A multiyear study was conducted using an enzyme-linked immunosorbent assay to measure antibody to address the application of the test to the diagnosis of aspergillosis in avian species. In general serostudies (n  =  1314), four avian groups (psittaciform, raptor, penguin, and zoo) were found to have samples with antibody reactivity. Penguin, raptor, and zoo groups were found to have higher levels of antibody to Aspergillus than the psittaciform group. Additional clinical information was collected on 303 cases, which resulted in the definition of presumptive normal, probable, and confirmed infection groups. Although the confirmed group was more likely to have antibody reactivity, the mean antibody index was not found to be significant between presumptive normal and probable or confirmed cases.

We have hypothesized that stress-induced subclinical infection of turkeys with Listeria monocytogenes may be an overlooked source of processing plant contamination, and we have shown that concurrent Escherichia coli challenge can increase L. monocytogenes colonization. The objective of this study was to determine the effects of dexamethasone (Dex) immunosuppressive treatment and transport stress on the isolation of L. monocytogenes in an E. coli–L. monocytogenes challenge model. Thirteen-week-old male turkeys housed in floor pens were either nonchallenged (NCH) or challenged (CH) by environmental exposure to E. coli and L. monocytogenes Scott A, using both a coarse spray and feed inclusion. One group of both NCH and CH birds was not stressed (NCH-Con and CH-Con, respectively), a second group was treated with Dex during challenge (NCH-Dex and CH-Dex, respectively), and a third group was subjected to a 12-hour transport (Trans) stress protocol at 15 wk of age (NCH-Trans and CH-Trans, respectively). All birds were bled and necropsied the morning after transport. Dexamethasone treatment increased mortality and disease incidence. The CH-Con, CH-Trans, and CH-Dex birds, as well as the NCH-Dex birds, had lowered body weights compared to the NCH-Con. The relative liver and heart weights were increased, and the relative bursal weights were decreased by both NCH-Dex and CH-Dex treatments. The heterophil:lymphocyte ratio, a measure of the stress response, was increased by CH-Trans, NCH-Dex, and CH-Dex. Total erythrocyte counts and hematocrit were decreased by NCH-Trans, CH-Trans, NCH-Dex and CH-Dex. The challenge strain of E. coli was isolated from the organs of a significant number of CH-Dex birds using direct plating and occasionally from CH-Trans birds. Listeria monocytogenes was not isolated from significant numbers of birds using direct plating, but was isolated from the knee or hip synovial tissues of a significant percentage of CH-Dex birds using pre-enrichment cultural methods, and from CH-Trans and CH-Dex birds using real-time PCR detection. These data suggest that L. monocytogenes colonization of processing-age turkeys can be increased by stress, and the organism may be harbored within inapparent infections of turkey synovial tissue.

West Nile virus (WNV) causes morbidity and mortality in humans, horses, and in more than 315 bird species in North America. Currently approved WNV vaccines are designed for parenteral administration and, as yet, no effective oral WNV vaccines have been developed. WNV envelope (E) protein is a highly antigenic protein that elicits the majority of virus-neutralizing antibodies during a WNV immune response. Leghorn chickens were given three vaccinations (each 2 wk apart) of E protein orally (20 µg or 100 µg/dose), of E protein intramuscularly (IM, 20 µg/dose), or of adjuvant only (control group) followed by a WNV challenge. Viremias were measured post-WNV infection, and three new enzyme-linked immunosorbent assays were developed for quantifying IgM, IgY, and IgA-mediated immune response of birds following WNV infection. WNV viremia levels were significantly lower in the IM group than in both oral groups and the control group. Total WNV E protein-specific IgY production was significantly greater, and WNV nonstructural 1-specific IgY was significantly less, in the IM group compared to all other treatment groups. The results of this study indicate that IM vaccination of chickens with E protein is protective against WNV infection and results in a significantly different antibody production profile as compared to both orally vaccinated and nonvaccinated birds.

The use of Flinders Technology Associates (FTA®) filter cards to quantify Marek's disease virus (MDV) DNA for the diagnosis of Marek's disease (MD) and to monitor MD vaccines was evaluated. Samples of blood (43), solid tumors (14), and feather pulp (FP; 36) collected fresh and in FTA cards were analyzed. MDV DNA load was quantified by real-time PCR. Threshold cycle (Ct) ratios were calculated for each sample by dividing the Ct value of the internal control gene (glyceraldehyde-3-phosphate dehydrogenase) by the Ct value of the MDV gene. Statistically significant correlation (P < 0.05) within Ct ratios was detected between samples collected fresh and in FTA cards by using Pearson's correlation test. Load of serotype 1 MDV DNA was quantified in 24 FP, 14 solid tumor, and 43 blood samples. There was a statistically significant correlation between FP (r  =  0.95), solid tumor (r  =  0.94), and blood (r  =  0.9) samples collected fresh and in FTA cards. Load of serotype 2 MDV DNA was quantified in 17 FP samples, and the correlation between samples collected fresh and in FTA cards was also statistically significant (Pearson's coefficient, r  =  0.96); load of serotype 3 MDV DNA was quantified in 36 FP samples, and correlation between samples taken fresh and in FTA cards was also statistically significant (r  =  0.84). MDV DNA samples extracted 3 days (t₀) and 8 months after collection (t₁) were used to evaluate the stability of MDV DNA in archived samples collected in FTA cards. A statistically significant correlation was found for serotype 1 (r  =  0.96), serotype 2 (r  =  1), and serotype 3 (r  =  0.9). The results show that FTA cards are an excellent media to collect, transport, and archive samples for MD diagnosis and to monitor MD vaccines. In addition, FTA cards are widely available, inexpensive, and adequate for the shipment of samples nationally and internationally.

An experimental study was conducted to determine the duration of growth depression and virus shedding in turkey poults after oral inoculation with intestinal contents from birds affected with poult enteritis syndrome (PES). Poults at day 14 of age were divided into four groups (groups A, B, C, and D) of 40 poults each and inoculated orally with unfiltered supernatant, filtered supernatant, sediment suspended in phosphate-buffered saline (PBS), or PBS alone (control), respectively. The poults were observed daily for clinical signs, and their growth response, pathology, and pathogen shedding were examined at 10, 20, 30, 40, and 50 days postinoculation (DPI). Body weights of eight poults in each group were recorded at each of these intervals followed by euthanasia. Dullness, depression, and diarrhea were observed in birds inoculated with supernatant or sediment suspension. All three treatments significantly reduced body weight gain of poults compared with the control group; average weight loss was 14%. Gross pathologic changes consisted of pale distended intestines with watery contents and distended ceca with frothy and watery contents. Astrovirus and rotavirus were detected in the inoculum by reverse transcription (RT)-PCR, whereas Salmonella was identified on bacterial isolation. Both viruses were detected in treated poults by RT-PCR for up to 10 and 40 DPI, respectively. Of the three treatments, sediment suspension caused maximal decrease in weight gain as well as greatest pathologic lesions followed by unfiltered supernatant and filtered supernatant. These findings suggest a role for bacteria in increasing the severity of PES. Lower weight gain in treated poults (compared with controls) at 9 wk of age also indicates that PES-affected poults may not reach normal weight at marketing, leading to economic losses for the producer.

Myocarditis associated with reovirus was diagnosed in 17-day-old, male turkey poults, based on virus isolation, reverse transcriptase-polymerase chain reaction (RT-PCR), demonstration of reovirus antigen in the cytoplasm of mononuclear inflammatory cells and myocytes in the heart by immunohistochemistry (IHC), and reovirus particles in the endoplasmic reticulum of myocytes by transmission electron microscopy (TEM). Clinical signs in the poults included anorexia, growth depression, and increased mortality. Gross lesions in the six poults examined were increased pericardial fluid, mild-to-moderate dilation of right ventricles, pale-yellow myocardium, and ascites. Other lesions in a few birds included mild pulmonary edema, congestion, and pale serosa of the small intestine that had watery contents in their lumens. Microscopically, in the heart, there was mild-to-severe necrosis of myocytes and infiltration of primarily lymphocytes mixed with a few heterophils, macrophages, and occasionally, plasma cells and multinucleated giant cells. There was mild-to-moderate lymphoid depletion in the bursa of Fabricius. Reovirus was isolated from the heart of the turkey poults in chicken-embryo liver cells and was confirmed by RT-PCR, IHC, and TEM. A retrospective search of the laboratory database for cases of myocarditis associated with reovirus in turkeys revealed that this condition has occurred sporadically in California turkey flocks since 1991. This is the first documentation of myocarditis in turkey poults associated with reovirus.

Anaerobic intestinal spirochetes (genus Brachyspira) include several species that are recognized as pathogens of poultry. Surveys undertaken in Europe and Australia have shown that layer and breeder flocks are often colonized by the pathogenic species Brachyspira intermedia and Brachyspira pilosicoli, but similar surveys have not been conducted in the United States. In the current study, DNA was extracted from fecal samples (n  =  50) collected from each of 21 flocks of laying hens >40 wk of age in Pennsylvania, and this material was tested for B. intermedia and B. pilosicoli using a duplex PCR. Negative samples also were tested using a Brachyspira genus-specific PCR. The consistency of the feces was observed, and manure handling systems and medication histories were recorded. Brachyspira intermedia was detected in 662 (63.1%) samples from 17 (81%) flocks, with a within-flock prevalence of 10%–100%. Brachyspira pilosicoli was detected in 112 (10.7%) samples from 5 flocks (23.8%), with a within-flock prevalence of 8%–82%. Four of the flocks had both pathogenic species present, three had no pathogenic species detected, and two had no Brachyspira species detected. Nine flocks had many fecal samples with a wet appearance and/or a caramel color, and all of these were colonized with one or the other of the two pathogenic species. Nine of 12 flocks with manure that was mainly dry also were colonized. Differences in colonization rates between flocks with or without wet manure were not significant. Colonization with pathogenic Brachyspira species, and particularly B. intermedia, occurs very commonly in layer flocks >40 wk of age in Pennsylvania. The significance of this high rate of colonization requires further investigation.

In the present study, pulsed-field gel electrophoresis (PFGE) and vlhA gene sequence analysis were applied and verified for typing the Mycoplasma synoviae live vaccine MS-H strain and field isolates from diseased chickens in Japan. The previously published PFGE protocol using SmaI digestion could not allow the discrimination of two of the 11 M. synoviae field isolates from the vaccine strain and had relatively low discrimination power (D  =  0.885). On the other hand, our new PFGE protocols using BlnI and BamHI digestions as well as the vlhA sequence analysis allowed the discrimination of all 11 M. synoviae field isolates from the vaccine strain. In addition, these PFGE protocols using BlnI and BamHI digestions generated unique fragment patterns in epidemiologically unrelated isolates, including those with identical SmaI-digested patterns or vlhA gene sequences (D  =  0.987 and 1.000, respectively), and generated indistinguishable or closely related patterns in epidemiologically related isolates. Therefore, we believe that they would be useful tools to determine whether M. synoviae clinical isolates from diseased chickens are derived from the vaccine strain or wild-type strain and to further elucidate the epidemiology of M. synoviae infection.

The prevalence of Escherichia coli, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis isolated from the feces of wild European starlings (Sturnus vulgaris) humanely trapped at a feedlot in central Kansas was assessed. All E. coli and Salmonella isolates recovered were tested for antimicrobial susceptibility using National Antimicrobial Resistance Monitoring System panels and the E. coli isolates were classified as to their content of genes associated with pathogenic E. coli of birds and cattle, including cvaC, iroN2, ompTp, hlyF2, eitC, iss, iutA, ireA, papC, stxI, stxII, sta, K99, F41, and eae. Escherichia coli O157:H7 and Mycobacterium avium subsp. paratuberculosis were not detected and Salmonella was isolated from only three samples, two of which displayed antimicrobial resistance. Approximately half of the E. coli isolates were resistant to antimicrobial agents with 96% showing resistance to tetracycline. Only one isolate was positive for a single gene associated with bovine pathogenic E. coli. An interesting finding of this study was that 5% of the E. coli isolates tested met the criteria established for identification as avian pathogenic E. coli (APEC). Thus these findings suggest that starlings are not a significant source of Salmonella spp., Mycobacterium avium subsp. paratuberculosis, E. coli O157, or other shiga toxin–producing E. coli in this feedlot. However, they may have the potential to spread APEC, an important pathogen of poultry and a potential pathogen to human beings.

The pathogenesis, virus shedding, and serologic response in specific-pathogen-free (SPF) chickens and commercial turkeys against H4, H6, and H9 type low pathogenic avian influenza viruses (LPAI) from wild birds was examined. Four-week-old chickens and three-week-old turkeys were given 1 × 10⁶ EID₅₀ of LPAI per bird, intrachoanally, and examined for clinical signs for 3 wk. Oropharyngeal and cloacal swabs, and fecal samples, were collected at 2, 4, and 7 days postinoculation (PI) for virus detection by real-time RT-PCR. Serum was collected at 7, 14, and 21 days PI and examined for antibodies against avian influenza virus (AIV) by the enzyme-linked immunosorbant assay (ELISA) and hemagglutination inhibition tests. Tissue samples for histopathology were collected from three birds per group at 3 days PI. The hemagglutinin genes of the viruses were sequenced, and phylogenetic analysis was conducted. Clinical signs ranged from no clinical signs to moderate depression, decreased activity, and decreased food and water consumption. Based on virus detection results, SPF chickens were generally found to be shedding more virus from both the oropharynx and cloaca than were commercial turkeys. Microscopic lesion results in both species showed the predominance of lesions in the respiratory and gastrointestinal tract, which is consistent with the fact that these viruses are of low pathogenicity. In chickens and turkeys, oropharyngeal shedding strongly correlated with the lesions found in the upper respiratory tract. Turkeys had fewer lesions in the respiratory tract and more lesions in the gastrointestinal tract compared to chickens. Thirteen LPAI viruses caused seroconversion in commercial turkeys, whereas only 6 LPAI viruses caused seroconversion in SPF chickens. Phylogenetic analysis of the HA genes showed that the H4, H6, and H9 viruses evaluated here represented the full genetic diversity of North American AIVs of their respective subtypes. This data is important for surveillance and control because some of the LPAI viruses (of wild bird origin and examined in this study) that can infect and be shed by chickens and turkeys would be difficult to detect in commercial poultry. Specifically, detection is difficult because these viruses did not cause overt clinical disease or mortality, but only induced mild microscopic lesions and exhibited poor seroconversion.

In iron-limiting environments, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium synthesize and secrete several types of siderophore to trap trivalent ferric ions; these bacteria then express siderophore receptors called iron-regulated outer membrane proteins (IROMPs). In this study, we experimentally reproduced iron-limiting environments using a divalent metal chelator. IroN, one of the IROMPs, was purified by affinity chromatography with an anti-IroN-MAb-immobilized column. Thirty-day-old chickens were immunized intramuscularly with purified IroN from Salmonella Typhimurium mixed with Freund's incomplete adjuvant; the chickens were then challenged intravenously with Salmonella Enteritidis. The mortality rate of immunized chickens was 10%. On the other hand, that of control chickens was 80%. By Western blot analysis, specific IgG antibody responses against IroN of Salmonella Enteritidis were identified in chickens immunized with purified IroN. These results indicate that IroN might be promising as an important vaccine component against Salmonella infection in chickens.

Recently a novel avian bornavirus has been described that has been suggested to be the possible etiological agent for proventricular dilatation disease or macaw wasting disease. This article describes two macaws that shed avian bornaviral RNA sequences and demonstrated anti-avian bornavirus antibodies as revealed by reverse transcriptase polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot, yet are free of outward clinical signs of the disease.

An outbreak of vaccinal infectious laryngotracheitis (LT) began in 2005 involving 57 ranches of two broiler companies in California. Standard biosecurity, and cleaning and disinfection programs along with vaccination, did not stop the outbreak. Due to the close proximity and number of birds in the same geographic area, the decision was made by both companies to attempt a joint regional and zonal depopulation strategy. The strategy involved extended downtime between flock placements on ranches located within close proximity to one another. This extended downtime on each ranch ranged from 30 to 91 days. An extensive biosecurity audit, with more than 70 items, was implemented. Briefly, this included heating all houses to 37 C for 100 hr, removing the litter, cleaning and disinfecting everything on the ranches, then again heating the houses to 37 C for 100 hr. Used litter was spread on crops away from poultry, or was sent to a litter processor for pasteurization. Extensive surveillance for LT at 28, 35, and 42 days of age was performed on the initial flocks, which had been placed immediately after the extended downtime. Since completion of this plan in early 2008, LT was diagnosed on only two of the previously 57 affected ranches. Those two ranches, and those within close proximity, went through the extended downtime program and biosecurity audit a second time. Currently, both companies are free of LT. This program lends credence to the importance of cooperation between companies to consider all the ranches within close proximity as the population at risk. In the control of LT in broilers, the program also highlights the necessity for extended downtime and enhanced biosecurity auditing of all flocks.

Between 2003 and 2008, more than 600 white stork (Ciconia ciconia) nestlings in the German federal state of Brandenburg were ringed and examined for influenza A viruses. With the spread of highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 among wild birds in Germany in spring 2006, dead wild birds, including 88 white storks, were tested for infection with HPAIV. Furthermore, fresh fecal samples were examined by RT-PCR to monitor the occurrence of HPAIV in adult storks. While the monitoring of nestlings and adult white storks failed to yield evidence of influenza A virus infections in these birds, two storks found dead in April 2006 in the same location tested positive for HPAIV H5N1. Sequence analysis revealed that the virus isolated from one of the storks belonged to clade 2.2, which was commonly found in wild birds in the north of Germany and other European countries during the epidemic in 2006. Despite these two cases, white storks seemed to serve as neither a vector nor as a reservoir for HPAIV in Germany. The risk of white storks transmitting HPAIV to domestic poultry and humans is low.

Here we report the development and application of an enzyme-linked immunosorbent assay (ELISA) to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus vectors. In baculovirus recombinant-infected Sf9 cells, the chicken parvovirus (ChPV) structural viral protein 2 (VP2) was detected as an abundant protein, and the 60-kDa VP2 strongly reacted with parvovirus-infected chicken serum in Western blot. A semipurified VP2 was then used in capture ELISA. Sera from chickens experimentally infected with ChPV and sera from uninfected chickens were tested to evaluate the assay. The ELISA was 93.3% sensitive and 100% specific in detecting ChPV-infected birds. Subsequent assays identified IgG type ChPV-specific maternally acquired antibodies in day-old chickens and demonstrated the production of virus-specific antibodies in young birds following infection with ChPV. In our study, a specific antibody response of infected chickens was observed starting with IgM production between 14 and 21 days postinfection (DPI) and switching into a predominant IgG response by 32 DPI. The availability of an ELISA for detection of virus-specific antibodies and its ability to differentiate between maternally acquired antibodies and antibodies produced following acute infection could prove to be a valuable tool to characterize pathobiological properties and immunogenicity of ChPV.

An outbreak of infectious bursal disease (IBD) in two California layer flocks resulted in the isolation of two infectious bursal disease viruses designated rA and rB. Increased mortality plus gross and histopathology in the layer flocks suggested rA and rB could be very virulent infectious bursal disease virus (vvIBDV). Preliminary studies indicated that high mortality resulted when bursa homogenates from the layer farms were used to inoculate specific-pathogen-free (SPF) chicks. In addition, rA and rB contained VP2 amino acid sequences typically seen in vvIBDV. Molecular and in vivo studies were conducted to more thoroughly identify and characterize the rA and rB viruses. Nucleotide sequence analysis demonstrated that rA and rB had identical sequences across the hypervariable VP2 (hvVP2) and segment B regions examined, and their amino acid sequences in the hvVP2 region were identical to the vvIBDV type strains UK 661, OKYM, and Harbin. Furthermore, the genome segment B nucleotide sequences examined for rA and rB were a 98.1% match with vvIBDV and only an 88.0% match with classic IBDV strains. Phylogenetic analysis placed the rA and rB viruses with other vvIBDV and confirmed these viruses were close genetic descendants of vvIBDV seen around the world. Pathogenicity studies in 4-wk-old SPF chicks demonstrated that at a high dose (10^5.5 50% egg infective dose [EID₅₀]) and a low dose (10^2.0 EID₅₀) of rA and rB, mortality ranged from 91% to 100%. A pathogenic classic virus, standard challenge (STC), at similar doses did not cause mortality in the SPF chicks. In addition, mortality occurred in three out of four SPF birds exposed by direct contact to rA and rB inoculated chicks. Serum from convalescent birds inoculated with rA had high titers to IBDV but were negative for antibodies to infectious bronchitis virus, avian influenza virus, chicken anemia virus, Newcastle disease virus, Mycoplasma gallisepticum, and Mycoplasma synoviae. Virus isolation attempts on the rA and rB bursa homogenate inocula also indicated that no contaminating microorganisms contributed to the high mortality and pathology observed in the SPF chicks. In one experiment, broilers with maternal immunity to IBDV were protected from infection and disease when they were challenged with 10² EID₅₀ and 10⁵ EID₅₀ of the STC virus. When challenged with 10² EID₅₀ of the rA virus, the maternally immune broilers were protected from disease but not infection as evidenced by a positive reverse transcription–polymerase chain reaction (RT-PCR) assay for the virus. When the broilers were challenged with 10⁵ EID₅₀ of the rA virus, they had typical gross and histopathologic signs of IBD but no mortality by 7 days postinoculation. It was concluded that the rA and rB viruses meet the genotypic and phenotypic characteristics of a vvIBDV.

Kirby–Bauer tests were used to analyze the antibiotic resistance of 224 isolates of Riemerella anatipestifer isolated between 1998 and 2005. Among the 36 antibiotics tested, 50% of the analyzed isolates were resistant to ampicillin, ceftazidime, aztreonam, cefazolin, cefepime, cefuroxime, oxacillin, penicillin G, rifampin, and trimethoprim/sulfamethoxazole. Higher levels of resistance were detected for aztreonam, cefepime, oxacillin, penicillin G, ceftazidime, and trimethoprim/sulfamethoxazole (87.8%, 64.3%, 88.6%, 86.9%, 75.9%, and 79.2% resistance, respectively). The lowest resistance rates were observed for amikacin (9.5%), cefoperazone (7.2%), imipenem (3.2%), and neomycin (9.5%). Four isolates were found to be resistant to 29 different antimicrobials. Riemerella anatipestifer drug resistance profiles changed over time, and the only consistent patterns observed were the resistance of R. anatipestifer to cefoperazone, piperacillin, spectinomycin, and aztreonam. In addition to determining the antibiotic-resistance profiles of R. anatipestifer isolates, we also examine the therapeutic efficacy of these antibiotics against lethal R. anatipestifer infection in ducks in vivo. According to these data, we have extrapolated an antibiotic treatment approach for veterinarians attending flocks of ducks. These data suggest that disk-diffusion analyses can be extrapolated to predict in vivo efficacy, thereby facilitating the identification of effective antibacterial treatments and potentially diminishing the irresponsible use of antibiotics.

West Nile virus (WNV) was identified from domestic psittacine birds by inoculating embryonated chicken eggs. Most of the embryos died 5 days postinoculation; flavivirus was detected in some by negative-staining electron microscopy. Immunohistochemistry performed on the embryos and their supporting structures detected the WNV antigen mainly in the chorioallantoic membrane, regardless of the inoculation route or passage number. WNV antigen was also found in the embryonic muscle (both skeletal and smooth muscles) and in multifocal areas of the skin. WNV was not detected in the viscera of the embryo or yolk sac. This study provides evidence of isolation and identification of WNV via embryonated chicken eggs.

In this study, the sequence of the H5 and PB1 genes of the low-pathogenic avian influenza virus (LPAI) A/Black Duck/NC/674-964/06 isolate were determined for replikin peptides and used to design and chemically synthesize a vaccine. The vaccine was used to immunize specific-pathogen-free (SPF) leghorn chickens held in Horsfall isolation units, by the upper respiratory route, at 1, 7, and 14 days of age. The birds were challenged at 28 days of age with 1 × 10⁶ 50% embryo infective dose of the LPAI Black Duck/NC/674-964/06 H5N1 virus per bird. Oropharyngeal and cloacal swabs were collected at 2, 4, and 7 days postinoculation (PI) for virus detection by real-time RT-PCR. Serum was collected at 7, 14, and 21 days PI and examined for antibodies against avian influenza virus by the enzyme-linked immunosorbent assay and hemagglutination inhibition (HI) tests. Tissue samples for histopathology were collected from three birds per group at 3 days PI. The experimental design consisted of a negative control group (not vaccinated and not challenged) and a vaccinated group, a vaccinated and challenged group, and a positive control group (challenged only). None of the nonchallenged birds, the vaccinated birds, or the vaccinated and challenged birds showed overt clinical signs of disease during the study. A slight depression was observed in the nonvaccinated challenged birds on day 2 postchallenge. Although the numbers of birds per group are small, no shedding of the challenge virus was detected in the vaccinated and challenged birds, whereas oropharyngeal and cloacal shedding was detected in the nonvaccinated and challenged birds. HI antibodies were detected in the vaccinated and nonchallenged group as well as in the vaccinated and challenged group, but rising antibody titers, indicating infection with the LPAI challenge virus, were not detected. Rising HI titers were observed in the nonvaccinated and challenged group. In addition, no antibodies were detected in the nonchallenged birds. Noteworthy microscopic lesions were not observed in the vaccinated and challenged birds, whereas nonvaccinated-challenged birds had microscopic lesions consistent with infection with LPAI viruses. Taken together, these data indicate that a replikin peptide vaccine, specifically made against the H5N1 Black Duck/NC/674-964/06 isolate, and administered three times to the upper respiratory tract, was capable of protecting chickens from infection and from shedding of the homologous virus, which is extremely important because reduced virus shedding and transmission decreases the potential for H5 LPAI viruses to become HPAI viruses. The study is also important because it shows that the vaccine can be effectively mass-delivered to the upper respiratory tract.

Infectious laryngotracheitis (ILT) is a respiratory disease of poultry caused by an alphaherpesvirus (ILTV). To evaluate differential detection of ILTVs belonging to the two types, wild-type or vaccine-type, both causing clinical signs, five PCRs were evaluated to detect wild-type and vaccine-type ILTV in clinical samples. By directly sampling the organs, we aimed to avoid changes in the virus genome and to facilitate a fast diagnosis. The samples were tracheal and spleen homogenates and feather shafts. The latter are easy to collect, nonlethal for the bird, and advantageous for monitoring purposes. We investigated the time interval for vaccine virus detection following commercial vaccination by the vent application, which is successfully practiced in Israel. The study indicated that ILTV amplification from feather shafts was possible in clinical cases for about a one-month period after vaccination. Vaccine strains were identified by nested PCR for the ILTV-gE gene and differed from wild-type ILTV strains by two criteria: 1) While avirulent vaccines could be detected for about a month after the vent application, wild-type virus could be detected, in conjunction with clinical signs, for an unlimited time period; and 2) The ILTV vaccine was present in the bird in minute quantities compared to the wild-type virus. We assessed the virus type that appeared in conjunction with the clinical signs and determined that the clinical signs appeared in conjunction with both molecular forms of ILTV. The vaccine virus-type and the wild-type ILTV differed by their distinct restriction pattern when using the HaeIII restriction enzyme digestion of the nested amplification product.

The use of viral vectors for transgenic expression of immunogenic proteins is a current trend in the poultry industry. The objective of this work was to assess the protection against the variant E of infectious bursal disease virus (IBDV), conferred by day-one vaccination with a commercial recombinant herpesvirus of turkey (HVT) vaccine (VAXXITEK®) expressing the immunogenic viral protein 2 from a classical IBDV. In separate trials, 1-day-old specific-pathogen-free (SPF) or broiler chickens were vaccinated by the subcutaneous route and challenged with the variant E strain at 18 or 28 days of age. Bursa/body weight ratio and bursal histopathology were assessed as protection criteria. Protection was demonstrated at both challenge points, bursal indexes in vaccinated SPF and broiler groups were significantly higher than in the challenged controls. The commercial vaccine protected against bursal damage as indicated by significantly lower bursal lesion scores in the vaccinated birds. These experimental results indicate that a single dose of the recombinant HVT-IBDV confers protection against variant E challenge even though the VP2 expressed by the recombinant herpes virus belongs to a standard strain.

Avian tuberculosis was diagnosed via histopathology, microbiology, and molecular biology in two of six pheasants from a local sanctuary bird house in Taiwan. Swinehoe's pheasant (Lophura swinhoii) is a near-threatened species in Taiwan. The infected birds showed clinical signs such as fatigue, inappetence, diarrhea, and fluffing of feathers. On postmortem, nonmineralized caseogranulomas were present in the brain, heart, lung, liver, spleen, costal membranes, and intestinal tracts. The presence of granulomas in the lungs of the infected pheasants may suggest that exposure to the infective agent was via the respiratory route rather than the alimentary route. Histopathologic findings were typical of avian tuberculosis, including acid-fast bacilli and centrally located caseous necrosis surrounded by epitheloid macrophages, lymphocytes, and multinucleated giant cells. Laboratory confirmation was made based on lesions and via Ziehl-Neelsen acid-fast stain, polymerase chain reaction, nucleic acid sequencing, and a reliable assay protocol for identification of diseases bioactive amplification with probing assay.

Warbler species of the families Sylviidae and Acrocephalidae occurring in the Danube river delta are frequently exposed to blood-sucking arthropods that transmit avian blood parasites. We investigated infections by three genera of hemosporidian parasites in blood samples from six warbler species. Altogether in 17 (32.6%) of 52 blood samples, a PCR product was amplified. The great reed warbler (Acrocephalus arundinaceus) had the highest prevalence, with 63.6% (7/11) infected individuals, whereas no infection was detected in marsh warbler (Acrocephalus palustris). The most common parasite genus was Haemoproteus, which was found in 15.4% (8/52) of individuals. Seven known parasite lineages (five Haemoproteus and two Plasmodium) and two new lineages were recorded (one Leucocytozoon and one Plasmodium).

A female, adult, pen-raised chukar (Alectoris chukar) was submitted for postmortem examination. The main gross findings were severe emaciation, coelomic cavity and pericardial edema, and a large, sharply demarcated area of necrosis in the liver. Histologically, the liver lesions were characterized by areas of severe necrosis and inflammation containing numerous protozoal organisms morphologically consistent with Histomonas meleagridis. There was necrotizing typhlitis, with few histomonads and scant Heterakis spp. worms, in the cecum. Numerous aphasmid organisms, consistent with capillarids, were present in the crop and esophageal mucosa. Histomonas meleagridis was identified from frozen samples of liver by polymerase chain reaction and nucleotide sequencing. Sequence analysis of the internal transcribed spacer (ITS)-1, 5.8S, and ITS-2 regions of the ribosomal RNA gene disclosed a 95% identity to a previously sequenced ITS-1, 5.8S, and ITS-2 region of H. meleagridis.