Pasteurella multocida is a Gram-negative bacterial pathogen that is the causative agent of a wide range of diseases in many animal species, including humans. A widely used method for differentiation of P. multocida strains involves the Heddleston serotyping scheme. This scheme was developed in the early 1970s and classifies P. multocida strains into 16 somatic or lipopolysaccharide (LPS) serovars using an agar gel diffusion precipitin test. However, this gel diffusion assay is problematic, with difficulties reported in accuracy, reproducibility, and the sourcing of quality serovar-specific antisera. Using our knowledge of the genetics of LPS biosynthesis in P. multocida, we have developed a multiplex PCR (mPCR) that is able to differentiate strains based on the genetic organization of the LPS outer core biosynthesis loci. The accuracy of the LPS-mPCR was compared with classical Heddleston serotyping using LPS compositional data as the "gold standard." The LPS-mPCR correctly typed 57 of 58 isolates; Heddleston serotyping was able to correctly and unambiguously type only 20 of the 58 isolates. We conclude that our LPS-mPCR is a highly accurate LPS genotyping method that should replace the Heddleston serotyping scheme for the classification of P. multocida strains. P asteurella multocida is the primary causative agent of a wide range of economically important diseases, including hemorrhagic septicemia in ungulates, atrophic rhinitis in pigs, fowl cholera in birds, snuffles in rabbits, and enzootic pneumonia and shipping fever in cattle, sheep, and pigs (1). P. multocida also causes opportunistic infections in humans, often following cat or dog bites, and plays a contributory role, together with other pathogens, in a range of lower respiratory tract infections and sporadic septicemias in ungulates (1).P. multocida strains have classically been differentiated using serological techniques. Strains can be classified into five capsular serogroups (A, B, D, E, and F) using an indirect hemagglutination test (2) and into 16 somatic or lipopolysaccharide (LPS) serovars (serotypes) using the Heddleston gel diffusion precipitin test (3). Both of these schemes have been widely used. Isolates are commonly assigned a combined designation, such as A:1 (capsular serogroup A and LPS serovar 1) or B:2 (capsular serogroup B and LPS serovar 2).P. multocida LPS is an immunodominant antigen critical for homologous protection stimulated by bacterin (killed-cell) vaccines (4). Furthermore, in the P. multocida strain VP161, a fulllength LPS molecule is essential for the ability to cause acute disease (5, 6). Heddleston serotyping is currently the only method used to differentiate P. multocida strains on the basis of LPS type. However, the accuracy of Heddleston serotyping has never been objectively tested, as the precise LPS structures produced by different strains have not been known. Indeed, there have been many informal as well as formal reports that the Heddleston system fails to type many isolates and lacks accuracy and reproducibilit...

Sixty-one Burkholderia cepacia isolates from patients with cystic fibrosis (CF) and four plant isolates were screened for production of the siderophores salicylic acid (SA), pyochelin, cepabactin, and ornibactins and fingerprinted by a PCR-based randomly amplified polymorphic DNA (RAPD) method. Of the 24 RAPD types determined, 22 (92%) were associated with isolates that produced SA, 21 (87%) were associated with isolates that produced ornibactins, 15 (60%) were associated with isolates that produced pyochelin, and 3 (12%) were associated with isolates that produced cepabactin. Of the 24 RAPD types plus 2 phenotypic variants of types 1 and 9, 3 were associated with isolates that produced all four siderophores, 8 were associated with isolates that produced three siderophores, 12 were associated with isolates that produced two siderophores, and 3 were associated with isolates that produced only one siderophore. These results suggest that the numbers and types of siderophores produced by CF isolates of B. cepacia correlate with RAPD type and that SA and ornibactins are the most prevalent siderophores produced.

The live attenuated influenza virus vaccine (LAIV) is preferentially recommended for use in persons 2 through 49 years of age but has not been approved for children under 2 or asthmatics due to safety concerns. Therefore, increasing safety is desirable. Here we describe a murine LAIV with reduced pathogenicity that retains lethality at high doses and further demonstrate that we can enhance safety in vivo through mutations within NS1. This model may permit preliminary safety analysis of improved LAIVs. Influenza A virus is a respiratory pathogen that infects through the upper airway and leads to pathology via replication in the lower airway (1). The temperature gradient between these two areas in people enabled the development of the cold-adapted, live attenuated influenza virus vaccine (LAIV [FluMist]) that replicates in the cooler upper respiratory tract to trigger a protective immune response but cannot damage the lower respiratory tract due to the elevated temperatures restricting replication (2). This temperature-sensitive (ts) attenuated (att) phenotype is imparted by five mutations within the viral replicative machinery: namely, PB2 N265S, PB1 K391E, D581G, and A661T, and NP D34G (3, 4). Although this vaccine has an overall acceptable safety profile, it is not approved for use in children under 2 years of age due to concerns about elevated hospitalizations due to wheezing (5, 6). For this reason, it is also not approved for use in asthmatics. Therefore, development of vaccines with increased safety over LAIV is desirable. Currently, no mouse model exists for the adequate assessment of the safety of the LAIV. In experimental animal studies with LAIV, elevated doses of LAIV do not elicit pathology, rendering determination of safety impossible (7-10). Here, we describe a model with which we can assess alterations in vaccine safety.The parental strain for our vaccine is a well-characterized, murine-lethal strain of influenza A virus (A/Puerto Rico/8/34 H1N1, PR8). We introduced four ts att mutations from LAIV into PR8 (NP D34G is natively present) via site-directed mutagenesis (Agilent) and rescued this virus using plasmid-based reverse genetics techniques (11). This ts att virus (referred to henceforth as "PR8 LAIV") has been previously characterized in cell culture, but its phenotype in mice was not demonstrated (8). PR8 wild-type (WT) virus has a 50% lethal dose (LD 50 ) in C57BL/6 (B6) mice of 10 to 25 PFU (12, 13). Thus, we sought to ascertain the LD 50 of PR8 LAIV (Fig. 1). Groups of mice (n ϭ 5) were intranasally

No abstract

We isolated serologically identical (by serovar determination and porin variable region [VR] typing) strains of Neisseria gonorrhoeae from an infected male and two of his monogamous female sex partners. One strain (termed 398078) expressed the L1 (Gal␣1 3 3Gal␤1 3 4Glc␤1 3 4HepI) lipooligosaccharide (LOS) structure exclusively; the other (termed 398079) expressed the lacto-N-neotetraose (LNT; Gal␤1 3 4GlcNAc␤1 3 3Gal␤1 3 4Glc␤1 3 4HepI) LOS structure. The strain from the male index case expressed both glycoforms and exhibited both immunotypes. Nuclear magnetic resonance analysis revealed that sialic acid linked to the terminal Gal of L1 LOS via an ␣2 3 6 linkage and, as expected, to the terminal Gal of LNT LOS via an ␣2 3 3 linkage. Insertional inactivation of the sialyltransferase gene (known to sialylate LNT LOS) abrogated both L1 LOS sialylation and LNT LOS sialylation, suggesting a bifunctional nature of this enzyme in gonococci. Akin to our previous observations, sialylation of the LNT LOS of strain 398079 enhanced the binding of the complement regulatory molecule, factor H. Rather surprisingly, factor H did not bind to sialylated strain 398078. LOS sialylation conferred the LNT LOS-bearing strain complete (100%) resistance to killing by even 50% nonimmune normal human serum (NHS), whereas sialylation of L1 LOS conferred resistance only to 10% NHS. The ability of gonococcal sialylated LNT to bind factor H confers high-level serum resistance, which is not seen with sialylated L1 LOS. Thus, serum resistance mediated by sialylation of gonococcal L1 and LNT LOS occurs by different mechanisms, and specificity of factor H binding to sialylated gonococci is restricted to the LNT LOS species.The lacto-N-neotetraose (LNT) lipooligosaccharide (LOS) of Neisseria gonorrhoeae can become sialylated when grown in medium supplemented with 5Ј-cytidinemonophospho-Nacetylneuraminic acid (CMP-NANA) (20). LOS sialylation renders strains that are otherwise susceptible to complementmediated killing, resistant to killing by nonimmune normal human serum (NHS) (26, 31). One explanation is the ability of gonococci bearing the sialylated LNT LOS to bind factor H (37), an important fluid-phase regulatory protein of the alternative pathway of complement (9,29,53,56). Gonococcal LOS undergoes significant phase variability in vivo, which may result in a shift of expression away from the "conventional" (terminal lactosamine of LNT) sialylation site (3, 12). LOS phase variation that results in the loss of the ability of an otherwise serum-sensitive gonococcal strain to sialylate its LOS may render such a strain highly susceptible to complement-mediated killing and therefore may be disadvantageous to an organism, necessitating a redundant strategy to evade killing by complement. The terminal Gal on meningococcal L1 LOS (Gal␣1 3 3Gal␤1 3 4Glc␤1 3 4HepI) can also be sialylated (51). The HepI hexose substitution of L1 LOS is structurally identical to the P K blood group antigen (19) and is commonly found in the LOS of Haemophilus and Moraxella ...