A probe-based droplet digital polymerase chain reaction assay for early detection of feline acute cytauxzoonosis.

Cytauxzoonosis is a tick-borne disease of domestic cats with high mortality and narrow therapeutic window, particularly in the southcentral and southeastern United States. The causative agent is the apicomplexan protozoal parasite Cytauxzoon felis and is primarily transmitted by Amblyomma americanum, the lone star tick. Currently there is no vaccine available to prevent cytauxzoonosis and treatment is often ineffective if not initiated early enough in the course of disease. Early diagnosis and therapeutic intervention are therefore crucial for the survival of infected cats. Several methods are available for diagnosis of cytauxzoonosis, with PCR being the most sensitive. However, current PCR assays, which employ double-stranded DNA intercalating dyes to detect C. felis infection, have inherent limitations such as the potential for false positive detection of non-specific amplification products and inability to provide absolute quantification of parasite load. The objective of this study was to develop a probe-based droplet digital PCR (ddPCR) assay capable of detection and quantification of C. felis load over time and during treatment. The C. felis ddPCR assay was able to (i) reliably detect and quantify C. felis DNA in clinical blood samples from cats with acute cytauxzoonosis and (ii) monitor clinical parasite load in response to anti-protozoal treatment through absolute quantification of C. felis DNA over time. When tested on blood samples from cats with experimental C. felis infection, the assay was able to detect infection in cats as early as 24 h prior to the development of clinical signs. In addition, we demonstrate that this probe-based design can be utilized in traditional real-time PCR systems, with similar detection capabilities as compared to ddPCR. The C. felis probe-based ddPCR was also able to detect infection in samples with lower parasite loads when compared to existing nested PCR assays, although these results were not significant due to small sample size. To the author's knowledge, this is the first reported probe-based ddPCR assay to detect Cytauxzoon felis infection in domestic cats.

Influence of rearing temperature on triacylglycerol storage in the pea aphid, Acyrthosiphon pisum.

Total fatty acids in the pea aphid reared at low temperatures increased significantly compared to that at high rearing temperatures. This change is reflected in a large increase of myristic acid, which occurs exclusively in triacylglycerols. When aphids were moved from 25°C to a lower rearing temperature at 10°C, saturated fatty acids accumulated over time, reaching a maximum at 16th day. When aphids were moved to 4°C, a temperature below the developmental threshold, those aphids did not accumulate saturated fatty acids. Similar results were observed when aphids were exposed to sequential decrease in rearing temperature. However, both total fatty acids and myristic acid in the aphids from the treatments of sequential decreasing rearing temperature were significantly higher compared to those in the aphids from the treatments of sudden decreasing rearing temperature. This result, therefore, supports the hypothesis that cold-adapted aphids can survive under threshold temperature for a longer period of time than noncold-adapted aphids. Acetyl-CoA carboxylase activity in the aphids at 25°C was twofold higher than that in the aphids at 10°C, whereas fatty acid synthase activities in the aphids reared at 25 and 10°C are similar. Aphids reared at 10°C showed a threefold reduction in reproduction rates. This reduced production of new nymphs reduces energy demand and would allow for accumulation of energy in the form of triacylglycerols. Therefore, the increased level of saturated fatty acids in aphids reared at low temperature is probably related to lower utilization of fatty acids rather than increased rates of biosynthesis.

Effect of precocene II on fatty acid metabolism in the pea aphid, Acyrthosiphon pisum, under cold stress.

Pea aphids, Acyrthosiphon pisum (Harris), reared at 10 degrees C contain higher levels of fatty acids than those reared at 25 degrees C. This is primarily the result of an accumulation of triacylglycerols containing myristic acid. When aphids reared at 25 degrees C were transferred to 10 degrees C there was a gradual increase in triacylglycerol content that reached a maximum at 16 days post-transfer. Treatment of aphids with precocene II prior to transfer to 10 degrees C blocked the accumulation of fatty acids including myristic acid. A single application of 2 microg precocene II/aphid or two applications of 0.5 microg precocene II/ aphid administered on consecutive days resulted in aphids moved to 10 degrees C maintaining the same fatty acid profile as aphids maintained at 25 degrees C. Aphids that were treated with precocene II and maintained at 25 degrees C did not show changes in fatty acid profiles. Rearing aphids at 10 degrees C resulted in lower rates of reproduction and lower total numbers of progeny with longer longevity. Treatment with precocene II significantly decreased the total number of progeny produced at both temperatures. Precocene II did not reduce life span of aphids reared at 25 degrees C, however, the life span of treated aphids reared at 10 degrees C was decreased. The mechanism by which precocene II prevents the accumulation of myristic acid in aphids reared at 10 degrees C remains to be determined.

The wheat (Triticum aestivum L.) leaf proteome.

The wheat leaf proteome was mapped and partially characterized to function as a comparative template for future wheat research. In total, 404 proteins were visualized, and 277 of these were selected for analysis based on reproducibility and relative quantity. Using a combination of protein and expressed sequence tag database searching, 142 proteins were putatively identified with an identification success rate of 51%. The identified proteins were grouped according to their functional annotations with the majority (40%) being involved in energy production, primary, or secondary metabolism. Only 8% of the protein identifications lacked ascertainable functional annotation. The 51% ratio of successful identification and the 8% unclear functional annotation rate are major improvements over most previous plant proteomic studies. This clearly indicates the advancement of the plant protein and nucleic acid sequence and annotation data available in the databases, and shows the enhanced feasibility of future wheat leaf proteome research.

A proteomics approach to characterizing tick salivary secretions.

The saliva of ticks contains a complex mixture of bioactive molecules including proteins that modulate host responses ensuring successful feeding. The limited amount of saliva that can be obtained from ticks has hampered characterization of salivary proteins using traditional protein chemistry. Recent improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize small amounts of protein. These methods were employed to characterize salivary proteins from Amblyomma americanum and Amblyvomma maculatum. Salivation was induced by injection of dopamine and theophylline. It was necessary to desalt and concentrate saliva before analysis by 2-D electrophoresis. Comparison of 1-D and 2-D gel patterns revealed that the major protein component of saliva did not appear on 2-D gels. Characterization of this protein showed that it was identical to the major protein present in the hemolymph of both tick species. Protein profiles obtained by 1-D and 2-D gel electrophoresis were similar for both tick species, however, higher concentrations of lower molecular weight proteins were present in A. maculatum. Protein analysis by MALDI-TOF mass spectrometry and western blot analysis showed that except for the most abundant protein with a molecular weight of 95 kDa, all of the proteins detected were of host origin. It is not known if this is an artifact of the collection method or has physiological significance. In either case, in these species of ticks, host proteins will have to be removed from saliva samples prior to 2-D analysis in order to characterize lower abundance proteins of tick origin.

A proteomics approach to characterizing tick salivary secretions.

The saliva of ticks contains a complex mixture of bioactive molecules including proteins that modulate host responses ensuring successful feeding. The limited amount of saliva that can be obtained from ticks has hampered characterization of salivary proteins using traditional protein chemistry. Recent improvements in two-dimensional gel electrophoresis, mass spectrometry, and bioinformatics provide new tools to characterize small amounts of protein. These methods were employed to characterize salivary proteins from Amblyomma americanum and Amblyvomma maculatum. Salivation was induced by injection of dopamine and theophylline. It was necessary to desalt and concentrate saliva before analysis by 2-D electrophoresis. Comparison of 1-D and 2-D gel patterns revealed that the major protein component of saliva did not appear on 2-D gels. Characterization of this protein showed that it was identical to the major protein present in the hemolymph of both tick species. Protein profiles obtained by 1-D and 2-D gel electrophoresis were similar for both tick species, however, higher concentrations of lower molecular weight proteins were present in A. maculatum. Protein analysis by MALDI-TOF mass spectrometry and western blot analysis showed that except for the most abundant protein with a molecular weight of 95 kDa, all of the proteins detected were of host origin. It is not known if this is an artifact of the collection method or has physiological significance. In either case, in these species of ticks, host proteins will have to be removed from saliva samples prior to 2-D analysis in order to characterize lower abundance proteins of tick origin.