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Molecular, Hematological and Biochemical Investigation of Trypanosoma spp. in Sheep

Enas M.M. Al-Eodawee1, Israa M. Essa1, Ola A. Aggar1, Hasanain A.J. Gharban1,*
1Department of Internal and Preventive Veterinary Medicine, College of Veterinary Medicine, University of Wasit, Wasit-52007, Iraq.

ABSTRACT

Background: Trypanosoma spp. is a flagellated unicellular protozoan parasite which caused a disease known as trypanosomiasis in various animals among many areas worldwide resulting in severe economic losses due to morbidities and mortalities. This study was conducted to estimate the prevalence of Trypanosoma spp. in sheep using the molecular assay with detection the impact of infection on various risk factors and blood markers.

Methods: Totally, 341 sheep were selected from different areas in Wasit province (Iraq) during June to September (2023) and subjected to draining the venous blood that tested molecularly by the conventional polymerase chain reaction (PCR). Status of different hematological, biochemical and mineral markers were further investigated.

Result: Targeting 16S sRNA gene, 7.92% animals were positively infected with trypanosomiasis. Relation to risk factors, positive infection and risk were significantly higher in females than males and in sheep aged >1-3 years than others. Hematological findings reported a significant reduction in RBCs and Hb; while biochemically, significant elevation was reported in concentration of urea. Regarding various minerals, insignificant alteration was seen in infected sheep when compared to healthy ones.

INTRODUCTION

Trypanosoma belongs to Trypanosomatida Family of Kinetoplastea Order, which first diagnosed in Indian camels by Griffith Evans in 1880 (Eyob and Matios, 2013; Borges et al., 2021). Then, many domestic animals were found to be incidentally infected with trypanosomiasis including T. congolense in goats, cattle, sheep, horses and dogs; T. vivax for cattle, horse, sheep and goats; and T. brucei and T. evansi for all mentioned animals as well as camels. Different studies were carried out attempting to clear the higher risk to reinfection with trypanosomiasis in an endemic farms (Auty et al., 2015; Vourchakbe et al., 2023). One of the most important key factors is the survival  mechanism that developed by this parasite to escape the host immune response is through antigenic variation and exhibiting on various membrane surface glycoproteins (Cardoso et al., 2016; Pech-Canul et al., 2017).
       
Consequently, this mechanism makes some of the animals in the infected herds act as a reservoir and reemerging of trypanosomiasis (Jilo et al., 2017). Whenever, the great risk of infection is primarily related to the existence of nearby wildlife reservoir such as bushbuck (Tragelaphus scriptus), warthog (Phacohoerus aethiopicus), bush pig (Potamochoerus porcus), lion (Panthera leo), white rhinoceros (Ceratotherium simum), leopard (Panthera pardus) and wild Equidae as well as hematophagous flies (stable flies, Tabanids and Stomoxys) that transmit the disease (Maharana et al., 2019; Eid et al., 2023; Agrawal et al., 2024).
       
The transmission of infection to various animals can occur via several routes; however, the most commonly is the cyclical transmission when the parasite is inoculated with saliva of infected tsetse flies (Abdi et al., 2017). Other routes are the mechanical transmission by biting insects especially when interrupting during feeding and intrauterine during partum by vertical transmission (Desquesnes et al., 2013). Oral transmission of trypanosomiasis may also occur particularly after birth with T. brucei when calved animal ingested contaminated blood and fluid during parturient; however, this route also reported in carnivores when fresh infected prey is eaten (Fagiolo et al., 2005; Salman and Steneroden, 2014). The high risk of re-infection and outbreak occurrence is usually differently related to the prevalence of vectors in the endemic area which depends mostly on the climate factor-like temperature and humidity in tropical regions that are favorable for vector breeding all year seasons (Liu and Zhou, 2015; Maharana et al., 2016). Also, the seasonal influence is related to activity of vectors climate changes outcome (Booth, 2018).
       
Since many factors may impact on the prevalence of trypanosomiasis including ecological zones, type of husbandry practices, the affected breeds and applied to control regulates (Meyer et al., 2016; Agrawal et al., 2020). In Iraq, sheep trypanosomiasis was largely neglected (Mahmood and Alobaidii, 2022); therefore, this study was conducted to estimate the prevalence of Trypanosoma spp. in sheep using the molecular assay with detection the impact of infection on various risk factors and blood markers.

MATERIALS AND METHODS

Samples
 
An overall 341 sheep of both sexes and different ages were selected to the current study at different areas of Wasit province (Iraq) during June to September (2023). Each study animal was subjected to collection 5 ml of jugular venous blood into an EDTA-K2 vacutainer plastic tube (AFCO, Jordan). At laboratory Unite (College of Veterinary Medicine, University of Wasit), blood samples were examined hematologically and then centrifuged (3000 rpm / 5 minutes) for biochemical and mineral markers.
 
Hematology
 
Automated hematological analysis was performed by Mythic 18-Vet Analyser (Orphee SA, Switzerland) to obtain the main hematological parameters (Gharban and Yousif, 2020). Total red blood corpuscles (RBCs) count, hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), total white blood cells (WBCs) count, lymphocytes (L), monocytes (M), neutrophils (N), eosinophils (E) and basophils (B) were measured. The steps of analysis were applied in based on manufacturer instruction. The values of lymphocytes/ neutrophils (L/N) ratios were measured manually.
 
Biochemical markers
 
Serum samples of all study camels were analyzed using of specified kits (Biomaghreb/Tunisia) to measurement the total protein, albumin, globulin, blood urea, creatinine, cholesterol, glucose and total bilirubin; while albumin/globulin (A/G) ratio was calculated based on obtained results.
 
Minerals
 
Iron (Fe) was determined by the atomic absorption spectrophotometery using a commercial available kit (SunLong Biotech/China); while other minerals including serum calcium (Ca), chloride (Cl), magnesium (Mg), phosphorus (P), sodium (Na) and potassium (K) were measured according to manufacturer instructions of each specific colorimetric assay kits (Linear / Spain).
 
Molecular study
 
Following the manufacturer instructions of the G-spinTM Total DNA Extraction Kit (Intron Biotechnology, South Korea), the frozen blood samples were thawed, processed and the extracted DNAs were tested for quantification of purity and concentration by the NanoDrop Spectrophotometer System (Thermo Scientific, USA). Targeting 18S rRNA gene, one set of primers was designed [F: (5'- GGG AAT ATC CTC AGC ACG TT-3') and R: (5'- ACT GGG CAG CTT GGA TCT C-3')] based on the GenBank Iranian isolate (Ref. No. KC427089.1), the MasterMix tubes of the AccupowerÒ PCR Premix Kit (Bioneer, South Korea) were prepared at 20 ml and subjected to Thermal Cycler conditions as following: 1 cycle initial denaturation (95°C, 5 minutes), 35 cycles of denaturation (95°C, 30 seconds), annealing (58°C, 30 seconds) and extension (72°C, 1 minute) and 1 cycle final extension (72°C, 5 minute). PCR products were analyzed by electrophoresis of agarose-gel stained with Ethidium Bromide at 80 Am and 100 Volt for 90 minutes. Using the UV Transilluminator System (Clinx, China), the positive samples were detected at a product size of 300 bp.
 
Statistical analysis
 
All study results of hematology, biochemical markers, minerals and coagulation factors were analyzed by the t-test (GraphPad Prism Software), to identify significant variation at P<0.05 (*) and P<0.01 (**) between the infected and healthy groups. Study values recorded as either percentage (%) or Mean ± Standard Error (M±SE), (Gharban, 2022).

RESULTS AND DISCUSSION

Molecular testing
 
Of 341 sheep selected, 37 (10.85%) animals were showed a positive result to Trypanosoma spp (Fig 1, 2). In endemic areas, Trypanosoma spp. considered as one the major veterinary problems, worldwide.
 

Fig 1: Total molecular results for testing 314 sheep by the conventional PCR assay.


 

Fig 2: Agarose-gel electrophoresis of some PCR products at 80 Am, 100 Volt for 90 minutes.


       
In this study, 10.85% of sheep were infected with Trypanosoma spp. using the conventional PCR assay; which comparatively less than reported recently in Iraq (21.04%) by Mahmood and Alobaidii (2022). Globally, the prevalence of sheep trypanosomiasis was 1.3-1.7% in Algeria (Benfodil et al., 2020), 2% in Ecuador (Coello-Peralta et al., 2020), 8.3% in Somalia (Hassan-Kadle et al., 2020), 24.3% in Chad (Vourchakbé et al., 2020) and 28.3% in Brazil (de Araújo-Neto et al., 2023). Random selection of study sheep grazing at different areas, ages and sexes might explain variation between the results of these studies. Also, the availability of suitable medication at some areas with the availability of awareness towards chemoprophylaxis and providing of good feeding might be considered as additional reasons for this difference. However, the susceptibility of sheep to Trypanosoma is conflicted between studies according to the species. Some authors mentioned that sheep are susceptible to T. vivax, T. evansi and T. equiperdum (Parra-Gimenez and Reyna-Bello, 2019); while others reported that sheep are susceptible to T. brucei (Vourchakbé et al., 2020) and T. cruzi (de Araújo-Neto et al., 2023).
 
Risk factors
 
Relation to sex, values of positivity and risk were significantly higher in females than males; while for age, values of positivity and risk were increased significantly in sheep of >1-3 years old when compared to others; 6 months, 6-12 months, >3-4 years and >4 years old (Table 1). The factors of age and sex remain the main factors known to be affected the prevalence of any infection among different animal species. Reducing of maternal immunity after 6-12 months, grazing of animals in pasture and exposure to vector/ contaminants, as well increasing of stress due to nutritional deficiency and maturity could explain the high incidence of infection after 1 year of age. The absence of positivity in study lambs of <6 months could result by either maternal immunity or high attention by owners due to the high economic benefits. Decreased susceptibility of adult population compared to younger could be caused by the chronic nature of disease that results in development of specific antibodies against Trypanosoma spp. and increasing resistance to concurrent parasitaemia.
 

Table 1: Association of PCR results to risk factors.


       
Although, both females and males are grazed out on same pastures, we showed that the parasite is more prevalent in females than males. This might be caused by either the low number of tested males or that females exposed to additional stresses due to pregnancy and lactation.
 
Hematology
 
In this study, infected sheep were showed a significant decrease (P<0.05) in values of RBCs and Hb only when compared to those of healthy ones (Table 2). Concerning leukocytes, insignificant differences were seen among infected and healthy sheep (Table 3).
 

Table 2: Association of PCR results to hematocrit.


 

Table 3: Association of PCR results to leukocytes.


       
Worldwide, hematology is becoming increasingly more importance in diagnostic and management tools in veterinary medicine. The blood picture of animal provides an opportunity to investigate the presence of abnormalities and help in distinguishing the normal state from diseased one (Roland et al., 2014). Decreased values of total RBCs and Hb of infected sheep with lack of other hematological alteration was seen in this study. Lower hematological values are suggestive an occurrence of anemia that considered the common feature of clinical trypanosomiasis. Boada-Sucre et al., (2016) confirmed experimentally that the parasite adhesion to RBCs was decreased significantly at late stage of infection due to the effect of immunity. However, various authors explain that the modification of trypanosomal antigens lead to erythrolysis and removing of infected RBCs (Rossi et al., 2017; Rao et al., 2021).
       
Fidelis Junior et al., (2016) revealed that leucopenia due to severe neutropenia and lymphopenia were appeared during the acute phase but not at chronic phase in experimentally infected cattle; whereas, Akpan et al., (2018) showed that significant leucocytosis due to lymphocytosis and neutrophilia was seen in experimentally infected sheep at 2nd week; however, no significant changes were seen in monocytes, esinophils and basophils. In camel, Derakhshanfar et al., (2010) reported that lymphocytosis occurs following chronic infection; while, Sivajothi et al., (2015) recognized that leuckocytosis due to lymphocytosis occurs in acute phase and followed by leucopenia during chronic phase.
 
Biochemical analysis
 
Data of serum biochemical markers revealed that no significant alteration (P>0.05) was identified in levels of all measured markers except urea that increased significantly (P<0.0356) in infected animals (Table 4). We thought that the type of infection (naturally or experimentally), species of Trypanosoma causes the infection and stage of disease have affected largely on the pathogenesis of trypanosomiasis and then physiological disorders occurred. However, the findings of increasing serum urea were observed by other studies in monkeys infected with T. rhoesiense (Sadun et al., 1973), human infected with T. gambiense (Awobode, 2006), rabbits infected with T. congolense (Ameen et al., 2012), sheep infected with T. vivax (Onasanya et al., 2018), cattle infected with T. theileri (Bittner et al., 2019) and camels infected with T. evansi (Al-Abedi et al., 2020). This might because that trypanosome infection in sheep leads to significant pathological and functional disorders in vital organs including liver and kidney.
 

Table 4: Association of PCR results to biochemical markers.


 
Mineral status
 
Regarding values of serum minerals, values of Ca, Cl, Fe, K, Mg, Na and P were showed insignificant alteration in their values when compare between the positively infected sheep with trypanosome and healthy ones (Table 5). Diseases such as trypansomiosis can affect mineral levels in serum of healthy sheep and other domestic animals (Masters, 2018). In Nigeria, Neils et al., (2006) detected that serum minerals responded actively in sheep infected experimentally with T. congolense suggesting the role of this species in the pathogenesis of disease. Da Silva et al., (2011) measured serum minerals in experimentally infected rabbits with T. evansi concluding that changes in circulating levels of these markers can be showed when the clinical signs of disease are observed. Hussain et al., (2018) mentioned that the pathophysiology or mechanism of trypanosomiosis in naturally infected buffaloes is complicated and multi-factorial in origin, indicating the effect of oxidative products in depletion of minerals. In Germany, Bittner et al., (2019) recorded a significant trace mineral deficiency in grazing cattle suggesting the importance of surveillance of mineral status in herds especially when forage quality is low.
 

Table 5: Association of PCR results to minerals.

CONCLUSION

In Iraq, information concerned the prevalence of Trypanosoma spp. in different animals remain very limited in particular in sheep. In this study, molecular PCR assay demonstrated a high efficacy in detection of Trypanosome infections in sheep; however, we recommended to application of phylogeny that can provide more information about the species of parasite and its identity to other global strains. Variation in different hematological and biochemical markers might depend on the Trypanosoma species, stage of infection (acute or chronic), type of infection (experimental or natural), species of parasite and animals. Hence, furthermore studies are greatly important to support our information about the prevalence of disease in other areas and in other animals.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

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