Analysis of a Secreted Aspartic Peptidase Disruption Mutant of Glomerella cingulata

Peptidases have been implicated in the pathogenicity of fungi that cause disease in plants. Expression of the secreted aspartic peptidase gene (gcsap), of a Glomerella cingulata isolate pathogenic on apples, is induced during appressorium formation. To determine whether the secreted aspartic peptidase (GcSAP) is essential to pathogenicity, gcsap was disrupted using a vector containing a 637 bp fragment of genomic DNA that encodes the sequence spanning the two active site aspartic acid (Asp) residues. To ensure that the truncated gcsap gene products could not have residual peptidase activity the codons for the active site residues Asp¹¹² and Asp²⁹⁷ were both mutated to histidine residues. Both PCR and Southern analysis confirmed disruption of gcsap. Neither gcsap mRNA nor GcSAP activity was detected in the disruption mutant. Pathogenicity tests on fruit from three apple cultivars showed that GcSAP was not required for pathogenicity. The disruption mutant grew on medium containing protein as the sole source of nitrogen because G. cingulata secretes a previously undetected peptidase(s). A serine peptidase that had a pH optimum between pH 7.0 and 8.0 and a K _m of 0.25 mM for the synthetic substrate succinyl-Ala–Ala–Pro–Phe-p-nitroanilide was identified.     

Investigation of Interspecies Interactions within Marine Micromonosporaceae Using an Improved Co-Culture Approach

With respect to bacterial natural products, a significant outcome of the genomic era was that the biosynthetic potential in many microorganisms surpassed the number of compounds isolated under standard laboratory growth conditions, particularly among certain members in the phylum Actinobacteria. Our group, as well as others, investigated interspecies interactions, via co-culture, as a technique to coax bacteria to produce novel natural products. While co-culture provides new opportunities, challenges exist and questions surrounding these methods remain unanswered. In marine bacteria, for example, how prevalent are interspecies interactions and how commonly do interactions result in novel natural products? In an attempt to begin to answer basic questions surrounding co-culture of marine microorganisms, we have tested both antibiotic activity-based and LC/MS-based methods to evaluate Micromonosporaceae secondary metabolite production in co-culture. Overall, our investigation of 65 Micromonosporaceae led to the identification of 12 Micromonosporaceae across three genera that produced unique metabolites in co-culture. Our results suggest that interspecies interactions were prevalent between marine Micromonosporaceae and marine mycolic acid-containing bacteria. Furthermore, our approach highlights a sensitive and rapid method for investigating interspecies interactions in search of novel antibiotics, secondary metabolites, and genes.     

Bordetella bronchiseptica-reactive antibodies in Canadian polar bears

Bordetella bronchiseptica is a promiscuous bacterium that infects a variety of species but has not been reported in free-ranging polar bears (Ursus maritimus). Sera from 385 polar bears from the western Hudson Bay region, 1986 to 2017, were tested for reactivity to B. bronchiseptica with enzyme-linked immunosorbent assays using anti-canine IgG and Streptococcus protein G as secondary reagents. Sera from bears had variable reactivity to B. bronchiseptica antigens, and there was no difference among bears that had a history of coming near the town of Churchill, Manitoba, and bears that did not. Although the sources of exposure were not determined, equivalent results in both groups suggest that potential exposure to humans (aside from handling during sampling) and their animals (dogs) was not an important co-factor in sero-positivity to B. bronchiseptica.     

Leiomyoma of the third eyelid in a dog

A 14‐year‐old neutered male Dachshund presented for the evaluation of oculus dexter (OD) third eyelid elevation ongoing for approximately 2 months. Complete ophthalmic examination revealed a large, nonpainful, well‐demarcated, soft mass at the base of the right third eyelid causing elevation and mild hyperemia. The mass was freely moveable with the third eyelid, and no right globe deviation was noted. No other abnormalities were noted on physical examination, routine blood chemistry, complete blood count, serum T4, urinalysis, or urine cortisol/creatinine ratio. Ocular B‐mode ultrasonography showed an anechoic, well‐demarcated, homogenous, soft tissue mass at the base of the third eyelid with no orbital extension. A leiomyoma was diagnosed after multiple punch biopsies were obtained from the palpebral surface of the mass. The right third eyelid was excised surgically. Histopathology confirmed a completely excised, nodular, unencapsulated, expansile mass within the third eyelid. Positive smooth muscle actin and negative S‐100 immunohistochemistry confirmed a leiomyoma. Bundles of normal smooth muscle were also present adjacent to the mass. The mass was compressing the adjacent lacrimal gland and associated with moderate dacryoadenitis. Twelve months postoperatively, the right globe position and motility remain normal with no evidence of mass regrowth. To the author's knowledge, this is the first reported case of a leiomyoma of the third eyelid in any species. In this case, the mass was completely excised and no regrowth has occurred twelve months after surgery. This case along with independently reviewed canine third eyelids clearly demonstrates the presence of smooth muscle within the canine third eyelid.     

Serum antibody responses to vaccinal antigens in lean and obese geriatric dogs

The immune responses in control dogs [1 to 4 years of age, body condition score (BCS): 4 to 5 out of 9] were compared to those of aging dogs (based on breed and body size) either categorized as lean (BCS: 4 to 5 out of 9) or obese (BCS: 8 to 9 out of 9). Of interest were the serum titers to the following common agents found in vaccines, canine parainfluenza virus (CPIV), canine parvovirus (CPV), canine distemper virus (CDV), canine respiratory coronavirus (CRCoV), and Bordetella bronchiseptica. There were no statistical differences in the antibodies to CPIV, B. bronchispetica, and CRCoV, among the age/weight categories, nor among the age/weight categories and the time, in days, between the date of sample collection and the date of the last recorded vaccination for CPIV, B. bronchiseptica, CPV, and CDV. For CPV, the control dogs had significantly (P < 0.002) higher serum neutralization (SN) titers than the lean geriatric dogs and the obese geriatric dogs. For CDV SN titers, the only statistically significant (P = 0.01) difference was that the control dogs had higher SN titers than the lean geriatric dogs.     

Retrospective assessment of tolerability and efficacy of zoledronate in the palliative treatment of cancer-bearing dogs

Zoledronate is a bisphosphonate frequently used for the treatment of hypercalcaemia of malignancy and tumour-associated bone pain in dogs, however, there is a paucity of information regarding its use in veterinary medicine. The aim of this retrospective study was to report the tolerability of zoledronate in the palliative treatment of cancer-bearing dogs and secondarily to to assess the efficacy of zoledronate for the treatment of hypercalcaemia of malignancy. Thirty-seven dogs (22 with tumour-associated bone pain and 15 with hypercalcaemia of malignancy) that received 114 zoledronate infusions were included. Tolerability was assessed by the absence of post-zoledronate hypocalcaemia or other adverse events as defined by Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events criteria. Efficacy was assessed by comparison of available ionized calcium levels before and after zoledronate administration in hypercalcaemic dogs. In 79% of zoledronate infusions, no adverse events were reported. The majority of adverse events which occurred in the other 21% of infusions could be attributed to concurrent chemotherapy or the underlying neoplastic disease. There was a small but significant increase in creatinine following treatment with zoledronate, however, none of the dogs developed clinically significant renal disease. In eight hypercalcaemic dogs with available ionized calcium following zoledronate administration, ionized calcium decreased rapidly within 7 days following treatment with zoledronate. Zoledronate is well-tolerated with few recorded adverse events, however, monitoring of serum creatinine is advised. Zoledronate seems to be effective in the treatment of hypercalcaemia of malignancy.     

Comparative Genomics for Gene Isolation in Legumes

The similarity in gene order between closely related taxa suggests that genomic information from model systems should facilitate gene isolation and characterization in target crops. If this is the case, a great deal of effort and investment can be saved by focusing attention on a few model systems that have appropriate applicability. Pea （Pisura sativum） provides a good test case： its genome is large and the insertion sites for repetitive elements, which comprise the bulk of its genome, are highly polymorphic （Jing et al., 2005; Vershinin et al., 2003）, so we expect a great deal of structural polymorphism between the genomes of Pisum lines. Yet the genetic map of Pisum is essentially coUinear with Medicago （Choi et al., 2004; Kalo et al., 2004）.