Acylation Enhances, but Is Not Required for, the Cytotoxic Activity of Mannheimia haemolytica Leukotoxin in Bighorn Sheep.

Mannheimia haemolytica causes pneumonia in domestic and wild ruminants. Leukotoxin (Lkt) is the most important virulence factor of the bacterium. It is encoded within the four-gene lktCABD operon: lktA encodes the structural protoxin, and lktC encodes a trans-acylase that adds fatty acid chains to internal lysine residues in the protoxin, which is then secreted from the cell by a type 1 secretion system apparatus encoded by lktB and lktD. It has been reported that LktC-mediated acylation is necessary for the biological effects of the toxin. However, an LktC mutant that we developed previously was only partially attenuated in its virulence for cattle. The objective of this study was to elucidate the role of LktC-mediated acylation in Lkt-induced cytotoxicity. We performed this study in bighorn sheep (Ovis canadensis) (BHS), since they are highly susceptible to M. haemolytica infection. The LktC mutant caused fatal pneumonia in 40% of inoculated BHS. On necropsy, a large number of necrotic polymorphonuclear leukocytes (PMNs) were observed in the lungs. Lkt from the mutant was cytotoxic to BHS PMNs in an in vitro cytotoxicity assay. Flow cytometric analysis of mutant Lkt-treated PMNs revealed the induction of necrosis. Scanning electron microscopic analysis revealed the presence of pores and blebs on mutant-Lkt-treated PMNs. Mass spectrometric analysis confirmed that the mutant secreted an unacylated Lkt. Taken together, these results suggest that acylation is not necessary for the cytotoxic activity of M. haemolytica Lkt but that it enhances the potency of the toxin.

A sensitive & specific multiplex PCR assay for simultaneous detection of Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei & Brucella species.

BACKGROUND & OBJECTIVES: Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei and Brucella species are potential biowarfare agents. Classical bacteriological methods for their identification are cumbersome, time consuming and of potential risk to the handler. METHODS: We describe a sensitive and specific multiplex polymerase chain reaction (mPCR) assay involving novel primers sets for the simultaneous detection of B. anthracis, Y. pestis, B. pseudomallei and Brucella species. An additional non-competitive internal amplification control (IAC) was also included. RESULTS: The mPCR was found to be specific when tested against closely related organisms. The sensitivity of the assay in spiked blood samples was 50 colony forming units (cfus)/25 µl reaction, for the detection of B. anthracis, Y. pestis and Brucella species; and 150 cfus/25 µl reaction, for B. pseudomallei. The assay proved useful in correctly and promptly identifing the clinical isolates of the targeted agents recovered from patients, compared to the gold standard culture methods. INTERPRETATION & CONCLUSION: The assay described in this study showed promise to be useful in application as a routine detection cum diagnostic method for these pathogens.

Glypican-3-Specific CAR T Cells Coexpressing IL15 and IL21 Have Superior Expansion and Antitumor Activity against Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death in the world, and curative systemic therapies are lacking. Chimeric antigen receptor (CAR)-expressing T cells induce robust antitumor responses in patients with hematologic malignancies but have limited efficacy in patients with solid tumors, including HCC. IL15 and IL21 promote T-cell expansion, survival, and function and can improve the antitumor properties of T cells. We explored whether transgenic expression of IL15 and/or IL21 enhanced glypican-3-CAR (GPC3-CAR) T cells' antitumor properties against HCC. We previously optimized the costimulation in GPC3-CARs and selected a second-generation GPC3-CAR incorporating a 4-1BB costimulatory endodomain (GBBz) for development. Here, we generated constructs encoding IL15, IL21, or both with GBBz (15.GBBz, 21.GBBz, and 21.15.GBBz, respectively) and examined the ability of transduced T cells to kill, produce effector cytokines, and expand in an antigen-dependent manner. We performed gene-expression and phenotypic analyses of GPC3-CAR T cells and CRISPR-Cas9 knockout of the TCF7 gene. Finally, we measured GPC3-CAR T-cell antitumor activity in murine xenograft models of GPC3+ tumors. The increased proliferation of 21.15.GBBz T cells was at least in part dependent on the upregulation and maintenance of TCF-1 (encoded by TCF7) and associated with a higher percentage of stem cell memory and central memory populations after manufacturing. T cells expressing 21.15.GBBz had superior in vitro and in vivo expansion and persistence, and the most robust antitumor activity in vivo These results provided preclinical evidence to support the clinical evaluation of 21.15.GPC3-CAR T cells in patients with HCC.

Leukotoxin of Bibersteinia trehalosi Contains a Unique Neutralizing Epitope, and a Non-Neutralizing Epitope Shared with Mannheimia haemolytica Leukotoxin.

Bibersteinia trehalosi and Mannheimia haemolytica, originally classified as Pasteurella haemolytica biotype T and biotype A, respectively, under Genus Pasteurella has now been placed under two different Genera, Bibersteinia and Mannheimia, based on DNA-DNA hybridization and 16S RNA studies. While M. haemolytica has been the predominant pathogen of pneumonia in ruminants, B. trehalosi is emerging as an important pathogen of ruminant pneumonia. Leukotoxin is the critical virulence factor of these two pathogens. While the leukotoxin of M. haemolytica has been well studied, the characterization of B. trehalosi leukotoxin has lagged behind. As the first step towards addressing this problem, we developed monoclonal antibodies (mAbs) against B. trehalosi leukotoxin and used them to characterize the leukotoxin epitopes. Two mAbs that recognized sequential epitopes on the leukotoxin were developed. One of them, AM113, neutralized B. trehalosi leukotoxin while the other, AM321, did not. The mAb AM113 revealed the existence of a neutralizing epitope on B. trehalosi leukotoxin that is not present on M. haemolytica leukotoxin. A previously developed mAb, MM601, revealed the presence of a neutralizing epitope on M. haemolytica leukotoxin that is not present on B. trehalosi leukotoxin. The mAb AM321 recognized a non-neutralizing epitope shared by the leukotoxins of B. trehalosi and M. haemolytica. The mAb AM113 should pave the way for mapping the leukotoxin-neutralizing epitope on B. trehalosi leukotoxin and the development of subunit vaccines and/or virus-vectored vaccines against this economically important respiratory pathogen of ruminants.

ß-Hemolysis May Not Be a Reliable Indicator of Leukotoxicity of Mannheimia haemolytica Isolates.

Mannheimia (Pasteurella) haemolytica causes bronchopneumonia in domestic and wild ruminants. Leukotoxin is the critical virulence factor of M. haemolytica. Since β-hemolysis is caused by a large number of leukotoxin-positive M. haemolytica isolates, all β-hemolytic M. haemolytica isolates are considered to be leukotoxic as well. However, conflicting reports exist in literature as to the leukotoxic and hemolytic properties of M. haemolytica. One group of researchers reported their leukotoxin-deletion mutants to be hemolytic while another reported their mutants to be non-hemolytic. The objective of this study was to determine whether β-hemolysis is a reliable indicator of leukotoxicity of M. haemolytica isolates. Ninety-five isolates of M. haemolytica were first confirmed for presence of leukotoxin gene (lktA) by a leukotoxin-specific PCR assay. Culture supernatant fluids from these isolates were then tested for presence of leukotoxin protein by an ELISA, and for leukotoxic activity by a cytotoxicity assay. All isolates were tested for β-hemolysis by culture on blood agar plates. Sixty-two isolates (65%) produced leukotoxin protein while 33 isolates (35%) did not. Surprisingly, 18 of the 33 isolates (55%), that did not produce leukotoxin protein, were hemolytic. Of the 62 isolates that produced leukotoxin, 55 (89%) were leukotoxic while 7 (11%) were not. All except one of the 55 leukotoxic isolates (98%) were also hemolytic. All seven isolates that were not leukotoxic were hemolytic. Taken together, these results suggest that β-hemolysis may not be a reliable indicator of leukotoxicity of M. haemolytica isolates. Furthermore, all M. haemolytica isolates that possess lktA gene may not secrete active leukotoxin.

Immunization of bighorn sheep against Mannheimia haemolytica with a bovine herpesvirus 1-vectored vaccine.

Mannheimia haemolytica is an important pathogen of pneumonia in bighorn sheep (BHS), consistently causing 100% mortality under experimental conditions. Leukotoxin is the critical virulence factor of M. haemolytica. In a 'proof of concept' study, a vaccine containing leukotoxin and surface antigens of M. haemolytica induced 100% protection in BHS, but required multiple booster doses. Vaccination of wildlife is difficult. BHS, however, can be vaccinated at the time of transplantation, but administration of booster doses is impossible. A vaccine that does not require booster doses, therefore, is ideal for vaccination of BHS. Herpesviruses are ideal vectors for development of such a vaccine because of their ability to undergo latency with subsequent reactivation which obviates the need for booster administration. The objective of this study was to evaluate the potential of bovine herpesvirus 1 (BHV-1) as a vector encoding M. haemolytica immunogens. As the first step towards this goal, the permissiveness of BHS for BHV-1 infection was determined. BHS inoculated with wild-type BHV-1 shed the virus following infection. The lytic phase of infection was superseded by latency, and treatment of latently-infected BHS with dexamethasone reactivated the virus. A recombinant BHV-1-vectored vaccine encoding a leukotoxin-neutralizing epitope and an immuno-dominant epitope of the outer membrane protein PlpE was developed by replacing the viral glycoprotein C gene with a leukotoxin-plpE chimeric gene. Four of six BHS vaccinated with the recombinant virus developed significant leukotoxin-neutralizing antibodies at day 21 post-vaccination, while two of six BHS developed significant surface antigen antibodies at day 17 post-vaccination. These antibodies, however, were inadequate for protection of BHS against M. haemolytica challenge. These data indicate that BHV-1 is a suitable vector for immunization of BHS, but additional experimentation with the chimeric insert is necessary for development of a more efficacious vaccine.

A chimeric protein comprising the immunogenic domains of Mannheimia haemolytica leukotoxin and outer membrane protein PlpE induces antibodies against leukotoxin and PlpE.

Mannheimia haemolytica is a very important pathogen of pneumonia in ruminants. Bighorn sheep (BHS, Ovis canadensis) are highly susceptible to M. haemolytica-caused pneumonia which has significantly contributed to the drastic decline of bighorn sheep population in North America. Pneumonia outbreaks in wild BHS can cause mortality as high as 90%. Leukotoxin is the critical virulence factor of M. haemolytica. In a 'proof of concept' study, an experimental vaccine containing leukotoxin and surface antigens of M. haemolytica developed by us induced 100% protection of BHS, but required multiple booster injections. Vaccination of wild BHS is difficult. But they can be vaccinated at the time of transplantation into a new habitat. Administration of booster doses, however, is impossible. Therefore, a vaccine that does not require booster doses is necessary to immunize BHS against M. haemolytica pneumonia. Herpesviruses are ideal vectors for development of such a vaccine because of their ability to undergo latency with subsequent reactivation. As the first step towards developing a herpesvirus-vectored vaccine, we constructed a chimeric protein comprising the leukotoxin-neutralizing epitopes and the immuno-dominant epitopes of the outer membrane protein PlpE. The chimeric protein was efficiently expressed in primary BHS lung cells. The immunogenicity of the chimeric protein was evaluated in mice before inoculating BHS. Mice immunized with the chimeric protein developed antibodies against M. haemolytica leukotoxin and PlpE. More importantly, the anti-leukotoxin antibodies effectively neutralized leukotoxin-induced cytotoxicity. Taken together, these results represent the successful completion of the first step towards developing a herpesvirus-vectored vaccine for controlling M. haemolytica pneumonia in BHS, and possibly other ruminants.

Fusobacterium necrophorum in North American Bighorn Sheep ( Ovis canadensis ) Pneumonia.

Fusobacterium necrophorum has been detected in pneumonic bighorn sheep (BHS; Ovis canadensis ) lungs, in addition to the aerobic respiratory pathogens Mannheimia haemolytica , Bibersteinia trehalosi , Pasteurella multocida , and Mycoplasma ovipneumoniae . Similar to M. haemolytica , F. necrophorum produces a leukotoxin. Leukotoxin-induced lysis and degranulation of polymorphonuclear leukocytes (PMNs) and macrophages are responsible for acute inflammation and lung tissue damage characteristic of M. haemolytica -caused pneumonia. As one approach in elucidating the role of F. necrophorum in BHS pneumonia, we determined the frequency of the presence of F. necrophorum in archived pneumonic BHS lung tissues, and susceptibility of BHS leukocytes to F. necrophorum leukotoxin. A species-specific PCR assay detected F. necrophorum in 37% of pneumonic BHS lung tissues (total tested n=70). Sequences of PCR amplicons were similar to the less virulent F. necrophorum subsp. funduliforme. Fusobacterium necrophorum leukotoxin exhibited cytotoxicity to BHS PMNs and peripheral blood mononuclear cells. As with the M. haemolytica leukotoxin, F. necrophorum leukotoxin was more toxic to BHS PMNs than domestic sheep PMNs. It is likely that F. necrophorum enters the lungs after M. haemolytica and other aerobic respiratory pathogens enter the lungs and initiate tissue damage, thereby creating a microenvironment that is conducive for anaerobic bacterial growth. In summary, Fusobacterium leukotoxin is highly toxic for BHS leukocytes; however, based on the PCR findings, it is unlikely to play a direct role in the development of BHS pneumonia.

Role of carriers in the transmission of pneumonia in bighorn sheep (Ovis canadensis).

In the absence of livestock contact, recurring lamb mortality in bighorn sheep (Ovis canadensis) populations previously exposed to pneumonia indicates the likely presence of carriers of pneumonia-causing pathogens, and possibly inadequate maternally derived immunity. To investigate this problem we commingled naïve, pregnant ewes (n=3) with previously exposed rams (n=2). Post-commingling, all ewes and lambs born to them acquired pneumonia-causing pathogens (leukotoxin-producing Pasteurellaceae and Mycoplasma ovipneumoniae), with subsequent lamb mortality between 4-9 weeks of age. Infected ewes became carriers for two subsequent years and lambs born to them succumbed to pneumonia. In another experiment, we attempted to suppress the carriage of leukotoxin-producing Pasteurellaceae by administering an antibiotic to carrier ewes, and evaluated lamb survival. Lambs born to both treatment and control ewes (n=4 each) acquired pneumonia and died. Antibody titers against leukotoxin-producing Pasteurellaceae in all eight ewes were 'protective' (>1:800 and no apparent respiratory disease); however their lambs were either born with comparatively low titers, or with high (but non-protective) titers that declined rapidly within 2-8 weeks of age, rendering them susceptible to fatal disease. Thus, exposure to pneumonia-causing pathogens from carrier ewes, and inadequate titers of maternally derived protective antibodies, are likely to render bighorn lambs susceptible to fatal pneumonia.

PCR assay detects Mannheimia haemolytica in culture-negative pneumonic lung tissues of bighorn sheep (Ovis canadensis) from outbreaks in the western USA, 2009-2010.

Mannheimia haemolytica consistently causes severe bronchopneumonia and rapid death of bighorn sheep (Ovis canadensis) under experimental conditions. However, Bibersteinia trehalosi and Pasteurella multocida have been isolated from pneumonic bighorn lung tissues more frequently than M. haemolytica by culture-based methods. We hypothesized that assays more sensitive than culture would detect M. haemolytica in pneumonic lung tissues more accurately. Therefore, our first objective was to develop a PCR assay specific for M. haemolytica and use it to determine if this organism was present in the pneumonic lungs of bighorns during the 2009-2010 outbreaks in Montana, Nevada, and Washington, USA. Mannheimia haemolytica was detected by the species-specific PCR assay in 77% of archived pneumonic lung tissues that were negative by culture. Leukotoxin-negative M. haemolytica does not cause fatal pneumonia in bighorns. Therefore, our second objective was to determine if the leukotoxin gene was also present in the lung tissues as a means of determining the leukotoxicity of M. haemolytica that were present in the lungs. The leukotoxin-specific PCR assay detected leukotoxin gene in 91% of lung tissues that were negative for M. haemolytica by culture. Mycoplasma ovipneumoniae, an organism associated with bighorn pneumonia, was detected in 65% of pneumonic bighorn lung tissues by PCR or culture. A PCR assessment of distribution of these pathogens in the nasopharynx of healthy bighorns from populations that did not experience an all-age die-off in the past 20 yr revealed that M. ovipneumoniae was present in 31% of the animals whereas leukotoxin-positive M. haemolytica was present in only 4%. Taken together, these results indicate that culture-based methods are not reliable for detection of M. haemolytica and that leukotoxin-positive M. haemolytica was a predominant etiologic agent of the pneumonia outbreaks of 2009-2010.