Assiut University web-site: www.aun.edu.eg

CLINICAL, PARASITOLOGICAL, SEROLOGICAL, AND MOLECULAR DIAGNOSIS OF TRYPANOSOMIASIS IN CAMELS (CAMELUS DROMEDARIUS)

ADEL E.A.M.; ALSAGHER O.A.A.; HASSAN Y.A.H.M. and MONA A.S.A.

Department of Animal Medicine, Faculty of Veterinary Medicine, South Valley

 University, Qena City, Qena 83523, Egypt, 2018

Received: 26 December 2018;     Accepted: 31 January 2019

INTRODUCTION

Historically, at 1880 in India was the first detection of Trypanosoma evansi in blood of camels and horses by Evans, (Leadbeater et al., 2000). The Trypanosomiasis distributed throughout tropical and subtropical regions of the world. In Africa, camels were the most vital host, while in Central and South America the steed was primarily influenced. In Asia, a much more extensive scope of the hosts, including dairy cattle, wild ox and pigs. And the incubation period of surra varies from 5 - 60 days and may extend to 3 months and the parasite appears in the blood before the 14^thday of infection. (Eyob et al., 2013).The Salivated trypanosomes were passed to   the   recipient   in  the  saliva  of  the   tsetse   fly

Corresponding author: MONA A.S.A.

E-mail address: monaazab2006@yahoo.com

Present address: Department of Animal Medicine, Faculty of Veterinary Medicine, South Valley University, Qena City, Qena 83523, Egypt, 2018

(Glossina spp.). (Ianet al., 2004), and transmitted mechanically by biting insects, (Desquesnes et al., 2013). Clinically camels manifested by fever, anorexia, edema and die; the chronic form was characterized by severe emaciation, intermittent fever, marked generalized muscular atrophy, pale mucous membranes and occasionally abdominal edema, and Sweat odor of urinary ketone, (Higgin, 1983). Anemia appears to be a major component of the pathology of Surra disease, and its continuation initiate anoxic conditions (Enwezor and Sackey, 2005).

The diagnosis of camel Trypanosomiasis still unsatisfactory up to date, because of the non-pathognomonic signs, (Dargantes et al., 2005). The routine microscopic examination requires sensitivity, and serological examination requires specificity or sensitivity, and the use the molecular detection PCR test is a more trusted technique for diagnosis and epidemiological studies, (Gonzales et al., 2007).

Various techniques have been utilized to identify non-particular humoral antibodies present in animals infected with trypanosomiasis. Like formol-gel, mercuric chloride precipitation test, and thymol turbidity test, and are thought to be old technique despite the fact that the formol-test may even now have some utilization in the field since it is easy to proceed. most tests rely on the high amount of serum globulins after taking the infection. (OIE, 2012).

RoTat 1.2 VSG "variable surface glycoprotein" is available in all T. evansi however not in      T. brucei (Claes et al., 2004). T. evansi are contained homogeneous DNA mini-circles, and they loss a part of the kDNA maxi-circles which described as T. evansi type B and a special primers to give positive results by the molecular detection, and all the results assures the presence of T. evansi type B isolates, T. brucei, and T. evansi type A without RoTat 1.2 gene in Kenyan isolates. (Njiru et al., 2006).

The clinical and non-clinical cases were positive by the DNA genome (TBR) while the clinical cases only detected by ITS and RoTat primers. (Elhaig et al., 2013).

Determination of the hematological and biochemical changes, (Hb %), PCV and RBCs count were significantly decreased, TLC, lymphocytes, and Monocytes demonstrated a significant increase. (Hilali et al., 2006), and Hematological analysis revealed a significant decrease the TEC, Hb% and PCV, However, the TLC and differential Leucocytes did not differ except for Eosinophils value which was significantly increased in Trypanosoma infected camels.  (Eljalii et al., 2015).

Liver function tests uncovered a significant increase in the action of lactate dehydrogenase catalyst (LDH), globulin, total bilirubin, and indirect bilirubin while alkaline phosphatase enzyme (ALP) indicated a significant decrease. Kidney function tests showed a marked decrease of both creatinine and urea. (Hilali et al., 2006). The Plasma total protein, albumin, globulin, cholesterol and urea concentrations were insignificantly decreased. (Omer et al., 2007). This study aimed to Investigate Trypanosoma evansi affecting camels in early stages, to estimate the haemato-biochemical values of camels infested and suspected with Trypanosomiasis, and also to evaluate a simple PCR-based technique for field diagnosis of Trypanosoma infection in camels, at Aswan governorate.

MATERIALS AND METHODS

1.  Animals and samples:

A total number of (106) one humped camels (Camelus dromedarius) from 4 localities in Aswan (Daraw, Aswan west Village, Sohil west Village, and Abu elreish village.) was carried outFrom January 2017 to December 2017.

Table 1: Showing number of examined camels in each area.

Camels in Daraw imported from Sudan and these animals not resident and sold, While camels in Aswan west village, Sohil west village, and Abu elreish village were resident and used to ride tourists, camels in villages grazing with other animals like sheep and goat.

 Age of the examined camels were 5 to 8 years old except 7 camels were 3 years old.

Whole blood samples of camels were collected by puncturing jugular vein into 2ml Ethylene Diamine tetra-acetic acid (EDTA) coated vacutainers tubes. Then kept in a cooler box and transported for lab activities. And another one in plain test tube to obtain serum.

METHODES

Clinical examination:

Camels were subjected to clinical examination according to the methods described by (Higgin, 1983).

Microscopic examination of blood smears:

It was carried out according to (Ijaz et al., 1998)

Formol gel test:

The formol gel test (FGT) was carried according to. (OIE, 2012) by adding two drops of concentrated formalin solution (37 % formaldehyde) to 1 ml of serum

Molecular diagnosis: (PCR TEST)

The test carried out at the ARRI (animal reproduction research Institute) bio- technology unit.

All chemicals and reagents were molecular biology grade and reagents were prepared according to (Sambrook and Russell, 2001). Finally, the DNA amplification carried out according to (Sarataphan et al., 2007). (Masiga et al., 1992) (Konnai et al., 2009), (Salim et al., 2011).

Heamatological and biochemical parameters:

Total Erythrocyte Count (TEC) and Total Leukocyte Count (TLC) by Neubauer’s haemocytometer and differential leucocytic count two blood smears were taken from each blood sample and stained with Giemsa (Bancroft et al., 1996). Whereas the concentrations of serum total protein (Biuret Method) and hemoglobin concentration (Hb), albumin concentrations, ALT, AST by using commercial test kits (spectrum-diagnostic) and spectrophotometer according to (Brit, 1967), (Kaplan et al., 1983), (IFCC, 1986), (ECCLS, 1989), (Tietz, 1990), (Young, 1990), (Tietz     et al., 1994), (Young, 1995), (Tietz et al., 1995),  (Burtis, 1999), (Young, 2001).

Statistical Analysis

The Significance of the results was evaluated using Analysis of variance (ANOVA) using Statistical Package for Social Science (SPSS) computer programs (2002).

RESULTS

Examination of 106 camels in this study revealed 88 (83 %) camels were apparently healthy, and 18 (17%) camels showed clinical abnormalities, loss of condition, weakness, depression, and a rough coat., pale mucous membrane and anemia, emaciation and thinning of the hump and prominent ribs, some cases show a rise in body temperature and watery eye, diarrhea with the hindquarters soiled with feces. As in photo (1).

By microscopic examination founded 7 (6.6%) positive Smears 6 of them were from the 18 clinically suspected camels and one from the 88 apparently healthy camels as in photo (2).

By using FGT result revealed 13 (12.26%) serum sample give positive result by Formol gel test, 2 serum samples were positive from the 18 clinically manifested camels and the 11 serum samples from the 88 apparently healthy camels. And there was only one sample give positive result by both microscopic examination and Formol gel test.

Table 2: Comparative evaluation of the techniques used for diagnosis of Trypanosoma during different seasons.

15 samples includes (5 positive samples by microscopic examination including the only one positive sample by both microscopic examination and formol gel test and 10 samples were negative by microscopic examination and 4 of them were positive by formol gel test,), examined for PCR total genomic DNA of camels blood samples were amplified using TBR1, and RoTat1.2 primers. A DNA fragment of 164 bp., and 151 bp. was amplified. The TBR-PCR revealed that 13 (86.7%) samples were positive for infection photo (3). All samples which were positive to T. evansi infection by blood film (5 samples including the positive sample by FGT) were positive by PCR while the remaining positives by PCR (8 samples) were negative by blood film, and 2 samples of them were positive by FGT While Amplification of the RoTat 1.2 primer (in the same 15 samples) was carried out. The results revealed all samples were negative (0 %) photo (4).

Table 3: Comparative evaluation of the techniques used for diagnosis of Trypanosoma.

43 samples (including the 5 positive samples by blood film) were subjected to the determination of TEC, TLC, Leuckocytic count, and Hb%. The data of the mean standard error of the mean for TEC, TLC differential leuckocytic count and Hb % in suspected and healthy camels with Trypanosoma evansi in the table (4), revealed that decrease in Hb%, TEC, and increase in TLC, Neutrophils and Eosinophils.

Table 4: Heamatological parameters in suspected camels.

Biochemical findings: The same 43 samples were subjected to the determination of serum proteins components (μg/dl) and liver enzymes (AST, ALT) and blood glucose level. The data of the mean ± standard error of the mean for serum proteins components (μg/dl) and liver enzymes (AST, ALT) and blood glucose in healthy and suspected camels with Trypanosoma evansi in the table (5), the result revealed a decrease in blood glucose level, AST and increase in ALT level, total protein, albumin, and globulin amounts.

Table 5: Biochemical parameters in suspected camels.

Photo 1: Trypanosoma evansi by microscopic examination.

Photo 2: Animal clinically suffering from trypanosomiasis.

Photo 3: TBR- PCR results: Agarose gel electrophoresis of amplified T. evansi DNA (164 bp PCR products). M: 100 bp DNA ladder as a standard marker, Lane (1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 14 and 15) Positive PCR products. Lane (8, and 9) Negative PCR products .

Photo 4: RoTat 1.2-PCR results: Agarose gel electrophoresis of amplified T. evansi DNA (151 bp PCR products). M: 100 bp DNA ladder as a standard marker, all samples were negative (giving several non- specific bands) including the 5 positive samples with microscopical examination and all the TBR primer positive samples.

DISCUSSION

These clinical signs were similar to those described by (Higgin, 1983).

Microscopic examination of stained blood smear revealed that the total infected camels was 7 (6.6%) similar to (Abd-Elmaleck et al., 2014), differ to (Mahmoud M., 2008), It is important to mention that because of microscopic low prevalence (6.6%), detection of Trypanosoma evansi in the blood of examined animal is suitable only in acute infection where the number of trypanosomes in blood is very high (parasitaemia). On the other hand in chronic infection trypanosomes in blood are scanty or completely absent, so the microscopic examination is not successful at this stage due to the result revealed that TBR-PCR high prevalence (86.6%) similar opinion was mentioned by (Chaudhary et al., 2010, and Shahzad et al., 2010).

All the microscopic confirmed camels samples were negative (except one were positive) in the Formol Gel Test, this serological test showed poor to slight accordance with any other diagnostic test for surra applied in the present study. Similar to (Chappuis, 2005), (Aslam     et al., 2010). The apparently poor specificity of the FGT is explained by the fact that it detects, in a non-specific way, high concentrations of plasma immunoglobulins that can be due to any acute or chronic infection causing hyper gammaglobulinemia. (Tehseen et al., 2015).

The highest infection rate of Trypanosoma in the study area were in spring and autumn which may be had an occasional variety during the rainy season and in the early dry season due to the rapid spread of insects similar to results mentioned by (Higgin, 1983), (Mahran, 2004), (Mahmoud M., 2008), When using molecular method (PCR) in this study TBR-PCR (86.6%) was the more specific and sensitive method of the all methods used during this study, TBR primers can be utilized routinely as an indication for early detection of animal reservoirs in the acute and chronic phases, that helps in rapid treatment of diseased animals and/or disposal of reservoirs of infection (Fernández et al., 2009, Pruvot et al., 2010).

However, this study revealed that many camels were negative by blood examination but positive by TBR-PCR, which may be related to low parasitaemia and/or low sensitivity of the stained blood smear technique and indicates that low parasitaemia might be due to early infections, chronic infection and/or lower strain virulence. The high proportion of non-clinical infection of T. evansi among the investigated camels was in agreement with the findings in the Upper Egypt of (Mahran, 2004).

Generally, the percentage of detection and the sensitivity of the PCR is variable depending upon the primers employed, which are determined by the number of copies and the homology of the primers with the target sequence (Fernández et al., 2009).

The negative samples RoTat1.2 PCR targets in this study agreed with some previous studies (Elhaig et al., 2013), and these negative results may be due to the trypanosomes may be Trypanosoma evansi type B (lacks of RoTat genome)which detected in Sudan (Salim et al., 2011) or may the sequence assorted variety of the RoTat1.2 gene in Egyptian trypanosome detected from camels, especially in relationship with parasite long perseverance in camels because of the chronic period of the disease (Amer et al., 2011).

The suspected camel showed significant decreases in total erythrocytes count (TEC), hemoglobin (Hb), and a non significant increase in total leucocyte count (TLC), There was a significant increase in Alanine Amino Transferase (ALT), Total protein, globulin show a significant increase while glucose, Aspartate Amino Transferase (AST) level didn’t differ significantly in suspected camels than healthy ones. The hematological and biochemical parameters changes are similar to (Hilali et al., 2006), (Eljalii et al., 2015), and differ to (Omer et al., 2007).

CONCLUSION

Finally, according to this study we can say that trypanosomiasis in camels was prevalent in Aswan, either by clinical and non-clinical cases and therefore, reliable diagnosis should be used for rapid treatment or control of the disease., the clinical examination of camels suspected with trypanosoma not a reliable method, the Microscopic examination frequently used for diagnosis of trypanosoma infections had low sensitivity. and depends on the acuteness of the case, high parasitaemia and the good technician skills, while the use of FGT as a field test for diagnosis of trypanosomiasis in camels, it is easy to apply, but it is non-specific technique because it depends on the presence of high amount immunoglobulin's which may occur in any other chronic disease. The TBR-PCR test is the more reliable and easy technique for diagnosis of Trypanosoma evansi (more specific and sensitive method). The all negative samples obtained by using RoTaT1.2- PCR test revealed presence of Trypanosoma evansi type B which lack to VSG which recently detected in Kenya, Ethiopia, and Sudan, or it may be any other trypanosomes except T. evansi type A. and Camel blood is consider as a mirror of the physiological or adverse conditions the heamatological and biochemical parameters changed due to the infection camels by trypanosomiasis. And recommended that Should Use of a simple PCR-based technique for routine field diagnosis of Trypanosoma for early detection, treatment, and control. Need for more investigation of T. evansi by using of suitable PCR test primers to determine the presence of T. evansi type B isolates, T. b. brucei and presence of T. evansi type A without RoTat 1.2 gene in Egyptian isolates. 

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