Seroprevalence of IgG and IgM antibodies and associated risk factors for toxoplasmosis in cats and dogs from subtropical arid parts of Pakistan

Tropical Biomedicine 31(4): 777 784 (2014) Seroprevalence of IgG and IgM antibodies and associated risk factors for toxoplasmosis in cats and dogs from subtropical arid parts of Pakistan Ahmad, N. 1*, Ahmed, H. 2, Irum, S. 2 and Qayyum, M. 1 1 Department of Zoology, Pir Mehr Ali Shah, Arid Agriculture University, Rawalpindi, Pakistan 2 Department of Biosciences, COMSATS Institute of Information Technology (CIIT), Park Road, Chakhshazad, Islamabad * Corresponding author email: nisarahmed11@gmail.com Received 12 December 2013; received in revised form 30 March 2014; accepted 20 May 2014 Abstract. Pet cats and dogs are an important source of human toxoplasmosis because of their intimate relationship with humans. Present study was designed to determine the prevalence and risk factors of toxoplasmosis in cats and dogs in northern sub-tropical arid region of Pakistan where no such work has been previously conducted. For this study 420 cats and 408 dogs visiting different pet clinics and veterinary hospitals were screened for the presence of anti-toxoplasma IgG and IgM antibodies using ELISA technique. Epidemiological information regarding age, sex, area, outdoor access and hunting practice was obtained from the owners by questionnaire interview. Overall seroprevalence in cats and dogs was 26.43% (111/420) and 28.43% (116/408) respectively. IgG antibodies were found in 23.33% (98) cats and 25.49% (104) dogs while IgM antibodies were found in 3.57% (15) cats and 3.92% (16) dogs. Seroprevalence was significantly high in cats and dogs older than one year. No significant difference was recorded between males and females. Cats and dogs from rural areas showed higher prevalence. Dogs which had access to outside also showed high seroprevalence. The present study indicates that Toxoplasma gondii is widespread in pet animals in Pakistan which may have important implication for public health. INTRODUCTION The role of felids including domestic cat in the spread of toxoplasmosis is very critical as they serve as definitive hosts of the parasite where oocysts are produced. All other mammals including human beings and birds serve as intermediate hosts (Dubey, 2004). Millions of oocysts are shed by cats after getting infected (Dabritz et al., 2007). These oocysts when ingested along with food and water can affect humans and livestock animals resulting in abortions, neonatal deaths and other complications in both humans and animals (Afonso et al., 2008). Although the infection can also be transmitted congenitally or by eating meat of T. gondii infected animals, it has been reported by many studies that T. gondii infection is not maintained in the environment in the absence of cats (Dubey et al., 1997). Seroprevalence of T. gondii in humans has been associated with close contact with cats and having pet cats at home (Pereira et al., 1992). Serological survey of T. gondii antibodies in cats is very much needed as it also assess the degree of oocysts contamination of the environment (Miro et al., 2004). Pet dogs are often regarded as closest faithful and intimate friends of humans. There are also some reports of T. gondii oocysts shedding by dogs because oocysts ingested via food and water in dogs can pass through intestinal tract and are excreted in the feces (Lindsay et al., 1997). The presence of pet dog in household has been found to be a risk factor for T. gondii infection in humans (Sroka et al., 2010). Dogs may facilitate the 777

transmission of the disease to humans by facilitating human exposure to oocysts present in the environment by rolling in contaminated cat faeces or excreting oocysts after ingesting oocyst infected food (Frenkel et al., 1995). Presence of dogs in household indicates a potential risk of toxoplasmosis to humans. Keeping in view the importance of the infection in cats and dogs, the present study was designed to workout the prevalence and associated risk factors in cats and dogs in sub-tropical arid parts of northeastern Pakistan. MATERIALS AND METHODS Study Area Present study was conducted in four districts of Potohar plateau region namely Rawalpindi, Jhelum, Chakwal and Attock. Potohar plateau is located between 32 30' to 34 northern latitudes and 71 45' to 73 45' eastern latitudes in northern parts of Punjab province and has a distinct arid sub-tropical climate as compared to other parts of the country (Fig. 1). Consisting of a population of 7.5 million individuals, the plateau covers an area of 23,160 square kilometers. The area is usually arid and agriculture mainly depends upon rainfall which varies between 350 mm to 500 mm annually. Average temperature is around 7.9 C in winter and 30.6 C in summers. Study Animals and Sampling Present study included pet cats and dogs visiting different pet clinics and veterinary hospitals in the study area. A total of 420 cats and 408 dogs were tested for the presence or absence of T. gondii infection from January to December 2012. The blood samples were collected from jugular or sephanous vein and were quickly transported to laboratory where they were centrifuged at 3000 rpm for 15 minutes to obtain serum. Epidemiological Information Additional epidemiological information regarding age, access to outside, area and hunting practice was obtained on questionnaire by interview to the owner. Climatic Data In order to workout the seasonal distribution of toxoplasmosis, the positive result from Figure 1. Map of Pakistan showing study area (shaded) 778

acute disease phase (IgM positive cases) were compared with climatic data. Daily climatic data (maximum temperature, minimum temperature, rainfall and relative humidity) was collected from the meteorological department of Pakistan. Serodiagnosis Commercial ELISA Kits (ID Screen Toxoplasmosis Indirect ID-VET Company, France) were used for the detection of IgG and IgM antibodies to Toxoplasma. Results were interpreted according to the manufacturer s instructions. Statistical Analysis Univariate and multivariate logistic regression models were used to find out the association between toxoplasmosis and various risk factors i.e. locality, sex, age, area (urban or rural), access to outside and hunting practice by using SPSS version 11.5. Crude and adjusted odds ratios were calculated by comparing non-infected and infected animals. Chi square test was performed by using Instat version 3.36. Results were considered significant at P<0.05. RESULTS A total of 111 cats and 116 dogs were true positive while 12 cats and 5 dogs were doubtful in serodiagnosis. All the doubtful samples were found negative in subsequent analysis. Overall prevalence was 26.43% and 28.43% in cats and dogs respectively. Prevalence of IgG antibodies in cats was 23.33% as 98 cats were found positive for Toxoplasma-specific IgG antibodies while prevalence of IgM antibodies was 3.57% as 15 cats were found positive for IgM antibodies. Two cats were found positive for both IgG and IgM antibodies. Similarly IgG antibodies were found in 104 dogs giving IgG prevalence of 25.49% while IgM antibodies were found in 16 dogs giving IgM prevalence of 3.92%. Four dogs had both IgG and IgM antibodies (Table 1). Despite dogs showing a little higher seroprevalence as compared to cats, there was no significant difference in seroprevalence in both species (P>0.05, χ 2 =0.4). No significant difference was observed in prevalence in cats (P>0.05, χ 2 =3.8) and dogs (P>0.05, χ 2 =3.0) from four districts. Seroprevalence was higher in older cats as compared to younger ones. No sex specific difference of seroprevalence was observed in cats. Prevalence was little high in female cats as compared to males but the difference was statistically non-significant (P>0.05, χ 2 =0.1). Access to outside also did not contribute in disease prevalence in cats (P>0.05, χ 2 =0.4). Similarly hunting practice was also a non-significant factor in the spread of the disease in cats (P>0.05, χ 2 =0.7). However, prevalence varied highly significantly in cats with respect to urban and rural areas. Infection in cats belonging to rural areas was significantly high as compared to urban cats (P<0.01, χ 2 =12.6). Table 1. Number of IgG and IgM positive or negative pet animals Categories Cats Dogs N % N % IgG (-) and IgM (-) 309 73.57 292 71.57 IgG (+) and IgM (-) 96 22.86 100 24.51 IgG (-) and IgM (+) 13 3.10 12 2.94 IgG (+) and IgM (+) 2 0.48 4 0.98 Total IgG (+) 98 23.33 104 25.49 Total IgM (+) 15 3.57 16 3.92 Overall Positives 111 26.43 116 28.43 Total Samples 420 408 779

In dogs, age was also a significant contributing factor in disease prevalence. Dogs older than one year had higher prevalence as compared to dogs younger than one year (P<0.01, χ 2 =15.0). Prevalence also did not vary with respect to sex in dogs (P>0.05, χ 2 =1.6). However, female dogs showed 31.13% prevalence as compared to 25.51% prevalence in males. Access to the outside also rendered dogs more seropositive (P<0.01, χ 2 =8.4). Seroprevalence was 31.13% in dogs which had access to outside as compared to 25.51% in those which had no access. Rural and urban areas also showed variation in dog seroprevalence. Rural dogs had significantly higher prevalence as compared to urban dogs (P<0.01, χ 2 =9.2). Hunting practice did not show any significant variation in seroprevalence in dogs (P>0.05, χ 2 =0.8). The comparison of climatic data collected during different months and acute infection (IgM positive cases) in cats and dogs during the same period did not show significant variations. However, we were able to observe an increasing trend of the acute infection in period having high monsoon rainfall and moist warm conditions (July to September) (Fig. 2 and 3). This trend was not observed in case of comparison of chronic infection with climatic data. Figure 2. Correlation between prevalence of acute toxoplasmosis and weather parameters in cats in northern Punjab, Pakistan Figure 3. Correlation between prevalence of acute toxoplasmosis and weather parameters in dogs in northern Punjab, Pakistan 780

Table 2. Univariate and multivariate analysis of risk factors associated with toxoplasmosis in cats Risk Factors Category Total Infected Prevalence % (95% C.I.) Univariate Multivariate Odds Ratio Adjusted Odds P (95% C.I.) Ratio (95% C.I.) P Locality Rawalpindi 100 24 24.00 (16.69 33.23) Reference N.C. Attock 121 26 21.49 (15.11 29.63) 0.87 (0.46 1.63) 0.65 Chakwal 108 32 29.63 (21.84 38.82) 1.33 (0.72 2.47) 0.36 Jhelum 91 29 31.87 (23.20 42.01) 1.48 (0.78 2.80) 0.22 Age < 1 year 146 23 15.75 (10.73-22.53) Reference Reference > 1 year 274 88 32.12 (26.87-37.86) 2.53 (1.52-4.22) <0.001 2.12 (1.64 3.88) <0.001 Sex Male 222 57 25.68 (20.38-31.81) Reference N.C. Female 198 54 27.27 (21.54-33.86) 1.09 (0.70-1.68) 0.71 Access to No 269 74 27.51 (22.52-33.13) Reference N.C. Outside Yes 151 37 24.50 (18.33-31.94) 0.86 (0.54-1.35) 0.50 Area Urban 132 20 15.15 (10.03-22.24) Reference Reference Rural 288 91 31.60 (26.50-37.18) 2.59 (1.51-4.42) <0.001 2.20 (1.44 5.52) <0.01 Hunting No 373 101 27.08 (22.82-31.81) Reference N.C. Yes 47 10 21.28 (11.99-34.91) 0.73 (0.35-1.52) 0.39 N.C. = Not calculated Table 3. Univariate and multivariate analysis of risk factors associated with toxoplasmosis in dogs Risk Factors Category N (+) Prevalence % (95% C.I.) Univariate Multivariate Odds Ratio P Adjusted Odds P (95% C.I.) Ratio (95% C.I.) Locality Rawalpindi 128 34 26.56 (19.67 34.81) Reference N.C. Attock 68 21 30.88 (21.17 42.63) 1.24 (0.65 2.36) 0.52 Chakwal 107 36 33.64 (25.39 43.02) 1.40 (0.80 2.46) 0.24 Jhelum 105 25 23.81 (16.68 32.79) 0.86 (0.48 1.57) 0.63 Age < 1 year 128 20 15.63 (10.35 22.91) Reference Reference > 1 year 280 96 34.29 (28.98 40.03) 2.82 (1.65 4.82) <0.001 2.23 (1.56 4.56) <0.001 Sex Male 196 50 25.51 (19.92 32.04) Reference N.C. Female 212 66 31.13 (25.28 37.65) 1.32 (0.86 2.04) 0.21 Access to No 256 60 23.44 (18.67 29.00) Reference Reference Outside Yes 152 56 36.84 (29.58 44.74) 1.91 (1.23 2.95) <0.001 1.78 (1.12 2.96) <0.01 Area Urban 106 18 16.98 (11.02 25.25) Reference Reference Rural 302 98 32.45 (27.42 37.92) 2.35 (1.34 4.12) <0.01 2.04 (1.22 3.45) <0.01 Hunting No 320 96 30.00 (25.24 35.23) Reference N.C. Yes 88 22 25.00 (17.13 34.96) 0.78 (0.45 1.33) 0.36 N.C. = Not calculated DISCUSSION Diagnosis of T. gondii infection in cats and dogs is always necessary not only because of their role in transmission of the infection to humans but also their diagnosis determines oocyst contamination of the environment (Miro et al., 2004). As T. gondii infection cannot be diagnosed according to clinical signs and symptoms, so serodiagnosis offers a good and reliable method of detection of the infection in cats and dogs. Toxoplasma gondii infection can also be diagnosed by inspection of various tissues but this practice 781

require killing of the animals (Al-Qassab et al., 2009). Simultaneous measurement of IgG and IgM antibodies give more reliable results as it estimates chronic as well as acute phase of the infection (Dubey et al., 1990). In other parts of Pakistan, Ahmad et al. (2001) have found 50% and 60% seroprevalence in dogs and cats respectively from Faisalabad district which was quite high as compared to the current study. However they used very small sample size of only 10 cats and 40 dogs. Seroprevalence in cats has been found in many studies conducted in different parts of the world including those conducted in Japan (Maruyama et al., 2003), Spain (Miro et al., 2004), France (Afonso et al., 2006), Brazil (Pena et al., 2006), Czech Republic (Sedlak & Bartova, 2006), China (Zhang et al., 2009), Korea (Lee et al., 2010) and Iran (Raeghi et al., 2011). Similarly, seroprevalence in dogs was also reported from Nigeria (Ogunrinade, 1978), Sri Lanka (Silva et al., 1997), Czech Republic (Sedlak & Bartova, 2006), Iran (Hosseininejad et al., 2011) and China (Wu et al., 2011). The differences in seroprevalence in cats and dogs in different regions of the world might be due to difference of stray versus pet animals, survival of the oocysts in environment and diagnostic tests used (Dubey, 2004). Higher prevalence found in cats and dogs older than one year may be attributed to increase in the possibility of the contact with parasite with age (Cabral et al., 1998). Higher seroprevalence in older cats and dogs is also reported by Pena et al. (2006) and Wu et al. (2011) respectively. Higher seroprevalence found in cats and dogs from rural areas is due to the fact that rural areas harbour more oocysts in environment as compared to urban areas because of two facts. Firstly, the number of domestic and wild felids is usually high in rural areas. Secondly, soil exposure is less common in urban areas which reduce the chances of coming closer with oocysts that are more frequently found in soil. Present study is also in agreement with Gyroke et al. (2011) and Opsteegh et al. (2012) in this respect as both studies found significantly higher seroprevalence in rural cats. The current study found a significant increase in seroprevalence in pet cats which had access to outside. However no such difference was observed in the case of dogs. This may be explained by the fact that pet dogs get access to outside only with owner. The owner can check the consumption of food by dog. Cats also get frequent access to outside without owner and can consume oocyst contaminated food and T. gondii infected birds and rodents. Presence of other cats in vicinity related to toxoplasmosis in cats has been found by many researchers including Gauss et al. (2003) and Lopes et al. (2008). Gyorke et al. (2011) also found outdoor access as significant risk factor for T. gondii infection in cats. Toxoplasma gondii infection may be transmitted to carnivorous mammals by ingesting infected prey such as birds and rodents. However, present study did not find any significant association of the infection with hunting practice in cats and dogs. Unlike present study Opsteegh et al. (2012) associated hunting practice as significant contributing risk factor for transmission of the infection in cats. Lack of association of hunting practice with T. gondii infection in present study may be attributed to low prevalence of infection in prey animals. High prevalence of acute toxoplasmosis observed from July to September may be attributed to higher monsoon rainfalls during the period as high rainfall, moist conditions and warm temperature increases the survival of the oocysts in the environment (Fayer, 1981) (Fig. 2 and 3). The time period from July to September is ideal for oocyst survival in Potohar region due to warm moist temperature and high percentage of relative humidity. Our study not only found that T. gondii infection is widespread in cats and dogs in northeastern parts of Pakistan but also worked out possible risk factors contributing to the transmission of infection in these pet animals. This information may serve as a road map for making control strategies to minimize the risk of this important zoonosis to humans. 782

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