Aggregation of cryopreserved mid-hindgut endoderm for more reliable and reproducible hPSC-derived small intestinal organoid generation.

A major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.

Outbreak of hepatitis B and hepatitis C virus infections associated with a cardiology clinic, West Virginia, 2012-2014.

OBJECTIVE: To stop transmission of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in association with myocardial perfusion imaging (MPI) at a cardiology clinic. DESIGN: Outbreak investigation and quasispecies analysis of HCV hypervariable region 1 genome. SETTING: Outpatient cardiology clinic. PATIENTS: Patients undergoing MPI. METHODS: Case patients met definitions for HBV or HCV infection. Cases were identified through surveillance registry cross-matching against clinic records and serological screening. Observations of clinic practices were performed. RESULTS: During 2012-2014, 7 cases of HCV and 4 cases of HBV occurred in 4 distinct clusters among patients at a cardiology clinic. Among 3 case patients with HCV infection who had MPI on June 25, 2014, 2 had 98.48% genetic identity of HCV RNA. Among 4 case patients with HCV infection who had MPI on March 13, 2014, 3 had 96.96%-99.24% molecular identity of HCV RNA. Also, 2 clusters of 2 patients each with HBV infection had MPI on March 7, 2012, and December 4, 2014. Clinic staff reused saline vials for >1 patient. No infection control breaches were identified at the compounding pharmacy that supplied the clinic. Patients seen in clinic through March 27, 2015, were encouraged to seek testing for HBV, HCV, and human immunodeficiency virus. The clinic switched to all single-dose medications and single-use intravenous flushes on March 27, 2015, and no further cases were identified. CONCLUSIONS: This prolonged healthcare-associated outbreak of HBV and HCV was most likely related to breaches in injection safety. Providers should follow injection safety guidelines in all practice settings.

Generation of CRISPR-Edited Rodents Using a Piezo-Driven Zygote Injection Technique.

Direct modification of the genome of the zygotes (i.e., one-cell embryos) by the CRISPR/Cas9-editing reagents, followed by embryo transfer to pseudopregnant females for live birth, has been the most effective method to generate laboratory rodent models for research. The method relies on proper delivery of the editing reagents into zygotes, which is commonly achieved by a standard or slightly modified pronuclear microinjection technique. In this chapter, we describe in detail an alternative delivery method, named piezo-driven cytoplasmic microinjection, which offers a superior embryo survival and birth rate. Because this method uses a much wider injection needle than that in pronuclear injection, it allows a larger volume of the editing materials to be transported into the zygotes, leading to an increase in the targeting efficiency. This also eliminates the clogging issues seen regularly in pronuclear injection. Moreover, Cytochalasin B that is used to soften zygotes during piezo-driven microinjection has been suggested a role in improving the knockin efficiency, which provides an additional benefit to use this injection method.

Reactive microglia and macrophage facilitate the formation of Müller glia-derived retinal progenitors.

In retinas where Müller glia have been stimulated to become progenitor cells, reactive microglia are always present. Thus, we investigated how the activation or ablation of microglia/macrophage influences the formation of Müller glia-derived progenitor cells (MGPCs) in the retina in vivo. Intraocular injections of the Interleukin-6 (IL6) stimulated the reactivity of microglia/macrophage, whereas other types of retinal glia appear largely unaffected. In acutely damaged retinas where all of the retinal microglia/macrophage were ablated, the formation of proliferating MGPCs was greatly diminished. With the microglia ablated in damaged retinas, levels of Notch and related genes were unchanged or increased, whereas levels of ascl1a, TNFα, IL1ß, complement component 3 (C3) and C3a receptor were significantly reduced. In the absence of retinal damage, the combination of insulin and Fibroblast growth factor 2 (FGF2) failed to stimulate the formation of MGPCs when the microglia/macrophage were ablated. In addition, intraocular injections of IL6 and FGF2 stimulated the formation of MGPCs in the absence of retinal damage, and this generation of MGPCs was blocked when the microglia/macrophage were absent. We conclude that the activation of microglia and/or infiltrating macrophage contributes to the formation of proliferating MGPCs, and these effects may be mediated by components of the complement system and inflammatory cytokines.

The reactivity, distribution and abundance of Non-astrocytic Inner Retinal Glial (NIRG) cells are regulated by microglia, acute damage, and IGF1.

Recent studies have described a novel type of glial cell that is scattered across the inner layers of the avian retina and possibly the retinas of primates. These cells have been termed Non-astrocytic Inner Retinal Glial (NIRG) cells. These cells are stimulated by insulin-like growth factor 1 (IGF1) to proliferate, migrate distally into the retina, and become reactive. These changes in glial activity correlate with increased susceptibility of retinal neurons and Müller glia to excitotoxic damage. The purpose of this study was to further study the NIRG cells in retinas treated with IGF1 or acute damage. In response to IGF1, the reactivity, proliferation and migration of NIRG cells persists through 3 days after treatment. At 7 days after treatment, the numbers and distribution of NIRG cells returns to normal, suggesting that homeostatic mechanisms are in place within the retina to maintain the numbers and distribution of these glial cells. By comparison, IGF1-induced microglial reactivity persists for at least 7 days after treatment. In damaged retinas, we find a transient accumulation of NIRG cells, which parallels the accumulation of reactive microglia, suggesting that the reactivity of NIRG cells and microglia are linked. When the microglia are selectively ablated by the combination of interleukin 6 and clodronate-liposomes, the NIRG cells down-regulate transitin and perish within the following week, suggesting that the survival and phenotype of NIRG cells are somehow linked to the microglia. We conclude that the abundance, reactivity and retinal distribution of NIRG cells can be dynamic, are regulated by homoestatic mechanisms and are tethered to the microglia.

A chick model of retinal detachment: cone rich and novel.

BACKGROUND: Development of retinal detachment models in small animals can be difficult and expensive. Here we create and characterize a novel, cone-rich retinal detachment (RD) model in the chick. METHODOLOGY/PRINCIPAL FINDINGS: Retinal detachments were created in chicks between postnatal days 7 and 21 by subretinal injections of either saline (SA) or hyaluronic acid (HA). Injections were performed through a dilated pupil with observation via surgical microscope, using the fellow eye as a control. Immunohistochemical analyses were performed at days 1, 3, 7, 10 and 14 after retinal detachment to evaluate the cellular responses of photoreceptors, Müller glia, microglia and nonastrocytic inner retinal glia (NIRG). Cell proliferation was detected with bromodeoxyuridine (BrdU)-incorporation and by the expression of proliferating cell nuclear antigen (PCNA). Cell death was detected with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). As in mammalian models of RD, there is shortening of photoreceptor outer segments and mis-trafficking of photoreceptor opsins in areas of RD. Photoreceptor cell death was maximal 1 day after RD, but continued until 14 days after RD. Müller glia up-regulated glial fibriliary acidic protein (GFAP), proliferated, showed interkinetic nuclear migration, and migrated to the subretinal space in areas of detachment. Microglia became reactive; they up-regulated CD45, acquired amoeboid morphology, and migrated toward outer retina in areas of RD. Reactive NIRG cells accumulated in detached areas. CONCLUSIONS/SIGNIFICANCE: Subretinal injections of SA or HA in the chick eye successfully produced retinal detachments and cellular responses similar to those seen in standard mammalian models. Given the relatively large eye size, and considering the low cost, the chick model of RD offers advantages for high-throughput studies.

The chicken cornea as a model of wound healing and neuronal re-innervation.

PURPOSE: The cornea is the major refractive component of the eye and serves as a barrier to the external environment. Understanding how the cornea responds to injury is important to developing therapies to treat vision disorders that affect the integrity and refractive properties of the cornea. Thus, investigation of the wound healing responses of the cornea to injury in a cost-effective animal model is a valuable tool for research. This study characterizes the wound healing responses in the corneas of White Leghorn chicken. METHODS: Linear corneal wounds were induced in post-natal day 7 (P7) chicks and cellular proliferation, apoptosis and regulation of structural proteins were assessed using immunohistochemical techniques. We describe the time course of increased expression of different scar-related markers, including vimentin, vinculin, perlecan and smooth muscle actin. RESULTS: We find evidence for acute necrotic cell death in the corneal region immediately surrounding cite of incision, whereas we failed to find evidence of delayed cell death or apoptosis. We find that the neuronal re-innervation of SV2-positive axon terminals within the corneal stroma and epithelium occurs very quickly after the initial scarring insult. We describe an accumulation of cells within the stroma immediately underlying the scar, which results, at least in part, from the local proliferation of keratocytes. Further, we provide evidence for scar-induced accumulations of CD45-positive monocytes in injured corneas. CONCLUSIONS: We conclude that the chick cornea is an excellent model system in which to study wound healing, formation of scar tissue, and neuronal re-innervation of sensory endings.

Evidence for a new avian paramyxovirus serotype 10 detected in rockhopper penguins from the Falkland Islands.

The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons.

Heterogeneity of glia in the retina and optic nerve of birds and mammals.

We have recently described a novel type of glial cell that is scattered across the inner layers of the avian retina [1]. These cells are stimulated by insulin-like growth factor 1 (IGF1) to proliferate, migrate distally into the retina, and up-regulate the nestin-related intermediate filament transition. These changes in glial activity correspond with increased susceptibility of neurons to excitotoxic damage. This novel cell-type has been termed the Non-astrocytic Inner Retinal Glia-like (NIRG) cells. The purpose of the study was to investigate whether the retinas of non-avian species contain cells that resemble NIRG cells. We assayed for NIRG cells by probing for the expression of Sox2, Sox9, Nkx2.2, vimentin and nestin. NIRG cells were distinguished from astrocytes by a lack of expression for Glial Fibrilliary Acidic Protein (GFAP). We examined the retinas of adult mice, guinea pigs, dogs and monkeys (Macaca fasicularis). In the mouse retina and optic nerve head, we identified numerous astrocytes that expressed GFAP, S100beta, Sox2 and Sox9; however, we found no evidence for NIRG-like cells that were positive for Nkx2.2, nestin, and negative for GFAP. In the guinea pig retina, we did not find astrocytes or NIRG cells in the retina, whereas we identified astrocytes in the optic nerve. In the eyes of dogs and monkeys, we found astrocytes and NIRG-like cells scattered across inner layers of the retina and within the optic nerve. We conclude that NIRG-like cells are present in the retinas of canines and non-human primates, whereas the retinas of mice and guinea pigs do not contain NIRG cells.

A novel type of glial cell in the retina is stimulated by insulin-like growth factor 1 and may exacerbate damage to neurons and Müller glia.

Recent studies have demonstrated that insulin can have profound affects on the survival of neurons within the retina. The purpose of this study was to determine how insulin-like growth factor 1 (IGF1) influences retinal cells; in particular, the glial cells. We identify a novel type of glial cell in the avian retina and provide evidence that these cells can respond to acute damage and IGF1. In normal retinas, we found a distinct cell-type, scattered across the ganglion cell and inner plexiform layers that express Sox2, Sox9, Nkx2.2, vimentin, and transitin, the avian homologue of mammalian nestin. These glial cells have a unique immunohistochemical profile, morphology, and distribution that are distinct among other known types of retinal glia, including microglia, oligodendrocytes, astrocytes, and Muller glia. We termed these cells nonastrocytic inner retinal glia-like (NIRG) cells. We found that the NIRG cells may express the IGF1 receptor and respond to IGF1 by proliferating, migrating distally into the retina, and upregulating transitin. In addition, IGF1 stimulated microglia to become reactive and upregulate lysosomal membrane glycoprotein and CD45. With microglia and NIRG cells stimulated by IGF1 there were elevated levels of cell death and numerous focal detachments across the retina in response to excitotoxic damage. Cell death was prominent within the areas of detachment coinciding with a stark loss of Müller glia and accumulation of NIRG cells. We conclude that NIRG cells are a novel type of retinal glia that is sensitive to IGF1 and whose activity may impact the survival of neurons and Müller glia.

Mitogen-activated protein kinase-signaling regulates the ability of Müller glia to proliferate and protect retinal neurons against excitotoxicity.

The purpose of this study was to investigate whether insulin, fibroblast growth factor (FGF), and mitogen-activated protein kinase (MAPK) pathways protect retinal neurons against excitotoxicity and regulate the proliferation of Müller glia. We found that intraocular injections of insulin or FGF2 had variable effects upon the phosphorylation of ERK1/2, p38 MAPK, and CREB, and the expression of immediate early genes, cFos and Egr1. Accumulations of pERK1/2, p38 MAPK, pCREB, cFos and Egr1 in response to insulin or FGF2 were confined to Müller glia, whereas retinal neurons did not seem to respond to growth factors. Unlike FGF2, insulin stimulated microglia-like cells to upregulate the intermediate filament transitin and lysosomal membrane glycoprotein (LMG). With microglia-like cells and Müller glia stimulated by insulin or FGF2 there were profound effects upon numbers of dying neurons in response to excitotoxic damage. Although FGF2 significantly reduced numbers of dying neurons, insulin significantly increased numbers of dying neurons. In addition to neuroprotective affects, FGF2 also "primed" the Müller glia to proliferate following retinal damage, whereas insulin had no effect upon glial proliferation. Further, we found that FGF receptor isoform 1 (FGFR1) and FGFR3 were prominently expressed in the retina, whereas the insulin receptor and FGFR2 are not expressed, or are expressed at very low levels. We conclude that MAPK-signaling through FGF receptors stimulates Müller glia to become more neuroprotective and progenitor-like, whereas insulin acting on Müller and microglia-like cells through unidentified receptors had the opposite effect.

Mitogen-activated protein kinase-signaling stimulates Müller glia to proliferate in acutely damaged chicken retina.

Müller glia in the mature retina have the capacity to become progenitor-like cells in a many different vertebrate classes. The cell-signaling pathways that control the ability of mature Müller glia to become progenitor-like cells remain uncertain. The purpose of this study was to investigate the roles of the Mitogen-Activated Protein Kinase (MAPK) pathway in regulating the activity of Müller glia in the chicken retina. In response to acute retinal damage, we found that Müller glia accumulated phosphorylated ERK1/2 and phospho-CyclicAMP Response Element Binding-protein (pCREB), and transiently expressed immediate early genes, cFos and Egr1, that are known to be downstream of MAPK-signaling. Egr1 and pCREB were normally expressed by retinal progenitors in the circumferential marginal zone (CMZ), whereas cFos and pERK1/2 were not. In addition, small molecule inhibitors of MEK (UO126) and the FGF-receptor (SU5402) suppressed the proliferation of Müller glia-derived progenitor-like cells. These inhibitors suppressed the accumulation of Egr1 and pCREB, whereas levels of cFos were unaffected in the glial cells. These findings suggest that Egr1 and pCREB are downstream of the signaling cascade activated by FGF-receptors and ERK1/2. Further, our findings suggest that Egr1 and pCREB may promote glial proliferation. We propose that activation of both the FGF-receptor and ERK1/2-pathway is required for the proliferation and transdifferentiation of Müller glia into progenitor-like cells.

Bullwhip neurons in the retina regulate the size and shape of the eye.

Bullwhip and mini-bullwhip cells are unconventional types of retinal neurons that utilize the neuropeptides glucagon, glucagon-like peptide 1 (GLP1) and substance P. These cells have been implicated in regulating the proliferation of neural progenitors in the circumferential marginal zone (CMZ) of the chicken retina. The purpose of this study was to investigate the roles of the bullwhip cells in regulating ocular size and shape. We found that intravitreal delivery of colchicine at postnatal day 7 destroys the vast majority (approximately 98%) of the bullwhip and mini-bullwhip cells and their peptidergic terminals that are concentrated in the CMZ near the equator of the eye. Interestingly, colchicine-treatment resulted in excessive ocular growth that involved the expansion of equatorial diameter, but not axial length. Intraocular injections of glucagon completely prevented the equatorial expansion that occurs with colchicine-treatment. In eyes with undamaged retinas, exogenous glucagon suppressed equatorial eye growth, whereas glucagon receptor antagonists caused excessive equatorial growth. Furthermore, visual stimuli that increase or decrease rates of ocular growth caused a down- or up-regulation, respectively, of the immediate early gene Egr1 in the bullwhip cells; indicating that the activity of the bullwhip cells is regulated by growth-guiding visual cues. We found that the glucagon receptor was expressed by cells in the fibrous and cartilaginous sclera in equatorial regions of the eye. Taken together, these findings suggest that glucagon peptide released from the terminals of the bullwhip and mini-bullwhip cells regulates the growth of the equatorial sclera in a vision-dependent manner. Although the bullwhip and mini-bullwhip cells are not abundant, less than 1000 cells per retina, their influence on the development of the eye is substantial and includes vision-guided ocular growth.

Transient expression of LIM-domain transcription factors is coincident with delayed maturation of photoreceptors in the chicken retina.

In the retina of warm-blooded vertebrates, photoreceptors are specified many days before the onset of synaptogenesis and the expression of photopigments. The factors that regulate the maturation of photoreceptors in the developing retina remain unknown. We report here that photoreceptors transiently express LIM-domain transcription factors during the development of the chicken retina. We examined the differentiation of photoreceptors through the normal course of embryonic development and at the far periphery of the postnatal retina, where the differentiation of photoreceptors is slowed and persists across a spatial gradient. In the embryonic retina, we find visinin-positive photoreceptors that transiently express Islet2 and Lim3 starting at E8 and ending around E15, but persisting in far peripheral regions of the retina through the first 2 weeks of postnatal development. During early stages of photoreceptor maturation, there is coincident and transient expression of the LIM-domain factors with axonin1, a cell surface glycoprotein that is a member of the immunoglobulin superfamily. Coincident with the downregulation of Islet2 and Lim3, we find the upregulation of calbindin, red/green opsin, rhodopsin, and a synaptic marker in the outer plexiform layer (OPL; dystrophin). In the periphery of the postnatal retina, photoreceptors that express Islet2, Lim3, and axonin1 do not overlap with photoreceptors that express calbindin, red/green opsin, rhodopsin, and dystrophin. We propose that Islet2 and Lim3 may promote the expression of genes that are involved in the early stages of differentiation but may suppress the expression of genes that are required in the mature photoreceptors.

Collagen XI chain misassembly in cartilage of the chondrodysplasia (cho) mouse.

Molecular mechanisms controlling the assembly of cartilage-specific types II, IX and XI collagens into a heteropolymeric network of uniformly thin, unbanded fibrils are not well understood, but collagen XI has been implicated. The present study on cartilage from the homozygous chondrodysplasia (cho/cho) mouse adds support to this concept. In the absence of alpha1(XI) collagen chains, thick, banded collagen fibrils are formed in the extracellular matrix of cho/cho cartilage. A functional knock-out of the type XI collagen molecule has been assumed. We have re-examined this at the protein level to see if, rather than a complete knock-out, alternative type XI chain assemblies were formed. Mass spectrometry of purified triple-helical collagen from the rib cartilage of cho/cho mice identified alpha1(V) and alpha2(XI) chains. These chains were recovered in roughly equal amounts based on Coomassie Blue staining of SDS-PAGE gels, in addition to alpha1(II)/alpha3(XI) collagen chains. Using telopeptide-specific antibodies and Western blot analysis, it was further shown that type V/XI trimers were present in the matrix cross-linked to each other and to type II collagen molecules to form heteropolymers. Cartilage from heterozygous (cho/+) mice contained a mix of alpha1(V) and alpha1(XI) chains and a mix of thin and thick fibrils on transmission electron microscopy. In summary, the results imply that native type XI collagen molecules containing an alpha1(XI) chain are required to form uniformly thin fibrils and support a role for type XI collagen as the template for the characteristic type II collagen fibril network of developing cartilage.

Prolyl 3-hydroxylase 1 deficiency causes a recessive metabolic bone disorder resembling lethal/severe osteogenesis imperfecta.

A recessive form of severe osteogenesis imperfecta that is not caused by mutations in type I collagen has long been suspected. Mutations in human CRTAP (cartilage-associated protein) causing recessive bone disease have been reported. CRTAP forms a complex with cyclophilin B and prolyl 3-hydroxylase 1, which is encoded by LEPRE1 and hydroxylates one residue in type I collagen, alpha1(I)Pro986. We present the first five cases of a new recessive bone disorder resulting from null LEPRE1 alleles; its phenotype overlaps with lethal/severe osteogenesis imperfecta but has distinctive features. Furthermore, a mutant allele from West Africa, also found in African Americans, occurs in four of five cases. All proband LEPRE1 mutations led to premature termination codons and minimal mRNA and protein. Proband collagen had minimal 3-hydroxylation of alpha1(I)Pro986 but excess lysyl hydroxylation and glycosylation along the collagen helix. Proband collagen secretion was moderately delayed, but total collagen secretion was increased. Prolyl 3-hydroxylase 1 is therefore crucial for bone development and collagen helix formation.

Effects of a single intranasal dose of modified-live bovine respiratory syncytial virus vaccine on cytokine messenger RNA expression following viral challenge in calves.

OBJECTIVE: To characterize cytokine messenger RNA (mRNA) expression in intranasally vaccinated calves after bovine respiratory syncytial virus (BRSV) challenge. ANIMALS: Twelve 8- to 12-week-old calves. PROCEDURES: Calves received modified-live BRSV vaccine (vaccinated) or spent tissue culture medium (mock-vaccinated) intranasally, followed by challenge 30 days later with BRSV, or mock challenge with spent tissue culture medium (mock-challenge controls). Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA was measured in lungs, bronchoalveolar lavage (BAL) fluid cells, pharyngeal tonsils, and tracheobronchial lymph nodes, and tumor necrosis factor-alpha (TNF-alpha) mRNA was measured in lungs and BAL fluid cells by reverse transcriptase-competitive polymerase chain reaction assay. RESULTS: Resistance to clinical signs of disease was conferred in vaccinated calves. Expression of TNF-alpha mRNA in lungs and BAL fluid cells was higher in mock-vaccinated calves than control or vaccinated calves. In the lung, IL-4 mRNA expression was higher in vaccinated calves than control or mock-vaccinated calves. In pharyngeal tonsils, expression of mRNA for IL-4 and IFN-gamma was higher in mock-vaccinated calves than control calves. In tracheobronchial lymph nodes, IFN-gamma mRNA expression was higher in mock-vaccinated calves than vaccinated calves. CONCLUSIONS AND CLINICAL RELEVANCE: Although vaccinated calves had decreased clinical signs of disease after BRSV challenge, compared with mock-vaccinated calves, this difference was not related to a T helper type 1 bias, as determined by increased expression of interferon-gamma mRNA relative to interleukin-4 mRNA in lungs, BAL fluid cells, or tracheobronchial lymph nodes of vaccinated calves. Pulmonary inflammation was decreased in vaccinated calves as determined by decreased expression of TNF-alpha mRNA.

Effects of a single intranasal dose of modified-live bovine respiratory syncytial virus vaccine on resistance to subsequent viral challenge in calves.

OBJECTIVE: To determine whether a single intranasal dose of modified-live bovine respiratory syncytial virus (BRSV) vaccine protects calves from BRSV challenge and characterize cell-mediated immune response in calves following BRSV challenge. ANIMALS: 13 conventionally reared 4- to 6-week-old Holstein calves. PROCEDURES: Calves received intranasal vaccination with modified live BRSV vaccine (VC-group calves; n = 4) or mock vaccine (MC-group calves; 6) 1 month before BRSV challenge; unvaccinated control-group calves (n = 3) underwent mock challenge. Serum virus neutralizing (VN) antibodies were measured on days -30, -14, 0, and 7 relative to BRSV challenge nasal swab specimens were collected for virus isolation on days 0 to 7. At necropsy examination on day 7, tissue specimens were collected for measurement of BRSV-specific interferon gamma (IFN-gamma) production. Tissue distribution of CD3+ T and BLA.36+ B cells was evaluated by use of immunohistochemistry. RESULTS: The MC-group calves had significantly higher rectal temperatures, respiratory rates, and clinical scores on days 5 to 7 after BRSV challenge than VC-group calves. No difference was seen between distributions of BRSV in lung tissue of VC- and MC-group calves. Production of BRSV-specific IFN-gamma was increased in tissue specimens from VC-group calves, compared with MC- and control-group calves. Virus-specific IFN-gamma production was highest in the mediastinal lymph node of VC-group calves. Increased numbers of T cells were found in expanded bronchial-associated lymphoid tissue and airway epithelium of VC-group calves. CONCLUSIONS AND CLINICAL RELEVANCE: An intranasal dose of modified-live BRSV vaccine can protect calves against virulent BRSV challenge 1 month later.