kept for histological analysis.
Hematological analysis was carried out as described by [
The effect of Salmonella typhi inoculation in Wistar rats resulted in a decrease in WBC and neutrophil levels, and an increase in lymphocytes and monocytes that were not statistically significant when compared with the normal group. These changes were reversed on administration of the standard drug and the test extract, except for the lymphocytes. Administration of the standard drug and the test extract resulted in an increase in WBCs (
Group | Day 0 | Day 6 | Day 11 | Day 16 |
---|---|---|---|---|
Control | 7.10 ± 0.4 | 6.87 ± 0.18 | 6.93 ± 0.24 | 6.83 ± 0.15 |
Neg. Control | 5.63 ± 0.75 | 3.60 ± 0.26** | 3.20 ± 0.35** | 1.43 ± 0.28** |
Low Dose | 5.63 ± 0.76 | 5.73 ± 0.81*** | 5.80 ± 0.76*** | 6.00 ± 0.72*** |
Medium Dose | 5.63 ± 0.75 | 5.87 ± 0.72*** | 6.03 ± 0.72*** | 6.40 ± 0.30*** |
High Dose | 5.63 ± 0.75 | 6.13 ± 0.69*** | 6.27 ± 0.58*** | 6.87 ± 0.09*** |
Ciprofloxacin | 5.63 ± 0.75 | 5.90 ± 0.70*** | 6.10 ± 0.72*** | 6.60 ± 0.23*** |
* = Significant difference between normal and test groups; ** = Significant difference between normal and negative control; *** = Significant difference between and negative control and test groups; 1) Normal (Animals not exposed to any form of treatment but were fed ad libitum); 2) Negative Control (Animals inoculated with Salmonella typhi without treatment); 3) Low Dose (100 mg/kg of extract); 4) Medium Dose (200 mg/kg of extract); 5) High Dose (400 mg/kg of extract); 6) Ciprofloxacin (500 mg/70kg).
Group | Day 0 | Day 6 | Day 11 | Day 16 |
---|---|---|---|---|
Control | 0.21 ± 0.006 | 0.21 ± 0.05 | 0.21 ± 0.006 | 0.20 ± 0.06 |
Neg. Control | 0.14 ± 0.03 | 0.11 ± 0.01 | 0.13 ± 0.02 | 0.04 ± 0.02** |
Low Dose | 0.14 ± 0.03 | 0.16 ± 0.03 | 0.17 ± 0.04 | 0.18 ± 0.05 |
Medium Dose | 0.14 ± 0.03 | 0.16 ± 0.04 | 0.18 ± 0.05 | 0.20 ± 0.06*** |
High Dose | 0.14 ± 0.03 | 0.18 ± 0.05 | 0.19 ± 0.04 | 0.22 ± 0.01*** |
Ciprofloxacin | 0.14 ± 0.03 | 0.17 ± 0.04 | 0.19 ± 0.05 | 0.20 ± 0.6*** |
Group | Day 0 | Day 6 | Day 11 | Day 16 |
---|---|---|---|---|
Control | 6.13 ± 0.26 | 5.90 ± 0.49 | 6.13 ± 0.20 | 6.10 ± 0.46 |
Neg. Control | 7.16 ± 1.02 | 7.27 ± 0.13 | 7.53 ± 0.75** | 7.97 ± 0.09** |
Low Dose | 7.16 ± 1.02 | 7.16 ± 1.24 | 7.25 ± 1.01 | 7.32 ± 0.94 |
Medium Dose | 7.16 ± 1.02 | 7.20 ± 1.27 | 7.30 ± 0.17 | 7.39 ± 0.23 |
High Dose | 7.16 ± 1.02 | 7.24 ± 1.29 | 7.35 ± 0.33 | 7.42 ± 0.29 |
Ciprofloxacin | 7.16 ± 1.02 | 7.19 ± 1.26 | 7.38 ± 0.36 | 7.37 ± 0.32 |
Group | Day 0 | Day 6 | Day 11 | Day 16 |
---|---|---|---|---|
Control | 0.11 ± 0.02 | 0.11 ± 0.02 | 0.11 ± 0.02 | 0.11 ± 0.02 |
Neg. Control | 0.15 ± 0.03 | 0.17 ± 0.04 | 0.18 ± 0.03** | 0.18 ± 0.02** |
Low Dose | 0.15 ± 0.012 | 0.14 ± 0.02 | 0.13 ± 0.03 | 0.12 ± 0.01*** |
Medium Dose | 0.15 ± 0.012 | 0.13 ± 0.01 | 0.120.02 | 0.12 ± 0.01*** |
High Dose | 0.15 ± 0.012 | 0.12 ± 0.02 | 0.11 ± 0.01 | 0.11 ± 0.01*** |
Standard | 0.15 ± 0.012 | 0.13 ± 0.02 | 0.12 ± 0.01 | 0.10 ± 0.01*** |
monocytes. ANOVA comparison showed a significant difference (p < 0.05) between negative control and normal and between negative control and other treatment groups on days 6, 11 and 16 in WBC count; between negative control and normal and between negative control and other treatment groups on day 16 for neutrophils and monocytes. Inoculation of a single contagious dose of salmonella typhi caused a significant decrease (p < 0.05) in the mean levels of thrombocytes, red blood cells (RBC), hemoglobin concentration (Hgb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC), when compared to the normal rats. On the contrary, administration of the standard drug and extract reversed these changes leading to a gradual increase in all these parameters. ANOVA comparison showed a significant difference (p < 0.05) between negative control and normal in all these parameters throughout the study. Similarly, a significant difference was observed between the negative control and other treatment groups on days 11 and 16 for platelets and RBCs (
Spleen histo-architectural examination of normal control rats (Plate 1) revealed normal morphology with clearly defined red and white pulp and marked,
Group | Day 0 | Day 6 | Day 11 | Day 16 |
---|---|---|---|---|
Normal | 652.33 ± 4.10 | 652.33 ± 4.10 | 652.33 ± 4.10 | 652.33 ± 4.10 |
Neg. Control | 306.67 ± 7.22** | 273.00 ± 37.10** | 239.00 ± 30.01** | 217.00 ± 9.29** |
Low Dose | 306.67 ± 7.22* | 341.00 ± 28.92* | *437.33 ± 27.51*** | 545.67 ± 13.38*** |
Medium Dose | 306.67 ± 7.22* | 418.67 ± 13.13* | *504.00 ± 8.89*** | 627.00 ± 15.50*** |
High Dose | 306.67 ± 7.22* | 474.00 ± 21.66* | 595.67 ± 9.02*** | 667.67 ± 22.36*** |
Ciprofloxacin | 306.67 ± 7.22* | 427.00 ± 9.29* | *572.33 ± 17.84*** | 631.00 ± 11.85*** |
Group | Day 0 | Day 6 | Day 11 | Day 16 |
---|---|---|---|---|
Normal | 8.35 ± 0.64 | 8.35 ± 0.64 | 8.35 ± 0.64 | 8.35 ± 0.64 |
Neg. Control | 3.70 ± 0.29** | 3.57 ± 0.21** | 3.17 ± 0.26** | 2.97 ± 0.09** |
Low Dose | 3.70 ± 0.29* | 3.87 ± 0.24* | *4.93 ± 0.09*** | *5.27 ± 0.12*** |
Medium Dose | 3.70 ± 0.29* | 4.57 ± 0.23* | *5.67 ± 0.26*** | *6.37 ± 0.29*** |
High Dose | 3.70 ± 0.29* | 4.87 ± 0.33* | *6.10 ± 0.38*** | 8.60 ± 0.36*** |
Ciprofloxacin | 3.70 ± 0.29* | 4.20 ± 0.23* | *5.00 ± 0.17*** | *6.37 ± 0.29*** |
Group | Day 0 | Day 6 | Day 11 | Day 16 |
---|---|---|---|---|
Normal | 16.20 ± 1.15 | 16.20 ± 1.15 | 16.20 ± 1.15 | 16.20 ± 1.15 |
Neg. Control | 6.70 ± 0.79** | 6.63 ± 1.06** | 6.20 ± 0.75** | 5.47 ± 0.58** |
Low Dose | 6.70 ± 0.79* | *11.50 ± 0.23*** | 12.70 ± 0.12*** | 13.43 ± 0.26*** |
Medium Dose | 6.70 ± 0.79* | *12.50 ± 0.32*** | 14.73 ± 0.44*** | 15.50 ± 0.15*** |
High Dose | 6.70 ± 0.79* | *13.43 ± 0.15*** | 15.27 ± 0.35*** | 16.23 ± 0.29*** |
Ciprofloxacin | 6.70 ± 0.79* | *11.67 ± 0.15*** | 14.83 ± 0.09*** | 15.63 ± 0.18*** |
Plate 1. Histological sections of the spleen of normal rats with clearly defined red and white pulp. The picture does not show any germinal centres but the trabecular are marked (Normal-Day 6).
Group | Day 0 | Day 6 | Day 11 | Day 16 |
---|---|---|---|---|
Normal | 43.00 ± 1.15 | 43.00 ± 1.15 | 43.00 ± 1.15 | 43.00 ± 1.15 |
Neg. Control | 18.00 ± 2.08** | 17.00 ± 1.53** | 15.67 ± 1.20** | 14.00 ± 0.58** |
Low Dose | 18.00 ± 2.08* | *26.00 ± 1.15*** | *29.67 ± 0.88*** | *33.33 ± 1.45*** |
Medium Dose | 18.00 ± 2.08* | *30.33 ± 3.28*** | *32.67 ± 2.73*** | *38.67 ± 0.88*** |
High Dose | 18.00 ± 2.08* | *33.00 ± 1.73*** | 39.00 ± 2.65*** | 42.67 ± 1.76*** |
Ciprofloxacin | 18.00 ± 2.08* | *31.00 ± 3.79*** | *33.00 ± 2.65*** | 40.00 ± 1.73*** |
Group | Day 0 | Day 6 | Day 11 | Day 16 |
---|---|---|---|---|
Normal | 51.00 ± 2.08 | 51.67 ± 1.45 | 51.67 ± 1.45 | 51.67 ± 1.45 |
Neg. Control | 21.00 ± 2.31** | 19.67 ± 0.88** | 18.00 ± 0.58** | 16.33 ± 0.88** |
Low Dose | 21.00 ± 2.31* | *24.00 ± 3.46 | *31.67 ± 2.73*** | *39.33 ± 2.19*** |
Medium Dose | 21.00 ± 2.31* | *25.33 ± 2.73 | *34.00 ± 1.15*** | *43.00 ± 1.15*** |
High Dose | 21.00 ± 2.31* | *32.00 ± 1.15*** | *41.33 ± 0.88*** | 51.00 ± 2.65*** |
Ciprofloxacin | 21.00 ± 2.31* | *27.00 ± 1.15*** | *36.33 ± 1.45*** | *43.67 ± 1.76*** |
Group | Day 0 | Day 6 | Day 11 | Day 16 |
---|---|---|---|---|
Normal | 18.77 ± 1.03 | 18.83 ± 1.08 | 18.83 ± 1.02 | 18.80 ± 1.00 |
Neg. Control | 7.17 ± 1.31** | 6.67 ± 0.78** | 6.30 ± 0.58** | 6.37 ± 0.64** |
Low Dose | 7.17 ± 1.31* | *8.97 ± 0.67 | 11.33 ± 0.50*** | 14.07 ± 1.12*** |
Medium Dose | 7.17 ± 1.31* | *11.30 ± 0.64*** | 14.73 ± 1.18*** | 16.03 ± 1.07*** |
High Dose | 7.17 ± 1.31* | *14.40 ± 1.10*** | 16.70 ± 1.31*** | 17.77 ± 1.90*** |
Ciprofloxacin | 7.17 ± 1.31* | *11.97 ± 1.23*** | 15.47 ± 1.49*** | 16.37 ± 1.21*** |
trabecular while splenic tissues of rats infected with S. typhi without treatment after 5 days (Plate 2) showed numerous splenic venous stenosis in the red pulp. In
Group | Day 0 | Day 6 | Day 11 | Day 16 |
---|---|---|---|---|
Normal | 34.77 ± 1.13 | 35.13 ± 1.85 | 34.87 ± 2.07 | 35.17 ± 1.48 |
Neg. Control | 11.83 ± 1.22** | 11.33 ± 0.78** | 10.43 ± 1.13** | 9.23 ± 0.77** |
Low Dose | 11.83 ± 1.22* | *13.73 ± 1.20 | *15.83 ± 2.57 | *21.90 ± 4.14*** |
Medium Dose | 11.83 ± 1.22* | *17.37 ± 1.48*** | *24.53 ± 2.73*** | 29.60 ± 3.18*** |
High Dose | 11.83 ± 1.22* | *23.63 ± 2.00*** | 27.83 ± 2.34*** | 35.07 ± 0.09*** |
Ciprofloxacin | 11.83 ± 1.22* | *17.90 ± 1.20*** | 27.60 ± 2.95*** | 32.00 ± 1.37*** |
Plate 2. Photomicrograph of splenic tissues of rat infected with S. typhi without treatment after 6 days. Numerous splenic venous stenosis are seen in the red pulp (Negative control-Day 6).
animals treated with 100 mg/kg of the extract for 5 days (Plate 3), the splenic tissue showed central white pulp with germinal center and in the field was the presence of a central artery In animals treated with 100 mg/kg of the extract for 5 days (Plate 4), the splenic tissues of rats showed destroyed tissues, and in those animals treated for with 200 mg/kg of the extract for 5 days (Plate 5), the splenic tissues showed infected tissue with white pulp having germinal centers. For those treated with 400 mg/kg of C. odorata for 5 days (Plate 6), the slpeen showed non-specific tissue disruption. Plate 7 also showed normal histo-morphology of the spleen in animals of normal group after 10 days exposure to normal laboratory conditions, while plate 8 is the photo micrograph of the spleen in animals exposed to Salmonella typhi for 10 days; it showed numerous splenic venous stenosis and tissue disruption in the red pulp. In animals treated with 500 mg/70kg of ciprofloxacin for 10 days (Plate 9), the spleen revealed white pulp with the presence of germinal center, while for those animals treated with 100 mg/kg of extract for 10 days (Plate 10), the spleen histology still showed infected tissues. For those animals treated with 200 mg/kg of extract (Plate 11), the spleen tissues revealed a non-specific tissue disruption, while for those animals treated with 400 mg/kg of extract for 10 days (Plate 12), the spleen tissues shows normal morphology. Plate 13 also showed normal histo-morphology of the spleen in animals of normal group after 15 days exposure to normal laboratory conditions, while Plate 14 is the photo micrograph of the spleen in animals exposed to Salmonella typhi for 15 days, also showing
Plate 3. Photomicrograph of splenic tissues of rat infected with S. typhi and treated with 500 mg of ciprofloxacin for 5 days. The splenic tissue presented a central white pulp with germinal center and in the field was the presence of a central artery.
Plate 4. Photomicrograph of spleenic tissues of rat infected with S. typhi and treated with 100 mg/kg of C. odorata for 5 days, showing destroyed tissues.
Plate 5. Photomicrograph of splenic tissues of rat infected with S. typhi and treated with 200 mg/kg of C. odorata for 5 days, showing disruption and infected tissue and white pulp with germinal centres.
Plate 6. Photomicrograph of splenic tissues of rat infected with S. typhi and treated with 400 mg/kg of C. odorata for 5 days, showing non-specific tissue disruption.
Plate 7. Histological sections of the spleen of normal rats with clearly defined red and white pulp. The picture does not show any germinal centres but the trabecular are marked (Normal Day 11).
Plate 8. Photomicrograph of splenic tissues of rat infected with S. typhi without treatment after 12 days. Numerous splenic venous stenosis and tissue disruption are seen in the red pulp (Negative control-Day 11).
Plate 9. Photomicrograph of splenic tissues of rat infected with S. typhi and treated with 500 mg of ciprofloxacin for 10 days. White pulp shows the presence of germinal center.
Plate 10. Photomicrograph of splenic tissues of rat infected with S. typhi and treated with 100 mg/kg of C. odorata for 10 days, showing infected tissues and disruption.
Plate 11. Photomicrograph of splenic tissues of rat infected with S. typhi and treated with 200 mg/kg of C. odorata for 10 days, showing non-specific tissue disruption.
Plate 12. Photomicrograph of splenic tissues of rat infected with S. typhi and treated with 400 mg/kg of C. odorata for 10 days showing normal cells.
Plate 13. Histological sections of the spleen of normal rats with clearly defined red and white pulp. The picture does not show any germinal centres but the trabecular are marked (Normal Day 16).
Plate 14. Photomicrograph of splenic tissues of rat infected with S. typhi without treatment after 12 days. Numerous splenic venous stenosis and disruption are seen in the red pulp (Negative control-Day 16).
numerous splenic venous stenosis and tissue disruption in the red pulp. For animals treated with 500 mg/70kg of ciprofloxacin for 15 days (Plate 15), the Splenic tissue appeared normal with clearly defined white and red pulp, while animals treated with 100 mg/kg of extract for 15 days (Plate 16), still showed non-specific tissue disruption, while those treated with 200 mg/kg of extract (Plate 17) and 400 mg/kg of extract (Plate 18) showed tissues with normal morphology.
During typhoid fever infection, the common hematological changes include; anemia, monocytosis and lymphocytosis [
Plate 15. Photomicrograph of splenic tissues of rat infected with S. typhi and treated with 500 mg of ciprofloxacin for 15 days. Splenic tissue appeared normal with clearly defined white and red pulp. The germinal centers were clearly defined.
Plate 16. Photomicrograph of splenic tissues of rat infected with S. typhi and treated with 100 mg/kg of C. odorata for 15 days, with non-specific tissue disruption.
Plate 17. Photomicrograph of splenic tissues of rat infected with S. typhi and treated with 200 mg/kg of C. odorata for 15 days, showing normal tissues.
Plate 18. Photomicrograph of splenic tissues of rat infected with S. typhi and treated with 400 mg/kg of C. odorata for 15 days, showing normal tissues.
as well as a significant decrease in mean levels of RBC, Hb, PCV, MCV, MCH and MCHC [
In our study, the haematological profile following inoculation of animals with Salmonella typhi revealed a decrease in total white blood cell count (WBC) and neutrophil levels, and an increase in the lymphocytes and monocytes levels, which agrees with the findings of [
Infection of Wistar rats with Salmonella typhi caused numerous splenic venous stenosis in the red pulp which agrees with the reports of [
MLECO reversed the adverse hematological and pathological changes in the spleen induced by S. tyhi infection at 200 and 400 mg doses, with the 400 mg dose being more effective at reversing splenic changes than ciprofloxacin, suggesting that MLECO may have antibacterial activity against S. typhi.
Isirima, J.C. and Siminialayi, I.M. (2018) Effect of Chromolaena odorata Extracton Hematotoxicity and Spleen Histopathology Induced by Salmonella typhi in Wistar Rats. Pharmacology & Pharmacy, 9, 85-99. https://doi.org/10.4236/pp.2018.94007