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.
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.
In December 2000, an infectious disease spread through a captive breeding group of African wild dogs (Lycaon pictus) in Tanzania, killing 49 of 52 animals within 2 months. The causative agent was identified as Canine distemper virus (CDV) by means of histologic examination, virus isolation, reverse transcriptase-polymerase chain reaction analysis, and nucleotide sequencing. This report emphasizes the importance of adequate protection against infectious diseases for the successful outcome of captive breeding programs of endangered species.
Morbillivirus; canine distemper virus; African wild dogs
One of the greatest threats to the conservation of wild cat populations may be dogs or, at least, one of their viruses. Canine distemper virus (CDV), a single-stranded RNA virus in the Paramyxoviridae family and genus Morbillivirus, infects and causes disease in a variety of species, not just canids. An outbreak of CDV in wild lions in the Serengeti, Tanzania, in 1994 was a wake-up call for conservationists, as it demonstrated that an infectious disease could swiftly impact a previously healthy felid population. To understand how this virus causes disease in noncanid hosts, researchers have focused on specific mutations in the binding site of the CDV hemagglutinin gene. Now, Seimon et al. provide information on CDV in its latest feline victim, the endangered wild Amur tiger (Panthera tigris altaica) [T. A. Seimon et al., mBio 4(4):e00410-13, 2013, doi:10.1128/mBio.00410-13]. Their findings of CDV strains infecting tigers, in combination with recent information from other felids, paints a different picture, one in which CDV strains from a variety of geographic lineages and with a variety of amino acid residues in the hemagglutinin gene binding site can infect cats and cause disease. Although CDV has been known as a multihost disease since its discovery in domestic dogs in 1905, perhaps it is time to reconsider whether these noncanid species are not just incidental or “spillover” hosts but, rather, a normal part of the complex ecology of this infectious disease.
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.
Canine distemper; inclusions; blood cells
Historians and epidemiologists have only recently begun to explore the Nazi anti-tobacco movement. Germany had the world's strongest antismoking movement in the 1930s and early 1940s, encompassing bans on smoking in public spaces, bans on advertising, restrictions on tobacco rations for women, and the world's most refined tobacco epidemiology, linking tobacco use with the already evident epidemic of lung cancer. The anti-tobacco campaign must be understood against the backdrop of the Nazi quest for racial and bodily purity, which also motivated many other public health efforts of the era.
Dr Kottow in his paper Classical medicine v alternative medical practices (1) places the alternative/orthodox medicine debate within an historical context of anti-quackery literature. My paper explores the nature of science as it is applied to clinical practice and challenges the narrow view of the diagnostic process as outlined by Dr Kottow. Research methodologies more appropriate to 'whole person' medicine are suggested as having more ethical value than those based on the clinical trial.
Serum antibody titers for canine parvovirus type-2 (CPV-2), canine distemper virus (CDV) and canine adenovirus type-1 (CAV-1) were investigated in 1031 healthy adult household dogs (2 to 18 years old) given an annual inoculation in the previous 11 to 13 months. The number of dogs retaining significant titers of antibodies against CPV-2, CDV, and CAV-1 were 888 (86%), 744 (72%), and 732 (71%), respectively. There were no differences between males and females in antibody titers against the 3 viruses. Antibody titer for CPV-2 was significantly higher in younger dogs than in older dogs, CDV antibody was significantly higher in older dogs than in younger dogs, and CAV titer was not associated with age.
A radioimmunoprecipitation-polyacrylamide gel electrophoresis technique was applied to sera from canine distemper virus-infected dogs. Sera from fatally infected dogs precipitated only the nucleoprotein, the matrix protein, and trace amounts of fusion glycoprotein. Sera from normal convalescent dogs precipitated all five major polypeptides. In contrast, sera from persistently infected dogs were characterized by a modest overall response compared with sera from convalescent dogs and by no or little response to the matrix and phosphorylated proteins until 5 to 7 weeks after infection.
The objective of this study was to determine whether post-vaccination antibody titers vary according to body weight in adult dogs. Antibody titers against canine parvovirus type 2 (CPV-2), canine distemper virus (CDV), and canine adenovirus type 1 (CAdV-1) were measured for 978 domestic adult dogs from 2 to 6 y of age. The dogs had been vaccinated approximately 12 mo earlier with a commercial combination vaccine. The dogs were divided into groups according to their weight. It was found that mean antibody titers in all weight groups were sufficient to prevent infection. Intergroup comparison, however, revealed that CPV-2 antibody titers were significantly higher in the Super Light (< 5 kg) group than in the Medium (10 to 19.9 kg) and Heavy (> 20 kg) groups and were also significantly higher in the Light (5 to 9.9 kg) group than in the Heavy group. Antibody titers against CDV were significantly higher in the Super Light, Light, and Medium groups than in the Heavy group. There were no significant differences among the groups for the CAdV-1 antibody titers.
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.
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.
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.
In a single shot, the design of efficient multivalent plasmids will be very beneficial for DNA-based vaccination against numerous diseases.
Rabies; CDV; DNA Vaccine; Multivalent; Public health; Zoonose
Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.
A simple and rapid (24-h) assay for peripheral blood lymphocyte-associated immunity to canine distemper virus (CDV) is described. The test is based upon leukocyte-associated inhibition of CDV-induced syncytia formation in Vero cells. The technique quantitates the response morphologically, thereby eliminating the requirement for release of radiolabeled compounds. Positive results were determined from specific-pathogen-free and gnotobiotic dogs exposed to CDV via hyperimmunization, vaccination with a modified live virus vaccine, and after virulent virus infection. Preinoculation lymphocytes and lymphocytes from non-immune dogs did not inhibit CDV-induced syncytia formation. Maximum responses were observed 7 to 21 days after initial exposure and declined thereafter. The method can be used to further investigate the role of immune lymphocytes in the recovery from CDV infection.
A canine distemper virus (CDV), DESIGNATED R252, originally recovered from a dog with demyelinating encephalomyelitis was shown to reproduce this disease in gnotobiotic dogs with a high incidence as compared to other CDV strains, which produced an acute fatal infection. In this investigation, R252 was propagated for the first time in Vero cells and compared to two known strains of CDV, Snyder-Hill (SH) and Onderstepoort (Ond). The results of this study revealed that intracellular R252 accumulated more slowly than either SH or Ond. There was extensive destruction of Vero monolayers infected with either R252 or SH. Each virus induced the formation of intracytoplasmic and intranuclear inclusions. Ond infection resulted in minimal cytopathic changes and intracytoplasmic inclusions. Immunofluorescence studies indicated that the spread of R252 infection within the monolayers was intermediate between the rapidly spreading SH and slowly spreading Ond. R252-infected cells developed characteristic immunofluorescent cytoplasmic inclusions. Initially, each stained homogeneously and later appeared as a non fluorescent body surrounded by a fluorescent ring. This characteristic pattern of fluorescence was observed only infrequently in thelate stage of SH infection and was absent in Ond-infected cultures. Reciprocal neutralization studies indicated that the three strains are of one serotype. These findings suggest that R252-CDV has biological properties which differ from two other strains of CDV and which may have bearing upon the in vivo capability of this virus to produce demyelinating encephalomyelitis.
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.
Natural and immune cytolysis of canine distemper virus (CDV)-infected target cells in vitro is described. Lymphocytes expressing natural cytotoxicity were found in specific-pathogen-free beagle dogs and in beagle-coonhound crosses before vaccination with CDV and indefinitely after vaccination, when the ephemeral immune lymphocyte-mediated cytotoxicity (ILMC) had declined. In contrast to the natural lymphocyte-mediated cytotoxicity, the ILMC was genetically restricted, could not be blocked by CDV-specific antibody, and was effective against measles virus-infected as well as CDV-infectd target cells. Lymphocyte populations were depleted of Fc receptor and surface immunoglobulin-bearing cells by rosetting techniques and tested in comparison. An antibody-dependent cell-mediated cytotoxicity was demostrated against CDV-infected target cells that were preincubated with CDV antibody when Fc receptor-bearing lymphocytes were not removed. The ILMC was measurable for approximately 10 days beginning at 6 days post-vaccination. In contrast, CDV antibody measured by virus neutralization and humoral cytotoxicity was detectable by 6 days postvaccination and persisted at peak levels for at least 5 months.
Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected.
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.
Apoptosis; Canine distemper virus; Caspase; Cervical tumor; HeLa cell; HPV
Canine distemper is characterized mainly by respiratory, enteric, and nervous symptoms. Infection of the central nervous system results in demyelination, to which inflammation has been shown to contribute significantly. It has been proposed that macrophages play a major role as effector cells in this process. We report that cultured dog brain cells contain a population of macrophages capable of producing reactive oxygen species as measured by luminol-dependent chemiluminescence. In cultures infected with canine distemper virus, a burst of reactive oxygen is triggered by antiviral antibody. This response depends on the presence of viral antigens on the surfaces of infected cells and is mediated by the interaction of antigen-bound antibody with Fc receptors on the macrophages. Since there is no evidence in vitro or in vivo that oligodendrocytes, the cells forming myelin, are infected, our observation supports the hypothesis that "innocent bystander killing" is important in demyelination caused by canine distemper virus. Reactive oxygen species released from macrophages may contribute to destruction of myelin.
cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge.
The history of medicine this century is darkened by the downfall of the German medical profession, exposed during the doctors' trial at Nuremberg in 1946. Relying largely on documents published during 1933 in German medical journals, this paper examines two widely accepted notions of those events, metaphorically termed "slippery slope" and "sudden subversion." The first connotes a gradual slide over infinitesimal steps until, suddenly, all footing is lost; the second conveys forced take over of the profession's leadership and values. Both concepts imply that the medical profession itself became the victim of circumstances. The slippery slope concept is a prominent figure of argument in the current debate on bioethics. The evidence presented here, however, strongly suggests that the German medical community set its own course in 1933. In some respects this course even outpaced the new government, which had to rein in the profession's eager pursuit of enforced eugenic sterilizations. In 1933 the convergence of political, scientific, and economic forces dramatically changed the relationship between the medical community and the government. That same convergence is occurring again and must be approached with great caution if medicine is to remain focused on the preservation of physical and medical integrity.
We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a “quick response” in vaccine development against well-known and “new” viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems.
This article critically examines the 25 June 1998 decision by the House of Lords regarding the psychiatric admission of a man with autism.1 Mr L was able neither to consent to, nor refuse, that admission and the disposition of his case illuminates the current debate regarding best interests of vulnerable adults by the judiciary and the psychiatric profession. This article begins with the assumption that hospitalisation was not the optimum response to Mr L's condition, provides alternative approaches to the interpretation of best interest and examines principles of liberty, anti-discrimination, and equal protection.
Key Words: Informal admission • autism • discrimination
In 1988 and 2002, two major phocine distemper virus (PDV) outbreaks occurred in harbour seals (Phoca vitulina) in north-western European coastal waters, causing the death of tens of thousands seals. Here we investigated whether PDV is still circulating among seals of the Dutch coastal waters and whether seals have protective serum-antibodies against PDV. Therefore seal serum samples, collected from 2002 to 2012, were tested for the presence of PDV-neutralizing antibodies. Antibodies were detected in most seals in 2002 and 2003 while after 2003 antibodies were detected only in seals less than two month-old and adult seals that probably had survived the 2002 PDV-epizootic. We estimated the current proportion of seals with antibodies against PDV at 11%. These findings suggest that at present the vast majority of seals are not immune to PDV infection. PDV re-introduction in this area may cause a major epizootic with infection of >80% and mass-mortality of >50% of the population.
phocine distemper virus; seals; serology; epizootic
Canine distemper virus (CDV) infection of ferrets is clinically and immunologically similar to measles, making this a useful model for the human disease. The model was used to determine if parenteral or mucosal immunization of infant ferrets at 3 and 6 weeks of age with attenuated vaccinia virus (NYVAC) or canarypox virus (ALVAC) vaccine strains expressing the CDV hemagglutinin (H) and fusion (F) protein genes (NYVAC-HF and ALVAC-HF) would induce serum neutralizing antibody and protect against challenge infection at 12 weeks of age. Ferrets without maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 5) or ALVAC-HF (n = 4) developed significant neutralizing titers (log10 inverse mean titer ± standard deviation of 2.30 ± 0.12 and 2.20 ± 0.34, respectively) by the day of challenge, and all survived with no clinical or virologic evidence of infection. Ferrets without maternal antibody that were vaccinated intranasally (i.n.) developed lower neutralizing titers, with NYVAC-HF producing higher titers at challenge (1.11 ± 0.57 versus 0.40 ± 0.37, P = 0.02) and a better survival rate (6/7 versus 0/5, P = 0.008) than ALVAC-HF. Ferrets with maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 7) and ALVAC-HF (n = 7) developed significantly higher antibody titers (1.64 ± 0.54 and 1.28 ± 0.40, respectively) than did ferrets immunized with an attenuated CDV vaccine (0.46 ± 0.59; n = 7) or the recombinant vectors expressing rabies glycoprotein (RG) (0.19 ± 0.32; n = 8, P = 7 × 10−6). The NYVAC vaccine also protected against weight loss, and both the NYVAC and attenuated CDV vaccines protected against the development of some clinical signs of infection, although survival in each of the three vaccine groups was low (one of seven) and not significantly different from the RG controls (none of eight). Combined i.n.-parenteral immunization of ferrets with maternal antibody using NYVAC-HF (n = 9) produced higher titers (1.63 ± 0.25) than did i.n. immunization with NYVAC-HF (0.88 ± 0.36; n = 9) and ALVAC-HF (0.61 ± 0.43; n = 9, P = 3 × 10−7), and survival was also significantly better in the i.n.-parenteral group (3 of 9) than in the other HF-vaccinated animals (none of 18) or in controls immunized with RG (none of 5) (P = 0.0374). Multiple routes were not tested with the ALVAC vaccine. The results suggest that infant ferrets are less responsive to i.n. vaccination than are older ferrets and raises questions about the appropriateness of this route of immunization in infant ferrets or infants of other species.