Abstract
The aim of the present study was to determine seroprevalence of Toxoplasma gondii infection in stray cats and correlation with oocyst shedding and IFN-γ concentration. From April to August 2016, one hundred fifty-nine stray cats were captured from various localities in Mashhad area. The blood and fecal samples were collected from each cat. The serum samples were examined to detect antibodies against T. gondii infection by ELISA assay and the fecal samples were microscopically examined for T. gondii oocyst detection. The concentration changes of IFN-γ in serum samples of seropositive and seronegative cats were measured using ELISA kit. The results showed that 59.12% (94/159) of cats had antibodies against T. gondii infection. The seroprevalence of T. gondii infection in the adult cats above three years olds was higher than other groups. Regarding gender, month and region factors, the difference of seroprevalence of T. gondii infection was not significant. In this study, the Toxoplasma/Hammondia like oocyst (THLO) were detected in 2.56% (4/156) in fecal samples of one seropositive and three seronegative cats. Results also showed that the mean value for IFN-γ concentration in the seropositive cats was significantly higher than that of the seronegative cats. Based on the results, the high percentages of stray cats were infected with T. gondii in this area. The IFN-γ concentration of seropositive cats was higher than that of the seronegative cats.
Key Words: ELISA, Interferon gamma, Prevalence, Stray cat, Toxoplasma
Introduction
Toxoplasma gondii, one of the most common parasitic infections of man and other warm-blooded animals, is an obligate intracellular parasite and a member of the Apicomplexa phylum.1 The Felidae family play prominent roles in the epidemiology of T. gondii infection because they repel millions of oocysts in a short period of time, 1 to 2 weeks, in feces2 and hence, pollute soil, food and/or water. Oocysts of T. gondii have been detected in the feces of less than 1.00% of cats.3 Based on serological studies, about one third of the world's population has been exposed to this widespread zoonotic agent.1 It has caused a major public health concern due to the immuno-suppressive effects of HIV/AIDS.4
Toxoplasma gondii infection induces a powerful IFN-γ driven cell-mediated immune response in the mammalian hosts. IFN-γ plays an important role in alternation of tachyzoites to bradyzoites and blockage reactivation tachyzoites.5 This is a necessary response to elimination of acute infection and control of a chronic, latent infection in the CNS.6 Recently, some studies have showed that ELISA-based on IFN-γ assay could be used as useful diagnostic tool for acute and chronic T. gondii infection.7-9 Several diagnostic methods such as serological testes, fecal flotation technique and PCR are being used for determining T. gondii infection in cats. In Iran, large numbers of cats are found roaming in streets and can be an important potential source of transition of zoonotic diseases such as T. gondii infection. The results of the epidemiological studies showed a high prevalence of Toxoplasma infection in cats of Iran.10-15
There is a poor literature regarding prevalence of T. gondii infection in cat in Mashhad, Iran. The aim of the present survey was to determine the seroprevalence of T. gondii infection in stray cats in the Mashhad and the relationship of seropositive status of cat with oocyst shedding and the serum IFN-γ concentration.
Materials and Methods
Study area. The study was performed from April 2016 to August 2016 in Mashhad, the capital city of Khorasan Razavi province, located in the northeastern part of Iran at 36° 18' 38.5164'' N and 59° 35' 58.0452'' E, with an area of more than 328 km2. The climate is the northern temperature with a semi-arid climate with cold winters and moderate summer.
Ethics and animal experimentation. All animal experiments of this study, No. 3.40334, were performed in strict accordance with the guidelines approved by the Animal Ethics Committee of the University.
Sample size and sampling. The prevalence of T. gondii infection in stray cats was estimated 1.20% and 89.20% in the various regions of Iran.15 Based on expected proportion at 10.00%, the desired sample size was 159 stray cats, using a 95.00% level confidence and 5.00% desired absolute precision. In this study, Mashhad was divided into four regions, The north, south, east, and west, and the stray cats were captured by the traps. The trapped cats were transported to the Small Animal Clinic, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran. The stray cats with different ages and genders initially were physically examined for any clinical signs by a veterinarian. Then the blood and fecal samples were collected from cats. The blood samples in a plain test tube were centrifuged for 5 min at 800 g after clotting at room temperature for 2 hr. The serum was removed and stored at –20 ˚C till ELISA assay.
Parasitological method. Fecal samples of 159 cats were examined by fecal flotation technique methods.16 Briefly, 1.00 g of fecal sample of each stray cats were emulsified in sucrose solution, specific gravity 1.203, filtered through gauze and centrifuged in a 15 mL tube at 400 g for 10 min. The supernatant of solution was taken and examined microscopically for presence of T. gondii oocysts.
Toxoplasma gondii antibodies assay. Toxoplasma gondii antibodies were detected through indirect ELISA using a commercially available kit (ID.vet Innovative Diagnostics, Grabels, France) according to the manufacturer’s instructions. Briefly, 90.00 μL dilution buffer 2 was added to each well of microplate, followed by 10.00 μL of the negative control in wells A1 and B1, and 10.00 μL of the positive control in wells C1 and D1. The serum samples were thawed and 10.00 μL were dispensed into the remaining wells. Microplates were then incubated for 45 min at room temperature. The wells were washed thrice with 300 μL wash solution, then 100 μL of conjugate were added to wells. Microplate were then incubated for 45 min at room temperature. The wells were washed thrice with 300 μL wash solution, 100 μL substrate solution was added, followed by incubation in the dark for 15 min at room temperature. The reaction was stopped by adding 100 μL stop solution. The optical density (OD) of the samples and controls were measured at 450 nm and recorded using a microplate reader (ELx800 absorbance reader; BioTeK, Winooski, USA). The test was considered valid if the mean OD values of the positive control was greater than 0.350 (ODPC > 0.350), and if the ratio of the OD values of the positive and negative controls was greater than 3.5 (ODPC/ODNC > 3.5). The sample/positive (S/P) percentage was computed for each sample. The results for each sample were labeled as either negative (S/P < 40.00%), doubtful (40.00% < S/P < 50.00%), or positive (S/P > 50.00%).
IFN-γ concentration assay. The concentration of IFN-γ in the cat’s serum samples were measured using a commercially available ELISA kit (Shanghai Korain Biotech Co. Ltd, Shanghai, China). This kit uses enzyme-linked immune sorbent assay based on the biotin double antibody sandwich technology to assay the cat IFN-γ. The procedure of this test was done according to the manufacturer's recommendations. The OD of each well was read at 450 nm in the ELISA plate reader (BioTek). The standard curve was drawn based on the standard concentrations and the corresponding OD values were calculated. Then according to the OD value of samples, the concentration of the corresponding sample was calculated.
Statistical analysis. Cats were grouped based on age, sex, sampling month and geographic area and differences of seroprevalence T. gondii infection between among groups were analyzed by Chi-square test. The normality and differences of IFN-γ concentrations between seropositive and seronegative groups were determined using kolmogorov-smirnov test and Student t-test, respectively. The correlation, Coefficient R2, among the serum test, stool exam and levels of IFN-γ were analyzed with the Pearson correlation coefficient. The statistical analyses were carried out using SPSS software (version 21.0; IBM Corp., Chicago, USA). Differences were considered significant when p value < 0.05.
Results
Out of 159 serum samples of stray cats, 94 (59.12%) had antibodies against T. gondii. The Seroprevalence of T. gondii was significantly higher in adult cats above three years of age than compared to other age groups (p < 0.001), There was no significant difference between the seroprevalence of T. gondii infection in different months and in different sex and ages groups in sampled cats (p > 0.05), (Table 1).
Table 1.
Seroprevalence of Toxoplasma gondii infection in stray cats by different risk factors
| Risk factors | variables | Negative | Positive (%) | Total |
|---|---|---|---|---|
| Total | 65 | 94(59.11) | 159 | |
| Age | ||||
| < 6 months | 23 | 6 (20.68) | 29 | |
| 6 months–3 years | 31 | 21 (40.38) | 52 | |
| > 3 years | 11 | 67(85.89) | 78 | |
| Gender | ||||
| Male | 18 | 43(70.49) | 61 | |
| Female | 47 | 51(52.04) | 98 | |
| Sampling time (month) | ||||
| April | 19 | 17(47.22) | 36 | |
| May | 8 | 21(72.41) | 29 | |
| June | 2 | 11(84.62) | 13 | |
| July | 19 | 17(44.74) | 38 | |
| August | 15 | 28(65.12) | 43 | |
| Region | ||||
| North | 13 | 7(35.00) | 20 | |
| East | 17 | 33(66.00) | 50 | |
| West | 13 | 28(65.12) | 43 | |
| South | 22 | 24 (52.17) | 46 | |
In this study, IFN-γ concentrations were compared in two equal seropositive and seronegative groups, 41 samples. The statistical analyses showed a significant correlation between serology results and IFN-γ concentrations in sampled cats (R= – 0.29, p < 0.05; Table 2). Toxoplasma/Hammondia like oocyst (THLO) were detected in 2.56% (4/159) of fecal samples in the stray cats using fecal flotation technique (Fig. 1). In this study, three of the cats were seronegative.
Table 2.
The correlation between seroprevalence of Toxoplasma gondii infection, oocyst shedding and IFN-γ value in sampled cats
| Variables | Seroprevalence of T. gondii | IFN-γ value | Oocyst shedding | |
|---|---|---|---|---|
| Seroprevalence of T. gondii | Pearson Correlation | 1 | 0.298* | 0.113 |
| Sig. (2-tailed) | - | 0.007 | 0.311 | |
| No. | 82 | 82 | 82 | |
| IFN-γ value | Pearson Correlation | 0.298* | 1 | 0.120 |
| Sig. (2-tailed) | 0.007 | - | 0.282 | |
| No. | 82 | 82 | 82 | |
| Oocyst shedding | Pearson Correlation | 0.113 | 0.120 | 1 |
| Sig. (2-tailed) | 0.311 | 0.282 | - | |
| No. | 82 | 82 | 82 |
Correlation is significant at the 0.01 level (2-tailed).
Fig. 1.
The Toxoplasma/Hammondia like oocyst (THLO) in fecal sample of cat
Discussion
The present study was the first report on seroprevalence of T. gondii infection in stray cats in the Mashhad, Iran. The results showed that 59.12% of sampled cats were infected with T. gondii. Different serological methods have been used in epidemiological studies of feline toxoplasmosis in Iran.15
Based on the previous studies, the seroprevalence of T. gondii infection in cats was reported with 89.00 to 90.00% in Tehran (Iran),11 86.00% in Kashan (Iran),13 24.70% and 59.40% in Ahvaz (Iran),12,17 85.00% in Gorgan (Iran),18 44.20% in Kerman (Iran),14 35.30% in Urmia (Iran),19 32.00% in Isfahan (Iran),20 40.00% in Sari (Iran),10 90.00% in Saudi Arabia,21 66.00% in Iraq 22 and 73.90% in India,23 57.80% in China,24 34.40% in Turkey,25 40.00 and 58.00 in Brazil,26,27 45.20 % in Colombia,28 44.70% in Portugal,29 40.70% in Italy,30 and 91.60% in Ethiopia.31 It seems that variations in reports of the sero-prevalence of T. gondii infection in various regions of Iran and other countries might be due to the differences in type of cat population, the weather, the sampling time and the used serologic methods.
Many studies show that stray cats generally have higher seropositivity rates and are more capacity to get infection.19,26 Lifestyle of stray cats affect their daily contact with the possible sources of contamination from intermediate hosts like raw or undercooked meat of domestic animals, mice, birds, and reptiles which may harbor Toxoplasma cysts.27 In the present study the seroprevalence of T. gondii infection in adult cats above three years was higher than that of the young cats. The similar results were reported by in Iran.10,11,14,17,25 and other countries.28,29 It seems that elderly cats have more chance to get T. gondii infection. Unlike to current study, the higher seroprevalence of T. gondii infection was reported in stray cats in Iraq22 and Italy.30
In the present study, the difference in the frequency of T. gondii infection in male and female cats were not significant. The results of the present study were in agreement with the studies that conducted in Iran,10,11,13,14,17,19 and other countries,29,32 however, disagreed with some other the studies, indicating that the frequency of T. gondii infection were significantly gender-related in stray cat in Iran,18 Iraq,22 and Italy.30
According to the sampling period, no significant influence of month was evidenced in the present study. The results were in agreement with other studies that showed the seropositivity of sampled cat were the same in different months.10,18,29 The findings of the present study were contrary to other studies which found the increased infection rate occurring in the spring/summer.30
In this study, IFN-γ concentration was compared to seropositive and seronegative groups by cat IFN-γ ELISA. Results showed that IFN-γ concentration mean value in positive group was higher than that of seronegative group. Our results confirmed that the Interferon-gamma release assay (IGRA) in serum samples could be used to detect T. gondii infection in cats. Some studies have shown that the IRGA is a sensitive test to detect T. gondii infection in early stage of disease in human and animal.7-9
The Toxoplasma/Hammondia like oocyst (THLO) were found in four stray cats (2.56%) in our study. Similar results were reported in the previous studies in Iran19,33 and Ethiopia,31 however, some studies did not detect any Toxoplasma oocysts in fecal samples of cats.17,24,28,34,35 The shedding of Toxoplasma oocysts depends on the age, immune status of cat and climatic condition in each area.
In this study, 75.00% of the positive oocysts shedding cats were seronegative. Two reasons might have caused these results: 1) these cats may be infected only with Hammondia spp and 2) The sporogony stage of Toxoplasma of in the intestinal tissue has caused poor immune system stimulation with low antibody titer against Toxoplasma infection and was non-detectable by ELISA.
In conclusion, the high seroprevalence of T. gondii infection was detected among stray cats in Mashhad. The Toxoplasma/Hammondia like oocysts (THLO) were microscopically observed in fecal samples of a few stray cats. Furthermore, Interferon-gamma release assay (IRGA) showed that it could be used as sensitive diagnostic method for detection of T. gondii in cat.
Acknowledgments
We would like to thank Mr. Amin Bakhshani for his help during sampling. The research leading to these results was funded by a grant (No. 3.40334) from the Research Council of Ferdowsi University of Mashhad, Mashhad, Iran.
Conflict of interest
The authors declare that there is no conflict of interest.
References
- 1.Dubey JP, Beattie CP. Toxoplasmosis of animals and man. Boca Raton, USA: CRC Press; 1988. pp. 8–64. [Google Scholar]
- 2.Dubey JP, Lappine MR. Toxoplasmosis and neosporosis. In: Greene CE, editor. Infectious diseases of the dog and cat. 4th ed. Philadelphia, USA: Saunders ; 2012. pp. 806–827. [Google Scholar]
- 3.Dubey JP. The history of Toxoplasma gondii – the first 100 years. J Eukaryot Microbiol. 2008;55(6):467–475. doi: 10.1111/j.1550-7408.2008.00345.x. [DOI] [PubMed] [Google Scholar]
- 4.Lindstrom I, Kaddu-Mulindwa DH, Kironde F, et al. Prevalence of latent and reactivated Toxoplasma gondii parasites in HIV-patients from Uganda. Acta Tropica. 2006;100(3):218–222. doi: 10.1016/j.actatropica.2006.11.002. [DOI] [PubMed] [Google Scholar]
- 5.Bohne W, Heesemann J, Gross U. Induction of bradyzoite-specific Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages. Infect Immun. 1993;61(3):1141–1145. doi: 10.1128/iai.61.3.1141-1145.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Suzuki Y, Orellana MA, Schreiber RD, et al. Interferon-gamma: The major mediator of resistance against Toxoplasma gondii. Science. 1988;240(4851):516–518. doi: 10.1126/science.3128869. [DOI] [PubMed] [Google Scholar]
- 7.Mahmoudi S, Mamishi S, Suo X, et al. Early detection of Toxoplasma gondii infection by using an interferon gamma release assay. Exp Parasitol. 2017;172:39–43. doi: 10.1016/j.exppara.2016.12.008. [DOI] [PubMed] [Google Scholar]
- 8.Yin Q, El-Ashram S, Liu XY, et al. Early detection of Toxoplasma gondii-infected cats by interferon-gamma release assay. Exp Parasitol. 2015;157:145–149. doi: 10.1016/j.exppara.2015.08.015. [DOI] [PubMed] [Google Scholar]
- 9.Yin Q, El-Ashram S, Liu H, et al. Interferon-gamma release assay: An effective tool to detect early Toxoplasma gondii infection in mice. PLoS One. 2015;10(9):e0137808. doi: 10.1371/journal.pone.0137808. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Sharif M, Daryani A, Nasrolahei M, et al. Prevalence of Toxoplasma gondii antibodies in stray cats in Sari, northern Iran. Trop Anim Health Prod. 2009;41(2):183–187. doi: 10.1007/s11250-008-9173-y. [DOI] [PubMed] [Google Scholar]
- 11.Haddadzadeh H, Khazraiinia P, Aslani M, et al. Seroprevalence of Toxoplasma gondii infection in stray and household cats in Tehran. Vet Parasitol. 2006;138(3-4):211–216. doi: 10.1016/j.vetpar.2006.02.010. [DOI] [PubMed] [Google Scholar]
- 12.Hoghooghi-Rad N, Afraa M. Prevalence of toxoplasmosis in humans and domestic animals in Ahwaz, capital of Khoozestan Province, south-west Iran. J Trop Med Hyg. 1993;96(3):163–168. [PubMed] [Google Scholar]
- 13.Hooshyar H, Rostamkhani P, Talari S, et al. Toxoplasma gondii infection in stray cats. Iranian J Parasitol. 2007;2(1):18–22. [Google Scholar]
- 14.Akhtardanesh B, Ziaali N, Sharifi H, et al. Feline immunodeficiency virus, feline leukemia virus and Toxoplasma gondii in stray and household cats in Kerman, Iran: Seroprevalence and correlation with clinical and laboratory findings. Res Vet Sci. 2010;89(2):306–310. doi: 10.1016/j.rvsc.2010.03.015. [DOI] [PubMed] [Google Scholar]
- 15.Rahimi MT, Daryani A, Sarvi S, et al. Cats and Toxoplasma gondii: A systematic review and meta-analysis in Iran. Onderstepoort J Vet Res. 2015;82(1):823–832. doi: 10.4102/ojvr.v82i1.823. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Little SE, Lindsay DS. Laboratory diagnosis of protozoal infections. In: Greene CE, editor. Infectious diseases of the dog and cat. 4th ed. Philadelphia, USA: Saunders ; 2012. pp. 711–717. [Google Scholar]
- 17.Mosallanejad B, Avizeh R, Razi-Jalali MH, et al. A study on seroprevalence and coproantigen detection of Toxoplasma gondii in companion cats in Ahvaz area, south-western Iran. Iranian J Vet Res. 2011;12(2):139–144. [Google Scholar]
- 18.Namroodi S, Shariat Bahadory E. Analysis of feral cats role in dissemination of Toxoplasma gondii infection in rural area, Golestan province, north-east of Iran. Int J Epidemiol Res. 2015;2(4):190–196. [Google Scholar]
- 19.Raeghi S, Sedeghi S. Prevalence of Toxoplasma gondii antibodies in cats in Urmia, northwest of Iran. J Anim Plant Sci. 2011;21(2):132–134. [Google Scholar]
- 20.Saljoghiyan H. Survey on seroprevalence of toxoplasmosis in stray cats from Isfahan, Iran. In Proceedings: Congress of National Veterinary Pathobiology; Garmsar, Iran: 2011. 77 pp. [Google Scholar]
- 21.Hamdan I, Mohammed Al. Seroprevalence of Toxoplasma gondii Infection in cats, dogs and ruminant animals in Al-Ahsa area in Saudi Arabia. Res J Med Sci. 2011;5(4):190–192. [Google Scholar]
- 22.Dhamraa R, Alwan JM. Seropathological diagnosis of Toxoplasma gondii in stray cats in Baghdad province. Iraqi J Vet Med. 2014;38(2):92–98. [Google Scholar]
- 23.Dubey JP, Moura L, Majumdar D, et al. Isolation and characterization of viable Toxoplasma gondii isolates revealed possible high frequency of mixed infection in feral cats (Felis domesticus) from St Kitts, West Indies. Parasitology. 2009;136(6):589–594. doi: 10.1017/S0031182009006015. [DOI] [PubMed] [Google Scholar]
- 24.Qian W, Wang H, Su C, et al. Isolation and characterization of Toxoplasma gondii strains from stray cats revealed a single genotype in Beijing, China. Vet Parasitol. 2012;187(3-4):408–413. doi: 10.1016/j.vetpar.2012.01.026. [DOI] [PubMed] [Google Scholar]
- 25.Can H, Doskaya M, Ajzenberg D, et al. Genetic characterization of Toxoplasma gondii isolates and toxoplasmosis seroprevalence in stray cats of Izmir, Turkey. PLoS One. 2014;9(8):e104930. doi: 10.1371/journal.pone.0104930. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Meireles LR, Galisteo AJ, Pompeu E, et al. Toxoplasma gondii spreading in an urban area evaluated by seroprevalence in free-living cats and dogs. Trop Med Int Health. 2004;9(8):876–881. doi: 10.1111/j.1365-3156.2004.01280.x. [DOI] [PubMed] [Google Scholar]
- 27.Dubey JP, Navarro IT, Sreekumar C, et al. Toxoplasma gondii infections in cats from Parana, Brazil: Seroprevalence, tissue distribution, and biologic and genetic characterization of isolates. J Parasitol. 2004;90(4):721–726. doi: 10.1645/GE-382R. [DOI] [PubMed] [Google Scholar]
- 28.Dubey JP, Su C, Corte JA, et al. Prevalence of Toxoplasma gondii in cats from Colombia, South America and genetic characterization of T. gondii isolates. Vet Parasitol. 2006;141(1-2):42–47. doi: 10.1016/j.vetpar.2006.04.037. [DOI] [PubMed] [Google Scholar]
- 29.Waap H, Cardoso R, Leitao A, et al. In vitro isolation and seroprevalence of Toxoplasma gondii in stray cats and pigeons in Lisbon, Portugal. Vet Parasitol. 2012;187(3-4):542–547. doi: 10.1016/j.vetpar.2012.01.022. [DOI] [PubMed] [Google Scholar]
- 30.Papini R, Sbrana C, Rosa B, et al. Serological survey of Toxoplasma gondii infections in stray cats from Italy. Revue Med. Vet. 2006;157(4):193–196. [Google Scholar]
- 31.Dubey JP, Darrington C, Tiao N, et al. Isolation of viable Toxoplasma gondii from tissues and feces of cats from Addis Ababa, Ethiopia. J Parasitol. 2013;99(1):56–58. doi: 10.1645/GE-3229.1. [DOI] [PubMed] [Google Scholar]
- 32.Sumner B, Ackland ML. Toxoplasma gondii antibody in domestic cats in Melbourne. Aust Vet J. 1999;77(7):447–449. doi: 10.1111/j.1751-0813.1999.tb12089.x. [DOI] [PubMed] [Google Scholar]
- 33.Razmi GR. Prevalence of feline coccidia in Khorasan province of Iran. J Appl Anim Res. 2000;17:301–303. [Google Scholar]
- 34.Dubey JP, Zhu XQ, Sundar N, et al. Genetic and biologic characterization of Toxoplasma gondii isolates of cats from China. Vet Parasitol. 2007;145(3-4):352–356. doi: 10.1016/j.vetpar.2006.12.016. [DOI] [PubMed] [Google Scholar]
- 35.Advincula JK, Iewida SY, Cabanacan-Salibay C. Serologic detection of Toxoplasma gondii infection in stray and household cats and its hematologic evaluation. Sci Med. 2010;20(1):76–82. [Google Scholar]