Toxoplasmosis is an important zoonotic parasitic disease transmitted from fields to any animal or human through the contamination of pasture with T. gondii oocytes of infected cats’ faces . T. gondii oocytes shed from cat's faces from 4 to 14 days after infection . Goats and sheep feed on green pastures contaminated with T. gondii oocytes. The present study revealed that the high incidence of T. gondii in lactating goats’ and lactating sheep's milk, but not in lactating camel milk, is due to the difference between the types of pastures of small ruminates and the camels in different governorates as shown in Figs. 1, 2, and 3. These findings are in line with those of . It is reported that the camels feed mainly on the dry matter that is present in the desert, and this feed fairway is contaminated by cats’ feces, which is the main source of Toxoplasma oocytes. In the present study, the prevalence of T. gondii in serum samples of male and female camels, sheep, and goats was determined by ELISA. The rates of contamination, which were 15/31 (48.3%), and 18/31 (58%), were higher in males than in lactating and non-lactating females, 18/48 (37.5%), and 45/113 (39.8%), respectively. The rates of T. gondii antibody detection in the serum samples of camels were 5/27 (18.51%), 14/40 (35%), and 15/41 (36.58%) in males, non-LF, and LF, respectively, in different governorates in Egypt as shown in Figs. 1, 2, and 3. The prevalence reported in our study is slightly lower than the one found by Saad et al. . The prevalence rates in sheep and goats are lower than those detected in two separate studies (64.34% and 64.9%, respectively) conducted in 2009 [18,19,20]… Seropositivity in goats was found to be higher than in sheep, and the selective feeding habits of goats may be explained the high seroprevalence detected in this species. The prevalence identified in a similar study in goats in another country (75%) is significantly higher than the one we found in goats in our study .
The rates of T. gondii DNA detection by PCR in blood samples of camels were 15%, 15%, and 20% in males, non-LF, and LF, and similar results were obtained using the LAMP assay. These results are shown in Table 2 and are similar to those obtained by . The results found similar or slightly higher rates than those , who reported the seroprevalence of T. gondii in camels’ serum and milk samples. They reported that the seroprevalence of T. gondii in camels’ serum and milk samples was similar to that reported by , who stated that the prevalence of T. gondii antibodies in camel's milk was 3.12%. Also, they explained that the highest incidence of T. gondii occurrence in milk in goats and sheep, but not in camels, is due to the difference in the types of pastures of small ruminants that feed on green pastures contaminated by T. gondii oocytes. Camels feed in the desert dry fodder fairway of cats, which is the main source of Toxoplasma oocytes .
The prevalence of T. gondii in sheep and goat milk samples by ELISA was 40/117 (34.18%) and 26/77 (33.7%). While T. gondii antibody was detected in 12/41 (29.26%) of the examined camel milk samples, as shown in Table 3 and Figs. 1, 2, and 3, the positive samples were subjected to further examination using PCR for detection of T. gondii DNA in milk samples of different animals and found 2/41 (4.8%) in camel milk, 8/117 (6.83%) in sheep milk, and 6/77 (7.79%) in goat milk, which can be explained by the lower incidence of T. gondii DNA detected by PCR in comparison to T. gondii antibodies may be due to the fact that IgG antibodies are produced late in the infection, and the parasite is localized in the organs and tissues rather than circulated in the blood; so, it does get to the milk. These findings, which are presented in Tables 3 and 4, are consistent with those of previous studies [25, 26]. The molecular assays are more accurate, sensitive, and specific than serological techniques. LAMP was sensitive, simple, and fast for DNA amplification; it took less than 1 h under isothermal conditions at 66 °C. The LAMP method has been proven to be very specific for the detection of T. gondii. Moreover, the LAMP method was much more sensitive in detecting T. gondii than other methods, and it was also much cheaper. We evaluated the specificity of the LAMP assay by screening T. gondii genomic DNA from other parasites. No cross-reactivity with E. coli, Staphylococcus spp, Salmonella species, Giardia lamblia, or N. caninum DNA samples was detected. The detection limit of this LAMP assay was five tachyzoites, which was similar in sensitivity to the PCR target at the B1 gene as shown in Tables 2 and 3. Also, it used several primers that recognize six to eight regions of the target DNA that eliminate the nonspecific binding of primers and are easily detected from the negative amplifications without the need for any gel electrophoresis to judge results .
In the present study, we detected T. gondii DNA in milk and blood samples of different animals by LAMP. The positive reaction medium turned green under UV light after a fluorescent dye was added as shown in Fig. 4. The results were obtained without the need for any gel electrophoresis or gel-documentation system, which reduces the amount of time required to evaluate the results. While the LAMP reaction tube under daylight without UV light gave white turbidity in the end product, this may be due to the magnesium pyrophosphate produced during the reaction, which leads to the production of a visible white precipitate. These results and explanations are in line with those of previous studies [11, 16]. The obtained results revealed that the incidence of Toxoplasma gondii antibodies as detected by ELISA in sheep milk samples was 39.66%. Lower values were reported by many authors who showed that the prevalence of Toxoplasma gondii was 5.94% in ovine milk samples by an ELISA test in Iran, and they found that the prevalence of anti-Toxoplasma gondii IgG antibodies in the raw milk of sheep was 3.76%.
The results shown in Tables 3 and 4 demonstrate that the LAMP assay is highly sensitive in the detection of T. gondii DNA in blood samples (100%), is relatively easy, and does not require advanced technical skills or expensive equipment. This result is in agreement with those of previous studies [11, 19]. However, LAMP seems to be extremely sensitive to contamination; as such, it should be performed in a biological safety cabinet type I as a separate part for LAMP. The LAMP assay (AF-LAMP and RE-LAMP) and cPCR using the B1 gene primer are similar, and the LAMP assay was carried out according to the reported general reaction conditions. Several samples were judged as positive via our LAMP assay, indicating surprisingly high sensitivity and specificity values, which are by far the most similar to those of conventional PCR at 100%, as shown in Tables 3 and 4. In this study, T. gondii DNA was detected in camel milk samples at 2/41 (4.8%), the lowest detection rate of DNA in milk samples relative to whole blood samples among different animal species, indicating that all seropositive animals do not secrete T. gondii tachyzoites into their milk, which could be a limiting factor for PCR results as in Table 4, and this result is in agreement with the results of previous studies [22, 28]. The high seropositivity and the presence of parasite DNA in milk led to the conclusion that T. gondii infection is present in the sheep population and that there is a risk of the human population becoming infected due to the consumption of raw, natural milk. Hill and Dubey  found Toxoplasma gondii DNA in 6.05% and 2.7% of the raw goat milk samples using the PCR technique in Brazil. The prevalence of Toxoplasma gondii DNA in naturally infected milk samples n in Italy was 13% . However, Egyptian researchers estimated that Toxoplasma gondii DNA was present in 8% of goats from rural areas in Sharkia, Egypt. Meanwhile, a higher prevalence was recorded; the prevalence of Toxoplasma gondii DNA was 32.56% in goat milk from a farm in Slovakia. These variations in results may be attributed to the immune statuses of the animals, the timing of the infection, and the genetic compositions of the host and the organism. Also, the explanation is in line with those of several authors  who reported that the rate of occurrence of tachyzoites in the milk of naturally infected sheep was 6.5% of T. gondii DNA in milk samples. Observing turbidity with the naked eye or adding intercalating dyes to the reaction tube after amplification is generally used to identify LAMP findings. However, these procedures need some expertise or the use of UV light to evaluate the outcome. Products may be easily spotted by visual examination when using LAMP products, reducing the reaction time and allowing the assay to be utilized for field-testing. Furthermore, because it is introduced before amplification, the LAMP test reduces the likelihood of false positives due to aerosol contamination [22, 28]. As a result, the LAMP method proposed here looks to be a potential alternative to cPCR for T. gondii detection; hence, this approach appears to be a potential alternative to cPCR for the identification of T. gondii DNA in blood samples (100 percent) in various species. T. gondii DNA was identified in blood and milk samples, as reported by previous studies [11,12,13]. However, for the examination of blood and milk samples, LAMP and cPCR findings were in perfect agreement. Because blood appears to be the transport channel for tachyzoites between body tissues, molecular tests are valuable for the early diagnosis of T. gondii in asymptomatic animals. Finally, the LAMP assay can be considered as one of the most accurate, specific, sensitive, and rapid diagnostic assays for early T. gondii tachyzoite detection in milk and blood samples.