Toxoplasma gondii in lactating animals: potential risk to milk consuming population in Khyber Pakhtunkhwa

Abstract Toxoplasma gondii is an intracellular zoonotic protozoan parasite usually infects human and animal worldwide. This study aimed to analyze the sero-prevalence of T. gondii in blood of lactating animals and human living in close proximity and also to detect Toxoplasma DNA in unpasteurized milk of the studied animals. A total of 233 blood and milk samples were collected from lactating animals, and 735 blood samples were taken from humans in District Upper Dir, Khyber Pakhtunkhwa, Pakistan. The blood samples were analyzed through ELISA while the milk samples were analyzed by PCR for the presence of T. gondii DNA. A standard questionnaire was introduced to collect the data from the participants. In animals, the reported sero-prevalence was 32.18% for IgM, 17.16% for IgG, and 6.4% for both IgM and IgG. The reported positivity for T. gondii DNA in milk was 14.44%, 34.8%, 20%, and 26% in sheep, goats, cows, and buffaloes, respectively. In the human blood samples, 9.8% were found positive for IgM and 11.2% for IgG while none of the samples was found positive for both IgM and IgG. Overall sero-prevalence reported in females was significantly higher than the male (p<0.05) poor hygiene condition (p < 0.0001) were the significant risk factors associated with T. gondii infections in animals. In conclusion, T. gondii infection is prevalent in lactating animals and humans using their raw milk in the study area. It is suggested that raw milk should be considered as a vehicle for the transmission of T. gondii to humans. Proper pasteurization of milk is very useful in limiting the transmission of infection. Awareness and control programs should be implemented to prevent the infection.

Toxoplasma gondii in lactating animals: potential risk to milk consuming population in Khyber Pakhtunkhwa

Description of the study area
The study was conducted in Upper Dir District of Khyber Pakhtunkhwa, Pakistan, lies at latitude of 35° 9'55.89"N and 72° 2'48.54"E longitude having total areas of the district is 3,699 km 2 (Figure 1).People of the area mostly depend on domestic animals for the purpose of milk and different types of dairy products such as butter, cheese, yoghurt, and meat.These are the main sources of income for the people of the region.Basic educational and health facilities have also been provided by the Government to the people of the area.

Ethical considerations
The Advance Study Research Board (ASRB), University of Peshawar Khyber Pakhtunkhwa, Pakistan has approved the research Proposal.Special permission was obtained from the owner of the animals during sampling.Informed consent was obtained from all participants of the study.

Data collection
Data related risk factors of toxoxoplasma in animals (feeding habits, type of food, source of drinking water, hygienic conditions and age) and humans (age, contact with cats and gender) were collected through a predesigned questionnaire.

Collection of blood and milk samples from animal
Blood and milk samples were collected from 233 lactating animals including sheep (n=76), goats (n=69), cows (n=50) and buffaloes (n=38)], from five different tehsils (Proper Dir, Wari, Barawal, Shringal and Kalkot) in district Upper Dir Khyber Pakhtunkhswa Pakistan.Blood samples (5mL) were collected from the jugular vein of lactating animals.Milk samples (5mL) were taken manually after disinfection of the teats with 70% ethanol.All the blood and milk samples were immediately transported to the

Introduction
Toxoplasma gondii (T.gondii) is an obligate intracellular cyst forming apicomplexan parasite, infecting all warmblooded animals including humans, with a cosmopolitan distribution (Rouatbi et al., 2019;Sharif et al., 2009) and is a causative agent of the geographically omnipresent infection known as toxoplasmosis (Rahimi et al., 2015;Sugden et al., 2016).The lactating animals are the significant hosts of T. gondii and thus considered most important transmission route of T. gondii infection.Usually, T. gondii infection is passed to grazing animals when coming in contact with the soil contaminated with T. gondii oocysts.Then these infected animals transmit tachyzoites through unpasteurized milk to their offspring as well as to human beings (Khamsian et al., 2021;Camossi et al., 2011;Sharif et al., 2009).
The disease is noticeable in both medical as well as veterinary field, as it result a significantly serious drain on productivity and the economy by damaging animal husbandry due to stillbirths and neonatal mortality in animals and stress on the health systems (Rahimi et al., 2015, Sharif et al., 2009).Many epidemics responsible for high mortality and morbidity and were reported in livestock sector of Pakistan (Ahmed et al., 2016).Toxoplasma infection in dairy animals may illustrate a real risk for transmission of parasite to human (Van den Brom et al., 2020;Omonijo et al., 2022), causes different complications such as Toxoplasmic encephalitis, blindness, stillbirth, abortion, fetal abnormalities, or even prenatal death in congenital cases, pneumonia, and sometimes heart infection (Chan and Smith, 2018).Globally prevalence of T. gondii is variable, different countries ranging from zero to hundred percent prevalence rates depending upon their civilizations, societies, inhabitant's life styles, animal's age and climate conditions (Lashari et al., 2018;Thai et al., 2019).The broad dissemination of this parasite may be attributed to various factors such as its co-evolution with multiple hosts (Mukhopadhyay et al., 2020;Alzaheb, 2018;Ding et al., 2017;Sanchez and Besteiro, 2021), multiple transmission routes (Foroutan et al., 2018), presence of diverse T. gondii genotypes (Shapiro et al., 2019), long viability in the moderate environment (Galvan-Ramirez et al., 2012), longer infectious time for about 18 months (Furtado et al., 2011) and resistant to freezing, drying and even disinfection (Rahimi et al., 2015).Milk is a highly consumable complete source of food as it contains all required nutrients in good proportion.Peoples need hygienic quality of milk for daily use and they fulfill their need from domestic and farm animals.The milk of these animals represents a source of infection for humans, therefore required to monitor prevalence of infection, aimed to reduce infections in them as much as possible.Better information regarding the environmental contamination of T. gondii is critical and vital to holistic determinations of potential health risks (Aguirre et al., 2019) and highlights the areas for further investigation so to implement effective measures to prevent T. gondii infections (Stelzer et al., 2019).Due to the economic importance of toxoplasmosis, this study aimed to analyze the sero-prevalence of T. gondii in blood of lactating animals and human living in close proximity and also to detect Toxoplasma DNA in unpasteurized milk of the studied animals in District Upper Dir, Pakistan.Laboratory of Virology and Immunology, Department of Zoology, University of Peshawar.Serum was separated by centrifugation.All samples were stored at -20°C for serological and molecular assays.

Collection of blood samples from humans
A total of 735 blood samples (350 males and 385 females) were collected from the participants who live nearby the animals and consume their milk especially animal's owners and their family members.
All the blood samples were collected in 5 mL sterile test tubes and serum was separated and stored at -20°C for further serological examination.

Serological examination of T. gondii
The serum of human were analysed for T. gondii IgM antibodies and IgG antibodies using commercially available Toxo-IgM kit (BioCheck Inc.USA, Catalog Number: BC-1087) and Toxo-IgG kit (BioCheck, Inc.USA, Catalog Number BC-1085) according to the manufacturer's instructions.The microwell plate of ELISA was read by a Microplate reader with O.D at 450 nm (Microplate Reader, Model Dia 710 UK).The serum of animals were analysed by using Commercial ELISA Kits (ID Screen Toxoplasmosis Indirect®ID-VET Company, France) for the detection of Toxo-IgG and Toxo-IgM antibodies.

T. gondii DNA detection from milk
The extraction of DNA from milk was carried out by using a DNA isolation kit (Favorgen Tissue Genomic DNA Extraction Mini kit, USA).The extracted DNA was amplified by using primers of B1 gene for T. gondii following the protocol of Sadek et al. (2015).The reaction conditions consisted of one cycle of 95ºC for 5 minutes followed by 40 cycles of 94ºC for 30 seconds, 60ºC for 30 seconds, 72ºC for 1 minute, and final extension at 72ºC for 10 minutes, and then the amplified DNA were electrophoresed on 2% agarose gel.The amplified DNA 115 bp bands were visualized under UV-trans illuminator and compared with a 50 bp DNA ladder marker (thermoisher scientific, USA).

Data analysis
The data were analyzed by using SPSS version 20.Chi-square test (X 2 ) was used to assess the associations between Toxoplasma seropositive and the studied factors.
A p-value less than 0.05 was considered statistically significant.The frequency of Toxoplasma DNA in milk samples was analyzed in four age groups of animals (≤5 years, 6-9 years, 10-12 years, and ≥13 years.Age-wise sero-prevalence of T. gondii was analyzed in the different age groups of humans (5-20 years, 21-35 years, 36-50 years, and ≥51 years).
The distribution of positive antibodies in blood of animals and DNA of milk was showed in Table 2.The positive results of PCR (23.60%) were significantly higher than those of IgG (10.73%) but lower than IgM (25.75%).The proof of DNA in milk samples by PCR in both IgG positive animals and IgM positive animals were statistically significant (p=0.0002 and p=0.0092, respectively) in comparison to concurrent presence IgG and IgM.Goats were reported with high prevalence (34.8%) of T. gondii DNA in their milk as compared to other animals.The frequency of Toxoplasma DNA in studied animals in age groups ≤5 years, 6-9 years, 10-12 years, and ≥13 years was 20.48%, 25.55%, 27.02%, and 38.49%, respectively.Statistically, T. gondii DNA positivity increased non-significantly (p=1.195) with age in all animals studied (Table 3).
Different risk factors of acquiring the parasite related to the IgM in positive and negative cases were also analyzed.It was found that contact with cats (p =0.9282), drinking water source (p=0.0955) and poor hygiene condition (p =0.39551) were not significant risk factor while feeding habits (p < 0.0001) of animals was associated with T. gondii infections (Table 4).
The prevalence of T. gondii infection in the human population was given in Table 6.Results of this study indicated that toxoplasmosis infections significantly increase with age in both male (p=0.02) and female (p=0.01)populations.

Discussion
In the current study, the reported sero-prevalence of IgM was 32.18%, IgG was 17.16% and both IgM and IgG was 6.4% of the studied animals.The prevalence of T. gondii DNA in the milk of studied animals was 23.60%.The prevalence of T. gondii DNA in the milk samples varies in different age groups, ranging from 16.66% in animals aged ≤5 years, 11.76% in 6-9 years, and the prevalence increase with the age of the animals.The present study was consistent with a previous study from the United Kingdom that old age animals were less resistant to T. gondii infection than younger animals due to low immunity and high susceptibility to infection (Schares et al., 2017).
Several past studies reported low prevalence of T. gondii DNA in sheep milk samples than the present study; 3.4% prevalence rate reported from Italy (Fusco et al., 2007), 5% reported from Brazil (Camossi et al., 2011), 6.48% from Iran (Dehkordi et al., 2013), the difference between the present study and -mentioned studies might be due to variation in cat density, contaminated water and grasses with T. gondii oocysts, and geographic factors, etc.The current study supported result reported from Serbia that constant contact of sheep to the environment progressively increases the risk of T. gondii infection with the age of the sheep because the environment was contaminated with T. gondii oocysts  Toxoplasma gondii in lactating animals in Khyber Pakhtunkhwa (Stelzer et al., 2019).The sero-prevalence reported from Southern Punjab Pakistan in sheep was 19.88%, which was lower than the present study (Hanif and Tasawar, 2016).Likewise, sero-prevalence reported in sheep was 36% from Mohmand agency, Pakistan (Thai et al., 2019), and 31.45% from Ethiopia (Gebremedhin and Gizaw, 2014).
The molecular prevalence of T. gondii DNA reported in sheep from Egypt was10.71%(Camossi et al., 2011), which was similar to current work.
In lactating goats in this study, the sero-prevalence rate of IgM was 34.78%, IgG was 21.74% and both IgM and IgG were 8.6% by ELISA.DNA of T. gondii detected in raw milk by PCR was 34.78%.The age of the lactating goats were evaluated for the relationship with T. gondii occurrence.Similar results were also reported from Ethiopia in old age caprine have high toxoplasmosis (Teshale et al., 2007), which justified the present study.
Data reported by several investigators in lactating goat milk from different countries of the world; such as from Egypt, the prevalence of T. gondii DNA through PCR was 22.73% (Camossi et al., 2011), and from El-Fayoum Governorate, Egypt was 25% (Ghoneim et al., 2009), from Brazil was 28.9% (Bisson et al., 2000), from Thailand was 27.9% (Jittapalapong et al., 2005), from Pakistan was 25.4% (Ramzan et al., 2009), and from Slovakia was 32.56% T. gondii DNA reported in goats milk (Spišák et al., 2010), which showed coherence with the present study.
Likewise, the prevalence of T. gondii DNA reported by several authors through PCR technique from different countries such as 6.05% in raw milk of goat from Brazil (Bezerra et al., 2015), and 13% of T. gondii DNA recorded in naturally infected lactating goats from Italy (Mancianti et al., 2013).Data reported from Northwest Tunisia that consuming raw goats' milk represents a real risk factor for human toxoplasmosis (Amairia et al., 2016).The reported prevalence of T. gondii DNA from Northwest of Iran was 9.44% in the raw milk of goats (Tavassoli et al., 2013).The sero-prevalence reported in goats from Multan Pakistan was 52% (Tasawar et al., 2011), while from Zimbabwe the prevalence rate reported was 67.9% (Hove et al., 2005) and from Egypt was 59.4% (Barakat et al., 2009).Detection of toxoplasmosis in different lactating species (goats, sheep, buffalos, cattle, and camels) was recently studied in Iran with different techniques and reported that the raw milk of the mentioned animal contains T. gondii (Dehkordi et al., 2013).Therefore, the unpasteurized milk of the said animals is a risk factor for T. gondii transmission to humans.In the current study, the molecular prevalence of T. gondii DNA was detected higher in goat milk as compared to other lactating animals included in the study population.A similar result was also reported by other authors that the milk of infected goat contains more T. gondii tachyzoites as compared to its meat (Spišák et al., 2010;Bezerra et al., 2015).
The high prevalence of T. gondii in lactating goats in the present research may be due to different climatic factors such as high humidity, temperature change, the difference in agro-ecology, high density of dogs and cats.
In the current study, the examined sero-prevalence in lactating cows through ELISA; IgM was 24%, IgG was 10% and both IgM and IgG was 4%.DNA of T. gondii was detected in raw milk of cow by PCR with the prevalence of 20%.In the different age groups of cows showed that the prevalence of T. gondii DNA considerably increases with the age of the cow.The present research was coherent with the reported prevalence in cows from different countries such as 20% from Mohmand agency, Pakistan (Shah et al., 2013), 13.3% by ELISA from Sudan (Elfahal et al., 2013), 15.91% from Iran (Delgado et al., 2022), and all these support the present study.While the reported prevalence of T. gondii in cattle from Serbia the estimated prevalence of T. gondii was observed 76.3% in cattle (Klun et al., 2006), and from Brazil, 71% prevalence of T. gondii was reported in cattle (Santos et al., 2009) which was extremely higher than the present study.There are limited reports throughout the world on the sero-prevalence of T. gondii infection in buffaloes (Shah et al., 2013).Current study was supported by 27.2% sero-prevalence reported from Brazil (Oliveira et al., 2018), 7.8% reported from Trinidad (Persad et al., 2011), 8.8% from Iran (Anvari et al., 2018).Several risk factors including sex, diagnostic test, age, and climatic alterations probably take part in changing the prevalence in buffaloes throughout the globe (Ahmad and Qayyum, 2014).The high prevalence of T. gondii infection in the present study might be due to differences in agro-ecology, climate, cat and dog density in the study area, sample size, unhygienic condition, animal management, contaminated water with oocysts, transmission routes, age of the animals and type of serological tests applied, etc. Animals reared for dairy production and reproduction often lives much longer.Therefore, T. gondii infection was higher in the milk of adult cattle than in the young cattle mentioning the reason that animals with longer life expectancy have longer exposure to risk factors, thus more contaminated with T. gondii parasite (Abdallah et al., 2019;Stelzer et al., 2019), also supported current study.Moreover, in older age, animals are proven less resistant to pathogens due to low immunity and high susceptibility to infection than younger ones (Ahmad and Qayyum, 2014;Roberts et al., 2001;Ahmed et al., 2016).
The poor hygienic state may favor water and food to be contaminated with cat feces, hence it increased risk of T. gondii infection in the livestock.Because a small proportion of individuals acquired the infection, then pose a potential reservoir of infection.While the majority later infected by using contaminated food and drinking water.Thus good hygienic status and cleaning measures have a protective effect (Stelzer et al., 2019).
In this study, 738 humans tested for T. gondii, 9.80% and 11.80% showed seropositivity to IgM and IgG, respectively, corresponding to an overall prevalence rate of 20.95%.This showed that T. gondii infection is highly prevalent in the study area.This region is an agricultural region and the people kept domestic animals for economic purposes.Cat and other pet animals are also generally kept in-home or near home, unhygienic nutritional tradition and low education level contributes to infection.The prevalence rate of T. gondii infection throughout the globe, as well as within-country, is variable since its prevalence depends upon the environment (water quality, sanitation, coverage, etc.), climatic variations from one region to another, socioeconomic (hygiene, source of food, etc.), climatic factors (humidity, temperature), lifestyle of the inhabitants (dietary habits, method of cooking, hand washing, kind of food item cleaning, contact with cats and dogs or other domestic animals, contact with the soil, etc.) and difference in serological tests with variable specificity and sensitivity (Tenter et al., 2000;Robert-Gangneux and Dardé, 2012).However, a statistically significant correlation between positive serology for T. gondii in humans and ingestion of goat milk also had been reported in the literature (Sroka et al., 2017).

Conclusions
High prevalence of T. gondii in milk samples indicates a potential risk for the transmission of T. gondii to human through raw unpasteurized milk consumption.Therefore, awareness regarding the transmission and prevention of the disease is crucial.In general, integrated strategies and measures should be implemented to prevent and control T. gondii infection in dairy animals as well as in human in the study area.

Table 1 .
Prevalence of T. gondii antibodies (IgM and IgG) and DNA in milk of lactating animals in Upper Dir district.

Table 2 .
Comparative analysis of T. gondii antibodies and DNA in Upper Dir district.

Table 3 .
T. gondii DNA in lactating animals by PCR in Upper Dir district.

Table 4 .
Risk factors related prevalence for T. gondii in lactating animals in Upper Dir district.

Table 5 .
Overall seroprevalence of T.gondii in human population in Upper Dir district.The sero-prevalence reported in lactating buffalo was 26.31% for IgM, 10.53% for IgG, and 6.4% for both IgG and IgM by ELISA.DNA of T. gondii detected by PCR was 26.0% in unpasteurized milk of buffalo.In the different age groups of buffalo, the prevalence of T. gondii DNA ranges such as, the positivity of 10% was reported in buffalo of age group II, 26.66% in age group III while 38.66% in age group IV.The frequency significantly increases with the age of the buffalo (p<0.05).

Table 6 .
Age-wise sero-prevalence of T. gondii in human population in Upper Dir district.Toxoplasma gondii in lactating animals in Khyber Pakhtunkhwa Brazilian Journal of Biology, 2023, vol.83, e267369 7/9