伪狂犬病,动物造模 z-bio 2021-11-05 460

　　Background: Pseudorabies (also known as "pseudorabies") is an acutely fatal disease caused by the pseudorabies virus, which belongs to the subfamily herpes disease of the virus genus and the subfamily alpha herpesvirus. Pigs are the main host of PRV, but many mammals are also susceptible to infection. It is believed that dogs (sheep dogs and companion dogs) can contract the virus by eating contaminated raw pork and offal. The clinical symptoms of canine rabies are different from those of porcine pseudorabies. Local itching of the skin occurs in the early stages of dogs, with an incubation period of 2-9 days. Most infected dogs die within 48 hours of the onset of symptoms. The clinical symptoms of dogs are similar, including facial skin itching, difficulty breathing, vomiting, hemorrhagic diarrhea, edema, ataxia, and muscle cramps. However, some dogs have no typical symptoms after death. There are few reports of histological changes in dogs infected with the PRV virus, and histopathological studies are limited to the heart and nervous system. However, the pathological characteristics of the canine PRV infection system are still not very clear. Regardless of the cause of the death or damage of cardiomyocytes, cardiac troponin-I (cTn-I) is a marker for cardiac detection. Within a few hours of injury, cTn-1 was released from the injured cardiomyocytes into the circulatory system, reached a peak within 2 days, and continued to increase as the injury continued. The elevation of CTn-I in serum can be continuously and effectively detected, monitored and quantified to assess myocardial damage.

　　Since the beginning of 2011, the PRV infection rate in pig farms in northern China has increased, including pigs that have been previously vaccinated against PRV. The epidemic has affected nine states and cities, including Beijing. During this porcine pseudorabies outbreak, the incidence of PRV infection in dogs also increased. A total of 13 rabies dogs were detected in our laboratory, including dogs and pet dogs from Beijing urban and rural farms. The pathological changes of these 13 dogs naturally infected with PRV were studied, and a new system was provided for the pathological characteristics of PRV. We also conducted an experimental study to infect dogs with PRV under laboratory conditions, isolated the BJ-YT strain, and investigated the cause of PRV infection in dogs. Results: The dogs spontaneously infected with PRV were confirmed by PCR, immunohistochemistry, virus isolation and rabbit vaccination test to confirm PRV infection. Dogs are itchy (13/13, 100%), shortness of breath and difficulty gradually worsen (11/13, 85%), excessive salivation (10/13, 77%), hematemesis (4/13, 31%)), Shivering (4/13, 31%) and vomiting (2/13, 15%). , Chest X-ray observation (4/4, 100%) showed no obvious abnormalities. 9/13 (69%) of the dogs ate raw pork or offal. all

　　Abnormalities are recorded in the autopsy report. Heart malformations (11/13, 85%) usually include epicardial hemorrhage (5/13, 38%, Figure 1A) and endocardial hemorrhage (9/13, 69%, Figure 1B). , Heart bleeding (3/13, 23%; Figure 1C), heart thrombosis (3/13, 23%). Localized pulmonary hemorrhage and/or congestion (11/13, 85%; Figure 1D) are most common in the respiratory system. In the dog's performance, foamy liquid appeared in the dog's lung airways. Thymus hemorrhage (6/9, 67%; Figure 1E) The pathology of the spleen is particularly uniform, filled with a large number of dark red to black, raised, soft, and bleeding areas of various sizes (9/13, 100%). ). ) Thymus hemorrhage (11/11, 100%; two dogs were excluded due to age without thymus; Fig. 1G) Renal hemorrhage was also observed (2/13, 15%). Other serious lesions include subcutaneous edema (2/13, 15%) and chest hemorrhage (1/13, 8%). Due to limited literature knowledge on dog PRV and limited previous experience, only partial histological microscopy can be obtained from 13 dogs. Brainstem disease is very important. Non-purulent encephalitis (8/8, 100%; Figure 1H), mild to severe perivascular cuffs, gliosis, and neurophagy were observed in the brainstem. The lesions of the brain and cerebellum are limited, with only mild hyperemia (1/5; 20%). Mild and severe bleeding and inflammation, eosinophilic interstitial exudate (3/6, 50%; Figure 1J) are the most obvious cardiac changes. Moderate to severe pulmonary edema, congestion, bleeding, and/or were also observed (3/3, 100%; Figure 1K). The pathological changes of the liver include mild to severe hyperemia (6/6, 100%) and local necrosis (local necrosis). 3/6, 50%). The thymus showed diffuse moderate to severe bleeding (6/6, 100%) and lymphatic failure (3/6, 50%). Incomplete contraction of the spleen was also observed (3/4, 75%). Lymph nodes showed depletion (4/4, 100%) and bleeding (2/4, 50%). Acute nephritis is a typical symptom, ranging from moderate to severe interstitial congestion (5/5, 100%) and accumulation of protein fluid in the renal tubules and capsules (3/5, 80%). Dogs infected with PRV under experimental conditions: Under experimental conditions, five dogs were vaccinated with PRV, and the incubation period was 87-93 hours. All infected dogs died within 31 hours of onset. All dogs showed depression, loss of appetite, itchy skin and vocalization. Severe local itching at the injection site continued until death, and all infected dogs had spontaneous scars on their skin. Urinary incontinence (3/5), dyspnea progressively worsened, late dyspnea (2/5) and excessive salivation (1/5). The control group did not find any abnormalities during the study period.

　　General pathology: In the experimental group, the fifth dog was euthanized under intravenous anesthesia on the 4th day after infection (DPI), and the other four died on the 5th day. .. After all dogs in the PRV infection group were sentenced to death, dogs in the control group were sentenced to death. It was observed that three-fifths of dogs infected with PRV saw changes in heart disease. Both dogs (NOS 1 and 2) had different degrees of epicardial and endocardial petechiae (Figure 2A) and petechiae (Figure 2B). Two dogs (NOS 1 and 3) showed mitral valve bleeding (Figure 2C). A dog bleeds from the mitral valve. Bleeding spots were observed in the lungs of two dogs (No. 1 and No. 2; Figure 2D). The infected dog showed diffuse crimson discoloration of the gastric mucosa. The duodenum (3/5, Figure 2E) and jejunum (3/5) showed local or diffuse mucosal redness. A dark red raised area was found in the spleen (4/5; Figure 2F). In the experimental group, the mandible had subcutaneous edema (1/5) and there was obvious ascites in the abdominal cavity. In the experimental group, there were no obvious macroscopic lesions in other organs or tissue samples. The control group had no obvious visible lesions.

　　Histopathological examination: PRV infection group, moderate to severe non-suppurative neuritis, multiple necrosis of stellate ganglion, gliosis and neurotrophic phenomena (5/5; Figure 3A), hemorrhage (4/5; Figure 3b) and eosinophilic inclusions (2/5; Figure 3c) are characteristic. Non-suppurative neuritis (1/5) was also observed in the peritoneal ganglia. Under an electron microscope, three dogs infected with the PRV virus (NOS 1, 2, 5) showed cardiomyopathy. Extensive myocardial hemorrhage and necrosis were observed in two dogs (Figure 3D). The cytoplasm of the affected cardiomyocytes swelled and deposited, and eosinophils were deeply stained. The necrotic cardiomyocytes lose their streaks and become fragmented. These 5 dogs did not have mild inflammation. The dog (No. 5) accumulated and exuded eosinophilic fibrous secretions in the myocardial interstitium (Figure 3E). No bleeding was seen. Four dogs infected with the PRV virus showed mild to moderate pulmonary congestion. An exudation of red blood cells and plasma proteins was observed in the lungs of a dog (No. 2; Figure 3f). The most common observations of pseudorabies virus infection in the canine gastrointestinal tract are mild to moderate congestion of the small intestine and mild lymph nodes in the ileum (3/5; Figure 3G) and colon (4/5). Exhausted. ). In the ileum (3/5), there is a small amount of bleeding in the lymph nodes of the cecum (2/5; Figure 3). Gastric mucosal necrosis was observed in dogs infected with pseudorabies virus. The spleens of dogs infected with PRV show moderate to severe incomplete contractions. The PRV intervention tissue is normal, but the soft tissue that is not fully contracted is full of blood, and the red pulp of the contracted spleen is bloodless. There was mild to moderate hemorrhage in the thymus (4/5; Figure 3I). Compared with the control group, lymphocytes were significantly reduced (4/5; Figure 3j). The mandibular lymph nodes showed mild bleeding (4/5), and the mesenteric lymph nodes showed lymphatic depletion (5/5; Figure 3K). The adrenal endothelium of dogs infected with PRV showed mild to moderate hemorrhage (fascial zone and reticular zone) (5/5; Figure 3). There were no histopathological abnormalities in other organs or tissue samples in the PRV infection group. The control group had no obvious lesions. Histochemistry: Immunohistochemical staining confirmed that neurons in the nervous system were infected with PRV. Brain stem (4/5; Fig. 4a), cervical spinal cord (2/5; Fig. 4b), stellate ganglion (5/5; Fig. 4c), abdominal ganglia (5/5; Fig. 4d) and PRV antigen The intestine was detected in the mesenteric ganglion (4/5; Figure 4E). No viral antigens were detected in other tissues or organs. CTn-I analysis: Investigate the potential relationship between heart disease and respiratory symptoms and death in dogs infected with PRV, and evaluate the serum cTn-I levels of experimental animals. Three dogs infected by PRV with myocardial injury (group II, 1, 2, and 5) showed elevated cTn-I, but the infected group (groups 3 and 4) and control group without myocardial injury; serum The concentration of cTn-I (Group C) showed only slight fluctuations (Figure 5).

　　Discussion: Under natural and experimental conditions, systemic bleeding in PRV-infected dogs is the most prominent pathological finding. However, under experimental conditions, bleeding in many tissues and organs of PRV-infected dogs is lower than that of bleeding in naturally infected dogs. This result indicates that hypovolemic shock caused by systemic hemorrhage plays an important role in the pathogenesis of canine rabies and the mortality of dogs caused by PRV infection. In dogs infected with PRV in nature or in experiments, common neurological diseases are rarely seen. The histology of the central nervous system (CNSS) of dogs naturally infected with PRV is limited to the brainstem. This is consistent with previous reports about PRV dogs and other unnatural hosts (such as cats and foxes). However, dogs infected with PRV under experimental conditions have no central nervous system disease. In contrast, significant non-suppurative neuritis and PRV antigen were observed under all experimental conditions. Similar findings have also been reported in mouse peripheral nervous system ganglia (PNS). Unfortunately, this study did not detect peripheral ganglia in dogs with natural PRV infection. Some researchers believe that the observed increase in saliva production may be due to trigeminal neuritis. Itching is caused by infection of the trigeminal nerve and dorsal root ganglion. Animals infected with wild-type virus show severe peripheral neuropathy and will not die from brain infections. According to previous studies, PRV virus can induce pathological changes in PNS, damage sympathetic nerve function, and even cause organ dysfunction and even death in infected dogs. Consistent with other reports, finding heart disease and conducting experiments in dogs infected with the PRV virus under natural conditions is an excellent result. It is worth noting that dogs infected with PRV have progressive shortness of breath, which is usually clinically observed as heart disease during autopsy. Therefore, the cause of shortness of breath is believed to be heart damage. Since cTn-1 is a specific indicator of myocardial injury, we measured the level of canine serum troponin IcTn-1. In experimental PRV-infected dogs, cTn-I levels increase when three-fifths of the survival rate is damaged by the myocardium. Statistical analysis of the level of cTn-I showed that in dogs infected with PRV, cTn-I was significantly higher than that of dogs without myocardial injury (P\u003c0.05). The results of these studies indicate that myocardial damage in dogs infected with PRV is a very important specific disease, and the cTn-I index is an excellent reference index for dogs suspected of being infected with PRV. Under natural and experimental conditions, valve thrombosis was observed in dogs infected with PRV. Cardiac thrombosis is usually caused by endothelial damage, so the thrombosis observed in this study is likely to be secondary to valve damage and systemic bleeding. Myocardial neuritis caused by the destruction of stellate ganglia and endothelium is likely to cause excessive sympathetic nerve stimulation, arrhythmia, and even sudden death. It is worth noting that lung macrophages have been identified as the target cells of porcine pseudorabies virus, and virus replication induces a large influx of phagocytes. Necrosis also occurs in the lung tissue of infected pigs. It can severely damage tissues and cause respiratory symptoms such as sneezing, coughing, runny nose, and difficulty breathing. In the current study, the rapid deterioration of shortness of breath and dyspnea often occurs in the later stages of natural wild virus infection in dogs. Severe heart disease was observed in both experimentally infected dogs. However, no viral antigens and necrosis were detected in the lung tissue. No PRV was found in the motor neurons in the centers of the spinal cord and medullary respiratory center of PRV-infected mice. Therefore, acute death caused by non-natural host viruses may be due to respiratory failure of the nervous system. We believe that excessive sympathetic nerve stimulation, neuritis and heart damage can cause body cavity, pulmonary edema and congestion. This may be caused by heart failure and caused heart asthma. Therefore, the dog’s respiratory symptoms are probably not due to primary lung injury or respiratory infections, but due to cardiogenic diseases. This may also explain why dogs with progressive asthma and dyspnea that are infected with PRV have no obvious lung lesions on chest X-rays. Incomplete contraction of the spleen was observed in all naturally and experimentally infected dogs. In this study, the thymus and lymph node lymphocytes of natural and experimentally infected canine PRV-infected foxes were not observed, but lymphoid organ proliferation was observed. The lack of proliferation of the lymphatic system can cause extensive damage. Early researchers believed that porcine infectious lymphocytes provided another way to spread the PRV virus. However, no viral antigens were detected in the lymphatic organs or tissues of these dogs. Hepatic necrosis was also observed in natural PRV-infected dogs, but no PRV-infected dogs appeared under the experimental conditions. This may be due to the fact that in dogs, foreign bodies are mainly removed by Kupffer cells in the liver, and Kupffer cells in the liver produce excessive cytokines.

　　Therefore, natural dogs infected with PRV tend to damage the liver, possibly because they are exposed to multiple pathogens throughout their lives. Natural PRV-infected dogs often show acute nephritis, while experimentally-infected dogs do not. The reason for this difference is unclear. Nephritis seems to be caused by the deposition of immune complexes. Since immune complex nephritis is usually associated with the loss of filtration selectivity, the protein in the renal tubules dilates the lumen. Another possible explanation is that PRV cross-reacts with antigen. In this study, adrenal bleeding was limited to the cortex, especially the reticular tissue, and ACTH stimulation caused bleeding in this area. This phenomenon has also been reported in mink research. The immunohistochemical staining method to detect viral antigens also showed that many sympathetic postganglionic neurons (stellate, abdominal cavity and posterior mesenteric ganglion) were infected. However, even if the adrenal medulla parenchymal cells change sympathetic neurons, in the adrenal gland

　　No viral antigens were detected. Why is the adrenal cortex area sensitive and easy to bleed? do not know yet. Dogs are believed to be in contact with viruses, pork or offal or infected pigs or pig carcasses. However, in the current study, there are 13 wild dogs infected with the poison that have no history of direct contact with pigs or pig carcasses. Although this virus can be spread in pigs, it can still be spread in the air. Not reported in dogs.

　　Therefore, the dog may have been exposed to contaminated debris and food and contracted PRV through the injured gastrointestinal tract. The injury caused by chewing hard objects can increase the dog's sensitivity to PRV.

　　Conclusion: Based on our research results, we have concluded that the virus alone or together causes systemic bleeding, PNS damage and/or heart damage, leading to the death of dogs infected with PRV. Cardiogenic pathology has led to the observation of respiratory symptoms in dogs infected with PRV.