Prevalence of Toxoplasmosis in Sheep and Goats in Pakistan: A Systematic Review and Meta-Analysis

Toxoplasmosis, a parasitic disease caused by Toxoplasma gondii, results in congenital disorders and miscarriages among livestock and humans worldwide. This systematic review and meta-analysis were conducted to determine the prevalence of T. gondii infection in sheep and goats in Pakistan from 2000 to 2020. We searched the PubMed, Scopus, EMBASE, and Google Scholar databases and selected 17 publications that fulfilled our inclusion criteria. Eight studies were conducted in Southern Punjab, six in Khyber Pakhtunkhwa, two in Northern Punjab, and one in Central Punjab. The diagnostic tests used in the included articles to confirm toxoplasmosis were the latex agglutination test in 56% of the studies, the enzyme-linked immunosorbent assay in 38%, and the indirect hemagglutination assay in 6%. The infection rates were substantially higher among sheep > 1 year of age (37%) than among sheep ≤ 1 year old (19%). Statistically significant differences in infection rates were found between male and female sheep and goats. The overall infection rate by age was also significant among sheep and goats. Sex and age variability between sheep investigations were significant, and sex heterogeneity and age homogeneity were significant among goats. Hence, robust infection control protocols should be implemented to prevent infection in animals and humans.


Introduction
Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii (T. gondii), an obligate intracellular protozoan parasite. Toxoplasmosis affects domestic animals (including goats Our study consisted of 3630 sheep, of which 1124 were positive for toxoplasmosis, and a total of 3128 goats, of which 1112 were infected. Hence, the overall prevalence of toxoplasmosis was 35.5% among the goats and 30.9% among the sheep (Figure 1). The incidence of T. gondii infection among the sheep and goats varied geographically; the highest prevalence of infection among the sheep was observed in Charsadda, Khyber Pakhtoon Khwa (KPK), followed by various districts in Punjab province, including 60% in Jalalpur, 48% in Sargodha, and 45% in Shujbad. Infection among the goats was most prevalent in the district of Charsadda, followed by districts in Punjab province, such as 61% in Sargodha, 60% in Jalalpur, and 53% in Mohmand Agency, KPK (cf. Table 2) in Jalalpur, 48% in Sargodha, and 45% in Shujbad. Infection among the goats was most prevalent in the district of Charsadda, followed by districts in Punjab province, such as 61% in Sargodha, 60% in Jalalpur, and 53% in Mohmand Agency, KPK (cf.    Figure 1. The overall study design and graphs of the prevalence of toxoplasmosis among sheep and goats. Twelve of the fourteen included articles on sheep investigated the prevalence of toxoplasmosis by sex (Table 3). In some of the studies, the statistical analyses revealed a strong correlation between gender and toxoplasmosis, and in a few other studies, no significant difference was found between them. However, a small difference in infection rates by gender was found between male (29%) and female (31%) sheep, compared to a larger difference between male (24%) and female (47%) goats.
Significant differences in infection rates among sheep were reported in studies from different geographical areas, including Multan (females: 65%; males: 25%), Rahim Yar Khan (females: 17%; males: 4.5%), and Dera Ismail Khan, Multan, and Khanewal (females: 18%; males: 30%). Similarly, only a few of the included studies reported a significant difference in the rates of infection among male and female goats, such as Mohmand Agency (females: 69%; males: 38%); Multan (females: 55%; males: 25%); Bhalwal, Kotmomin, Sahiwal, Shahpur, Silanwali, and Sargodha (females: 50%; males: 23%); and Rahim Yar Khan (females: 37%; males: 62%) (cf. Table 3) Two of the ten studies on sheep were not included in the meta-analysis due to the variation in the data of their age groups. Therefore, eight studies were included, and their findings proved that the infection rates increased with age. In contrast, two of the seven articles on goats were not included in the meta-analysis due to the variation of the data on their age groups; thus, five studies were included ( Table 4). Four of the five studies conducted on goats reported a strong positive correlation with age. The findings indicated that the infection rates were significantly greater (37%) among sheep older than 1 year compared to the 19% infection rate among sheep-bearing age (younger than 1 year).
The results of the fixed-effects model showed a statistically significant difference in infection rates between male and female sheep (i.e., odds ratio (OR) 0.67, 95% confidence interval (CI) 0.56-0.82) and goats (OR 0.34, 95% CI 0.28-0.43) (cf. Tables 5 and 6). Similar findings were observed in the random effects model for both sheep (OR 0.64, 95% CI 0.41-0.99) and goats (OR 0.35, 95% CI 0.27-0.45) (cf. Tables 5 and 6). Extensive variation among the different studies in the prevalence estimates of infection among the sheep was observed in the analysis by sex, and the Q statistic was 35.67, p < 0.001, and I 2 = 69% ( Figure 2a). The Q statistic of 7.97, p = 0.54, and I 2 = 0% in Figure 2b revealed no significant variation in the prevalence estimates of infection among goats in the different studies.     Using the data analyzed by sex, a forest plot was drawn based on the proportion of infected cases among the male and female sheep (Figure 3a). The Q statistic of 294.09, p < 0.01, and I 2 = 96% show significant variation among the different studies in the prevalence estimates of infection among the sheep. The Q statistic of 275.10, p < 0.01, and I 2 = 97% (cf. Figure 3b) show significant variation among the different studies in the prevalence estimates of infection among the male and female goats.
We divided the animals into two groups, i.e., group 1 (≤1 year of age) and group 2 (>1 year of age) to analyze the data by age. The fixed-effects model showed a significant difference in the infection rates between the two age groups of the sheep (OR 0.32, 95%    We divided the animals into two groups, i.e., group 1 (≤1 year of age) and group (>1 year of age) to analyze the data by age. The fixed-effects model showed a significa difference in the infection rates between the two age groups of the sheep (OR 0.32, 95% 0. 26

Discussion
Over several decades, Pakistan's livestock industry has become a vital subsector of the country's agricultural sector. Goats and sheep are used for various purposes, including producing meat, milk, and other dairy products and breeding. Goat and sheep populations have increased substantially during the last 3 years, as have meat and milk production from sheep and goats. Goats and sheep are most frequently infected among livestock with toxoplasmosis [57], and the primary route of T. gondii infection is cat feces; however, it may be transported through the consumption of tissue cysts in raw foods and undercooked foods [73]. The prevalence of toxoplasmosis among livestock varies significantly worldwide, ranging from 0 to 100% in different nations [74,75], depending on the country's traditions, customs, lifestyle, meteorological conditions, farming practices, and the age of the animal [76]. Prevalence rates are related to the oocyst excretion of cats and the infection of animals and humans after sporulation [3].
After searching four databases, 17 articles, including 3630 sheep with a positive case count of 1124 and 3128 goats with a positive case count of 1112, were selected for the analysis. The data showed that the overall prevalence of infection with toxoplasmosis was 35% among goats and 30.9% among sheep. The peak incidence of T. gondii was 86.4%, which was reported in KPK (Shabqadar, Tangi, and Charsadda), and the lowest prevalence rate was 1.47% in the Bannu district [66,77]. The studies reported a significant association between toxoplasmosis infection and the ages of goats and sheep. The higher disease prevalence in animals older than 1 year may be caused by longer exposure throughout their lives (Tables 7 and 8) [47,57,60,61,63,67,68].
In countries other than Pakistan, the frequency of toxoplasmosis infection in goats and sheep varies. Prevalences of 33.62% for sheep and 36.41% for goats were observed in Iran [78], and a more recent study reported a prevalence of 14.4% among sheep and 8.8% among goats [35]. Seroprevalence, which was reported to be 52.6% among sheep and 24% among goats in Nazareth, Ethiopia, was confirmed by the modified agglutination test, and

Discussion
Over several decades, Pakistan's livestock industry has become a vital subsector of the country's agricultural sector. Goats and sheep are used for various purposes, including producing meat, milk, and other dairy products and breeding. Goat and sheep populations have increased substantially during the last 3 years, as have meat and milk production from sheep and goats. Goats and sheep are most frequently infected among livestock with toxoplasmosis [57], and the primary route of T. gondii infection is cat feces; however, it may be transported through the consumption of tissue cysts in raw foods and undercooked foods [73]. The prevalence of toxoplasmosis among livestock varies significantly worldwide, ranging from 0 to 100% in different nations [74,75], depending on the country's traditions, customs, lifestyle, meteorological conditions, farming practices, and the age of the animal [76]. Prevalence rates are related to the oocyst excretion of cats and the infection of animals and humans after sporulation [3].
After searching four databases, 17 articles, including 3630 sheep with a positive case count of 1124 and 3128 goats with a positive case count of 1112, were selected for the analysis. The data showed that the overall prevalence of infection with toxoplasmosis was 35% among goats and 30.9% among sheep. The peak incidence of T. gondii was 86.4%, which was reported in KPK (Shabqadar, Tangi, and Charsadda), and the lowest prevalence rate was 1.47% in the Bannu district [66,77]. The studies reported a significant association between toxoplasmosis infection and the ages of goats and sheep. The higher disease prevalence in animals older than 1 year may be caused by longer exposure throughout their lives (Tables 7 and 8) [47,57,60,61,63,67,68].
In countries other than Pakistan, the frequency of toxoplasmosis infection in goats and sheep varies. Prevalences of 33.62% for sheep and 36.41% for goats were observed in Iran [78], and a more recent study reported a prevalence of 14.4% among sheep and 8.8% among goats [35]. Seroprevalence, which was reported to be 52.6% among sheep and 24% among goats in Nazareth, Ethiopia, was confirmed by the modified agglutination test, and seroprevalence of 56% among sheep and 25.9% among goats were verified by the ELISA test in the same region [79]. The seropositivity of T. gondii in China was reported in 9.84% of sheep and 10.73% of goats [27], and another study reported it in 9.9% of goats [80].
The findings of the fixed-effects model indicated a significant difference in the infection rates between male and female sheep (OR 0.67, 95% CI 0.56-0.82) and goats (OR 0.34% CI 0.28-0.43) (cf. Tables 5 and 6). The results of the present study are consistent with those of previous studies [54,61,[81][82][83], although the results of two studies [69,84] that reported higher seropositivity in males than females were inconsistent with our findings. Females are more susceptible to protozoan parasites than males are. The stress of lactation and childbirth causes immunological suppression in female sheep and goats, predisposing them to toxoplasmosis [70,[85][86][87].
The included articles reported that infection rates were significantly higher among sheep more than 1 year of age (37%) compared to sheep less than 1 year (19%) of age (cf. Table 4). The risk factors for toxoplasmosis were more prevalent among older sheep than younger sheep, implying that animals have a greater probability of infection as they age, which is consistent with the study by Shah et al. [61]. The higher susceptibility of older animals than younger animals to infection is thought to be related to their longer exposure to risk factors for infection, and the observations of sheep and goats in other studies are consistent with those of our study [54,58]. However, our findings contradict those of Ramzan et al. [57]. Seroprevalence increases with age due to an increased risk of environmental contamination [88]. This increase could be related to the inability of animals 1 year and younger to retain adequate passive immunity transferred from their mothers. Therefore, the age of the animal is regarded as an essential risk factor for toxoplasmosis infection in animals [89].
In the current study, the overall prevalence of toxoplasmosis in Pakistan was 35.5% among goats and 30.9% among sheep. A higher T. gondii seropositivity was observed in goats than in sheep in Northern Punjab [59]. Similarly, the results of other studies indicated that goats are more vulnerable to toxoplasmosis than sheep, owing to increased mobility and migration [57,90], which may have increased their probability of coming into contact with contaminated sources. These correlations may be explained by the constant grazing of the many sheep flocks in the included articles, whereas the goat herds were confined to houses. As a result, the potential for contacting contaminated food and grasslands was higher among the sheep flocks throughout the grazing season. In Pakistan and other countries worldwide, there is significant variation in the occurrence of toxoplasmosis. The findings of our study indicate that toxoplasmosis is widespread in goats and sheep in Sargodha, Sahiwal, Bhalwal, Silanwali, and Shaahpur. Similar results have been reported in Pakistan's southern areas [54,57,58], KPK [61] and Iran, India, and China [71,72,91]. This substantial variation in the seroprevalence of toxoplasmosis between regions is attributed to differences in temperature, sanitary conditions, farming techniques, sample size, and diagnostic techniques [72].
There are some limitations in this systematic review and meta-analysis. In the different studies, the sex of the animals was not examined equally; hence, uneven samples from each sex were included in the analysis, which may have biased the results and conclusions of the study. Finally, age, which is a critical factor, was not investigated in all of the included articles.

Data Search Strategy
Our study was a detailed investigation of the prevalence of T. gondii infection among goats and sheep in Pakistan. We collected data from searches of four databases, including Google Scholar, Scopus, EMBASE, and PubMed, and analyzed relevant findings identified in the articles. Keywords, including "toxoplasmosis", "T. gondii", "prevalence", "sheep", "goat", and "Pakistan" were searched alone or in combination in the four databases.

Data Collection
Research articles written in English were selected for review. All the study's authors conducted the systematic review and meta-analysis of the included articles, gathered research reports, and defined the study's inclusion criteria.

Inclusion and Exclusion Criteria
The exclusion and inclusion criteria were evaluated, and the articles for inclusion were selected accordingly. Studies conducted in Pakistan that investigated the prevalence of toxoplasmosis in the country's sheep and goats were screened and evaluated for inclusion in the systematic review and meta-analysis. Irrelevant data, incomplete information, duplicate articles, case series, studies that did not examine the prevalence of toxoplasmosis, and studies without sheep and goats were excluded. A PRISMA flowchart of the selection of articles is presented in Figure 6.
A total of 17 articles were chosen based on the study's inclusion criteria. The following information about the included articles were collected: year of publication, country where the study was conducted, sample size, diagnostic tests, number of animals tested, number of cases of infected animals, and prevalence rates. The studies were then coded as per the defined parameters, and the data were entered into Microsoft Excel. "sheep", "goat", and "Pakistan" were searched alone or in combination in the four databases.

Data Collection
Research articles written in English were selected for review. All the study's authors conducted the systematic review and meta-analysis of the included articles, gathered research reports, and defined the study's inclusion criteria.

Inclusion and Exclusion Criteria
The exclusion and inclusion criteria were evaluated, and the articles for inclusion were selected accordingly. Studies conducted in Pakistan that investigated the prevalence of toxoplasmosis in the country's sheep and goats were screened and evaluated for inclusion in the systematic review and meta-analysis. Irrelevant data, incomplete information, duplicate articles, case series, studies that did not examine the prevalence of toxoplasmosis, and studies without sheep and goats were excluded. A PRISMA flowchart of the selection of articles is presented in Figure 6  A total of 17 articles were chosen based on the study's inclusion criteria. The following information about the included articles were collected: year of publication, country where the study was conducted, sample size, diagnostic tests, number of animals tested, number of cases of infected animals, and prevalence rates. The studies were then coded as per the defined parameters, and the data were entered into Microsoft Excel.

Meta-Analysis
The weighted prevalence of T. gondii infection was used in the analyses of sheep and goats by sex (i.e., male and female) and age group, i.e., group 1 (≤1 year of age) and group 2 (>1 year of age). We used ORs for the pooled effect sizes of both analyses; the Sidik-Jonkman estimator was used to estimate the variance, and 0.1 increments were added for a continuity correction of zero cells. However, for the proportions of the infected cases by sex and age group, we analyzed single proportions. We used the Freeman-Tukey double arcsine transformation for the analyses of the proportions, and the DerSimonian-Laird method to estimate the inverse variances. Cochran's Q test and the 2 Statistics were used

Meta-Analysis
The weighted prevalence of T. gondii infection was used in the analyses of sheep and goats by sex (i.e., male and female) and age group, i.e., group 1 (≤1 year of age) and group 2 (>1 year of age). We used ORs for the pooled effect sizes of both analyses; the Sidik-Jonkman estimator was used to estimate the variance, and 0.1 increments were added for a continuity correction of zero cells. However, for the proportions of the infected cases by sex and age group, we analyzed single proportions. We used the Freeman-Tukey double arcsine transformation for the analyses of the proportions, and the DerSimonian-Laird method to estimate the inverse variances. Cochran's Q test and the I 2 Statistics were used to determine heterogeneity between the studies, and forest plots with 95% Cls and effect sizes were used to present a graphical summary of the results.

Conclusions
Examining these findings contributes to an updated epidemiological assessment and geographic context in Pakistan. More surveys are recommended to monitor this infection continuously. Attention should be paid to farming and testing animals using techniques to control the disease before a contaminated product is obtained and consumed. Health measures and precautions should be taken to prevent and control the disease. Our data provide meaningful information and statistics on the prevalence of toxoplasmosis, which may aid in the disease's control and management. Additional investigations are needed to improve control strategies, reduce toxoplasmosis among goats and sheep, and continuously buffer Pakistan's community health, economy, and financial status against the societal damages caused by toxoplasmosis.