Characterization, distribution, antimicrobial resistance and resistance risk factors in staphylococci isolated from cats from 2001 to 2014.

Relatively few studies have been published describing the patterns of staphylococcal isolation and antimicrobial resistance over time in cats. The objective of this retrospective study was to determine the frequency, location, characteristics and antimicrobial resistance profiles of staphylococci isolated by the Louisiana Animal Disease Diagnostic Laboratory between the years 2001 and 2014. All feline staphylococcal isolates were classified phenotypically. Isolates corresponding to known or possibly pathogenic species (Staphylococcus intermedius group (SIG) and Staphylococcus aureus (SA)) as well as Staphylococcus epidermidis (SE) and non-speciated coagulase-negative staphylococci (CNS) were further evaluated to determine antimicrobial resistance patterns. A total of 519 staphylococci were isolated. The largest percentage of isolates was CNS, representing 39.3% of the total, while SIG, SE, SA and non-speciated coagulase positive staphylococci (CPS) represented 18.1%, 10.2%, 8.3% and 7.3%, respectively. Methicillin resistance (MR) was identified in 57.1% of SA and 20.5% of SIG. Resistance to 3 or more antimicrobial classes (multidrug resistance; MDR) was demonstrated in 54.5% of SA and 23.9% of SIG. The prevalence of MDR increased over time in both SIG and SA, while the prevalence of MR increased over time in SIG. An increase in mean antimicrobial resistance score over time was seen in SIG. This study demonstrates a high and increasing prevalence of MDR in SIG and SA, as well as increasing prevalence of MR in SIG isolated from cats.

Effect of West Nile virus DNA-plasmid vaccination on response to live virus challenge in red-tailed hawks (Buteo jamaicensis).

OBJECTIVE: To evaluate the safety and efficacy of an experimental adjuvanted DNA-plasmid vaccine against West Nile virus (WNV) in red-tailed hawks (Buteo jamaicensis). ANIMALS: 19 permanently disabled but otherwise healthy red-tailed hawks of mixed ages and both sexes without detectable serum antibodies against WNV. PROCEDURES: Hawks were injected IM with an experimental WNV DNA-plasmid vaccine in an aluminum-phosphate adjuvant (n = 14) or with the adjuvant only (control group; 5). All birds received 2 injections at a 3-week interval. Blood samples for serologic evaluation were collected before the first injection and 4 weeks after the second injection (day 0). At day 0, hawks were injected SC with live WNV. Pre- and postchallenge blood samples were collected at intervals for 14 days for assessment of viremia and antibody determination; oropharyngeal and cloacal swabs were collected for assessment of viral shedding. RESULTS: Vaccination was not associated with morbidity or deaths. Three of the vaccinated birds seroconverted after the second vaccine injection; all other birds seroconverted following the live virus injection. Vaccinated birds had significantly less severe viremia and shorter and less-intense shedding periods, compared with the control birds. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the WNV DNA-plasmid vaccine in red-tailed hawks was safe, and vaccination attenuated but did not eliminate both the viremia and the intensity of postchallenge shedding following live virus exposure. Further research is warranted to conclusively determine the efficacy of this vaccine preparation for protection of red-tailed hawks and other avian species against WNV-induced disease.

In vitro comparison of Staphylococcus pseudintermedius susceptibility to common cephalosporins used in dogs.

The in vitro activity of 10 cephalosporin antimicrobial agents against 75 isolates of methicillin-susceptible Staphylococcus pseudintermedius derived from dogs was assessed. The lowest minimal inhibitory concentration for 90% of strains (MIC90) values obtained were for cephalothin, cefovecin, and cefazolin (0.12 ug/mL), followed by ceftiofur and cefoxitin (0.25 ug/mL), cefpodoxime (0.5 ug/mL), and cefaclor and cefadroxil (1 ug/mL). The highest MIC90 values were found for cephalexin and cefixime (2 ug/mL). In this in vitro study, sensitivity to cephalothin was indicative of cephalexin susceptibility, although there were marked differences in MICs. Cephalothin susceptibility was not indicative of susceptibility to all tested cephalosporins, nor was there a clear trend in susceptibility based on cephalosporin generation.

Detection of West Nile virus RNA in mosquitoes and identification of mosquito blood meals collected at alligator farms in Louisiana.

Since 2001, alligator farms in the United States have sustained substantial economic losses because of West Nile virus (WNV) outbreaks in American alligators (Alligator mississippiensis). Once an initial infection is introduced into captive alligators, WNV can spread among animals by contaminative transmission. Some outbreaks have been linked to feeding on infected meat or the introduction of infected hatchlings, but the initial source of WNV infection has been uncertain in other outbreaks. We conducted a study to identify species composition and presence of WNV in mosquito populations associated with alligator farms in Louisiana. A second objective of this study was to identify the origin of mosquito blood meals collected at commercial alligator farms. Mosquitoes were collected from 2004 to 2006, using Centers for Disease Control light traps, gravid traps, backpack aspirators, and resting boxes. We collected a total of 58,975 mosquitoes representing 24 species. WNV was detected in 41 pools of females from 11 mosquito species: Anopheles crucians, Anopheles quadrimaculatus, Coquillettidia perturbans, Culex coronator, Culex erraticus, Culex nigripalpus, Culex quinquefasciatus, Mansonia titillans, Aedes sollicitans, Psorophora columbiae, and Uranotaenia lowii. The blood meal origins of 213 field-collected mosquitoes were identified based on cytochrome B sequence identity. Alligator blood was detected in 21 mosquitoes representing six species of mosquitoes, including Cx. quinquefasciatus and Cx. nigripalpus. Our results showed that mosquitoes of species that are known to be competent vectors of WNV fed regularly on captive alligators. Therefore, mosquitoes probably are important in the role of transmission of WNV at alligator farms.

Factors associated with mosquito pool positivity and the characterization of the West Nile viruses found within Louisiana during 2007.

BACKGROUND: West Nile virus (WNV) is an arbovirus of public health importance in the genus Flavivirus, a group of positive sense RNA viruses. The NS3 gene has a high level of substitutions and is phylogenetically informative. Likewise, substitutions in the envelope region have been postulated to enable viruses to subvert immune responses. Analysis of these genes among isolates from positive mosquitoes collected in Louisiana illustrates the variation present in the regions and provides improved insight to a phylogenetic model. Employing a GIS eco-regionalization method, we hypothesized that WNV pool positivity was correlated with regional environmental characteristics. Further, we postulated that the phylogenetic delineations would be associated with variations in regional environmental conditions. RESULTS: Type of regional land cover was a significant effect (p < 0.0001) in the positive pool prediction, indicating that there is an ecological component driving WNV activity. Additionally, month of collection was significant (p < 0.0001); and thus there is a temporal component that contributes to the probability of getting a positive mosquito pool. All virus isolates are of the WNV 2002 lineage. There appears to be some diversity within both forested and wetland areas; and the possibility of a distinct clade in the wetland samples. CONCLUSIONS: The phylogenetic analysis shows that there has been no reversion in Louisiana from the 2002 lineage which replaced the originally introduced strain. Our pool positivity model serves as a basis for future testing, and could direct mosquito control and surveillance efforts. Understanding how land cover and regional ecology effects mosquito pool positivity will greatly help focus mosquito abatement efforts. This would especially help in areas where abatement programs are limited due to either funding or man power. Moreover, understanding how regional environments drive phylogenetic variation will lead to a greater understanding of the interactions between ecology and disease prevalence.

Evaluation of surveillance methods for detection of West Nile virus activity in East Baton Rouge Parish, Louisiana, 2004-2006.

A 3-year study was conducted to determine if testing mosquitoes collected in modified sentinel chicken boxes for West Nile virus (WNV) or testing sentinel chickens for WNV antibody would detect WNV activity before onset of human cases in East Baton Rouge Parish, LA. In each year mosquitoes tested positive for WNV before the onset of human cases were detected, but seroconversions of sentinel chickens were detected after the human cases occurred. In 1 year we also compared the effectiveness of Centers for Disease Control and Prevention (CDC) light traps, gravid traps, and sentinel chicken box traps for collecting WNV-positive mosquitoes. Gravid traps collected more WNV-positive mosquitoes than CDC light traps or sentinel chicken box traps. However, WNV was detected earlier in mosquitoes collected from sentinel chicken box traps than in mosquitoes collected with gravid traps or CDC light traps. In total, 1,222 pools containing 19,353 mosquito specimens representing 18 species were tested for WNV. West Nile virus was detected in 59 mosquito pools from 4 species; 87% of the positive pools were detected from Culex quinquefasciatus, which was the most abundant species collected in all 3 years.

Complete genome analysis and virulence characteristics of the Louisiana West Nile virus strain LSU-AR01.

West Nile virus (WNV) is a member of the Flaviriridae family, which can cause significant morbidity and mortality in birds, horses, and humans. The WNV-LSU-AR01 strain was isolated from a dead blue jay in Louisiana in 2001. Phylogenetic analysis using 75 full WNV genomes revealed that the LSU-AR01 strain belongs to a distinct subclade among the North American strains. The LSU-AR01 strain differed from the NY-99 prototypic strain by 26 nucleotides causing six amino acid changes. An asparagine-to-lysine change was located immediately proximal to a known CD8(+)T cell epitope in NS4B, while a glutamine-to-lysine change was located within a predicted CD8(+)T cell epitope in NS5. The LSU-AR01 strain caused pronounced neuronal necrosis, perivascular cuffing and gliosis in comparison to the NY-99-infected mice. These results suggest that the previously identified Connecticut strains may contain highly neurovirulent strains such as the LSU-AR01 that have spread in North America.

Recombinant vesicular stomatitis virus-based west Nile vaccine elicits strong humoral and cellular immune responses and protects mice against lethal challenge with the virulent west Nile virus strain LSU-AR01.

Vesicular stomatitis virus (VSV) has been extensively utilized as a viral vector system for the induction of protective immune responses against a variety of pathogens. We constructed recombinant VSVs specifying either the Indiana or Chandipura virus G glycoprotein and expressing the West Nile virus (WNV) envelope (E) glycoprotein. Mice were intranasally vaccinated using a prime (Indiana)-boost (Chandipura) immunization approach and challenged with the virulent WNV-LSU-AR01. Ninety-percent (9 of 10) of the vaccinated mice survived as compared to 10% of the mock-vaccinated mice after WNV lethal challenge. Histopathological examination of brain tissues revealed neuronal necrosis in mock-vaccinated mice but not in vaccinated mice, and vaccinated, but not mock-vaccinated mice developed a strong neutralizing antibody response against WNV. Extensive immunological analysis using polychromatic flow cytometry staining revealed that vaccinated, but not mock-vaccinated mice developed robust cellular immune responses as evidenced by up-regulation of CD4(+) CD154(+) IFNgamma(+) T cells in vaccinated, but not mock-vaccinated mice. Similarly, vaccinated mice developed robust E-glycoprotein-specific CD8(+) T cell immune responses as evidenced by the presence of a high percentage of CD8(+) CD62L(low) IFNgamma(+) cells. In addition, a sizeable population of CD8(+) CD69(+) cells was detected indicating E-specific activation of mature T cells and CD4(+) CD25(+) CD127(low) T regulatory (T reg) cells were down-regulated. These results suggest that VSV-vectored vaccines administered intranasally can efficiently induce protective humoral and cellular immune responses against WNV infections.

Evaluation of microbial culture techniques for the isolation of Pythium insidiosum from equine tissues.

The purpose of this study was to evaluate the effects of sample handling, storage, and culture techniques on the isolation of Pythium insidiosum from infected equine tissues. Tissue and kunker samples obtained immediately posteuthanasia from a horse with subcutaneous pythiosis were used to assess the effects of sample type (kunkers vs. tissues), media type (selective vs. nonselective), storage technique, and storage time on P. insidiosum isolation rate. Overall, isolation rates were higher from fresh kunkers (94.6%) and stored kunkers (76.4%) than from fresh tissues (8.3%) or stored tissues (4.6%). Isolation of P. insidiosum also occurred more often on antibiotic-containing media than on nonselective media for both fresh and stored samples. For samples that were stored for 1-3 days prior to culture, P. insidiosum isolation rates were highest for the following techniques: kunkers stored at room temperature and plated on selective media (100%), kunkers stored at 4 C and then plated on either nonselective (91.7%) or selective (95.8%) media, kunkers stored on cold packs and then plated on either nonselective (93.8%) or selective (100%) media, kunkers stored in ampicillin solution and plated on selective media (100%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). For samples stored for 4-5 days, P. insidiosum isolation rates were highest for kunkers stored at 4 C and then plated on either nonselective (81.3%) or selective (87.5%) media, kunkers stored in ampicillin solution and then plated on selective media (87.5%), and kunkers stored in ampicillin/gentocin solution and plated on selective media (87.5%). Results of this study suggest that optimal isolation rates of P. insidiosum from infected equine tissues are achieved by culturing fresh kunkers on selective media. For samples that cannot be processed immediately, acceptable handling techniques include storage at room temperature for up to 3 days, refrigeration for up to 5 days, shipping on cold packs, and storage in antibiotic solution, each combined with subsequent inoculation on selective media.

Experimental Reproduction of Feline Mycobacterium fortuitum Panniculitis.

Resumen- Con el fin de lograr un modelo experimental de la infección por Mycobacterium fortuitum, causante de paniculitis en condiciones naturales en el gato, se inoculóM. fortuitum en el cojinte plantar de ratones o en el tejido adiposo inguinal de conejos y gatos. Los ratones manifestaron una dermatitis crónica y una linfadenitis granulomatosa necrotizante con localizatión intracelular del microorganismo. Los conejos manifestaron inflamaciones granulomatosas supurativas necrotizantes con microrganismos en vacuolas adiposas rodeadas por heterófilos macrófagos epitelioides y/o zonas de necrosis. Los cinco gatos adultos y una de las tres crias mostraron fistulas supurativas, úlceras puntuales o nódulos en el paniculo adiposo de la zona inguinal. La lesión en la región inguinal de estos seis animales consistia en una paniculitis granulomatosa; las otras dos crias presentaban una paniculitis piogranulomatosa necrotizante. Se identificaron bacilos en los cortes histológicos teñidos con Hematoxilina y Eosina en cuatro gatos adultos y en una de las crias. Se aislóMycobacterium fortunitum a partir del tejido adiposo en todos los gatos adultos y en una de las tres crias. La inoculación de 1.4 × 1010 M.fortuitum en el tejido adiposo subcutáneo inguinal en los gatos con grandes masas grasas en esas zonas causó una infección microbacteriana idéntica a la enfermedad felina en condiciones naturales. [Lewis, D. T., Hodgin, E. C, Foil, C. S., Cox, H. U., Roy, A. F., Lewis, D. D. Experimental reproduction of feline Mycobacterium fortuitum panniculitis. (Reproductión experimental de la paniculitis felina por Mycobacterium fortuitum). Abstract- In order to establish an animal model of the naturally occurring feline Mycobacterium fortuitum panniculitis an inoculum of M. fortuitum organisms was injected into the hindlimb footpad of mice or subcutaneous fat of the inguinal area of rabbits and cats. Mice developed chronic dermatitis and necrotizing granulomatous lymphadenitis with intracellular localization of the organism. Rabbits developed necrotizing suppurative granulomatous inflammation with organisms in heterophil-lined fat vacuoles, epithelioid macrophages and/or necrotic areas. All five adult cats and one of three kittens developed draining tracts, punctate ulcers or nodules in the panniculus adiposus of the inguinal area. A pyogranulomatous panniculitis characterized the inguinal region in these six animals; a necrotizing pyogranulomatous panniculitis was present in the remaining two kittens. Rod-shaped bacilli were present on hematoxylin and eosin stained tissue sections in four adult cats and one kitten. Mycobacterium fortuitum was isolated from the inguinal subcutaneous fat in all five adult cats and one of three kittens. Injection of 1.4 × 1010 M. fortuitum organisms into the subcutaneous fat of the inguinal area of cats with extensive inguinal fatpads produced a mycobacterial infection identical to the naturally occurring feline disease.