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Byline: F. A. Atif, M. S. Khan, T. Roheen, F. Muhammad, M. Younus, M. Avais and S. Ullah


The study was designed to investigate the seroprevalence of Anaplasma marginale among cattle from Sargodha, Khushab and Rawalpindi districts of the northern Punjab, Pakistan. A total of 1050 samples were collected from selected small holders and private livestock farms using multistage cluster random sampling technique. The overall seroprevalence of Anapalsma marginale infection was 31.05 percent % using competitive enzyme-linked immunosorbent assay (cELISA). The highest seroprevalence was recorded in Sargodha (37.14%) district followed by Khushab (31.43%) and Rawalpindi (24.57 %) districts. A significant relationship was found among different age groups and breed. The seroprevalence was significantly higher in small holders than private livestock farms in all study districts. It is concluded that small holder's crossbred cattle of more than four years of age from Sargodha district are more susceptible to Anaplasma marginale infection in summer season.

Key words: Seroprevalence, Anaplasma marginale, cattle, northern Punjab.


Anaplasmosis is one of the important globally distributed tick-borne disease of cattle with great economic impact (Minjauw and Mcleod, 2003). Bovine anaplasmosis (BA) is caused by obligate intraerythrocytic rickettsia of the order Rickettsiales, family Anaplasmataceae, genus Anaplasma (Dumler et al, 2001) characterized by fever, weight loss, decreased milk production, pale mucous membranes, severe anaemia, jaundice, brownish urine, hyper-excitability abortion and mortality without hemoglobinemia and hemoglobinuria during acute phase of the infection (Richey and Palmer, 1990).

Serological tests commonly used for the serodiagnosis of Anaplasma marginale including agglutination test, indirect fluorescent antibody test, complement fixation and indirect ELISA. There were problems of sensitivity, reproducibility, interpretation and non-specific reactions associated with these tests (OIE, 2004; OIE, 2012). Competitive ELISA has obvious advantage because of greater sensitivity 96% and specificity 95% (Urdaz-Rodriguez et al., 2009).

There exist very scanty information if ever available on the serological survey of Anaplasma marginale infection in cattle using competitive enzyme linked immunosorbent assay from northern, Punjab. Most of the earlier reports from Pakistan were based on the examination of stained blood smears (Atif et al., 2012).

In Pakistan, the prevalence of Anaplasma marginale has been recorded as 7.36-75.71 percentusing microscopic examination of blood smears (Khan et al., 2004, Afridi et al., 2005; Rajput et al., 2005). This technique usually fails to detect chronically infected carriers with low levels of parasitaemia (Kocan et al., 2010). The identification of carriers are important for epidemiological standpoint as well as for planning disease prevention and control strategies. Therefore, the present study was designed to investigate the prevalence of Anaplasma marginale infection from Sargodha, Khushab and Rawalpindi districts of the Punjab, Pakistan.


Study area: Epidemiological studies were conducted in Sargodha, Khushab and Rawalpindi districts of the Punjab, Pakistan. Sargodha district has average temperature ranging from 25-49oC in summer and 5-23oC in winter and annual rainfall of 526 millimeter. Khushab is the driest and hottest district. The temperature ranges from 25-48oC in summer and 19-29oC in winter with average annual precipitation of 521 millimeter. Rawalpindi district is categorized as arid high rainfall zone (PARC, 2012). The average temperature ranges from 23.5-35.5oC in summer and 10-17.5oC in winter with annual average rainfall of 1364 millimeter (GOP, 2012).

Sampling strategy: A serological survey on the prevalence of Anaplasma marginale was conducted at Sargodha, Khushab and Rawalpindi districts of the Punjab, Pakistan from indigenous and crossbred cattle during September, 2009 to August, 2010. A total of 1050 blood samples were collected from randomly selected small holders (n=90) and private livestock farms (n=12) using multistage cluster random sampling technique (Thrusfield, 2005) and serum was separated. A total of 30 union councils, 34 cattle farms (30 small holders and 4 livestock farms) and 350 animals were selected as primary, secondary and tertiary sampling units from each district. Animals were sampled in different age groups i.e. less than 1 year, 1-2 year, greater than 2-4 year and greater than 4 years.

The criteria for the selection of small holders and private livestock farms were: a) small holder having 1-10 cattle; (b) Livestock farm having [greater than or equal to] 50 cattle; (c) Distance between small holder farms [greater than or equal to] 5 kilometer; (d) Distance between livestock farms greater than10 kilometer.

Serological screening: Antibodies against A. marginale in serum were detected by MSP-5 competitive enzyme linked immunosorbent assay (cELISA) using commercially available Anaplasma Antibody Test Kit, cELISA (VMRD Inc., Pullman, WA, USA) validated previously by Urdaz-Rodriguez et al. (2009). The test was performed according to the manufacturer's instruction (VMRD Inc., Pullman, WA, USA).

An ELISA reader (Statfax(r) 2100 Microplate Reader, Awareness Technologies, Inc.) was used to measure the optical density at 620 nm wavelength. A cutoff of 30% inhibition was used to differentiate between positive and negative samples. Serum samples with [greater than or equal to]30% inhibition were considered positive and samples with less than30% inhibition were considered negative. The percent inhibition was calculated using formula: [Sample optical density Inhibition percentage = 100 ---------------------------x 100] [Mean negative control optical density] Prevalence was estimated using formula: P= d/n x 100; where P= Prevalence, d= No. of animals found positive, n= Total no. of animals sampled (Thrusfield, 1995).

Statistical analysis: The data was statistically analyzed by applying Chi square test using Statistical Package for Social Services (SPSS) version 13.0. A p-value less than0.05 was considered statistically significant.


Out of 1050 samples, 326 (31.05%) were found positive for Anaplasma marginale. Highest seroprevalence was recorded in Sargodha district (37.14%) followed by Khushab (31.43%) and Rawalpindi (24.57%) districts (Table 1). There was significant difference in seroprevalence of Anaplasma marginale among districts. Irrespective of the district under study, a significant (Pless than0.001) association among different age groups was recorded. The highest seroprevalence of Anaplasma marginale was found in greater than4 yearsage as compared to other age cohorts i.e. less than1, 1-2 and greater than2-4 years. Though seroprevalence was higher in females compared to males (Table 1); but statistically it was non-significant (Pgreater than0.05). Regardless of the district under study, seroprevalence of Anaplasma marginale was significant between breeds. The seroprevalence was higher in crossbred as compared to indigenous cattle.

Similarly, significant association was observed on the seroprevalence of Anaplasma marginale infection among small holders in all districts whereas, seroprevalence had non- significant association at livestock farms (Table 1). The highest (39.60%) seroprevalence of Anaplasma marginale was recorded in summer in all study districts. Seasonal prevalence was found significant in Sargodha and Khushab districts, while non-significant association was revealed in Rawalpindi district.

Table 1. Seroprevalence of Anaplasma marginale among cattle in Sargodha, Khushab and Rawalpindi districts of the Punjab, Pakistan from September, 2009 to August, 2010.



Age###less than1 year###47/318###14.78

1-2 years###42/198###21.21

greater than2-4 years###80/210###38.10

greater than4 years###157/324###48.46









Farm size###Small holder###157/450###34.89

Cattle farm###169/600###28.17




n= no. of tick infested cattle; N= total no of cattle examine; significant association, chi square p-value less than0.05; df= degree of freedom; Age: (kh2= 98.99, df= 3, P less than 0.001); Sex: (kh2= 1.215, df= 1, P greater than 0.05); Breed: (kh2= 12.02, df= 1, P less than 0.001). Seasonal prevalence: Sargodha (kh2= 12.95, df= 3, Pless than0.01); Khushab (kh = 8.82, df= 3, Pless than0.05); Rawalpindi (kh = 5.763, df= 3, Pgreater than0.05); Small holder (kh = 7.76, df= 2, Pless than0.05); Livestock farms (kh2= 5.552, df= 2, Pgreater than0.05); District: (kh2= 12.95, df= 2, Pless than0.05);


It was hard to find any previous reference regarding sero-prevalence of Anaplasma marginale infection in northern Punjab, Pakistan. There were variations in the distribution of A. marginale infection in different

geographical regions. The seroprevalence was statistically significant among all study districts. Major parts of study districts have distinct agro-ecological zone (PARC, 2012), justify the significant difference in seroprevalence among study districts. Moderate climate of Sargodha district favours the growth and multiplication of vector ticks. Moreover, higher prevalence of R. (Boophilus) microplus and stall feeding practices could have possibly accounted for higher prevalence of Anaplasma marginale infection at Sargodha. Marufu et al. (2010) have recorded the seroprevalence as 26percent in semi-arid, sweet and sour rangeland of South Africa using competitive inhibition ELISA. The seroprevalence is quite comparable with Rawalpindi and Khushab districts having arid environment.

The seroprevalence recorded in the present study was 31.05%, pointing the region is rather endemically unstable. Endemic stability most likely occurs in regions where serum antibodies prevail in 70% of animal population (Perry and Young, 1995; Peter et al., 1997). The competitive ELISA was used for the detection of serum antibodies as recommended by World Animal Health Organization for the serodiagnosis of anaplasmosis in cattle (OIE, 2004). Competitive ELISA based on major surface protein-5 has obvious advantage over other serological tests because of higher sensitivity 96% and specificity 95% for anaplasmosis (Urdaz- Rodriguez et al., 2009). The cELISA uses a 19-kDa antigen based on recombinant major surface protein (MSP5) which is highly conserved among Anaplasma species (Knowles et al., 1996).

Agreement was found on the age-wise prevalence of Anaplasma marginale with Swai et al. (2005) and Urdaz-Rodriguez et al. (2009). The significant relationship of different breeds on the prevalence of Anaplasma marginale was found. Similarly, Khan et al. (2004) mentioned higher prevalence of tick-borne disease in cross bred cattle (19.4%) than indigenous Red Sindhi (17%) and Dhanni (14%) breeds. Lower seroprevalence in indigenous cattle indicate inherent resistance to ticks which results in lower Anaplasma marginale infection (Swai et al., 2007). Tick resistance trait most likely contributed lower Anaplasma marginale infection. The European breeds are more susceptible to tick-borne diseasesdue to higher infestation of ticks (Bock et al., 1997).

Higher prevalence of Anaplasma marginale in small holders (34.89%) endorse the findings of Urdaz- Rodriguez et al. (2009) who also reported an increasing trend in seropositivity in small holders than medium and large livestock farms. Hugh-Jones et al. (1998) found no association between herd size and seroprevalence while Perez et al. (1994) documented that seroprevalence increased with increasing herd size in dairy animals. Swai et al. (2005) considered that higher seroprevalence in small holders might be associated with specific age group. The association of herd size with Anaplasma marginale seropositivity was found non-significant (Pgreater than0.05) in the present study. Poor management, lack of tick control practices and inadequate economic sustainability of poor resource small holder farmers for the implementation of proper management and animal health practices contributed the higher seroprevalence (Swai et al., 2005; Gralen, 2009).

It is concluded that Sargodha district's small holder crossbred cattle of more than four years of age are more susceptible to Anaplasma marginale infection in summer.


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Publication:Journal of Animal and Plant Sciences
Geographic Code:9PAKI
Date:Aug 31, 2013

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