Of more than 2500 identified Salmonella serotypes, only a small proportion are common in poultry flocks. However, there is an epidemiologically important connection between poultry products and human infections because many of the serotypes that are most prevalent in humans (such as Salmonella Typhimurium and Salmonella Enteritidis) are similarly common in poultry. The scope of food safety efforts for poultry products has been broadened in recent years to include more attention to animal production (or preharvest) issues. The goal of preharvest poultry food safety is to minimize opportunities for the introduction, persistence, and transmission of flock infections with Salmonella and other human pathogens. This objective can be pursued either by general strategies directed against all Salmonella serotypes (and in some instances against other pathogenic microorganisms as well) or by more specific strategies that are designed to act with precision against particular Salmonella serotypes with distinctive public health or economic significance. Risk assessment studies have recommended intervention at multiple steps in the farm-to-table continuum as the most productive overall approach. A comprehensive quality assurance strategy, encompassing both broadly based risk-reduction practices and targeted testing to detect pathogens of concern, has been associated with a lower incidence of Salmonella Enteritidis infections in both egg-laying flocks and humans in a number of countries. Although the emphasis in these types of programs is primarily on risk reduction, testing provides essential verification of the efficacy and cost-effectiveness of risk-reduction practices (and identifies flocks infected with uniquely problematic serotypes). Vaccination can enhance the short-term responsiveness of control programs to address problems involving specific serotypes of elevated significance.

Eleven strains of the avian pathogen Mycoplasma synoviae were evaluated for the presence of sialidase activity with the use of the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid and the sialidase inhibitor 2-deoxy-2,3-didehydro-N-acetylneuraminic acid. The kinetics of in vitro growth in modified Frey medium were also assessed for each strain. Five strains had been isolated from clinically symptomatic chickens, and strains WVU1853^T and K3344 have been demonstrated to be capable of reproducing disease in specific-pathogen-free chickens. All strains exhibited sialidase activity, although the amount varied 65-fold among strains (P < 0.0001) from 1.3 × 10⁻⁷ to 2.0 × 10⁻⁹ activity units per colony-forming unit. Strains originally isolated from clinically symptomatic birds had more (P < 0.05) sialidase activity than strains from asymptomatic birds. Strain WVU1853^T exhibited the most sialidase activity (P < 0.0001) and grew to the highest culture density (P < 0.0001) among strains, but across strains, the rank correlation of growth rate with sialidase activity was not significant. Negligible activity was detected in conditioned culture supernatant fluid. This is the first report of sialidase activity in pathogenic strains of M. synoviae, which suggests a potential enzymatic basis for virulence of the organism.

Necrotic enteritis is an enteric disease of avian species caused by the anaerobic bacterium Clostridium perfringens. The disease is regularly controlled in the broiler chicken industry with antimicrobials in feed but is reemerging in areas such as Europe where there is a ban on antimicrobials as growth promoters. To study prospective therapies, researchers must be able to reproduce this disease in a controlled environment, but this is not always possible because of differences in the pathogenicity of C. perfringens strains. Our objective was to test the potential of five isolates (SNECP43, 44, 47, 49, and 50), taken from field cases of necrotic enteritis, at recreating the disease in a controlled challenge experiment. SNECP43 and 50 were derived from a common clone, with SNECP50 passed in vivo and SNECP43 subcultured in vitro. Four hundred birds were divided into 16 pens, with three pens each receiving one of five treatments, with one control pen. Day-old birds were raised on a high wheat-based diet to promote necrotic enteritis development and were challenged with between 3.4 × 10⁹ and 3.2 × 10¹¹ colony-forming units (cfu) of C. perfringens in feed for a period of 24 hr starting on day 13 of the challenge experiment. Lesion scores were assessed on two birds per pen sacrificed on day 17 and on any dead birds during the 25-day study. Growth performance was assessed up to 25 days, and mortality recorded throughout. Only SNECP50 produced necrotic enteritis mortalities significantly different (P ≤ 0.05) from the control. The five isolates were also typed using pulsed-field gel electrophoresis to assess their genetic relatedness. All epidemiologically unrelated isolates were deemed genetically unrelated, whereas SNECP43 and 50 differed by only a single minor band. Toxin type was assessed using polymerase chain reaction (PCR), which was also used for the detection of the gene encoding the β2-toxin.

Five clinically normal chickens from three farms (farm A, farm B, and farm C), for a total of 15 clinically normal chickens, were examined bacteriologically. In a similar manner, five dead chickens with lesions of peritonitis from each of the same three commercial egg-laying operations were selected for bacterial culturing. Escherichia coli were isolated from the cloaca in 14 of 15 healthy chickens and from all 15 chickens with peritonitis. Oviducts of normal chickens did not contain E. coli (0/15) whereas oviducts from 13 of 15 hens with peritonitis were positive for this pathogen. No lesions and no E. coli (0/15) were found in the peritoneal cavity of healthy hens, but peritonitis lesions from 13 of 15 dead chickens yielded E. coli. On farm A and farm B, a flock consisted of all chickens within a single house and all chickens in each flock were of the same age and same genetic strain. In flock 1 from farm A, all five E. coli isolates from the oviduct and all five isolates from the peritoneal cavity were serogrouped as O78; contained the virulence genes iroN, sitA, iutA, tsh, and iss; and belonged to phylogenetic group A. In flock 2 from farm B, all four E. coli isolates from the oviduct and all four isolates from the peritoneal cavity were serogrouped as O111; contained virulence genes iroN, sitA, iutA, traT, iss, and ompT; and belonged to phylogenetic group D. These data suggest that all chickens with peritonitis in a single flock on farms A and B were likely infected by the same E. coli strain. Escherichia coli isolates from the magnum and peritoneum had the same serogroup, virulence genotype, and phylogenetic group, which is consistent with an ascending infection from the oviduct to the peritoneal cavity.

Twenty bursal samples were obtained from four infectious bursal disease virus (IBDV)–vaccinated layer flocks experiencing problems with immune suppression that was thought to be infectious bursal disease. All the samples were found to be positive for IBDV by reverse transcriptase polymerase chain reaction (RT-PCR). Restriction fragment length polymorphism analysis of the samples identified them as classic molecular group 3 and group 4 viruses. Two samples from each of the four flocks were sequenced, and within a flock, these sequences were identical; however, between flocks, some differences were observed. One virus from each of the four flocks was selected for further analysis. The VP2 hypervariable sequence region of samples GA-1, H-30, and CS-2-35 had nucleotide and amino acid similarities with the D78 and Vi Bursa G classic vaccines that were used in those flocks. The sequence of HPR-2 was similar to the Bursa Vac 4 vaccine used in that flock and the STC virulent classic IBDV strain. The deduced amino acid sequence of these isolates revealed that all the isolates had proline at position 222, which is characteristic of U.S. classic viruses. The phylogenetic analysis of these isolates on the basis of the VP2 hypervariable amino acid sequence clustered GA-1, H-30, and CS-2-35 with the D78 vaccine and HPR-2 with STC. The pathogenicity of these isolates was tested in specific-pathogen-free chickens. Bursa–body weight (B-BW) ratios and histopathologic lesion scores in the bursa were determined. Gross lesions were observed in the bursa, and the B-BW ratios of the birds infected with all four wild-type viruses were significantly different compared with the D78 vaccine and uninoculated control groups. Histopathology of the bursa from groups infected with GA-1, H-30, CS-2-35, and HPR-2 showed different degrees of follicular depletion and necrosis. A very slight lymphoid depletion was observed in the D78-infected group at 5 days postinoculation, and no microscopic lesions were observed in this group at 8 days postinoculation or at any time in the uninoculated control group. The bursa collected from the field virus and D78-infected birds at necropsy revealed the presence of IBDV via RT-PCR, and the VP-2 hypervariable nucleotide sequences of the GA-1, H-30, CS-2-35, HPR-2, and D78 samples were identical to the original viral isolates and vaccine, respectively.

The unique open reading frame 11 (LORF11) of Marek's disease virus (MDV) is present in all three serotypes of MDV and is located in the unique long region of the MDV genome. In the serotype 1 Md5 genome, LORF11 comprises 2711 nucleotides and encodes a predicted protein of 903 amino acids. In order to study the biological function of LORF11 we deleted it from the MDV cosmid A6 by using the RecA-assisted restriction endonuclease cleavage method. The recombinant cosmid, A6ΔLORF11, was transfected into duck embryo fibroblasts (DEF) in conjunction with parental SN5, P89, SN16, and B40 cosmid clones. Recombinant rMd5ΔLORF11 plaques were evident at 12–13 days after transfection. Polymerase chain reaction amplification of DEF cells infected with rMd5ΔLORF11 viruses confirmed the deletion of a 2.57-kb fragment resulting in a 296-bp fragment. Three rMd5ΔLORF11 mutants were generated and their biological functions were studied in vitro and in vivo. In vitro growth characteristics of rMd5ΔLORF11 viruses were similar to those of parental rMd5, indicating that LORF11 is not essential for replication in vitro. In vivo studies of rMd5ΔLORF11 mutants showed that they were impaired in viral replication in the lymphoid organs and had 100× lower viremia than chickens infected with the parental rMd5 virus. Furthermore, rMd5-infected chickens horizontally transmitted the virus to contact controls whereas no horizontal transmission occurred in rMd5ΔLORF11-infected chickens. Three independent deletion mutants were tested and showed the same phenotypes, so it is unlikely that the observed phenotype is because of any random mutation in the genome. Therefore the LORF11 gene of MDV is essential for normal virus replication in chickens and deletion of LORF11 renders an attenuated virus.

Various diagnostics techniques were compared for their ability to detect infectious laryngotracheitis (ILT) during an outbreak in chickens aged between 4 and 21 wk. Gross lesions ranged from excess mucus to accumulation of fibrinonecrotic exudate in the larynx and trachea. Syncytial cells with intranuclear inclusion bodies were found in sinus, conjunctiva, larynx, trachea, lung, and air sac. Virus isolation in chicken embryos was attempted in every case. Negative-stain electron microscopy detected herpesvirus in only 6% of the cases. Yet, isolation of ILT virus in the chorioallantoic membrane was presumed by histology in >20% of the samples and confirmed by fluorescent antibody (FA) in 35% of the embryos inoculated with conjunctivas or tracheas from affected birds. Overall, results from histology and FA tests were highly correlated. FA test has the advantage over histology of being diagnostically specific for ILT virus. Polymerase chain reaction was the most sensitive test and detected the viral DNA even in cases where histology and FA were negative. ILT virus DNA was quantified by real-time polymerase chain reaction (Re-Ti ILTV). Histologic and FA results from larynx and trachea were negative if the concentration of the viral DNA was ≤4 of log₁₀. A viral DNA concentration higher than log₁₀ 4, as determined by Re-Ti ILTV, was required for clinical ILT to be manifested.

During the period 2002–2005, 109 infectious bursal disease virus (IBDV) field strains were isolated from bird flocks located in various parts of Italy. Out of these strains, 91 were isolated from broilers, 12 from pullets, and six from backyard flocks. Forty-two IBDV strains were further investigated and characterized on the basis of the geographical origin, source, and clinical signs. Antigenic and genetic characterizations were carried out using a monoclonal antibody (MAb)–based antigen-capture (AC) enzyme-linked immunosorbent assay (ELISA) or a virus neutralization assay and a reverse transcription, amplification, and direct sequencing of a genome fragment encoding the VP2 variable domain. The viruses were compared with reference IBDV strains, F52/70 (classical, 1970), 89163 (typical very virulent [vv]IBDV, 1989), 91168 (antigenically modified vvIBDV, 1991) and 94432 (antigenically modified vvIBDV, 1994) among others. All 42 strains were genetically characterized, and the comparison of their nucleotide sequences revealed the presence of six clusters having 100% identity, named group 1, 2, 3, 4, 5, and 6. Twelve strains, representative of each molecular group and/or with interesting amino acid sequence, were also antigenically characterized. The antigenic characterization showed six strains—151573, 157185 (group1), 192294 (group 2), 77882 (group 3), 217 (group 4), and 192304—with the profile typical of vvIBDV (lack of binding of MAbs 3 and 4). Two strains, 77165 and 204875 (group 6), were also related to vvIBDV but did not react with MAb 5. Three isolates exhibited a profile of cell culture–adapted viruses and classical strains but with some differences: strain 157776 reacted with all MAbs; strain 168026 with all MAbs except MAb 4, which weakly neutralized it; and strain 72293 with all MAbs except MAb 9, which is rather unusual. The last strain, 213622, showed a very uncommon antigenic profile with missing or reduced binding of MAbs 3, 4, 5, 6, 8, and 9. Genetic characterization revealed 37 strains identified as vvIBDV viruses divided in 26 isolates (including groups 1, 2, 3, and 4) with the four amino acids residues typical of vvIBDV (222A, 256I, 294I, 299S) and 11 isolates (including groups 5, 6, and 213622) with some other amino acid exchanges. Four isolates (72293, 168026, 196783, and 222220) presented an amino acid sequence closely related to attenuated classical viruses whereas the last isolate (157776) exhibited a rather different sequence with some mutations typical of vvIBDV and others for cell culture–adapted viruses. Results of the antigenic and genetic characterization revealed that the majority of viruses (n  =  37) were related to vvIBDV strains but, among these, 11 strains presented antigenically and genetically modified characteristics and originated, in major part, from the area where viruses have been circulating for a long time. The remaining viruses (n  =  5) were related but not identical to attenuated classical viruses and came from areas where vaccination with intermediate strains is applied.

Highly pathogenic avian influenza (HPAI) H5N2 reemerged in ostriches in South Africa during 2006, and a low-pathogenic AI H5N2 virus was also isolated. Molecular and phylogenetic characterization was performed to determine whether the outbreak strains were genetically derived from the supposedly eradicated Eastern Cape ostrich outbreak HPAI H5N2 strain of 2004. It was demonstrated that although the 2004 and 2006 South African H5N2 strains shared a common ancestor, the two outbreaks were not related. Not only were extensive reassortments with wild bird viruses involved in the evolution of the 2006 strains, but the precursor HA molecule HA₀ cleavage site sequence of the 2006 HPAI H5N2 virus also contained fewer basic amino-acid insertions. Multiple transmission events occurred from wild birds to ostriches in 2006, and it appears that a reservoir of H5N2 with pathogenic potential for poultry is established in the South African wild duck population.

Three coated plant extracts (RepaXol® and two experimental formulations) were tested for their minimal lethal concentration for histomonads in vitro and the effect of those substances on the bacterial growth in the histomonadal culture. After 48 hr, RepaXol® and experimental formulation B were lethal to histomonads at a concentration of 1.25 µl/ml. Experimental formulation C was lethal at a concentration of 2.5 µl/ml. All products also decreased the growth of bacteria at concentrations inhibiting the growth of histomonads.

Data on the prevalence of antimicrobial resistant enterococci and staphylococci from the poultry production environment are sparse in the United States. This information is needed for science-based risk assessments of antimicrobial use in animal husbandry and potential public-health consequences. In this study, we assessed the susceptibility of staphylococci and enterococci isolated from poultry litter, recovered from 24 farms across Georgia, to several antimicrobials of veterinary and human health importance. Among the 90 Enterococcus isolates recovered, E. hirae (46%) was the most frequently encountered species, followed by E. faecium (27%), E. gallinarum (12%), and E. faecalis (10%). Antimicrobial resistance was most often observed to tetracycline (96%), followed by clindamycin (90%), quinupristin–dalfopristin (62%), penicillin (53%), erythromycin (50%), nitrofurantoin (49%), and clarithromycin (48%). Among the 110 staphylococci isolates recovered, only coagulase-negative staphylococci (CNS) were identified with the predominant Staphylococcus species being S. sciuri (38%), S. lentus (21%), S. xylosus (14%) and S. simulans (12%). Resistance was less-frequently observed among the Staphylococcus isolates for the majority of antimicrobials tested, as compared with Enterococcus isolates, and was primarily limited to clarithromycin (71%), erythromycin (71%), clindamycin (48%), and tetracycline (38%). Multidrug resistance (MDR) phenotypes were prevalent in both Enterococcus and Staphylococcus; however, Enterococcus exhibited a statistically significant difference in the median number of antimicrobials to which resistance was observed (median  =  5.0) compared with Staphylococcus species (median  =  3.0). Because resistance to several of these antimicrobials in gram-positive bacteria may be attributed to the shuttling of common drug-resistance genes, we also determined which common antimicrobial-resistance genes were present in both enterococci and staphylococci. The antimicrobial resistance genes vat(D) and erm(B) were present in enterococci, vgaB in staphylococci, and mobile genetic elements Tn916 and pheromone-inducible plasmids were only identified in enterococci. These data suggest that the disparity in antimicrobial-resistance phenotypes and genotypes between enterococci and staphylococci isolated from the same environment is, in part, because of barriers preventing exchange of mobile DNA elements.

The complete genomic sequence of very virulent infectious bursal disease virus (vvIBDV) Gx strain was determined, including the sequences of segment A, encoding the precursor polyprotein, and segment B, encoding the viral RNA polymerase (VP1) and 5′- and 3′-untranslating regions. Alignment of segment A of Gx with the sequences of 12 other vvIBDV strains showed 97.5% to 99.0% amino acid identity, whereas alignment of segment B of Gx with nine other vvIBDV strains revealed high sequence divergence, ranging from 10.3% to 11%. Phylogenetic analysis of segments A and B showed that they were in different branches, indicating that the reassortment occurred in this strain and that segment A and segment B derived from different pathotype strains. The mutant spectrum analysis of quasispecies virus demonstrated that the mean minimum mutation frequency in VP1 was 8.78-fold higher than in the polyprotein. The most frequent mutations were in the first 1986 nucleotides (nonsynonymous mutations) and the last 660 nucleotides (synonymous mutations), indicating that the 219 amino acid residues in the C-terminal of the VP1 form a functional region.

The pathogenesis of infection involving both infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) causes reproductive damage in hens after viral replication in the epithelium of the oviduct, resulting in loss of cilia and degeneration and necrosis of the epithelial and glandular cells. Although IBV has been indicated as a possible cause of the formation of calcium stones in the epididymus of roosters, a definitive association has not been confirmed. This report describes the detection of IBV and aMPV in the testes of roosters from a Brazilian poultry broiler breeder's flock with epididymal stones and low fertility. Samples of testis, trachea, and lungs from breeder males aged 57 wk were positive for IBV by reverse transcriptase–polymerase chain reaction (RT-PCR), and virus isolation and testis samples were also positive for aMPV by RT-PCR. The inoculation of testis samples into embryonated chicken eggs via the allantoic cavity resulted in curled, hemorrhagic, and stunted embryos typical of IBV infection. The allantoic fluid was positive by RT-PCR aimed to amplify the region coding for the S1 subunit of the IBV S gene, but it was not positive for aMPV. Sequence analysis of the amplified fragment revealed a close relationship with European IBV genotype D274, previously unreported in Brazil. These results indicate that IBV and perhaps aMPV are likely to have played a role in the pathogenesis of the testicular disease described and should be regarded as factors that can influence male fertility disease in chickens.

The aim of this study was to evaluate the replication of live attenuated infectious laryngotracheitis virus vaccines in selected tissues and their ability to transmit to contact-exposed birds. Four-week-old specific-pathogen-free chickens were eye drop–inoculated with tissue culture origin (TCO) and chicken embryo origin (CEO) vaccines. Contact-exposed chickens were housed in direct contact with eye drop–inoculated chickens from the first day postinoculation. Virus isolation and real-time polymerase chain reaction were used to detect the presence of live virus and viral DNA, respectively, in the trachea, trigeminal ganglia, eye conjunctiva, cecal tonsils, and cloaca from eye drop–inoculated and contact-exposed birds at days 2, 4, 5 to 10, 14, 18, 21, 24, and 28 postinoculation. No differences were observed in the ability of the TCO and CEO vaccines to replicate in the examined tissues. Both vaccines presented a localized replication in the eye conjunctiva and the trachea. Both vaccines were capable of transmitting to contact-exposed birds, attaining peaks of viral DNA as elevated as those observed in inoculated birds. The CEO vaccine replicated faster and reached higher viral genome copy number than the TCO vaccine in the conjunctiva and trachea of eye drop–inoculated and contact-exposed birds. The viral DNA from both vaccines migrated to the trigeminal ganglia during early stages of infection. Although the CEO and TCO vaccines were not recovered from the cecal tonsils and the cloaca, low levels of viral DNA were detected at these sites during the peak of viral replication in the upper respiratory tract.

Ten-week-old Hy-Line Commercial W-36 pullets were spray-vaccinated with MYCOVAC-L® at the manufacturer's recommended dosage (1×) or at 15 times that rate (15×). At 22 or 45 wk of age, subsets of 1×- and 15×-vaccinated pullets were challenged with the virulent Mycoplasma gallisepticum (MG) strain R_low. Percent hen-day egg production was determined through week 55. Analyses for treatment effects on overall (22–56 wk) percent hen-day egg production revealed no significant differences between nonchallenged 1× and nonchallenged 15× MYCOVAC-L® treatments. Among 1× MYCOVAC-L®–vaccinated groups, R_low challenge at 45 wk corresponded to significantly (P ≤ 0.01) lower overall egg production compared with the unchallenged 1×-vaccinated control (70.88% vs. 79.15%, respectively). Conversely, at the 15× MYCOVAC-L® dosage level, overall egg production was not significantly affected by virulent MG challenge at 45 wk compared with its unchallenged counterpart (84.09% vs. 81.03%, respectively) and could indicate increased protection from virulent MG challenge. Serologic monitoring indicated the virulent MG challenge was consistently (100%) associated with seroconversion. Comparisons among the nonchallenged experimental treatments indicated that vaccinations at the 15× MYCOVAC-L® dosage rate were associated with a greater seroconversion rate at weeks 21, 27, and 44, but not at week 50.

Growth rate in broiler birds has increased substantially in the last decade due to improvement in genetics, feed formulation, cleaner environment, and vaccine formulations. As a result, it has become necessary to review and revise prediction method for vaccination in chicks. This study was undertaken to determine the possible use of the rate of weight gain rather than age in predicting vaccination time. Two groups of 1-day-old broilers originating from old and young breeders, respectively, and with different levels of maternal antibodies against infectious bursal disease virus (IBDV) were used in this study. The chicks were divided into four groups and subjected to two feed regiments: groups A1 and B1 were fed broiler feed for normal growth rate, and groups A2 and B2 were fed breeder feed for slower growth rate. At 1, 4, 8, 12, 16, 22, 29, and 36 days of age, 22 chicks in each group were weighed, and blood samples were collected. Serum samples were tested for antibodies against IBDV by enzyme-linked immunosorbent assay (ELISA) and virus neutralization test. Maternal antibody decline curves for each group were plotted according to chick age and chick weight. Fast-growing birds in groups A1 and B1 showed a faster rate of antibody decline, whereas slow-growing birds in groups A2 and B2 had a slower rate of antibody decline. Based on the effect of weight gain on maternal antibody decline, a new way of predicting vaccination time for IBDV based on measuring maternal antibody titers at 4 days of age was proposed and tested. The predicted antibody decline was shown to correspond to the real ELISA titers measured in our experiments (R  =  0.9889), whereas a lower correlation (R  =  0.8355) was detected between real ELISA titers and the titers predicted by the current method using age-based Deventer formula.

The potential for infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) replication interference was evaluated using quantitative reverse transcription–polymerase chain reaction (qRT-PCR). Fourteen-day-old broiler chickens were inoculated via eyedrop with live commercial vaccine strains of IBV and NDV alone or in combination to directly evaluate IBV and NDV replication in the trachea at 1, 3, and 5 days after vaccination. Commercial NDV vaccine strains used were B1, VG/GA, and C2. The vaccine strains of IBV tested were Massachusetts (Mass) and Arkansas (Ark). The NDV Mass vaccines used were commercially manufactured combined products. The NDV Ark vaccines used were commercial vaccines manufactured as single entity products that were administered by eyedrop to opposite eyes of each chicken.

As measured by qRT-PCR, the replication of NDV strains B1, VG/GA, and C2 did not interfere with the growth of IBV Mass and Ark strain vaccines in the combined vaccine treatment groups. Combination vaccinations using B1 and VG/GA did not interfere with IBV immunity based on challenge or serum antibody production. In the C2 Mass vaccination trial, IBV immunity after challenge was reduced, but it did not seem to be a result of reduced Mass vaccine growth or the ability of the Mass vaccine to induce serum IBV antibody. In contrast, the replication of IBV strains Mass and Ark interfered with the growth of NDV strains B1, VG/GA, and C2 as measured by qRT-PCR. However, interference with NDV replication was not reflected in a reduction in Newcastle disease challenge of immunity findings when combination Mass NDV products manufactured by vaccine companies were tested. Moreover, NDV immunity was not compromised in two of three trials using single entity vaccines of NDV and Ark IBV vaccines manufactured separately but administered simultaneously. However, in one trial, NDV immunity was decreased where a NDV single entity product (C2) was given with an IBV single entity Ark vaccine. This finding emphasizes the importance of using manufactured combination vaccines whenever possible to avoid potential interference.

Several antigenic and pathogenic subtypes of infectious bursal disease virus (IBDV) have been identified worldwide. Simple and quick differential diagnostic assays are vital for implementing control and prevention strategies for infectious bursal disease (IBD). Currently reverse transcriptase–polymerase chain reaction/restriction fragment length polymorphism analysis (RT-PCR/RFLP) and real-time RT-PCR detection have been used. However, the RT-PCR/RFLP analysis is time consuming and not feasible for assaying large numbers of samples, and the real-time PCR is expensive. The reliable indicator for very virulent (vv) IBDV (vvIBDV) remains in vivo pathogenicity testing because of the lack of a virulence marker. In this study simple RT-PCR assays were developed for differentiating various types of IBDV. Two sets of primers specific for serotype 2 and vvIBDV were designed based on the sequences of segment A and B of IBDVs. Initially a total of 25 previously characterized virus strains including 11 serotype 1 classics, 4 serotype 1 variants, and 5 serotype 1 vv and 5 serotype 2 strains were used to validate the differential RT-PCR assays. The results indicated that primer set 1 specifically amplified a 415 bp RT-PCR product for the serotype 2 viruses, and primer set 2 specifically amplified a 715 RT-PCR product for vvIBDV except for two vv Taiwan strains. To further confirm the specificity of primer set 2 for the vvIBDVs, 20 field samples suspected to be vvIBDVs from different geographic locations around the world were tested. All but one suspected vv Korean strain (91108) tested positive by this primer set. To understand the molecular basis for the failure to detect these viruses with primer set 2, a complete VP1 gene of two vv Taiwan (PT and IL) strains along with two vv Turkey (OA and OE) and one vv Holland (Hol) strain was sequenced and phylogenetically analyzed with 11 other strains retrieved from GenBank. The results showed that PT and IL were closely related to the classic strain IM with nucleotide (nt) similarities of 98.6% and 97.7%, respectively. The two Taiwan strains clustered together with the classic and variant IBDVs in the unrooted neighbor-joining phylogenetic tree, indicating independent evolution of these strains from the rest of the vv isolates. The RT-PCR assays developed in this study could differentiate vvIBDVs from classic strains and serotype 1 from serotype 2 IBDVs with a high degree of sensitivity and specificity and are fast, simple, and inexpensive.

Avian leukosis subgroup J (ALV-J) causes a variety of tumors and mortality in meat-type chickens. Since its discovery in the late 1980s, ALV-J has spread to breeding stock produced by most primary breeding companies of North America, the European Union, and Asia. ALV-J seems to have been eradicated from elite breeding stock produced by most primary breeders, albeit ALV-J still circulates in some commercial poultry. This study was undertaken to examine the molecular epidemiology and evolution of ALV-J detected in breeding stock and broiler chickens representing eight primary breeding companies over a period of approximately 20 yr (1988–2007). The redundant transmembrane region of the envelope gene has been deleted in some isolates, suggesting that this region is dispensable for viral fitness. Within the 3′ untranslated region (3′ UTR), the direct repeat 1 was present in 100% of the ALV-J isolates studied. In contrast, the E element has undergone substantial deletions in >50% of the ALV-J proviruses studied. Overall, the unique region 3 was the least conserved within the 3′ long terminal repeat (LTR), albeit the transcriptional regulatory elements typical of avian retroviruses (CAAT, CArG, PRE, TATA, and Y boxes) were highly conserved. The direct repeat region of the LTR was identical in all of the proviruses, and the 3′ unique region 5 was relatively well conserved. Thus, the 3′ UTR of ALV-J has evolved rapidly, reflecting significant instability of this region. Some of the mutations in the 3′ UTR have resulted in the emergence of moderately distinct genetic lineages representing each primary breeding company from which ALV-J was isolated.

A method previously described by Jeffers and Bentley, involving calculation of a growth and survival ratio and optimal anticoccidial activity index, was used to investigate the sensitivity of 23 field isolates of Eimeria obtained from turkey flocks to the ionophorous antibiotic monensin. Isolates were obtained from litter and intestinal samples from several major turkey-growing regions of the United States, and in most cases contained at least two species of Eimeria. A mixture of strains that had been maintained in the laboratory for many years in the absence of exposure to anticoccidial drugs was found to be sensitive to monensin. Six of the field isolates were judged sensitive, seven partially resistant, and ten resistant to the drug, judged by the Jeffers and Bentley criteria. This is the first report of the acquisition of resistance to monensin in isolates of Eimeria from turkey flocks in the United States.

Although Salmonella remains one of the leading causes of foodborne illnesses in the United States, the Salmonella enterica serovars and genetic types associated with most infections appear to fluctuate over time. Recently, the Center for Disease Control and Prevention (CDC) has reported an increase in cases of salmonellosis caused by Salmonella 4,[5],12:i:−. Similarly, this unusual Salmonella serovar has been isolated from cattle and poultry in the state of Georgia. We examined the genetic relatedness of Salmonella 4,[5],12:i:−, isolated from several different poultry companies and dairy farms in Georgia, by pulsed-field gel electrophoresis (PFGE). Several Salmonella 4,[5],12:i:− isolates had PFGE patterns identical or similar to PFGE patterns of Salmonella Typhimurium isolated from numerous animal sources. We identified distinct PFGE patterns for Salmonella 4,[5],12:i:− and matching Salmonella Typhimurium PFGE patterns, identifying four “distinct” strains. We focused a more specific analysis on the poultry Salmonella 4,[5],12:i:− and Salmonella Typhimurium isolates and found that of these Salmonella 4,[5],12:i:− isolates, 32% lacked the entire phase 2 antigen gene, fljB; 61% contained partial deletion(s); and 4% had partial deletion(s) in fljB and an adjacent gene hin, 5′ to fljB. Thirteen percent contained smaller deletions or point mutations not identified by our DNA probes. The Salmonella 4,[5],12:i:− isolates were positive for several genes present in the Salmonella Typhimurium, including lpfE (100%), sseI (96%), and spvC (93%). Genetic analysis indicates independent, spontaneous mutations in fljB in at least four distinct Salmonella Typhimurium strains of animal origin circulating in nature.

Surveillance for avian influenza A viruses in wild bird populations is often limited by requirements for a cold chain from time of specimen collection, by availability of ultra–low temperature specimen storage within a few hours or days, and by laborious classical virologic procedures. Successful storage of specimens in preservatives at ambient temperature and subsequent detection of RNA by reverse transcriptase–polymerase chain reaction (RT-PCR) would assist in helping influenza surveillance efforts become more widespread in remote areas, as well as more timely and inexpensive. Here, we describe bird feces spiked with influenza A virus preserved with guanidine and commercial buffers or alcohols at ambient temperature and analyzed by RT-PCR protocols. Virus-specific RT-PCR products of, at most, 206 bp were recovered for samples preserved with alcohols and up to 521 bp for samples preserved with guanidine or commercial buffers. These results suggest that this approach is feasible in the field and that preserved specimens might be better assayed molecularly when preserved in guanidine or commercial buffers.

The cell-associated vaccine strain CVI 988, which is the active component of several commercial Marek's disease vaccines, normally is frozen and stored in liquid nitrogen. In order to ascertain good efficacy of the vaccine, it is crucial that the right procedures are followed for thawing and diluting of the virus. In the study presented here, ampoules containing the frozen product were taken from storage in liquid nitrogen and were thawed in a water bath at 27 C, which is similar to a lukewarm bath, and in a water bath at 37 C, with and without agitation. The effect of the thawing procedure on the live virus titer of the vaccine was investigated. Samples of thawed vaccine were diluted in diluent with different temperatures, and live virus titers were determined directly after dilution and after incubation of the diluted vaccine at different temperatures. The results show that directly after thawing in the water baths at 27 C and 37 C, with or without agitation, the live virus titers for CVI 988 were all in the same range. After incubation of the thawed virus at both temperatures for 15 min, the live virus titers were still in the same range. Decreases in live virus titers were observed after incubation for 4 hr. Live virus titration of the vaccine in diluent in different temperatures revealed that the highest titers were found with diluent at a temperature of 30 C to 37 C and the lowest titers in diluent at a temperature of 5 C. In addition, a combination product containing cell-associated CVI 988 and cell-associated herpesvirus of turkeys (HVT) was tested. For this combination product, the titers for HVT also were highest in diluent with a high temperature (i.e., 37 C), whereas the titers for CVI were highest in diluent at a temperature of 22 C. Both strains had relatively low titers in diluent at 5 C. After incubation of the diluted vaccine at the various temperatures for several hours, again, live virus titrations were done. Live virus titers were most stable with diluent at temperatures of 22 C. Vaccine virus diluted in diluent at 37 C could be stabilized by placing the diluted vaccine at 5 C directly after diluting. After evaluation of these data, the following is recommended. For thawing of the vaccine, a water bath at approximately 27 C, which is similar to a lukewarm bath, is preferred. For diluting the vaccine, diluent should be used at a temperature of 22 C or higher. If diluted in diluent at temperatures higher than 22 C, the diluted vaccine should be stored under cooling in order to avoid titer losses.

Infectious bronchitis virus (IBV) is the causative agent of avian infectious bronchitis, which is characterized by respiratory, reproductive, and renal signs. However, the role of IBV as an enteric pathogen in still controversial. In Brazil, antigenic groups of IBV divergent from the Massachusetts serotype used for vaccination schedules in that country have already been demonstrated. The present study aimed to assess the different genotypes of IBV in Brazilian commercial poultry flocks by partial sequencing of the S1 amino-terminus coding region using enteric contents as samples and examine their relationship with the vaccine serotype currently in use. Samples of enteric contents were taken as pools of five birds from each of 18 poultry farms (17 broiler and one laying farm) from five Brazilian states between 2002 and 2006. Birds were presenting watery diarrhea and poor general condition but were without respiratory, renal, or reproductive signs. Conventional antibacterial and anticoccidial therapies were unsuccessful and, furthermore, all samples proved negative for rotavirus, reovirus, and astrovirus. Eleven IBV samples were isolated in embryonated eggs and resulted in S1 sequences. Phylogenetic analysis showed that these segregated into an exclusive cluster, close to serotype D274, but distant from Massachusetts. Mean amino acid identity amongst these Brazilian strains was 94.07%; amongst these and serotypes D274, 4/91, and Massachusetts, mean amino acid identity was 77.17%, 69.94%, and 68.93%, respectively. In conclusion, the presence of genotype variant strains of IBV in Brazilian poultry flocks has been demonstrated and might be the reason for the unsuccessful control of IBV in Brazil. Furthermore, these results also strengthen the implications of IBV in enteric diseases of poultry.

A commercial live attenuated, freeze-dried avian metapneumovirus vaccine, Pneumomune^TM, was assessed for its viability at three different temperatures (5.6 C, 21 C, and 37 C). No significant reduction in virus titer was observed when the vaccine was stored at 5.6 C for a period of 24 hr. However, reductions in virus titer of 1 log₁₀ and 2 log₁₀ were observed after 24 hr at 21 C and 37 C, respectively. Batch-to-batch variation in virus reduction was also observed. The addition of a dye or a vaccine stabilizer to the vaccine preparation did not have any deleterious effect on the survival of vaccine virus.

An enzyme-linked immunosorbent assay (ELISA) was applied to evaluate the antibody response of commercial White Leghorn chickens to vaccination against Marek's disease (MD) at hatch (day 0) with serotype-1 (Rispens), -2 (SB-1), or -3 (turkey herpesvirus, HVT) vaccine virus and to challenge on day 21 with MD virus. Antigens for the test were whole chicken embryo fibroblast cells infected with Rispens, SB-1, or HVT. The chickens were progeny of stock that had been vaccinated with HVT, and on day 21 the nonvaccinated group had higher levels of maternal antibodies to HVT than to other antigens (P < 0.05). Only SB-1 vaccine had induced antibodies by day 21, and this was detected only against homologous antigens. On day 49, all three vaccines had induced higher levels of antibodies to homologous than to heterologous antigens. Marek's Disease virus (MDV) induced antibodies to all three antigens, but challenging vaccinated chicks did not significantly increase levels of antibodies on day 81 to any of the three antigens. It was concluded that an ELISA using whole cells as antigens would have potential value for monitoring the antibody response induced by MD vaccines and virulent MDV.

Dwyer medium is the most frequently employed culture medium for Histomonas meleagridis. Both for subculturing and for resuscitation of H. meleagridis from storage in liquid nitrogen, modified Dwyer medium with an increased rice powder concentration (0.8%) and no chicken embryo extract proved superior to Dwyer standard medium, with threefold (10^6.3vs. 10^5.8 histomonads/ml) to 10-fold (10^6.7vs. 10^5.8 histomonads/ml) higher concentrations of parasites after resuscitation or subculturing, respectively.

Eight captive-bred horned parakeets (Eunymphicus cornutus) and four captive-bred Major Mitchell cockatoos (Cacatua leadbeateri) from the same aviary tested positive for psittacine circovirus (PsCV) DNA in whole blood by nested–polymerase chain reaction (PCR). The chronic form of disease with feather fragility and loss was observed in three horned parakeets. Infection in other individuals was subclinical. Immunosuppression, either hematologically or as susceptibility to secondary infections, was not observed. Treatment consisted of the administration of β-(1,3/1,6)-d-glucan from oyster mushroom (Pleurotus ostreatus). Excluding two accidentally dead parakeets, four out of the original six horned parakeets, and all Major Mitchell cockatoos were negative for PsCV DNA in whole blood in 7–9 mo after the treatment was started. Even though the absence of PsCV DNA in blood does not signify elimination of the virus from the whole organism, these preliminary results indicate a possible effect of β-glucan in the treatment of PsCV infection. To the author's knowledge, this is the first report of PsCV in horned parakeets.

A red-bellied woodpecker (Melanerpes carolinus), clinically exhibiting intermittent seizures, was examined by the Southeastern Cooperative Wildlife Disease Study. Microscopically, the woodpecker had protozoal meningoencephalitis. Toxoplasma gondii was identified by immunohistochemistry and polymerase chain reaction. Sequence analysis of the SAG2 locus disclosed that the T. gondii detected was the Type III genotype. This is apparently the first report of clinical neural toxoplasmosis in any species in the order Piciformes.