The small subunit ribosomal RNA (SSU rRNA) genetic analysis showed that there are at least 26 subtypes (STs) of Blastocystis sp. [6]. However, different STs exhibit significant distribution differences in different animal hosts. For example, 10 STs including ST1–9 and ST12 can infect humans, among which ST1–4 are more common; at least 11 STs such as ST1–5, ST7, ST8, ST10, ST13, ST15, and ST19 can infect nonhuman primates; other STs are most observed in nonhuman primates [7–10]. In addition, the distribution of Blastocystis sp. also demonstrates geographical differences. In the case of zoonotic STs, ST3 is the most common in Europe, followed by ST4 and ST1, while ST3 is the most common in other regions, followed by ST1 and ST2 [11–13].

So far, no report has been disclosed about human and animal infection by Blastocystis sp. in Northern China [14]. Golden monkey as one of the unique species in China, has been introduced by the zoos in various provinces because of its great ornamental value and popularity. Thus, golden monkeys were considered as the subjects in this study. Given the lack of data about the life history, transmission path and transmission mechanism of Blastocystis sp., this study adopted molecular biology method based on SSU rRNA gene to investigate the Blastocystis sp. infection rates of both the golden monkeys and cockroaches living in the same environment. The genetic diversity was analyzed to understand the transmission path of Blastocystis sp., which will lay the foundation for epidemiological analysis of Blastocystis sp.

Thirty-seven feces samples of golden monkeys were collected from Shijiazhuang Zoo in Hebei Province, and 116 cockroaches were also collected from the living area of the golden monkeys from July to October 2019. The feces samples were immediately transferred to the disposable sterile centrifuge tube after defecation and stored in refrigerator. After dissection, the cockroach intestines were taken out and stored in the refrigerator. This study complied with the guidelines of the Regulations for the Administration of Affairs Concerning Humans and Experimental Animals and was approved by the Biomedical Ethical Committee of Hebei Normal University, Hebei, China (ID: 2019SC14).

DNA from feces was purified using the Stool Genomic DNA Extraction Kit (Beijing Solarbio Science & Technology Co., Ltd, Solarbio, Beijing, China). Polymerase chain reaction (PCR) amplification was performed according to the identification method of Blastocystis sp. based on SSU rRNA gene reported by Scicluna et al. [15]. The forward primer BhRDr (5′-GAGCTTTTTAACTGCAACAACG-3′) and the reverse primer RD5 (5′-ATCTGGTTGATCCTGCCAGT-3′) were used in a standard PCR reaction. Electrophoresis of PCR products was conducted in Goldview-containing agar gel, and the results were observed directly by UV gel imaging system. The amplified products with expected DNA fragment size were purified according to the specification of DNA Purification Kit (Solarbio^®) and sent to Shanghai Bioengineering Co., Ltd for sequencing.

PCR results showed that 18 golden monkey feces samples were successfully amplified with the expected size of the gene fragments, which were identified as Blastocystis sp. by sequencing. Thus, the infection rate for golden monkeys was 48.7% (18/37). In recent years, the infection rate of nonhuman primates by Blastocystis sp. in China has been always high. In 2010, Lv et al. [16] reported the infection rate of Rhinopithecus roxellana was 66.7% in Shanxi Province, China. In 2017, Zhao et al. [10] claimed that the infection rates of Rhinopithecus roxellana, Cercopithecus neglectus, Macaca mulatta and Presbytis francoisi were 89.1%, 80.0%, 96.6%, and 100.0%, respectively, in Qinling areas in China. In fact, He et al. found that a variety of non-human primates were infected by Blastocystis sp. in Guangzhou, China, and the highest rate could reach 78.9% as early as 1990. Although the infection rate of 40.9% for golden monkeys investigated in this paper is much lower than the above reports, the probability that other primates are infected with this parasite cannot be ignored, let alone the impact of this parasite on the animal health. In addition, although there have been no reports about infection of golden monkeys by Blastocystis sp., the infection of non-human primates by the parasite have been widely reported. For example, the infection rate of Thai long-tailed macaques was 41.9% [17]; that of Ecuadorian howler monkeys was 60.0% [18]; those of Tanzanian chimpanzees, grivet monkeys and guereza monkeys were 71.4%, 84.7%, and 83.7%, respectively [19]; that of Nepalese macaques was 100.0% [20]; and the overall infection rate of Australian nonhuman primates was 37.9% [21]. Above results indicated that nonhuman primates are susceptible animals easily infected by Blastocystis sp., which is very common.

Cockroach, as a common indoor arthropod, can transmit a variety of pathogens, such as Blastocystis sp. However, just a few reports have illustrated the transmission of Blastocystis sp. by cockroaches. In the study, 96 cockroach samples were identified as Blastocystis sp., revealing that the infection rate was 82.8% (96/116) (Table 1). In 1993, Zaman et al. [22] first found Blastocystis sp. in cockroaches collected from a sewage pool in Singapore, which exhibited a high infection rate; and the existence of cysts in their isolates indicated that cockroaches may be the transmission media of Blastocystis sp. However, the infection rates in the following studies are significantly different from each other. Suresh et al. [23] reported a low infection rate (10.0%) of cockroach in residential areas in Malaysia, while Farah Haziqah et al. [24] revealed an infection rate of 40.4% in cockroaches living in drainage systems in Malaysia after nearly a decade. The results in this study showed that the infection rate of cockroaches in the living area for golden monkeys was as high as 82.8%, which is in sharp contrast to previous reports. The difference may be related to the host animals grown in different geographical environments.

The DNA sequences of the SSU rRNA genes were subjected to correction prior to BLAST alignment (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The reference sequences (MK416177.1, KU361313.1, LT222069.1, KP055707.1, MN338075.1, KM374613.1, MF072955.1, KY610164.1, MN736506.1, KX234596.1 MK861933.1 and Proteromonas lacertae) used were from National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) and Blastocystis sp. subtypes identified in the present study are indicated using conventional subtype designations. All seauences were aligned using MEGA 6.06 and edited manually (available on request). Bayesian analysis was performed using MrBayes 3.0. The analysis of 18 positive golden monkey samples showed that there were three genotypes, i.e. ST1, ST2, and ST3, accounting for 38.9% (7/18), 44.4% (8/18), and 16.7% (3/18), respectively. There was no dominant ST as shown in Fig. 1 and Table 1. But all the genotypes in 96 positive cockroach samples were ST2 (Table 1; Fig. 1). ST2 exists in both golden monkeys and cockroaches and their relationship were near with each other. Although there are few reports about cockroaches as the transmission media of Blastocystis sp., golden monkeys and cockroaches share the same Blastocystis sp. ST2, which is the dominant ST as transmitted by cockroaches and the possibility of mutual transmission between the 2 animals cannot be excluded. The large-area activity of cockroaches may increase the transmission rate of Blastocystis sp. to other animals.

This study demonstrated that Blastocystis sp. as a zoonotic intestinal parasitic protozoa, exhibits different ST distribution in different hosts. Nonhuman primates with the closest genetic relationship with humans, are more likely to transmit the Blastocystis sp. to humans. Especially when the humans visited the zoo, the possibility will be increased too much for close contact between humans and the nonhuman primates, which will undoubtedly bring adverse effect on the human health. From the latest reports, the common zoonotic STs are ST1–ST9 and ST12 [25]. And ST2 is also considered as one of the more popular Blastocystis sp. STs in China, the role of cockroaches in the transmission cannot be ignored.