The incidence of Marek's disease (MD), an important neoplastic disease of chickens, suddenly increased in 1997 in Korea. Most MD cases of this country were detected in chickens over 20 wk of age. Five MD viruses were isolated from field flocks in which severe MD losses had occurred, and one of the viruses was studied to compare its pathotype with the prototype JM strain.

The isolate KOMD-IC induced severe depression not only in body weight but also in relative bursal weight, and the depression by KOMD-IC was more severe than that induced by JM strain. In addition, the incidence of MD tumor caused by KOMD-IC was higher than that caused by the JM strain.

The protective capacity of several MD vaccines was studied against challenge with KOMD-IC. The protective levels of several MD vaccines such as herpesvirus of turkeys (HVT), HVT plus SB1, and Rispens were usually lower against challenge with KOMD-IC than those challenged with JM strain, even if the chickens vaccinated with serotype 1 were not completely protected against challenge with KOMD-IC.

The above results indicate that the virulence of KOMD-IC isolated recently was increased, and the increase of MD outbreak in Korea may be related to the virulence increase of the virus.

Various MD vaccine programs were applied to reduce MD loss to a broiler breeder farm where severe MD loss had occurred. Serotype 1 vaccine could dramatically decrease the mortality due to MD, and the best results were obtained from the flocks vaccinated with bivalent vaccine of Rispens and HVT.

A group of 1-day-old commercial leghorn chickens was prophylactically treated with lymphokines obtained from lymphocyte cultures of chickens previously infected with Salmonella enteritidis (S. enteritidis-immune lymphokines [SE-ILK]) with the objective to investigate the effect of SE-ILK on development of Newcastle disease (ND) infection caused by Chimalguacan strain, a Mexican velogenic ND virus (vNDV). Clinical signs, histologic lesions, and hemagglutination-inhibition (HI) serum titers were compared with four other groups, namely, chickens without SE-ILK treatment with virus challenge; with SE-ILK without virus challenge; with nonimmune lymphokine (NILK) treatment and virus challenge; with lymphokine treatment and no virus challenge. SE-ILK was administered intraperitoneally in a dose of 0.5 ml/chicken and was followed 30 min later with the challenge of vNDV in a dose of 10^7.6 50% embryo lethal dose/ml per bird. Birds were observed during 21 days of postchallenge. Detection of histologic changes and virus isolation procedures were carried out on the third, seventh, 14th, and 21st postinoculation days. HI tests were performed first before treatment and later on the days of histologic sample collection except on the third postinoculation day. Results showed that SE-ILK administration conferred resistance to the chickens because: 1) it significantly diminished the severity of ND infection by inhibiting appearance of clinical signs (P < 0.001), lesions (P < 0.005), and histopathologic changes (P < 0.005); 2) it decreased vNDV isolation rate from the organs (P < 0.001), and 3) it potentialized and even accelerated (P < 0.005) primary immune response by antibodies in the presence of vNDV.

Campylobacter jejuni, a foodborne pathogen closely associated with market poultry, is considered to be the most frequent agent of human gastroenteritis in the United States. The pathways involved in the contamination of poultry flocks, vertical transmission and/or horizontal transmission, are unclear. In this study, Campylobacter isolates from two independent commercial broiler breeder flocks, as well as from their respective progeny, were characterized and compared by PstI ribotype analysis and by DNA sequence analysis of the short variable region (SVR) of the flaA gene (flaA SVR). Campylobacter isolates originating from one set of breeder hens and the feces from their respective progeny demonstrated identical ribotype patterns as well as identical flaA SVR DNA sequences, thereby suggesting that these isolates were clonal in origin. Ribotype analysis of Campylobacter isolates from the second set of breeder hens and processed carcasses from their offspring resulted in two patterns. Sequence analysis placed these isolates into two closely related groups and one distant group, similar to the ribotype analysis. These results demonstrate that Campylobacter isolates from commercial broiler breeder flocks and from the respective broiler progeny may be of clonal origin and that breeder hens can serve as a source for Campylobacter contamination in poultry flocks.

Three types of Bifidobacterium thermophilum extract were prepared and fed to 2-wk-old chickens to evaluate their usefulness in enhancing the defense activity of the chickens against pathogenic Escherichia coli. All three preparations resulted in significant reduction (P < 0.05) of E. coli numbers in the lungs of the treated chicken groups compared with the control nontreated group. Besides, improvement in the survival rate was observed in the treated chicken groups, especially the one administered the enzyme-digested B. thermophilum extract sample. Concanavalin A–stimulated lymphocytes from the latter group demonstrated significantly higher proliferation activity compared with those from the control group. These results suggest that oral administration of B. thermophilum preparations may be used to enhance the resistance of chickens against E. coli infection.

The hypothesis that an effective protection of progeny chickens against inclusion body hepatitis/hydropericardium syndrome (IBH/HP) can be achieved by dual vaccination of breeders with fowl adenovirus (FAV) serotype 4 and chicken anemia virus (CAV) was tested. Thus, 17-wk-old brown leghorn pullet groups were vaccinated by different schemes including single FAV (inactivated), single CAV (attenuated), FAV and CAV dually, or were not vaccinated (controls). Subsequent progenies of these breeders were challenged with the virulent strains FAV-341 and CAV-10343 following three strategies: 1) FAV-341 intramuscularly (i.m.) at day 10 of age (only FAV-vaccinated and control progenies); 2) FAV CAV i.m. simultaneously at day 10 of age (all progenies); 3) CAV i.m. at day 1 and FAV orally at day 10 of age (all progenies). The induction of IBH/HP in these progenies was evaluated throughout a 10-day period. Both breeder groups vaccinated against FAV and those vaccinated against CAV increased virus neutralizing specific antibodies. Challenge strategy 1 showed 26.6% mortality in control progeny chickens and 13.3% in the progeny of FAV-vaccinated breeders. Presence of lesions in the liver of these groups showed no significant differences (P > 0.05), suggesting a discreet protective effect of the vaccine. Challenge strategy 2 showed 29.4% mortality in controls and 94% of chickens showed hepatic inclusion bodies (HIB). Single CAV vaccination of breeders did not demonstrate a beneficial effect, with both mortality and liver lesions resembling the nonvaccinated controls. FAV vaccination of breeders significantly reduced both mortality (7.4%) and liver lesions (26% HIB) (P < 0.05), providing protection against this challenge strategy. Dual vaccination of breeders with FAV and CAV proved to be necessary to achieve maximum protection of the progeny (no mortality and 7% HIB). Challenge strategy 3 produced no mortality but consistent liver damage in controls (96% HIB). In this case, both CAV and FAV CAV-vaccinated breeders showed best protection results in terms of liver histopathology (8% and 0% HIB, respectively). FAV vaccination alone produced 24% HIB, similar to challenge strategy 2, demonstrating a lower protective effect.

Since 1997, avian pneumovirus (APV) has caused estimated annual losses of $15 million to the Minnesota turkey industry. In order to develop an attenuated live vaccine against APV, we serially passaged a Minnesota isolate of APV (APV/MN/turkey/1-a/97) in vitro in cell cultures for 41 passages. Laboratory experiments with this high-passage virus (P41) indicated that the attenuated virus provided immunogenic protection to turkeys against challenge with virulent APV, although some birds showed mild to moderate clinical signs after inoculation. To reduce the residual pathogenicity of P41, while maintaining its immunogenicity, we decided to vaccinate turkeys with P41 in the presence of an immunomodulator, S-28828 (1-n-butyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-4-amine-hydrochloride), which is a potent cytokine inducer. The combined inoculation of S-28828 (5 mg/kg body weight) and P41 resulted in a significant reduction in the incidence of virus-induced clinical signs in comparison with birds that received P41 without immunomodulator (P < 0.05). Only 17% of birds inoculated with S-28828 APV P41 showed mild respiratory symptoms at 5 days postinoculation as compared with 46% of the vaccinated turkeys that did not receive S-28828. Vaccination with either P41 or with P41 S-28828 protected turkeys against clinical signs and viral replication after challenge with virulent APV. These results indicate that immunomodulators, such as S-28828, may act as good vaccine adjuvants that can reduce the pathogenicity but maintain the immunogenicity of partially attenuated vaccines.

The biochemical phenotypes and antimicrobial susceptibility patterns of 105 clinical Escherichia coli isolates from flocks with colibacillosis in a turkey operation were compared with 1104 fecal E. coli isolates from 20 flocks in that operation. Clinical isolates and 194 fecal isolates with biochemical phenotypes or minimum inhibitory concentrations for gentamicin and sulfamethoxazole similar to clinical isolates were tested for somatic antigens and the potential virulence genes hylE, iss, tsh, and K1. The predominant biochemical phenotype of clinical isolates contained 21 isolates including 14 isolates belonging to serogroup O78 with barely detectable β-d-glucuronidase activity. Thirty-five fecal isolates had biochemical phenotypes matching common phenotypes of clinical isolates. Sixty-six (63%) clinical isolates exhibited intermediate susceptibility or resistance to gentamicin and sulfamethoxazole compared with 265 (24%) fecal isolates (P < 0.001). Seventy-seven clinical isolates reacted with O-antisera, of which 51 (66%) belonged to the following serogroups: O1, O2, O8, O25, O78, O114, and O119. In comparison, 8 of 35 (23%) fecal isolates subtyped on the basis of biochemical phenotype belonged to these serogroups and four of 167 (2%) fecal isolates subtyped on the basis of their antimicrobial resistance patterns belonged to these serogroups. Iss, K1, and tsh genes were detected more often among clinical isolates than these fecal isolates (P < 0.05). In summary, a small subgroup of E. coli strains caused most colibacillosis infections in this operation. These strains existed at low concentration in normal fecal flora of healthy turkeys in intensively raised flocks. The data suggest that colibacillosis in turkey operations may be due to endogenous infections caused by specialized pathogens.

Three glycoproteins of infectious laryngotracheitis virus (ILTV), gC, gE, and gp60, were expressed in Escherichia coli as fusion proteins with a 6-histidine tag at their amino termini. The proteins expressed, designated as r-gC, r-gp60, and r-gE, all retain their antigenicity, as revealed by Western blot with chicken antiserum against ILTV. However, only r-gp60 and r-gE, but not r-gC, were found to be soluble. The soluble r-gp60 and r-gE were purified by a nickel column and then used as the enzyme-linked immunosorbent assay (ELISA) antigen for detecting ILTV-specific antibodies. The diagnostic potential of r-gE and r-gp60 ELISA was assessed with the use of sera prepared from vaccinated or unvaccinated chickens of either specific-pathogen-free (SPF) or field origins. The result shows that r-gp60 and r-gE ELISA could discriminate vaccinated SPF chickens from unvaccinated ones 2 wk postvaccination. Moreover, r-gp60 and r-gE ELISA could also discriminate vaccinated field flocks from unvaccinated ones. This result indicates that r-gp60 and r-gE might serve as an alternative ELISA antigen for detecting ILTV-specific antibodies. Moreover, r-gp60 or r-gE ELISA might play an important role in the eradication of infectious laryngotracheitis (ILT) in the future when the gp60- or gE-deleted marker vaccine of ILT is available.

Criteria for evaluating genetic differences in resistance and susceptibility to infectious bursal disease (IBD) within a commercial broiler breeder line of chickens were compared. Line A broiler breeder chickens were challenged with graded doses of Animal and Plant Health Inspection Service (APHIS) strain IBD virus (IBDV) and evaluated at 2 time points, 3 days postinoculation (PI) and 10 days PI. Measures obtained at both time points included bursa to body weight, bursa histology, bursa lymphocyte count, and percentage of T cells in the bursa. Furthermore, viral load in the bursa was determined 3 days PI and anti-IBDV antibody titers, 10 days PI. A dose of 50 50% embryo infective dose caused IBD in about half the line A birds at the 10-day time point, and this dose was chosen for further studies. The data were analyzed for correlation among the various measures. Comparison of the 3-day- and 10-day-PI bursa lymphocyte counts indicated that birds challenged with low doses of virus suffered lymphocyte depletion at the 3-day time point, but many or all (depending on the dose) recovered by the 10-day time point. With a viral dose that caused bursal atrophy in about half the birds by 10 days PI, families segregating for 2 major histocompatibility complex (MHC) haplotypes were compared in terms of resistance to IBD. Results indicated that there was no difference among the 3 MHC genotypes in incidence of IBD by any of the disease measures.

Mycoplasma gallisepticum (MG) is a reproductive/respiratory disease in poultry implicated in suboptimum egg production and decreased hatchability. Commercial layer hens raised in a controlled environment were inoculated with the S6 strain of MG at 10 wk of age. Egg production and selected egg and egg quality parameters were quantitated over the entire lay cycle for inoculated and control birds. The S6 inoculation had no effect on bird weight, egg production, associated egg quality parameters, or histopathologic lesion scores. This study shows that in the absence of environmental stressors a prelay S6 MG inoculation does not produce detrimental effects on layer hen performance.

Subgroup J avian leukosis virus (ALV-J) causes serious economic losses in the commercial poultry industry. Measuring group-specific antigen (GSA) by enzyme-linked immunosorbent assay (ELISA) has been used to identify chickens infected with this virus. However, the inability of ELISA to discriminate the GSA from endogenous ALV (subgroup E ALV [ALV-E]) or ALV-J infection has limited its usage. The purpose of the present study was to develop a method to discriminate between uninfected flocks having ALV-E and ALV-J–infected flocks by ELISA. The GSA and anti-ALV-J antibody in the plasma samples from chickens at different ages in three grandparent farms were measured by ELISA. Infected flocks were confirmed by reverse transcription–polymerase chain reaction with different subgroup-specific primers and sequence analysis. The results indicated that the GSA of ALV-J–infected flocks increased, but that of the uninfected flocks decreased during young ages. The anti-ALV-J antibody of infected flocks was higher and increased earlier than that of uninfected flocks. Thus, measuring GSA in blood at the ages of 1 and 6 wk by ELISA is suitable to discriminate between ALV-J–infected flocks and uninfected flocks having ALV-E.

A total of 100 poultry farms in northern and middle areas of Jordan were sampled to investigate the bacteria associated with airsacculitis in broiler chickens. Of 170 bacterial isolates, 88.2% were identified as Escherichia coli, 8.8% as Ornithobacterium rhinotracheale, and 3% as Bordetella avium. Fourteen serotypes of E. coli were identified among 66 typeable isolates and the remainder were untypeable. The most prevalent serotypes were O1, O8, and O78. The main serotype of O. rhinotracheale was serotype A. Experimental inoculation of O. rhinotracheale via intravenous, intratracheal, and intra-air sac routes resulted in growth retardation, thickening in the air sacs, arthritis, and liver necrosis. Reisolation of O. rhinotracheale from the air sacs, liver, trachea, heart, and spleen at day 7 postinoculation confirmed its role. In vitro susceptibility testing revealed that E. coli isolates were sensitive to gentamicin and colistin, O. rhinotracheale to tetracyline, and B. avium to most of the nine antibiotics examined.

A multivalent in ovo vaccine (MIV) was tested for safety and efficacy in a commercial broiler complex. The MIV comprised five replicating live viruses including serotypes 1, 2, and 3 of Marek's disease virus (MDV), an intermediate infectious bursal disease virus (IBDV) and a recombinant fowl poxvirus (FPV) vector vaccine containing HN and F genes of Newcastle disease virus (NDV). The performance of MIV-vaccinated broilers was compared with that of hatchmates that received turkey herpesvirus (HVT) alone (routinely used in ovo vaccine in the broiler complex). The chickens that hatched from the MIV-injected and HVT-injected eggs were raised under commercial conditions in six barns. Barn 1 housed 17,853 MIV-vaccinated chickens and each of the barns 2–6 housed 18,472–22,798 HVT-vaccinated chickens. The HVT-vaccinated chickens were given infectious bronchitis virus (IBV) and NDV vaccines at hatch and at 2 wk of age. The MIV-vaccinated chickens received IBV vaccine at hatch and IBV NDV at 2 wk of age. The relative values of hatchability of eggs, livability and weight gain of chickens, and condemnation rates at processing were comparable between the MIV and the HVT groups (P > 0.05). Chickens from the MIV- and the HVT-vaccinated groups were challenged with virulent viruses under laboratory conditions. The resistance of vaccinated chickens against Marek's disease could not be assessed because of high natural resistance of unvaccinated commercial broilers to virulent MDV. The relative resistances of the MIV- and the HVT-vaccinated groups, respectively, against other virulent viruses were as follows: IBDV, 100% for both groups; NDV, 81% vs. 19%; FPV, 86% vs. 0%. The successful use of MIV under field conditions expands the usefulness of the in ovo technology for poultry.

A live attenuated Mycoplasma gallisepticum vaccine, ts-11, has been used for control of M. gallisepticum in several countries. The rapid serum agglutination test is usually used as an indicator of flock response to vaccination; however, in some flocks, the detected response may be weak or absent. We investigated whether the low level, or lack, of systemic antibodies in ts-11-vaccinated flocks is correlated with susceptibility to infection after challenge with a virulent M. gallisepticum strain.

Birds from 2 separate ts-11-vaccinated commercial flocks with no, or weak, rapid serum agglutination responses (at 11 or 14 wk postvaccination) were randomly selected and subjected to aerosol challenge with either M. gallisepticum strain Ap3AS or sterile mycoplasma broth. A group of nonvaccinated specific-pathogen-free chickens at similar age were also exposed to aerosolization with M. gallisepticum strain Ap3AS and used as positive controls. Postmortem examination of the birds, performed 2 wk after challenge, revealed no significant difference in microscopic tracheal lesions or mucosal thicknesses between the ts-11-vaccinated field birds irrespective of their aerosolization treatment. However, both microscopic tracheal lesions and tracheal mucosal thicknesses of nonvaccinated challenged birds were significantly greater than those of ts-11 vaccinates. Hence, broiler breeders vaccinated in the field showed significant protection against virulent M. gallisepticum challenge even when no serum antibody was detected by rapid serum agglutination test. These results reveal that seroconversion detected by rapid serum agglutination test after ts-11 vaccination is not a reliable predictor of protection against M. gallisepticum infection. The possible significance of local antibody response and cell-mediated immunity against M. gallisepticum infection is discussed.

Experimental infection models are useful tools for understanding how Salmonella enteritidis is deposited in eggs and for testing potential strategies to control eggborne transmission of disease to humans. Oral inoculation of laying hens is presumed to provide the closest simulation of naturally occurring infections, but alternatives such as intravenous or aerosol inoculation have sometimes been recommended as options to induce higher incidences of egg contamination. The present study compared the frequency, level, and location of S. enteritidis deposition in egg contents after experimental inoculation by three different routes. In two replicate trials, specific-pathogen-free laying hens were infected with an S. enteritidis culture mixture prepared to optimize invasive behavior. Groups of hens received either an oral dose of 10⁹ S. enteritidis, an aerosol dose of 10⁹ S. enteritidis, or an intravenous dose of 10⁵–10⁷ S. enteritidis. Oral inoculation led to the highest incidence of fecal shedding of S. enteritidis, whereas intravenous inoculation produced the highest specific antibody titers. Eggs laid during the first 21 days postinoculation were cultured to detect and enumerate S. enteritidis in the yolk and albumen. No significant differences were observed among the three inoculation routes in the frequencies of isolation of S. enteritidis from either yolk or albumen. For all three routes of administration, S. enteritidis was recovered more often from yolk (at frequencies ranging from 4% to 7%) than from albumen (0 to 2%). Over 73% of contaminated eggs harbored fewer than 1 colony-forming unit (CFU) of S. enteritidis per milliliter, and only 3% of such eggs contained more than 100 CFUs/ml. Significantly higher levels of S. enteritidis contaminants were associated with intravenous inoculation than with the other routes. No advantage of using aerosol or intravenous administration of S. enteritidis as an alternative to oral inoculation for inducing the production of contaminated eggs was evident in this study.

Nitric oxide (NO), a free radical produced by the enzyme NO synthase (NOS), is a potent antiviral agent in addition to having immune regulating functions. Recently, it was reported that chickens resistant (N2a, MHC: B²¹B²¹) to the development of Marek's disease (MD) had a greater potential to produce NO than MD-susceptible chickens (P2a, MHC: B¹⁹B¹⁹). This difference was shown by measuring NO levels in chick embryo fibroblast cultures obtained from these chickens after treatment with lipopolysaccharide and recombinant chicken interferon-gamma (IFN-γ). To extend these results, the levels of NO in blood plasma from N2a and P2a chickens inoculated with the nonattenuated JM-16 strain of MD virus (MDV) were examined. In four out of five experiments, N2a chickens had increased NO levels at 7 days postinoculation (DPI). In contrast, P2a chickens challenged with JM-16 had a significant increase in NO in only one of four experiments, and in that experiment the increase was delayed (10 DPI) compared with N2a chickens. Attenuation abrogated MDV-induced NO in chickens. Inoculation with MDV strains ranging from mild to very virulent plus showed that the more virulent strains induced the highest level of NO in blood plasma, suggesting a role of NO in the pathogenesis of MD with more virulent strains. On the basis of quantitative real-time reverse transcription–polymerase chain reaction (RT-PCR) assays for analysis of mRNA expression, IFN-γ does not appear to be the primary inducer of inducible (i)NOS gene expression during MDV infection. iNOS gene expression and NO production are mediated during the cytolytic phase of MDV infection on the basis of real-time RT-PCR assays with primers specific for glycoprotein B, a late gene expressed only during the cytolytic phase of MDV infection. These findings implicate NO as a factor potentially involved in increasing virulence of MDV, possibly through immune suppression.

The pathogenicity, transmissibility, tissue distribution, and persistence of avian pneumovirus (APV) in turkey poults were investigated in three experiments. In the first experiment, we inoculated 2-wk-old commercial turkey poults oculonasally with APV alone or in combination with Bordetella avium. In the dually infected group, clinical signs were more severe, the virus persisted longer, the bacteria invaded more respiratory tissues, and the birds had higher antibody titer than the group exposed to APV or B. avium alone.

In the second experiment, we studied the distribution of APV in different tissues in experimentally inoculated 2-wk-old commercial turkey poults. Only samples from sinuses, tracheas, and lungs were positive for APV by both reverse transcriptase–polymerase chain reaction and virus isolation.

In the third experiment, we studied the ability of APV to spread among birds in 1-wk-old commercial turkey poults inoculated oculonasally. The virus was isolated and the viral RNA was detected in the inoculated and direct contact birds. The virus was not isolated, viral RNA was not detected, and no antibodies were detected in the indirect contact birds. These birds were placed in different cages in the same room where the airflow was directed from the infected toward the uninfected indirect contact group.

We investigated the interaction between Newcastle disease virus (NDV) and Escherichia coli in cell cultures, embryonated eggs, and 8-wk-old chickens. We measured the interactions on the basis of bacterial adherence and NDV hemagglutination titer in chickens, chicken embryos, and chicken embryo cell culture.

Depending on the inoculation order of E. coli, a significant alteration of the growth of NDV was observed in both chickens and chicken embryos. When certain strains of E. coli were given before NDV exposure, the virus titers were lowered. In chickens, the mean virus titer was significantly (P < 0.05) lowered in the crop, the proventriculus, the gizzard, and the jejunum. However, there were no significant differences (P < 0.05) between the two groups for NDV titers in the duodenum, ileum, and cecum. In chicken embryos, when E. coli serotypes O78 and O119:B14 were inoculated before NDV exposure, the mean NDV titers were significantly (P < 0.5) lowered. However, there were no significant differences (P < 0.05) in NDV titer between the two groups when E. coli serotypes O78:K80:NM and O1ab:K:NM were inoculated 24 hr before NDV exposure.

When NDV was given prior to E. coli exposure, NDV titer was higher in both chickens and chicken embryos. In chickens, when NDV was given 48 hr before E. coli inoculation, NDV was detected in the proventriculus, gizzard, jejunum, ileum, and cecum, whereas no virus was detected in the control groups (NDV only). In the crop, NDV was detected at a significantly (P < 0.05) higher titer in the E. coli-inoculated group when compared with the control group that received NDV alone. In chicken embryos, virus titer was significantly (P < 0.05) higher when NDV was given 24 hr before E. coli inoculation for all three NDV strains used (Ulster and V4 strains).

Adherence of E. coli to chicken embryo kidney (CEK) cells was significantly higher (P < 0.05) when the CEK cells were infected first with NDV and then by E. coli. The mean bacterial count per microscopic field in NDV-uninfected monolayers was eight compared with 112 for the NDV-infected monolayers. In approximately 10% of the fields in NDV-infected monolayers, the bacteria were too numerous to count.

The immune response to four cell surface antigens of avian pathogenic Escherichia coli (APEC) was investigated as the first step in identifying vaccine candidates. F1 pilus adhesin, P pilus adhesin, aerobactin receptor protein, and lipopolysaccharide (LPS) from an O78 E. coli (strain EC99) were used as antigens. The proteins were purified as 6xhistidine-tagged recombinant proteins and LPS was purified from a phenol/water extract. Groups of 12 broiler chickens were vaccinated intranasally with the EC99 strain and challenged with the same strain 10 days later via the intra-air sac route. The chickens that survived were euthanatized 10 days postchallenge. Scores were assigned to infected chickens on the basis of lesions and recovery of the challenge E. coli. The immunoglobulin (Ig) IgG, IgA, and IgM antibodies to the four antigens were measured in serum and air sac washings in an enzyme-linked immunosorbent assay. Among the chickens that were not vaccinated prior to challenge, two died and three of the survivors were ill, whereas, of the chickens that were vaccinated prior to challenge, one died and one of the survivors became ill. After the intranasal vaccination, high antibody activity against all four antigens was associated with each Ig isotype in serum and air sac washings. IgG was the predominant isotype of Ig in air sac washings as detected by radial immunodiffusion. Chickens that were not ill after challenge had greater IgG, IgA, and IgM antibody activity against all four antigens in serum and air sac washings than did sick chickens. Thus, all of the antigens tested appear to be suitable candidates for a vaccine to protect chickens from respiratory tract infections caused by APEC.

In situ hybridization and immunohistochemistry were utilized to identify tissues infected in ovo with infectious bronchitis virus (IBV). Chicken embryos were inoculated in ovo (chorioallantoic sac) with the Arkansas (Ark) serotype of IBV at 18 days of age. At 24, 48, 72, and 120 hr postinfection (HPI), bursa, lung, spleen, heart, and thymus were collected, fixed in 10% neutral buffered formalin, and paraffin embedded. The digoxigenin-labeled antisense S1 riboprobe detected viral mRNA in the cytoplasm of respiratory epithelial cells in the primary bronchus at 24, 48, and 72 HPI. Viral mRNA was detected in bursa samples collected at 48 hr. Immunohistochemistry detected viral antigens in epithelial cells of the parabronchi and bursal tissues at 24 and 48 hr, respectively. No viral mRNA or antigen was detected by in situ hybridization or immunohistochemistry, respectively, in heart, thymus, or spleen at any time after inoculation. On the basis of these data, IBV apparently initially infects lung tissue, then migrates to and infects cells of the bursa. These results indicate that in situ hybridization can be useful in detection of IBV-infected chickens and in understanding the pathogenesis and virulence of IBV infection.

Ten gram-negative, pleomorphic, rod-shaped isolates from coryza-like, respiratory diseased laying and broiler chickens were identified as Ornithobacterium rhinotracheale. All O. rhinotracheale isolates showed typical biochemical and enzymatic characteristics. Also, all isolates showed hemagglutinating activity with glutaraldehyde-fixed erythrocytes. On the basis of this property, a rabbit-raised antiserum was produced for an isolate. All isolates were identified by antiserum by hemagglutination-inhibition tests. No cross-reactions were observed when O. rhinotracheale isolates were tested with Haemophilus paragallinarum antisera, and vice versa. Mild respiratory signs, including mild nasal discharge, slight rales, and sneezing, were observed in challenged chickens. At postmortem examination, multifocal pneumonia, airsacculitis, and foamy exudate in abdominal cavity were observed. Furthermore, because bacterial adherence is regarded as an essential step in the infection process, in vitro adherence of O. rhinotracheale isolates to chicken tracheal epithelial cells was tested. All isolates showed positive adherence. Obtained results indicate that O. rhinotracheale is a pathogenic agent present in the Mexican poultry.

A multiplex polymerase chain reaction (PCR) was developed and optimized to simultaneously detect 6 avian respiratory pathogens. Six sets of specific oligonucleotide primers for infectious bronchitis virus (IBV), avian influenza virus (AIV), infectious laryngotracheitis virus (ILTV), Newcastle disease virus (NDV), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS) were used respectively in the test. With the use of agarose gel electrophoresis for detection of the PCR-amplified DNA products, the sensitivity of detection was between 10 pg for IBV, AIV, MG, and ILTV and 100 pg for NDV and MS after 35 cycles of PCR. Similar sensitivity of these primers was achieved with chickens experimentally infected with respiratory pathogens. In experimental infections, the multiplex PCR was able to detect all the infected chickens in each group at 1 and 2 wk postinfection as compared with serologic tests at 2 wk postinfection that confirmed the presence of specific antibodies. The multiplex PCR was also able to detect and differentiate coinfections with two or more pathogens. No specific DNA amplification for respiratory avian pathogens was observed among noninoculated birds kept separately as a negative control group.

Two different whole-virus enzyme-linked immunosorbent assays (ELISAs), developed in Ohio (OH) with APV/Minnesota/turkey/2a/97 and in Minnesota (MN) with APV/Colorado/turkey/97, and the virus neutralization (VN) test were used to test 270 turkey serum samples from 27 Minnesota turkey flocks for avian pneumovirus (APV) antibodies. In addition, 77 turkey serum samples and 128 ostrich serum samples from Ohio were tested. None of the turkey samples from Ohio had antibodies to APV by the VN test and OH ELISA. The ostrich samples were only tested with the VN test and were all negative for antibodies to APV. For the Minnesota serum samples, 107, 115, and 120 were positive by the VN test, the OH ELISA, and the MN ELISA, respectively. The Kappa values of 0.938 and 0.825 showed excellent agreement between the VN test and the OH ELISA and the MN ELISA, respectively, for detection of antibodies to the APV. The OH ELISA and MN ELISA had sensitivities of 1.0 and 0.953, specificities of 0.950 and 0.889, and accuracies of 0.970 and 0.914, respectively. Our results indicate that the 3 methods are sensitive and specific for diagnosis of the APV infection.

In this study, we assessed the pathogenic potential of Escherichia coli associated with a commercial competitive exclusion (CE) product by examining the phenotypic characteristics associated with E. coli virulent for humans and domestic animals. Most E. coli isolates were capable of proliferating in iron-deplete chicken sera. Interestingly, none of the E. coli isolates from the commercial CE product contained the bacterial adhesin Tsh characteristic of avian pathogenic E. coli associated with airsacculitis and colisepticemia. In terms of virulence potential for humans, most E. coli isolates (78%) were sensitive to killing by 12.5% human sera. Because of their sensitivity to human sera, the E. coli in the CE product are not likely to cause a serious systemic infection in humans and, therefore, do not present a risk of causing septicemia in humans. Because these isolates also lack the gene tsh, they are also less likely to cause systemic disease or airsacculitis in poultry than pathogenic strains commonly isolated from diseased birds.

Serologic testing by the serum plate agglutination (SPA) procedure was performed to detect the presence of cross-reacting antibodies to Mycoplasma meleagridis, Mycoplasma synoviae, and Mycoplasma gallisepticum in lesser prairie-chickens (Tympanuchus pallidicinctus) trapped over a 2-yr period in Finney and Kearny counties of southwestern Kansas. Sera examined from birds (n = 50) obtained in March–April 2000 tested positive for M. meleagridis, M. synoviae, and M. gallisepticum at levels of 6%, 10%, and 10%, respectively, for the population examined. Mycoplasma meleagridis antibodies were detected in 3 samples (2.7%), M. synoviae antibodies in 2 samples (1.7%), and M. gallisepticum antibodies in 3 samples (2.7%) from birds (n = 112) collected in March–April 2001. Data obtained by SPA can result in false positives and should be verified by additional procedures such as the hemagglutination-inhibition test. Low amounts of sera prohibited this additional testing. Thus, the positive SPA results should be considered presumptive for the presence of Mycoplasma antibodies. Although Mycoplasma antibodies have been detected in wild turkeys (Meleagris gallopavo) from Kingman and Butler counties in Kansas, this report is the first of possible mycoplasmosis in Finney and Kearny counties, Kansas. All birds testing positive by this procedure should be considered as potential carriers of Mycoplasma and should not be used in relocation efforts.

In this study, we determined the occurrence of the tsh gene among 305 Escherichia coli isolates from chickens by means of the polymerase chain reaction and agglutination of chicken erythrocytes; 200 of those isolates were obtained from chickens with colisepticemia, 52 isolates were from lesions of cellulitis, and 53 were from feces of normal chickens. The tsh gene was found in 79 (39.5%) isolates from colisepticemia, in 10 (19%) cellulitis-derived E. coli isolates, and in two (3.8%) fecal isolates. Among the tsh strains, 68 (86%) isolates from colisepticemia and nine (90%) from cellulitis agglutinated chicken erythrocytes in the presence of mannose, after growing the strains on colonization factor antigen agar plates at 26 C, which confirms a correlation between mannose-resistant hemagglutination and expression of hemagglutinin Tsh. These results show, for the first time, the presence of the gene tsh in cellulitis-derived E. coli isolates; the high frequency of this gene among avian pathogenic E. coli isolates in Brazil indicates that its putative role as a virulence factor should be studied more thoroughly.

Pooled semen samples from 12 groups of mature commercial broiler breeder roosters were analyzed for the presence of Campylobacter. Each of the 12 groups was comprised of eight individuals and was sampled weekly for five consecutive weeks. Once a day, roosters were allowed to have a restricted amount of feed after the semen samples were collected by abdominal massage. This feeding schedule reduced the amount of fecal contamination in and around the vent as well as in the semen sample. For replications 1, 2, and 3, the numbers of Campylobacter-positive groups were 8, 5, and 5, respectively, out of 12. For replications 4 and 5, 6 of 8 and 6 of 11 groups were positive, respectively. Only two groups were positive for Campylobacter at all sampling times, two groups were negative each time, and eight groups produced variable results. Also, fecal droppings, external swabs of the genitalia, and semen samples were taken from individual roosters between 49 to 65 wk of age. Of the total 275 semen samples collected, 9.47% contained naturally occurring Campylobacter, whereas 9.6% of 114 fecal droppings and 7.9% of the 114 genital swabs were positive. Levels of the organism present in the fecal samples ranged from 1.0 to 4.2 log colony-forming units (CFU)/g with an average of 2.9 log CFU/g feces. For semen, the levels ranged from as low as enrichment recovery only to as high as 3.1 log CFU/ml of semen with an average of 1.2 log CFU/ml. For swabs of genitalia, the levels of Campylobacter were so low that recovery was achieved only through enrichment. These data suggest that rooster semen may serve as a vehicle for transmission of Campylobacter to the reproductive tract of the hen and subsequently to the fertile egg.

DNA-DNA hybridization, cultured cell lines, and transmission electron microscopy were used to study pathogenicity traits of 64 Escherichia coli isolated from apparently healthy chickens from 18 small-scale farms in Thika District, Kenya. A total of 39 (60.9%) isolates hybridized with the eae gene probe for enteropathogenic E. coli (EPEC) whereas another 16 (25%) hybridized with the lt and st gene probes and were categorized as enterotoxigenic E. coli. Electron microscopic examination of the eae probe-positive E. coli cultures with the HT-2919A cell line confirmed that they were able to attach intimately and produced effacement typical of EPEC. In addition, negative stain electron microscopy showed that the EPEC strains produced pili that have previously been associated with increased virulence of E. coli infections in chickens. This study has also demonstrated that apparently healthy chickens may carry enteropathogenic E. coli strains.

Earlier findings from our laboratory based on analysis of nucleotide and predicted amino acid sequence identities of 15 avian pneumoviruses (APVs) isolated from the United States (subgroup C) demonstrated that the viruses were phylogenetically separated from the European subgroup A and subgroup B viruses. Here, we investigated whether viruses from the three subgroups were cross-reactive by testing field sera positive for each of the APV subgroups in an enzyme-linked immunosorbent assay (ELISA) test with recombinant matrix (M) and nucleoprotein (N) proteins generated from a Minnesota APV isolate (APV/MN2A). Sera from turkeys infected with APV subgroup A, B, or C reacted with recombinant M protein derived from APV/MN2A. In contrast, recombinant N protein from APV/MN2A virus was reactive with sera from subtypes A and C viruses but not from subtype B virus. The results illustrate that viruses from the three APV subtypes share antigenic homology, and the M protein—based ELISA is adequate for monitoring APV outbreaks but not for distinguishing between different subtypes.

A 10-page questionnaire on biosecurity practices was mailed to 187 growers on the Delmarva Peninsula in October 2000. The growers were selected by three broiler integrators on the basis of flock performance and were classified as cases (bottom 10% performers) and controls (top 10% performers). After two mailings, 71 growers (38%) responded to the survey, of which 47 (66.2%) represented good performers and 24 (33.8%) represented poor performers. Univariate and multivariate statistical analyses revealed that increased frequency of sanitizing water lines was statistically significantly associated with good flock performance (P = 0.0449). The presence of wild birds inside poultry houses during grow-out showed a slightly statistically significant association with poor flock performance (P = 0.0633).

Several cases of elevated mortality with neurologic signs in 14-to-16-day-old broilers were presented to the Poultry Diagnostic and Research Center from one local integrated company. Suspected of “spiking mortality” associated with hypoglycemia, blood glucose levels were <150 mg/dl overall, with several birds with blood glucose levels as low as 30 mg/dl. Tissues, submitted for histopathology, revealed rickets in 50% of the birds. Virus isolation and serology for reovirus and infectious bursal disease virus were negative. After evaluation of these cases, a brief investigation was conducted to determine the effects of feed restriction on the induction of rickets and resulting hypoglycemia. One-hundred 1-day-old broilers were randomly assigned to three treatment groups of: 1) ad libitum feed, 2) 25% restriction, and 3) 50% restriction. Restriction began on the fifth day of age and continued to 21 days of age. Samples collected during the course of the study included whole blood for blood glucose measurements and proximal tibiotarsus for histopathologic examination for rickets. We were unable to reproduce the clinical signs of spiking mortality, neurologic changes, or hypoglycemia experimentally. Histopathology of the growth plates of the proximal tibiotarsus did indicate mild changes consistent with rickets, but the changes were not significant between treatment groups.

We investigated reports of stunted growth and high mortality occurring among young poultry in 38 small privately owned flocks in Vermont and New Hampshire. Tests for infectious agents including avian influenza and Newcastle disease viruses were negative, as were toxicologic tests for mycotoxins, heavy metals, pesticides, and monensin in feed samples. Analysis of 6 samples of implicated feed revealed 330–870 ppm of sodium (median level 350 ppm), whereas the recommended minimum level for chick feed is 1200–2000 ppm. A case-control study included 38 affected and 23 unaffected flocks. Statistical analysis showed that affected flocks were 29 times more likely to have consumed a particular brand of poultry feed (odds ratio = 29.2, 95% confidence interval = 3.2–675.8) but did not show any association between clinical signs and hatchery, location of chick purchase, chick purchase date, or years of producer experience.

Commercial white leghorn egg layer flocks being used to produce fertile eggs for human vaccine production exhibited dramatically low peaks in egg production, two to four times higher than normal weekly mortality, and high numbers of cull, nonlaying birds after the onset of sexual maturity. These lower production characteristics could not be associated with management-related problems. Gross lesions of cull and fresh dead birds necropsied showed approximately 60% lacked ovarian activity and had lesions of a bacterial bursitis or synovitis, whereas the other 40% had tumors of the viscera but not of the bursa of Fabricius. Histologic examination of tumor-containing tissues showed lesions typical of myelocytomatosis. The diagnosis of myeloid leukosis was confirmed by the isolation of a recombinant avian leukosis virus (ALV) containing the LTR of subgroup J and the envelope of subgroup B ALV. A positive polymerase chain reaction with primers specific for the 3′ untranslated region LTR confirmed the presence of LTR of ALV-J. The source of infection with this recombinant ALV was not determined; however, it is likely that commingling of the day-old egg-type chicks with ALV-J–infected meat-type chicks in a common hatchery had contributed to this outbreak.

Sixty-three Escherichia coli strains isolated from broilers with respiratory problems were examined for virulence factors, hemolysin synthesis ability, motility, hemagglutination capacity, operon pap presence, colicin production, and serum resistance. The capacity to hemagglutinate guinea pig erythrocytes was found in 53 (84.1%) of the samples, but only 30 (47.6%) agglutinated chicken erythrocytes. d-mannose-sensitive hemagglutination against guinea pig erythrocytes was found in 19 (30.2%) samples and against chicken erythrocytes, in 15 (23.8%) samples, whereas the d-mannose-resistant hemagglutination with guinea pig erythrocytes was found in 34 (54%) samples, and 13 of these (20.6%) showed this characteristic against chicken erythrocytes. Operon pap, P fimbria codifier, was detected in 26 samples in a total of 34 d-mannose-resistant samples. Colicin production was observed in 55 (87.3%) of the strains, and 41.8% presented V colicin production. Of the samples analyzed, 56 (88.9%) presented serum resistance, six (9.5%) were intermediate, and only one (1.6%) was sensitive to the action of the complement. The diversity of virulence profiles detected in the samples in this study explains in part the multifactorial characteristics of avian colibacillosis.

A 5-mo-old great rhea (Rhea americana) gradually became emaciated over a 1-wk period and died. Necropsy revealed several small yellow nodules in the lungs. Microscopically, the nodules consisted of granulomas containing numerous thin, 4-µm-diameter, septate, branching fungal hyphae. Aspergillus fumigatus grew readily on Sabouraud dextrose agar. This report appears to be the first of mycotic pneumonia in great rheas.

This study involved 14 ostriches of both sexes between 3 and 24 mo of age. Some hematologic and biochemical parameters were studied in animals with stomach impaction.

Clinical examination of the birds revealed anorexia, emaciation, decreased defecation, listlessness, separation from the flock, and recumbence. The total number of leukocytes (10.4 × 10³ mm³), the concentrations of serum glucose (166 mg/dl), and total protein (2.4 g/dl) decreased; serum creatine phosphokinase (1240 U/L) and alkaline phosphatase (598 U/L) increased; whereas no changes were found in the concentrations of calcium, phosphorus, magnesium, zinc, and copper.

Medical treatment was initiated in 13 animals; six of them recovered and seven birds died. Necropsy revealed edema, erosions, and hemorrhagic ulcers in the stomach of the dead birds. Foreign bodies such as sand; stone; pieces of wood, glass, and plastic; and metallic objects were encountered in the stomach at the necropsy. In one bird, metallic foreign body was diagnosed and the bird was referred to surgery.

Under stress factors, ostriches tend to eat foreign material; therefore, adequate housing, nutrition, and care are crucial in prevention of stomach impaction.