Dataset Information

RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China.

ABSTRACT: Toxoplasma gondii, which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. T. gondii causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for T. gondii infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect T. gondii. The lowest limit of detection of the assay was 31 copies/μL for the T. gondii B1 gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify T. gondii within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of T. gondii in stray cats and dogs in Zhejiang province, Eastern China. The positive rates of T. gondii infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of T. gondii, showing promise for on-site surveillance.

Importance

Toxoplasma gondii is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of T. gondii are distributed worldwide, and cats and dogs are common hosts of T. gondii. Therefore, rapid diagnosis of early T. gondii infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of T. gondii without false-negative results. Moreover, we detected the prevalence of T. gondii in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of T. gondii and could be applied in prevalence surveys of T. gondii in stray dogs and cats.

SUBMITTER: Sun H

PROVIDER: S-EPMC11218441 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China.

Sun Hao H Fan Jiyuan J Chu Hongkun H Gao Yafan Y Fang Jiawen J Wu Qinli Q Ding Haojie H Zhuo Xunhui X Kong QingMing Q Lv HangJun H Zheng Bin B Lu Shaohong S

Microbiology spectrum 20240529 7

Toxoplasma gondii, which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. T. gondii causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for T. gondii infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polyme ...[more]

PMID: 38809001

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Project description:BackgroundToxoplasma gondii is prevalent in most areas of the world and may cause abortions or neonatal complications in humans. As the only definitive host, cats play an important role in the epidemiology of the disease. Infection rates in cats, especially stray or free-living cats, are considered to be the best sentinels of the level of T. gondii in the environment. The T. gondii infection can be diagnosed in different ways with different methods depending on the target. However, little information on T. gondii infection in cats was available in Shanghai, China. Moreover reports on prevalence of circulating antigens, antibodies and DNA of T. gondii in the same study are rare.MethodsIn the present study, the presence of antibodies (Ab), circulating antigens (CA), and/or DNA of Toxoplasma gondii in samples from 145 stray or unwanted cats from 6 animal shelters in Shanghai (China) was determined in order to estimate the prevalence of T. gondii infection, by Ab-ELISA, CA-ELISA, and nested-PCR, respectively.ResultsThe positive rates for the antibodies, circulating antigen and DNA of T. gondii were 11.7% (17 of 145), 5.5% (8 of 145) and 5.71% (2 of 35), respectively. No cat tested was positive by both the Ab-ELISA and the CA-ELISA, but the results of the PCR were consistent with the CA-ELISA assay. Therefore, the overall estimated prevalence of toxoplasmosis was 17.2% (25 of 145). According to our results, the positive rates of specific antibodies and circulating antigen of T. gondii were significantly different between adult cats (>1 year old) and juvenile cats (≤1 year old); the former was 13.5% versus 3.9% by Ab-ELISA, while the latter was 1.7% versus 23.1% by CA-ELISA. From the results obtained with all three detection methods used in this study, the rate of infection was not significantly different between male and female cats (P ≥0.05); and the overall rate was 17.9% for males versus 16.4% for females.ConclusionsThe results suggest that detection of circulating antigens (CA) is necessary in surveys of T. gondii infection, especially for juvenile cats. Our investigation revealed that the prevalence of T. gondii infection in stray cats in Shanghai is high. Control programs are needed for stray cat populations in order to reduce the risk of zoonotic transmission of toxoplasmosis to other domestic animals and humans, especially females.

Project description:Toxoplasma gondii and Toxocara spp. are the most critical parasites common between humans and cats. The close association of cats with humans in urban areas persuaded us to investigate the prevalence of these parasites in stray and household cats and their possible role in the owners' infection. Herein, 132 and 33 fecal samples of stray and household cats, respectively, and 33 blood samples of their owners were collected in Tehran, Iran. The prevalence of T. gondii was determined by targeting the B1 gene in the feces of stray and household cats and the blood of cat owners. Furthermore, genotypes of T. gondii were identified based on the multilocus genotyping of BTUB, GRA6, SAG3, and APICO loci. Toxocara spp. were detected by targeting the second internal transcribed spacer (ITS-2) of the ribosomal DNA of these parasites in the cats' feces and the humans' blood. Also, Toxocara IgG was assessed in the human serum samples. The B1 gene amplification showed that 15.2% of stray cats, 18.2% of household cats, and 51.5% of cat owners were infected with T. gondii. The multilocus sequence analysis revealed the predominance of genotype I of T. gondii in stray cats and genotype II of T. gondii in household cats and cat owners. The amplifying of ITS-2 revealed a high prevalence of T. cati infection (47.0%) in stray cats, whereas no infection was found in the feces of household cats or the serum of cat owners. Likewise, Toxocara IgG was not detected in the serum of humans. The lower prevalence of T. gondii in stray/household cats than in the cat owners indicates the limited impact of close contact with infected cats in human toxoplasmosis. However, the high prevalence of T. cati infection in stray cats can cause contamination of the environment by excreting eggs that may lead to infecting humans through soil or water. Therefore, public health education in urban management planning is necessary for routine urban cat deworming programs and for training the healthcare workers to prevent, control, and treat these infections.

Dataset Information

RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect Toxoplasma gondii in stray dogs and cats in Zhejiang province, China.

Importance

Publications

RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect <i>Toxoplasma gondii</i> in stray dogs and cats in Zhejiang province, China.

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