Pinworm infection in laboratory mice in southern China

Abstract

The present investigation examined the prevalence and intensity of pinworm infection in laboratory mice from three laboratory animal centres in China's southern Guangdong Province between August 2008 and April 2009. A total of 301 laboratory mice representing BALB/c, Kunming (KM) and C57BL/6J breeds were examined and the worms were counted and identified according to existing keys and descriptions, and their specific identity was ascertained by polymerase chain reaction (PCR) amplification of the second internal transcribed spacer (ITS-2) of ribosomal DNA. While the BALB/c and C57BL/6J mice were not found to be infected with pinworms, postmortem examination of the KM mice revealed a prevalence of 46.7%. PCR amplification of ITS-2 rDNA and subsequent sequencing confirmed that the mice were infected with Syphacia obvelata. The results of the present investigation demonstrated that the prevalence of pinworm infection in laboratory mice was high in the Guangdong province, and integrated control strategies and measures should be implemented to prevent and control pinworm infection in laboratory mice.

Three pinworms of the family Oxyuridae, namely Syphacia obvelata, Syphacia muris and Aspiculuris tetraptera, are important parasites of laboratory mice, rats and hamsters. ¹ Several characteristics, including their simple life cycle, short development period and high resistibility of eggs in vitro, make pinworms infect laboratory mice, rats and hamsters with a high prevalence even in well-managed colonies. ^2–4

Although pinworms usually infect laboratory animals with no obvious clinical signs, they have deleterious effects on research. ^5–7 While there have been a few investigations of pinworm infections in laboratory animals internationally, ^8,9 there is little information about the prevalence of pinworm infection in laboratory animals in mainland China under the present management practices. ¹⁰ In order to provide a foundation for the improved control of pinworm infection in laboratory mice, the objective of the present investigation was to estimate the prevalence and the intensity of pinworm infection in laboratory mice in China's southern Guangdong province.

The present survey took place between August 2008 and April 2009. A total of 301 laboratory mice from three laboratory animal centres in Guangzhou, the Capital of Guangdong province were surveyed for infection with pinworms. Their breeds, gender and prevalence and intensity of pinworm infection are given in Table 1. These animals were treated humanely, according to the Animal Ethics Procedures and Guidelines of the People's Republic of China, and the study was approved by South China Agricultural University.

Table 1

Prevalence of pinworm infection in mice of different breeds and genders from three laboratory animal centres in China's southern Guangdong province

		Centre A				Centre B				Centre C
Breeds		Examined number	Infected number	Infection rate (%)	Infection intensity	Examined number	Infected number	Infection rate (%)	Infection intensity	Examined number	Infected number	Infection rate (%)	Infection intensity
KM mice	M	33	32	97.0	1–35	33	15	45.5	1–12	59	0	0	0
	F	63	40	63.5	1–64	24	12	50.0	1–13	–	–	–	–
BALB/c	M	–	–	–	–	–	–	–	–	–	–	–	–
	F	–	–	–	–	77	0	0	0	–	–	–	–
C57BL/6J	M	–	–	–	–	–	–	–	–	4	0	0	0
	F	–	–	–	–	–	–	–	–	8	0	0	0

‘–’ means no mice were examined, ‘M’ represents male mice and ‘F’ represents female mice

The small and large intestines of laboratory mice euthanized by cervical dislocation were separated, opened, washed and examined for the presence of pinworms. Worm counts were performed on all washings and the intensity of infection (the ranges of numbers of worms) are shown in Table 1. Nematode specimens were fixed in warm ethanol 70% (v/v) and stored in this percentage of ethanol containing 5% glycerol before being cleared in lactophenol and identified according to existing descriptions and keys. ¹¹ The mean prevalence for pinworms was calculated by dividing the number of infected laboratory mice by the total number of laboratory mice examined, and was expressed as a percentage.

In order to ascertain the specific identity of the collected pinworms, genomic DNA was extracted from one to two pinworms randomly selected from each infected mouse using the SDS/proteinase K treatment, column-purified (Wizard^® SV Genomic DNA Purification System, Promega, Madison, WI, USA) and eluted into 35 µL elution buffer according to the manufacturer's recommendations.

The partial second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) was then amplified from these pinworm DNA samples by species-specific polymerase chain reaction (PCR) assays for S. obvelata, S. muris and A. tetraptera, respectively, using primers and amplification conditions developed previously. ¹ One S. obvelata DNA sample confirmed by sequencing of the entire ITS rDNA was used as a positive control. ¹ Negative control (no-DNA control) was included in each amplification run, and in no case was amplicon detected in the no-DNA controls. Each amplicon (4 µL) was examined by agarose gel (1.5%) electrophoresis to validate amplification efficiency. Three representative PCR products were sequenced directly to confirm their specific identity.

Postmortem examination of the Kunming (KM) mice revealed a pinworm prevalence of 46.7%, and the intensities of infection (worm counts) were 1–64, whereas the BALB/c and C57BL/6J mice were not found to be infected with pinworms (Table 1). The collected pinworms were identified as Syphacia sp. based on their morphological features.

The specific identity of 171 randomly selected pinworms from examined mice was determined by specific PCR assays. ¹ While no PCR product was amplified from these pinworms by species-specific PCR assays for S. muris and A. tetraptera, a PCR product of approximately 350 bp was amplified from each of the examined pinworms by species-specific PCR assay for S. obvelata (Figure 1), which is consistent with the expected length of the specific PCR product for S. obvelata. ¹ Subsequent sequencing of three representative PCR products revealed that the amplified partial ITS-2 rDNA represented S. obvelata, with 100% identity.

Figure 1

Representative agarose gel showing an amplified partial ITS-2 fragment from individual pinworms collected from laboratory animal centres in Guangdong province, China. Lane 1 represents a Syphacia obvelata DNA sample. Lanes 2–15 represent representative pinworm samples. Lane 16 represents no-DNA control. M represents a DNA size marker (ordinate values in bp). ITS: internal transcribed spacer

In this survey, we examined a significantly greater number of KM mice, but examined only a limited number of BALB/c and C57BL/6J mice, because the KM mice are the most widely and commonly used laboratory mice in China, are much cheaper than the BALB/c and C57BL/6J mice and are readily available. The KM mouse was imported from Hoffkine Institute, India in 1944, and is an inbred strain, coming from the Swiss mice. The three mouse strains examined in the present investigation are not in contact in the three respective facilities.

The present investigation revealed that infection rates and intensities of KM mice with pinworms were different among different laboratory animal centres. KM mice from laboratory animal centre A had an overall infection rate of 75%, KM mice from laboratory animal centre B had an infection rate of 47.4%, whereas no examined KM mice from laboratory animal centre C were infected with S. obvelata. The difference in the prevalence may be related with the difference in susceptibility of different breeds of mice to pinworms, as well as differences in management practice of different laboratory animal centres.

The present investigation demonstrated that the prevalence and intensity of pinworm infection in laboratory KM mice in Guangdong province are high, and therefore, integrated control strategies and measures should be implemented to prevent and control pinworm infection in laboratory mice.