MOLECULAR CHARACTERIZATION OF SPECIES FROM CHOLISTANI CAMELS, CATTLE, GOATS, SHEEP AND EQUINES BY USING SPECIES SPECIFIC PRIMERS

A Thesis Submitted

In Partial Fulfillment of the requirement for the

Degree of Master of Science (MS) in

ZOOLOGY

By

SOBIA MALIK

Roll No. 1

Department Of Life Sciences

The Islamia University of Bahawalpur

Session 2013-2015

IN THE NAME OF ALLAHA the MOST BENEFICENT AND the MOST MERCIFUL

Contents

Chapters Title Page no.

List of tables ii

List of figures iii-iv

Abstract. 1-2

1 Introduction. 3-15

2 Review of Literature. 16-22

3 Material and Methods. 23-30

4 Results. 31-49

5 Discussion. 50-55

6 References. 56-69

7 Appendix 70-73

List of Tables

Table Title Page no. no.

1 List of primers used. 28

2 Reaction mixture for PCR. 29

3 Prevalence of Trypanosomiasis in Camels through different tests. 44

4 Prevalence of Trypanosomiasis in Cattle through different tests. 45

5 Prevalence of Trypanosomiasis in Goats through different tests. 46

6 Prevalence of Trypanosomiasis in Sheep through different tests 47

7 Prevalence of Trypanosomiasis in Donkeys through different 48 tests.

8 Prevalence of Trypanosomiasis through PCR by using three 49

species specific pimers

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List of Figures

Figure Title Page no. no.

1 Worldwide distribution of animal trypanosomiasis 6

2 General morphological construction of 8

3 Map showing sampling sites. 23

4 Giemsa stained thin smear slides. 25

5 The extracted genomic DNA. 27

6 Showing positive giemsa stained thin smear slide. 31

7 Showing positive and negative samples through Formol gel test. 32

8 Showing packed cell volume (PCV) through microhaemotocrit centrifuge 33

Confirmation of trypanosome in camels samples through PCR by sequence 9 34 specific primer TBR.

Confirmation of trypanosome in camels samples through PCR by sequence 10 35 specific primer TRYP4.

Confirmation of trypanosome in cattle samples through PCR by sequence 11 36 specific primer TBR.

Confirmation of trypanosome in cattle samples through PCR by sequence 12 36 specific primer ROTAT.

Confirmation of trypanosome in cattle samples through PCR by sequence 13 37 specific primer TRYP4.

Confirmation of trypanosome in goats samples through PCR by sequence 14 38 specific primer TBR.

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Confirmation of trypanosome in goats samples through PCR by sequence 15 38 specific primer ROTAT.

Confirmation of trypanosome in goats samples through PCR by sequence 16 39 specific primer TRYP4.

Confirmation of trypanosome in sheep samples through PCR by sequence 17 40 specific primer TBR.

Confirmation of trypanosome in sheep samples through PCR by sequence 18 40 specific primer ROTAT.

Confirmation of trypanosome in sheep samples through PCR by sequence 19 41 specific primer TRYP4.

Confirmation of trypanosome in donkeys samples through PCR by sequence 20 42 specific primer TBR.

Confirmation of trypanosome in donkeys sample through PCR by sequence 21 42 specific primer ROTAT.

Confirmation of trypanosome in donkeys samples through PCR by sequence 22 43 specific primer TRYP4.

23 Comparison of different diagnostic tests from camels samples 44

24 Comparison of different diagnostic tests from cattle samples 45

25 Comparison of different diagnostic tests from goats samples 46

26 Comparison of different diagnostic tests from Sheep samples 47

27 Comparison of different diagnostic tests from donkeys samples 48

28 Overall prevalence of trypanosomiasis through different techniques 49

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MOLECULAR CHARACTERIZATION OF TRYPANOSOMA

SPECIES FROM CHOLISTANI CAMELS, CATTLE, GOATS,

SHEEP AND EQUINES BY USING SPECIES SPECIFIC

PRIMERS

ABSTRACT

Trypanosoma is the blood-dwelling protozoan parasites that cause a disease known as

Trypanosomiasis which is common in livestock species like cattle, sheep, goats, horses and camels. Basically this disease spread through a vector known as tsetse fly commonly but rarely by other blood sucking insects. Many cases of trypanosomiasis were reported from Cholistan region of Pakistan but there molecular characterization was not done before. The current study was designed to identify the existing trypanosoma species in animals of Cholistan region and estimate most recent rate of infection in animals. Total 272 animals were bled and these include 61 camels, 52 donkeys, 59 cattle, 50 goats and 50 sheep. Blood samples were collected from the jugular vein of animals and further divided into three parts. From first part, thin smear microscopy, micro haematocrit, PCV and DNA was extracted, from second part serum was separated and formol gel test was performed. The last and third part of blood was preserved in cryo preservative tubes. The PCR test was performed with three different set of primers like TBR, TRYP 4 and ROTAT. TBR and ROTAT are genus specific primers and TRYP4 is species specific primer. From camels samples 4 animals were positive on thin smear microscopy (6.55%), 34 animals were positive on formol gel test (55.7%), 11 animals were positive on PCV (18.03%) and 24 animals were confirmed positive by PCR (39.3%). Similarly, in case of cattle 2 animals were positive on thin smear microscopy (3.38%), 15 animals were positive on formol gel

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(25.42%), 47 animals were confirmed positive on PCV(79.6%) and 10 animals were positive by PCR (16.9%). In case of goats 2 animals were positive on thin smear microscopy (4.0%), 34 animals were positive on formol gel (68.0%), 37 were positive on PCV (74.0%) and 22 were confirmed positive on PCR (36.6%). In case of sheep 3 animals were positive on thin smear microscopy (6.0%), 30 animals were positive on formol gel (60.0%), 45 animals were positive on PCV (90.0%) and 24 animals were confirmed positive on PCR (48.0%). In case of donkeys 6 animals were positive on thin smear microscopy (11.5%), 26 animal were positive on formol gel (50.0%), 9 animals were positive on PCV (17.3%) and 20 animals were confirmed positive on

PCR (38.4%). Overall 17 /272 (6.25%) animals were found positive from microscopy,

139 / 272 (51.1%) samples were found positive by formol gel test, 149 /272 (54.77%) samples were positive from PCV and 100/272 (36.76%) animals were confirmed positive from PCR. So it was concluded that formol gel and PCV were non-specific tests while thin smear microscopy and PCR were specific i.e. on the basis of PCR 100 animals were confirmed positive (36.76%).

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Chapter 1

INTRODUCTION

1.1. Trypanosomiasis

In general trypanosomiasis is a deep-rooted evolving infection which is typically lethal if suitable action is not taken. Trypanosomiasis, which is most extensively dispersed of the pathogenic animal protozoan infectivity, is caused by dissimilar species of trypanosomes that includes , Trypanosoma evansi, Trypanosoma congolense, Trypanosoma equiperdum,and Trypanosoma vivax etc. aside from horses, the disease affects an extensive series of additional foremost hosts including, camels, cattle, buffaloes, donkeys, dogs, elephants, pigs, cats, tapirs, capybaras, deer; and specific blood nourishing invertebrates like vectors (Hamilton et al., 2007). The medical depiction of trypanosomiasis is manifested as fever, anemia, oedema of limbs as well as petechial and genitalia hemorrhages in conjunctiva

(Urquhart, 1985).

Trypanosomosis in animals due to T.evansi is a chief parasitic sickness recognize as “surra”. On the other hand the name T. evansi was prevalent, at the same time in several areas it was recognized with other names like T. kirdanii and T. annamense. Evans in 1880 in India first described T. evansi, but surra most likely has been upsetting the domestic animals of Asian and North Africa from the ancient times

(Hoare, 1972). “Surra” comes from the India which means “Rotten” (Vittoz, 1955); which particularly related to camels evolving disease. Generally in Asia the name

“surra” is used while a numbers of other names were also employed, like purana

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(chronic or older), tibarsa (a three-year ailment) and dubla (thin) (Luckins, 1988) or makhi ki bimari (a horse-fly infection) (Gill, 1977).

Economically consequence of surra is frequently under estimated due to fewer infection exposure rates. It needs to put into practice and support wide-reaching projects to study T. evansi and as an initial step for studies and observation of T. evansi, FAO along with WAHO/ Office International des Epizooties stressed (OIE) out the demand in favor of consistent investigative tools into sequence to gather trustworthy or analogous information (FAO - Food and Agriculture

Organization/IAEA, 1993).

Diagnostic tool such as PCR proved to be very sensitive and specific for T. evansi detection (Wuyts et al., 1994) and from 1990 parasitological techniques for detection of infections are improved by such tools (Masiga et al., 1992). T. evansi infection diagnosis largely depends on blood microscopic examination or by the micro he-matocrit test (Herrera et al., 2004; Baticados et al., 2011). Though, the parasitaemia level is repeatedly low and changeable, especially for the duration of the chronic stage, and therefore the occurrence of the trypanosome could stay undetected by inadequately sensitive methods (Woo, 1970; Murray, 1989; Monzon et al., 1990).

1.2. Trypanosma ─ The Past

Trypanosoma evansi is the foremost pathogenic mammalian trypanosome in the world, in 1880, via Griffith Evans, within the blood of Indian dromedaries and equines (Hoare, 1972). Their main host is formerly the camel however it is present in dromedaries, horses and other Equidea plus in other large number of hosts. T. evansi thought to be the derivative of T. brucei (intermittently transmitted by tsetse flies),

4 although it is no longer capable to undertake its cycle in Glossina owing to the loss of the maxicircles of kinetoplastic mitochondrial DNA (Lai et al., 2004); (Borst et al.,

1987).

At the present time the geological division of T. evansi is continuous from the North Africa through the Middle East to South-East Asia. While it is impracticable to found the early spread of T. evansi to east side, so their chronological data records suggests that surra was previously present in India ever since time immemorial, as a minimum VIII centuries B.C., and so that livestock have got to suffered from surra in the deficiency of treatment (Hoare, 1972); (Vittoz, 1955).

T. evansi incessantly present eastwards in the Arabian peninsula, counting

Saudi Arabia Kingdom, UAE, Hashemite kingdom of Jordan, the Lebanon Republic,

Israel, Iraq, Oman, Syria, Turkey and also in Bulgaria as one infrequent record; also present in Mediterranean and Sub-Saharan climates but it can be found in temperate regions and in addition to this also occurred in semiarid steppes and arid deserts. In addition to this, the protozoan parasite is observed from Kazakhstan as well as in

Afghanistan and Pakistan (Desquesnes and Dia, 2003; Desquesnes et al., 2009; Hasan et al., 2006; Srivastava et al., 1984) as shown in Figure 1.

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Fig. 1: Worldwide distribution of animal trypanosomiasis (Woo, 1977).

1.3. The Concept of a Species

Subgenus Trypanozoon belong to genus Trypanosoma which is divided into three species i.e.T. evansi, T. brucei and T. equiperdumin while T. brucei separated into three species (T. b. brucei , T. b. rhodesiense and T. b. gambiense ) which were distinct through disease causing capacity, distribution and host range (Hoare, 1972).

Bloodstreams of these three species of trypanosomes are morphologically impossible to differentiate. In lab isolates of T. brucei the characteristic of pleomorphism can be missing and they then turn out to be identical from the single species i.e. Trypanosoma evansi or Trypanosoma equiperdum. Pleomorphism on the practical stage reveals Trypanosoma brucei capacity to build up in their vector moreover as a result reliant on control of complete with purposeful genes deposit

6 proposed for mitochondrial method. In the maxicircle DNA of T. brucei the mitochondrial genome is enclosed simultaneously with mini-circle encoded genes set which are obligatory in favor of cutting maxicircle copies, so transformed properly. Following attributes describe Trypanosoma brucei, and their non- appearance describes Trypanosoma evansi or Trypanosoma equiperdum.

Either Trypanosoma evansi or Trypanosma equiperdum is episodically transmittable by tsetse, and undeniably, no species is competent of cyclic growth.

Mitochondrial genome absent in T. evansi and its kinetoplast is full of homogeneous mini-circles. A small number of Trypanosoma equiperdum isolates also have absent kinetoplast DNA (Lun et al., 1992).

1.4. Trypanosoma evansi (T. evansi)

Trypanosomes are blood and occasionally tissue parasites which fit in to order

Kinetoplastida, family Trypanosomatidae and genus Trypanosoma which are transmitted by stinging and biting insects in which the majority of them go through a biological cycle (Coura and Borges-Pereira, 2010).

T. evansi typically show willowy morphology and morphometry when observed in fresh blood samples. It has small size, slim posterior edge having free flagellum presenting active movements although producing some degree of displacements below microscope through very limited polymorphism and lacking any uniqueness qualifying at species level, and furthermore having vastly detectable rising and falling membrane which traps the light (light can come into view to be go into at one end/side of parasite and transferred to the other end/side to be released) (Hoare,

1972).

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These protozoan parasites show a discrepancy in dimensions equally in lengthwise measurement as well as in breadth seeing that 18 - 36µm in length and 1.5 - 2.5µm into width (Pathak and Singh, 2005) as shown in Figure 2.

Fig. 2: General morphological construction of Trypanosoma evansi.

(Vickerman, 1970)

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1.5. Medical Signs inside Host Animals

Diagnosis in host animal is based on finding an anemic animal in deprived state within an endemic area. Brutality of disease varies with the species of trypanosome concerned and with species and age of the animal infected.

1.5.1. Camels

In camels (Camelus dromedarius and C. bactrianus) surra perhaps severe by soaring fever, weakness, anemia, and fatality; it is also often mortal from time to time within a few months; on the other hand it is more frequently chronic than in horses and know how to normally last two to three years (as well called as Tibersa) (Parsani et al., 2008).

Illness signs become visible with sporadic fever (41°C), roughly about a week; the animals show dullness and matte and grow to be gradually weaker with stare hair, abortion, oedema of ventral parts, udder or scrotum, and sheath; anemia with light mucous membrane, failure to desire for food and loss of weight; and ecchymotic or petechial hemorrhages. In camels surra commonly starts happening abruptly after weaning. Sometimes nervous symptoms are observed for instance episodic convulsions. Camel owners detect a precise smell of the urine in their animal, which is resourceful for diagnose the infection. It is thought that if the animal survives more than three years they will then recover. (Stephen, 1986)

1.5.2. Cattle

Different workers worldwide reported the incidence of cattle haemoparasitic infections (Laha et al., 1989; Thach et al., 1996). In Pakistan there are 32.7 million

Buffaloes (Anonymous, 2011); in which 65 % of the world„s well-known Nili Ravi

9 total buffalo population of country is present in Punjab Province (Livestock Census,

2006). In dairy cattle, decreased milk production and fever, abortion are frequently reported (Pholparket al., 1999; Kashiwazakiet al., 1998) while in beef cattle, when surra happen for the first time in a latest area, sky-scraping mortalities can be recorded (Chobjit et al., 2006).

When T. vivax infect the cattle it might be wholly asymptomatic or may show evidence of rigorous hematological alterations causing whiting of mucous membranes, miscarriage along with progressive weight loss (Silva et al., 1999). The disease is manifested by means of pyrexia, in a straight line connected with parasitaemia, mutually through increasing anemia, loss of sluggishness (Luckins,

2004).

Trypanosomiasis reduces calving levels, milk production, off take and oxen work competence while calf death is enlarged in endemic vicinities (Swallow, 2000).

Also surra infectivity cause loss in meat production along with loss in draught power, most regularly for the period of chronic development which can show the way to entirely wasted animals and occasionally the evolution may be acute, promptly leading to death (Payne et al., 1993). Sometimes nervous signs are recorded by meningoencephalitis (Sudarto et al., 1990).

1.5.3. Equines

In equines trypanosomiasis due to Trypanosoma evansi prevalently known as

“Mal de cadeiras” in Brazil, “Derrengadera” in Venezuela, “Mal de caderas” in

Argentina, “Murrina” in Central America, and “Surra” in India and worldwide (Brun et al., 1998; Garcia et al., 2003).

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Trypanosomiasis symptoms in horses come out after incubation period of one to two weeks or up to eight weeks along with this changeable fever (41.5°C up to 44°C) with high levels with parasitaemia, anemia, brutal weight loss, weakness, laziness, transitory local or common cutaneous outbreak also petechial hemorrhages on the eyelids predominantly the nictitating membrane (which may change yellow when getting the icteric phase), vaginal plus vulvar mucosa, abortion furthermore amendment of locomotion along with nervous signs characteristically describe within horses for example “They may stagger at the forelegs and pull the hind legs”

(Stephen,1986) , which most likely called “Mal de Caderas” in addition to this called as oedema of sub maxillary, testicle, sheath or udder, legs, briskets, and abdomen which appear after a little instance.

Evans described as “Living skeleton” in which staring hair and rapidly weight loss as well rather a conserved appetite can be observed, in addition to this other authors talk about appetite loss(Silva et al., 1995) and emaciation is over and over again accompanied as a result of exceedingly colored urine moreover jaundice

(Stephen, 1986). In enzootic regions horses possibly will reveal a certain confrontation with chronic or else subclinical cases with fit carriers. Donkeys moreover mules display the matching symptoms nevertheless milder than those in horses.

1.5.4. Goats & Sheep

In goats there is often low down defenselessness of trypanosomiasis (Rottcher,

1987; Jacquiet et al., 1993) although in experimental infections goats demonstrate meek symptoms by a few incidents of fever in untimely infection and showed arthritis within six months while low parasitaemia still continual (Gutierrez, 2004).

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Experimental infections in the Philippines lead to the observation of progressive emaciation, variable fever, testicular extension, anemia, coughing moreover diarrhea nevertheless not in all animals (Dargantes et al., 2005) but in further reports reveal reasonable (Ngeranwa, 1993) however sometimes strict or serious infections with fever, loss of hunger, lachrymation, salivation, and nervous warning signs like paroxysm and trembling afterward hypothermia and bereavement (Youssif et al.,

2008). Ocular lacerations have also been verified (Morales, 2006). On the other hand in goats under natural circumstances numerous reports declare mild clinical signs due to T.evansi (Stephen, 1986) (Gutierrez, 2006).

In sheep natural infection is generally well thought-out as asymptomatic or gentle (Boehringer and Prosen, 1961). But in a few cases experimental infections can be still fail but in others they can show the way to clinical signs such as fever (40°C), be short of appetite, and anemia. In addition to this for the duration of hyperthermia variation of behavior such as exhaustion or unexpected ferociousness has been experiential and anemia can move away after two months while parasitaemia is normally low and decreases until undetectable for more than a few months yet under definite circumstances such as food limitation or transport anxiety, parasites can go back into the blood and clinical signs re-emerge (Desquesnes, 1997).

1.6. Transmission

Mechanically trypanosomiasis transmission is effected by a variety of blood- sucking insects like flies which belong to the family Tabanidae commonly called as horse flies and flies of Stomoxys spp. During getting a blood meal start on an infected animal and finished on a healthy animal, these biting insects may possibly carry trypanosomes. This type of transmission is the rule for T. evansi which reproduce

12 simply by means of asexual reproduction and divided via binary fission in blood flow of host animal (Gill, 1997). As a consequence we conclude so as to transmission needs just that blood having infectious trypanosomes which can be transferred from one animal to a new animal.

1.7. Control

To manage animal trypanosomiasis make use of chemotherapy along with vector control are two frequent strategies as well as action is feasible on diverse features of the epizootiological cycle of the infection parasites within host animals plus vectors.

The Melarsominedihydrochloride (Cymelarsan) drug is the most recent well- known trypanocide which is used at a dosage rate of 0.15mg/kg for controlling surra in camels by means of deep intramuscular inoculation. Another chemical/ compound called as Suramine which was utilized in horses and camels via intravenous way furthermore it was awfully effectual alongside infectivity of T. evansi, but it is no more accessible within Pakistan. Quinapyramine sulphate and chloride is another medicine used against horse trypanosomiasis which offers resilient defense by its long-term special effects (Gillingwater et al., 2009). If equines trypanosomiasis can not treat by means of trypanocidal drugs i.e. diminazeneaceturate, isometamidium chloride, quinapyramine, suramin, or cymelarsan, this disease can go ahead to death during two to eight weeks in which animals be able to either pass away abruptly and unpredictably or reveal signs of hallucination and move violently for hours earlier than they die of tiredness (Sudarto, 1990).

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The single trypanocides in cattle that is homidium chloride or bromide and isometamidium chloride which has jointly disease curing along with preventing/protecting properties while diminazeneaceturate which simply has restorative characteristics. For trypanosomiasis vaccines have proved indefinable so therefore individual control greatly relies on chemoprophylaxis and chemotherapy.

Further control procedures in opposition to trypanosomiasis include marking the vector flies but their effectiveness requires performing at an area-wise and not individual level (Jordan, 1992; Holmes & Torr, 1988; Leach &Roberts, 1981).

Vector population control is an additional technique to control trypanosomiasis.

It can be made through various manners including use of steeped screens and traps or by means of insecticides applying on livestock group but insecticide sprays was found efficient barely in undersized congested deforested vicinities (Desquesnes et al.,

2013).

Effective controls of vector-borne diseases have need of suitable handling to keep away from livestock losses through weakness from lengthened sickness and fatality. Trypanosomiasis which is also a vector-borne disease does not constantly a reason of serious medical signs or else takes life of lot of animals, however animal‟s general states are influence by this and it reduces animal production and reproducing ability (Stephen, 1986). This production loss distress farmers source of revenue and is reasonably destructive since its non-dramatic character fallout in farmer‟s failure to take some steps to organize it. Farmers not have comprehensive knowledge of infection inducement and indications so their outcome of controlling disease performances predominantly trypanocides usage is usually badly chosen (Machila et al., 2003).

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1.8. Objectives

Keeping in view the importance of camel, cattle, equines, goats and sheep in

Cholistan region and economic losses due to trypanosomiasis infections in these animals, the present study was designed with following objectives:

1) To determine the overall prevalence of disease in camel, cattle, equines, goats

and sheep from different localities of Cholistan desert.

2) To characterize the trypanosome species associated with these animals by using

specie specific primers.

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