The deposition characteristics of Schistosoma japonicum eggs and its application in hatching method

Schistosomiasis is an important zoonotic parasitic disease, which a major public in china. However, the detection of schistosomiasis in the eld is still based on the traditional faecal hatching method, which is tedious and time-consuming. Therefore, method for detecting schistosomiasis in the eld needs to be improved. New Zealand rabbits articially infected with S. japonicum cercariae were used as animal models to study the deposition characteristics of Schistosoma japonicum eggs. The distributions of eggs in the intestinal wall at 42 d and 60 d post-infection were compared. The distributions of eggs in rabbit faecal samples were also observed. Goat faeces were used to compare the conventional faecal hatching method and the simplied direct immersion faecal hatching method.


Results
The distribution of eggs in the intestinal wall in the animal model at 42 d post-infection was as follows: the number of eggs per gram (EPG) was the highest (42780.13 ± 4789.81 eggs/g) in the rectum. The caecum had the largest proportion (42.97%) of eggs deposited. At 60 d post-infection, the rectum still had the highest EPG (117868.20 ± 67232.80 eggs/g). However, instead of the caecum, the lower colon had the largest proportion (64.90%). Moreover, 42.20% of eggs occupied the periphery of rabbit faeces. In the comparison between the conventional faecal hatching method and the simpli ed direct immersion faecal hatching method, the direct faecal hatching method was simpler, and the results were similar to those of the conventional faecal hatching method.

Conclusion
The deposition characteristics of eggs and their distributions in faecal samples suggest that the direct faecal hatching method can be used to simplify routine faecal hatching detection.

Background
Schistosomiasis is an important zoonotic parasitic disease, and transmission has been reported in 78 countries [1][2][3]. More than 40 animals can be naturally infected with Schistosoma japonicum [4]. Livestock, the main reservoir host and source of infection of schistosomiasis japonicum, has been a key schistosomiasis control points in China [5].
In schistosomiasis-endemic areas, buffaloes and goats are the main livestock. Studies in the early period of schistosomiasis control in China indicated that buffaloes and goats were the most important reservoirs of disease transmission [6][7][8][9]. In recent years, goat breeding has expanded, though the rearing method still comprises traditional breeding of scattered populations. As goats are schistosomiasis infection sources, the prevalence rate and number of positive cases in goats have shown increasing trends. An improved detection method will facilitate rapid disease diagnosis and play an important role in schistosomiasis control [10,11]. However, current techniques for the detection of the disease have limitations.
The detection of schistosomiasis in the eld is still based on the traditional faecal hatching method [12][13][14]. Goat faeces have typical granular morphological characteristics. The conventional faecal hatching method requires faecal samples to be soft so that miracidia can emerge. Therefore, faecal samples are soaked until they are soft, and external force is subsequently applied to stir and mix the samples. After sieving, water is added to remove any faecal residue. This method is tedious and time-consuming. Therefore, method for detecting schistosomiasis in the eld needs to be improved.
In this study, New Zealand rabbits, which produce faeces with morphological characteristics similar to those of goat faeces, were used as an animal model to investigate the distribution of S. japonicum eggs in the intestinal wall and faecal samples. A comparative experiment was conducted to elucidate the difference between the results of the direct faecal hatching method and the conventional hatching method. Revealing the distribution of schistosomiasis eggs in faeces will provide guidance for optimizing the hatching method.

Materials And Methods
Host animals, parasites and samples Six healthy male New Zealand rabbits, each weighing approximately 4 kg, were purchased from Shanghai Slake Experimental Animals. S. japonicum-infected snails were kept in the laboratory. Goat faeces were collected from 6 goat breeds in endemic areas that were positive for S. japonicum according to serology.

Animal infections
Six rabbits were xed on a board, and their abdominal hair was shaved. The cercariae of S. japonicum were counted under an optical microscope. Each rabbit was infected with 1000 cercariae. Rabbits were divided into two groups: A and B. Group A was sacri ced and dissected after 42 days, and group B was sacri ced and dissected after 60 days.

Sampling
Faecal collection: Rabbit faeces were collected from the rectal segment and stored at 4℃ for subsequent analysis.
Intestinal wall samples: The whole intestine of rabbits was harvested and the external tissues, such as blood vessels and the mesangium, attached to the intestinal wall were separated and removed. According to intestinal function, the small intestine (including the duodenal jejunum and ileum) was divided into the caecum, colon and rectum. The small intestine was subdivided into upper, middle and lower segments. The colon was equally divided into upper and lower sections. The above samples were dissected; the intestinal contents were removed and sieved, and all the intestinal mucosal tissues were scraped with glass slides. The processed samples were placed in 50 ml centrifuge tubes for weighing, and normal saline was added for homogenization.

Intestinal tissue egg counts
The above 800 µl homogenate was digested with 10% NaOH at 37℃ for 2 h, and 100 µl was subsequently removed to count eggs under an optical microscope. Three biological replicates were performed for each sample, and 3 replicates were counted for each sample. The egg density and total number of eggs from each segment of the intestine were calculated.

Frozen sectioning and observation of eggs in faecal samples
The faecal pellets were frozen and sliced into sections with a thickness of 40 µm. Dye containing malachite green was added, and the sample was covered with a cover glass. After incubation for 0.5-1 hour at 37℃, the distribution of eggs in the faecal samples was observed, recorded and photographed. Five hundred micrometres from the edge of the faecal pellet was de ned as the periphery of the faecal pellet, while the remaining area was de ned as the interior of the faecal pellet. The numbers of eggs in the faecal samples (periphery or interior faecal sphere) were counted, and the percentages were calculated.

Egg hatching assay
The collected faeces from schistosomiasis-positive goats were randomly assigned to four groups containing different goat faeces numbers (10, 20, 40 and 80). Four samples of faeces were incubated directly or mixed after being crushed. Four replicates were conducted for each group for each method.
The protocols are as follows.
Direct hatching method: A certain number of faecal pellets were placed directly into a 500 ml hatching ask, and dechlorinated water was added to the height of the bottleneck. Loose absorbent cotton was placed on the water surface in the bottle. Then, 10 ml of distilled water was added slowly. The hatching ask was transferred into an incubator and incubated at 30 °C for 6 hours. During this period, the liquid above the cotton was collected every 2 hours; this step was repeated three times. After sampling, dechlorinated water was supplemented to the previous volume and eggs were allowed to continue to hatch.
Mixed hatching method: A certain number of faecal pellets were placed in a beaker, and dechlorinated water was added. The samples were fully mixed and subsequently mashed into a homogenate. After ltering the homogenate with a 60-mesh copper sieve, water was added until no obvious residual coarse bre could be seen. The lter residue was resuspended in water and left for 20 min at room temperature for separation. The upper liquid phase was removed, and the residue was transferred into a 500 ml hatching ask. The remaining steps were the same as those in the direct hatching method.

Counts of S. japonicum miracidia
The collected liquid was combined according to the different hatching methods. Formaldehyde was added and the solution was left overnight to x the S. japonicum miracidia in the liquid. After the liquid was centrifuged at 4000 g for 10 min, the supernatant was discarded, and the precipitate was collected. The miracidia were sequentially stained with 1-2 drops of iodine. The total number of miracidia was counted under an optical microscope.
Statistical analysis SPSS 16.0 was used to analyse the experimental data. The arithmetic mean and standard deviation (`x ± s) of each indicator were calculated. Student's t test was employed to determine the statistical signi cance of the differences between the two hatching methods. According to the number of eggs per gram of tissue (EPG) in each intestinal segment, the proportions of eggs per intestinal segment were calculated (Fig. 2). Comparison of egg hatching methods in goat faecal samples collected from the eld Faecal samples from goats infected with S. japonicum were collected on-site to compare the hatching rate between the direct hatching method and mixed hatching method. With the increase in the number of in faecal samples, the number of miracidia detected by the two methods showed an increasing trend, and there was no signi cant difference between the two methods (P = 0.127) ( Table 1). Table 1 The two methods were compared for the hatching detection of goat faecal in the eld

Discussion
S. japonicum mainly parasitizes the mesenteric vein and portal vein of the host. Reproductive success was investigated approximately 24 days after the host was infected with cercariae. Schistosoma eggs are mainly deposited in the liver and intestines, with only a small amount deposited in the mesenteric lymph nodes, pulmonary system, spleen and central nervous system [15,16]. Aetiological diagnosis of S. japonicum in domestic animals is important in the diagnosis of schistosomiasis, and the faecal hatching method is the basic and common method for aetiological diagnosis [17,18]. Some faecal samples from domestic animals, especially herbivorous livestock such as rabbits and goats, have special characteristics. The small intestine of domestic animals exhibits peristaltic movement, namely, segmented movement, which is mainly accomplished by circular muscle relaxation and contraction. After the absorption of water by the colon and rectum, a spherical faecal sample is formed. In the eld of pathogen detection, there are differing opinions on the distribution of S. japonicum eggs in faeces [19][20][21][22][23]. Therefore, the challenge of how to analyse spherical faecal pellets has also become a controversial problem. The controversy revolves around whether it is necessary to crush the faecal pellet and incubate it. Some people think that crushing the pellet is required because eggs need to fully contact the hatching water to successfully hatch; however, others do not recommend crushing goat faeces, as there are many disadvantages after soaking. First, it increases the operation time. Second, it causes some eggs to be lost or hatch before bottling. Third, it increases the in uence of faecal impurities in the hatching water, and fourth, it increases the turbidity of the hatching water, which affects the observation of miracidia.
The results showed that there was no signi cant difference in the hatching rate among 10 to 80 directincubated faecal pellets and the same amount of crushed faeces. However, the number of miracidia detected by the direct incubation method was higher than that detected by the conventional faecal hatching method.
An arti cially infected S. japonicum animal model were employed to investigate the possible causes. During the necropsy of rabbits at 60 d post-infection, we also found that injuries to the rectal wall were more serious than those to the small intestine wall. Erosion, ulceration, irregular haemorrhage in the rectum, paving stone-like changes in the rectum and typical yellowish-grey nodules of S. japonicum eggs were observed. The faecal pellet can also cause mechanical friction, which leads to bleeding and allows eggs to enter the intestinal cavity.
According to the investigation of the distribution of schistosome eggs in the intestines of experimental rabbits, it was found that the egg density in the large intestine, especially the rectum, was the highest and the pathological damage was the most severe. The eggs in each segment of the intestine enter the faeces via the effect of intestinal peristalsis on chyme and digestive juice mixing. However, the spherical shape of the faeces is formed in the large intestine, especially the rectum; therefore, the eggs in the blood vessels or nodules are unlikely to be transferred to the inside of the faecal pellet; rather, they remain on the periphery of the faecal pellet. Considering the substantial total number of eggs in the rectal segment, it is concluded that it is feasible to hatch eggs in the faeces directly in water.
To observe the distributions of eggs in the faecal pellets, we used the section technique. The results showed that the distribution of eggs in a faecal pellet was consistent with that the distribution of eggs in the intestinal wall. There was no signi cant difference between the number of eggs the periphery and interior of the faecal pellet. Section observation revealed that although the surface of the spherical faecal pellet was smooth and uniform, the interior of the faecal pellet was loose and porous. It is speculated that during the incubation process, water easily permeates the interior of the faecal pellet so that eggs inside the faecal pellet can hatch successfully and the miracidia can emerge into the water.
It was demonstrated that the direct hatching method can be used for aetiological detection of schistosomiasis in spherical faecal samples. This method reduces the number of operational steps, such as mixing and crushing the faecal samples, and avoids the loss of eggs in the operational process. Moreover, the contamination of hatching water by pigments or toxins released after crushing the faecal samples can also be avoided with this method; this improves the observation of miracidia and thus has a prominent effect on detection. Therefore, this method is suitable for the qualitative detection of goat schistosomiasis on-site.

Conclusions
The distribution of schistosomiasis eggs in faeces was elucidated, and the hatching method was optimized. The detection results of the direct faecal hatching method were basically consistent with those of the mixed hatching method (P=0.127). The simplicity of this method makes it suitable for on-site goat schistosomiasis detection.

Consent for publication
Not applicable Availability of data and material The datasets used or analysed in the current study are available from the corresponding author upon reasonable request.
Competing interests Infection of 60 d.