A Bacterium-like Particle Vaccine Displaying Envelope Proteins of Canine Distemper Virus Can Induce Immune Responses in Mice and Dogs.

Canine distemper virus (CDV) can cause fatal infections in giant pandas. Vaccination is crucial to prevent CDV infection in giant pandas. In this study, two bacterium-like particle vaccines F3-GEM and H4-GEM displaying the trimeric F protein or tetrameric H protein of CDV were constructed based on the Gram-positive enhanced-matrix protein anchor (GEM-PA) surface display system. Electron microscopy and Western blot results revealed that the F or H protein was successfully anchored on the surface of GEM particles. Furthermore, one more bacterium-like particle vaccine F3 and H4-GEM was also designed, a mixture consisting of F3-GEM and H4-GEM at a ratio of 1:1. To evaluate the effect of the three vaccines, mice were immunized with F3-GEM, H4-GEM or F3 and H4-GEM. It was found that the level of IgG-specific antibodies and neutralizing antibodies in the F3 and H4-GEM group was higher than the other two groups. Additionally, F3 and H4-GEM also increased the secretion of Th1-related and Th2-related cytokines. Moreover, F3 and H4-GEM induce IgG and neutralizing antibodies' response in dogs. Conclusions: In summary, F3 and H4-GEM can provoke better immune responses to CDV in mice and dogs. The bacterium-like particle vaccine F3 and H4-GEM might be a potential vaccine candidate for giant pandas against CDV infection.

Ferret Pediatrics.

Ferrets are bred to be pets, utilized for hunting, and as laboratory models. Despite the fact that ferrets in some areas of the world are neutered by the breeder before entering the pet trade, the importance of pediatric management should not be overlooked. Pregnant, whelping, and lactating jills should be closely monitored and kept in a quiet, stress-free environment. Hand-rearing baby kits is very challenging due to their requirement for ferret milk. Minimizing maternal stress and disease can prevent the need to hand rear kits. Infectious diseases in juvenile ferrets include canine distemper virus, rotavirus, coccidiosis, feline panleukopenia virus (experimental only), and Toxoplasma-like disease. All juvenile ferrets should be vaccinated against canine distemper and rabies. Congenital diseases are reported to affect the auditory, ocular, cardiovascular, urogenital, central nervous, and musculoskeletal systems. Early detection of these diseases is important to prevent the progression of curable diseases.

Canine distemper virus (CDV)-neutralizing activities of an anti-CDV canine-derived single-chain variable antibody fragment 4-15 (scFv 4-15) screened by phage display technology.

Canine distemper virus (CDV) is a highly contagious pathogen that causes severe diarrhea, fever and vomiting in domestic dogs, posing a serious threat to the dog breeding industry. Currently, there are no effective therapeutic agents for emergency treatment despite the availability of vaccines against CDV infection. Single-chain fragment variable (scFv) antibody has been demonstrated to effectively inhibit virus infections, suggesting a potential candidate as a therapeutic agent for canine distemper. In this study, a phage-displayed scFv library was constructed from the peripheral blood lymphocytes of dog immunized intramuscularly with live-attenuated CDV vaccine, and was subjected to four rounds of pannings against CDV. Subsequent indirect enzyme-linked immunosorbent assay screening revealed high-affinity scFv antibodies specific to CDV, and indirect immunofluorescence assay screening revealed CDV-neutralizing activity of scFv antibodies. Our results showed that a scFv antibody 4-15 (scFv 4-15) with high-affinity binding to CDV and neutralizing activity against CDV was obtained, which displayed effective therapeutic potential in vivo for dogs challenged with a lethal dose of CDV. Conclusively, the scFv 4-15 with high-affinity binding and neutralizing activity to CDV that was obtained by phage display technology provides a promising candidate for the therapeutic agents against CDV infection.

Post-vaccinal distemper-like disease in two dog litters with confirmed infection of vaccine virus strain.

Modified live canine distemper virus (CDV) vaccines are widely used and considered both safe and effective. Although there are occasional literature reports of suspected vaccine-induced disease, there are none where the vaccine strain has been identified in affected tissues. Here we describe two such cases in different litters. In litter A, five of ten puppies presented with fever, anorexia, vomiting, and diarrhea a few days post-vaccination. Four puppies died or were euthanized, and autopsy revealed atypical necrosis of the lymphoid tissue. In litter B, two of five puppies developed typical neurological signs some months post-vaccination and autopsy revealed encephalitis. In all cases, affected organs tested positive for CDV on immunohistochemistry, and CDV RNA extracted from the lesions confirmed the presence of vaccine strain. Since multiple puppies from each litter were affected, it cannot be excluded without further studies that some undiagnosed inherited immunodeficiency disorder may have been involved.

DNA Vaccine Co-Expressing Hemagglutinin and IFN-γ Provides Partial Protection to Ferrets against Lethal Challenge with Canine Distemper Virus.

Canine distemper (CD), caused by canine distemper virus (CDV), is a highly contagious and lethal disease in domestic and wild carnivores. Although CDV live-attenuated vaccines have reduced the incidence of CD worldwide, low levels of protection are achieved in the presence of maternal antibodies in juvenile animals. Moreover, live-attenuated CDV vaccines may retain residual virulence in highly susceptible species and cause disease. Here, we generated several CDV DNA vaccine candidates based on the biscistronic vector (pIRES) co-expressing virus wild-type or codon-optimized hemagglutinin (H) and nucleocapsid (N) or ferret interferon (IFN)-γ, as a molecular adjuvant, respectively. Apparently, ferret (Mustela putorius furo)-specific codon optimization increased the expression of CDV H and N proteins. A ferret model of CDV was used to evaluate the protective immune response of the DNA vaccines. The results of the vaccinated ferrets showed that the DNA vaccine co-expressing the genes of codon-optimized H and ferret IFN-γ (poptiH-IRES-IFN) elicited the highest anti-CDV serum-neutralizing antibodies titer (1:14) and cytokine responses (upregulated TNF-α, IL-4, IL-2, and IFN-γ expression) after the third immunization. Following vaccination, the animals were challenged with a lethal CDV 5804Pe/H strain with a dose of 105.0 TCID50. Protective immune responses induced by the DNA vaccine alleviated clinical symptoms and pathological changes in CDV-infected ferrets. However, it cannot completely prevent virus replication and viremia in vivo as well as virus shedding due to the limited neutralizing antibody level, which eventually contributed to a survival rate of 75% (3/4) against CDV infection. Therefore, the improved strategies for the present DNA vaccines should be taken into consideration to develop more protective immunity, which includes increasing antigen expression or alternative delivery routes, such as gene gun injection.

Clinical assessment of a point-of-care assay to determine protective vaccinal antibody titers to canine viral diseases.

Guidelines recommend that dogs are vaccinated for canine distemper virus (CDV), canine parvovirus (CPV), and canine adenovirus (CAV) every 3 years. Alternatively, their antibody titers are measured and vaccines given when titers fall below a protective threshold. In this study, a point-of-care (POC) assay was compared to hemagglutination inhibition (for CPV) and virus neutralization (for CAV and CDV) assays to predict the need for revaccination Ninety-two dogs presented for vaccination were enrolled. The POC assay indicated protective titers against CDV in 79/80, CPV in 89/90, and CAV in 91/91 dogs with reference standard antibody measurements that were over a protective threshold. The sensitivity of the POC assay for to detect protective concentrations of CDV antibodies was 99% (95% confidence interval [CI 95%], 93.3-99.9%). Ten dogs were falsely considered protected against CDV by the POC assay with a specificity of 17% (CI 95%, 3.0-44.8%). The sensitivity of the POC assay for protective concentrations of CPV titers was 99% (CI 95%, 93.9-99.9%). The sensitivity of the POC assay to detect protective concentrations of CAV antibodies was 100% (CI 95%, 95.9-100%). Only classifying high-positive CDV and CPV titers on the POC assay as protective improved assay specificity to 100%, but sensitivity decreased to 51% and 76% respectively. This POC assay had a high sensitivity for the detection of protective antibody titers; however, some dogs were falsely categorized as protected, especially for CDV.

SEROLOGIC RESPONSE AND ADVERSE EFFECTS OF RECOMBINANT CANINE DISTEMPER VACCINATION IN THREE AQUARIUM-HOUSED WALRUSES (ODOBENUS ROSMARUS).

Fatalities have been associated with phocine and canine distemper viruses in marine mammals, including pinnipeds. No data are available regarding distemper disease or vaccination in walruses. This study evaluates seroconversion and clinical adverse effects following administration of a canarypox-vectored recombinant distemper vaccination (two 1-ml doses, 3 wk apart) in three adult aquarium-housed walruses. Serum antibodies to distemper were measured using seroneutralization on blood samples collected under operant conditioning prior to and for 12 mon after vaccination or until titers were <32. All walruses seroconverted. Medium positive titers (64-128) were detected for 4 to 9.5 mon in two of three individuals. Interindividual variability was noted, with one individual displaying only low positive titers. Major swelling at the site of injection and lameness for a week following injection occurred in all three walruses. Further studies on dosing amount and interval are needed to make vaccine recommendations in this species.

LRP6 Is a Functional Receptor for Attenuated Canine Distemper Virus.

Wild-type canine distemper virus (CDV) is an important pathogen of dogs as well as wildlife that can infect immune and epithelial cells through two known receptors: the signaling lymphocytic activation molecule (SLAM) and nectin-4, respectively. Conversely, the ferret and egg-adapted CDV-Onderstepoort strain (CDV-OP) is employed as an effective vaccine for dogs. CDV-OP also exhibits promising oncolytic properties, such as its abilities to infect and kill multiple cancer cells in vitro. Interestingly, several cancer cells do not express SLAM or nectin-4, suggesting the presence of a yet unknown entry factor for CDV-OP. By conducting a genome-wide CRISPR/Cas9 knockout (KO) screen in CDV-OP-susceptible canine mammary carcinoma P114 cells, which neither express SLAM nor nectin-4, we identified low-density lipoprotein receptor-related protein 6 (LRP6) as a host factor that promotes CDV-OP infectivity. Whereas the genetic ablation of LRP6 rendered cells resistant to infection, ectopic expression in resistant LRP6KO cells restored susceptibility. Furthermore, multiple functional studies revealed that (i) the overexpression of LRP6 leads to increased cell-cell fusion, (ii) a soluble construct of the viral receptor-binding protein (solHOP) interacts with a soluble form of LRP6 (solLRP6), (iii) an H-OP point mutant that prevents interaction with solLRP6 abrogates cell entry in multiple cell lines once transferred into recombinant viral particles, and (iv) vesicular stomatitis virus (VSV) pseudotyped with CDV-OP envelope glycoproteins loses its infectivity in LRP6KO cells. Collectively, our study identified LRP6 as the long sought-after cell entry receptor of CDV-OP in multiple cell lines, which set the molecular bases to refine our understanding of viral-cell adaptation and to further investigate its oncolytic properties. IMPORTANCE Oncolytic viruses (OV) have gathered increasing interest in recent years as an alternative option to treat cancers. The Onderstepoort strain of canine distemper virus (CDV-OP), an enveloped RNA virus belonging to the genus Morbillivirus, is employed as a safe and efficient vaccine for dogs against distemper disease. Importantly, although CDV-OP can infect and kill multiple cancer cell lines, the basic mechanisms of entry remain to be elucidated, as most of those transformed cells do not express natural receptors (i.e., SLAM and nectin-4). In this study, using a genome-wide CRISPR/Cas9 knockout screen, we describe the discovery of LRP6 as a novel functional entry receptor for CDV-OP in various cancer cell lines and thereby uncover a basic mechanism of cell culture adaptation. Since LRP6 is upregulated in various cancer types, our data provide important insights in order to further investigate the oncolytic properties of CDV-OP.

Proteomic analysis of canine vaccines.

OBJECTIVE: To use proteomic analysis to identify qualitatively and quantitatively mammalian protein components of commercial veterinary vaccines against canine distemper, leptospirosis, borreliosis, and rabies. SAMPLE: 25 licensed veterinary vaccines (from 4 different manufacturers) against canine distemper and leptospirosis, borreliosis, and rabies (3-year and 1-year durations of immunity). PROCEDURES: Duplicate samples from a single-lot vial of each vaccine were prepared by acetone precipitation and proteolysis with trypsin and Lys-C protease mix. Peptides mixtures (1 µg) were analyzed by liquid chromatography-tandem mass spectrometry using an Orbitrap Fusion Lumos mass spectrometer. Liquid chromatography-tandem mass spectroscopy data were searched against a Bos taurus protein database using MaxQuant to identify and quantify mammalian proteins in the vaccines. Identified proteins were classified by function and network analysis to visualize interactions. RESULTS: The largest number of mammalian proteins was identified in 3-year rabies vaccines (median, 243 proteins; range, 184 to 339 proteins) and 1-year rabies vaccines (median, 193 proteins; range, 169 to 350 proteins). Borrelia and leptospirosis-distemper (L&D) vaccines had the lowest number of proteins. Rabies vaccines had the highest number of identified proteins in common (n = 316); 33 were unique to 1-year products and 44 were found in 3-year products. Borrelia and L&D vaccines had 16 and 22 uniquely identified proteins, respectively. The protein classifications were primarily modulators of protein-binding activity, enzymes, transfer-carrier proteins, cytoskeletal proteins, defense-immunity proteins, calcium-binding proteins, and extracellular matrix proteins. CLINICAL RELEVANCE: This study demonstrates proteomics application to evaluate quality differences among different vaccines, identifying potential stimulants of desirable and undesirable immune responses.

Effect of maternally derived antibodies on two commercial vaccines in changes of serum antibody titres against distemper in puppies.

BACKGROUND: Maternally derived antibodies (MDA) have protection against canine distemper virus (CDV) in the first weeks of puppies' life. However, MDA decreases with age. The most important and effective factor on immunization is timely vaccination. But in recent years, there were some outbreaks of CDV among puppies in Iran and this problem could be related to vaccine failure. OBJECTIVES: The aim of this study is to evaluate the possible effect of MDA titre on vaccines against CDV and the efficacy of two commercial vaccines by using the enzyme-linked immunosorbent assay (ELISA). METHODS: In this regard, 24 healthy 8-week old terrier puppies were selected and divided into three identical groups based on a randomized, double-blind comparative trial. The control group was injected normal saline, and group A was vaccinated by the vaccine namely Biocan L (Bioveta, Czech Republic), and the group B was vaccinated by the other vaccine called Duramune Max 5 + LCI/GP (Fort Dodge Animal Health, USA). The vaccines were used for the puppies between 8 and 16 weeks of age and in every 4 weeks. RESULTS: The results showed that the response of both vaccines was satisfactory, and no significant difference was observed between them. Moreover, the MDA in the control group reached an unprotective level in all puppies prior to their 14 weeks of age. In the vaccinated groups, after the second vaccine, all puppies in both groups reached protective levels. DISCUSSION: This is the first study on evaluation of two commercial vaccines in changes of serum antibody titres against distemper in puppies in Iran. CONCLUSION: It is recommended that veterinarians during consulting use the ELISA to measure antibody titres to optimize the vaccination schedule and reduce the cost of vaccination failure. This is of paramount importance for puppies.

A novel and highly divergent Canine Distemper Virus lineage causing distemper in ferrets in Australia.

Canine distemper virus (CDV) causes a highly contagious systemic infection in an array of animal species. In this study we report an outbreak of distemper in ferrets in two research facilities in Australia, caused by a novel lineage of CDV. While the CDV strain caused mainly mild symptoms in ferrets, histopathology results presented a typical profile of distemper pathology, with multi-system virus replication. Through the development of a discriminatory PCR, paired with full genome sequencing, we revealed that the outbreak was caused by a novel lineage of CDV. The novel CDV lineage was highly divergent, with less than 93% similarity across the H gene to other described lineages, including the vaccine strain, and diverged approximately 140-400 years ago. Enhanced surveillance to determine the prevalence of CDV in ferrets, dogs and other at-risk species is critical to better understand the presence and diversity of CDV in Australia currently.

A point-of-care dot blot ELISA assay for detection of protective antibody against canine adenovirus, canine parvovirus, and canine distemper virus is diagnostically accurate.

OBJECTIVE: To determine diagnostic accuracy of a point-of-care antibody-screening test by determining sensitivity, specificity, and overall accuracy when compared to reference standard tests for antibody against core vaccine viruses canine adenovirus (CAV), canine parvovirus (CPV), and canine distemper virus (CDV). A further aim was to provide the practitioner with information to guide selection of vaccinal antibody testing methods. SAMPLES: Canine sera from across North America were submitted to a fee-for-service titer-testing laboratory. Samples came from healthy pet dogs with known core vaccination history (n = 431) as well as unvaccinated dogs held in isolation (132). This study examined a total of 563 samples for CDV/CPV and 183 for CAV. PROCEDURES: Serum virus neutralization assays determined antibody titers for CDV and CAV. Hemagglutination inhibition assay determined antibody titers against CPV. All sera were also tested by point-of-care dot blot ELISA (index test). RESULTS: For all 3 viral antigens, the index test provided sensitivity ranging from 96.03% to 96.75% and specificity ranging from 87.50% to 94.33%. Overall accuracy ranged from 93.43% to 95.91%. CLINICAL RELEVANCE: The index test correlates well with reference standard tests and is a reliable, rapid screening test for detection of protective vaccinal antibody against CAV, CDV, and CPV in healthy dogs over 20 weeks of age. An accurate assessment of immunity allows clinicians to administer core vaccines appropriately as needed, avoiding unnecessary risk of adverse vaccine events.

Contributions of distemper control and habitat expansion to the Amur leopard viability.

The Amur leopard (Panthera pardus orientalis) is a critically endangered top predator that struggles on the brink of extinction due to threats such as canine distemper virus (CDV), habitat loss, and inbreeding depression. Here we develop a viability analysis metamodel that combines a traditional individual-based demographic model with an epidemiological model to assess the benefits of alternative population management actions in response to multiple distinct threats. Our results showed an extinction risk of 10.3%-99.9% if no management actions were taken over 100 years under different levels of inbreeding depression. Reducing the risk of CDV infection in Amur leopards through the low-coverage vaccination of leopards and the management of sympatric domestic dogs could effectively improve the survival probability of the leopard population, and with habitat expansion added to these management measures, the population expanded further. Our findings highlight that protecting the Amur leopard necessitates a multifaceted synergistic effort, and controlling multiple threats together may significantly escalate overall viability of a species, especially for small-isolated threatened population. More broadly, our modeling framework could offer critical perspectives and scientific support for conservation planning, as well as specific adaptive management actions for endangered species around the world.

A Single Oral Immunization with Replication-Competent Adenovirus-Vectored Vaccine Induces a Neutralizing Antibody Response in Mice against Canine Distemper Virus.

Canine Distemper Virus (CDV) is a fatal and highly contagious pathogen of multiple carnivores. While injectable vaccines are very effective in protecting domestic animals, their use in the wild is unrealistic. Alternative vaccines are therefore needed. Adenovirus (AdV) vectors are popular vaccine vectors due to their capacity to elicit potent humoral and cellular immune responses against the antigens they carry. In parallel, vaccines based on live human AdV-4 and -7 have been used in U.S. army for several decades as replicative oral vaccines against respiratory infection with the same viruses. Based on these observations, the use of oral administration of replication competent AdV-vectored vaccines has emerged as a promising tool especially for wildlife vaccination. Developing this type of vaccine is not easy, however, given the high host specificity of AdVs and their very low replication in non-target species. To overcome this problem, the feasibility of this approach was tested using mouse adenovirus 1 (MAV-1) in mice as vaccine vectors. First, different vaccine vectors expressing the entire or part H or F proteins of CDV were constructed. These different strains were then used as oral vaccines in BALB/c mice and the immune response to CDV was evaluated. Only the strain expressing the full length CDV H protein generated a detectable and neutralizing immune response to CDV. Secondly, using this strain, we were able to show that although this type of vaccine is sensitive to pre-existing immunity to the vector, a second oral administration of the same vaccine is able to boost the immune response against CDV. Overall, this study demonstrates the feasibility of using replicating AdVs as oral vaccine vectors to immunize against CDV in wildlife carnivores.

Transcriptome Analysis Reveals the Alternative Splicing Changes in the Immune-Related Genes of the Giant Panda (Ailuropoda melanoleuca), in Response to the Canine Distemper Vaccine.

Canine distemper virus (CDV) is a highly fatal virus to the giant panda (Ailuropoda melanoleuca). Although vaccination is a key preventative measure in captive giant pandas, the immune response of giant pandas after vaccination remains unclear. Therefore, this study focuses on differential alternative splicing (DAS) events of giant pandas before and after vaccination to investigate the role of alternative splicing in the immune response of giant pandas after CDV vaccination. In this study, we identified 1113 DAS genes, which had 1288 DAS events. The KEGG functional enrichment analysis of DAS genes showed enrichment of some DNA damage repair and immune-related pathways. In the combined analysis of DAS and differentially expressed genes (from our previous research), we identified 66 differentially expressed genes with a DAS event, and found that some important immune-related genes, such as IL15, IL18, IL18RAP, CHUK, IFI44, CD40, and CD46 underwent DAS events and were involved in the immune response of giant pandas after CDV vaccination. We describe here the alternative splicing events of giant pandas after CDV vaccination for the first time and show that the results indicated that alternative splicing has an important role in regulating the immune response of giant pandas after vaccination.

Evaluation of Intrathecal Injection of Modified Live Newcastle Disease Virus Vaccine in Dogs with Canine Distemper Encephalitis.

The neurological form of canine distemper virus (CDV) infection can occur concurrently with systemic signs or develop following apparent recovery. There are no specific antiviral or immunomodulatory therapies recognized for treatment of CDV infections, and the neurological form typically carries with it a high mortality rate. The intrathecal injection of a modified live Newcastle disease virus vaccine (NDV-MLV) has been proposed for the treatment of the neurological forms of CDV infections. Thirteen dogs confirmed to have canine distemper infections by polymerase chain reaction testing and with neurological signs consistent with CDV infection were treated with an intrathecal injection of NDV-MLV and were scheduled for re-evaluation 3-4 mo later. Six dogs survived to follow-up and four dogs survived long term (>3 yr). Cerebrospinal fluid cytokines were measured and reported. Changes in cerebrospinal fluid cytokines and long-term survival could not be attributed to the intrathecal injection of NDV-MLV, and this therapy cannot be recommended for treatment of dogs with neurological forms of CDV infection.

Compatibility between a rabies vaccine and two canine combined vaccines against canine distemper, adenovirosis, parvovirosis, parainfluenza virus disease and leptospirosis, with or without canine coronavirus.

In many countries, vaccination programs still require dogs to be vaccinated against rabies in addition to Canine distemper virus (CDV), adenovirus (CAV), parvovirus (CPV), parainfluenza virus (CPiV), Leptospira (L) or Canine coronavirus (CCV= Cv). Few vaccines containing all these antigens are commercially available and, unless compatibility between the vaccines was demonstrated, concurrent administration of a DAPPi-L(Cv) vaccine and a vaccine against rabies should not be recommended. This may be of concern for practitioners who wish to vaccinate dogs with all components on the same day. This study aimed at evaluating immunological compatibility between a monovalent rabies vaccine (Rabisin™) and two large combination vaccines against CDV, CAV, CPV, CPiV with 2 leptospira components +Cv (Recombitek® C6/Cv) or with 4 Leptospira components (Recombitek® C8), when injected concomitantly at two separate injection sites. Fourteen days after administration of the rabies vaccine, with or without concomitant administration of combo vaccines, all dogs had seroconverted against rabies and maintained protective titers over the duration of the study. In addition, 100% of the puppies vaccinated with one or the other combo vaccines seroconverted against CDV, CAV, CPV, CPiV (CCV) and Leptospira, whatever the vaccination group. Lack of immunological interference between Rabisin™ and all components of the Recombitek® C6/Cv or Recombitek® C8 Combo vaccines was demonstrated by non-inferiority analysis, except for CDV in the Recombitek®C8+ Rabisin™ group. Based on these results, a concomitant administration of Rabisin™ with Recombitek® C6/Cv or Recombitek® C8 can be recommended in daily practice, which can be essential for facilitating vaccination compliance.

RECOMBITEK CANINE DISTEMPER VACCINE AS AN ALTERNATIVE FOR PUREVAX DISTEMPER VACCINE IN ENDANGERED BLACK-FOOTED FERRETS (MUSTELA NIGRIPES).

Black-footed ferrets (Mustela nigripes) are an endangered species in North America that are highly sensitive to canine distemper virus (CDV) infections and any exposure could be devastating to species recovery. The U.S. Fish and Wildlife Service recovery program has safely used a recombinant DNA (rDNA) canarypox-vectored CDV vaccine, Purevax® Ferret Distemper (PFD), to vaccinate black-footed ferrets. Because of a PFD shortage in 2015, an rDNA vaccine labeled for use in dogs, Recombitek® CDV (rCDV), was chosen to vaccinate black-footed ferrets. Our goal was to compare the serum neutralizing (SN) titers after vaccination of 17 captive and 18 wild black-footed ferrets with rCDV or PFD, respectively, considering ≥1:128 as a protective titer. Both vaccines produced comparable 1 yr postvaccination protective titers in captive and wild black-footed ferrets. In wild black-footed ferrets, one PFD vaccination produced SN titers similar to two PFD vaccinations at 1 yr postvaccination. One year after vaccination with rCDV, SN titers in captive black-footed ferrets were higher than in wild ferrets. These results indicate rCDV may be an effective alternative CDV vaccine in captive black-footed ferrets and PFD should be prioritized for wild ferrets because one dose was effective for animals that can be difficult to recapture.

Agreement between In-Clinics and Virus Neutralization Tests in Detecting Antibodies against Canine Distemper Virus (CDV).

Core vaccinations and specific antibody titer evaluations are strongly recommended worldwide by all the vaccination guidelines. Virus neutralization (VN) is considered the gold standard for measuring antibody titer against canine distemper virus, but it is complex and time consuming, and the use of in-clinics tests would allow to obtain quicker results. The aim of this study was to evaluate the agreement of the commercial in-clinics VacciCheck test compared to VN. A total of 106 canine sera were analyzed using both methods. The best agreement was obtained using a protective threshold of ≥1:32. VacciCheck showed 95.5% sensitivity, 87.2% specificity, and 92.5% accuracy. The Cohen's kappa coefficient between methods was 0.84 (CI 95% 0.73 to 0.95), revealing an optimal agreement between the two methods (p = 0.0073). The evaluation of discordant results reveal that most samples had less than 1.5 dilution difference, and that usually did not affect the classification as protected or non-protected. Results also suggest that, in dubious cases, especially when a protective result is expected, retesting is advisable. In conclusion, VacciCheck may be considered as a reliable instrument that may help the clinician in identifying the best vaccine protocol, avoiding unnecessary vaccination, and thus reducing the incidence of adverse effects.

Self-assembling ferritin nanoparticles coupled with linear sequences from canine distemper virus haemagglutinin protein elicit robust immune responses.

BACKGROUND: Canine distemper virus (CDV), which is highly infectious, has caused outbreaks of varying scales in domestic and wild animals worldwide, so the development of a high-efficiency vaccine has broad application prospects. Currently, the commercial vaccine of CDV is an attenuated vaccine, which has the disadvantages of a complex preparation process, high cost and safety risk. It is necessary to develop a safe and effective CDV vaccine that is easy to produce on a large scale. In this study, sequences of CDV haemagglutinin (HA) from the Yanaka strain were aligned, and three potential linear sequences, termed YaH3, YaH4, and YaH5, were collected. To increase the immunogenicity of the epitopes, ferritin was employed as a self-assembling nanoparticle element. The ferritin-coupled forms were termed YaH3F, YaH4F, and YaH5F, respectively. A full-length HA sequence coupled with ferritin was also constructed as a DNA vaccine to compare the immunogenicity of nanoparticles in prokaryotic expression. RESULT: The self-assembly morphology of the proteins from prokaryotic expression was verified by transmission electron microscopy. All the proteins self-assembled into nanoparticles. The expression of the DNA vaccine YaHF in HEK-293T cells was also confirmed in vitro. After subcutaneous injection of epitope nanoparticles or intramuscular injection of DNA YaHF, all vaccines induced strong serum titres, and long-term potency of antibodies in serum could be detected after 84 days. Strong anti-CDV neutralizing activities were observed in both the YaH4F group and YaHF group. According to antibody typing and cytokine detection, YaH4F can induce both Th1 and Th2 immune responses. The results of flow cytometry detection indicated that compared with the control group, all the immunogens elicited an increase in CD3. Simultaneously, the serum antibodies induced by YaH4F and YaHF could significantly enhance the ADCC effect compared with the control group, indicating that the antibodies in the serum effectively recognized the antigens on the cell surface and induced NK cells to kill infected cells directly. CONCLUSIONS: YaH4F self-assembling nanoparticle obtained by prokaryotic expression has no less of an immune effect than YaHF, and H4 has great potential to become a key target for the easy and rapid preparation of epitope vaccines.