SUMMARY. The possibility of genomic recombination among different strains of infectious bronchitis virus (IBV) was examined in ovo by coinfecting specific pathogen free embryonating chicken eggs with commonly used, embryo-adapted vaccine strains of IBV (Arkansas, Massachusetts, and Connecticut), and a Delaware-072-like field virus isolated from a layer farm in Minnesota. Recombination was observed between the Massachusetts and the Delaware-072-like strains of the virus. The recombination event was assessed by reverse transcriptase–polymerase chain reaction (RT-PCR) using a combination of specific primers designed to flank a known recombination hot spot of the viral genomic sequence that codes for the S1 subunit of the spike envelope protein. The use of these primers allowed the detection of viruses that have undergone recombination around this hot spot. Cloning and sequencing of the RT-PCR product obtained was performed to confirm these results.

SUMMARY. To determine whether or not exposure to chronic hypoxia and subsequent development of pulmonary hypertension syndrome (PHS) induce alterations in endothelial nitric oxide (NO) production in broiler's pulmonary vascular bed of broilers, we studied the expression of nitric oxide synthase enzyme in pulmonary endothelial cells by a nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemical staining reaction. For this purpose, 60 broilers of three different ages (17, 30, and 42 days) were used. The animals were distributed in two groups: a) 30 healthy (nonhypertensive) broilers and b) 30 chicks with PHS. All broilers in group b had fewer NADPH-diaphorase-positive endothelial cells in arterioles than did the nonhypertensive broilers. These differences were highly significant (P < 0.01). These results demonstrate for, the first time in broilers, that hypoxia-induced pulmonary hypertension is associated with a decrease of endothelial-derived NO expression in pulmonary vessels.

SUMMARY. An infectious bronchitis virus (IBV) was isolated from commercial broilers from the state of California exhibiting respiratory distress, inflamed tracheas, airsaculitis, and edematous lungs. After reverse transcriptase–polymerase chain reaction (RT-PCR), the California isolate exhibited an identical restriction fragment length polymorphism (RFLP) pattern to some isolates obtained from California, known as California 99 isolates. Commercial Mass–Conn and Mass–Ark vaccines were used to vaccinate commercial broiler chickens via eye drop once at 1 or 10 days of age or twice at 1 and 10 days of age. At 27 days of age the birds were challenged via eye drop with the isolated IBV California 99 strain. Protection was measured by failure to reisolate the challenge virus from tracheas 5 days postchallenge and complemented with the tracheal and epithelium thickness scores. When the Mass–Ark vaccine was included in the vaccination programs, there was protection against challenge with the IBV California 99 isolate. The Mass–Conn vaccine conferred protection when used once at 1 day of age and twice at 1 and 10 days of age. However, no total protection was achieved when used as the only vaccine at 10 days of age, since one of the replicates was positive for virus isolation. Significant differences (P < 0.05) in the epithelium thickness and tracheal scores were observed between the unvaccinated–unchallenged group and the groups vaccinated once or twice with the Mass–Conn vaccine. Based on these results, all chickens were protected against the California 99 isolate when the IBV Arkansas type was used as a vaccine.

SUMMARY. The objective of the present study was to investigate the feasibility of a DNA vaccine and CpG oligodeoxynucleotide (ODN) to protect chickens against infectious bursal disease virus (IBDV) infection. Two plasmids DNA carrying VP2 genes of the very virulent (vv) strain of IBDV were constructed with reverse transcription–polymerase chain reaction and designated as pcDNA3.1-VP2 and pCI-VP2. The VP2 protein expressed in COS-7 cells transfected with the plasmid was confirmed by indirect immunofluorescence assay. Seven-day-old chickens were intramuscularly injected with the plasmids alone or plus commercial attenuated infectious bursal disease (IBD) vaccine or synthetic CpG ODN twice at weekly intervals. Chickens at 5 wk old were orally inoculated with vvIBDV strain 99J1 and observed for 7 days after challenge. Immunization with plasmid plus commercial attenuated IBD vaccine or CpG ODN conferred protection for 70%–80% of chickens, as evidenced by the absence of clinical signs, mortality, and atrophy in the cloacal bursa. About 25%–45% of chickens vaccinated with commercial attenuated IBD vaccine or pcDNA3.1-VP2 or pCI-VP2 plasmid alone had clinical signs and died after challenge. Furthermore, there were significantly different histopathologic lesion scores in the clocal bursae between the pcDNA3.1-VP2 or pCI-VP2 plus CpG or live vaccine and pcDNA3.1-VP2, pCI-VP2, or live vaccine vaccinated group. Enzyme-linked immunosorbent assay antibody titers in chickens vaccinated the constructs DNA plus live vaccine or CpG ODN were significantly higher than in those inoculated with the constructs or the live vaccine alone. These results suggest that coadministration of the constructed plasmid pcDNA3.1-VP2 or pCI-VP2 with CpG ODN or commercial attenuated IBD vaccine could protect chickens efficiently from direct vvIBDV challenge.

SUMMARY. In a 2 × 2 factorial study, a broiler starter ration was amended for vitamin A (control, C; deficient, A) and probiotic status (−, P) to investigate their modulatory effects on the host immune system. Birds were inoculated orally with Eimeria acervulina (EA) oocysts, and disease susceptibility was evaluated by assessment of fecal oocyst shedding. Humoral and local cellular mediated immunity were assessed by evaluation of antibody and cytokine (interferon-γ [IFN-γ] and interleukin-2 [IL-2]) levels in sera and intestinal secretions on a 3-day interval after inoculation. Fecal oocyst shedding was highest (P < 0.05) in A− birds, followed by AP, C−, and CP birds. Feeding the probiotic reduced shed oocysts by 20% in A fed birds and by 26% in C fed birds. Intestinal IFN-γ was relatively constant in all treatment groups except for A−, where it declined steadily and was lower (P < 0.05) from day 6 on. Serum IFN-γ levels fluctuated within each treatment and over time were not revealing. Intestinal IL-2 was highest in CP birds at 3 and 9 days postinfection (DPI) and lowest in A− birds at 3, 9, and 12 DPI (P < 0.05); no difference between treatments was found at 6 DPI (P > 0.05). Eimeria-specific intestinal antibody (Ab) level was constant (P > 0.05) in C− birds but increased with time (P < 0.05) in A−, AP, and CP birds. Serum Ab levels were also constant in A− and CP birds but increased (P < 0.05) in C− and AP birds after 6 DPI. The data demonstrate for the first time a probiotic-enhanced immunity in vitamin A deficient birds. This study is also the first to demonstrate the probiotic effect on local cell-mediated immunity of chickens, best manifested by apparent lower intestinal invasion and development by EA, on the basis of higher IL-2 secretion and lower EA oocyst production.

SUMMARY. Eight Chinese field strains of subgroup J avian leukosis viruses (ALV-J) were isolated from broilers or parent stocks during January 1999 to April 2001. One strain, SD9902, was an acute transforming virus, able to induce typical myelocytomatosis in 22–38 days after inoculation of 1-day-old meat-type chicks. The envelope protein and 3′-untranslated region (UTR) of the eight field strains were compared with the U.K. prototype HPRS-103 and several U.S. field strains isolated in 1993–97. All Chinese strains shared an almost identical deletion with the U.S. strain 4817 in the E element region of 3′-UTR when compared with the prototype HPRS-103, indicating that they have a very close phylogenic relationship. Every year, China has to import grandparent stocks of meat-type chickens, mainly from the United States. Chinese isolates should represent a part in the phylogenic tree of U.S. ALV-J evolution. Envelope protein gp85 amino acid sequence analysis demonstrated that, interestingly, all recent Chinese isolates were more closely related to HPRS-103 and the earliest U.S. isolates but not to the late U.S. isolates. The result implies that envelope gp85 may not have diverged from prototype and older strains. It is also possible that some recently imported birds could have been infected by the older viruses that were introduced in the late 1990s.

SUMMARY. Lymphocyte proliferation and interleukin (IL)-2 and IL-6 levels in serum were measured as indicators of cell-mediated immunity after immunization of chickens with a commercial killed Salmonella enteritidis (SE) vaccine or experimental subunit vaccines of crude protein (CP) extract or the outer membrane protein (OMP). Significantly increased proliferative responses to SE flagella, but not lipopolysaccharide, porin, CP, or OMP, were observed at 1 wk postimmunization in the three vaccination groups. The responses to flagella were specific because flagella-induced proliferation was not seen in chickens immunized with adjuvant alone. Of the three immunization protocols, use of the killed SE vaccine appeared most effective because it induced higher flagella-stimulated lymphocyte proliferation at 1 and 2 wk postvaccination compared with the CP- and OMP-vaccinated groups. Significantly increased IL-2 and IL-6 levels in serum were seen at 1 wk postimmunization in the three vaccination groups compared with adjuvant alone, but there were no differences between the killed vaccine and the subunit vaccines at this time, and the levels of both lymphokines returned to baseline at 2 wk postimmunization. We conclude that cell-mediated immunity to SE after vaccination with the killed bacterial vaccine or subunit vaccines is transient and mainly limited to flagella.

SUMMARY. The immunopathologic effects induced by two attenuated chicken anemia virus (CAV) isolates, known as cloned isolate 34 (CI 34) and cloned revertant isolate 18 (CRI 18), that were derived from highly passaged pools of Cux-1 CAV isolate, were compared with those induced by a pathogenic, molecularly cloned, low-passage Cux-1 isolate (CI Cux). This comparison involved the intramuscular inoculation of 1-day-old specific-pathogen-free chicks with each of the viruses and investigation of birds at selected days postinoculation for gross pathology and depletions in the thymic T-cell populations as determined by flow cytometry. Whereas infection with the pathogenic CI Cux produced severe anemia and pronounced bone marrow and thymus lesions, infections with the attenuated CRI 18 and CI 34 isolates produced no anemia, no or mild lesions, respectively, and moderate T-cell depletion. The results suggest that, with CAV, reduced pathogenicity for 1-day-old chicks correlates with reduced depletion of T-cell populations in the thymus and with reduced severity of lesions in the thymus and bone marrow.

SUMMARY. The effect of lysine deficiency on chicken immune function was evaluated using broiler chickens fed a diet with lysine at 67% of the control diet (1.24% lysine). The evaluation of humoral immune function was conducted by measuring the antibody production to a live Newcastle disease virus (NDV) vaccination using the hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA). The cellular immune function was evaluated through the use of cutaneous basophil hypersensitivity test. The antibody response to NDV vaccination was reduced in broiler chickens fed a lysine-deficient diet when measured by ELISA but not when measured by HI. The cell-mediated immune response was also reduced by lysine deficiency.

SUMMARY. Salmonella in birds is a concern because of the human foodborne illness associated with the consumption of poultry meat and eggs. One of the methods of transmission of Salmonella within a flock can be by the air. Therefore, we used reduction of transmission of Salmonella to monitor the effectiveness of the electrostatic space charge system (ESCS). During the average broiler breeder laying cycle of 40 wk, a large amount of dust becomes airborne and accumulates on walls, ceiling, and equipment. Many microorganisms adhere to these dust particles, making dust an excellent vector for horizontal disease transmission between birds. We used two environmentally controlled rooms containing commercial broiler breeders to evaluate the effectiveness of an ESCS that produced a strong negative electrostatic charge to reduce airborne dust and, subsequently, microorganism levels. The ESCS caused the dust to become negatively charged, therefore moving to the grounded floor in the treatment room.

The use of the ESCS resulted in a significant reduction (P < 0.0001, 61% reduction) in airborne dust concentration levels, which resulted in a significant reduction (P < 0.0001, 76% reduction) in total airborne bacteria and gram-negative bacteria (48% reduction) in the treatment room. Significant reductions (P < 0.05) of gram-negative bacteria (63% reduction) on the egg collection belts were also recorded in the treatment room, which resulted in a significant reduction (P < 0.0001) of gram-negative bacteria (28% reduction) on the eggshell surface. The ESCS treatment resulted in fewer Salmonella enteritidis–positive hens and their progeny from the treatment room due to reductions of dust and airborne bacteria. In addition, this significant reduction in bacteria on the eggshell surface should result in less bacteria in the day-old chicks, therefore better early chick livability. There was no significant difference (P > 0.05) in egg production, male or female body weights, mortality, or reproductive performance in the ESCS room compared with the control room.

SUMMARY. Two experiments determined the influence of an experimental reovirus-antibody complex vaccine on Marek's disease virus (MDV) vaccine when used in ovo. Designs were the same except that specific-pathogen-free (SPF) broiler eggs were used in Experiment 1 and commercial broiler eggs with maternal antibodies against reovirus were used in Experiment 2. At 18 days of incubation, embryos were separated into four groups and inoculated with either diluent, MDV vaccine, reovirus-antibody complex vaccine, or a combination of reovirus-antibody complex and MDV vaccine. At 5 days of age, half the chickens in each group were challenged with MDV. At 7 wk old, all were euthanatized, weighed, and examined. At 7 days of age, remaining chickens in each group were challenged with reovirus. At 21 days old, chickens were euthanatized and weighed. No vaccine adversely affected hatchability or posthatch mortality in SPF or commercial chickens. There were no significant differences in protection against reovirus challenge when vaccines were used separately or in combination, and lesion scores were nearly identical in all vaccinated groups in both experiments. However, percentage of protection against reovirus was lower in Experiment 2, indicating an adverse effect of maternal immunity on efficacy of the reovirus vaccine. There were no significant differences in protection against MDV when the vaccines were used separately or combined. Severity of MDV lesions was nearly identical in all vaccinated groups in both experiments. However, the combination of vaccines gave numerically lower protection against MDV than MDV vaccine alone. Use of a larger number of birds, as in field conditions, may result in statistically lower protection for the vaccine combination. Large field trials are needed to determine the potential of the reovirus-antibody complex vaccine.

SUMMARY. The individual and combined effects of feeding fumonisin B₁ (FB₁; 0, 100, 200 mg FB₁/kg) and moniliformin (M; 0, 100, 200 mg M/kg) were evaluated using a 3 × 3 factorial arrangement of treatments. Significant mortality (P < 0.05) occurred in chicks fed all diets containing 200 mg M/kg (50%–65%). Compared with controls and chicks fed FB₁, both feed intake and body weight gain were decreased (P < 0.05) in chicks fed diets containing 100 mg M/kg. Chicks fed M had heavier heart weights (P < 0.05) than control chicks or chicks fed FB₁. Compared with controls, chicks fed diets containing 200 mg M/kg or a combination of 200 mg FB₁/kg and 100 mg M/kg had increased kidney and liver weights (P < 0.05). Significant FB₁ by M interactions (P < 0.05) were observed for serum total protein and aspartate aminotransferase. Mild to moderate periportal extramedullary hematopoiesis and mild focal hepatic necrosis were observed in chicks fed FB₁ alone. An increased incidence of large pleomorphic cardiomyocyte nuclei, loss of cardiomyocytes, and mild focal renal tubular mineralization were observed in chicks fed M alone. Both cardiac and renal lesions were observed in chicks fed combinations of FB₁ and M. Data indicate FB₁ and M, alone or in combination, can adversely affect chick performance and health at these dietary concentrations. The interactive effects of FB₁ and M were not synergistic and were less than additive in nature. At the dietary concentrations studied, M is much more toxic to broilers than FB₁.

SUMMARY. In this study we compared protection by DNA vaccination with the F (pCMV-F) or N (pCMV-N) gene from avian metapneumovirus (aMPV) in turkeys. One-week-old turkey poults received two intramuscular injections 2 wk apart. Birds were challenged with a turkey-embryo-adapted aMPV at 5 wk of age. Birds vaccinated with pCMV-F had decreased clinical signs of disease as well as significantly reduced virus load in tracheal swabs compared with birds vaccinated with pCMV-N or unvaccinated control birds. Serum neutralizing antibodies were significantly higher in birds receiving pCMV-F compared with all other groups. These results indicate that DNA vaccination with the F, but not N, gene of aMPV can induce significant protection against aMPV infection.

SUMMARY. The complete nucleotide sequences of two plasmids from avian isolates of Pasteurella multocida that caused outbreaks of fowl cholera in Taiwan were determined. The entire sequences of the two plasmids, designated as pJR1 and pJR2, were 6792 bp and 5252 bp. Sequence analysis showed that the plasmid pJR1 contained six major genes: the first gene (sulII) encoded a type II sulfonamide resistant dihydropteroate synthase, the second gene (tetG) encoded a tetracycline resistance protein, the third gene (catB2) encoded a chloramphenicol acetyltransferase, the fourth gene (rep) encoded a replication protein, and the fifth and sixth genes (mbeCy and ΔmbeAy) encoded proteins involved in the mobilization of plasmid. The plasmid pJR2 contained five major genes: the first gene (ΔintI1) encoded a truncated form of a type I integrase, the second gene (aadA1) encoded an aminoglycoside adenylyltransferase that confers resistance to streptomycin and spectinomycin, the third gene (blaP1) encoded a beta-lactamase that confers resistance to ampicillin and carbenicillin, and the fourth and fifth genes might encode proteins involved in the plasmid replication or segregation. Sequence comparisons showed that the antibiotic resistance genes found in pJR1 and pJR2 exhibited a high degree of sequence homology to the corresponding genes found in a great variety of gram-negative bacteria, including Escherichia coli, Salmonella enterica Typhimurium DT104, Pseudomonas spp., P. multocida, Mannheimia spp., and Actinobacillus pleuropneumoniae, which suggests that these resistance genes were disseminated in these bacteria. Although sulII and tetG genes were found previously in P. multocida or Mannheimia spp., this is the first report on the presence of catB2, aadA1, and blaP1 genes in bacteria of the family Pasteurellaceae. Moreover, the aadA1 and blaP1 genes found in pJR2 were organized into an integron structure, which is a site-specific recombination system capable of capturing and mobilizing antibiotic resistance genes. This is also the first report on the presence of an integron in bacteria of the family Pasteurellaceae. The presence of a P. multocida integron might facilitate the spreading of antibiotic resistance genes between P. multocida and other gram-negative bacteria.

SUMMARY. The purpose of this experiment was to characterize a lesion of the rhamphotheca associated with tryptophan (TRP) deficiency, search for other histological abnormalities, and determine whether bird size and housing conditions are contributing factors to these lesions. Day-old broiler chicks (Ross × Ross 308) were placed in either floor pens with fresh pine shavings or Petersime battery brooders with two pens of 10 chicks each per treatment. Broiler chicks from 0 to 21 days of age were fed adequate (0.24%) and deficient (0.09%) levels of TRP in diets based on corn, corn gluten meal, and gelatin. Separate groups of control chicks were pair fed daily with the deficient chicks. Deficient chicks grew less efficiently than did the pair-fed controls. Upon gross examination, a lesion of the maxillary rhamphotheca in the vicinity of the nares was observed in 61% of TRP-deficient birds housed in the battery and 13% of the birds housed in floor pens. A similar gross lesion was only observed in one control bird. These lesions were located along the upper portion of the beak between the nares and appeared as a crusty or scab-like area on gross examination, composed of detritus, heterophils, and plasma protein. Inflammation occasionally was observed at the dermoepidermal junction. The incidence of lesions was reduced in floor pens compared to battery brooders, but similarly sized birds did not exhibit the lesion. The number of lesions seen grossly and histologically in TRP-deficient birds, as compared to control birds, supports the hypothesis that TRP deficiency is the primary cause of these lesions around the nares of broilers. Secondary environmental factors, perhaps coprophagy, also influence the incidence of the lesion.

SUMMARY. A bacteriophage to a serotype 02, nonmotile Escherichia coli was isolated from municipal waste treatment facilities and poultry processing plants. A study was conducted to determine the efficacy of multiple vs. single intramuscular (i.m.) injections of bacteriophage to treat a severe E. coli respiratory infection. The birds were challenged at 7 days of age by injection of 6 × 10⁴ colony-forming units (cfu) of E. coli into the thoracic air sac followed by an i.m. injection into the thigh with either heat-killed or active bacteriophage. There were 16 treatments with three replicate pens of 10 birds. There were four control treatments, which included untreated birds, birds injected with either heat-killed or active bacteriophage, and birds challenged only with E. coli. In the remaining treatments, birds were injected with heat-killed or active bacteriophage either once immediately after E. coli challenge or immediately after challenge and at 8 and 9 days of age, once at 8 days of age or at 8, 9, and 10 days of age, and once at 9 days of age or at 9, 10, and 11 days of age. Mortality was significantly decreased from 57% to 13% in the birds given a single i.m. injection of bacteriophage immediately after E. coli challenge, and there was complete recovery in birds treated immediately after challenge and at 8 and 9 days of age, which was a significant improvement from the single injection treatment. There was a significant reduction in mortality from 57% to 10% in the birds treated with bacteriophage once at 8 days of age and those birds treated at 8, 9, and 10 days of age, with no difference between single or multiple treatments. The mortality in the single or multiple phage treated birds that started at 9 days of age was reduced from 57% to 28% and 27%, respectively, but was not statistically different from the control. These data suggest that bacteriophage can be an effective treatment when administered early in this experimental E. coli respiratory disease and that early multiple treatments are better than a single treatment. The efficacy of bacteriophage treatment diminishes as it is delayed, with no difference between single or multiple treatments. Bacteriophage may provide an effective alternative to antibiotics, but like antibiotic therapy, the effectiveness of phage to rescue animals decreases the longer treatment is delayed in the disease process.

SUMMARY. Diagnosis of avian mycobacteriosis, caused by Mycobacterium genavense or species belonging to the Mycobacterium avium complex (MAC), is problematic. Polymerase chain reaction (PCR) offers rapid and sensitive detection of minute quantities of DNA, and conventional protocols have been used for evaluating avian specimens. The recent development of real-time PCR offers several advantages over conventional PCR.

In attempts to improve diagnosing avian mycobacteriosis, a real-time TaqMan^® PCR assay was developed targeting the 65-kD heat shock protein gene of M. genavense and MAC spp. Nineteen reference isolates, 16 clinical isolates, and 32 avian tissue samples were used to evaluate the assay. When sufficient amplicons were produced, the species of mycobacteria was determined by standard sequencing of TaqMan^® PCR products and compared with results from commercial mycobacteriology laboratories and/or standard sequencing of conventional PCR products.

The TaqMan^® PCR detected DNA from reference isolates of M. genavense, MAC spp., and Mycobacterium tuberculosis complex spp. Of the clinical isolates, the TaqMan^® PCR detected DNA from 10 of 12 Mycobacterium avium avium isolates and two of three Mycobacterium avium intracellulare isolates. For the tissue samples, the TaqMan^® PCR amplified DNA in six of nine samples that were identified by sequencing of conventional PCR products and/or by commercial mycobacteriology laboratories as being MAC spp. positive and three of four samples that were positive for M. genavense. There was some disagreement between speciation results from the TaqMan^® PCR and those from commercial mycobacteriology laboratories or conventional PCR or both. This disagreement was suspected to be because of relatively small numbers of base pairs in the TaqMan^® PCR products. The TaqMan^® PCR may provide a useful tool for evaluating clinical samples for DNA from mycobacteria species that most commonly infect birds; however, further refinement is needed in order to improve sensitivity and provide more accurate speciation.

SUMMARY. A mixture of viruses with variations in virulence is likely present within a low-pathogenicity avian influenza virus (LPAIV) population. An H6N1 AIV was isolated from a field case showing 3.8% weekly mortality and a 33% egg production drop in Taiwan. The pathologic lesions included proventricular hemorrhage and urate deposition in the kidneys and on visceral organs. From the field isolate, a clone (2838N) that caused no lesions or death was obtained using limit dilution in chicken embryos and a clone (2838V) that caused renal lesions and death was obtained using contact infection in chicks. Both clones were inoculated intranasally in 4-week-old specific pathogen free (SPF) chickens to test their virulence. Renal urate deposition was found in chickens inoculated with 2838V but not in chickens inoculated with 2838N. In situ hybridization, polymerase chain reaction, and virus isolation were used to confirm the spread of 2838V from the respiratory tract to the renal tissue. We found that contact infection in chickens is a good method to obtain a more virulent clone from a heterogeneous virus population.

SUMMARY. A commercial reovirus vaccine alone or experimental reovirus vaccine plus antibody complex were inoculated into 18-day-old specific pathogen free (SPF) broiler embryos at 0.1 of the recommended chick dose. The following groups were used: group 1A was not vaccinated or challenged; group 1B was not vaccinated, but was challenged with virulent reovirus; group 2 received the vaccine complexed with 1/4 dilution of antiserum; group 3 received the vaccine with 1/8 dilution of antiserum; group 4 received the vaccine with 1/16 dilution of antiserum, and group 5 received vaccine alone. At 1, 3, 6, 9, and 12 days of age, serum was collected and antibody against avian reovirus was analyzed by enzyme-linked immunosorbent assay (ELISA). At the same times, spleens were collected and vaccine virus detected by inoculating chicken embryo fibroblasts (CEFs) and examining for cytopathic effect. At 15 days of age, chickens in groups 2–5 were challenged with reovirus. At 22 days of age, birds were euthanatized and weighed. Efficacy of the vaccines was based on safety, percent protection, and antibody response.

In ovo vaccination with the commercial or experimental vaccines did not adversely affect hatchability of SPF chickens. The vaccine complexed with antibody resulted in significantly less posthatch mortality (3.7%) when compared to mortality of chickens that received vaccine alone (17%). Both vaccine virus recovery and antibody response were delayed at least 3 days in birds receiving the experimental vaccines. In ovo administration of reovirus antibody complex vaccines provided at least 70% protection. The experimental reovirus–antibody complex vaccines were safe and efficacious when given in ovo to SPF broiler embryos.

SUMMARY. Campylobacter jejuni and Campylobacter coli strains were isolated from feces of dairy cattle at farms with no known problem due to campylobacteria. Farms were located in the northeast, desert southwest, and Pacific west. Twenty isolates were identified by ribotyping with a RiboPrinter.^® The ability of these bovine isolates to colonize the ceca of chicks was determined by challenge inoculation and reisolation of the challenge strain from the ceca at 1 and 2 wk after challenge. Isolates recovered from chick ceca were examined by ribotyping to assure they matched the challenge strain. One hundred percent of the bovine-derived challenge strains were capable of colonizing chicks. These results indicate that dairy cattle may be asymptomatic Campylobacter carriers and potential sources of campylobacteria contamination of poultry facilities.

SUMMARY. An adenovirus was isolated from intestinal samples of two long-tailed ducks (Clangula hyemalis) collected during a die-off in the Beaufort Sea off the north coast of Alaska in 2000. The virus was not neutralized by reference antiserum against known group I, II, or III avian adenoviruses and may represent a new serotype. The prevalence of the virus was determined in live-trapped long-tailed ducks at the mortality site and at a reference site 100 km away where no mortality was observed. Prevalence of adenovirus antibodies in serum samples at the mortality site was 86% compared to 10% at the reference site. Furthermore, 50% of cloacal swabs collected at the mortality site and only 7% of swabs from the reference site were positive for adenoviruses. In 2001, no mortality was observed at either of the study areas, and virus prevalence in both serum and cloacal samples was low, providing further evidence that the adenovirus was linked to the mortality event in 2000. The virus was used to infect long-tailed ducks under experimental conditions and resulted in lesions previously described for avian adenovirus infections and similar to those observed in long-tailed duck carcasses from the Beaufort Sea. The status of long-tailed ducks has recently become a concern in Alaska due to precipitous declines in breeding populations there since the mid-1970s. Our findings suggest that the newly isolated adenovirus is a disease agent and source of mortality in long-tailed ducks, and thus could be a contributing factor in population declines.

SUMMARY. Colibacillosis caused by Escherichia coli infections account for significant morbidity and mortality in the poultry industry. Yet, despite the importance of colibacillosis, much about the virulence mechanisms employed by avian E. coli remains unknown. In recent years several genes have been linked to avian E. coli virulence, many of which reside on a large transmissible plasmid. In the present study, a multiplex polymerase chain reaction (PCR) protocol to detect the presence of four of these genes is described. Such a protocol may supplement current diagnostic schemes and provide a rapid means of characterizing the E. coli causing disease in poultry. The targets of this procedure included iss, the increased serum survival gene; tsh, the temperature sensitive hemagglutinin gene; cvi, the ColV immunity gene; and iucC, a gene of the aerobactin operon. Organisms, known for their possession or lack of these genes, were used as a source of the template DNA to develop the multiplex PCR protocol. Identity of the amplicons was confirmed by size, DNA:DNA hybridization with specific gene probes, and DNA sequencing. When the multiplex PCR protocol was used to characterize 10 E. coli isolates incriminated in avian colibacillosis and 10 from the feces of apparently healthy birds, nine of the isolates from apparently healthy birds contained no more than one gene, while the 10th contained all four. Also, eight of the isolates incriminated in colibacillosis contained three or more genes, while the remaining two contained two of the target genes. Interestingly, the isolates of sick birds containing only two of the targeted genes killed the least number of embryos, and the isolate of healthy birds that contained all the genes killed the most embryos among this group. These genes were not found among the non–E. coli isolates tested, demonstrating the procedure's specificity for E. coli. Overall, these results suggest that this protocol might be useful in characterization and study of avian E. coli.

SUMMARY. One-day-of-age pheasant chicks were treated orally with the competitive exclusion product Broilact^® in three replicate trials. The following day the treated chicks and untreated control chicks were challenged likewise with approximately 10³ colony-forming units (cfu) of Salmonella infantis. Five days after challenge the cecal contents of the birds were examined quantitatively and by enrichment for S. infantis. In all three trials Broilact^® effectively reduced colonization of the challenge organism, the mean infection factor (IF) value (the logarithmic number of colony forming units of salmonella organisms per gram of cecal contents) for the treated groups being 2.9 and that for the salmonella control groups 8.4. Mortality during the 1 week rearing period was 5.0% in the Broilact^® treated groups and 8.5% in the salmonella control groups.

SUMMARY. The reverse transcriptase–polymerase chain reaction/restriction fragment length polymorphism (RT-PCR/RFLP) technique was used for identification and characterization of Egyptian field strains of infectious bursal disease viruses (IBDVs) that caused severe outbreaks with (30%–60%) mortality rate.

Twenty-four bursal samples collected from 24 field outbreaks in commercially reared chicken flocks experiencing signs typical of infectious bursal disease (IBD) were used. Ten of the bursal samples examined were determined to contain IBDV as evidenced by amplification of a 743-bp region of the VP2 gene of IBDV by RT-PCR. The RT-PCR products of the detected viruses were characterized by digestion with three restriction enzymes, BstN, MboI, and SspI. Three different RT-PCR/RFLP profiles were observed. Seven of the detected viruses had RFLP profiles identical to the very virulent European strains of IBDV (vvIBDVs). One virus had a RFLP profile identical to the U.S. classic vaccine strain, and one virus had a unique RFLP profile. The clinical history of the outbreaks and the presence of the SspI site in the 743-bp RT-PCR fragment were the criteria for designating the viruses as belonging to the very virulent (vv) phenotype.

SUMMARY. Thirty-one outbreaks of Marek's disease (MD) were reported in the Netherlands and retrospectively analyzed. The outbreaks occurred mostly in vaccinated commercial layer and a few breeder flocks of several breeds; however, the cause of the outbreaks could not be established. Therefore, in a prospective study, the occurrence of true vaccine failures was assessed on five hatcheries. The plaque-forming units (PFU) of MD vaccine per chicken dose were determined through in vitro assays on vaccine ampoules (2 to 5 per hatchery) and samples of reconstituted vaccine (approximately 22 per hatchery). All forty reconstituted vaccine samples of hatcheries 1 and 4 showed PFU doses <10³. In hatchery 4, 14 samples showed extreme low PFU (≤10 PFU). In hatcheries 2, 3, and 5, the numbers of MD vaccine suspensions with a titer ≥10³ PFU, which is the standard required, were 1 (5%), 17 (77%), and 3 (14%), respectively. Some vaccine ampoules showed <10³ PFU per chicken dose. This study shows the usefulness to assess the PFU per chicken dose of reconstituted MD vaccine and vaccine ampoules to unravel true vaccine failures, which could result in disease outbreaks in the field.

SUMMARY. Chicken anemia virus (CAV) can cause a disease syndrome characterized by severe anemia, bone marrow atrophy, and severe immunosuppression in young chicks. Maternal antibodies prevent these clinical signs but do not prevent infection, transmission of the virus, or immunosuppression. The clinical disease is rare today because of the widespread practice of vaccinating breeders, but the subclinical form of the disease is ubiquitous. The dynamics of CAV infection, CAV antibody responses, relative lymphoid organ weights, and associated lesions were studied in two broiler flocks from a commercial producer. Both groups had detectable CAV antibodies at hatch, which waned over the first 3 wk of life. Both groups had detectable CAV DNA in both thymi and bursae over the same period. At 35 days of age, virus was detectable by polymerase chain reaction in 16 of 20 chickens, and 7 of 20 had detectable antibodies. By 42 days of age, virus was detectable in 18 of 20 chickens, and 18 of 20 had antibodies to CAV. We observed a decrease in relative thymic weights beginning at 35 days of age, coincidental with the detection of CAV in the thymus. Bursal sizes began to decrease at 28 days of age, coincidental with a rise in antibody titers to infectious bursal disease virus. In this study, we demonstrated that under typical field conditions CAV infections in broilers have unique dynamics unlike those reported in egg laying strains of chickens managed under specific-pathogen-free conditions.

SUMMARY. A series of experiments was undertaken to investigate the infection dynamics of various doses of S. typhimurium in day-old and 14-day-old broiler chickens kept in isolators. The infections were followed quantitatively in ceca and ileum by enumerating the colony forming units (cfu) of the challenge strain. It was found that the inoculation of 10⁷ cfu of S. typhimurium to day-old chickens established stable cecal infection in all the animals for 35 days. For 14-day-old chickens, stable and lasting infections were seen with inoculation of 10⁹ cfu. Lower doses yielded more variable results, and the bacteria were rapidly eliminated from most birds, especially in 14-day-old inoculated chickens. Salmonella was found in spleen and liver 2–3 days postinoculation. Salmonella was cleared from both organs or reduced to very low numbers within 3 weeks.

SUMMARY. Viscera of 49 wild turkeys (Meleagris gallopavo) collected in the spring of 2001 and 23 wild turkeys collected in the fall and winter of 2001–02 from 12 counties in eastern Kansas were examined for enteric helminths. Four cestode species, two trematode species, one nematode species, and one acanthocephalan species were identified. Two cestode and two trematode species present in the spring sample also were present in the fall and winter sample. Parasite prevalence was similar to previous studies of enteric helminths of wild turkeys except for the low numbers of nematode species and individuals recovered in the present study.

SUMMARY. Mycobacteriosis is an avian disease that is most commonly caused by Mycobacterium avium or Mycobacterium genavense. In order to optimize molecular laboratory tests for diagnosing mycobacteriosis in birds, we compared four methods of rapid DNA extraction with isolates of M. avium, M. genavense, and Mycobacterium fortuitum. DNA extraction methods included enzymatic lysis, boiling for 30 min followed by enzymatic lysis, four cycles of freezing and thawing followed by enzymatic lysis, and bead beating followed by enzymatic lysis. The DNA yield and purity for the four methods were evaluated by spectrophotometry and compared. The bead beating with enzymatic lysis technique yielded significantly purer and higher concentrations of extracted DNA compared with other DNA extraction methods. All four methods yielded extraction products for all three organisms that were successfully amplified by polymerase chain reaction (PCR) for a fragment in the 65-kD heat shock protein gene. Subjectively, the PCR amplification products were most abundant for samples extracted by bead beating with enzymatic lysis.

SUMMARY. Fowl cholera (FC) caused by Pasteurella multocida was diagnosed in waterfowl, Baikal teals (Anas formosa), submitted to the National Veterinary Research and Quarantine in Korea. The total number of mortalities was 13,228 out of approximately 100,000 birds that wintered in Cheonsoo Bay, the most important habitat area of Baikal teals in the world. Clinical signs were detected in only a few birds because of sudden death. Grossly, the dead Baikal teals had lesions consistent with FC, including multifocal necrotic foci in the liver with enlargement, petechial or ecchymotic hemorrhages on the heart, and mucoid exudates in the duodenal mucosa. Microscopically, there were hepatocytic necrosis with bacterial colonization, hemorrhage and necrosis in the myocardium, and hemorrhagic enteritis. Pasteurella multocida was isolated from the liver and the heart of all birds examined, and the isolate (P-627) was the serotype 1 × 12 × 13 by the agar gel immunodiffusion test. In order to estimate the virulence of P-627, 5-wk-old commercial ducks were exposed intramuscularly or intratracheally to the bacterium. On the basis of mortality rate, the isolate, P-627, was found to be highly virulent. This is the first report of an outbreak of FC in Baikal teals in Korea.

SUMMARY. Neural signs (torticollis, drowsiness) and mortality were observed in five chickens of a native chicken flock (reared for meat) that included 450 male birds on a farm that had 2300 native chickens and 1120 layers. Histologic lesions were observed in the medulla oblongata, optic lobe, cerebellum, and spinal cord of the affected birds. The lesions, which were most severe in the medulla oblongata, were massive abscesses with rarefaction (demyelination and malacia) of the parenchyma with gram-positive bacteria. The degenerative and necrotic areas were characterized by fibrin thrombosis, hemorrhages, and congestion in the blood vessels. Immunohistochemically, the bacteria positive for L. monocytogenes antigen were observed in the medulla oblongata, cerebellum, and spinal cord. Ultrastructurally, the small rod-shaped and thin-cell-walled bacteria were observed in the parenchyma of the medulla oblongata. Listeria monocytogenes (serotype 4b) was isolated from the medulla oblongata and spinal cord. The pathogenesis of listerial encephalitis in chickens was discussed.