Can Bacteriotherapy Using Commercially Available Probiotics, Prebiotics, and Organic Acids Ameliorate the Symptoms Associated With Runting-Stunting Syndrome in Broiler Chickens?

Runting-stunting syndrome (RSS) in poultry has been known for more than 40 years, but the precise etiology remains unknown and a licensed vaccine is consequently not currently available. In order to mitigate the symptoms associated with RSS, a series of experiments was performed to investigate whether a combined bacteriotherapeutic treatment consisting of probiotics, prebiotics, and organic acids could influence the outcome of this disease. Initially two groups of commercial broiler chickens were either left uninoculated or inoculated with filtrate from homogenized intestines of RSS-affected broiler chickens. One group from each of these two challenge groups was treated, with a bacteriotherapeutic regimen. After 12 days chickens were euthanatized, the body weight was measured, and duodenal lesions were enumerated. Five consecutive broiler chicken flocks were then raised either on litter from RSS-affected birds or on fresh wood shavings. Treatment had no beneficial effect on the number and severity of intestinal lesions. There appeared to be a significant build-up of RSS agent(s) in poultry litter, with each consecutive flock placement, independent of bacteriotherapeutic treatment, as more individuals exhibited intestinal lesions on built-up litter in RSS-affected houses (28.9% vs. 44%). While treatment did not appear to consistently reduce intestinal lesions, it did significantly improve the mean body weights (P<0.05) and uniformity of 12-day-old chickens placed on reused litter in houses in which RSS-infected birds were previously raised. A combination of litter management and bacteriotherapy may be needed to ameliorate the adverse effects of RSS on intestinal health and body weight in broiler chickens.

Comparative full genome analysis of four infectious laryngotracheitis virus (Gallid herpesvirus-1) virulent isolates from the United States.

Gallid herpesvirus-1 (GaHV-1), commonly named infectious laryngotracheitis (ILT) virus, causes the respiratory disease in chickens known as ILT. The molecular determinants associated with differences in pathogenicity of GaHV-1 strains are not completely understood, and a comparison of genomic sequences of isolates that belong to different genotypes could help identify genes involved in virulence. Dideoxy sequencing, 454 pyrosequencing and Illumina sequencing-by-synthesis were used to determine the nucleotide sequences of four genotypes of virulent strains from GaHV-1 groups I-VI. Three hundred and twenty-five open reading frames (ORFs) were compared with those of the recently sequenced genome of the Serva vaccine strain. Only four ORFs, ORF C, U(L)37, ICP4 and U(S)2 differed in amino acid (aa) lengths among the newly sequenced genomes. Genome sequence alignments were used to identify two regions (5' terminus and the unique short/repeat short junction) that contained deletions. Seventy-eight synonymous and 118 non-synonymous amino acid substitutions were identified with the examined ORFs. Exclusive to the genome of the Serva vaccine strain, seven non-synonymous mutations were identified in the predicted translation products of the genes encoding glycoproteins gB, gE, gL and gM and three non-structural proteins U(L)28 (DNA packaging protein), U(L)5 (helicase-primase) and the immediate early protein ICP4. Furthermore, our comparative sequence analysis of published and newly sequenced GaHV-1 isolates has provided evidence placing the cleavage/packaging site (a-like sequence) within the inverted repeats instead of its placement at the 3' end of the U(L) region as annotated in the GenBank's entries NC006623 and HQ630064.

Characterization of infectious bursal disease viruses isolated in 2007 from Delmarva commercial broiler chickens.

A study was performed in 2007 to isolate and characterize infectious bursal disease viruses (IBDVs) in commercial broilers grown in the Delmarva (DMV) Peninsula region of the United States. Bursae of Fabricius were collected weekly from 1 to 4 wk of age from broilers on 10 farms with a history of poor performance. Microscopic pathology was used to determine the infectious bursal disease (IBD) status of the broilers. Bursae from 1- and 2-wk-old broilers did not show IBD microscopic lesions. Moreover, broilers on 1 of the 10 farms were IBD lesion free at 3 and 4 wk of age. However, 3 of 9 and 9 of 9 farms yielded broilers with IBD-affected bursae from 3- and 4-wk-old commercial broilers, respectively. Ten IBDV isolates were recovered from 3 of 3 lesion-positive bursal pools at 3 wk of age and 7 of 9 lesion-positive bursal pools at 4 wk of age. Analysis of the viral protein (VP) 2 genes identified all isolates as serotype 1 Delaware (Del) variant viruses. Five field isolates, each representing different molecular clades of the Delaware variant viruses, were selected for further study. Experimental infection of specific-pathogen-free white leghorn chickens with isolates DMV/4813/07, DMV/4947/07, DMV/4955/07, DMV/5038/07, and DMV/5041/07 produced gross and microscopic pathology of the bursa consistent with Delaware variant infection. Monoclonal antibody testing showed DMV/4813/07, DMV/4947/07, DMV/ 4955/07, and DMV/5041/07 to be similar to previous recognized variant viruses. However, DMV/5038/07 was found to be unreactive with the monoclonal antibodies that typically recognize reference strains STC, Del E, GLS, RS593, and AL2. In a challenge of immunity study, 10-day-old progeny from breeders immunized with a commercially available inactivated IBDV vaccine containing the Del E and classic strains were protected to a lesser degree against isolate DMV/5038/07 compared to Del E challenge based on microscopic lesion scores (P < 0.01) of the bursa. This result suggests the virus is antigenically different from the Del E strain contained in the vaccine. Collectively, the monoclonal antibody and progeny challenge of immunity findings suggest DMV/5038/07 is antigenically different from the Del E strain contained in the vaccine.

Molecular characterization of avian astroviruses.

Astroviruses are frequently associated with enteric diseases in poultry, being isolated from cases of runting-stunting syndrome (RSS) of broiler chickens, poult enteritis complex (PEC), and poult enteritis mortality syndrome (PEMS) of turkeys. Currently, five types of avian astrovirus have been identified: turkey astroviruses 1 and 2 (TAstV-1, TAstV-2), avian nephritis virus (ANV), chicken astrovirus (CAstV) and duck astrovirus (DAstV). The objective of this study was to molecularly characterize the different types of avian astroviruses circulating in commercial poultry. Sequence analysis of a region of ORF2, which encodes the capsid precursor protein associated with serotype and viral pathogenesis, revealed extensive variation in amino acid sequence within each subtype: TAstV-2 (81.5%-100%), ANV (69.9%-100%), and CAstV (85.3%-97.9%). However, this region was more conserved in TAstV-1's (96.2%-100%). Furthermore, a novel astrovirus was detected in chicken samples and found to be <64% similar to ANV and <30.6% similar to CAstV. The results of this study underline the great genetic variability of avian astroviruses and indicate that there are most likely multiple serotypes of each avian astrovirus circulating in commercial poultry.

Variability of NS1 proteins among H9N2 avian influenza viruses isolated in Israel during 2000-2009.

The main aims of the present study were to characterize NS1 protein from H9N2 avian influenza viruses (AIVs) isolated in Israel and to investigate the possibility to use NS1-based indirect ELISA. To achieve these purposes, the non-structural gene (NS1) of 79 AIVs of the H9N2 subtype isolated in Israel in 2000-2009 was sequenced and genetically analyzed. The phylogenetic analysis demonstrated that four distinct introductions of H9N2 occurred in Israel during this period. Analysis of the inferred amino acid sequences of the NS1 proteins showed high, about 10%, differences between viruses of the 3rd and 4th introductions. Antibodies against NS1 protein in immune sera were tested by means of indirect ELISA using recombinant NS1 as antigen. Immune sera were obtained from experimentally H9N2-infected chicken after infection on 4, 7, 10, 14, and 21 days. All sera from chickens experimentally infected with 3rd- or 4th-introduction AIV contained anti-NS1 antibodies that were detected by enzyme-linked immunosorbent assay (NS1-ELISA) even though the recombinant NS1 used as antigen for NS1-ELISA differed significantly in its amino acid sequences from the NS1 protein of AIV that caused infection in experimental birds. These findings indicate that the sites of the NS1 protein by which viruses belonging to 3rd and 4th introduction are out of antigenic epitope positions were responsible for the results of NS1-based iELISA.

Development and evaluation of an avian influenza, neuraminidase subtype 1, indirect enzyme-linked immunosorbent assay for poultry using the differentiation of infected from vaccinated animals control strategy.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed using baculovirus, purified, recombinant N1 protein from A/chicken/Indonesia/PA7/2003 (H5N1) virus. The N1-ELISA showed high selectivity for detection of N1 antibodies, with no cross-reactivity with other neuraminidase subtypes, and broad reactivity with sera to N1 subtype isolates from North American and Eurasian lineages. Sensitivity of the N1-ELISA to detect N1 antibodies in turkey sera, collected 3 wk after H1N1 vaccination, was comparable to detection of avian influenza antibodies by the commercial, indirect ELISAs ProFLOK AIV Plus ELISA Kit (Synbiotics, Kansas City, MO) and Avian Influenza Virus Antibody Test Kit (IDEXX, Westbrook, ME). However, 6 wk after vaccination, the Synbiotics ELISA kit performed better than the N1-ELISA and the IDEXX ELISA kit. An evaluation was made of the ability of the N1-ELISA to discriminate vaccinated chickens from subsequently challenged chickens. Two experiments were conducted, chickens were vaccinated with inactivated H5N2 and H5N9 viruses and challenged with highly pathogenic H5N1 virus, and chickens were vaccinated with recombinant poxvirus vaccine encoding H7 and challenged with highly pathogenic H7N1 virus. Serum samples were collected at 14 days postchallenge and tested by hemagglutination inhibition (HI), quantitative neuraminidase inhibition (NI), and N1-ELISA. At 2 days postchallenge, oropharyngeal swabs were collected for virus isolation (VI) to confirm infection. The N1-ELISA was in fair agreement with VI and HI results. Although the N1-ELISA showed a lower sensitivity than the NI assay, it was demonstrated that detection of N1 antibodies by ELISA was an effective and rapid assay to identify exposure to the challenge virus in vaccinated chickens. Therefore, N1-ELISA can facilitate a vaccination strategy with differentiation of infected from vaccinated animals using a neuraminidase heterologous approach.

Establishment of a competitive ELISA (cELISA) system for the detection of influenza A virus nucleoprotein antibodies and its application to field sera from different species.

A recombinant baculovirus (RBV) encoding the nucleoprotein (NP) of avian influenza virus (AIV) was generated and the appropriate protein was expressed in Sf9 cells. Purified recombinant NP and the NP-specific monoclonal antibody HB65 were used to establish a competitive ELISA (cELISA) system for the detection of NP-specific antibodies in sera of ducks, geese and wild birds. Tests to evaluate this method were carried out using sera of ducks experimentally infected with AIV, pre-immune duck and chicken sera, and poultry field sera, which tested negative in the haemagglutination inhibition (HI) assay, and field sera of several poultry species experimentally infected with other viruses. The evaluation of the test demonstrated a high sensitivity and specificity of this method. Tests carried out using field sera of duck and goose flocks revealed widely corresponding results obtained by HI assay and cELISA indicating that this test is applicable for flock diagnosis. Differing results were obtained for individual samples. It can be assumed that for the most part this was because of a better recognition of the conserved NP antigen by serum antibodies, although some results remained unclear.

Neurotropism of highly pathogenic avian influenza virus A/chicken/Indonesia/2003 (H5N1) in experimentally infected pigeons (Columbia livia f. domestica).

This investigation assessed the susceptibility of experimentally infected pigeons to the highly pathogenic avian influenza virus (HPAIV) H5N1 that caused recent outbreaks of avian influenza in birds and humans in several countries of Asia. For this purpose 14 pigeons were infected ocularly and nasally with 10(8) EID50 and clinical signs were recorded and compared with five chickens infected simultaneously as positive controls. The chickens demonstrated anorexia, depression, and 100% mortality within 2 days postinoculation. Three of the pigeons died after a history of depression and severe neurological signs consisting of paresis to paralysis, mild enteric hemorrhage, resulting in a mortality of 21%. Gross lesions in these pigeons were mild and inconsistent. Occasionally subcutaneous hyperemia and hemorrhage and cerebral malacia were observed. Microscopic lesions and detection of viral antigen were confined to the central nervous system of these pigeons. In the cerebrum and to a minor extent in the brain stem a lymphohistiocytic meningoencephalitis with disseminated neuronal and glial cell necrosis, perivascular cuffing, glial nodules, and in one bird focally extensive liquefactive necrosis could be observed. The remaining nine pigeons showed neither clinical signs nor gross or histological lesions associated with avian influenza, although seroconversion against H5 indicated that they had been infected. These results confirm that pigeons are susceptible to HPAIV A/chicken/Indonesia/2003 (H5N1) and that the disease is associated with the neurotropism of this virus. Although sentinel chickens and most pigeons did not develop disease, further experiments have to elucidate whether or not Columbiformes are involved in transmission and spread of highly pathogenic avian influenza.

Complete nucleotide sequences of 5'- and 3'-noncoding regions of both genome segments of different strains of infectious bursal disease virus.

The terminal sequences of both genomic dsRNA segments of different strains of infectious bursal disease virus were determined. Analysis of the linear sequences revealed in the 5'-noncoding region of both segments the presence of a 32-nucleotide sequence with motifs conserved between segments and serotypes. The 3'-noncoding regions showed a high degree of conservation within segment A or B, but differed between segments. Different inverted terminal repeats were detected in both segments. A sequence of 13 nucleotides found in the 5'-noncoding regions of both segments could function as a binding site for chicken 18 S rRNA. Secondary structure prediction of the different noncoding regions showed a high degree of similarity. Surprisingly, the predicted secondary structure of the 5'-noncoding region of segment A differed between serotypes. A possible influence of these different RNA structures on viral replication is discussed.

Synthetic transcripts of double-stranded Birnavirus genome are infectious.

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines.

VP5 of infectious bursal disease virus is not essential for viral replication in cell culture.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, encodes in its bisegmented double-stranded RNA genome four structural virion proteins, VP1, VP2, VP3, and VP4, as well as a nonstructural protein, VP5. Recently, the establishment of an infectious cRNA system for IBDV has been described (E. Mundt and V. N. Vakharia, Proc. Natl. Acad. Sci. USA 93:11131-11136, 1996). Here, we report the isolation of a VP5- IBDV mutant constructed by site-directed mutagenesis of the methionine start codon of VP5, followed by cRNA transfection. The resulting virus mutant was replication competent in cell culture, which indicates that VP5 is not required for productive replication of IBDV. Absence of VP5 expression was verified by lack of reactivity with newly established anti-VP5 monoclonal antibodies and polyclonal sera. VP5- IBDV exhibited a delay in replication in chicken embryo cells compared to the VP5+ parental virus. However, final yields were similar. Our results thus show that VP5 is nonessential for IBDV replication, which makes it a prime candidate for the construction of deleted, marked vaccines.

Identification of a novel viral protein in infectious bursal disease virus-infected cells.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae, specifies two genomic double-stranded RNAs, segment A and segment B. Segment A encodes a 110 kDa polyprotein which is processed into virus proteins VP2, VP3 and VP4. A second open reading frame (ORF), designated ORF A-2, immediately preceding and partially overlapping the 110 kDa protein gene has also been described. After prokaryotic expression of this ORF and immunization of rabbits with the expressed protein we obtained reagents that allowed the identification of the ORF A-2 gene product in IBDV-infected cells. The ORF A-2 protein exhibits an apparent molecular mass of 21 kDa which is larger than the size of 16.5 kDa calculated from the deduced amino acid sequence. Immunofluorescence studies demonstrated the presence of the ORF A-2 protein in bursa samples from IBDV-infected chicken. In summary, the IBDV ORF A-2 product represents the fifth IBDV protein described. Therefore, we propose to designate it as IBDV VP5.

A non-canonical lon proteinase lacking the ATPase domain employs the ser-Lys catalytic dyad to exercise broad control over the life cycle of a double-stranded RNA virus.

We have identified a region related to the protease domain of bacterial and organelle ATP-dependent Lon proteases in virus protein 4 (VP4) of infectious bursal disease virus strain P2 (IBDVP2), a two-segmented double-stranded RNA virus. Unlike canonical Lons, IBDVP2 VP4 possesses a proteinase activity though it lacks an ATPase domain. Ser652 and Lys692 of IBDVP2 VP4 are conserved across the Lon/VP4 family and are essential for catalysis. Lys692 has the properties of a general base, increasing the nucleophilicity of Ser652; a similar catalytic dyad may function in the other Lons. VP4 can cleave in trans and is responsible for the interdomain proteolytic autoprocessing of the pVP2- VP4-VP3 polyprotein encoded by RNA segment A. VP2, which is later derived from pVP2, and VP3 are major capsid proteins of birnaviruses. Results of the characterization of a range of the IBDVP2 VP4 mutants in cell cultures implicate VP4 in trans-activation of the synthesis of VP1, putative RNA-dependent RNA polymerase encoded by RNA segment B, and in cleavage rate-dependent control of process(es) crucial for the generation of the infectious virus progeny.

Complete genomic sequence of viral hemorrhagic septicemia virus, a fish rhabdovirus.

The complete nucleotide sequence of the fish rhabdovirus viral hemorrhagic septicemia virus (VHSV) has been determined. The genome comprises 11158 bases and contains six long open reading frames encoding the nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G, nonstructural viral protein NV, and polymerase L. Genes are arranged in the order 3'-N-P-M-G-NV-L-5'. The exact 3' and 5' ends were determined after RNA-oligonucleotide ligation or RACE. They show inverse complementarity as in other rhabdovirus genomes. Nucleotide and deduced amino acid sequences exhibit significant homology to corresponding sequences in the related fish rhabdovirus infectious hematopoietic necrosis virus.

Pseudorabies virus UL37 gene product is involved in secondary envelopment.

Herpesvirus envelopment is a two-step process which includes acquisition of a primary envelope resulting from budding of intranuclear capsids through the inner nuclear membrane. Fusion with the outer leaflet of the nuclear membrane releases nucleocapsids into the cytoplasm, which then gain their final envelope by budding into trans-Golgi vesicles. It has been shown that the UL34 gene product is required for primary envelopment of the alphaherpesvirus pseudorabies virus (PrV) (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063-10073, 2000). For secondary envelopment, several virus-encoded PrV proteins are necessary, including glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). We show here that the product of the UL37 gene of PrV, which is a constituent of mature virions, is involved in secondary envelopment. Replication of a UL37 deletion mutant, PrV-DeltaUL37, was impaired in normal cells; this defect could be complemented on cells stably expressing UL37. Ultrastructural analysis demonstrated that intranuclear capsid maturation and budding of capsids into and release from the perinuclear space were unimpaired. However, secondary envelopment was drastically reduced. Instead, apparently DNA-filled capsids accumulated in the cytoplasm in large aggregates similar to those observed in the absence of glycoproteins E/I and M but lacking the surrounding electron-dense tegument material. Although displaying an ordered structure, capsids did not contact each other directly. We postulate that the UL37 protein is necessary for correct addition of other tegument proteins, which are required for secondary envelopment. In the absence of the UL37 protein, capsids interact with each other through unknown components but do not acquire the electron-dense tegument which is normally found around wild-type capsids during and after secondary envelopment. Thus, apposition of the UL37 protein to cytoplasmic capsids may be crucial for the addition of other tegument proteins, which in turn are able to interact with viral glycoproteins to mediate secondary envelopment.

Alteration of amino acids in VP2 of very virulent infectious bursal disease virus results in tissue culture adaptation and attenuation in chickens.

Reverse genetics technology offers the possibility to study the influence of particular amino acids of infectious bursal disease virus (IBDV) on adaptation to tissue culture. Genomic segments A and B of the very virulent (vv) IBDV field strain UK661 were completely cloned and sequenced, and the strain was rescued from full-length cDNA copies of both segments (UK661rev). Using site-directed mutagenesis, alteration of a single amino acid in the segment A-encoded VP2 (A284T) resulted in a limited capacity of UK661 to replicate in tissue culture. Additional alteration of a second amino acid (Q253H) increased replication efficiency in tissue culture. The second mutant (UK661-Q253H-A284T) was used to infect chickens and results were compared with UK661 and UK661rev. Whereas UK661 and UK661rev induced 100% morbidity and 50-80% mortality, UK661-Q253H-A284T proved to be strikingly attenuated, producing neither morbidity nor mortality. Moreover, UK661-Q253H-A284T-infected animals were protected from challenge infection. Thus, alteration of two specific amino acids in the VP2 region of IBDV resulted in tissue culture adaptation and attenuation in chickens of vvIBDV. The data demonstrate that VP2 plays a decisive role in pathogenicity of IBDV.

A second form of infectious bursal disease virus-associated tubule contains VP4.

Preparations of density gradient-purified infectious bursal disease virus (IBDV) were found to contain full and empty icosahedral virions, type I tubules with a diameter of about 60 nm, and type II tubules 24 to 26 nm in diameter. By immunoelectron microscopy we demonstrate that virions and both types of tubular structures specifically react with anti-IBDV serum. In infected cells intracytoplasmic and intranuclear type II tubules reacted exclusively with an anti-VP4 monoclonal antibody, as did type II tubules in virion preparations. The immunofluorescence pattern with the anti-VP4 antibody correlated with electron microscopical findings. Neither purified extracellular nor intracellular virions were labeled with the anti-VP4 MAb. Our data show that the type II tubules contain VP4 and suggest that VP4 is not part of the virus particle.

Chimeras in noncoding regions between serotypes I and II of segment A of infectious bursal disease virus are viable and show pathogenic phenotype in chickens.

Two serotypes, I and II, have been identified for infectious bursal disease virus (IBDV), a member of the family BIRNAVIRIDAE: Here, the generation by reverse genetics of IBDV chimeras in segment A of the bisegmented genome is reported. The 5- and 3'-noncoding regions (NCRs) of a serotype II strain were exchanged with the NCRs of a full-length cDNA clone of segment A of a serotype I strain. Isolated chimeric viruses were characterized in cell culture and susceptible chickens. The results show that IBDV chimeras in segment A were able to replicate in cell culture and that VP1 encoded by a serotype I segment B is functionally active with serotype I NCRs as well as with serotype II NCRs. Chimeric viruses infected susceptible chickens and caused mild depletion of bursal cells. Thus, the noncoding regions of segment A are not responsible for the different pathotypes of IBDV serotypes I and II.