Abstract. Clinical and Laboratory Standards Institute interpretive breakpoints for in vitro susceptibility tests that predict mecA-mediated oxacillin resistance in Staphylococcus pseudintermedius isolates from animals have been changed twice in the past decade. Moreover, there are no counterpart recommendations for human isolates of S. pseudintermedius. Individual medical and veterinary laboratories variably use interpretive breakpoints identical to those recommended for use with Staphylococcus aureus or identical to those recommended for use with coagulase-negative staphylococci. The purpose of the current study was to examine correlations between oxacillin disk diffusion, oxacillin gradient diffusion, oxacillin microbroth dilution, and cefoxitin disk diffusion tests used to predict mecA-mediated resistance in S. pseudintermedius and to retrospectively estimate, from disk diffusion zone diameter measurements, the prevalence and rate of increase of oxacillin resistance among canine S. pseudintermedius isolates submitted to a veterinary teaching hospital laboratory. Oxacillin disk diffusion zone diameters of #17 mm and oxacillin minimum inhibitory concentrations of $0.5 mg/ml were highly correlated with detection of mecA in canine S. pseudintermedius isolates by polymerase chain reaction. MecA-mediated resistance among S. pseudintermedius isolates from dogs increased from less than 5% in 2001 to near 30% in 2007. More than 90% of the methicillin-resistant S. pseudintermedius isolates in 2006 and 2007 were also resistant to representatives of $4 additional antimicrobial drug classes. Cefoxitin disk diffusion with the resistance breakpoint set at #24 mm significantly underestimated the presence of mecA in S. pseudintermedius.

The ability of convalescent serum to passively protect calves against Haemophilus somnus-induced pneumonia was studied. Preimmune and convalescent serum were obtained from calves before or after recovery from experimental chronic H. somnus pneumonia. Passive protection was assessed in another group of calves by intrabronchial inoculation of H. somnus that had been incubated with preimmune or convalescent serum. Each calf was inoculated with each treatment in alternating caudal lung lobes. Twenty-four hours after inoculation almost no pneumonia was present in lungs inoculated with bacteria incubated with convalescent serum, whereas severe pneumonia was present in lungs inoculated with bacteria incubated with preimmune serum. Quantitation of calf pneumonia in both treatment groups indicated a significantly different protective capacity between convalescent serum and preimmune serum (P < 0.0005). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting of purified H. somnus lipopolysaccharide resulted in intense reactivity with convalescent serum, but no reactivity was detected with preimmune serum. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of H. somnus outer membrane-enriched fractions, Western blots with convalescent serum gave intense reactions against H. somnus outer membrane antigens with apparent molecular masses of 78 and 40 kilodaltons and weaker reactions with 60-, 34-, 31-, 29-, 18-, and 15-kilodalton outer membrane antigens. No reactivity was detected with preimmune serum. Antibodies eluted from H. somnus after adsorption of convalescent serum reacted almost identically to unadsorbed convalescent serum in Western blots against bacterial outer membrane-enriched fractions. Thus, most of the antigens recognized by convalescent serum are likely to be on the bacterial surface and accessible to antibody. Surface antigens recognized by protective convalescent serum are candidate antigens for a subunit vaccine against H. somnus pneumonia.

Polymyxin nonapeptides, proteolytic derivatives of polymyxin antibiotics, are less toxic than their parent compounds but retain some of their antibacterial activities. To confirm and expand observations that polymyxin nonapeptides have anti-endotoxin activity, we studied the ability of colistin nonapeptide to bind to bacterial lipopolysaccharide (LPS) LPS-induced [3H]thymidine uptake by splenic lymphocytes, but its activity was less than 1/10 that of colistin. We conclude that colistin nonapeptide binds to LPS and possesses antiendotoxin activity. However, the anti-endotoxin activity of the nonapeptide is considerably less than that of its parent compound, colistin.The polymyxins are a family of polycationic amphipathic antibiotics that have a wide gram-negative spectrum yet are generally too toxic for human use. These compounds consist of a hydrophilic polypeptide ring and a hydrophobic fatty acid chain and are believed to act by binding to the bacterial cell membrane and functioning as a cationic detergent to produce cytoplasmic leakage and cell death (5, 15, 21). The polymyxins also bind tightly to bacterial lipopolysaccharide (LPS) (14,16) and are able to block a number of its biological activities in vitro (2, 7, 13) and in vivo (1,(9)(10)(11)17). One of the polymyxins, colistin, can be degraded to a nonapeptide that lacks the fatty acid moiety and is approximately 15-fold less toxic to mice (6) than is the parent compound (3). Unlike the parent compound, colistin nonapeptide has little direct antibacterial activity (6). Recently, a similar polymyxin derivative, a nonapeptide made by using polymyxin B as a parent material, has been shown to render gram-negative organisms susceptible to the action of hydrophobic antibiotics, complement, and antibody, apparently by acting as an outer membrane disorganizer and allowing increased access of these substances to their site of action (22)(23)(24)(25)(26).In these studies we wished to determine whether polymyxin nonapeptide derivatives bound to LPS in a similar manner as did the parent compounds and whether they could inhibit the biological effects of LPS. We chose to evaluate colistin nonapeptide because it has been tested for acute toxicity (6) and because colistin sulfate, the parent compound, is less toxic for humans than is polymyxin B (3). Parke, Davis & Co., Morris Plains, N.J. Crude ficin was obtained from Sigma Chemical Co., St. Louis, Mo., and partially purified as described by Chihara et al. (6). Ficin (500 mg) was combined with 3.3 g of colistin sulfate in 330 ml of 0.1 M phosphate buffer (pH 7.0), activated (12) by the addition of 2-mercaptoethanol at a final concentration of 0.007 M, and incubated for 48 h at 37°C. The reaction was terminated by boiling, and the resulting suspension was centrifuged to remove particulate matter, filtered for sterility through a filter (pore size, 0.2 ,um), and lyophilized. MATERIALS AND METHODSColistin assay. A standard curve for colistin concentration was made by using a disk sensitivity technique (Fig. 1). The te...

Abstract. Feline coronavirus (FCoV) is an important pathogen of domestic and nondomestic Felidae. Investigation into the prevalence of FCoV in exotic Felidae has relied primarily on serology. The usefulness of genetic detection of FCoV using reverse transcription and nested polymerase chain reaction (RT/nPCR) for viral screening was investigated. Seventy-five biologic samples, primarily feces, from captive felids from 11 institutions were tested using PCR. Serum samples collected from all but 12 of these animals were tested for antibodies to type I and type II FCoV by indirect immunofluorescence. Twenty-four animals were positive using RT/nPCR for virus. Twenty-nine animals were seropositive to type I and/or type II FCoV. From serologic data, infection with a virus antigenically related to FCoV type I occurred most commonly. Serology did not correlate with virus shedding because 13 animals were seronegative to FCoV type I and II but positive using RT/nPCR for virus. Conversely, 20 animals were seropositive but negative using RT/nPCR for FCoV. Some of the populations in which virus was detected had experienced health problems, including feline infectious peritonitis (FIP), necrotizing colitis, and mild enteritis. In addition to its role in FIP, this virus may play a role in gastrointestinal diseases of infected animals. This study demonstrates that FCoV is a significant infectious agent of captive felids because over half of the animals tested were positive by viral genetic detection, serology, or both. Dependence upon one method for detection of infection is unreliable.Infectious diseases are potentially devastating to wildlife conservation. In particular, large carnivore populations are susceptible to serious consequences because they are already threatened by restricted ranges, habitat destruction, and overexploitation of themselves and their prey. 9 In addition, extenuating circumstances such as inbreeding, stress, or malnutrition can worsen the outcome of infection with a pathogen. 9 Feline coronavirus is a contagious and serious pathogen of Felidae. It is associated with mild to severe enteritis and is the etiologic agent of feline infectious peritonitis (FIP), a fatal disease. 2,6 In domestic cat populations, FIP is a sporadic disease though the virus is ubiquitous. 10 In contrast, outbreaks of FIP have been reported in several nondomestic species. 1,3,13 In addition, feline coronavirus (FCoV) enteritis has resulted in mild to severe chronic diarrhea in several felid species and has been associated with vague signs of disease including weight loss, depression, and inappetence. 3,8 Control of this pathogen is complicated by the occurrence of persistent infections, with carriers serving as an important source of the virus in felid populations. 8 The vulnerability of exotic felid populations and the significant exposure rate emphasize the need for effective screening methods. The usefulness of FCoV genomic detection using reverse transcription and nested polymerase chain reaction (RT/nPCR) was investigated. V...

The ability of concentrated antibody against the 78or 40-kilodalton (kDa) outer membrane protein (OMP) of Haemophilus somnus to passively protect calves against H. somnus-induced pneumonia was determined. The 78-and 40-kDa OMPs were evaluated in passive protection experiments, because results of previous studies demonstrated their (i) immunogenicity for cattle, (ii) intense reactivity with convalescent-phase sera which passively protected calves against experimental H. somnus pneumonia, (iii) surface location and accessibility to antibody, and (iv) conservation among a wide range of H. somnus isolates obtained from animals with different diseases and from different geographic locations. The specificity of the two antisera evaluated in this study was verified by (i) immunoblots in which reactivity against the 78or 40-kDa OMP was present in postimmunization but not preimmunization serum and (il) immunoblots in which affinity-purified, surface-reactive antibodies in each antisera were used, which demonstrated that essentially only antibody to the 78or 40-kDa OMP was reactive with the surface of H. somnus. In enzyme-linked immunosorbent assays, the antiserum against the 40-kDA OMP contained immunoglobulin Gl (IgGl), IgG2, and IgM against H. somnus, while the antiserum against the 78-kDa OMP contained IgGl and IgM but no IgG2 against H. somnus. The antiserum against the 40-kDa OMP contained IgGl and IgG2 specific for the 40-kDa OMP, as determined by Western blot analysis. Slight reactivity against H. somnus lipopolysaccharide was detected by enzyme-linked immunosorbent assay but not by Western blot analysis. In passive protection experiments, preincubation of bacteria with antibody against the 40-kDa OMP protected calves (P < 0.025) against H. somnus pneumonia, while antibody against the 78-kDa OMP failed to protect calves against H. somnus pneumonia. Determination of the potential protective capacity of the 78-kDa OMP awaits resolution of the role of anti-78-kDa IgG2 in protection against H. somnus pneumonia. The 40-kDa OMP is, however, a good candidate antigen for evaluation of protective * Corresponding author.

Staphylococcus pseudintermedius is the primary cause of canine pyoderma and has been associated with diseases in other animals, including human beings. A high prevalence of methicillin and multidrug resistance has been reported in this bacterium in some geographic regions of the United States. Multilocus sequence type (MLST) 68 was implicated, initially, as the major clonal genotype based on a limited number of samples. The objectives of this study were to determine the population genetics of S. pseudintermedius isolated from a cross-section of the United States using a seven-locus multilocus sequence typing method, to identify clonal complexes (CCs), and to correlate sequence types with antimicrobial susceptibility profiles. A total of 190 S. pseudintermedius with 86 different MLSTs were detected and the constituents of three major CCs of methicillin-resistant S. pseudintermedius (MRSP), CC68, CC71, and CC84, were identified. Different patterns of resistance were associated with each CC. CC71 from the United States had notable differences with CC71 studied on other continents with chloramphenicol, tetracycline, and trimethoprim/sulfamethoxazole resistance. Some isolates with resistance to the broadest range of drugs tested, including that to chloramphenicol, had STs unrelated to the major CCs, suggesting the potential for the emergence of new clonal populations of MRSP that are resistant to most therapeutically useful antimicrobials.

To quantify the neutralization of bacterial lipopolysaccharide (LPS) by human plasma, dilutions of Escherichia coli 0113 LPS were incubated with plasma, followed by the addition of Limulus amebocyte lysate (LAL). The reaction between the LPS and LAL was monitored spectrophotometrically, and the concentration of LPS resulting in 50% lysate response (LR50) was determined. 'Analysis of 145 outdated plasma samples yielded a range of LR50 between 6 and 1,500 ng/ml. Pools of plasma with high and low LRs0 were prepared.

ABSTRACT:Free-ranging caribou and moose populations in some regions of Alaska undergo periodic declines in numbers. Caribou and moose are managed by the state as valuable resources for not only sustenance and subsistence, but also for cultural heritage. Incidence and prevalence of diseases that may impact herd health and recruitment from year to year are relevant to management decisions aimed to protect the long-term viability of these herds. Neospora caninum and Toxoplasma gondii are two apicomplexan parasites that can cause neurologic disease and abortions in their intermediate hosts and less frequently cause disease in their definitive hosts. The definitive hosts of N. caninum and T. gondii are canids and felids, respectively, and prevalence in the environment is in part dependent on maintenance of the life cycle through the definitive hosts. Serum samples from caribou (Rangifer tarandus, n5453), wolf (Canis lupus, n5324), moose (Alces alces, n5201), black-tailed deer (Odocoileus hemionus, n555), coyote (Canis latrans, n512), and fox (Vulpes vulpes, n59) collected in Alaska were assayed for N. caninum-and T. gondii-reactive antibodies with an immunofluorescent antibody test (IFAT) and a modified agglutination test (MAT), respectively. Seroprevalence of N. caninum was greater in caribou (11.5%) than in wolves (9.0%), moose (0.5%), or black-tailed deer (0%). Seroprevalence of T. gondii was greater in wolves (17.8%) than in caribou (0.4%), moose (0%), or black-tailed deer (0%). Seroprevalence of N. caninum and T. gondii were 16.7% and 0.0% in coyotes and 0.0% and 12.5% in fox, but small sample sizes prevented further analysis. Antibodies to N. caninum in young caribou compared to adult caribou suggest that vertical transmission may be an important component of new infections in Alaskan caribou. The spatial distribution of antibody-positive individuals across Alaska may reflect differences in frequency of definitive hosts and alteration of predation patterns among regions.