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Escherichia coli (E. coli) are typically present as commensal bacteria in the gastro-intestinal tract of most animals including poultry species, but some avian pathogenic E. coli (APEC) strains can cause localized and even systematic infections in domestic poultry. Emergence and re-emergence of antimicrobial resistant isolates (AMR) constrain antibiotics usage in poultry production, and development of an effective vaccination program remains one of the primary options in E. coli disease prevention and control for domestic poultry. Thus, understanding genetic and pathogenic diversity of the enzootic E. coli isolates, particularly APEC, in poultry farms is the key to designing an optimal vaccine candidate and to developing an effective vaccination program. This study explored the genomic and pathogenic diversity among E. coli isolates in southern United States poultry. A total of nine isolates were recovered from sick broilers from Mississippi, and one from Georgia, with epidemiological variations among clinical signs, type of housing, and bird age. The genomes of these isolates were sequenced by using both Illumina short-reads and Oxford Nanopore long-reads, and our comparative analyses suggested data from both platforms were highly consistent. The 16 s rRNA based phylogenetic analyses showed that the 10 bacteria strains are genetically closer to each other than those in the public database. However, whole genome analyses showed that these 10 isolates encoded a diverse set of reported virulence and AMR genes, belonging to at least nine O:H serotypes, and are genetically clustered with at least five different groups of E. coli isolates reported by other states in the United States. Despite the small sample size, this study suggested that there was a large extent of genomic and serological diversity among E. coli isolates in southern United States poultry. A large-scale comprehensive study is needed to understand the overall genomic diversity and the associated virulence, and such a study will be important to develop a broadly protective E. coli vaccine., (© 2023. The Author(s).)

Effects of dietary Original XPC (XPC) on 17 selected blood variables in commercial layer pullets challenged with the virulent, low-passage R strain of Mycoplasma gallisepticum (R low MG) were investigated. Hy-Line W-36 pullets sourced from M. gallisepticum-clean layer breeders were fed a basal diet with XPC (1.25 kg/metric ton) or without from hatch until 12 wk of age (woa). At 8 and 10 woa, half of the birds in each dietary treatment were challenged with R low MG. Blood samples were taken immediately before the initial R low MG challenge at 8 woa and again at 12 woa (4 wk after challenge). At 8 woa, blood pH was lower and glucose concentration was higher in the preassigned challenge treatment groups. At 12 woa, the concentration of oxygen dissolved in the blood was significantly lower in the R low MG-challenged group than the unchallenged group of birds regardless of dietary treatment. The R low MG challenge significantly increased blood carbon dioxide partial pressure, calcium, sodium, anion gap, osmolality, glucose, and corticosterone levels but significantly decreased blood oxygen partial pressure, oxyhemoglobin concentration, concentration of oxygen dissolved in the blood, chloride, and pH levels. Because blood pH and glucose concentration at 8 woa were examined before challenge, their baseline values were biased with respect to challenge treatment before treatment was applied. However, the lack of a significant main effect due to diet at 8 woa for blood pH and glucose concentration, along with the other 15 blood variables, indicate that the baseline data with respect to dietary treatment were unbiased, allowing for real dietary effects to be accurately assessed. In conclusion, layer pullets challenged with R low MG undergo a stress response associated with changes in various physiological blood variables, and a decrease in pH and increase in carbon dioxide partial pressure, in association with a lack of change in bicarbonate, indicates that the stress response caused by the R low MG challenge was associated with respiratory acidosis. Nevertheless, feeding XPC did not influence the effects of challenge treatment on these postchallenge physiological blood values., (Copyright © 2020. Published by Elsevier Inc.)

Effects of dietary Original XPC (XPC) in commercial layer pullets challenged with the virulent, low passage R strain of Mycoplasma gallisepticum (R low MG) were investigated. Hy-Line W-36 pullets sourced from MG-clean breeders were fed a basal diet with or without (CON) XPC (1.25 kg/metric ton) from hatch until 12 wk of age (woa). At 8 and 10 woa, half of the birds in each dietary treatment were challenged with R low MG. Body weight was recorded at 3, 8, and 12 woa, and ovary, ceca, and bursa weights were recorded at 3 and 12 woa. Blood samples were taken immediately before the initial R low MG challenge at 8 woa and again at 12 woa to test for IgM and IgG antibody production against MG. All birds were evaluated for MG lesion scores at 12 woa. Regardless of challenge, inclusion of XPC in the diet did not significantly alter BW at 3 or 8 woa or relative organ weights at 3 or 12 woa. However, at 12 woa, BW of XPC-fed birds, regardless of challenge was significantly (P = 0.0038) heavier than CON by 25.7 g. All birds tested negative for MG antibodies before the 8 woa challenge. Respective percentage serum plate agglutination and ELISA positive birds at 12 woa were 0 and 0% (CON, nonchallenged), 1.4 and 0% (XPC, nonchallenged), 100 and 47.2% (CON, challenged), and 100 and 50.0% (XPC, challenged). Diet did not significantly affect ELISA titers, but they were significantly (P < 0.0001) increased due to challenge. Furthermore, lesion scores were significantly higher for R low MG-challenged birds (P = 0.0012), and dietary treatment with XPC in challenged birds numerically reduced MG lesion scores from 0.278 to 0.194. In conclusion, although dietary XPC did not significantly alter the humoral immune response, antibody titer levels, or severity of MG lesions in layer pullets that were or were not challenged with R low MG, it led to an increase in their rate of growth through 12 woa., (Copyright © 2020. Published by Elsevier Inc.)

The chicken bursa of Fabricius is a primary lymphoid tissue important for B-cell development. Our long-term goal is to understand the role of bursal microenvironment in an early B-cell differentiation event initiating repertoire development through immunoglobulin gene conversion in the chick embryo. We hypothesize that early bursal B-cell differentiation is guided by signals through cytokine receptors. Our theory is based on previous evidence for expression of the receptor tyrosine kinase superfamily members and interleukin receptors in unseparated populations of bursal B-cells and bursal tissue. Knowledge of the expressed genes that are responsible for B-cell differentiation is a prerequisite for understanding the bursal microenvironment's function. This project uses transcriptomic analysis to evaluate gene expression across early B-cell development. RNA-seq was performed with total RNA isolated from bursal B-cells at embryonic day (ED) 16 and ED 19 (n = 3). Approximately 90 million high-quality clean reads were obtained from the cDNA libraries. The analysis revealed differentially expressed genes involved in the Jak-STAT pathway, Wnt signaling pathway, MAPK signaling pathway, metabolic pathways including tyrosine metabolism, Toll-like receptor signaling pathway, and cell-adhesion molecules. The genes predicted to encode surface receptors, signal transduction proteins, and transcription factors identified in this study represent gene candidates for controlling B-cell development in response to differentiation factors in the bursal microenvironment., (© The Author(s) 2019. Published by Oxford University Press on behalf of Poultry Science Association.)

The chicken as a research model has a disadvantage compared with the mouse and the human because of the low number of available antibodies against gene products of interest. The goal of this study was to identify the antigen recognized by monoclonal antibody (mAb) GIIF3, which is a 42 kDa protein that appears in follicle-associated epithelium of the guinea hen as well as in different muscle types during chicken embryonic development. The 42 kDa protein, immunoprecipitated from chicken gizzard protein lysates, was evaluated by mass spectrometry. Mass spectrometry analysis revealed peptides specific for the chicken β- or γ-actin isoforms. The mAb GIIF3 can be used as a new research tool for smooth muscle cell and bursa of Fabricius developmental studies., (© 2016 Poultry Science Association Inc.)

Besides humans, H3 subtypes of influenza A viruses (IAVs) can infect various animal hosts, including avian, swine, equine, canine, and sea mammal species. These H3 viruses are both antigenically and genetically diverse. Here, we characterized the antigenic diversity of contemporary H3 avian IAVs recovered from migratory birds in North America. Hemagglutination inhibition (HI) assays were performed on 37 H3 isolates of avian IAVs recovered from 2007 to 2011 using generated reference chicken sera. These isolates were recovered from samples taken in the Atlantic, Mississippi, Central, and Pacific waterfowl migration flyways. Antisera to all the tested H3 isolates cross-reacted with each other and, to a lesser extent, with those to H3 canine and H3 equine IAVs. Antigenic cartography showed that the largest antigenic distance among the 37 avian IAVs is about four units, and each unit corresponds to a 2 log&thinsp;2 difference in the HI titer. However, none of the tested H3 IAVs cross-reacted with ferret sera derived from contemporary swine and human IAVs. Our results showed that the H3 avian IAVs we tested lacked significant antigenic diversity, and these viruses were antigenically different from those circulating in swine and human populations. This suggests that H3 avian IAVs in North American waterfowl are antigenically relatively stable.

Currently available ELISAs used to diagnose Mycoplasma hyopneumoniae infection in pigs have high specificity but low sensitivity. To develop more sensitive assays, the kinetics of specific serum IgG and respiratory mucosal sIgA responses against three M. hyopneumoniae antigens, namely, P97R1 (an adhesin protein), P46 (a membrane protein), and P36 (a cytosolic protein), were characterised over 133 days following experimental infection. Immunoglobulin G against the three proteins remained at high concentrations from 28 to 133 days post-infection (dpi), although IgG against P97R1 was detected earlier and was more reactive than the other two antigens under assessment. Mucosal sIgA appeared earlier than serum IgG but did not persist as long; sIgA concentrations against P97R1 were the highest. Seroconversion was detected 2 weeks earlier with the P97R1-based ELISA than with a commercially available ELISA. On analysis of serum samples from five pig farms that did not use a M. hyopneumoniae vaccine, the P97R1-based IgG ELISA demonstrated a 73.6% coincidence rate with the commercial kit. Moreover, this more specific P97R1-based ELISA detected more positive samples than the commercial kit (52.8% vs. 39.2%). It was concluded that the systemic immune response to M. hyopneumoniae infection in pigs was delayed in onset but persistent whereas the mucosal response developed more rapidly but was less sustained. The P97R1 antigen was identified as a suitable serological marker for diagnosing M. hyopneumoniae infection in pigs, particularly early stage infection., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mycoplasma hyopneumoniae (M. hyopneumoniae) causes a chronic respiratory disease with high morbidity and low mortality in swine, and has been presented as a major cause of growth retardation in the swine industry. Aerosol vaccination presents a needle free, high throughput, and efficient platform for vaccine delivery, and has been widely applied in poultry vaccination. However, aerosol vaccines have rarely been used in swine vaccination primarily because the long and curving respiratory track of swine presents a barrier for vaccine particle delivery. To develop an effective M. hyopneumoniae aerosol vaccine, three major barriers need to be overcome: to optimize particle size for aerosol delivery, to maintain the viability of mycoplasma cells in the vaccine, and to optimize the environmental conditions for vaccine delivery. In this study, an aerosol mycoplasma vaccine was successfully developed based on a conventional live attenuated M. hyopneumoniae vaccine. Specifically, the Pari LCD nebulizer was used to produce an aerosol vaccine particle size less than 5 μm; and a buffer with 5% glycerol was developed and optimized to prevent inactivation of M. hyopneumoniae caused by aerosolization and evaporation. Before nebulization, the room temperature and relative humidity were control to 20-25 °C and 70-75%, respectively, which helped maintain the viability of aerosol vaccine. Animal experiments demonstrated that this newly developed aerosol vaccine was effectively delivered to swine low respiratory track, being confirmed by nested-PCR, in situ hybridization and scanning electron microscope. Moreover, M. hyopneumoniae specific sIgA secretion was detected in the nasal swab samples at 14 days post-immunization. To our knowledge, this is the first report on a live M. hyopneumoniae aerosol vaccine., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Two simultaneous trials were conducted using a commercially available, live, F strain Mycoplasma gallisepticum (FMG) vaccine (trial 1) or 2 inocula of layer complex-derived MG strains (LCD-MG; trial 2). In each of the 2 trials, 4 commercial turkeys were housed in each of 2 adjoining pens immediately adjacent to air inlets. The turkeys (8/trial) were inoculated in the right eye with either a 1× dose of FMG (trial 1) or with 0.02 mL of 1 of 2 actively growing LCD-MG inocula (4 turkeys/inoculum; trial 2). In each of the 2 trials, one pen housing 4 inoculated turkeys was maintained without the addition of other poultry, whereas 16 MG-free broilers and 4 MG-free layers were added to the other pen of 4 inoculated turkeys. Within each of the trials and at increasing intervals, either 4 layers (3 pens) or 4 turkeys (3 pens) were placed down-airstream from the inoculated pens. The distance of the first pen from the inoculated turkeys was separated by the width of one pen that was empty. Succeeding down-airstream pens were situated such that the empty distance (absence of any poultry) between pens that contained poultry doubled from one pen to the next such that the final pen that contained poultry had 4 empty pens between it and the next up-airstream pen that also contained poultry. At 106 d postinoculation, all poultry were bled, swabbed for MG from the choanal cleft, and then euthanized and necropsied. No commingled poultry in trial 1 (FMG), whether inoculated (turkeys) or commingled (layers and broilers), died during the course of the trial, and 5 of the 8 FMG-vaccinated turkeys exhibited serological but not cultural evidence of mycoplasmosis. In trial 2 (LCD-MG), 2 commingled broilers died and no inoculated turkeys exhibited either serological or cultural evidence of mycoplasmosis. In both trials, no poultry housed down-airstream from the inoculated poultry showed evidence of clinical signs of mycocplasmosis and none showed either serological or cultural evidence of mycoplasmosis.

Different from most other host-specific mycoplasmas, Mycoplasma gallinarum has been isolated from various hosts, such as poultry, pig, cattle, and sheep. The wide distribution among different hosts, the low pathogenesis, and the weak host immunological responses suggest this mycoplasma has a unique host adaptation mechanism. In this study, we applied two-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (2D LC-MS/MS) to characterize the protein profiling of M. gallinarum. Our results suggest that M. gallinarum possesses homologs of cytadhesin proteins found in other mycoplasmas lacking an organized tip organelle. Our results showed that there are possibly multiple aminopeptidase gene homologs present in M. gallinarum, which might be involved in nutrient acquisition of M. gallinarum. The information present here would be useful for future studies to identify genes responsible for the colonization and host adaptation properties of M. gallinarum., ((c) 2010 Elsevier B.V. All rights reserved.)

Vaccination of multi-age layer operations, wherein one million plus commercial layer chickens are housed, has been spurious until the development of a self-propelled, constant-speed spray vaccinator. Still, even with its use, live Mycoplasma gallisepticum (MG) vaccinations have been questionable in terms of seroconversion. Using the vaccinator as a research tool over the past 5 yr, factors have been elucidated which impact seroconversion to one live MG vaccine in particular, the F strain of MG (FMG). These factors include the type of nozzle used to spray the vaccine, the temperature of the water used to rehydrate and administer the vaccine, and the pH and osmolarity of the fluid used to apply the vaccine. In the present study, one farm was monitored for its seroconversion rates over 4 1/2 yr, during which time the FMG vaccination protocol was amended as factors were identified that enhanced seroconversion rates. The results of this study showed that implementation and inclusion of the optimized factors into the vaccination protocol for FMG enhanced seroconversion rates because they went from an initial 50%-55% positive seroconversion rate to a consistent 100% positive seroconversion rate over the 56-mo study period.

The prevalence of and risk factors associated with the shedding of cryptosporidial oocysts were determined for domestic cats in northeastern Mississippi and northwestern Alabama. Cryptosporidial oocysts were found in a single fecal samples from 30 of 250 cats using a centrifugal concentration technique followed by an immunofluorescent assay. The odds of a cat shedding oocysts were increased when another cat shedding oocysts was present in the household. Logistic regression analysis showed only concurrent shedding of Giardia cysts to be significantly associated with the presence of Cryptosporidium oocysts in the feces. Oocysts from 12 cats were successfully genotyped, based on sequencing of a fragment of the 18S rRNA gene, all isolates were Cryptosporidium felis.

The complete annotation of the cattle genome allows reliable protein identification by tandem mass spectrometry (MS(2)) and greatly facilitates proteomics. Previously, we reported that differential detergent fractionation (DDF) analysis of bovine monocytes reveals proteins related to antigen pattern recognition, uptake and presentation to immunocompetent lymphocytes. Here we have identified 47 bovine proteins, involved in immune function of professional antigen-presenting cells (APC) that have been significantly altered after cytopathic (cp) Bovine Viral Diarrhea Virus (BVDV) infection. In particular, proteins related to immune responses such as cell adhesion, apoptosis, antigen uptake, processing and presentation, acute phase response proteins, MHC class I- and II-related proteins and other molecules involved in immune function of professional antigen presentation have been significantly altered after BVDV infection. Our data suggest that cp BVDV, while promoting monocyte activation and differentiation, is inhibiting their antigen presentation to immunocompetent T cells, thus resulting in the uncontrolled inflammation mediated by activated macrophages, enhanced viral spread, and impaired anti-viral defense mechanisms in the host.

We have used noncytopathic (ncp) and cytopathic (cp) Bovine Viral Diarrhea Viruses (BVDV) to determine the expression levels of TLR genes, type I IFN, pro-inflammatory and Th1/Th2 cytokine gene expression in bovine monocytes. In general, both BVDV strains had similar effects. However, we found some significant differences that could be due to biological differences between cp and ncp BVDV strains. TLR3 was significantly up-regulated in 1h ncp, but not in cp BVDV- infected monocytes, whereas TLR7 expression dominated in 24h infection with both BVDV strains. Type I IFN and IL-12 gene expression was also significantly up-regulated in 1h ncp, but not cp BVDV infection that correlated with the enhanced TLR3 gene expression. Both BVDV biotypes suppressed pro-inflammatory cytokines TNF-alpha, IL-1beta, and IL-6, co-stimulatory molecules CD80 and CD86, but did not change Th1 type cytokine IL-12 and INF-gamma, gene expression after 24h infection. We hypothesize that BVDV may escape immune responses by altering the expression of TLR 3 and 7 and their signaling pathways.

Background: Giardia duodenalis is an intestinal flagellated protozoan that affects many mammalian species often causing severe diarrheal disease. Several different genotypes have been identified (Assemblages A-G). Most isolates recovered from domestic cats have been assigned to either Assemblage A, the zoonotic form of the parasite, or Assemblage F, identified thus far only in cats. Genotypic variation within G. duodenalis may influence clinical presentation and course of disease. Therefore, host-adapted genotypes may not be responsible for diarrheal disease (eg, Assemblage F in cats)., Hypothesis: Multiple Giardia genotypes will be present in domestic cats, including Assemblage F, which will not be correlated with clinical signs., Animals: 250 domestic cats from eastern Mississippi and northwestern Alabama., Methods: Prevalence survey. Fecal samples evaluated for cysts using a centrifugation concentration technique and a commercially available direct immunoflourescent antibody kit. Giardia isolates were characterized by PCR amplification and sequencing of the glutamate dehydrogenase gene., Results: Both Assemblage A-I (6/17) and Assemblage F (11/17) were identified. Although Assemblage was significantly associated with age and housing, no association was detected between Assemblage and a variety of other factors including the presence of gastrointestinal signs (acute vomiting, diarrhea, and constipation)., Conclusions and Clinical Importance: The presence of diarrhea in domestic cats with Giardia cannot be used as a predictor of the presence of zoonotic genotypes in animals within the study area. Although Assemblage A was associated with age and housing, veterinarians should consider any isolation of Giardia from domestic cats as potentially zoonotic.

The prevalence of cats shedding Giardia cysts (13.6%) in the present study was found to be higher than previously reported (1% to 11%) and may reflect a higher sensitivity for the diagnostic test used. The presence of Cryptosporidium spp. oocysts, coccidial oocysts, and a clinical history of chronic (>2 weeks) gastrointestinal signs were significantly associated with the presence of Giardia spp. cysts in the feces. There were no associations between the presence of Giardia spp. cysts and type of housing, acute gastrointestinal signs, vomiting, gender, source of cat (i.e., animal shelter versus private breeder), or gastrointestinal parasites other than Cryptosporidium spp. and intestinal coccidial agents.

While advances in proteomics have improved proteome coverage and enhanced biological modeling, modeling function in multicellular organisms requires understanding how cells interact. Here we used the chicken bursa of Fabricius, a common experimental system for B cell function, to model organ function from proteomics data. The bursa has two major functional cell types: B cells and the supporting stromal cells. We used differential detergent fractionation-multidimensional protein identification technology (DDF-MudPIT) to identify 5198 proteins from all cellular compartments. Of these, 1753 were B cell specific, 1972 were stroma specific and 1473 were shared between the two. By modeling programmed cell death (PCD), cell differentiation and proliferation, and transcriptional activation, we have improved functional annotation of chicken proteins and placed chicken-specific death receptors into the PCD process using phylogenetics. We have identified 114 transcription factors (TFs); 42 of the bursal B cell TFs have not been reported before in any B cells. We have also improved the structural annotation of a newly sequenced genome by confirming the in vivo expression of 4006 "predicted", and 6623 ab initio, ORFs. Finally, we have developed a novel method for facilitating structural annotation, "expressed peptide sequence tags" (ePSTs) and demonstrate its utility by identifying 521 potential novel proteins from the chicken "unassigned chromosome".

Four experiments were conducted to evaluate valine (Val) requirements in Ross 508 broilers from 3 to 6 weeks of age. Common diets were fed to broilers until 3 weeks of age. Growth and carcass measurements were taken in all experiments. Immune responsiveness measurements were taken in Experiments 1 and 2. Birds given 7.2 g Val/kg of diet in Experiment 1 had more abdominal fat than birds given 8.2 g Val/kg of diet, but there were no differences in growth or other carcass measurements. Because growth performance was not reduced in birds given 7.2 g Val/kg of diet, Val concentration was reduced to 6.4 g Val/kg of diet in Experiments 2 and 3. Increasing Val from 6.4 to 8.7 g Val/kg of diet resulted in linear increases for BW gain, feed efficiency and Val intake in male birds, and Val intake in female birds. Quadratic responses to increasing dietary Val were not observed in any experiment. There were no effects of Val on innate or adaptive immunity. A nonessential amino acid mixture containing the same nitrogen content as the L-Val additions in Experiment 4 was added and tends to support the idea that the responses to Val were specific and not due to increases in total nitrogen. Dose responses to Val resulted in male, but not female, birds given 7.3 g Val/kg of diet having improved BW gain and feed efficiency compared with birds receiving 6.4 g Val/kg of diet.

Professional antigen presenting cells (APC), dendritic cells (DC) and their myeloid progenitors, monocytes/macrophages are critical controllers of innate and adaptive immunity. Here we show that differential detergent fractionation (DDF) analysis of bovine monocytes reveals proteins related to antigen pattern recognition, uptake and presentation to immunocompetent lymphocytes. We identify 53 bovine proteins involved in immune function of professional APC. In particular, 13 adhesion molecules, three toll-like receptors (TLR1, 6 and 8), three antigen uptake-related proteins (including mannose receptor [MR] precursor), and eight actin-like proteins involved in active endocytosis were identified. In addition, MHC class I and II-related proteins, cytokines, active substances and growth factors have been identified. We conclude that the DDF approach can provide interpretable and meaningful functional information concerning protein expression profiles associated with monocyte activation, transformation into macrophages and/or immature DC, and maturation of monocyte-derived DC in the presence of multiple bovine pathogens.

Differential detergent fractionation (DDF), which relies on detergents to sequentially extract proteins from eukaryotic cells, has been used to increase proteome coverage of 2D-PAGE. Here, we used DDF extraction in conjunction with the nonelectrophoretic proteomics method of liquid chromatography and electrospray ionization tandem mass spectrometry. We demonstrate that DDF can be used with 2D-LC ESI MS2 for comprehensive cellular proteomics, including a large proportion of membrane proteins. Compared to some published methods designed to isolate membrane proteins specifically, DDF extraction yields comprehensive proteomes which include twice as many membrane proteins. Two-thirds of these membrane proteins have more than one trans-membrane domain. Since DDF separates proteins based upon their physicochemistry and subcellular localization, this method also provides data useful for functional genome annotation. As more genome sequences are completed, methods which can aid in functional annotation will become increasingly important.

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

Broilers fed diets with reduced amino acid levels may be limiting in isoleucine. Because research addressing daily Ile needs for broiler immunity is sparse, Ile responses for immunity in female broilers were evaluated in 2 experiments in broilers from 30 to 42 d of age. Cellular and humoral immunity were evaluated in diets limiting in Ile and diets varying in Ile from deficient to adequate. Pen was the experimental unit in both experiments. Treatments in experiment 1 consisted of 2 levels of Ile (0.42 vs. 0.72% total of diet) and 3 strains of broilers, Arbor Acres+, Ross 508, Ross 708 (6 treatments; 5 pens each). In experiment 1, measurements consisted of: a cutaneous basophil hypersensitivity test to phytohemagglutinin-P (PHA-P) on d 37 and 38; cell quantification of CD4+, CD8+, and BU-1+ lymphocytes at d 41 and 42; and relative immune organ weights at 42 d. No Ile x strain interaction occurred. Feeding an Ile-deficient diet to broilers suppressed the cell mediated response to PHA-P, and reduced thymus weight and the percentage of CD8+ T cells. There were no significant differences between strains. In experiment 2, gradations of Ile (0.42, 0.50, 0.58, 0.66, 0.74, and 0.82% total of diet) were fed to one strain (Ross 508) of female broilers (7 pens per diet). A control diet containing 0.70% Ile (6 pens) was compared with an Ile surfeit concentration. Measurements in experiment 2 consisted of a hypersensitivity test to PHA-P on d 35 and 36; a primary antibody response to SRBC from 35 to 42 d; cell quantification of CD8+ alpha, beta, and T cell receptor (TCR)-1 (delta/gamma) lymphocytes on d 41 and 42; and immune organ weights at 42 d. Immunity measurements in birds fed surfeit Ile in the titration diets were equal to birds fed the control diet. A linear response to increasing Ile was obtained for relative bursa, but no Ile quadratic responses were noted for other measurements in experiment 2. Although feeding broilers a diet deficient in Ile suppressed some immune criteria, it does not appear that a marginal Ile deficiency will compromise immunity in growing female broilers.

A clinical study was conducted on commercial layers housed in biological isolation units, within which exogenous stress factors potentially affecting bird performance were minimized. This set-up was devised in order to assess how a pre-lay inoculation of S6 strain Mycoplasma gallisepticum affects the leukocytic properties of laying chickens. Previous studies have demonstrated relative decreases in lymphocyte and relative increases in heterophil percentages in birds infected with other strains of Mycoplasma gallisepticum. However, current results showed that the differential percentages of lymphocytes were decreased, whereas those of heterophils were increased, in both sham-inoculated control birds and birds inoculated with S6 Mycoplasma gallisepticum between 19 and 26 wk of age. This study clearly shows that a pre-lay inoculation of S6 Mycoplasma gallisepticum alone had no apparent effect on the leukocyte profile of commercial layers housed in biological isolation units.

Aminopeptidases (APN) may play a role in host colonization of M. gallinarum. Characterization of endogenous APN activity suggests that the leucine APN (LAP) of M. gallinarum is a metallo-aminopeptidase activated by Mn2+ and is present in the cytosol and possibly associated with the inner leaflet of the membrane. A 1.36-kb open reading frame (ORF) identified from overlapping genomic phage clones showed 68% nucleotide identity and 51% amino acid identity with the M. salivarium LAP gene. This ORF is expressed as a 1.5-kb monocistronic transcript and is present as a single copy in M. gallinarum. This gene sequence was modified to account for codon usage, and expression in E. coli produced a 51-kDa protein, which compares well with the product predicted from the ORF. This ORF is a strong candidate for contributing the LAP activity of M. gallinarum protein extracts.

Fatty liver hemorrhagic syndrome (FLHS) was observed in each of three trials in which commercial layers were utilized to determine the effect of Mycoplasma gallinarum (MGn) on egg and eggshell quality parameters and egg production. In each of three trials, FLHS occurred 31-54 days later in MGn-inoculated hens as compared with the Mycoplasma-clean (control) hens. In trials 1 and 2, no therapeutic intervention was initiated to ameliorate FLHS. In trial 3, therapeutic intervention was instituted and consisted of the addition of 1 pound of choline chloride/ton of feed. Total mortality recorded throughout the duration of each trial and attributable to FLHS was not significantly different between the control and the MGn-inoculated treatment. However, FLHS-associated mortality in each of the three trials was numerically greater for the control treatment.

Mycoplasma gallisepticum (MG), a reproductive/respiratory pathogen in poultry, has been implicated in suboptimum egg production and decreased hatchability. Commercial layer hens raised in a controlled environment were inoculated with the S6 strain of MG at 20 wk of age. The S6 inoculation had no effect on bird weight, egg production, digestive tract weight and length, or histopathologic lesion scores, although significant differences were noted in the lengths and weights of various portions of the reproductive tract. This study shows that S6MG inoculation does not detrimentally affect layer hen performance when in the absence of environmental stressors customary to a caged layer facility.

Feline aminopeptidase N (fAPN) has been shown to serve as a receptor for feline, canine, porcine and human coronaviruses. Our objective was to determine if fAPN can serve as a receptor for infectious bronchitis virus (IBV). Feline kidney cells that express fAPN and hamster kidney fibroblasts that do not express fAPN were inoculated with IBV and monitored for replication by indirect fluorescent assay and confocal microscopy and in chicken embryonated eggs. The results showed that the feline cells were permissive to IBV but the hamster cells were not. The hamster cells became permissive to IBV after transfection with a fAPN cDNA suggesting that the feline APN molecule plays a role in IBV entry.

In each of two trials, 80 commercial leghorn-type pullets were separated into two treatments with four replicates of 10 chickens in each treatment. Forty pullets were designated as controls and received no inoculation, whereas the remaining 40 pullets received the 6/85 vaccine strain of Mycoplasma gallisepticum (MG) at 10 wk of age. Hen-day egg production, egg weight, eggshell strength, Haugh unit score, pimpling incidence, and blood/meat spot incidence were monitored and recorded weekly in each trial through an entire laying cycle of 43 wk. Further, eggs from all treatments were collected daily, Monday-Thursday, and individually weighed. No significant difference was observed between the treatments for 43-wk means for hen-day egg production, for any of the monitored egg or eggshell quality parameters, or for the number of extra large, large, medium, small, pee wee, or undergrade egg sizes. A significant (P < or = 0.05) difference was observed for the number of jumbo-sized eggs between the two treatments. Results of this study suggest that vaccination of commercial layer chickens at 10 wk of age with 6/85 strain MG does not detrimentally impact egg production, egg size distribution, or ovary/oviduct function as evidenced by selected egg parameters monitored in this study.

The purpose of this study was to determine whether bovine immunodeficiency virus (BIV) is vertically transmitted in naturally infected dairy cattle. Twenty-two dam/calf pairs from a Mississippi Agriculture and Forestry Experiment Station dairy were the study group. Blood samples were collected following delivery of calves, the peripheral blood leukocytes were purified from these samples, and the leukocyte DNA was used in polymerase chain reactions targeting the pol gene region of the BIV provirus. Southern blotting and hybridization were used to confirm the BIV specificity of the amplified fragments. BIV provirus was detected in 14 of 22 calves (64%), demonstrating vertical transmission. Eight of the calves were disqualified from the final interpretation of transplacental transfer because they may have nursed their mothers prior to blood collection, allowing the possibility of lactogenic transfer of the virus. Transplacental transmission of BIV was identified in 6 of 22 calves (27%).

Cryptosporidium sp. was found in 3 out of 49 caribou (Rangifer tarandus) from northern Alaska. Segments of both the 18S ribosomal RNA and the heat shock protein genes were amplified from the caribou isolate and compared with that obtained from an isolate from a wild white-tailed deer (Odocoileus virginianus) in Virginia as well as other species and isolates available from GenBank. Analyses showed the white-tailed deer isolate to be identical with the C. parvum cattle genotype; however, the caribou isolate represents a new genotype closely related to C. serpentis, C. muris, and C. andersoni. Giardia sp. was not detected in any of the caribou samples nor was Cryptosporidium sp. or Giardia sp. detected in any of the 42 moose (Alces alces) samples examined.

Live Mycoplasma gallisepticum (MG) vaccines have been USDA approved and licensed for use in commercial layer chickens since 1988; however, egg production and egg quality data exist only for the F strain of MG. Information pertinent to the effects of ts-11 MG on egg and eggshell quality parameters, as well as egg size distribution, is lacking. In this study, pullets were inoculated at 10 wk of age with ts-11 strain MG and placed in biological isolation units at 10 birds/unit. Hen-day egg production, eggshell strength, Haugh unit score, pimpling incidence, and blood/meat spot incidence were monitored and recorded in each trial through a 45-wk production cycle. Further, eggs from all treatments were collected daily, Monday-Thursday, and individually weighed. Results of this study indicate that no significant difference was observed between the treatments for the parameters measured or for egg size distribution. Therefore, these data should lessen producers' concerns pertaining to the impact of ts-11 strain MG on egg production, egg and eggshell quality parameters, and egg size distribution.

An experiment was conducted to determine the effects of age at inoculation and induced molt on the reisolation of Mycoplasma gallisepticum (MG) from commercial leghorn hens that had been eyedrop-inoculated with F strain MG at either 10 or 66 wk of age. Chickens were maintained in biological isolation units from 10 wk of age through 78 wk of age. At 70 wk of age (premolt), hens were swabbed, cultured for MG, and molted. Swabs were taken both at the end of molt (postmolt [74 wk]) and again 4 wk later (postmolt+4 [78 wk]). A significant (P < or = 0.05) decrease in MG isolations was observed in the postmolt swabs as compared with the premolt swabs of hens inoculated at either 10 or 66 wk of age. A significant (P < or = 0.05) increase in isolations was observed in the postmolt+4 swabs as compared with the postmolt swabs of hens inoculated at either 10 or 66 wk of age. For the hens inoculated at 10 wk, no significant difference was found in premolt as compared with postmolt+4 MG isolations; however, for hens inoculated at 66 wk, a significant (P < or = 0.05) decrease was observed between premolt and postmolt+4 isolations. Significantly (P < or = 0.05) fewer MG isolations were obtained from the premolt swabs of hens inoculated at 10 wk as compared with hens inoculated at 66 wk. No significant difference in MG isolations was observed in either the postmolt or postmolt+4 swabs between hens inoculated at either 10 or 66 wk.

In each of two trials, 160 commercial pullets were separated into four treatments with four replicates of 10 chickens in each treatment. Forty pullets were designated as controls and received no inoculation; 40 pullets received F strain Mycoplasma gallisepticum (FMG); an additional 40 pullets received Mycoplasma synoviae (MS); and the final 40 pullets were inoculated with both FMG and MS (dual). All inoculations occurred at 10 wk of age. Eggs from all treatments were collected daily, Monday-Thursday, and individually weighed. No significant difference was observed among the treatments for percentages of jumbo, extra-large, medium, small, peewee, or undergrade eggs. As a percentage of eggs laid for the 4 days of each week over the 44-wk laying cycle of each trial, the FMG hens laid significantly fewer large size eggs (43.2%) as compared with either controls (51.17%) or dual-infected hens (49.95%). No significant difference was found in percentage of large eggs laid by FMG hens when compared with MS hens.

Genetic variability is a salient feature of lentiviruses, contributing to the pathogenesis of these viruses by enabling them to persist in the host and to resist anti-retroviral treatment. Bovine immunodeficiency virus (BIV), a lentivirus of unknown pathology, infects cattle in the United States and worldwide. Genetic diversity of BIV that is associated with naturally infected cattle is not well studied. We examined the genetic diversity and natural selection of a segment of the BIV pol gene amplified from the leukocyte DNA of naturally infected cattle. A portion of the reverse transcriptase domain (183 bp) of the pol region was targeted for amplification by PCR. PCR products were sequenced directly and aligned. When compared to the sequences of BIV R29-127, a molecular clone of the original BIV R29 isolate, all isolates were greater than 91% identical in nucleotide sequences and 77% identical in amino acid sequences. Pol genotypes were polymorphic at 14% of the nucleotide sites. The ratio of nonsynonymous to synonymous nucleotide substitutions (relative to the number of respective sites, Ka/Ks) was 0.16, indicating that this region of the BIV genome, like that of HIV-1, is subject to purifying selection. Based on the McDonald-Kreitman analysis, this region also was under positive Darwinian selection as HIV-1 and BIV diverged from a common progenitor. Phylogenetic analysis revealed that genotypes were geographically distinct, possibly indicating a common source of infection for animals within a herd., (Copyright 1999 Academic Press.)

cDNA was obtained from the bursae of Fabricius of chickens from six B-congenic lines developed at this laboratory and studied for expression of class II B-LB genes. Following cDNA amplification, cloning and sequencing, genes were assigned to B-LB loci based on characteristic DNA sequences, amino acid relatedness to characterized genes, and level of expression. Genes from the B-LBI, B-LBII, and B-LBVI loci were differentially expressed in chickens with the B2, B5, B13, B15, or B21 haplotypes. Chickens of all haplotypes expressed a B-LBII gene. Additional B-LB genes expressed included: B-LBI genes in the B5 and B19 haplotypes; a B-LBI/VI recombinant gene in the B2 haplotype; and a B-LBVI gene in the B13 haplotype. The B-congenic lines have demonstrable differences in resistance to Marek's disease (MD), and in responses to MD viral vaccines. This variability in disease resistance may be correlated with polymorphisms in the expressed B-LB genes, or with differential expression of genes at different loci.

Two strains of chickens selected for differential Marek's disease (MD) resistance or susceptibility were studied for the presence of the recently described Rfp-Y major histocompatibility complex (Mhc-like) haplotypes. MD resistant chickens from line N were fixed for the classical Mhc B21 haplotype, whereas MD susceptible line P chickens were fixed for the B19 haplotype. The Rfp-Y haplotypes were identified by restriction fragment polymorphism (RFP) analysis using enzymes and Mhc probes for B-G, B-L beta II, and B-FIV. In addition an Rfp-L beta III clone was developed that differentiated Rfp-L beta from B-L beta genes. Three Rfp-Y haplotypes, defined for both class I and class II Mhc-like loci, were identified in line N (Rfp-Y5, Rfp-Y7, and Rfp-Y8) and in line P (Rfp-Y5, Rfp-Y8, and Rfp-Y9), respectively. The Rfp-Y7, Rfp-Y8, and Rfp-Y9 haplotypes have not been described previously. The Rfp-Y5 haplotype was most frequent (0.70) in line N, but existed in low frequency (approximately 0.04) in line P; the Rfp-Y9 haplotype was most frequent in line P (0.63), but was absent in line N. The Rfp-Y haplotypes-segregated in a Mendelian fashion in each line based on analysis of progeny from Rfp-Y heterozygous matings. The frequency of recombination between the Rfp-Y F and L loci was estimated to be less than 0.25%.

Marek's disease (MD) is a lymphoproliferative disease caused by a member of the herpesvirus family, and the best understood genetic resistance to MD involves the chicken major histocompatibility complex (MHC) B-complex. Preliminary observations have suggested that MHC-like Rfp-Y genes might also influence the incidence of MD. This study describes the differentiation and definition of unique Rfp-Y genes in inbred lines 6(3) and 7(2), lines that possess identical B-complex genes, but that are resistant or susceptible to MD, respectively. To assess if Rfp-Y genes affect susceptibility to MD, 265 6(3) x 7(2) F2 chickens were challenged with the JM strain of MD virus at 1 week of age and were evaluated for MD lesions at up to 10 weeks of age. Genotyping of the F2 chickens for Rfp-Y haplotypes was performed by restriction fragment length polymorphism analysis of genomic DNA using TaqI and a B-FIV probe. Analysis of variance and interval mapping procedures were used to determine association between the Rfp-Y haplotypes and the phenotypic MD values of the F2 chickens. The cosegregation analysis of 265 F2 chickens indicated that there was no association between Rfp-Y haplotypes and MD susceptibility. Furthermore, the fact that the Rfp-Y haplotypes fit the 1:2:1 segregation ratio and the Rfp-Y allele frequencies did not differ significantly from 0.5 in the full population or in selected subpopulations (of either 40 MD-resistant or 39 MD-susceptible chickens) also indicated that Rfp-Y haplotypes do not significantly influence MD susceptibility. We conclude that Rfp-Y haplotypes do not play a major role in determining the genetic susceptibility to MD in 6(3) x 7(2) F2 White Leghorn chickens.

In each of two trials, 160 commercial pullets were separated into four treatments with four replicates of 10 chickens in each treatment. Forty pullets were designated as controls and received no inoculation; 40 other pullets received F strain Mycoplasma gallisepticum (FMG); an additional 40 pullets received Mycoplasma synoviae (MS); and the final 40 pullets were inoculated with both FMG and MS (dual). Hen-day egg production, egg weight, eggshell strength, Haugh unit score, pimpling incidence, and blood/meat spot incidence were monitored and recorded in each trial through an entire laying cycle. No significant difference was observed among the treatments for hen-day egg production, egg weight, eggshell strength, or Haugh unit scores. Significant differences were observed for pimpling incidence among controls (1.63%), Mycoplasma gallisepticum (MG)-infected (2.09%), and dual-infected hens (2.41%). A significant difference in blood/meat spot incidence was observed between MG-infected hens (0.27%) and dual-infected hens (0.45%). Histopathologic examination of the ovary and all segments of the oviduct revealed no significant differences among the treatments. These results suggest that the majority of the hen reproductive tract functions similarly in FMG-vaccinated, MS-infected, or dual-infected hens as compared with Mycoplasma-clean hens.

In each of three trials, 150 day-old broiler chicks were eyedrop inoculated with 0.04 ml of high-passage F strain Mycoplasma gallisepticum (MG) and housed in biological isolation units at 10 chicks per unit. At 4 wk of age, 50 chickens were designated as controls and remained on tap water (pH 7.30), 50 chickens were provided tap water containing 0.63% ammonium chloride (NH4Cl, pH 6.91), and 50 chickens were provided tap water containing 1.26% sodium bicarbonate (NaHCO3, pH 8.17). Fluids were supplied for ad libitum consumption. At 5 wk of age, all chickens were swabbed from the choanal cleft for MG isolation and bled from the left cutanea ulnea vein for pH determination. As a percent of total swabs obtained, significantly fewer chickens that consumed water containing NH4Cl (38.3%) were positive for MG by culture compared with either the NaHCO3 group (61.3%) or the control group (67.6%). Nonmycoplasmal swab contamination was significantly higher for chickens that consumed water containing NH4Cl (59.7%) compared with either controls (31.8%) or NaHCO3-treated chickens (38.7%). When contaminated cultures were discarded, MG isolations from the tap water group were not significantly different from MG isolations from either the NH4Cl or NaHCO3 group. However, MG isolations from the NH4Cl group (95%) were significantly less compared with the NaHCO3 group (100%). Mortality was significantly higher in chickens that consumed water containing NaHCO3 (8.7%) compared with either controls (1.3%) or the NH4Cl-treated chickens (0.7%). Blood pH values were lower for the NH4Cl group (7.927), higher for the NaHCO3 group (8.093), and intermediate for controls (8.035). Results of this study suggest that water containing NH4Cl hinders the bacteriological recovery of MG from the choanal cleft.

Development of B cells in chickens proceeds via a series of discrete developmental stages that includes the maturation of committed B cell progenitors in the specialized microenvironment of the bursa of Fabricius. The bursa has been shown to be required for the amplification of the B cell pool and selects for cells with productive immunoglobulin rearrangement events. Other events regulating chicken B cell development such as lymphocyte trafficking and apoptosis are just beginning to be elucidated. Within the bursa, the variable regions of immunoglobulin genes of B cell progenitors are diversified by a process of intrachromosomal gene conversion, where blocks of sequence information are transferred from pseudo-V regions to the recombined variable regions of the immunoglobulin genes. Recently gene conversion has been determined to play a role in the diversification of the immune repertoire in other species. In this review we focus on the current understanding and recent advances of B cell development in the chicken.

Major histocompatibility complex (Mhc) genes influencing transplantation rejections were first described in mice within the H2 complex and secondly in chickens within the B complex. In chickens, Rfp-Y haplotypes have recently been identified which contain class I and class II Mhc-like genes that assort independently of the B complex. Three Rfp-Y haplotypes have been defined in a closed breeding flock of line N chickens. In this study, progeny were obtained from line N Rfp-Y heterozygous matings to establish the role of Rfp-Y in transplantation immunity. Rfp-Y incompatibility did not induce significant one-way mixed lymphocyte responses. However, Rfp-Y-incompatible skin grafts were rejected more frequently and at a faster rate than Rfp-Y-compatible grafts by two-week-old chicks. The control Mhc B-incompatible grafts were rejected faster than the Rfp-Y-incompatible grafts; the latter were rejected at speeds that resemble rejection of minor histocompatibility antigens. We conclude that Rfp-Y class I and II Mhc-like genes are linked to the expression of minor histocompatibility antigens in chickens.

Conditioned medium containing immune interferon (IFN) activity was prepared by stimulating spleen lymphocytes obtained from inbred SC chickens with 10 micrograms concanavalin A (Con A) for 48 h. Pretreatment of spleen cells with monoclonal antibody against CD4, but not CD8, abrogated IFN production suggesting that CD4+ lymphocytes are responsible for immune IFN production. Immune IFN was purified 25-fold from Con A conditioned medium using controlled-pore glass column chromatography resulting in an increase in specific antiviral activity from 7 to 3290 units mg-1. Partially purified immune IFN retained antiviral and macrophage-activating factor (MAF)-like activities. Normal peripheral blood macrophages, when cultured in the presence of partially purified immune IFN, showed a dose-dependent increase in cell surface major histocompatibility complex Class II antigen expression by flow cytometry. Northern blot analysis of mRNA obtained from IFN-treated macrophages showed a concomitant increase in Class II gene expression. This effect was more obvious in cells induced for 48 h than in those induced for 24 h. These results strongly suggest that existence of an avian homologue of the MAF-like activity.

A chicken MHC class I (B-F) cDNA from SPAFAS line 11 embryonic liver tissue was isolated and characterized by nucleotide sequencing. Comparing this sequence with previously described B-F cDNAs highlights clustered nucleotide substitutions in exon 3, encoding amino acids located on the alpha-helical region of the alpha 2 domain.

Chickens of the B2, B5, B15, B19, and B21 B-congenic haplotypes differ in disease resistance. Complementary DNA from B-congenic chicken strains have been analyzed for allelic diversity of expressed Class II MHC genes. The predicted amino acid sequences of eight genes from five haplotypes were subjected to Wu-Kabat variability analysis. The B-L gene polymorphic regions and conserved regions are highly similar to the human leukocyte antigen Class II genes. Therefore, the present analysis reveals candidate polymorphisms important in determining the spectrum of antigenic peptides presented to T helper cells, and allelic differences possibly important in resistance to avian disease.

An avian equivalent of the mammalian thyroid isthmus appeared in thiouracil-treated (.1% in feed nd hatching to 6 and 8 weeks of age) chickens. In two experiments in which chickens were fed a diet containing thiouracil, thyroid tissue was found connecting the thyroid glands. Histological examination of the connecting tissue revealed that it was of the same cellular composition as the thyroid glands.

An avian equivalent of the mammalian thyroid isthmus appeared in thiouracil-treated (.1% in feed from hatching to 6 and 8 weeks of age) chickens. In two experiments in which chickens were fed a diet containing thiouracil, thyroid tissue was found connecting the thyroid glands. Histological examination of the connecting tissue revealed that it was of the same cellular composition as the thyroid glands.