We designed this study to compare the replication potential of turkey coronavirus (TCV) and its effect in chickens and turkeys and to study the effect of single and combined infection of turkey poults with TCV and astrovirus. We studied the pathogenicity of TCV in experimentally inoculated turkey poults and chickens by observing the clinical signs and gross lesions. Two trials were conducted with 1-day-old and 4-wk-old specific-pathogen-free turkey poults and chickens. One-day-old turkey poults developed diarrhea at 48 hr postinoculation. Poults euthanatized at 3, 5, and 7 days postinoculation had flaccid, pale, and thin-walled intestines with watery contents. The 4-wk-old turkeys had no clinical signs or gross lesions. One-day-old and 4-wk-old chicks developed no clinical signs or gross lesions although the TCV was detected in gut contents of the birds throughout the experimental period (14 days). In another experiment, mean plasma d-xylose concentrations in 3-day-old turkey poults inoculated with TCV, turkey astrovirus, or a combination of both viruses were significantly lower than in the uninoculated controls.

An outbreak of Mycoplasma gallisepticum (MG) in commercial turkeys involving very mild clinical signs was difficult to confirm by routine methods. In the first part of this study (trial A), we conducted a bioassay to increase the likelihood of detecting MG. Susceptible turkeys were inoculated with sinus exudates from four different affected commercial turkey flocks. Turkeys were evaluated for clinical signs, as well as by serology and culture of tracheal swabs, at 21 and 42 days postchallenge. An MG isolate from one of the sinus exudates used for inoculation, designated K5054, was very similar to isolates from house finches when characterized by random amplified polymorphic DNA analysis as well as DNA sequence analysis of portions of the phase-variable putative adhesin protein (pvpA) gene, a lipoprotein gene, and the cytadhesin gapA/mgc1 gene. The turkeys inoculated with the K5054 sinus exudate seroconverted in the absence of severe clinical signs. There was a single reisolation of K5054 from these turkeys 42 days postchallenge. Susceptible contact turkeys were commingled with the K5054-inoculated turkeys at 49 days postchallenge. We found no evidence of transmission of MG to the contacts by culture or serology at 7, 21, or 35 days after commingling. In the second part of this study (trial B), we challenged the contacts and K5054 sinus exudate–inoculated turkeys from trial A with virulent R strain 88 days after the K5054 sinus exudate inoculation. On necropsy 10 days postchallenge, the evaluation of gross and microscopic lesions, serology, and culture showed that the turkeys previously inoculated with K5054 sinus exudate were protected against disease and reinfection.

We conducted a processing plant survey to determine the prevalence of cellulitis (enlarged sternal bursa) and focal ulcerative dermatitis (FUD) in Ontario turkeys during the summer and to establish whether the two conditions were associated. A total of 11,772 birds from 24 different farms were observed at three processing plants in Ontario between June and August 2001. Each bird was examined for the presence of cellulitis and FUD. The severity and location of lesions were also noted. The overall prevalence of severe cellulitis (requiring either extensive trimming or condemnation of the bird) was 8.8% (95% confidence interval [CI] = 6.7, 10.9) and the overall prevalence for FUD was 22.6% (19.3, 25.9). The Pearson correlation coefficient between the prevalences of the two lesions was 0.56 (P = 0.0043), suggesting farms with a high prevalence of FUD in their birds also had a high prevalence of cellulitis. More specifically, birds with FUD were, on average, 10.8 times more likely to have cellulitis than birds without FUD (Mantel–Haenszel summary odds ratio = 10.8; 95% CI = 9.5, 12.3). Our observations suggest FUD may predispose birds to cellulitis, in which case, preventing FUD may reduce the occurrence of cellulitis condemnations in turkeys.

We conducted a retrospective study to determine the prevalence of cellulitis condemnations in the Ontario chicken industry and the relative proportion of variation attributable to factors that vary between processors, producers, and lots and over time. The time span studied was April 1998 to April 2001. We obtained condemnation data randomly from the Chicken Farmers of Ontario and analyzed the data with a generalized mixed model. The (weighted) average prevalence of cellulitis in Ontario between April 1998 and 2001 was 0.94% (0.87%, 1.03%). The prevalence of cellulitis ranged from 0% to 14.9%, with one outlier at 30% and 95% of the data between 0 and 2.58%. The final mixed model we used to describe the variation in the prevalence of cellulitis between lots included random effect terms, the plant where the birds were processed, the producer, the quota period when the birds were processed, and the interaction term quota period by processing plant, as well as fixed effects terms, the type of inspection system and the average weight of the birds. The final model containing these variables explained approximately 78% of the total variation in the data. Our findings indicate all three random effects variables accounted for a significant amount of variation in the cellulitis data; however, the greatest source of variation was ascribed to the plants where the birds were processed. Some of the variation in cellulitis associated with processing plants was explained by the type of inspection system used by the plant, but even after controlling for this factor, there remained a relatively large amount of variation between processing plants (approximately 30%). These findings suggest there may be discrepancies in the diagnoses of the condition. Some of the variation in the prevalence of cellulitis (∼13%) was also attributed to the producer; however, more of the variation in the data was attributed to differences in lot-specific factors (∼22%). Therefore, future control efforts for cellulitis should focus on standardizing the classification of cellulitis at processing plants and identifying lot specific factors that may increase the risk of the condition.

Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB₁, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB₁, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody–positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB₁ (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB₁UL32 (46%), rFPV/gB₁gEgI (72%), and rFPV/gB₁gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB₁ and rFPV/gI were comparable with that of HVT. Only gB₁ and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB₁gEgI (72%) and rFPV/gB₁gEgIUL32(70%) against Marek's disease was significantly enhanced compared with a single gB₁ or gI gene (40%). This protective synergism between gB₁ and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB₁gIgEUL32 HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB₁gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement.

We investigated the ability of a high virulence (STR 357) and a low virulence (STR 598) strain of Streptococcus gallolyticus to attach to the intestinal tract of pigeons. For that purpose, first of all, two groups of six pigeons were anesthetized and ligatures were placed at the beginning of duodenum, jejunum, ileum, and colon. The obtained intestinal loops of the birds of the first and second group were injected with S. gallolyticus strains STR 357 and STR 598, respectively. At 15, 30, and 60 min postinoculation, two pigeons of each group were euthanatized and the various intestinal loops were sampled for histologic, immunohistochemical, and electron microscopic examination. Both the high and low virulence strains were able to adhere to the intestinal mucosa. Indeed, all samples clearly showed numerous coccal-shaped bacteria that stained positively with S. gallolyticus antiserum and were lining up against the intestinal epithelium. Likewise, on electron microscopic examination, cocci were seen in the mucus covering the intestinal epithelium. Second, the association of S. gallolyticus strains of differing virulence with the intestinal tissue was determined quantitatively. Experiments were performed as described above. The number of S. gallolyticus bacteria that adhered to the intestinal epithelium was determined by plating out 10-fold serial dilutions of the segments. No significant differences in the number of adhered bacteria were found between the strains of high and low virulence.

Atherosclerosis is a common disease among parrots, but little is known about possible risk factors. Important risk factors in humans are an elevated plasma cholesterol concentration and increased platelet aggregation; high intakes of polyunsaturated fatty acids have beneficial effects. In this study, we tried to establish a relationship, if any, between dietary fatty acids and the severity of atherosclerosis in parrots. We collected dead parrots and scored the degree of atherosclerosis in the beginning of the aorta and the brachiocephalic arteries. It was not possible to assess the intake of fatty acids with food questionnaires so fatty acid composition of adipose tissue and breast muscle had to be used as an index of the dietary fatty acid composition. In all, 202 birds were collected. Gender was not related with atherosclerosis, but the degree of atherosclerosis increased with age and among the various species; African grey parrots appeared to be the most susceptible. The contents of linoleic acid in breast muscle or adipose tissue were not associated with the severity of atherosclerosis. For the relative percentage of α-linolenic acid in either breast muscle (P = 0.09; n = 175) or adipose tissue (P = 0.056; n = 21), a borderline significant relation with the degree of atherosclerosis was found. Parrots without atherosclerosis had significantly higher levels of α-linolenic acid than did the other animals. On the basis of these data, we suggest tentatively that a high dietary intake of α-linolenic acid protects against the development of atherosclerosis in parrots.

In an effort to protect chickens against Newcastle disease (ND), a nonreplicating virosome vaccine was produced by solubilization of Newcastle disease virus (NDV) with Triton X-100 followed by detergent removal with SM2 Bio-Beads. Biochemical analysis indicated that the NDV virosomes had similar characteristics as the parent virus and contained both the fusion and hemagglutinin-neuraminidase proteins. To target the respiratory tract, specific-pathogen-free chickens were immunized intranasally and intratracheally with the NDV virosome vaccine. This vaccine was compared with a standard NDV (LaSota) live-virus vaccine for commercial poultry. Seroconversion (≥ four fold increase in hemagglutination inhibition [HI] antibody titers) was achieved in all birds vaccinated with the virosome vaccine. Upon lethal challenge with a velogenic NDV strain (Texas GB), all birds receiving either vaccination method were protected against death. Antibody levels against NDV, as determined by enzyme-linked immunosorbent assay and HI titer, were comparable with either vaccine and increased after virus challenge. These results demonstrate the potential of virosomes as an effective tool for ND vaccination.

Antimicrobial resistance in nearly all human and animal pathogens is on the increase. In poultry, Ornithobacterium rhinotracheale has been identified as a newly emerging respiratory bacterial pathogen that has caused significant economic losses to the poultry industry. In this study, we examined in vitro antibiotic resistance profiles of 125 isolates of O. rhinotracheale isolated from turkeys in Minnesota during 1996–2002. A majority of isolates was sensitive to clindamycin, erythromycin, spectinomycin, and ampicillin. Resistance against sulfachloropyridiazine decreased from 1996 to 2002, but an increase in resistance was seen against gentamicin, ampicillin, trimethoprim sulfa, and tetracycline. The annual trend slopes for these antibiotics were 7.36%, 3.02%, 2.43%, and 1.95%, respectively. The resistance against penicillin remained constant from year to year with a trend slope of only 0.54% per year. These results emphasize the need for continued monitoring of O. rhinotracheale isolates for antibiotic resistance and establishment of baseline resistance pattern data for this organism. These data can then be used to design and evaluate local epidemiological interventions.

Avian infectious bronchitis virus (IBV) causes a highly contagious and economically significant disease in chickens. Establishment of a carrier state in IBV infection and the potential for the persistent virus to undergo mutations and recombination in chicken tissues have important consequences for disease management. Nevertheless, whether chickens can maintain persistent IBV infection in the absence of reinfection from exogenous sources or the presence of antibody in the host can modulate virus persistence remains unclear. Indeed, whether or not IBV genome can undergo genetic changes during in vivo infection has not been demonstrated experimentally.

In the present study, IBV shedding and tissue persistence were monitored in individual chickens maintained under strict isolation that precluded reinfection from exogenous sources. In the first of two experiments, intranasal exposure of 6-wk-old antibody-free chickens to IBV vaccine virus resulted in intermittent shedding of the virus from both trachea and cloaca of individual birds for up to 63 days. Also, the virus was recovered from the internal organs (spleen, gonad, kidney, lung, cecal tonsil, and cloacal bursa) of six of eight birds killed at various intervals between 27 and 163 days postinoculation (DPI). In the second experiment, IBV exposure of 1-day-old maternal antibody–positive chicks led to periodic virus shedding from the trachea and cloaca in all chickens until 77 days; however, internal organs (lungs and kidneys) of only one of seven birds (killed at 175 DPI) were virus positive, suggesting that presence of antibody at the time of infection protects internal organs from IBV infection. When the lung and kidney isolates of IBV from the latter experiment were compared with the parent-vaccine virus, no changes in their antigenicity, tissue tropism, or the nucleotide sequence of the S1 glycoprotein gene were observed. These findings indicate that, unlike the mammalian coronaviruses, propensity for frequent genetic change may not be inherent in the IBV genome.

Since the initial report of West Nile virus in the northeastern United States in 1999, the virus has spread rapidly westward and southward across the country. In the summer of 2002, several midwestern states reported increased cases of neurologic disease and mortality associated with West Nile virus infection in various native North American owl species. This report summarizes the clinical and pathologic findings for 13 captive and free-ranging owls. Affected species were all in the family Strigidae and included seven snowy owls (Nyctea scandiaca), four great-horned owls (Bubo virginianus), a barred owl (Strix varia), and a short-eared owl (Asio flammeus). Neurologic signs identified included head tilt, uncoordinated flight, paralysis, tremors, and seizures. Owls that died were screened for flaviviral proteins by immunohistochemical staining of formalin-fixed tissues, followed by specific polymerase chain reaction assay to confirm West Nile virus with fresh tissues when available. Microscopic lesions were widespread, involving brain, heart, liver, kidney, and spleen, and were typically nonsuppurative with infiltration by predominantly lymphocytes and plasma cells. Lesions in owls were much more severe than those previously reported in corvids such as crows, which are considered highly susceptible to infection and are routinely used as sentinel species for monitoring for the presence and spread of West Nile virus. This report is the first detailed description of the pathology of West Nile virus infection in Strigiformes and indicates that this bird family is susceptible to natural infection with West Nile virus.

We used Mycoplasma meleagridis (MM) to infect the RP-9 cells and the eggshell membranes and scanning electron microscopy (SEM) and confocal microscopy to study the interactions between the organisms and the cell surfaces. The surface of the RP-9 cells contained numerous projections. After 24 hr of infection with MM, those projections were either lost or aggregated to the side; MM-like particles could be seen on the surface of the cells, and surface fluorescence could be detected by confocal microscopy. On the surface of MM-infected shell membranes were necrotic fibrous tissues and cells detected by SEM and an intense surface fluorescence detected by confocal microscopy. These results indicate that MM infection of the cell surface can result in cellular damage.

The effects of avian leukosis virus subgroup J (ALV-J) infection on meat-type chickens reared in a simulated commercial setting were evaluated. Each of three ALV-J isolates was evaluated with both simulated horizontal transmission (SHT) and simulated vertical transmission (SVT). Mortality, morbidity, disease condemnations, and feed conversions were increased and body weights at processing were decreased in ALV-J infected birds as compared to sham inoculated hatch mates. The adverse effects of ALV-J infection were more severe in birds exposed by SVT than in birds exposed by SHT.

At 8 weeks of age response to vaccination for infectious bronchitis virus and Newcastle disease virus or prior exposure to a pathogenic reovirus was assessed in the ALV-J and sham inoculated broiler chickens by challenge studies. Although not statistically significant, an overall trend of decreased protection to challenge after vaccination, or prior exposure, was observed in the ALV-J inoculates as compared to sham inoculated hatch mates. Differences in vaccine response were most evident in groups inoculated with ALV-J by the SVT route.

In 1998, novel strains of infectious bronchitis virus (IBV) were identified in chickens from the southeastern United States and classified as a new serotype designated Georgia 98 (GA98). Because of the widespread nature of the GA98 virus in the southeastern United States and the lack of adequate protection with the DE072 vaccine, we developed a specific vaccine for the GA98 serotype. The GA98/0470/98 isolate of IBV was passaged in embryonating chicken eggs 70 times, and attenuation of the virus was determined in specific-pathogen-free chicks. Pass 70 of the GA98/0470/98 strain of IBV when given at 1 day of age by coarse spray and at 14 days of age in the drinking water at 1 × 10^4.5 50% embryo infectious dose/bird protected against the homologous GA98 challenge as well as provided good protection against the DE072-type virus. In addition, the vaccine was shown to be adequately attenuated and safe at a 10× dosage.

Experimental infection models are valuable tools for understanding and preventing the deposition of Salmonella enteritidis inside eggs. Oral inoculation is believed to closely simulate naturally occurring S. enteritidis infections of chickens, but oral infection studies have often generated relatively low frequencies of egg contamination. The present study assessed whether repeated in vivo passage of an S. enteritidis strain could affect its ability to cause egg contamination in experimentally infected hens. The incidence of egg contamination was determined in groups of hens inoculated orally with either a phage type 13a S. enteritidis strain or derivatives of this parent strain that were obtained by three successive rounds of passage and reisolation from tissues of infected hens. Passaged S. enteritidis isolates recovered from ovaries and oviducts induced a significantly higher incidence of egg contamination (16.97%) than was attributed to the parent strain (8.27%). However, passaged S. enteritidis isolates recovered from livers and spleens were not associated with a significantly increased frequency of deposition in eggs. By either inducing or selecting for the expression of relevant microbial properties, passage of S. enteritidis through reproductive tissues of chickens may be useful for improving the efficiency at which experimental infection models produce egg contamination.

Mycoplasma gallisepticum, a major pathogen of chickens and turkeys, has caused significant declines in house finch (Carpodacus mexicanus) populations in the eastern United States since it was first observed in this species in 1994. There is evidence that M. gallisepticum infection is now endemic among eastern house finches, although disease prevalence has declined, suggesting an evolving host–parasite relationship. Studies based on randomly amplified polymorphic DNA (RAPD) have documented the presence of a single, unique RAPD profile in house finch M. gallisepticum isolates, suggesting a single point source of origin, which agrees with the known epidemiologic observations. In the present study, we evaluated the molecular variability of 55 house finch isolates as well as 11 chicken and turkey isolates including reference strains of M. gallisepticum. Molecular variability was evaluated by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) analysis and nucleotide sequencing of the pvpA gene, which encodes for the putative cytadhesin protein PvpA. Three different RFLP groups and 16 genotypes were evident from the 55 house finch isolates evaluated. Sequence analysis of pvpA gene PCR products showed that although most house finch M. gallisepticum isolates clustered more closely to each other, others clustered more closely to either turkey or chicken field isolates. These findings suggest that house finch isolates are more polymorphic than previously recognized by RAPD studies. This feature may allow us to learn more about the molecular evolution and epidemiology of this emerging disease host–parasite relationship.

Pasteurella multocida group B, serotype 3, was isolated from sinusitis-affected khaki Campbell ducks. To study the role of P. multocida in sinusitis, commercial khaki Campbell ducks were experimentally infected with P. multocida alone or combined with Escherichia coli. In Expt. 1, experimental ducks were infected with P. multocida intranasally or ocularly. A comparison was done by intranasal inoculation with pooled nasal discharge from the affected ducks or phosphate-buffered saline. The ducks intranasally inoculated with the nasal discharge or P. multocida showed sinusitis. In Expt. 2, E. coli alone or a combination of P. multocida and E. coli was intranasally inoculated into experimental ducks. The ducks intranasally inoculated with the combination of P. multocida and E. coli had sinusitis, the same as found in the field but less severe than that of the field cases. Pasteurella multocida was already present in litter/floor of duck farms. We concluded that P. multocida played a role in induction of sinusitis. However, the sinusitis in ducks may be initiated by poor management, especially in the brooding period of ducks.

Previous studies demonstrated that Salmonella enteritidis infections in hens undergoing molt via feed withdrawal were more severe than in full fed hens. We conducted two trials to determine if immunizing specific-pathogen-free, Salmonella-culture-negative hens via aerosol exposure to MeganVac1™, a commercially available attenuated Salmonella typhimurium vaccine, would reduce transmission of S. enteritidis from infected hens to uninfected but contact-exposed hens during a molt. In trial 1, one group of hens received two aerosol doses of vaccine 2 wk apart whereas a second group of hens remained nonvaccinated. In trial 2, the vaccinated group received only one dose of vaccine. Two weeks after the final immunization, feed was removed from all the hens, and on day 4, the center hen in rows of 11 hens received a dose of 3 × 10⁵ (trial 1) or 1.3 × 10⁶ (trial 2). Transmission to the unchallenged hens was followed 3, 10, 17, and 24 days later. Vaccination reduced the horizontal spread of S. enteritidis in vaccinated hens compared with their nonvaccinated counterparts, with vaccinated hens shedding significantly less S. enteritidis on day 10 postchallenge in trial 1 and on days 3, 10, 17, and 24 in trial 2. Recovery of S. enteritidis from ovaries was significantly reduced in the vaccinated hens in trial 1 and from livers/spleens, ovaries, and cecum in trial 2. These studies indicate that immunization of hens with a live S. typhimurium vaccine could help reduce S. enteritidis problems during a molt situation.

Chicken infectious anemia virus (CIAV) is a ubiquitous and highly resistant virus of chickens that causes anemia and death in chicks less than 3 wk of age and immunosuppression in chickens older than 3 wk of age. The production of specific-pathogen-free eggs free of CIAV is essential for research and vaccine production. Currently, flocks are screened for CIAV by antibody tests to ensure freedom from CIAV infection. Recent evidence, however, indicates that chickens may carry and vertically transmit CIAV DNA independently of their antibody status. In this study, we tested embryos and eggshell membrane residues by nested polymerase chain reaction (PCR) as a sensitive method of detecting CIAV DNA. CIAV DNA could be detected in the blastodisks and semen obtained from antibody-positive and -negative chickens. Examination of different tissues between 18 and 20 days of incubation indicated that many but not all organs of individual embryos were positive. The lymphoid organs and gonads had the highest incidence of CIAV DNA, which was significantly different (P < 0.05) from the incidence in the liver. Eggshell membrane samples from embryos or newly hatched chicks were an excellent noninvasive source for the detection of CIAV DNA, identifying significantly more positive embryos than did pooled lymphoid organs. The use of dexamethasone injections as a method to improve the detection of carrier birds did not result in an increase of vertical transmission or cause seroconversion in the treated hens. A combination of testing eggshell membrane residues at hatch and periodic testing of blood DNA by nested PCR can be used to identify chickens carrying CIAV DNA and may be used to eradicate carrier birds.

In previous studies, the embryo lethality assay (ELA) was able to discriminate between virulent and avirulent avian Escherichia coli isolates and to predict percent mortality of the embryos resulting from an isolate based on three traits. The abilities to resist host complement, presence of Colicin V activity, and presence of the increased serum survival gene cluster (iss), were used together in a logistic regression analysis to predict the percentage of embryo deaths resulting from each of 20 avian E. coli isolates used in the ELA. In the present study, the same 20 isolates are used in an intravenous chicken challenge model in an effort to determine whether the ELA could be used to replace chicken challenge studies. Correlations between the mortality and a combination of mortality and morbidity (the survivors at trial termination with lesions suggestive of colibacillosis) and the previous ELA results and with selected isolate traits were performed. Additionally, resulting body weights in surviving chickens were compared between groups. The highest positive correlations were observed between the ELA and the combined mortality/morbidity of the intravenous challenge (r = 0.861, P < 0.0001 for the first ELA challenge, and r = 0.830, P < 0.0001 for the second ELA challenge). The IV challenge combined mortality/morbidity results had the highest correlation coefficients with the presence of iss (r = 0.864, P < 0.0001) and the expression of ColV (r = 0.878, P < 0.0001). The presence of tsh was slightly correlated with mortality (r = 0.465, P = .0389) but demonstrated a higher correlation with the combined mortality and morbidity of the IV challenge (r = 0.558, P = 0.0106). Even though the ELA results in a higher number of nonspecific deaths, the two challenge methods exhibit similar results and a high correlation with each other. Interestingly, some of the isolates showed differences in their ability to cause mortality between the ELA and the IV challenge model. Furthermore, some isolates reflected significant differences in body weights of surviving birds at IV trial termination.

Acute necrotic proventriculitis is a naturally occurring disease of broiler chickens that causes proventricular rupture during routine evisceration. Although infectious bursal disease virus (IBDV) has been implicated, it has not been proven to be a direct cause of this disease. To further study the role of IBDV in proventriculitis, proventriculi and bursas were collected during both acute and chronic phases of naturally occurring proventriculitis and from chickens experimentally infected with seven different IBDV strains. All tissues were examined for IBDV by light microscopy, immunohistochemistry (IHC), and real time reverse transcriptase (RT)–polymerase chain reaction (PCR) and for apoptosis by the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling method (TUNEL). Tissues from naturally occurring proventriculitis had bursal and proventricular lesions. Two out of four bursas had no IHC-stainable IBDV antigen or RT-PCR detectable IBDV sequences. No proventriculus had IBDV detectable by any of these methods. Bursas from chickens experimentally infected with IBDV had microscopically evident lesions, IBDV was detectable by IHC and RT-PCR, and strong IHC staining for apoptosis was present. Proventriculi from these experimentally exposed chickens had no lesions, low levels of IBDV detectable by IHC or RT-PCR, and very little IHC-stainable apoptosis. We conclude that naturally occurring proventriculitis can occur in the absence of IBDV and that the IBDV strains tested do not directly produce proventriculitis or induce increased proventricular apoptosis.

In order to assess ochratoxin A (OA) and T-2 toxin (T-2) binding ability of two commercial sorbents, both in vitro and in vivo trials with broilers were performed. Crude OA and T-2 extracts from contaminated grain were used to assess in vitro binding ability of two sorbents (Zeotek™ [Zk] and Mycofix™ [Mx]), by quantifying free mycotoxin through an enzyme-linked immunosorbent assay (ELISA) test. For in vivo trial, a 3 × 2 × 2 factorial arrangement was used for this experiment, being the factors: adsorbents (none, Zk, and Mx), OA (0 and 567 parts per billion [ppb]) and T-2 (0 and 927 ppb). OA and T-2 contaminated wheat and corn, respectively, were added to sorghum–soybean meal diets to meet 567 ppb of OA and 927 ppb of T-2. Mycotoxins were fed alone or combined in treatments. After 21 days, blood chemistry, gross, and histological evaluations were performed. Relative weights of liver, kidney, and bursa of Fabricius were obtained. Zk had the highest OA and T-2 in vitro binding ability (100% and 8.67%, respectively). Chickens fed OA with or without sorbents had a lower body weight and feed intake reduction. However, those birds fed T-2 were partly protected by a sorbent. Birds fed both toxins showed toxic additive effects, and no protection of any adsorbent was observed. A significant reduction in plasma proteins, albumin, and globulins was a characteristic observed in all birds fed diets with OA both with or without adsorbents. Uric acid level in blood was increased in all chickens fed OA-contaminated diets. Histological findings observed in birds fed OA-contaminated diets were necrosis of kidney tubular cells, swollen and necrotic hepatocytes, bile ducts hyperplasia, and increased diameter of proventriculus glands. In birds that received T-2 alone, only the liver, with the same kind of lesions, was affected.

According to these results, it can be concluded that there is not a relation between in vitro and in vivo trials. OA toxic effects could not be counteracted by any sorbent. T-2 toxicity could be partially counteracted by an adsorbent used in this research.

Avian pneumovirus (APV) causes respiratory tract infection in turkeys and was first seen in the United States in Colorado in late 1996. In early 1997, the disease was recognized in Minnesota and caused estimated losses of up to $15 million per year. This virus has not been reported in the other turkey producing states. We here report the seroprevalence of APV in Minnesota from August 1998 to July 2002. The average rate of seroprevalence has been 36.3% (range = 14.2%–64.8%). A seasonal bias was observed, with peak incidences in the fall and spring. A higher rate of seropositivity was observed in counties with the highest concentration of turkeys.

Clostridium perfringens has been shown to be widespread in the broiler chicken hatchery, grow-out, and processing operations. In a previous study, ribotypes of certain strains of C. perfringens isolated from processed chicken carcasses were shown to match ribotypes isolated from paper pad lining trays used to transport commercial chicks from the hatchery to the grow-out facility on the farm. These results suggest that C. perfringens contaminating the processed product could originate from facilities in the integrated poultry operation prior to grow out. In this study, samples were collected from the breeder farm, hatchery, previous grow-out flock, during grow out and after processing. In the first trial, C. perfringens was recovered from the breeder farms, the hatchery, previous grow-out flock, grow-out flock at 3 weeks of age, grow-out flock at 5 weeks of age, from processed carcasses, and from the breeder farm after processing in 4%, 30%, 4%, 0%, 2% and 16%, and 4% of the samples, respectively. In the second trial, the incidence of C. perfringens in samples collected from breeder farms, the hatchery, previous grow-out flock, grow-out flock at 3 weeks of age, grow-out flock at 5 weeks of age, and from processed carcasses was 38%, 30%, 32%, 8%, 4%, and 8%, respectively. The genetic relatedness of the isolated strains as determined by ribotyping suggests that C. perfringens may be transmitted between facilities within the integrated broiler chicken operation.

In this work, we describe a rapid detection procedure for Mycoplasma gallisepticum from chicken tracheal swabs by real-time polymerase chain reaction (PCR) by LightCycler system, where we were able to monitor the amplification of the newly synthesized M. gallisepticum–specific PCR product as a proportionally increasing fluorescent signal by using the double-stranded DNA binding dye SYBR Green I and have identified M. gallisepticum–specific PCR products by DNA melting curve analysis by plotting the first negative derivative (−d[F1]/dT) of fluorescence over temperature. Detection limits of the PCR were found to be 3 and 3000 colony-forming units ml⁻¹ with pure culture of M. gallisepticum and artificially spiked samples, respectively. Out of 96 tracheal swabs, 68 were taken from live chickens and 28 were taken by scraping the mucosal surface of the trachea (SMST) of necropsied chickens. All of the 18 PCR-positive results were from the swabs taken by the SMST method, whereas all of the samples taken from live chickens were negative. Thus, the PCR with the SMST method had a sensitivity and a specificity of 64.2% (18 of 28 chickens) and 100%, respectively. The total time required for template preparation from tracheal swab samples and real-time PCR was approximately 65 min. These results indicate that real-time PCR with the LightCycler technology is a rapid and sensitive test to identify M. gallisepticum–infected flocks if a proper sampling is applied.

SUMMARY. In this report, we describe a real-time reverse transcriptase–polymerase chain reaction (RRT-PCR) diagnostic test for infectious bronchitis virus (IBV) with the use of fluorescence resonance energy transfer (FRET) technology. Two primers that amplify a 383-base pair product between nucleotide positions 703 and 1086 relative to the start codon for the S1 gene of the Massachusetts 41 virus were designed and used to amplify the Beaudette, Massachusetts 41, Florida 18288, Connecticut, Iowa 97, Arkansas DPI, CA/NE95/99, DE/072/92, and GA/0470/98 strains of IBV. The primers were specific and did not amplify New Castle disease virus, Mycoplasma spp., or infectious laryngotracheitis virus. For RRT-PCR by FRET, an anchor probe conjugated to fluorescein and a detection probe conjugated to a red fluorophore were designed to anneal to a hypervariable region within the 383-base pair product. The level of sensitivity was 1 × 10⁴ RNA molecules used as starting template. After amplification, a melting curve analysis was conducted to specifically identify IBV types. Because of sequence differences in the annealing position of the detection probe, the Arkansas, Connecticut, Beaudette, and Massachusetts 41 strains could be differentiated. No fluorescence was observed for the DE/072/92 and GA/0470/98 viruses with the anchor and detection probes. When the Beaudette strain was examined, two melting peaks were observed at 44 C and 51 C, indicating a quasispecies in that laboratory strain of IBV. Routine typing of vaccine strains of IBV was possible with this technology, but high standard deviations associated with the melting curve analysis of the FRET probes described herein made it difficult to use this test reliably for routine typing of IBV field isolates.

We used slot blot hybridization of the hypervariable regions of the S1 subunit of spike peplomer gene to identify and characterize infectious bronchitis virus (IBV) strains. Template DNA was created from six reference strain IBVs of different serotypes and immobilized on a nitrocellulose membrane. We synthesized digoxigenin-labeled probes from reference and unknown field viruses and hybridized them to template DNA. All reference strains could be distinguished and isolates identified by serotype if they were at least 95% identical to a reference strain. This slot blot hybridization procedure was specific and reproducible, and strain typing was consistent with the S1 sequencing of the IBV genome. This study thus provides a simple and rapid method for typing of IBV.

In this investigation, we determined the prevalence of the Ornithobacterium rhinotracheale (ORT) infection in broilers and broiler breeders in southern Brazil. We also correlated the presence of antibodies in broilers with performance. Sera from 1550 broilers from 50 flocks were collected during the slaughter time in nine companies with federal veterinary inspection of the state of Rio Grande do Sul. Sera from 480 meat-type breeders of 40 flocks from 14 companies in southern Brazil were also analyzed by enzyme-linked immunosorbent assay, and the prevalence of antibodies was determined. The prevalence of ORT antibodies in broiler flocks was 63.83%, but in each individual flock only 6.52% of the birds were positive. The prevalence in broiler breeder flocks was 100.00%, and in each individual flock 94.62% of the birds were positive. There was a positive correlation between the presence of antibodies to ORT and decreased body weight in broilers. There was no significant correlation between presence of antibodies to ORT and age, lineage, efficiency index, feed conversion, and mortality. There was a positive correlation between the presence of respiratory signs and antibodies to ORT, although the reverse correlation was not significant. These results confirm that ORT is present and widespread in broilers and broiler breeders in southern Brazil.

We used real-time reverse transcriptase (RT)–polymerase chain reaction (PCR) to detect infectious bursal disease virus (IBDV) strains. The LightCycler (Roche) and hybridization probe system (Roche, Molecular Biochemicals) were used. A mutation probe labeled with fluorescein and an anchor probe labeled with Red-640 dye were prepared for each of the STC, Del E, D78, and Bursine 2 viral sequences. The mutation probes were designed to hybridize to nucleotides that encode the hydrophilic B region of VP2 for each virus. The anchor probes were designed to a relatively conserved region immediately downstream from the mutation probes. When hybridized to the RT-PCR product, a mutation and anchor probe pair produced fluorescence resonance energy transfer that was detected by the LightCycler instrument. Because they were designed to have a lower melting temperature (Tm), the mutation probes dissociated from the template before the anchor probes. The Tm values of the four mutation probes for each of their homologous viruses (exact sequence match) were STC, 69.3 ± 1.2 C; D78, 67.8 ± 0.9 C; Del E, 65.5 ± 0.6 C; and Bursine 2, 71.7 ± 0.4 C. These values were compared with the Tm values observed for a particular probe and heterologous virus. If the Tm values observed for heterologous viruses were within two standard deviations of the Tm for the probe and its homologous virus, the nucleotide sequences of the viruses were considered to be similar. If they were below two standard deviations, they were considered to have one or more nucleotide mutations. The results indicated that the STC and Variant Vax BD viruses have similar genetic sequences at the hydrophilic B region. Likewise, Bursine 2, Bursine, Bursine , BioBurs, BioBurs W, BioBurs AB, and IBDV Blen have similar nucleotide sequences in this region. The Tm values obtained for the D78 and Del E mutation probes with heterologous viruses indicated that none of the viruses tested had nucleotide sequences that matched these probes. Because the mutation probes were designed to bind to a region that encodes a neutralizing epitope, viruses with similar sequences were expected to have antigenically similar epitopes.

Genetic and molecular methods to investigate the pathogenesis of the poultry respiratory pathogen Mycoplasma gallisepticum are quite limited. Therefore, the objective of this study was to design and evaluate a functional genomics approach to identify M. gallisepticum genes involved in colonization of the poultry respiratory tract. To serve as a transcriptional reporter, a promoterless lacZ gene from Escherichia coli was cloned into the Tn4001 transposon. The transposon was used to randomly mutagenize the chromosome of the M. gallisepticum S6 strain, and a bank of 1386 transposon mutants containing lacZ fusions to mycoplasma chromosomal DNA was assembled. Each mycoplasma clone containing the lacZ reporter was independently screened in the chicken tracheal ring organ culture (TROC) model system for increased production of β-galactosidase. A twofold or greater increase in β-galactosidase was consistently observed for eight mutants. In one of the mutants, the transposon was inserted in a pMGA gene encoding a cell surface adhesin involved in hemagglutination. Therefore, these data indicate that screening of a M. gallisepticum transposon reporter bank with a chicken TROC model is useful for the identification of genes induced during poultry colonization and virulence.

Sixteen Chilean flamingos, Phoenicopterus chilensis, and 10 red-tailed hawks, Buteo jamacensis, were vaccinated in the pectoral muscle with 0.2 ml of a commercially produced killed West Nile virus vaccine intended for use in horses. Half the birds of each species received a booster vaccination 3 weeks after the first injection. Three weeks after the booster vaccination, none of 13 birds surveyed had detectable antibody to West Nile virus.

Campylobacter jejuni cells entered the viable but nonculturable (VBNC) state upon suspension in sterile water. Cell viability was determined with tetrazolium violet. VBNC cells suspended in water for 7, 10, or 14 days were given, by gavage, to day-of-hatch leghorn chickens. The ceca of control and challenged birds were examined for the presence of campylobacteria by conventional microbiological methods at 1 wk and 2 wk after challenge inoculation and by polymerase chain reaction methods at 1 wk after challenge. We did not find culturable Campylobacter cells in the ceca. Neither was Campylobacter DNA found in cecal samples. Therefore, VBNC cells did not revert to the culturable colonizing form, nor did VBNC cells persist within the cecal environment.

Two experiments were conducted to determine the effect of a β-mannanase feed enzyme on the performance of broiler chicks subject to a necrotic enteritis disease challenge model involving oral inoculation of Eimeria sp. and Clostridium perfringens. β-Mannanase is known to improve productive performance when added to poultry and swine diets. In both experiments, disease challenge in the absence of feed additives demonstrated significant reductions in performance as measured by weight gain, feed conversion, and the incidence of coccidial lesion scores. Significant mortality was also observed in challenged groups in Experiment 1. The disease challenge model was therefore judged as highly effective. Additions of a commonly used antibiotic, bacitracin methylene disalicilate (BMD), and coccidiostat, salinomycin, were highly effective in partially counteracting negative effects of the disease challenge. In both experiments, addition of β-mannanase significantly improved performance and reduced lesion scores in disease-challenged groups. The degree of improvement was somewhat less than that afforded by a combination of BMD and salinomycin in Experiment 1 but was not different from that afforded by BMD alone in Experiment 2. We conclude that the β-mannanase enzyme can play a role in circumstances where the use of antibiotics is not desired.

The survival of avian pneumovirus (APV) in turkey litter was studied at different temperature (room temperature, [approximately 22–25 C], 8 C, and −12 C) conditions. Built-up turkey litter from a turkey breeder farm known to be free of APV was obtained and was divided into two portions. One portion was sterilized by autoclaving and the other portion was kept nonautoclaved. Both samples were inoculated with a Vero cell–propagated Minnesota isolate of APV subtype C (APV/MN2A) with a titer of 10⁵ 50% tissue culture infective dose at 1% level. These samples were then stored at three different temperatures: −12 C, 8 C, and room temperature (20–25 C). The samples were tested for the presence of viral RNA by reverse transcriptase–polymerase chain reaction and for the presence of live virus by virus isolation in Vero cells at the intervals of 1, 2, 3, 7, 14, 30, 60, and 90 days. Our studies revealed the presence of APV RNA even after 90 days in the autoclaved litter samples kept at −12 C and at 8 C. The virus was isolated from the autoclaved litter kept at −12 C up to 60 days. From the nonautoclaved litter, viral RNA was detected up to 60 days and virus was isolated up to 14 days. The present study indicated that APV could survive in built-up turkey litter up to 60 days postinoculation at a temperature of −12 C.

Thirty-three live 9-day-old broiler chicks were submitted for laboratory evaluation because of blindness. Blindness was observed in up to 1% of 25,000 birds from four different houses. All the chicks were from the same breeder source. Blindness was apparent when the chicks were 2–3 days old. Clinically, the blind chicks were smaller than their counterparts, were unable to find feed and water, and wandered aimlessly. Necropsy did not reveal any lesions. Two chicks examined clinically at 14 days of age lacked pupillary reflexes after light stimulation, and the anterior and posterior segments of the eye were normal. Microscopically, degeneration of photoreceptor cells characterized by vacuoles was evident at 9 days of age, and rosette formation of the retina, disorganization of retinal layers, synechia of the retina with mild inflammation in the choroid, and proliferation of the retinal pigment epithelial cells were evident by 15 days of age. Because the chicks came from the same breeder source and the incidence of blindness was low, a probable genetic etiology is suspected as the cause of blindness in this flock.

In this case report, we describe a tawny owl chick (Strix aluco) coming from a Wild Fauna Recovery Center with multiple congenital malformations in the limbs. The animal was unable to fly and showed marked malnutrition and poor general appearance. Physical, radiologic, and anatomic examinations showed osseous malformations including dislocation of radius and carpometacarpus with abnormal nonfunctional fixation of ligamentum propatagialis, absence of most parts of the bones of the manus in both wings, and twisted nonfused left tarsometatarsus with marked griphosis of digits. Routine toxicologic and pathologic examinations did not reveal a specific etiology.

A total of 120 ring-necked pheasants from a 3000-bird flock in Zeeland, MI, died over a 3-day period. Clinical signs included sudden death, diarrhea, and limping. At necropsy, hepatomegaly with multifocal cream-colored foci randomly distributed throughout the parenchyma was observed in diseased birds. Additionally, the spleen was enlarged up to three times its normal size and had a marbled appearance. Microscopically, there was multifocal splenic and hepatic necrosis with intralesional rod-shaped bacteria. Pasteurella multocida serotype 3/4 was isolated from liver and spleen. In this paper, we report an outbreak of acute fowl cholera in ring-necked pheasants.