Establishment of a special pathogen free Chinese Wuzhishan Minipigs Colony

Animal use protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Guangdong Laboratory Animal Monitoring Institute in accordance with the Guide for the Care and Use of Laboratory Animals [11]. Animal work was conducted using institutional operating procedures and policies in a barrier facility with approval of an oversight by the Institutional Environmental Health and Safety Office.

Eight adult candidate sows were strictly selected with clear background and free of porcine reproductive and respiratory syndrome virus, swine fever virus, pseudorabies, brucella bacteria, and toxoplasma. After fertilization, pregnant sows were transferred to clean delivery room at day 100 of gestation, and close observation and care followed to be performed. At 111 ± 2 days of gestation, hysterectomy procedures were carried out as described previously [12]. Feeds and water were forbidden for at least 18 h before caesarean sections of pregnant sows. Obtained piglets were bred in positive isolators with high efficiency particulate air filter for 40-45d (Figure 1A, B). During this period, piglets were fed humanly with milk powder at first and additional feeds after two weeks. Then all survival piglets were transferred to barrier environment and fed with standard feeds only. In order to establish a closed SPF colony, strong piglets should be chosen from different sows to form original SPF colony. And the minipigs were used to reproduce for generations in the established original SPF colony when they were 6–8 months old.

Figure 1. The facility and habitats of SPF WZSP colony. A. researchers were operating the isolator; B. piglets were fed with milk power by the breastfeeding frames in the isolator; C. the corridor of the barrier system; D. SPF piglets in the barrier system.

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According to the CNS, barrier and isolation environment are suited to raise SPF laboratory animals [13]. Positive isolators were used to breed suckling piglets for reducing the risk of infection, and barrier environment to breed weaned pigs for gaining more space for activities (Figure 1). The parameters of positive isolators and barrier environment were listed in Table 1, which met the demands of the CNS. The synthetic milk powder was sterilized by gamma irradiation (dosage: 20 kGy), and the complete formula feed was sterilized by autoclave sterilization (121°C, 30 min). The drinking water was sterilized by autoclave (121°C, 30 min) in the isolation environment, and the acidified water (pH = 3.0) was used in the barrier environment. Other instruments and reagent were all sterilized by physical or chemical disinfection methods before entering the barrier or isolation environment.

The minipigs were weighed routinely at 1 week interval from 0 to 4 weeks old, 4 weeks interval from 1 to 6 months old, and 8 weeks interval from 6 to 12 months old. And the results of body weight (BW) were compared with each other among SPF F0, F1 and CV WZSP.

Reproductive performances were routinely recorded for 14 SPF sows and 45 CV sows at birth and at weaning. And the differences between SPF and CV sows were analyzed by student’s t-test.

About 2.0 mL blood samples were obtained via popliteal venipuncture for clinical hematology and blood biochemistry analysis after fasting for 12 h. Haematological indices were determined by automatic hematology analyzer (Sysmex XT-2000iv), and blood biochemistry indices were determined by automatic biochemical analyzer (Hitachi 7020).

The sera, dander and hair of animals were collected for pathogen detection. The sampled minipigs included 8 pregnant sows for cesarean, 9 SPF F0 minipigs (4 males and 5 females), 2–3 months of age, 10 SPF F0 minipigs (5 males and 5 females), 14–15 months of age, and 20 SPF F1 minipigs (10 males and 10 females), 2–3 months of age. The methods for pathogens detection are described in Table 2. All detections were performed by commercial kits according to manufacturer’s instructions. The kits of PRV-gE, PRV-Ab, and PRRSV-Ab were purchased from Beijing IDEXX Yuanheng Laboratories Co.,Ltd. PPV, HPS and JEV kits were purchased from Wuhan Keqian Animal Biological Products Co.,Ltd. CSFV-Ab and CSFV-Ag kits were purchased from Korea JBT INC. Toxoplasma and chlamydia kits were purchased from Lanzhou veterinary research institute of Chinese academy of agricultural sciences. PCV2-Ab kit, FMDV-Ab, Brucella and IV kits were purchased from Ringpu (Baoding) biological pharmaceutical Co.,Ltd., Beijing jinnuo baxter technology Co., Ltd., and China institute of veterinary drugs control and Beijing wantai biological pharmacy enterprise Co., Ltd respectively. TGEV, PEDV, TPm, MH and APP kits were from South China Agricultural University.

All numerical parameters were expressed as mean ± SD. The data were statistically assessed by Student’s t-test. Significance was judged at the p < 0.05 level.

Sixty-one piglets were obtained from 8 candidate sows after aseptic surgery and hysterectomy, 55 out of them survived after cardio pulmonary resuscitation, and 49 piglets were survival at weaning. During the artificial lactating period, a breastfeeding frame was designed to reduce manpower (Figure 1B). After weaning, piglets were fed with autoclaved feeds and acidified water which were determined to be sterile repeatedly. The original SPF colony was formed by 35 survival F0 piglets from 8 candidate sows. In the end, we gained 14 litters of SPF F1 minipigs by reproducing in the original SPF colony.

Growth curve of BW was shown in Figure 2. As a result, the dynamic changes of BW in SPF F0 minipigs were different from CV minipigs demonstrating a slower increase before weaning and a sharper increase after weaning (p < 0.05). However, SPF F1 minipigs showed similar increase to CV minipigs before weaning (p > 0.05), higher increase to CV minipigs (p < 0.05) and similar increase to SPF F0 minipigs (p > 0.05) after weaning.

Figure 2. Growth curve for BW of different ages. The BW of SPF F0 minipigs showed a slower increase before weaning and a sharper increase after weaning compared to that of CV minipigs (P < 0.05). The BW of SPF F1 minipigs showed similar increase before weaning and higher increase after weaning compared to that of CV minipigs.

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Reproductive performances recorded for 8 candidate sows, 14 SPF colony sows and 45 CV sows were shown in Table 3 and compared with each other. Reproductive behavior parameters of SPF WZSP, including estrus cycle, estrus duration, gestation period, were similar to CV WZSP (p > 0.05) [14]. The average survival rate of piglets at weaning of 8 candidate sows (80.3%) was lower than that of SPF F0 sows (82.2%) and CV sows (85.8%), but there were no statistical differences (p > 0.05). In a word, there were no significant differences in litter performances among the three kinds of sows analyzed by student’s t-test (p > 0.05).

Hematology and blood biochemistry indices play an important role in evaluation of minipigs’ homeostasis and disease diagnosis. Tables 4 and 5 present the background data of hematology and blood biochemistry for SPF WZSP and comparison of SPF WZSP with CV WZSP.

In 15 hematological parameters, HGB, HCT, MCH, MCHC, NEUT, MONO, and BASO showed significant differences between SPF and CV WZSP (p < 0.05). Five indices out of 12 blood biochemical parameters showed significant differences between SPF and CV WZSP (p < 0.05), which were ALT, ALP, ALB, GLB, and CREA.

Pathogen detection results of CV candidate sows and SPF WZSP were listed in Table 6. For 8 candidate sows, 3 kinds of pathogens were detected out with detection rate of 50% (4/8), 100% (8/8) and 100% (8/8) for HPS, IV and PPV. However, 18 kinds of detected pathogens were successfully forbidden in F0 piglets by hysterectomy procedures. The F1 piglets were keeping free of those pathogens by repeated detection, indicating a satisfy SPF minipigs colony and effective SPF control system.