Immune responses in mice vaccinated with a suicidal DNA vaccine expressing the hemagglutinin glycoprotein from the peste des petits ruminants virus

Peste des petits ruminants (PPR), an acute and highly contagious disease, affects sheep, goats, and some small ruminants. The hemagglutinin (H) glycoprotein of the PPR virus (PPRV) is considered important for inducing protective immune responses. In this study, a suicidal DNA vaccine based on the Semliki Forest virus (SFV) replicon was constructed and tested for its ability to induce immunogenicity in a mouse model. For this, the H gene of PPRV was cloned and inserted into pSCA1, an SFV replicon vector. The resultant plasmid named pSCA1-H was then transfected into BHK-21 cells following which the antigenicity of the expressed protein was confirmed by Western blotting and immunofluorescence. The pSCA1-H plasmid was then injected intramuscularly into BALB/c mice thrice at 2-week intervals. To evaluate the immunogenicity of pSCA1-H, specific antibodies and neutralizing antibodies against PPRV-H were measured using an indirect enzyme-linked immunosorbent assay and a microneutralization test, respectively. Cell-mediated immune responses were also examined using a lymphocyte proliferation assay. The results showed that pSCA1-H could express the H protein in BHK-21 cells. Specific antibodies, neutralizing antibodies, and lymphocyte proliferation responses were all induced in mice. Thus, this suicidal DNA vaccine could be a promising new approach for vaccine development against PPR.     

Oral immunization against influenza virus

Summary Mice and ferrets were immunized by oral, intranasal or intraperitoneal route with attenuated or inactivated influenza A₂/Aichi/68. Production of antibodies in groups immunized orally was lower than in groups immunized intranasally or intraperitoneally when attenuated virus was administered. When inactivated virus was inoculated the serum antibody response was high following intraperitoneal injection, low following intranasal injection and nil following oral administration.Deaths occurred between the 3rd and the 11th day after intranasal immunization with partially attenuated virus, showing that immunization by the intranasal route necessitates more attenuated strains than by oral or by intraperitoneal routes. Higher doses of vaccine were required by oral than intranasal or intraperitoneal route to confer an equivalent degree of protection.This work was partially supported by a grant from National Research Council (915).     

Rapid competitive enzyme-linked immunosorbent assay for detection of antibodies to peste des petits ruminants virus

Peste des petits ruminants (PPR) is a contagious viral disease of small ruminants that is of economic importance in Africa, the Middle East, and Asia. We developed a rapid competitive enzyme-linked immunosorbent assay (rapid c-ELISA) for the diagnosis and surveillance of PPR. This assay detects PPR virus (PPRV) antibodies in serum samples by quantifying the amount of monoclonal antibody (MAb) P-3H12 after 30 min of incubation of a serum-MAb conjugate mixture on plates coated with a PPRV recombinant nucleocapsid protein (rPPRV-N). We tested 249 PPRV-positive serum samples and 733 PPRV-negative serum samples from field ruminants. The threshold of percent inhibition (PI) was determined to be <50 on the basis of the mean PI plus 3 standard deviations for sera from PPRV-negative ruminants. The relative specificity and sensitivity of the rapid c-ELISA were 98.5% (722 of 733 serum samples) and 93.4% (234 of 249 serum samples), respectively. The rapid c-ELISA sensitively detected PPRV antibodies in hyperimmune sera (virus neutralization test [VNT] titer, >512), even at dilutions > or = 512 in normal goat serum, and as early as 6 to 13 days postinfection from 12 goats, each of which was infected with one of the four PPRV lineages. Hyperimmune sera from animals experimentally vaccinated with rinderpest virus gave positive results by the rapid c-ELISA when the rinderpest virus VNT titers were >512, although the rapid c-ELISA titers were very low (2 to 16). However, the rapid c-ELISA was negative when the rinderpest virus VNT titer was < or = 128. The rapid c-ELISA developed in the present work provides a short turnaround time and could be a useful tool for the diagnosis of PPR and screening for PPRV in the field.     

Monkey CV1 cell line expressing the sheep-goat SLAM protein: a highly sensitive cell line for the isolation of peste des petits ruminants virus from pathological specimens

Peste des petits ruminants (PPR) is an important economically transboundary disease of sheep and goats caused by a virus which belongs to the genus Morbillivirus. This genus, in the family Paramyxoviridae, also includes the measles virus (MV), canine distemper virus (CDV), rinderpest virus (RPV), and marine mammal viruses. One of the main features of these viruses is the severe transient lymphopaenia and immunosuppression they induce in their respective hosts, thereby favouring secondary bacterial and parasitic infections. This lymphopaenia is probably accounted for by the fact that lymphoid cells are the main targets of the morbilliviruses. In early 2000, it was demonstrated that a transmembrane glycoprotein of the immunoglobulin superfamily which is present on the surface of lymphoid cells, the signalling lymphocyte activation molecule (SLAM), is used as cellular receptor by MV, CDV and RPV. Wild-type strains of these viruses can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. The present study has demonstrated that monkey CV1 cells expressing goat SLAM are also highly efficient for isolating PPRV from pathological samples. This finding suggests that SLAM, as is in the case for MV, CDV and RPV, is also a receptor for PPRV.     

Determination of prevalence and risk factors for infection with <Emphasis Type="Italic">Babesia ovis</Emphasis> in small ruminants from Turkey by polymerase chain reaction

In this study, PCR and thin blood smear-based diagnostic methods were used to assess the frequency of Babesia infection in small ruminants. A total of 300 sheep and 100 goats from 37 randomly selected herds located in eight locations of eastern Turkey were examined for the presence of Babesia infection and any tick species on the body of the animals. Of 400 blood samples examined, 6 (1.5%) were positive for Babesia spp. piroplasms upon microscopic examination, whereas 33 (8.25%) were positive for the presence of B. ovis by PCR. The prevalence of babesiosis in small ruminants detected by PCR was significantly higher than obtained in microscopic examination of thin blood smears (P < 0.05). Thirty-three animals produced the DNA fragment specific for Babesia ovis of which 32 (10.66%) were sheep and 1 (1%) was goat. The difference between the prevalence of Babesia infection in sheep and goats were statistically significant (P < 0.05). The prevalence of Babesia infection in different age groups of sheep were statistically non-significant (P > 0.05). The frequency of B. ovis infection was higher in herds with tick burden than no tick burden (P < 0.05). Seven amplicons (six from sheep and one from goat) were sequenced. The resulting sequences were identical to the recently reported nucleotide sequence of B. ovis. A total of 510 ticks belonging to the Rhipicephalus spp. were collected from sheep. Ticks were identified to be R. bursa and R. turanicus on the basis of morphological features. Nucleotide sequences data reported in this paper are available in EMBL, GenBank and DDJB databases under the accession numbers: DQ409332, DQ409333, DQ409334, DQ409335, DQ409336, DQ409337, DQ409338.     

Rapid Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Peste des Petits Ruminants Virus

Peste des petits ruminants (PPR) is a contagious viral disease of small ruminants that is of economic importance in Africa, the Middle East, and Asia. We developed a rapid competitive enzyme-linked immunosorbent assay (rapid c-ELISA) for the diagnosis and surveillance of PPR. This assay detects PPR virus (PPRV) antibodies in serum samples by quantifying the amount of monoclonal antibody (MAb) P-3H12 after 30 min of incubation of a serum-MAb conjugate mixture on plates coated with a PPRV recombinant nucleocapsid protein (rPPRV-N). We tested 249 PPRV-positive serum samples and 733 PPRV-negative serum samples from field ruminants. The threshold of percent inhibition (PI) was determined to be <50 on the basis of the mean PI plus 3 standard deviations for sera from PPRV-negative ruminants. The relative specificity and sensitivity of the rapid c-ELISA were 98.5% (722 of 733 serum samples) and 93.4% (234 of 249 serum samples), respectively. The rapid c-ELISA sensitively detected PPRV antibodies in hyperimmune sera (virus neutralization test [VNT] titer, >512), even at dilutions ≥512 in normal goat serum, and as early as 6 to 13 days postinfection from 12 goats, each of which was infected with one of the four PPRV lineages. Hyperimmune sera from animals experimentally vaccinated with rinderpest virus gave positive results by the rapid c-ELISA when the rinderpest virus VNT titers were >512, although the rapid c-ELISA titers were very low (2 to 16). However, the rapid c-ELISA was negative when the rinderpest virus VNT titer was ≤128. The rapid c-ELISA developed in the present work provides a short turnaround time and could be a useful tool for the diagnosis of PPR and screening for PPRV in the field.     

Effect of immunosuppression on pathogenesis of peste des petits ruminants (PPR) virus infection in goats

In this study an attempt to address the effects of immunosuppression on pathogenesis of peste des petits ruminants (PPR) virus infection was undertaken. Cyclophosphamide and dexamethasone were used to immunosuppress the animals. The drug treated animals exhibited severe leukopaenia and lymphopaenia; one of the indicators of immunosuppression. Experimental peste des petits ruminants virus (PPRV) infection was then given to both drug-induced immunosuppressed and non-immunosuppressed goats and observed their effects. Findings indicated that, the immunosuppressed goats had a short period of viremia, more extensive and severe disease advancement and higher mortality rate than the non-immunosuppressed goats. PPRV antigen distribution in both ante-mortem and post-mortem materials was extensive and diffused in immunosuppressed animals than that of non-immunosuppressed. Some of the atypical organ(s)/tissues like liver, kidney, heart etc showed more antigen load than non-immunosuppressed group. Histopathological and immunohistochemical studies of tissues from the two groups showed that pathological changes in the non-immunosuppressed animals were confined only to gastrointestinal tract, whereas in the immunosuppressed animals histopathological changes and PPRV antigen distribution were more extensive and diffused. The present study indicated that immunosuppression increased the extent and severity of the pathological lesions associated with peste des petits ruminants virus infection.     

Occurrence of <Emphasis Type="Italic">Cryptosporidium parvum</Emphasis> and <Emphasis Type="Italic">Giardia duodenalis</Emphasis> in healthy adult domestic ruminants

To determine the prevalence and intensity of infection of Cryptosporidium spp. and Giardia duodenalis in healthy adult domestic ruminants, faecal samples were collected from 379 cattle of between 3 and 13 years old, 446 sheep and 116 goats selected at random from 60 dairy farms and 38 and 20 herds, respectively, in Galicia (NW Spain). Cryptosporidium spp. oocysts were detected in 32 cows (8.4%), 24 sheep (5.3%) and in nine goats (7.7%) from, respectively, 48.3% of the farms and 34.2 and 30.0% of the herds. The intensity of infection in cows ranged between 25 and 5,924 oocysts per gram of faeces (OPG), whereas in sheep and goats, the number of oocysts shed ranged from 8–515 OPG and from 17–782 OPG, respectively. Parasitization by Cryptosporidium spp. was significantly higher (P < 0.05) in cows than in sheep and goats. G. duodenalis cysts were identified in 101 cows (26.6%), 86 sheep (19.2%) and 23 goats (19.8%) from, respectively, 96.6% of the farms and 92.1 and 90% of the herds. The number of cysts shed by cows ranged between 15 and 3,042 cyst per gram of faeces (CPG), whereas the intensity of infection in sheep and goats ranged from 16–3010 CPG and from 15–1845 CPG, respectively, and was significantly lower (P < 0.05) than in cows and sheep. The number of Cryptosporidium spp. oocysts isolated from sheep and goats was insufficient for successful polymerase chain reaction analysis. Nevertheless, gene sequence analysis of the hsp70 and 18SrRNA genes of Cryptosporidium revealed the presence of only C. parvum in faecal samples from cows. Genotyping studies of the β-giardin and glutamate dehydrogenase genes of G. duodenalis revealed mainly assemblage E of Giardia in cows, sheep and goat faecal samples. Assemblage B of G. duodenalis was also detected in one sheep sample. These animals should be considered as a possible source of cryptosporidiosis and giardiosis, thereby maintaining the infections on farms and in herds.     

Effect of a live attenuated intranasal vaccine on latency and shedding of feline herpesvirus 1 in domestic cats

Summary.  A prospective study was conducted that evaluated duration of virus shedding through acute and experimentally-induced recurrent disease episodes in 12 cats, and tissue distribution of latent infections, following intranasal vaccination with a temperature sensitive (ts) mutant strain of feline herpesvirus 1 (FHV1). Six of these cats were challenged with a virulent field strain of the agent to assess the extent to which vaccination affected subsequent shedding of virus and the establishment of latent infections. Virus isolation (VI) tests were done in parallel with a polymerase chain reaction (PCR) assay to compare the performance of each diagnostic method. The PCR confirmed that all 12 cats shed virus throughout the periods of vaccination, challenge or mock-challenge, and a cyclophosphamide-dexamethasone stress protocol to reactivate latent infections. Shedding to the tsFHV1 was documented by VI for up to 25 days following vaccination and for up to 15 days following challenge, but not after experimental stress. Overall, FHV1 was present in 144 of 300 (48%) cat-days of testing by PCR compared to 32 of 300 (11%) by VI. The frequency and distribution of latent FHV1 detected in neurologic, ophthalmic, and other tissues by PCR were identical for vaccine-only and vaccine-challenge groups, thereby disproving previous hypotheses that tsFHV1 mutants administered by this route protect against latency. Accepted July 30, 1997 Received July 3, 1997     

Mucosal Immunization with PsaA Protein,Using Chitosan as a Delivery System,Increases Protection Against Acute Otitis Media and Invasive Infection by Streptococcus pneumoniae

As infection with Streptococcus pneumoniae (mainly via the mucosal route) is a leading cause of acute otitis media, sinus and bacterial pneumonia, the mucosal immunity plays an important role in the prevention of pneumococcal diseases. Therefore, intranasal vaccination may be an effective immunization strategy, but requires appropriate mucosal vaccine delivery systems. In this work, chitosan was used as a mucosal delivery system to form chitosan–PsaA nanoparticles based on ionotropic gelation methods and used to immunize BALB/c mice intranasally. Compared to mice immunized with naked PsaA, levels of IFN‐γ, IL‐17A and IL‐4 in spleen lymphocytes, the systemic (IgG in serum) and mucosal (IgA in mucosal lavage) specific antibodies were enhanced significantly in mice inoculated with chitosan–PsaA. Furthermore, increased protection against acute otitis media following middle ear challenge with pneumococcus serotype 14, and improved survival following intraperitoneal challenge with pneumococcus serotype 3 or serotype 14, was found in the mice immunized with chitosan–PsaA nanoparticles. Thus, intranasal immunization with chitosan–PsaA can successfully induce mucosal and systemic immune responses and increase protection against pneumococcal acute otitis media and invasive infections. Hence, intranasal immunization with PsaA protein, based on chitosan as a delivery system, is an efficient immunization strategy for preventing pneumococcal infections.     

Genetic analysis of the M RNA segment of Crimean-Congo hemorrhagic fever virus strains in Turkey

Summary Crimean-Congo hemorrhagic fever (CCHF) virus is member of the genus Nairovirus of the family Bunyaviridae. All members of the family Bunyaviridae are enveloped viruses containing tripartite, negative polarity, single-stranded RNA. CCHF is characterized by high case mortality, occurring in Asia, Africa, the Middle East and Europe. During recent years, outbreaks have been reported in Turkey. However, little information is available on the genetic diversity of CCHF virus in Turkey. In this study, a total of 1227 adult ticks were collected from domestic ruminants (796 specimens from cattle, 399 specimens from goats and 32 specimens from sheep). The presence of the M segment of CCHF virus was determined in 4 of 40 (10%) Hyalomma marginatum marginatum pools, in 2 of 38 (7.89%) Rhipicephalus bursa pools, and in 1 of 7 (7%) Boophylus annulatus pools. Hyalomma anatolicum anatolicum pools gave negative RT-PCR result against CCHF virus. Serum samples from seven patients infected with CCHF were selected and subjected to RT-PCR to amplify partial M segment of CCHF virus. This report introduces the first data on partial nucleotide sequences of M RNA segments of CCHF virus strains circulating in Turkey, isolated from ticks. Correspondence: Aykut Ozdarendeli, Department of Virology, Firat University College of Veterinary Medicine, 23119 Elazig, Turkey     

Pathological aspects of immunization of mice against influenza virus infection.

Groups of mice were immunized against influenza Ao/NWS virus by a single intranasal administration of inactivated homologous virus, by 2 intranasal doses of vaccine separated by an interval of 2 weeks, or by 2 intraperitoneal doses of the same vaccine. When subjected 2 weeks later to a standard challenge of 6 x 10(5) egg infecting units Ao/NWS virus instilled intranasally, mortality fell significantly from 64% in unimmunized mice to 39% in mice given a single intranasal dose of vaccine and to 29% in animals which received double intranasal vaccine. The best protection was conferred by double intraperitoneal immunization, after which mortality was 10%. Immunity waned with time, since the mortality of mice doubly immunized by the respiratory route and challenged 30 weeks later was 49%. Intrapulmonary lymphoid tissue developed in large amounts in a proportion of mice immunized by all methods and challenged after an interval of 2 weeks. Attention is drawn to this reaction as a possible unfavourable consequence of vaccination. There were no lesions in the lungs or central nervous system after immunization without subsequent challenge. The importance of histopathology in vaccine trials in experimental animals is emphasized by the consistently higher detection rate of lesions in lungs by histological examination than by visual inspection alone.     

The occurrence of vacuolated neurons in the brains of hamsters affected with subacute sclerosing encephalitis following measles or Langat virus infection.

Hamsters infected by the intranasal route with either hamster adapted Langat virus passaged at least 10 times in hamsters and the HNT strain of measles virus passaged 149 times in hamsters, developed subacute sclerotic lesions in the pyriform cortex, and beginning from 2 months after infection, were accompanied by neuronal vacuolation with ballooning of the cytoplasm, especially in parts of the brain in close proximity of the sclerotic lesions. The vacuolated neurons resembled those seen in scrapie, especially of sheep and goats, suggesting a similarity between the effects of slow and subacute viral infections.     

Inflammatory responses to influenza vaccination at the extremes of age

Age affects the immune response to vaccination, with individuals at the extremes of age responding poorly. The initial inflammatory response to antigenic materials shapes the subsequent adaptive response and so understanding is required about the effect of age on the profile of acute inflammatory mediators. In this study we measured the local and systemic inflammatory response after influenza vaccination or infection in neonatal, young adult and aged mice. Mice were immunized intramuscularly with inactivated influenza vaccine with and without the adjuvant MF59 and then challenged with H1N1 influenza. Age was the major factor affecting the inflammatory profile after vaccination: neonatal mice had more interleukin‐1α (IL‐1α), C‐reactive protein (CRP) and granulocyte–macrophage colony‐stimulating factor (GMCSF), young adults more tumour necrosis factor‐α (TNF), and elderly mice more interleukin‐1 receptor antagonist (IL‐1RA), IL‐2RA and interferon‐γ‐induced protein 10 (IP10). Notably the addition of MF59 induced IL‐5, granulocyte colony‐stimulating factor (G‐CSF), Keratinocyte Chemotractant (KC) and monocyte chemoattractant protein 1 (MCP1) in all ages of animals and levels of these cytokines correlated with antibody responses. Age also had an impact on the efficacy of vaccination: neonatal and young adult mice were protected against challenge, but aged mice were not. There were striking differences in the localization of the cytokine response depending on the route of exposure: vaccination led to a high serum response whereas intranasal infection led to a low serum response but a high lung response. In conclusion, we demonstrate that age affects the inflammatory response to both influenza vaccination and infection. These age‐induced differences need to be considered when developing vaccination strategies for different age groups.     

Molecular characterization of peste des petits ruminants virus from the Karamoja region of Uganda (2007-2008)

Antibodies against peste des petits ruminants virus (PPRV) were first detected in goats in East Africa in 1995 without any clinical disease. It was not until during the years 2006 and 2007 that the disease outbreaks were first reported in Kenya and Uganda, respectively. This study was carried out to detect and characterize PPRV from a suspected outbreak in sheep and goats in the Karamoja region in 2007-2008. Oculo-nasal and blood samples were tested using F-gene-based primers, and their genetic relationships to other sequences in the GenBank database were investigated. A total of 383 samples suspected to contain PPRV were randomly collected and tested. Sixty-seven (17.5%) were positive when F protein gene primers were used. During the years 2007 and 2008, 38.1% (26/67) and 13.0% (41/316) of samples were positive by PCR, respectively. The 2007 sequences clustered with Asian sequences in lineage 4 and Cote d’Ivoire 86 (ICV 86) in lineage 2, while all of the 2008 samples clustered in lineage 1. Over the years, the implicated strains were genetically close (88%–91%) to the vaccine strain (Nig 75/1). Based on this study, the circulating PPR strains in Uganda are heterogeneous, and therefore, the disease may have been introduced from different sources.     

Species composition and morphology of protostrongylids (Nematoda: Protostrongylidae) in ruminants from Bulgaria

Lungs of 52 ruminants from different regions of Bulgaria, 16 from goats (Capra aegagrus f. domestica L.), 15 from sheep (Ovis ammon f. domestica L.), 11 from mouflons (Ovis musimon L.), and 10 from chamois (Rupicapra rupicapra L.), were investigated. The aim of the study was to determine the species composition of small lungworms in these hosts. The obtained results are summarized with those of previous studies, and a picture of the present status of the species composition of protostrongylids in ruminants from Bulgaria is forwarded. Morphometric data about the species Muellerius capillaris, Cystocaulus ocreatus, Neostrongylus linearis, Protostrongylus brevispiculum, and Protostrongylus rufescens are presented. The data on the morphology of these five species are supplied for the first time both for Bulgaria and the south-east part of the European continent.     

Intranasal administration of RSV antigen-expressing MCMV elicits robust tissue-resident effector and effector memory CD8+ T cells in the lung

Cytomegalovirus vectors are promising delivery vehicles for vaccine strategies that aim to elicit effector CD8+ T cells. To determine how the route of immunization affects CD8+ T-cell responses in the lungs of mice vaccinated with a murine cytomegalovirus vector expressing the respiratory syncytial virus matrix (M) protein, we infected CB6F1 mice via the intranasal or intraperitoneal route and evaluated the M-specific CD8+ T-cell response at early and late time points. We found that intranasal vaccination generated robust and durable tissue-resident effector and effector memory CD8+ T-cell populations that were undetectable after intraperitoneal vaccination. The generation of these antigen-experienced cells by intranasal vaccination resulted in earlier T-cell responses, interferon gamma secretion, and viral clearance after respiratory syncytial virus challenge. Collectively, these findings validate a novel approach to vaccination that emphasizes the route of delivery as a key determinant of immune priming at the site of vulnerability.     

Pathogenicity and immunogenicity for mice of temperature-sensitive mutants of vesicular stomatitis virus

Temperature-sensitive (ts) mutants of vesicular stomatitis (VS) virus were tested for their pathogenicity and immunogenicity in weanling mice. Compared with the wild-type virus (ts(+)), ts mutants representing genetic complementation groups I, II, and IV were considerably less pathogenic for mice infected by the intracerebral route and caused few deaths after intranasal inoculation. Mice were completely resistant to ts(+) and ts mutants by the intraperitoneal route. Resistance to intracerebral challenge with ts(+) VS virus was only minimal in mice vaccinated intraperitoneally with ts(+) or ts mutants and only moderate in mice vaccinated intranasally with three ts mutants. Intranasal vaccination, particularly with group IV mutants, resulted in solid immunity within 3 days to intranasal challenge with ts(+) virus. VS viral neutralizing antibody was present in the bronchial secretions of mice by 12 h after intranasal inoculation of mutant ts IV44; the bronchial antibody titers declined to undetectable levels between 3 and 7 days after vaccination. Neutralizing antibody was detected in the serum of mice by the third day after intranasal vaccination with ts IV44 and persisted at high level for at least 11 days. Certain classes of ts mutants would appear to be promising candidates for use as attenuated, live virus vaccines.     

Experimental foot-and-mouth disease in sheep and goats: An epizootiological model

Summary Observations were made on the appearance and spread of foot-and-mouth disease (FMD) in groups of sheep and goats after either intranasal inoculation of virus or contact with an infected steer. Viremia was used as the primary indicator of the spread of infection. Of 91 goats exposed to virus, 88 (97%) became infected and 92% of the infected ones had demonstrable viremia. Comparable figures for sheep were 33 of 43 (77%) infected and 72% of these with viremia. It was concluded that goats, being less expensive than cattle to purchase and house, could be suitable experimental subjects for studies of the epizootiology of FMD under laboratory conditions.