The purpose of this work was to develop a live, attenuated vaccine strain to protect chickens against colonization by group C Salmonella. We constructed two candidate vaccines: a Δcya Δcrp derivative and a ΔphoP derivative of Salmonella hadar. White Leghorn chickens were vaccinated at day of age and at 2 wk with one of the two strains. A nonvaccinated group served as a control. At 4 wk of age, all birds were challenged with wild-type S. hadar and necropsied 6 days later. Numbers of S. hadar in the ceca were determined. Enzyme-linked immunosorbent assay–derived serum immunoglobulin G responses against S. hadar lipopolysaccharide indicated that both strains induced a serum antibody response. The average optical density₄₅₀ for birds vaccinated with the ΔphoP or Δcya Δcrp derivatives was 0.456 and 0.881, respectively. Although the Δcya Δcrp derivative induced higher levels of serum antibody, it did not provide an immune response protective against colonization by S. hadar. Conversely, birds vaccinated with the ΔphoP strain showed significant protection against S. hadar challenge. Seventy percent of the nonvaccinates, 60% of the Δcya Δcrp vaccinates, and 15% of ΔphoP vaccinates were positive for S. hadar in tissues. In a second experiment, birds were vaccinated with either the ΔphoP strain or buffer and challenged with a 10-fold higher dose than in the first experiment. After challenge, all of the birds in both groups were colonized. The geometric mean number of cecal S. hadar isolated from the control group was 1.0 × 10⁶ colony-forming units (CFU)/g, and from the vaccinated group, this value was 32 CFU/g, indicating a four to five log reduction in colonization by the challenge strain.

Investigations for detection and differentiation of nine avian poxviruses (APVs) were carried out by the use of a polymerase chain reaction (PCR) combined with restriction enzyme analysis (REA) and further nucleotide sequence analysis. With one primer set, which framed a region within the fowl poxvirus 4b core protein gene, we were able to detect APV-specific DNA from 19 tested strains and isolates belonging to five defined Avipoxvirus species and four previously undefined isolated species. PCR results revealed no recognizable differences in size of amplified fragments among the different APVs. REA of PCR products with MseI and EcoRV allowed us to differentiate most of the tested avipox species. Nucleotide sequence analysis of the amplified fragments showed a nucleotide similarity of 72%–100% among the different species. Phylogenetic analysis documented five distinguishable sequence clusters in accordance with results obtained by REA. PCR in combination with REA and sequencing of the amplified fragments is a rapid and effective diagnostic system, and it is a new approach to refine epidemiologic studies of APV infections.

The present study was aimed at elucidating the role of heterophil granulocytes during the initial infection with Pasteurella multocida subsp. multocida in chickens. Chickens (17 and 19 wk old) were depleted of their heterophil granulocytes by 5-fluorouracil treatment. When the heterophil blood counts were significantly reduced, the birds were inoculated intratracheally with 1.8–4.3 × 10⁴ colony-forming units of P. multocida. Twelve, 24, or 48 hr postinoculation, the birds were euthanatized and examined for macroscopic and histologic lesions in the lungs. Bacterial invasion was determined by culture of P. multocida from the spleen. Recruitment of heterophils into the respiratory tract during infection was found to contribute considerably to the lung lesions in chickens and was found to mediate tissue damage, possibly allowing a more rapid systemic spread of P. multocida. However, during progression of the infection, the heterophil-mediated necrosis in chickens seemed to stimulate giant cell demarcation of infected lung tissue, which coincided with the clearance of P. multocida from the spleen, thus hampering further invasion. Consequently, heterophil activation plays a dual role for the outcome of a P. multocida infection in chickens, where it initially seems to promote invasion and systemic spread but subsequently helps limit the infection by giant cell formation and bacterial clearance.

Marek's disease (MD) is caused by Marek's disease virus (MDV), a highly cell-associated alphaherpesvirus. MD is primarily characterized by lymphocyte infiltration of the nerves and the development of lymphomas in visceral organs, muscle, and skin. MDV encodes two phosphoproteins, pp24 and pp38, that are highly expressed during lytic infection. These proteins were initially identified in MDV-induced tumors but are now known to be linked primarily to MDV lytic infection. Despite the recent characterization of a pp38 deletion mutant MDV, the functions of these phosphoproteins remain unknown. The goal of this work was to construct recombinant MDVs having direct fusions of a marker gene, the green fluorescent protein (GFP), to pp38 in order to study the expression patterns and localization of this protein during stages of MDV infection. We report the construction of two recombinant viruses, one having the enhanced green fluorescent protein (eGFP) fused in-frame to the pp38 open reading frame (ORF) (RB1Bpp38/eGFP) and the other having soluble-modified GFP (smGFP) downstream but out-of-frame with pp38 (RB1Bpp38/smGFP).

During construction of RB1Bpp38/eGFP, an ORF located downstream of pp38 (LORF12) was partially deleted. In RB1Bpp38/smGFP, however, LORF12 and its immediate 5′ upstream sequence was left intact. This report describes the construction, cell culture, and in vivo characterization of RB1Bpp38/eGFP and RB1Bpp38/smGFP. Structural analysis showed that the virus stocks of RB1Bpp38/eGFP and RB1Bpp38/smGFP had incorporated the GFP cassette and were free of contaminating parent virus (RB1B). Moreover, RB1Bpp38/eGFP and RB1Bpp38/smGFP contained two and three and four and five copies of the 132-bp repeats, respectively. Expression analysis showed that the transcription of genes in RB1Bpp38/eGFP-and RB1Bpp38/smGFP-infected chicken embryo fibroblasts (CEFs) were similar to RB1B-infected CEFs, with the notable exception of deletion of a LORF12-specific transcript in RB1Bpp38/eGFP-infected cells. In CEFs, RB1Bpp38/eGFP and RB1Bpp38/smGFP showed comparable one-step growth kinetics to parental virus (RB1B). RB1Bpp38/eGFP and RB1Bpp38/smGFP, however, showed quite distinct growth characteristics in vivo. Two independent clones of RB1Bpp38/eGFP were highly attenuated, whereas RB1Bpp38/smGFP exhibited pathogenesis similar to parent virus and retained oncogenicity. Our results suggest that the RB1Bpp38/eGFP phenotype could be due to an interference with an in vivo-specific pp38 function via GFP direct fusion, to the deletion of LORF12, or to a targeting of the immune response to eGFP. Because deletion of pp38 was recently found not to fully attenuate very virulent MDV strain MD-5, it is possible that deletion of LORF12 may be at least partially responsible for the attenuation of RB1Bpp38/eGFP. The construction of these viruses and the establishment of cell lines from RB1Bpp38/smGFP provide useful tools for the study of MDV lytic infection in cell culture and in vivo, in studies of the reactivation of MDV from latency, and in the functional analysis of LORF12.

Mycoplasma gallisepticum (MG) was used to expose chicken peripheral blood lymphocytes (PBLs), red blood cells (RBCs), heterophils, and chicken tumor cells (MSB-1 and HD-11 cells). Incubation of PBLs with MG for 3 hr resulted in extensive clumping of lymphocytes. Incubation of the MSB-1 cells with MG also caused clumping of the cells, with many of the cells showing perforations and others showing capping of the surface projections. Incubation of RBCs with MG resulted in an altered cell surface morphology, a decrease in cell size, and perforation. There were no discernible changes on the surface of the heterophils and the HD-11 cells. However, the HD-11 cells appeared to have a decreased ability to attach to the surface of the plastic and to have a decreased ability to respond to chemoattractant fMLP after 24 hr of incubation. These results suggest that, under the conditions used, MG caused certain damage to peripheral blood cells and a significant decrease in chemotactic response in the HD-11 cells.

The events during the pathogenesis of chicken anemia virus (CAV) infection following intramuscular (IM) and oral inoculation were further elucidated and compared by sequential clinical, pathologic, and morphometric histopathologic evaluations, and by sequential determination of CAV genome concentrations in different organs. Specific-pathogen-free chickens were inoculated by IM or oral routes with the same dose (2 × 10⁶ mean tissue culture infective dose [TCID₅₀]) of CAV isolate 03-4876 at 1 day of age. Weights and hematocrits were obtained at 7, 10, 14, 18, 21, 25, and 28 days postinoculation (DPI). Seven birds from each group were necropsied at 7, 10, 14, and 28 DPI, and samples of thymus, Harderian gland, and cecal tonsils (CT) were obtained for histopathologic examination and CAV genome quantification by real-time polymerase chain reaction. Peak CAV genome concentrations were detected in the thymus at 10 and 14 DPI in the IM and orally infected chickens, respectively. High CAV DNA concentrations were maintained throughout the experimental period until 28 DPI, despite specific seroconversion occurring by 14 DPI in the IM-inoculated chickens. CAV was isolated from both orally and IM-infected chickens 28 DPI. Peak CAV genomes in the thymuses of IM and orally infected chickens coincided with peak lymphocyte depletion in these organs. Lymphocyte repopulation of the thymus occurred by 28 DPI in spite of the presence of the virus in the organs of both infected chicken groups. CAV genomes were detected in the CT, but histopathologic changes were not observed. Compared with the IM route of infection, orally infected chickens did not show apparent signs of illness. Clinical parameters, including reduction of weight gains and hematocrits, and gross and histopathologic changes were delayed and less severe in the orally inoculated chickens. This was concurrent with a delay in accumulation of CAV genomes in the thymus of these chickens.

Mycoplasma meleagridis (MM) has the ability to cause bone deformity in turkey poults. However, few pathological lesions have been described and no evidence of MM-induced damage to the bones has been shown. In this study, 17-day-old turkey embryos were inoculated with MM into the allantoic cavity. On the 27th day, eight of the 22 embryos presented with curved toes. Scanning electron microscopy of the tarsometatarsal joints showed fissures in the cartilage. Histological sections of the joints revealed only the infiltration of cells with eosinophilic granules. Immunohistochemical staining (IHS) showed the presence of MM in the aggregates of the bone marrow cells and the cells with eosinophilic granules. Some of these cells were harvested by laser capture microdissection (LCM), lysed, and used as template DNA. With a pair of MM-specific primers in a conventional polymerase chain reaction (PCR), a gene product was amplified, and it comigrated with the MM DNA, which indicates that these captured cells contained MM DNA. Thus, this research shows that inoculation of MM into the turkey embryos produced joint lesions and caused cellular infiltration within the bones.

To understand the genetic variations between the field strains of waterfowl parvoviruses and their attenuated derivatives, we analyzed the complete nucleotide sequences of the viral protein 1 (VP1) genes of nine field strains and two vaccine strains of waterfowl parvoviruses. Sequence comparison of the VP1 proteins showed that these viruses could be divided into goose parvovirus (GPV) related and Muscovy duck parvovirus (MDPV) related groups. The amino acid difference between GPV- and MDPV-related groups ranged from 13.1% to 15.8%, and the most variable region resided in the N terminus of VP2. The vaccine strains of GPV and MDPV exhibited only 1.2% and 0.3% difference in amino acid when compared with their parental field strains, and most of these differences resided in residues 497-575 of VP1, suggesting that these residues might be important for the attenuation of GPV and MDPV. When the GPV strains isolated in 1982 (the strain 82-0308) and in 2001 (the strain 01-1001) were compared, only 0.3% difference in amino acid was found, while MDPV strains isolated in 1990 (the strain 90-0219) and 1997 (the strain 97-0104) showed only 0.4% difference in amino acid. The result indicates that the genome of waterfowl parvovirus had remained highly stable in the field.

Cellulitis has emerged as an economically important disease of broiler chickens. The impact of environmental risk factors on the incidence of cellulitis has not been evaluated in the United States. Escherichia coli (E. coli), the causative agent, is introduced through skin scratches during the grow out. Our previous work suggested that the litter was an important reservoir for cellulitis-associated E. coli. We hypothesized that factors contributing to a positive environment for E. coli growth would increase the opportunity for exposure of a broiler to an infectious dose of E. coli, capable of initiating a cellulitis lesion. This prospective study of 304 flocks on five farms from two integrated broiler companies was conducted to determine the effect of environmental factors on the prevalence of cellulitis in California broiler flocks. Environmental variables included temperature, wind velocity, and relative humidity (RH) at the litter surface. Litter variables measured included E. coli and total gram-negative bacteria load (colony forming units/g dry matter), water activity, and pH. Management variables such as clean out, the number of flocks reared on the same litter (litter run, LR), and downtime (DT) between flocks were also evaluated. Cellulitis ranged from 0.197% to 6.04%. Significant associations were identified using linear regression between farm, LR, DT, ambient temperature during the brooding period, gram-negative bacteria load in the litter during the brooding period, RH mid–grow out, and E. coli load late in the grow out. The significant variation in the rate of cellulitis between farms combined with the strong association of LR and DT with cellulitis demonstrated that management choices were highly influential in this disease syndrome. Based on these data and our previous findings, managers would be advised to increase DT between flocks and perform a total clean out of the house when a flock processes with a high incidence of cellulitis.

A multiplex reverse transcription–polymerase chain reaction (RT-PCR) assay was developedfor the simultaneous detection of two enteric viruses of poultry: turkey enteric coronavirus (TCV) and turkey astrovirus (TAstV). PCR primers were designed to conserved regions within the nucleocapsid gene of TCV and to the polymerase gene of TAstV-2. The primer pairs were successfully used in a multiplex RT-PCR to detect nucleic acid of TAstV-2 and TCV. The test was optimized for use with intestines/feces from naturally infected turkeys. The primers were specific and did not amplify other common RNA or DNA avian viruses. The detection limit was determined to be 10 ng of RNA used as starting template. The use of this specific test allows the rapid and early diagnosis of two financially costly viruses affecting the commercial turkey industry.

Very virulent infectious bursal disease viruses (vvIBDVs) were detected in phenol inactivated bursal samples obtained from Brazil, the Dominican Republic, and Venezuela.After nucleotide sequence analysis of the hypervariable region of VP2 gene, the vvIBDVs from Brazil and Venezuela exhibited all of the 14 nucleotide changes that are conserved in the European UK-661 and most other vvIBDV strains. However, the vvIBDV from the Dominican Republic presented 11 nucleotide changes that are conserved in vvIBDV strains. After phylogenetic analysis, the Latin American strains were found to be related to other vvIBDV strains from Europe, Asia, and Africa. However, Brazilian and Dominican vvIBDVs clustered in two separate subgroups, while the vvIBDVs from Venezuela were closely related to other strains from other parts of the world. By deduced amino acid sequence, the three conserved amino acid residues in vvIBDV strains (222 Ala, 256 Ile, and 294 Ile) were confirmed in the Latin American viruses, and one amino acid change (300 Ala) was unique to all vvIBDVs from the Dominican Republic. The occurrence of this change in the Dominican vvIBDVs may have an impact in their antigenic makeup. Results of this study indicate that the vvIBDVs detected in Latin America are genetically similar to IBDV strains from other parts of the world. However, vvIBDVs from Venezuela were more similar to the vvIBDV strains from Europe and Asia. Of all the samples analyzed, vvIBDVs from Brazil and the Dominican Republic exhibited more genetic changes. These changes may have emerged as a result of the different management practices and environmental conditions present in each particular geographic area.

This cross-sectional, double-blind study reports the prevalence of Salmonella enterica serotype enteritidis (SE) on California egg layer premises using single vs. pooled manure drag swabs and presents a description of egg production and management systems in the state and an initial analysis of risk factors for SE. The study included 91% of all known eligible egg premises in California, representing the majority of eggs produced in the state. The overall prevalence of SE on California egg layer premises was 10.5%, while 1.1% of all rows sampled were positive for SE. The percentage of positive rows for SE on any premises never exceeded 25% of the 16 swabs collected per premises. A description of egg production and management on California egg layer premises is presented. Statistically significant associations for SE were not evident and were limited because of sample size and the low prevalence of SE on California egg layer premises. Several biological and management factors, such as flock health, stage of production, manure management, ventilation, and watering systems, show trend associations with premises positive for SE and require further investigation. Manure drag swabs serve as a useful tool to validate the core components of an egg quality assurance program for SE based on process control principles.

Mycoplasma gallisepticum was isolated from several turkey flocks at different locations in the United States that were clinically affected with respiratory disease. Five of these isolates from four series of outbreaks had patterns similar to the 6/85 vaccine strain of M. gallisepticum by random amplified polymorphic DNA (RAPD) analysis using three different primer sets, whereas with a fourth primer set (OPA13 and OPA14), only two of the isolates were similar to 6/85. Results obtained by sequencing portions of the pvpA, gapA, and mgc2 genes and an uncharacterized surface lipoprotein gene indicated that the field isolates had DNA sequences that ranged from 97.6% to 100%, similar to the 6/85 results. In some of the outbreaks there was an indirect association with the presence of commercial layers in the area that had been vaccinated with this vaccine strain, but there was no known close association with vaccinated birds in any of the outbreaks. Turkeys were challenged with two of the field isolates and with 6/85 vaccine strain. Turkeys challenged with the field isolates developed respiratory disease with airsacculitis and a typical M. gallisepticum antibody response, whereas birds challenged with 6/85 developed no respiratory signs or lesions and developed only a weak antibody response. Although these isolates were very similar to the 6/85 vaccine strain, it was not possible to prove that they originated from the vaccine strain—it is possible that they could be naturally occurring field isolates.

Carcasses of 13 red-tailed hawks (RTHAs) and 11 Cooper's hawks (COHAs) were tested for West Nile virus (WNV) using WNV-specific reverse transcriptase–polymerase chain reaction (RT-PCR) on fresh brain tissue and WNV-specific immunohistochemistry (IHC) on various organs. Ten COHAs (91%) and 11 RTHAs (85%) were positive for WNV RNA by RT-PCR. All 11 COHAs (100%) and 10 RTHAs (77%) were positive for WNV antigen by IHC. A triad of inflammatory lesions, including chronic lymphoplasmacytic and histiocytic encephalitis, endophthalmitis, and myocarditis, was common in both species. In COHAs, the heart (54%), cerebrum (50%), and eye (45%) were the organs that most commonly contained WNV antigen. The amount of WNV antigen was usually small. In RTHAs, the kidney (38%), cerebrum (38%), cerebellum (38%), and eye (36%) were the organs most commonly containing WNV antigen. Unlike COHAs, larger amounts of WNV antigen were present in the cerebrum of RTHAs. WNV antigen was detected in similar cell populations in both species, including neurons of brain, spinal cord, and retina, pigmented epithelial cells of the retina, epithelial cells of renal medullary tubules, cardiomyocytes, endothelial cells and smooth muscle cells of arteries, dendritic cells of splenic lymph follicles, exocrine pancreatic cells, adrenal cells, and keratinocytes of the skin. The study presents strong evidence that WNV can cause a chronic fatal disease in RTHAs and COHAs. The lesion distribution of WNV infection in both species is variable, but inflammatory lesions are common, and a triad of lesions including encephalitis, myocarditis, and endophthalmitis is indicative of WNV infection in both species.

The disease caused by infectious bronchitis virus (IBV) produces great economic loss for the poultry industry. The purpose of this study is to investigate the molecular epidemiology of IBV in Taiwan. An old IBV strain isolated in 1964 and another 31 strains isolated from 1991 to 2003 were selected for N-terminal S1 gene analysis. Based on their phylogenetic tree, 13 strains were selected for sequencing the entire S1 and partial nucleocapsid (N) genes. The results indicated that Taiwanese IBV strains could be divided into two distinct lineages, Taiwan Group I and Taiwan Group II, with one Massachusetts strain and one Chinese strain. No recombination was found between H120 and the Taiwanese strains in the S1 gene. However, the S1 gene showed a noticeably higher divergence than the N gene. The phylogenetic trees constructed from the S1 and N genes indicate that intergenic recombination has occurred. Since most local strains are in Taiwanese clusters, developing vaccines from local strains is necessary for IBV control in Taiwan.

Between the summer of 1998 and the winter of 2000, Salmonella analysis was performed on 2128 single and 532 pooled manure drag swabs obtained from 133 California commercial egg laying farms. The isolation of Salmonella from all rows and from all flocks using single or pooled swabs was 80% and 92%, respectively. Hence, there was no statistical difference between single vs. pooled swabs in terms of identifying Salmonella on a row or flock basis. A total of 14 serogroups comprising 44 serotypes were isolated from 123 of 133 farms. When the top 10 serotypes were considered, there was no significant difference in the range of serotypes isolated by the two culturing methods. The overall S. enteritidis prevalence for California flocks was 10.5% (14/133). The overall row prevalence for S. enteritidis for all the farms was 1.1% (24/2128), and the overall pool prevalence was 2.4% (13/532). Sixty percent (12/20) of the S. enteritidis isolates from the positive farms were phage type 4, and 40% (8/20) represented five other phage types (1, 6B, 7, 8, and 28).

Avian pathogenic Escherichia coli strains are associated with a variety of extraintestinal poultry diseases, including airsacculitis, colisepticemia, and cellulitis. A number of E. coli serotypes are associated with these diseases, although the most prevalent serotype is O78. Fimbrial proteins expressed by these strains appear to be important virulence factors, including type 1 fimbriae, P fimbriae, and curli. We have been working to develop an effective vaccine to protect chickens against these diseases. We have previously shown that an attenuated Salmonella typhimurium strain expressing O78 lipopolysaccharide provides protection against challenge with an O78 avian pathogenic E. coli strain. In this work, we have constructed an attenuated S. typhimurium that expresses both the O78 lipopolysaccharide and E. coli–derived type 1 fimbriae. In these studies, chickens were vaccinated at day of hatch and again at 2 wk of age. Birds were challenged at 4 wk of age. We found that the vaccine candidate provided significant protection against airsacculitis as compared to untreated controls or birds vaccinated with an attenuated S. typhimurium that did not express any E. coli antigens. In a separate experiment, challenged vaccinates showed significant weight gain compared to challenged nonvaccinates. We were not able to demonstrate protection against E. coli O1 or O2 serotype challenge, nor against challenge with wild-type S. typhimurium.

Mycoplasma synoviae is a major pathogen of chickens and turkeys, causing economic losses to the poultry industry worldwide. In this study, we validated and applied polymerase chain reaction (PCR) and DNA sequence analysis on the N-terminal end of the hemagglutinin encoding gene vlhA as an alternative for the detection and initial typing of field strains of M. synoviae in commercial poultry. PCR primers were tested against isolates of M. synoviae from various sources along with other avian mycoplasma and other bacterial species. The vlhA gene–targeted PCR assay was highly specific in the identification of M. synoviae, with a detection limit of 4.7 × 10² color changing units /ml. DNA sequence analysis of amplified products was also conducted to validate the potential for typing M. synoviae strains using the N-terminal region of the vlhA gene. To evaluate the test, we applied the PCR assay to tracheal swabs collected from chickens challenged with M. synoviae strain K1968 and compared the results to the serologic detection. The PCR assay was also evaluated directly on tracheal samples collected from commercial layers. Overall, this vlhA gene–targeted PCR is a useful tool for detection and initial typing of M. synoviae and can be applied in the preliminary identification of M. synoviae isolates directly from clinical samples.

A reproducible and original method for the preparation of chicken intestine epithelial cells from 18-day-old embryos for long-term culture was obtained by using a mechanical isolation procedure, as opposed to previous isolation methods using relatively high concentrations of trypsin, collagenase, or EDTA. Chicken intestine epithelial cells typically expressed keratin and chicken E-cadherin, in contrast to chicken embryo fibroblasts, and they increased cell surface MHC II after activation with crude IFN-γ containing supernatants, obtained from chicken spleen cells stimulated with concanavalin A or transformed by reticuloendotheliosis virus. Eimeria tenella was shown to be able to develop until the schizont stage after 46 hr of culture in these chicken intestinal epithelial cells, but it was not able to develop further. However, activation with IFN-γ containing supernatants resulted in strong inhibition of parasite replication, as shown by incorporation of [³H]uracil. Thus, chicken enterocytes, which are the specific target of Eimeria development in vivo, could be considered as potential local effector cells involved in the protective response against this parasite.

Campylobacter is one of the most commonly reported bacterial causes of human foodborne infections in the United States, and epidemiologic evidence indicates that a significant proportion of human infections result from the improper preparation of poultry products. Campylobacter frequently colonizes the avian intestinal tract, but recent research indicates that this organism can also colonize the avian reproductive tract and possibly contaminate eggs and subsequent offspring. The present studies were undertaken to determine the prevalence of Campylobacter in the reproductive systems of commercial turkeys. In the first study, pooled semen samples from seven commercial turkey farms were randomly collected by abdominal massage over a period of 13 wk. The pooled semen samples were serially diluted, and 0.1 ml of each dilution was plated on Campy-Line agar and incubated at 42 C for 48 hr in a microaerophilic environment for enumeration of Campylobacter. Campylobacter was isolated from 57 of the 59 pooled semen samples, and levels ranged from below the limit of detection (<10¹) to 1.6 × 10⁶ cfu/ml of semen. In the second study, the reproductive tracts of 11 hens and 17 toms were aseptically excised, and the segments (female: vagina, shell gland, isthmus, magnum, and infundibulum; male: ductus deferens and testes) were swabbed with a dry cotton sterile swab. The swabs were incubated for 24 hr in Campylobacter enrichment broth, and 0.1 ml of the enriched sample solution was streaked onto Campy-Line agar plates and incubated at 42 C for 48 hr in a microaerophilic environment. Of the 11 hens sampled, Campylobacter was isolated from the vagina (10/11), the shell gland (7/11), the isthmus (8/11), the magnum (6/11), and the infundibulum (4/11). Of the 17 toms sampled, Campylobacter was isolated from the ductus deferens (8/17) and the testes (2/17). Campylobacter is present in the reproductive tracts and semen of commercial turkeys and may lead to vertical transmission of Campylobacter from the hen to the chick.

The effects of diclazuril and monensin, when included in the feed of turkeys from 0 to 10 wk, upon performance and development of immunity to Eimeria species was investigated. Birds were initially inoculated with a low dose of oocysts of three species of Eimeria at 3, 5, 7, and 9 days of age in order to simulate a natural infection. Weight gain and feed intake from 0 to 6 wk of age was significantly greater in medicated birds compared with those that received no anticoccidial medication. Weight gain and feed intake from 6 to 10 wk was greater in birds that received diclazuril than in unmedicated birds. No differences in performance were evident after drug withdrawal from 10 to 16 wk. Immunity to Eimeria species developed by 10 wk in birds that received no anticoccidial medication but did not develop in those given diclazuril or monensin.

A novel infectious bronchitis variant, designated as IS/885/00, associated with nephritis, was isolated from outbreaks in 23 broiler farms in Israel. The virus was first identified by reverse transcriptase–polymerase chain reaction and showed a distinct restriction fragment length polymorphism pattern from previously described Israeli isolates. Sequence analysis of the S1 gene and the deduced amino acid sequence revealed 97.2% protein similarity to genotype IS/720/99 and 71.6% similarity to the vaccine strain H120, the only strain permitted for use in this country. A database search in GenBank revealed a closely related isolate from Egypt, Egypt/Beni-Seug/01, with 96.6% similarity. Other published nephropathogenic infectious bronchitis virus strains/isolates shared less than 77% similarity with IS/885/00. A vaccine protection test in specific-pathogen-free chicks indicated 91% protection to the trachea and only 25% protection to the kidneys in vaccinated birds challenged with IS/885/00.

The objective of this research was to evaluate the safety of the 6/85 strain vaccine strain of Mycoplasma gallisepticum in turkeys by backpassing the vaccine strain up to 10 times by contact infection in turkeys and challenging turkeys with the resulting backpassaged strain. The vaccine strain, however, did not spread to in-contact turkeys, and it was necessary to reisolate the organism before challenging turkeys for the next passage. The challenge strain, therefore, was one that had been backpassaged four times in turkeys, with a total in vivo time in turkeys of 66 days. The backpassaged 6/85 vaccine strain was no different in pathogenicity than the original vaccine strain, except that at 10 days postchallenge, it was isolated in higher numbers from air sacs. Both the original 6/85 vaccine strain and the backpassaged strain were apathogenic in turkeys, except for a slightly increased diameter of the tracheal mucosa at 10 days postchallenge; at 20 days postchallenge the tracheal mucosal thickness was no different from that of controls.

Campylobacter jejuni cells are able to enter a viable but nonculturable (VBNC) state when they are suspended in water. In the present experiments we inoculated day-of-hatch leghorn and broiler chicks with normal gut microflora and subsequently challenged these with high doses of VBNC C. jejuni. The objective was to determine if the pre-establishment of a normal gut flora would enable VBNC Campylobacter to recover, revert to the vibrionic form, and colonize the cecum. Day-of-hatch leghorn and broiler chicks were gavaged through the esophagus with 0.75 ml of a continuous-flow culture of normal cecal organisms. Two days after gavage, the same chicks were gavaged with 0.75 ml (greater than 10⁹ colony-forming units) of a VBNC suspension of C. jejuni. Seven days later, cecal contents were collected, serially diluted, and examined for the presence of viable culturable C. jejuni. Our results demonstrated that the VBNC C. jejuni cells were unable to revert to a vibrionic culturable form capable of colonizing the cecum.

The open reading frame of the S3 segment encoding the σ2 protein of four turkey reovirus field isolates was analyzed for sequence heterogeneity. The turkey reoviruses we present here have a 97% amino acid identity to turkey NC 98. The S3 nucleotide and amino acid sequence similarity was ≤61% and 78%–80%, respectively, when compared to the chicken reovirus isolates. Comparison of amino acid sequences from chickens and turkeys with that of a duck isolate revealed a 53% and 55% similarity, respectively. Phylogenetic analyses, based on both nucleotide and amino acid sequence, resulted in three major groups among the avian reoviruses; these groups were clearly separated by species. The results of this study provide further evidence, based on the deduced σ2 sequence, that turkey reoviruses form a distinct, separate group relative to chicken and duck isolates. In addition, as a result of the limited sequence identity with their avian counterparts, turkey reoviruses could potentially be considered a separate virus species within subgroup 2 of the Orthoreovirus genus.

The purpose of the present study was to compare the ability of enrofloxacin, oxytetracycline, and sulfadimethoxine to reduce morbidity and mortality caused by Escherichia coli (colibacillosis) in broiler chickens.

The chickens were raised in 80 pens (20 birds per pen) with 20 pens representing each treatment group under simulated commercial conditions that produced a colibacillosis challenge scenario. Each group of 20 randomized pens (replicates) was given one of four water treatments. Chickens that received enrofloxacin had significantly less mortality (P < 0.01), lower average gross pathology (colibacillosis) scores (P < 0.01), and better feed-conversion ratios (P < 0.05) than did chickens that received either oxytetracycline or no medication. Chickens that received enrofloxacin had significantly less mortality and lower pathology scores than those that received sulfadimethoxine and numerically lower feed conversion than the sulfadimethoxine group.

Results from the present study show that enrofloxacin is superior to oxytetracycline and sulfadimethoxine for the control of morbidity and mortality caused by E. coli in broiler chickens. Our findings will help veterinarians choose and prescribe the most efficacious antimicrobial when treating colibacillosis.

Transgenic plants represent a safe, effective, and inexpensive way to produce vaccines. The immunogenicity of VP2 protein of an infectious bursal disease (IBD) virus variant E isolate expressed in transgenic Arabidopsis thaliana was compared with a commercial vaccine in specific-pathogen-free broiler chickens. The VP2 coding sequence was isolated and integrated into A. thaliana genome by Agrobacterium tumefaciens–mediated transformation. Soluble VP2 expressed in transgenic plants was used to immunize chickens. Chickens receiving oral immunization with plant-derived VP2 at 1 and 3 wk of age had an antibody response using enzyme-linked immunosorbent assay and 80% protection against challenge infection at 4 wk. Chickens primed with a commercial vaccine at 1 wk followed by an oral booster with VP2 expressed in plants at 3 wk of age showed 90% protection. Chickens immunized with a commercial vaccine at 1 and 3 wk showed 78% protection. Results supported the efficacy of plant-produced VP2 as a vaccine against IBD.

During the first weeks of 2003, after exotic Newcastle disease (END) was confirmed in commercial layer flocks in Southern California, it became apparent that the virus survival information in the literature varied widely and was difficult to extrapolate to current local conditions. The END Task Force used the information available in the literature and the recommendations of research scientists to establish protocols for safely handling manure from infected and depopulated premises. In an attempt to gain more applicable knowledge in the management of contaminated poultry manure in the course of the END outbreak, this virus survival study was designed and implemented. Environmental drag swabs were tested for END virus from two of the early-infected commercial ranches that consisted of several houses following immediate removal of the infected flocks. A total of 293 samples, composed of 168 manure drag swab pools, 72 dropping board swab pools, and 38 compost swab pools from 3 houses (ranch 1), and 180 manure belt scraper swab pools from ranch 2 were analyzed for ND virus isolation and characterization for 21 consecutive days postdepopulation. Thirteen manure drag swab pools (from houses 1 and 3) and two manure dropping board swab pools (from house 3) collected from ranch 1 were positive for END virus at 0, 1, 2, 3, 4, 7, 10, 12, and 16 days postdepopulation. No END virus was isolated after the 16th day following depopulation from any of the samples. All samples from ranch 2 were negative during the entire observation period.

A new facility was designed to hold 1.8 million birds in 10 houses; chickens were placed in five of the houses, and the remaining five houses were under construction when this outbreak occurred. An increase in mortality was reported in five houses; however, mortality in house 7 was quite high. Well-fleshed birds were suddenly found dead without a significant drop in egg production. The middle and distal intestines were distended with gas, congested, thin walled, atonic, and bluish or pale in color with sloughed mucosa in some places. Necrotic enteritis was diagnosed as the cause of increased mortality. The ingesta in the crop occasionally contained flies. The 4-wk mortality in house 7 was 6.55% with a loss of 10,898 chickens. The 4-wk mortality rate in the other houses ranged from 0.54% to 1.98%.

The houses affected with necrotic enteritis were treated for coccidiosis with amprolium because low numbers of the oocysts were present in the intestinal specimens of some of the chickens. Household bleach was added to the water at a dilution of one part bleach to 1040 parts water to control bacterial contamination.

The fly (Musca domestica) population was out of control. Clostridium perfringens was isolated from the alcohol-washed macerated flies caught from houses 4 and 7. Dead flies were often seen in the feed troughs. The chickens may possibly have had C. perfringens infection as a result of consumption of dead flies or their secretions/excretions. The alcohol-washed, macerated, clarified fly extract from the affected houses caused death in 11 inoculated mice and paralysis in one mouse. Similarly, illness and mortality were present in four mice inoculated with clarified intestinal contents. The bacterium isolated on anaerobic culture was identified as C. perfringens by polymerase chain reaction.

The disease was brought under control after straw was added and mixed in with the litter. As a result, the litter temperature increased, causing a decrease in the fly population. This study suggests that flies in the poultry houses acted as mechanical transmitters of C. perfringens and that the development of necrotic enteritis was by ingestion of bacteria present in the flies and their secretions/excretions.

Two flocks of turkey breeders experienced an increased mortality and high culling rate in the first weeks of egg production. The majority of dead and culled hens had cheesy core in the cloaca and vagina. Postmortem examination revealed fibrinous pseudomembranes in the vagina and cloaca. The thickness of these membranes posed an obstruction to egg passage leading to internal laying and egg peritonitis. Swabs from cloaca and vagina produced numerous colonies of only E. coli. Investigations of this unusual vaginitis showed that these two flocks had a higher number of immature hens with present hymens, and insemination crews mistakenly inseminated all hens in which they were able to evert the cloaca. Breaking the hymen with an insemination pipette created a wound and developed extensive infection with E. coli bacteria.

A defect in the pupil shape of rosecomb bantam chickens was noticed by a breeder. The pupil in affected birds appeared to be elongated at the lower margin and consistent with a coloboma of the iris. Upon examination of all birds in the flock, the defect was found in 26% of females and 9% of males. Examination of breeders older than 1 yr led to the discovery of colobomas, although there were not enough birds to accurately determine the prevalence of the problem for previous generations. Affected birds were examined for other lesions, and none were found. Specific-pathogen-free (SPF) eggs were incubated and hatched with eggs from the affected flock as sentinels. No SPF chicks developed colobomas of the iris, while 37% of the bantams in the same hatch were affected. This experiment eliminated many possible infectious agents and incubator or brooder malfunction as causes of this eye lesion. An analysis of the flock pedigree suggested that the defect might be inherited and might be related either to feather color or to gender. No statistically significant correlation could be made between feather color and the iris colobomas. However, the trait was twice as likely to occur in females as in males, which suggests that it is a sex-influenced trait. Based on the determination that this was an inherited trait, a breeding strategy to reduce the prevalence of the lesion in which affected birds were removed from the breeding flock was developed and followed for 2 yr. The prevalence of colobomas was 22% in females and 2.9% in males after the first year and 7.8% and 0.8% in females and males, respectively, after the second year.

Clinical signs and gross lesions observed in poultry submitted for postmortem examination (PME) from the first five infected poultry flocks preceding the detection of the primary outbreak of highly pathogenic avian influenza (HPAI) of subtype H7N7 during the 2003 epidemic in the Netherlands are described. The absence of HPAI from the Netherlands for more than 75 yr created a situation in which poultry farmers and veterinary practitioners did not think of AI in the differential diagnosis as a possible cause of the clinical problems seen. Increased and progressive mortality was not reported to the governmental authorities by farmers or veterinary practitioners. It took 4 days from the first entry of postmortem material to notify the governmental authorities of a strong suspicion of an AI outbreak on the basis of a positive immunofluoresence test result. The gross lesions observed at PME did not comply with the descriptions in literature, especially the lack of hemorrhagic changes in tissues, and the lack of edema and cyanosis in comb and wattles is noted. The following lessons are learned from this epidemic: a) in the future, increased and progressive mortality should be a signal to exclude AI as cause of disease problems on poultry farms; b) intensive contact between the veterinary practitioner in the field and the veterinarian executing PME is necessary to have all relevant data and developments at one's disposal to come to a conclusive diagnosis; c) in an anamnesis, reporting of high or increased mortality should be quantified in the future (number of dead birds in relation to the number of birds brought to the farm to start production, together with the timing within the production cycle), or else this mortality cannot be interpreted properly; d) if clinical findings such as high mortality indicate the possibility of HPAI, the pathologist should submit clinical samples to the reference laboratory, even if PME gives no specific indications for HPAI; e) the best way to facilitate early detection of an HPAI outbreak is to have the poultry farmer and/or veterinary practitioner immediately report to the syndrome-reporting system currently in operation the occurrence of high mortality, a large decrease in feed or water intake, or a considerable drop in egg production; f) in order to detect low pathogenic avian influenza infections that could possibly change to HPAI, a continuous serologic monitoring system has been set up, in which commercial poultry flocks are screened for antibodies against AI virus of subtypes H5 and H7.

An outbreak of infectious catarrhal enteritis, associated with the flagellated protozoan Spironucleus meleagridis (syn. Hexamita meleagridis), is reported from a commercial flock of chukar partridges in California. The disease affected birds between the ages of 4 and 6 wk and resulted in diarrhea, listlessness, depression, and high mortality. Concurrent infection with other intestinal pathogens, including Cryptosporidia, group E Salmonella, long-segmented filamentous microorganisms (LSFMOs), and Rotavirus-like virus particles, was found in some but not all affected birds. Dermatitis of the face, shanks, and feet, suggestive of B-complex vitamin deficiency, was present in most affected birds as well. Flagellated protozoan parasites could be found in the lumen of the duodenum and jejunum and in the intestinal crypts. In some cases the flagellates were wedged between epithelial cells or were located intracellularly within cells of the mucosal epithelium and the intestinal lamina propria.

Histomoniasis was diagnosed in a flock of 6-wk-old commercial chickens. Clinical signs included depression, stilted gait, inappetence, and a slight increase in mortality. At necropsy, there were pale-yellow to dark-gray circular and depressed necrotic lesions in the liver. The ceca were enlarged and impacted with caseous cores. Cecal worms were not observed either at necropsy or on histopathology. Histomonads were demonstrated microscopically within the bursa of Fabricius in addition to the liver, ceca, and spleen. This is the first report of the presence of histomonads in the bursa of Fabricius in commercial chickens.

From January 2001 to December 2002, 543 ostrich eggs were submitted for bacteriologic investigation. The eggs were laid by 387 domesticated ostriches that suffered fertility disorders and that came from 44 farms located in different areas of Northern and Central Italy. Microbiologic investigations showed bacterial isolation in 105 (19.3%) of 543 eggs examined, with a high prevalence of enterobacteria from albumen and yolk. In only a few cases did bacterial isolation result from yolk or albumen alone. An antibiotic sensitivity test was conducted on isolates by the Kirby–Bauer disc diffusion method. This is the first report regarding the microbiologic status of eggs from ostrich farms located in different Italian regions.