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1.  ‘Saving the lives of our dogs’: the development of canine distemper vaccine in interwar Britain 
This paper examines the successful campaign in Britain to develop canine distemper vaccine between 1922 and 1933. The campaign mobilized disparate groups around the common cause of using modern science to save the nation's dogs from a deadly disease. Spearheaded by landed patricians associated with the country journal The Field, and funded by dog owners and associations, it relied on collaborations with veterinary professionals, government scientists, the Medical Research Council (MRC) and the commercial pharmaceutical house the Burroughs Wellcome Company (BWC). The social organization of the campaign reveals a number of important, yet previously unexplored, features of interwar science and medicine in Britain. It depended on a patronage system that drew upon a large base of influential benefactors and public subscriptions. Coordinated by the Field Distemper Fund, this system was characterized by close relationships between landed elites and their social networks with senior science administrators and researchers. Relations between experts and non-experts were crucial, with high levels of public engagement in all aspects of research and vaccine development. At the same time, experimental and commercial research supported under the campaign saw dynamic interactions between animal and human medicine, which shaped the organization of the MRC's research programme and demonstrated the value of close collaboration between veterinary and medical science, with the dog as a shared object and resource. Finally, the campaign made possible the translation of ‘laboratory’ findings into field conditions and commercial products. Rather than a unidirectional process, translation involved negotiations over the very boundaries of the ‘laboratory’ and the ‘field’, and what constituted a viable vaccine. This paper suggests that historians reconsider standard historical accounts of the nature of patronage, the role of animals, and the interests of landed elites in interwar British science and medicine.
doi:10.1017/S0007087413000344
PMCID: PMC4014013  PMID: 24941736
2.  Characteristics of a Canine Distemper Virus Outbreak in Dichato, Chile Following the February 2010 Earthquake 
Simple Summary
A disease outbreak in domestic dogs was reported by a coastal Chilean community following the February 2010 earthquake and tsunami. Using clinical exams and diagnostic testing, canine distemper virus was confirmed. Most dogs seen had never been vaccinated, and the majority of those with positive results were recorded in dogs less than two years of age. There were no facilities to contain or treat dogs locally, and no plan to shelter free-roaming dogs. This observational study demonstrates the great need for disaster preparedness planning in developing countries that includes companion animals.
Abstract
Following the earthquake and tsunami disaster in Chile in February 2010, residents of Dichato reported high morbidity and mortality in dogs, descriptions of which resembled canine distemper virus (CDV). To assess the situation, free vaccine clinics were offered in April and May. Owner information, dog history and signalment were gathered; dogs received physical examinations and vaccines protecting against CDV, and other common canine pathogens. Blood was collected to screen for IgM antibodies to CDV. In total, 208 dogs received physical exams and vaccines were given to 177. IgM antibody titres to CDV were obtained for 104 dogs. Fifty-four dogs (51.9%) tested positive for CDV at the cut off titre of >1:50, but a total of 91.4% of dogs had a detectable titre >1:10. Most of the positive test results were in dogs less than 2 years of age; 33.5% had been previously vaccinated against CDV, and owners of 84 dogs (42.2%) reported clinical signs characteristic of CDV in their dogs following the disaster. The presence of endemic diseases in dog populations together with poor pre-disaster free-roaming dog management results in a potential for widespread negative effects following disasters. Creation of preparedness plans that include animal welfare, disease prevention and mitigation should be developed.
doi:10.3390/ani3030843
PMCID: PMC4494441  PMID: 26479537
free-roaming dogs; Chile; canine distemper virus; disaster preparedness; natural disaster; companion animals; disaster response
3.  Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs 
PLoS Neglected Tropical Diseases  2015;9(7):e0003914.
Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.
Author Summary
More than 1 million new cases of leishmaniasis occur throughout the world every year. Leishmaniasis typically presents as one of two clinical forms, either cutaneous or visceral. Dogs harboring Leishmania act as reservoirs, and are closely associated with human infections in South America and southern Europe. Therefore, immunization of dogs with effective vaccines against Leishmania will also effectively prevent Leishmania infection in humans. In this study, we have evaluated the utility of recombinant canine distemper viruses (CDVs) that express Leishmania antigen as effective polyvalent candidate vaccines against CDV and cutaneous Leishmania infections. The results indicated that recombinant CDV completely protected against challenge with a virulent strain of CDV. Furthermore, mixed immunization with three recombinant CDVs that express different antigens that mediate distinct immune responses, significantly reduced the nodule size after Leishmania major challenge. These results strongly suggest that a cocktail of multiple antigens confers more effective immunity throughout the life cycle of Leishmania. We propose that a combination of recombinant CDV-based vaccines expressing different antigens has utility as a polyvalent vaccine for the prevention of leishmaniasis epidemics by inhibiting the transmission of the parasites through dogs.
doi:10.1371/journal.pntd.0003914
PMCID: PMC4498809  PMID: 26162094
4.  Knowledge, Attitudes and Practices of Animal Bite Victims Attending an Anti-rabies Health Center in Jimma Town, Ethiopia 
PLoS Neglected Tropical Diseases  2015;9(6):e0003867.
Background
Rabies is an important but preventable cause of death in Ethiopia. We assessed the knowledge, attitudes and practices of animal bite victims attending an anti-rabies health center in Jimma Town, Ethiopia.
Methodology/Principal Findings
Between July 2012 and March 2013 a cross-sectional questionnaire was administered to 384 bite victims or their guardians in the case of minors (aged <15 years). Factors associated with knowledge, attitudes and practices were evaluated using generalized linear models. Almost all participants (99%) were aware that rabies was transmitted by the bite or lick of a rabid dog, however only 20.1% identified “germs” as the cause of disease. A majority of participants stated rabies could be prevented by avoiding dog bites (64.6%) and confining dogs (53.9%); fewer (41.7%) recognized vaccination of dogs/cats as an important preventive strategy. Regarding attitudes, most (91.1%) agreed that medical evaluation should be sought as soon as possible. However, most (75.0%) also believed that traditional healers could cure rabies. Rural residence (adjusted odds ratio [OR] = 2.1, p = 0.015) and Protestant religion (OR = 2.4, p = 0.041) were independently associated with this belief. Among 186 participants who owned dogs, only 9 (4.8%) had ever vaccinated their dog and more than 90% of respondents indicated that their dog was free-roaming or cohabitated with the family. Only 7.0% of participants applied correct first aid following exposure, and the majority (47.7%) reported that the animal was killed by the community following the incident. Female sex and Muslim religion were independently associated with higher and lower practices scores, respectively, due largely to differences in animal management practices following the incident.
Conclusions/Significance
Although respondents demonstrated reasonably sound knowledge of rabies and its transmission, attitudes and practices were inconsistent with rabies prevention. Culturally- and gender-sensitive activities that promote proper first aid and healthcare seeking behavior as well as appropriate animal management, particularly in rural areas, are needed to prevent deaths associated with rabies in this setting.
Author Summary
Rabies is an important but preventable cause of death in Ethiopia. We assessed the knowledge, attitudes and practices of animal bite victims attending an anti-rabies health center in Jimma Town, Ethiopia. We found generally high levels of knowledge about rabies. Participants recognized domestic dogs as the source and identified a range of appropriate preventive measures, including avoidance of bites and the need for dog confinement. Despite this reasonable level of knowledge, attitudes and practices that were not consistent with rabies prevention were identified. In particular, a belief that rabies could be cured by traditional healers (herbal medicine) was common among the participants, especially among those who lived in rural areas or who were of Protestant faith. Further, few of the dog owners actually confined their dogs and practiced vaccination. First aid following a suspect rabid bite was inadequate in the majority of participants and, contrary to established guidelines, most animals were killed rather than placed under quarantine following a bite incident. Female bite victims were more likely to report improved animal management practices while those of Muslim faith tended to report unfavorable animal management practices. These findings highlight the need for culturally- and gender-sensitive awareness raising programs that improve first aid and healthcare seeking behavior as well as appropriate animal management in order to prevent rabies-related deaths in Jimma Zone, Ethiopia.
doi:10.1371/journal.pntd.0003867
PMCID: PMC4482645  PMID: 26114573
5.  SUBCLINICAL INFECTION OF DOGS BY CANINE-ADAPTED MEASLES VIRUS EVIDENCED BY THEIR SUBSEQUENT IMMUNITY TO CANINE DISTEMPER VIRUS 
Journal of Bacteriology  1961;82(5):702-705.
Moura, Roberto A. (Chas. Pfizer and Company, Inc., Terre Haute, Ind.) and Joel Warren. Subclinical infection of dogs by canine-adapted measles virus evidenced by their subsequent immunity to canine distemper virus. J. Bacteriol. 82:702–705. 1961.—Young dogs were inoculated with virulent measles virus which had been adapted to canine kidney or human amnion cell culture. None of the animals showed any clinical symptoms nor could virus be isolated from the blood, although measles-neutralizing and complement-fixing antibodies developed during convalescence. All dogs failed to develop antibody to canine distemper. However, when these and normal control animals were subsequently inoculated intracerebrally with virulent distemper virus, each of the controls succumbed to typical symptoms, whereas all of the measles-immune dogs survived. These results suggest that the cross-protection conferred by measles against canine distemper virus infection involves factors other than humoral antibody. The immunity persists for a considerable length of time.
PMCID: PMC279238  PMID: 14476677
6.  Clinical and molecular investigation of a canine distemper outbreak and vector-borne infections in a group of rescue dogs imported from Hungary to Switzerland 
BMC Veterinary Research  2015;11:154.
Background
Canine distemper virus (CDV) is a major pathogen of dogs and wild carnivores worldwide. In Switzerland, distemper in domestic dogs is rarely reported. In recent years, the import of dogs from Eastern Europe to Switzerland has steadily increased. In the present study, we describe a distemper outbreak in 15 rescue dogs that were imported from Hungary to Switzerland by an animal welfare organisation. The data on vaccination and medical history were recorded (14 dogs), and the samples were collected to investigate CDV and vector-borne infections (13 dogs) and canine parvovirus infection (12 dogs). The dogs were monitored for six months.
Results
One dog was euthanised directly after import. Thirteen dogs showed clinical signs after arrival, i.e., diarrhoea (57 %), coughing (43 %) and nasal and/or ocular discharge (21 %); radiographic findings that were compatible with bronchopneumonia were present in four dogs. CDV infection was diagnosed in 11 dogs (85 %); 10 dogs (91 %) tested PCR-positive in conjunctival swabs. Vector-borne infections (Babesia spp., Leishmania infantum, Dirofilaria immitis) were found in 4 dogs (31 %). Three dogs were hospitalized, and six dogs received ambulatory therapy for up to two months until recovery. None of the dogs developed neurological disease. CDV shedding was detected for a period of up to four months. Because dogs were put under strict quarantine until CDV shedding ceased, CDV did not spread to any other dogs. The CDV isolates showed 99 % sequence identity in the HA gene among each other and belonged to the Arctic-like lineage of CDV.
Conclusions
The present study highlights the imminent risks of spreading contagious viral and vector-borne infections through the non-selective import of sick dogs and dogs with incomplete vaccination from Eastern Europe. CDV shedding was detected for several months after the cessation of clinical signs, which emphasised the roles of asymptomatic carriers in CDV epidemiology. A long-term follow-up using sensitive PCR and strict quarantine measures is of upmost importance in preventing the spread of infection. Dog owners and animal welfare organisations should be educated regarding the importance of complete vaccinations and the impact of dog imports on the spread of viral and vector-borne pathogens.
doi:10.1186/s12917-015-0471-0
PMCID: PMC4504088  PMID: 26179635
Canine distemper virus; Outbreak; Domestic dog; Import; Vector-borne infections; Phylogenetic analysis; Vaccination; Arctic-like lineage
7.  Protection against canine distemper virus in dogs after immunization with isolated fusion protein. 
Journal of Virology  1986;58(2):536-541.
Canine distemper virus attachment (hemagglutinin [H] equivalent) and fusion (F) antigens were purified by affinity chromatography with monoclonal antibodies. The purified antigens were used to immunize groups of three dogs. Radioimmune precipitation assays with sera from these animals showed that the F antigen preparation was pure and induced only an F polypeptide-specific antibody response but that the H antigen preparation had a slight contamination by the F antigen. Immunized animals were challenged with virulent canine distemper virus. Two animals in each group developed pronounced humoral and cellular immune responses after challenge. Among these infected animals, only the dogs immunized with H antigen developed symptoms, albeit mild. In contrast, three nonimmunized control animals developed severe disease, with a fatal outcome in two cases. The complete resistance against challenge in two dogs was interpreted to reflect in one case anti-F immunity and in the other case most likely a high level of anti-H immunity. It is suggested that the F antigen may be of particular interest for the development of morbillivirus and possibly other paramyxovirus subunit or synthetic vaccines, because it can induce immunity capable of blocking virus infection and in situations of virus replication prevent the emergence of symptoms.
Images
PMCID: PMC252942  PMID: 3754590
9.  Detection of Canine Distemper Virus Nucleoprotein RNA by Reverse Transcription-PCR Using Serum, Whole Blood, and Cerebrospinal Fluid from Dogs with Distemper 
Journal of Clinical Microbiology  1999;37(11):3634-3643.
Reverse transcription-PCR (RT-PCR) was used to detect canine distemper virus (CDV) nucleoprotein (NP) RNA in serum, whole blood, and cerebrospinal fluid (CSF) samples from 38 dogs with clinically suspected distemper. Results were correlated to clinical findings, anti-CDV neutralizing antibody titers, postmortem findings, and demonstration of CDV NP antigen by immunohistochemistry. The specificity of the RT-PCR was ensured by amplification of RNA from various laboratory CDV strains, restriction enzyme digestion, and Southern blot hybridization. In 29 of 38 dogs, CDV infection was confirmed by postmortem examination and immunohistochemistry. The animals displayed the catarrhal, systemic, and nervous forms of distemper. Seventeen samples (serum, whole blood, or CSF) from dogs with distemper were tested with three sets of primers targeted to different regions of the NP gene of the CDV Onderstepoort strain. Expected amplicons were observed in 82, 53, and 41% of the 17 samples, depending upon the primer pair used. With the most sensitive primer pair (primer pair I), CDV NP RNA was detected in 25 of 29 (86%) serum samples and 14 of 16 (88%) whole blood and CSF samples from dogs with distemper but not in body fluids from immunohistochemically negative dogs. Nucleotide sequence analysis of five RT-PCR amplicons from isolates from the field revealed few silent point mutations. These isolates exhibited greater homology to the Rockborn (97 to 99%) than to the Onderstepoort (95 to 96%) CDV strain. In summary, although the sensitivity of the RT-PCR for detection of CDV is strongly influenced by the location of the selected primers, this nucleic acid detection system represents a highly specific and sensitive method for the antemortem diagnosis of distemper in dogs, regardless of the form of distemper, humoral immune response, and viral antigen distribution.
PMCID: PMC85712  PMID: 10523566
10.  Rapid and Sensitive Detection of Immunoglobulin M (IgM) and IgG Antibodies against Canine Distemper Virus by a New Recombinant Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay 
Journal of Clinical Microbiology  1999;37(4):1049-1056.
Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups. Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in Heliothis virescens larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (κ = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of ≥50% showed very good inter-rater agreement (κ = 0.968) with V-NA titers of ≥1/100 50% neutralizing dose (ND50) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of ≥1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR. The recombinant capture-sandwich ELISA detecting N-specific antibodies of the IgG class provided superior sensitivity and specificity and thus represents a rapid and cost-effective alternative to classical CDV V-NA. By detection of specific IgM antibodies, the ELISA will be complementary to RT-PCR and V-NA in the diagnosis of acute distemper infections.
PMCID: PMC88648  PMID: 10074525
11.  Prevalence and risk factors for viral exposure in rural dogs around protected areas of the Atlantic forest 
Background
Despite the crucial role of domestic dogs as reservoirs for zoonosis and some of the most threatening diseases for wild carnivores such as distemper and parvovirosis, little is known about the epidemiological features and the risk factors involved in pathogen exposure of dogs that live in human/wildlife interfaces and actually contacts wildlife. Through a cross-sectional serological approach and questionnaire survey, we assessed the prevalence along with individual and environment-associated risk factors for four important viral diseases of rural dogs living in households around six Atlantic Forest fragments in southeast Brazil.
Results
Widespread exposure to canine parvovirus (97 %), canine distemper virus (15 %) and canine adenovirus (27 %) was detected, but none for canine coronavirus. Dogs from small private reserves were more exposed to parvovirus and canine distemper virus than those from larger state parks. Exposure was associated with dog sex and age, lack of health care and the number of people in the households. Remarkably, factors linked to free-ranging behaviour of dogs were associated with the exposure for all pathogens detected.
Conclusions
According to identified associations, reducing viral pathogen exposure in dogs will require inhibiting dog’s movements and access to nearby forests and villages and improving veterinary assistance. Promoting dog vaccination and population control through sterilization around protected areas is also necessary. The study provides support for preventive management actions aimed to protect the health of rural dogs, and consequently of Atlantic Forest’s wild carnivores.
doi:10.1186/s12917-016-0646-3
PMCID: PMC4730773  PMID: 26822375
Atlantic Forest; Canis familiaris; Epidemiology; Virus exposure; Risk factor; Human/wildlife interface
12.  Molecular and serological surveillance of canine enteric viruses in stray dogs from Vila do Maio, Cape Verde 
Background
Infections caused by canine parvovirus, canine distemper virus and canine coronavirus are an important cause of mortality and morbidity in dogs worldwide. Prior to this study, no information was available concerning the incidence and prevalence of these viruses in Cape Verde archipelago.
Results
To provide information regarding the health status of the canine population in Vila do Maio, Maio Island, Cape Verde, 53 rectal swabs were collected from 53 stray dogs during 2010 and 93 rectal swabs and 88 blood samples were collected from 125 stray dogs in 2011. All rectal swabs (2010 n = 53; 2011 n = 93) were analysed for the presence of canine parvovirus, canine distemper virus and canine coronavirus nucleic acids by quantitative PCR methods. Specific antibodies against canine distemper virus and canine parvovirus were also assessed (2011 n = 88).
From the 2010 sampling, 43.3% (23/53) were positive for canine parvovirus DNA, 11.3% (6/53) for canine distemper virus RNA and 1.9% (1/53) for canine coronavirus RNA. In 2011, the prevalence values for canine parvovirus and canine coronavirus were quite similar to those from the previous year, respectively 44.1% (41/93), and 1.1% (1/93), but canine distemper virus was not detected in any of the samples analysed (0%, 0/93). Antibodies against canine parvovirus were detected in 71.6% (63/88) blood samples and the seroprevalence found for canine distemper virus was 51.1% (45/88).
Conclusions
This study discloses the data obtained in a molecular and serological epidemiological surveillance carried out in urban populations of stray and domestic animals. Virus transmission and spreading occurs easily in large dog populations leading to high mortality rates particularly in unvaccinated susceptible animals. In addition, these animals can act as disease reservoirs for wild animal populations by occasional contact. Identification of susceptible wildlife of Maio Island is of upmost importance to evaluate the risk of pathogen spill over from domestic to wild animals in Cape Verde and to evaluate the associated threat to the wild susceptible species.
doi:10.1186/1746-6148-10-91
PMCID: PMC4005843  PMID: 24755118
Canine coronavirus; Canine distemper virus; Canine parvovirus; Cape verde; Molecular surveillance
13.  Associations between biosecurity and outbreaks of canine distemper on Danish mink farms in 2012–2013 
Background
During 8 months from July 2012 to February 2013, a major outbreak of canine distemper involving 64 mink farms occurred on the Danish peninsula of Jutland. The canine distemper outbreak was associated with exposure of farmed mink to infected wild carnivores and could represent a deficit in biosecurity on the mink farms. The aim of this study was to investigate the extent and association of specific biosecurity measures with the outbreak. The study was carried out in an epidemiological case–control design. The case group consisted of the 61 farms, which had a confirmed outbreak of canine distemper from July 2012 to February 2013. The control group included 54 farms without an outbreak of canine distemper in 2012 or 2013, selected as the closest geographical neighbour to a case farm.
Results
The results showed that significantly more control than case farms had vaccinated their mink against canine distemper virus. Mortality was only assessed on the case farms, and there was a non-significantly lower mortality on vaccinated farms than on the non-vaccinated farms. Furthermore, the proportion of farms with observations of wild red foxes (Vulpes vulpes) inside the farm enclosures were larger for case farms, indicating that the control farms had a better biosecurity or were not equally exposed to canine distemper virus. Generally, all farms had very few specific precautions at the gate entrance in respect to human visitors as well as animals. The use of biosecurity measures was very variable in both case and control farms. Not using plastic boot covers, presence of dogs and cats, presence of demarcated area for changing clothes when entering and leaving the farm area and presence of hand washing facilities significantly lowered the odds of the farm having a canine distemper virus outbreak.
Conclusions
The results of the study indicate that consistent use of correct vaccination strategies, implementation of biosecurity measures and limiting human and animal access to the mink farm can be important factors in reducing the risk for canine distemper outbreaks.
Electronic supplementary material
The online version of this article (doi:10.1186/s13028-015-0159-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13028-015-0159-2
PMCID: PMC4589036  PMID: 26423523
Canine distemper virus; Biosecurity; Mink; Neovison vison
14.  Canine distemper virus induces apoptosis in cervical tumor derived cell lines 
Virology Journal  2011;8:334.
Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV) induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi), by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control.
doi:10.1186/1743-422X-8-334
PMCID: PMC3141686  PMID: 21718481
Apoptosis; Canine distemper virus; Caspase; Cervical tumor; HeLa cell; HPV
15.  Design of different strategies of multivalent DNA-based vaccination against rabies and canine distemper in mice and dogs 
Virology Journal  2012;9:319.
Background
During the vaccination campaigns, puppies younger than 3 months old are not targeted and remain unvaccinated for at least the first year of their lives. Almost half of the reported rabid dogs are 6 months or younger. Hence, we should recommend the vaccination against rabies of young puppies. Unfortunately, owing to the exposure of puppies to infections with either canine parvovirus (CPV) or distemper virus (CDV) after the intervention of the vaccinators, owners are reluctant to vaccinate puppies against rabies. Therefore, it is necessary to include the CPV and CDV valences in the vaccine against rabies. Multivalent DNA-based vaccination in dogs, including rabies and distemper valences, could help in raising vaccine coverage.
Methods
We have designed monovalent and multivalent DNA-based vaccine candidates for in vitro and in vivo assays. These plasmids encode to the rabies virus glycoprotein and/or the canine distemper virus hemagglutinin. The first strategy of multivalent DNA-based vaccination is by mixing plasmids encoding to a single antigen each. The second is by simply fusing the genes of the antigens together. The third is by adding the foot and mouth disease virus (FMDV) 2A oligopeptide gene into the antigen genes. The last strategy is by the design and use of a bicistronic plasmid with an “Internal Ribosome Entry Site” (IRES) domain.
Results
The monovalent construct against canine distemper was efficiently validated by inducing higher humoral immune responses compared to cell-culture-derived vaccine both in mice and dogs. All multivalent plasmids efficiently expressed both valences after in vitro transfection of BHK-21 cells. In BALB/c mice, the bicistronic IRES-dependant construct was the most efficient inducer of virus-neutralizing antibodies against both valences. It was able to induce better humoral immune responses compared to the administration of either cell-culture-derived vaccines or monovalent plasmids. The FMDV 2A was also efficient in the design of multivalent plasmids.
Conclusions
In a single shot, the design of efficient multivalent plasmids will be very beneficial for DNA-based vaccination against numerous diseases.
doi:10.1186/1743-422X-9-319
PMCID: PMC3547725  PMID: 23270301
Rabies; CDV; DNA Vaccine; Multivalent; Public health; Zoonose
16.  Improvement Control System of Rabies in Ukraine 
Objective
The purpose of the research was to find out the reasons of rabies antropurgisation in Ukraine.
Introduction
In Ukraine in spite of considerable financial expenses on oral immunization of foxes and parenteral immunization of dogs and cats, it is not succeeded to reach considerable results in the fight with rabies. Unfortunately there was a negative tendency to increasing a part of dogs and cats in the structure of rabies disease which are the main source of rabies in people.
Methods
Analysis of 228 anamnesis data of rabies infected dogs during 2008–2012. Research of 234 samples of the blood serum from dogs on existence of antibodies to the rabies virus by the ELISA method.
Results
Analysis of animal morbidity on rabies in Ukraine in period of 2006–2011 found out the changes of structure of morbidity in animal species that means decreasing a part of wild animals (from 49,0 % in 2006 to 38,7 % in 2011) and increasing a part of dogs (from 18,3 % in 2006 to 23,2 % in 2011) and cats (from 19,8 % in 2006 to 25,0 % in 2011) in the general amount of animals which perished from rabies (Fig. 1).
A lot of Ukrainian scientists and doctors of veterinary medicine consider that the main reason of spreading the rabies is a great number of homeless animals which factually are the reservoirs of infection in towns and villages.
However, in our opinion spreading of rabies shows the insufficient level of measures of control of rabies among home animals. It was confirmed with conducted analysis that only 26 (12,9 %) dogs were stray, others 202 (87,1 %) had owners, but didn’t get necessary protective rabies vaccination.
According to Ukrainian instruction “Preventive measures against rabies of animals”, all the dogs must be vaccinated against rabies, but it actually appears it is quite not so.
At research of 234 samples of the blood serum of dogs on existence of antibodies to the rabies virus it was determined that the level of population immunity in dogs is 36,6 % in Ukrainian towns, but the protective level of antibodies was found in 9,1 % of village dogs. We consider that the main reason of this is imperfection of the Ukrainian legislation in the questions of responsibility of proprietors of animals.
For breaking of epizootic chain it is necessary the percent of vaccinated animals (in this case dogs) to be on a high level – more than 50%. Nowadays for reaching epidemic welfare in rabies it is important within the framework of the registered epidemic incidents dogs to be brought over to the ecocycles of infection sporadically without taking part in circulation of exciter, stay their biological deadlock and have low epidemic potential.
Conclusions
The conducted analysis expressly demonstrates that at present problems the eradication of rabies in Ukraine is impossible considering the low level of dogs’ protection from rabies. Obtained results were sent to the State committee of veterinary medicine of Ukraine and will be the argument for enhancement of control after conducting rabies vaccination of dogs.
Rabies cases in the years 2006–2011.
PMCID: PMC3692804
population rabies immunity; rabies vaccination; stray dogs
17.  Contact with Domestic Dogs Increases Pathogen Exposure in Endangered African Wild Dogs (Lycaon pictus) 
PLoS ONE  2012;7(1):e30099.
Background
Infectious diseases have contributed to the decline and local extinction of several wildlife species, including African wild dogs (Lycaon pictus). Mitigating such disease threats is challenging, partly because uncertainty about disease dynamics makes it difficult to identify the best management approaches. Serious impacts on susceptible populations most frequently occur when generalist pathogens are maintained within populations of abundant (often domestic) “reservoir” hosts, and spill over into less abundant host species. If this is the case, disease control directed at the reservoir host might be most appropriate. However, pathogen transmission within threatened host populations may also be important, and may not be controllable by managing another host species.
Methodology/Principal Findings
We investigated interspecific and intraspecific transmission routes, by comparing African wild dogs' exposure to six canine pathogens with behavioural measures of their opportunities for contact with domestic dogs and with other wild dogs. Domestic dog contact was associated with exposure to canine parvovirus, Ehrlichia canis, Neospora caninum and perhaps rabies virus, but not with exposure to canine distemper virus or canine coronavirus. Contact with other wild dogs appeared not to increase the risk of exposure to any of the pathogens.
Conclusions/Significance
These findings, combined with other data, suggest that management directed at domestic dogs might help to protect wild dog populations from rabies virus, but not from canine distemper virus. However, further analyses are needed to determine the management approaches – including no intervention – which are most appropriate for each pathogen.
doi:10.1371/journal.pone.0030099
PMCID: PMC3253127  PMID: 22238695
18.  Effect of owner-controlled acaricidal treatment on tick infestation and immune response to tick-borne pathogens in naturally infested dogs from Eastern Austria 
Parasites & Vectors  2013;6:62.
Background
Tick-borne infections resulting from regular tick infestation in dogs are a common veterinary health problem all over the world. The application of repellent and acaricidal agents to prevent transmission of pathogens is a major protection strategy and has been proven to be highly effective in several trials under laboratory and natural conditions in dogs. Despite such promising results, many dog owners still report tick infestation in their dogs although acaricidal agents are used. Information about the current infection status and changes of the infection status regarding tick-borne diseases (TBD) in dogs treated by the owner’s controlled acaricide application is lacking.
Methods
In this study 30 dogs were each treated with permethrin, fipronil + S-methoprene, or served as untreated controls. Application of the acaricide was performed by the owner who decided when and how often to use the spot on preparation. Over a period of 11 months, dogs were clinically examined and sampled for antibody responses against Babesia canis, Anaplasma phagocytophilum, Borrelia burgdorferi s. l., and TBE virus before the study started, 6 months later and at the end of the investigation period.
Results
The permethrin acaricide was applied on average 3.40 times within the examination period, whereas the fipronil + S-methoprene medication was applied 3.03 times. Approximately 2/3 of all dogs, independent of the group, had a positive immune response to one or more pathogens. Three dogs developed clinical symptoms of canine babesiosis, all other dogs remained healthy. Individual number of ticks per dog or number of infections per dog did not correlate with the application rate, and the number of ticks per dog did not influence the number of infections per dog. As owners did not apply the acaricides regularly no influence on the number of infections could be documented although the number of ticks was clearly reduced by the application of the spot-on drugs.
Conclusions
Clinical disease in dogs exposed to tick-borne pathogens is rare, although a humoral immune response reflecting infection is common. More educational training for dog owners is necessary to make the application of acaricides effective regarding the prevention of tick-borne diseases.
doi:10.1186/1756-3305-6-62
PMCID: PMC3623908  PMID: 23497548
Tick infestation; Tick-borne pathogens; Immune response; Acaricides; Efficacy
19.  Review on Dog Rabies Vaccination Coverage in Africa: A Question of Dog Accessibility or Cost Recovery? 
PLoS Neglected Tropical Diseases  2015;9(2):e0003447.
Background
Rabies still poses a significant human health problem throughout most of Africa, where the majority of the human cases results from dog bites. Mass dog vaccination is considered to be the most effective method to prevent rabies in humans. Our objective was to systematically review research articles on dog rabies parenteral vaccination coverage in Africa in relation to dog accessibility and vaccination cost recovery arrangement (i.e.free of charge or owner charged).
Methodology/Principal Findings
A systematic literature search was made in the databases of CAB abstracts (EBSCOhost and OvidSP), Scopus, Web of Science, PubMed, Medline (EBSCOhost and OvidSP) and AJOL (African Journal Online) for peer reviewed articles on 1) rabies control, 2) dog rabies vaccination coverage and 3) dog demography in Africa. Identified articles were subsequently screened and selected using predefined selection criteria like year of publication (viz. ≥ 1990), type of study (cross sectional), objective(s) of the study (i.e. vaccination coverage rates, dog demographics and financial arrangements of vaccination costs), language of publication (English) and geographical focus (Africa). The selection process resulted in sixteen peer reviewed articles which were used to review dog demography and dog ownership status, and dog rabies vaccination coverage throughout Africa. The main review findings indicate that 1) the majority (up to 98.1%) of dogs in African countries are owned (and as such accessible), 2) puppies younger than 3 months of age constitute a considerable proportion (up to 30%) of the dog population and 3) male dogs are dominating in numbers (up to 3.6 times the female dog population). Dog rabies parenteral vaccination coverage was compared between “free of charge” and “owner charged” vaccination schemes by the technique of Meta-analysis. Results indicate that the rabies vaccination coverage following a free of charge vaccination scheme (68%) is closer to the World Health Organization recommended coverage rate (70%) than the achieved coverage rate in owner-charged dog rabies vaccination schemes (18%).
Conclusions/Significance
Most dogs in Africa are owned and accessible for parenteral vaccination against rabies if the campaign is performed “free of charge”.
Author Summary
Rabies is one of the most fatal diseases in both humans and animals. A bite by a rabid dog is the main cause of human rabies in Africa. Parenteral mass dog vaccination is the most cost-effective tool to prevent rabies in humans. Our main objective was to review research articles on the parenteral dog rabies vaccination coverage in Africa. We aimed to review published research articles on percentage of dogs owned and percentage of dogs vaccinated against rabies, and on the relation between vaccination coverage and cost recovery. We followed the standard procedures of a systematic literature review resulting in a final review of 16 scientific articles. Our review results indicate that only a small percentage of African dogs is ownerless. Puppies younger than 3 months of age constitute a considerable proportion of the African dog population. There are considerably more male dogs than female dogs present within the dog population. The dog rabies parenteral vaccination coverage following a “free of charge” vaccination scheme (68%) is closer to World Health Organization recommended threshold coverage rate (70%) compared to the coverage rate achieved in “owner-charged” dog rabies vaccination schemes (18%). In conclusion, most dogs in Africa are owned and accessible for vaccination once the necessary financial arrangements have been made.
doi:10.1371/journal.pntd.0003447
PMCID: PMC4315526  PMID: 25646774
20.  Myelin-Specific Autoantibodies Associated with Central Nervous System Demyelination in Canine Distemper Virus Infection 
Infection and Immunity  1973;8(5):819-827.
Sera from dogs with spontaneously occurring and experimentally produced canine distemper virus-associated demyelinating encephalitis were examined for antibodies to central nervous system myelin by the complement fixation and indirect immunofluorescent methods. Complement-fixing immunoglobulin M antibodies and non-complement-fixing immunoglobulin G antibodies were found in 97% of the spontaneous cases. In comparison, only 28% of control sera contained these antibodies; furthermore, mean antibody titers in the control groups were significantly lower (P < 0.005) when compared to the distemper group. Complement-fixing antimyelin antibodies were also demonstrated in gnotobiotic dogs with experimentally induced distemper virus-associated demyelination. The antibody response could be correlated with clinicopathological features of the disease produced. Results of this study indicate that demyelination in canine distemper may proceed by immune mechanisms.
Images
PMCID: PMC422933  PMID: 4584054
21.  Glucose-6-phosphate dehydrogenase deficiency and the risk of malaria and other diseases in children in Kenya: a case-control and a cohort study 
The Lancet. Haematology  2015;2(10):e437-e444.
Summary
Background
The global prevalence of X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency is thought to be a result of selection by malaria, but epidemiological studies have yielded confusing results. We investigated the relationships between G6PD deficiency and both malaria and non-malarial illnesses among children in Kenya.
Methods
We did this study in Kilifi County, Kenya, where the G6PD c.202T allele is the only significant cause of G6PD deficiency. We tested the associations between G6PD deficiency and severe and complicated Plasmodium falciparum malaria through a case-control study of 2220 case and 3940 control children. Cases were children aged younger than 14 years, who visited the high dependency ward of Kilifi County Hospital with severe malaria between March 1, 1998, and Feb 28, 2010. Controls were children aged between 3–12 months who were born within the same study area between August 2006, and September 2010. We assessed the association between G6PD deficiency and both uncomplicated malaria and other common diseases of childhood in a cohort study of 752 children aged younger than 10 years. Participants of this study were recruited from a representative sample of households within the Ngerenya and Chonyi areas of Kilifi County between Aug 1, 1998, and July 31, 2001. The primary outcome measure for the case-control study was the odds ratio for hospital admission with severe malaria (computed by logistic regression) while for the cohort study it was the incidence rate ratio for uncomplicated malaria and non-malaria illnesses (computed by Poisson regression), by G6PD deficiency category.
Findings
2863 (73%) children in the control group versus 1643 (74%) in the case group had the G6PD normal genotype, 639 (16%) versus 306 (14%) were girls heterozygous for G6PD c.202T, and 438 (11%) versus 271 (12%) children were either homozygous girls or hemizygous boys. Compared with boys and girls without G6PD deficiency, we found significant protection from severe malaria (odds ratio [OR] 0·82, 95% CI 0·70–0·97; p=0·020) among G6PD c.202T heterozygous girls but no evidence for protection among G6PD c.202T hemizygous boys and homozygous girls (OR 1·18, 0·99–1·40; p=0·056). Median follow-up for the mild disease cohort study was 2·24 years (IQR 2·22–2·85). G6PD c.202T had no effect on other common diseases of childhood in heterozygous girls (incidence rate ratio 0·98, 95% CI 0·86–1·11; p=0·82) or homozygous girls or hemizygous boys (0·93, 0·82–1·04; p=0·25), with the sole exception of a marginally significant increase in the incidence of helminth infections among heterozygous girls.
Interpretation
Heterozygous girls might be the driving force for the positive selection of G6PD deficiency alleles. Further studies are needed to definitively establish the mechanisms by which G6PD deficiency confers an advantage against malaria in heterozygous individuals. Such studies could lead to the development of new treatments.
Funding
Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health (as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative).
doi:10.1016/S2352-3026(15)00152-0
PMCID: PMC4703047  PMID: 26686045
22.  Viral Oncolysis — Can Insights from Measles Be Transferred to Canine Distemper Virus? 
Viruses  2014;6(6):2340-2375.
Neoplastic diseases represent one of the most common causes of death among humans and animals. Currently available and applied therapeutic options often remain insufficient and unsatisfactory, therefore new and innovative strategies and approaches are highly needed. Periodically, oncolytic viruses have been in the center of interest since the first anecdotal description of their potential usefulness as an anti-tumor treatment concept. Though first reports referred to an incidental measles virus infection causing tumor regression in a patient suffering from lymphoma several decades ago, no final treatment concept has been developed since then. However, numerous viruses, such as herpes-, adeno- and paramyxoviruses, have been investigated, characterized, and modified with the aim to generate a new anti-cancer treatment option. Among the different viruses, measles virus still represents a highly interesting candidate for such an approach. Numerous different tumors of humans including malignant lymphoma, lung and colorectal adenocarcinoma, mesothelioma, and ovarian cancer, have been studied in vitro and in vivo as potential targets. Moreover, several concepts using different virus preparations are now in clinical trials in humans and may proceed to a new treatment option. Surprisingly, only few studies have investigated viral oncolysis in veterinary medicine. The close relationship between measles virus (MV) and canine distemper virus (CDV), both are morbilliviruses, and the fact that numerous tumors in dogs exhibit similarities to their human counterpart, indicates that both the virus and species dog represent a highly interesting translational model for future research in viral oncolysis. Several recent studies support such an assumption. It is therefore the aim of the present communication to outline the mechanisms of morbillivirus-mediated oncolysis and to stimulate further research in this potentially expanding field of viral oncolysis in a highly suitable translational animal model for the benefit of humans and dogs.
doi:10.3390/v6062340
PMCID: PMC4074931  PMID: 24921409
canine distemper virus; measles virus; tumor treatment; viral oncolysis
23.  Canine Distemper Viral Inclusions in Blood Cells of Four Vaccinated Dogs 
The Canadian Veterinary Journal  1985;26(12):368-372.
Four cases of canine distemper were detected by the presence of numerous cytoplasmic inclusions in various circulating blood cells. Fluorescent antibody techniques and electron microscopy confirmed the identity of the viral inclusions. The cases occurred in the same geographic area and within a short time span. All four dogs had been vaccinated against canine distemper, but stress or other factors may have compromised their immune status. The possibility of an unusually virulent virus strain was also considered.
Images
PMCID: PMC1680115  PMID: 17422596
Canine distemper; inclusions; blood cells
24.  Sand Fly Salivary Proteins Induce Strong Cellular Immunity in a Natural Reservoir of Visceral Leishmaniasis with Adverse Consequences for Leishmania 
PLoS Pathogens  2009;5(5):e1000441.
Immunity to a sand fly salivary protein protects against visceral leishmaniasis (VL) in hamsters. This protection was associated with the development of cellular immunity in the form of a delayed-type hypersensitivity response and the presence of IFN-γ at the site of sand fly bites. To date, there are no data available regarding the cellular immune response to sand fly saliva in dogs, the main reservoirs of VL in Latin America, and its role in protection from this fatal disease. Two of 35 salivary proteins from the vector sand fly Lutzomyia longipalpis, identified using a novel approach termed reverse antigen screening, elicited strong cellular immunity in dogs. Immunization with either molecule induced high IgG2 antibody levels and significant IFN-γ production following in vitro stimulation of PBMC with salivary gland homogenate (SGH). Upon challenge with uninfected or infected flies, immunized dogs developed a cellular response at the bite site characterized by lymphocytic infiltration and IFN-γ and IL-12 expression. Additionally, SGH-stimulated lymphocytes from immunized dogs efficiently killed Leishmania infantum chagasi within autologous macrophages. Certain sand fly salivary proteins are potent immunogens obligatorily co-deposited with Leishmania parasites during transmission. Their inclusion in an anti-Leishmania vaccine would exploit anti-saliva immunity following an infective sand fly bite and set the stage for a protective anti-Leishmania immune response.
Author Summary
Leishmaniasis is a neglected infectious disease with a global distribution encompassing 88 countries, 350 million people at risk, and an annual incidence of 2 million cases. Leishmaniasis is a vector-borne disease transmitted by sand fly bites where parasites are co-deposited with saliva into the wound. Our group has demonstrated that distinct molecules in the saliva of various sand fly species drive an immune response that protects experimental rodent models from self-healing cutaneous and fatal visceral leishmaniasis. Here we show for the first time that dogs, natural reservoirs of visceral leishmaniasis, develop a strong immune response to two salivary proteins from the natural vector sand fly. Blood from immunized dogs contained immune cells that produced molecules (IFN-γ) typically associated with protection from Leishmania parasites. This response efficiently recruited appropriate immune cells to the site of sand fly bites in the skin and had an adverse effect on Leishmania parasites in an experimental assay. These findings suggest that inclusion of these salivary molecules in anti-Leishmania canine vaccines would enhance their efficiency in protecting dogs from visceral leishmaniasis. A successful anti-Leishmania canine vaccine would not only protect dogs from a fatal disease but could have a considerable effect on reducing human infections.
doi:10.1371/journal.ppat.1000441
PMCID: PMC2677456  PMID: 19461875
25.  Efficacy of RTS,S malaria vaccines: individual-participant pooled analysis of phase 2 data 
The Lancet Infectious Diseases  2013;13(4):319-327.
Summary
Background
The efficacy of RTS,S/AS01 as a vaccine for malaria is being tested in a phase 3 clinical trial. Early results show significant, albeit partial, protection against clinical malaria and severe malaria. To ascertain variations in vaccine efficacy according to covariates such as transmission intensity, choice of adjuvant, age at vaccination, and bednet use, we did an individual-participant pooled analysis of phase 2 clinical data.
Methods
We analysed data from 11 different sites in Africa, including 4453 participants. We measured heterogeneity in vaccine efficacy by estimating the interactions between covariates and vaccination in pooled multivariable Cox regression and Poisson regression analyses. Endpoints for measurement of vaccine efficacy were infection, clinical malaria, severe malaria, and death. We defined transmission intensity levels according to the estimated local parasite prevalence in children aged 2–10 years (PrP2–10), ranging from 5% to 80%. Choice of adjuvant was either AS01 or AS02.
Findings
Vaccine efficacy against all episodes of clinical malaria varied by transmission intensity (p=0·001). At low transmission (PrP2–10 10%) vaccine efficacy was 60% (95% CI 54 to 67), at moderate transmission (PrP2–10 20%) it was 41% (21 to 57), and at high transmission (PrP2–10 70%) the efficacy was 4% (−10 to 22). Vaccine efficacy also varied by adjuvant choice (p<0·0001)—eg, at low transmission (PrP2–10 10%), efficacy varied from 60% (95% CI 54 to 67) for AS01 to 47% (14 to 75) for AS02. Variations in efficacy by age at vaccination were of borderline significance (p=0·038), and bednet use and sex were not significant covariates. Vaccine efficacy (pooled across adjuvant choice and transmission intensity) varied significantly (p<0·0001) according to time since vaccination, from 36% efficacy (95% CI 24 to 45) at time of vaccination to 0% (−38 to 38) after 3 years.
Interpretation
Vaccine efficacy against clinical disease was of limited duration and was not detectable 3 years after vaccination. Furthermore, efficacy fell with increasing transmission intensity. Outcomes after vaccination cannot be gauged accurately on the basis of one pooled efficacy figure. However, predictions of public-health outcomes of vaccination will need to take account of variations in efficacy by transmission intensity and by time since vaccination.
Funding
Medical Research Council (UK); Bill & Melinda Gates Foundation Vaccine Modelling Initiative; Wellcome Trust.
doi:10.1016/S1473-3099(13)70005-7
PMCID: PMC3771416  PMID: 23454164

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