Seroprevalence of Toxoplasma gondii, Neospora caninum and Trichinella spp. in Pigs from Cairo, Egypt

Simple Summary Toxoplasma gondii and Trichinella species are parasites of major public health importance because they cause severe clinical consequences in infected humans. In addition, infections of Neospora caninum in livestock are reported to cause substantial losses to the economy because of adverse effects on animal production and reproduction. In Egypt, the breeding of pigs has particular characteristics regarding the targeted consumers (tourists and Christians), breeding places (Cairo and Giza), and licensed abattoirs (only El-Bassatin in Cairo). Herein, we sought to provide a comprehensive investigation of the major parasites of pigs, including T. gondii, Trichinella species, and N. caninum, a parasite of high priority in ruminants. This study revealed variable seropositive rates against all tested parasites in pigs from Cairo, Egypt. Seropositivity was the highest for T. gondii (45.8%), followed by N. caninum (28.0%), mixed infection by both parasites (18.7%), and Trichinella spp. (1.2%). We also found the location was a predisposing factor for seropositivity for T. gondii, while location and sex were identified as predisposing factors for N. caninum seropositivity in pigs. The provided information in this study presents valuable information on the seroprevalence of T. gondii and Trichinella, and novel information on N. caninum existence among pigs in Cairo, Egypt. Abstract Pork production is a niche economy in Egypt, and pigs are typically raised as backyard animals with no sanitary control, potentially exposing them to various pathogens. Commercially available ELISAs were used to detect specific antibodies to the food-borne zoonotic parasites Toxoplasma gondii and Trichinella spp., as well as to Neospora caninum, in serum samples of pigs slaughtered at Egypt’s only licensed pig abattoir, the El-Bassatin abattoir in Cairo. Among the tested sera (n = 332), seroreactivity for T. gondii was 45.8% (95% confidence interval: 40.4–51.3), N. caninum was 28.0% (95% CI: 23.3–33.2), and Trichinella spp. was 1.2% (95% CI: 0.4–3.3). Mixed infection was only detected for T. gondii and N. caninum (18.7%; 95% CI: 14.7–23.4). The seroprevalence of T. gondii was significantly higher (p = 0.0003) in animals collected from southern Cairo (15 May city slum) than in eastern Cairo (Ezbet El Nakhl slum). Seroprevalence for N. caninum was higher in western (Manshiyat Naser slum; p = 0.0003) and southern Cairo (15 May city slum; p = 0.0003) than in that of eastern Cairo (Ezbet El Nakhl slum; p = 0.0003). Moreover, female pigs exhibited a higher rate of N. caninum antibodies than male ones (p < 0.0001). This study provides the first seroprevalence data for N. caninum in pigs in Egypt, and updates the prevalence of the zoonotic parasites Trichinella spp. and T. gondii.


Introduction
A lack of food security is one of the issues plaguing the world's rapidly growing human population, and the global prevalence of malnutrition increased dramatically between 2019 and 2020, owing mostly to the COVID-19 pandemic [1].The population in Egypt is growing fast; the estimated population in 2021 was 109.3 million, with a population change rate of 1.59% [2].Egypt is primarily a Muslim country; however, it has lately been estimated that approximately 10% of the Egyptian population are Christians [3].The swine/pigs stock number in the country was estimated as 11,000 head by the year 2021 [4].Pigs in Egypt are typically reared as backyard animals and fed on human garbage [5,6].Consequently, it is critical to investigate pigs for parasites that are either of zoonotic or veterinary importance.
With a global distribution, Toxoplasma gondii infects nearly all warm-blooded animals, including humans, livestock, and marine mammals [7,8].The definitive hosts are felids, which produce and shed environmentally-resistant oocysts [8,9].Pigs, like humans and other animals, become infected with T. gondii after ingesting sporulated oocysts shed by cats or tissue cysts in the meat of other intermediate hosts [10].Natural T. gondii infection in pigs is a public health issue, as transmission to humans by consumption of raw or undercooked pork meat or offal can occur [11].Infections in pigs are usually asymptomatic or with non-pathognomonic symptoms, and severe clinical toxoplasmosis is rare [11].Human primary infection with T. gondii normally manifests as mild flu-like symptoms in immune-competent hosts; however, it can induce life-threatening infections in immune-compromised persons.Furthermore, T. gondii transplacental transmission during pregnancy might result in significant complications such as abortion, stillbirth, or congenital anomalies [8].
The pooled global T. gondii seroprevalence in pigs (148,092 pigs from 47 countries) was estimated to be 19% based on different serological methods, among which enzymelinked immunosorbent assays (ELISA) were used most frequently [12].In Egypt, the reported seroprevalence in pigs varies between 14% and 80.4% [13][14][15][16][17][18][19][20][21].Additionally, viable T. gondii was isolated from the heart of a healthy seropositive pig in Egypt.Many cases of human toxoplasmosis have been reported in Egypt and reviewed previously.The reported seroprevalence of T. gondii in the Egyptian human population ranges from 2.5% to 67.4%, and toxoplasmosis is commonly regarded by Egyptian physicians as a cause of abortions and complications in pregnant women; nonetheless, the published studies are not well-structured, and there is a lack of definitive diagnoses [20][21][22].
Neosporosis is a disease caused by the heteroxenous cyst-forming parasite Neospora caninum, an intracellular apicomplexan protozoan that infects a wide range of domestic and wild animals [23].The definitive hosts of N. caninum are domestic and wild dogs, coyotes, and grey wolves [24,25].Cattle are the most common intermediate hosts of N. caninum; however, sheep, water buffaloes, bison, and white-tailed deer have also been identified as intermediate hosts [26].Abortions and newborn mortality are serious complications of neosporosis in livestock, particularly in cattle [23,24].
Anti-N.caninum antibodies have been reported in domestic and feral pigs from various countries [24], and, more recently, parasite DNA has been detected in the brains of domestic pigs in China [27], indicating natural infection.However, the pathogenesis of neosporosis Vet.Sci.2023, 10, 675 3 of 13 and its consequences in pigs remains unclear [28,29].The detection of N. caninum infection in live animals is based mainly on the identification of anti-N.caninum antibodies using a variety of serological tests, the most frequently used of which is ELISA [30,31].In Egypt, N. caninum infections have been detected in domestic animals such as cattle, buffaloes, and camels, but there have been no reports in pigs.
Trichinella nematodes are zoonotic parasites with a global distribution [32].Pork is the most common source of human trichinellosis, which can cause clinical symptoms such as myalgia, diarrhea, fever, facial edema, and headaches, and may even be fatal [33].Trichinella spiralis is the only species reported in Egypt so far [32], and it has been identified in pigs [34,35], rodents [36], and dogs [34,37].In Egypt, the reported infection rate in slaughtered pigs and pork products ranges from 0% to 6% [19,35,[38][39][40][41].Only one old report on the seroprevalence of Trichinella spp. in pigs in Egypt (35.6%) was found in a literature search, and it used an immunofluorescent antibody test (IFAT) [42].Nevertheless, ELISA is the recommended assay for epidemiologic serosurveillance of pigs for Trichinella infection [43].Human trichinellosis linked to the consumption of pork has recently been reported from Egypt based on ELISA, and the seroprevalence rate of T. spiralis (IgG) was 10% (9/90) [35] and 60.9% (56/92) [40] in the examined humans.
Based on the above-mentioned facts, there have been a few reports in Egypt on the prevalence of T. gondii and Trichinella spp. in pigs, but none on N. caninum.We therefore opted to update the information on the seroprevalence of T. gondii and Trichinella spp., as well as to provide baseline data for the seroprevalence of N. caninum infections, in pigs in Egypt.Our presented data will serve the public health and veterinary sectors in minimizing the health and economic hazards of these parasites in Egypt.

Description of the Animals and Regions of the Study
Blood samples were collected from male and female pigs (n = 332) during slaughtering in the El-Bassatin abattoir in the Cairo governorate.All pigs were raised in backyard pens containing 50 to 100 pigs in the slums of the Cairo governorate by garbage collectors as one of their main sources of income.These rearing pens and slums lack many procedures ensuring sanitary conditions or biosecurity and pigs are fed mainly on garbage.This condition allows for high rates of contact with free-roaming animals such as dogs, cats, rodents, and birds, increasing the incidence of infection with many biohazardous agents.In addition, other livestock animals, especially sheep, are also reared with these pigs.The majority of pigs are reared for fattening and are slaughtered at the age of 5 months.They are typically sold to pig merchants who collect animals from different small-scale farmers and ship them to the abattoir.Pigs used in this study originated from the following slums: Manshiyat Naser, Cairo's largest pig farming slum in the western region (30.0362 of them male) in the southern Cairo region (Figure 1).The pigs from various herds were held together in abattoir pens before slaughtering without identification.
Blood was collected in plain tubes without anticoagulants directly from the slaughtered animals during bleeding according to the El-Bassatin abattoir regulations for pig slaughtering and pork processing.All samples were labeled and transferred on ice to the Reference Laboratory at the Animal Health Research Institute (AHRI), Giza.Upon arrival at the laboratory, blood samples were kept overnight for serum separation and then centrifuged at 900× g for 10 min.Serum samples were aliquoted and kept at −20 • C until testing.Aliquots from these samples were sent on ice to the Faculty of Veterinary Medicine, South Valley University, Qena, for ELISA testing.
testing.Aliquots from these samples were sent on ice to the Faculty of Veterinary Medicine, South Valley University, Qena, for ELISA testing.

ELISAs for Screening Antibodies to T. gondii, N. caninum, and Trichinella Species in Pigs
Regarding T. gondii, the serum samples were tested using an indirect multi-species ELISA for toxoplasmosis (ID.vet,Grabels, France) according to the manufacturer's instructions.Serum samples and controls were diluted 1:10.The optical density (OD) obtained was used to determine the percentage of sample (S) to positive (P) ratio (S/P %) for each of the test samples using the following formula: S/P (%) = (OD sample -OD negative control)/(OD positive control − OD negative control) × 100.Samples with an S/P% less than 40% were regarded as negative, an S/P% between 40% and 50% was regarded as doubtful, and the test was considered positive if the S/P% was more than 50%.
For N. caninum, serum samples were analyzed using a competitive multi-species ELISA for neosporosis (ID.vet,Grabels, France).Serum samples and controls were diluted 1:2.The ODs obtained were used to calculate the percentage of sample (S) to negative (N) ratio (S/N %) for each of the test samples according to the following formula S/N (%) = OD sample/OD negative control × 100.Samples with an S/N% more than 60% were regarded as negative, an S/N% between 50% and 60% was regarded as doubtful, and the test was considered positive if the S/N% was less than 50%.
Serum samples were analyzed for Trichinella species using an indirect multi-species ELISA for trichinellosis (ID.vet,Grabels, France).Serum samples and controls were diluted 1:20.The ODs obtained were used to determine the percentage of sample (S) to positive (P) ratio (S/P %) for each of the test samples using the following formula: S/P (%) = (OD sample -OD negative control)/(OD positive control -OD negative control) × 100.Samples with an S/P% less than 50% were regarded as negative, an S/P% between 50% and 60% was regarded as doubtful, and the test was considered positive if the S/P% was greater than or equal to 60%.
The ODs of all ELISA results were read at 450 nm using an Infinite ® F50/Robotic ELISA reader (Tecan Group Ltd., Männedorf, Switzerland).See Table 1 for additional information on the ELISAs used in this study.

ELISAs for Screening Antibodies to T. gondii, N. caninum, and Trichinella Species in Pigs
Regarding T. gondii, the serum samples were tested using an indirect multi-species ELISA for toxoplasmosis (ID.vet,Grabels, France) according to the manufacturer's instructions.Serum samples and controls were diluted 1:10.The optical density (OD) obtained was used to determine the percentage of sample (S) to positive (P) ratio (S/P %) for each of the test samples using the following formula: S/P (%) = (OD sample -OD negative control)/(OD positive control − OD negative control) × 100.Samples with an S/P% less than 40% were regarded as negative, an S/P% between 40% and 50% was regarded as doubtful, and the test was considered positive if the S/P% was more than 50%.
For N. caninum, serum samples were analyzed using a competitive multi-species ELISA for neosporosis (ID.vet,Grabels, France).Serum samples and controls were diluted 1:2.The ODs obtained were used to calculate the percentage of sample (S) to negative (N) ratio (S/N%) for each of the test samples according to the following formula S/N (%) = OD sample/OD negative control × 100.Samples with an S/N% more than 60% were regarded as negative, an S/N% between 50% and 60% was regarded as doubtful, and the test was considered positive if the S/N% was less than 50%.
Serum samples were analyzed for Trichinella species using an indirect multi-species ELISA for trichinellosis (ID.vet,Grabels, France).Serum samples and controls were diluted 1:20.The ODs obtained were used to determine the percentage of sample (S) to positive (P) ratio (S/P %) for each of the test samples using the following formula: S/P (%) = (OD sample − OD negative control)/(OD positive control − OD negative control) × 100.Samples with an S/P% less than 50% were regarded as negative, an S/P% between 50% and 60% was regarded as doubtful, and the test was considered positive if the S/P% was greater than or equal to 60%.
The ODs of all ELISA results were read at 450 nm using an Infinite ® F50/Robotic ELISA reader (Tecan Group Ltd., Männedorf, Switzerland).See Table 1 for additional information on the ELISAs used in this study.$ All ELISA kits were manufactured by ID.vet Innovative Diagnostics, Grabels, France.* The sensitivity and specificity of the diagnostic kits were provided by the manufacturer of the kits.# detects antibodies to T. spiralis, T. pseudospiralis, T. britovi and T. nativa.

Statistical Analysis
The significance of the differences in the prevalence rates and risk factors were analyzed using Fisher's exact test, 95% confidence intervals (95% CI) (including a continuity correction), and odds ratios (OR) using an online statistical website www.vassarstats.net(accessed on 22 July 2023).From this website, calculator 3 was selected from the clinical research calculators.Then, in squares of data entry, the numbers of negative (absent) and positive (present) samples from group 1 (regarded as the reference group) were added.Similarly in the lower squares, the numbers of negative and positive samples were added.In these tests, we used the two-tailed p values of Fisher's exact test.p-values and odds ratios were confirmed with GraphPad Prism version 5 (GraphPad Software Inc., La Jolla, CA, USA) [44,45].

Discussion
Despite the presence of a considerable pig population and pig consumers in Egypt, there has been relatively little effort to conduct parasite epidemiological studies.Herein, we targeted pigs as sentinel animals for infection with three parasites: T. gondii, N. caninum, and Trichinella species.All animals were admitted to El-Bassatin abattoir, Cairo, the only licensed place for pig slaughtering in Egypt.The highest seroprevalence was recorded for T. gondii (45.8%), followed by N. caninum (28.0%) and T. gondii and N. caninum co-infection (18.7%), and the lowest was for Trichinella spp.(1.2%).
A variety of techniques and antigens are available for the detection of distinct immunoglobulin isotypes against T. gondii [46,47], among which ELISA has been the most frequently used method in the past century [48].In Egypt, few serological studies have been published for T. gondii infections in pigs, and prevalence rates between 14% and 80.4% have been reported [13][14][15][16][17][18][19][20][21].These studies were conducted over a large time span , and a multitude of test types were used, such as indirect fluorescent antibody test (IFAT) [13,16], indirect hemagglutination assay (IHA) [19,20], dye test (DT) [14], modified agglutination test (MAT) [15], and ELISA [17,18].In the present study, we opted for commercially available ELISAs, which have the advantage of good repeatability, robustness, and reportedly very good sensitivity and specificity.The detected seroprevalence of 45.8% for T. gondii in the assessed backyard pigs indicates a potential risk to the health of pig fatteners, abattoir workers, and consumers.A similar finding was published by Barakat et al. [16], who reported a seroprevalence for T. gondii of 74.7% in pigs reared entirely on waste feed in Cairo, and a seroprevalence of 37.7% (48 of 127) in the workers at these pig farms.
Neospora caninum infection in Egyptian animals is underestimated, with a few seroprevalence reports in cattle, buffaloes, sheep, goats, camels, dogs, and cats, as well as limited information on clinical neosporosis.In our previous reports using the same ELISA as in the current study, we demonstrated seroprevalence rates for N. caninum of 3.9% in camels [44], 15.5% and 5% in sheep and goats, respectively [45], 5.8% in dogs, 3.4% in cats [49], and 24.6% in cattle [50].Nonetheless, serum samples from aborted cows revealed N. caninum infections in addition to Coxiella burnetii [51] or Bovine Viral Diarrhoea Virus [52].Furthermore, the number of N. caninum seropositive camels increased among females with an abortion history [53].On the other hand, the pathogenesis of neosporosis and its consequences in pigs remains unclear [28,29].Neospora caninum experimental infection in pigs can be transferred through the placenta and produce reproductive problems such as mummified fetuses, particularly in the first and second gestational thirds [28].
To the best of our knowledge, the current study is the first serological study of N. caninum infection in pigs in Egypt.Thus, we compared our recorded prevalence (28%) with the rates in other animals in different regions of Egypt.Although N. caninum might not be a serious parasite for the pig industry, its detection in pigs might constitute a major risk for ruminants and other animals because of multiple species being raised in the same place.The seroprevalence in pigs was most similar to rates reported in cattle (24.6% [50], 29% [51], and 30.2% [52]) and higher than the rates reported in all other animal species, with the exception of one study in buffaloes (Table 5).Furthermore, in comparison with the seroprevalence rate of N. caninum in domestic pigs from other countries, our infection rate seemed higher.For example, a rate of 3% was reported in the Czech Republic [54]; 1.9% in Hunan province, China [27]; 5.2% in Brazil [55]; and 6.7% in Italy [56] for N. caninum infections in pigs.Only one study found a higher seroprevalence rate (36.4%) in Qinghai-Tibetan plateau, China [57].Overall, these findings can best be explained by the sanitary conditions in which the pigs were raised.They were garbage-fed backyard pigs and fattened as an additional income by inhabitants of the slums of Cairo.Both parasites can be transmitted through the feeding of uncooked infected meat from other species or via oocysts shed by cats for T. gondii or by dogs for N. caninum [8,23].Slums typically harbor a large population of stray cats, dogs, and other animals that may serve as sources of infection for the pigs.
Little is known about the oocyst-shedding rates of cats and dogs in Egypt.The presence of T. gondii-like oocysts in the feces of cats in Egypt may have been overestimated, where 1432 cats were examined, revealing a pooled prevalence of 11.9% [64].Neospora caninum-like oocysts were found in the feces of one of 78 dogs tested in a single study (Dakahlia governorate), and it is conceivable that certain small-sized oocysts detected in prior studies from Egypt are N. caninum [65].Furthermore, N. caninum-like oocysts were found in the feces of five of nine puppies fed the placentas and brains of nine aborted bovine fetuses in Egypt [66].
In this study, we analyzed the effects of sex and breeding locations on the seroprevalence of T. gondii and N. caninum infection.The breeding location was identified as a risk factor for T. gondii where 15 May city had a significantly higher seroprevalence than the two other places.For N. caninum, both sex and location significantly affected the prevalence rate; females had a higher rate than males, and meanwhile, 15 May and Manshiyat Naser had higher rates than Ezbet El Nakhl.Notably, 15 May city recorded the highest prevalence for both T. gondii and N. caninum.This might be related to the more recent establishment of 15 May city, which has a larger desert area compared to the other two locations, and thus a higher chance of the presence of wild canines and felines.Another reason might be its distant location from the crowded areas of Cairo, which rendered it a main place for garbage collection.However, because this is the first study that investigated such factors in Egypt, other investigations are required to confirm these results and reveal the cause of variations.Meanwhile, numerous studies have found that location is a risk factor for the seroprevalence of T. gondii in pigs or other animals [21].Concerning N. caninum in pigs, the available data are very scanty, both in Egypt and globally.Nevertheless, the location is considered a potential risk factor for N. caninum in other animal species [45,50,67,68].Similarly, and taking into account the limited data concerning pigs, sex is regarded as a risk factor for T. gondii and N. caninum in cases of other animal species [50,69].Age is usually also a risk factor for seropositivity to T. gondii or N. caninum, but this could not be assessed in our study, as all pigs were slaughtered at approx. 5 months old.
Trichinella spp.transmission primarily occurs in poor areas where veterinary services are unavailable, as well as in traditional small-scale "backyard" rearing of pigs for household and local use, particularly by the feeding of food waste [70,71].Trichinella infection in pigs can be prevented through management practices such as grain feeding or cooking the feed garbage, indoor production, and removing rat carcasses from the premises [72][73][74][75].
In Egypt, larvae of Trichinella spp.have infrequently been found by trichinoscopy and/or digestion methods (Table 6).Interestingly, data on 33,812 slaughtered pigs were collected through a standard form from the El-Bassatin abattoir archives during one year of screening, and 359 (1.06%) were found to be infected [35].However, Mohammed et al. [35] additionally tested a subsample of pigs with the digestion method and found a higher prevalence of 3.3%.Using artificial digestion, a prevalence rate of 13.3% was determined in rodents from Alexandria, and high infection rates were identified in rodents taken from and surrounding abattoirs [36].Based on the isoenzyme electrophoretic patterns, Trichinella spiralis was identified in two stray dogs [37], two isolates from domestic pigs, and one isolate from a domestic dog from Egypt [34], and by PCR in domestic pigs [35].Furthermore, human trichinellosis linked to the consumption of pork has been reported in Egypt [35,40,[76][77][78].Although current serodiagnostic approaches cannot replace traditional meat inspection for this zoonotic nematode, ELISA can be useful for surveillance programs and generally has an increased sensitivity compared to direct detection of larvae [79].ELISA testing of 14,579 sera from pigs in 21 studies indicated an overall prevalence of 4.3% worldwide [75].The seroprevalence rate obtained for Trichinella infection in pigs in our study was quite low (1.2%) and very similar to the infection rates determined by trichinoscopy at slaughter.Experimental studies have shown that Trichinella species have a different larval burden and immunological response in pigs at 30-70 days post-infection that can last up to 40 weeks [80][81][82][83].Thus, the low seroprevalence of Trichinella spp.might be related to the younger age of tested pigs that are slaughtered at an age of 5 months.Likewise, the seroprevalence of Trichinella spp. in Vietnam increased with the pigs' age: it was 1.1%, 1.2%, 32.9%, and 55.6% for pigs < 2 months, 2-8 months, 9-36 months, and >36 months, respectively [84].Larger studies including different age groups and combining serological and direct methods should be envisaged to determine the infection rate in Egyptian pigs.
In conclusion, this study revealed variable seropositive rates against all tested parasites in pigs from Cairo, Egypt.Seropositivity was highest in T. gondii (45.8%) followed by

Figure 1 .
Figure 1.Geographical locations of sample origins.Serum samples were collected from pigs during slaughtering in the El-Bassatin abattoir, Cairo.These animals originated from three main slums of pig raising in three parts of Cairo, the capital governorate of Egypt: Ezbet El Nakhl (eastern Cairo), Manshiyat Naser (western Cairo), and 15 May city (southern Cairo).

Figure 1 .
Figure 1.Geographical locations of sample origins.Serum samples were collected from pigs during slaughtering in the El-Bassatin abattoir, Cairo.These animals originated from three main slums of pig raising in three parts of Cairo, the capital governorate of Egypt: Ezbet El Nakhl (eastern Cairo), Manshiyat Naser (western Cairo), and 15 May city (southern Cairo).

Table 1 .
Commercially available ELISA test kits used for detecting antibodies against T. gondii, N. caninum, and Trichinella species # .

Table 2 .
Seroprevalence of T. gondii, N. caninum, Trichinella spp.and mixed infections in pigs from Egypt.

Table 3 .
Risk factors for T. gondii antibodies in pigs, Cairo, Egypt.Odds ratio and 95% confidence interval calculated by http://vassarstats.net/(accessed on 22 July 2023).# p value was evaluated by Fisher's exact test using online statistics software http://vassarstats.net/(accessed on 22 July 2023) and GraphPad Prism version 5. Ref. is the value used as a reference. *

Table 4 .
Risk factors for N. caninum antibodies in pigs, Cairo, Egypt.
* Odds ratio and 95% confidence interval calculated by http://vassarstats.net/(accessed on 22 July 2023).# p value was evaluated by Fisher's exact test using online statistics software http://vassarstats.net/(accessed on 22 July 2023) and GraphPad Prism version 5. Ref. is the value used as a reference.

Table 5 .
Seroprevalence of N. caninum antibodies in animals in Egypt.

Table 6 .
Prevalence of Trichinella sp.larvae in pigs in Egypt.
* Data were collected through a standard form from the Al-Basateen abattoir archives.# Trichinella spiralis was identified based on PCR of the GAPDH gene.