Despite improved biosecurity and vaccination programs in recent years, infectious laryngotracheitis continues to emerge in the field on a regular basis in poultry producing states. Evidence is mounting that most field outbreaks are caused by viruses indistinguishable from chicken-embryo–origin vaccine strains and, for that reason, broiler outbreaks are often referred to in the field as “vaccinal laryngotracheitis” (VLT). Cooperative industry programs are described, in which the poultry industry, along with government, laboratories, and other sectors involved with poultry live production work together to control and contain VLT outbreaks. These programs take into account many epizootiologic aspects of the disease and, when diligently followed, are successful in most instances at keeping the numbers of cases low and the outbreaks under control. The programs include the rapid diagnosis of the disease, the use of geographic information system technology, biosecurity, vaccination, and communications between all stakeholders.

Specific-pathogen-free chickens inoculated with H5N1 highly pathogenic avian influenza (HPAI) viruses isolated in Japan in 2004 were investigated pathologically. The chickens inoculated intravenously with the viruses died within 26 hr after inoculation. Macroscopically, minimal necrosis of the tip of the comb, and hemorrhages of the palpebral conjunctiva, liver, cerebellum, and muscles were rarely observed. Histologically, dead chickens had minimal focal necrosis of hepatocytes with fibrinous thrombi in sinusoids, mild necrosis of splenic ellipsoids with fibrinous exudation, minimal necrosis of the brain, mild necrosis of epidermal cells of the comb with congestion of the lamina propria, and hemorrhages and edema of the lamina propria of the conjunctiva. Virus antigens were seen in the sinusoidal endothelial cells and hepatocytes in the liver, the capillary endothelial cells of the spleen, the capillary endothelial cells and cardiac myocytes in the heart, the capillary endothelial cells and necrotic nerve cells in the brain, the capillary endothelial cells in the lamina propria of the comb, the renal tubular epithelial cells, and the pancreatic acinar cells. The chickens inoculated by natural infectious routes died within 1–4 days after inoculation. Macroscopically, some chickens had hemorrhages in the conjunctiva, edematous swelling of the face and wattles, hydropericardium, hemorrhages of the proventriculus and bursa of Fabricius, increased secretion of tracheal mucus, and congestion and edema of lungs. Histologic lesions by natural infectious routes were similar to those by intravenous inoculation, except for the pancreatic necrosis. This study suggests H5N1 HPAI viruses isolated in Japan in 2004 cause pathologic conditions similar to natural cases.

The incidence of necrotic enteritis (NE) due to Clostridium perfringens (CP) infection in commercial poultry has been increasing at an alarming rate. Although pre-exposure of chickens to coccidia infections is believed to be one of the major risk factors leading to NE, the underlying mechanisms of CP virulence remain undefined. The objectives of this study were to utilize an experimental model of NE produced by Eimeria maxima (EM) and CP coinfection to investigate the pathologic and immunologic parameters of the disease. Broilers coinfected with EM plus CP exhibited more severe gut pathology compared with animals given EM or CP alone. Additionally, EM/CP coinfection increased the numbers of intestinal CP bacteria compared with chickens exposed to an identical challenge of CP alone. Coinfection with EM and CP repressed nitric oxide synthase gene expression that was induced by EM alone, leading to lower plasma NO levels. Intestinal expression of a panel of cytokine and chemokine genes following EM/CP coinfection showed a mixed response depending on the transcript analyzed and the time following infection. In general, IFN-α, IFN-γ, IL-1β, IL-2, IL-12, IL-13, IL-17, and TGF-β4 were repressed, whereas IL-8, IL-10, IL-15, and LITAF were increased during coinfection compared with challenge by EM or CP alone. These results are discussed in the context of EM and CP to act synergistically to create a more severe disease phenotype leading to an altered cytokine/chemokine response than that produced by infection with the individual pathogens.

Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is an extraintestinal disease that causes great economic loss to the poultry industry each year. APEC must overcome host defenses, such as immune system components found in serum, in order to establish infection; however, the mechanism of such serum resistance has been elusive. In the present study, a proteomic approach was used to evaluate APEC proteins that were differentially expressed after exposure to chicken serum to identify specific proteins that may be involved in serum resistance of APEC isolates. Proteins were isolated and separated by two-dimensional (2D) gel electrophoresis, and 10 protein spots corresponding to differentially expressed proteins were chosen for sequencing using electrospray ionization tandem mass spectrometry. Eight proteins were identified among the spots, some of which have previously been associated with the virulence of E. coli. Significantly, an outer-membrane protein previously associated with serum resistance, OmpA, was among those proteins identified, further indicating that differential regulation of this protein may be involved in serum resistance. This study opens the door to future research using a proteomic approach to identify the key players in serum resistance of APEC.

Studies were performed to determine if mucosal vaccination with inactivated avian metapneumovirus (aMPV) subtype C protected turkey poults from clinical disease and virus replication following mucosal challenge. Decreases in clinical disease were not observed in vaccinated groups, and the vaccine failed to inhibit virus replication in the tracheas of 96% of vaccinated birds. Histopathologically, enhancement of pulmonary lesions following virus challenge was associated with birds receiving the inactivated aMPV vaccine compared to unvaccinated birds. As determined by an enzyme-linked immunosorbent assay (ELISA), all virus-challenged groups increased serum immunoglobulin (Ig) G and IgA antibody production against the virus following challenge; however, the unvaccinated aMPV-challenged group displayed the highest increases in virus-neutralizing antibody. On the basis of these results it is concluded that intranasal vaccination with inactivated aMPV does not induce protective immunity, reduce virus shedding, or result in decreased histopathologic lesions.

Experiments were carried out to establish an infection and disease model for Clostridium perfringens in broiler chickens. Previous experiments had failed to induce disease and only a transient colonization with challenge strains had been obtained. In the present study, two series of experiments were conducted, each involving four groups of chickens with each group kept in separate isolators. A coccidial vaccine given at 10 times the prescribed dosage was used to promote the development of necrotic enteritis. In the first experiment, cultures of C. perfringens were mixed with the feed at day 9, 10, 11, and 12, and the coccidial vaccine was given at day 10, whereas in the second experiment, C. perfringens cultures were mixed with the feed at day 17, 18, 19, and 20, and the coccidial vaccine was given at day 18. Chickens were examined at day 9, 11, 12, and 15 (Experiment 1), and at day 17, 18, 20, and 24 (Experiment 2). There was no mortality in any of the groups; however, chickens in the groups receiving both coccidial vaccine and C. perfringens developed the subclinical form of necrotic enteritis, demonstrated by focal necroses in the small intestine, whereas chickens in control groups or groups receiving only coccidial vaccine or only C. perfringens cultures developed no necroses. The results underline the importance of predisposing factors in the development of necrotic enteritis.

The Asian H5N1 highly pathogenic avian influenza (HPAI) virus causes a systemic disease with high mortality of poultry and is potentially zoonotic. In both chickens and ducks, the virus has been demonstrated to replicate in both cardiac and skeletal muscle cells. Experimentally, H5N1 HPAI virus has been transmitted to chickens through the consumption of raw infected meat. In this study, we investigated virus replication in cardiac and skeletal muscle and in the trachea of chickens after experimental intranasal inoculation with the H5N1 HPAI virus. The virus was detected in tissues by real-time reverse transcription–polymerase chain reaction (RRT-PCR) and virus isolation, and in the trachea by RRT-PCR and a commercial avian influenza (AI) viral antigen detection test. A modified RNA extraction protocol was developed for rapid detection of the virus in tissues by RRT-PCR. The H5N1 HPAI virus was sporadically detected in meat and the tracheas of infected birds without any clinical sign of disease as early as 6 hr postinfection (PI), and was detected in all samples tested at 24 hr PI and later. No differences in sensitivity were seen between virus isolation and RRT-PCR in meat samples. The AI viral antigen detection test on tracheal swabs was a useful method for identifying infected chickens when they were sick or dead, but was less sensitive in detecting infected birds when they were preclinical. This study provides data indicating that preslaughter tracheal swab testing can identify birds infected with HPAI among the daily mortality and prevent infected flocks from being sent to processing plants. In addition, the modified RNA extraction and RRT-PCR test on meat samples provide a rapid and sensitive method of identifying HPAI virus in illegal contraband or domestic meat samples.

Among winter migratory waterfowl, Northern pintails (Anas acuta), in one of the largest flocks in Tohoku district, northeast Japan, were surveyed for influenza A viruses at five wintering sites in three prefectures, viz., Aomori, Akita, and Miyagi. A total of 38 influenza A viruses were isolated from 2066 fecal samples collected during November 2006 through March 2007. The overall isolation rate was 1.84%. Eleven different subtypes were isolated, including nine H5N2, seven H6N8, seven H10N1, four H4N6, three H6N1, three H11N9, and one each of H1N1, H6N2, H6N5, H10N9, H11N1. Only the H4N6 subtype was detected during two successive months, November and December, from Lake Ogawara of Aomori prefecture. One wintering site, Lake Izunuma of Miyagi prefecture, was negative for virus isolation throughout the study period. During the sampling period, the highest virus isolation rate was in December (4.90%) followed by November (2.18%), January (0.91%), and February (0.30%). Virus isolation was negative for samples collected in March 2007. These results suggest that influenza viruses are introduced by Northern pintail when they migrate into Japan, but the viruses are not maintained in the flocks, most likely because the birds are not breeding during the winter. We believe that this relatively large data set creates a strong foundation for future studies of avian influenza virus (AIV) prevalence, evolution, and ecology in wintering sites, along with the role of Northern pintails in the spread of AIV during their migration from northern Russia and Asia to Japan.

A molecular technique based on the restriction fragment length polymorphism of the 16S ribosomal genes amplified by a polymerase chain reaction (PCR), referred to as amplified 16S ribosomal DNA restriction analysis (ARDRA), was designed to identify 19 Avibacterium paragallinarum strains isolated from infraorbital sinus and nasal turbinate bone samples of broiler chickens, breeders, and laying hens from different regions of Peru. The 16S rDNA was amplified by PCR using a pair of bacterial universal primers and restriction analysis of 16S rDNA sequences was done to select endonucleases with the highest number of cutting points inside the 16S rDNA. The DNA patterns with DdeI and RsaI endonucleases were identical for the 19 A. paragallinarum strains, but differed from those obtained for Ornithobacterium rhinotracheale, a bacterium with a high genetic and phenotypic resemblance to A. paragallinarum, as well as from Escherichia coli, a bacterium associated with infectious coryza. The ARDRA method could prove to be valuable for molecular identification of A. paragallinarum, a microorganism implicated in respiratory diseases in commercial birds.

Infectious laryngotracheitis (ILT) is a highly contagious, acute respiratory disease of chickens, of worldwide distribution, that affects growth and egg production and leads to significant economic losses during periodic outbreaks of the disease. Live attenuated vaccines (chicken embryo origin [CEO] and tissue-culture origin [TCO]) have been widely used to control the disease in the United States. It is believed that most of the outbreaks in the United States are caused by vaccine-related isolates that persist in the field and spill over into naïve poultry populations. The objective of this study was to utilize the previously developed polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis to genotype recent ILT virus (ILTV) isolates from commercial poultry. Forty-six samples were collected during January 2006 to April 2007 from five poultry production regions of the United States and were characterized within PCR-RFLP groups III–VI. Sixty-three percent of the samples analyzed were categorized as closely related to the vaccine strains (groups III–V), whereas 33% were categorized as group VI viruses that differed in six and nine PCR-RFLP patterns from the CEO and TCO vaccines; a mixture of group IV and V viruses was detected in two samples (4%). In general, groups V and VI were the most prevalent viruses, found in 52% and 33% of the samples tested respectively. Both types of viruses were detected in vaccinated and nonvaccinated flocks. Although genetically different, both viruses produced severe disease in the field.

Three different lytic bacteriophages (BPs) were isolated from the sewage system of commercial chicken flocks and used to reduce Salmonella Enteritidis (SE) colonization from experimental chickens. Ten-day-old chickens were challenged with 9.6 × 10⁵ colony-forming units (CFU)/ml of a SE strain and treated by coarse spray or drinking water with a cocktail of the three phages at a multiplicity of infection (MOI) of 10³ plaque-forming units (PFU) 24 hr prior to SE challenge. Chickens were euthanatized at day 20 of age for individual SE detection, quantitative bacteriology, and phage isolation from the intestine and from a pool of organs. SE detection was performed by both bacteriologic culture and genome detection by polymerase chain reaction (PCR). Qualitative bacteriology showed that aerosol-spray delivery of BPs significantly reduced the incidence of SE infection in the chicken group (P  =  0.0084) to 72.7% as compared with the control group (100%). In addition, SE counts showed that phage delivery both by coarse spray and drinking water reduced the intestinal SE colonization (P < 0.01; P < 0.05, respectively). BPs were isolated at 10 days postinfection from the intestine and from pools of organs from BP-treated chickens. We conclude that the phage treatment, either by aerosol spray or drinking water, may be a plausible alternative to antibiotics for the reduction of Salmonella infection in poultry.

Between January 2004 and December 2005, cloacal swabs from essentially healthy chickens and silky chickens from live birds markets in Guangdong and Hunan provinces in southeastern China were screened for chicken anemia virus (CAV) by polymerase chain reaction. Phylogenetic analysis of the major structural protein VP1 sequences showed no clear genotype cluster and no correlation with the geographic origin of CAV strains. Virus evolution at the amino acid level was very slow, which corresponds to a strong negative selection of the VP1 gene in China and worldwide. A high proportion (87%) of birds was CAV positive, suggesting that many farms in the region were infected. Further investigations are necessary to evaluate the economic losses caused by CAV and the cost–benefit of vaccination.

The objective of this study was to isolate and identify a hypothetical Chlamydiaceae pathogen from laying hens with an oviduct cyst, and to characterize its potential causal relation with decreased egg production. Our clinical survey showed that cystic oviducts were prevalent at rates of 10% and 15.1% in breeder and commercial hen flocks, respectively. Chlamydial antigens were detected in 20 of 50 pharyngeal swabs (40%) and in 17 of 20 oviduct tissues (85%) using enzyme-linked immunosorbent assay (ELISA) antigen detection kits. The isolated pathogen was identified as Chlamydophila psittaci via complement fixation test, PCE-ELISA, and immunofluorescence assay. Avian influenza virus, Newcastle disease virus, and infectious bronchitis virus were excluded after oviduct tissues were inoculated onto the chorioallantoic membrane of embryonating eggs. The nucleotide sequence of the omp1 gene (accession no. EF202608) from the isolate was similar to that of C. psittaci avian type C (accession no. L25436). Typical cystic oviducts were observed in specific-pathogen-free hens inoculated intraperitoneally with the isolate. The high presence of chlamydial antigen is consistent with the cystic oviducts and poor egg production. We conclude that the isolated C. psittaci is most likely associated with cystic oviducts in laying hens.

The crop immune response against Salmonella Enteritidis (SE) challenge in eight commercial egg-layer strains (five white-egg layer and three brown-egg layer) and specific-pathogen-free (SPF) White Leghorn (WL) hens was investigated. Pre- and post-SE challenge mucosal immune responses within the crops were evaluated. Commercial layers and SPF WL hens were orally challenged with 10⁸ CFU/ml SE PT13a and SE nalR PT13, respectively. Crop lavage samples were collected at weekly intervals from day 0 (pre-challenge) to day 25–27 postinfection (PI), and bacteriological examination was performed to monitor progression of SE infection. Crop lavage samples were analyzed for SE-lipopolysaccharide (LPS)–specific IgA using enzyme-linked immunosorbent assay (ELISA). H&E-stained slides of crop sections from day 34 PI and uninfected controls were assessed for lymphoid tissue via light microscopy. Lymphoid areas were graded based on morphology, size, and cellularity using a score 0 to 5 scale. The 0 to 5 (low to high) numerical values represented progressive increases in size and cellular density of lymphoid tissue. Bacterial culture results showed the highest percentage of SE-positive crop lavage samples from all hen groups at day 5–6 PI and day 11–12 PI. A progressive decline in percentage of SE-positive crop lavage samples did occur as time PI lengthened; however, at day 25–27 PI SE persisted in crop lavage samples from SPF WL hens and three commercial white-egg layer strains. A marked increase in SE-LPS-specific IgA was measured in crop lavage samples between day 0 and day 11–12 PI for all hen groups. Crop SE-LPS-specific IgA response remained elevated above day 0 baseline for the duration of the experiment. Well-defined score 3 to 5 lymphoid tissue aggregates were observed in crop tissue sections harvested at day 34 PI. Comparison of crop sections determined a 1.2–4.0 times increase in ratio of lymphoid tissue in day 34 PI SE-challenged hens vs. uninfected control hens.

The objective of this study was to identify and understand the regular distribution pattern and primary penetration site for Salmonella Enteritidis (SE) in the gastrointestinal tract of ducks. An assay based on the serovar-specific DNA sequence of SE from GenBank, a serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction, was developed for the detection of SE. We used this assay to detect genomic DNA of SE in the blood and gastrointestinal tract, including duodenum, jejunum, ileum, cecum, rectum, esophagus, stomach muscularis, and stomach glandularis, from ducks after oral challenge at different time points. The results showed that SE was consistently detected in all segments of the gastrointestinal tract. The jejunum and ileum were positive 8 hr postinoculation (PI). The organism was detected in blood 12 hr PI, while the final organ to show a positive result was the stomach at 24 hr PI. The copy number of SE DNA in each tissue reached a peak at 24–36 hr PI, with the jejunum, ileum, and cecum containing high concentrations of SE, whereas the blood, duodenum, rectum, stomach, and esophagus had low concentrations. SE populations began to decrease and were not detectable at 2 days PI, but were still present up to 9 days PI in the jejunum, ileum, and cecum without causing apparent symptoms. By 3 days PI the cecum had significantly higher numbers of SE than any of the other areas (P < 0.01), and this appeared to reflect its function as a repository for SE. In conclusion, the results provided significant data for understanding the life cycle of SE in the gastrointestinal tract and showed that the jejunum, ileum, and cecum were the primary sites of invasion in normal ducks after oral challenge. This study will help to understand the mechanisms of action of SE infection in vivo.

Mycoplasmas are pathogens of different avian species, but the role of Mycoplasma in raptors is not yet completely determined. As Mycoplasma isolation and identification present several difficulties, species-specific polymerase chain reactions (PCRs) for the detection of mycoplasmas found in birds of prey (Mycoplasma buteonis, Mycoplasma corogypsi, Mycoplasma falconis, and Mycoplasma gypis) were established. The specificity of the PCR methods were investigated using known avian Mycoplasma reference strains and isolates as well as related bacteria and was found to be specific. Amplificons obtained with these PCRs from field samples showed no false-positive results in restriction enzyme analysis and sequencing. The sensitivities of the different PCR assays varied between 50 fg and 1 pg DNA. Twenty-five tracheal swabs from healthy captive birds of prey were investigated by culture and immunobinding assay as comparison to the PCRs. Mycoplasmal DNA was detected in 88% of the samples, with negative results only from vultures. Mycoplasma falconis and M. buteonis were regularly found in falcons, and M. gypis was found in a common buzzard. Mycoplasma corogypsi was not demonstrated. Several isolates could not be differentiated using an immunobinding assay as well as the described PCR methods.

Wild birds are suspected to play a role in the spread of avian influenza H5N1; however, much remains unknown about the ecology and epidemiology of H5N1 in wild birds. Lake Constance is an important wetland area and was a focus for surveillance of dead wild birds between February and June 2006. Dead wild birds collected from the lake and surrounding regions were tested for avian influenza H5. This article provides a descriptive and spatial analysis of the data collected during this period and includes discussion of the strengths and limitations of this type of surveillance. The sampling of dead birds may provide a rapid and cost-effective means of detecting the presence of H5N1; however, such sampling is prone to certain biases and lacks sensitivity in detecting asymptomatic infections. The benefit of such surveillance will be enhanced by detailed ornithologic information, greater accuracy of spatially referenced data, and quantification of surveillance effort.

A low pathogenic avian influenza virus (AIV) serotype H9N2 affected many commercial flocks in the Middle East in late 1990s and early 2000s. Due to the varying pathogenicity of AIV H9N2 reported in previous studies, this study was carried out to determine the pathogenicity of a Jordanian isolate of H9N2 in broiler and specific-pathogen-free (SPF) chickens. Mild tracheal rales were observed in the broilers but not in the SPF birds starting 3 days postinfection (DPI) and until the end of the experiment at 16 DPI. Infected chickens had gross and histologic changes limited to the respiratory system (sinuses, trachea, lungs, and air sacs) characterized by congestion and lymphoplasmacytic inflammation. However, the lesions in the broiler chickens were more severe than those in the SPF chicks. Furthermore, the virus caused significant (P  =  0.004) reduction (230 g) in average body weight of the infected broiler group compared with the uninfected broiler group. Both broiler and SPF-infected groups seroconverted, and they had a geometric mean titer of 2^8.2 and 2^9.3, respectively, on the hemagglutination inhibition test at 16 DPI. Cloacal virus shedding was not detected by 9 DPI and 15 DPI in broiler and SPF-infected groups, respectively. This study demonstrated the pathogenic nature of the local Jordanian H9N2 isolate and the variation from what it has been reported in other countries of the region. Regional effort should be directed to start an eradication program of this disease because of its pathogenicity for chickens, wide distribution, and possible interference with surveillance for H5N1 serotype.

A recombinant fowlpox virus (rFPV) coexpressing the Newcastle disease virus (NDV) fusion and hemagglutinin-neuraminidase genes and infectious laryngothracheitis virus (ILTV) glycoprotein B gene was constructed. This virus was then evaluated for its ability to protect specific-pathogen-free (SPF) chickens against clinical symptoms and death after challenge by virulent NDV and ILTV. SPF chickens were grouped and vaccinated with the rFPV and commercial NDV (La Sota) and ILTV attenuated live vaccine (Nobilis ILT), respectively. After challenge with NDV 10 days postvaccination, 70% of chickens vaccinated with rFPV were protected from death, whereas 100% of the commercial NDV-vaccinated chickens were protected from death. In contrast, 100% of the unvaccinated chickens died after challenge. After challenge with ILTV, both the rFPV and commercial ILTV-vaccinated chickens were completely protected from death and 70% of chickens were protected from respiratory signs. In comparison, 100% of the unvaccinated chickens developed severe respiratory disease and 10% of chickens died. The protective efficacy was also measured by the antibody responses and isolation of challenge viruses. Results showed that this rFPV could be a potential vaccine for preventing NDV and ILTV by a single immunization.

RESUMEN. Protección de aves contra la enfermedad de Newcastle y la laringotraqueítis infecciosa con un virus recombinante de viruela aviar coexpresando los genes F, HN del virus de la enfermedad de Newcastle y el gen gB del virus de laringotraqueítis infecciosa.

Six disinfectant chemicals were tested individually for effectiveness against low pathogenic avian influenza virus (LPAIV) A/H7N2/Chick/MinhMa/04. The tested agents included acetic acid (C₂H₄O₂), citric acid (C₆H₈O₇), calcium hypochlorite (Ca(ClO)₂), sodium hypochlorite (NaOCl), a powdered laundry detergent with peroxygen (bleach), and a commercially available iodine/acid disinfectant. Four of the six chemicals, including acetic acid (5%), citric acid (1% and 3%), calcium hypochlorite (750 ppm), and sodium hypochlorite (750 ppm) effectively inactivated LPAIV on hard and nonporous surfaces. The conventional laundry detergent was tested at multiple concentrations and found to be suitable for inactivating LPAIV on hard and nonporous surfaces at 6 g/L. Only citric acid and commercially available iodine/acid disinfectant were found to be effective at inactivating LPAIV on both porous and nonporous surfaces.

Many commercial enzyme-linked immunosorbent assays (ELISAs) are unable to differentiate antibody responses to different avian influenza virus (AIV) subtypes. Developing an ELISA for specifically detecting the H5 antibody is the purpose of this study. Four monoclonal antibodies (Mabs) were raised using A/duck/Yunlin/04 (H5N2). They were confirmed as being specific to H5. Two of these antibodies showed hemagglutination inhibition (HI) activity using the HI test. Using immunodot blot assays, three Mabs recognized both Eurasian and American H5, whereas the other Mab recognized only the tested Eurasian H5 virus. When testing denatured H5 antigen, one of the Mabs lost its antigen binding activity using Western blotting. For detecting the H5 humoral response in serum, one monoclonal antibody was purified and labeled with horseradish peroxidase to set up a blocking ELISA. Chicken sera that blocked H5 Mab binding by >29% were considered H5 antibody positive. Inhibition percentages for sera from chickens infected with other AIV subtypes, H1 to H15, were <29%. This blocking ELISA was used for 478 field chicken serum samples. The results showed that the sensitivity and specificity of this ELISA were 98.3% (232/236) and 95.9% (232/242), respectively. This blocking ELISA could be used specifically for detecting the H5 humoral responses in chickens.

As part of ongoing ecological studies of Humboldt penguins (Spheniscus humboldti) at Punta San Juan, Ica Department, Peru, health surveys were conducted in November 1992, 1993, and 1994. In the three surveys, 98 birds in total were handled for examination, and blood was collected for laboratory analysis from 90 of these birds. All birds seemed to be in good condition. Body weights of females were significantly lower in 1994 than in the other years. Fleas (Parapsyllus humboldti) and ticks (Ornithodoros amblus) were found on the penguins and in their nests. Females had significantly higher plasma calcium and phosphorus levels, and they had lower weights than males. No other differences were found between the sexes. Hematology, plasma chemistries, and plasma mineral levels varied between years. Positive antibody titers for Chlamydophila psittaci (62%), avian adenovirus (7%; 1994 only), paramyxovirus-2 (7%; 1993 only), and Salmonella Pullorum (7%) were found. Plasma chemistry and mineral levels differed between individuals testing positive vs. negative on serologic tests for avian adenovirus and Salmonella Pullorum. Serologic tests for antibodies to avian influenza A virus, avian encephalomyelitis virus, infectious bronchitis virus, avian reovirus, duck viral enteritis virus, equine encephalitis (eastern, western, and Venezuelan) viruses, infectious bursal disease virus, infectious laryngotracheitis virus, Aspergillus sp., and paramyxovirus-1 and -3 were negative. All chlorinated pesticide and polychlorinated biphenyl analyses were below detectable limits.

Lyophilized Mycoplasma gallisepticum (MG) vaccines are generally rehydrated and diluted with distilled or chlorine-free water as per manufacturer recommendations. However, as mycoplasma species lack a cell wall, this can lead to decreased viability of live vaccine during administration. The ability of phosphate-buffered saline (PBS) to prevent losses in live vaccine viability was examined. It was shown that a concentration of 1× PBS prevented the two–fourfold decrease in MG viability seen when the vaccines were diluted with water alone.

Because of recent interest in bacteriophage therapy in poultry, information regarding the interaction of bacteriophages and potential host bacteria in the environment should be collected. The present studies were initiated with a rather typical commercial broiler integrator within the south-central United States to examine environmental Salmonella levels in two broiler complexes, attempt to isolate Salmonella-lytic bacteriophages, and elucidate a possible reason for differing apparent Salmonella prevalence. Significantly (P < 0.05) less Salmonella was isolated from houses in complex 1 (15/44 [34%] Salmonella-positive drag swabs) as compared to houses in complex 2 (22/24 [92%]). A total of seven Salmonella-lytic bacteriophages were isolated from Salmonella-positive environments, and two bacteriophages were isolated from a single Salmonella-negative house. During the initial bacteriophage isolation, individual bacteriophages did not replicate in the Salmonella host isolated from the same environment, and lysis of additional Salmonella hosts relied on high numbers of bacteriophage to be present. This suggests that the presence of these bacteriophages in the environment of a commercial broiler house had little to no effect on the presence of Salmonella. This study highlights the need to find additional bacteriophage sources, more effective isolation methods, and more innovative approaches to using bacteriophages to treat enteric disease.

The effect of a Lactobacillus spp.–based probiotic (FM-B11^TM) on Salmonella enterica serovar Enteritidis (SE) recovery was evaluated in liquid (Expt. 1) and lyophilized (Expt. 2) forms in two separate experiments with two trials each. For each trial, 80 broiler chicks were randomly allocated into two treatments: control and probiotic culture. All chicks were challenged with SE (∼10⁴ colony-forming units [cfu]) upon arrival at our laboratory. In both experiments, probiotic culture was administered in the drinking water for 3 consecutive days at a final concentration of approximately 10⁶ cfu/ml, beginning 1 hr after SE challenge. Cecal tonsils were aseptically removed at 24 and 72 hr postchallenge, followed by enrichment and plating on xylose lactose deoxycholate (XLD) agar for the presence or absence of Salmonella-typical colonies. In Expt. 1, a significant reduction (P < 0.05) in SE-positive samples was observed in both trials at 24 and 72 hr postchallenge. Additionally, in Expt. 2, the lyophilized probiotic decreased (P < 0.05) SE recovery at both 24 and 72 hr postchallenge compared with the control group in trial 1. In trial 2, SE evaluation was performed only at 72 hr after challenge and fewer (P < 0.001) treated samples were positive for SE. Results showed that application of either liquid or lyophilized probiotic culture in the drinking water for 3 consecutive days can help to reduce SE recovery from young birds, although further research is needed to elucidate the mechanism of this response.

Fowl cholera continues to be of concern to the poultry industry, especially for turkey growers. This disease costs the turkey industry millions of dollars annually. In order to develop improved live attenuated vaccines or subunit vaccines, the outer-membrane proteins of Pasteurella multocida were examined with the use of proteomics. Of the 11 proteins total present in an outer-membrane subfraction of P. multocida, four additional proteins were identified, completing the composition of the detergent-soluble cross-protective protein fraction. These additional four proteins include protective bacterial surface antigen, OMA87 (Accession no. 15603857); heme–hemopexin receptor, HemR (Accession no. 15602441); lactate permease, LctP (Accession no. 15603717); and heptosyl transferase F, RfaF (Accession no. 15603709). Both the Oma87 and the HemR proteins would be of interest for subunit and modified live vaccine studies, respectively, because of their purported roles as virulence factors for P. multocida.

Ten representative isolates of Newcastle disease virus (NDV) obtained from outbreaks in waterfowl (geese and ducks) in China since 1997 were characterized both pathotypically and genotypically. The mean death time and intracerebral pathogenicity index were used to evaluate the virulence of the isolates. Pathogenicity tests showed that all 10 isolates were velogenic strains. The main functional region of the F gene made up of 535 nucleotides was amplified by reverse transcription-polymerase chain reaction and sequenced. The deduced amino acid sequence of the fusion protein cleavage site in all 10 isolates was 112RRQKRF117, which is a typical sequence of velogenic strains and is in agreement with the results of in vivo pathogenicity tests. For genotyping, a phylogenetic tree based on nucleotides 47–435 of the F gene was constructed. Phylogenetic analysis showed that most of the isolates were of the genotype VII virus. Only one strain, WG, was found to be of the genotype IX virus. This strain was closest to F48E9, which was isolated in China in 1946 and has been used as a standard challenge strain in vaccine evaluation in China. So, genotype IX virus still causes sporadic infections in geese in China. Further phylogenetic analyses on the genotype VII strains found that all these strains can be subdivided into 5 subgenotypes, and most of the isolates (8 strains) were classified as VIId, a predominant genotype responsible for most Newcastle disease (ND) outbreaks since the end of the past century in China. Only 1 strain, NDV03-053, was shown to be of genotype VIIc virus. Results indicate that the strains of genotype VIId NDV have been the major pathogen, responsible for most epizootic ND outbreaks in waterfowl in China since 1997.

Fowl typhoid is a disease of adult chickens and is caused by Salmonella Gallinarum infection via the alimentary tract. The experimental reproduction of fowl typhoid per os (PO) requires artificial conditions to minimize the effect of gastric acid, and several Salmonella serovars have been known to be transmitted via the respiratory route. Therefore, we have hypothesized the existence of a respiratory route for Salmonella Gallinarum infection and have attempted to reproduce fowl typhoid via intratracheal challenge. In accordance with our hypothesis, the intratracheal challenges of Salmonella Gallinarum reproduced exactly same lesions as fowl typhoid and induced higher mortality and morbidity than those of the PO challenge. Therefore, this study represents the first reproduction of fowl typhoid via respiratory route, and our findings may be useful for understanding the transmission of Salmonella Gallinarum in the field.

During the past years surveillance for avian influenza has been conducted in the live bird markets (LBMs) in New York as well as other states along the east coast. Repeated attempts to eradicate H5 and H7 influenza from the New York markets have focused efforts on the LBMs themselves. Despite repeated mandatory market closures accompanied by cleaning and disinfecting (C/D) procedures, avian influenza virus continued to be isolated. In an effort to assess the adequacy of the C/D procedure, samples were collected in temporal proximity to the depopulation and C/D. Comparison of the pre-C/D (83% virus positive), at C/D approval (1.6% positive) and post-C/D testing (33% positive) indicate that the current procedures of C/D can be effective at eliminating these influenza viruses. However, reinfection via introduction of influenza-virus–positive birds can occur shortly after the market reopens.

The aims of this study were 1) to determine the prevalence of Salmonella in clinically ill birds in aviaries in Ankara, Turkey, and 2) to compare conventional culture and polymerase chain reaction (PCR) for detection of Salmonella in feces from clinically ill pet birds. In the study, 185 fecal samples (feces and/or swabs) collected from the pet birds kept in the seven different aviaries in the city of Ankara were investigated for the existence of Salmonella spp. by bacterial isolation and PCR. The conventional isolation and identification methods were performed for Salmonella isolation from fecal cultures. Suspected colonies were confirmed with the Salmonella polyvalent O antiserum and serogrouped with Salmonella group-specific antiserum. PCR was performed after the fecal swabs were incubated for 18 hr in 10 ml of tetrathionate broth. Three (1.63%) out of 185 fecal samples were found to harbor Salmonella spp. by conventional identification tests and were found to belong to serogroup B. Five (2.7%) swab samples were found to harbor Salmonella DNA by PCR tests. As a conclusion, PCR following incubation of clinical samples in pre-enrichment broth seemed to be a fast and practicable method for Salmonella spp. diagnosis when compared to protracted labor-intensive conventional culture techniques.

Histomonosis (syn. histomoniasis) is a parasitic disease which affects predominately turkeys but also other avian species. Concurrent with the ban of therapeutic and prophylactic substances, the disease, caused by the flagellated protozoon Histomonas meleagridis, is more frequently reported. Due to somewhat diverse results reported in the past, a well-characterized culture was used in the present study to investigate the possible influence of certain parameters on the outcome of the disease. For this study, turkeys were infected with different doses of the mono-eukaryotic culture Histomonas meleagridis/Turkey/Austria/2922-C6/04 using birds of both sexes at various ages. All study groups consisted of 14 birds, of which 10 birds were directly infected via the cloacal route and four birds were kept as in-contact birds. This scheme was used to investigate the pathogenicity of the cloned isolate in 1-day-old and 14-day-old turkeys. In 8-week-old turkeys, only eight birds out of 12 were infected. When 1-day-old and 8-week-old turkeys were infected with 10⁴ histomonads per bird, all turkeys died between 11 and 21 days postinfection or had to be euthanatized due to their poor condition. In a group of 14 poults, infective doses of either 10 histomonads (100 histomonads among 10 birds) or 10³ histomonads per bird had hardly any influence on the first notification of clinical signs. However, even though the onset of clinical signs and mortality was delayed with the lower dose, none of the birds survived the infection. As a consequence, no differences were noticed between male and female turkeys using the mono-eukaryotic culture of Histomonas meleagrigis/Turkey/Austria/2922-C6/04 in the current experimental setting.

The morphogenesis of the new type gosling viral enteritis virus (NGVEV) and the characteristic ultrastructural changes in the duck embryo fibroblasts (DEFs) were investigated by ultrathin sectioning and transmission electron microscopy after monolayer DEFs were experimentally infected with a virulent NGVEV strain. The investigation demonstrated that typical NGVEV particles were round, with a diameter ranging from 75 nm to 90 nm and that they were present in both the nucleus and cytoplasm of the infected DEFs. The mature virions contained nucleocapsids and nucleic acids. The virion penetrated the DEF, replicated, and matured in the nucleus, and they were finally released into the extracellular space via budding and disruption of the cytoplasmic membrane. With the appearance of progeny NGVEV, certain virus-related structures that were densely electron stained, which were circular, U-shaped, or irregular in appearance, could be observed in the cytoplasm of the infected DEFs. In this research, we first detected three types of intracytoplasmic inclusion bodies during the NGVEV infection, which always contained a number of NGVEV particles. Furthermore, we detected that NGVEV could induce apoptosis in DEFs, which had not been reported previously. The morphologic changes of apoptosis included shrinking of the apoptotic cells, chromatin condensation and margination, appearance of vacuoles on the cytoplasmic membrane, and the formation of apoptotic bodies. The mitochondria were ultracondensed and aggregated into compact clusters during apoptosis.

Improper cleaning of the water storage tank resulted in a toxic concentration of sodium in drinking water in a commercial turkey flock. Within the first week after placement 40% of the birds in the flock died. Clinically, poults were depressed and weak, huddled together, and reluctant to walk. At necropsy the birds had crops and gizzards filled with rice hulls, moderately swollen livers, distended gall bladders, and congested lungs. Neither ascites nor round heart was observed. The major microscopic lesion was multifocal symmetrical malacia of brain and spinal cord. Laboratory results revealed a high concentration of sodium in water (2340 mg/liter). The concentration of sodium in brain and liver ranged from 1870 to 2680 (mean  =  2185; SD  =  321.5) mg/liter wet weight and from 1810 to 2360 (mean  =  2191.67; SD  =  193.2) mg/liter wet weight, respectively, whereas the normal expected sodium concentration in the brain and liver tissues from young turkeys (<7 days old) that were submitted for other causes averaged 1233 and 983 mg/liter wet weight, respectively. Based on the histological and toxicological results, a diagnosis of salt poisoning was made. This case investigation demonstrated that sodium analysis of brain and liver are diagnostically useful when confirming sodium poisoning in young turkeys.

Streptococcus gallolyticus subsp. gallolyticus causes endocarditis in humans and acute septicemia in domestic birds. We describe here the infective endocarditis caused by the bacterium found among clinically healthy broilers at two abattoirs in Japan. The chickens were thought to be healthy because of the lack of clinical symptoms and normal levels of mortality before slaughtering. At the time of inspection, some chickens were condemned because of organ disorders characterized by vegetative valvular endocarditis as well as focal necrosis in heart, liver, and spleen. Streptococcus gallolyticus subsp. gallolyticus was isolated from the organs as a pure culture, indicating that the bacterium probably was the causative agent of the disorders. Amplified fragment length polymorphism analysis of the isolates collected at the abattoirs from chickens grown in nine different farms indicated that the isolates were different variants of the same clonal lineage and may have been derived from the same ancestor. These results suggest that S. gallolyticus subsp. gallolyticus causes infectious endocarditis in chickens and that healthy chickens may possess the bacterium in their normal flora as an opportunistic pathogen.

Sarcocystosis in psittaciform birds occurs in several different presentations, making ante-mortem diagnosis challenging without specific laboratory tests. This study followed the course of 11 birds diagnosed with sarcocystosis by serologic analysis and/or post-mortem examinations during a 10-month period in 2006–07. The disease presented in three different clinical forms: an acute pulmonary disease (three birds), muscular disease (five), and neurological disease (three). Early diagnosis of sarcocystosis was possible through the combined used of plasma protein electrophoresis and indirect fluorescent antibody serology in birds presented with the neurological and muscular forms of the disease. In three of these birds the plasma electrophoretic patterns revealed marked hypergammaglobulinemia. All of the birds that presented with the acute pulmonary form developed similar gross and microscopic lesions. Definitive diagnosis was ultimately made by microscopic observation of intravascular pulmonary schizonts containing merozoites. Schizonts were identified in the cerebellum and brainstem in two birds with the neurological form of disease. Those birds that initially presented with severe lethargy and weakness were considered to suffer from the muscular form of disease if they had extreme elevations of muscle enzyme activities (creatine phosphokinase, aspartate aminotransferase) and beta and gamma globulins concentrations, and were seropositive for antibodies to Sarcocystis falcatula. In this group the progression of the disease varied. Two birds recovered completely, and secondary aspergillosis was diagnosed in three birds. The histopathological lesions observed are discussed and interpreted in light of earlier findings from experimental infections in budgerigars, which provide insights into the natural course of sarcocystosis in psittaciform birds.