SOME STUDIES ON TRYPANOSOMIASIS IN IMPORTED CAMELS

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

SOME STUDIES ON TRYPANOSOMIASIS IN IMPORTED CAMELS

AHMED M.A. ZAITOUN¹; SAFAA S. MALEK¹; KHALED A.S. EL-KHABAZ¹ and

SALHEEN G. ABD-EL-HAMEED²

¹ Infectious Diseases, Department of Animal Medicine, Faculty of Veterinary Medicine, Assiut University

² Field Practitioner

Received: 15November 2016;       Accepted:20 December 2016

INTRODUCTION

Trypanosomiasis caused by Trypanosoma evansi is the most important single cause of morbidity and mortality in camels. The disease transmitted non-cyclically by haematophagus flies (eg. Tabanus), and is endemic in Africa, Asia, central and South-America. Because of the wide spread of the disease, its control has attracted international attention, with focus on formulating and implementing effective strategies aimed at increasing productivity and achieving decrease in morbidity and mortality Obihiro, (1998). Trypanosomiasis is an acute/chronic disease of camel results progressive anaemia, anoxic condition and immunosuppression which later develops and predisposes the animal to other infections and death if untreated. It causes economic losses     as   a    result     of     reduced    productivity,

Corresponding author: Dr. SAFAA S. MALEK

E-mail address: safaamalek80@yahoo.com

Present address: Infectious Diseases, Department of Animal Medicine, Faculty of Veterinary Medicine, Assiut University.

abortion in all age groups of pregnancy period, and drop in milk and meat yield morbidity up to 30% and mortality of a round 3%. In Egypt, the instability of local enzootic situation may be come into view through the massive inflow of imported Sudanese camels that may act as a continuous source of exotic Trypanosoma evansi infection El-Said et al. (1998). The accurate identification of camels infected by Trypanosoma evansi is a key factor in the success of any epidemiological surveillance or control program for Trypanosoma evansi infection among camels. The diagnosis of Trypanosoma evansi infection in camels follows the classical diagnostic methods for trypanosomoses involving clinical diagnosis, parasitological, serological as well as molecular techniques Tizard et al. (1979). The specific clinical diagnosis of trypanosomosis is difficult due to non-specific clinical signs coupled with intermittent fever and low parasitaemias FAO, (2000). Diagnosis using PCR could offer a very precise method for detecting infection and discriminating between infected and non-infected animals. The aim of this study to determine the prevalence of Trypanosoma evansi infection among imported camels by using conventional thin blood film and evaluate Polymerase Chain Reaction.

MATERIALS AND METHODS

AREA OF STUDY AND ANIMALS:

Abu-Simbel Veterinary quarantine in Abu-Simbel city at southern of Aswan Governorate which representing the South border of Egypt and considered the major point of entry of the imported camel (Camelusdromedarius) particularly from Sudan and Ethiopia into Egypt. A total number of 396 imported camels (Camillus dromedarius) were studied at the period from September, 2014 till September, 2015. All imported Camels were male in relation to sex. All of camels were subjected to clinical examination according to Kohler- Rollesfon et al. (2001). They classified to clinically suspected cases that show abnormal clinical signs (Emaciation, Diarrhea, High body temperature, Mucous Membrane Paleness, Dullness, Edema and Rough coat), and clinically healthy, As shown in (Table 1).

Thin blood smear:

Ten ml of blood was collected from each animal into a tube with the anticoagulant (EDTA) by jugular venipuncture. Thin blood smears were prepared from each sample soon after collection, fixed with methanol, stained with Giemsa, and microscopically examined at 100× under oil following the procedures described by Sengupta et al. (2010).

DNA EXTRACTION FROM BLOOD SAMPLES AND PCR AMPLIFICATION:

DNA was extracted from whole blood samples according to Mahony et al. (2000). The PCR mixture (25 µl) contained 12.5 µl GoTaq® Green Master Mix (Lot No: 0000062929, Promega, USA). 1.0 µl Upstream Primer TBR-1, 1.0 µl Downstream Primer TBR-2, 5.0 µl DNA templates and 5.5 µl of nuclease free water. PCR was carried out foramplification of 164 bpbyusingahighly repeated sequenceofmini-chromosomesatel-lite DNATBR1/2(TBR1:5 –GAATATTAAACAATGCGCAG-3 and TBR2:5 -CCATTTATTAGCTTTGTTGC-3) (Masiga et al., 1992; Muieed et al., 2010 and Pruvot et al., 2010). The primer set (TBR1 & TBR2) was specific for Trypanosomabrucei, but it would be useful especially outside the tsetse belt in which the kinetoplastic /or akinetoplastic Trypanosoma evansi were widely prevalent but not for Trypanosomabrucei as reported by Inoue et al. (1998). TBR1 & TBR2 found in nucleus not in kinetoplast making results independent of the parasite kinetoplastic state and avoid the problem of failure targeting kDNA(Ventura et al., 2000 and Gonzales et al., 2003). The PCR amplification was performed as follows: initialcycle at 94◦C for 1 min and then 30 cycles of denaturation at 94◦C for 30s, annealing at 60◦C for 1 min and extension at 72◦C for 30s, and finallyonecycle at 72◦C for 2min. Desquesnes et al. (2001). The PCR products were analyzed by electrophoresis Through 1.5% agarose gel containing ethidium bromide (0.5 g/ml) and the image of the amplified DNA was captured using a gel documentation system (Biospectrum UVP, UK).

RESULTS

During the period of investigation from September, 2014 till September, 2015, Total number of 396 imported camels (Camillus dromedarius) were inspected and clinically examined during the quarantine measurements at Abu-Simbel Veterinary Quarantine Station. These imported camels were male in sex.

Out of 396 examined camels, 189 (47.73%) cases showed clinical abnormalities and the remained cases (52.27%) appeared to be clinically healthy. Blood samples of all camels with clinical abnormalities were microscopically examined for detection of Trypanosoma evansi infection and found that 23 (12.17%) cases were harbored infectionas shown in photo no. (10). The remained cases (n=166, 87.83%) were trypanosoma free by using blood film proceduresas showed in table (2). Approximately, 75.30% (n=125) of the clinically suspected cases with negative results of blood film examination were subjected to molecular detection of Trypanosoma evansi infection using PCR based TBR 1/2 primer. It was found that 60 (48%) were positive and 65 (52%) were negative to Trypanosoma evansi infection. On the other hand, all clinically healthy cases (n= 207) of the examined camels (n= 396) were subjected to blood film technique and they were trypanosoma free. Twenty five cases of the clinically healthy were randomly selected and subjected to PCR procedure. It was found that 5 (20%) cases were positive and 20 (80%) cases were negative to Trypanosomaevansi infection as showed in table (3).

The clinical signs of Trypanosoma evansi infection noticed were in acute form which representing 21.74% (5/23 microscopically positive and clinically suspected). The chronic form was representing 78.26% (18/23 microscopically positive and clinically suspected) indicating that the chronic form is more common than the acute form of the disease.

Signs of acute form of Trypanosoma evansi infection were:

Poor body condition with muscular fatigue that interference standing-up and inability to walk to long distance with frequent recumbency. Regarding to the morning at 6.00 O'clock. Rectal temperature of the infected camels, there were rising of body temperature up to (38.81 ± 0.05^°C), it was higher than that recorded for the healthy camels (36.71 ± 0.05^°C). Hyper-lacrimation with congestion of ocular mucous membranes during feverous condition in 8.68 % (2/23) cases, highly characteristic. Edematous swelling in the lower parts of legs was 4.3% (1/23) cases as shown in photo no. (1).

Signs of chronic form of Trypanosoma evansi infection were:

General debility and severe emaciation (disappearance of the hump and the hump appeared as a case filled with extra-soft gelatinous material. Remarkable appearance of the ribs (the ribs were well demarcated from the intercostal spaces), Atrophy of the thigh muscles) as shown in photo no. (2a&b). The hump was bent to one side as shown in photo no. (3). Pale mucous membrane of conjunctivae. Lacrimation and there were gap between the eye ball and bone when press the tears down in 26.09% (6/23) cases. Faeces were normal, or were sometime coated with mucus; and generally tapered faeces were noticed. The skin was remarkably dried, harsh and scurfy, as shown in photo no. (4). They were dullness, stretching of their neck and not response well to external stimulus. The camel was yawning and the tongue was pale or slightly bluish. Enlargement of lymph nodes particularly superficial cervical lymph nodes at the base of the neck in 21.74% (5/23) cases; sometimes surrounded with edematous area, as shownin photos no. (5a&b). Edematous swelling was noticed in 30.43 % (7/23) cases:

a- Edematous swelling at the base of neck in 13.04 % (3/23) cases as shown in photo no. (6).

 b- Edematous swelling in scrotal sacs with enlargement of testicles and the skin covering genital organs were rough (keratinized) and superficially fissured in 17.39 % (4/23) cases, as shown in photo no. (8a&b). Edematous and Prolapsed Penis with disappearance of the characteristic hook shaped tip of the penis by closely examination and obvious signs of Balanoposthitis were observed in 13.04 % (4/23) cases. The outer covering of the edematous penis were eroded and partially sloughed and there were grumbling with signs of pain during palpation, as shown in photo no. (7). Long hair of the humps was breaking-off or sloughed. 34.78% (8/23) of the microscopically positive (Blood film) camels have hard ticks at different sites of animal body particularly genital system, as shown in photo no. (9). Hard ticks may play an outstanding role in spread of Trypanosoma evansi infection.

Regarding the Prevalence of Trypanosoma evansi infection of the examined camels by thin blood smear. Out of 396 examined camels, 189 (47.73%) cases showed clinical abnormalities and the remained cases (52.27%) appeared to be clinically healthy. Blood samples of all camels with clinical abnormalities were microscopically examined for detection of Trypanosoma evansi infection and found that 23 (12.17%) cases were harbored infection. The remained cases (n=166, 87.83%) were trypanosoma free by using blood film procedures. On the other hand, all clinically healthy cases (n= 207) of the examined camels (n= 396) were subjected to blood film technique and they were trypanosoma free.

Approximately, 75.30% (n=125) of the clinically suspected cases with negative results of blood film examination were subjected to molecular detection of Trypanosoma evansi infection using PCR based TBR 1/2 primer. It was found that 60 (48%) were positive as shown in photo no. (11), and 65 (52%) were negative to Trypanosoma evansi infection. Twenty five cases of the clinically healthy were randomly selected and subjected to PCR procedure. It was found that 5 (20%) cases were positive and 20 (80%) cases were negative to Trypanosomaevansi infection.

On the current work the prevalence of Trypanosoma evansi infection using blood film technique was 5.81% of all tested camels (12.17% among clinically suspected camels). Whereas, the prevalence of Trypanosoma evansi infection using TBR 1/2 primer-based PCR was jumped up to 43.3% of all examined camels (48% among clinically suspected camels and 20% among apparently healthy camels). This may indicate that every case shows positive to infection by blood film technique opposites 7.5 cases positive to infection by TBR 1/2 primer-based PCR technique (1:7.5).

Table 1: The number of samples in relation to clinical state of examined camels:

Table 2: Prevalence of Trypanosoma evansi infection of the examined camels by thin blood smear:

*166 representing clinically suspected and hematologically negative cases. 

**207 representingclinically healthy and hematologically negativecases 

Table 3: Prevalence of Trypanosoma evansi infection by PCR:

*125 representing 75.30 % of the clinically suspected cases and hematologically negative Cases (n= 166).

**25 representing 12.08 % of clinically healthy and hematologically negative (n = 207).

DISCUSSION

Exportation of camels is one of important economic resources for many African countries where camels were present in large numbers, Egypt import camels from different African countries mainly Sudan. Trypanosomiasis, Pulmonary hydatiosis and gastroin test in alnematodiasis appears to be the most common endoparasitic diseases of camels in Upper Egypt Abdel-Rady, (2014). Trypanosoma evansi was regarded as a major constrain for Camel health and productivity in all camel rearing areas of the World Atarhouch, (2003). Procedures including diagnosis, curative or preventive treatment and quarantine should be established to insure the status of the camels moving from one country to another.

Concerning the observed clinical signs on trypanosomes infected camels in the current study. Trypanosoma evansi occurred in chronic and acute forms. The chronic form of the disease was the most common. Similar previous results were reported by (Gutierrez et al., 2000 and Njiru et al., 2004). Signs of acute form of Trypanosoma evansi infection were poor general condition. Trypanosoma evansi by itself was one of the major causes resulting in poor body condition Röttcher et al. (1987). On the other hand, camels with poor body conditions were more likely to be positive in Giemsa stained thin smear compared to those with good body condition.

Regarding to the morning rectal temperature of the infected camels, there were rising of body temperature up to (38.81 ± 0.05°C), it was higher than that recorded for the healthy animals (36.71 ± 0.05°C). Higher elevation of body temperature is associated with parasitaemia which is an important sign of camels infected with Trypanosoma evansi Tehseen et al. (2015).

Edematous swellings in 34.78% of camels infected with Trypanosoma evansi occur in the lower parts of legs, scrotal sacs and testicles and in the base of neck. Similar results were reported by Higgins, (1983)who reported that edema may develop along the neck and abdomen, Kaufmann et al. (1996) recorded edema of the feet, brisket, under belly and eyelids, Mottelib et al. (2005) stated that edematous swelling at some parts of the body especially hind limbs. Abdel-Rady, (2008) reported edematous swelling in testicles. Trypanosoma evansi was more virulent and devastating in its effect on reproductive performance of dromedary bulls due to formation and precipitation of immune complexes at the site of junctional complexes of the seminiferous tubules, pituitary dysfunction and testicular degeneration leading to infertility and sterility Al-Qarawi et al. (2004). About 13.04% of camels infected with Trypanosoma evansi showed edematous with Prolapsed Penis with disappearance of the characteristic hook shaped tip of the penis; These signs could be attributed to the chronic form of Trypanosoma evansi infection was likely to be associated with secondary infection due to immuno-suppression Tekle and Abebe (2001). Signs of chronic form of Trypanosoma evansi infection were general debility, severe emaciation, pale mucous membrane of conjunctiva, disappearance of the hump, projection of the ribs atrophy of the thigh muscles, the skin was harsh and scurfy, enlargement of lymph nodes and lacrimation. Similar signs were reported by (Kohler-Rollefson et al., 2001; Getachew, 2005; Saleh et al., 2009 and Tehseen et al., 2015).

Approximately 33% (34.78%) (Microscopically positive and clinically suspected camels) had hard ticks at different sites of camel's body particularly genital system. Ticks may play a role in transmission of Trypanosoma evansi Uilenberg, (1995). Higgins (1983) concluded that camels infected with Trypanosoma evansi were highly susceptible to ectoparasites such as mange and ticks.

On current study, the prevalence of Trypanosoma evansi infection among the examined camels with clinical abnormalities was 12.17%. However, the overall prevalence was 5.81%, the low percent of prevalence rate by thin blood smear may attribute to the chronic nature of the disease. In chronic phase of Trypanosoma evansi, there was a tendency for the parasite to invade the tissues, which may lead to either scanty or totally absent of Trypanosoma evansi in the blood of infected camels Chaudhri et al. (1996). Furthermore, in chronic infection, parasitaemia is very low; therefore, diagnosis of Trypanosoma evansi by thin blood smear is easily procedure, cheap and useful in acute stage of the disease; but it is insufficient in chronic stage Pathak et al. (1997).

The prevalence rate (5.81%) by thin blood smear was coincided with the previous results of (El-Sawalhey and Ebeid 1994), Abu-Zeid, (2003), Mottelib et al. (2005) and Zayed et al. (2010). They reported that prevalence of Trypanosoma evansi in Egypt was 5%, 5.82%, 5.82%, 5.67%, respectively, and, 5.3% (Delafosse and Doutoum, 2004) in Chad, 6% (Ibrahim, (2008) in Sudan and 5.15% Aregawi et al. (2015) in Ethiopia. On the other hand, the present prevalence rate was lower than previous results of Mahran (2004) and Elhaig et al. (2013), they reported that prevalence of Trypanosoma evansi in Egypt was 11.5% and 12%%, respectively, and 13.3% El-Amin, (1997) in Western Sudan, 12.12% Hagos et al. (2009) in Ethiopia and 14% Bennoune et al. (2013) in Algeria. Moreover, the results in the present study were higher than previous reports of Abdel-Rady, (2008) and Abd Elmaleck et al. (2014) who concluded that the prevalence of the disease in Egypt was 4.1% and 3.06%, respectively, and 1.3% Dia et al. (1997) in Mauritania, 2.3% Ngaira et al. (2002) in Kenya, 2% Fikru et al. (2015) in Ethiopia. The results in the present study were higher than previous reports may attribute to drug-resistant infections of Trypanosoma evansiZhang et al. (1993), Ng'ayo et al. (2005) and Salim et al. (2011).

The variation in prevalence rate of Trypanosoma evansi may attributed to the fact that trypanosoma parasites circulate within a wide and diverse host community, number of the examined camels, variations in the density of mechanical vectors, movements of camels from area to area increase the risk of infection, local herd management system and control interventions practiced by competent authorities.

In Egypt, Mahmoud et al. (2008); Barghash et al. (2014) and El-Hewairy et al. (2014) indicated prevalence rates of Trypanosoma evansi by thin blood smears about 27.6%, 20.9% and 16.9%, respectively. Their results were higher than the present results. This may attributed to the occurrence of high levels of newly and active infections, the close contact of camels with other carrier animals, such as sheep and goats act as a serious source of infection and transmission.

PCR detection of Trypanosoma evansi has known a great expansion during the last 20 years, but primer sets are often insufficiently assessed and compared Pruvot et al. (2010). PCR detected acute and chronic animals and the following of drug treated case. TBR1/2 that amplified 164 bp DNA fragment provided valuable tools to study the epidemiology of Trypanosoma evansi infection in camels' population in Egypt Ashour et al. (2013). Currently, PCR depends on identifying portions (base pairs) of DNA, from the nucleus or from the kinetoplast, which are specific for Trypanosoma evansi. The target sequences for TBR1 &TBR2 found in nucleus not in kinetoplast making results independent of the parasite kinetoplastic state and avoid the problem of failure targeting kDNA as reported by Gonzales et al. (2003). Pruvot et al. (2010) indicated that TBR primers showed the highest sensitivity and specificity for the detection of Trypanosoma evansi and were able to detect 0.01 pg of purified Trypanosoma evansi DNA and a parasitemia below one parasite per ml of blood without false positive reactions of samples tested. The high detection capacity of TBR primers was attributed to highly repeated sequences.

The prevalence of Trypanosoma evansi in the present study by TBR 1/2 primer-based PCR revealed that Trypanosoma evansi infection was 43.3 %. Higher prevalence of Trypanosoma evansi detected by PCR may attribute to the higher sensitivity of the molecular technique. This result was in agreement with Elhaig et al. (2013) who reported that the prevalence of clinical and sub-clinical Trypanosoma evansi infection among camels in Egypt was 46% by TBR 1/2 primer-based PCR technique. On the other hand, the result recorded in the present study were higher than result of Tehseen et al. (2015) who indicated that the prevalence of Trypanosoma evansi infection among 1005 dromedary camels in Pakistan about 31.9 % with TBR 1/2 primer-based PCR. Moreover, the obtained result was lower than result of Barghash et al. (2014) who reported that the prevalence rate of Trypanosoma evansi infection of 249 camels in Northern-West Coast, Egypt was 74.7% with TBR 1/2 primer-based PCR. These variations in the prevalence rate may be attributed to geographic and climatic conditions. The obtained result of PCR indicate high proportion of sub-clinical infection of Trypanosoma evansi among the investigated camels and this result is in agreement with the findings of Mahran, (2004) and Elhaig et al. (2013).

Interestingly, on the current work the prevalence of Trypanosoma evansi infection using blood film technique was 5.81% of all tested camels (12.17% among clinically suspected camels). Whereas, the prevalence of Trypanosoma evansi infection using TBR 1/2 primer-based PCR was reached to 43.3% including (48% among clinically suspected camels and 20% among apparently healthy camels). This may indicate that every case shows positive to infection by blood film technique opposites 7.5 cases positive to infection by TBR 1/2 primer-based PCR technique (1:7.5). This may indicate high sensitivity of TBR 1/2 primer-based PCR technique than thin blood smear. Many clinically suspected camels were negative by thin blood smear examination and found to be positive by TBR 1/2 primer-based PCR technique which may indicate high proportion of sub-clinical infection of Trypanosoma evansi among the investigated camels, Approximately 20% of apparently healthy camels were positive by TBR 1/2 primer-based PCR and negative by blood film technique referring to the carrier state of Trypanosoma evansi infection in camels imported from Sudan.

The results obtained by Delafosse and Doutoum (2004) and Ibrahim, (2008) indicated that the proportion between thin blood technique and PCR technique was 1:13 and 1:11, respectively. The most microscopic techniques are poorly sensitive and Giemsa stained thin smear examination revealing the lower detection limit is greater than 500,000 trypanosomes/ml of blood (OIE, 2012). Furthermore, it was reported that the detection of less than 2.5 × 106 trypanosomes per ml in blood samples by microscopy is not functional Desquesnes, (2004). In contrast, PCR based assay is a highly sensitive and specific for the detection of T evansi present in the blood of different animals and vector Abdel-Rady, (2008) and Baticados et al. (2011).

CONCLUSION

The obtained results in the present study indicate the spread of Trypanosoma evansi infection among camel population imported from Sudan. Consequently, it is recommended that good management and hygienic precautions should be carried out immediately to minimize the entrance of infectious diseases from Sudan to Egypt, establish a modern laboratory unit containing PCR in Abu-Simbel Veterinary Quarantine for camel diseases to reveal up the infectious diseases among camels and other animals.

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