Serodiagnosis and Haematological Effect of Anaplasmosis in Goats and Sheep of District Mardan, Khyber Pakhtunkhwa, Pakistan

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1 World Journal of Zoology 11 (2): 67-80, 2016 ISSN IDOSI Publications, 2016 DOI: /idosi.wjz Serodiagnosis and Haematological Effect of Anaplasmosis in Goats and Sheep of District Mardan, Khyber Pakhtunkhwa, Pakistan Nasreen, Kausar Saeed, Adil Khan, Sadaf Niaz and Naveed Akhtar Department of Zoology, Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa, Pakistan Abstract: Anaplasmosis is globally distributed tick-borne disease of livestock with great economic importance in cattle industry. A total of 360 blood samples were randomly collected from three localities; Tehsils Mardan, Katlang and Lund khwar during the study period from January 2014 to December Microscopic examination of the Giemsa stained blood smears revealed an overall prevalence of blood parasites as 13.89% and 8.33% in sheep and goats respectively. The overall seroprevalence of anaplasmosis in sheep using celisa was 23.33%. Females (sheeps) were found to be more susceptible 28/114(24.56%) than males 15/66 (22.72%). The prevalence was significantly higher 29/119 (24.36%) in above one year sheep than the younger ones14/61 (22.95%) (P<0.05). The seroprevalence in sheep was significantly (P<0.01) higher in summer. The overall seroprevalence of anaplasmosis in goats using celisa was 20.56%. Females (goats) were found to be more susceptible 24/110(21.81%) than males 13/70 (18.56%). The prevalence was significantly higher 26/112 (23.21%) in above one year goats than the younger ones11/68 (16.18%) (P<0.05). It was concluded that anaplasmosis is widely distributed in district Mardan, Khyber Pakhtunkhwa, Pakistan. There is a need for country wide epidemiological studies on ticks and TBDs using advanced serological and molecular techniques. Key words: Anaplasmosis ELISA Seroprevalence Mardan INTRODUCTION because this disease increases not only the costs of the veterinary care but also reduces production of milk, body Rickettsia causing diseases transmitted by ticks weight of animal, causes the abortions in females and cause great economic loss to livestock managing mostly leads to death [10-12]. worldwide [1, 2]. The Rickettsiales bacteria have been In 1912, the first report on anaplasmosis in sheep identified as zoonotic agents in the Americas, Africa and was presented in Zimbabwe [13]. The causes of ovine especially in Europe over the last several decades [3]. anaplasmosis is mostly Anaplasma ovis and The tick borne diseases that affect the livelihood of poor Anaplasma marginale [14] in tropical and subtropical farming people were ranked high in the developing areas [15, 16]. Small ruminants (Goats and sheep) are countries [4, 5]. The surveillance on species of rickettsial mostly parasitized by A. ovis and its presence in most bacteria which circulate in animals and humans has been region of the world both in wild and farm animals has increasing considerably. As a result research has been been confirmed [17]. increased which aim to identify and characterize strains Many tick species transmit anaplasmosis. The six at molecular level in various countries worldwide [6]. genera (Ermacentor, Rhipicephalus, Argas, Ixodes, Tick-borne diseases (TBDs) are generally prevalent in Hyalomma and Ornithodoros) have twenty such countries located in tropics and subtropics in which species which have been experimentally proved to Pakistan is also included [7, 8]. transmit A. marginale infection [18]. Anaplasmosis in sheep and goats is a rickettsial Anaplasmosis is endemic in countries located on disease [9] and which is caused by various Anaplasma tropic and subtropic and mostly detected in temperate species. For animal breeders it is an important concern regions of the world [19, 20]. Corresponding Author: Nasreen, Department of Zoology, Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa, Pakistan. 67

2 Tick borne diseases are commonly diagnosed by the TakhtBhai and Katlang. The District is divided into 231 microscopic examination of Giemsa stained blood smear. local councils including 178 villages. Most of the people This method is relatively economical and require less are farmers in profession in villages. time but this method has low sensitivity and specificity. The hottest season of the year is summer season. The effectiveness of this method is linked with experience In the month of June, the temperature reaches to its and ability of the person [21]. maximum. Other months with high recorded temperature In ruminants like sheep and goat antibody can be are May, June, July, August and September. December detected by a number of serological tests and the most and January are the coldest months. January, December, widely used of these tests is the indirect fluorescent July and august are high rainy months. antibody test which make use of cell culture schizont antigen. Whole parasite lysates or specific antigens Sample Collection: A total of 360 blood samples were isolated by monoclonal antibodies enzyme-linked assays collected from sheep and goats from different localities of have been developed [22]. Further, many investigators Mardan district. An amount of 5 ml blood was collected have shown that ELISA is a high sensitive and specific directly from jugular vein of goats and sheep using sterile diagnostic test [23, 24]. syringe in gel-added tubes. Samples were collected from The epidemiology of Anaplasmosis is not fully animals of different sex and age. Age, sex and locality of known because for screening large numbers of animals each sample were noted. Thin blood smears were prepared practical diagnosing assay is not available, serological at the time of blood collection for microscopical methods would be suitable for this [25]. The Commercially examination of the parasite. The samples were kept in available (competitive ELISA) kit was recently proved vaccutainer and then placed in ice jar for further analysis. very effective for the detection of anaplasmosis in small The samples were, then, brought to the laboratory of ruminants [26, 27]. Zoology Department AWKUM for further analysis. It is expected that competitive enzyme-linked The work was started from January to December immunosorbentassay (celisa), will improve our ability to detect infected animals, thus exact surveys of Staining Procedure: The blood smears were prepared and Anaplasma spp. would be possible. Further, then fixed by using methanol. Giemsa stain (1:10) ratio hematological changes in naturally infected animals as diluted were added for minutes. To remove excess compared to healthy animals will be analyzed to arrive at stain these smears were rinsed three to four times using the conclusion regarding the pathogenicity induced by tap water and then air-dried. These slides were then Anaplasma spp. observed at 100X magnification under oil immersion lens Keeping in view the importance of the disease due to of microscope. To detect Anaplasma species 20 economic losses to the livestock industry, the present microscopic fields were carefully observed research work is design to carry out the following objectives. Serum Separation: Collected blood samples were centrifuged for 5 minutes at 3500 rpm. With the help of Prevalence of Anaplasma spp. in sheep and goats of pasture pipette the supernatant was aspirated into a district Mardan Khyber Pakhtunkhwa, Pakistan. sterile plastic dropper. Then the serum was stored at To analyze the haematological changes in infected 20 C until analysis. animals to arrive at the conclusion regarding the pathogenicity induced by Anaplasma spp. Enzyme Linked Immuno-Sorbent Assay (ELISA) Principle of Kit: The serum Anaplasma antibodies in MATERIALS AND METHODS positive serum sample inhibit the binding of horseradish peroxidase labeled monoclonal antibody to Anaplasma Study Area: Mardan is situated near rashakie inter change coated micro-titer plate wells. The inhibition or lack of at distance of 34 Km from Peshawar with total area 1632 horseradish peroxidase labeled monoclonal antibody km. It is surrounded by district Swabi, Buner, Charsadda, conjugate is detected by the addition of enzyme Nowshera and Malakand protected area. substrate. The development of strong color indicates The district of Mardan is administratively subdivided negative test result. On the contrary, the weak or no color into five tehsils that is Lund Khwar, Mardan, Rustam, reveal the presence of Anaplasma antibodies. 68

3 Preparation of Reagents and Plates: The serum samples, Area-Wise Prevalence (%) Using Microscopy: High plates and reagents before the start of the test were prevalence was noted in Katlang (16.67%) followed by brought to room temperature. Plates (Antigen coated and Takhtbhai (13.33%) and lowest in Mardan (11.67%). When adsorption) were accordingly labeled. By adding antibody analyzed statistically all areas showed non-significant peroxidase conjugate with conjugate diluting buffer in difference (Table 1). 1:99 ratio the conjugate was prepared. To prepare the wash solution the wash solution and deionized water were Season Wise Prevalence (%) Using Microscopy: In all added in 1:9 ratio. tehsils of district Mardan the highest prevalence was noted in summer (25 %) followed by spring and autumn Hematological Studies: A total of 10 blood samples 13.33% while the lowest prevalence was recorded during were collected from infected goats and sheep and 10 winter 6.67%.When analyzed statistically significant non-infected goat and sheep. The haematological difference (P<0.01) was noted in summer while spring and changes were studied including total erythrocyte count, autumn showed non-significant (P >0.05) difference total leukocyte count, packed cell volume, hemoglobin, compare to winter (Table 1). mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration were analyzed in infected and Age Wise Prevalence Using Microscopy Technique: non- infected sheep and goats using hematology It was evident from the table 1 that prevalence was higher analyzer. in adult sheep (15.97 %) of age group (0-1 year) than young sheep (> 1 year) (9.83%). Statistically no Statistical Analysis: The data was analyzed statistically difference was noted in the prevalence of two age groups by using statistical software. (Table 1). RESULTS Gender Wise Prevalence Using Microscopy Technique: Female sheep (15.79%) were more vulnerable to Total 360 blood samples were taken from sheep and Anaplasmosis as compared to male (10.60%). goats of three tehsils (Mardan, Takhtbhai and Katlang) of Statistically no difference was recorded in female and male district Mardan during the period of January 2014 to (Table 1). December The seasons and climate of these tehsils were similar as they are located in one district but Prevalence by Month and Area (%): The highest variations were noted in management and cleanliness of prevalence (%) was noted in July from Takhtbhai, Katlang the three tehsils. The prevalence (%) of anaplasmosis in and Mardan i.e. 40%, 40% and 20% respectively and the sheep and goats was recorded. lowest prevalence in December i.e.0% from all tehsils (Table 2). Anaplasmosis in Sheep of District Mardan: Overall Prevalence (%) by using microscopy technique. Prevalence by Season and Area (%): The highest An overall prevalence (%) of anaplasmosis in sheep prevalence was reported in summer (23.33%), then spring by using microscopy technique was found to be 13.89% (13.33%), autumn (13.33%) and lowest during winter (25/180) in district Mardan from January 2014 December season (6.67%) (Table 2) (Table 1). Prevalence by Area and Gender (%): Overall Month Wise Prevalence (%) by Using Microscopy: gender wise prevalence was higher in females of The highest and the lowest prevalence was observed in Katlang, Mardan and Takhtbhai (20%, 15.38% and July (33.33%) and December (0%) respectively. When 12.5%) than males (12%, 4.76% and 15% respectively) analyzed statistically all months showed non-significant (Table 2). difference as compared to prevalence (%) of December except June and July. July and June showed significant Prevalence by Age and Area (%): In all tehsils the differences (P<0.01 and P<0.05 respectively) compared to prevalence (%) was high in above one years old sheep as December (Table 1). compared to less than one year sheep (Table 2). 69

4 Table 1: Prevalence of Anaplasmosis in Sheep of District Mardan by using Microscopy Technique during January 2014-December 2014 Factors Total No. of observed samples Total No. of infected samples Prevalence (%±S.E) Months January ±6.67 February ±6.67 March ±6.67 April ±6.67 May ±0 June ±6.67* July ±6.67** August ±0 September ±6.67 October ±6.67 November ±6.67 December ±0 Season Winter ±2.89 Spring ±3.33** Summer ±0** Autumn ±0 Gender Male ±3.04 Female ±2.18 Age 0-1 year ±2.61 >1year ±1.01 Area Mardan ±5.15 Katlang ±3.75 Takhtbhai ±4.33 Total ±1.45 Statistics applied: t-test: P<0.05=*; P<0.01=** Table 2: Overall Prevalence of Anaplasmosis in Sheep by using Microscopy Technique during January 2014-December 2014 Mardan Katlang Takhtbhai Factors Inf./Total Prevalence (%) Inf./Total Prevalence (%) Inf./Total Prevalence (%) Season Winter 0/20 0 2/ /20 5 Spring 1/ / /10 10 Summer 5/ / /20 25 Autumn 1/ / /10 10 Age 0-1year 1/ / / >1year 6/ / / Gender Male 1/ / /20 15 Female 6/ / / Months January 0/5 0 1/5 20 0/5 0 February 0/5 0 0/5 0 1/5 20 March 0/5 0 1/5 20 1/5 20 April 1/5 20 1/5 20 0/5 0 May 1/5 20 1/5 20 1/5 20 June 2/5 40 1/5 20 1/5 20 July 1/5 20 2/5 40 2/5 40 August 1/5 20 1/5 20 1/5 20 September 0/5 0 1/5 20 1/5 20 October 1/5 20 0/5 0 0/5 0 November 0/5 0 1/5 20 0/5 0 December 0/5 0 0/5 0 0/5 0 Total 7/ / /

5 Table 3: Prevalence of Anaplasmosis in Sheep of District Mardan by using ELISA Technique during January 2014-December2014 Factors Total No. of observed samples Total No. of infected samples Prevalence (%±S.E) Months January ±6.67 February ±0 March ±6.67 April ±6.67* May ±6.67* June ±6.67* July ±6.67* August ±6.67 September ±4.50 October ±6.67 November ±6.67 December ±6.67 Season Winter ±1.67 Spring ±5.77 Summer ±3.33** Autumn ±3.33 Gender Male ±1.30 Female ±1.93 Age 0-1 year ±0.65 >1year ±0.54 Area Mardan ±4.79 Katlang ±8.00 Takhtbhai ±5.54 Total ±1.47 Statistics applied: t-test: * = P<0.05, ** = P<0.01 Overall Prevalence by Using ELISA Technique: An overall prevalence (%) of Anaplasmosis in sheep by using ELISA technique was found to be 23.89% (43/180) in district Mardan from January 2014 December 2014 (Table 3). Month Wise Prevalence (%) by Using ELISA: The highest prevalence was recorded in June (46.67%) and the lowest prevalence was noted in December (6.67%). Statistically significant differences were noted from April, May, June and July when compared to December (Table 3). Season Wise Prevalence by Using ELISA: The data showed that summer has highest prevalence (35%) while the lowest prevalence was in winter (13.33%). When analyzed statistically prevalence in summer was significantly high as to winter (Table 3). Area-wise Prevalence (%) by Using ELISA: In Katlang (26.67%) prevalence was noted followed by Takhtbhai (23.33%) and in Mardan (21.65%) the prevalence was low. The prevalence in all three areas was nonsignificant (P>0.05) statistically (Table 3). Age Wise Prevalence Using ELISA Technique: It was evident from the table 3 that prevalence was higher in adult sheep (24.36%) of age group (<1 year) than young sheep ( 1 year) (22.95%). Statistically the sheep below one year and above one year old were not different (Table 3). Gender Wise Prevalence Using ELISA Technique: Female sheep showed higher prevalence (24.56%) than that of males (22.72%). Statistically the male and female sheep showed no difference (Table 3). Prevalence by Month and Area (%) by Using ELISA: In June prevalence in three tehsils were high that iskatlang60%, Mardan (40%) and Takhtbhai (40%) while the lowest prevalence was recorded during December in Mardan, Katlang and Takhtbhai (0%), (0%) and (20%) (Table 4). Prevalence by Season and Area (%) Using ELISA: In summer prevalence of anaplasmosis in all three tehsils was highest and in winter it was the lowest (Table 4). 71

6 Table 4: Overall Prevalence of Anaplasmosis in Sheep of Three Tehsils of District Mardan by using ELISA Technique during January 2014-December2014 Mardan Katlang Takhtbhai Factors Inf./Total Prevalence(%) Inf./Total Prevalence(%) Inf./Total Prevalence(%) Season Winter 2/ / /20 15 Spring 3/ / /10 20 Summer 6/ / /20 40 Autumn 2/ / /10 20 Age 0-1year 4/ / / > 1year 9/ / / Gender Male 4/ / /20 25 Female 9/ / / Months January 1/5 20 1/5 20 0/5 0 February 1/5 20 1/5 20 1/5 20 March 1/5 20 2/5 40 1/5 20 April 2/5 40 2/5 40 1/5 20 May 1/5 20 2/5 40 2/5 40 June 2/5 40 3/5 60 2/5 40 July 1/5 20 2/5 40 2/5 40 August 2/5 40 1/5 20 1/5 20 September 1/5 20 1/5 20 1/5 20 October 1/5 20 0/5 0 1/5 20 November 0/5 0 1/5 20 1/5 20 December 0/5 0 0/5 0 1/5 20 Total 13/ / / Table 5: Prevalence of Anaplasmosis in Goats of District Mardan by using Microscopy Technique during January 2014-December2014 Factors Total No. of observed samples Total No. of infected samples Prevalence(%±S.E) Months January ±0 February ±0 March ±6.67 April ±667 May ±6.67 June ±6.67 July ±6.67* August ±6.67 September ±6.67 October ±0 November ±0 December ±0 Season Winter ±0 Spring ±3.33 Summer ±2.89** Autumn ±3.33 Gender Male ±1.19 Female ±1.19 Age 0-1 year ±0.17 >1year ±1.38* Area Mardan ±3.75 Katlang ±5.91 Takhtbhai ±4.79 Total ±0.96 Statistics applied t-test: P<0.05 =*; P<0.01 =** 72

7 Table 6: Overall Prevalence of Anaplasmosis in Goats of Three Tehsils of District Mardan by using Microscopy Technique during January 2014-December2014 Mardan Katlang Takhtbhai Factors Inf./Total Prevalence(%) Inf./Total Prevalence(%) Inf./Total Prevalence(%) Season Winter 0/20 0 0/20 0 0/20 0 Spring 1/ /10 0 1/10 10 Summer 3/ / /20 20 Autumn 0/10 0 1/ /10 0 Age 0-1year 1/ / / > 1year 3/ / / Gender Male 1/ /25 8 1/ Female 3/ / / Months January 0/5 0 0/5 0 0/5 0 February 0/5 0 0/5 0 0/5 0 March 1/5 20 0/5 0 0/5 0 April 0/5 0 0/5 0 1/5 20 May 0/5 0 1/5 20 1/5 20 June 1/5 20 1/5 20 1/5 20 July 1/5 20 2/5 40 1/5 20 August 1/5 20 1/5 20 1/5 20 September 0/5 0 1/5 20 0/5 0 October 0/5 0 0/5 0 0/5 0 November 0/5 0 0/5 0 0/5 0 December 0/5 0 0/5 0 0/5 0 Total 4/ / / Prevalence by Age and Area (%) Using ELISA: In all (10%) and the lowest in Mardan (5%). Nonsignificant tesils the prevalence (%) was high in sheep below one difference (P >0.05) was observed statistically (Table 5). year old than above one year old (Table 4). Gender Wise Prevalence Using Microscopy Technique: Anaplasmosis in Goats Female goats (10%) showed high prevalence than males Overall Prevalence (%) by Using Microscopy Technique: (5.71%). Statistically the difference was nonsignificant An overall prevalence (%) of anaplasmosis in goats by (Table: 5). using microscopy technique was found to be 8.33% (15/180) in district Mardan from January 2014 December Age Wise Prevalence Using Microscopy Technique: 2014, (Table 5). Prevalence in above one year old goats (10.71%) was high than young goats (0-1year old) (4.41%). The difference Month Wise Prevalence (%) by Using Microscopy: It was significant statistically (Table 5). was evident from the data that highest prevalence was present in July (26.67%) and the lowest was in January, Prevalence by Month and Area (%): The high prevalence February, March, October, November and December (0%). (%) was noted during July in Katlang, Mardan and July showed statistically significant difference comparing Katlang 40%, 20% and 20% respectively while the lowest to the lowest prevalence in January, February, March, during January, February, March, October, November and October, November and December (Table 5). December (0%)i.e.0% in all tehsils (Table 6). Season Wise Prevalence (%) Using Microscopy: Summer Prevalence by Season and Area (%): During summer and winter showed the highest and lowest prevalence highest prevalence was observed in Katlang, Takhtbhai (20%) and (0%) respectively. Significant difference was and Mardan 25%, 20% and 15% respectively while the noted in summer as compared to winter when analyzed lowest during winter season 0% in all the areas (Table 6). statistically (Table 5). Prevalence by Area and Age (%): Goats of above one Area Wise Prevalence (%) Using Microscopy: The year age have high prevalence than those of less than one highest degree of prevalence was recorded in Katlang year old when data was analyzed (Table 6). 73

8 Table 7: Prevalence of Anaplasmosis in Goats of District Mardan by using ELISA technique during January 2014-December2014 Factors Total No. of observed samples Total No. of infected samples Prevalence (%±S.E) Months January ±6.67 February ±6.67 March ±6.67 April ±6.67 May ±6.67 June ±6.67* July ±0** August ±6.67 * September ±0.00 October ±6.67 November ±6.67 December ±6.67 Season Winter ±2.89 Spring ±3.33 Summer ±1.667 ** Autumn ±3.33* Gender Male ±0.99 Female ±2.09 Age 0-1 year ±1.75 >1year ±0.98* Area Mardan ±4.79 Katlang ±5.40 Takhtbhai ±6.61 Total ±1.11 Statistics applied: t-test: P<0.05=*, P<0.01=** Table 8: Overall Prevalence of Anaplasmosis in Goats of Three Tehsils of District Mardan by using ELISA technique during January 2014-December2014 Mardan Katlang Takhtbhai Factors Inf./Total Prevalence(%) Inf./Total Prevalence(%) Inf./Total Prevalence(%) Season Winter 2/ / /20 5 Spring 1/ / /10 20 Summer 6/ / /20 35 Autumn 2/ / /10 30 Age 0-1year 3/ / / > 1year 8/ / /36 25 Gender Male 4/ / / Female 7/ / /36 25 Months January 0/5 0 1/5 20 0/5 0 February 1/5 20 1/5 20 0/5 0 March 1/5 20 1/5 20 0/5 0 April 1/5 20 0/5 0 1/5 20 May 2/5 40 1/5 20 1/5 20 June 1/5 20 2/5 40 2/5 40 July 2/5 40 2/5 40 2/5 40 August 1/5 20 2/5 40 2/5 40 September 1/5 20 1/5 20 1/5 20 October 1/5 20 1/5 20 2/5 40 November 0/5 0 1/5 20 1/5 20 December 1/5 20 0/5 0 0/5 0 Total 11/ / /

9 Table 9: Microscopy and ELISA Comparison for the Diagnosis of Anaplasmosis in Sheep in 3 Tehsils of District Mardan from January 2014 to December 2014 Microscopy ELISA Factors Total observed samples No. of positive Prevalence (%) No. of positive Prevalence (%) Months January February March April May June July August September October November December Season Winter Spring Summer Autumn Gender Male Female Age 0-1 year >1year Area Mardan Katlang Takhtbhai Total Prevalence by Area and Gender (%): High prevalence Season Wise Prevalence by ELISA: All three tehsils had was noted in female goats than males in all the areas. high prevalence in summer andlowest prevalence in winter (Table 6). (Table 7). When analyzed statistically summer and autumn showed significant high prevalence than winter. Over All Prevalence by Using ELISA Technique: An overall prevalence (%) of Anaplasmosis in goats by using Gender Wise Prevalence Using ELISA Technique: ELISA technique was found to be 20.56% (37/180) in Female goats showed (21.81%) were more susceptible to district Mardan from January 2014 December 2014, anaplasma infection than males (18.57%). Statistically no (Table 7). difference was present in male and female goats (Table 7). Month Wise Prevalence (%) by Using ELISA: In July Age Wise Prevalence Using ELISA Technique: It was (40%) prevalence was observed which was the evident from the data that prevalence was higher in young highestand January and December showed lowest goats (23.07%) of age group (0-1 year) than adult sheep prevalence (6.67%). Statistically July and august (> 1 year) (18.36%). Statistically significant difference was reflected significantly high prevalence compared to noted when analyzed (Table 7). January and December which showed lowest prevalence (Table 7). Prevalence by Month and Area (%) Using ELISA: All the three tehsils showed high prevalence in july (40%), during Area-Wise Prevalence (%) Using ELISA: The highest june and august in Katlang and Takhtbhai (40%), during degree of prevalence was recorded in Takhtbhai and May in Mardan and during october in Takhtbhai ( 40%), Katlang (21.67%) and the lowest in Mardan (18.33%). The while the lowest during December in Katlang and prevalence showed non-significant differences (P>0.05) Takhtbhai 0% and during January in Mardan and when analyzed statistically (Table 7). Takhtbhai (Table 8). 75

10 Table 10: Microscopy and ELISA Comparison for the Diagnosis ofanaplasmosis in Goats in 3 Tehsils of District Mardan from January December 2014 Microscopy ELISA Factors Total observed samples No. of positive Prevalence (%) No. of positive Prevalence (%) Months January February March April May June July August September October November December Season Winter Spring Summer Autumn Gender Male Female Age 0-1 year >1year Area Mardan Katlang Takhtbhai Total Prevalence by Season and Area (%) Using ELISA: The 2 two diagnostic methods ( = 5.87, df = 1; P = in highest and lowest prevalence was recorded during sheep and( 2 = 10.88, df = 1; P = in goats) (Table 9 summer and winter respectively in all three tehsils and 10). (Table 8). Month Wise Comparison: In month wise data microscopy Prevalence by Area and Age (%): Age wise prevalence shown highest prevalence in July i.e %, while ELISA was recorded higher in adults in Mardan, Katlang and method shown highest prevalence in June i.e % Takhtbhai (21.62%, 23.08% and 25%) than young ones while lowest prevalence was shown in December by both (13.04%, 19.05% and 16.67% respectively) (Table 8). the diagnostic methods in sheep (Table 9). In goats the high prevalence was in July that is Prevalence by Gender and Area (%): High prevalence 26.67% and 40% in microscopical examination and ELISA was observed in female goats in Katlang and respectively while lowest in October, November, Takhtbhai (22.86%, 25%) than males (20%, 16.67% December, January and February (0%) and 6.67% in respectively. While in Mardan male goats have higher December and January by microscopy and ELISA prevalence than females i.e % and 17.95% respectively (Table 10). respectively (Table 8). Season Wise Comparison: Through both microscopy and Microscopy and ELISA Comparison for Diagnosis of ELISA the highest prevalence in sheep was associated Anaplasmosis with summer (23.337%) and (35%) and lowest in winter Overall Comparison: Overall prevalence of Anaplasmosis (6.67%) and (13.33%) respectively (Table 9). diagnosed through microscopical examination and ELISA In goats the highest prevalence was observed in test was observed as 13.89% and 23.89% in sheep and summer (20% and 33.33%) and the lowest prevalence was 8.833% and 20.56% in goats respectively. When analyzed noted in winter (0% and 10%) by both microscopy and statistically significant difference was noted between the ELISA respectively (Table 10). 76

11 Table 11: Specificity and Sensitivity of ELISA Factors Specificity Sensitivity Sheep Goats Table 12: Effect of Anaplasmosis on hematology of sheep Parameters Non-infected Infected 12 RBC (10 /L 9.44± ± 0.15 * Hemoglobin (g/dl) 11.84± ± 1.20* Packed cell volume% 31.20± ± 0.17* MCHC(mg/dl) 37.10± ± 0.45* MCH (pg) 12.69± ± 1.09* MCV (fl) 35.71± ± WBC (10 /L) 8.97± ± 0.60* Table 13: Effect of Anaplasmosis on hematology of goats Parameters Non-infected Infected 12 RBC (10 /L ± ±1.01* Hemoglobin (g/dl) ± ±0.83* Packed cell volume% ± ±0.99* MCHC(mg/dl) ± ±1.68* MCH (pg) 9.90 ± ±2.10 MCV (fl) ± ±1.48* 9 WBC (10 /L) 9.70 ± ±374.2* Age Wise Comparison: Sheep above 1 year showed higher rate of prevalence (15.97%), (24.36%) than less than 1 year (9.83%), (22.95%) by microscopic examination and ELISA respectively (Table 9). Goats above 1 year showed higher rate of prevalence (10.71%) and (23.21%) than less than 1 year (4.41%), (16.18%) by microscopy and ELISA respectively (Table 10). Gender Wise Comparison: Female sheep showed higher susceptibility (15.79% and 24.56%) than males as (10.60% and 22.72%) respectively by Microscopic examination and ELISA (Table 9).Similarly female goats showed higher susceptibility (10% and 21.81%) than males as (5.71% and 18.57%) respectively by microscopic examination and ELISA (Table 10). Area Wise Comparison: In sheep higher prevalence was recorded in Katlang both by microscopy and ELISA i.e % and respectively (Table 9). While the lowest prevalence was recorded in Mardan by microscopy and ELISA technique i.e.6.66% and 21.67% (Table 9). In goats the higher prevalence was recorded in Katlang both by microscopy and ELISA i.e. 10% and respectively (Table 9). While the lowest prevalence was recorded in Mardan by microscopy (6.67%) and ELISA (18.33%) (Table 10). Specificity and Sensitivity of ELISA: Specificity and sensitivity of ELISA was 100% and >88% respectively for sheep as well as goats (Table 11). Hematological Studies: Hematological parameters of infected sheep and goats showed a decrease in all blood indices except WBCs which showed an increase. When analyzed statistically all blood parameters showed significant decrease except WBCs which showed significant increase (Tables 12 and 13). DISCUSSION The research on sheep and goats anaplasmosis is rare and little literature is available. This study was designed to report the prevalence of anaplasmosis in goats and sheep in district Mardan of Khyber Pakhtunkhwa province, Pakistan. In this study, 360 blood samples from goats and sheep were collected from January 2014 to December 2014 from three tehsils of district Mardan to identify seroprevalence of anaplasmosis. Moreover, hematological analysis of the infected and non infected animals was done to study the effect of anaplasmosis on blood profile of the infected animals. The present study showed some epidemiological aspects of ovine and caprine anaplasmosis. The occurrence of anaplasmosis at three regions i.e Mardan, Takhtbhai and Katlang is influenced by various factors like environment and presence of vector. In current study the anaplasmosis by microscopy was prevalent in 13.89% sheep. The present results were similar to that of Talat et al. [28] and Azza et al. [29]. In this study, the prevalence of anaplasmosis in goats by microscopy was 8.33%. The present results were similar to that of Okaiyeto et al. [30]. In different countries several authors recorded different infection rates like [31] recorded 47.5 % in sheep and 80.3 % in goats, Boo-Hong et al. [32] 71.7 %, 20.1 % in goats and Usman et al. [33] 33.3% in sheep. In present research, the prevalence (%) of anaplasmosis in sheep by ELISA was 23.89%. The present results were similar to that of Kashif et al. [34]. In present research, the prevalence of anaplasmosis in goats by ELISA was 20.56% which is higher than recorded by Ramos et al. [35] 11.93% and lower than recorded by Shompole et al. [36], % and Mbati et al. [37] 100 %. 77

12 In current research, the highest prevalence was recorded in summer 23.33% and 20% in sheep and goats respectively whereas the lowest during winter 6.67% in sheep and 0% in goats by using microscopy technique. Similarly the highest seasonal wise prevalence by ELISA was noted during summer 35% and 33.33% in sheep and goats respectively whereas the lowest prevalence was recorded in winter 13.33% in sheep and 10% in goats. In this study the relatively high infection observed in summer could be due to the favorable environmental conditions for tick vectors proliferation and survival which are responsible for the transmission of the anaplasmosis. The present results were similar to that of Azza et al. [29], Razmi et al. [31], Adejinmi et al. [38] and Kyung-Ok et al. [39] inversely [40] reported that in animals greater than one year of age anaplasmosis is predominantly a disease of autumn and winter. Tick vector population in district Mardan was low although tick vectors were noted in some sheep and goats during sample collection and this may be the reason of comparatively low prevalence of the infection in this region. Hematological parameters of infected sheep and goats showed a decrease in all blood indices except WBCs which showed an increase, the reason may be the extravascular hemolytic effect of this infection and phagocytosis of infected erythrocytes by reticuloendothelial cells. Similar findings were recorded by others [30, 32, 38, 41, 42]. This research is the first epidemiological study of Anaplasma infection in sheep and goats from Mardan Khyber Pakhtunkhwa Pakistan and it is concluded that anaplasmosis is prevalent in district Mardan. Haemolyticanaemia is the main haematological finding of anaplasmosis. In sheep and goats the competitive ELISA seems best for use in Anaplasmosis diagnosis Further country wide studies are needed to know the epidemiology of Anaplasma spp. infection in sheep and goats in Pakistan by using advanced serological and molecular techniques, mainly to define which species is involved. CONCLUSION It is concluded that Anaplamosis is widely distributed in district Mardan, KPK, Pakistan. Hemolytic anemia is the major hematological finding of Anaplasmosis. The findings of the present study would help in planning prevention and control strategies for bovine Anaplasmosis in Pakistan. REFERENCES 1. Uilenberge, G., General review of tick-borne diseases of sheep and goats worldwide. Parasitologia, 39: Bekker, C.P., S. De Vos, A. Taoufik, O.A. Sparagano and F. Jongejan, 2002.Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichiaruminantiumin Amblyommavariegatum ticks by reverse line blot hybridization. Veterinary Microbiology, 89: Parola, P., B. Davoust and D. Raoult, Tick- and flea-borne rickettsial emerging zoonoses. Veterinary Research, 36: Perry, B.D., T.F. Randolph, J.J. Mcdermott, K.R. Sones and P.K. Thornton, 2002.Investing in animal health research to alleviate poverty. International Livestock Research Institute, Nairobi, Kenya. 5. Minjauw, B. and A. Mcleod, Tick-borne diseases and poverty. The impact of ticks and tick-borne diseases on the livelihoods of small-scale and marginal livestock owners in India and eastern and southern Africa. Research report, DFID Animal Health Programme, Centre for Tropical Veterinary Medicine, University of Edinburgh, UK. 6. Nicholson, W.L., K.E. Allen, J.H. McQuiston, E.B. Breitschwerdt and S.E. Little, The increasing recognition of rickettsial pathogens in dogs and people. Trends in Parasitology, 26: Jongejan, F. and G. Uilenberg, The global importance of ticks. Parasitology, 129: Khan, M.Q., A. Zahoor, M. Jahangir and M.A. Mirza, Prevalence of blood parasites in cattle and buffaloes. Pakistan Veterinary Journal, 24: Theiler, A., Further investigations into anaplasmosis of South African cattle. First Report of the Director of Veterinary Research Union of South Africa, pp: Splitter, E.J., M.J. Twiehaus and E.R. Castro, Anaplasmosis in sheep in the United State. Journal of the American Veterinary Medical Association, 127: Sainz, A., I. Amusategui and M.A. Tesouro, Ehrlichia platys infection and disease in dogs in Spain. Journal of Veterinary Diagnostic Investigation, 11:

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