Epitope mapping and a candidate vaccine design from canine distemper virus.

Background: Canine distemper (CD) is a worldwide spread disease that has been described in 12 families of mammals, especially in the Carnivora order, being better studied in domestic canines where vaccination represents the best means of control. CD is controlled by vaccination, but many cases of the disease still occur in vaccinated animals. Aim: The aim of this work was to study antigen-specific epitopes that can subsidize the development of a new vaccine approach. Methods: Mapping of T cell reactive epitopes for CD virus (CDV) was carried out through enzyme-linked immunospot assays using 119 overlapped synthetic peptides from the viral hemagglutinin protein, grouped in 22 pools forming a matrix to test the immune response of 32 animals. Results: Evaluations using the criteria established to identify reactive pools, demonstrated that 26 animals presented at least one reactive pool, that one pool was not reactive to any animal, and six pools were the most frequent among the reactive peptides. The crisscrossing of the most reactive pools in the matrix revealed nine peptides considered potential candidate epitopes for T cell stimulation against the CDV and those were used to design an in-silico protein, containing also predicted epitopes for B cell stimulation, and further analyzed using immune epitope databases to ensure protein quality and stability. Conclusion: The final in silico optimized protein presents characteristics that qualify it to be used to develop a new prototype epitope-based anti-CDV vaccine.

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.

Persistent and Severe Viral Replication in PBMCs with Moderate Immunosuppression Served an Alternative Novel Pathogenic Mechanism for Canine Morbillivirus.

Measles virus and canine distemper virus (CDV) cause lethal infections in their respective hosts characterized by severe immunosuppression. To furtherly acknowledge the attenuated mechanisms of the regionally ongoing epidemic CDV isolates and provide novel perspectives for designing new vaccines and therapeutic drugs, a recombinant CDV rHBF-vacH was employed with a vaccine hemagglutinin (H) gene replacement by reverse genetics based on an infectious cDNA clone for the CDV wild-type HBF-1 strain. Interestingly, unlike previously published reports that a vaccine H protein completely changed a pathogenic wild-type CDV variant to be avirulent, rHBF-vacH was only partially attenuated by alleviating the degree of viral immunosuppression, and still caused 66.7% lethality in ferrets with a prolonged period of disease. Further comparisons of pathogenic mechanisms proved that the weaker but necessary invasions into peripheral blood mononuclear cells (PBMCs) of rHBF-vacH, and subsequently persistent viral replications in PBMCs and multiple organs, together contributed to its 66.7% mortality. In addition, despite significantly higher titers than the parent viruses, rHBF-vacH would not be a suitable candidate for a live vaccine, with great invasion and infection potentials of PBMCs from 16 tested kinds of host species. Altogether, sustained and severe viral replication in PBMCs with moderate immunosuppression was first proven to be an alternative novel pathogenic mechanism for CDV, which might help us to understand possible reasons for CDV fatal infections among domestic dogs and the highly susceptible wild species during natural transmission. IMPORTANCE Despite widespread vaccine campaigns for domestic dogs, CDV remained an important infectious disease in vaccinated carnivores and wild species. In recent years, the regionally ongoing epidemic CDV isolates have emphasized conservation threats to, and potentially disastrous epidemics in, endangered species worldwide. However, little is known about how to deal with the CDV variants constantly regional epidemic. In this study, we employed a recombinant CDV rHBF-vacH with a vaccine H gene replacement in a CDV wild-type HBF-1 context to attenuate the epidemic CDV variant to design a new vaccine candidate. Interestingly, rHBF-vacH was only partially attenuated by alleviating the degree of viral immunosuppression, and still caused 66.7% lethality in ferrets by weaker but necessary invasions into PBMCs, and subsequently persistent and severe viral replications in PBMCs. Significantly higher virus titers of rHBF-vacH in vitro might indicate the rapid cell-to-cell spreads in vivo that indirectly contribute to fatal infections of rHBF-vacH in ferrets.

Prevalence of Neutralizing Antibodies to Canine Distemper Virus and Response to Vaccination in Client-Owned Adult Healthy Dogs.

Re-vaccinations against canine distemper virus (CDV) are commonly performed in 3-year intervals. The study's aims were to determine anti-CDV antibodies in healthy adult dogs within 28 days of vaccination against CDV, and to evaluate factors associated with the presence of pre-vaccination antibodies and with the antibody response to vaccination. Ninety-seven dogs, not vaccinated within 1 year before enrollment, were vaccinated with a modified live CDV vaccine. A measurement of the antibodies was performed before vaccination (day 0), on day 7, and 28 after the vaccination by virus neutralization. A response to vaccination was defined as a ≥4-fold titer increase by day 28. Fisher's exact test was used to determine factors associated with a lack of antibodies and vaccination response. In total, 94.8% of the dogs (92/97; CI 95%: 88.2-98.1) had antibodies (≥10) prior to vaccination. A response to vaccination was not observed in any dog. Five dogs were considered humoral non-responders; these dogs neither had detectable antibodies before, nor developed antibodies after vaccination. Young age (<2 years) was significantly associated with a lack of pre-vaccination antibodies (p = 0.018; OR: 26.825; 95% CI: 1.216-1763.417). In conclusion, necessity of re-vaccination in adult healthy dogs should be debated and regular vaccinations should be replaced by antibody detection.

Natural distemper infection in stone martens (Martes foina): From infection to neutralizing antibodies.

We report an outbreak of canine distemper virus (CDV) among stone martens (Martes foina) in Italy. After being rescued in Northern Italy between April and June 2018, six subjects were kept in a wildlife and exotic animal rescue center in Bologna province. Subjects have been monitored for 15 months in captivity. Within this time-lapse, two subjects died, while among the remaining four, only one showed clinical symptoms referable to distemper. Surviving subjects have been regularly tested for CDV by means of reverse transcriptase-PCR from conjunctival and oropharyngeal swabs for eleven months. The identified viruses belonged to the Wildlife-Europe CDV genetic subgroup. Neutralizing antibodies were detected at the end of the eleven months, when all subjects tested reverse transcriptase-PCR negative. Our findings confirm the circulation of the Wildlife-Europe CDV genetic subgroup (Europe 1/South America 1 lineage) within the Italian wildlife, and improve knowledge on viral infection in stone martens.

Comparison of four commercially available point-of-care tests to detect antibodies against canine distemper virus in dogs.

Pre-vaccination antibody testing to determine dogs' immunity against canine distemper virus (CDV) is increasingly used. Four point-of-care tests (POC A-D) are available in Europe, but their diagnostic accuracy has not been compared. The study evaluated the diagnostic accuracy and usability of these tests. Sera of client-owned dogs (n = 198; healthy n = 22; unhealthy dogs n = 176) and specific pathogen-free (SPF) dogs (n = 40) were included. Virus neutralisation (VN) was performed as the reference standard. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and overall accuracy (OA) were determined. McNemar's test was used to determine significant differences between specificity and sensitivity of the tests and Cohen's kappa was used to assess agreement. The prevalence of anti-CDV antibodies by VN was 80% in client-owned dogs overall, with 100% prevalence in healthy dogs, and 0% in SPF dogs. POC-C and POC-D were considered easiest to perform. Specificity of all tests was high using sera from SPF dogs (88-100%). In healthy dogs, sensitivity was variable (45-98%). Specificity was low in all four POC tests when using sera from acutely ill dogs (6-53%) and clinically healthy dogs with chronic disease (5-77%). In client-owned dogs, including healthy and unhealthy dogs, agreement was poor between tests. All POC tests had a low specificity when investigating sera from ill client-owned dogs and usefullness of these tests especially in dogs that are acutely ill or have chronic disease is not supported by this study.

Diagnóstico de cinomose canina por meio de teste imunocromatográfico e sua correlação com achados clínicos e hematológicos no semiárido da Paraíba / Diagnosis of canine distemper by immunochromatographic test and its correlation with clinical and hematological findings in the semiarid region of Paraíba

Objetivou-se através deste trabalho, determinar a prevalência de cinomose canina no semiárido da Paraíba, através de testes rápidos imunocromatográficos, correlacionando-a com os principais achados clínicos e hematológicos. Foram analisadas 67 fichas de animais em que foram realizados testes rápidos para pesquisa de antígeno em amostras nasais e oculares no período de janeiro a dezembro de 2019. Observou-se que 47% (32/67) dos cães analisados foram positivos para cinomose canina. As variáveis que apresentaram diferença estatística significativa (p<0,05) para a infecção foram animais sem raça definida 60% (21/35), animais não vacinados 70% (29/42), e período seco do ano, sendo o mês de agosto (40%; 13/32), com maior ocorrência. Os principais sistemas afetados foram o respiratório 61% (17/28), oftalmológico 70% (22/31), nervoso 69% (13/19), dermatológico 45% (9/20), e gastrintestinal 42% (6/14). As principais alterações hematológicas foram anemia 66% (23/32), leucopenia 76% (19/25) e linfopenia 48% (15/31). Concluiu-se que foi elevada a ocorrência de cinomose canina em animais com suspeita clínica no Semiárido Paraibano, e animais sem raça definida, não vacinados, no período seco do ano foram mais diagnosticados com a enfermidade.

The objective of this study was to determine the prevalence of distemper canine distemper vírus (CDV) infection in the semi-arid region of Paraíba, using rapid immunochromatographic tests, correlating it with the main clinical and hematological findings. 67 records of animals were analyzed in which rapid tests were performed for antigen research in nasal and ocular from January to December 2019. It was observed that 47% (32/67) of compulsory dogs were positive for canine distemper. The variables that defined difference difference (p <0.05) for infection were mixed breed animals 60% (21/35), unvaccinated animals 70% (29/42), and dry period of the year, being the August (40%; 13/32), with greater occurrence. The main affected systems were the respiratory 61% (17/28), ophthalmological 70% (22/31), nervous 69% (13/19), dermatological 45% (9/20), and gastrointestinal 42% (6/14 )) The main changes were hematological, anemia 66% (23/32), leukopenia 76% (19/25) and lymphopenia 48% (15/31). It was concluded that the occurrence of canine distemper in animals with clinical suspicion in the Semiarid Paraibano was high, and non-vaccinated mixed-breed animals in the dry period of the year were more diagnosed with the disease.

Canine distemper in neotropical procyonids: Molecular evidence, humoral immune response and epidemiology.

Canine Distemper Virus (CDV) can produce a fatal multisystem disease in carnivores and other mammals and is an important threat for wildlife conservation. However, integrative and comparative studies in wild carnivores are scarce and some areas of the world lack of genetic studies. We explore the dynamic of host-CDV in a procyonid community during an outbreak. This study reports for the first time an index case occurred in a common raccoon (Procyon lotor) and for which a complete CDV diagnosis was performed. The long-term epidemiological analysis in two sympatric populations of common raccoons and white-nosed coatis (Nasua narica) was achieved through seroneutralization, RT-PCR and direct immunofluorescence assays. Additionally, hematologic analyses were performed and phylogenetic reconstruction of CDV was done using molecular data from this study. Overall prevalence for white-nosed coatis was 19.6 % and for common raccoons was 25.3 % by seroneutralization, and 13.3 % and 17.3 % by RT-PCR. Antibodies titer average for white-nosed coatis was 1:512 and 1:156 for common raccoons. Significant difference in prevalence between white-nosed coatis and common raccoons was detected during one season (summer 2013). White-nosed coatis showed differences in erythrocytes and monocytes counts between positives and negative animals. A 100 % similarity was found between CDV of white-nosed coati and CDV of common raccoon and is a new CDV sequence not previously described; this sequence is close to Asian and European lineage. An endemic state of distemper in both species was observed but showed different dynamics over time per host species. Differences in cellular and humoral responses were also detected between procyonids. The evidence found here may have serious implications for CDV understanding in wild carnivores, it reveals clear differences in the response over time to the same CDV strain, in two close related carnivore species.

Vaccination and monitoring strategies for epidemic prevention and detection in the Channel Island fox (Urocyon littoralis).

Disease transmission and epidemic prevention are top conservation concerns for wildlife managers, especially for small, isolated populations. Previous studies have shown that the course of an epidemic within a heterogeneous host population is strongly influenced by whether pathogens are introduced to regions of relatively high or low host densities. This raises the question of how disease monitoring and vaccination programs are influenced by spatial heterogeneity in host distributions. We addressed this question by modeling vaccination and monitoring strategies for the Channel Island fox (Urocyon littoralis), which has a history of substantial population decline due to introduced disease. We simulated various strategies to detect and prevent epidemics of rabies and canine distemper using a spatially explicit model, which was parameterized from field studies. Increasing sentinel monitoring frequency, and to a lesser degree, the number of monitored sentinels from 50 to 150 radio collared animals, reduced the time to epidemic detection and percentage of the fox population infected at the time of detection for both pathogens. Fox density at the location of pathogen introduction had little influence on the time to detection, but a large influence on how many foxes had become infected by the detection day, especially when sentinels were monitored relatively infrequently. The efficacy of different vaccination strategies was heavily influenced by local host density at the site of pathogen entry. Generally, creating a vaccine firewall far away from the site of pathogen entry was the least effective strategy. A firewall close to the site of pathogen entry was generally more effective than a random distribution of vaccinated animals when pathogens entered regions of high host density, but not when pathogens entered regions of low host density. These results highlight the importance of considering host densities at likely locations of pathogen invasion when designing disease management plans.

Contribution of astrocytes and macrophage migration inhibitory factor to immune-mediated canine encephalitis caused by the distemper virus.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that is produced by many cell types in situations of homeostasis or disease. One of its functions is to act as a proinflammatory molecule. In humans, several studies have shown that MIF levels become elevated in the serum, urine, cerebrospinal fluid and tissues of patients with chronic inflammatory diseases (systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, sepsis, atheromas, diabetes and cancer). In dogs, distemper is a viral infectious condition that may lead to demyelination and inflammation in the central nervous system (CNS). In addition to the action of the virus, the inflammatory process may give rise to lesions in the white matter. Therefore, the objectives of the present study were to evaluate the role of MIF in the encephalitis that the canine distemper virus causes and to compare this with immunodetection of major histocompatibility complex-II (MHC-II), CD3 T lymphocytes, MMP-9 and glial fibrillary acidic protein (GFAP; astrocytes) in demyelinated areas of the encephalon, in order to ascertain whether these findings might be related to the severity of the encephalic lesions. To this end, a retrospective study on archived paraffinized blocks was conducted, in which 21 encephala from dogs that had been naturally infected with the canine distemper virus (infected group) and five from dogs that had been free from systemic or CNS-affecting diseases (control group) were used. In the immunohistochemical analysis on the samples, the degree of marking by GFAP, MHC-II, MMP-9 and MIF was greater in the demyelinated areas and in the adjacent neuropil, and this was seen particularly in astrocytes. Detection of CD3 was limited to perivascular cuffs. In areas of liquefactive necrosis, Gitter cells were positive for MMP-9, MIF and MHC-II. Hence, it was concluded that activated astrocytes influenced the afflux of T lymphocytes to the encephalon (encephalitis). In the more advanced phases, activated phagocytes in the areas of liquefactive necrosis (Gitter cells) continued to produce inflammatory mediators even after the astrocytes in these localities had died, thereby worsening the encephalic lesions. Distemper virus-activated astrocytes and microglia produce MIF that results in proinflammatory stimulus on glial cells and brain-infiltrating leukocytes. Therefore, the effect of the inflammatory response is potentiated on the neuropil, resulting in neurological clinical signs.

Prevalence and risk factors for the presence of serum antibodies against canine distemper, canine parvovirus, and canine adenovirus in communities in mainland Ecuador.

The purpose of this study was to estimate the apparent prevalence and identify risk factors for antibody levels (AL) against canine distemper virus (CDV), canine parvovirus (CPV), and canine adenovirus (CAV) in three communities in the metropolitan area of Quito, Ecuador that have limited access to regular veterinary care. Whole blood samples were collected from 154 dogs presenting to three veterinary field clinics in mainland Ecuador and tested for AL against CDV, CPV, and CAV by a commercially available point-of-care ELISA. Potential risk factors for the presence of AL were analyzed. A majority of dogs had AL against CDV (66%, 95% CI = 58-73%), CPV (95%, 95% CI = 91-98%) and CAV (60%, 95% CI = 52-67%). Dogs had significantly greater odds of AL against CDV if they were >2 years of age, from an urban community, and had previously received veterinary care. Dogs had significantly greater odds of AL against CAV if they were male, >2 years of age, and had previously received veterinary care. Results provide baseline estimates of AL within each community and allow for the targeting of future veterinary services to communities and dogs most at risk.

Neurotoxic potential of reactive astrocytes in canine distemper demyelinating leukoencephalitis.

Canine distemper virus (CDV) causes a fatal demyelinating leukoencephalitis in young dogs resembling human multiple sclerosis. Astrocytes are the main cellular target of CDV and undergo reactive changes already in pre-demyelinating brain lesions. Based on their broad range of beneficial and detrimental effects in the injured brain reactive astrogliosis is in need of intensive investigation. The aim of the study was to characterize astrocyte plasticity during the course of CDV-induced demyelinating leukoencephalitis by the aid of immunohistochemistry, immunofluorescence and gene expression analysis. Immunohistochemistry revealed the presence of reactive glial fibrillary acidic protein (GFAP)+ astrocytes with increased survivin and reduced aquaporin 4, and glutamine synthetase protein levels, indicating disturbed blood brain barrier function, glutamate homeostasis and astrocyte maladaptation, respectively. Gene expression analysis revealed 81 differentially expressed astrocyte-related genes with a dominance of genes associated with neurotoxic A1-polarized astrocytes. Accordingly, acyl-coA synthetase long-chain family member 5+/GFAP+, and serglycin+/GFAP+ cells, characteristic of A1-astrocytes, were found in demyelinating lesions by immunofluorescence. In addition, gene expression revealed a dysregulation of astrocytic function including disturbed glutamate homeostasis and altered immune function. Observed findings indicate an astrocyte polarization towards a neurotoxic phenotype likely contributing to lesion initiation and progression in canine distemper leukoencephalitis.

Interferon-Stimulated Genes-Mediators of the Innate Immune Response during Canine Distemper Virus Infection.

The demyelinating canine distemper virus (CDV)-leukoencephalitis represents a translational animal model for multiple sclerosis. The present study investigated the expression of type I interferon (IFN-I) pathway members in CDV-induced cerebellar lesions to gain an insight into their role in lesion development. Gene expression of 110 manually selected genes in acute, subacute and chronic lesions was analyzed using pre-existing microarray data. Interferon regulatory factor (IRF) 3, IRF7, signal transducer and activator of transcription (STAT) 1, STAT2, MX protein, protein kinase R (PKR), 2'-5'-oligoadenylate synthetase (OAS) 1 and interferon-stimulated gene (ISG) 15 expression were also evaluated using immunohistochemistry. Cellular origin of STAT1, STAT2, MX and PKR were determined using immunofluorescence. CDV infection caused an increased expression of the antiviral effector proteins MX, PKR, OAS1 and ISG15, which probably contributed to a restricted viral replication, particularly in neurons and oligodendrocytes. This increase might be partly mediated by IRF-dependent pathways due to the lack of changes in IFN-I levels and absence of STAT2 in astrocytes. Nevertheless, activated microglia/macrophages showed a strong expression of STAT1, STAT2 and MX proteins in later stages of the disease, indicating a strong activation of the IFN-I signaling cascade, which might be involved in the aggravation of bystander demyelination.

Canine distemper virus increased the differentiation of CD4+CD8+ T cells and mRNA expression of inflammatory cytokines in peripheral blood lymphocyte from canine.

BACKGROUND: Canine distemper virus (CDV) can cause a highly contagious disease to canid. However, how CDV affects peripheral blood lymphocyte (PBL) remains unclear. METHODS: In this study, CDV infected PBL was cultured to investigate the effect of CDV on the differentiation of lymphocytes and the mRNA expression of inflammatory cytokines in PBL. RESULTS: The results showed that CDV changed the phenotype of lymphocytes and increased the percentage of CD4+CD8+ T cells. To explore the effect of immune response of lymphocytes to CDV, the mRNA expression of pro- and anti-inflammatory cytokines was examined. Interleukin (IL-6, IL-12B), and tumor necrosis factor (TNF)-α mRNA expression was significantly increased at 12-48 h after CDV infection. IL-10 mRNA expression was dramatically enhanced at 12-36 h after CDV infection. However, IL-4 and transforming growth factor (TGF-ß) were not response to CDV infection. These results indicated that PBL differentiated intoCD4+CD8+ T cells and improved the inflammatory response to CDV infection. CONCLUSIONS: After CDV infection, PBL differentiated into CD4+CD8+ T cells and initiated inflammatory response.

A recombinant canine distemper virus expressing interleukin-7 enhances humoral immunity.

Canine distemper (CD) causes gastrointestinal and respiratory and/or neurological signs and results in high morbidity and mortality, remaining a threat to carnivores around the world. Live-attenuated vaccines have been widely used to reduce the number of CD outbreaks, but efforts are still needed to improve immune efficiency. Interleukin-7 (IL-7) has been reported to boost host immunity by recruiting follicle helper T (TFH) or germinal center (GC) B cells. Here, we constructed a recombinant canine distemper virus (rCDV) by reverse genetics and evaluated the properties of six intergenic sites for insertion of a foreign gene. We found that the P/M intergenic region was the optimal site to insert a foreign gene into the CDV genome. The effect of overexpressing IL-7 on rCDV immunogenicity was then evaluated in a mouse model. We found that mice immunized with rCDV-IL7 could not significantly enhance the maturation of dendritic cells (DCs) but significantly facilitated the generation of TFH cells, GC B cells and plasma cells (PCs), as well as the formation of GCs, consequently enhancing the production of CDV-specific neutralizing antibodies and total IgG. Together, these results suggested that the overexpression of IL-7 by rCDV could enhance humoral responses by activating the TFH-GC B-PC axis, which will help to improve vaccines for CD.

The serological response in dogs inoculated with canine distemper virus vaccine at the acupuncture point governing vessel-14: A randomized controlled trial.

The improvement of immunity to vaccination has historically focused on manipulation of antigen presentation rather than the host. Immune modulation by stimulating specific acupuncture points along the Meridian System has been practiced in Traditional Chinese Medicine. The purpose of this study was to quantitatively determine whether acupoint vaccination, in which vaccine is administered at an acupuncture point in dogs, has the potential to enhance the immune response. A randomized controlled trial was conducted to compare the effectiveness of acupoint vaccination versus a conventional method, based on humoral immune response in dogs given Canine Distemper Vaccine (CDV). One hundred client-owned dogs were admitted to the study with following characteristics: (1) passed a routine physical exam, (2) aged between 1 and 10 years old, (3) had no history of chronic disease, and (4) were not on immunomodulating medications. Dogs were randomly assigned to either the Acupuncture group inoculated at the acupoint Governing Vessel (GV)-14, or to the Control group inoculated conventionally at a non-acupuncture site. Mean changes from Day0 to Day14 of the response to CDV vaccination, measured by serum neutralization (SN) titers with log-transformation for reducing outlier effects, were compared between groups. No significant difference was found between groups in age, weight, or sex (all p >0.2). Both groups had significant increases of CDV SN titer post-vaccination (p < 0.001). The mean increase in Acupuncture group (0.72; SD = 0.79) was significantly greater than that of the Control group (0.36; SD = 0.67); p = 0.019. Inference on percentage of change in raw SN titer data further revealed that the effects in the Acupuncture group was significantly greater than the Control group (242% vs. 83%; p = 0.02). This study demonstrated that Acupoint vaccination at GV-14 resulted in a significantly elevated humoral immune response to CDV vaccine compared to Controls, which suggests the potential of acupoint vaccination to enhance the immune response.

Vaccination at different anatomic sites induces different levels of the immune responses.

This study was to evaluate the effects of anatomical sites for vaccination on the immune responses. In experiment A, rats were subcutaneously (s.c.) immunized with a quintuplet vaccine twice at houhai acupoint, underjaw, popliteal fossa or back with a two weeks interval. The serum specific antibody levels were determined 2, 4 and 6 weeks after second immunization. Splenocytes were separated for detection of lymphocyte proliferation and cytokine mRNA expression. In experiment B, 10 female Rottweiler puppies at their age of 34 ±â€¯2 days were subcutaneously injected with a bivalent vaccine Nobivac® Puppy DP containing live attenuated canine distemper virus (CDV) and parvovirus (CPV) for primary vaccination, and a quadrivalent vaccine Nobivac® DHPPI containing live attenuated canine distemper virus (CDV), adenovirus type 2 (CAV-2), parvovirus (CPV) and parainfluenza virus (CPIV) for subsequent vaccination at houhai acupoint (4 dogs), the shoulder (3 dogs) or the nape (3 dogs) region. Blood samples were collected at 0, 2, 4 and 6 weeks after vaccination for determination of serum specific antibody responses by ELISA. The results showed that injection of a vaccine in houhai acupoint induced the highest antibody responses in both rats and dogs. When a vaccine was injected in houhai acupoint, significantly increased proliferative responses to Con A and LPS as well as mRNA expression of IL-4, IL-10, IL-12 and IFN-γ of splenocytes were detected in rats. Therefore, houhai acupoint is recommended for injection of a vaccine to improve the immune response in dogs.

Evaluation of the humoral immune response induced by vaccination for canine distemper and parvovirus: a pilot study.

BACKGROUND: Canine Distemper Virus (CDV) and Canine Parvovirus (CPV) lead to infections with high mortality rates in dogs. These viruses affect unvaccinated dogs or dogs with incomplete vaccination protocols. Vaccination plays an important role in reducing death rates, preventing clinical cases and controlling the spread of virus However, the efficacy of vaccination might be affected by different factors including vaccine scheduling and the neutralization of the vaccine targets by maternal antibodies. In face of these factors, the main goals of this study are (i) to investigate the antibody responses of puppies undergoing different primary vaccination protocols against CPV and CDV and (ii) to estimate the time until seroreversion in adult dogs unvaccinated for at least 3 years. RESULTS: Antibody protection against CDV and CPV was evaluated in a total of 20 dogs: 5 puppies that initiated immunization at 6 weeks after birth (group A), 8 animals that started vaccination between 8 and 12 weeks of age (group B), and 7 adult dogs that have not been vaccinated for at least 3 years (group C). Blood samples were collected from each animal, with 3 to 4 weeks apart. Antibody responses were measured using indirect ELISA. In the second immunization point, no significant differences were found between the seroconversion of groups A and B for each viral infection (p = 0.81 and 0.20 for CDV and CPV, respectively). In the third immunization, there was evidence for a shorter time to achieve a protective titer against CPV in group B when compared to group A (p = 0.015). Similar evidence was not found for CDV (p-value = 0.41). In Group C, the average time until seroveversion was estimated at 2.86 years and 7.63 years for CDV and CPV, respectively. CONCLUSION: Vaccine response to CDV and CPV is specific in each individual. Effective immune protection in primary vaccination depends mainly on the initial titer of maternal antibodies acquired by the neonate. Other factors such as environmental exposure, immunization schedules and immune system activity influence the duration of immunity in adult dogs. The variability found reinforces the need to determine individual humoral immunity levels in order to assess vaccine efficacy.

Antibodies against canine distemper virus, parvovirus and Ehrlichia spp. in wild captive carnivores in midwestern Brazil / Anticorpos contra o vírus da cinomose canina, parvovírus e Ehrlichia spp. em carnívoros selvagens cativos no centro-oeste do Brasil

The occurrence of antibodies against canine distemper virus (CDV), parvovirus and Ehrlichia spp. in wild captive carnivores was evaluated in a zoological park in midwestern Brazil. Serum samples were collected between 2007 and 2014 from 45 carnivores. Antibodies were evaluated by virus neutralization assay for CDV, hemagglutination inhibition test for parvovirus, indirect immunofluorescent and Enzyme-linked immunosorbent assay for Ehrlichia spp. Antibodies against CDV and parvovirus were detected in 75% of Canidae and Felidae. Procyonidae were negative for CDV, although one Mustelidae was positive. TwoCanidae presented antibodies reactive to E. canis antigens. The high antibodies rates to CDV and parvovirus suggest the contact with both pathogens, however since no clinical history of disease are registered in the Zoo-UFMT, we can presume that carnivores have responded satisfactorily against the antigens. The low serological rates observed against Ehrlichia spp. may be resulted to the low occurrence of ticks among carnivores.(AU)

A ocorrência de anticorpos contra o vírus da cinomose canina (CDV), parvovírus e Ehrlichia spp. em carnívoros selvagens em cativeiro foi avaliada em um parque zoológico do centro oeste do Brasil. As amostras de soro foram coletadas entre 2007 e 2014 de 45 carnívoros. Os anticorpos foram avaliados por ensaio de neutralização de vírus para CDV, teste de inibição de hemaglutinação para parvovírus, imunofluorescência indireta e ensaio imunoenzimático ligado à enzima para Ehrlichia spp. Anticorpos contra CDV e parvovírus foram detectados em 75% de canídeos e felídeos. Procionídeos foram negativos para CDV, embora um mustelídeo fora positivo. Dois canídeos apresentaram anticorpos reativos aos antígenos de E. canis. As altas taxas de anticorpos para CDV e parvovírus sugerem o contato com ambos os patógenos, entretanto desde que nenhuma história clínica de doença está registrada no Zoo-UFMT, podemos presumir que os carnívoros têm respondido satisfatoriamente contra os antígenos. As baixas taxas serológicas observadas contra Ehrlichia spp. pode ser resultado da baixa ocorrência de carrapatos entre os carnívoros.(AU)

Effects of subclinical inflammation on C-reactive protein and haptoglobin levels as well as specific humoral immunity in dogs vaccinated against canine distemper and parvovirus.

BACKGROUND: The aim of the present study was to assess the effects of subclinical inflammation on specific humoral immunity in dogs vaccinated with Nobivac® DHP based on serum levels of CRP and Hp. Dogs from the group I were administered Nobivac® DHP, the vaccine against distemper, infectious hepatitis and parvovirus whereas group II animals received subcutaneous turpentine oil to induce subclinical inflammation, followed by Nobivac® DHP after 24 h. Animals in group III received only turpentine oil in the way and amount identical to that as in group II. RESULTS: Nobivac DHP relatively poorly induced the immune inflammatory response showing good immunogenic properties, which was evidenced by only a double increase in mean CRP and Hp levels associated with antigenic stimulation in group I. In group II, serum neutralization (SN) and haemagglutination inhibition (HI) results were quite closely correlated with serum levels of CPR and Hp. CONCLUSIONS: Our findings suggest that the efficacy of vaccinations in dogs can be significantly affected by subclinical inflammations, which is indicated by a correlation between serum CRP and Hp levels versus antibody titres for canine distemper and parvovirus in both experimental groups of dogs (group I and II). The correlation of mean CRP and Hp values in dogs with subclinical inflammation and after vaccination with the kinetics of increasing antibody titres against distemper and parvovirus in group II dogs reflects the severity of inflammatory response and the extent of specific humoral immunity. Routine determinations of serum CRP and Hp levels as the indices of inflammation severity can be the essential biochemical markers for assessment of dogs' health in the period preceding specific immunoprophylaxis and efficacy of the vaccine.