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Canine distemper outbreak and laryngeal paralysis in captive tigers (Panthera tigris)

BMC Veterinary Research volume 21, Article number: 33 (2025) Cite this article

Abstract

The canine distemper virus (CDV) could infect various wildlife species worldwide. The viral infection in large felids directly impacts wildlife conservation. This study aimed to understand better the burden of CDV outbreaks in captive tiger populations in Thailand and a novel discovery of their clinical signs with a history of CDV exposure. We followed up on their infection from May 2016 to October 2020 with laboratory testing and veterinary medical records. The cumulative morbidity and mortality rates were relatively high. Moreover, 50% of the tigers survived at 2 years after infection. All suspected and confirmed cases of CDV infections were significantly associated with laryngeal inflammation, which developed into paralysis in almost 50% of cases. Altogether, 50% of all tiger cases with chronic infection developed stridor at 314 days after virus infection [95% CI: 302–320]. Therefore, laryngeal paralysis may result from CDV infection and degeneration, potentially affecting the peripheral and central nervous systems. This condition could pose a life-threatening risk to tigers. The virus could spread quickly by contact with bodily excretion among tigers and fomite contamination once it affects a specific population. Implementation of biosecurity measures and vaccination is essential to mitigate the risk of disease spread and infection rates in tiger populations.

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Introduction

Canine distemper virus (CDV), an enveloped single-strand RNA virus, is classified to the genus Morbillivirus, which is part of the subfamily Orthoparamyxovirinae within the family Paramyxoviridae [1]. The virus causes a life-threatening disease that initially infected domestic dogs (Canis familiaris) [2, 3]. However, it can wildly infect numerous wildlife carnivores, including felids, hyaenids, procyonids, ailurids, ursids, mustelids, viverrids, and marine mammals [4,5,6]. In felids, CDV infection was previously reported in many countries, including Russia, the United States, Japan, and China [6,7,8,9], and could affect Siberian tigers [2,3,4,5,6], Bengal tigers [10, 11], lions [7, 12, 13], and so on. Most evidence of morbidity and mortality during the outbreak in tigers was reported only by the number of affected tigers rather than as morbidity or mortality rates [6,7,8]. The spread of CDV among domestic dogs, tigers, and other wildlife is a significant concern. This transmission threatens tiger conservation, potentially leading to population decline and decreased genetic diversity [6]. Domestic dogs are the important reservoirs of CDV, especially in urban ecosystems, where the virus can spill over from domestic animals and lead to outbreaks in wild species. Clinical signs reported in domestic dogs include catarrhal and respiratory symptoms, gastrointestinal symptoms such as diarrhea and vomiting, neurological symptoms like lethargy, gait incoordination, ataxia, myoclonus, tremor, nystagmus, and myoclonus, as well as non-neurological signs like conjunctivitis, hyperkeratosis of digital cushions or nose [14, 15]. The clinical characteristics of infected dogs can be categorized into acute and chronic stages. The acute phase occurs shortly after infection, with viremia and widespread viral activity, leading to observed clinical signs in various systems. The virus can be found in all bodily secretions. The chronic stage follows and is marked by persistent neurological issues. Demyelinating leukoencephalitis is a significant characteristic of the chronic phase, which causes long-term brain damage and neurological deficits. Although the virus may be cleared from most tissues, it can persist in certain organs, such as the central nervous system and lymphoid tissue, leading to ongoing symptoms or lifelong neurological problems [16]. These symptoms are also observed in infected wild felids. CDV-infected felids may present with seizures, behavioral changes, and respiratory and gastrointestinal signs [7, 8].

In May 2016, systemic infections with respiratory and gastrointestinal symptoms were identified in captive tiger populations in two centers, Centers A and B in the western part of Thailand. Following this outbreak, the number of tigers showing signs of stridor increased consistently. Furthermore, there have been reports of sudden deaths among these tigers since 2017. A series of captive tiger deaths from those populations showed, particularly a large number of Siberian tigers (Panthera tigris altaica).

Laryngeal paralysis has been sporadically found in captive tigers in the last 10 years in Thailand, with services by the Faculty of Veterinary Science, Mahidol University. Both bilateral and unilateral laryngeal paralyzes occurred and required surgery [17]. Laryngeal paralysis cases in tigers increased continuously, particularly in those tiger populations as mentioned above, while the disease’s cause was still unclear. Further disease investigation and diagnosis were conducted until October 2020 (Additional File 1) because it was suspected that the aforementioned disease might lead to unexplained laryngeal inflammation and paralysis. Additionally, this study aimed to explore the impact of CDV outbreaks on captive tiger populations while unveiling a novel discovery concerning their clinical signs, which offers valuable insights into virus infection.

Materials and methods

The study presented our findings using secondary data obtained from disease investigations and laboratory results conducted by our team from the Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals (MoZWE). This ensured ethical practices and adherence to the ARRIVE guidelines for transparent animal research reporting, and the details are provided as follows.

Outbreak investigation

The center A and B are situated in rural areas near protected areas and local communities. Surrounding both centers are facilities for rescuing and rehabilitating various wild animals. The number of tigers in Centers A and B was 91 and 65 at the beginning time, respectively. At Centers A and B, 85 out of 91 and all 65 tigers were confiscated animals, respectively. The outbreaks of the two centers occurred after a large amount of animal translocation. All tiger species included the Malayan, Siberian, Indochinese, and unspecified tigers. Unspecified tigers refer to those that were not tested to identify their species. Additionally, the history of vaccination for those tigers was unclear.

The initial phase of an outbreak in the tiger population was observed by veterinarians at Center A. The tigers’ health history was extracted from the veterinary medical records, and observations from veterinarians and keepers. The veterinary medical records from May 2016 to October 2020 were retrospectively reviewed and analyzed. The tigers exhibited systemic clinical signs, including diarrhea, melena, and nasal discharge. Breathing difficulties and stridor were detected after the appearance of these systemic signs, with the first case being identified in June 2016. The consecutive deaths of tigers in that population began in early 2017. Additionally, there was a lack of clear information regarding vaccinations for these tiger populations.

Some clinical signs observed in this tiger population were not previously reported in other tiger populations. Furthermore, not all tigers were diagnosed in the laboratory due to limited resources such as financial constraints, veterinary specialists, etc. As a result, the case definition was established to identify additional cases within tiger populations, enabling early detection and effective disease control. A ‘suspected case’ included all generations of purebred/crossbred Indochinese, Malayan, Siberian, and unidentified-species tigers with the following clinical signs: serous nasal discharge, dyspnea, stridor, panting, diarrhea/melena, vomiting, weight loss, and neurological signs such as facial muscle atrophy, opisthotonos, seizure, and death without investigation during the consecutive death period in the tiger populations having CDV confirmed cases. Also, the animal directly cohabited with confirmed cases and was cared for by the same keepers and shared equipment. Subsequently, a ‘confirmed case’ was defined as a suspected case with CDV confirmed by Reverse transcription polymerase chain reaction (RT-PCR) and sequencing, and/or immunohistochemistry (IHC), and/or immunofluorescence assay (IFA), and/or viral isolation.

The active outbreak phase was observed in 2017, showing a peak in consecutive tiger deaths until the end of 2019. In 2020, the total number of tiger deaths gradually declined, marking the post-outbreak phase.

Sample collection

Fecal samples were collected from 101 tigers from the two centers. Ocular and nasal swabs were also conducted on restraint tigers. Whole blood and serum were collected from some sick animals for biochemical analysis. Urine samples on the concrete floor were also collected when the outbreak was identified. The environmental specimens in the cages and surrounding areas, such as water in the bathtub, were sampled. The water exhibited an orange-red coloration, and the presence of dark reddish skin hair from tigers was noted. Given the suspicion of bleeding or hematuria in the tigers, hemoglobin testing was subsequently performed. We also collected the feces from other animals residing on the premises, including two Asian black bear (Ursus thibetanus), seven pig-tailed macaques (Macaca nemestrin), a small Indian civet (Viverricula indica), three Indochinese leopards (Panthera pardus delacouri), one smooth-coated otter (Lutrogale perspicillata), one oriental small-clawed otter (Aonyx cinereus), one Leopard cat (Prionailurus bengalensis), one long-tailed macaque (Macaca fascicularis), one fox (Golden Jackal, Canis aureus), and one White Handed gibbon (Hylobates lar) lived near sick tigers in Center A.

Altogether, 88 dead tigers were necropsied on sites of the outbreak areas and at the Pathological Unit of the Faculty of Veterinary Science, Mahidol University. We then performed gross and microscopic examinations. The specimens obtained from internal organs were collected for histopathological testing. IHC, IFA, and real-time RT-PCR were also performed on those specimens.

Laboratory diagnosis

Histopathology, immunohistochemistry and immunofluorescence assay for CDV detection

All collected tissues were fixed in 10% neutral buffered formalin and embedded in paraffin wax. The tissue sections were stained with hematoxylin and eosin. For IHC and IFA, the paraffin sections of the tissue samples were applied to the primary antibody using a CDV (DV2-12) mouse monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA, sc-57660) raised against nucleoprotein of CDV origin. For IHC, the tissue section was incubated with an anti-mouse IgG secondary antibody. The tissue slides were then applied with diaminobenzidine and counterstained with hematoxylin. For IFA, the tissues were stained with the secondary antibody labeled with fluorescence [18]. The omitted primary antibody and tissue of the tiger infected with avian influenza were used as negative controls in both techniques.

Polymerase chain reaction and sequencing for pathogen detection

We conducted various polymerase chain reaction (PCR) tests to identify potential pathogens that may cause infections and co-infections in tiger populations, while also ruling out certain other diseases. The differential diagnoses included canine distemper virus, feline herpes virus, feline hemobartonellosis, canine adenovirus, rabies, Newcastle disease, and avian influenza. The methods for detecting these pathogens, with the exception of canine distemper virus (CDV), are detailed in Additional File 2.

RNA extraction

Total RNA was extracted from organ tissues, feces, and nasal and ocular swabs using the Total RNA Mini Kit (Geneaid) according to the manufacturer’s protocol. Approximately 25 mg of organ tissues and 100 mg of feces were used for the extraction. To enhance RNA yield, Carrier RNA (Qiagen) was added at the lysis step at a concentration of 5 µg per sample. For both tissue and feces samples, lysis involved a combination of mechanical homogenization using a micropestle to grind the tissue, followed by chemical lysis. The extraction process adhered to the column-based method outlined in the manufacturer’s protocol.

Real-time reverse transcription-polymerase chain reaction for canine distemper virus detection

The real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) is beneficial for detecting viral RNA in various specimen types, including tissues, feces, and nasal swabs. The primers and hydrolysis probe of real-time RT-PCR, targeting an 87-base pair (bp) region of the nucleoprotein gene, were described in a previous study [19]. The RNA extracted from the specimens was used for CDV detection through real-time RT-PCR. The primers CDV-F and CDV-R and hydrolysis probe (CDV-P) of real-time RT-PCR targeting an 87-base pair (bp) region of a nucleoprotein gene described in a previous study [19], were used in this study (Table 1).

Table 1 Sequences of the primers and probe of the real-time reverse transcription polymerase chain reaction for canine distemper virus detection
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Real-time RT-PCR was amplified using the SuperScript™ III OneStep RT-PCR System with Platinum® Taq DNA Polymerase kit (Invitrogen, US). The 20 µl reaction mixture contained 5 µl RNA sample, 10 µl 2X Reaction mix, 0.4 µl SuperScript™ III RT/Platinum® Taq Mix, 0.4 µl probe (10 µM), and 0.4 µl of each primer (20 µM). The thermal profile was performed with 50 °C for 30 min and 95 °C for 2 min, followed by 40 cycles with 10 s at 95 °C, 15 s at 48 °C, and 1 min at 60 °C. The threshold cycle (CT) data for each sample was assigned [19].

Sequencing for canine distemper virus detection

The partial nucleoprotein sequences of CDV were obtained from amplicons of semi-nested RT-PCR. The RT-PCR step was amplified using primers CDV-NP-238 F and CDV-NP-708R utilizing the QIAGEN® OneStep RT-PCR kit (QIAGEN, Germany) (Table 1). The mixture contained 1X QIAGEN OneStep RT-PCR reaction buffer, 400 µM of each dNTP, 0.5 µM of each primer, 1 µl QIAGEN OneStep RT-PCR Enzyme Mix, and 5 µl of the RNA template in a final volume of 25 µl. The RT-PCR condition comprised a reverse transcription step at 50 °C for 30 min and an initial denaturing step at 95 °C for 15 min, followed by 35 cycles at 95 °C for 15 s, 50 °C for 30 s, 72 °C for 1 min, and a final extension step of 72 °C for 10 min.

In the semi-nested RT-PCR step, the products of the RT-PCR step were amplified using primers CDV-NP-357 F and CDV-NP-708R. The PCR reaction was carried out using the i-Taq™ plus DNA Polymerase kit (iNtRON Biotechnology, Korea), 1X i-Taq™ plus PCR buffer, 3.5 mM MgCl2, 0.25 mM of each dNTP, 0.5 µM of each primer, 1.25 U of i-Taq™ DNA Polymerase, and 2 µl of RT-PCR product. Nuclease-free water was added to adjust the real-time RT-PCR reaction volume to a final volume of 25 µl. The PCR product sizes for the RT-PCR and semi-nested RT-PCR steps were 470 bp and 351 bp, respectively. The product sizes of the RT-PCR and semi-nested RT-PCR steps were 470 and 351 bp, respectively.

The hemagglutinin (H) gene sequences of CDV were obtained from amplicons generated through semi-nested RT-PCR. The initial RT-PCR step was performed using the primers CDV-HC-F and CDV-HC-R with the QIAGEN® OneStep RT-PCR kit (QIAGEN, Germany). For the semi-nested RT-PCR step, primers CDV-HF and CDV-HR (Table 1) were used with the i-Taq™ plus DNA Polymerase kit (iNtRON Biotechnology, Korea). The PCR conditions followed the protocol outlined in Table 1.

The fragments of the semi-nested RT-PCR step were purified using the QIAquick Gel Extraction kit (QIAGEN, Germany) and sent to a DNA sequencing laboratory for nucleotide sequencing.

The nucleotide sequences were used to construct a phylogenetic tree employing the neighbor-joining (NJ) method [18] with bootstrapping (1000 replicates) using MEGA 11 software [21]. Before constructing the phylogenetic tree, MUSCLE alignment was performed within the MEGA software to compare the nucleotide sequence from this study with other canine distemper virus sequences in the GenBank database. A study investigated the relationships between CDV isolates across different species and countries. The analysis was performed on CDV’s hemagglutinin (H) and nucleoprotein (N) gene sequences. An outgroup for the tree was established using a phocine distemper virus.

Fecal examination

Fresh fecal samples were collected from tigers, and fecal smear, flotation, and sedimentation were performed to detect intestinal parasites. Fecal swabs were collected in Cary Blair Transport Medium to preserve them to isolate Clostridium perfringens [22] (Additional File 2).

Blood profile and urinalysis

The blood profiles of 11 out of 156 tigers with suspected laryngitis and laryngeal paralysis were rechecked. The complete blood count, along with the levels of alanine transferase, alkaline phosphatase (ALP), blood urea nitrogen, creatinine, and creatine kinase, was measured. A total of 5–10 ml of urine samples for urinalysis were collected from the concrete floor to avoid the use of invasive techniques that would necessitate anesthesia.

Environmental sample testing

We investigated the other animals sharing the tiger habitat. We monitored them and examined the feces for CDV infection by real-time RT-PCR [19]. We collected 50 ml of water samples from the water basin inside the cages and tested the presence of hemoglobin by spectrophotometry. Moreover, the 50 ml of tap water samples for cage cleaning were examined as the control samples [23].

Statistical analysis

Descriptive, Fisher’s exact test, logistic regression, and survival analyses were applied to describe these situations. All analyses were performed using the programming language R version 4.3.2 (R Development Team, Vienna, Austria).

Results

Outbreak investigation

Table 2 displayed the demographic details of the sampled tigers, providing insight into the locations of Centers A and B.

Table 2 Tigers’ demographic data
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Based on our case definitions, we identified 101 suspected cases out of 156 tigers. Out of these, 26 cases were confirmed in the laboratory as CDV positive from a group of 113 tigers tested using methods such as RT-PCR, IFA, or IHC (Table 3).

Table 3 Number of tigers affected by canine distemper virus
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From May 2016 to October 2020, the overall cumulative morbidity, mortality, and case fatality rates from all suspected and confirmed cases were estimated in Table 4. All suspected and confirmed cases of tigers are adults. We divided them into two age groups using the median age range: (1) tiger with more than or equal to 7 years old, and (2) tiger with lower than 7 years old. There was evidence that older tigers are at a higher risk of illness and death compared to younger and adult tigers. Additionally, male tigers were significantly more likely to develop symptoms and have a higher risk of mortality than female tigers (Table 4).

Table 4 Impact of canine distemper virus on tigers demographics
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The completed history data was the first center (center A) that emerged from this outbreak. At the first center, the most common signs of all cases were serous nasal discharge [91.21% (83/91)], diarrhea [51.65% (47/91)], melena [24.17 (22/91)], and stridor [68.13% (62/91)]. The most common clinical signs were observed in all cases from the second center were diarrhea [43.07% (28/65)], melena [26.15% (17/65)], vomiting [7.69% (5/65)], and stridor [40.00% (26/65)]. Of the cases, 56.41% (88 out of 156) developed stridor after the systemic symptoms. Among this cohort, 32.95% (29 out of 88) were subsequently diagnosed with laryngeal paralysis. The tigers with laryngeal edema and vocal cord dysfunction were confirmed by surgery to supportive therapy or necropsy. Among the tigers, 1.92% (3/156) had seizures, and 15.38% (10/65) had opisthotonos and facial muscle atrophy. However, decerebrate rigidity and ataxia were observed, but formal records were lacking. The clinical manifestations of the tigers from the two centers are presented in Fig. 1.

Fig. 1
figure 1

The clinical manifestation of the tigers from two centers

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Additionally, 10 tigers with CDV infection presented with rough and dark reddish skin hair. The color of their bathtub water became orange-red, indicating the presence of hemoglobin based on the spectrophotometry results.

The majority of the cases in center A were identified between May and September 2016. The average incubation period could be > 1 month. Data collection on the clinical appearance of the CDV-infected animals was discontinued after the first two cases were detected in Center A on week 9 of 2016. It then was limited by the medical record that started recording most animals in week 22 of 2016 (Fig. 2).

Fig. 2
figure 2

Epidemic curve of detecting clinical signs of canine distemper virus in Center A

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Association between canine distemper virus infection and laryngeal paralysis

The stridor was exhibited a continuous increase in the number of affected animals. The presence of stridor sounds in all confirmed and suspected cases showed a statistically significant association with a p-value of less than 0.001. Conversely, the association between stridor sounds and only confirmed cases yielded a p-value of 0.17. Furthermore, the association between laryngeal paralysis and the combined total of confirmed and suspected cases demonstrated significance, with a p-value of 0.03. However, the association between laryngeal paralysis and the confirmed cases yielded an insignificant result, with a p-value of 0.25. Among 91 tigers in Center A, 50% of the confirmed cases, suspected cases, and all cases had stridor after developing an infection at 308 days [95% CI at 229 days as a minimum], 315 days [95% CI: 291–321 days], and 314 days [95% CI: 302–320 days], respectively (Fig. 3a and c). Moreover, 29 out of 62 tigers (46.77%) with stridor were eventually diagnosed with laryngeal paralysis when surgical treatment and showing severe respiratory distress with loud stridor sound.

In case of death was the end of tiger survival, the median survival time of the tigers was 841 days or 2.3 years [95% CI: 527–941 days] after detection of the CDV infection, whereas the median survival time of the tiger still alive until 2020 was 4.34 years [95% CI: 4.29–4.37 years] (Fig. 3d).

Fig. 3
figure 3

Disease progression characterized by stridor and laryngeal paralysis after developing a canine distemper virus infection in (a) confirmed cases, (b) suspected cases, (c) all cases, and (d) Survival time of tigers until death after developing a canine distemper virus infection

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Laboratory results

Out of 113 tigers tested, 26 were positive for the virus using all detection methods. Specifically, 22 out of 109 tigers were positive for CDV using real-time RT-PCR. All 16 tigers tested with the IFA test were also positive for CDV, and both tigers tested were positive by IHC. The types of positive specimens were described in Table 5 using different techniques. In total, positive specimens were detected by real-time RT-PCR, IFA, and IHC at 5.15% (13/252), 56.04% (56/92), and 69.23% (9/13), respectively (Table 5).

Table 5 Laboratory detection of canine distemper virus in different types of samples in tigers
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Macroscopic findings

The necropsy results showed less amount of serosanguineous exudates in the external snares, lung congestion with diffuse hemorrhage, frothy exudates in the tracheal and bronchial lumens, and necrosis on hepatic parenchyma and pancreas. Some tigers presented congestion, and hemorrhagic lesions throughout the brain, and meningitis. Kidneys showed swelling and necrosis in certain cases. The larynx showed edema with or without inflammation. The tonsils were also found to be hemorrhagic. Moreover, reddish skin hair was often detected in tigers with severe clinical signs. The mortality and pathology findings, classified by type of cases, are presented in Additional File 3.

Microscopic finding

The microscopic findings included unusual histological patterns in various organs. Severe hyperemia, edema, and hemorrhage in the lung parenchyma were also observed. The alveolar septae also showed hyperemia with accentuated syncytial and dysplastic cells. Additionally, eosinophilic intranuclear inclusion bodies were also detected in the pneumocytes. The liver biopsy specimens showed hepatic necrosis with sinusoidal congestion, whereas eosinophilic intranuclear inclusions were present in most necrotic hepatocytes. The pancreatic cells showed necrosis with eosinophilic intracellular inclusions in multifocal regions. The spleen and lymph nodes showed lymphoid depletion with hemorrhage in the subcapsular area.

Furthermore, renal tubular cells underwent hydropic degeneration, cell swelling, and necrosis. The epithelial cells covering the villi of the gastric mucosa displayed hyperemia and necrosis. The other organs had no significant lesions.

Immunohistochemistry and immunofluorescence assay

The CDV-positive organs detected by IHC or IFA included the brain, heart, lung, larynx, liver, kidney, gastrointestinal organ, spleen, and lymph node (Table 5; Fig. 4). The antibody detected the CDV antigen on both nuclear and cytoplasm of each organ. In the antigen detection test, a greenish appearance was observed from the IFA samples (Fig. 4). In the IFA method, the lymph node and larynx were the most common site of CDV detection, followed by the lung, gastrointestinal organs, liver, brain, kidney, and heart (Table 5; Fig. 4). The positive results were consistent with the IHC and pathologic findings. Many antigens were also detected in the hepatic cells (Fig. 4). The IFA and IHC methods detected positive results in 18 out of 28 tigers tested. However, 61 dead tigers were not diagnosed by IFA and IHC tests from tissue carcasses.

Fig. 4
figure 4

The CDV-infected organs stained by the immunofluorescence technique. (a) Tonsil. (b) Larynx. (c) Lung. (d) Brain. (e) Liver. The positive organs expressed CDV protein markers (green); DAPI staining (blue) was used to detect nuclei. (Leica DMi8 inverted fluorescence microscope and images captured with an attached Leica DFC7000 camera: 40×, and scale bar 20 μm)

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Real-time reverse transcription polymerase chain reaction

The presence of CDV was mainly confirmed by real-time RT-PCR testing and sequencing, as mentioned above. It’s important to note that fecal samples were tested only once using real-time RT-PCR. Those were collected in 2017 after the initial phase of outbreak (Table 5).

The amplification of the H gene was unsuccessful; therefore, the N gene of CDV, consisting of 331 base pairs, was utilized to construct the phylogenetic tree. Our results indicated a strong correlation between the Canine Distemper Virus (CDV) in each isolated and a higher occurrence of clusters within the same region or country. The CDV that was isolated from the tiger, with the accession number OR975333, is available in the GenBank database (https://www.ncbi.nlm.nih.gov/nucleotide) in this study. It had a close relationship with the tiger and CDV Asia-4 genotypes. These genotypes were previously identified in Thailand using the GenBank database. Also, the closest cluster includes the virus isolated from the Mongolian dog, CDV Asia-4 isolated from two dogs, and an Asian palm civet in Thailand (as presented in Fig. 5).

Fig. 5
figure 5

Phylogenetic tree for a partial nucleoprotein (N) gene of canine distemper virus. The CDV sequence from a tiger in this study (OR975333, marked by a black square) was compared with other CDV sequences from the GenBank database. The tree was constructed using a Neighbor-Joining method and a Tamura-3-parameter substitution model. Phocine distemper virus served as the outgroup. Bootstrap support values, representing the percentage of replicate trees in which the associated branches are clustered together, were derived from 1000 replications, with values ≥ 50% shown

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Blood profile and urinary analysis

Altogether, all tigers presented relatively low hematocrit levels and mean corpuscular volume but relatively high plasma protein; however, 36.36% (4/11) of the tigers had thrombocytopenia, 18.18% (2/11) had giant platelets, and 18.18% (2/11) had platelet clumping.

For the urinalysis, urine samples were collected from the concrete floor. Among the 15 tigers, the urinalysis revealed that 73.33% (11/15) of the tigers had proteinuria, 26.67% (4/15) had urinary tract infections, and 13.33% (2/15) had renal failure.

Fecal examination

Among the 56 fecal samples, Trichostrongylus eggs were found in one tiger, and Strongylus eggs were found in the other two animals. Also, Clostridium perfringens type A was isolated from all tested fecal samples (4/4).

Environmental finding

We found 0.258 mg/ml of hemoglobin in the water within the cage compared to the controlled water samples. The limit of detection should be < 0.15 mg/ml hemoglobin [23]. The animals sharing a habitat with the tigers were examined and monitored for CDV infection.

In Center A, CDV was found in the feces of two out of nine (22.22%) macaques and one out of two (50%) Asiatic black bears. Their clinical signs were not detected in those animals. However, our laboratory tests on their feces samples did not reveal any positive CDV results in other animals living near sick tigers, including one palm civet, three Indochinese leopards, one smooth-coated otter, one oriental small-clawed otter, one Leopard cat, one long-tailed macaque, one fox, and one gibbon. Moreover, none of the animals died during our outbreak investigation.

Disease control

After disease detection, antibiotics and supplements (e.g. multivitamins and immunomodulators) were administered to the studied population. All biosecurity on the sites were strict and restricted animal translocation from the other sites. Surgery was performed as supportive therapy for cases with laryngeal paralysis.

Discussion

CDV can affect various species. Its cumulative morbidity and mortality rates were relatively high, the same as some wildlife species [2]. In our study, male tigers presented higher infection and mortality rates than female tigers, contrasting with the findings of some studies involving other species that showed a higher infection rate in females [24]. The higher prevalence of CDV infection in male dogs compared to females has also been reported in previous studies [25] This difference may be related to variations in sex steroids. A decrease in progesterone concentration levels in the cerebellum has been recorded in cases of CDV infection [26]. Therefore, the relevance of CDV infection and sex was observed, as low levels of progesterone in males have been linked to the development and worsening of brain damage caused by CDV infection. Older tigers, aged 7 years or older, had higher infection and mortality rates than adult tigers under 7 years old (Table 4). This aligns with previous studies indicating that the virus can impact tigers of various ages [7, 8]. The clinical signs observed in the tigers were similar to those of domestic dogs and wildlife species with systemic infections and neurological symptoms [2, 5, 27,28,29,30]. However, stridor could develop in tigers and may serve as a typical sign that appeared after the onset of systemic symptoms (Fig. 3a and c). Therefore, tigers with laryngeal paralysis or stridor may have long-term CDV infection. Since the tigers could survive for > 2 years (Fig. 3d) and may present with only laryngeal paralysis, this infection may not be recognized. Although our study found no significant association between the presence of stridor or laryngeal paralysis and confirmed CDV cases, a significant association was observed when both confirmed and suspected cases were included. This discrepancy arose from challenges in diagnosing laryngeal paralysis due to financial limitations preventing population-wide diagnosis, restricted carcass quality hindering confirmatory diagnosis using IFA and IHC, and the risk of underreporting. Furthermore, all fecal samples were collected at the outset of a series of consecutive tiger deaths in 2017, rather than during the initial phase of the outbreak when evidence of gastrointestinal and respiratory signs would have been present. As a result, we may have missed some positive cases during the later stages of disease progression. These factors contributed to a small sample size for both laryngeal paralysis and confirmed cases, which may have caused the change in the p-value from significant results to non-significant results, as we observed [31]. Further data collection is necessary to validate this hypothesis.

Our study validated the effectiveness of real-time RT-PCR in detecting CDV RNA across various specimen types, including tissues (brain, gastrointestinal organs, liver) and feces. These findings was consistent with Saltik and Kale (2023), who emphasized the sensitivity and reliability of real-time RT-PCR targeting the nucleoprotein gene region for diagnosing CDV infections. Notably, rectal swabs and fecal samples were especially suitable for this diagnostic method [32]. Most of the virus was still detected in the feces by real-time RT-PCR and sequencing after the outbreak in several months, 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. 6).

Fig. 6
figure 6

Diagram of disease transmission and pathogenesis of CDV in tigers

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CDV was detected in many organs, including lymphoid tissue, lung, larynx, stomach, intestines, liver, kidney, brain, and heart (Table 5). The tigers who died due to CDV infection were reported intermittently after the initial outbreak. Chronic infection could lead to tissue damage, inflammation, and organ failure due to the immune system’s response. We then observed severe lesions in many organs of necropsy [33]. In systemic infections, viruses could initially infect internal organs and invade the nervous system later [34]. 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 might be divided into acute and chronic phases.

The tigers diagnosed with laryngeal paralysis received supportive treatment by surgery. It expressed both unilateral and bilateral paralysis of the larynx’s vocal cords. There was no evidence to support how it occurred. Because CDV can infect epithelial, lymphoid, and neurological cells, causing cell damage and inflammation. Furthermore, infected cells then travel to local draining lymph nodes, initiating primary viremia and spreading to secondary lymphoid organs, subsequently affecting the immune system [16]. Therefore, the infected epithelial cells lining the larynx may lead to inflammation and laryngitis. Weak immune systems and secondary bacterial infections can worsen symptoms, while stress and poor habitat conditions contribute to respiratory issues. Also, aging tigers may experience nerve decline, resulting in laryngeal paralysis.

Additionally, 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 [35, 36]. 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 [37, 38]. 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.

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, where the virus can infect the epithelial cells of the urinary system [5], as well as from kidney diseases, as shown in our diagnostic results. It 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 isolation of the canine distemper virus (CDV) was limited due to our study’s lack of suitable cell resources, such as SLAM or Vero cells [5, 6, 8, 39]. As a result, comprehensive genetic sequencing could not be performed. Moreover, a sequence analysis of the hemagglutinin (H) gene is needed to identify the different genotypes. However, the amplification of the H gene was unsuccessful in our study, which presents a limitation. It is possible that the virus in those tiger populations was genetically distinct. However, the CDV nucleoprotein (N) gene sequence by the PCR product could provide some information on genetic variation. The close relationship of CDV within the same cluster suggests the potential for interspecies transmission such as the CDV Asia-4 virus originating from dogs (Fig. 5). Also, it highlighted the capability of CDV to spread extensively among different animal species, particularly the transmission between domestic animals and wildlife. A prior investigation documented a fatal outbreak of CDV Asia-4 in caged wild-caught civets in Thailand. Also, utilizing evolutionary and synonymous codon usage bias analysis of the hemagglutinin (H) gene suggested that CDV likely originated and was adapted from dogs [40]. Moreover, it is important to investigate the connection between all the reported tiger populations in our study and another tiger site that detected the virus (Fig. 5) in Thailand to determine the mode of virus spread. In order to tackle the issue at hand and avoid any harm to the tiger population, we need to conduct extensive research to comprehend the transmission of the virus to the tigers.

Co-infections with C. perfringens could worsen clinical symptoms in animals already infected with canine distemper virus (CDV). C. perfringens is particularly associated with gastrointestinal complications, which may become more severe in such cases [40], as detected in our study. Several terrestrial carnivores and non-carnivores are susceptible to the CDV virus [4, 12, 34], and bears [41] and monkeys [27] can shed it through their feces. Domestic dogs [42, 43], and other wild animals, such as civet [44], raccoon dogs, and weasel [45], can be viral reservoirs in Asia, whereas the virus can cause fatality in red pandas [46]. These CDV strains were detected in the Asia-1, Asia-4, or Asia-6 lineages. Therefore, it is important for humans, animals, and vehicles to implement risk assessments to investigate and prevent the introduction of the virus to wildlife stations. 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. Separating the positive animals from the others is imperative to complement these efforts. Wildlife stations should enhance biosecurity by understanding the route of disease transmission to prevent virus contamination from the keepers, equipment, and environment to other susceptible tigers and animal reservoirs [47]. To effectively manage animal infections and mitigate viral spread among animals in captive wildlife stations, it’s essential to closely monitor records, implement systematic observations, and conduct laboratory diagnostics, such as sampling feces samples on the ground in their cage, which will enable the keepers to identify and separate sick animals from the population early [48]. Regularly cleansing cages with disinfectants and implementing enhanced biosecurity measures, such as separating keepers and equipment, are crucial [47]. Additionally, providing supportive treatment to aid in animal recovery is necessary. Considering effective vaccination for healthy animals living in the same habitat is also advisable [6, 10, 49, 50]. Furthermore, having a contingency plan in place will aid the station in effectively addressing any potential future outbreaks.

Our findings have significant implications for understanding the impact of CDV on tigers across various species. The observation of stridor sound and laryngeal paralysis in tigers within captive wildlife stations and zoos suggests potential long-term exposure or a chronic stage of CDV infection. Effective management strategies should prioritize preventing disease transmission to other tigers. Furthermore, this information contributes to the disease knowledge of the large felids and enhances surveillance efforts for non-captive wild tigers. It underscores the importance of studying disease transmission among wildlife species sharing the same habitat and the need for more comprehensive investigations into the pathogenic effects of this infection on the nervous system.

Data availability

The datasets generated and analyzed during the current study are available in the GenBank repository, [https://www.ncbi.nlm.nih.gov/nucleotide], with accession number OR975333.

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Acknowledgements

This work was successfully completed because of the support of many individuals and organizations. We are grateful to Banpot Maleehual and the official staff of the Department of National Park, Wildlife, and Plant Conservation for the help with data collection. We are also grateful to Asst. Prof. Dr. Tawewan Tansatit, Asst. Prof. Dr. Duangthip Chatchaisak, Asst. Prof. Phingphol Charoonrut, Asst. Prof. Dr. Namphung Suemanotham, and Dr. Tanit Kasantikul for providing for the expert assistance with result interpretation. Nevertheless, we are thankful to Asst. Prof. Dr. Boonrat Chantong, Dr. Ladawan Sariya, Mr. Parut Suksai, and Ms. Rassameepen Phonarknguen for the laboratory support.

Funding

This research did not receive any specific grant from funding agencies. We gathered relevant data from the routine operations of the Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals (MoZWE) at the Faculty of Veterinary Science, Mahidol University, and used it for our research.

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Authors and Affiliations

  1. Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Salaya, Nakhon Pathom, Thailand

    Sarin Suwanpakdee, Anuwat Wiratsudakul, Ruangrat Buddhirongawatr & Natharin Ngamwongsatit

  2. The Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals, Faculty of Veterinary Science, Mahidol University, Salaya, Nakhon Pathom, Thailand

    Sarin Suwanpakdee, Anuwat Wiratsudakul, Nattarun Chaisilp, Benjaporn Bhusri, Chalisa Mongkolpan, Ruangrat Buddhirongawatr, Jarupa Taowan, Peerawat Wongluechai, Witthawat Wiriyarat & Nareerat Sangkachai

  3. Department of National Parks, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Chatuchak, Bangkok, Thailand

    Luxsana Prasittichai, Anurux Skulpong & Patarapol Maneeorn

  4. Department of Pre-clinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, Salaya, Nakhon Pathom, Thailand

    Nlin Arya, Parin Suwannaprapha & Witthawat Wiriyarat

  5. Laboratory of Bacteria, Veterinary Diagnostic Center, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, 73170, Thailand

    Natharin Ngamwongsatit

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Contributions

SS, AW, and NS conceived and designed the study. SS, AW, NS, LP, AS, RB, and PW investigated and collected the data. NC, BB, CM, JT, NA, PS, NN, and WW performed laboratory investigations. PM, LP, and SS collaborated between organizations. WW contributed reagents and materials. SS, AW, and NS analyzed the data and wrote the paper. All authors reviewed the manuscript.

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Correspondence to Nareerat Sangkachai.

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Ethics approval and consent to participate

During our routine investigation of Zoonotic Diseases in Wildlife and Exotic Animals at the Monitoring and Surveillance Center, Mahidol University, live animals were examined. The study protocol was approved by the Committee on Animal Care and Use, Faculty of Veterinary Science, Mahidol University (FVS-MU-IACUC) under COA No. MUVS-2021-05-23, designating it as an exemption protocol for utilizing information gathered during our organization’s routine investigation for data analysis. Notably, the FVS-MU-IACUC did not need to consider the routine practices of any organization. Our team adhered to the protocols outlined in the AZA Tiger Species Survival Plan, 2016 (https://assets.speakcdn.com/assets/2332/tiger_care_manual_2016.pdf), and the WOAH Terrestrial Manual 2008 (https://www.woah.org/fileadmin/Home/eng/Animal_Health_in_the_World/docs/pdf/1.1.01_COLLECTION.pdf) while investigating disease outbreaks and collecting samples. All methods employed in this study are reported in accordance with the ARRIVE guidelines.

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Suwanpakdee, S., Wiratsudakul, A., Chaisilp, N. et al. Canine distemper outbreak and laryngeal paralysis in captive tigers (Panthera tigris). BMC Vet Res 21, 33 (2025). https://doi.org/10.1186/s12917-025-04490-9

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