Generation and screening of a comprehensive \u3ci\u3eMycobacterium avium\u3c/i\u3e subsp. \u3ci\u3eparatuberculosis\u3c/i\u3e transposon mutant bank

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s Disease in ruminants. This enteritis has significant economic impact and world wide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay developent. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P\u3e95% )was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped up stream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated

Learning Temporal Patterns of Risk in a Predator-Diverse Environment

Predation plays a major role in shaping prey behaviour. Temporal patterns of predation risk have been shown to drive daily activity and foraging patterns in prey. Yet the ability to respond to temporal patterns of predation risk in environments inhabited by highly diverse predator communities, such as rainforests and coral reefs, has received surprisingly little attention. In this study, we investigated whether juvenile marine fish, Pomacentrus moluccensis (lemon damselfish), have the ability to learn to adjust the intensity of their antipredator response to match the daily temporal patterns of predation risk they experience. Groups of lemon damselfish were exposed to one of two predictable temporal risk patterns for six days. “Morning risk” treatment prey were exposed to the odour of Cephalopholis cyanostigma (rockcod) paired with conspecific chemical alarm cues (simulating a rockcod present and feeding) during the morning, and rockcod odour only in the evening (simulating a rockcod present but not feeding). “Evening risk” treatment prey had the two stimuli presented to them in the opposite order. When tested individually for their response to rockcod odour alone, lemon damselfish from the morning risk treatment responded with a greater antipredator response intensity in the morning than in the evening. In contrast, those lemon damselfish previously exposed to the evening risk treatment subsequently responded with a greater antipredator response when tested in the evening. The results of this experiment demonstrate that P. moluccensis have the ability to learn temporal patterns of predation risk and can adjust their foraging patterns to match the threat posed by predators at a given time of day. Our results provide the first experimental demonstration of a mechanism by which prey in a complex, multi-predator environment can learn and respond to daily patterns of predation risk

Feeding Preferences and the Nutritional Value of Tropical Algae for the Abalone Haliotis asinina

Understanding the feeding preferences of abalone (high-value marine herbivores) is integral to new species development in aquaculture because of the expected link between preference and performance. Performance relates directly to the nutritional value of algae – or any feedstock – which in turn is driven by the amino acid content and profile, and specifically the content of the limiting essential amino acids. However, the relationship between feeding preferences, consumption and amino acid content of algae have rarely been simultaneously investigated for abalone, and never for the emerging target species Haliotis asinina. Here we found that the tropical H. asinina had strong and consistent preferences for the red alga Hypnea pannosa and the green alga Ulva flexuosa, but no overarching relationship between protein content (sum of amino acids) and preference existed. For example, preferred Hypnea and Ulva had distinctly different protein contents (12.64 vs. 2.99 g 100 g−1) and the protein-rich Asparagopsis taxiformis (>15 g 100 g−1 of dry weight) was one of the least preferred algae. The limiting amino acid in all algae was methionine, followed by histidine or lysine. Furthermore we demonstrated that preferences can largely be removed using carrageenan as a binder for dried alga, most likely acting as a feeding attractant or stimulant. The apparent decoupling between feeding preference and algal nutritive values may be due to a trade off between nutritive values and grazing deterrence associated with physical and chemical properties

Quantitative Analysis of Levels of Serum Immunoglobulin G against Botulinum Neurotoxin Type D and Association with Protection in Natural Outbreaks of Cattle Botulism

The recent outbreaks of cattle botulism in vaccinated Israeli dairy cattle prompted us to determine vaccine efficacy and reasons for vaccine failure. Analysis of clinical signs, feeding practice, vaccination history, and epidemic curves enabled us to define a study population in two outbreaks, where high doses of Clostridium botulinum neurotoxin type D (BoNT/D) were evenly consumed by the affected animal groups. Attack rates among unvaccinated 6- to 24-month-old heifers were 96% (55/57) and 85% (53/62). The attack rates in vaccinated parity 1, 2, and ≥3 cows were 40.4% (21/52), 14.3% (4/28), and 5.6% (3/54), respectively. Vaccine efficacies for these cow groups were 52.5%, 83.2%, and 93.4%, respectively. In younger, unvaccinated 2- to 6-month-old calves, presumably protected by maternal antibodies, the attack rate was 24% (17/71). These differences correlated with significant differences in levels of specific anti-BoNT/D antibody in serum by an enzyme-linked immunosorbent assay (ELISA). The ELISA performance for predicting protection was analyzed by receiver operating characteristic analysis and was found to be highly significant, with an area under the curve of 0.941 (standard error, 0.034; 95% confidence interval, 0.875 to 1.008; P < 0.000). No animals with serum ELISA unit levels above 0.33 were affected in these exposed groups. At this cutoff level, the specificity of the ELISA was 100%, sensitivity was 67%, and accuracy was 92%. We concluded that botulinum toxoids can confer adequate protection against natural exposure to lethal doses of BoNT/D; however, the vaccination protocols should be optimized. Our in-house ELISA system will enable us to optimize vaccination protocols in the animal population