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Development of a Multiplex Quantitative PCR Assay for Eyeworm ( Oxyspirura petrowi ) and Caecal worm ( Aulonocephalus pennula ) Detection in Northern Bobwhite quail ( Colinus virginianus ) of the Rolling Plains Eco-Region, Texas
... Nonlethal procedures are made even more difficult given no reported evidence of eyeworm or caecal worm expulsion in the feces of bobwhite for morphological identification. Consequently, Kalyanasundaram et al. [9] successfully developed an accurate and precise multiplex quantitative PCR (qPCR) method to detect eyeworm and caecal worm egg presence in bobwhite feces, which has been utilized in a mobile research laboratory [10]. This progress has provided an opportunity to nonlethally assess parasitic infection in bobwhite and also provides a platform for Regular surveillance of parasitic infection is vital as it can track the efficiency of mitigation efforts after rounds of treatment [11,12]. ...
... While feces is the best sample type when using qPCR to detect eyeworms and caecal worms, it has been suggested that cloacal swabs are more efficient to collect in field applications [9,10,17]. However, cloacal swab efficiency in determining infection may be dependent on the amount of feces present in the swab and thus, may affect the outcome of qPCR results. ...
... DNA extraction protocols followed procedures outlined in Kalyanasundaram et al. [9] and Kistler et al. [17]. Fecal samples were weighed to 180-220 mg prior to extraction. ...
Over the last few decades, there has been a decline in Northern bobwhite quail (Colinus virginianus) throughout their native range. While there are various factors that may be influencing this decline, it is suggested that parasites should be taken into consideration as a potential contributor in the Rolling Plains Ecoregion. High prevalence of the eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) in bobwhite of this region, coupled with a continuous decline, creates a need to assess infection through alternative methods for regional surveillance. Previous studies have developed a qPCR method and mobile research laboratory as an
option for nonlethal procedures. However, there is still a need for standardization of these techniques. Therefore, this study builds on previous protocols to develop an application that considers factors that may influence qPCR results. In this study, cloacal swabs are collected from bobwhite in three locations throughout the Rolling Plains and scaled based on amount of feces present on the swab. This data is compared to qPCR standards as a limit of quantification for both eyeworm and caecal worm to define parasitic infection levels. Binary logistic regressions confirm that the probability of detection increases for both eyeworm (Odds Ratio: 2.3738; 95% Confidence
Interval: [1.7804, 3.1649]) and caecal worm (Odds Ratio: 2.8516; 95% Confidence Interval: [2.2235, 3.6570]) as swab score increases. Infection levels for eyeworm and caecal worm are based on the generated cycle threshold value averages of qPCR standards. Based on the results of this study, this method can be applied in the mobile research laboratory to quantitatively assess regional parasitic infection in bobwhite throughout the Rolling Plains.
... Quantitative PCR (qPCR) can be used to indicate parasite reproduction based on parasite egg presence in fecal matter (Kalyanasundaram et al., 2018b) whereas necropsies can provide worm counts for parasite intensity. In this study, we use fecal samples, cloacal swabs, and average worm counts per bobwhite collected between March and October of 2014 through 2017 to perform predictive analyses on parasite reproduction and intensity in relation to temperature and precipitation. ...
... Feces samples weighing 0.18-0.22 g and cloacal swabs were snapfrozen in liquid nitrogen and then extracted using the QIAamp DNA Stool Mini Kit (Germany) following manufacturer's protocol as outlined in Kistler et al. (2016a) with a final elution step of 50 μL per sample followed from Kalyanasundaram et al. (2018b). Quantitative PCR techniques also followed Kalyanasundaram et al. (2018b) methods with 20 μL mastermix volumes as follows: 10 μL Taqman Fast Advanced Mastermix (Applied Biosystems), 0.4 μL of forward and reverse eyeworm primers and eyeworm probe, 0.2 μL of forward and reverse caecal worm primers and caecal worm probe, 0.2 μL of forward and reverse quail primers and quail probe, 0.1 μL bovine serum albumin (BSA), and 7.66 μL of molecular-grade water. ...
... g and cloacal swabs were snapfrozen in liquid nitrogen and then extracted using the QIAamp DNA Stool Mini Kit (Germany) following manufacturer's protocol as outlined in Kistler et al. (2016a) with a final elution step of 50 μL per sample followed from Kalyanasundaram et al. (2018b). Quantitative PCR techniques also followed Kalyanasundaram et al. (2018b) methods with 20 μL mastermix volumes as follows: 10 μL Taqman Fast Advanced Mastermix (Applied Biosystems), 0.4 μL of forward and reverse eyeworm primers and eyeworm probe, 0.2 μL of forward and reverse caecal worm primers and caecal worm probe, 0.2 μL of forward and reverse quail primers and quail probe, 0.1 μL bovine serum albumin (BSA), and 7.66 μL of molecular-grade water. Primer and probe sequences used in this study are outlined in Table 1. ...
The northern bobwhite quail (Colinus virginianus) is a popular gamebird in the Rolling Plains Ecoregion of West Texas. However, there has been a population decline in this area over recent decades. Consistent reports indicate a high prevalence of the eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula), which may be of major influence on the bobwhite population. While research has suggested pathological consequences and genetic relatedness to other pathologically significant parasites, little is known about the influence of climate on these parasites. In this study, we examined whether seasonal temperature and precipitation influences the intensity of these parasites in bobwhite. We also analyzed quantitative PCR results for bobwhite feces and cloacal swabs against temperature and precipitation to identify climatic impacts on parasite reproduction in this region. Multiple linear regression analyses were used for parasite intensity investigation while binary logistic regression analyses were used for parasite reproduction studies. Our analyses suggest that caecal worm intensity, caecal worm reproduction, and eyeworm reproduction are influenced by temperature and precipitation. Temperature data was collected 15, 30, and 60 days prior to the date of collection of individual bobwhite and compared to qPCR results to generate a temperature range that may influence future eyeworm reproduction. This is the first preliminary study investigating climatic influences with predictive statistics on eyeworm and caecal worm infection of northern bobwhite in the Rolling Plains.
... Mobile laboratories have previously been executed for various facets of research including atmospheric pollutant measurements (Bukowiecki et al., 2002), Marburg virus research in Angola (Grolla et al., 2011), and human specimen collection and processing for biosafety level two research in Germany (Lermen et al., 2014). Using cloacal swabs in lieu of euthanasia and a highly sensitive multiplex quantitative PCR (qPCR) assay developed by Kalyanasundaram et al. (2018b) and Kistler et al. (2016), the WTL's mobile laboratory can utilize the presence of parasite eggs detected in bobwhite fecal matter to analyse when reproduction is occurring. It is a useful technique that allows large sample sizes to be tested in a time-efficient and cost-effective manner. ...
... The qPCR protocol for this study follows Kalyanasundaram et al. (2018b). Standards for O. petrowi and A. pennula were used from Kistler et al. (2016) and Kalyanasundaram et al. (2018b), respectively. ...
... The qPCR protocol for this study follows Kalyanasundaram et al. (2018b). Standards for O. petrowi and A. pennula were used from Kistler et al. (2016) and Kalyanasundaram et al. (2018b), respectively. Standard concentrations used in this study ranged from 10 5 to 10 1 . ...
Northern bobwhite quail ( Colinus virginianus ), a popular gamebird among hunters, have been declining over recent decades in the Rolling Plains ecoregion. Investigations in the past few years have revealed a high prevalence of eyeworms ( Oxyspirura petrowi ) and caecal worms ( Aulonocephalus pennula ) in this ecoregion, prompting a need to better understand their host–parasite interaction and other factors that influence infection. In this study, the efficiency of a mobile laboratory was tested by deploying it to three field sites in the Rolling Plains between July and August of 2017 and collecting cloacal swabs from bobwhites. The DNA was extracted from swabs for quantitative PCR and was run in the mobile and reference laboratory to specifically detect A. pennula and O. petrowi infection. When compared with the Wildlife Toxicology's reference laboratory, the mobile laboratory had a 97 and 99% agreement for A. pennula and O. petrowi , respectively. There were no significant differences in infection levels between field sites. Due to its efficiency, it is proposed that the mobile laboratory would be an effective way to monitor infection levels, in addition to factors that may affect infection such as climate, diapause, and intermediate host populations.
... DNA from cloacal swabs was extracted using Qiagen Fast Stool mini kits following Kistler et al. (2016). A quantitative polymerase chain reaction (qPCR) was then used to estimate eyeworm and cecal worm infection following Kalyanasundaram et al. (2018) with modifications in Leach et al. (in review). Infection intensity was then assigned according to Blanchard et al. (2019). ...
Northern bobwhite quail (Colinus virginianus) are an economically significant gamebird that has experienced continued general decline in the Rolling Plains ecoregion of Texas. Habitat loss and changing environmental conditions have been cited as major contributors to this decline, with factors such as parasites being considered inconsequential. To better assess the impacts of parasite infections on bobwhite populations in the Rolling Plains, bobwhite abundance was monitored in response to anthelminthic treatment. With the prevalence of Oxyspirura petrowi and Aulonocephalus pennula infections in quail from Mitchell County, Texas confirmed by previous studies, the anthelminthic agent Fenbendazole was introduced as a means of parasite control in 2014–2015. Bobwhite abundance was determined through a series of call counts which provided an index of bobwhite populations and were conducted throughout the course of the study. In 2016, call counts revealed a significant increase of bobwhite in the area subject to Fenbendazole treatment, while untreated areas showed no changes in abundance. Fall populations of bobwhite in the treated zone approached 300% of those in untreated areas, these findings suggest that parasites may have a more significant impact on quail populations in the Rolling Plains than previously suspected. With the importance of bobwhite as a game bird in the Rolling Plains, the potential impacts of parasites must be taken into consideration as a factor contributing to bobwhite declines. Further research into the long-term effects these parasites have on quail populations in the ecoregion may aid landowners in developing affordable and effective conservation strategies.
... Fecal samples were collected from all birds for fecal float [34] and DNA extraction to ensure experimental birds were parasite-free. Primers and qPCR methods reported in Kalyanasundaram et al. [35] were used to screen for O. petrowi and caecal worm, Aulonocephalus pennula infections. After birds were confirmed parasite free, male and female birds were separated equally into two groups (Group A & B) of 10 birds each. ...
Background
The Northern bobwhite (Colinus virginianus) is an economically important, and popular game bird in North America. Northern bobwhites have experiencing declines of > 3.5% annually in recent decades due to several factors. The eyeworm Oxyspirura petrowi is a nematode parasite frequently found in the eyes of bobwhites. Although reported frequently in wild bobwhites, there is no research to understand the host-parasite mechanism. Hence, it is important to investigate mechanisms of eyeworm invasion and immune modulation in bobwhite. Cytokine gene expression using RT-PCR is widely used to identify the innate immune response of a host to an infection.
Methodology
In this study, we evaluated ten reference genes (HMBS, RPL19, RPL32, RPS7, RPS8, TATA, SDHA, YWHAZ, GAPDH, and ACTB) for their stability across three tissues (liver, spleen, and caecal tonsils) of control and O. petrowi infected Northern bobwhites. Primer efficiency and reference genes stability were assessed using GeNorm, NormFinder, and BestKeeper.
Results
Expression of these reference genes with respect to O. petrowi infection in bobwhites showed RPL32 and HMBS were the most stable genes in the liver, HMBS and SDHA were the most stable genes in the spleen, and HMBS and YWHAZ were equally stable reference genes in the caecal tonsils.
Conclusion
Based on the geometric mean of all three analyses, our results indicate that the combination of RPL32 and HMBS for the liver, HMBS and SDHA for the spleen, and YWHAZ and HMBS for caecal tonsils might be used as reference genes for normalization in gene expression investigations on Northern bobwhites.
... Nevertheless, a great deal of progress has been made to pave the way for future work investigating the interactions between bobwhite and parasites. Molecular techniques have been developed in order to nonlethally assess parasitic infection in bobwhite via a cloacal swab or feces sample that is evaluated by quantitative PCR (qPCR) and can detect the DNA for as little as one egg (Kistler et al., 2016b;Kalyanasundaram et al., 2018c). These methods were then adapted for use at a regional scale (Blanchard et al., 2018), as they are considered an effective form of parasite monitoring (Gray et al., 2012) that provides a valuable supplement to traditional methods (Archie et al., 2009). ...
The potential of parasites to affect host abundance has been a topic of heated contention within the scientific community for some time, with many maintaining that issues such as habitat loss are more important in regulating wildlife populations than diseases. This is in part due to the difficulty in detecting and quantifying the consequences of disease, such as parasitic infection, within wild systems. An example of this is found in the Northern bobwhite quail (Colinus virginanus), an iconic game bird that is one of the most extensively studied vertebrates on the planet. Yet, despite countless volumes dedicated to the study and management of this bird, bobwhite continue to disappear from fields, forest margins, and grasslands across the United States in what some have referred to as “our greatest wildlife tragedy”. Here, we will discuss the history of disease and wildlife conservation, some of the challenges wildlife disease studies face in the ever-changing world, and how a “weight of evidence” approach has been invaluable to evaluating the impact of parasites on bobwhite in the Rolling Plains of Texas. Through this, we highlight the potential of using “weight of the evidence” to better understand the complex effects of diseases on wildlife and urge a greater consideration of the importance of disease in wildlife conservation.
The chapter will focus on the ophthalmology of members of the superorder Galloanserae (fowl), which includes both those that are strictly terrestrial and those that spend time in water. Landfowl (Galliformes) include the brushturkey and scrubfowl (Megapodiidae), the guans and curassows (Cracidae), guineafowl (Numididae), quail (Odontophoridae), and the chickens, turkeys, pheasants, partridges, and grouse (Phasianidae). Waterfowl (Anseriformes) include the screamers (Anhimidae), the magpie goose (Anseranatidae), and the ducks, geese, and swans (Anatidae). The term “poultry” refers to members of the Galloanserae that have potential commercial use for its meat, eggs, offal, feathers, and manure, directly or indirectly entering the human food chain, regardless of the actual use of individual birds of each species. Poultry largely consist of chickens (Gallus gallus), quails (Coturnix coturnix), turkeys (Meleagris gallopavo), pheasants (Phasianus colchicus), and ducks (Anas platyrhynchos). The largest part of this chapter is about chickens (Gallus gallus domesticus), a subspecies of the red jungle fowl, a Southeast Asian cousin of pheasants that was domesticated more than 7,000 years ago. Chickens are technically considered domestic animals, although there are places where wild (or feral) chickens are common, such as in Hawaii and parts of the world where jungle fowl are still wild. When these patients are kept as pets and are brought to the veterinarian, usually avian veterinarians will examine them, not poultry or farm animal veterinarians.
Aulonocephalus pennula is a nematode living in the caeca of the wild Northern bobwhite quail (Colinus virginianus) present throughout the Rolling Plains Ecoregion of Texas. The cytochrome oxidase 1 (COX 1) gene of the mitochondrial genome was used to screen A. pennula in wild quail. Through BLAST analysis, similarity of A. pennula to other nematode parasites was compared at the nucleotide level. Phylogenetic analysis of A. pennula COX1 indicated relationships to Subuluridae, Ascarididae, and Anisakidae. This study on molecular characterization of A. pennula provides new insight for the diagnosis of caecal worm infections of quail in the Rolling plains Ecoregion of Texas.
Parasitic nematodes that infect quail have been understudied and long been dismissed as a problem in quail management. Within the Rolling Plains ecoregion of Texas, an area that has experienced quail population “boom and bust” cycles and ultimately a general decline, the need to determine why Northern bobwhite (Colinus virginianus) populations are diminishing has increased in priority. Previously, caecal parasites have been documented to cause inactivity, weight loss, reduced growth, inflammation to the caecal mucosa, and even death. The caecal worm Aulonocephalus pennula is an intestinal nematode parasite that is commonly found within the caecum of quail, as well as many other avian species. In the Rolling Plains ecoregion, A. pennula has been documented to have as high as a 98% prevalence in bobwhite quail samples; however, the effect it has on its host is not well understood. The present study documents A. pennula causes no pathological changes within the caeca of the Northern bobwhite. However, there is concern for disruption of digestion and the possible implications of infection for wild bobwhite quail survival are discussed.
Oxyspirura petrowi is a parasitic nematode that infects wild birds. This parasite has a broad host range, but has recently been reported in high prevalences from native Galliformes species in the United States. In order to better understand the impact O. petrowi has on wild bird populations, we developed a quantitative PCR protocol to detect infections in wild northern bobwhites (Colinus virginianus). We used paired fecal and cloacal swab samples from wild caught and experimentally infected northern bobwhites and matching fecal float data from experimentally infected birds to validate our assay. Overall we detected more positive birds from fecal samples than the paired cloacal swabs and there was strong agreement between the qPCR results from fecal samples and from fecal flotation (84%; κ = 0.69 [0.53–0.84 95% CI]). We also detected O. petrowi DNA in ten replicates of samples spiked with one O. petrowi egg. This qPCR assay is an effective assay to detect O. petrowi infections in wild birds. Our results suggest that fecal samples are the most appropriate sample for detecting infections; although, cloacal swabs can be useful for determining if O. petrowi is circulating in a population.
Debilitating ocular diseases are often reported in avian species. By and large, helminth parasites have been overlooked in avian diseases and regarded as inconsequential. The decline of Northern bobwhite quail (Colinus virginianus) in the Rolling Plains ecoregion of Texas has prompted an investigation of the factors influencing their disappearance. Infection by the eyeworm (Oxyspirura petrowi) has been documented in many avian species; however, the effect it has on its host is not well understood. Heavy eyeworm infection has been documented in Northern bobwhites throughout this ecoregion, leading to eye pathology in this host species. The present study further documents and supports the pathological changes associated with O. petrowi in bobwhites.
Northern bobwhite (Colinus virginianus) and scaled quail (Callipepla squamata) have experienced chronic declines within the Rolling Plains ecoregion of Texas. Parasitic infection, which has long been dismissed as a problem in quail, has not been studied thoroughly until recently. A total of 219 northern bobwhite and 101 scaled quail from Mitchell County, Texas were captured and donated from 2014-2015, and examined for eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) infections. In 2014, bobwhites averaged 19.6±1.8 eyeworms and 98.6±8.2 caecal worms and 23.5±2.1 eyeworms and 129.9±10.7 caecal worms in 2015. Scaled quail averaged 4.8±1.0 eyeworms and 50±6.8 caecal worms in 2014 and 5.7±1.3 eyeworms and 38.1±7.1 caecal worms in 2015. This study expands the knowledge of parasitic infection in quail inhabiting the Rolling Plains of Texas. A significant difference was documented in O. petrowi infection between species but there was no significant difference in A. pennula between quail species. No significant difference was detected in parasite infection between the sexes of both northern bobwhite and scaled quail. This study also documented the highest reported O. petrowi infection in both species of quail. Additional research is needed on the life history and infection dynamics of O. petrowi and A. pennula infections to determine if there are individual and/or population level implications due to parasitic infection. .
Abstract We captured 36 Northern Bobwhites (Colinus virginianus) in Mitchell County, Texas in June-September 2013, and examined them for the eyeworm Oxyspirura petrowi. We recovered 334 eyeworms from 28 of 29 adult bobwhites (97%); infections ranged from 1-40 worms and mean (±SD) abundance of 11.9±13.0. Three of seven juveniles were infected, and those infected had one eyeworm each. Prevalence of eyeworms was similar among months. However, mean abundance of eyeworms peaked in July and August (3.3±2.1, 13.5±15.0, and 16.9±15.5), and decreased in September (6.3±3.0). We suggest that several previous studies may underreport prevalence and abundance because in those studies only the eye surface and nictitating membrane were examined, and not eye-associated tissue, ducts, glands, or sinuses.
Although some parasites have obvious pathogenic effects, others appear to have subtle, indirect effects that are poorly understood, particularly in natural populations. Indirect effects may result from parasites altering host metabolic rate and hence host energy needs, yet no experimental studies have shown this to be the case for non-laboratory hosts. We report the results of a long-term field experiment designed to test the impact of parasites on host energetics. We measured the energetics of feral rock doves (Columba livia) with populations of feather-feeding lice, traditionally considered to have little or no effect on host fitness. The lice reduced feather mass leading to increased thermal conductance and metabolic rate, as well as a steady reduction in host body mass over the course of the nine-month study. Our results demonstrate that even classically `benign' parasites such as feather lice can reduce host condition through the accumulation of subtle energetic costs over time. We argue that experimental manipulations are a prerequisite for documenting such effects.
This review considers in a selective way the literature on diapause in parasitic nematodes, concentrating on four species of animal parasites and three species of plant parasites. We define diapause as a developmental arrest which is temporarily irreversible, so development will not resume, even under favourable conditions, until some intrinsic changes have been completed. Our analysis recognises four stages in diapause. The first is induction, typically brought about by environmental signals (although diapause may be genetically programmed independently of the environment). These environmental signals typically do not have an immediate effect on development, but we recognise a second phase, which we call the diapause pathway, in which worms have been induced to enter diapause at a later developmental stage. Surprisingly, entry into the diapause pathway may under some circumstances be reversible. The third stage is diapause development, a period during which development is suspended, but some ill-understood process must be completed prior to the fourth stage, emergence from diapause. Although diapause development is complete, resumption of development may be further delayed because of conditions in the host or in the environment: the worm is once more capable of development, but development is prevented by unfavourable conditions extrinsic to the worm. These may include the immune state of the host or the total parasite burden in animal hosts.
The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the
detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital
were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional
modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR
results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained
fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species.
The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four
most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity
demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice
for enteric protozoan parasites.
PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance.
Advances in the biological sciences and technology are providing molecular targets for diagnosing and treating cancer. Current classifications in surgical pathology for staging malignancies are based primarily on anatomic features (e.g., tumor-node-metastasis) and histopathology (e.g., grade). Microarrays together with clustering algorithms are revealing a molecular diversity among cancers that promises to form a new taxonomy with prognostic and, more importantly, therapeutic significance. The challenge for pathology will be the development and implementation of these molecular classifications for routine clinical practice.
This article discusses the benefits, challenges, and possibilities for solid-tumor profiling in the clinical laboratory with an emphasis on DNA-based PCR techniques.
Molecular markers can be used to provide accurate prognosis and to predict response, resistance, or toxicity to therapy. The diversity of genomic alterations involved in malignancy necessitates a variety of assays for complete tumor profiling. Some new molecular classifications of tumors are based on gene expression, requiring a paradigm shift in specimen processing to preserve the integrity of RNA for analysis. More stable markers (i.e., DNA and protein) are readily handled in the clinical laboratory. Quantitative real-time PCR can determine gene duplications or deletions. Furthermore, melting curve analysis immediately after PCR can identify small mutations, down to single base changes. These techniques are becoming easier and faster and can be multiplexed. Real-time PCR methods are a favorable option for the analysis of cancer markers.
There is a need to translate recent discoveries in oncology research into clinical practice. This requires objective, robust, and cost-effective molecular techniques for clinical trials and, eventually, routine use. Real-time PCR has attractive features for tumor profiling in the clinical laboratory.
Entamoeba histolytica, Giardia lamblia, and Cryptosporidium are three of the most important diarrhea-causing parasitic protozoa. For many years, microscopic examination of stool samples
has been considered to be the “gold standard” for diagnosis of E. histolytica, G. lamblia, and C. parvum infections. Recently, more specific and sensitive alternative methods (PCR, enzyme-linked immunosorbent assay, and direct
fluorescent-antibody assay) have been introduced for all three of these parasitic infections. However, the incorporation in
a routine diagnostic laboratory of these parasite-specific methods for diagnosis of each of the respective infections is time-consuming
and increases the costs of a stool examination. Therefore, a multiplex real-time PCR assay was developed for the simultaneous
detection of E. histolytica, G. lamblia, and C. parvum in stool samples. The multiplex PCR also included an internal control to determine efficiency of the PCR and detect inhibition
in the sample. The assay was performed on species-specific DNA controls and a range of well-defined stool samples, and it
achieved 100 percent specificity and sensitivity. The use of this assay in a diagnostic laboratory would provide sensitive
and specific diagnosis of the main parasitic diarrheal infections and could improve patient management and infection control.
The Rolling Plains have historically provided some of the best opportunities to hunt northern bobwhite (Colinus virginianus) populations anywhere. Historically, scaled quail (Callipepla squamata) have been common to abundant over much of the Rolling Plains, but the populations decreased dramatically in the late 1980s and have been slow to reclaim their historic range. Copyright © 2007 by Leonard Alfred Brennan Manufactured in the United States of America All rights reserved.
SUMMARY Over the past few decades, nucleic acid-based methods have been developed for the diagnosis of intestinal parasitic infections. Advantages of nucleic acid-based methods are numerous; typically, these include increased sensitivity and specificity and simpler standardization of diagnostic procedures. DNA samples can also be stored and used for genetic characterization and molecular typing, providing a valuable tool for surveys and surveillance studies. A variety of technologies have been applied, and some specific and general pitfalls and limitations have been identified. This review provides an overview of the multitude of methods that have been reported for the detection of intestinal parasites and offers some guidance in applying these methods in the clinical laboratory and in epidemiological studies.
The ceca, intestinal outpocketings of the gut, are described, classified by types, and their occurrence surveyed across the Order Aves. Correlation between cecal size and systematic position is weak except among closely related species. With many exceptions, herbivores and omnivores tend to have large ceca, insectivores and carnivores are variable, and piscivores and graminivores have small ceca. Although important progress has been made in recent years, especially through the use of wild birds under natural (or quasi-natural) conditions rather than studying domestic species in captivity, much remains to be learned about cecal functioning. Research on periodic changes in galliform and anseriform cecal size in response to dietary alterations is discussed. Studies demonstrating cellulose digestion and fermentation in ceca, and their utilization and absorption of water, nitrogenous com- pounds, and other nutrients are reviewed. We also note disease-causing organisms that may be found in ceca. The avian cecum is a multi-purpose organ, with the potential to act in many different ways-and depending on the species involved, its cecal morphology, and ecological conditions, cecal functioning can be efficient and vitally important to a bird's physiology, especially during periods of stress. Received 14 Feb. 1994, accepted 2 June 1994. The digestive tract of most birds contains a pair of outpocketings that project from the proximal colon at its junction with the small intestine (Fig. 1). These ceca are usually fingerlike in shape, looking much like simple lateral extensions of the intestine, but some are complex in struc- ture. Within the Class Aves, ceca range in size from very long to very short, or they may be entirely absent (Table 1). Unlike the case in almost all mammals, most avian ceca are paired and of approximately equal length, with separate lateral or ventrolateral openings into the colon (rec- tum). In some species the openings are dorsal or ventral, and a few paired ceca share a common orifice, but the great majority open into the lateral colon opposite one another (McLelland 1989). There has been confusion over how the passage of material through cecal openings is controlled, but it is now believed that the interdigitating meshwork of villi at the cecal entrance act as a filter, excluding large particles and allowing only fluid and fine particles to be separated and pushed from the colonic con- tents into the ceca by colonic antiperistalsis (Duke 1986a). This villous meshwork exists even in species, such as the House Sparrow (Passer domesticus), with very small, possibly nonfunctional, ceca (Klem et al. 1983). In the few species with larger ceca that have been studied histo- logically in detail (domestic chickens, G&us, other galliforms, and ducks, Anus; Calhoun 1954, Clarke 1978, Fenna and Boag 1974b, Mahdi and
Standard techniques for counting parasites are often time-consuming, difficult and inaccurate, and occasionally unpleasant. Real-time quantitative polymerase chain reaction has recently been applied to parasitology, specifically Plasmodium, Toxoplasma, Leishmania and Neospora. These techniques are truly quantitative, give results over a range of 6-7 orders of magnitude, are quick to perform and require no manipulations post-amplification. They can be used to count genome numbers and to study levels of gene expression. The advantages and limitations of existing thermocyclers and applicable detection systems are discussed here, and promising new developments are highlighted.
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