CDV can affect various species. Its cumulative morbidity and mortality rates were relatively high, the same as some wildlife species 1. In our study, male tigers presented a higher infection rate than female tigers, contrasting with the findings of some studies involving other species that showed a higher infection rate in females 14,15. The clinical signs observed in the tigers were similar to those of domestic dogs and wildlife species with systemic infections and neurological symptoms 1,4,16,17. However, stridor could develop, and in tigers, laryngeal paralysis could be a typical sign that occurs after the development of systemic symptoms during the chronic phase (Fig. 3b). Therefore, the tigers with laryngeal paralysis or stridor may have had long-term CDV infection. Since the tigers could survive for > 2 years (Fig. 3a) and may present with only laryngeal paralysis, this infection may not be recognized.
Most of the virus was still detected in the feces by real-time RT-PCR and sequencing after the outbreak, indicating that the virus was still shed during the outbreak until the chronic phase. The main transmission routes could be contact with bodily excretions among tigers, such as feces and urine, and fomite contamination. Aerosol, direct contact, and food contamination were also possible disease transmission routes (Fig. 5).
CDV was detected in many organs, including lymphoid tissue, lung, larynx, stomach, intestines, liver, kidney, brain, and heart (Table 1). The tigers who died due to CDV infection were reported intermittently after the initial outbreak. IHC and IFA detected CDV in the lungs and larynx in the cases, which could have caused the death by respiratory failure. The gross lesions of many tigers were found in the swelling and inflammatory/non-inflammatory larynxes. Some tigers had atrophy of the larynx. Therefore, the disease stage may be divided into acute and chronic phases. The tigers diagnosed with laryngeal paralysis received supportive treatment by surgery. It expresses both unilateral and bilateral paralysis of the larynx's vocal cords. There is no evidence to support how it occurs. Our laboratory results indicated that CDV may infect both the peripheral and central nerves and cause laryngeal paralysis. The central nerve, the vagal nerve arising from a part of the brainstem, controls muscle movement of the larynx 18. Substantial evidence has shown that CDV infected the astrocytes that regulate blood flow and maintain extracellular homeostasis in the brain, which could lead to myelin loss 19,20. The degeneration of the myelin sheath will reflect the deterioration of the information transmission between neurons. The peripheral nerve, for example, at the larynx, could deteriorate consequently. Then, the infection may result in laryngeal paralysis.
Seizure episodes characterized by opisthotonos may indicate meningoencephalitis 21, whereas decerebrate rigidity may indicate the presence of midbrain lesions [30]. Ataxia also indicates the presence of cerebellar lesions 22. It is concordant with the lesion of the whole brain. As in previous studies, viruses could be through the blood-brain barrier to the brain via infected mononuclear cells, cerebrospinal fluid, and infection of endothelial cells 23. Viruses invade the nervous system after developing infections in the other internal organs, as observed in systemic infections 24. Therefore, the virus might not be detected by real-time RT-PCR easily in the chronic phase. The IFA and IHC may be used for viral detection in tissue specimens in cases in the chronic stage, as they can detect the remaining viral protein after virion release. Chronic infection can cause tissue damage from the immune system, resulting in inflammation and organ failure 25. We then observed severe lesions in many organs of necropsy. The abnormal blood profile may be affected by hemorrhage in some organs, such as the lungs, brains, kidneys, and so on. Hematuria may be due to urinary tract infections and may be characterized by hemoglobin in the bathtub water. Moreover, the animals' reddish skin hair may be dyed with hemoglobin in the bathtub water.
The present study has some limitations. The isolation of CDV was limited in our study. Moreover, the pathogen strain was not described, although the difference in genetic variation detected in large felids has been reported previously 5–7. The specimens were not fresh for the viral isolation, whereas the incubation period of the viral replication in the cell culture may need longer for the protocol. Other cell culture types, such as CD150 (SLAM) cells, Vero cells, and so on, should be considered 4,5,7,26. Moreover, a sequence analysis of the hemagglutinin (H) gene is needed to identify the different genotypes. Those will be a limitation of our study.
The presence of co-infections, such as those by Clostridium perfringens in species with CDV infections, may increase the severity of their clinical signs, particularly gastrointestinal lesions, as detected in our study. Various terrestrial carnivores and non-carnivores could be infected by the virus 4,12,27, and bears 28 and monkeys 17 were found to shed the virus via their feces. Although there was unclear evidence of the outbreak's origin in this animal population, contact with bodily excretions among tigers and fomite contamination was suspected as the cause of the spread of the pathogen among their population. A vaccination program was recommended for disease prevention in some countries 5,9,29,30. Moreover, the restriction of biosecurity became a high concern in captive wildlife in Thailand.