Sarfraz-ur-Rahman  ( Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan. )
Aqib Saeed  ( Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan. )
Sakandar Khan  ( Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan. )
Zia-ul-Rehman Khalid  ( Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan. )
Muhammad Imran Rashid  ( Department of Parasitology, University of Veterinary and Animal Sciences (UVAS) Lahore, Pakistan )
Haroon Akbar  ( Department of Parasitology, University of Veterinary and Animal Sciences (UVAS) Lahore, Pakistan )

Objective: To review the seroprevalence of toxoplasmosis in Pakistan.

 

Method: The systematic review comprised search on Science Direct, Google Scholar, PubMed and Scopus databases for studies related to the seroprevalence of toxoplasmosis in Pakistan published between 2006 and 2020 which used serological diagnostic tests to detect Toxoplasma gondii. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were used throughout the review and statistical analysis was done using forest plot and random effect model.

 

Results: Of the 7093 human studies initially found, 20(0.28%) were reviewed. Of the 16,432 animal studies, 16(0.09%) were selected for detailed review. The pooled seroprevalence of toxoplasmosis in humans, calculated in this review was found as (76%) (95% confidence interval: 69-83%). Seroprevalence of human toxoplasmosis was higher in Khyber Pakhtunkhwa (31.7%) than Punjab (20.4%). Pooled seroprevalence in animals calculated in this review was found as (69%) (95% confidence interval: 64-74%). Seroprevalence in animals was higher in Khyber Pakhtunkhwa (44.7%) than Punjab (29.4%).

 

Conclusions: The seroprevalence of toxoplasmosis in both humans and animals should be studied it other parts of Pakistan as well.

 

Key Words: Livestock, Humans, Random-effect model, Toxoplasma gondii, Seroprevalence. DOI: 10.47391/JPMA.5699

 

Submission completion date: 12-01-2022

Acceptance date: 11-11-2022

 

Introduction

 

Toxoplasmosis is a zoonotic disease caused by parasite Toxoplasma (T.) gondii. The modes of transmission of the parasite include the consumption of the meat of sheep, cattle, goat, and pigs having bradyzoites of T. gondii, and the consumption of milk of goat, sheep and cattle, or contaminated chicken eggs.¹ More commonly, humans acquire infection through unintentional ingestion of unwashed fruits, vegetables, water contaminated with oocysts excreted by infected felines.² The principal reservoir for T. gondii is wild and domesticated cats, and the oocysts are produced in cats through sexual stages of their lifecycle. During pregnancy, T. gondii can cause abortion in the pregnant host, an infection of the placenta and in the foetus, leading to miscarriage, foetal mummification and or death in all types of mammalian hosts and among animals, especially in sheep and cattle.³ T. gondii has a huge zoonotic potential and causes infection in humans across the globe. High seroprevalence in the population leads to higher ratio of infections.⁴ It can cause different neurological disorders in immunocompromised or human immunodeficiency virus/acquired immunod- eficiency syndrome (HIV/AIDS) patients.⁵

Pooled seroprevalence of toxoplasmosis in Pakistan has been determined by compiling data.

It has been found that 30-50% of human population is infected with T. gondii, worldwide.⁶ Almost 20 million new cases of congenital and acquired toxoplasmosis were reported in 2010.⁷ On the basis of public and animal health, interaction of microorganisms and environment, horticulture, gardening, trade effects as well as socio-economic impacts, T. gondii is graded as one of the top 10 food-borne parasites.⁸ T. gondii is identified through direct methods, like microscopy and detection of deoxyribon-ucleic acid (DNA) by polymerase chain reaction (PCR), while the segregation of T. gondii is done by various bioassays.⁹ T. gondii is also identified through indirect methods, like the detection of antibodies against T. gondii in the serum of humans and animals.¹⁰ Various serological techniques, such as Sabin-Feldman Dye test, enzyme-linked immunosorbent assay (ELISA), latex agglutination test (LAT), indirect haemagglutination test (IHAT) and rapid diagnostic test strip (RDTS), have been employed for the identification of T. gondii antibodies in humans and animals.¹¹ In Pakistan, various epidemics cause morbidity and death in the livestock sector. Due to these diseases, Pakistan faces an estimated loss of around PKR79 billion.¹² Toxoplasmosis is one of the cosmopolitan parasitic diseases of the animals and humans, and it is ranked third in terms of the rate of global spread. Approximately 2.34 billion people are assumed to have been infected with T. gondii worldwide.^13,14 The current systematic review was planned to evaluate the seroprevalence of toxoplasmosis in humans and animals in Pakistan.

 

Materials and Methods

 

The systematic review comprised search on Science Direct, Google Scholar, PubMed and Scopus databases for studies related to the seroprevalence of toxoplasmosis in Pakistan published between 2006 and 2020 which used serological diagnostic tests to detect T. gondii. The Cochrane and the European Food Safety Authority (EFSA)  instructions¹⁵ as well as Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed.¹⁶

The key words used included “Toxoplasmosis”, “Seroprevalence”, “Humans”, “Livestock”, “Sheep and Goat”, “Cattle and Buffalo”, “Poultry”, “Pakistani”, and “Pakistan”. Vivid studies, case reports, reviews, letters to editors, studies not representing the target population, duplicates, and studies not using immunological assays were excluded.

The studies found were screened and data was retrieved from the ones that were shortlisted for detailed review. Data was extracted separately if an article contained multiple studies, like with different animals.

Seroprevalence was approximated with 95% confidence interval (CI). Further, the random effect model of forest plot, I2 and Cochrane Q statistics were used, as appropriate.

 

Results

 

Of the 7093 human studies initially found, 20(0.28%) were reviewed (Figure 1).

 

 

Of them, 11(55%) were from Khyber Pakhtunkhwa (KP) and 9(45%) were from Punjab. A total of 7145 individuals were part of these studies, and 1813(25.37%) were seropositive for toxoplasmosis. A broad difference was seen in the estimation of seroprevalence among various studies (Q=863.34; df=19; p<0.001; I2=97.8%; 95% CI: 97.5-98.1%) The pooled seroprevalence of toxoplasmosis was determined as (76%) (95% CI: 69-83%) (Figure 2). Seroprevalence was higher in KP (31.7%) compared to Punjab (20.42%)  (Figure 3). The seroprevalence was detected using LAT in (10%) studies (Table 1).

 

 

 

 

Of the 16,432 animal studies, 16(0.09%) were selected for detailed review (Figure 4).

 

 

Of them, 4(25%) were done in KP and 12(75%) in Punjab. A total of 8,519 animals were part of these studies, and 2,647(31%) of them were seropositive. A wide range of difference was seen in the seroprevalence estimation among the studies (Q=504.5; df=19; p<0.001; I2=95.4%; 95% CI: 94.5-96.1%). The pooled seroprevalence of toxoplasmosis in animals was determined as 69% (95% CI: 64-74%) (Figure 5). The seroprevalence of toxoplasmosis in animals was higher in KP (44.7 (%)) compared to Punjab (29.4 (%)) (Figure 6).

 

 

 

LAT, ELISA and IHAT were used for the diagnosis of toxoplasmosis in animals (Table 2).

 

 

Discussion

 

Besides causing a high disease load in humans, T. gondii also causes considerable economic loss to the livestock sector.¹⁷ The pooled seroprevalence of toxoplasmosis in humans and animals determined in the current systematic review was found as 76% and 69%, respectively. The findings are like those in Central America, Latin America, Europe and Asia (75-85%), while northern Europe and the United States have shown low seroprevalence.¹⁸

In Pakistan, the average seroprevalence of toxoplasmosis in humans was 20% between 2006 and 2020. No data were reported from the Sindh, Balochistan, Gilgit-Baltistan and Azad Jammu and Kashmir (AJK) in this regard. Thereafter, the seroprevalence of toxoplasmosis in humans has ranged between 1.3-65.7%.^19,20

Also, in Pakistan, the average seroprevalence of toxoplasmosis in animals was 31% between 2006 and 2020. In other studies, it has ranged from 10% to 55.4%.²¹

Seroprevalence of toxoplasmosis in the region has been reported in humans and animals to be 39.3% and 31%, respectively, in Iran, and 12.5% and 23.7%, respectively, in China.^22,23

Toxoplasmosis is associated with numerous risk factors.²⁴ Abundance of cats is one of the vital sources of the spread of T. gondii infection in various regions of Pakistan. Cats can discharge millions of oocysts after eating even one tissue cyst containing bradyzoites.^14,25 In addition, the sporulated oocysts can live for months and even for years in the soil. Moreover, earthworms, flies and beetles can escalate the oocysts and pass these on to the food chain.²⁶

In Pakistan, such high rate of seropositivity of T. gondii may be due to lack of awareness, lack of education among people, and improper hygienic conditions of the population. Lack of awareness about the infection, its sources of spread, its routes of transmission and ignorance of the outcomes of the diseases are important factors in the risk of infections.²⁷ The chances of infections can be reduced by increasing the level of education and spreading awareness among the people.²⁸

Various serological methods are regularly used for the identification of T. gondii antibodies.²⁸ The current review noted that the most common methods used were LAT and ELISA. However, Toxoplasma immunoglobulin M (IgM) chemiluminescence ELISA (CLIA) is a robotic test that is performed in a very short time but is not a regular method used in either private or public laboratories in Pakistan.

Epidemiological investigations reckon important information for devising precautions for the control of toxoplasmosis. Awareness of seroprevalence of Toxoplasma, disease problems and precautionary methods can lead to potent incidence curtailment.²⁹

The current systematic review has some limitations, like missing information about the seroprevalence of toxoplasmosis in different regions of Pakistan because of the absence of relevant studies, use of different methods for the identification of toxoplasmosis with variable levels of sensitivities and specificities, and diverse epidemiological outcomes. All these factors can have an influence on the overall seroprevalence rate mentioned in the review.

 

Conclusion

 

There is a need to create awareness among the masses to control toxoplasmosis in Pakistan. The relevant authorities should execute a screening programme in all provinces using standardised methods in order to estimate the actual prevalence and risk factors of toxoplasmosis in the country. Finally, there is a dire need to perform studies in Balochistan, Sindh, GB, and AJK regarding the seroprevalence of toxoplasmosis in human and animal populations

 

Acknowledgements: We are grateful to Mr Ubaid Ullah and Mr Hasham Zaheer, of the University of Veterinary and Animal Sciences (UVAS), Lahore, Pakistan, for editorial assistance, and to Prof (Dr) Muhammad Azam, of UVAS, for statistical assistance.

 

Disclaimer: None.

 

Conflict of Interest: None.

 

Source of Funding: None.

 

References

 

1.      Pinto-Ferreira F, Caldart ET, Pasquali AKS, Mitsuka-Breganó R, Freire RL, Navarro IT. Patterns of transmission and sources of infection in outbreaks of human toxoplasmosis. Emerg Infect Dis. 2019; 25:2177-82. doi:  10.3201%2Feid2512.181565.

2.      Lass A, Ma L, Kontogeorgos I, Zhang X, Li X, Karanis P. First molecular detection of Toxoplasma gondii in vegetable samples in China using qualitative, quantitative real-time PCR and multilocus genotyping. Sci Rep. 2019; 9:1-11. doi:10.1038/s41598-019-54073-6.

3.      Stelzer S, Basso W, Silván JB, Ortega-Mora LM, Maksimov P, Gethmann J, et al. Toxoplasma gondii infection and toxoplasmosis in farm animals: Risk factors and economic impact. Food Waterborne Parasitol. 2019; 15:1-32. doi: 10.1016/j.fawpar.2019.e00037.

4.      Shwab EK, Saraf P, Zhu XQ, Zhou DH, McFerrin BM, Ajzenberg D, et al. Human impact on the diversity and virulence of the ubiquitous zoonotic parasite Toxoplasma gondii. Pro Nat Acad Sci USA. 2018; 115:E6956-E63. doi:  10.1073/pnas.1722202115.

5.      Laksemi D, Suwanti LT, Mufasirin M, Suastika K, Sudarmaja M. Opportunistic parasitic infections in patients with human immunodeficiency virus/acquired immunodeficiency syndrome: A review. Vet World. 2020; 13:716-25. doi:  10.14202%2Fvetworld.2020.716-725.

6.      Abugri DA, Jaynes JM, Witola WH. Anti-Toxoplasma activity of Sorghum bicolor-derived lipophilic fractions. BMC Res Notes. 2019; 12:1-7. doi:  10.1186/s13104-019-4732-z.

7.      Cissé G. Food-borne and water-borne diseases under climate change in low-and middle-income countries: Further efforts needed for reducing environmental health exposure risks. Acta Tropica. 2019; 194:181-8. doi:  10.1016/j.actatropica.2019.03.012.

8.      Hazards EPoB, Koutsoumanis K, Allende A, Alvarez‐Ordóñez A, Bolton D, Bover‐Cid S, et al. Public health risks associated with food‐borne parasites. EFSA J. 2018; 16:1-113. doi: 10.2903/j.efsa.2018.5495.

9.      Chalmers RM, Robertson LJ, Dorny P, Jordan S, Kärssin A, Katzer F, et al. Parasite detection in food: Current status and future needs for validation. Trends Food Sci Tech. 2020; 99:337-50. doi10.1016/j.tifs.2020.03.011.

10.    Sharma R, Parker S, Al-Adhami B, Bachand N, Jenkins E. Comparison of tissues (heart vs. brain) and serological tests (MAT, ELISA, and IFAT) for detection of Toxoplasma gondii in naturally infected wolverines (Gulo gulo) from the Yukon, Canada. Food Waterborne Parasitol. 2019; 15:1-8. doi: 10.1016/j.fawpar.2019.e00046.

11.    Rostami A, Karanis P, Fallahi S. Advances in serological, imaging techniques and molecular diagnosis of Toxoplasma gondii infection. Infection. 2018; 46:303-15. doi:  10.1007/s15010-017-1111-3.

12.    Abbas Q, Han J, Adeel A, Ullah R. Dairy Production under climatic risks: Perception, perceived impacts and adaptations in Punjab, Pakistan. Int J Environ Res Public Health. 2019; 16:4036-47. doi:  10.3390/ijerph16204036.

13.    Belluco S, Simonato G, Mancin M, Pietrobelli M, Ricci A. Toxoplasma gondii infection and food consumption: a systematic review and meta-analysis of case-controlled studies. Crit Rev Food Sci Nut. 2018; 58:3085-96. doi:  10.1080/10408398.2017.1352563.

14.    Hill DE, Dubey JP. Toxoplasma gondii.  Foodborne parasites. [Online] [Cited 2018 January 26]. Available from: URL: http://www.sciepub.com/reference/355760.

15.    Farrah K, Young K, Tunis MC, Zhao L. Risk of bias tools in systematic reviews of health interventions: an analysis of PROSPERO-registered protocols. Syst Rev. 2019; 8:1-9. doi:10.1186/s13643-019-1172-8.

16.    Selçuk AA. A guide for systematic reviews: PRISMA. Turk Arch Otorhino. 2019; 57:57-8. doi:10.5152%2Ftao.2019.4058.

17.    Mose JM, Kagira JM, Kamau DM, Maina NW, Ngotho M, Karanja SM. A review on the present advances on studies of toxoplasmosis in eastern Africa. BioMed Res Int. 2020; 2020:1-12. doi:  10.1155/2020/7135268.

18.    Al-Kadassy AM, Baraheem OH, Bashanfer SA. Prevalence of Toxoplasma gondii infection in women of child-bearing age in faculty of Medicine and health sciences Hodeida City, Yemen. Phar Inn J. 2018; 7:256-61. doi:  10.1155/2020/7135268.

19.    Aleem U, Ullah S, Qasim M, Suliman M. Seroprevalence of Toxoplasmosis in Pregnant Women in Matta, Upper Swat, Khyber Pakhtunkhwa, Pakistan. J Saidu Med Col Swat. 2018; 2018:8-19. doi:  10.52206/jsmc.2018.8.2.%25p.

20.    Shoukat T, Awan UA, Mahmood T, Afzal MS, Wasif S, Ahmed H, et al.  Pathogens. Epidemiology of Toxoplasmosis among the Pakistani Population: A Systematic Review and Meta-Analysis. Pathogens. 2022; 11:675-92. doi:  10.3390/pathogens11060675.

21.    Lashari MH, Farooq U, Mubeen S, Hassan W, Azhar MF, Shahida S, et al. Seroprevalence of Toxoplasma gondii and associated haematological alterations in small ruminants of DG Khan district of Southern Punjab, Pakistan. Arqu Brasi de Med Vet Zootec. 2020; 72:1698-704. doi:10.1590/1678-4162-11723.

22.    Dong H, Su R, Lu Y, Wang M, Liu J, Jian F, et al. Prevalence, risk factors, and genotypes of Toxoplasma gondii in food animals and humans (2000–2017) from China. Front Microbiol. 2018; 2108:1-10. doi:10.3389/fmicb.2018.02108.

23.    Foroutan M, Dalvand S, Daryani A, Ahmadpour E, Majidiani H, Khademvatan S, et al. Rolling up the pieces of a puzzle: a systematic review and meta-analysis of the prevalence of toxoplasmosis in Iran. Alex J Med. 2018; 54:189-96. doi:  10.1016/j.ajme.2017.06.003.

24.    Pleyer U, Gross U, Schlüter D, Wilking H, Seeber F. Toxoplasmosis in Germany: epidemiology, diagnosis, risk factors, and treatment. Deut Ärzte Int. 2019; 116:435-44. doi:10.3238%2Farztebl.2019.0435.

25.    Hill DE, Dubey JP. Toxoplasma gondii as a parasite in food: analysis and control. Microbiol Spectr. 2016; 4. doi:  10.1128/microbiolspec.PFS-0011-2015.

26.    Shah SSA, Khan A. One health and parasites.  Global Applications of One Health Practice and Care. Germany: IGI Global, 2019; pp-82-112.

27.    Shoukat T, Awan UA, Mahmood T, Afzal MS, Wasif S, Ahmed H, et al. Epidemiology of Toxoplasmosis among the Pakistani Population: A Systematic Review and Meta-Analysis. Pathogens. 2022; 11:675-92. doi:   10.3390/pathogens11060675.

28.    Eroglu S, Asgin N. Awareness, knowledge, and risk factors of Toxoplasma gondii infection among pregnant women in the Western Black Sea region of Turkey. J Obstet Gynaecol. 2021; 41:714-20. doi:10.1080/01443615.2020.1789954.

29.    Guegan H, Robert-Gangneux F. Toxoplasma gondii in Solid Organ and Stem Cell Transplant: Prevention and Treatment. Emerg Transpl Infect. 2020:1-29. doi:  10.1111/j.1469-0691.2008.02091.x.

30.    Ali S, Amjad Z, Khan TM, Maalik A, Iftikhar A, Khan I, et al. Occurrence of Toxoplasma gondii antibodies and associated risk factors in women in selected districts of Punjab province, Pakistan. Parasitology. 2020; 147:1133-9. doi:  10.1017/S0031182020000967.

31.    Rehman F, Shah M, Ali A, Rapisarda AMC, Cianci A. Seroprevalence and risk factors of Toxoplasma gondii infection in women with recurrent foetal loss from the province of Khyber Pakhtunkhwa, Pakistan. J Neonatal Perinatal Med. 2020; 14:115-21. doi:10.3233/NPM-190323.

32.    Ahmad N, Khan IA, Iqbal Z, Naseem AA, Kayani AR, Afshan K, et al. Seroepidemiology of Toxoplasmosis in Human Population with Reference to Its Zoonotic Potential in Sub-Tropical Areas of Pakistan. Pak Vet J. 2019; 39: 2074-7764. doi: 10.29261/pakvetj/2019.017.

33.    Sadiqui S, Shah SRH, Almugadam BS, Shakeela Q, Ahmad S. Distribution of Toxoplasma gondii IgM and IgG antibody seropositivity among age groups and gestational periods in pregnant women. F1000Res. 2018; 7:1823. doi: 10.12688/f1000research.15344.3.

34.    Khan F, Rooman M, Rehman H, Rab A, Khan A, Rehman A, et al. Prevalence of Toxoplasma gondii antibodies among pregnant women in District Bannu, Khyber Pakhtunkhwa, Pakistan. Int J Biosci. 2018; 12:235-39. doi:10.12692/ijb/12.5.235-8.

35.    Khan F, Rooman M, Hameed UR, Rab A, Faiz UR, Abid UR. Seroprevalence of toxoplasmosis in human (Homo sapien) population of District Bannu Khyber Pakhtunkhwa (KP), Pakistan. Int J Biosci. 2018; 12:255-64. doi: 10.12692/ijb/12.5.255-8.

36.    Faisal S, Jan H, Haroon M, Khan MT, Jan FU, Iqbal T, et al. Seroprevalence of human Toxoplasma gondii infection among pregnant women in Charsadda, KP, Pakistan. J Parasit Dis. 2018; 42:554-8. doi: 10.1007/s12639-018-1033-8.

37.    Aleem U, Ullah S, Qasim M, Suliman M. Seroprevalence of Toxoplasmosis in Pregnant Women in Matta, Upper Swat, Khyber Pakhtunkhwa, Pakistan. J Saidu Med College. 2018; 8:11-23. doi:10.52206/jsmc.2018.8.2.%25p.

38.    Shah N, Khan A, Khisroon M, Adnan M, Jawad SM. Seroprevalence and risk factors of toxoplasmosis in pregnant women of district Swabi. Pure and Applied Biology (PAB). 2017; 6:1306-13. doi:10.19045/bspab.2017.600139.

39.    Nazir MM, Akhtar M, Maqbool A, Waheed A, Sajid MA, Ali MA, et al. Antibody Prevalence and Risk Factors for Toxoplasma gondii Infection in Women from Multan, Pakistan. Zoono Pub Health. 2017; 64:537-42. doi:10.1111/zph.12336.

40.    Hussain Shah MS, Naz F, Jan A, Ullah R, Khan SFA, Haseeb A, et al. Seroprevalence and Risk Factors of Toxoplasmosis among Women in District Chitral, Khyber Pakhtunkhwa, Pakistan. World J Zoo. 2016; 11:135-40. doi:  10.5829/idosi.wjz.2016.135.140.

41.    Shah M, Zahid M, Sthanadar AA, Ali PA. Seroprevalence of Toxoplasma gondii Infection in Human Population of Mohmand Agency Khyber Pakhtunkhwa, Pakistan. Pak J Zoo. 2014; 46:1169-72.

42.    Khan MZ, Rahman SU, Gul N, Khan AA. Toxoplasmosis; seroprevalence, comparative analysis of diagnostic techniques and identification of risk factors in humans in Malakand Agency, Khyber Pakhtunkhwa, Pakistan. Int J Biosci. 2014; 5:1-6. doi:10.12692/ijb/5.4.1-8.

43.    Hayat S, Tasawar Z, Akhtar T. Seroprevalence of human toxoplasmosis in kallarwali, Muzaffar garh, Pakistan. Gomal J Med Sci. 2014; 12:129-32.

44.    Tasawar Z, Raza AA, Aziz F, Lashari MH. Prevalence of human toxoplasmosis in district Muzaffargarh, Punjab, Pakistan. Gomal J Med Sci. 2012; 10: 37-41.

45.    Tasawar Z, Aziz F, Lashari MH, Shafi S, Ahmad M, Lal V, et al. Seroprevalence of Human toxoplasmosis in southern Punjab, Pakistan. Pak J Life Soc Sci. 2012; 10:48-52.

46.    Ahmad M, Maqbool A, Mahmood-ul-Hassan M, Mushtaq-ul-Hassan M, Anjum A. Prevalence of Toxoplasma gondii antibodies in human beings and commensal rodents trapped from Lahore, Pakistan. J Anim Plant Sci. 2012; 22:51-3.

47.    Tasawar Z, Nawaz S, Lashari MH, Aziz F, Hayat S. Seroprevalence of human toxoplasmosis in Dera Ghazi Khan, Punjab. Gomal J Med Sci. 2011; 9:83-5.

48.    Khan SN, Khan S, Ayaz S, Jan AH, Jehangir S, Attaullah S, et al. Seroprevalence and risk factors of toxoplasmosis among pregnant women in District Kohat, Khyber Pakhtunkhwa, Pakistan. World Applied Sci J. 2011; 14:1032-36. doi: 10.3233/NPM-190323.

49.    Chaudhary Z, Ahmed R, Hussain S, Shakoori A. Detection of Toxoplasma gondii infection in butchers and buffaloes by polymerase chain reaction and latex agglutination test. Pak J Zoo. 2006; 38:333-6.

50.    Fatima T, Mehnaz S, Wang M, Yang J, Sajid MS, Shen B, et al. Seroprevalence of Toxoplasma gondii in one-humped camels (Camelus dromedarius) of Thal and Cholistan deserts, Punjab, Pakistan. Parasitol Res. 2019; 118:307-16. doi: 10.1007/s00436-018-6124-z.

51.    Ullah M, Awais M, Akhtar M, Anwar M, Navid M, Khan I, et al. Seroprevalence, associated risk factors and hematological impacts of toxoplasmosis in small ruminants of Multan, Punjab-Pakistan. Trop Biomed. 2018; 35:1028-40.

52.    Khan MT, Din JU, Ali S, Kamal A, Hussain A, Yar A, et al. Seroprevalence of Toxoplasma gondii infection in cows and goats of district Charsadda, Khyber Pakhtunkhwa, Pakistan. Int J Fauna Biol Stud. 2018; 5:18-22.

53.    Hanif M, Tasawar Z. Seroprevalence and risk factors associated with toxoplasmosis in sheep in Multan and Khanewal districts of Punjab (Pakistan). JAPS: J Ani & Plan Sci. 2016; 26:1620-27.

54.    Ahmed H, Malik A, Arshad M, Mustafa I, Khan MR, Afzal MS, et al. Seroprevalence and Spatial Distribution of Toxoplasmosis in Sheep and Goats in North-Eastern Region of Pakistan. Korean J Parasitol. 2016; 54:439-46. doi: 10.3347/kjp.2016.54.4.439.

55.    Ahmad S, Tasawar Z. Seroprevalence of Toxoplasmosis in Small Ruminants from Cholistan Desert and Agricultural Areas of Rahim Yar Khan and Rajan Pur (Punjab) Pakistan. Pak J Zool. 2016; 48:1003-8.

56.    Saqib M, Hussain MH, Sajid MS, Mansoor MK, Asi MN, Fadya AA, et al. Sero-epidemiology of equine toxoplasmosis using a latex agglutination test in the three metropolises of Punjab, Pakistan. Trop Biomed. 2015; 32:276-85.

57.    Ahmad N, Iqbal Z, Mukhtar M, Mushtaq M, Khan KM, Qayyum M. Seroprevalence and associated risk factors of toxoplasmosis in sheep and goats in Pothwar region, Northern Punjab, Pakistan. Pak J Zool. 2015; 1:161-7.

58.    Chaudhry UN, Ali AA, Ashraf S, Khan T, Nadeem SM, Ashraf K. Seroprevalence of Toxoplasma gondii infection in camels (Camelus dromedarius) in and around Bahawalpur region of Pakistan. J Inf Mol Biol. 2014; 2:16-8. Doi: 10.14737/jimb.2307-5465/2.1.16.18.

59.    Awais MA, Ahmed A, Muhammad M, Muhammad A, Saleemi K, Ashraf K, et al. Seroprevalence of Toxoplasma gondii in the Backyard Chickens of the Rural Areas of Faisalabad Punjab Pakistan. [Cited 2014 June 11]. Available from: URL: http://www.fspublishers.org/published_papers/488.pdf.

60.    Ahmad N, Qayyum M. Seroprevalence and risk factors for toxoplasmosis in large ruminants in northern Punjab, Pakistan. J Infect Dev Ctries. 2014; 8:1022-8. doi:10.3855/jidc.4405.

61.    Shah M, Zahid M, Asmat P, Alam A, Sthanadar A. Seroprevalence of Toxoplasma gondii in goats and sheep of district Mardan, Pakistan. Int J Biosci. 2013; 7:90-7. doi: 10.12692/ijb/3.7.90-97.

62.    Tasawar Z, Lashari MH, Hanif M, Hayat C. Seroprevalence of Toxoplasma gondii in domestic goats in Multan, Punjab, Pakistan. Pak J Sci. 2011; 9:24-7.

63.    Lashari MH, Tasawar Z. Seroprevalence of toxoplasmosis in sheep in Southern Punjab, Pakistan. Pak Vet J. 2010; 30:1225-9. doi: 10.1007/s11250-009-9304-0.

64.    Ramzan M, Akhtar M, Muhammad F, Hussain I, HiszczynskaSawicka E, Haq AU, et al. Seroprevalence of Toxoplasma gondii in sheep and goats in Rahim Yar Khan (Punjab), Pakistan. Trop Anim Health Prod. 2009; 41:1225-9. doi: /10.1007/s11250-009-9304-0

65.    Ahmad S, Tasawar Z. Seroprevalence of Toxoplasma gondii in Four Ovine Breeds of Cholistan Desert of Pakistan. Pak J Life Soc Sci. 2015; 13:91-6.

66.    Azeem S, Sharma B, Shabir S, Akbar H, Venter E. Lumpy skin disease is expanding its geographic range: A challenge for Asian livestock management and food security. Vet J. 2022; 279:1-7. doi: 10.1016/j.tvjl.2021.105785.