Pullorum Disease: Evolution of the Eradication Strategy.

The history of pullorum disease is closely intertwined with the history of avian health research and that of the poultry industry. The seriousness of the disease galvanized the attention and brought together, for the first time, the pioneers of poultry health research to work cooperatively on different aspects of the disease. Control of the disease made it possible for intensive poultry production to develop as the basis for the modern poultry industry. During the early 1900s, bacillary white diarrhea (BWD) was a devastating disease of young chickens threatening the developing poultry industry. Dr. Leo F. Rettger isolated and described the bacterial pathogen, Salmonella enterica serotype Pullorum, for the first time in 1900. BWD was renamed pullorum disease in 1929. In subsequent years, Rettger and coworkers were able to reproduce the disease and fulfill Koch's postulates. Rettger et al. also showed that Salmonella Pullorum was vertically transmitted, which was the first time that a pathogen was shown to be vertically transmitted. The development of serologic tests was of crucial importance because it led to the development of effective eradication methods to identify carrier birds and to exclude these birds from the breeder flocks. The negative impact of pullorum disease on the poultry industry ultimately was one of the major reasons that the National Poultry Improvement Plan (NPIP) was developed by scientists, the poultry industry, and the United States Department of Agriculture (USDA). Needless to say, the work of the pioneering researchers formed the basis for the control of the disease. The NPIP started in 1935, with 34 states participating in testing 4 million birds representing 58.2% of the birds hatched. The program rapidly expanded to 47 states by 1948 and tested more than 30 million birds. In 1967, all commercial chicken hatcheries participating in the NPIP were 100% free of pullorum and typhoid disease caused by Salmonella enterica serotype Gallinarum. This historical overview of pullorum disease describes in some detail the progress made, especially during the early years, toward controlling this disease using methodologies that were often very basic but nonetheless effective. One has to admire the ingenuity and persistence of the early researchers leading to their achievements considering the research tools that were available at the time.

Artículo histórico­Pulorosis: Evolución de las estrategias de erradicación La historia de la pulorosis está estrechamente relacionada con la historia de la investigación en salud aviar y de la industria avícola. La severidad de la enfermedad despertó la atención y reunió, por primera vez a los pioneros de la investigación en salud avícola para trabajar de manera cooperativa en diferentes aspectos de la enfermedad. El control de la enfermedad hizo posible que la producción avícola intensiva se desarrollara como base de la industria avícola moderna. A principios de la década de los 1900, la diarrea blanca bacilar (con las siglas en inglés BWD) era una enfermedad devastadora de pollos jóvenes que amenazaba la industria avícola en desarrollo. El Dr. Leo F. Rettger aisló y describió el patógeno bacteriano, Salmonella enterica serotipo Pullorum, por primera vez en 1900. La diarrea blanca bacilar pasó a llamarse pulorosis (pullorum disease) en 1929. En los años siguientes, Rettger y sus colaboradores pudieron reproducir la enfermedad y cumplir los postulados de Koch. Rettger y col. también mostraron que Salmonella Pullorum se transmitía verticalmente, y fue la primera vez que se demostró que un patógeno se transmitía verticalmente. El desarrollo de pruebas serológicas fue de crucial importancia porque condujo al desarrollo de métodos de erradicación efectivos para identificar aves portadoras y eliminar a estas aves de las parvadas reproductoras. El impacto negativo de la pulorosis en la industria avícola fue, en última instancia, una de las principales razones por las que los científicos, la industria avícola y el Departamento de Agricultura de los Estados Unidos (USDA) desarrollaron el Plan Nacional de Mejoramiento Avícola (NPIP). Es importante decir que el trabajo de los investigadores pioneros formó la base para el control de la enfermedad. El Plan Nacional de Mejoramiento Avícola comenzó en año 1935, con 34 estados participando en el análisis de 4 millones de aves que representaban el 58.2% de las aves producidas. El programa se expandió rápidamente a 47 estados en 1948 y evaluó a más de 30 millones de aves. En 1967, todas las plantas incubadoras de pollos comerciales que participaban en el Plan Nacional de Mejoramiento Avícola estaban 100% libres de pulorosis y tifoidea aviar causada por Salmonella enterica serotipo Gallinarum. Esta reseña histórica de la pulorosis describe con cierto detalle el progreso realizado, especialmente durante los primeros años, hacia el control de esta enfermedad utilizando metodologías que a menudo eran muy básicas no obstante efectivas. Es admirable el ingenio y la persistencia de los primeros investigadores que los llevaron a sus logros considerando las herramientas de investigación que estaban disponibles en ese momento.

Methyltransferase-defective avian metapneumovirus vaccines provide complete protection against challenge with the homologous Colorado strain and the heterologous Minnesota strain.

UNLABELLED: Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis virus, is the causative agent of turkey rhinotracheitis and is associated with swollen head syndrome in chickens. Since its discovery in the 1970s, aMPV has been recognized as an economically important pathogen in the poultry industry worldwide. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at guanine N-7 (G-N-7) and ribose 2'-O positions. In this study, we generated a panel of recombinant aMPV (raMPV) Colorado strains carrying mutations in the S-adenosyl methionine (SAM) binding site in the CR VI of L protein. These recombinant viruses were specifically defective in ribose 2'-O, but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of specific-pathogen-free (SPF) young turkeys. Importantly, turkeys vaccinated with these MTase-defective raMPVs triggered a high level of neutralizing antibody and were completely protected from challenge with homologous aMPV Colorado strain and heterologous aMPV Minnesota strain. Collectively, our results indicate (i) that aMPV lacking 2'-O methylation is highly attenuated in vitro and in vivo and (ii) that inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for aMPV and perhaps other paramyxoviruses. IMPORTANCE: Paramyxoviruses include many economically and agriculturally important viruses such as avian metapneumovirus (aMPV), and Newcastle disease virus (NDV), human pathogens such as human respiratory syncytial virus, human metapneumovirus, human parainfluenza virus type 3, and measles virus, and highly lethal emerging pathogens such as Nipah virus and Hendra virus. For many of them, there is no effective vaccine or antiviral drug. These viruses share common strategies for viral gene expression and replication. During transcription, paramyxoviruses produce capped, methylated, and polyadenylated mRNAs. Using aMPV as a model, we found that viral ribose 2'-O methyltransferase (MTase) is a novel approach to rationally attenuate the virus for vaccine purpose. Recombinant aMPV (raMPV) lacking 2'-O MTase were not only highly attenuated in turkeys but also provided complete protection against the challenge of homologous and heterologous aMPV strains. This novel approach can be applicable to other animal and human paramyxoviruses for rationally designing live attenuated vaccines.

Prevalence of parvoviruses in commercial turkey flocks.

Turkey parvovirus belongs to the family Parvoviridae, subfamily Parvovirinae, Genus parvovirus. Since the initial report on turkey parvovirus in the United States appeared in 1983, there had been no further reports of parvovirus in turkeys until 2008. The aims of our study were to determine the prevalence of parvovirus in commercial turkey flocks using PCR; to determine their genetic relationship to previous strains identified in North America and Europe; and to test samples for enteric viruses by transmission electron microscopy (TEM). A total of 169 fecal samples collected from 42 turkey farms in four different states within the United States between 2000 and 2010 were examined. We found that the most frequently detected viruses by TEM were small round viruses, accounting for 52% of the examined samples; however, the PCR detected parvoviruses in 71% of the samples. The phylogenetic analysis of partial nonstructural gene sequences showed a certain degree of variability among the turkey samples tested in the study. Moreover, there was a clear dichotomy in the phylogenetic tree between chicken and turkey samples, with the exception of one turkey isolate from 2000, which clustered together with the chicken group.

Detection of influenza A viruses in eggs laid by infected turkeys.

Studies are limited on evaluating the potential of influenza viruses for egg-borne dissemination. In our previous studies, experimental infection of breeder turkeys with A/turkey/Ohio/313053/04 resulted in drastic declines in egg production, and we confirmed high levels of virus replication and an abundant distribution of avian-specific alpha2,3 sialic acid-gal receptors in the oviduct of these turkeys. In the present study, following experimental inoculation of A/turkey/Ohio/313053/04 in breeder turkeys, we detected these viruses in the albumin of eggs using real-time RT-PCR (RRT-PCR) and virus isolation in embryonated chicken eggs. Swabs from egg shells were also found positive by RRT-PCR. This is the first report of the detection of low pathogenic influenza viruses from internal egg contents following experimental infection. The possibility of hatchery contamination by egg-borne influenza viruses, and the spread of virus during movement of contaminated cracked eggs and egg flats, pose concerns regarding viral dissemination of influenza.

The high susceptibility of turkeys to influenza viruses of different origins implies their importance as potential intermediate hosts.

Several previous reports and our studies show that waterfowl-origin influenza viruses can be more easily transmitted to domestic turkeys than chickens. Similarly, studies indicate turkeys to be better hosts for low pathogenic avian influenza viruses isolated from commercial poultry operations and live bird markets in comparison to chickens. Low 50% infectious-dose titers of wild bird as well as poultry-adapted viruses for turkeys further suggest that turkeys can be easily infected following a low-dose exposure. Also, interspecies transmission of swine influenza viruses to turkeys occurs frequently. These findings suggest the role of turkeys as suitable intermediate hosts that can be easily infected with influenza viruses of different origins and that turkeys can act as source of infection for other land-based poultry or even mammals.

Pathobiological characterization of low-pathogenicity H5 avian influenza viruses of diverse origins in chickens, ducks and turkeys.

We undertook one of the most comprehensive studies on the replication and intraspecies transmission characteristics of low-pathogenicity avian influenza viruses in ducks, chickens and turkeys. Our results indicated that most of these isolates could replicate and be transmitted in poultry without inducing clinical disease. However, differences in transmission to contact control birds were noted, emphasizing the importance of having contact control cage mates in biological characterization experiments. Ducks supported the replication of viruses of wild aquatic bird origin in their respiratory and digestive tracts equally well. The viruses from wild aquatic birds were not effectively transmitted among chickens. In contrast, the wild-bird isolates and viruses of domestic bird origin from live-bird markets and commercial poultry operations replicated and were transmitted more efficiently in turkeys than in chickens or ducks. We also found a lower minimal infectious dose requirement for infection of turkeys compared to chickens and ducks. Our data support an important role of turkeys as being more susceptible hosts for avian influenza viruses than domestic ducks and chickens. These results highlight the role of turkeys as intermediate or bridging hosts in the transmission of influenza viruses from wild birds to land-based domestic poultry or among different land-based bird species.

Characterization of triple reassortant H1N1 influenza A viruses from swine in Ohio.

An H1N1 influenza A virus, A/swine/Ohio/24366/07, was isolated from pigs in an Ohio county fair. Twenty-six people who came in contact with the infected pigs developed respiratory disease and two of these people were laboratory confirmed as H1N1 by the Centers for Disease Control and Prevention (CDC). The A/swine/Ohio/24366/07 virus we isolated from swine was shown at the CDC to have 100% identical genome sequence to the human virus associated with the county fair. This prompted us to characterize three swine and two human origin H1N1 influenza A viruses isolated at different time points in the State of Ohio. The three swine viruses were shown to be triple reassortant viruses harboring genes of human (PB1), swine (HA, NA, NP, M, and NS), and avian (PB2 and PA) lineage viruses. Although viruses evaluated in this study were isolated during a short time interval (3 years), genetic drift was observed within the HA and NA genes, including changes at the receptor binding and antigenic sites of HA1 protein. Nevertheless, all viruses exhibited antigenic similarity as evaluated with hemagglutination inhibition and virus neutralizing tests. Internal genes were similar to other reassortant viruses of various subtypes currently circulating in the United States. Interestingly, two of the swine viruses including the 2007 isolate replicated well in human airway epithelial cells, however, another virus isolated in 2006 showed very little replication.

Pathobiology of triple reassortant H3N2 influenza viruses in breeder turkeys and its potential implication for vaccine studies in turkeys.

Triple reassortant (TR) H3N2 influenza viruses have been isolated from turkeys in the United States since 2003. These TR H3N2 virus infections have been associated with drastic declines in egg production in breeder turkeys although co-infection with multiple agents could have been responsible for exacerbating the clinical signs. In this study, we experimentally confirmed that TR H3N2 influenza virus alone can cause drastic reduction/complete cessation of egg production and pathology of the reproductive tract in 26-week-old breeder turkeys. We confirmed high levels of virus replication and abundant distribution of avian specific alpha2,3 sialic acid-galactose receptors in the oviduct of these turkeys. Although 2-6-week-old turkeys are routinely used for pathogenicity and vaccine protection studies, the low levels of viral shedding and asymptomatic infections in this age group often pose difficulty in interpretation of results. Our study shows that breeder turkeys should be used to assess the potential pathogenicity of TR H3N2 viruses and the viral titers and pathology of the oviduct as well as egg production data can be good measures of protection following in vivo challenge in vaccine efficacy studies.

Characterization of influenza virus variants with different sizes of the non-structural (NS) genes and their potential as a live influenza vaccine in poultry.

From a stock of A/turkey/Oregon/71-delNS1 (H7N3) virus, which has a 10 nucleotide deletion in the coding region of the NS1 gene, we found that several variants with different sizes of NS genes could be produced by passaging the virus in 10- and 14-day-old embryonating chicken eggs (ECE), but not in 7-day-old ECE or Vero cells. We were able to rescue the reassortant virus that has different sizes of the NS genes and confirmed that those NS genes are genetically stable. By conducting in vivo studies in 2-week-old chickens, we found two plaque purified variants (D-del pc3 and pc4) which can be used as a potential live-attenuated vaccine. The variants were highly attenuated in chickens and did not transmit the virus from infected chickens to uninoculated cage mates. At the same time, the variants induced relatively high antibody titers which conferred good protection against a high dose heterologous virus challenge. Our study indicates that naturally selected NS1 deletion variants might be useful in the development of live-attenuated influenza vaccines in poultry. Furthermore, deletion in the NS1 protein can be potentially useful as a negative marker for a differentiating infected from vaccinated animals (DIVA) approach.

Genetic and antigenic relatedness of H3 subtype influenza A viruses isolated from avian and mammalian species.

In 2004, we isolated triple reassortant H3N2 influenza viruses from turkey breeder hens in Ohio and Illinois. The Illinois flock was vaccinated twice with an inactivated H3N2 vaccine containing a swine origin virus before the outbreak. Additionally, a commercial inactivated vaccine containing an H3N4 virus of duck origin is being used in some turkey breeders. This prompted us to initiate a comparative study on the antigenic and genetic relatedness of various H3 subtype influenza viruses isolated from turkeys, ducks, pigs and humans. The antigenic relatedness between the different viruses was evaluated with the Archetti and Horsfall formula, while nucleotide genetic similarities were calculated using pairwise alignments. Results obtained indicated a high degree of antigenic (>90%) and genetic (>99%) similarities among the turkey-origin H3N2 viruses. However, the turkey viruses were antigenically distantly related to the swine-origin vaccine virus (<30%), although they had approximately 95% genetic similarity in the HA1 gene. Additionally, major genetic and antigenic changes were observed between the turkey viruses and the H3N4 duck vaccine virus as well as the H3N2 human virus. Such genetic and antigenic differences between the turkey-origin viruses and other H3 subtype viruses including vaccine strains could be the reason for the failure in protection in the Illinois turkey breeders vaccinated with swine origin virus. This also emphasizes the importance of using viruses for vaccines that are antigenically similar to the field strains.

Development of differential RT-PCR assays and molecular characterization of the complete VP1 gene of five strains of very virulent infectious bursal disease virus.

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.

Detection of antibodies against serotypes 1 and 2 infectious bursal disease virus by commercial ELISA kits.

Two distinct serotypes of infectious bursal disease virus (IBDV) are recognized in chicken and turkey flocks in the United States. Serologic testing of chicken flocks for serotype 1 viruses is routinely performed to monitor disease status and vaccination. Earlier studies indicated that enzyme-linked immunosorbent assay (ELISA) test detects antibodies to both serotypes of the virus, while the virus neutralization (VN) test is serotype specific. It is useful to evaluate currently available commercial ELISA kits for their ability to differentiate between antibodies elicited by the two serotypes. Three trials were performed in which chickens were orally inoculated with either a high or a low dose of serotype 1 STC or serotype 2 OH strains of IBDV. Sera collected at 0, 7, 14, and 21 days from these chickens and antisera procured from naturally infected broiler (n=20) and layer (n=30) flocks were tested with five different commercial ELISA kits and by VN. All ELISA kits detected different levels of antibodies elicited against serotype 1 of the virus and moderate and high levels of antibodies against serotype 2 virus. A correlation existed between the ELISA and the VN titers of experimentally infected chickens. All serum samples tested from the commercial layer flocks and 65% of the broiler flocks had antibodies against the OH strain. However, no correlation between the VN titers and ELISA titers was observed for the commercial broilers and layers sera by the majority of the kits. The results indicated that currently available commercial ELISA kits detect antibodies elicited by the two serotypes of IBDV. Hence, the prevalence of serotype 2 antibodies in the flocks should be considered while determining antibody profiles of the flocks against serotype 1 viruses.

Pathogenicity of turkey astroviruses in turkey embryos and poults.

The pathogenicity of turkey astrovirus 2001 (TAstV2001) and turkey astrovirus 1987 (TAstV1987) in specific-pathogen-free (SPF) turkey embryos and commercial poults was investigated. The virus shedding in poults was monitored using electron microscopy (EM) and reverse transcription-polymerase chain reaction (RT-PCR) during the 14-day experimental period. Both viruses caused enteritis and growth depression in SPF turkey embryos and poults. The TAstV2001 did not induce macroscopic or microscopic lesions in thymuses and bursas of embryos or poults. No macroscopic changes were observed in thymuses and bursas of embryos and poults inoculated with TAstV1987, and no statistically significant differences in bursa weight/ body weight ratios (P > 0.05) were detected. However, TAstV1987 infection resulted in microscopic lesions in bursas but not in thymuses of infected embryos and poults. Both TAstV2001 and TAstV1987 were shed during the whole 14-day experimental period as detected by EM and RT-PCR. These findings indicated that both TAstV1987 and TAstV2001 are etiologic agents of turkey enteritis. In addition, TAstV1987 might cause impairment of the immune system of infected poults. The pathogenicity of TAstV1987 is somewhat different from TAstV2001.

Development of antigen-capture enzyme-linked immunosorbent assay and RT-PCR for detection of turkey astroviruses.

Turkey astrovirus (TAstV) is an important agent of poult enteritis. The diagnosis of astroviruses has been dependent mainly on electron microscopy (EM) or immune EM (IEM). To develop other simple, rapid, and reliable diagnostic assays, two antigen-capture enzyme-linked immunosorbent assays (AC-ELISAs), polyclonal AC-ELISA and monoclonal AC-ELISA, were developed in this study. Monoplex and multiplex reverse transcription-polymerase chain reactions (RT-PCRs) were also developed using nondegenerate primer sets specific to the capsid region and degenerate primer pairs specific to the polymerase area of two TAstV. EM was included for comparison. Fecal or intestinal contents samples from naturally and experimentally infected poults with enteritis were examined using the developed assays. The polyclonal AC-ELISA had higher sensitivity and wider detection spectrum than the monoclonal AC-ELISA with group-specific monoclonal antibody (MAb), whereas the monoclonal AC-ELISA had very high specificity but lower sensitivity, which was estimated at 0.06 microg of viral proteins. Small round viruses (SRV) that could be astroviruses or other small viruses were detected in 34.4% of the samples examined by EM. The monoplex RT-PCR results amplified with primers SRV-1-3 and SRV-1-5 revealed that the positive rate of astroviruses was 45.3%, which was 10.9% higher than that of EM even if other SRVs were not excluded. Multiplex RT-PCR with SRV-1-3 and SRV-1-5 and AFCP-F1 and AFCP-R1 and the monoplex RT-PCR with degenerate primers verified that the positive rate of astroviruses was 59.4%, which was 25% higher than that of EM. Both RT-PCRs showed good specificity and wider detection spectrum compared with earlier published data.

Isolation and characterization of H3N2 influenza A virus from turkeys.

Five 34-wk-old turkey breeder layer flocks in separate houses of 2550 birds each in a single farm in Ohio experienced a drop in egg production from late January to early February 2004. Tracheal swabs (n = 60), cloacal swabs (n = 50), and convalescent sera (n = 110) from the flocks were submitted to the laboratory for diagnostics. Virus isolation was attempted in specific-pathogen free embryonating chicken eggs and Vero and MDCK cells. Virus characterization was performed using agar gel immunodiffusion, the hemagglutination test, the hemagglutination inhibition test, the virus neutralization test, reverse transcription-polymerase chain reaction, sequencing, and phylogenetic analysis. A presumptive influenza virus was successfully propagated and isolated on the first passage in MDCK cells, but initially not in Vero cells or specific-pathogen free chicken embryos. After two passages in MDCK cells, it was possible to propagate the isolate in specific-pathogen free chicken embryos. Preliminary sequence analysis of the isolated virus confirmed that it was influenza A virus with almost 100% (235/236) identity with the matrix gene of a swine influenza A virus, A/Swine/Illinois/100084/01 (H1N2). However, it was not possible to subtype the virus using conventional serotyping methods. The results of genetic characterization of the isolated virus showed that it was the H3N2 subtype and was designated as A/Turkey/OH/313053/04 (H3N2). Phylogenetic analysis of the eight gene segments of the virus showed that A/Turkey/OH/313053/04 (H3N2) isolate was most closely related to the triple-reassortant H3N2 swine viruses [A/Swine/WI/14094/99 (H3N2)] that have been circulating among pigs in the United States since 1998, which contains gene segments from avian, swine, and human viruses. The A/Turkey/OH/313053/04 (H3N2) isolated from turkeys in this study was classified as a low pathogenic avian influenza A virus because it only caused a drop in egg production with minor other clinical signs and no mortality.