This document summarizes research on Hematodinium, a parasitic dinoflagellate that causes bitter crab disease (BCS) in various crab species. It infects the hemolymph of crabs, proliferates and alters hemolymph chemistry, leading to metabolic exhaustion and death. A qPCR assay was developed to more sensitively detect and quantify Hematodinium infections than previous methods. Testing in Southeast Alaska found increasing prevalence of infections from previous years. Further research aims to better understand transmission and impacts on crab populations.
Perspectives of predictive epidemiology and early warning systems for Rift Va...ILRI
Presentation by MO Nanyingi, GM Muchemi, SG Kiama, SM Thumbi and B Bett at the 47th annual scientific conference of the Kenya Veterinary Association held at Mombasa, Kenya, 24-27 April 2013.
Background
Influenza A viruses are medically significant pathogens responsible for higher mortality and morbidity throughout the world. Swine influenza is known to be caused by influenza A subtypes H1N1, H1N2, and H3N2, which are highly contagious, and belongs to the family Orthomyxoviridae. Efficient and accurate diagnosis of influenza A in individuals is critical for monitoring of a constantly evolving pandemic. A rapid result is important, because timely treatment can reduce disease severity and duration. Rapid antigen tests were among the first-line diagnostic tools for the detection of pandemic H1N1 (2009) virus infection during the initial outbreak. Current study focuses on the significant approach of the usage of molecular method utilizing real-time PCR for the detection of type A influenza virus (H1N1 subtype) in humans.
Methods
A total of 2000 mixed nasal/throat swab specimens collected in commercial viral transport from Apollo hospitals, Hyderabad were submitted to Institute of Preventive Medicine for molecular testing by reverse transcriptase polymerase chain reaction (RT-PCR) from 2009 to 2015 from its affiliated primary care clinics.
Results
Among the 2000 samples collected, 700 samples were positive for Human Inf A, swine Inf A, and Swine Inf H1 (fourth table in the article). One thousand two hundred samples were negative for Human Inf A, swine Inf A, and Swine Inf H1, and 100 samples were positive for Influenza A only.
Conclusion
The molecular testing of H1N1 patients helped the clinicians in timely diagnosis and treatment of these patients during the pandemic surveillance. The RT-PCR test has higher sensitivity and specificity; hence it is considered to be the best tool to use during the pandemic surveillance, as compared to the any other commercial antigen-based tests, which show a variable performance, with the sensitivities of tests from different manufacturers ranging from 9 to 77%.
Genomic surveillance of the Rift Valley fever: From sequencing to Lineage ass...ILRI
Poster prepared John Juma, Vagner Fonseca, Samson Limbaso, Peter van Heusden, Kristina Roesel, Bernard Bett, Rosemary Sang, Alan Christoffels, Tulio de Oliveira and Samuel Oyola for the Kenya One Health Online Conference, 6-8 December 2021
Presented by Etienne de Villiers at the African Swine Fever Diagnostics, Surveillance, Epidemiology and Control Workshop, Nairobi, Kenya, 20-21 July 2011
Perspectives of predictive epidemiology and early warning systems for Rift Va...ILRI
Presentation by MO Nanyingi, GM Muchemi, SG Kiama, SM Thumbi and B Bett at the 47th annual scientific conference of the Kenya Veterinary Association held at Mombasa, Kenya, 24-27 April 2013.
Background
Influenza A viruses are medically significant pathogens responsible for higher mortality and morbidity throughout the world. Swine influenza is known to be caused by influenza A subtypes H1N1, H1N2, and H3N2, which are highly contagious, and belongs to the family Orthomyxoviridae. Efficient and accurate diagnosis of influenza A in individuals is critical for monitoring of a constantly evolving pandemic. A rapid result is important, because timely treatment can reduce disease severity and duration. Rapid antigen tests were among the first-line diagnostic tools for the detection of pandemic H1N1 (2009) virus infection during the initial outbreak. Current study focuses on the significant approach of the usage of molecular method utilizing real-time PCR for the detection of type A influenza virus (H1N1 subtype) in humans.
Methods
A total of 2000 mixed nasal/throat swab specimens collected in commercial viral transport from Apollo hospitals, Hyderabad were submitted to Institute of Preventive Medicine for molecular testing by reverse transcriptase polymerase chain reaction (RT-PCR) from 2009 to 2015 from its affiliated primary care clinics.
Results
Among the 2000 samples collected, 700 samples were positive for Human Inf A, swine Inf A, and Swine Inf H1 (fourth table in the article). One thousand two hundred samples were negative for Human Inf A, swine Inf A, and Swine Inf H1, and 100 samples were positive for Influenza A only.
Conclusion
The molecular testing of H1N1 patients helped the clinicians in timely diagnosis and treatment of these patients during the pandemic surveillance. The RT-PCR test has higher sensitivity and specificity; hence it is considered to be the best tool to use during the pandemic surveillance, as compared to the any other commercial antigen-based tests, which show a variable performance, with the sensitivities of tests from different manufacturers ranging from 9 to 77%.
Genomic surveillance of the Rift Valley fever: From sequencing to Lineage ass...ILRI
Poster prepared John Juma, Vagner Fonseca, Samson Limbaso, Peter van Heusden, Kristina Roesel, Bernard Bett, Rosemary Sang, Alan Christoffels, Tulio de Oliveira and Samuel Oyola for the Kenya One Health Online Conference, 6-8 December 2021
Presented by Etienne de Villiers at the African Swine Fever Diagnostics, Surveillance, Epidemiology and Control Workshop, Nairobi, Kenya, 20-21 July 2011
African Swine Fever (ASF) virus genomics and diagnosticsILRI
Presented by Richard Bishop and Cynthia Onzere at the Closing workshop of the BecA‐ILRI‐CSIRO‐AusAID project on Understanding ASF epidemiology as a basis for control, Nairobi, Kenya, 2‐3 October 2013
Dr. Stephanie Rossow - Applications of Next Generation SequencingJohn Blue
Applications of Next Generation Sequencing - Dr. Stephanie Rossow, College of Veterinary Medicine, University of Minnesota, from the 2016 Allen D. Leman Swine Conference, September 17-20, 2016, St. Paul, Minnesota, USA.
More presentations at http://www.swinecast.com/2016-leman-swine-conference-material
Dr. X.J. Meng - Designing PRRSV Vaccines for Heterologous ProtectionJohn Blue
Designing PRRSV Vaccines for Heterologous Protection - Dr. X.J. Meng, VA-MD College of Veterinary Medicine, Virginia Tech, from the 2015 North American PRRS Symposium, December 4 - 5, 2015, Chicago, IL, USA.
More presentations at http://www.swinecast.com/2015-north-american-prrs-symposium
Presentation during IITA R4D week 2015 (23 - 27 Nov. 2015). By: Ndyetabula, S. Merumba, S. Jeremiah, S. Kasele, G. Mkamilo, F. Kagimbo, C. Chuwa and J. Legg.
UTMB: Chikungunya Vector Relationships and Prospects for Control in Americas ...Donna Ramirez
Presented at ICAAC 2015 by Scott C. Weaver, Institute for Human Infections and Immunity and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston
Dr. Hanchun Yang - Pathogenesis and control of Chinese highly pathogenic Porc...John Blue
Pathogenesis and control of Chinese highly pathogenic Porcine Reproductive & Respiratory Syndrome (PRRSV) - Dr. Hanchun Yang, China Agricultural University, from the 2016 North American PRRS Symposium, December 3‐4, 2016, Chicago, Illinois, USA.
More presentations at http://www.swinecast.com/2016-north-american-prrs-symposium
African Swine Fever (ASF) virus genomics and diagnosticsILRI
Presented by Richard Bishop and Cynthia Onzere at the Closing workshop of the BecA‐ILRI‐CSIRO‐AusAID project on Understanding ASF epidemiology as a basis for control, Nairobi, Kenya, 2‐3 October 2013
Dr. Stephanie Rossow - Applications of Next Generation SequencingJohn Blue
Applications of Next Generation Sequencing - Dr. Stephanie Rossow, College of Veterinary Medicine, University of Minnesota, from the 2016 Allen D. Leman Swine Conference, September 17-20, 2016, St. Paul, Minnesota, USA.
More presentations at http://www.swinecast.com/2016-leman-swine-conference-material
Dr. X.J. Meng - Designing PRRSV Vaccines for Heterologous ProtectionJohn Blue
Designing PRRSV Vaccines for Heterologous Protection - Dr. X.J. Meng, VA-MD College of Veterinary Medicine, Virginia Tech, from the 2015 North American PRRS Symposium, December 4 - 5, 2015, Chicago, IL, USA.
More presentations at http://www.swinecast.com/2015-north-american-prrs-symposium
Presentation during IITA R4D week 2015 (23 - 27 Nov. 2015). By: Ndyetabula, S. Merumba, S. Jeremiah, S. Kasele, G. Mkamilo, F. Kagimbo, C. Chuwa and J. Legg.
UTMB: Chikungunya Vector Relationships and Prospects for Control in Americas ...Donna Ramirez
Presented at ICAAC 2015 by Scott C. Weaver, Institute for Human Infections and Immunity and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston
Dr. Hanchun Yang - Pathogenesis and control of Chinese highly pathogenic Porc...John Blue
Pathogenesis and control of Chinese highly pathogenic Porcine Reproductive & Respiratory Syndrome (PRRSV) - Dr. Hanchun Yang, China Agricultural University, from the 2016 North American PRRS Symposium, December 3‐4, 2016, Chicago, Illinois, USA.
More presentations at http://www.swinecast.com/2016-north-american-prrs-symposium
Presentation 8: Vibrio parahaemolyticus: a versatile pathogen that can adapt ...ExternalEvents
http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/
International Technical Seminar/Workshops on Acute hepatopancreatic necrosis disease (AHPND)
In this presentation we provide the evidence that dengue fever is transmissible through blood transfusions. A discussion can be found at The Brazilian Blood Journal.
Ascaris lumbricoides and other Gastrointestinal Helminthic Parasites among Qe...iosrjce
IOSR Journal of Dental and Medical Sciences is one of the speciality Journal in Dental Science and Medical Science published by International Organization of Scientific Research (IOSR). The Journal publishes papers of the highest scientific merit and widest possible scope work in all areas related to medical and dental science. The Journal welcome review articles, leading medical and clinical research articles, technical notes, case reports and others.
Dr. Paul Fey - Livestock-associated Staphylococcus aureus: Recent TrendsJohn Blue
Livestock-associated Staphylococcus aureus: Recent Trends - Dr. Paul Fey, Professor and Medical Director of Clinical Microbiology Laboratory, University of Nebraska Medical Center, from the 2013 NIAA Symposium Bridging the Gap Between Animal Health and Human Health, November 12-14, 2013, Kansas City, MO, USA.
More presentations at http://www.trufflemedia.com/agmedia/conference/2013-niaa-antibiotics-bridging-the-gap-animal-health-human-health
Dr. Thomas J. DeLiberto - Highly Pathogenic Avian Influenza: The Role of Wild...John Blue
Highly Pathogenic Avian Influenza: The Role of Wild Birds - Dr. Thomas J. DeLiberto, NWRC Assistant Director, Wildlife Services, APHIS, USDA, from the 2016 NIAA Annual Conference: From Farm to Table - Food System Biosecurity for Animal Agriculture, April 4-7, 2016, Kansas City, MO, USA.
More presentations at http://www.trufflemedia.com/agmedia/conference/2016_niaa_farm_table_food_system_biosecurity
Melanoma epidemiology, etiopathogenesis and prevention - Professor Torello L...VR Foundation
Melanoma incidence has continued to increase significantly during the last half of 20th century wherever available data exist.
The incidence of malignant melanoma appears to be lower and stable in dark-skin individuals (Africans, Native Americans, Asians, and Hispanics).
Decreased incidence reported from some countries is probably partly due to an influx of low risk immigrants.
Melanoma epidemiology, etiopathogenesis and prevention - Professor Torello L...
2009 NSA
1. L.M. CROSSON*, C.S. FRIEDMAN. School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195. J.F. MORADO. National Oceanic & Atmospheric Administration, AFSC, 7600 Sand Point Way NE, Seattle, WA 98115.
2. BCS is a fatal disease caused by an undescribed parasitic dinoflagellate of the genus Hematodinium Hematodinium proliferates in hemolymph – alters hemolymph chemistry metabolic exhaustion death Infects numerous species of decapods & method of infection is unknown Left : Heavily infected Chionoecetes bairdi Right : Hematodinium infected (top) versus healthy (bottom) C. bairdi
16. Design primers Conventional PCR Insert amplicon into plasmid Clone multiple copies of plasmid Purify plasmid Quantify plasmid copy number Create dilution set Create standard curve with QPCR Graphic courtesy of Nate Wight
17. 30M 3M 300K 30K 3K 300 30 3 Comparison of cPCR to QPCR. Conventional PCR can reliably detect 300 or more copies, while QPCR can detect as low as 3 copies indicating that QPCR is 100 times more sensitive than cPCR cPCR QPCR
18. Dissociation curve analysis with a single peak illustrating the presence of a single DNA fragment amplified by the QPCR test
21. Port Camden Holkham Bay/ Stephens Passage Glacier Bay Icy Strait Douglas Island 0% Port Camden 8% Holkham Bay 10% Thomas Bay 0% Glacier Bay & Icy Strait 10-20% Douglas Island Thomas Bay
22. 2006 Glacier Bay = 0% 2005 Holkham Bay = 8% Stephens Passage Glacier Bay Blood Smears 40% 2% QPCR 64% 12%
BCS is a fatal disease caused by an undescribed parasitic dinoflagellate of the genus hematodinium. The dinoflagellate proliferates in the hemolymph or blood and alters the hemolymph chemistry leading to metabolic exhaustion of the crab and ultimately death. Hematodinium is known to infect over 20 different species of decapods however the method of infection has yet to be determined.
Some commercially important species effected by this disease are snow and tanner crabs, blue crabs, and the norwegian lobster. The map shows worldwide reports of hematodinium related disease. The distribution of this disease is global, having severe impacts in AK, Canada, the eastern US, Europe and even Australia. It is also important to note the majority of new infections have occurred in commercially important decapods in northern temperate zones that are experiencing increased warmer temperatures.
Some signs that are indicative of hematodinium infection are that the animal becomes lethargic with drooping limbs and mouthparts, its exoskeleton will have a cooked appearance as shown by the infected crabs highlighted in yellow, the animals hemolymph will be milky white in color, and the arthrodial membranes will turn from translucent to opaque. While hematodinium is not known to cause harm to humans, it does however give the animals meat a chalky texture and bitter taste deeming it unmarketable.
Prior to 1985 there were 7 decapod species reported to harbor hematodinium infections. Currently the disease has been reported in over 20 species worldwide with some areas in SE alaska, such as lynn canal, show prevalences as high as 95% of the population sampled. Mortalities and fishery impacts associated with hematodinium epidemics and the potential for this pathogen to impact affected commercial decapod abundance and distribution patterns are significant. Economic losses attributed to this parasite in the tanner crab fishery alone are estimated to be at least 3 million dollars.
While epidemics and mortalities of commercial size decapods are significant, evidence suggests that hematodinium associated disease is more prevalent in juvenile decapods than in adults and that a high percentage of small infected crustaceans die before they can recruit into the fishery. The parasite is also more prevalent in female than male crab providing additional potential for population and fishery impacts. For example strong bering sea snow crab cohorts in 2000,2001,2002 did not materialize and recruit into the fishery. A potential cause for these failures is Hematodinium. The highest prevalences of infection are found July through October with peak mortalities occurring in August and September. Cooler winter temperatures are thought to inhibit parasite reproduction.
This is a simplistic diagram of the life cycle of a parasitic dinoflagellate. For most dinoflagellates, the dinospore is the infectious stage and once entering a host will begin to grow as a trophont, then become a mobile plasmodia, and reproduce to form new spores.
Here are some typical parasite morphologies. Note the elongated motile plasmodia stage is very easy to discriminate. However, the trophont stage of the parasite looks very similar to the actual hemocytes of the host which can make diagnosis of this disease by microscopy alone particularly difficult and can lead to underestimation of disease prevalence.
To date only 2 species of hematodinium have been fully described from life history stages encountered in infected crabs. Others have not because few differentiating morphological characteristics exist and not all parasitic forms are encountered in infected hosts. Hematodinium from Tanner crab may be a new species however without additional characterization of the established and novel strains, the number of species and the extent of host specificity remains unknown.
Disease transmission is thought to occur during the post-molt period and may be enhanced by cannibalism or mating behavior. As I mentioned earlier for many parasitic dinoflagellates the dinospore is the infectious stage, whether this is true for hematodinium awaits further examination. We also do not know if prey species or seawater may possibly be serving as parasite vectors or reservoirs.
So far, in vitro transmission of the disease has not been successful due to loss of infectivity and only partial life histories have been described from infected tissues. In order to resolve the mode of transmission external life history stages require further characterization.
The traditional technique used for pathogen detection is histology. Histology allows you to detect the pathogen and indicate infection status, it is semi quantitative and can be used for downstream analysis such as ISH however the processes is very expensive, time consuming and takes a well trained eye. It also cannot be used to look for pathogen vectors. A molecular techniques that is more sensitive and allows for higher throughput is cPCR but with cPCR you are limited to only presence-absence data. It is not quantitative.
The goal of my project is to develop a molecular tool with high sensitivity and specificity to accurately quantify parasite loads. Being able to quantify parasite loads allows for many new questions to be addressed such as what is the tolerance level of infection before an animal becomes symptomatic? At what infection level is the parasite fatal to its host? Is there a temperature or depth refuge for the host? Do seawater or plankton act as reservoirs for the parasite? Theses are all important questions that we can address using QPCR to help track the proliferation of hematodinium and also to detect very low levels of infection to avoid underestimation of disease prevalence.
QPCR enables the user to quantify loads in a reproducible and high throughput manner. The Taqman probe chemistry employed detects and measures fluorescence as PCR product is produced with each cycle of amplification unlike endpoint detection via cPCR. The probe requires specific hybridization therefore the target sequence must be present to generate fluorescence.
I designed my QPCR assay by first aligning all known 18S rDNA sequences of Hematodinium and related genera using clustalW. I selected sequence areas unique to only hematodinium where I designed primer and probe combinations using Primer3 software. I was then able to develop a hematodinium plasmid standard containing the amplified segment of the parasite genome amplified by my primers. After quantifying the plasmid copy number, a dilution set of known copy numbers was prepared to create a standard curve.
Here is a nice schematic of the steps: I designed my primers, ran a cPCR, took the PCR product or amplicon, inserted it into a plasmid, cloned the plasmid, purified it, quantified it, created a dilution set of known copy numbers, and generated a standard curve with QPCR which I can now use to determine copy numbers of hematodinium DNA in my preserved blood samples.
In Oct of 2006, I was fortunate enough to join ADF&G for their Tanner crab survey where I sampled ~ 60 tanner crab from each location to provide 95% confidence of detecting infections with a prevalence of >5%. Hemolymph was extracted from the arthrodial membrane using a sterile syringe and preserved in ethanol. For every crab sampled gross characteristics were recorded and hemolymph smears were taken for microscopy which will be used as a gold standard against our QPCR assay. Seawater was also sampled, filtered, and preserved from each location to look for parasite vectors.
The prevalences of crabs with visible signs of infection ranged from 0% in Port Camden to ~8% in Holkham Bay and ~10% in crabs from Thomas Bay. These prevalences are similar to those observed on previous cruises with nearly no Hematodinium observed in crabs from Glacier Bay and Icy Strait and ~10-20% in those collected near Douglas Island.
A lot of difference in the zero ……..realtive concordinance in a log linear relationship X blood smear intensity of hemat Y qpcr copy number
In the process of assessing the best way to coordinate data taking into consideration the polymorphic nature of parasite Validating diagnostic and analysitcal sensitivity and specificity.