The Investigation of Canine Distemper Virus in Different Diagnosis Materials of Dogs using Molecular and Pathological Methods, Northeastern Turkey

Canine distemper (CD) is a major viral infection commonly observed in domestic and wild carnivores (McCarthy et al., 2007). The infection is very common worldwide and has high transmission and mortality rates, especially in immunologically compromised populations. In 1906, Carré determined that the causative agent of this disease, known since the 16^th century, is a virus (Loots et al., 2017). Canine distemper virus (CDV), belonging to the Morbillivirus genus in the Paramyxoviridae family, has a very close genetic and antigenic relationship with other members of this genus (Mochizuki et al., 1999, von Messling et al., 2001). The viral genome has a total of 6 gene regions, namely, the nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H) and polymerase protein (L) gene regions (Kodi et al., 2021). The H protein is one of the two surface proteins of the CDV and allows the virus to attach to cellular receptors. Moreover, the H protein is the major determinant of viral tropism and cytopathogenicity. Based on phylogenetic analyses of the H gene, CDV strains cluster in 12 different lineages, namely, America 1, America 2, Asia 1, Asia 2, Europe/South America 1, Europe wildlife, South America 2 and 3, Arctic, Rockborn-like, Africa 1 and Africa 2 (Bhatt et al., 2019). The N protein, found in both the virion and infected cell, is involved in shaping the viral genome and protecting the nucleic acid. Furthermore, the N protein is involved in viral replication and transcription (Lee et al., 2010). The target of the CDV is ymphoid tissue and mucous membrane cells throughout the body. Due to viral affinity, some respiratory, digestive and central nervous system cases develop an acute, subacute or chronic infection accompanied by skin rashes and hyperkeratosis (Mochizuki et al., 1999, Deem et al., 2000).
       
Even if clinical findings suggest CDV infection, a definitive diagnosis requires laboratory testing. A number of testing methods are used to diagnose CDV infection. The ELISA test is widely used in serological studies (Gençay et al., 2004, Kelly et al., 2005). Virus isolation (Maclachlan and Dubovi, 2011), immunohistochemical and immunofluorescence assays (Çomakli et al., 2020, Zhang et al., 2020), sandwich-dot enzyme-linked immunosorbent assay (dot-ELISA) (Li et al., 2013, Zhang et al., 2020) and in situ hybridization (Zurbriggen et al., 1993) have been used for the detection of the CDV antigen. However, most of these methods are laborious, time-consuming and not very productive. Today, the methods frequently used to diagnose CDV infection are the rapid test, RT-PCR and nucleotide sequencing (Maganga et al., 2018; Oguzoglu et al., 2018; Costa et al., 2019; Çomakli et al., 2020, Zhang et al., 2020, Kodi et al., 2021). Molecular methods can be performed only in equipped laboratories, but are highly sensitive and useful. Organ materials such as lymph nodes, the spleen, liver and lungs as well as oral and nasal swabs, serum, whole blood and CSF samples can be used in PCR tests to identify CDV nucleic acid (Frisk et al., 1999; Shin et al., 2004; Lan et al., 2006; Kim et al., 2006; Saito et al., 2006).
 
This study was aimed at investigating the presence of CDV nucleic acid in different clinical samples and organ materials, from naturally infected dogs, by RT-PCR and to molecularly characterize distemper strains according to the partial N gene sequence. Furthermore, tissue samples underwent histopathological examination for distemper infection.

Ethics statement
 
This research was conducted pursuant to the approval of the Animal Testing Local Ethics Council of Kafkas University (Approval Number: KAU HADYEK-2019-006).
 
Sampled animals and sample collection
 
The dogs sampled in this study were obtained from Kafkas University Veterinary Faculty, Clinics and Pathology Department between 2020 and 2021 in the Kars region of Turkey. In total, 60 dogs of varying age and breeds, showing signs of high fever, respiratory, digestive and/or nervous system infections, were sampled. A total of 202 materials (37 leuokocyte, 34 nasal and 31 rectal swab samples, 30 cerebellum, 18 lung, 18 kidney, 18 spleen, 11 CSF and 5 footpad) were collected from dogs with clinically suspected distemper. 9 out of 60 dogs were vaccinated and the other dogs were unvaccinated.
 
RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR)
 
RNA was extracted from the samples using a High Pure Viral RNA Kit (Roche, Mannheim, Germany) and complementary DNA (cDNA) synthesis was performed using a RevertAid first-strand cDNA synthesis kit (Thermo Fisher Scientific, USA), according to the manufacturers’instructions. RT-PCR was performed using the method and primer (PP-1) described by Frisk et al., (1999). The formation of PCR products of the expected size (287 bp) was analysed by DNA gel electrophoresis. The primer sequences used in this study are shown in Table 1.
 

 
Sequencing and phylogenetic analyses
 
Seven PCR products with clean and good PCR bands as a result of RT-PCR were subjected to sequence reaction via service procurement (DNA Lab, Ýstanbul, Turkey). The raw sequencing data were aligned using BioEdit version 7.0.5 (Hall, 1999) with the Clustal W algorithm. The phylogenetic map of the aligned sequences was established using MEGA version 6.0 (Tamura et al., 2013). Partial genome sequences of CDV, deposited in GenBank under the following accession numbers, were used: MH496779 (CDV6_TH/2014), KJ466106 (CDV_SY), EU716337 (164071), KJ123771 (17139113), AY386315 (5804), MF437053 (Gabon), MF041963 (CDV06), AF378705 (Ondersteport), GU138403 (SnyderHill/Vaccine), KJ747371 (13_1941), KJ747372 (13_2262), KX774415 (PT61/Pt/2004), KF914669 (CDV2784/2013), KY971532 (WT02SA), KY971528 (W01SA), MT149210 (CDV-CP8-TH/2017), MH496777 (CDV8_TH/2014), AB474397 (007Lm), AB475097 (M25CR), MH484613 (Kiki), KM280689 (Uy251) (Fig 1).
 

 
Histopathology
 
For histopathological examination, tissue samples were taken at systemic necropsies, fixed in 10% buffered formaldehyde solution, embedded in paraffin wax, sectioned at 5 μm and stained with Mayer’s haematoxylin-eosin (H&E). The sections were examined and photographed under a light microscope.

Clinical findings
 
In this study, 46 out of 60 suspicious animals were evaluated as positive by PCR.
       
The evaluation of the distribution of CDV infection by age, in 4 different age groups, revealed 23 cases (50%) in the 0-3 months age group, 12 cases (26%) in the 3-6 months age group, 10 cases (21.8%) in the 6-12 months age group and 1 case (2.2%) in the group over 12 months of age.
       
No reliable information could be obtained on the vaccination status of the vaccinated cases (completed or single dose), as either the animals were abandoned at the clinic or the animal owners were not informed.
       
Based on anamnesis and clinical findings, 4 cases (8.7%) had respiratory system infection and another 4 cases (8.7%) had nervous system infection alone. Some animals had developed multisystemic infections. Infections of the respiratory and digestive systems in 7 cases (15.2%), respiratory and nervous systems in 11 cases (24%), digestive and nervous systems in 11 cases (24%) and respiratory, digestive and nervous systems in 9 cases (19.6%) were found to coexist. Skin lesions characterized by rash along with systemic infection findings were detected in 5 cases (11%). In these cases, hyperkeratosis had developed on the footpad and tip of the nose.
 
RT-PCR
 
CDV nucleic acid was detected in 112 of 202 materials by RT-PCR with PP-I primer pairs. The 287 bp-DNA product of CDV was observed in all positive samples. According to RT-PCR results, positivity rates of 88.2% (30/34), 72.2% (13/18), 60% (3/5), 55.5% (10/18), 55.5% (10/18), 51.6% (16/31), 45.5% (5/11), 37.8% (14/37) and 36.7% (11/30) were detected in the nasal swab, lung, footpad, kidney, spleen, rectal swab, CSF, leuokocyte and cerebellum samples, respectively.
 
Sequencing and phylogenetic analyses
 
Seven samples yielded 287 bp-amplicons upon RT-PCR with NP gene specific primers. The amplicons were sequenced and their GenBank accession numbers are TR-KARS-CDV-1 (MW921485), TR-KARS-CDV-2 (MW921486), TR-KARS-CDV-3 (MW921487), TR-KARS-CDV-4 (MW921488), TR-KARS-CDV-5 (MW921489), TR-KARS-CDV-6 (MW921490) and TR-KARS-CDV-7 (MW921491). The strains obtained in this study were 97.7-100% similar to each other and 94.5-98.1% similar to the reference strains. According to the phylogenetic tree, the strains identified in this study showed a higher affinity to the European strains (MF437053 and AY386315), but were genetically distant to the vaccine strains (AF378705 and GU138403) (Fig 1).
 
Histopathology
 
Macroscopic results
 
Hyperkeratosis was detected on both sides of the nose and on footpads in Dogs 38, 43, 46, 52 and 53. The lungs were not collapsed, hyperemic or edematous. The spleen and liver were enlarged and haemorrhagic. Mild catarrhal enteritis was observed, especially in the ileum. While the serosal surface of the urinary bladder had a normal structure, hyperemic areas were detected on the mucosal surface.
 
Microscopic results
 
Generally, the cases with respiratory symptoms displayed interstitial pneumonia. Mononuclear cell infiltration existed in the bronchi and bronchioles and the interalveolar septum had thickened due to lymphocyte infiltration. The most conspicuous histological findings were eosinophilic cytoplasmic and nuclear inclusion bodies. Purulent bronchopneumonia was accompanied by interstitial pneumonia in Dogs 3, 14, 26 and 45 (Fig 2 a-c). Intranuclear inclusion bodies were observed in the epithelial cells of the renal pelvis. The urinary bladder was locally hyperemic and intranuclear and intracytoplasmic inclusion bodies were detected in the mucosal epithelial cells (Fig 3 a-b). While demyelinated areas were absent in the pons and medulla oblangata, necrosis and neuronophages were observed in the neurons. Significant demyelination was detected in the cerebellum, near the 4th ventricle and cerebral peduncle. Also, demyelinated areas were seen in different regions of the subtantia alba in the cerebellum. Intranuclear eosinophilic inclusion bodies existed in astrocytes localized to the demyelinated areas. Perivascular mononuclear cell infiltrations were detected in the cerebellum and brain stem (Fig 4 a-d).
 

 

 

       
RT-PCR, used to diagnose CDV infection, is a fast and reliable diagnostic method. This viral nucleic acid assay can be applied to samples of both living and dead animals, regardless of the form of the disease, immune response and viral antigen distribution (Frisk et al., 1999). RT-PCR is a fast and sensitive method that allows the detection of small amounts of viral RNA even in the advanced stages of the disease (Barrett, 1999). PCR’s increased effectiveness depends on many factors, such as the number of cycles, quality of the starting material, length of the target DNA and variability of the annealing and elongation temperatures. Using RT-PCR, Frisk et al., (1999) detected CDV RNA in 25 of 29 (86%) serum samples and 14 of 16 (88%) whole blood and CSF samples from 38 dogs with clinically suspected distemper. In the present study, materials including nasal swabs, lungs, footpads, kidneys, spleens, rectal swabs, CSF, leuokocytes and cerebella were used to diagnose CDV infection with RT-PCR. The materials that were used were of high diagnostic value. Positivity rates for nasal swabs, lungs, footpads, kidneys, spleens, rectal swabs, CSF, leuokocytes and cerebella were 88.2% (30/34), 72.2% (13/18), 60% (3/5), 55.5% (10/18), 55.5% (10/18), 51.6% (16/31), 45.5% (5/11), 37.8% (14/37) and 36.7% (11/30), respectively. Viral nucleic acid was detected at higher rates in the nasal swabs, compared to the other samples. The high positivity rates detected in the nasal swabs and the respiratory system infection findings observed were consistent with each other. Kim et al., (2006) evaluated the presence of viral nucleic acid in nasal and conjunctival swabs, leukocytes and urine samples with RT-PCR in the early stages of the disease in dogs experimentally infected with CDV. They reported that nucleic acid was detected 1-14 days after infection in the conjunctival swabs and 3-14 days after infection in the nasal swabs, at varying levels. Lan et al., (2005) determined that viral nucleic acid could be determined with RT-PCR in nasal swabs of dogs experimentally infected with CDV, up to the 28_th day post-infection. Kim et al., (2006) reported that the detection of viral nucleic acid in leukocyte samples was more difficult and less practical, because the virus is eliminated from the blood faster than from other tissues.
       
In agreement with the report of Murphy et al., (1999), CD infection was most common in animals aged 0-6 months in this study. Recent studies on cellular receptors used by the CDV suggest that especially the infection of young animals may be related to the virus-specific receptors in the immune system (Tatsuo et al., 2001).
       
The most common clinical findings were respiratory system infection accompanied by cough and nasal and ocular discharge. In parallel, a high positivity rate was detected in nasal swabs with RT-PCR. Anamnesis revealed that a short-term gastrointestinal infection developed approximately 15 days before acute clinical infection in 5 dogs (Dogs 3, 10, 20, 26 and 45). The development of infection during this period was attributed to the secondary viral proliferation stage following viremia (Bertus and Duprex, 2006). Acute infections were associated with nervous system findings such as nervous tics, abnormal gait and chewing movements of the jaw, all resulting from CNS infections. Studies have shown that the clinical findings and pathological lesions of CNS infection vary with the CDV strain (Summers et al., 1984).
       
Dogs 38, 43, 46, 52 and 53 showed hard pad disease, coinciding with the histopathological findings of CDV infection. Hard pad disease is a dermatological sign of CDV infection and is characterised by hyperkeratosis, a pathognomonic sign of the disease (Headley and Sukura, 2009). We detected CDV nucleic acid in the footpads of Dogs 38, 43 and 52. Although inclusion bodies were not identified within the epithelia of the footpads, CDV RNA was detected molecularly.
       
Macroscopic lesions were similar to those reported in previous studies. For example, lungs with foci of consolidation, as well as enlarged spleens and livers, have been reported in dogs (Viana et al., 2020, Headley et al., 2018). Microscopically, the lungs displayed interstitial pneumonia and cytoplasmic and intranuclear inclusion bodies were observed in the bronchiolar and alveolar cells. The pathological findings of the respiratory system were consistent with previous studies (Viana et al., 2020, Headley et al., 2018). Neurological signs are common in animals with CDV and were observed both clinically and pathologically in our study. Intranuclear eosinophilic inclusion bodies existed in the astrocytes localized to the demyelinated areas. Perivascular mononuclear cell infiltrations were detected in the cerebellum and brain stem. The microscopic findings of the nervous system agreed with the results of previous studies (Klemens et al., 2019, Çomaklý et al., 2020).
       
Ozkul et al., (2004) reported that the CD viruses circulating in Turkey were similar to the European strains. Similarly, the strains detected in this study were genetically related to viruses of the European lineage. Although there are not many CDV isolates deposited in the gene bank at the N gene level, phylogenetic analysis shows that the European strains form 2 clusters. While AY386315 and TR-KARS-CDV- 1, 2, 3, 4, 6 are located in the same cluster, MF437053 and TR-KARS-CDV- 5 and 7 are located in the other cluster. Therefore, even if studied in a small geographical region, differences detected between strains in the same region are important. This points out to the necessity for more detailed molecular epidemiological studies. The strains detected in this study were only 94.5-95.4% similar to the vaccine strains and were genetically quite different from the vaccine strains which located in America 1 lineage. In this study, CDV infection was also detected in vaccinated animals. The occurrence of CDV infection in the vaccinated animal may be due to many factors (inactivaton of the vaccine virus, not being vaccinated at the right time, insufficient vaccination, etc.), as well as the genetic difference of the vaccine strain can be considered.

RT-PCR is very sensitive in the diagnosis of CD infection. Necropsy samples are valuable diagnostic material and nasal swabs have the highest diagnostic value in the detection of CDV with RT-PCR. Turkish CDV strains are independent from vaccine viruses and have been found to be more closely related to the European lineage. CDV infection was also detected in vaccinated animals in present study. The occurrence of CDV infection in the vaccinated animal may be due to many factors (inactivaton of the vaccine virus, not being vaccinated at the right time, insufficient vaccination, etc.), as well as the genetic difference of the vaccine strain can be considered.

This project was supported by the Commission for Scientific Research Projects of Kafkas University (2019-TS-27).