Diagnosis Of A Case Of Porcine Hemagglutinating Encephalomyelitis

Porcine hemagglutinating encephalomyelitis( PHE) is a highly contagious disease in piglets caused by porcine hemagglutinating encephalomyelitis virus(PHEV), which mainly infects the central nervous system of piglets and it’s mortality rate varies from 20 % to 100 %, while the adult pigs could rarely be infected.According to the clinical manifestation, route of transmission, epidemiology features and histopathologic changes of PHE, the disease was divided into two categories, one was vomiting and wasting disease(VWD), which is characterized by vomiting and exhaustion. And the other one is encephalomyelitis. The former was the original type of PHE and it often breaks out acutely. Serological surveys revealed that PHEV infections in pigs occurred and prevalent in the worldwide such as China, Canada,Britain, Northern Ireland, Japan and the United States. In China, PHEV was first reported in 1986 in Beijing, then, several outbreaks were reported in some other provinces. Recently, PHEV infections were widespread in the northeast of China,serological surveys revealed a high positive rate of PHEV infections in Jilin and Liaoning provinces.In recent years, the incidence of PHE has been reported in many large-scale pig farms. In August 2015, the breeding sows began to farrow in the Beisihuanyuquan farm of Jilin province. No miscarriage, weak production or stillbirths in sows were observed. And the newborn piglets were very lively and no abnormalities. However,the piglets at about 10 days of age began to onset(individuals were 2 days of age)which appeared convulsion, posterior paresis, and ataxia and so on. Some piglets showed vomiting and diarrhea, and some weaned pigs even also were infected. Until the middle of August, a total of 128 piglets produced by 10 sows died(100%mortality). In the study, we performed the research from the pathology and etiology aspects respectively in order to make a definitive diagnosis.1 Pathological diagnosisThe gross pathological examinations were performed in sick and dead pigs.There are no obvious pathological changes were observed on visceral organs except edema and congestion of the cerebral meninges and contents accumulated in the gastrointestine. And then, the spleen, kidney, brain, spinal cord and other tissues were collected, which were used for preparation of the paraffin sections. The observation results of microscopy are as follows: Serious venous congestion of the hepatic portal area and granular degeneration in liver were observed. Splenic corpuscle lymphocyte cells showed loosing structure and the periarteriolar lymphoid sheath in white pulp got thickened. In addition, the nucleus of few lymphocytes occurred concentration.The neurons were surrounded by some glial cells to form satellitosis in the spinal gray.The brain showed neuronophagia, which was the typical feature of nonsuppurative encephalitis. In addition, a part of renal tubular epithelial cells got degeneration and necrosis.2 Etiology diagnosisRT-PCR were performed using PHEV specific primers to amplify the expected gene fragments which were extracted from some tissues and organs of the infected piglets by PHEV. In order to exclude the infections of other pathogens, we performed the differential diagnosis using the specific primers of PHEV, PRV, PEDV, PDCo V,Po RV and TGEV. The amplification results were negative of all the other viruses except for the PHEV. And the detection result of bacteriology was also negative. Then the tissue samples of PHEV infection were grinded and the supernatant was inoculated into the PK-15 cells to isolate the virus. The typical CPE such as cell rounding, integrated and cluster were observed after PHEV inoculation The typical PHEV particles with envelope were observed by negative staining electron microscope observation. Finally, animal regression experiment was performed by inoculated Balb/c mice at the age of 18-20 days with the virus isolate. At the same time, the negative and positive groups were respectively established. The results showed that the inoculated mice in the experiment group appeared the obvious neurological signs and died at the 7th days after PHEV inoculation. Neuronophagia was observed on the brain of mice inoculated with PHEV. In addition, the results of RT-PCR amplification of brain and spleen from the died mice were positive.According to the above results, the outbreak of the disease was confirmed due to PHEV infection. The M gene and N gene of the PHEV isolatewere respectively amplified and sequenced, and the phylogenetic trees based on the M gene and F gene were respectively constructed. The results showed that the PHEV isolate had high homology with PHEV-JT06 isolated from Jilin in 2014 and the PHEV-Canada isolated in 2002.The study not only provides an important technological method for the rapid diagnosis of PHEV, but also establishs an important material foundation for the development of PHEV vaccines. Furthermore, this study provides a theoretical basis for the development of effective prevention and control measures of PHEV.