Insertion-duplication mutagenesis of neisseria: use in characterization of DNA transfer genes in the gonococcal genetic island.

We created plasmids for use in insertion-duplication mutagenesis (IDM) of Neisseria gonorrhoeae. This mutagenesis method has the advantage that it requires only a single cloning step prior to transformation into gonococci. Chromosomal DNA cloned into the plasmid directs insertion into the chromosome at the site of homology by a single-crossover (Campbell-type) recombination event. Two of the vectors contain an erythromycin resistance gene, ermC, with a strong promoter and in an orientation such that transcription will proceed into the cloned insert. Thus, these plasmids can be used to create insertions that are effectively nonpolar on the transcription of downstream genes. In addition to the improved ermC, the vector contains two copies of the neisserial DNA uptake sequence to facilitate high-frequency DNA uptake during transformation. Using various chromosomal DNA insert sizes, we have determined that even small inserts can target insertion mutation by this method and that the insertions are stably maintained in the gonococcal chromosome. We have used IDM to create knockouts in two genes in the gonococcal genetic island (GGI) and to clone additional regions of the GGI by a chromosome-walking procedure. Phenotypic characterization of traG and traH mutants suggests a role for the encoded proteins in DNA secretion by a novel type IV secretion system.

Exophthalmos secondary to aspergillosis in a cat.

Clinical signs of orbital disease include exophthalmos, strabismus, or, less commonly, enophthalmos. Fungal orbital disease is uncommon. A case of nasal, frontal sinus, and orbital aspergillosis in a cat is described. Diagnostics for exophthalmos and therapy for retrobulbar abscesses are discussed.

Pediatric ocular emergencies.

There are few ocular emergencies that are unique to the pediatric patient. Most ocular emergencies are traumatic in origin, and the prognosis is often determined by the extent of the injury. Some congenital anomalies that may present as ocular emergencies are also discussed. The focus of this article is recognition and initial therapy for the more common pediatric ocular emergencies.

Surgical reconstruction of severe cicatricial ectropion in a puppy.

A three-month-old chow chow mixed-breed puppy was presented with severe cicatricial ectropion of the upper eyelids after being burned maliciously. The burn wounds healed by second intention with contracture, causing eversion, elevation, and immobilization of the upper eyelids. The puppy was unable to blink or close the upper eyelids due to cicatrix (i.e., scar) formation. Surgical repair using tissue-relaxing procedures was successful in reducing corneal exposure and improving the puppy's appearance.

Traumatic ocular proptoses in dogs and cats: 84 cases (1980-1993).

Eighteen eyes of 66 dogs were visual on reevaluation of traumatic proptosis. Twenty-one eyes were enucleated, and 4 dogs were euthanatized. In 18 cats, no eyes regained vision after traumatic proptosis: 12 cats had the affected eye enucleated, 2 had an eye that was considered blind, and 4 cats were euthanatized. Affected eyes of 45 dogs and 2 cats underwent surgical replacement and temporary tarsorrhaphy. Favorable prognostic indicators for eyes undergoing surgical replacement included proptosis in a brachycephalic dog, positive direct or consensual pupillary light response, normal findings on posterior segment examination, and a proptosed eye that had vision on initial examination. Unfavorable prognostic indicators included proptosis in a nonbrachycephalic dog, proptosis in cats, hyphema, no visible pupil, facial fractures, optic nerve damage, and avulsion of 3 or more extraocular muscles.

Effects of mycobactin J and lactoferrin supplementation of drinking water on the in vivo multiplication of Mycobacterium paratuberculosis in gnotobiotic mice.

In this study the effect of supplementation of drinking water with mycobactin J or lactoferrin on the multiplication of Mycobacterium paratuberculosis in gnotobiotic mice was investigated. The results indicated that neither mycobactin J nor lactoferrin, at the doses used, appeared to have any effect on fecal shedding or tissue burdens of M. paratuberculosis.

Mycobacterium paratuberculosis monoassociated nude mice as a paratuberculosis model.

In this study, a paratuberculosis (Johne's disease) model was developed by intragastrically dosing gnotobiotic athymic nude mice with Mycobacterium paratuberculosis. The mice infrequently shed bacilli from their intestinal tracts during the first 4 months after inoculation. Following this time, increasing numbers of M. paratuberculosis (greater than 4.0 log10 bacilli per fecal pellet by 40 weeks) were recovered from the feces of the 12 mice that remained in the isolator. A similar pattern of recovery of M. paratuberculosis was obtained from the ileum, cecum, colon, and liver. Histopathologic lesions and acid-fast bacilli were rare during the first 4 months of infection and then, with time, increased in prevalence and severity. Mice maintained for 7 months or longer exhibited severe granulomatous inflammation and large numbers of acid-fast bacilli in the gastrointestinal tract and liver (up to 10(8) log10 colony forming units per gram wet weight). Five mice maintained for 7 months or more developed clinical signs consistent with those seen in paratuberculosis (weight loss, chronic diarrhea); three of these mice eventually died or became moribund and were euthanatized. M. paratuberculosis monoassociated mice released increased levels of tumor necrosis factor activity into their sera, as compared to uninfected control mice, when they were injected with bacterial lipopolysaccharide. The clinical signs, fecal shedding of M. paratuberculosis, granuloma formation, and progressive bacillary multiplication observed with these mice are consistent with naturally occurring M. paratuberculosis infection of ruminants (Johne's disease). This model will be useful for future studies of immunoregulation and antimicrobial therapy of paratuberculosis.

Intestinal multiplication of Mycobacterium paratuberculosis in athymic nude gnotobiotic mice.

In this study gnotobiotic mice were inoculated with a human isolate of Mycobacterium paratuberculosis (strain Linda; ATCC 43015) in an attempt to investigate the pathogenesis of intestinal paratuberculosis. Mycobacterium paratuberculosis-monoassociated nu/+ mice developed a persistent low-level intestinal infection but did not support progressive bacillary multiplication. In contrast, monoassociated nu/nu mice eventually harbored approximately 10(7) M. paratuberculosis per g of intestinal tissue. Acid-fast bacilli and granulomas were observed in the intestinal mucosa and livers of nu/nu but not nu/+ mice. Similar results were obtained after intragastric inoculation of M. paratuberculosis into nu/+ and nu/nu flora-defined mice. These observations suggest that the presence of an intact cellular immune system is important for limiting intestinal multiplication of M. paratuberculosis. The results of this study may be relevant to our understanding of the pathogenesis of Johne's disease in ruminants and of human inflammatory bowel diseases that have a mycobacterial etiology (e.g., some cases of Crohn's disease and Mycobacterium avium-Mycobacterium intracellulare enteritis in patients with acquired immunodeficiency syndrome).

Ingestion and killing of Pasteurella haemolytica A1 by bovine neutrophils in vitro.

In this study, various parameters affecting the ability of bovine neutrophils to ingest and kill a virulent strain of Pasteurella haemolytica A1 in vitro were examined. Ingestion of P. haemolytica was serum dependent (optimal serum concentration 10%) and was mediated principally by heat-stable opsonins, presumably antibodies, that could be removed by absorption with formalin-killed P. haemolytica. Ingested P. haemolytica were killed by neutrophils within 1-4 h incubation; the magnitude of killing being directly dependent on the number of neutrophils present. The number of viable P. haemolytica was reduced by approximately 1.5 log at bacterial concentrations of 0.01-100 P. haemolytica per neutrophil; a concomitant reduction in neutrophil viability was observed at the highest bacterial concentration (100:1). Bovine neutrophils underwent a vigorous luminol-enhanced chemiluminescence response after ingesting opsonized P. haemolytica, thus indicating that reactive oxygen intermediates were being formed that could have contributed to the intracellular killing of P. haemolytica.

Cultured bovine monocytes exhibit decreased release of superoxide anion and increased levels of lysosomal enzymes but do not secrete detectable lysozyme activity.

In this study we determined the effects of in vitro maturation on the phagocytic activity, lysosomal enzyme content and oxidative response of bovine monocytes. Intracellular levels of the lysosomal enzymes acid phosphatase, beta glucuronidase, and beta glucosaminidase increased as bovine monocytes matured in vitro. However, in marked contrast to the mononuclear phagocytes of other mammalian species, lysozyme activity was undetectable in the culture supernatants and cell lysates of adherent bovine blood monocytes cultured for one to fifteen days. In vitro maturation of bovine monocytes also increased their phagocytic activity as determined by the ingestion of opsonized yeast. A greater percentage of monocyte-derived macrophages that were stimulated with opsonized yeast and phorbol myristic acetate (PMA) reduced nitroblue tetrazolium than did similarly treated monocytes. Monocyte-derived macrophages stimulated with PMA released significantly less superoxide anion than did PMA-stimulated monocytes. Bovine monocytes and macrophages also failed to bind the monoclonal antibody Mac-1, which binds to human and mouse macrophages. Bovine monocytes demonstrated both similarities and differences with other mammalian mononuclear phagocytes, thus making them a useful model for further study of the comparative and developmental biology of mononuclear phagocytes.

Bovine neutrophils ingest but do not kill Haemophilus somnus in vitro.

Phagocytosis of Haemophilus somnus by bovine blood neutrophils required opsonization of the bacteria with antibodies against H. somnus. Few bacteria were phagocytosed in the absence of serum; in addition, absorption of immune serum with Formalin-killed H. somnus significantly reduced ingestion of H. somnus. Heat inactivation of antiserum (56 degrees C for 30 min) to eliminate complement activity had little effect on its ability to opsonize H. somnus for uptake by bovine neutrophils. Antiserum from an H. somnus-immunized calf and autologous sera from adult cattle supported equivalent phagocytosis of H. somnus by bovine neutrophils, suggesting that normal, healthy cattle may contain sufficient antibodies in their sera to facilitate phagocytosis of H. somnus. Although bovine neutrophils readily ingested H. somnus, they were unable to kill the bacterium in vitro. These same neutrophils readily killed opsonized Escherichia coli and Staphylococcus epidermidis, suggesting that H. somnus is able to survive and perhaps multiply within bovine neutrophils. Because bovine neutrophils stimulated with opsonized H. somnus demonstrated a reduced oxidative burst (as measured by chemiluminescence) compared with neutrophils stimulated by opsonized E. coli, we suggest that reduced production of reactive oxygen intermediates during the phagocytosis of H. somnus may account, in part, for the enhanced survival of H. somnus in bovine neutrophils.