Canine distemper virus (CDV) represents one of the main causes of a viral systemic pathology with lethal outcome in domestic and wild carnivores (mainly canids and mustelids), but also in numerous other wild species [1
]. The main factors that favor the spread of the virus are genetic variability, the broad host spectrum, and uncontrolled animal movements of stray and domestic dogs. Vaccination still remains the main measure for disease prevention today.
CDV, the etiological agent of Canine Distemper (CD), is a member of the genus Morbillivirus that belongs to the Paramyxoviridae
family; its genome is a single strand RNA, with negative polarity. Like other paramyxoviruses, the virus contains six structural proteins that are the nucleocapsid (N), matrix (M), fusion (F), hemagglutinin (H), polymerase (L) and phosphoprotein (P) [1
The circulating CDV strains are divided into different groups in relation to their geographical distribution and genetic characteristics, and, in particular, relative to the gene encoding for the H protein. Indeed, the study of the phylogenetic and molecular evolutionary analysis of the CDV revealed that the appearance of the disease in new species is correlated to mutations affecting the H protein-binding site for virus entry receptors [3
]. There are numerous reports of CDV in Italy, describing also the different viral circulating strains belonging to distinct genetic lineages [6
]. Some studies additionally report pathogenetic differences between the different strains responsible for CDV infections, such as for tissue tropism inducing an assortment of histopathological lesions [9
In Italy, the availability and administration of effective vaccines allow the control of CDV infections. However, outbreaks still occasionally occur, which are generally related to the illegal trade of infected dogs, especially from Eastern Europe [4
]. The last major CDV reported epidemic in Italy occurred in 2013 in Abruzzo, a region characterized by the presence of numerous natural parks with a significant animal biodiversity, and was caused by a strain belonging to the Arctic lineage (prototype CDV2784/2013), detected for the first time in the wild population in Europe [4
]. In this outbreak, CDV caused clinical disease in unvaccinated domestic dogs, Appennine wolves (Canis lupus
) and other wild carnivores present in Abruzzo, as well as in the neighboring regions, such as Molise. CDV circulation was also detected in the following years in wild animals of the same area [2
Since 2018, an increase in the presence of CDV in foxes (Vulpes vulpes) and badgers (Meles meles) found dead was detected through passive surveillance in Northern Italy (Region of Friuli Venezia Giulia).
In Italy, the circulation of strains Europe 1, Europe wildlife and Arctic [4
] has been reported. In this study, we investigated the circulation of CDV, and the characteristics of the circulating strains detected through passive surveillance in the regions of Latium and Tuscany, as these data are not available for these areas. The present study was conducted on wild and domestic animals submitted to Istituto Zooprofilattico Sperimentale del Lazio e della Toscana (IZSLT) for post-mortem examinations between 2011 and 2019, and tests were carried out within a research project funded by the Italian Ministry of Health.
2. Materials and Methods
2.1. Animals Examined
From March 2017 to October 2019, CDV susceptible wild species found dead or hunted, exotic dead animals of a Tuscany zoo and domestic dogs that died with a clinical history of neurological signs and other symptoms and/or lesions attributable to CD, were submitted to IZSLT for post-mortem examination and sampled for CDV diagnosis within a research project funded by the Ministry of Health; in this period, we received a complete set of biological samples [14
] consisting of brain and lung, conjunctival, nasal and rectal swabs but also intracardiac clot and urine or bladder swabs collected from a group of 122 subjects. To obtain a broader picture of the CDV circulating strains, we extended our investigation to 181 similar cases of dogs and wild species submitted to our Institute for post-mortem examination between 2011 and 2018, for which only some of the target specimens (lung and brain) were available stored at −20 °C, that were analysed by PCR for CDV.
2.2. Sample Collection and Stastistical Analysis of the Results
Within the research project funded by the Ministry of Health (March 2017–October 2019), a set of samples were collected when possible at necropsy from each subject: brain, lung, conjunctival, nasal and rectal swabs, urine (or swab from bladder) and intracardiac clot.
Portions of brain and lung were immediately fixed in 10% neutral buffered formalin for histological and immunohistochemical analysis. The rest of the samples were stored at −20 °C for CDV diagnosis and subsequent biomolecular analysis. The same organs and swabs were also examined for the presence of CDV vaccine strains [15
For the retrospective study, samples of brain and lung collected from 2011 to 2018, were submitted to biomolecular analysis; in case of PCR positive cases, fresh sections were cut from brain and lung samples, previously included in paraffin and stained for histological examination and for immunohistochemistry.
Fisher’s test (p
-value < 0.05) was used [16
] to analyse the results obtained for the different biological samples of positive animals to identify the most suitable biological sample for CDV diagnosis.
2.3. RNA Extraction
Total RNA was extracted by homogenizing 20–30 mg tissue by using high speed shaking in Eppendorf tubes with stainless steel beads (5 mm diameter in the TissueLyser II (QIAGEN). Homogenates were centrifuged at 20,000× g for 5 min at room temperature and 200 µL of the supernatants were submitted to RNA extraction with QIAamp cador Pathogen Mini Kit (QIAGEN) according to the manufacturer’s instructions. The same protocol was employed to extract total RNA from 200 µL of urine and from swabs (200 µL of vortexed 1 mL of transport medium in which the swabs are immersed); medium used for swabs was Dulbecco’s modified Eagle’s medium (D-MEM) added with Fetal Bovine Serum (2%), penicillin (500 IU/mL) and stremptomycin (500 µg/mL). RNA was eluted from each sample in a final volume of 60 µL of elution buffer using QIAgen columns.
2.4. Reverse Transcription and cDNA Synthesis
Reverse transcription was carried out using a volume of 30 µL of RNA with the addition of the following reagents: 6 µL of 10× random hexamer primers, 6 µL 10× RT-Buffer, 2.4 µL dNTP mix 100 mM, 3 µL of 5U Multi Scribe Reverse Transcriptase and 12.6 µL of RNase-free water by High Capacity cDNA Reverse Transcription kit (Applied Biosystems™, ThermoFisher Scientific); cDNA synthesis was carried out using Gene Amp® PCR System 9700 (Applied Biosystems) using the following thermal amplification profile: 25 °C for 10 min, 37 °C for 45 min and 85 °C for 5 min.
2.5. Real Time PCR for Field CDV Strain Diagnosis
For field CDV strain diagnosis, the specific primer pair (CDVF4 5′-GTCGGTAATCGAGGATTCGAGAG-3′ and CDVR 5′-GCCGAAAGAATATCCCCAGTTAG-3′) and the TaqMan Probe (3CDV 5′-6-FAM-ATCTTCGCCAGAATCCTCAGTGCT-MGB-3′), designed on a highly conserved region of the P gene, were used [17
] in the Real Time PCR using the AgPATH-ID™ One-step RT-PCR Kit (Applied Biosystems™, ThermoFisher Scientific) with the following composition: 12.5 µL of 2X RT-PCR Buffer, 1 µL of 25X RT-PCR Enzyme mix, 600 nM of forward primer, 600 nM of reverse primer, 300 nM of probe, 5 µL of RNA and 4.75 µL RNase-free water for a 25 µL total final volume. The Real Time PCR was carried out using System QuantStudio 7 Flex (Applied Biosystems) and the conditions were 45 °C for 10 min, 95 °C for 10 min, and 50 cycles of 95 °C for 15 s, 60° C for 1 min and 10 s. The thermal cycles were optimized following the manufacturer’s instructions.
All data were analysed using the System QuantStudio 7 Flex Sequence Detection System SDS software package (Applied Biosystems, Foster City, CA, USA).
2.6. Real Time PCR for Vaccine CDV Strains Diagnosis
For CDV vaccine strain diagnosis, the primer pair used was (CDV Vaccine Fw 5′-ATAATGATGTTATCATCAGYGATGAT-3′ and CDV Vaccine Rv 5′-CTTGGTCCGATAATGATCAACC-3′) together with the TaqMan Probe (CDV probe AM1 5′-FAM-CTTAGTAGCAYTGCCCAAGATCCCTTGATC-BHQ1-3′), designed in a 249 bp portion of the CDV M gene and M-F intergenic region of the Onderstepoort vaccine strain. This Real Time PCR protocol was developed to specifically identify the following vaccine strains: Onderstepoort, Duramune, Snyder Hill, Nobivac [15
]. For the Real Time PCR vaccine CDV strains, the TaqMan®
Universal Master Mix kit (Applied Biosystems™, ThermoFisher Scientific) was used with the following composition: 12.5 µL of TaqMan®
2X Universal PCR Master Mix, 600 nM of forward primer, 600 nM of reverse primer, 300 nM of probe, 5 µL of cDNA and 5.75 µL RNase-free water for a 25 µL total final volume. The Real Time PCR for vaccine CDV strains was carried out using Quant Studio 7 Flex (Applied Biosystems) and the conditions were: 50 °C for 2 min, 95 °C for 10 min, and 50 cycles of 95 °C for 15 s, 58 °C for 30 s and 60 °C for 1 min. All data were analysed using the Quant Studio 7 Flex Detection System SDS software package (Applied Biosystems, Foster City, CA, USA). All the examined samples were also tested in parallel with the Wilkes protocol to exclude any positivity from being linked to vaccinations.
2.7. PCR for the CDV Strains Characterization
For the samples resulting positive in Real Time PCR, we proceeded with the genetic characterization of the CDV strain. The PCR protocol amplified a 287 bp fragment located on the conserved N terminus of the morbillivirus NP gene and used primers (MvF 5′-ACAGGATTGCKGAGGACCTAT-3′ and MvR 5′-VARGATAACCATGTACGGTGC-3′) degenerated in position 779 (forward primer) and 1055 and 1053 (reverse primer), respectively [18
]. The Master Mix was carried out on a final volume of 50 µL and consisted of: 5 µL Platinum Taq Buffer 10×, 2 µL dNTPmix 10 mM (at 0.4 mM final concentration), 2.5 µL MgCl2 50 mM (at 2.5 mM final concentration), 0.33 µL for both the MvF 30 µM and MvR30 µM (each to the final concentration of 0.2 µM), 0.5 µL Platinum™ Taq DNA Polymerase 5 U/µL (Invitrogen™, ThermoFisher Scientific) (at 0.05 U/µL final concentration), 5 µL cDNA template and 34.34 µL of RNase-free water. The reaction was performed in a Gene Amp®
PCR System 9700 (Applied Biosystems) with the following PCR conditions: 95 °C for 10 min, 40 cycles of 95 °C for 1 min, 59.5 °C for 1 min, 72 °C for 1 min; 72 °C for 10 min. PCR products were visualized in 1.5% agarose gel electrophoresis by staining with GelRed dye (Biotium, Hayward, CA, USA).
2.8. Sequencing for CDV Strain Characterization
The amplicons for CDV strain characterization were purified with the QIAquick PCR Purification kit (Qiagen, Hilden, Germany) and sequenced using the PCR primers MvF/MvR as described in point 2.7 with the BigDye Terminator Cycle Sequencing Ready Reaction kit, version 3.1 (PerkinElmer, Applied Biosystems, Foster City, CA, USA) in an automated sequencer (ABI Prism 310 DNA sequencer, Applied Biosystems, Foster City, CA, USA). Alignments were obtained with the multiple program DNASTAR (DNASTAR Inc., Madison, WI, USA) using Clustal W. The nucleotide sequences obtained were analysed using Basic Local Alignment Search Tool (BLAST) by comparing them to sequences from reference strains of different CDV virus accessed on NCBI GenBank (http://www.ncbi.nlm.nih.gov/
(accessed on April 2020). The samples were considered as belonging to a particular strain when sequence identity and query cover were between 98 and 100%.
2.9. Histological and Immunohistochemical Exams
Portions of brain and lung of PCR positive animals, previously fixed in formalin, were embedded in paraffin, cut at thickness of 5 µm and routinely stained with hematoxylin and eosin.
Immunohistochemistry was carried out on newly prepared unstained sections of both organs, using the following procedure: blocking of endogenous peroxidase with 3% H2O2 at room temperature for 30 min, antigen retrieving with trypsin at 37 °C for 30 min, overnight incubation at 4 °C with a monoclonal antibody anti-Canine Distemper Virus (clone 8-1) (LSBio). A positive reaction was detected using 3, 3′- diaminobenzidine (EnVision Plus kit, Dako) as chromogen with a 3-min development at room temperature and counterstained with hematoxylin.
The analysis of the detected sequences, starting from the samples received at the IZSLT for diagnostic purposes from 2011 to 2019, highlighted the circulation of the strain CDV 12254/2015 (KX024709.1) belonging to the “Arctic” lineage [20
] in dogs coming from provinces of Latium and Tuscany. Starting from 2017, genetic analyses conducted on samples from wild animals (fox and wolf) received at our Institute for diagnostic or research purposes detected the circulation, in Latium and Tuscany regions, of the strain CDV599/2016 (KX545421.1) belonging to the “Europe wildlife” lineage, and, in a dog coming from Rome, of strain CDV/BJ16C9 (MF926604.1). These outcomes confirm the co-circulation of the “Europe wildlife” and “Arctic” lineages in Central Italy, as already noted by Di Sabatino [4
], while, in Italy, the circulation of at least three separate CDV lineages was confirmed by other authors, including Europa-1 [8
], which has not yet been detected in our area of study.
The circulation of the Arctic strain in wild animals already reported in Central Italy highlights the possible role of reservoir that these species can have, with the possibility of passing the strain to unvaccinated domestic animals and the occurrence of subsequent epidemic events. The data obtained in the study carried out indicate two different and separate cycles for CDV distribution in wild and in domestic animals in the area under study, and the characterization of different strains in the two different populations leads to the hypothesis that, for these Regions, there exist distinct epidemiological cycles that are kept separated by ecological barriers.
To date, the CDV/BJ16C9 strain (MF926604.1) that belongs to the cluster of America-1 CDV strains closely related to the Onderstepoort vaccine strain [21
] is the first report of its circulation in our territory. As none of the samples tested positive to the Wilkes protocol [15
], the hypotheses explaining the presence of this vaccine strain could be: (a) an antigenic escape; (b) a genetic recombination with wild-type strains; (c) an environmental adaptation; (d) an evolution of the CDV [24
]. The small number of positives identified requires further investigation, which we are carrying out to identify more positive subjects and characterize their strains, to strengthen the data obtained and to confirm the simultaneous circulation in the area under study of different strains in wild and domestic populations.
The animals detected positive were dogs, foxes and wolves, which are, however, the most represented species. Not having had a relevant number of positive subjects, it was impossible for us to define the histolesive characterization of the isolated strains.
The importance of taking the complete set of conjunctival, nasal and rectal swabs to maximize the sensitivity of the diagnostic system in the event of a suspicion of distemper in both living and dead subjects—associated also, in the latter case, with the brain and lung—is highlighted. In particular, the rectal swab is apparently the most appropriate sample, but due to the limited number of positive subjects, further samples are required to confirm this.
It will be interesting to continue to monitor domestic dogs and other wild species with symptoms attributable to CDV and verify the different circulating field strains in central Italy, which could provide important information on the evolution of the virus and employing different vaccine strains. The use of the two PCR methods selected for the respective detection of CDV field and vaccine strains allows the advantage of being able to exclude positivity related to vaccination.
For the genetic characterization, the NP gene, as also employed by other authors [25
], was chosen because it contains a conserved region that allows the detection of the known CDV strains circulating in mammals (wild and otherwise). The choice of the NP gene for identification and sequencing of the strains was made by selecting a region of the nucleoprotein (NP) of the CDV, which also shows a great homology among morbillivirus strains, as reported by Verna [18
] in reference to previous papers in which the NP region appears as an appropriate candidate for the analysis of circulating strains of CDV [28
]. The genetic study of the circulating strains by the laboratories that carry out the diagnosis provides information on the evolution of the virus that could be useful for updating vaccine strains. Collaboration between laboratories and vaccine manufacturers would be desirable to produce effective immunization products.