Introduction

T. gondii is an important coccidian with worldwide distribution, which causes a serious disease, toxoplasmosis, in human and animals. The stray and feral cat’s definitive hosts can just excrete million oocysts by their feces. Cysts can remain infective for many months in sultry climates and spread in the environment or anything mixed with the feces. Stray cats can be infected by ingesting tissue cyst from intermediate hosts, such as wild rodents (Verma et al. 2018; Wang et al. 2018c).

There are a lot of tests for detecting positive infection with T. gondii including ELISA, indirect hem agglutination assay (IHA), ELISA Avidity and PCR for different genes. The LAT, ELISA and ELISA avidity are currently applicable for serological diagnosis of the infection; however the ELISA avidity with 97% sensitivity is a highly user-friendly method to detect toxoplasmosis infection (Hecker et al. 2018; Hou et al. 2018; Oh et al. 2018).

The aim of this study was to investigate the new prevalence of T. gondii among rodents and stray cats using ELISA avidity and PCR methods in Northeast of Iran. This investigation was necessary to assess the prevalence of Toxoplasmosis, using both serological and molecular methods.

Materials and methods

Study area

Golestan province is the main reservoir of the rodents and stray cats as toxoplasmosis circulating agents. The region is located next to the Caspian Sea, and has very sultry and rainy climate. This study was performed in 2017.

Sampling

We collected 286 heart tissues and blood samples of rodents and 210 heart tissues and blood samples of stray cats simultaneously from Golestan forest, where rodents and stray cats live. The samples were collected using intravenous blood sampling with syringe; then the animals were killed to isolate their heart tissues. The samples were transferred to a laboratory to examine nested-PCR, using GRA6 gene for detecting toxoplasmosis. Finally, IgG avidity was assessed in serum samples of the infected hosts, using the ELISA avidity method for evaluation of the infection time. The study was based on the prevalence and randomized abundance analysis. Tissue sample examination, processing and lysis were conducted before DNA extraction and nested-PCR.

Animal species were Rattus Rattus, Rattus norvegicus, Mus musculus, Rombomys opimus and stray cat.

DNA extraction

Extraction of genomic DNA was conducted using sodium DNG/proteinase K method. The material was purchased from Sinacolon Company and eluted into 50 Âµl DDH2O, according to the manufacturer’s instructions. A nested-PCR for T. gondii GRA6 gene was applied to confirm probable infection.

We cut 3 g of the sample tissue into small pieces and put them in a 1.5 ml sterile tube. 180 µl lyse buffer was added for homogenization and 400 Âµl DNG solution was also added and homogenized once again; then, 20 µl proteinase K was added.

We mixed them immediately by shaking for 20 s and incubated at 60 °C for 1 h to lyse the sample. If tissue resists to lysing, the incubation time would increase to 2–3 h. The samples were shacked or inverted in 10–15 min intervals. Then, we added 300 Âµl Isopropanol to DNA precipitation. After 5–10 min the tubes were washed by 70% ethanol. Finally, we eluted the DNA by deionized H2o. The eluted DNA was preserved at − 20 °C using a nested-PCR method for detection of infection. The positive control sample in this study was Tehran strain of T. gondii. The PCR kit was purchased from the Pishgam Company-Iran.

Nested-PCR analysis for T. gondii GRA6 gene

Nested-PCR analysis was performed to detect Toxoplas-ma GRA6 gene as a diagnostic gene. The primers were designed according to Pishgam Corporation (Table 1).

Table 1 Primer sequences
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Amplification of the GRA6 gene was completed in a 5-min cycle at 95 °C for initial denaturation, followed by 30 one-min cycles at 95 °C, 1 min at 62 °C, and 3 min at 74 °C. The best annealing temperature for PCR 1 was 58 °C and for PCR 2 was 57. 5 °C.The segment lengths were 546 bp in PCR1 and 344 bp in PCR2 (Table 2). The reason for choosing this gene was generalization of this molecular marker in various strains of T. Gondii and this gene was used in many studies.

Table 2 PCR substrate
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Then, we evaluated the serum of the infected rodents and stray cats to detect IgG avidity, using tachyzoite enzyme-linked immune assay method.

Tachyzoite based ELISA avidity test

ELISA avidity test has been used for detection of the Toxoplasma IgG in blood serum of the rodents and stray cats, according to the Euro immune ELISA avidity kit with specific conjugation. The positive control in the avidity ELISA test was embedded in the commercial kit. The ELISA avidity procedure was performed using Euro Immune Kit-France, according to manufacturer’s instructions.

The formula of ELISA avidity (semi quantitative):

$$ \frac{{{\text{OD patients }}\left( {\text{serum samples}} \right)}}{\text{OD calibrator 2}} $$

The result > 1.1 represents positive toxoplasmosis.

The formula of quantitative ELISA avidity (qIgG):

$$ \frac{{{\text{OD }}\left( {\text{serum samples}} \right){\text{ in urea buffer}}}}{{{\text{OD }}\left( {\text{serum samples}} \right){\text{ in phosphate buffer}}}} $$

The result × 100 = RIA (relative index avidity); the RIA value below 40% is in low avidity, between 40 and 60% is in intermediate avidity and oer 60% is in high avidity.

Statistical analysis

In this study, one-sample and two-sample t-tests, correlation table, frequency charts, high-low diagram and SPSS 19 software were used for data analysis. IgGq was nearly equal to the quantitative IgG ELISA avidity.

Results

In this study, 246/286 (86.01%) of rodents and 180/210 (85.71%) of stray cats were positive based on the Toxoplasma IgG ELISA avidity method. It was also found that 68 rodent samples and 38 stray cat samples were positive in presence of GRA6 gene. In rodents, 38 samples were Ratus ratus, 10 samples were Ratus norvegicus, 10 samples were Mus musculus and 10 samples were Rombumys opiums. These samples were positive in 344 bp location. The primer designing was conducted using BLAST software—NCBI site. Among 131 Rattus Rattus samples, 38 were positive (29%), among 45 Rattus norvegicus samples, 10 were positives (22%), among 60 Mus musculus samples, 10 were positive (16%), among 50 Rombomys opimus samples,10 were positive (20%) and finally among 210 stray cat’s samples,38 were positive (18%) (Fig. 1).

Fig. 1
figure 1

Nested-PCR result using GRA6 gene

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In this survey, we analyzed the prevalence study to show the important correlation between Toxoplasma PCR results (68/286 samples of rodents and 38/210 samples of stray cats) and positivity of Toxoplasma IgG ELISA Avidity (246/286 samples of rodents and 180/210 samples of stray cats). However, we found that 246/286 (86.01%) of rodents and 180/210(85.71%) of stray cats were positive, using IgG ELISA avidity method but just 68/286 of the rodents and 38/210 of the stray cats showed positive band in 344 bp location of GRA6 gene, using the nested PCR method.

Tables 3 and 4 show the t test results, and analysis of the infected rodents and stray cats to compare serum levels of semi quantitative IgG and quantitative IgG (IgGq), with 95% confidence interval. Figure 2 shows the frequency histogram tables of the serum level of Toxoplasma IgG avidity among the rodents and stray cats. The results were 2–2.5 (toxoplasmosis positive) among 246/286 rodents and 180/210 stray cats, with 95% confidence interval. Figure 3 shows the frequency histogram tables of the serum levels of Toxoplasma IgG quantitative avidity among the rodents and stray cats. The results were about 30% with 95% confidence interval, respectively. In this study, we showed that Toxoplasma GRA6 gene was detected only in rodents and stray cats with Toxoplasma intermediate ELISA avidity (40–60% relative index avidity).

Table 3 One-sample statistics
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Table 4 One-sample t-test
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Fig. 2
figure 2

a and b Histogram table of Toxoplasma IgG avidity in rodents and stray cats

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Fig. 3
figure 3

a and b Histogram table of Toxoplasma IgGq avidity in rodents and stray cats

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Figure 4 shows the high/low bars of Toxoplasma IgG and IgGq (quantitative) positive rodents and stray cats. The results of IgG avidity were in 2–2.5(semi quantitative ELISA) among these animals with 95% confidence interval. These results were in about 30% among 68/286 rodents and 38/210 stray cats (quantitative ELISA) with 95% confidence interval. Table 5 shows the comprehensive data achieved through analysis of Toxoplasma IgG and IgGq rodents and stray cats. According to this table, the mean of Toxoplasma IgG was in 2.20 for rodents and 2.30 for stray cats or the mean of Toxoplasma IgG q (quantitative) was 31.43 among the rodents and 31.01 among the stray cats. The proved intermediate avidity in these infected hosts and the SD of Toxoplasma IgG was 1.099 among the rodents and 1.10 among the stray cats. In addition, the SD of Toxoplasma IgGq (quantitative) was 11.31 among the rodents and 11.82 among the stray cats. Evaluating the prevalence analysis of the two methods for detecting toxoplasmosis in Northeast of Iran revealed a statistical significance. The results of nested-PCR were just applied for approving the existence of parasite, also semi quantitative ELISA avidity detected the presence of IgG antibody; however quantitative ELISA avidity method was used to evaluate the acuteness of the infection. In this study, we found that these animals with Toxoplasma GRA6 gene had acute infection, based on the quantitative ELISA avidity, but the hosts with chronic infection reported negative for Toxoplasma GRA6 gene. It means that GRA6 gene was detected only in quantitative ELISA avidity positive animals.

Fig. 4
figure 4

a and b High low bars of Toxoplasma IgG and IgGq avidity in rodents and stray cats. S sample, C control, q quantitative

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Table 5 Data analyze of Toxoplasma IgG and IgG quantitative avidity rodents and stray cats
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Discussion

One of the main difficulties of this study was simultaneous sampling of the rodents and stray cats. Golestan province located in Northeast of Iran with a sultry climate. In this study, the serologic and molecular methods have been used simultaneously for the first time to test the serum and tissue samples of the rodents and stray cats in the Golestan province. The results of this study revealed that the prevalence of toxoplasmosis with ELISA avidity procedure was higher than that in the polymerase chain reaction method in Northeast of Iran. Golestan province is the main reservoir of the rodents and stray cats as disseminators of toxoplasmosis. Rodents are the main reservoir host for toxoplasmosis that infects stray cats. Climate characteristic of region is highly suitable for toxoplasmosis. Many studies have been conducted on the occurrence of T. gondii among the cats and rodents in Iran. Sharif et al. 2009 have reported prevalence of forty percent T. gondii antibodies among the stray cats in Sari, Northern Iran. They conducted T. gondii antibodies test with latex agglutination on 100 serum samples collected from stray cats in five urban areas of Sari. Mostafavi et al. detected the differences in T. gondii infection between the male and female stray cats. Khademvatan et al. 2013 showed the existence of birds toxoplasmosis in Southwest of Iran (Mostafavi and Jalali Monfared 2012).

In recent years, there were no study to detect rodent and stray cat’s toxoplasmosis in Golestan area. The present study showed the important role of the rodents and stray cats in dissemination of toxoplasmosis in sultry areas. It also revealed that the GRA6 gene was a critical marker to detect the abundance of toxoplasmosis and that the heart tissue was one of the main tissues to follow up GRA6 gene from Toxoplasma parasite. Moreover, the rodents and stray cats were important agents of spreading toxoplasmosis in the region. The parasite forms tissue cysts with bradyzoites in various tissues (also in heart tissue), so the detected parasitic DNA (GRA6 gene) comes from bradyzoites more probably than from tachyzoites. The result of the recent studies revealed that toxoplasmosis among the rodents and stray cats of the Golestan province is about 85%. Regarding the climate of the Golestan province, which is theoretically highly suitable for T. gondii growth, detection of high rate of seropositive samples is probable. T. gondii parasite is responsible for toxoplasmosis as one of the most important diseases that causes ecological problems in rodents and stray cats’ environment. Serological test can be applied as an important instrument to diagnose the infection among the herds. This test detects specific anti-T.gondii antibodies, and involves techniques such as indirect fluorescence antibody test (IFAT), toxoplasma agglutination test (TAT), enzyme-linked immunosorbent assay (ELISA), rapid immune chromatographic test (RIT), and the immunoblot assay. T.gondii is an inevitable intracellular coccidian parasite and an important zoonotic pathogen that causes severe diseases in congenitally infected rodents and strays cats and in immune-compromised patients. Rodents and stray cats are the main reservoir for toxoplasmosis in Northeast of Iran. Early detection of the toxoplasmosis is very important to follow up the disease. The intrinsic factors of oocyst dispersal from the initial Drop-off locations of the infected feces may also be a crucial factor contributing to the widespread distribution of oocysts in soil. Antibodies to T.gondii was found in the cat, in 5/28 black rats (18%), and 2/18 brown rats, from Brazil (11%)(Silva et al. 2017; Simon et al. 2017).

In these hosts, the incidence of transmission elevates thereafter to more than 90%, when congenital infection occurs during the late weeks before delivery, which is more likely to be asymptomatic, yet may proceed to neurologic disorders later in childhood or in adolescence. One-third of the mother rodents, who acquire a primary T.gondii infection during pregnancy, transmit the infection to their fetuses and disseminate the infection to the environment (Bastien et al. 2018; Brouat et al. 2018; Foronda et al. 2015; Gennari et al. 2015; Mikhail et al. 2017; Normaznah et al. 2015; Wang et al. 2018d).

One of the reasons for using ELISA avidity method in rodents and stray cat’s studies was to find out the proper time for the host to be tested for Toxoplasma parasites, because finding this time and that the host is at what stage of infection are efficient in the severity of the infection. The Rodents were divided into four groups and stray cats in our study included in the group with the total hosts involved with Toxoplasma parasites. According to the results of this study, 246/286 rodents and 180/210 stray cats developed positive antibodies. Moreover, the results revealed that there were 68 positive T. gondii GRA6 gene rodents and 38 positive T. gondii GRA6 gene cats among the samples. Rodents and cats cannot develop positive antibodies and more probably ELISA test can be positive because of the presence of antibodies against T.gondii in the serum of the infected animals. Prevention and treatment of this parasitic infection in animals is not common, even if they are diagnosed in the early acute stage. The preventive measures are only used to avoid infection in domestic animals and human.Toxoplasmosis of rodents and stray cats may not create environmental and health problems for human, in Northeast of Iran. Despite the efforts of veterinarians to eradicate this disease in northeastern Iran, the infection remains as one of the unresolved problems. In Golestan region, especially in its tourism areas, people are in contact with these animals, and in rare cases, they can lead to severe infections or even epidemics. Early detection of the toxoplasmosis in these animals can be helpful to prevent the spread of infection to human and other hosts. A simple ELISA method used in toxoplasmosis analysis detects presence of the antibodies in these hosts. The molecular method is positively associated with the ELISA avidity method and can contribute to the health of the region (Jung et al. 2017; Must et al. 2017; Pena et al. 2017; Wang et al. 2018a, b, d; Wang et al. 2017).

Prevention of the congenital toxoplasmosis in rodents and stray cats has been based mainly on serological tests for anti-Toxoplasma antibodies. Many serological tests, including the ELISA and ELISA avidity methods have been used to detect the antibodies against T.gondii (Flegr 2017; Liang et al. 2016; Macaluso et al. 2018; Poulle et al. 2016; Spada et al. 2012; Teimouri et al. 2018). However, infection should be diagnosed at the early acute stages, when treatment is more effective. Therefore, the ELISA avidity method is useful in detection of the congenital toxoplasmosis among the rodents and stray cats and its results will be reliable, if we use the designed ELISA avidity kit from Toxoplasma ES antigens (tachyzoite based ELISA avidity) (Ahmadpour et al. 2017; Felin et al. 2017; Naghili et al. 2017; Patel et al. 2017; Pishkari et al. 2017).Toxoplasmosis is not critical disease in Iran but it is necessary to prevent it from spreading animals to humans.

Conclusion

According to the results of this study, the Northeast of Iran is a very important region for following up the toxoplasmosis infection, specifically among the animals such as rodents and stray cats (their main reservoir is Golestan). Moreover, quantitative IgG avidity (qIgG) results were at the intermediate level among the rodents and stray cats with T.gondii GRA6 positive gene in the region. We also concluded that the prevalence rate of toxoplasmosis in serology study was higher than that in the molecular detection.