Heather R. Shive

T Cells Are Essential for Bacterial Clearance, and Gamma Interferon, Tumor Necrosis Factor Alpha, and B Cells Are Crucial for Disease Development in Coxiella burnetii Infection in Mice

ABSTRACT Coxiella burnetii, the etiological agent of Q fever, has two phase variants. Phase I has a complete lipopolysaccharide (LPS), is highly virulent, and causes Q fever in humans and pathology in experimental animals. Phase II lacks an LPS O side chain, is avirulent, and does not grow well in immunocompetent animals. To understand the pathogenicity of Q fever, we investigated the roles of immune components in animals infected with Nine Mile phase I (NM I) or Nine Mile phase II (NM II) bacteria. Immunodeficient mice, including SCID mice (deficient in T and B cells), SCIDbg mice (deficient in T, B, and NK cells), nude mice (deficient in T cells), muMT mice (deficient in B cells), bg mice (deficient in NK cells), mice deficient in tumor necrosis factor alpha (TNF-α−/− mice), and mice deficient in gamma interferon (IFN-γ−/− mice), were compared for their responses to infection. SCID, SCIDbg, nude, and IFN-γ−/− mice showed high susceptibility to NM I, and TNF-α−/− mice showed modest susceptibility. Disease caused by NM I in SCID, SCIDbg, and nude mice progressed slowly, while disease in IFN-γ−/− and TNF-α−/− mice advanced rapidly. B- and NK-cell deficiencies did not enhance clinical disease development or alter bacterial clearance but did increase the severity of histopathological changes, particularly in the absence of B cells. Mice infected with NM II showed no apparent clinical disease, but T-cell-deficient mice had histopathological changes. These results suggest that T cells are critical for clearance of C. burnetii, either NM I or NM II, that IFN-γ and TNF-α are essential for the early control of infection, and that B cells are important for the prevention of tissue damage.

Coxiella burnetii isolates cause genogroup-specific virulence in mouse and guinea pig models of acute Q fever.

ABSTRACT Q fever is a zoonotic disease of worldwide significance caused by the obligate intracellular bacterium Coxiella burnetii. Humans with Q fever may experience an acute flu-like illness and pneumonia and/or chronic hepatitis or endocarditis. Various markers demonstrate significant phylogenetic separation between and clustering among isolates from acute and chronic human disease. The clinical and pathological responses to infection with phase I C. burnetii isolates from the following four genomic groups were evaluated in immunocompetent and immunocompromised mice and in guinea pig infection models: group I (Nine Mile, African, and Ohio), group IV (Priscilla and P), group V (G and S), and group VI (Dugway). Isolates from all of the groups produced disease in the SCID mouse model, and genogroup-consistent trends were noted in cytokine production in response to infection in the immunocompetent-mouse model. Guinea pigs developed severe acute disease when aerosol challenged with group I isolates, mild to moderate acute disease in response to group V isolates, and no acute disease when infected with group IV and VI isolates. C. burnetii isolates have a range of disease potentials; isolates within the same genomic group cause similar pathological responses, and there is a clear distinction in strain virulence between these genomic groups.

Simultaneous development of vocal and physical object combinations by a Grey parrot (Psittacus erithacus): bottle caps, lids, and labels.

On the basis of primarily behavioral data, researchers (e.g., P. M. Greenfield, 1991) have argued (a) that parallel development of communicative and physical object (manual) combinatorial abilities exists in young children; (b) that these abilities initially have a common neural substrate; (c) that a homologous substrate in great apes allows for similar, if limited, parallel development of these 2 abilities; and (d) that such abilities thus may indicate a shared evolutionary history for both communicative and physical behavior (J. Johnson-Pynn, D. M. Fragaszy, E. M. Hirsh, K. E. Brakke, & P. M. Greenfield, 1999). The authors of the present study found a comparable, if limited, parallel combinatorial development in a Grey parrot (Psittacus erithacus). Given the evolutionary distance between parrots and primates, the authors suggest that the search for and arguments concerning responsible substrates and common behavior should be approached with care and should not be restricted to the primate line.

Replication, neurotropism, and pathogenicity of avian paramyxovirus serotypes 1-9 in chickens and ducks.

Avian paramyxovirus (APMV) serotypes 1–9 have been isolated from many different avian species. APMV-1 (Newcastle disease virus) is the only well-characterized serotype, because of the high morbidity, mortality, and economic loss caused by highly virulent strains. Very little is known about the pathogenesis, replication, virulence, and tropism of the other APMV serotypes. Here, this was evaluated for prototypes strains of APMV serotypes 2–9 in cell culture and in chickens and ducks. In cell culture, only APMV-1, -3 and -5 induced syncytium formation. In chicken DF1 cells, APMV-3 replicated with an efficiency approaching that of APMV-1, while APMV-2 and -5 replicated to lower, intermediate titers and the others were much lower. Mean death time (MDT) assay in chicken eggs and intracerebral pathogenicity index (ICPI) test in 1-day-old SPF chicks demonstrated that APMV types 2–9 were avirulent. Evaluation of replication in primary neuronal cells in vitro as well as in the brains of 1-day-old chicks showed that, among types 2–9, only APMV-3 was neurotropic, although this virus was not neurovirulent. Following intranasal infection of 1-day-old and 2-week-old chickens, replication of APMV types 2–9 was mostly restricted to the respiratory tract, although APMV-3 was neuroinvasive and neurotropic (but not neurovirulent) and also was found in the spleen. Experimental intranasal infection of 3-week-old mallard ducks with the APMVs did not produce any clinical signs (even for APMV-1) and exhibited restricted viral replication of the APMVs (including APMV-1) to the upper respiratory tract regardless of their isolation source, indicating avirulence of APMV types 1–9 in mallard ducks. The link between the presence of a furin cleavage site in the F protein, syncytium formation, systemic spread, and virulence that has been well-established with APMV-1 pathotypes was not evident with the other APMV serotypes.

brca2 in zebrafish ovarian development, spermatogenesis, and tumorigenesis

Humans with inherited mutations in BRCA2 are at increased risk for developing breast and ovarian cancer; however, the relationship between BRCA2 mutation and these cancers is not understood. Studies of Brca2 mutation by gene targeting in mice are limited, given that homozygous Brca2 mutation typically leads to early embryonic lethality. We established a zebrafish line with a nonsense mutation in brca2 exon 11 (brca2Q658X), a mutation similar in location and type to BRCA2 mutations found in humans with hereditary breast and ovarian cancer. brca2Q658X homozygous zebrafish are viable and survive to adulthood; however, juvenile homozygotes fail to develop ovaries during sexual differentiation. Instead, brca2Q658X homozygotes develop as infertile males with meiotic arrest in spermatocytes. Germ cell migration to the embryonic gonadal ridge is unimpaired in brca2Q658X homozygotes; thus, failure of ovarian development is not due to defects in early establishment of the embryonic gonad. Homozygous tp53 mutation rescues ovarian development in brca2Q658X homozygous zebrafish, reflecting the importance of germ cell apoptosis in gonad morphogenesis. Adult brca2Q658X homozygous zebrafish are predisposed to testicular neoplasias. In addition, tumorigenesis in multiple tissues is significantly accelerated in combination with homozygous tp53 mutation in both brca2Q658X homozygous and brca2Q658X heterozygous zebrafish. These studies reveal critical roles for brca2 in ovarian development and tumorigenesis in reproductive tissues.

Long-Term Follow-up of Foamy Viral Vector-Mediated Gene Therapy for Canine Leukocyte Adhesion Deficiency

The development of leukemia following gammaretroviral vector-mediated gene therapy for X-linked severe combined immunodeficiency disease and chronic granulomatous disease (CGD) has emphasized the need for long-term follow-up in animals treated with hematopoietic stem cell gene therapy. In this study, we report the long-term follow-up (4-7 years) of four dogs with canine leukocyte adhesion deficiency (CLAD) treated with foamy viral (FV) vector-mediated gene therapy. All four CLAD dogs previously received nonmyeloablative conditioning with 200 cGy total body irradiation followed by infusion of autologous, CD34(+) hematopoietic stem cells transduced by a FV vector expressing canine CD18 from an internal Murine Stem Cell Virus (MSCV) promoter. CD18(+) leukocyte levels were >2% following infusion of vector-transduced cells leading to ongoing reversal of the CLAD phenotype for >4 years. There was no clinical development of lymphoid or myeloid leukemia in any of the four dogs and integration site analysis did not reveal insertional oncogenesis. These results showing disease correction/amelioration of disease in CLAD without significant adverse events provide support for the use of a FV vector to treat children with leukocyte adhesion deficiency type 1 (LAD-1) in a human gene therapy clinical trial.

Evaluation of the Contributions of Individual Viral Genes to Newcastle Disease Virus Virulence and Pathogenesis

ABSTRACT Naturally occurring Newcastle disease virus (NDV) strains vary greatly in virulence. The presence of multibasic residues at the proteolytic cleavage site of the fusion (F) protein has been shown to be a primary determinant differentiating virulent versus avirulent strains. However, there is wide variation in virulence among virulent strains. There also are examples of incongruity between cleavage site sequence and virulence. These observations suggest that additional viral factors contribute to virulence. In this study, we evaluated the contribution of each viral gene to virulence individually and in different combinations by exchanging genes between velogenic (highly virulent) strain GB Texas (GBT) and mesogenic (moderately virulent) strain Beaudette C (BC). These two strains are phylogenetically closely related, and their F proteins contain identical cleavage site sequences, 112RRQKR↓F117. A total of 20 chimeric viruses were constructed and evaluated in vitro, in 1-day-old chicks, and in 2-week-old chickens. The results showed that both the envelope-associated and polymerase-associated proteins contribute to the difference in virulence between rBC and rGBT, with the envelope-associated proteins playing the greater role. The F protein was the major individual contributor and was sometimes augmented by the homologous M and HN proteins. The dramatic effect of F was independent of its cleavage site sequence since that was identical in the two strains. The polymerase L protein was the next major individual contributor and was sometimes augmented by the homologous N and P proteins. The leader and trailer regions did not appear to contribute to the difference in virulence between BC and GBT. IMPORTANCE This study is the first comprehensive and systematic study of NDV virulence and pathogenesis. Genetic exchanges between a mesogenic and a velogenic strain revealed that the fusion glycoprotein is the major virulence determinant regardless of the identical virulence protease cleavage site sequence present in both strains. The contribution of the large polymerase protein to NDV virulence is second only to that of the fusion glycoprotein. The identification of virulence determinants is of considerable importance, because of the potential to generate better live attenuated NDV vaccines. It may also be possible to apply these findings to other paramyxoviruses.

Experimental infection of hamsters with avian paramyxovirus serotypes 1 to 9

Avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds throughout the world and are separated into nine serotypes (APMV-1 to -9). Only in the case of APMV-1, the infection of non-avian species has been investigated. The APMVs presently are being considered as human vaccine vectors. In this study, we evaluated the replication and pathogenicity of all nine APMV serotypes in hamsters. The hamsters were inoculated intranasally with each virus and monitored for clinical disease, pathology, histopathology, virus replication, and seroconversion. On the basis of one or more of these criteria, each of the APMV serotypes was found to replicate in hamsters. The APMVs produced mild or inapparent clinical signs in hamsters except for APMV-9, which produced moderate disease. Gross lesions were observed over the pulmonary surface of hamsters infected with APMV-2 & -3, which showed petechial and ecchymotic hemorrhages, respectively. Replication of all of the APMVs except APMV-5 was confirmed in the nasal turbinates and lungs, indicating a tropism for the respiratory tract. Histologically, the infection resulted in lung lesions consistent with bronchointerstitial pneumonia of varying severity and nasal turbinates with blunting or loss of cilia of the epithelium lining the nasal septa. The majority of APMV-infected hamsters exhibited transient histological lesions that self resolved by 14 days post infection (dpi). All of the hamsters infected with the APMVs produced serotype-specific HI or neutralizing antibodies, confirming virus replication. Taken together, these results demonstrate that all nine known APMV serotypes are capable of replicating in hamsters with minimal disease and pathology.

Zebrafish Models for Human Cancer

For decades, the advancement of cancer research has relied on in vivo models for examining key processes in cancer pathogenesis, including neoplastic transformation, progression, and response to therapy. These studies, which have traditionally relied on rodent models, have engendered a vast body of scientific literature. Recently, experimental cancer researchers have embraced many new and alternative model systems, including the zebrafish (Danio rerio). The general benefits of the zebrafish model for laboratory investigation, such as cost, size, fecundity, and generation time, were quickly superseded by the discovery that zebrafish are amenable to a wide range of investigative techniques, many of which are difficult or impossible to perform in mammalian models. These advantages, coupled with the finding that many aspects of carcinogenesis are conserved in zebrafish as compared with humans, have firmly established a unique niche for the zebrafish model in comparative cancer research. This article introduces methods for generating cancer models in zebrafish and reviews a range of models that have been developed for specific cancer types.

Liposarcoma in the Nasal Cavity of a Cow

Liposarcomas are rare neoplasms in domestic animals, but have been reported to occur in many species. in humans, liposarcoma is one of the most common malignant mesenchymal tumors. classification of liposarcomas in humans has been well established and categorization by type can be of prognostic value; no such unique classification scheme has been established for liposarcomas in animals. Liposarcoma of the head and neck in humans are uncommon, and are rarely reported in the nasal cavity, sinuses, and nasopharynx. To our knowledge, a liposarcoma has never been reported in the nasal cavity of a domestic animal. In this report we describe a liposarcoma that developed in the nasal cavity of a cow, with local invasion into the oral cavity.