Days Postinoculation Research Articles

First Report of Oregon Grape (Mahonia aquifolium) as an Alternate Host for the Wheat Stripe Rust Pathogen (Puccinia striiformis f. sp. tritici) Under Artificial Inoculation.

As the primary host of the stripe rust pathogen, Puccinia striiformis f. sp. tritici (Pst), wheat can be infected by both aeciospores and urediniospores, and later is the host that gives rise to urediniospores and teliospores. Barberry species (e.g., Berberis vulgaris) can be infected by basidiospores, produced from the teliospores of wheat plants, and later gives rise to pycniospores and aeciospores, which has been demonstrated through artificial inoculation (3). Oregon grape (Mahonia aquifolium), closely related to Berberis, is a native evergreen shrub that is also grown as an ornamental plant in the Pacific Northwest. To determine if M. aquifolium can also serve as an alternate host for Pst, we conducted artificial inoculations under controlled conditions. Seeds of M. aquifolium collected from Pullman, WA, were sown in pots filled with soil mixture, and plants were grown in a greenhouse under wheat-growing conditions (1). In the first experiment, conducted in May to June 2011, the inoculum was telia collected from artificially inoculated wheat cv. Avocet S with urediniospores of isolate 09-134 (race PST-127) from the greenhouse. In the second experiment, conducted in July to August 2011, the inoculum was telia collected from naturally infected wheat cv. Nugaines with urediniospores from isolate 11-292 (race PST-127) from an experimental field near Pullman. For each experiment, mature teliospores of 60 telia from a single wheat plant were suspended in 1.0 ml of distilled water and inoculated with a fine paint brush onto the leaves of seven or eight 10- to 15-day-old plants of M. aquifolium. Plants were incubated initially in a dew chamber at 10°C for 72 h in darkness, then transferred to a growth chamber with a diurnal temperature cycle of 10 to 24°C and a 16 h light/8 h dark cycle (1). Reddish pycnia with nectar appeared on adaxial surfaces of inoculated leaves at 12 days post-inoculation (DPI), and reddish aecia were produced on the baxial surface at 16 DPI. All 15 M. aquifolium leaves of the 15 plants inoculated with teliospores produced pycnia and aecia. Seedlings of Nugaines and Avocet S, wheat cultivars that are susceptible to all Pst races (1), were then inoculated with a water suspension of aeciospores of 30 aecia collected from the M. aquifolium plants. Wheat plants were incubated as described above for M. aquifolium. Uredinia appeared at 15 DPI, and telia were produced after an additional 15 days. From these uredinia that formed on inoculated wheat, a total of 30 single-uredinium isolates were obtained using the standard procedure (1). Virulence tests were carried out on 20 wheat differentials for 10 randomly selected urediniospore isolates, revealing six virulence patterns. When tested with four selected Pst SSR markers (PstP001, PstP003, PstP005, PstP029) (2) and compared to other race PST-127 isolates, all 10 progeny isolates were homozygous, as were the parental isolates (09-134, 11-292). The virulence tests and marker genotypes verified that the urediniospore isolates resulted from infection by aecia, produced by parental isolate 09-134 through its sexual cycle on M. aquifolium. The study exhibited the completed sexual lifecycle of Pst through the five spore stages on wheat and M. aquifolium in a controlled setting, and suggests that under appropriate weather conditions, M. aquifolium may serve as an alternate host for Pst. Due to the wide distribution of M. aquifolium, further studies are needed to determine if the species can be infected by Pst under natural conditions.

Evaluation of Genetic and Environmental Parameters Determining Antibody Response Induced by Vaccination Against Foot and Mouth Disease

Factors determining the variation in antibody response post foot and mouth disease virus (FMDV) vaccine were investigated in 395 sera samples of crossbred cattle maintained at the institute farm located in the temperate Himalayan region of India. Pre and post sera were tested using liquid phase blocking enzyme linked immunosorbent assay (LPBE) specific for serotypes O, A, and Asia1. All the pre-vaccination samples were unprotected and increase in the antibody titer was seen 28-day post-vaccination. All the samples were negative for infection status. Among the environmental determinants, cohort, season, and vaccination number proved to be significant sources of variation in the vaccine response (P 1 year) were found to be significantly more protected than the younger animals (P < 0.05). Cows in the first trimester of lactation showed reduced protection by vaccine that might be attributed to the weakening of immune system consequent to the stress of production and calving as well as increased metabolic demands typical of a postpartum cow. The estimates of heritability (±SE) for FMDV vaccine response for serotypes O, A, and Asia1 were 0.17 ± 0.07, 0.03 ± 0.06, and 0.05 ± 0.06, respectively, indicating little scope of improvement after mass selection.

Microdialysis measurement of intratumoral temozolomide concentration after cediranib, a pan-VEGF receptor tyrosine kinase inhibitor, in a U87 glioma model

Combining anti-angiogenesis agents with cytotoxic agents for the treatment of malignant gliomas may affect the cytotoxic drug distribution by normalizing the blood-brain barrier (BBB). This study examines the intratumoral concentration of temozolomide (TMZ) in the presence and absence of the pan-VEGF receptor tyrosine kinase inhibitor, cediranib. Seven nude rats bearing U87 intracerebral gliomas had a microdialysis probe centered within the tumor. Ten-days after tumor implantation, TMZ (50 mg/kg) was given orally. The extracellular fluid (ECF) concentrations of TMZ within the tumor were assessed via microdialysis for 6 h following TMZ administration. Cediranib (6 mg/kg) was then given orally, and 12 h later, TMZ was re-administered with subsequent microdialysis collection. A subset of animals also underwent functional MRI to assess angiogenesis in vivo at post-inoculation days 12 and 21, before and after the cediranib treatment. After dosing of oral TMZ only, ECF-TMZ mean-C(max) and area under the concentration curve(AUC(0-∞)) within the tumor were 0.59 μg/mL and 1.82 μg h/mL, respectively. Post-cediranib, ECF-TMZ mean-C(max) and AUC(0-∞) were 0.83 μg/mL and 3.72 ± 0.61 μg h/mL within the tumor, respectively. This represented a 1.4-fold (p = 0.3) and 2.0-fold (p = 0.06) increase in the ECF-TMZ C(max) and AUC(0-∞), respectively, after cediranib administration. In vivo MRI measurements of the various vascular parameters were consistent with a BBB "normalization" profile following cediranib treatment. In the U87 intracerebral glioma model, within the first day of administration of cediranib, the intratumoral concentrations of TMZ in tumor ECF were slightly, but not statistically significantly, increased when compared to the treatment of TMZ alone with radiographic evidence of a normalized BBB.

Transition from acute to persistent infection: Antigen persistence and cytotoxic T cells response in infectious bursal disease virus infected SPF chickens (P6129)

Abstract Described by Cosgrove about a half century ago, infectious bursal disease (IBD) is as an acute, highly contagious viral infection of young chickens, which last for 7 to 10 days and leads to immunosuppression. Here, we are reporting for the first time that in IBD virus (IBDV) -infected, specific pathogen free (SPF) chickens bursa, the virus antigen, macrophages, CD4+, CD8+ cells, Fas, Fas ligand (FasL), and caspase-3 were consistently detected by immunohistochemistry up to 56 days post inoculation (DPI). Two-week-old SPF chickens were inoculated intraocularly with standard challenge strain (STC) using a 104embryo infectious dose. Histopathology of bursal tissues revealed severe lymphoid follicular depletion at DPI 5-28. A distinctive pattern of bursal follicles in the form of small and large follicles was detected from DPI 35 to 56. At 42 and 56 DPI a characteristic clustered pattern of IBDV antigen, macrophages, CD4+, CD8+ cells, Fas, FasL, and caspase-3 were detected in or around the small bursal follicles as compared to large bursal follicles. This is the first report on the persistence of IBDV antigen causing a latent infection, in bursal tissues of SPF chicken. Detection of persistent IBDV infection is highly significant in the elucidation of virus pathogenesis, immunology and epidemiology and it raises many questions that need to be answered in future studies.

Kinetics of the porcine reproductive and respiratory syndrome virus (PRRSV) humoral immune response in swine serum and oral fluids collected from individual boars.

BackgroundThe object of this study was to describe and contrast the kinetics of the humoral response in serum and oral fluid specimens during acute porcine reproductive and respiratory syndrome virus (PRRSV) infection. The study involved three trials of 24 boars each. Boars were intramuscularly inoculated with a commercial modified live virus (MLV) vaccine (Trial 1), a Type 1 PRRSV field isolated (Trial 2), or a Type 2 PRRSV field isolate (Trial 3). Oral fluid samples were collected from individual boars on day post inoculation (DPI) -7 and 0 to 21. Serum samples were collected from all boars on DPI −7, 0, 7, 14, 21 and from 4 randomly selected boars on DPI 3, 5, 10, and 17. Thereafter, serum and oral fluid were assayed for PRRSV antibody using antibody isotype-specific ELISAs (IgM, IgA, IgG) adapted to serum or oral fluid.ResultsStatistically significant differences in viral replication and antibody responses were observed among the three trials in both serum and oral fluid specimens. PRRSV serum IgM, IgA, and IgG were first detected in samples collected on DPI 7, 10, and 10, respectively. Oral fluid IgM, IgA, and IgG were detected in samples collected between DPI 3 to 10, 7 to 10, and 8 to 14, respectively.ConclusionsThis study enhanced our knowledge of the PRRSV humoral immune response and provided a broader foundation for the development and application of oral fluid antibody-based diagnostics.

Experimental infection with a Thai reassortant swine influenza virus of pandemic H1N1 origin induced disease

BackgroundFollowing the emergence of the pandemic H1N1 influenza A virus in 2009 in humans, this novel virus spread into the swine population. Pigs represent a potential host for this virus and can serve as a mixing vessel for genetic mutations of the influenza virus. Reassortant viruses eventually emerged from the 2009 pandemic and were reported in swine populations worldwide including Thailand. As a result of the discovery of this emergent disease, pathogenesis studies of this novel virus were conducted in order that future disease protection and control measures in swine and human populations could be enacted.MethodsThe pandemic H1N1 2009 virus (pH1N1) and its reassortant virus (rH1N1) isolated from pigs in Thailand were inoculated into 2 separate cohorts of 9, 3-week-old pigs. Cohorts were consisted of one group experimentally infected with pH1N1 and one group with rH1N1. A negative control group consisting of 3 pigs was also included. Clinical signs, viral shedding and pathological lesions were investigated and compared. Later, 3 pigs from viral inoculated groups and 1 pig from the control group were necropsied at 2, 4, and 12 days post inoculation (DPI).ResultsThe results indicated that pigs infected with both viruses demonstrated typical flu-like clinical signs and histopathological lesions of varying severity. Influenza infected-pigs of both groups had mild to moderate pulmonary signs on 1-4 DPI. Interestingly, pigs in both groups demonstrated viral RNA detection in the nasal swabs until the end of the experiment (12 DPI).ConclusionThe present study demonstrated that both the pH1N1 and rH1N1 influenza viruses, isolated from naturally infected pigs, induced acute respiratory disease in experimentally inoculated nursery pigs. Although animals in the rH1N1-infected cohort demonstrated more severe clinical signs, had higher numbers of pigs shedding the virus, were noted to have increased histopathological severity of lung lesions and increased viral antigen in lung tissue, the findings were not statistically significant in comparison with the pH1N1-infected group. Interestingly, viral genetic material of both viruses could be detected from the nasal swabs until the end of the experiment. Similar to other swine influenza viruses, the clinical signs and pathological lesions in both rH1N1 and pH1N1 were limited to the respiratory tract.

Attempt to develop live attenuated bacterial vaccines by selecting resistance to gossypol, proflavine hemisulfate, novobiocin, or ciprofloxacin

In an attempt to develop attenuated bacteria as potential live vaccines, four chemicals (gossypol, proflavine hemisulfate, novobiocin, and ciprofloxacin) were used to modify the following four genera of bacteria through chemical-resistance strategy: (1) Aeromonas hydrophila (9 isolates); (2) Edwardsiella tarda (9 isolates); (3) Streptococcus iniae (9 isolates); and (4) S. agalactiae (11 isolates). All bacteria used in this study were able to develop high resistance to gossypol. However, only some bacteria were able to develop resistance to proflavine hemisulfate, novobiocin, or ciprofloxacin. When the virulence of resistant bacteria was tested in tilapia or catfish, none of the gossypol-resistant isolate was attenuated, whereas majority of the proflavine hemisulfate-resistant isolates were attenuated. However, all proflavine hemisulfate-attenuated bacteria failed to provide significant protection to fish. Eight novobiocin- or ciprofloxacin-resistant Gram-positive bacteria (S. agalactiae and S. inaie) were found to be attenuated. However, none of them offered protection higher than 70%. Of seven attenuated novobiocin- or ciprofloxacin-resistant Gram-negative isolates (A. hydrophila and E. tarda), only one (novobiocin-resistant E. tarda 30305) was found to safe and highly efficacious. When E. tarda 30305-novo vaccinated Nile tilapia were challenged by its virulent E. tarda 30305, relative percent of survival of vaccinated fish at 14- and 28-days post vaccination (dpv) was 100% and 92%, respectively. Similarly, E. tarda 30305-novo offered 100% protection to channel catfish against challenges with virulent parent isolate E. tarda 30305 at both 14- and 28-dpv. Our results suggest that the development of live attenuated bacterial vaccines that are safe and efficacious is challenging, although it is feasible.

Efficacy of rifampicin in the treatment of experimental acute canine monocytic ehrlichiosis

To assess the efficacy of rifampicin in achieving clinical and haematological recovery and clearing infection in dogs with experimentally induced acute monocytic ehrlichiosis. Five Ehrlichia canis-infected Beagle dogs were treated with rifampicin (10 mg/kg/24 h orally for 3 weeks), nine E. canis-infected dogs received no treatment (infected untreated dogs) and two dogs served as uninfected controls. Clinical score, platelet counts, immunofluorescent antibody titres and PCR detection of E. canis-specific DNA in blood, bone marrow and spleen aspirates were evaluated on post-inoculation days 21 (start of rifampicin), 42 (end of rifampicin) and 98 (end of the study). By day 21 post-inoculation, all infected dogs became clinically ill and thrombocytopenic, seroconverted and were PCR positive in at least one tissue. Clinical scores and antibody titres did not differ between the treated and infected untreated dogs throughout the study. The rifampicin-treated dogs experienced an earlier resolution of their thrombocytopenia (Kaplan-Meier survival plot, P=0.048), and the median platelet counts were significantly higher in the treated compared with the infected untreated dogs on post-inoculation days 42 (P=0.0233) and 98 (P=0.0195). At the end of the study, three treated and six untreated infected dogs remained PCR positive in one tissue each. The rifampicin treatment regimen applied in this study hastened haematological recovery, but was inconsistent in eliminating the acute E. canis infection.

Chicken Parvovirus–Induced Runting-Stunting Syndrome in Young Broilers

Previously we identified a novel parvovirus from enteric contents of chickens that were affected by enteric diseases. Comparative sequence analysis showed that the chicken parvovirus (ChPV) represented a new member in the Parvoviridae family. Here, we describe some of the pathogenic characteristics of ChPV in young broilers. Following experimental infection, 2-day-old broiler chickens showed characteristic signs of enteric disease. Runting-stunting syndrome (RSS) was observed in four of five experimental groups with significant growth retardation between 7 and 28 days postinoculation (DPI). Viral growth in small intestine and shedding was detected at early times postinoculation, which was followed by viremia and generalization of infection. ChPV could be detected in most of the major tissues for 3 to 4 wk postinoculation. Immunohistochemistry staining revealed parvovirus-positive cells in the duodenum of inoculated birds at 7 and 14 DPI. Our data indicate that ChPV alone induces RSS in broilers and is important determinant in the complex etiology of enteric diseases of poultry.

The pathogenicity of avian metapneumovirus subtype C wild bird isolates in domestic turkeys

BackgroundAvian metapneumovirus subtype C (aMPV/C) causes severe upper respiratory disease in turkeys. Previous report revealed the presence of aMPV/C in wild birds in the southeast regions of the U.S.MethodsIn this study, aMPV/C positive oral swabs from American coots (AC) and Canada geese (CG) were passaged three times in the respiratory tract of specific pathogen free (SPF) turkeys and used as aMPV/C P3 virus isolates in subsequent studies.ResultsWild bird P3 isolates showed similar growth characteristics when compared to virulent aMPV/C in chicken embryo fibroblast ( CEF) cell cultures and their glycoprotein G gene sequence was closely related to the G gene of aMPV/C Colorado reference virus. Three-day-old commercial or SPF turkeys were inoculated oculonasally with wild bird aMPV/C P3 isolates. At 5 and 7 days post-inoculation (DPI), severe clinical signs were observed in both of the AC and CG virus-exposed groups. Viral RNA was detected in tracheal swabs by reverse transcriptase polymerase chain reaction (RT-PCR). In addition, immunohistochemistry showed virus replication in the nasal turbinate and trachea. All virus-exposed turkeys developed positive antibody response by 14 DPI.ConclusionsOur data demonstrate that aMPV/C wild bird isolates induced typical aMPV/C disease in the domestic turkeys.

Efficacy of ‘indigenous vaccine’ using native ‘Indian bison type’ genotype of Mycobacterium avium subspecies paratuberculosis for the control of clinical Johne’s disease in an organized goat herd

Therapeutic efficacy of a new 'Indigenous vaccine' prepared from native highly pathogenic 'Indian Bison Type' genotype of Mycobacterium avium subspecies paratuberculosis (MAP) of goat origin has been evaluated with respect to control of clinical Johne's disease in naturally infected Mehsana breed of goat in North Gujarat. Fifty goats from Sheep and Goats Research Station, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar, were randomly divided into 2 groups viz.,'Vaccinated'(n = 35) and 'Control'(n = 15). After vaccination, goats were monitored for physical condition, morbidity, mortality, body weights, shedding of MAP in feces, internal condition, gross lesions and humoral immune responses up to 120 days (at each interval of 30 days). At the end of 120 days trial, there was marked overall improvement in physical condition and body weights of vaccinated goats as compared to 'Control' goats. Vaccinated goats gained significantly (P < 0.05) higher body weights, hardly exhibited any lesions characteristic of JD, had significantly higher (P < 0.01) antibody titers and shedding of MAP was significantly (P < 0.01) reduced. Few of the vaccinated goats were positive for MAP DNA in faecal PCR and blood PCR before vaccination. However, all were found as negative at 120 days post vaccination (DPV). Overall vaccine exhibited effective in restriction of MAP infection and significant improvement in production parameters and reduction in mortality and morbidity due to JD. The trial in the herd will be continued.

A proposal for an alternative quality control test procedure for inactivated vaccines against food-and-mouth disease virus

Foot-and-mouth disease (FMD) control in Brazil includes a strict mandatory vaccination program with vaccines produced in certified laboratories subject to inspection by the Brazilian Ministry of Agriculture, Livestock, and Food Supply (MAPA). The FMD vaccine's potency is tested through antibodies titration against structural viral proteins in sera from cattle that have not had any exposure to food-and-mouth disease virus (FMDV), at 28 days post-vaccination. Biological product testing using large animals is expensive and unwieldy. Thus, alternative testing procedures using laboratory animals have been proposed for quality control of these products. Such biological methods for vaccine evaluation using animals from vivarium facilities can have a significant impact through reduced costs, easier handling, and shorter testing times. The present study was designed to access Balb/C mice's humoral immune responses to a FMDV experimental vaccine, the composition of which contains three virus serotypes of FMDV (O1 Campos, A24 Cruzeiro, and C3 Indaial). Balb/C mice were immunized at doses that were 5% and 10% of the vaccine volume administered in cattle. Immunized mice had their antibody titers probed at 14, 21, and 28 DPV (days post vaccination). The results obtained were compared to those previously known from cattle's immune responses to the FMDV vaccine. An adequate immune response to the vaccine was seen with 10% formulation at 21 DPV. The study results are encouraging and indicate that the mouse model can be used for quality control in experimental vaccine testing.

Optical detection of downy mildew in grapevine leaves: daily kinetics of autofluorescence upon infection.

A 15-day survey of autofluorescence has been conducted upon infection by downy mildew [Plasmopara viticola (Berk. & M.A. Curtis) Berl. & de Toni] of leaves of a susceptible grapevine genotype. Different autofluorescence signals were followed from the cellular to the whole-leaf level by using four types of devices for fluorosensing: a macroscope, a spectrofluorimeter, a portable field optical sensor (the Multiplex 3), and a field fluorescence sensor prototype with 335nm excitation. It was shown for the first time, by the three different techniques and at three different scales, that the stilbene-dependent violet–blue autofluorescence (VBF) had a transitory behaviour, increasing to a maximum 6 days post-inoculation (DPI) and then decreasing to a constant lower level, nevertheless significantly higher than in the control leaf. This behaviour could be sensed from both sides of the leaf. On the abaxial side, VBF could discriminate the presence of infection from 1 DPI, and on the adaxial side from 3 DPI. There was a constant increase in blue-excited green fluorescence starting from 8 DPI, concomitant with a decrease in leaf chlorophyll content sensed by one reflectance and two fluorescence indices available on the Multiplex 3 sensor. These results show that a pre-symptomatic and symptomatic sensing of downy mildew is possible by autofluorescence-based sensors, and this is potentially applicable in the field.

Efficacy of single dose of an inactivated porcine circovirus type 2 (PCV2) whole-virus vaccine with oil adjuvant in piglets

BackgroundPost-weaning multisystemic wasting syndrome (PMWS) associated with PCV2 is one of the most costly diseases currently faced by the swine industry. The development of effective vaccines against PCV2 infection has been accepted as an important strategy in the prophylaxis of PMWS.MethodsIn the present study, a PK-15 cell-adapted formalin-inactivated prototype vaccine candidate was prepared using a strain of PCV2 from China. Inactivation of the virus was accomplished using a standard formalin inactivation protocol. The protective properties of the inactivated PCV2 vaccine were evaluated in piglets. Ten 28-day-old pigs were randomly assigned to two groups, each with five. Group 1 was vaccinated intramuscularly with the inactivated virus preparation; Group 2 received sterile PBS as a placebo. By 28 days post-vaccination (DPV), Groups 1 and 2 were challenged intranasally and intramuscularly with 5 × 107 TCID50 of a virulent PCV2 isolate.ResultsThe vaccinated pigs seroconverted to PCV2 and had high levels of serum antibodies to PCV2 at 28 days after vaccination, whereas the control pigs remained seronegative. No significant signs of clinical disease were recorded following the challenge with PCV2, but moderate amounts of PCV2 antigen were detected in most lymphoid organs of the control pigs. PCV2 was detected in two out of the five vaccinated pigs. Furthermore, pathological lesions and viremia were milder in the vaccinated group.ConclusionsThe obtained results indicate that the inactivated PCV2 virus vaccine with an oil adjuvant induce an immunological response in pigs that appears to provide protection from infection with PCV2. The vaccine, therefore, may have the potential to serve as a vaccine aimed to protect pigs from developing PMWS.

Hematological and serum biochemical analyses in experimental caprine coccidiosis

In order to provide information on hematological and biochemical changes in Eimeria arloingi infection, eighteen newborn kids were allocated to three equal groups. Two groups, A and B were inoculated with a single dose of 1×10(3) and 1×10(5) sporulated oocysts of E. arloingi, respectively. The third group C remained uninfected as control. Blood samples were collected from jugular vein before inoculation as control and at 7, 14, 21, 28, 35 and 42days post inoculation (DPI). Total erythrocyte and total leukocyte counts, hemoglobin (Hb), packed cell volume (PCV) and differential leukocyte counts were determined. Serum biochemical parameters including total protein (TP), albumin, chloride (Cl(-)), sodium (Na(+)), potassium (K(+)), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma glutamyltransferase (GGT) were measured. All infected animals showed symptoms of clinical coccidiosis including diarrhea and oocyst excretion after the prepatent period that varied from 16 to 18days after inoculation. The oocysts output did not stop until the end of the experiment. The diarrhea was associated with a reduction in ALP activity, increases in PCV and Hb and decreases in Na(+), Cl(-) and K(+). No significant differences were found in AST, ALT, GGT, albumin and TP during 42 DPI. There was no hepatic damage.

Enhanced replication of swine influenza viruses in dexamethasone-treated juvenile and layer turkeys

Frequent transmission of swine influenza viruses (SIVs) to turkeys has been reported since 1980s. Experimental studies also showed that SIVs can infect turkeys with varying replication and transmission efficiency depending on the strain. However, host factors involved in infection/replication efficiency remain unclear. To investigate whether the immune status of turkeys might play a role in the susceptibility of turkeys to SIVs, we studied the replication efficiency of two recent SIVs (human-like H1N2 and triple reassortant (TR) H3N2) in dexamethasone-treated turkeys. The viruses were inoculated intranasally in both dexamethasone-treated and untreated control juvenile and layer turkeys. Amount of virus shedding was monitored at 2, 4, and 7 days post inoculation (DPI). Additionally, passage of both viruses was attempted in dexamethasone-treated 4-week-old turkeys. In both juvenile and layer turkeys, we were able to detect human-like H1N2 SIV only from dexamethasone-treated turkeys and no virus was detected in untreated birds. The virus shedding of the TR H3N2 SIV was also consistently higher (≈1Log10EID50/ml) in dexamethasone-treated birds in both tracheal and cloacal swabs compared to untreated birds. Virus passage in dexamethasone-treated turkeys was successful up to the second passage and no virus was recovered from the third passage. These results show that potential immunosuppression due to dexamethasone treatment may enhance the transmission and adaptation of SIVs in turkeys through enhancement of virus replication, prolonged virus shedding, and possible decrease of infectious dose required to initiate infection.

Detection of Influenza A Virus Nucleoprotein Antibodies in Oral Fluid Specimens From Pigs Infected Under Experimental Conditions Using a Blocking ELISA

In commercial swine populations, influenza is an important component of the porcine respiratory disease complex (PRDC) and a pathogen with major economic impact. Previously, a commercial blocking ELISA (FlockChek(™) Avian Influenza Virus MultiS-Screen(®) Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME, USA) designed to detect influenza A nucleoprotein (NP) antibodies in avian serum was shown to accurately detect NP antibodies in swine serum. The purpose of this study was to determine whether this assay could detect NP antibodies in swine oral fluid samples. Initially, the procedure for performing the NP-blocking ELISA on oral fluid was modified from the serum testing protocol by changing sample dilution, sample volume, incubation time and incubation temperature. The detection of NP antibody was then evaluated using pen-based oral fluid samples (n=182) from pigs inoculated with either influenza A virus subtype H1N1 or H3N2 under experimental conditions and followed for 42days post inoculation (DPI). NP antibodies in oral fluid were detected from DPI 7 to 42 in all inoculated groups, that is, the mean sample-to-negative (S/N) ratio of influenza-inoculated pigs was significantly different (P<0.0001) from uninoculated controls (unvaccinated or vaccinated-uninoculated groups) through this period. Oral fluid versus serum S/N ratios from the same pen showed a correlation of 0.796 (Pearson's correlation coefficient, P<0.0001). The results showed that oral fluid samples from influenza virus-infected pigs contained detectable levels of NP antibodies for ≥42 DPI. Future research will be required to determine whether this approach could be used to monitor the circulation of influenza virus in commercial pig populations.

INTESTINAL EXCRETION OF A WILD BIRD-ORIGIN H3N8 LOW PATHOGENIC AVIAN INFLUENZA VIRUS IN MALLARDS (ANAS PLATYRHYNCHOS)

Mallards (Anas platyrhynchos) and other dabbling ducks in the genus Anas are an important component of the wild bird reservoir for avian influenza (AI) virus; these viruses are maintained in migratory duck populations through a fecal-oral transmission route. We provide a detailed characterization of intestinal viral shedding in Mallards infected with a wild bird-origin low pathogenic (LP) AI virus. Five of eight, 1-mo-old Mallards inoculated with a high dose of an H3N8 LP AI virus became infected as determined by reisolation and seroconversion. Infected birds excreted high concentrations of virus for up to 14 days postinoculation (DPI) without exhibiting overt clinical signs of disease. The pattern of viral shedding was relatively consistent between individual birds, with peak shedding on 2-3 DPI and a progressive decline over the remainder of infection. Detection of viral shedding varied depending on sample type (excrement sample or cloacal swab) and diagnostic test (virus isolation or real-time quantitative reverse transcription polymerase chain reaction). Our data provide detailed insights into the intestinal excretion of an H3N8 LP AI virus in Mallards and the performance of diagnostic assays commonly used in wild bird surveillance. Such information is valuable for estimating potential risks for spillover of LP AI viruses from Mallards to domestic animals, developing accurate transmission models for Mallard populations and facilitating the interpretation and comparison of surveillance results from different studies.

CD4+ CD25− FoxP3+ regulatory cells are the predominant responding regulatory T cells after human rotavirus infection or vaccination in gnotobiotic pigs

The distribution and dynamic changes of CD4(+) CD25(+) FoxP3(+) and CD4(+) CD25(-) FoxP3(+) regulatory T (Treg) cells induced by human rotavirus (HRV) infection and vaccination were examined in neonatal gnotobiotic pigs infected with virulent HRV (VirHRV) or vaccinated with attenuated HRV (AttHRV). Subsets of gnotobiotic pigs in the AttHRV and control groups were challenged with VirHRV at post-inoculation day (PID) 28. We demonstrated that VirHRV infection or AttHRV vaccination reduced frequencies and numbers of tissue-residing Treg cells, and decreased the frequencies of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) producing CD4(+) CD25(-) Treg cells in ileum, spleen and blood at PID 28. The frequencies of IL-10 and TGF-β producing CD4(+) CD25(-) Treg cells in all sites at PID 28 were significantly inversely correlated with the protection rate against VirHRV-caused diarrhoea (r = -1, P < 0.0001). Hence, higher frequencies of functional CD4(+) CD25(-) Treg cells can be an indicator for poorer protective immunity against rotavirus. Our results highlighted the importance of CD4(+) CD25(-) Treg cells over CD4(+) CD25(+) Treg cells in rotavirus infection and immunity. AttHRV vaccination (induction of immune effector responses) reduced the expansion of CD4(+) CD25(-) Treg cells in ileum seen in the challenged naive pigs during the acute phase of VirHRV infection and preserved normal levels of intestinal TGF-β producing Treg cells post-challenge. The reduced suppressive effect of Treg cells in AttHRV-vaccinated pigs would unleash effector/memory T-cell activation upon challenge. Preserving TGF-β producing CD4(+) CD25(-) Treg cells is important in maintaining homeostasis. Based on our findings, a model is proposed to depict the dynamic equilibrium course of Treg and effector T-cell responses after primary rotavirus infection/vaccination and challenge.

Serum and Egg Yolk Antibody Detection in Chickens Infected with Low Pathogenicity Avian Influenza Virus

Surveillance for low pathogenicity avian influenza virus (LPAIV) infections has primarily relied on labor-intensive collection and serological testing of serum, but for many poultry diseases, easier-to-collect yolk samples have replaced serum for surveillance testing. A time-course LPAIV infection study in layers was performed to evaluate the utility of antibody detection in serum vs. egg yolk samples. Layers inoculated with the LPAIV A/Bobwhite Quail/Pennsylvania/20304/98 (H7N2) were tested for antibody levels in the serum and egg yolk by using the agar gel immunodiffusion test (AGID), hemagglutination-inhibition test (HI), and a commercially available enzyme-linked immunosorbent assay (ELISA). Anti-influenza specific antibodies were detected in the serum as early as 7 days postinoculation (DPI), and the majority of the hens remained positive until 42 DPI. Antibodies in the egg yolk were first detected by AGID at 7 DPI, which was also the first day of detection in serum. However, the majority of the eggs were positive by all techniques at 11 DPI and remained positive until 42 DPI, at which time the number of AGID+ and HI+ samples declined slightly as compared to ELISA+ samples. These results suggest that egg yolk can be an alternative to serum for flock serological surveillance against LPAIV infections, and the three methods (AGID, HI, and ELISA) will give similar results for first 42 days after infection, although AGID may give earlier positive response.