The systems approach and project management in the Naval Laboratory

http://archive.org/details/systemsapproachp00qur

The Entomopathogenic Bacterial Endosymbionts Xenorhabdus and Photorhabdus: Convergent Lifestyles from Divergent Genomes

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points

Data for: Molecular Surveillance of Novel Tick-borne Parasites in Madagascar’s Lemurs

PCR results for 4 different lemur species testing for 3 different tick-borne parasites. Information on the lemurs' location, sex and age are included

Vector‐borne disease and its relationship to hematologic abnormalities and microalbuminuria in retired racing and show‐bred greyhounds

Abstract Background Reference intervals for platelets and white blood cell (WBCs) counts are lower in greyhounds than other breeds. Proteinuria is common. Vector‐borne diseases (VBD) cause thrombocytopenia, leukopenia, and proteinuria. Racing greyhounds are commonly exposed to vectors that carry multiple organisms capable of chronically infecting clinically healthy dogs. Hypothesis/Objectives Vector‐borne disease prevalence is higher in retired racing greyhounds than in show‐bred greyhounds. Occult infection contributes to breed‐related laboratory abnormalities. Animals Thirty National Greyhound Association (NGA) retired racing and 28 American Kennel Club (AKC) show‐bred greyhounds. Methods Peripheral blood was tested for Anaplasma, Babesia, Bartonella, Ehrlichia, hemotropic Mycoplasma, and Rickettsia species using PCR. Antibodies to Anaplasma, Babesia, Bartonella, Ehrlichia, and Rickettsia species and Borrelia burgdorferi were detected using immunofluorescence and ELISA assays. Complete blood counts, semiquantitative platelet estimates, and microalbuminuria concentration were determined. Results Seven of 30 NGA and 1/28 AKC greyhounds tested positive for ≥1 VBD (P = .05). More positive tests were documented in NGA (10/630) than in AKC dogs (1/588; P = .02). Exposure to Bartonella species (3/30), Babesia vogeli (2/30), Ehrlichia canis (1/30), and infection with Mycoplasma hemocanis (3/30) occurred in NGA dogs. Platelet counts or estimates were >170 000/μL. White blood cell counts  .99; 1/8 VBD positive; 8/51 VBD negative, P = .99) and microalbuminuria (10/21 AKC; 5/26 NGA, P = .06; 1/8 VBD positive; 14/25 VBD negative, P = .41) were not associated with VBD. Conclusions and Clinical Importance The prevalence of thrombocytopenia and B. vogeli exposure was lower than previously documented. Larger studies investigating the health impact of multiple VBD organisms are warranted

Targeted Next-Generation Sequencing for Comprehensive Testing for Selected Vector-Borne Pathogens in Canines

The standard for detecting vector-borne pathogens is real-time PCR (rtPCR). However, this requires many individual tests to obtain an accurate diagnosis. The purpose of this study was to develop and validate a targeted next-generation sequencing (NGS) assay for vector-borne pathogens. Pathogen target regions were amplified via PCR using two primer pools that were developed in conjunction with ThermoFisher Scientific, and barcoded DNA libraries were prepared and sequenced with the Ion Torrent S5 system. Data were assembled using SPAdes and mapped to a reference file containing sequences from the pathogens. The raw reads were analyzed to confirm the results. Test feasibility and analytical specificity were evaluated with type strains or validated positive clinical samples from dogs. The analytical sensitivity of the method was compared to Ct values obtained by rtPCR testing. Diagnostic sensitivity and specificity were assessed with a set of known positive and negative clinical samples based on rtPCR testing. Positive and negative percent agreements and Cohen’s kappa were calculated. The primer sets were specific for the intended targets, based on sequence analysis of the amplified products, and the method detected 17 different pathogens. Analytical sensitivity was equivalent to an rtPCR Ct value of approximately 35–36. The positive percent agreement was 92%, and the negative percent agreement was 88%. Cohen’s kappa was 0.804, which indicates almost perfect agreement between the rtPCR assays and the targeted NGS assay. Using a targeted method reduces the costs associated with NGS sequencing and allows for a 2–3 day turn-around time, making this a viable method for detection of vector-borne pathogens in canine whole blood samples

Detection of Anaplasma phagocytophilum in an inflammatory pericardial effusion of a dog

Abstract An 11‐year‐old female spayed German Wirehaired Pointer with a 1‐week history of lethargy, hyporexia, diarrhea, and coughing presented with pericardial effusion causing cardiac tamponade. An echocardiogram revealed no structural cause for pericardial effusion. The pericardial effusion was an exudate with mixed macrophagic and neutrophilic inflammation. Morulae occasionally were found within neutrophils. The pericardial fluid and blood were qPCR and cPCR positive for Anaplasma phagocytophilum (NC State University, Vector‐borne Disease Diagnostic Laboratory, Raleigh, NC). The dog's blood was negative by ELISA (Vetscan Flex4 Rapid Test, Zoetis, Parsippany, NJ) for A. phagocytophilum antibodies at initial presentation and subsequently positive (SNAP4DxPlus, IDEXX, Westbrook, ME) 7 days later. After pericardiocentesis and administration of doxycycline (5 mg/kg PO q12h for 14 days), a repeat echocardiogram performed 1 month later showed no recurrence of pericardial effusion

RT-PCR-based tyrosine kinase display profiling of canine melanoma: IGF-1 receptor as a potential therapeutic target

Canine malignant melanoma (CMM) resembles human malignant melanoma in terms of metastatic behavior, refractoriness to standard therapy, and tumor antigen expression but it is largely unknown how CMM resembles human melanoma with regard to molecular pathogenesis and cellular signaling. No attempt has been made to systematically define the repertoire of tyrosine kinases (TKs) expressed in CMM. This study used a reverse transcription-PCR display technique to evaluate the expression of multiple TKs in the 17CM98 CMM cell line. RT-PCR was performed using degenerate primers coding for highly conserved regions flanking the kinase domains of many TKs and the repertoire of TKs expressed was determined using standard molecular cloning techniques. Sequencing 46 clones yielded canine homologs of insulin-like growth factor-1 receptor (IGF-1R) (50%), JAK1 (17%), PDGFR-a (11%), FGFR1 (9%), Axl (7%), Abl (4%), and PTK2 (2%). Interestingly, IGF-1R, JAK1, and Axl were detected in human melanoma using similar techniques, supporting the cross-species validity of this assay. Given the abundance of IGF-1R clones, we determined the biological effect of rhIGF-1 in 17CM98 cells. IGF-1 stimulated cell proliferation and vascular endothelial growth factor production in 17CM98, and addition of the IGF-1R inhibitor ADW742 abrogated IGF-1-induced phenotypic changes. Expression of IGF-1R mRNA was detected in five of five additional CMM cell cultures, and IGF-1R protein was detected in five of six primary tumors evaluated, suggesting that IGF-1R expression may be common in CMM and may provide a novel target for future therapy. In conclusion, this study suggests that similar TKs are expressed in human and canine melanoma, and shows potential antitumor effects of IGF-1R inhibition in CMM

Serological and molecular analysis of feline vector-borne anaplasmosis and ehrlichiosis using species-specific peptides and PCR

Abstract Background With the exception of Bartonella spp. or Cytauxzoon felis, feline vector-borne pathogens (FVBP) have been less frequently studied in North America and are generally under-appreciated as a clinical entity in cats, as compared to dogs or people. This study investigated selected FVBP seroreactivity and PCR prevalence in cats using archived samples. Methods Feline blood samples submitted to the Vector Borne Diseases Diagnostic Laboratory (VBDDL) at North Carolina State University College of Veterinary Medicine (NCSU-CVM) between 2008 and 2013 were tested using serological assays and PCR. An experimental SNAP® Multi-Analyte Assay (SNAP® M-A) (IDEXX Laboratories, Inc. Westbrook, Maine, USA) was used to screen all sera for antibodies to Anaplasma and Ehrlichia genus peptides and A.phagocytophilum, A.platys, B.burgdorferi, E.canis, E.chaffeensis, and E.ewingii species-specific peptides. PCR assays were used to amplify Anaplasma or Ehrlichia DNA from extracted ethylenediaminetetraacetic acid (EDTA)-anti-coagulated blood samples. Amplicons were sequenced to identify species. Results Overall, 7.8 % (56/715) of cats were FVBP seroreactive and 3.2 % (13/406) contained Anaplasma or Ehrlichia DNA. Serologically, B.burgdorferi (5.5 %) was the most prevalent FVBP followed by A.phagocytophilum (1.8 %). Ehrlichia spp. antibodies were found in 0.14 % (12/715) of cats with species-specific seroreactivity to E.canis (n = 5), E.ewingii (n = 2) and E.chaffeensis (n = 1). Of seropositive cats, 16 % (9/56) were exposed to more than one FVBP, all of which were exposed to B.burgdorferi and either A.phagocytophilum (n = 7) or E.ewingii (n = 2). Based upon PCR and DNA sequencing, 4, 3, 3, 2, and 1 cat were infected with A.phagocytophilum, A.platys, E. ewingii, E. chaffeensis and E.canis, respectively. Conclusions Cats are exposed to and can be infected with vector-borne pathogens that commonly infect dogs and humans. To our knowledge, this study provides the first evidence for E.chaffeensis and E.ewingii infection in naturally-exposed cats in North America. Results from this study support the need for regional, serological and molecular FVBP prevalence studies, the need to further optimize serodiagnostic and PCR testing for cats, and the need for prospective studies to better characterize clinicopathological disease manifestations in cats infected with FVBP