Commercial hatcheries typically infuse hydrogen peroxide or formaldehyde gas into hatching cabinets to reduce airborne pathogens that may lead to disease transmission during the hatch. A nonchemical option, an electrostatic space charge system (ESCS), was customized for full-sized commercial hatching cabinets and was tested extensively in broiler hatcheries. The ESCS cleans air by transferring a strong negative electrostatic charge to dust and microorganisms that are aerosolized during the hatch and collecting the charged particles on grounded plates or surfaces. In studies with three poultry companies, the ESCS resulted in significant (P < 0.0001) reductions of airborne dust of 77%–79%, in Enterobacteriaceae and fungus levels not significantly different (P ≥ 0.05) from those with formaldehyde, and in 93%–96% lower Enterobacteriaceae than with no treatment or with hydrogen peroxide treatment (P < 0.01). The ESCS significantly (P < 0.05) reduced airborne Salmonella by 33%–83% compared with no treatment or hydrogen peroxide treatment.

Results of this study suggest that the ESCS is a viable alternative to chemical treatment for reducing airborne pathogens in full-sized commercial hatchers, and it also provides dust control and containment, which should be helpful in reducing cross contamination and loading of ventilation ducts within different areas of the hatchery.

Macrorhabdus ornithogaster is a newly described anamorphic ascomycetous yeast that has been reported to cause a chronic, debilitating disease in many species of birds, including poultry. Study of this organism is complicated by the limited ability to grow M. ornithogaster in vitro. In this study, we showed that the chicken can be used to amplify this organism and as a model to study its pathogenicity. An infection rate of 100% was achieved in day-old chicks orally inoculated with 10⁵ M. ornithogaster derived from the budgerigar (Melopsittacus undulatus). The organism was also determined to increase in number by greater than 10-fold 14 days after oral inoculation in these chicks. Chickens infected with M. ornithogaster demonstrated no sign of illness but had decreased feed conversion efficiency and consistent and characteristic histopathologic lesions in the proventriculus and isthmus of the stomach, suggesting that M. ornithogaster may represent a potential threat to the poultry industry.

The efficacy of four different commercial live vaccines (vaccines A, B, C, and D) against the infectious laryngotracheitis virus (ILTV) was assessed in specific-pathogen-free (SPF) chickens. SPF chickens were vaccinated intraocularly at 6 wk old with ILTV live vaccines and were challenged intratracheally with the N91B01 strain of virulent Korean ILTV 2 wk after vaccination. The immunity against ILTV live vaccines was assessed by the incidence of latent infection by the challenge virus in the chickens' tracheas and trigeminal ganglia, the reisolation rate of the challenge virus, and the clinical signs in the chickens challenged with the N91B01 strain of ILTV. The latent infection in chickens was assessed by nested polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP). Our data showed that the clinical signs and challenge virus isolation were negative in all chickens receiving four different commercial ILTV live vaccines. The viral DNA of the vaccine strain, but not that of the challenge virus, was detected in chickens vaccinated with vaccine A by nested PCR-RFLP. The viral DNAs of both the vaccine and challenge strains were detected from chickens vaccinated with vaccines B, C, and D. This study showed that only vaccine A can protect chickens from latent infection with the field virulent ILTV. We speculate that the efficacy of infectious laryngotracheitis live vaccines to protect chickens from latent infection with virulent ILTVs can be assessed by nested PCR-RFLP analysis.

We have constructed a DNA vaccine (pDKArkS1-DPI) expressing the S1 glycoprotein (Arkansas DPI) of infectious bronchitis virus (IBV) to examine protective immunity after in ovo and intramuscular DNA immunization. Birds receiving in ovo DNA followed by live virus vaccination at 2 wk of age were 100% protected from clinical disease. Birds receiving only live virus vaccine or only in ovo DNA vaccination were ≤80% protected. IBV was detected up to 10 days postchallenge in unvaccinated control groups, whereas birds receiving in ovo DNA and live virus vaccination cleared IBV from tracheal samples before day 5 postchallenge. Transcription of the S1 gene was confirmed in lung tissue after in ovo vaccination by an antisense riboprobe, and the S1 protein was detected by immunohistology in the heart and bursa. In a separate experiment, birds were injected intramuscularly with either 50, 100, or 150 µg of the DNA vaccine at 1 day of age and then again with either 100, 200, or 300 µg of the DNA vaccine, respectively, at 14 days of age. At 10 days postchallenge, no clinical signs were observed and no challenge virus was reisolated from the birds vaccinated with 150 µg and 300 µg of DNA. Between DNA-vaccinated birds and nonvaccinated control birds, no statistical differences were observed for IBV-specific serum antibodies as detected by enzyme-linked immunosorbent assay or the virus neutralization test. These data indicate that DNA vaccination with the S1 gene either in ovo or intramuscularly can provide birds with some protection against clinical disease after homologous IBV challenge.

Because of the limited analysis of fowl poxvirus (FPV) promoters, expression of foreign proteins by recombinant FPV has usually been directed by heterologous vaccinia virus or synthetic poxvirus promoters. Thus, the impact of completely homologous recombinant virus vaccines has yet to be realized by the poultry industry. In an effort to increase the availability of such transcriptional regulatory elements, the modulation of gene expression by six previously uncharacterized FPV late promoters was examined. To simplify this comparison, each promoter region was separately coupled to the same reporter gene (lacZ) in individual plasmid constructs, and their activities in transfected, virus-infected cells were monitored. In each of the four selected unidirectional transcriptional regulatory elements as well as a 30-base pair representative of the bidirectional promoter region, the predicted temporal specificity of expressing at late stages of virus replicative cycle was verified. Stable lacZ gene transcripts arising from each plasmid varied less than threefold in quantity, whereas the amounts of β-galactosidase product ranged within a 130-fold interval. Only the promoter that naturally regulates expression of the A type inclusion body protein gene directed production of β-galactosidase at a level comparable with that associated with the strong vaccinia virus P11 promoter. Because one of the remaining unidirectional transcriptional regulatory elements, P174, was only 2.4-fold less efficient, both of these promoters, P174 and P190, should be satisfactory for directing the expression of poultry pathogen genes inserted into the genomes of FPV recombinant vaccines.

Marek's disease virus (MDV), a herpesvirus, and avian leucosis virus subgroup J (ALV-J), a retrovirus, were used for experimental coinfection of chickens. Chimeric molecules having sequences of both viruses were detected by the hotspot–combined polymerase chain reaction (HS-cPCR) system. The detection of chimeric molecules provided evidence for avian retroviral inserts in the herpesvirus genome. The persistence of chimeric molecules on in vivo passage served to indicate the infectivity of the recombinant virus. The evaluation of formation and persistence of the chimeric molecules was performed in two trials involving three in vivo passages. The chimeric molecules were identified according to the primer sets, their product length, and pattern. The persistence of chimeric molecules on in vivo passages served as an indication of their ability to replicate in and infect chickens.

In the first experimental passage, MDV and ALV-J prototype strains, MD11 and HC-1, were intraperitoneally (IP) injected into 1-day-old chicks. The second trial included two passages. Passage II chicks were injected IP and passage III chickens were in contact with the chickens of passage II. For passage II, enriched white blood cells from blood samples of chickens from the first trial that had chimeric molecules were injected IP into 1-day-old chicks. For passage III, uninfected chicks were included together with the infected chicks.

Synthesis evidence for the various species of chimeric molecules was assessed in the tissues of birds of the second trial. DNA was extracted from blood and feathers and analyzed by the hotspot–combined PCR and by pulsed field gel electrophoresis. To overcome the limits of detection, three amplification assays followed by hybridization of the products to specific viral probes were conducted. A variety of chimeric molecules were detected in low concentrations. Five species of chimeric molecules were characterized in blood, tumors, and feathers. Chimeric molecules were detected in 18 of 36 dually infected birds from the first trial and in 14 of 21 dually infected birds from the second trial. The findings show that, in four out of seven groups of the second trial, the chimeric molecule species persisted on passage.

A cDNA library was constructed with Eimeria necatrix merozoite mRNA and immunologically screened by chicken sera against this parasite. One of the positive clones containing an insert of 879 nucleotides, pNP19, showed similarity to part of a published gene expressed in E. tenella merozoite by the homology search system. The inserted DNA was subcloned into baculovirus, and a 35-kD protein was expressed, purified, and used for the antigen in enzyme-linked immunosorbent assay (ELISA). Antibodies from the chickens vaccinated with the E. necatrix attenuated strain, Nn-P125, were detected from 14 days after vaccination by ELISA. The mean absorbance increased rapidly to a peak around 21 days after vaccination; thereafter, it began to decline. Even though some of the vaccinated chickens showed very low levels of antibody response to the recombinant protein 56 days after vaccination, they were protected against challenge with virulent strain of E. necatrix. The mean absorbances in sera from both vaccinated and nonvaccinated chickens highly increased 14 days after challenge. On the other hand, the antibody was not detected in ELISA when chickens were exposed to other Eimeria species such as E. tenella, E. acervulina, and E. maxima. These results demonstrate that this recombinant protein is suitable for detecting the specific antibody in chickens infected with both attenuated and virulent strains of E. necatrix.

The pathogenesis of six Newcastle disease virus (NDV) isolates recovered from chickens (Ckn-LBM and Ckn-Australia) and wild (Anhinga) and exotic (YN parrot, pheasant, and dove) birds was examined after the isolates had been passaged four times in domestic chickens. Groups of 10 4-wk-old specific-pathogen-free white leghorn chickens were inoculated intraconjunctivally with each one of the isolates. The infected birds were observed for clinical disease and were euthanatized and sampled at selected times from 12 hr to 14 days postinoculation or at death. Tissues were examined by histopathology, by immunohistochemistry (IHC) to detect viral nucleoprotein (IHC/NP), and by in situ hybridization to detect viral mRNA and were double labeled for apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling ([TUNEL] or IHC/caspase-3) and viral nucleoprotein (IHC/NP). Birds infected with the three low virulence viruses (Ckn-LBM, YN parrot, and Ckn-Australia) did not develop clinical disease. Microscopic lesions were observed only at the inoculation site and in organs of the respiratory system. The detection of viral nucleoprotein (N) was restricted to the inoculation site. The pheasant and dove isolates were highly virulent for chickens with marked tropism for lymphoid tissues, confirmed by the presence of large numbers of cells positive for viral N protein and viral mRNA. Viral N protein was detected early in the cytoplasm of cells in the center of the splenic ellipsoids. The apoptosis assays (TUNEL and IHC/caspase-3) showed increased apoptosis in the splenic ellipsoids as well. Apparently, apoptosis is an important mechanism in lymphoid depletion during NDV infection.

Thirteen infectious laryngotracheitis virus (ILTV)-specific monoclonal antibodies (MAbs) were isolated after immunization of mice with purified infectious laryngotracheitis virions. On the basis of their reactions in western blot analyses of ILTV-infected cells, the MAbs were assigned to five different virus proteins or protein groups. Two of the viral target proteins could be identified after transient expression of cloned ILTV genes in eucaryotic cells. The MAbs of group II detected a 60-kD protein that was shown to be the ILTV homologue of herpes simplex virus type 1 (HSV-1) glycoprotein (g)C. The MAbs of group I reacted with the positional homologue of HSV-1 gJ, which is encoded by the open reading frame (ORF) 5 gene within the unique short genome region of ILTV. The ORF 5 gene product of ILTV was previously described as a 60-kD glycoprotein (gp60), whereas multiple protein bands with apparent molecular masses of 85, 115, 160, and 200 kD were identified in the present study. Immunoelectron microscopy revealed that both gC and gJ of ILTV are localized in the envelope of virus particles, whereas the 15-kD protein detected by the MAbs of group III presumably represents a tegument component. Immunofluorescence analyses of infected cells demonstrated that the epitopes of the gC- and gJ-specific MAbs are conserved in all tested ILTV isolates originating from different parts of the world and that these MAbs are also suitable for in situ antigen detection in tissues of ILTV-infected chickens. The remaining ILTV-specific MAbs recognized viral proteins of 22 kD (group IV) and 38 kD (group V) that were not further characterized up to now.

Evidence of the widespread occurrence of reticuloendotheliosis virus (REV) sequence insertions in fowl poxvirus (FPV) genome of field isolates and vaccine strains has increased in recent years. However, only those strains carrying a near intact REV provirus are more likely to cause problems in the field. Detection of the intact provirus or REV protein expression from FPV stocks has proven to be technically difficult. The objective of the present study was to evaluate current and newly developed REV and FPV polymerase chain reaction (PCR) assays to detect the presence of REV provirus in FPV samples. The second objective was to characterize REV insertions among recent “variant” FPV field isolates and vaccine strains. With REV, FPV, and heterologous REV-FPV primers, five FPV field isolates and four commercial vaccines were analyzed by PCR and nucleotide sequence analysis. Intact and truncated REV 5′ long terminal repeat (LTR) sequences were detected in all FPV field isolates and vaccine strains, indicating heterogeneous REV genome populations. However only truncated 3′ LTR and envelope sequences were detected among field isolates and in one vaccine strain. Amplifications of the REV envelope and 3′ LTR provided strong evidence to indicate that these isolates carry a near intact REV genome. Three of the four FPV vaccine strains analyzed carried a solo complete or truncated 5′ LTR sequence, indicating that intact REV provirus was not present. Comparison of PCR assays indicated that assays amplifying REV envelope and REV 3′ LTR sequences provided a more accurate assessment of REV provirus than PCR assays that amplify the REV 5′ LTR region. Therefore, to differentiate FPV strains that carry intact REV provirus from those that carry solo 5′ LTR sequences, positive PCR results with primers that amplify the 5′ LTR should be confirmed with more specific PCR assays, such as the envelope, or the REV 3′ LTR PCR.

The live attenuated temperature-sensitive (ts ) Mycoplasma synoviae (MS) strain, MS-H, is used as a vaccine in a number of countries to control virulent MS infection in commercial chicken flocks. Nine out of 50 isolates made from flocks vaccinated with MS-H were found to have lost the ts phenotype of the original vaccine strain. In order to examine the influence of the ts⁻ phenotype on virulence of the isolates, four of the ts⁻ isolates, the MS-H vaccine, and the vaccine parent strain 86079/7NS were administered by aerosol in conjunction with infectious bronchitis virus to 3-wk-old specific-pathogen-free chickens. The four ts⁻ clones induced only minimal air sac lesions that were not different in severity from those caused by MS-H vaccine; however, the vaccine parent strain 86079/7NS caused air sac lesions that were significantly greater than those of MS-H and all ts⁻ clones. The vaccine parent strain 86079/7NS and two of the ts⁻ clones were recovered from the air sacs of the respectively infected chickens whereas the MS-H vaccine and two other ts⁻ clones were not. Three of the ts⁻ isolates caused increased tracheal mucosal thicknesses that were significantly greater than those from birds inoculated with MS-H, and one caused increased tracheal mucosal thicknesses that were significantly less than those from birds inoculated with 86079/7NS.

In conclusion, unlike the MS-H vaccine, the MS-H ts⁻ clones were associated with minor changes in tracheal mucosa; however, unlike the vaccine parent strain, they did not induce lesions in the air sacs. These results suggest that factors other than ts phenotype are involved in the attenuation of the MS-H vaccine.

A monoclonal antibody (MAb)-based dot-enzyme-linked immunosorbent assay (ELISA) has been developed that detected the epitopes specifically associated with avian influenza virus (AIV). The dot-ELISA detected the antigens of AIV directly from clinical and field specimens. Data obtained from experimentally AIV-infected specific-pathogen-free chickens and also the 2001/02 AIV outbreak of serotype H7N2 positive flocks in Pennsylvania indicated that the mean sensitivity (Se) of the dot-ELISA ranged between 45% and 68% and the mean specificity (Sp), between 85% and 90%. The values were derived from various clinical and field specimens when compared with virus isolation with embryonating chicken eggs. On routine AIV surveillance samples, the dot-ELISA achieved a 92%–100% Sp on the basis of testing over 1500 AIV surveillance samples that were confirmed negative by virus isolation. The dot-ELISA detected AIV antigens with a 5-µl allantoic fluid sample that contained a concentration of 0.4 hemagglutinating units. Furthermore, the dot-ELISA retained its specificity for AIV because no cross-reactions were obtained with various other avian viruses. The findings in this study indicated that the dot-ELISA was highly sensitive and specific and comparable with the commercial Directigen® test in the detection of AIV obtained from clinical and field specimens.

Differentiating between virulent and avirulent avian Escherichia coli isolates continues to be a problem for poultry diagnostic laboratories and the study of colibacillosis in poultry. The ability of a laboratory to conduct one simple test that correlates with virulence would simplify studies in these areas; however, previous studies have not enabled researchers to establish such a test. In this study, the occurrence of certain phenotypic and genotypic traits purported to contribute to avian E. coli virulence in 20 avian E. coli isolates was correlated with the results of embryo challenge studies. This analysis was undertaken in an effort to determine which trait(s) best identified each avian E. coli isolate as virulent or avirulent. Traits selected were complement resistance, production of colicin V (ColV), motility, type F1 pili expression, presence of the temperature-sensitive hemagglutinin gene (tsh), and presence of the increased serum survival genetic locus (iss). ColV production, complement resistance, and presence of the iss genetic element were the three traits most highly correlated with high embryo lethality. A logistic regression model was used to predict the embryo lethality results on the basis of the most frequent isolate characteristics. Results indicate that ColV, complement resistance, and iss are significant predictor variables for the percentage of embryo lethality resulting from challenge with a specific avian E. coli isolate. However, no single trait has the ability to predict virulent isolates 100% of the time. Such results suggest the possibility that the embryo lethality assay may prove to be the one test needed to determine if an avian E. coli isolate is virulent.

Detection of Salmonella by bacteriologic methods is known to be time consuming. Therefore, we have developed a real-time probe-specific polymerase chain reaction (PCR) to rapidly detect Salmonella invA gene–based PCR products from chicken feces and carcasses by a fluorescence resonance energy transfer assay. The sensitivity and the specificity of this system were determined as 3 colony-forming units ml⁻¹ and 100%, respectively. Overnight tetrathionate broth enrichment cultures of chicken feces and carcass samples were used in template preparation for PCR. Also, a standard bacteriology was performed (National Poultry Improvement Plan–U.S. Department of Agriculture, Bacteriological Analytical Manual–Food and Drug Administration Center for Food Safety and Applied Nutrition) for confirmation. Seventy-two cloacal swab, 147 intestine, and 50 carcass (neck) samples were examined. Thirteen (8.8%) and 25 (17%) of the intestinal samples were found to harbor Salmonella by bacteriology and PCR, respectively. Forty-five of 50 (90%) carcass samples were Salmonella positive by both methods. Salmonella was not detected from cloacal swab samples. Results indicate that this assay has the potential for use in routine monitoring and detection of Salmonella in infected flocks and carcasses.

The phase 1 (fliC) and phase 2 (fljB) Salmonella flagella genes were analyzed by restriction fragment length polymorphism (RFLP)–polymerase chain reaction (PCR) to aid in the identification of different Salmonella serotypes. Twenty-four phase 1 flagellin and eight phase 2 flagellin genes could be differentiated among each other with restriction endonucleases Sau3A and HhaI in RFLP-PCR analysis. These flagellin genes comprise the major antigenic formulas for 52 serotypes of Salmonella sp., which include the common serotypes found in poultry and other important food animal species. With the knowledge of the O antigen composition determined from conventional O serotyping, 90% of the Salmonella serotypes could be identified by this double restriction enzyme RFLP analysis of fliC and fljB genes. This RFLP-PCR flagellar typing scheme was successfully applied to the identification of serotype for 112 Salmonella isolates obtained from poultry environment. There was a significant correlation between RFLP-PCR and conventional serotyping (chi-square, P < 0.001). Overall, PCR-RFLP proved to be a fast, accurate, and economical alternative approach to serotyping Salmonella sp.

In a previous study, turkey coronavirus (TCV) and enteropathogenic Escherichia coli (EPEC) were shown to synergistically interact in young turkeys coinfected with these agents. In that study, inapparent or mild disease was observed in turkeys inoculated with only TCV or EPEC, whereas severe growth depression and high mortality were observed in dually inoculated turkeys. The purpose of the present study was to further evaluate the pathogenesis of combined TCV/EPEC infection in young turkeys and determine the role of these agents in the observed synergistic interaction. Experiments were conducted to determine 1) effect of EPEC dose, with and without concurrent TCV infection, and 2) effect of TCV exposure, before and after EPEC exposure, on development of clinical disease. Additionally, the effect of combined infection on TCV and EPEC shedding was determined.

No clinical sign of disease and no attaching and effacing (AE) lesions characteristic of EPEC were observed in turkeys inoculated with only EPEC isolate R98/5, even when turkeys were inoculated with 10¹⁰ colony forming units (CFU) EPEC (high dose exposure). Only mild growth depression was observed in turkeys inoculated with only TCV; however, turkeys inoculated with both TCV and 10⁴ CFU EPEC (low dose exposure) developed severe disease characterized by high mortality, marked growth depression, and AE lesions. Inoculation of turkeys with TCV 7 days prior to EPEC inoculation produced more severe disease (numerically greater mortality, significantly lower survival probability [P < 0.05], increased frequency of AE lesions) than that observed in turkeys inoculated with EPEC prior to TCV or simultaneously inoculated with these agents. Coinfection of turkeys with TCV and EPEC resulted in significantly increased (P < 0.05) shedding of EPEC, but not TCV, in intestinal contents of turkeys. These findings indicate that TCV infection predisposes young turkeys to secondary EPEC infection and potentiates the expression of EPEC pathogenicity in young turkeys.

Campylobacter is considered to be the leading bacterial etiologic agent of acute gastroenteritis in humans. Evidence implicates poultry as a major source of the organism for human illness; however, the pathways involved in Campylobacter contamination of poultry flocks, horizontal transmission and/or vertical transmission, remain unclear. Recent evidence implicates breeders as a potential source for Campylobacter contamination of the subsequent broiler offspring. In this investigation, Campylobacter isolated from feces, cloacal swabs, ceca, semen, and vas deferens of 12 breeder broiler roosters were genotyped by both flagellin A short variable region (flaA SVR) DNA sequence analysis and repetitive element (rep)–polymerase chain reaction (PCR). In 9 of 12 roosters, Camplylobacter was isolated from multiple sites sampled. Comparison of multiple isolates obtained from individual roosters revealed variable results. In five of the nine roosters, all Campylobacter isolated demonstrated closely related flaA SVR DNA sequences as well as rep-PCR patterns; isolates from these roosters were collected from both the gastrointestinal and the reproductive tracts or from the gastrointestinal tract alone. The remaining four roosters had Campylobacter that were distinct by both typing methods. Isolates from two of these four roosters originated from both the gastrointestinal and the reproductive tracts. Isolates from the remaining two roosters originated from only the reproductive tract. Comparisons of all Campylobacter isolates recovered from a distinct sample type within either the reproductive tract or the gastrointestinal tract (feces, semen, cloaca, vas deferens, or ceca) were quite diverse. No relationship between the genotypes and the sample type could be ascertained. Further investigation is needed to determine the route of contamination and if the presence of Campylobacter within the rooster leads to contamination of the broiler offspring via the fertilized egg.

A study was conducted to evaluate the effects of ochratoxin A (OA) on Escherichia coli–challenged broiler chickens. Day-old broiler chicks were separated into two groups of 92 chicks each, with one group fed a control mash diet, and the other fed a mash diet containing 2 ppm OA. On day 14, each group was further separated into two groups, with one group inoculated with E. coli O78 (1 × 10⁷ colony-forming units/0.5 ml), whereas the other group was not inoculated with E. coli. After E. coli inoculation on day 14, four birds from each group were euthanatized at 1, 2, 3, 5, 7, 10, 14, and 21 days postinoculation. Escherichia coli infection caused dullness, depression, huddling, and diarrhea. Mortality was 14.3% in chicks infected with E. coli but fed no OA. Mortality increased to 35.7% in chicks fed OA and infected with E. coli. Decreased body weight and reduced feed intake were observed in chicks fed OA, and the effects were more pronounced in chicks fed OA and infected with E. coli. Increased serum levels of aspartate aminotransferase, alanine aminotransferase, uric acid, and creatinine and decreased levels of total proteins, albumin, globulins, calcium, and phosphorus were observed in OA-fed birds. Escherichia coli infection did not cause significant alteration in any of the serum biochemical parameters. The presence of OA in poultry rations increased mortality and the severity of an E. coli infection.

We have recently described the isolation and molecular characteristics of two recombinant avian leukosis subgroup J viruses (ALV J) with an avian leukosis virus subgroup A envelope (r5701A and r6803A). In the present study, we examined the role of the subgroup A envelope in the pathogenesis of these recombinant viruses. Chickens of line 151₅ × 7₁ were inoculated at 1 day of age with r5701A, r6803A, Rous-associated virus type 1 (RAV-1), or strain ADOL-Hcl of ALV-J. At 2, 4, 10, 18, and 32 wk postinoculation (PI), chickens were tested for avian leukosis virus (ALV)-induced viremia, shedding, and neutralizing antibodies. All except one chicken inoculated with the recombinant viruses (98%) developed neutralizing antibodies by 10 wk PI compared with only 16% and 46% of the ADOL-Hcl and RAV-1–inoculated birds, respectively. ALV-induced tumors and mortality in the two groups inoculated with recombinant viruses were different. The incidence of tumors in groups inoculated with r5701A or RAV-1 was 100% compared with only 9% in the groups inoculated with r6803A or ADOL-Hcl. The data suggest that differences in pathogenicity between the two recombinant viruses might be due to differences in the sequence of the 3′ untranslated region (presence or absence of the E element), and, therefore, not only the envelope but also other elements of the viral genome play an important role in the pathogenesis of ALV.

Mycobacterial infections are an important cause of morbidity and mortality in birds and a considerable diagnostic challenge until the disease is advanced. In order to develop more clinically useful antemortem tests, a biological model was created that replicated naturally occurring disease.

Japanese quail (Coturnix coturnix japonica; n = 8) were inoculated intravenously with Mycobacterium avium. Two additional birds served as uninoculated controls. Mean survival time of the inoculated birds was 68 ± 13 days postinoculation (PI). Seven of the eight inoculated birds died naturally. Clinical and postmortem abnormalities in inoculated birds were characteristic of naturally occurring mycobacteriosis. Abnormal clinical findings included decreased activity, feather erection, and sudden death. Mean body weight and packed cell volume declined and mean total white blood cells (primarily heterophils, bands, and monocytes) increased from 28 days PI onward.

Similar to birds that are naturally infected with mycobacteriosis, the inoculated birds were thin and had severe hepatosplenomegaly on postmortem examination. All eight birds had lesions in the liver, spleen, intestine, lung, gonads, and serosa. Less commonly affected tissues included bone marrow, thymus, gizzard, heart, pancreas, and brain. Lesions were invariably severe in the liver and spleen. These gross postmortem findings were consistent with natural infections of avian mycobacteriosis.

Mycobacterium avium was isolated from the liver, spleen, and intestine of all inoculated birds. Both control birds remained disease free and culture negative. This inoculation protocol is a reliable and practical means of inducing avian mycobacteriosis for further study.

In this study we compared culture, acid-fast stains, and polymerase chain reaction (PCR) for the detection of acid-fast organisms in fecal and tissue samples from Japanese quail (Coturnix coturnix japonica) that were experimentally inoculated intravenously with Mycobacterium avium. For culture, three different culture media (modified Herrold egg yolk with mycobactin; Lowenstein–Jensen [L-J]; and L-J with cyclohexamide, naladixic acid, and lincomycin) were tested to determine which medium had the greatest success in isolating mycobacteria. Acid-fast staining methods included Ziehl–Neelsen (Z-N) and Truant. The PCR assay detected mycobacterial DNA with primers specific for the 65-kD heat shock protein gene.

Culture was considered the “gold standard.” Compared with other culture media, L-J yielded more positive cultures and greater numbers of colonies on positive tubes, and incubation times were shorter. Mycobacterium avium was isolated from all of the harvested tissue samples (liver, spleen, and intestine) of inoculated birds. Mycobacteria were isolated from 53% (69/130) of fecal samples from inoculated birds. As the disease advanced, fecal culture was positive on more culture days, indicating that the culture-positive rate was higher later in the course of the disease.

Compared with culture, all of the laboratory methods had 100% specificity for the tissue samples. Sensitivities for the tissue samples were 82.6% (Z-N), 95.7% (Truant), and 100% (PCR). For the fecal samples, the specificity was >95% for all methods. Sensitivities compared with fecal culture were 7.2% (Z-N), 30.4% (Truant), and 20.3% (PCR). Tissue and fecal samples from the two control birds were negative for acid-fast organisms by any method.

These results were comparable with clinical cases of avian mycobacteriosis where culture and PCR of tissue samples seem to be the most sensitive and specific laboratory tests and evaluation of fecal samples still remains challenging. On the basis of the results of this study, identification of mycobacteria in fecal samples from Japanese quail can be optimized by repeated cultures and Truant acid-fast staining of fecal smears.

Antibody-forming cells (AFCs) against Newcastle disease virus (NDV) and their immunoglobulin (Ig) class were demonstrated by a double immuno-enzyme histochemical technique. The AFCs were stained and quantified in spleen sections of chickens euthanatized at day 7 postexposure to the Roakin strain of NDV. The sections were incubated with NDV to determine the specificity of the AFCs. Bound virus was subsequently visualized with a primary monoclonal antibody (MAb), a secondary horseradish peroxidase–conjugated MAb, and 3-amino-9-ethylcarbazole as substrate. IgM and IgA were stained with MAbs and an alkaline phosphatase (AP)-conjugated secondary antibody. IgG class antibodies were demonstrated with an AP-conjugated rabbit serum. The final substrate for the three Igs was naphthol AS-MX-phosphate and fast blue BB. About 64–159/mm² AFCs against NDV were detected. Of these virus-binding cells, about 55% produced IgM, 37% produced IgG, and the remainding 8% produced IgA.

Fatty liver hemorrhagic syndrome (FLHS) was observed in each of three trials in which commercial layers were utilized to determine the effect of Mycoplasma gallinarum (MGn) on egg and eggshell quality parameters and egg production. In each of three trials, FLHS occurred 31–54 days later in MGn-inoculated hens as compared with the Mycoplasma-clean (control) hens. In trials 1 and 2, no therapeutic intervention was initiated to ameliorate FLHS. In trial 3, therapeutic intervention was instituted and consisted of the addition of 1 pound of choline chloride/ton of feed. Total mortality recorded throughout the duration of each trial and attributable to FLHS was not significantly different between the control and the MGn-inoculated treatment. However, FLHS-associated mortality in each of the three trials was numerically greater for the control treatment.

SUMMARY. A 42-day broiler floor pen study was conducted comparing the anticoccidial efficacy of toltrazuril (Baycox) as a stand alone treatment and as an additional treatment to in-feed anticoccidial programs. Toltrazuril was administered on days 18 and 19 in the drinking water at 7 mg/kg of body weight. The treatments were 125 ppm nicarbazin (days 0–14) to 66 ppm salinomycin (SAL) (days 15–35) with and without toltrazuril, SAL (days 0–35) with and without toltrazuril, nonmedicated (NM) to SAL with toltrazuril, and NM with and without toltrazuril. The controls were NM noninfected and infected. The treatments were replicated in five blocks of eight pens each in a randomized complete block design. All withdrawal feed was nonmedicated. On day 14, birds, except noninfected, were exposed to coccidial oocysts (Eimeria acervulina, Eimeria maxima, and Eimeria tenella) seeded litter. On days 21, 28, 35, and 42, birds and feed were weighed, four birds per pen were coccidial lesion scored, and litter oocyst counts were performed. The coccidial infection in the NM infected treatment caused a significant (P < 0.05) coccidiosis infection. Coccidiosis was moderately controlled in the anticoccidial treatment birds without toltrazuril. Performance in the NM with toltrazuril was equal to or better (P < 0.05) than the anticoccidial programs without toltrazuril. Toltrazuril was equal to the noninfected birds in performance. Toltrazuril most completely eliminated all coccidial lesions and dramatically reduced oocyst shedding. The performance data, lesion scores, and oocyst counts showed that a 2-day treatment with toltrazuril successfully controlled the coccidiosis with no relapse of infection. Toltrazuril can thus be used for supplemental control with in-feed anticoccidials or as a primary anticoccidial with nonmedicated feed.

The purpose of this pilot study was to determine if pigeons (Columba livia) are susceptible to infection with Mycobacterium bovis by either oral or intratracheal inoculation and to assess their possible role in the lateral transmission of bovine tuberculosis. Six pigeons were orally inoculated with 1.3 × 10⁵ colony-forming units of M. bovis, six pigeons were intratracheally inoculated with the same dose, and six pigeons served as noninoculated controls. The study continued for 90 days postinoculation (PI), with groups of birds necropsied at 30-day intervals, and fecal samples and tissues were collected for mycobacterial culture. Two pigeons, one intratracheally inoculated and one orally inoculated, shed M. bovis in their feces at 1 day PI, and one intratracheally inoculated bird shed M. bovis in its feces 60 days PI. Whereas no illness or weight loss was present during the course of the study, 2 of 12 inoculated birds exhibited microscopic lesions of mycobacteriosis, and the organism was isolated from tissues of three inoculated birds. Pigeons are susceptible to infection with M. bovis after high dose inoculation and can shed the organism in their feces for up to 60 days PI; intratracheally inoculated birds appear more likely to become active fecal shedders of M. bovis. Although these were high dose inoculations under experimental conditions, pigeons may potentially play a role in the lateral transmission of bovine tuberculosis between infected and uninfected mammalian hosts.

As part of the basic characterization of Ornithobacterium rhinotracheale, the minimal inhibitory concentrations of 10 antimicrobial drugs were determined for reference strains and Mexican isolates by a broth microdilution method. For optimal growth of the organisms, a supplemented brain–heart infusion broth was used. The susceptibility of O. rhinotracheale to amoxicillin, enrofloxacin, and oxytetracycline was variable. However, consistent higher minimal inhibitory concentrations values were obtained for gentamicin, fosfomycin, trimethoprim, sulfamethazine, sulfamerazine, sulfaquinoxaline, and sulfachloropyridazine. Obtained results among Mexican isolates indicate a marked antimicrobial drug resistance trend.

Four avian pneumovirus (APV) isolates from chickens clinically diagnosed with swollen head syndrome were genetically characterized as to the subtypes of the virus in Japan. The results of reverse transcriptase–polymerase chain reactions based on subtype-specific primers and direct sequence analysis of G genes indicated subtypes A and B but not C or D of APV were present in Japan. Several routes or sources are conceivable for APV to invade into Japan.

Incubation of Mycoplasma gallisepticum with washed chicken red blood cells for 1 hr or 5 hr resulted in altered red blood cell surface morphology and perforations of the cells.

The lateral transmission of Histomonas meleagridis in turkeys was studied in floor pens without the presence of Heterakis gallinarum. Battery-reared poults (120) were transferred at 2 wk of age to concrete-floored floor pens with fresh pine shavings litter (40/group). One group received no exposure. In other groups, either 10% or 25% of the birds were inoculated per cloaca with cultured H. meleagridis (200,000/bird) and placed in the pens as seeder birds. Inoculated birds died at 10–18 days postinfection (PI) showing typical liver and cecal lesions of histomoniasis. Birds in the high-exposure group died of histomoniasis beginning 16 days PI and continuing to 100% mortality by day 23 PI. Birds in the low-exposure (LE) group died beginning on day 19 PI and continuing through day 31 PI. All but one LE bird alive on day 31 PI had severe liver and cecal lesions of histomoniasis at necropsy. There was no evidence of histomoniasis in unexposed birds. No cecal worms (H. gallinarum) were found at necropsy of dead birds or in unexposed birds at the end of the experiment. Even though H. gallinarum is the only known reservoir for H. meleagridis, these results suggest that lateral transmission of histomoniasis through a flock can occur readily through normal contact between uninfected birds and infected birds and their droppings in the total absence of cecal worms.

During August 2001, a syndrome characterized by acute lethargy and dyspnea was observed in a population of 45 lorikeets and lories in an open-air zoologic exhibit. The first death occurred on August 10, and within the next 12 days, nine more birds died (22% mortality rate). Hepatomegaly, reddening and congestion of the lungs, and injection of the serosal surface of the intestines were the common gross findings. Histologic changes, including fibrinonecrotic hepatitis and splenitis, bacterial emboli (liver, spleen, lung, kidney, proventriculus), pulmonary congestion and hemorrhage, and enteritis, were indicative of an acute, overwhelming bacterial septicemia. Salmonella typhimurium, with the same antibiogram, was isolated from four birds. Several birds had attacked and killed a snake on July 24, and Salmonella serogroup B (untypeable) was isolated from intestine and kidney samples of a garter snake caught in the open-air exhibit on August 28. Salmonella was also isolated from environmental samples of the exhibit but not from food preparation areas. After antimicrobial therapy, Salmonella spp. was not isolated from the surviving birds. The source of Salmonella in this outbreak remains unknown, but infection either directly or indirectly from snakes in the exhibit is possible. Contact between captive psittacine populations and reptiles should be avoided to prevent the risk of salmonellosis.

Eighty-three-week-old table egg layers with swollen sinuses were presented with a history of increased mortality. Serology revealed positive titers to Mycoplasma gallisepticum (MG). The birds were part of a flock in which some birds had been vaccinated with 6/85 live MG vaccine at 18 wk of age. Tracheal cultures were obtained from both vaccinated and unvaccinated birds within the flock. The cultures were indistinguishable from 6/85 vaccine by both random amplified polymorphic DNA analysis and DNA sequence analysis. Challenge studies were performed to compare the field isolates with 6/85 vaccine and the R strain of MG. The field isolates produced a greater antibody response by serum plate agglutination than did the 6/85 vaccine. The isolates effectively colonized the trachea without increasing the tracheal mucosal thickness; however, they did not extensively colonize the air sacs or cause airsacculitis in the experimental birds.

Ten out of 42 (23.8%) white storks (Ciconia ciconia) admitted to two rehabilitation centers in central Spain had lesions caused by the trematode Chaunocephalus ferox in the small intestinal wall. Fourteen of the examined birds were adults, five were subadults, and 23 were chicks of various ages. Parasitation was 32% (n = 8) in chicks and 13% (n = 2) in adult birds, whereas no juvenile bird was affected. Among dead birds, stork chicks affected by C. ferox lesions had a lower body weight (2196.1 g, SD = 814.2) than storks without lesions (2965.8 g, SD = 742.9, P < 0.05). Two chicks were additionally infected with Salmonella subspecies I serotype enteritidis 1,9,12: g, m:1, 7. Prevalence of the parasite in the examined birds was lower than in a population of Asian open-billed storks (Anastomus oscitans), in which it was pathogenic due to the destruction of the tunica muscularis and formation of large granulomatous lesions in the wall of the postduodenal portion of the small intestine. Pathogenic alterations caused by C. ferox are presumed to be related to numbers of adults present. Because storks admitted to rehabilitation centers suffer stress due to various reasons that may lower their immune response and exacerbate existing infections, the analysis of fecal sediments of white storks admitted for rehabilitation is recommended.