Direct detection and characterization of foot-and-mouth disease virus in East Africa using a field-ready real-time PCR platform.

Effective control and monitoring of foot-and-mouth disease (FMD) relies upon rapid and accurate disease confirmation. Currently, clinical samples are usually tested in reference laboratories using standardized assays recommended by The World Organisation for Animal Health (OIE). However, the requirements for prompt and serotype-specific diagnosis during FMD outbreaks, and the need to establish robust laboratory testing capacity in FMD-endemic countries have motivated the development of simple diagnostic platforms to support local decision-making. Using a portable thermocycler, the T-COR™ 8, this study describes the laboratory and field evaluation of a commercially available, lyophilized pan-serotype-specific real-time RT-PCR (rRT-PCR) assay and a newly available FMD virus (FMDV) typing assay (East Africa-specific for serotypes: O, A, Southern African Territories [SAT] 1 and 2). Analytical sensitivity, diagnostic sensitivity and specificity of the pan-serotype-specific lyophilized assay were comparable to that of an OIE-recommended laboratory-based rRT-PCR (determined using a panel of 57 FMDV-positive samples and six non-FMDV vesicular disease samples for differential diagnosis). The FMDV-typing assay was able to correctly identify the serotype of 33/36 FMDV-positive samples (no cross-reactivity between serotypes was evident). Furthermore, the assays were able to accurately detect and type FMDV RNA in multiple sample types, including epithelial tissue suspensions, serum, oesophageal-pharyngeal (OP) fluid and oral swabs, both with and without the use of nucleic acid extraction. When deployed in laboratory and field settings in Tanzania, Kenya and Ethiopia, both assays reliably detected and serotyped FMDV RNA in samples (n = 144) collected from pre-clinical, clinical and clinically recovered cattle. These data support the use of field-ready rRT-PCR platforms in endemic settings for simple, highly sensitive and rapid detection and/or characterization of FMDV.

Molecular epidemiology identifies only a single rabies virus variant circulating in complex carnivore communities of the Serengeti.

Understanding the transmission dynamics of generalist pathogens that infect multiple host species is essential for their effective control. Only by identifying those host populations that are critical to the permanent maintenance of the pathogen, as opposed to populations in which outbreaks are the result of 'spillover' infections, can control measures be appropriately directed. Rabies virus is capable of infecting a wide range of host species, but in many ecosystems, particular variants circulate among only a limited range of potential host populations. The Serengeti ecosystem (in northwestern Tanzania) supports a complex community of wild carnivores that are threatened by generalist pathogens that also circulate in domestic dog populations surrounding the park boundaries. While the combined assemblage of host species appears capable of permanently maintaining rabies in the ecosystem, little is known about the patterns of circulation within and between these host populations. Here we use molecular phylogenetics to test whether distinct virus-host associations occur in this species-rich carnivore community. Our analysis identifies a single major variant belonging to the group of southern Africa canid-associated viruses (Africa 1b) to be circulating within this ecosystem, and no evidence for species-specific grouping. A statistical parsimony analysis of nucleoprotein and glycoprotein gene sequence data is consistent with both within- and between-species transmission events. While likely differential sampling effort between host species precludes a definitive inference, the results are most consistent with dogs comprising the reservoir of rabies and emphasize the importance of applying control efforts in dog populations.

Evaluation of Two Lyophilized Molecular Assays to Rapidly Detect Foot-and-Mouth Disease Virus Directly from Clinical Samples in Field Settings.

Accurate, timely diagnosis is essential for the control, monitoring and eradication of foot-and-mouth disease (FMD). Clinical samples from suspect cases are normally tested at reference laboratories. However, transport of samples to these centralized facilities can be a lengthy process that can impose delays on critical decision making. These concerns have motivated work to evaluate simple-to-use technologies, including molecular-based diagnostic platforms, that can be deployed closer to suspect cases of FMD. In this context, FMD virus (FMDV)-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time RT-PCR (rRT-PCR) assays, compatible with simple sample preparation methods and in situ visualization, have been developed which share equivalent analytical sensitivity with laboratory-based rRT-PCR. However, the lack of robust 'ready-to-use kits' that utilize stabilized reagents limits the deployment of these tests into field settings. To address this gap, this study describes the performance of lyophilized rRT-PCR and RT-LAMP assays to detect FMDV. Both of these assays are compatible with the use of fluorescence to monitor amplification in real-time, and for the RT-LAMP assays end point detection could also be achieved using molecular lateral flow devices. Lyophilization of reagents did not adversely affect the performance of the assays. Importantly, when these assays were deployed into challenging laboratory and field settings within East Africa they proved to be reliable in their ability to detect FMDV in a range of clinical samples from acutely infected as well as convalescent cattle. These data support the use of highly sensitive molecular assays into field settings for simple and rapid detection of FMDV.

Growth of rat mammary adenocarcinoma cells in semisolid clonogenic medium not correlated with spontaneous metastatic behavior: heterogeneity in the metastatic, antigenic, enzymatic, and drug sensitivity properties of cells from different sized colonies.

Using rat 13762NF mammary tumor cell clones of varying spontaneous metastatic potentials and biochemical properties and known phenotypic stabilities we studied the relationship between cell colony growth in a clonogenic assay and the biological and biochemical properties of cells derived from different cell colonies. The spontaneous metastatic potential of in vivo or in vitro grown 13762NF tumor cells was not related to their in vitro cloning efficiencies; cells of both low and high metastatic potential formed colonies of various sizes and shapes during 14 days of growth in 0.3% or 0.6% semisolid agarose. A highly metastatic cell clone of relatively low growth potential in agarose was examined further. Individual tumor cell colonies derived from this cell clone were removed from agarose and their properties determined. Cells from small (less than 100-microns-diameter) or large (greater than 500-microns-diameter) agarose colonies had similar self-renewal capacities in agarose and formed variously sized cell colonies when replated in agarose medium. Metastatic potential, drug sensitivity parameters, and expression of a high Mr mucin-like glycoprotein antigen and type IV collagenolytic activity known to be associated with spontaneous metastasis of 13762NF tumor cells were dissimilar in cells from different colonies, and these characteristics were independent of original tumor cell colony size in agarose. In contrast, the expression of cell surface proteins of Mr less than 300,000 were similar among cells derived from different agarose colonies. The data indicate that heterogeneity exists in the ability of 13762NF adenocarcinoma cells of different biochemical and metastatic potentials and drug sensitivities to grow in semisolid agarose. In addition, the cells that grow in agarose to form detectable colonies (greater than 50 cells) are not necessarily those with a high potential of metastasizing spontaneously to distant sites.

Transfection of activated c-H-rasEJ/pSV2neo or pSV2neo genes into rat mammary cells: rapid stimulation of clonal diversification of spontaneous metastatic and cell-surface properties.

We examined whether the conversion of benign rat mammary cells to metastatic cells by transfection of c-H-rasEJ/pSV2neo or control pSV2neo genes results in rapid stimulation of diversification of cellular phenotypes. Transfection of c-H-rasEJ into twice-cloned, stable (greater than 1 year) rat mammary MTC.4 cells, followed quickly by cell cloning, revealed differences in transfected oncogene copy numbers and expression of p21rasEJ. No correlation between c-H-rasEJ copy numbers or the cellular amounts of p21rasEJ or total p21ras and spontaneous metastatic potentials was found. By subcloning transfected cells as soon as possible after gene transfer, we found some rearrangements and amplifications of c-H-rasEJ and heterogeneous spontaneous metastatic potentials. In addition, the expression of the mammary tumor metastasis-associated cell-surface glycoprotein gp580 on untransfected and transfected MTC.4 cells indicated that the cell populations of higher metastatic potential were also more diverse in their cell-to-cell antigen expression than untransfected or non-metastatic, transfected MTC.4 cells. In contrast, the expression of a putative metastasis-suppressor gene, nm23, was unchanged after transfection and subcloning. Control pSV2neo transfections or calcium phosphate treatment alone also resulted in the generation of cellular heterogeneity, although at an apparently lower frequency than c-H-rasEJ transfections, suggesting that transfection of activated, dominantly acting oncogenes, or in some cases control genes, can result in destabilization of transfected cells, rapid diversification and generation of heterogeneity in growth rate, spontaneous metastatic potential and antigen expression.

Lack of correlation between intercellular junctional communication, p21rasEJ expression, and spontaneous metastatic properties of rat mammary cells after transfection with c-H-rasEJ or neo genes.

The loss of intercellular junctional communication between rat 13762NF mammary carcinoma cells and their spontaneous metastatic potentials from the mammary fat pad show a high degree of correlation. We examined a stable, benign, completely junctionally coupled cell clone (MTC.4) of this system after calcium phosphate-mediated transfection with c-H-rasEJ/pSV2neo and control pSV2neo-containing plasmids. There was a good correlation between the copy numbers of c-H-rasEJ incorporated into MTC.4 cells and their contents of p21rasEJ; however, there was not always a correspondence between spontaneous metastatic potential and copy number of c-H-rasEJ or amount of p21rasEJ. After c-H-rasEJ/pSV2neo transfection, some MTC.4 cells lost intercellular junctional communication and became spontaneously metastatic, although some nonmetastatic transfectants also had low percentages of junctionally coupled cells. One of the control pSV2neo transfectants also became metastatic and lost intercellular junctional coupling, and calcium phosphate treatment itself resulted in increased growth rates at mammary fat pad sites and a marginal increase in incidence of spontaneous metastases, both of which preceded loss of intercellular junctional coupling in some cells. Examination of 12 subclones derived from two cloned transfectants, however, revealed a poor correlation between spontaneous metastatic potential and intercellular junctional coupling. The results suggest that loss of junctional communication between cells is often but not always associated with the progression of cells from benign to metastatic states.

Differential tumor growth of blood-borne B16 melanoma variants in cerebral dura mater is related to tumor-host cell reactions.

Intracarotid injection of B16-B14b or B16-B15b melanoma cells, previously established from B16-F1 melanoma by in vivo selection fourteen- or fifteen-times, respectively, for brain surface colonization, preferentially produced tumor nodules in mice at brain surface sites, most frequently in the dura mater, followed by the leptomeninges and cerebral cortex. There was a marked difference, however, in tumor growth at these sites using the two B16 sublines. Intracarotid injection of B16-B14b cells rarely produced visible tumors, whereas B16-B15b cells formed deeply pigmented tumors up to 7 mm in diameter in the brain menings of almost all mice examined. Histologic and electron microscopic investigation revealed that B16-B14b tumors evoked dramatic immunocyte cell infiltration and granulomatous reactions, while B16-B15b tumors were accompanied by much less tumor-host cell reactions. Splenectomy or laparotomy 1-2 weeks before or after intracarotid injection of B16-B14b cells dramatically enhanced tumor growth in the dura mater without extensive tumor-host cell reactions. The results suggest that the differential growth of B16-B14b and B16-B15b tumor cells in the cerebral dura mater is based, in part, on the abilities of these melanoma cells to elicit host cell reactions.

Brain meninges tumor formation by in vivo-selected metastatic B16 melanoma variants in mice.

The murine melanoma subline B16-F1 of low brain- and lung-colonizing potential has been used to obtain brain-colonizing variant sublines by sequential selection in vivo for their abilities to form brain meningeal tumors. After fourteen and fifteen selections in syngeneic C57BL/6 mice sublines B16-B14b and B16-B15b, respectively, were established in culture. These were then assayed in vivo by injection of single tumor cell suspensions into the tail vein (i.v.), left ventricle of the heart (i.c.) or left common carotid artery (i.a.), and the resulting tumors were examined histologically. Injection of subline B16-B14b or B16-B15b resulted in brain meningeal tumor formation in the dura mater and leptomeninges with invasion into underlying brain parenchyma and also some brain ventricular tumors at the sites of i.a. injection. Lung colonization ability remained in the brain-selected sublines, although it was remarkably reduced in i.a. tumor cell-injected animals. The brain meningeal tumors that formed were of three types: intravascular, nodular or infiltrative. Injection of tumor cells i.v. resulted mainly in the establishment of the intravascular type of brain meningeal tumors with eventual perivascular invasion, while injection i.a. or i.c. resulted mostly in nodular or infiltrative type brain meningeal tumors. The B16-B14b brain meningeal tumors formed were small (less than 1 mm in diameter) and usually nonpigmented, while B16-B15b tumors were generally large (up to 7 mm in diameter) and pigmented. Host reactions towards B16-B14b and B16-B15b tumors at meningeal sites differed. The former B16 subline was characterized by extensive fibrosis with some immunocytic cell infiltration in and around the meningeal tumors, while the latter subline did not elicit such host reactions. In contrast, tumors in brain parenchyma failed to evoke observable host reactions, and there was little evidence of immunocyte cell infiltration or glial cell alterations.

Anti-hyperalgesic effect of octreotide in patients with irritable bowel syndrome.

BACKGROUND: Octreotide has been found to be beneficial in the treatment of chronic pain, although the mechanisms underlying its therapeutic effect are incompletely understood. AIMS: To assess the effect of octreotide on perceptual responses to rectal distension in irritable bowel syndrome patients and healthy controls at baseline and following the experimental induction of rectal hyperalgesia. METHODS: In study 1, rectal perception thresholds for discomfort were determined in seven irritable bowel syndrome patients and eight healthy controls on three separate days using a computer-controlled barostat. Subjects received saline, low-dose and high-dose octreotide in a random double-blind fashion. In study 2, perceptual responses to rectal distension were obtained in nine irritable bowel syndrome patients and seven controls before and after repetitive high-pressure mechanical sigmoid stimulation. RESULTS: Octreotide increased the discomfort thresholds in irritable bowel syndrome patients, but not in controls, without changing rectal compliance. Repetitive sigmoid stimulation resulted in decreased rectal discomfort thresholds in the patient group only. In irritable bowel syndrome patients, octreotide prevented the sensitizing effect of repetitive sigmoid stimulation on rectal discomfort thresholds. CONCLUSIONS: Octreotide effectively increased discomfort thresholds in irritable bowel syndrome patients, but not in controls, at baseline and during experimentally induced rectal hyperalgesia. These findings suggest that octreotide exerts primarily an anti-hyperalgesic rather than analgesic effect on visceral perception.

A case of pancreatic carcinoma causing massive bronchial fluid production and electrolyte abnormalities.

A 39-year-old man developed massive bronchorrhea (2 to 3.5 L/d) with electrolyte and volume depletion about 2 years after undergoing a Whipple's procedure for pancreatic carcinoma. An open lung wedge biopsy specimen was consistent with metastatic adenocarcinoma with extensive growth along preexisting pulmonary architecture. Chemical analysis of the bronchial fluid revealed markedly elevated levels of amylase confirming the pancreatic origin of the tumor. The mechanism of massive bronchorrhea is not known. Chemical analysis of bronchial fluid in comparison to serum and the temporary response to chemotherapy are most consistent with secretory and transudative mechanisms.

Postirradiation sarcomas in Sprague-Dawley rats.

A series of radiation-induced neoplasms occurred in Sprague-Dawley rats 4-8 months after irradiation of a single hind leg with 60Co gamma rays. The rats were exposed to fractionated cumulative doses that ranged from 0 to 106 Gy. Osteosarcomas, malignant fibrous histiocytomas and fibrosarcomas developed in the radiation fields of a number of the rats in the higher-dose groups. Tumors did not develop throughout an 8-month observation period in rats that received doses of only 0 or 46 Gy. The most common postirradiation sarcomas in humans are osteosarcoma, malignant fibrous histiocytoma and fibrosarcoma. The Sprague-Dawley rat may serve as a good animal model in studying the development of sarcoma in humans after regional radiotherapy.

Effects of the corticotropin-releasing factor (CRF) on rectal afferent nerves in humans.

Corticotropin-releasing factor (CRF) released in the gastrointestinal mucosa from immune cells or enterochromaffin cells may play a role in the modulation of rectal afferent function. In the current study we evaluated the effects of peripherally administered CRF on afferent mechanisms in the human rectum. We used rectal balloon distention in seven healthy volunteers to evaluate the effect of CRF (1 microgram/kg) on visceral afferents originating in the rectum which are involved in the following functions: thresholds and intensity of conscious perception, receptive relaxation, reflex inhibition of internal anal sphincter and a viscerosomatic reflex. Rectal mechanoreceptors were stimulated either by distending the rectum using a volume ramp (40 and 400 mL/min), or by intermittent phasic distention. CRF decreased the thresholds and increased the intensity for the sensation of discomfort in response to both ramp and phasic distention. During slow ramp distention, CRF also lowered the stool threshold. CRF increased rectal compliance during slow ramp distention without affecting the rate of receptive relaxation or the inflection point of the compliance curve. CRF had no effect on viscerosomatic referral patterns, or on the rectoanal inhibitory reflex. These findings are consistent with a dual effect of CRF on afferent pathways mediating perception of aversive rectal sensations, and on rectal smooth muscle.

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by kappa-opioids.

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca(2+)](i)) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by kappa-opioids. [Ca(2+)](i) responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca(2+)](i) increases which were abolished in Ca(2+)-free solution, but unaffected by lanthanum (25 microM) or tetrodotoxin (10(-6) M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd(3+); 250 microM) and amiloride (100 microM) abolished the [Ca(2+)](i) transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca(2+)](i) transients. The kappa-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca(2+)](i) transients but had little effect on capsaicin-induced [Ca(2+)](i) transients. The inhibitory effect of U50,488 was abolished by the kappa-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30-100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca(2+)](i) transients in small diameter DRG somas are mediated by influx of Ca(2+) through a Gd(3+)- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca(2+) transients are sensitive to kappa-opioid agonists.

Approaches to the modulation of abdominal pain.

Despite their high prevalence and significant economic impact on the health care system, functional gastrointestinal disorders have evaded successful therapy. Conventional medical therapies are based on inadequate disease models, and the great majority of published treatment trials are flawed in their design, permitting no conclusions to be drawn about the true efficacy of any particular treatment. During the past several years, a new, comprehensive disease model based on alterations in brain-gut interactions has rapidly evolved. Even though the precise mechanisms and sites underlying these alterations remain incompletely understood, plausible targets for the development of effective pharmacological treatments are receptors on peripheral terminals of visceral afferent nerves (opioids and serotonin), ion channels and receptors on dorsal horn neurons within the spinal cord (opioids, glutamate, calcitonin gene-related peptide and neurokinin-1), and supraspinal targets in the brainstem within the limbic system and in the prefrontal cortex (serotonin, catecholamines, dopamine and acetylcholine). Regardless of the primary pathophysiology underlying functional gastrointestinal disorders (ie, central versus peripheral), different pharmacological strategies targeted at different sites in the periphery or within the central nervous system may become effective therapies in the future.

Iatrogenic Horner's syndrome in an experimental pig.

An adult domestic female pig (Sus scrofa) exhibited clinical signs of right-sided Horner's syndrome after experimental placement of a woven aortic stent followed by aortic catheterization. The clinical signs included a miotic pupil, ptosis of the upper eyelid, prolapse of the nictitating membrane, and enophthalmos. Necropsy revealed a large mass in the right midcervical region that encased or was in contact with the carotid artery, internal jugular vein, and vagus nerve. Closer evaluation of the mass revealed that it was a small piece of surgical suture material that was embedded within the lumen of the carotid artery. This extrinsic material served as a nidus for an inflammatory reaction involving the vagus nerve.

[24-hour blood pressure: effects of sports in aged subjects]. / La pressione arteriosa di 24-h: effetto dello sport in soggetti anziani.

The present study deals with the blood pressure (BP) and heart rate (HR) 24-h pattern in aged people of both sexes ranging in age from 66 to 75 years. 20 subjects were active in agonistic sports (running and swimming), 20 others had sedentary habits. BP and HR were studied by non-invasive monitoring along with chronobiometric analysis. The BP and HR 24-h pattern was seen to preserve its circadian rhythm in both aged athletes and sedentary elderly. The analysis reveals that the aged athletes show a higher mesor for systolic BP and a lower mesor for diastolic BP and HR. The daily pressure load, as the integral of the BP 24-h values multiplied by the HR mesor, is lower in aged people practising sport as compared to age-matched individuals with sedentary habits. The spectral analysis reveals that physical exercise acts to change the time structure of BP and HR 24-h patterns. The conclusion is drawn that active sport plays beneficial effects in aged people because of a lower pressure load.

Rabies control in rural Africa: evaluating strategies for effective domestic dog vaccination.

Effective vaccination campaigns need to reach a sufficient percentage of the population to eliminate disease and prevent future outbreaks, which for rabies is predicted to be 70%, at a cost that is economically and logistically sustainable. Domestic dog rabies has been increasing across most of sub-Saharan Africa indicating that dog vaccination programmes to date have been inadequate. We compare the effectiveness of a variety of dog vaccination strategies in terms of their cost and coverage in different community settings in rural Tanzania. Central-point (CP) vaccination was extremely effective in agro-pastoralist communities achieving a high coverage (>80%) at a low cost (US$5/dog) and inadequate (<20% coverage); combined approaches using CP and either house-to-house vaccination or trained community-based animal health workers were most effective with coverage exceeding 70%, although costs were still high (>US$6 and >US$4/dog, respectively). No single vaccination strategy is likely to be effective in all populations and therefore alternative approaches must be deployed under different settings. CP vaccination is cost-effective and efficient for the majority of dog populations in rural Tanzania and potentially elsewhere in sub-Saharan Africa, whereas a combination strategy is necessary in remote pastoralist communities. These results suggest that rabies control is logistically feasible across most of the developing world and that the annual costs of effective vaccination campaigns in Tanzania are likely to be affordable.

Diagnosis and management of fecal incontinence in elderly patients.

Fecal incontinence is a physically and psychologically disabling condition that affects millions of Americans, especially those over the age of 65 years. The pathophysiology is often multifactorial, with decreased anorectal sensation, reduced rectal compliance. anal sphincter dysfunction, altered stool consistency and immobility playing significant roles. A detailed history and a thorough physical examination are always necessary in patients with fecal incontinence and physiologic tests, including anorectal manometry, cinedefecography and electromyography, may be required for proper diagnosis and treatment. In most patients fecal incontinence is initially treated with conservative measures, such as biofeedback training or alteration of the stool consistency (if appropriate). If conservative management fails, surgical intervention, such as sphincteroplasty or gracilis muscle transposition, may be considered.

GERD progressing to diffuse esophageal spasm and then to achalasia.

The pathophysiology of achalasia is not completely understood. Several reports have suggested that esophageal motility disorders may progress from one type to another. We report a patient with symptoms and esophageal motility findings consistent with gastroesophageal reflux who subsequently developed a diffuse esophageal spasm and then achalasia. We believe this to be the first report showing such a progression in esophageal motility.