Multilocus Sequence Typing of Eimeria maxima in Commercial Broiler Flocks.

About 35% of all broiler flocks in the United States receive an anticoccidial vaccine, but it is not possible to easily differentiate Eimeria vaccine strains from Eimeria field isolates. Being able to do that would allow using vaccines in a more targeted way. The objective of this study was to collect Eimeria maxima isolates from broiler flocks that received anticoccidial feed additives and flocks that had been vaccinated against coccidia and then test them with a multilocus sequencing typing (MLST) scheme developed for this study. Fecal samples were obtained from commercial broiler flocks in Alabama and Tennessee. Oocyst counts in samples tended to be lower in flocks receiving anticoccidial feed additives and higher in vaccinated flocks. Selected samples were screened for presence of E. maxima by quantitative PCR, and Eimeria spp. composition was investigated by next-generation amplicon sequencing (NGAS) in 37 E. maxima positive samples. Other detected Eimeria spp. besides E. maxima were Eimeria acervulina in 35 samples, Eimeria praecox in 23 samples, Eimeria mitis or Eimeria mivati in 17 samples, and Eimeria necatrix or Eimeria tenella in 10 samples. Six partial E. maxima genes (dnaJ domain containing protein, 70-kDa heat shock protein, prolyl endopeptidase, regulator of chromosome condensation domain containing protein, serine carboxypeptidase, and vacuolar proton-translocating ATPase subunit) of 46 samples were sequenced. The MLST scheme was able to differentiate two vaccines from each other. Three of 17 samples from vaccinated flocks differed from the vaccine used in the flock, while 16 of 29 samples from unvaccinated flocks differed from the vaccine. However, there was also a large number of low-quality, ambiguous chromatograms and negative PCRs for the selected genes. If and when more advanced, possibly next-generation sequencing-based methods will be developed, the genes should be considered as targets.

Tipificación por secuenciación multilocus de Eimeria maxima en parvadas comerciales de pollos de engorde. Alrededor del 35% de todas las parvadas de pollos de engorde en los Estados Unidos recibe una vacuna anticoccidial, pero no es posible diferenciar fácilmente las cepas vacunales de Eimeria de los aislados de campo de Eimeria. La posibilidad de diferenciar entre cepas vacunales y de campo permitiría usar vacunas de una manera más específica. El objetivo de este estudio fue recolectar aislamientos de Eimeria maxima de parvadas de pollos de engorde que recibieron aditivos alimenticios anticoccidiales y parvadas que habían sido vacunadas contra coccidia y luego analizarlos con un esquema de tipificación por secuenciación multilocus (MLST) desarrollado para este estudio. Las muestras fecales se obtuvieron de parvadas comerciales de pollos de engorde en Alabama y Tennessee. Los conteos de ooquistes en las muestras tendieron a ser más bajos en las parvadas que recibieron aditivos alimenticios anticoccidiales y más altos en las parvadas vacunadas. Las muestras seleccionadas se examinaron para determinar la presencia de E. maxima mediante PCR cualitativa, y Eimeria spp. la composición se investigó mediante secuenciación de amplicones de próxima generación (NGAS) en 37 muestras positivas de E. maxima. Además de E. máxima, otras Eimeria spp detectadas, fueron Eimeria acervulina en 35 muestras, Eimeria praecox en 23 muestras, Eimeria mitis o Eimeria mivati en 17 muestras, y Eimeria necatrix o Eimeria tenella en 10 muestras. Se secuenciaron seis genes parciales de E. maxima (proteína que contiene al dominio dnaJ, proteína de choque térmico de 70 kDa, prolil endopeptidasa, proteína que contiene al regulador del dominio de condensación cromosómica, serina carboxipeptidasa y la subunidad de ATPasa vacuolar translocadora de protones) de 46 muestras. El esquema MLST pudo diferenciar dos vacunas entre sí. Tres de 17 muestras de parvadas vacunadas diferían de la vacuna utilizada en la parvada, mientras que 16 de 29 muestras de parvadas no vacunadas diferían de la vacuna. Sin embargo, también hubo una gran cantidad de cromatogramas ambiguos y de baja calidad y PCR negativos para los genes seleccionados. En Cuando se desarrollen métodos más avanzados, posiblemente de próxima generación, basados en la secuenciación, estos genes deben considerarse como objetivos.

Evaluation of field and laboratory protocols used to detect avian influenza viruses in wild aquatic birds.

Careful selection and observance of standard field and laboratory protocols are critical for successful detection and characterization of avian influenza viruses (AIV) from wild birds. Cloacal swabs were collected from hunter-killed or nesting waterfowl and shorebirds from wildlife refuges in Alabama, Georgia, and Florida during 2006 to 2008. Swab samples were inoculated into embryonated eggs followed by hemagglutination (HA) test to determine the presence of hemagglutinating agents. Antigen capture-ELISA (AC-ELISA) and real-time reverse transcription-PCR (RRT-PCR) were used to detect AIV from both allantoic fluids (AF) and swab specimens of HA-positive samples. Hemagglutination inhibition test was used to detect Newcastle disease virus, another hemagglutinating virus common in wild birds. The HA-positive AF were sent to the National Veterinary Services Laboratory for subtyping of the isolates. Out of 825 samples tested, 19 AIV and 3 avian paramyxovirus subtypes were identified by the National Veterinary Services Laboratory. Without egg passage, AC-ELISA did not detect virus, whereas matrix gene of 13 AIV were detected using RRT-PCR. When testing was done on AF, 14 were positive for influenza A by AC-ELISA and 20 by RRT-PCR. Antigen capture-ELISA did not detect influenza A when the HA titer was lower than 125, whereas RRT-PCR detected AIV from AF with HA titer as low as 4. The highest isolation rate was from Florida, where out of 109 samples analyzed, 14 AIV were detected by RRT-PCR from AF. Real-time reverse transcription-PCR was more sensitive, specific, and cost-effective than AC-ELISA. However, to avoid false-negative results, testing should be performed on AF and not directly from cloacal swabs. Our procedures to detect AIV directly from cloacal swabs need further optimization for improved sensitivity.

Detection and characterization of avian influenza and other avian paramyxoviruses from wild waterfowl in parts of the southeastern United States.

Cloacal swabs were taken from migratory hunter-killed, nonmigratory, nesting waterfowl and migratory shorebirds from wildlife refuges in Alabama, Georgia, and Florida during 2006 to 2008. Samples were processed in embryonated eggs followed by hemagglutination (HA), Directigen, and real-time reverse transcription-PCR tests. Sequence analysis of the hemagglutinin (H) gene of the H10N7 Alabama isolate revealed that it was closely related (98%) to recent isolates from Delaware and Canada, but only 90% related to an H10N7 isolated 30 yr ago. Four isolates had 94 to 97% similarity to published H1N1 isolates including one from swine. No H5 or H7 isolates were found. One sample was highly pathogenic in embryos, produced a high HA titer, and was positive for both avian influenza (AIV) and Newcastle disease virus or avian paramyxovirus (APMV)-1. In recent (2008) sampling, more (14%) AIV, APMV, or both were isolated than in 2006 to 2007 (1% isolation rate). The higher isolation rate during 2008 may be attributed to optimized sample collection, storage in dry ice, new egg incubator, healthier eggs, time or habitat for isolation, species sampled, migratory status of birds, and more experience with detection procedures. An additional egg passage resulted in increased viral titer; however, no HA-negative samples became HA positive. The chance of transmission of APMV or low-nonpathogenic AIV from wild waterfowl to commercial poultry is possible. However, the chance of transmission of H5 or H7 AIV isolates from waterfowl to commercial farms in Alabama, Georgia, or Florida is unlikely. Therefore, continual testing of these birds is justified to ensure that H5 or H7 AIV are not transmitted to commercial poultry.

Molecular and phenotypic characterization of infectious bursal disease virus isolates.

Two infectious bursal disease viruses (IBDVs 1174 and V1) were isolated from IBDV-vaccinated broiler flocks in California and Georgia. These flocks had a history of subclinical immunosuppression. These isolates are commonly used in IBDV progeny challenge studies at Auburn, AL, as well as vaccine manufacturer's vaccine efficacy studies, because they come from populated poultry-producing states, and are requested by poultry veterinarians from those states. Nested polymerase chain reaction (PCR) generated viral genome products for sequencing. A 491-bp segment from the VP2 gene, covering the hypervariable region, from each isolate was analyzed and compared with previously sequenced isolates. Sequence analysis showed that they were more closely related to the Delaware (Del) E antigenic variant than they are to the Animal Health Plant Inspection Service (APHIS) standard, both at the nucleotide level (96%, 97%) and at the amino acid level (94%, 97%). Both isolates had the glutamine to lysine shift in amino acid 249 which has been reported to be critical in binding the virus neutralizing Mab B69. Phenotypic studies showed that both isolates produced rapid atrophy of the bursae and weight loss, without the edematous bursal phase, in 2-wk-old commercial broilers having antibody against IBDV. A progeny challenge study showed both isolates produced more atrophy of the bursae (less percentage of protection) than the Del E isolate. Molecular and phenotypic data of these important IBDV isolates help in the improved detection and control of this continually changing and important viral pathogen of chickens.

Transmissible proventriculitis in broilers.

This article reviews transmissible proventriculitis in poultry from 1971 to 2006. The disease is important in commercial broilers worldwide, resulting in reduced profits. The aetiology of this disease is unknown and different clinical presentations often result in a confused or complicated diagnosis. The lesion of enlarged proventriculus is often referred to as proventriculitis. However, the term proventriculitis can only be used correctly when there is microscopic evidence of inflammation of the proventriculus glands. Infectious and non-infectious causes of proventriculitis, with major emphasis on the infectious or transmissible causes, are reviewed.

Effect of in ovo administered reovirus vaccines on immune responses of specific-pathogen-free chickens.

We investigated the effect of in ovo administered reovirus vaccines on the immune responses of specific-pathogen-free chickens. T-cell mitogenic responses to concanavalin A were numerically lower at 9 and 12 days of age and significantly lower at 6 days of age in birds vaccinated with a commercial reovirus vaccine compared with unvaccinated birds or birds vaccinated with an experimental reovirus-antibody complex vaccine. There were no significant differences in proportions of subpopulations of helper (CD4+CD8-) or cytotoxic (CD4-CD8+) T cells except at 12 days of age, when the percentages of CD4-CD8+ cells in the two vaccinated groups were statistically higher than in the nonvaccinated group. B-cell populations were not different among vaccine groups except at 9 days of age, when the vaccinated groups had the highest level of B cells. This commercial reovirus vaccine should not be given in ovo to embryos having little or no maternal antibody, otherwise immunosuppression may occur in the chicks. The addition of the antibody complex to the vaccine prevented this T-cell immunosuppression.

Influence of a reovirus-antibody complex vaccine on efficacy of Marek's disease vaccine administered in ovo.

Two experiments determined the influence of an experimental reovirus-antibody complex vaccine on Mareks disease virus (MDV) vaccine when used in ovo. Designs were the same except that specific-pathogen-free (SPF) broiler eggs were used in Experiment 1 and commercial broiler eggs with maternal antibodies against reovirus were used in Experiment 2. At 18 days of incubation, embryos were separated into four groups and inoculated with either diluent, MDV vaccine, reovirus-antibody complex vaccine, or a combination of reovirus-antibody complex and MDV vaccine. At 5 days of age, half the chickens in each group were challenged with MDV. At 7 wk old, all were euthanatized, weighed, and examined. At 7 days of age, remaining chickens in each group were challenged with reovirus. At 21 days old, chickens were euthanatized and weighed. No vaccine adversely affected hatchability or posthatch mortality in SPF or commercial chickens. There were no significant differences in protection against reovirus challenge when vaccines were used separately or in combination, and lesion scores were nearly identical in all vaccinated groups in both experiments. However, percentage of protection against reovirus was lower in Experiment 2, indicating an adverse effect of maternal immunity on efficacy of the reovirus vaccine. There were no significant differences in protection against MDV when the vaccines were used separately or combined. Severity of MDV lesions was nearly identical in all vaccinated groups in both experiments. However, the combination of vaccines gave numerically lower protection against MDV than MDV vaccine alone. Use of a larger number of birds, as in field conditions, may result in statistically lower protection for the vaccine combination. Large field trials are needed to determine the potential of the reovirus-antibody complex vaccine.

Safety and efficacy of an experimental reovirus vaccine for in ovo administration.

A commercial reovirus vaccine alone or experimental reovirus vaccine plus antibody complex were inoculated into 18-day-old specific pathogen free (SPF) broiler embryos at 0.1 of the recommended chick dose. The following groups were used: group 1A was not vaccinated or challenged; group 1B was not vaccinated, but was challenged with virulent reovirus; group 2 received the vaccine complexed with 1/4 dilution of antiserum; group 3 received the vaccine with 1/8 dilution of antiserum; group 4 received the vaccine with 1/16 dilution of antiserum, and group 5 received vaccine alone. At 1, 3, 6, 9, and 12 days of age, serum was collected and antibody against avian reovirus was analyzed by enzyme-linked immunosorbent assay (ELISA). At the same times, spleens were collected and vaccine virus detected by inoculating chicken embryo fibroblasts (CEFs) and examining for cytopathic effect. At 15 days of age, chickens in groups 2-5 were challenged with reovirus. At 22 days of age, birds were euthanatized and weighed. Efficacy of the vaccines was based on safety, percent protection, and antibody response. In ovo vaccination with the commercial or experimental vaccines did not adversely affect hatchability of SPF chickens. The vaccine complexed with antibody resulted in significantly less posthatch mortality (3.7%) when compared to mortality of chickens that received vaccine alone (17%). Both vaccine virus recovery and antibody response were delayed at least 3 days in birds receiving the experimental vaccines. In evo administration of reovirus antibody complex vaccines provided at least 70% protection. The experimental reovirus-antibody complex vaccines were safe and efficacious when given in ovo to SPF broiler embryos.

Safety and efficacy of in ovo administration of infectious bursal disease viral vaccines.

In ovo vaccination against Marek's disease virus and infectious bursal disease virus (IBDV) in commercial broilers in the United States is common. Little information exists as to the safety and efficacy of intermediate IBDV vaccines given in ovo. Experiments were initiated to determine the safety and efficacy of three commercially available live intermediate IBDV vaccines by in ovo route. Commonly used vaccines were given at 18 days of embryonation to specific-pathogen-free (SPF) broiler embryos (first and second study) or to commercial broiler embryos (third study) that had maternal antibody against IBDV. When any of the antigenic standard vaccines was given at full dose to SPF embryos, embryonic and 3-wk posthatch mortality increased. Vaccines also caused significant microscopic lesions in the bursa of Fabricius at 1 and 3 wk posthatch. In contrast, there was no adverse effect on embryonic or posthatch mortality when vaccines were given at half dose to SPF or commercial broiler embryos. However, significant microscopic lesions were evident at 1 and 3 wk posthatch in the bursae of SPF embryos given the vaccines at half dose. When vaccines were given at half dose to commercial broiler embryos, lesions were evident at 1 but not 3 wk of age. In the third study, in ovo vaccinated chickens were challenged with either a virulent standard (APHIS) or antigenic variant (variant E) IBDV virus at 3 wk of age. All vaccines produced at least 87% protection against the standard and 60% protection against the variant challenge IBDV, as measured by bursal weight to body weight ratios. This study was the first to examine the safety and efficacy of the three commonly used intermediate IBDV vaccines given in ovo in protection against standard and antigenic variant IBDV challenge viruses.

Detection of infectious bursal disease vaccine viruses in lymphoid tissues after in ovo vaccination of specific-pathogen-free embryos.

Control of infectious bursal disease virus (IBDV) by vaccination is important for poultry production worldwide. Two vaccines, an IBDV immune complex (ICX) vaccine and an IBDV-2512 vaccine, were administered at 100 mean embryo infectious dose to specific-pathogen-free 18-day-old broiler embryos in ovo. At 3, 6, 9, 15, and 21 days post in ovo vaccination (PIOV), bursa, spleen, and thymus tissues were collected and analyzed for virus protein by antigen capture chemiluminescent enzyme-linked immunosorbent assay (ELISA). Chicks were bled and antibody titers were determined by the antibody ELISA. At 21 days PIOV, chickens were challenged with a 1:500 dilution of an antigenic standard IBDV strain. At 28 days PIOV, birds were euthanatized and bursa weight:body weight ratios were determined. Embryos vaccinated with either vaccine exhibited 92% hatchability; however, within 1 wk of hatch, birds vaccinated with IBDV-2512 showed 56% mortality, whereas those given IBDV-ICX had only 3.2% mortality. Both IBDV-ICX and IBDV-2512 vaccines were detected in bursa, spleen, and thymus at day 3 PIOV. A 5-day delay in virus replication was observed with IBDV-ICX vaccine. By day 15 PIOV, the IBDV-ICX was no longer detectable in the bursa and spleen but persisted in the thymus. The IBDV-2512 vaccine persisted in the spleen and thymus on day 15 PIOV. By day 21 PIOV, neither vaccine virus was detected in any lymphoid organ. This assay can be useful in the early detection of vaccine virus in the tissues of chickens vaccinated via the in ovo route. Both vaccines caused bursal atrophy at all times PIOV. The IBDV-2512 caused splenomegaly at day 6 PIOV, whereas splenomegaly was not seen in IBDV-ICX-vaccinated birds until day 9 PIOV. Thymus atrophy was observed in IBDV-2512-vaccinated chicks from day 3 PIOV, whereas this occurred on day 15 PIOV in IBDV-ICX-vaccinated birds. Bursa weight: body weight ratios in IBDV-ICX-vaccinated unchallenged and vaccinated challenged birds were not different (P < 0.05).

Simplified sample processing combined with a sensitive nested polymerase chain reaction assay for detection of infectious bursal disease virus in the bursa of Fabricus.

A rapid and sensitive protocol for the detection and amplification of infectious bursal disease virus (IBDV) RNA in the bursa of Fabricius was developed. By digestion with proteinase K and subsequent extraction with phenol and chloroform, IBDV RNA was efficiently released from a single bursa. IBDV RNA extraction time was shortened to 4 hr, compared with 24 hr with the traditional method, and only one bursa was needed instead of five. This more simplified procedure resulted in a significant reduction in costs due to less labor and the reduction in expensive chemicals and reagents. Four primers were selected from the sequence of a hypervariable region in VP2 genes. For the amplification of genomic IBDV RNA, the product (643 bp) of reverse transcriptase-polymerase chain reaction (RT-PCR) was reamplified and double checked by a nested PCR amplifying a 491-bp cDNA. The sensitivity of nested PCR was at least 100 times greater than RT-PCR as determined by dilution of the bursal homogenate. The fidelity of the nested PCR product was confirmed by Southern blotting, demonstrating specificity to the VP2 gene of IBDV. The simplified sample processing, shortened procedure time, and technical ease of this nested PCR render it more suitable for implementation in routine RT-PCR with restriction fragment length polymorphism testing for the detection and differentiation of IBDV RNA, especially for studies of IBDV infections of individual chickens.

Sequence comparisons of the variable VP2 region of eight infectious bursal disease virus isolates.

The VP2 gene is part of the genomic segment A of infectious bursal disease virus (IBDV). It has been identified as the major host-protective antigen of IBDV and is known to contain conformationally dependent protective epitopes. A 643-base pair segment covering the hypervariable region of this gene from three recent serologic variant IBDV isolates from the southeastern United States, two variants from the Delmarva Peninsula, and three serologic standard viruses were amplified and sequenced using the reverse transcription polymerase chain reaction and cycle sequencing techniques. This was done to determine the molecular similarity among isolates that differ antigenically and pathologically. Sequence analysis suggested that the Arkansas (Ark) and Mississippi (Miss) isolates evolved closely and separately from the Delmarva variants (GLS and DELE), in contrast to the other southeastern variant Georgia (Ga), which is more closely related (98.32%) to Delaware E (DELE). All variants, except for Miss, underwent a shift in amino acid number 222 from proline to threonine. The sequence of Univax BD virus, a commercially available intermediate vaccine, was markedly different, evolving from a separate lineage than the others. Restriction enzyme sites could differentiate most isolates. Except for Miss, variants do not have EcoRII site at the larger hydrophilic domain. All variants lost their HaeIII, StuI, and StyI cutting sites with a change in base number 856. The TaqI site is in DELE, whereas the SpeI site is absent in the standard vaccine viruses. The SWASASGS heptapeptide is conserved in all virulent viruses, including APHIS, but not in the attenuated (Univax BD and Bursa Vac 3) and published (D78 and PBG98) vaccines.

Detection of genetic variations in serotype I isolates of infectious bursal disease virus using polymerase chain reaction and restriction endonuclease analysis.

Reverse transcription with polymerase chain reaction (PCR) followed by restriction endonuclease analysis detected genetic variations among serotype I isolates of infectious bursal disease virus (IBDV). Using a set of synthetic primers derived from the large genome segment of APHIS-IBDV, the hypervariable region (AccI-SpeI fragment) located in the VP2 gene was amplified. With all strains, a cDNA fragment of approximately 643 bp was amplified, indicating that there were no apparent deletions or insertions in this region among isolates. Fragments amplified from 9 isolates were digested with 14 restriction enzymes. Restriction fragment profiles generated by restriction enzymes NaeI, StuI, TaqI, and SacI, showed genetic variations among isolates. This study provided a simple and sensitive method for detection of genetic variations among isolates that are closely related serologically and could not be differentiated using current serologic methods.

Coarse-spray immunization of one-day-old broilers against enteric reovirus infections.

Coarse-spray (CS) administration of a commercial S1133 reovirus vaccine was evaluated in 1-day-old specific-pathogen-free broilers for prevention of clinical infection induced by intratracheal challenge with two enteric reovirus isolates. In Expt. 1, chickens were challenged at 4 days of age with either the 2408 or CO8 isolate. In Expt. 2, chickens were challenged at 7 days of age with either isolate. In Expt. 3, chickens were challenged at 3, 5, or 7 days of age with the 2408 isolate. In Expt. 1, vaccinated birds showed significant protection against challenge with either isolate at 4 days of age as measured by morbidity, mortality, gross lesions, and body weight. In Expt. 2, vaccinated birds showed greater protection against challenge at 7 days of age. In Expt. 3, resistance in vaccinated birds increased with time between vaccination and challenge. Vaccinated birds challenged at 3 days of age showed no significant protection, whereas vaccinated birds challenged at 5 or 7 days of age had increased resistance. This vaccine did not induce a drop in weight gain, morbidity, mortality, or microscopic lesions in the tendons.