Differentiation of porcine dendritic cells by granulocyte-macrophage colony-stimulating factor expressed in Pichia pastoris

Dendritic cells (DC) are potent inducers of acquired immunity due to their ability to present antigens in the context of a costimulatory environment and consequently serve an essential role in vaccine efficacy. Strategies to enhance their function, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 treatment to induce DC differentiation from peripheral blood monocytes, may therefore be useful as vaccine adjuvants. We now have evaluated the effect of recombinant GM-CSF on the differentiation of DC in swine. GM-CSF mRNA was readily detected in porcine splenocytes, with increased levels following treatment of the cells with ConA and LPS. Porcine GM-CSF was cloned and expressed in the methylotrophic yeast, Pichia pastoris, as a glycosylated protein that induced proliferation of porcine bone marrow cells. P. pastoris-derived GM-CSF induced expression of antigen presenting (MHC class II) and costimulatory (CD80-CD86) molecules and enhanced antigen presenting cell (APC) function consistent with the induction of functional DC. Thus, recombinant GM-CSF produced by P. pastoris may be a potent adjuvant for swine vaccines.     

Candida albicans infection in free-living populations of hihi (stitchbird; Notiomystis cincta)

Abstract

AIM: To describe the occurrence of candidiasis in hihi (stitch-bird; Notiomystis cincta) nestlings, and investigate the carriage and impact of Candida albicans infection in a free-living population of hihi.

METHODS: Mortality of nestlings was investigated in a reintroduced population of the endangered, endemic hihi at Zealandia: Karori Sanctuary, Wellington, New Zealand. Oral and faecal samples were collected from live hihi nestlings, for microbiological examination, between October 2008 and April 2009. All hihi that died and could be recovered were submitted to the New Zealand Wildlife Health Centre (NZWHC) at Massey University, for post-mortem examination. The results were compared with data obtained retrospectively from the National Wildlife Mortality (NWM) database for two other reintroduced populations of hihi on Mokoia and Tiritiri Matangi Islands.

RESULTS: Fifty chicks fledged from 82 eggs hatched during the 2008–2009 breeding season at Zealandia: Karori Sanctuary. Thirty-four live nestlings were sampled from 11 nests, and C. albicans was isolated from gastrointestinal swabs of 13 live nestlings from four nest sites. Eight (62%) of those nestlings survived to fledge, compared with 17/21 (81%) of those that tested negative (p=0.254; Fisher's exact test). Of the 32 hihi nestlings that died during the period of the study, 25 were recovered for necropsy. Histopathological examination revealed candidiasis was a factor in the deaths of four nestlings. An adult hihi that died during the period of the study at Zealandia: Karori Sanctuary was also found to have candidiasis. Retrospective analysis of data from the NWM database revealed candidiasis was also a factor in the deaths of five nestlings aged between 1 and 10 days from Mokoia Island, and of three nestlings <5 days old and one adult from Tiritiri Matangi Island.

CONCLUSIONS: Candida albicans was isolated from 38% of hihi nestlings sampled in this study, and vertical transmission of this organism from parent to offspring is likely to occur. Some colonised nestlings developed ventriculitis associated with Candida spp., but survival to fledging was not significantly different between nestlings that tested positive or negative, although the fate of birds following fledging was unknown.     

Retention of ingested porcine reproductive and respiratory syndrome virus in houseflies

OBJECTIVE: To evaluate retention of porcine reproductive and respiratory syndrome virus (PRRSV) in houseflies for various time frames and temperatures. SAMPLE POPULATION: Fifteen 2-week-old pigs, two 10-week-old pigs, and laboratory-cultivated houseflies. PROCEDURE: In an initial experiment, houseflies were exposed to PRRSV; housed at 15 degrees, 20 degrees, 25 degrees, and 30 degrees C; and tested at various time points. In a second experiment to determine dynamics of virus retention, houseflies were exposed to PRRSV and housed under controlled field conditions for 48 hours. Changes in the percentage of PRRSV-positive flies and virus load per fly were assessed over time, and detection of infective virus at 48 hours after exposure was measured. Finally, in a third experiment, virus loads were measured in houseflies allowed to feed on blood, oropharyngeal washings, and nasal washings obtained from experimentally infected pigs. RESULTS: In experiment 1, PRRSV retention in houseflies was proportional to temperature. In the second experiment, the percentage of PRRSV-positive houseflies and virus load per fly decreased over time; however, infective PRRSV was found in houseflies 48 hours after exposure. In experiment 3, PRRSV was detected in houseflies allowed to feed on all 3 porcine body fluids. CONCLUSIONS AND CLINICAL RELEVANCE: For the conditions of this study, houseflies did not support PRRSV replication. Therefore, retention of PRRSV in houseflies appears to be a function of initial virus load after ingestion and environmental temperature. These factors may impact the risk of insect-borne spread of PRRSV among farms.     

Effects of xylazine administration on serum amylase, pancreas-specific isoamylase and pancreatic polypeptide in normal dogs.

Changes in serum total amylase, pancreas-specific isoamylase and pancreatic polypeptide activities were measured in six dogs following multiple intramuscular injections of xylazine at 0.5 mg/kg of body weight. The activities of those analytes did not change over 24 hours of study.     

Use of BACTEC radiometric culture method and polymerase chain reaction for the rapid screening of faeces and tissues for Mycobacterium paratuberculosis

SUMMARY The BACTEC radiometric culture method for detection of Mycobacterium paratuberculosis was evaluated on faeces from cattle on a farm in quarantine for Johne's disease. A multiplex polymerase chain reaction (PCR) based on the IS900 sequence specific for M paratuberculosis and a genus specific 16S rRNA region was developed and used to test cultures showing evidence of mycobacterial growth in the BACTEC liquid radiometric culture medium. Using the BACTEC - PCR combination, confirmation of M paratuberculosis from faeces and tissue of clinically affected animals was achieved within 2 to 4 weeks and 1 week, respectively, a substantial improvement on traditional culture and identification methods. The PCR provided rapid exclusion of M paratuberculosis when other Mycobacterium spp were grown. The radiometric culture medium proved to be very sensitive for culturing Mycobacterium spp.     

Suspected immune-mediated megakaryocytic hypoplasia or aplasia in a dog

Megakaryocytic hypoplasia or aplasia causing thrombocytopenia was diagnosed in a 3-year-old Miniature Poodle with scleral hemorrhage, melena, ecchymoses, and blood-loss anemia. Immunosuppressive, cytotoxic, and lithium carbonate therapy resulted in successful correction of the dog's disease. It is important to recognize megakaryocytic hypoplasia or aplasia as the cause of thrombocytopenia, since the response to therapy will be slower when there is stem cell injury.     

Cerebellar abiotrophy in crossbred cattle

Cerebellar abiotrophy affected 9 of 74 calves sired by a Poll Hereford bull over 2 successive calving seasons. The disease was characterised by episodes of recumbency and ataxia, with hypermetria and wide base stance. Clinical signs commenced between birth and 8 months of age. Two calves which were affected first at 8 months of age recovered clinically 9 months later. Histological lesions were found in the cerebellar cortex of 7 calves and consisted of segmental degeneration and loss of Purkinje cells, and axonal swellings. The clinical signs and pathological findings were consistent with bovine familial convulsions and ataxia, which has not been described previously in Australia. The clinical signs were not attributable to the lesions observed in the cerebellum and an underlying electrophysiological abnormality is proposed. The aetiology of the condition is probably genetic and appears to have a multifactorial basis.