A p37-based ELISA used to monitor anti- Mycoplasma hyorhinis IgG in serum from pigs immunized with inactivated M. hyorhinis vaccines.

Mycoplasma hyorhinis is an important pathogen of swine that can often occur as a respiratory coinfection with viral pathogens, but can also cause arthritis and polyserositis in infected animals. To date, no assay is available to assess the serologic response to M. hyorhinis vaccines, to our knowledge. We used recombinantly expressed M. hyorhinis p37 protein to monitor the magnitude of the IgG response in vaccinated animals. The assay was able to distinguish animals vaccinated with M. hyorhinis from those vaccinated with the other important Mycoplasma species: M. hyopneumoniae and M. hyosynoviae. When formulated with an ideal adjuvant, inactivated vaccines designed to protect animals against M. hyorhinis induced a measurable and dose-dependent antibody response against the p37 protein. Additionally, the protein appears to be highly conserved between strains of M. hyorhinis isolated in the United States. The specificity of the assay as well as the conservation and immunogenicity of the p37 protein make it an ideal candidate antigen for use in measuring the immune response against M. hyorhinis after vaccination in weaned pigs.

Loss of Immunization-Induced Epitope-Specific CD4 T-Cell Response following Anaplasma marginale Infection Requires Presence of the T-Cell Epitope on the Pathogen and Is Not Associated with an Increase in Lymphocytes Expressing Known Regulatory Cell Phenotypes.

We have shown that in cattle previously immunized with outer membrane proteins, infection with Anaplasma marginale induces a functionally exhausted CD4 T-cell response to the A. marginale immunogen. Furthermore, T-cell responses following infection in nonimmunized cattle had a delayed onset and were sporadic and transient during persistent infection. The induction of an exhausted T-cell response following infection presumably facilitates pathogen persistence. In the current study, we hypothesized that the loss of epitope-specific T-cell responses requires the presence of the immunizing epitope on the pathogen, and T-cell dysfunction correlates with the appearance of regulatory T cells. In limited studies in cattle, regulatory T cells have been shown to belong to γδ T-cell subsets rather than be CD4 T cells expressing forkhead box protein P3 (FoxP3). Cattle expressing the DRB3*1101 haplotype were immunized with a truncated A. marginale major surface protein (MSP) 1a that contains a DRB3*1101-restricted CD4 T-cell epitope, F2-5B. Cattle either remained unchallenged or were challenged with A. marginale bacteria that express the epitope or with A. marginale subsp. centrale that do not. Peripheral blood and spleen mononuclear cells were monitored for MSP1a epitope F2-5B-specfic T-cell proliferative responses and were stained for γδ T-cell subsets or CD4(+) CD25(+) FoxP3(+) T cells before and during infection. As hypothesized, the induction of T-cell exhaustion occurred only following infection with A. marginale, which did not correlate with an increase in either CD4(+) CD25(+) FoxP3(+) T cells or any γδ T-cell subset examined.

Comparative genomic analysis of seven Mycoplasma hyosynoviae strains.

Infection with Mycoplasma hyosynoviae can result in debilitating arthritis in pigs, particularly those aged 10 weeks or older. Strategies for controlling this pathogen are becoming increasingly important due to the rise in the number of cases of arthritis that have been attributed to infection in recent years. In order to begin to develop interventions to prevent arthritis caused by M. hyosynoviae, more information regarding the specific proteins and potential virulence factors that its genome encodes was needed. However, the genome of this emerging swine pathogen had not been sequenced previously. In this report, we present a comparative analysis of the genomes of seven strains of M. hyosynoviae isolated from different locations in North America during the years 2010 to 2013. We identified several putative virulence factors that may contribute to the ability of this pathogen to adhere to host cells. Additionally, we discovered several prophage genes present within the genomes of three strains that show significant similarity to MAV1, a phage isolated from the related species, M. arthritidis. We also identified CRISPR-Cas and type III restriction and modification systems present in two strains that may contribute to their ability to defend against phage infection.

Genome-Wide Association Studies of Virulent and Avirulent Haemophilus parasuis Serotype 4 Strains.

Haemophilus parasuis is a normal commensal of the upper respiratory tract of healthy pigs. However, in conjunction with stress and/or viral infections, or in immunocompromised animals, H. parasuis can transform into a pathogen causing Glasser's disease, which is typically characterized by fibrinous polyserositis, polyarthritis, meningitis, and sometimes acute pneumonia and septicemia. H. parasuis serotype 5 is highly virulent and more frequently isolated from respiratory and systemic infection in pigs. Recently Newport Laboratories isolated highly virulent H. parasuis serotype 4 strains from the tissues of diseased pigs. This study was undertaken to identify the genes responsible for H. parasuis serotype 4 virulence. To achieve this objective we performed genome-wide association studies (GWAS) across two virulent and three avirulent H. parasuis serotype 4 strains.

Genome Sequences of Seven Mycoplasma hyosynoviae Strains Isolated from the Joint Tissue of Infected Swine (Sus scrofa).

Mycoplasma hyosynoviae is a Gram-negative bacterium that can cause debilitating arthritis in swine. Currently, there are no M. hyosynoviae genome sequences in the GenBank database, which makes it impossible to understand its pathogenesis, nutrition, or colonization characteristics, or to devise an effective strategy for its control. Here, we report the genome sequences of seven strains of M. hyosynoviae. Within each genome, several virulence factors were identified that may prove important in the pathogenesis of M. hyosynoviae-mediated arthritis and serve as potential virulence markers that may be critical in vaccine development.

Genome sequences of porcine epidemic diarrhea virus: in vivo and in vitro phenotypes.

Since the outbreak of porcine epidemic diarrhea virus (PEDV) in May 2013, U.S. swine producers have lost almost five million baby pigs. In an attempt to understand the evolution of PEDV in the United States and possibly develop a control strategy, we compared the genome sequences of a PEDV strain isolated from an infected piglet against its in vitro adapted version. The original PEDV strain was grown in Vero cells and passed 10 times serially in a MARC145 cell line. The sequence analysis of the native PEDV strain and in vitro passaged virus shows that the cell culture adaptation specifically modifies PEDV spike protein whereas the open reading frame 1a/b (ORF1a/b)-encoded polyprotein, the nucleoprotein, NS3B (ORF3), and membrane and envelope proteins remain unchanged.

Genome Sequence of a Presumptive Mannheimia haemolytica Strain with an A1/A6-Cross-Reactive Serotype from a White-Tailed Deer (Odocoileus virginianus).

Mannheimia haemolytica is a Gram-negative bacterium and the principal etiological agent associated mostly with bovine respiratory disease complex. However, we report here the sequence of a strain with the novel A1/A6-cross-reactive serotype, strain PKL10, isolated from white-tailed deer. PKL10 was isolated from the spleen of farmed white-tailed deer showing clinical signs of pneumonia. The genome structure of PKL10 is dramatically different from that of previously sequenced isolates, which was demonstrated by genome alignments. In addition, the coding sequences in PKL10 share approximately 86% sequence identity with the coding sequences in other fully sequenced M. haemolytica strains. This suggests that PKL10 is a novel Mannheimia species.

CD4 T cell antigens from Staphylococcus aureus Newman strain identified following immunization with heat-killed bacteria.

Staphylococcus aureus is a commensal bacterium associated with the skin and mucosal surfaces of humans and animals that can also cause chronic infection. The emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA) and strains causing chronic intramammary infections (IMI) in cows results in severe human and livestock infections. Conventional approaches to vaccine development have yielded only a few noneffective vaccines against MRSA or IMI strains, so there is a need for improved vaccine development. CD4 T lymphocytes are required for promoting gamma interferon (IFN-γ) mediated immunoglobulin isotype switching in B lymphocytes to produce high-affinity IgG antibodies and IFN-γ-mediated phagocyte activation for an effective resolution of bacterial infection. However, the lack of known CD4 T cell antigens from S. aureus has made it difficult to design effective vaccines. The goal of this study was to identify S. aureus proteins recognized by immune CD4 T cells. Using a reverse genetics approach, 43 antigens were selected from the S. aureus Newman strain. These included lipoproteins, proteases, transcription regulators, an alkaline shock protein, conserved-domain proteins, hemolysins, fibrinogen-binding protein, staphylokinase, exotoxin, enterotoxin, sortase, and protein A. Screening of expressed proteins for recall T cell responses in outbred, immune calves identified 13 proteins that share over 80% sequence identity among MRSA or IMI strains. These may be useful for inclusion in a broadly protective multiantigen vaccine against MRSA or IMI.

A virion-associated protein kinase induces apoptosis.

Apoptosis and inhibition of host gene expression are often associated with virus infections. Many viral polypeptides modulate apoptosis by direct interaction with highly conserved apoptotic pathways. Some viruses induce apoptosis during late stages of the infection cycle, while others inhibit apoptosis to facilitate replication or maintain persistent infection. In previous work, we showed that Chilo iridescent virus (CIV) or CIV virion protein extract induces apoptosis in spruce budworm and cotton boll weevil cell cultures. Here, we characterize the product of a CIV gene (iridovirus serine/threonine kinase; istk) with signature sequences for S/T kinase and ATP binding. ISTK appears to belong to the superfamily, vaccinia-related kinases (VRKs). The istk gene was expressed in Pichia pastoris vectors. Purified ISTK (48 kDa) exhibited S/T kinase activity. Treatment with ISTK induced apoptosis in budworm cells. A 35-kDa cleavage product of ISTK retaining key signature sequences was identified during purification. Pichia-expressed 35-kDa polypeptide, designated iridoptin, induced apoptosis and inhibition of host protein synthesis in budworm and boll weevil cells. A mutation in the ATP-binding site eliminated both kinase and apoptosis activity of iridoptin, suggesting that kinase activity is essential for induction of apoptosis. Analysis with custom antibody confirmed that ISTK is a structural component of CIV particles. This is the first demonstration of a viral kinase inducing apoptosis in any virus-host system and the first identification of a factor inducing apoptosis or host protein shutoff for the family Iridoviridae.

Concerted action of two avirulent spore effectors activates Reaction to Puccinia graminis 1 (Rpg1)-mediated cereal stem rust resistance.

The barley stem rust resistance gene Reaction to Puccinia graminis 1 (Rpg1), encoding a receptor-like kinase, confers durable resistance to the stem rust pathogen Puccinia graminis f. sp. tritici. The fungal urediniospores form adhesion structures with the leaf epidermal cells within 1 h of inoculation, followed by hyphae and haustorium formation. The RPG1 protein is constitutively expressed and not phosphorylated. On inoculation with avirulent urediniospores, it is phosphorylated in vivo within 5 min and subsequently degraded. Application of arginine-glycine-aspartic acid peptide loops prevented the formation of adhesion structures for spore attachment, the phosphorylation of RPG1, and germination of the viable spores. Arginine-glycine-aspartic acid affinity chromatography of proteins from the ungerminated avirulent rust spores led to the purification and identification of a protein with fibronectin type III and breast cancer type 1 susceptibility protein domains and a vacuolar protein sorting-associated protein 9 with a coupling of ubiquitin to endoplasmic reticulum degradation domain. Both proteins are required to induce in vivo phosphorylation and degradation of RPG1. Combined application of both proteins caused hypersensitive reaction on the stem rust-resistant cultivar Morex but not on the susceptible cultivar Steptoe. Expression studies indicated that mRNA of both genes are present in ungerminated urediniospores and are constitutively transcribed in sporelings, infected leaves, and haustoria in the investigated avirulent races. Evidence is presented that RPG1, in yeast, interacts with the two protein effectors from the urediniospores that activate cooperatively the stem rust resistance protein RPG1 long before haustoria formation.

Co-expression of ovine LPS receptor CD14 with Mannheimia haemolytica leukotoxin receptor LFA-1 or Mac-1 does not enhance leukotoxin-induced cytotoxicity.

Leukotoxin (Lkt) and LPS are the major virulence determinants of Mannheimia haemolytica that contribute to the pathogenesis of bovine and ovine pneumonic pasteurellosis. We have previously identified bovine and ovine CD18 as the functional receptor for Lkt. LPS complexes with Lkt resulting in increased thermal stability and enhanced cytotoxic activity of Lkt. Cellular recognition of LPS involves several different molecules including CD14. We hypothesized that expression of ovine CD14 together with LFA-1 or Mac-1 would enhance Lkt-induced cytotoxicity. Ovine cDNA for CD14 was amplified by PCR and cloned into mammalian expression vectors. The 1122 bp cDNAs for bighorn sheep (BHS) and domestic sheep (DS) CD14 encode 373 amino acids which exhibit 99% identity with each other. Ovine CD14 plasmids were transfected either into HEK-293 cells, or previous HEK-293 transfectants stably expressing ovine LFA-1 or Mac-1. Flow cytometric analysis of transfectants confirmed the cell surface expression of CD14. The transfectants expressing LFA-1 or Mac-1 and the transfectants co-expressing CD14 with LFA-1 or Mac-1 did not show any significant difference in Lkt-induced cytotoxicity when incubated with LPS complexed Lkt. In contrast, incubation of the LFA-1 or Mac-1 and LFA-1/CD14 or Mac-1/CD14 transfectants with Lkt which lacks LPS, resulted in reduced cytotoxicity. None of the above transfectants showed any difference in [Ca²+](i) elevation when incubated with both types of Lkt preparations. Lkt did not induce any cytotoxicity or [Ca²+](i) elevation in ovine CD14 transfectants or parent HEK-293 cells. Based on these findings, we conclude that expression of CD14 together with LFA-1 or Mac-1 does not enhance Lkt-induced cytotoxicity, whereas LPS enhances cytotoxicity by complexing with Lkt.

The role of two f-box proteins, SLEEPY1 and SNEEZY, in Arabidopsis gibberellin signaling.

The SLEEPY1 (SLY1) F-box gene is a positive regulator of gibberellin (GA) signaling in Arabidopsis (Arabidopsis thaliana). Loss of SLY1 results in GA-insensitive phenotypes including dwarfism, reduced fertility, delayed flowering, and increased seed dormancy. These sly1 phenotypes are partially rescued by overexpression of the SLY1 homolog SNEEZY (SNE)/SLY2, suggesting that SNE can functionally replace SLY1. GA responses are repressed by DELLA family proteins. GA relieves DELLA repression when the SCF(SLY1) (for Skp1, Cullin, F-box) E3 ubiquitin ligase ubiquitinates DELLA protein, thereby targeting it for proteolysis. Coimmunoprecipitation experiments using constitutively expressed 35S:hemagglutinin (HA)-SLY1 and 35S:HA-SNE translational fusions in the sly1-10 background suggest that SNE can function similarly to SLY1 in GA signaling. Like HA-SLY1, HA-SNE interacted with the CULLIN1 subunit of the SCF complex, and this interaction required the F-box domain. Like HA-SLY1, HA-SNE coimmunoprecipitated with the DELLA REPRESSOR OF GA1-3 (RGA), and this interaction required the SLY1 or SNE carboxyl-terminal domain. Whereas HA-SLY1 overexpression resulted in a decrease in both DELLA RGA and RGA-LIKE2 (RGL2) protein levels, HA-SNE caused a decrease in DELLA RGA but not in RGL2 levels. This suggests that one reason HA-SLY1 is able to effect a stronger rescue of sly1-10 phenotypes than HA-SNE is because SLY1 regulates a broader spectrum of DELLA proteins. The FLAG-SLY1 fusion protein was found to coimmunoprecipitate with the GA receptor HA-GA-INSENSITIVE DWARF1b (GID1b), supporting the model that SLY1 regulates DELLA through interaction with the DELLA-GA-GID1 complex.

A three-way comparative genomic analysis of Mannheimia haemolytica isolates.

BACKGROUND: Mannhemia haemolytica is a Gram-negative bacterium and the principal etiological agent associated with bovine respiratory disease complex. They transform from a benign commensal to a deadly pathogen, during stress such as viral infection and transportation to feedlots and cause acute pleuropneumonia commonly known as shipping fever. The U.S beef industry alone loses more than one billion dollars annually due to shipping fever. Despite its enormous economic importance there are no specific and accurate genetic markers, which will aid in understanding the pathogenesis and epidemiology of M. haemolytica at molecular level and assist in devising an effective control strategy. DESCRIPTION: During our comparative genomic sequence analysis of three Mannheimia haemolytica isolates, we identified a number of genes that are unique to each strain. These genes are "high value targets" for future studies that attempt to correlate the variable gene pool with phenotype. We also identified a number of high confidence single nucleotide polymorphisms (hcSNPs) spread throughout the genome and focused on non-synonymous SNPs in known virulence genes. These SNPs will be used to design new hcSNP arrays to study variation across strains, and will potentially aid in understanding gene regulation and the mode of action of various virulence factors. CONCLUSIONS: During our analysis we identified previously unknown possible type III secretion effector proteins, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated sequences (Cas). The presence of CRISPR regions is indicative of likely co-evolution with an associated phage. If proven functional, the presence of a type III secretion system in M. haemolytica will help us re-evaluate our approach to study host-pathogen interactions. We also identified various adhesins containing immuno-dominant domains, which may interfere with host-innate immunity and which could potentially serve as effective vaccine candidates.

Transmission of Mannheimia haemolytica from domestic sheep (Ovis aries) to bighorn sheep (Ovis canadensis): unequivocal demonstration with green fluorescent protein-tagged organisms.

Previous studies demonstrated that bighorn sheep (Ovis canadensis) died of pneumonia when commingled with domestic sheep (Ovis aries) but did not conclusively prove that the responsible pathogens were transmitted from domestic to bighorn sheep. The objective of this study was to determine, unambiguously, whether Mannheimia haemolytica can be transmitted from domestic to bighorn sheep when they commingle. Four isolates of M. haemolytica were obtained from the pharynx of two of four domestic sheep and tagged with a plasmid carrying the genes for green fluorescent protein (GFP) and ampicillin resistance (AP(R)). Four domestic sheep, colonized with the tagged bacteria, were kept about 10 m apart from four bighorn sheep for 1 mo with no clinical signs of pneumonia observed in the bighorn sheep during that period. The domestic and bighorn sheep were then allowed to have fence-line contact for 2 mo. During that period, three bighorn sheep acquired the tagged bacteria from the domestic sheep. At the end of the 2 mo of fence-line contact, the animals were allowed to commingle. All four bighorn sheep died 2 days to 9 days following commingling. The lungs from all four bighorn sheep showed gross and histopathologic lesions characteristic of M. haemolytica pneumonia. Tagged M. haemolytica were isolated from all four bighorn sheep, as confirmed by growth in ampicillin-containing culture medium, PCR-amplification of genes encoding GFP and Ap(R), and immunofluorescent staining of GFP. These results unequivocally demonstrate transmission of M. haemolytica from domestic to bighorn sheep, resulting in pneumonia and death of bighorn sheep.

Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia.

Mycoplasma ovipneumoniae has been isolated from the lungs of pneumonic bighorn sheep (BHS). However experimental reproduction of fatal pneumonia in BHS with M. ovipneumoniae was not successful. Therefore the specific role, if any, of M. ovipneumoniae in BHS pneumonia is unclear. The objective of this study was to determine whether M. ovipneumoniae alone causes fatal pneumonia in BHS, or predisposes them to infection by Mannheimia haemolytica. We chose M. haemolytica for this study because of its isolation from pneumonic BHS, and its consistent ability to cause fatal pneumonia under experimental conditions. Since in vitro culture could attenuate virulence of M. ovipneumoniae, we used ceftiofur-treated lung homogenates from pneumonic BHS lambs or nasopharyngeal washings from M. ovipneumoniae-positive domestic sheep (DS) as the source of M. ovipneumoniae. Two adult BHS were inoculated intranasally with lung homogenates while two others received nasopharyngeal washings from DS. All BHS developed clinical signs of respiratory infection, but only one BHS died. The dead BHS had carried leukotoxin-positive M. haemolytica in the nasopharynx before the onset of this study. It is likely that M. ovipneumoniae colonization predisposed this BHS to fatal infection with the M. haemolytica already present in this animal. The remaining three BHS developed pneumonia and died 1-5 days following intranasal inoculation with M. haemolytica. On necropsy, lungs of all four BHS showed lesions characteristic of bronchopneumonia. M. haemolytica and M. ovipneumoniae were isolated from the lungs. These results suggest that M. ovipneumoniae alone may not cause fatal pneumonia in BHS, but can predispose them to fatal pneumonia due to M. haemolytica infection.

Ovis aries CR4 is involved in Mannheimia haemolytica leukotoxin-induced cytotoxicity.

Pneumonia caused by Mannheimia haemolytica is an important disease of domestic sheep (DS, Ovis aries) and cattle (BO). M. haemolytica is a normal commensal of the upper respiratory tract in ruminants, but during stress and viral infection it breaches the host innate mucosal defense and descents into lungs causing fibrinous pleuropneumonia. Leukotoxin (Lkt) produced by M. haemolytica is cytolytic to all subsets of ruminant leukocytes. Earlier, we and others have shown that DS and BO LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) can mediate Lkt-induced cytolysis. It is not clear whether CR4 (CD11c/CD18), which is involved in chemotaxis, phagocytosis and regulates host immune response can also mediate Lkt-induced cytolysis in ruminants. The host innate immune response to M. haemolytica is poorly understood and the involvement of CR4 in M. haemolytica pathogenesis is one of the most understudied. This problem is further compounded by the lack of cd11c genes from any ruminant species. Therefore, the objectives of this study were to clone cd11c and determine whether CR4 can serve as a receptor for Lkt. In this direction we cloned two alleles of cd11c gene from leukocytes isolated from DS blood by RT-PCR. Transfectants developed expressing functional DS CR4 were found to be cytotoxic to Lkt from four different isolates of M. haemolytica. This is the first report confirming the ability of a recombinant ovine CR4 to bind to M. haemolytica Lkt and mediate concentration-dependent lysis of host cells, thus, confirming their role in M. haemolytica pathogenesis. This is a critical step in understanding host innate immunity and the management of pneumonia in sheep.

Genome sequences of Mannheimia haemolytica serotype A2: ovine and bovine isolates.

This report describes the genome sequences of Mannheimia haemolytica serotype A2 isolated from pneumonic lungs of two different ruminant species, one from Ovis aries, designated ovine (O), and the other from Bos taurus, designated bovine (B).

Mannheimia haemolytica serotype A1 exhibits differential pathogenicity in two related species, Ovis canadensis and Ovis aries.

Mannheimia haemolytica causes pneumonia in both bighorn sheep (BHS, Ovis canadensis) and domestic sheep (DS, Ovis aries). Under experimental conditions, co-pasturing of BHS and DS results in fatal pneumonia in BHS. It is conceivable that certain serotypes of M. haemolytica carried by DS are non-pathogenic to them, but lethal for BHS. M. haemolytica serotypes A1 and A2 are carried by DS in the nasopharynx. However, it is the serotype A2 that predominantly causes pneumonia in DS. The objectives of this study were to determine whether serotype A1 exhibits differential pathogenicity to BHS and DS, and to determine whether leukotoxin (Lkt) secreted by this organism is its primary virulence factor. Three groups each of BHS and DS were intra-tracheally administered either 1 x 10(9)cfu of serotype A1 wild-type (lktA-Wt group), Lkt-deletion mutant of serotype A1-(lktA-Mt group), or saline (control group), respectively. In the lktA-Wt groups, all four BHS died within 48h while none of the DS died during the 2-week study period. In the lktA-Mt groups, none of the BHS or DS died. In the control groups, one DS died due to an unrelated cause. Necropsy and histopathological findings revealed that death of BHS in the lktA-Wt group was due to bilateral, fibrinohemorrhagic pneumonia. Although the A1-Mt-inoculated BHS were clinically normal, on necropsy, lungs of two BHS showed varying degrees of mild chronic pneumonia. These results indicate that M. haemolytica serotype A1 is non-pathogenic to DS, but highly lethal to BHS, and that Lkt is the primary virulence factor of M. haemolytica.

beta(2) integrin Mac-1 is a receptor for Mannheimia haemolytica leukotoxin on bovine and ovine leukocytes.

Pneumonia caused by Mannheimia haemolytica is an important disease of cattle (BO), domestic sheep (DS, Ovis aries) and bighorn sheep (BHS, Ovis canadensis). Leukotoxin (Lkt) produced by M. haemolytica is cytolytic to all leukocyte subsets of these three species. Although it is certain that CD18, the beta subunit of beta(2) integrins, mediates Lkt-induced cytolysis of leukocytes, whether CD18 of all three beta(2) integrins, LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18) and CR4 (CD11c/CD18), mediates Lkt-induced cytolysis of BO, DS and BHS leukocytes remains a controversy. Based on antibody inhibition experiments, earlier studies suggested that LFA-1, but not Mac-1 and CR-4, serves as a receptor for M. haemolytica Lkt. PMNs express all three beta(2) integrins, and they are the leukocyte subset that is most susceptible to Lkt. Therefore we hypothesized that all three beta(2) integrins serve as the receptor for Lkt. The objective of this study was to determine whether Mac-1 of BO, DS and BHS serves as a receptor for Lkt. cDNAs for CD11b of BO, DS and BHS were transfected into a Lkt-non-susceptible cell line along with cDNAs for CD18 of BO, DS and BHS, respectively. Transfectants stably expressing BO, DS or BHS Mac-1 specifically bound Lkt. These transfectants were lysed by Lkt in a concentration-dependent manner. Increase in intracellular [Ca(2+)](i) was observed in transfectants following exposure to low concentrations of Lkt indicating signal transduction through secondary messengers. Collectively, these results indicate that Mac-1 from these three species serves as a receptor for M. haemolytica Lkt.

CD11b of Ovis canadensis and Ovis aries: molecular cloning and characterization.

Leukotoxin (Lkt) is the primary virulence factor secreted by Mannheimia haemolytica which causes pneumonia in ruminants. Previously, we have shown that CD18, the beta subunit of beta(2) integrins, mediates Lkt-induced cytolysis of ruminant leukocytes. CD18 associates with four distinct alpha subunits giving rise to four beta(2) integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 (CR4), and CD11d/CD18. It is not known whether all the beta(2) integrins serve as a receptor for Lkt. Since PMNs are the leukocyte subset that is most susceptible to Lkt, and Mac-1 expression on PMNs exceeds that of other beta(2) integrins, it is of interest to determine whether Mac-1 serves as a receptor for Lkt which necessitates the cloning of CD11b and CD18. In this study, we cloned and sequenced the cDNA encoding CD11b of Ovis canadensis (bighorn sheep) and Ovis aries (domestic sheep). CD11b cDNA is 3455 nucleotides long encoding a polypeptide of 1152 amino acids. CD11b polypeptides from these two species exhibit 99% identity with each other, and 92% with that of cattle, and 70-80% with that of the non-ruminants analyzed.