Qianbei-Pockmarked goats used in this study were cared for as outlined in the Guide for the Care and Use of Animals in Agricultural Research and Teaching Consortium [8]. Sample collections in animals were approved by the Institute of Zoology, Chinese Academy of Sciences, Institutional Animal Care and Use Committee (Project A0066).

Between April and November 2017, detailed investigations on the epidemiology of Ruanguzheng disorder were carried out in affected pastures. The data collected included ascertaining the history, incidence, character and regularity of the disorder, the natural ecological condition, and the effects on local animal husbandry. Data on the ecological and environmental conditions, and on the effects of diseases were obtained from local records and annual reports provided by the local government. Clinical signs were recorded by direct observation while following herds on the pastures. Upon clinical examination, body temperatures, pulse rates, respiratory rates, lymph nodes, and conjunctivae were normal. Upon further inspection, the extremities were flexed due to the relaxation of joint ligaments and the animals had an abnormal posture. Additionally, hooves were dry and back and animals had difficulties walking and standing. The weight was carried primarily on the heels.

On August 1, 2017, ten herbage samples were collected from five affected pastures, and two samples from each pasture in an affected area in Xishui County, China. Ten herbage samples were also collected from healthy pastures in Zengyi County, China. To reduce soil contamination, forage samples were cut 1 cm above the ground [9]. At the same locations, twenty soil samples were taken from the surface layer (0 to 20 cm) using a 30 mm diameter cylindrical corer in affected and healthy areas. Each soil sample was composited by four soil cores collected at the site. Soil and herbage samples were dried at 60°C to 80°C for 48 h and passed through a 2 mm sieve.

On August 10, 2017, 20 Qianbei-Pockmarked goats with an average live weight of 21.65±1.31 kg, were selected for the study. Of the 20 goats, 10 goats aged 1 to 2 years showed symptoms of lameness and were picked from an affected area (multiple grazing animals in this area showed obvious clinical signs, including lameness, weakness, muscular relaxation, stiffness of the extremities, and abnormal curvatures in the long bones) in Xishui County of Guizhou Province, China. The other 10 goats, aged 1 to 2 years, were selected from healthy pastures in Zengyi County in Guizhou Province, where the disorder had not been reported. Clinical examination showed that all Qianbei-Pockmarked goats were in good health, and those animals were used as the control group.

Hair samples were taken from the neck of the Qianbei-Pockmarked goats, washed and degreased, and kept on silica gel in a desiccator until analysis [10]. Blood samples were obtained from the jugular vein using 1% sodium heparin as an anticoagulant, and stored at −4°C until analysis of mineral elements. Serum samples for biochemical analyses were separated by centrifugation (G: 10,000 to 15,000, time: 10 min, and plastic tube type: EF9976) and stored at −4°C in a vial [11].

After the experimental animal and the control animal were slaughtered, we conducted routine post-mortem pathological examinations by visually observing the tissues. Samples of hips, ribs and teeth were collected from animals to determine mineral elements. Humeri, femurs, and ribs were collected from a goat aged 1 year for pathological pictures, while radiographic images were obtained from the forelegs.

Serum electrophoretic studies of total protein, albumin, and globulin were performed using cellulose acetate [11]. Inorganic phosphorus (IP), lactate dehydrogenase (LDH), γ-glutamyl transferase, alkaline phosphatase (AKP), creatinine (CRT), and calcium (Ca) contents were determined using an automatic biochemical analyzer (OLYMPUS AU 640, Olympus Optical Co., Tokyo, Japan) [12]. Quality control serum was used to validate the blood biochemistry data (Shanghai Biochemical Co, China). All biochemical serum values were measured at 20°C [13].

Iron (Fe), copper (Cu), manganese, zinc, and Ca contents were determined using a Perkin-Elmer AAS5000 atomic absorption spectrophotometer (Perkin-Elmer, Norwalk, CT, USA) [12,14]. Molybdenum (Mo) contents were determined using flameless atomic absorption spectrophotometry (Perkin-Elmer 3030 graphite furnace with a Zeeman background correction) [6,11]. Fluorine was measured using ion chromatography (Metrohm MIC-7 Advanced, Herisau, Switzerland). Phosphorus was determined by spectrophotometry. The accuracy of the analytical values was verified by reference to certified values of elements in the National Bureau of Standards (bovine liver SRM 1577a) [6,15].

Twenty affected Qianbei-Pockmarked goats were selected from affected areas in Xishui County for a treatment experiment. Ten affected Qianbei-Pockmarked goats (two males, three lambs, and five females) were administered disodium hydrogen phosphate (Na₂HPO₄) orally at a dose of 60 g per Qianbei-Pockmarked goat and were allowed to graze in a fenced pasture. The treatment was repeated once per week from August to October 2017. The remaining affected Qianbei-Pockmarked goats grazed in the affected pasture without treatment. Clinical signs were recorded by direct observation of goat activities in the pasture.

Data were analyzed using the statistical package for the social sciences (SPSS, version 20.0, Inc., Chicago, IL, USA), and presented as the mean±standard error. Significant differences between groups were assessed using the Student’s t-test with least significant differences of 1% (p<0.01) or 5% (p<0.05) [7].

Disease mainly occurred in lambs and mature ewes throughout the year, with a peak incidence between July and October. Post-partum and pregnant ewes were most commonly affected by the disorder. Clinical symptoms were less obvious in mature males. In severe areas, 28.33% of Qianbei-Pockmarked goats were affected and mortality reached 47.14%. Aside from the symptoms described above, the long bones of affected Qianbei-Pockmarked goats were frequently broken without apparent stress. However, the respiratory rates, body temperatures, and heart rates of affected animals were healthy (Table 1).

Visual autopsy examinations showed that the gross bone lesions of affected mature ewes and lambs were similar to those in affected Qianbei-Pockmarked goats. Almost all bones, particularly the scapula, mandible, ilium, ribs, and hip bones, were affected. The affected bones were brittle, porous, light, susceptible to fracturing, and easily cut and sawn. The marrow cavity was enlarged and extended into the epiphysis, and the cortex was spongy, thin, and soft. Spontaneous fractures frequently occurred on the pelvises and ribs of affected Qianbei-Pockmarked goats.

Proximal humeri showed flattening of the humeral head and separation of articular cartilage from the collapsed subchondral bone (Figure 1). Segmental thickening of the physes, thickened metaphyseal trabeculae, and thickened cortices in proximal humeri were also observed (Figure 1). Distal femura showed segmental physeal thickening (Figure 2). Impaired provisional calcification of cartilage at sites of endochondral ossification led to the accumulation of hypertrophic chondrocytes, resulting in thickened and irregular growth plates with islands and tongues of chondrocytes extending into the metaphyses (Figure 3). The enlargement of joints with an apparent bowing of the long bones and a broadening of the epiphyses were also typical. Irregular ulcers were also observed on the surface of joints in affected Qianbei-Pockmarked goats (Figure 4). Lesions were most severe in the fastest-growing bones, including the radius, tibia, metacarpals, and metatarsals. Radiographic analyses revealed that widening of the physeal growth plates was the most archetypal change. Other abnormalities observed upon radiographic analysis included metaphyseal flaring, thinning of the cortex, poor mineralization of the skeleton, and pathological fractures (Figure 4). Similar changes occurred beneath the articular epiphyseal cartilage complexes in the expanding epiphyses of young goats. Other microscopic changes included the development of thick osteoid seams lining trabeculae and the disorganization or absence of the primary spongiosa. Hemorrhage and signs of trauma were observed in the metaphyses and primary spongiosa due to damage to the weakened trabeculae of poorly mineralized bone.

Serum levels of CRT, LDH, and AKP from affected Qianbei-Pockmarked goats were markedly higher than those in the healthy goats (p<0.01), while IP values were approximately half of those in the control group. The AKP levels of affected Qianbei-Pockmarked goats were approximately two times higher than that of healthy goats (Table 2). Similarly, the levels of serum α-globulin and β-globulin in affected Qianbei-Pockmarked goats were markedly higher than those of the control group (p<0.01). Serum levels of γ-globulin in affected Qianbei-Pockmarked goats were markedly lower than those in the control group (p<0.01) (Table 3). There were no differences in other biochemical values between healthy and affected Qianbei- Pockmarked goats.

Phosphorus contents of the herbage and soil in the affected pastures were markedly lower than those in the healthy pastures (p<0.01) (Table 4). Phosphorus contents in the herbage in healthy pastures were 5.56 times higher than those in the affected area. The Ca:P ratio in herbage of the affected pastures was approximately 13:1. Other mineral contents were within the healthy ranges. Phosphorus contents in wool and blood samples and in hips, ribs, and teeth from affected Qianbei-Pockmarked goats were approximately half of those in healthy animals (Tables 5, 6).

Affected Qianbei-Pockmarked goats treated with dibasic sodium phosphate (Na₂HPO₄) recovered gradually within 15 to 20 d. Appetites improved rapidly and the signs of lameness that were observed in most affected Qianbei-Pockmarked goats improved within 5 to 15 d following treatment. However, foreleg deformations recovered slowly and required prolonged treatments. Lambs and mature ewes were more vulnerable than treated and healthy male Qianbei-Pockmarked goats. Ten treated Qianbei-Pockmarked goats survived. Among the 10 untreated Qianbei-Pockmarked goats, two lambs and three mature females survived, while two females and three lambs eventually died.

The phosphorus contents in blood were significantly increased in the treatment group (p<0.01), and reached a healthy value at 10 day. The same result was not observed in the control group. No significant changes in the concentration of other elements were observed in the blood of the treatment group (Table 7).