| Abstract|| |
Introduction: Toxoplasma gondii; is extreme zoonotic intracellular protozoan, exceeding human medical impact due congenital and cerebral toxoplasmosis. Diabetes is characterized by hyperglycemia resulting from perturbation in insulin secretion, insulin action or both, possible through its long depilating term to flare up the intrinsic (latent) stage of T. gondii. Objective: Is focused on studying the latent toxoplasmosis, based on identifying levels of both average brain parasite load and immunoglobulin titers in diabetic induced rat. Materials and Methods: A total number of 60 Wistar male rats (120-180 g) were divided into four groups, 15 each and experimented as follow; un-infected-normoglycaemic, un-infected-diabetic, infected-normoglycaemic and infected-diabetic groups. Blood samples were taken from rats for glucose determination and for serological assay, and at the same time brain tissue samples for parasite load estimation were collected from three sacrificed rats in each group at sequence days; 1, 15, 30, 45 and 60 cycle to days post diabetes, with the same compatible days at 45, 60, 75, 90 and 105 corresponding to days post infection. Results: Showed significance higher in both blood glucose levels and in cysts number in infected diabetic group than the uninfected-diabetic one. The elevated brain parasite load and IgM titer were always exceeding higher progression all over the course and parallel to the elevated glucose levels in infected diabetic group. Conclusion: Hyperglycemic factor was confirmed to opportunist higher T. gondii brain parasite load and elevate immunoglobulin titer in latent infected and diabetes induced rat, possible match with T. gondii sero-positive diabetic patient, and reflects the importance of serological monitoring in particular uncontrolled hyperglycemic ones.
Keywords: Diabetes, opportunistic cerebral toxoplasmosis, parasite load, rat
|How to cite this article:|
Hassanain MA, El-Fadaly HA, Hassanain NA. Toxoplasma gondii parasite load elevation in diabetic rats as latent opportunistic character. Ann Trop Med Public Health 2014;7:110-5
|How to cite this URL:|
Hassanain MA, El-Fadaly HA, Hassanain NA. Toxoplasma gondii parasite load elevation in diabetic rats as latent opportunistic character. Ann Trop Med Public Health [serial online] 2014 [cited 2021 Apr 14];7:110-5. Available from: https://www.atmph.org/text.asp?2014/7/2/110/146396
| Introduction|| |
Toxoplasmosis is an ideal latent opportunistic zoonoses caused by Toxoplasma gondii, which is obligate intracellular tissue cyst-forming protozoan, with a global distribution in humans, warm-blooded animals, and birds. Human primary infection mainly occurs through consumption of either under cooked meat harboring viable tissue cysts or via environmental oocysts, plus to placental diffusion.  The acute tachyzoite stage is rapidly attacks the nucleated cell via active invasion, proliferate, and destroy the host cells. Latent cystogenic strains (cyst-forming) types II and III are equally predominant (75%) in cerebral and in extra-cerebral human toxoplasmosis forming brain cyst. 
Diabetes is one of the most global chronic hyperglycemic syndromes, due to decrease in either circulating pancreatic insulin (insulin deficiency) or in insulin response of peripheral tissues (insulin resistance). The disease reduces cellular and humeral immune status through their deplorable long hyperglycemic course and possible stimulate latent opportunistic pathogens. ,,
Due to unfit host immune condition, the latent bradyzoites have the fitness to transmutation with powerful opportunistic variability to revert acute blood phase through tachyzoites re-conversion, and order to immunological and physiological stressors or progression to intrinsic stimulus or chronic disease.  But, the actual stimulating dynamic of opportunistic toxoplasmosis is still vague.  Consequently, latent opportunistic human toxoplasmosis possibly consequence to diabetes, the re-converted tachyzoites revert acute blood phase through parasitemia, accordingly initiate both higher brain parasite load and elevated immunoglobulin (Ig) titer specially IgM.  Therefore, these findings suggest that diabetics patients those harboring T. gondii brain cysts are more susceptible to neural signs or encephalitis than the normoglycemic ones. Hence, diabetes may elevate T. gondii brain parasite load in about 5% of the world population whose suffer from diabetes, and may be create miss-diagnostic symptoms or even exploit signs of diabetes. 
The estimated world-prevalence of human toxoplasmosis rises up to a third of the world's population.  Furthermore, the prevalence of T. gondii seropositive patients in the general hospitals in Daejeon was 6.6% with major morbidities, and confirmed diabetes next to malignant neoplasms.  Hence, severe cerebral toxoplasmosis ranks among the 10 most commonly latent opportunistic pathogens, , and proofed the third leading cause of death among immune-suppressed individuals.
The present study aims to clarify the relationship between T. gondii brain parasite loads and IgM/IgG titers in long-term alloxan-induced diabetic rats.
| Materials|| |
Alloxan (2, 4, 5, 6-pyrimidinetetrone) was obtained from Sigma Chemical Co. (St. Louis, MO, USA), as alloxan hydrate in aqueous solution. Alloxan is an oxygenated pyrimidine derivative; it is a toxic glucose analog, creates superoxide radicals, which liberate hydrogen peroxide; selectively destroys insulin-producing cells in rodents and many other animal species.  The alloxan induces long-lasting insulin-dependent diabetes, with characteristics similar to type-1 diabetes in humans.
The T. gondii ME-49 virulent cystogenic tachyzoites strain was secured in Zoonotic Diseases Department, National Research Center, Egypt [Figure 1]a and maintained by an oral or intraperitoneal cycle inoculation of brain cysts [Figure 1]c in mice every 2 months. Under sterile conditions, mice brain was homogenized in a 20-ml Potter's tube, and the bradyzoites [Figure 1]b were counted and diluted as necessary (5-20 factor dilution), ready for oral or intraperitoneal injection for continuous passage in mice. 
|Figure 1: (a) The original number of ME-49 cystogenic strain tachyzoites collected from mice exudates after intraperitoneal serial passage every 72 h interval (×400). (b) ME-49 bradyzoites from brain emulsion (×1500). (c) ME-49 mice intact brain cyst at 45 days postinfection (×400)|
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A total number of 60 pathogen free Wistar male rats of average weight of about (120-180 g) was obtained from Laboratory Animals House, National Research Center, Egypt is used in the experiment. The animals were housed in standard environmental conditions of temperature (24°C) and relative humidity (50%) with a 12:12 light: Dark cycle with free access to a standard commercial diet and water. Experiments were performed according to the Guide for the Care and Use of Laboratory Animals and Ethical Approval of animal rights.
Blood samples and sera were collected for glucose estimation and serological assay, respectively. The collected samples were kept at −20°C until measurements. Furthermore, brain tissue samples were collected for parasite load estimation from three sacrificed rats in each group at 1, 15, 30, 45, and 60 sequence to days postdiabetes (DPD), with the same compatible days at 45, 60, 75, 90, and 105 corresponding to days postinfection (DPI).
| Methods|| |
A total number of 60 rats was divided into four groups; 15 each: Uninfected normoglycemic (UN), uninfected diabetic (UD), infected normoglycemic (IN), and infected diabetic (ID) groups.
Chronic toxoplasmosis induced rats
Rats of both (IN and ID) groups were infected intraperitoneal (IP) by 10 3 of mice brain cysts of T. gondii cystogenic ME-49 virulent strain at the day 45 prior to the onset of diabetic induction, and at the same time animals in IN group are still normoglycemic, the animals in ID group are alloxan-induced diabetic. Three rats were sacrificed and brain tissue was removed sequence to; 45, 60, 75, 90, and 105 corresponding to DPI. At the same time, animals in UN and UD groups are considered control uninfected groups. Chronic infected rats are confirmed by detection T. gondii brain cysts at 45 DPI. 
Diabetes induced rats
Diabetes was induced in rats in UD and ID by IP injection of alloxan monohydrate in a dose of 120 mg/kg body weight, dissolved in 0.9% w/v of NaCl (physiological saline) in overnight fasted rats at 45 DPI.  Blood glucose levels (BGLs) were checked using one touch glucometer, and hyperglycemia confirmed 72 h after alloxan application. Alloxan dose was repeated every 3 days up to 60 DPD to maintain hyperglycemic levels as long as the term of study. BGLs were judged in three sacrificed rats sequence at 1, 15, 30, 45, and 60 DPD.  With the same compatible days at 45, 60, 75, 90, and 105 corresponding to DPI. Normal fasting blood glucose for rat is around 110 mg/dL (~6 mmol/L). Animals present glucose levels lower than 250 mg/dl glucose level were rejected, and only rats exceeding blood glucose than 300 mg/dl considered as diabetic and used for the experiment. 
Immunoglobulin M/immunoglobulin G ELISA antibody titers
The sera were immediately separated by centrifugation of rat blood samples at 3000 rpm for 10 min, sera were kept at -20°C until examined by ELISA using IgM/IgG ELISA diagnostic kits (VIRO, Germany), sensitivity and specificity of kits for IgM and IgG were (100% and 98.1%) and (100% and 99.1%), respectively. ,
Parasite load estimation
Brain tissue emulation was prepared by homogenization with an equal volume of PBS, pH 7.4 and passed through 16-gauge needle 10 times by means of a syringe to release tissue cysts. One drop of the brain homogenate was spread on a slide and the parasite was microscopically counted in average of every 10 ml using hemocytometer. The average parasite load (APL) (10 mg/brain) was estimated by calculating the average number of tissue cysts per brain multiplying in the average number of bradyzoites per cyst. Thus, the practical evaluation of parasite load was done by counting the total number of cyst-free bradyzoites plus in cysts bradyzoites/g/brain in all groups. Furthermore, the elevated parasite load (EPL) (10 mg/brain) were recorded by subtracted the APL in between the IN and ID groups. 
Data were statistically analyzed using the SPSS is a product developed by IBM Corporation (MSTAT and STATISTICA (6.0) computer programs). The average and standard deviation among the different parameters were determined as well.  Only differences with a probability of error of <0.05 were considered significant.
The work is approved ethically by the Medical Research Ethics Committee, National Research Center, Al Tahrir St. Dokki, Giza, Egypt under registration number 1-2/0-2-1.2012.
| Results|| |
Blood glucose levels in four groups are concise with the sequence of both postdiabetic and postinfection days pattern. The ID group shows significance higher BGLs than the UD group during the course of the experiment. Furthermore, significance higher levels were recorded in IN group than the UN control one [Table 1], [Figure 2].
|Figure 2: Compare blood glucose levels (BGLs) (mg/dl) in four groups: The infected diabetic group shows significance higher BGLs than the uninfected diabetic group during the course of the experiment. Furthermore, significance higher levels were recorded in infected normoglycemic group than the uninfected normoglycemic control one|
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|Table 1: Variations of parasite load, ELISA IgM/IgG assay, and BGLs in long-term alloxan-diabetic-induced rats harboring latent T. gondii brain cysts|
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The average cysts number (10 mg/brain) and the average bradyzoites number/cyst in both infected groups (IN and ID) are correlated with the postdiabetic and postinfection days; the higher cysts numbers were recorded in ID, and higher significance than the IN. The curve of cysts number in ID shows continuous progression all over the course and decline at 45 DPD and/or 90 DPI, while the both curves of bradyzoites/cyst show semi-equivalent points all over the course, except at 90 DPD and/or 105 DPI where the bradyzoites number in ID started to elevate corresponding to decline values in IN [Table 1], [Figure 3].
ELISA sero-monitoring concerning IgM and IgG titer in both infected groups (IN and ID) links to both postdiabetic and postinfection days. The IgM curve of ID proves superior progression values in comparison with IgM of IN in a sequence decline. The IgG curves in both (IN and ID) groups always exceed higher than the corresponding IgM curves up to 45 DPD where sero-conversion of IgM was recorded superior values than the corresponding IgG at 60 DPD [Table 1], [Figure 4].
|Figure 3: Compare both average cysts number and bradyzoites number/cyst in infected groups: The curve of cysts number in infected diabetic (ID) shows continuous progression all over the course and decline at 45 days postdiabetes (DPD) and/or 90 days postinfection (DPI), while the both curves of bradyzoites/cyst show semi-equivalent points all over the course, except at 90 DPD and/or 105 DPI where the bradyzoites number in ID starts to elevate corresponding to decline values in infected normoglycemic|
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|Figure 4: Compare immunoglobulin M (IgM) and IgG in infected groups: The IgM curve of infected diabetic (ID) proves superior progression values in comparison with IgM of infected normoglycemic (IN) in a sequence decline. The IgG curves in both (IN and ID) groups always exceed higher than the corresponding IgM curves up to 45 days postdiabetes|
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The APL (cysts number/brain multiplying in bradyzoites number/cyst plus the cyst-escaping bradyzoites) was estimated in 10 mg/brain for both infected groups (IN and ID) related to postdiabetic and postinfection days; the parasite load curve of IN group declined all over the long-term of study in contrast to parasite load curve of ID group which proved superior progression values. The highest values (946 parasite/10 mg brain) at 15 DPD and/or 60 DPI revert higher at 60 DPD and/or 105 DPI [Table 1].
The EPL (bouncy estimation of the APL between the IN and ID groups) was estimated in 10 mg/brain and coupled to diabetic and infection series days, and is compared with the hyperglycemic sequences in the same days and ID group. The result values always exceed higher progression all over the course and parallel to the elevated glucose levels; up to sharp parasite elevation at the beginning of 45-60 DPD and/or 90-105 DPI, with continuous altitude even with the beginning of decline hyperglycemic curve [Table 2], [Figure 5].
|Figure 5: Compare parasite load with hyperglycemic levels in infected diabetic group: The elevated parasitic load curve always exceeds higher progression all over the course and parallel to the elevated glucose levels; up to sharp parasite elevation at the beginning of 45-60 days postdiabetes and/or 90-105 days postinfection|
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| Discussion|| |
Toxoplasma gondii is a zoonotic opportunistic protozoan, characterize higher brain parasite load in immune-compromised personnel, associated with neurological manifestations. The prevalence rates of diabetes and toxoplasmosis are exceeding all over the world. Where, the estimated world-prevalence of human toxoplasmosis was up to a third of the world's population  and 60% of diabetic patients were positive for the T. gondii antibody. Diabetic patients are more susceptible to toxoplasmic encephalitis than normoglycyemic individuals.  Thus, the question is how extent diabetic patient can stimulating opportunistic cerebral toxoplasmosis and elevate brain parasite load.
In the present study, we have succeeded in induce 2 months long-lasting diabetes and maintain hyperglycemic levels (≥300 mg/dl) in diabetic rat groups (UD and ID) with higher levels than the control one UN, which still within un-diabetic levels. The elevated BGLs within the ID group were higher than the UD one, as long as the 2 months study. The significance higher of new cysts number in ID group concise with the decline cyst number in IN group with 60 days course denoted the progression of hyperglycemic levels, exciting the latent in-cyst bradyzoites to opportunist and forming novel cysts, which confirmed through the little number of dividing zoites than the original cysts in IN group.
The uppermost cyst number in ID group was recorded at 45 DPD; symbolize the end of opportunistic out-cyst dividing phase, followed by decrease cyst numbers sequence to the decline in hyperglycemic values at the begging of in-cyst dividing phase. The results indicated long-term hyperglycemia in diabetic rats possible to opportunist higher T. gondii brain cyst, this is due to diabetes eventually induces production of reactive oxygen and nitric oxide which is the most stimulant initiating opportunist intracellular pathogens  associated with benign or sever encephalitis course due to inflammatory reaction of migrated tachyzoites.  The number of bradyzoites/cyst was little or semi-equivalent values in ID than the IN groups as long as the end of opportunistic out-cyst dividing phase up to 45 DPD, where bradyzoites/cyst in ID group were prevail over than the IN one sequence to in-cyst dividing phase.
In the present study, ELISA showed IgM and IgG variant titers in the two infected groups: IN and ID. Usually, IgM begins hours after primary infection and sooner than IgG, followed by sequence decline parallel with sequence elevated IgG titers, therefore, the IgM titer normally do not exceed the matching IgG titer except at the starting of both primary or opportunistic infection.  Hence, in the present study, the sharp elevated IgM titer recorded superior values than the analogous IgG in ID group at 45-60 days of diabetic duration, signifies powerful success of tachyzoites stage re-conversion, able to excite blood phase relapse, and reverts acute parasitemia, which was confirmed by the augmented titer of IgM.
The APL of ID group established higher significance progression values in contrast to decline values in IN group, and reflect the effectual hyperglycemic factor for elevating APL. Furthermore, the EPL is always exceeds higher progression with the vital dynamic hyperglycemia in ID group; clearing that the exceeding in EPL is irreversible even with decline BGLs in rats, and denoted the possible particular hyperglycemic values in brain of diabetic patients. The average number of bradyzoites in ID rats group exceeds 35.7% than the count in IN group. According to this average statistics, we have expected that the brain in ID patient possible opportunist a huge number of bradyzoites, may produce brain inflammatory response with neuro-psychiatrics alterations particularly toward dopamine.  The nervous signs develop in T. gondii seropositive diabetics, may be via tachyzoites brain migration or diabetic peripheral neuritis. Until now, it is not known even diabetes stimulate higher T. gondii brain cysts or the invading tachyzoites have a role for stimulating diabetes susceptibility, it may be excited progression to pancreatities due to T. gondii acute relapse, which could directly damage β cells  and enhance diabetes. Furthermore, patient's death due to acute toxoplasmic pancreatitis has been reported, and the disseminated tachyzoites in pancreas has been described in other clinical and pathological studies. ,,
The current study revealed that normoglycemic level is very important factor for controlling extra brain parasite load, and diminishes nervous signs in diabetic patients, where the fit normoglycemic diabetic will be successfully interacting with the outside escaping tachyzoites.
| Conclusion|| |
Diabetes and toxoplasmosis have two directions; possible stimulate or maximize the nervous signs and complications of each other. Therefore, studying of T. gondii latent opportunity and diabetes should be supported by the difference of both parasite load and Ig titers and BGLs among the ID groups. Furthermore, validate the value of periodical check and symptomatic differential diagnostic of diabetics particularly with immune-compromised, elderly, pregnant diabetics, and who suffering nervous signs.
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Mohey A Hassanain
Prof. of Parasitology and Zoonotic Diseases, Department of Zoonotic Diseases, Tahrir Street, Dokki, Giza
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]