Coccidiosis is a ubiquitous intestinal protozoan infection of poultry seriously impairing the growth and feed utilization of infected animals. Conventional disease control strategies rely heavily on chemoprophylaxis, which is a tremendous cost to the industry. Existing vaccines consist of live virulent or attenuated Eimeria strains with limited scope of protection against an ever-evolving and widespread pathogen. The continual emergence of drug-resistant strains of Eimeria, coupled with the increasing regulations and bans on the use of anticoccidial drugs in commercial poultry production, urges the need for novel approaches and alternative control strategies. Because of the complexity of the host immunity and the parasite life cycle, a comprehensive understanding of host-parasite interactions and protective immune mechanisms becomes necessary for successful prevention and control practices. Recent progress in functional genomics technology would facilitate the identification and characterization of host genes involved in immune responses as well as parasite genes and proteins that elicit protective host responses. This study reviews recent coccidiosis research and provides information on host immunity, immunomodulation, and the latest advances in live and recombinant vaccine development against coccidiosis. Such information will help magnify our understanding of host-parasite biology and mucosal immunology, and we hope it will lead to comprehensive designs of nutritional interventions and vaccination strategies for coccidiosis.

In an experiment with broilers infected with a rifampicin-resistant Salmonella Typhimurium strain at 15 days of age, it was demonstrated that whole wheat feeding influenced the course of infection. The pH in the contents of the gizzard decreased as the amount of whole wheat in the diet increased over time. Following infection, lower numbers of Salmonella Typhimurium were found in the gizzard and ileum of birds receiving whole wheat compared to pellet-fed birds. However, there was no difference with respect to the numbers of Salmonella Typhimurium in the ceca and rectum between the two experimental groups. Quantitative cultivation of different groups of bacteria belonging to the natural intestinal microflora showed that whole wheat feeding significantly reduced the numbers of Clostridium perfringens in the intestinal tract of the birds. The present results indicate that the gizzard has an important function as a barrier organ, one that prevents pathogenic bacteria from entering the distal digestive tract.

In Denmark, increased problems associated with streptococci and enterococci have been observed in broilers and broiler parent flocks, resulting in increased mortalities, uneven flocks, and subsequent downgrading and increased condemnations. Postmortem lesions associated with recent outbreaks due to Enterococcus hirae have been accompanied or dominated by septicemia and endocarditis. As a result of infection at an early age and relatively low mortality rates, outbreaks are not always clearly defined and may go unnoticed or may be attributed to poor chick quality. For the same reasons, the pathogenesis and epidemiology of observed outbreaks has only remained speculative. Four separate outbreaks associated with E. hirae infections in broiler flocks occurring between 1998 and 2002 have been investigated. Two of the outbreaks indicated evidence of two separate clones, with 89% and 79% of isolates involved in the individual outbreaks belonging to a single pulsed-field gel electrophoresis (PFGE) profile, respectively. Another outbreak (outbreak 4) demonstrated clear clonality, with all isolates demonstrating affinity to one of two PFGE profiles that differed by only two bands. However, all three outbreaks demonstrated a different clone. The remaining outbreak was nonclonal, with isolates distributed between six separate PFGE profiles. One of the outbreaks (outbreak 4) was descended from a parent broiler flock previously associated with an outbreak of Streptococcus gallinaceus; the flock also exhibited septicemia and endocarditis. Initial indications suggested the possibility of vertical transmission of S. gallinaceus to the current broiler flock, causing infection. By extended phenotypic characterization and subsequent genetic characterization, including 16S rRNA sequencing, all strains from the four outbreaks were confirmed as E. hirae. This investigation highlights the problems associated with characterizing enterococci infections in broiler flocks.

We investigated the feasibility of using Flinders Technology Associates (FTA^®) filter cards for the storage of allantoic fluid containing an infectious bronchitis virus (IBV), such as Arkansas-DPI, Connecticut, and Massachusetts, and for their identification by reverse transcriptase (RT)-polymerase chain reaction (PCR) and characterization by restriction fragment length polymorphism (RFLP) or nucleotide sequencing. FTA^® paper is a cotton-based cellulose membrane containing lyophilized chemicals that lyses many types of bacteria and viruses. IBV was inactivated upon contact with the FTA^®, as shown by the inability of the virus to be propagated in embryonating chicken eggs. RT-PCR of the S1 gene showed that viral RNA in allantoic fluid remained stable after storage on FTA^® filter cards and that the stability was time and temperature sensitive for the large (1700 base pair [bp]) but not the small (383 bp) PCR products. Analysis of the amplified products showed that molecular characterization is feasible in allantoic fluid stored on FTA^® under nonfavorable environmental conditions (41 C) for at least 15 days. The use of FTA^® cards for the collection, transport, and storage of IBV–containing samples is safe, inexpensive, and adequate for molecular diagnosis. We propose that specimens coming from overseas on FTA^® cards would be first analyzed by RT-PCR with primers yielding a 1700-bp product followed by RFLP of the positive cases. Negative cases would be analyzed with primers yielding a 383-bp product (to exclude detrimental effect of the storage conditions) followed by nucleotide sequencing of the positive cases.

Three air contaminants that may have serious health consequences to humans and poultry are ammonia, dust, and aerosolized bacteria. This study measured ammonia concentrations, dust concentrations, and the presence or absence of aerosolized Salmonella spp. and Escherichia coli inside and outside five commercial layer facilities. The average outside ammonia concentration measurements decreased as the distance from the facility increased from 10 to 40 ft. The measurements at 10 ft from the facilities were consistently higher than the average concentrations inside the facilities. The ammonia measurement trends inside of the facilities were affected by the temperature-dependent ventilation systems. Average dust concentrations inside the five facilities were consistently below 2 mg/m³. Three facilities also experienced average outside dust concentrations at all measured distances below 2 mg/m³. Two facilities had relatively high average dust measurements at 10 ft from ventilation fans (32.12 mg/m³ and 75.18 mg/m³). Escherichia coli and Salmonella were isolated from the air inside all five facilities and outside the facilities up to 40 ft from the ventilation fans. In conclusion, dust concentrations may pose the largest risk to human and animal health at 10 ft away from the poultry facilities; risks associated with ammonia inhalation are greatest inside facilities during the coolest months of the year; and aerosolized bacteria are found inside and outside poultry facilities, but further work is needed to quantify the bacteria to further assess the health risk related to this issue.

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, and malabsorption syndrome. The P10 protein is a viroporin and induces cell fusion, whereas the biological function of P17 protein is completely unknown. In this study, the nucleotide sequences of the P10- and P17-encoding genes from 17 field isolates and vaccine strains of ARV isolated over a 23-year period from distinct geographic locations were analyzed to define phylogenetic profiles and to study sequence variability and genetic evolution. These genes displayed the signs of a high level of sequence divergence and have evolved into five distinct lineages, respectively. The P17-encoding gene showed higher sequence divergence than that of P10-encoding gene. Our results indicated that synonymous substitutions predominate over nonsynonymous substitutions in both genes. Comparison of P10 and P17 gene phylograms with those of S-class genes revealed distinct evolutionary patterns, indicating that P10 and P17 evolve in an independent manner. Comparative sequence analysis also showed extensive sequence divergence between ARV and other orthoreoviruses. The phylogenetic analysis of P10- and P17-encoding genes revealed that diversity within both genes is neither dependent of viral serotypes nor correlated with the disease states caused by avian reovirus.

Amplified fragment length polymorphism (AFLP) was used to type 34 strains of Mycoplasma gallisepticum (MG) including vaccine strains ts-11, 6/85, and F. Using AFLP, a total of 10 groups, with 30 distinguishable AFLP typing profiles, were generated in the analysis. The AFLP method was able to identify and differentiate both MG field strains from recent outbreaks and those that were epidemiologically related. The AFLP procedure will provide assistance in identifying the sources of mycoplasma infections. Vaccine strains were also differentiated from other field strains, which will be useful in the evaluation of vaccination programs. The AFLP discrimination potential was compared to other molecular typing techniques such as gene-targeted typing by DNA sequence analysis of the MG cytadhesin-like protein encoding gene, mgc2, and random amplified polymorphic DNA assay on the same MG isolates. The three assays correlated well with one another, with AFLP analysis having a much higher discriminatory power and reproducibility.

The morphogenesis of duck enteritis virus (DEV) and distribution in vivo were observed by electron microscopy after ducks were infected experimentally with DEV virulent strain. The investigation showed that a few typical herpesvirus virions and nucleocapsids were first observed in the spleen, thymus, and bursa of Fabricius (BF), and many nucleocapsids, mature viruses, and viral inclusion bodies could be found in the nucleus and cytoplasm of infected liver, small intestine, spleen, thymus, and BF when the ducks died. Nucleocapsids assembled both in nucleus and cytoplasm and could be divided into four different types according to their structures. Typical herpesvirus, light particles (L-particles), and virions without tegument could be observed at the same time. With the replication, assembly, and maturation of the viruses, intracytoplasmic and intranuclear inclusion bodies, electron-density particles, microtubules, hollow tubes, and coated electron-density bodies were observed in infected cells.

Molecular changes of cell culture–adapted Newcastle disease virus (NDV) were studied by adapting a velogenic NDV isolated from commercial layer chicken-to-chicken embryo fibroblast (CEF) cells. The isolate was passaged 50 times in CEF cells. At every 10th passage the virus was characterized conventionally by mean death time analysis, intracerebral pathogenicity index, and virus titration. As the passage level increased, a gradual reduction in the virulence of the virus was observed. Molecular characterization of the virus included cloning and sequencing of a portion of the fusion gene (1349 bp) encompassing the fusion protein cleavage site (FPCS), which was previously amplified by reverse transcription–polymerase chain reaction. Sequence analysis revealed a total of 134 nucleotide substitutions, which resulted in the change of 41 amino acids between the parent and the 50th passage virus. Pathogenicity studies conducted in 20-wk-old seronegative chickens revealed gross and histopathologic changes in the chickens injected with the parent virus and absence of the lesions in chickens injected with the adapted virus. The 50th passage cell culture virus was back-passaged five times in susceptible chickens and was subjected to virulence attribute analysis and sequence analysis of the FPCS region, with minor differences between them.

Based on the hypothesis that bacteria with minimal embryo lethality might be good candidates for vertical transmission, 103 lactose-positive Escherichia coli isolates were collected from different broiler-related conditions (sources) and analyzed using a variety of in vitro assays: biochemical profiles, sensitivity to antimicrobials, and the presence of plasmids in the 2000- to 16,000-base pair range. The results of these assays were analyzed to determine if they were associated with, or could be used as predictors of, the degree of lethality these isolates produced in 12-day-old embryos. In addition, the in vitro assay results were analyzed to determine if there was any correlation between any particular pair of factors. On the basis of biochemical profiles, the isolates were classified into 17 different groups; however, only a limited number of biochemical reactions separated a majority of the isolates. The isolates varied considerably in the number and size of plasmids they contained and in their sensitivity to the antimicrobials evaluated. The isolates also varied in their ability to kill chicken embryos—killing from 0% to 100% of those inoculated—yet significant differences were detected in lethality based on source and biochemical profile of the isolate. In addition, a predictive model for embryo lethality was constructed and evaluated based on three characteristics of these 103 isolates, namely, their ability to ferment raffinose and sorbose and their sensitivity to gentamicin.

An experiment was conducted to investigate the effects of polysaccharide extracts (E) of two mushrooms, Lentinus edodes (LenE) and Tremella fuciformis (TreE), and an herb, Astragalus membranaceus (AstE), on the immune responses of chickens infected with Eimeria tenella. A total of 180 broiler chickens were assigned to nine groups: three groups were fed with each of the extracts (LenE, TreE, and AstE), three groups were fed with the extracts and immunized with live oocyst vaccine (LenE V, TreE V, and AstE V), a group was immunized with the vaccine only, and there were two controls (E. tenella–infected and noninfected groups). The oocyst vaccine was given at 4 days of age, and the extracts (1 g/kg of the diet) were supplemented from 8 to 14 days of age. At 18 days of age, all birds except those in the noninfected group were infected with 9 × 10⁴ sporulated oocysts. The results showed that at 7 days postinfection (p.i.), birds fed the extracts without vaccination had lower body weight (BW) gain than those given the vaccine only. However, the extracts in conjunction with the vaccine significantly enhanced BW gain of the infected chickens compared with the vaccine group. Of the three extracts, LenE and TreE showed a better growth-promoting effect. The extracts largely increased oocyst excretion of droppings during the primary response postvaccination. The cecal peak oocyst output and lesion scores measured at 7 days p.i. were higher in the groups fed the extracts than in the group immunized with the vaccine only, whereas those of the groups fed with the extracts and immunized with the vaccine were not significantly different from the vaccine group. Of the three extracts, both LenE- and AstE-fed groups showed lower cecal oocyst output. Thus, as compared with the extracts, the live, attenuated vaccine showed better results with significantly increased immune response in coccidial infected birds. The polysaccharide extracts may prove useful against avian coccidiosis, and, particularly when they are used in conjunction with vaccine, they have shown preliminary promise against the experimental coccidial infection.

The autoinducer-2 molecule, AI-2, is considered to be a universal signal for regulating a wide variety of physiological processes in bacteria by modulating gene expression. Studies were conducted to observe how Escherichia coli cells would respond to subtherapeutic tetracycline concentrations under continuous culture (chemostat) conditions, to observe AI-2 activity within the probiotic chicken microbial consortium (Preempt® CF3; MS Bioscience, Dundee, IL) under in vitro conditions simulating a chicken cecum, and to observe the AI-2 activity in vivo within a chicken cecum as a function of exposure to subtherapeutic levels of chlortetracycline, tylosin, and vancomycin. The AI-2 activity in the E. coli continuous culture showed a 20-fold increase over baseline conditions for up to 24 hours. When the E. coli culture was subsequently exposed to pulses of chlortetracycline additions at subtherapeutic concentrations (2 μg/ml), AI-2 activity increased with increasing levels of tetracycline additions. The probiotic Preempt CF3 culture, however, did not exhibit any AI-2 activity in Viande Levure (VL) medium in the presence or absence of subtherapeutic levels of tetracycline. In vivo studies in the cecum of poultry chicks demonstrate that though AI-2 activity increased initially in the presence of vancomycin, there was no significant increase in AI-2 activity in the presence of tetracycline or tylosin. These results indicate that detectable levels of AI-2 activity are not evident within the probiotic culture (CF3) or within the chicken cecum. Understanding the relationships between AI-2 activity and microbial consortia characteristics could provide clues regarding the vulnerability of poultry chicks to enteric bacterial pathogen colonization.

Two low-pathogenicity (LP) and two high-pathogenicity (HP) avian influenza (AI) viruses were inoculated into chickens by the intranasal route to determine the presence of the AI virus in breast and thigh meat as well as any potential role that meat could fill as a transmission vehicle. The LPAI viruses caused localized virus infections in respiratory and gastrointestinal (GI) tracts. Virus was not detected in blood, bone marrow, or breast and thigh meat, and feeding breast and thigh meat from virus-infected birds did not transmit the virus. In contrast to the two LPAI viruses, A/chicken/Pennsylvania/1370/1983 (H5N2) HPAI virus caused respiratory and GI tract infections with systemic spread, and virus was detected in blood, bone marrow, and breast and thigh meat. Feeding breast or thigh meat from HPAI (H5N2) virus-infected chickens to other chickens did not transmit the infection. However, A/chicken/Korea/ES/2003 (H5N1) HPAI virus produced high titers of virus in the breast meat, and feeding breast meat from these infected chickens to other chickens resulted in AI virus infection and death. Usage of either recombinant fowlpox vaccine with H5 AI gene insert or inactivated AI whole-virus vaccines prevented HPAI virus in breast meat. These data indicate that the potential for LPAI virus appearing in meat of infected chickens is negligible, while the potential for having HPAI virus in meat from infected chickens is high, but proper usage of vaccines can prevent HPAI virus from being present in meat.

Poult enteritis complex (PEC) is an economically important disease of young turkeys characterized by diarrhea, poor weight gain, and, in some cases, high mortality. Although PEC is considered to be a polymicrobial disease, numerous viruses, including turkey coronavirus (TCV), turkey astrovirus type 2 (TAstV-2), and avian reoviruses (ARVs), have been associated with PEC-like disease. Real-time reverse transcription–polymerase chain reaction (RRT-PCR), a highly sensitive and specific detection method for viral RNA, was developed in a multiplex format for the simultaneous detection of TAstV-2 and TCV and for the detection of two genetic types of ARV. Assay sensitivity was determined using in vitro transcribed RNA and varied by target between 150 gene copies for TAstV-2 alone and 2200 gene copies for TCV when multiplexed. Virus detection was evaluated with samples collected from poults inoculated at 1 day of age with each of the viruses. Cloacal swabs and intestinal samples were obtained at 1, 2, 3, 4, 6, 9, 14, 17, and 21 days after inoculation, processed, and tested for virus detection by RRT-PCR. Cloacal swabs from TAstV-2– and TCV-infected poults were shown to have sensitivity for virus detection similar to that of intestinal samples when compared directly. ARV detection by RRT-PCR was compared with virus isolation and had similar sensitivity.

Pigeon paramyxovirus-1 (PPMV-1) was isolated from pigeons from east-central Alabama and used in association with chicken anemia virus (CAV), infectious bursal disease virus (IBDV), or finch Mycoplasma gallisepticum (MG) in specific-pathogen-free chickens to assess clinical disease and pathology. PPMV-1 infection in all groups was conducted at day 10 of age via the ocular route. The low passage PPMV-1 isolate was inoculated into chickens in different groups at 10 days post–CAV infection, 6 days post–IBDV infection, and 6 days post–finch MG infection, respectively. Additionally, to obtain information on the status of paramyxovirus infection in the wild bird population of the region, we used a multispecies competitive enzyme-linked immunosorbent assay kit to assess serum samples from 180 wild birds representing 24 species obtained throughout 2001. Mild respiratory signs characterized by sneezing were observed in PPMV-1–infected chicks. In the brain, PPMV-1 caused disseminated vasculitis in the neuropile and meninges, sometimes with small foci of gliosis. Most brains had only mild lesions. In the upper respiratory tract, lesions were confined to the larynx and proximal trachea as hyperplasia of laryngeal mucosa-associated lymphoid tissue. In the lung, PPMV-1 caused minimal to moderate multifocal interstitial pneumonia. Lymphocytic expansion occurred in the interstitium of the Harderian gland. PPMV-1 in the spleen caused expansion of the white pulp as a result of hypertrophy of the macrophages in the periarteriolar sheaths accompanied by lymphocytic hyperplasia at the periphery. No severe aggravation of either signs or lesions could be attributed to any of the avian pathogens used in association with PPMV-1. The serologic survey in wild birds showed antibody levels that were considered negative or doubtful. Interestingly, significantly (P < 0.05) higher mean titers were observed during the months of October and November 2001, following closely multiple PPMV-1 episodes of mortality in wild collard doves in northwestern Florida.

Infectious bursal disease virus is a contagious, immunosuppressive disease of young chickens that is controlled by vaccination. Cross-protection occurs between different strains of the virus as a result of shared neutralizing epitopes. However, interactions between two antigenically similar strains (a mild and a pathogenic) coinfecting the same host have not been investigated. Groups of specific-pathogen-free chickens were inoculated with a mild strain followed by a pathogenic strain at 0, 16, 24, or 48 hr postinoculation (PI) with a mild strain. Virus persistence and the predominant strain of the virus were determined by reverse transcriptase–polymerase chain reaction and restriction fragment length polymorphism analysis, respectively, in bursas at 2, 4, 8, 14, and 21 days PI with the pathogenic strain. Severity of infection was assessed by the bursa/body weight ratios and histopathologic lesion scores. The mild virus interfered with replication of the pathogenic virus. The greatest interference was observed when the pathogenic strain was inoculated 24 hr PI with the mild strain. The interference phenomenon observed might be due to competition for host receptor sites or production of cytokine(s). This interference phenomenon could have practical implications for vaccine usage and protection.

Riemerella anatipestifer is a gram-negative bacteria that can cause disease in a wide variety of wild and domesticated birds, especially waterfowl. The infection can be peracute, acute, or chronic. Although various routes of transmission have been proposed, to date, there is little information on the effects of route of transmission and challenge dosage on R. anatipestifer infection. Hence, the objective of this study was to determine the effect of route of inoculation and challenge dosage on R. anatipestifer infection and pathology. To achieve this objective, one hundred forty-seven 14-day-old white Pekin ducks (Anas platyrhynchos) were equally divided into 13 experimental groups (12 challenge and 1 control group). Each challenge group had 11 ducks. The control group had 15 ducks. Four routes of inoculation were evaluated (intranasal, oral, subcutaneous, and intravenous). Three dosage levels were evaluated for each inoculation route (10², 10⁴, and 10⁶ colony forming units [CFU]/ml). At the 10⁶ CFU/ml dosage level, mortality was most associated with the subcutaneous (91%) and intravenous (82%) routes, followed by the nasal (18%) and oral (9%) routes. A unique pathologic lesion was found in the bursa of Fabricius and spleen of affected birds. Within the spleen and bursa of Fabricius, there were varying degrees of lymphoid depletion and necrosis within the cortical and medullary regions. These pathologic lesions have not been previously reported in ducks with R. anatipestifer infection.

Strains of the bacterium Ornithobacterium rhinotracheale (ORT), a causal agent of respiratory diseases in birds, were microbiologically isolated, identified, and molecularly characterized. Blood-enriched culture media and biochemistry tests were used for microbiologic identification. Polymerase chain reaction (PCR) and repetitive extragenic palindromic PCR (rep-PCR) techniques were used for molecular identification and characterization, respectively, of the microorganism. ORT strains were isolated in enriched media from the trachea and air sacs of broilers, breeders, and layers from several geographic zones of Peru. Of the original 75 strains isolated from 75 clinical samples from which ORT was recovered during 1998–2000, 25 were selected for further study based on ORT as the primary pathogenic isolate (no other pathogens were detected). Selected isolates were molecularly identified and characterized by PCR using specific primers designed from the conserved zones of the 16S ribosomal genes. Primers used for the identification of ORT produced a specific fragment of 784 base pair (bp), which did not appear in Haemophilus paragallinarum or Pasteurella multocida, microorganisms with similar morphologic and biochemical characteristics that produce clinical signs identical to those of ORT. All 25 strains of ORT tested with rep-PCR had a genetic profile similar to that of ORT American Type Culture Collection 51463, indicating the presence of only one genotype in the ORT strains studied.

A cloned Eimeria acervulina gene (3-1E) was used to vaccinate chickens in ovo against coccidiosis, both alone and in combination with genes encoding interleukin (IL)-1, IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or interferon (IFN)-γ. Vaccination efficacy was assessed by increased serum anti–3-1E antibody titers, reduced fecal oocyst shedding, and enhanced body weight gain following experimental infection with E. acervulina. When used alone, anti–3-1E antibody titers were transiently, but reproducibly, increased at 2 wk and 3 wk posthatching in a dose-dependent manner. Similarly, significantly reduced oocyst shedding and increased weight gain were observed at relatively high-dose 3-1E vaccinations (≥25 μg/egg). Combined immunization with the 3-1E and IL-1, IL-2, IL-15, or IFN-γ genes induced higher serum antibody responses compared with immunization with 3-1E alone. Following parasite infection, chickens hatched from embryos given the 3-1E gene plus the IL-2 or IL-15 genes displayed significantly reduced oocyst shedding compared with those given 3-1E alone, while 3-1E plus IL-15 or IFN-γ significantly increased weight gain compared with administration of 3-1E alone. Taken together, these results indicate that in ovo immunization with a recombinant Eimeria gene in conjunction with cytokine adjuvants stimulates protective intestinal immunity against coccidiosis.

Salmonella-specific bacteriophages (BP) and competitive exclusion (CE) were used to reduce Salmonella colonization in experimentally infected chickens. A “cocktail” of distinct phage (i.e., phage showing different host ranges and inducing different types of plaques on Salmonella Typhimurium [ST] cultures) was developed. The killing activity of the selected BPs on ST cultures differed significantly, as determined in in vitro killing assays. BPs were administered orally to the chickens several days prior and after ST challenge but not simultaneously. BPs were readily isolated from the feces of the BP-treated chickens approximately 48 hr after administration. A CE product consisting of a defined culture of seven different microbial species was used either alone or in combination with BP treatment. CE was administered orally at hatch. Salmonella counts in intestine, ceca, and a pool of liver/spleen were evaluated in Salmonella-challenged chickens treated with BP or with BP and CE. In both trials 1 and 2, a beneficial effect of the phage treatment on weight gain performance was evident. A reduction in Salmonella counts was detected in cecum and ileum of BP-, CE-, and BP CE-treated chickens as compared with nontreated birds. In trial 1, BP treatment reduced ST counts to marginal levels in the ileum and reduced counts sixfold in the ceca. A reduction of Salmonella counts with BP, CE, and BP CE treatments was evident in chickens from trial 2. Both CE and BP treatments showed differences in the reduction of Salmonella counts after challenge between specimens obtained at days 4 and 14 postchallenge in ceca, liver/spleen, and ileum. The preliminary data presented in this report show that isolation and characterization of Salmonella-specific BP is uncomplicated and feasible on a larger scale. Results indicate a protective effect of both Salmonella-specific BPs and a defined competitive exclusion product against Salmonella colonization of experimentally infected chickens. These results are encouraging for further work on the use of BP as an effective alternative to antibiotics to reduce Salmonella infections in poultry.

Four generic Mycoplasma gallisepticum (MG) polymerase chain reactions (PCRs) (16S rRNA PCR, three newly developed PCR methods that target surface protein genes [mgc2, LP (nested) and gapA (nested)]) were compared for analytical specificity and sensitivity and for diagnostic sensitivity (Se) and specificity of detection from tracheal swabs. The licensed MG DNA Test Kit Flock Chek test (IDEXX, Laboratories, Inc., Westbrook, ME) was as well evaluated for the diagnostic specificity and sensitivity of detection from tracheal swabs. Analytical specificity was evaluated for the four generic PCR methods using a panel of DNA samples from microorganisms that may be isolated from the trachea of commercial poultry and other fowl. PCR methods mgc2, nLP, and ngapA only amplified DNA from MG, whereas 16S rRNA PCR amplified DNA from MG and Mycoplasma imitans. The analytical sensitivity of the four generic PCR methods expressed in color-changing units (CCU)/amplification reaction was estimated for each PCR method and ranged from 4 to 400 CCU/reaction; the sensitivities of single PCR methods 16S rRNA and mgc2 were estimated at 40 CCU/reaction, the nLP at 400 CCU/reaction, and the ngapA at 4 CCU/reaction. The diagnostic sensitivity and specificity of MG detection from tracheal swab pools, as compared to isolation from choanal cleft swabs, was evaluated for the five PCR methods using three groups of birds exposed to vaccine strains ts-11 and 6/85 and to challenge strain R. All PCR methods were able to detect the vaccine strains and the challenge strain R directly from tracheal swabs, indicating that PCR primers from the different methods amplified divergent MG strains. Isolation and PCR results correlated satisfactorily among the three experimentally infected groups, with agreement values (k) ranging from 0.52 to 1.00. The ngapA, IDEXX, and mgc2 PCRs showed the best sensitivity (Se) ratios for detection of M. gallisepticum strains as compared to isolation. Compared to the ngapA and IDEXX PCR methods, the mgc2 PCR has a faster turnaround time, since this test consists of a single amplification reaction and the amplification product is detected by gel electrophoresis. Therefore, among the PCR methods evaluated in this study, the mgc2 PCR is the method of choice to further validate in the field.

To determine the Mycoplasma gallisepticum (MG) rapid serum plate agglutination (RSPA) test response of broiler breeders after ts-11 strain vaccination, 55 Cobb pullets derived from a nonvaccinated, MG-negative, commercial, broiler breeder grandparent flock were monitored from 8 to 20 wk of age (over a 12-wk trial period). To evaluate the effect of lateral spread of the ts-11 vaccine strain on RSPA test results from commingled and adjacently penned birds, treatment groups included (A) birds vaccinated with ts-11strain MG at 8 wk of age, (B) commingled nonvaccinates in the same pen as the vaccinated birds, (C) nonvaccinates in a second pen separated from the first pen by a distance of 2 m, and (D) birds vaccinated with ts-11 strain MG at 8 wk of age and kept in a separate room. Rapid serum plate agglutination tests were performed once a week for 6 wk and then every 2 wk for 6 more wk, postvaccination. A polymerase chain reaction (PCR) assay specific for ts-11 strain MG was used to confirm vaccination, and a second PCR specific for non-ts-11 strain MG was used to confirm the absence of field infection. Seroconversion was first detected by the RSPA test 2 wk postvaccination and attained maximum positive rates of 58% at 12 wk postvaccination in treatment A and 60% at 8 wk postvaccination in treatment D. Seroconversion rates in nonvaccinated, commingled pullets was 10% at 5 wk and 30% at 12 wk after the vaccination of pen mates. The ts-11-specific PCR detected the vaccine strain in 80%–100% of the vaccinated birds 2 wk after vaccination. One of 15 nonvaccinated birds penned 2 m from vaccinated birds yielded ts-11 by PCR assay 12 wk after vaccination, which indicates that the spread of ts-11 over short distances may be possible in situations in which there is a common caretaker. PCR on tracheal swabs taken 12 wk postvaccination detected ts-11 in 50% and 60% of the vaccinated birds in treatments A and D, respectively; in 30% of the commingled nonvaccinates; and in 6.6% of the separately penned nonvaccinates. In contrast, choanal swabs collected from vaccinated birds at 12 wk were 21% and 40% PCR positive for ts-11 strain MG, while those from nonvaccinates were negative. All samples were PCR negative for field strain MG. The pattern of seroconversion as measured by RSPA test in small groups of broiler breeders was different from that previously reported for leghorns. Lateral spread of the ts-11 strain to commingled pen mates occurred rapidly, causing RSPA seroconversion patterns that mimicked those of the vaccinated pen mates.

Trophozoites and cysts of Spironucleus (Hexamita) meleagridis were detected in the intestinal fluid and mucus of pheasant poults with spironucleosis (hexamitiasis, hexamitosis) following staining with Heidenhain iron hematoxylin (HIH) and the Romanowsky-type stain Hemacolor®. Their morphology was consistent with that of flagellates of the genus Spironucleus, and bright-field morphologic observations were confirmed by transmission electron microscopy. Cysts occurred mostly within intestinal mucus, which was firmly compressed between microscope slides prior to staining. The internal structures of cysts were similar to those of trophozoites, allowing them to be confidently recognized. Hemacolor provided differential color staining of trophozoites and cysts, allowing accurate identification of S. meleagridis life cycle stages, even in smears in which there was heavy background staining. While HIH often produced clearer and more detailed staining of protozoan structures, in the context of a diagnostic laboratory its use was outweighed by the ease of use, rapidity of results, and differential color staining provided by Hemacolor. The possible significance of a resistant cystic stage in the life cycle of S. meleagridis is discussed.

The purpose of this study was to investigate whether mallard ducks (Anas platyrhynchos) are susceptible to infection with Mycobacterium bovis by either oral or intratracheal inoculation and to assess their potential role in the spread of bovine tuberculosis. Six ducks were orally inoculated with 1.0 × 10⁵ colony-forming units of M. bovis, six ducks were intratracheally inoculated with the same dose, and six ducks served as sham-inoculated controls. The study length was 90 days postinoculation, with samples of two birds from each group necropsied at 30-day intervals. Both fecal and tissue samples were collected for mycobacterial culture. None of the inoculated ducks shed M. bovis in their feces at any culture point (days 1, 30, and 60) during the study. No evidence of illness or weight loss was present during the course of the study, and only one duck had M. bovis isolated from any tissue, although there were no associated microscopic lesions. Mallard ducks were highly resistant to infection with M. bovis following high-dose inoculation and did not shed the organism in their feces. This study was conducted using high-dose inoculation; therefore, it appears that ducks are unlikely to play any significant role in the transmission of M. bovis between infected and uninfected mammalian hosts.

Vaccination of commercial layer chickens is labor intensive and often results in poor rates of seroconversion, which, in turn, generally correlate with decreased flock uniformity and performance. Attempts to improve the vaccination process include numerous variations of individual shop-built vaccinators in use by the layer sector of the poultry industry. Each of these vaccinators has limitations that contribute to poor vaccinations. Major problems include the nonuniform speed of the applicator system and pressure fluctuations at the spray nozzles, which contribute to sporadic dispersion of the vaccine as the vaccinator is pushed or carried past the cages. A battery-powered, self-propelled, constant-speed vaccinator was designed and constructed that operates with constant nozzle pressure. In field use, this vaccinator has resulted in both labor savings (reduction of manpower from five to one to vaccinate 75,000 chickens) and time savings (from 45 min to 7.5 min/poultry house) as well as improved vaccination results (higher positive seroconversion rates) against the poultry pathogen Mycoplasma gallisepticum (MG), a bacterium associated with losses of 15.7 eggs/hen over a 45-wk laying period in MG-infected layers as compared with layers maintained free from infection with MG.

Boyden-type chemotactic chambers were used to demonstrate that Mycoplasma gallisepticum (MG) was capable of migrating into chemotactic membranes. Scanning electron microscopy was used to confirm that MG could penetrate the membranes. To further demonstrate the invasive ability of MG, MG was deposited on the shell membranes of 9-day-old chicken embryos, and after 6 days of incubation, the presence of MG DNA in the allantoic fluids was detected by polymerase chain reactions. These results indicate that MG can penetrate cellular membrane, possibly by going through the porous cellular surface.

Day-old broiler chicks (n = 30) were obtained from a commercial hatchery and inoculated, either orally or intracloacally, with a characterized strain of Campylobacter jejuni. At 1 hr, 1 day, and 1 wk after inoculation, broilers (n = 5) from the orally and intracloacally inoculated groups along with control birds (n = 4) were humanely killed by cervical dislocation. The broilers from the control and treatment groups were aseptically opened, and the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca were aseptically removed and individually analyzed for C. jejuni. Overall, C. jejuni was isolated after oral inoculation from 13% (10/75), 17% (13/75), and 28% (14/50) of the 1-hr, 1-day, and 1-wk samples, respectively. Campylobacter jejuni was isolated from 10% (4/40), 8% (3/40), 10% (4/40), 25% (10/40), and 40% (16/40) of the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca samples, respectively. Following the intracloacal route of inoculation, C. jejuni was recovered from 32% (24/75), 8% (6/75), and 16% (8/50) of the 1-hr, 1-day, and 1-wk samples, respectively. Campylobacter jejuni was isolated from 5% (2/40), 5% (2/40), 5% (2/40), 45% (18/40), and 40% (16/40) of the thymus, spleen, liver/gallbladder, bursa of Fabricius, and ceca samples, respectively, for all sampling periods. Campylobacter spp. were not recovered from sample sites examined from the control broilers from trial one, trial two, or trial three samples examined after 1 hr and 1 day. However, one control sample was positive from the 1-wk sampling from repetition three; therefore, those data were omitted. The rapid movement of Campylobacter to internal organs following both oral and intracloacal inoculation may be significant, particularly if it persists in these organs as reservoirs throughout the 65-wk life cycle of breeding birds.

Hammerhead ribozymes are catalytic RNA molecules that specifically cleave a target RNA molecule. Herein, we report the design, synthesis, and in vitro analysis of a hammerhead ribozyme targeted to the infectious bronchitis virus (IBV) nucleocapsid mRNA. At a concentration of 0.5 or 10 μM, the ribozyme, designated IBV-N-Rz, effectively cleaved target RNAs in trans (37 C, 10 mM MgCl₂, 50 mM Tris). Cleavage products were visualized by agarose gel analysis. The time course of the ribozyme reaction was monitored by agarose gel analysis and relative quantitative reverse transcription–polymerase chain reaction. The amount of target RNA continually declined over a 5-hr period, indicating that the ribozyme was truly catalytic. Although stability and delivery problems must be overcome, a hammerhead ribozyme targeted to the IBV nucleocapsid mRNA most likely has antiviral activity and may be an effective therapeutic/prophylactic reagent in the future.

A liver from a hunter-harvested wild turkey (Meleagris gallopavo) from Kansas was evaluated by the Southeastern Cooperative Wildlife Disease Study. Grossly, the liver contained several grayish-white masses ranging from 0.5–3 cm in diameter. These masses were scattered throughout the parenchyma and bulged from the capsular surface. Histologic examination revealed multifocal to coalescing granulomas with low numbers of acid-fast bacilli within multinucleated giant cells at the periphery of the granulomas. Culture of the liver yielded Mycobacterium avium subspecies avium and low numbers of Staphylococcus intermedius.

Two adult ostriches developed anorexia, prostration, and severe hemorrhagic diarrhea, dying 24 hr after the onset of clinical signs. On postmortem examination, the cecal mucosa showed locally extensive areas of hemorrhages and fibrino-necrotic typhlitis with a white-yellowish material covering the mucosal surface. Multiple serosal petequial hemorrhages and fibrinous peritonitis were present. Histologic examination revealed an intense mononuclear infiltration in the lamina propria and submucosa of the cecum and extensive superficial necrosis associated with fibrin and serocellular deposits. Several gram-negative bacterial colonies were observed within the necrotic areas. Samples from intestinal lesions were collected, and pure growth of Escherichia fergusonii was obtained. Escherichia fergusonii is a member of Enterobacteriaceae, closely related to Escherichia coli and Shigella sp., established as a new species of the genus Escherichia in 1985. In veterinary medicine, E. fergusonii has been reported in calves and sheep from clinical cases suggestive of salmonellosis. To our knowledge, this report represents the first description of E. fergusonii associated with enteritis in ostrich.

We report the isolation and characterization of a paramyxovirus from geese in South China during 1997. The isolate, designated as goose paramyxovirus/QingYuan 1997-1 (GPMV/QY97-1), showed pathogenicity to geese and could agglutinate chicken erythrocytes. Its hemagglutinating activity was inhibited by antiavian paramyxovirus serotype 1 (APMV-1) serum. The F gene of isolate was amplified by reverse transcription polymerase chain reaction, and sequence analysis proved that its sequence conformed to that reported in the literature, encoding an F₀ protein of 553 amino acids with 13 cysteine residues and 6 potential glycosylation sites. It also contained multiple basic amino acids at the deduced cleavage site of the fusion protein, which is a typical feature of highly virulent APMV-1 strains. Sequences analysis of the F gene of GPMV/QY97-1 revealed a homology with other APMV-1 isolates, with its identity ranging from 84.1% to 99.9% on a nucleotide basis and from 88.8% to 99.6% on an amino acid basis. Phylogenetic analysis of the APMV-1 isolates showed that this isolate most closely resembled the reference APMV-1 strain GD/1/98/Go, which was originally isolated from geese in 1998.