Ultrastructural studies of the zygote and oocyst wall formation of Eimeria truncata of the lesser snow goose

Zygote development and oocyst wall formation of Eimeria truncata occurred in epithelial cells in renal tubules and ducts of experimentally infected lesser snow geese (Anser c. caerulescens). Post-fertilization stages were present throughout the kidneys beginning nine days post-inoculation. Initially, a single plasmalemma enclosed the zygote, and type 1 wall-forming bodies (WF1) became labyrinthine and moved toward the surface. There, WF1 degranulated and formed the outer layer of the oocyst wall between the plasmalemma and a newly formed second subpellicular membrane. Several WF2 fused and formed the inner layer of the oocyst wall between the third and fourth subpellicular membranes. Six subpellicular membranes were observed during wall formation. Other features of oocyst development were similar to those of other eimerian species.     

Response of American lobsters Homarus americanus to infection with a field isolate of Aerococcus viridans var. homari (Gaffkemia): survival and haematology

American lobsters Homarus americanus were inoculated with a field isolate of the Gram-positive bacterium Aerococcus viridans var. homari, causative agent of gaffkemia, at 1 x 10(6), 1 x 10(4) or 1 x 10(2) colony forming units (CFU) kg(-1) or with sterile 3% NaCl and maintained at 10 or 15 degrees C until they died or were euthanised. Progression of disease in individual animals was monitored daily by total haemocyte count (THC) and haemolymph culture. Post-mortem examinations were performed on all lobsters. Effects of both ambient temperature and infective dose on survival time were observed. Marked bacteraemia occurred in all mortalities. Haemocytopenia (THC < 10 x 10(9) cells l(-1)) preceded death in most, but not all, mortalities.     

Evaluation of the 16S and 12S rRNA genes as universal markers for the identification of commercial fish species in South Africa

The development of DNA-based methods for the identification of fish species is important for fisheries research and control, as well as for the detection of unintentional or fraudulent species substitutions in the marketplace. The aim of this study was to generate a comprehensive reference database of DNA sequences from the mitochondrial 16S and 12S ribosomal RNA (rRNA) genes for 53 commercial fish species in South Africa and to evaluate the applicability of these genetic markers for the identification of fish at the species level. The DNA extracted from all target species was readily amplified using universal primers targeting both rRNA gene regions. Sequences from the 16S and 12S rRNA genes were submitted to GenBank for the first time for 34% and 53% of the fish species, respectively. Cumulative analysis of the 16S rRNA gene sequences revealed mean conspecific, congeneric and confamilial Kimura two parameter (K2P) distances of 0.03%, 0.70% and 5.10% and the corresponding values at the 12S level were 0.03%, 1.00% and 5.57%. K2P neighbour-joining trees based on both sequence datasets generally clustered species in accordance with their taxonomic classifications. The nucleotide variation in both the 16S and 12S sequences was suitable for identifying the large majority of the examined fish specimens to at least the level of genus, but was found to be less useful for the explicit differentiation of certain congeneric fish species. It is recommended that one or more faster-evolving DNA regions be analysed to confirm the identities of closely-related fish species in South Africa.     

Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide

Aims: The detection of viable Enterobacter sakazakii cells is important due to the association of this pathogen with outbreaks of life-threatening neonatal infections. The aim of this study was to optimize a PCR-based method for selective detection of only viable Ent. sakazakii cells in the presence of dead cells, utilizing propidium monoazide (PMA) or ethidium bromide monoazide (EMA). Methods and Results: PMA or EMA was added to suspensions of viable and/or dead Ent. sakazakii cells at varying concentrations (10, 50 or 100 μg ml⁻¹) prior to DNA isolation and PCR with Ent. sakazakii-specific primers. At concentrations of 50 and 100 μg ml⁻¹, PMA completely inhibited PCR amplification from dead cells, while causing no significant inhibition of the amplification from viable cells. PMA was also effective in allowing selective PCR detection of only viable cells in mixtures of varying ratios of viable and dead cells. EMA was equally effective in preventing amplification from dead cells, however, it also inhibited DNA amplification from viable cells. Conclusions: This study demonstrated the efficiency of PMA for viable and dead differentiation of Ent. sakazakii, as well as the lack of selectivity of EMA for this purpose. Significance and Impact of the Study: PMA-PCR, in particular, will be useful for monitoring the resistance, survival strategies and stress responses of Ent. sakazakii in foods and the environment.     

Classification of Homarus americanus hemocytes and the use of differential hemocyte counts in lobsters infected with Aerococcus viridans var. homari (Gaffkemia)

Hemocytes of the American lobster (Homarus americanus H. Milne Edwards) were classified after examination of Wright-Giemsa stained cytocentrifuge preparations by brightfield light microscopy. Eleven hemocyte types were identified using morphologic criteria. The classification system was then used to monitor changes in the differential hemocyte count (DHC) of lobsters infected with the Gram positive coccus Aerococcus viridans var. homari, etiologic agent of gaffkemia. The appearance of less mature hemocytes in the DHCs of lobsters in the late stages of infection was similar to the 'left shift' of vertebrate inflammation. Results from this study suggest that DHCs can be used to assess and characterize inflammation in H. americanus and possibly other crustaceans.     

Use of injectable potassium chloride for euthanasia of American lobsters (Homarus americanus)

Potassium chloride (KCl: 330 mg/ml) was assessed as an euthanasia agent in American lobsters (Homarus americanus). Two groups of 10 lobsters (408.2 to 849.9 g) were maintained at 11.9 to 12.1 degrees C ('warm') and 1.5 to 2.5 degrees C ('cold') to evaluate the possible effect of ambient temperature on response to KCl. Death was defined as time of cardiac arrest, as viewed and measured by use of ultrasound. The KCl solution was injected (100 mg of KCl/100 g of body weight) at the base of the second walking leg to flood the hemolymph sinus containing the ventral nerve cord with potassium. Disruption of this 'central nervous system' was immediate, followed by cardiac arrest within 60 to 90 seconds. Group median ( +/- SD) baseline heart rate was 42 +/- 14 'warm' and 36 +/- 5 'cold' beats per minute. Time until cardiac arrest ranged from 35 to 90 (57 +/- 18) seconds in the 'warm' group and from 40 to 132 (53 +/- 34) seconds in the 'cold' group. There was no significant difference between group medians for either parameter. Histologic lesions were limited to mild to moderate acute degeneration, characterized by cell swelling, loss of contraction bands, and occasional mild cytoplasmic vacuolation of skeletal muscle at the injection site. Injectable KCl solution was an effective, reliable method for euthanasia of H. americanus.     

Microheterogeneity and coevolution: an examination of rDNA sequence characteristics in Neoparamoeba pemaquidensis and its prokinetoplastid endosymbiont

Neoparamoeba pemaquidensis, the etiological agent of amoebic gill disease, has shown surprising sequence variability among different copies of the 18S ribosomal RNA gene within an isolate. This intra-genomic microheterogeneity was confirmed and extended to an analysis of the internal transcribed spacer (ITS) region. High levels of intra-genomic nucleotide diversity (Pi=0.0201-0.0313) were found among sequenced ITS regions from individual host amoeba isolates. In contrast, the ITS region of its endosymbiont revealed significantly lower levels of intra-genomic nucleotide diversity (Pi=0.0028-0.0056) compared with the host N. pemaquidensis. Phylogenetic and ParaFit coevolution analyses involving N. pemaquidensis isolates and their respective endosymbionts confirmed a significant coevolutionary relationship between the two protists. The observation of non-shared microheterogeneity and coevolution emphasizes the complexity of the interactions between N. pemaquidensis and its obligate endosymbiont.