Michael J. Dark

SCID in Jack Russell Terriers: A New Animal Model of DNA-PKcs Deficiency

We recently described the incidence of a SCID disease in a litter of Jack Russell terriers. In this study, we show that the molecular defect in these animals is faulty V(D)J recombination. Furthermore, we document a complete deficit in DNA-dependent protein kinase activity that can be explained by a marked diminution in the expression of the catalytic subunit DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We conclude that as is the case in C.B-17 SCID mice and in Arabian SCID foals, the defective factor in these SCID puppies is DNA-PKcs. In mice, it has been clearly established that DNA-PKcs deficiency produces an incomplete block in V(D)J recombination, resulting in “leaky” coding joint formation and only a modest defect in signal end ligation. In contrast, DNA-PKcs deficiency in horses profoundly blocks both coding and signal end joining. Here, we show that although DNA-PKcs deficiency in canine lymphocytes results in a block in both coding and signal end joining, the deficit in both is intermediate between that seen in SCID mice and SCID foals. These data demonstrate significant species variation in the absolute necessity for DNA-PKcs during V(D)J recombination. Furthermore, the severity of the V(D)J recombination deficits in these three examples of genetic DNA-PKcs deficiency inversely correlates with the relative DNA-PK enzymatic activity expressed in normal fibroblasts derived from these three species.

scAAV-Mediated Gene Transfer of Interleukin 1-Receptor Antagonist to Synovium and Articular Cartilage in Large Mammalian Joints

With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for adeno-associated virus (AAV)-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1-receptor antagonist (hIL-1Ra) or green fluorescent protein was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 1011 vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA.

Multistrain genome analysis identifies candidate vaccine antigens of Anaplasma marginale

Anaplasmosis in domestic livestock is an impediment to animal health and production worldwide, especially in developing countries in Africa, Asia, and South America. Vaccines have been developed and marketed against the causative organism, Anaplasma marginale; however, these have not been widely used because of breakthrough infections caused by heterologous strains and because of the risk of disease induced by live vaccine strains themselves. Recently, molecular studies have enabled progress to be made in understanding the causes for breakthrough infections and in defining new vaccine targets. A. marginale has a system for antigenic variation of the MSP2 and MSP3 outer membrane proteins which are members of the pfam01617 gene superfamily. In this study, we used high throughput genome sequencing to define conservation of different superfamily members in ten U.S. strains of A. marginale and also in the related live vaccine strain A. marginale subspecies centrale. The comparisons included the pseudogenes that contribute to antigenic variation and other superfamily-encoded outer membrane proteins. Additionally, we examined conservation of other proteins proposed previously as vaccine candidates. These data showed significantly increased numbers of SNPs in A. marginale subspecies centrale when compared to all U.S. A. marginale strains. We defined a catalog of 19 conserved candidate vaccine antigens that may be suitable for development of a multi-component recombinant vaccine. The methods described are rapid and may be suitable for other prokaryotes where repeats comprise a substantial portion of their genomes.

Evidence for persistent Bovine viral diarrhea virus infection in a captive mountain goat (Oreamnos americanus).

Bovine viral diarrhea (BVD) viruses are pestiviruses that have been isolated from domestic and wild ruminants. There is serologic evidence of pestiviral infection in more than 40 species of free-range and captive mammals. Vertical transmission can produce persistently infected animals that are immunotolerant to the infecting strain of Bovine viral diarrhea virus (BVDV) and shed virus throughout their lives. Seven species (white-tailed deer, mouse deer, eland, domestic cattle, alpaca, sheep, and pigs) have been definitively identified as persistently infected with BVDV. This study provides serological, molecular, immunohistochemical, and histological evidence for BVDV infection in 2 captive mountain goats from a zoological park in Idaho. The study was triggered by isolation of BVDV from tissues and immunohistochemical identification of viral antigen within lesions of a 7-month-old male mountain goat (goat 1). Blood was collected from other mountain goats and white-tailed and mule deer on the premises for BVDV serum neutralization, viral isolation, and reverse transcription polymerase chain reaction. One 3-month-old mountain goat (goat 2) was antibody negative and BVDV positive in serum samples collected 3 months apart. This goat subsequently died, and though still antibody negative, BVDV was isolated from tissues and identified by immunohistochemistry within lesions. Sequencing and phylogenetic analysis identified the isolates as BVDV-2. These findings provide evidence of persistent infection in a mountain goat, underscoring the need for pestivirus control strategies for wild ruminants in zoological collections.

Whole-genome sequencing in bacteriology: state of the art

Over the last ten years, genome sequencing capabilities have expanded exponentially. There have been tremendous advances in sequencing technology, DNA sample preparation, genome assembly, and data analysis. This has led to advances in a number of facets of bacterial genomics, including metagenomics, clinical medicine, bacterial archaeology, and bacterial evolution. This review examines the strengths and weaknesses of techniques in bacterial genome sequencing, upcoming technologies, and assembly techniques, as well as highlighting recent studies that highlight new applications for bacterial genomics.

Autosomal recessive severe combined immunodeficiency of Jack Russell Terriers

Because of unexplained mortality among 33 sibling offspring of a single pair of dogs, a family of Jack Russell Terriers was investigated. Twelve pups, 5 male and 7 female, died between 8 and 14 weeks of age. Six of those animals died in the field within 50 hours following vaccination with modified live vaccines. Subsequent histopathologic examination revealed the absence of splenic white pulp in 4 dogs and hepatic inclusions diagnostic for adenoviral infection in 2 dogs. Two additional litters yielded 2 pups with the same splenic and hepatic lesions. These observations led to a detailed study of 7 siblings whelped specifically for this investigation. Four of these 7 siblings had a profound lymphopenia and a decrease in serum immunoglobulins. Six of these dogs were necropsied at 7 weeks of age, and 4 of them had marked hypoplasia of all lymphoid tissue. The affected pups had an 86% decrease in mean thymic weight, with poor corticomedullary differentiation, and very few CD3-positive (T cell) thymocytes were detected immunohistochemically. However, the affected thymic tissue stained intensely with a immunochemical stain for cytokeratin. The other affected lymphoid tissues were identified histologically only by stromal architectural characteristics. Lymph nodes lacked both CD3 and CD79a (B cell) positive cells. The analyzed breeding data were consistent with an autosomal recessive mode of inheritance. This canine severe combined immunodeficiency has immunologic and pathologic features similar to those observed in immunodeficient C.B-17 mice and Arabian horses.

Comparative genomics and transcriptomics of trait-gene association

BackgroundThe Order Rickettsiales includes important tick-borne pathogens, from Rickettsia rickettsii, which causes Rocky Mountain spotted fever, to Anaplasma marginale, the most prevalent vector-borne pathogen of cattle. Although most pathogens in this Order are transmitted by arthropod vectors, little is known about the microbial determinants of transmission. A. marginale provides unique tools for studying the determinants of transmission, with multiple strain sequences available that display distinct and reproducible transmission phenotypes. The closed core A. marginale genome suggests that any phenotypic differences are due to single nucleotide polymorphisms (SNPs). We combined DNA/RNA comparative genomic approaches using strains with different tick transmission phenotypes and identified genes that segregate with transmissibility.ResultsComparison of seven strains with different transmission phenotypes generated a list of SNPs affecting 18 genes and nine promoters. Transcriptional analysis found two candidate genes downstream from promoter SNPs that were differentially transcribed. To corroborate the comparative genomics approach we used three RNA-seq platforms to analyze the transcriptomes from two A. marginale strains with different transmission phenotypes. RNA-seq analysis confirmed the comparative genomics data and found 10 additional genes whose transcription between strains with distinct transmission efficiencies was significantly different. Six regions of the genome that contained no annotation were found to be transcriptionally active, and two of these newly identified transcripts were differentially transcribed.ConclusionsThis approach identified 30 genes and two novel transcripts potentially involved in tick transmission. We describe the transcriptome of an obligate intracellular bacterium in depth, while employing massive parallel sequencing to dissect an important trait in bacterial pathogenesis.

Evaluation of the effect of formalin fixation on skin specimens in dogs and cats.

Skin and subcutaneous tissues are the origin of most common neoplasms affecting dogs, accounting for approximately one third of all tumors encountered in the species. Surgical excision is frequently the best chance for a cure; determining factors influencing the success of excision are vital for surgical management of cases. This work examined the shrinkage of skin of various lengths from three sites in formalin for both dogs and cats. Tissues were measured on the animal (initial measurement), at the time of excision (post-removal), and after formalin fixation (post-fixation). While shrinkage after tissue removal was found in samples from the thorax, abdomen, and rear leg in dogs and from the rear leg in cats, no significant shrinkage due to formalin fixation was detected in any sample except for the thoracic samples from the dog. Therefore, when determining where to make incisions to effect a surgical cure, initial measurements should take into account tissue shrinkage effects.

Determining the Repertoire of Immunodominant Proteins via Whole-Genome Amplification of Intracellular Pathogens

Culturing many obligate intracellular bacteria is difficult or impossible. However, these organisms have numerous adaptations allowing for infection persistence and immune system evasion, making them some of the most interesting to study. Recent advancements in genome sequencing, pyrosequencing and Phi29 amplification, have allowed for examination of whole-genome sequences of intracellular bacteria without culture. We have applied both techniques to the model obligate intracellular pathogen Anaplasma marginale and the human pathogen Anaplasma phagocytophilum, in order to examine the ability of phi29 amplification to determine the sequence of genes allowing for immune system evasion and long-term persistence in the host. When compared to traditional pyrosequencing, phi29-mediated genome amplification had similar genome coverage, with no additional gaps in coverage. Additionally, all msp2 functional pseudogenes from two strains of A. marginale were detected and extracted from the phi29-amplified genomes, highlighting its utility in determining the full complement of genes involved in immune evasion.

GASTROINTESTINAL LEIOMYOSARCOMA IN A PYGMY SPERM WHALE (KOGIA BREVICEPS)

An adult male pygmy sperm whale (Kogia breviceps) was stranded within a tidal pool on Fernandina Beach on the north Florida Atlantic coast (USA) and expired soon after discovery. Necropsy findings included a small intestinal mass markedly expanding the intestinal wall and partially obstructing the lumen. This finding likely led to the malnutrition and ultimately the stranding of this whale. The differential diagnoses for the mass based on gross evaluation included a duodenal adenocarcinoma, leiomyoma/sarcoma, gastrointestinal stroma tumor, and benign/malignant peripheral nerve sheath tumor, previously referred to as neurofibromas or schwannomas. The mass was presumptively diagnosed as a leiomyosarcoma via routine histopathology and confirmed by immunoreactivity for desmin and smooth actin (SMA). KIT, a gene name for CD 117, was negative, excluding a gastrointestinal stromal tumor (GIST). Leiomyosarcomas have been reported within numerous wild and domestic species, although this is the first reported case of any neoplasm in a pygmy sperm whale (K. breviceps).