B cell heterogeneity in the teleost kidney: Evidence for a maturation gradient from anterior to posterior kidney

The fish immune system is quite different from the mammalian system because the anterior kidney forms the main site for hematopoiesis in this species. Using transcription factor-specific Abs derived from the murine system, together with anti-trout Ig Abs and Percoll gradient separation, we analyzed B cells from trout kidney sections and compared them to those from spleen and blood. For this study, immune cells were separated by Percoll gradients, and the resulting subpopulations were defined based on expression of B cell-specific transcription factors Pax-5 and B lymphocyte-induced maturation protein-1, as well as proliferative and Ig-secreting properties. Comparison of kidney, blood, and spleen B cell subsets suggest that 1) the anterior kidney contains mostly proliferating B cell precursors and plasma cells; 2) posterior kidney houses significant populations of (partially) activated B cells and plasmablasts; and 3) trout blood contains resting, non-Ig-secreting cells and lacks plasma cells. After LPS induction of resting B cells in vitro, the kidney and spleen have a high capacity for the generation of plasma cells, whereas the blood has virtually none. Our results indicate that trout B cell subsets are profoundly different among blood, anterior kidney, posterior kidney, and spleen. We hypothesize that developing B cells mature in the anterior side of the kidney and then migrate to sites of activation, either the spleen or the posterior kidney. Lastly, our data support the notion that the trout kidney is a complex, multifunctional immune organ with the potential to support both hemopoiesis as well as humoral immune activation

Exploration of Machine Learning and Statistical Techniques in Development of a Low-Cost Screening Method Featuring the Global Diet Quality Score for Detecting Prediabetes in Rural India.

BACKGROUND: The prevalence of type 2 diabetes has increased substantially in India over the past 3 decades. Undiagnosed diabetes presents a public health challenge, especially in rural areas, where access to laboratory testing for diagnosis may not be readily available. OBJECTIVES: The present work explores the use of several machine learning and statistical methods in the development of a predictive tool to screen for prediabetes using survey data from an FFQ to compute the Global Diet Quality Score (GDQS). METHODS: The outcome variable prediabetes status (yes/no) used throughout this study was determined based upon a fasting blood glucose measurement ≥100 mg/dL. The algorithms utilized included the generalized linear model (GLM), random forest, least absolute shrinkage and selection operator (LASSO), elastic net (EN), and generalized linear mixed model (GLMM) with family unit as a (cluster) random (intercept) effect to account for intrafamily correlation. Model performance was assessed on held-out test data, and comparisons made with respect to area under the receiver operating characteristic curve (AUC), sensitivity, and specificity. RESULTS: The GLMM, GLM, LASSO, and random forest modeling techniques each performed quite well (AUCs >0.70) and included the GDQS food groups and age, among other predictors. The fully adjusted GLMM, which included a random intercept for family unit, achieved slightly superior results (AUC of 0.72) in classifying the prediabetes outcome in these cluster-correlated data. CONCLUSIONS: The models presented in the current work show promise in identifying individuals at risk of developing diabetes, although further studies are necessary to assess other potentially impactful predictors, as well as the consistency and generalizability of model performance. In addition, future studies to examine the utility of the GDQS in screening for other noncommunicable diseases are recommended

The humoral immune response of Lates calcarifer to Streptococcus iniae

This study characterises various aspects of barramundi (Lates calcarifer) humoral immunity, including ontogeny, temperature modulation and kinetics following challenge with Streptococcus iniae. It was discovered that Staphylococcal protein A (SpA) was able to efficiently isolate antibody from serum, and that all barramundi Ig found in serum is tetrameric with a weight of approximately 800 kDa. This tetramer is composed of 8 heavy chains (72 kDa) and 8 light chains (28 kDa). Denaturing, non-reducing electrophoresis demonstrated differential disulfide polymerization (redox forms) of the tetrameric Ig which was consistent with those observed with other species. Polyclonal and monoclonal antibodies were produced against the protein A purified barramundi Ig, and various ELISA formats were developed. These serological tools were used to investigate aspects of barramundi humoral immunity. Examination of ontogeny of humoral immunity, revealed that barramundi possess minimal maternal antibody (<10 μg/ml wet weight) post-hatch, which is depleted rapidly (within 3 days). By day 8 systemic Ig is able to be detected, which continues to increase over the following months. However, it is not until seven week post-hatch that barramundi fingerlings are able to mount a prolonged immune response following vaccination with S. iniae. Environmental temperature was also found to significantly impact the ability of barramundi to respond to vaccination with S. iniae. Barramundi maintained at low temperatures (<230C) displayed a diminished, delayed and highly variable humoral immune response following vaccination, with many of the experimental animals failing to respond to primary vaccination. These responses could be mediated by either administering a booster vaccine or by elevating the environmental temperature. This study also demonstrated that there was a relationship with specific serum antibody and protection against S. iniae, with fish possessing high levels of specific Ig being protected from lethal challenge, while those with low titres being more susceptible to disease. Specific antibody in barramundi could be generated through natural exposure to the bacterium from the environment or through vaccination. Thus bath vaccination of fish (50,000) held at two facilities resulted in elevated systemic antibody levels and lower observed mortality, when compared to the unvaccinated control fish. Infections due to S. iniae were determined to be associated with elevated water temperatures. Laboratory trials and field data indicated that water temperatures between 24 and 280C resulted in the highest barramundi mortality. A weak association was also determined with low pH and mortality, with fish exposed to low pH’s (<6.0) being more susceptible to infection. No association was observed with mortality and salinity. Four monoclonal antibodies (Mab’s) were also generated against a 21 kDa protein from cell wall of S. iniae. The Mab’s displayed a high level of specificity for S. iniae, including those from Australia, Israel and America, and minimal cross-reactivity with other bacterial species tested. The Mab’s were used in an immunohistochemical study that confirmed the neurotropic nature of S. iniae infections, as well as demonstrating the presence of the bacterium in the intestine of infected fish

The humoral immune response of Lates\ud calcarifer to Streptococcus iniae

This study characterises various aspects of barramundi (Lates calcarifer)\ud humoral immunity, including ontogeny, temperature modulation and kinetics\ud following challenge with Streptococcus iniae. It was discovered that\ud Staphylococcal protein A (SpA) was able to efficiently isolate antibody from\ud serum, and that all barramundi Ig found in serum is tetrameric with a weight\ud of approximately 800 kDa. This tetramer is composed of 8 heavy chains (72\ud kDa) and 8 light chains (28 kDa). Denaturing, non-reducing electrophoresis\ud demonstrated differential disulfide polymerization (redox forms) of the\ud tetrameric Ig which was consistent with those observed with other species.\ud Polyclonal and monoclonal antibodies were produced against the protein A\ud purified barramundi Ig, and various ELISA formats were developed. These\ud serological tools were used to investigate aspects of barramundi humoral\ud immunity.\ud Examination of ontogeny of humoral immunity, revealed that barramundi\ud possess minimal maternal antibody (<10 μg/ml wet weight) post-hatch, which\ud is depleted rapidly (within 3 days). By day 8 systemic Ig is able to be\ud detected, which continues to increase over the following months. However, it\ud is not until seven week post-hatch that barramundi fingerlings are able to\ud mount a prolonged immune response following vaccination with S. iniae.\ud Environmental temperature was also found to significantly impact the ability\ud of barramundi to respond to vaccination with S. iniae. Barramundi\ud maintained at low temperatures (<230C) displayed a diminished, delayed and\ud highly variable humoral immune response following vaccination, with many of\ud the experimental animals failing to respond to primary vaccination. These\ud responses could be mediated by either administering a booster vaccine or by\ud elevating the environmental temperature.\ud This study also demonstrated that there was a relationship with specific\ud serum antibody and protection against S. iniae, with fish possessing high levels of specific Ig being protected from lethal challenge, while those with\ud low titres being more susceptible to disease. Specific antibody in barramundi\ud could be generated through natural exposure to the bacterium from the\ud environment or through vaccination. Thus bath vaccination of fish (50,000)\ud held at two facilities resulted in elevated systemic antibody levels and lower\ud observed mortality, when compared to the unvaccinated control fish.\ud Infections due to S. iniae were determined to be associated with elevated\ud water temperatures. Laboratory trials and field data indicated that water\ud temperatures between 24 and 280C resulted in the highest barramundi\ud mortality. A weak association was also determined with low pH and\ud mortality, with fish exposed to low pH’s (<6.0) being more susceptible to\ud infection. No association was observed with mortality and salinity.\ud Four monoclonal antibodies (Mab’s) were also generated against a 21 kDa\ud protein from cell wall of S. iniae. The Mab’s displayed a high level of\ud specificity for S. iniae, including those from Australia, Israel and America, and\ud minimal cross-reactivity with other bacterial species tested. The Mab’s were\ud used in an immunohistochemical study that confirmed the neurotropic nature\ud of S. iniae infections, as well as demonstrating the presence of the bacterium\ud in the intestine of infected fish

Environmental factors affecting the susceptibility of barramundi to Streptococcus iniae

A long-term environmental study was conducted to evaluate the significance of water temperature, pH and salinity on the prevalence of Streptococcus iniae mortalities in barramundi sea-cage facilities. It was determined that there is a strong association between temperature and increased mortality, specifically between 25 and 28 °C. Temperatures outside of that range result in decreased mortalities attributed to S. iniae. There was no statistical significance between pH or salinity and S. iniae induced mortality (pN0.05), although acidic conditions (bpH 6) occurring due to anthropogenic disturbances did result in acute mortalities and S. iniae was recovered from approximately 70% these fish. Laboratory challenge studies confirmed the temperature dependence of S. iniae infections as well as the increased susceptibility of barramundi to S. iniae during acid water conditions

Experimental bacteriophage-mediated virulence in strains of Vibrio harveyi

Vibriosis is a major disease problem in prawn aquaculture. Until now there has been no clear explanation why some strains of Vibrio are pathogenic, while others are not. This study demonstrated that the presence of the bacteriophage V. harveyi myovirus like (VHML) may confer virulence to V. harveyi Strain 642. This was demonstrated by infecting naïve avirulent V. harveyi Strains 12, 20, 45 and 645 with the bacteriophage and converting them into virulent strains. The previously naïve strains of Vibrio infected with Bacteriophage VHML from V. harveyi Strain 642 demonstrated up-regulation of haemolysin, up-regulation of protein excretion, additional proteins which were recognised as toxic proteins from Strain 642 by monoclonal antibodies specific to the exotoxin sub-units, and a significant increase in mortality of larval Penaeus monodon. It was concluded that Bacteriophage VHML conferred virulence to V. harveyi Strains 12, 20, 45 and 645 and that Bacteriophage VHML either fully or partly confers virulence in V. harveyi Strain 642

Quantification of coral heat shock proteins from individual coral polyps

Author Posting. © Inter-Research, 2009. This article is posted here by permission of Inter-Research for personal use, not for redistribution. The definitive version was published in marine Ecology Progress Series 376 (2009): 123-132, doi:10.3354/meps07812.The induction and regulation of heat shock proteins (hsps) is a significant defense mechanism that can preserve metabolic function and foster recovery from short-term stress events. Present coral sampling methodologies that involve hsp analysis often require the harvesting of large samples of live coral colonies that may already be stressed or in poor health. In the present study, 3 novel protocols were developed to: (1) extract single coral polyps, minimizing colony trauma; (2) purify protein from single coral polyps (approximately 12 mm3); and (3) develop a more sensitive protein quantification method. The preliminary testing of 5 separate protein preparation methods resulted in a range of total protein yields from 47 to 77 µg coral polyp–1. The optimized methods were able to recover, on average, 44 ± 12 µg protein polyp–1 (n = 20). Subsequent SDS-PAGE and immunoblotting analysis of single coral polyps resolved as little as 87 pg hsp70 coral polyp–1. This minimally invasive sampling protocol reduces coral damage and, thus, reduces stress and diseases caused by sampling.This research was funded by a grant from the National Oceanic and Atmospheric Administration’s Undersea Research Center at the University of North Carolina at Wilmington pursuant to NOAA Award Number NA03OAR4300088

A newly developed ELISA showing the effect of environmental stress on levels of hsp86 in Cherax quadricarinatus and Penaeus monodon

The induction of hsps by stress in Cherax quadricarinatus and Penaeus monodon was investigated using SDS-PAGE, Western blotting and ELISA techniques. Western blotting showed the presence of an immuno-reactive protein to mouse α-human hsp70 IgG1 monoclonal antibody at a mass of 86 kDa (hsp86) in pleopod samples but was not sensitive enough to detect differences in response to stress. An enzyme-linked immunosorbent assay (ELISA) was developed using this antibody for the detection of hsp86 in the pleopods of C.quadricarinatus and P. monodon. Using this assay, significantly higher levels of hsp86 were detected in hyperthermally stressed C. quadricarinatus (21 to 70 g) and P. monodon (14 to 32 g) and hypoosmotically stressed P. monodon (14 to 32 g). Male C. quadricarinatus and P. monodon were thermally stressed with an increase in temperature from 24 to 33 °C for a period of 2 h then a recovery period of 6 h. SDS-PAGE gels of thermally stressed C. quadricarinatus and P. monodon samples revealed an increase in protein band intensity at 97 kDa (C. quadricarinatus) and 43 and 35 kDa in P. monodon. A 25 kDa mass protein was induced in C. quadricarinatus when thermally stressed. P. monodon were osmotically stressed with a decrease from 31 to 15 ppt for 2 h with a recovery of 6 h. SDS-PAGE gels revealed increased intensity of bands at 35 and 43 kDa and a 100 kDa band was induced demonstrating a wide range response of protein profile to stress in these species. SDS-PAGE gels of both species investigated also revealed an apparent reduction in band intensity of the haemocyanin subunits in stressed samples. The ELISA described here constitutes the first quantitative assay for the detection of a hsp in crustaceans and the following investigations are believed to be the first to describe the response of hsps to stress in C. quadricarinatus and P. monodon. In doing so, they provide a sound basis for future studies of the role of hsps in physiological functions in commercially cultured crustaceans

Use of staphylococcal protein A in the analysis of teleost immunoglobulin structural diversity

Staphylcoccal protein A (SpA) adsorption and sephacryl-S300 filtration were employed to isolate Ig from the sera of six aquaculturally important teleost species; Morone saxatilis (striped bass), Lates calcarifer (barramundi), Oreochromis mossambicus (Mozambique tilapia) and Oreochromis niloticus (Nile tilapia), Salmo salar (Atlantic salmon), and Oncorhynchus mykiss (rainbow trout). While both gel filtration (S300) and SpA adsorption could purify the 800 kDa tetrameric Ig, SpA demonstrated species-specific variability in the amount retrieved. Virtually 100% of this high molecular weight Ig could be isolated from Mosambique tilapia serum, while 84, 17, 10.7 and 0.5% could be isolated from barramundi, striped bass, Nile tilapia, and Atlantic salmon, respectively. Significant amounts of Ig could not be isolated (<0.1%) from rainbow trout (O. mykiss) serum. All SpA-isolated proteins were approximately 800 kDa in molecular weight and were solely composed of equimolar concentrations of H ( approximately 75 kDa) and L ( approximately 25 kDa) chains. Denaturing, non-reducing electrophoresis demonstrated differential disulfide polymerization (redox forms) consistent with those observed with other teleost species; however, SpA exhibited less affinity for Ig possessing completely polymerized tetramers than the more reduced forms, with the exception of Mossambique tilapia. The existence of three different molecular weight H chains (75, 85, 95 kDa) in Nile tilapia was also observed. Each redox form of Nile tilapia Ig incorporated only one size of H chain