Mem Inst Oswaldo Cruz, Rio de Janeiro, VOLUME 115 | JUNE 2020
The impact of imported malaria by gold miners in Roraima: characterizing the spatial dynamics of autochthonous and imported malaria in an urban region of Boa Vista
1Universidade Federal de Roraima, Boa Vista, RR, Brasil
2Secretaria de Saúde de Roraima, Coordenação Geral de Vigilância em Saúde, Boa Vista, RR, Brasil
3Secretaria de Vigilância em Saúde do Ministério da Saúde, Brasil
4Fundação Oswaldo Cruz-Fiocruz, Instituto Nacional de Infectologia Evandro Chagas, Rio de Janeiro, RJ, Brasil
5Temple University, Institute for Genomics and Evolutionary Medicine, Department of Biology, Philadelphia, PA, United States of America
6Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Imunoparasitologia, Rio de Janeiro, RJ, Brasil
BACKGROUND The number of malaria cases in Roraima nearly tripled from 2016 to 2018. The capital, Boa Vista, considered a low-risk area for malaria transmission, reported an increasing number of autochthonous and imported cases.
OBJECTIVES This study describes a spatial analysis on malaria cases in an urban region of Boa Vista, which sought to identify the autochthonous and imported cases and associated them with Anopheles habitats and the potential risk of local transmission.
METHODS In a cross-sectional study at the Polyclinic Cosme e Silva, 520 individuals were interviewed and diagnosed with malaria by microscopic examination. Using a global positional system, the locations of malaria cases by type and origin and the breeding sites of anopheline vectors were mapped and the risk of malaria transmission was evaluated by spatial point pattern analysis.
FINDINGS Malaria was detected in 57.5% of the individuals and there was a disproportionate number of imported cases (90.6%) linked to Brazilian coming from gold mining sites in Venezuela and Guyana.
MAIN CONCLUSIONS The increase in imported malaria cases circulating in the west region of Boa Vista, where there are positive breeding sites for the main vectors, may represent a potential condition for increased autochthonous malaria transmission in this space.
Malaria continues to be a significant public health problem worldwide, causing about 228 million new cases, with approximately 405,000 deaths per year, especially in Africa.(1) Although the global incidence rate decreased by 18% between 2010 and 2016, there was a substantial increase between 2014 and 2015 in the Americas, primarily due to a surge of malaria cases in Brazil and Venezuela.(2) In Brazil, the number of malaria cases increased from 143,748 in 2015 to 193,837 in 2018 after seven years of decline, and in Venezuela (Bolivarian Republic of Venezuela) increased from 91,918 in 2015 to 414,527 in 2017, mainly in the Bolivar State in the southeast of the country bordering with Guyana and the Brazilian State of Roraima.(3) Also, the region has gold mining areas where working and living conditions are considered a problem for malaria control. Indeed, Bolivar State is a regional hot-spot from which malaria has spilled over to other neighboring countries overloading health care services in border municipalities of Brazil and Colombia that receive imported cases of malaria, including Plasmodium falciparum.(4)
Roraima is the Brazilian State in the extreme north of the country that shares international borders with Guyana and Venezuela. In that space, human mobility across borders was usually intense but temporary. However, since 2017, Roraima has experienced the impact of an unprecedented unidirectional migration of Venezuelans due to the economic and political crisis in their country.(3) According to UNICEF and migration authorities in Brazil, an estimate of 178,000 Venezuelans has crossed into Roraima, with at least 32,000 settlings in Boa Vista, the State capital, located approximately 100 km distant from Guyana and 200 km from Venezuela.(5) Certainly, the increase in border population movements between Brazil, Venezuela, and Guyana have affected malaria control measures and contributed to the spread of the disease in Roraima State.(6)
Data from the Ministry of Health, SIVEP-Malaria (Epidemiologic Surveillance Information System ― Malaria), show that the number of reported cases in Roraima nearly tripled from 8,969 cases in 2016 to 23,369 in 2018.(7) Malaria is endemic in many municipalities which have various degree of endemicity. However, 39% of the cases were imported from other states in Brazil (3,625 cases) and other countries (5,513), mainly from Venezuela (4,478 cases) and Guyana (610 cases).(10) P. falciparum infections accounted for 26% of the imported and 9.8% of the autochthonous case.(7)
Boa Vista is the municipality reporting most of the malaria imported cases from neighboring countries. In 2010 the number of reported cases were 5,948 and only 6% were autochthonous.(7) In 2011, the number of imported cases dropped to 4,354 and the autochthonous to 248 cases. This tendency to fall was maintained until reaching its lowest value in 2016, with about 2,357 imported cases and 12 autochthonous. However, in the years 2017 to 2018 there was a new increase in imported cases.(7)
In 2018, Boa Vista reported 5,713 cases, 168 (3%) autochthonous, 2,805 (49%) imported from other municipalities/states in Brazil, and 2,740 (48%) from other countries, mainly Venezuela (n = 2,115) and Guyana (n = 533). It is also noteworthy that, in 2018, cases of P. falciparum infection from Venezuela (n = 481) and Guyana (n = 97) represent 80% of the notifications of P. falciparum registered in Boa Vista (722 cases).(7) Studies of malaria vectors in Roraima performed more than ten years ago found that Anopheles albitarsis and An. darlingi were infected with Plasmodium spp.(8, 9)
Despite being considered a low-risk area for malaria transmission, the consistent numbers of imported malaria cases, which are a possible source of infection, together with the presence of competent vectors in urban areas, make possible outbreaks and epidemics in Boa Vista.(10) A sustained increase in local transmission would be a significant setback for malaria elimination efforts in Boa Vista.
Considering the multiple factors that may drive the increase in malaria observed in Brazil, disentangling their differential contribution will guide how to better deploy interventions. Therefore, this study aimed to understand the epidemiology of the imported malaria cases and the potential risk of increased transmission in an urban region of Boa Vista.
SUBJECTS AND METHODS
Study site - The study was carried out in Boa Vista, Roraima State, the only Brazilian State capital located entirely in the northern hemisphere, between Latitude 02º49’12” N and Longitude 60º40’23”W. Boa Vista occupies an area of 5,687,037 Km2, concentrates 63.4% of the state’s population and has a demographic density of 49.99 inhabitants per Km2 (Instituto Brasileiro de Geografia e Estatística - IBGE, 2016). Savanna is the predominant ecological environment in Boa Vista and the climate presents two distinct seasons: a rainy season between April and November with high rainfall indices during the months of June and July and the dry season between December and March. The geographical position of the municipality allows easy access to international borders thus positioned: in the north part of the state in the municipality of Pacaraima, which borders Santa Elena do Uairén in Venezuela, 231.5 km from Boa Vista, a land route (BR- 174). The municipality of Bonfim bordering Lethem, Cooperative Republic of Guyana, 133.3 km from Boa Vista via BR-401. To the South, the State is bordered by Amazonas State and Pará, Fig. 1.
Study population and sample collection - Samples and research data were collected from March 2016 to September 2018 at the Polyclinic Cosme e Silva. It is a health unit of the Unified Health System (SUS) that has a high demand for various medical specialties. This Polyclinic is strategically located in the west area of the city of Boa Vista, which has a population of 152,062 inhabitants (IBGE - 2016), distributed in 33 (thirty-three) neighborhoods. According to data from Ministry of Health, SIVEP - Malaria, among the 13 health units that offer malaria diagnosis in Boa Vista, the Polyclinic was the one that registered the highest number of cases in the last 5 years, as follow: in 2011, 2,354 cases; in 2012, 1,424 cases; in 2013, 1,713 cases; in 2014, 1,384 cases; and 966 cases in 2015.(10) Taking in account the total number of reported cases in Boa Vista in the same period (2011-2015), the Polyclinic Cosme e Silva was responsible for 51%, 44%, 42%, 54% and 48.8% of the reported cases, respectively.(7) Therefore, all individuals seeking a malaria diagnosis in the Public Health Unit Polyclinic Cosme e Silva, were eligible to participate in the study.
A questionnaire, completed by each participant, was used to collect demographic data, travel history, and epidemiological data. Blood was drawn from each participant by venipuncture and malaria infection was diagnosed by microscopic examination of Giemsa stained blood smears. The parasitological evaluations were performed by examination of 200 fields at 1,000-fold magnification under oil immersion. Parasite densities were estimated by counting the number of parasites per microliter of blood (all species and stages) per 200 leukocytes in thick blood films, multiplying this by the number of individual leukocytes, and dividing this by 200.
All positive individuals were treated with antimalarial drugs, according to the protocol of the Brazilian Ministry of Health. All the questionnaire data were stored in Epi Info™ 2002 (CDC, Atlanta, GA, USA). This protocol was approved by the Federal University of Roraima Ethical Committee (CAAE: 44055315.0.0000.5302).
Case definition - All malaria cases were classified as autochthonous or imported depending on the most likely place of acquisition as per the questionnaire. In this study, imported malaria was defined as malaria acquired outside the municipality of Boa Vista but diagnosed within the municipality. This was determined by the individual history of staying in an endemic municipality, state or country during the last one month. Autochthonous cases were defined as malaria acquired in the municipality of Boa Vista in persons without travel history outside the municipality in the last one month.
Breeding sites and larval collection - Twenty-three breeding sites were identified and georeferenced using Garmin GPS by technicians of the entomology service of the General Coordination of Health Surveillance of Roraima. The breeding sites were chosen based on the location of autochthonous cases in previous 3 years (2014 to 2016). These breeding sites have been monitored by the entomology service of the General Coordination of Health Surveillance every two months between 2017 and 2018 and only qualitatively classified as positive or negative for the presence of Anopheles spp larvae. The larvae collections were performed using standard entomological scoops (BioQuip, Ranch Dominguez, CA, USA). At each breeding site, nine scopes were made, three to the right, three to the front, and three to the left, following the Ministry of Health standard.(10) Due to the great difficulty in rearing Anopheles larvae stage in the laboratory, Anopheles larvae from 1st and 2nd was used to determine if the breeding place were positive for Anopheles spp. Larvae (third and fourth-instar) and adults were identified using the keys of Consoli and Lourenço-de-Oliveira and Forattini.(11, 12) The larvae collected were transferred to plastic bags, labeled with the breeding site code, date and collector. All collected material was transferred to the state entomology laboratory to be screened and identified. The breeding site was considered positive if in at least one collection per year presented Anopheles spp larvae.
Spatial analysis - The spatial dynamics of malaria in Boa Vista were analysed considering three situations: mapping of individuals with malaria, positive breeding sites for Anopheles and circulation time between first symptoms and treatment initiation. To perform spatial analysis, the locations of disease occurrence was recorded based on the address of malaria patients (Street and Neighborhood) and geographic coordinates were obtained, in decimal degrees geographic coordinate system, via Google Maps geographic location service.
The data were processed in the QGIS 2.14.17 software, executed from the Windows platform, to generate maps.
The basic cartography set was georeferenced using the decimal degrees geographic coordinate system and the World Geodetic System-1984 reference system (WGS84). All geographic data were processed in the Landscape Metrics Laboratory of the Department of Geography of the Federal University of Roraima (UFRR), which has available satellite images and cartographic basis of Boa Vista for the preparation of thematic maps.
Statistical analysis and spatial point pattern analysis - Demographic, epidemiological and parasitological data were entered into a database created using the Epi Info software (Epi Info™, Atlanta, GA, USA). For statistical purpose, the following variables were taken into consideration: gender, age, residence, nationality, occupation, place of infection, symptoms, days since first symptoms and past and present malaria infections. Proportions were compared using 2x2 contingency tables with either chi-squared tests, adjusted by Yates’ continuity correction or Fishers exact tests, as appropriate. Odds ratios (OR) analysis were used to quantify the strength of the association between variables. The statistical significance threshold was p < 0.05, with 95% confidence intervals (95% CI) for all hypothesis tests. These analyses were performed using GraphPad InStat, version 3 (GraphPad Software, San Diego, CA, USA).
Kolmogorov-Smirnov test was used to verify the hypothesis of risk for malaria transmission in the western region of Boa Vista due to the existence of breeding sites and individuals with imported malaria, part of which with gametocytes. This test was applied in order to assess whether the distribution of the points follows a homogeneous Poisson process or whether the points are distributed according to a clustering model. For the interpretation of the values, consider a 5% level of significance in the results (95% CI). In summary, the null hypothesis to be tested is that the distribution of points throughout the region under study follows a homogeneous Poisson process.(13, 14)
In order to verify the hypothesis of complete spatial randomness, the analysis of specific processes was carried out by evaluating the graph of the G, F, K and L functions, to verify whether the points are distributed randomly or in the form of grouping throughout the region under study. The G function represents the distance from the nearest event (breeding sites, malaria and gametocytes cases), the plotted results of this function can be used as an exploratory method to check if there is evidence of interaction between the events; F function aims to check the risk rate, since it measures the distribution of all distances from an arbitrary point (cases, breeding sites and gametocytes) to the point closest to it; the K function represents the Ripley’s function, that allows the detection of the spatial pattern at different distance scales simultaneously; while the L function consists of the Besag transformation function of the Ripley’s function.(15) In the horizontal axis of the graphs it is shown distances between the points (individuals with malaria, breeding sites and gametocytes), so that in the vertical axis we have the density values based on the estimators of the functions G, F, K and L, respectively. The black lines mean the observed values, while the red lines correspond to the theoretical values of the function. The gray value range consists of the 95% CI, considering 99 simulations. The analyzes were performed using the software R v. 3.6.2.
Characteristic of the studied population - During the study period, 520 individuals participated in the study, 299 (57.5%) were positive for Plasmodium in the thick blood smear. The characteristics of the study population are presented in Table I. The majority of the individuals were male (74.6%), the mean age was 36 years and the nationality was predominantly Brazilians with permanent residence in Boa Vista, Roraima State. However, their main occupation was gold mining or mining activities in Venezuela and Guyana. Overall, 468 individuals declared a history of malaria episodes (90%); the majority reported more than five episodes (74.4%) and 10% did not recall past infection in their lifetime. Ninety-six percent of the asymptomatic individuals (n = 19), 68.4% had a positive diagnosis for malaria while 31.6% were negative. No difference was observed in the nationality, place of residence, occupation, and number of malaria episodes in the last three years between positive and negative individuals. However, proportion of malaria positives individuals decreases in the age groups 36-44 (OR = 0.59, 95% CI 0.35 - 0.98; p = 0.04) and > 44 (OR = 0.36, 95% CI 0.21 - 0.6; p < 0.01), as compared to groups of younger ages.
Breeding sites - Boa Vista is located in an area with large rivers, streams and lakes, characteristics that favors the existence of breeding sites, in addition to the climate favorable to the presence of the main malaria vectors. The breeding sites were lakes, streams and natural permanent rivers, partially shaded and sunny with the presence of marginal vegetation, mainly in the dark and still waters. All 23 breeding sites mapped mostly murky and standing waters. All 23 were larval habitats for Anopheles spp larvae. An. darlingi and An. albitarsis were the species found in all investigated breeding sites, however, other species were as also found, An. Nuneztovari s.l., An. Mattogrossensis, An. Triannulatus and An. Evansae.
Autochthonous and imported malaria cases - Of the 299 individuals positive for malaria infection during the studied period, 294 (98.3%) were imported and only five (1.7%) were autochthonous from the municipality of Boa Vista. Among these 172 were infected with P. vivax, 109 with P. falciparum, and 13 with mixed (P. falciparum + P. vivax). P. vivax was the most prevalent Plasmodium species found in 100% of the autochthonous cases and 57.5% of the imported cases. The observed P. falciparum and mixed (P. falciparum + P. vivax) infections were imported, Table II.
Brazilian were the majority of the cases (94.6%) followed by Venezuelan (5.1%) and Guyanese (0.3%). A large number of the imported cases were acquired in Venezuela (58.8%) and Guyana (32.3%). The cases from French Guyana and Suriname corresponded to 1%) and 7.7% came from other municipalities of Roraima or other states in Brazil. Individuals with P. falciparum had, on average, a greater parasitemia than individuals affected by P. vivax and mixed infections. On average patients infected with P. falciparum and/or mixed infections sought diagnosis after about five days of onset of symptoms.
Spatial distribution of infecting malaria species and vectors - The available addresses of 271 imported and five autochthonous cases were used to plot the spatial distribution of the cases by species, Fig. 2. Only two out of five autochthonous P. vivax cases reported their residence; the other three reported the place of infection as the neighborhood where they work. All individuals reported not leaving the municipality in the last 1 month.
A radius of 1 km was established from the identified breeding sites as the mosquito’s flight coverage area (green region of the map).(16, 17) An area at risk of malaria transmission was delimited on the map (pink region of the map) considering the location of autochthonous and imported cases and their spatial relations with the breeding sites. In Fig. 2, the majority plotted points were of P. vivax malaria (151 imported and five autochthonous), P. falciparum malaria corresponds to 88 georeferenced points, while 32 points on the map represent mixed malaria. The map includes the 23 georeferenced breeding sites in the city. We observed that the majority of cases were concentrated in the west of the city. Several breeding sites were also observed in the east side of the city (green area in Fig. 2).
Spatial distribution of gametocytes carriers and breeding sites - The residence of individuals positive for gametocyte forms of P. falciparum and P. vivax and their relationship with breeding sites was plotted, Fig. 3. We observed that gametocyte positive individuals were found within the radius of action of the Anopheles established at 1 km (green area) from the larval collection points in the streams and rivers positive for both An. darlingi and An. albitarsis. The highest concentration of these individuals was in the south and west of the municipality. The latter has the largest number of neighborhoods, 33, and it is the most populous with 152,062 inhabitants. Most of these neighborhoods are in the city outskirts and are within the breeding places range (pink color on the map of Fig. 3). Noteworthy, 73% of the gametocyte’s carriers sought treatment 48 h after the symptoms had started and these individuals were living in areas at risk of malaria transmission (pink color on the map) in several districts of Boa Vista, including the most populous zone of the municipality.
Risk of local malaria transmission - According to the test results [Kolmogorov-Smirnov = 0.2376 (p-value < 0.001 in 95% CI)], there are specific locations that tend to have a higher frequency of malaria cases. It is to say that the circulation of individuals with malaria along the west zone of the municipality of Boa Vista increases the risk of malaria transmission.
Results in Fig. 4 (G function) show that the observed values, that is, the distance between the cases of malaria and individuals carrying gametocytes from breeding sites (black line), are outside and above the confidence interval given by the gray area. Therefore, the circulation of individuals with the disease close to breeding sites is a risk factor for local malaria transmission. Analysis in a wider area, Fig. 4 (function F), the observed values, that is, the risk rate (black line), are outside and below the confidence interval given by the gray area. Thus, by the behavior of the function graph, the presence of individuals with malaria in this part of the municipality poses risk of local malaria transmission. Indeed, in Fig. 4. The reduced second order measures (K and L functions), endorse risk of local malaria transmission indicated in function G and K. Therefore, the spatial distribution and proximity of malaria cases, individuals carrying gametocytes and malaria vector breeding sites favors local malaria transmission.
Roraima State has suffered an increase in the number of malaria cases. Its capital, Boa Vista, is the municipality with the highest number of imported cases in the State.(3) Imported malaria is considered a critical impediment to elimination in many countries including Brazil. Indeed, this study shows that most of the cases reported in the Health Unit in the urban area of Boa Vista, during the time of this study, were imported from Venezuela (58.8%) and Guyana (32.3%) with only few autochthonous cases (1.7%), as previously observed.(7) Autochthonous infections were all P. vivax while imported infections were attributed mainly to P. vivax and to a lesser extent to P. falciparum. Despite its relatively low number, falciparum/vivax mixed infections were detected in the study’s site. Moreover, the vast majority of these imported cases (90.4%) were Brazilians coming from gold mining sites (90.4%) in Venezuela and Guyana rather than migrants from these countries. Overall, gold miners are at higher risk of contracting malaria making them a “hot pop” or subpopulation at higher risk of Plasmodium infection.(18)
Brazilian miners spend part of their time between the gold mines abroad and Boa Vista, where they live (98.3%) and sell the products of this labor, generating high mobility between endemic areas in Venezuela and Guyana to Boa Vista. Indeed, the massive flow of miners in this health unit reflects the high risk of malaria in the mining sites.(7, 19) Consistent with these observations, imported malaria in recent years has increased exponentially as gold mining activities increase in 2018 and gold was the second most exported product in the state.(19, 20, 21) There are currently 1,097 illegal mining points located in Venezuela, Guyana and Brazil (Yanomami indigenous lands) producing high mobility of job seekers.(22) Although these mining regions are generally unhealthy and dangerous, it provides an economic incentive that motivates the return of these individuals in search of survival, while still at risk of malaria. Overall, the situation observed in Roraima is consistent with reports emerging from other endemic areas in the Americas.(23, 24)
Results from our study indicate that there are several positive breeding sites for An. darlingi and An. albitarsis in Boa Vista. Also, we found asymptomatic individuals (4.3%) and the “time to start treatment of patients after the date of first symptoms” was over 48 h for 60% of patients. These factors could increase the risk of local transmission after malaria has been introduced. This situation requires increasing surveillance as evidenced by an increase of 1.300% in autochthonous cases in Boa Vista in 2018.
Importantly, the Boa Vista health services attract migrants from other municipalities and border cities seeking malaria treatment and the location of the city favors the movement of people and goods. These characteristics create a continuous flow between the mining areas and Boa Vista. The relationship between people’s mobility and imported malaria has been pointed out in several studies as a concern for malaria control and elimination. In this case, mobility is linked to gold miners.
The risk of local malaria transmission observed by the spatial point pattern analysis, even if it is limited to an area of Boa Vista, deserves attention from health surveillance agencies, as it is a populous area where most neighborhoods are concentrated, in addition to housing farms and water collection. It is possible that this risk may extend to other areas of the city given the similarity of the characteristics observed in the urban and peri-urban perimeter of the municipality. Therefore, the expansion of this study to other areas of the city is important in order to assess this risk for the city of Boa Vista as a whole.
In Boa Vista, during the study period, the main malaria control measures were passive case detection and treatment and other measures follow a specific demand focused on the occurrence of autochthonous cases. Active case detection of infected individual’s, insecticide spraying of households and health education are limited to the probable place of infection of confirmed autochthonous case and neighborhood. Occasionally, identification of breeding sites and Anopheles larvae collection are performed. Therefore, implementation of control measures to reduce the risk of autochthonous transmission such as continuous surveillance of active case detection, monitoring Anopheles breeding sites associated with insecticide spraying of households and health education targeting the hot pop, especially gold miners should be strongly considered in Boa Vista.(25, 26)
It is worth noting that we did not observe any autochthonous P. falciparum infections, although our number was low (n = 5). However, data from the Ministry of Health, SIVEP-Malaria have indicated 14 P. falciparum imported cases in Boa Vista in the last three years.(7) Imported P. falciparum cases present an unusual distribution in other studies, affecting about two out of five individuals examined, a cause for concern as there is a risk of introducing and spreading P. falciparum resistant strains from neighboring countries in Brazil.(27, 28) In a macro-policy scenario, advocated by the Ministry of Health, through the elimination plan, Brazil aims to eliminate falciparum malaria by 2022.(28)
There is no bilateral cooperation between Brazil and Venezuela such as that between Andean countries (Venezuela, Peru, Colombia, and Ecuador) for malaria containment.(28) However, since Brazilians miners are the ones introducing malaria, this may reveal weaknesses in border control measures, and poses a challenge for malaria control and, particularly, for P. falciparum elimination goals. Given the relatively good communication between Bolivar State and Roraima, and the gold mining activities in Bolivar State, the Brazilian-Venezuelan border could be regarded as a malaria corridor where the disease will likely remain resilient to uncoordinated control efforts across international borders.(29)
The data reported in this research are important, however limited, as they represent only a certain area of the city of Boa Vista. In this study, participation of Brazilians with malaria was significantly higher than Venezuelans, with the majority of imported malaria coming from the mining region. As this study was limited to just one health unit, it is possible that other health centers provided assistance to Venezuelans with malaria.
Despite this limitation, it is important to note that in regions with low malaria transmission, the distribution of autochthonous cases has become more and more irregular and heterogeneous, capable of modifying local epidemiology, as observed by the increase of autochthonous cases in Boa Vista (1.300%).
In conclusion - Imported malaria in an urban area of Boa Vista is driven by Brazilians working on gold mines traveling across international borders. It seems that the origin of imported malaria in the west region of Boa Vista is related to mining and not to the migratory movement. Regardless of the motivation, malaria represents the universe of individuals at social risk. Despite the low number of autochthonous cases, the influx of imported infected travelers into areas with suitable vectors breeding sites, there is a potential risk for an increase in autochthonous malaria in the studied area, including the risk of introducing P. falciparum malaria. Therefore, early detection of malaria cases is necessary to limit their potential negative impact in achieving the goals of elimination plan proposed by the Brazilian Ministry of Health.
To Rispah Abdhallah, Naomi W Lucchi, and Venkatachalam Udhayakumar, CDC. We thank Pedro Rufino da Silva, Valdenor Alves Macedo and Cleomar Oliveira da Silva for assistance with the breeding places mapping and Secretary of Health of Roraima ― General Coordination for Health Surveillance for fieldwork support.
JOF, JL and AAE conceived the idea and participated in the study design; JL, NCVA and JLPA were responsible for sample collection; JOF, JL, NCVA, JLPA, TMC, JC carried out the experiments and analysed the data; JOF, JL and TMC drafted the manuscript and JOF and AAE revised. All authors read, corrected and approved the final manuscript. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.


Development of the mammary gland requires the coordinated action of proteolytic enzymes during two phases of remodelling. Firstly, new ducts and side-branches thereof need to be established during pregnancy to generate an extensive ductal tree allowing the secretion and transport of milk. A second wave of remodelling occurs during mammary involution after weaning. We have analysed the role of the cell surface protease aminopeptidase N (Anpep, APN, CD13) during these processes using Anpep deficient and Anpep over-expressing mice. We find that APN deficiency significantly delays mammary gland morphogenesis during gestation. The defect is characterised by a reduction in alveolar buds and duct branching at mid-pregnancy. Conversely over-expression of Anpep leads to accelerated ductal development. This indicates that Anpep plays a critical role in the proteolytic remodelling of mammary tissue during adult mammary development.


Automated amplicon design suitable for analysis of DNA variants by melting techniques
BMC Research Notes volume 8, Article number: 667 (2015)
The technological development of DNA analysis has had tremendous development in recent years, and the present deep sequencing techniques present unprecedented opportunities for detailed and high-throughput DNA variant detection. Although DNA sequencing has had an exponential decrease in cost per base pair analyzed, focused and target-specific methods are however still much in use for analysis of DNA variants. With increasing capacity in the analytical procedures, an equal demand in automated amplicon and primer design has emerged.
We have constructed a web-based tool that is able to batch design DNA variant assay suitable for analysis by denaturing gel/capillary electrophoresis and high resolution melting. The tool is developed as a computational workflow that implements one of the most widely used primer design tools, followed by validation of primer specificity, as well as calculation and visualization of the melting properties of the resulting amplicon, with or without an artificial high melting domain attached. The tool will be useful for scientists applying DNA melting techniques in analysis of DNA variations. The tool is freely available at http://meltprimer.ous-research.no/.
Herein, we demonstrate a novel tool with respect to covering the whole amplicon design workflow necessary for groups that use melting equilibrium techniques to separate DNA variants.
Friedrich Miescher discovered the nucleic acid in 1868–69 (reviewed by R. Dahm) [1, 2]. This may be defined as the starting point of a research field that has expanded into a huge area of medical and biological research. Many important methodological advances have been made in order to facilitate analysis of DNA. The polymerase chain reaction (PCR), first published by Kleppe et al. , which opened for mass amplification of DNA amplicons, is still a key method in modern molecular analysis of DNA. PCR amplification has the ability to amplify specific target sequences, as well as whole genomes up to a factor of 1011. In 1977, a DNA sequencing approach introduced by Sanger used dideoxy nucleotides to terminate enzymatic amplification of single stranded DNA . This method was refined and eventually used to sequence almost the entire human genome. PCR amplification and Sanger sequencing, and variations of these core techniques, are still very important parts in present day DNA research. Following the development of these and other molecular methods, techniques enabling separation and visualization of amplified DNA have taken place, from the starting point of using radioactively labeled DNA and gel electrophoresis, through laser-induced fluorescence capillary electrophoresis, until various high throughput-sequencing platforms with the capacity to determine up to 5G bases/day.
One set of methods that was developed to detect unknown DNA variants was based on differential migration velocities of mutant single-stranded sequences or wild type/mutant heteroduplexes drawn through a macromolecular matrix by an electric field [5–7]. Of these, denaturing gel electrophoresis when performed in capillaries (constant denaturing capillary electrophoresis, CDCE) under optimized conditions has been demonstrated to comprehensively detect any point mutation, including single base insertions and deletions, in target sequences of ~70–140 bp [8, 9]. Methods employing CDCE have been reported with sensitivities to detect and identify mitochondrial and nuclear point mutations at levels at or above 2 × 10−6 mutations per gene copy in human cells, tissues and pooled blood samples [10–12]. Under a given concentration of chemical denaturant, such as urea, multiple capillary runs were required to define the generally narrow temperature range (~0.1 °C) that would separate heteroduplexes containing any of a wide variety of single deletion or single substitution mutants from wild type homoduplexes. CDCE has been adapted to commercial multi-capillary instruments, thus increasing the throughput of the method [13, 14]. A second improvement of the method was the introduction of oscillating temperature. By rapidly changing the denaturing condition in the capillaries, multiple DNA target sequences could be scanned simultaneously in the same run [13, 15–17]. One limitation of melting gel techniques results from DNA sequences rich on GC content, which can lose the resolution power due to complete strand dissociation at elevated temperatures. However, data from the complete melting map of the human genome indicates that the melting gel method can be applied to about 90 % of the human genome . Because melting gel techniques are still much in use (more than 700 articles published in 2013), we have created a computational workflow that selects PCR primers , validates primer specificity in the genome of interest , and computes the DNA melting profile  with an artificial high temperature melting domain . This web application, which is embedded in the Galaxy framework [23–25], will simplify amplicon design and increase the throughput of the method when amplicons are analyzed in multi-capillary instruments. Additionally, the amplicons designed in this web application, are also suitable for high resolution melting. This is a method that emerged after the introduction of thermocycler with fluorescent detection systems enabling visualization of the PCR amplification of DNA in real-time [26–30].
Results and discussion
The amplicon design interface is available at the following URL: http://meltprimer.ous-research.no/. Currently, the Variant Melting Profile tool supports design for DNA variants in the human genome (hg19) and the mouse genome (mm10). Upon request to the authors, additional genomes from other organisms can be added to the server. We have used the human genome as a model to test the Variant Melting Profile tool. The tool can run in single or batch mode, depending on the need to design amplicons for a single variant or a set of variants. There are two ways of entering variants of interest, either by using dbSNP reference IDs (e.g. rsIDs), or by specifying custom positions using a chromosome number, base pair position on the specific chromosome, and entering the relevant reference and variant alleles manually (chromosome:position:reference_allele:alternative_allele). An example of valid input is shown in Fig. 1. The lower variants entered in Fig. 1 demonstrate the two different ways of presenting the same genomic position. The resulting primers for these two ways of entering will be identical. The default values in the interface comprise standard primer length, annealing temperatures range and amplicon length intervals. These settings can be adjusted as needed. Depending on the allele discrimination method to be used downstream from the design, a high-temperature melting domain (GC-clamp) can be added to amplicons of interest. The GC-clamp is selected through a drop-down menu (Fig. 1). After the genomic position or reference numbers have been entered in the entry field, the workflow checks each position towards the latest build provided by NCBI. Un-recognized rs numbers will be called invalid and must be checked by the user, and invalid rs numbers must be removed prior to execution of the workflow. The execute button will start the selection of primers using Primer3, which is the most widely used open-source tool for selecting primers [19, 31]. From the primer list generated for each amplicon, the first candidate set is checked by an in silico PCR instance within the software for its specificity in the genome. Primer pairs resulting in more than one genomic region being amplified will be rejected. The column “ID” reports unspecific primers as “No primers found” (Fig. 2). The reason for this stringent setting is that amplicons with multiple hits in the in silico PCR are most likely to fail in the chemical PCR reaction. An example of a variant that will give multiple hits in the in silico PCR is chr9:66783838:C:T. The primers suggested, 5′AGACAGAAGCATTCTCAGGAACT3′ and 5′CCTGGTCTATCAAAAGAAAGGT3′, will result in 188 theoretical amplifications. Thus, the amplicon design tool rejected the primers and the amplicon.
From the results list, useful information regarding amplicon length, sequence, mutation position and primer annealing temperature can be obtained. Figure 3 represents part of the results list when designing amplicons around variants in the TP53 gene (all data not shown). Of the 725 rs numbers in the input list, a total of 68 were found invalid and had to be removed before amplicon design could be performed. The computing time for primer selection, check of primer specificity, and melting curve calculation took 102 min for these amplicons combined. On average, one DNA melting assay is computed in 9.3 s. Importantly, manual design of primer sets is an error-prone process due to the large number of steps involved, especially in batch design settings. The default “orientation” of the GC-clamp is based on the amplicon melting profile. The clamp is simulated on the side of the amplicon with the highest average melting temperature, thus facilitating a decrease in melting temperature towards the side of the amplicon without the GC-clamp (Figs. 4, 5). Information on clamp position is found in the column “Clamp pos” (Figs. 2, 3). The melting temperature of amplicons with small insertions/deletions are always plotted from 5′–3′, with the GC-clamp positioned at the 5′ end. Consequently, the difference in melting profile of the deletion/insertion will be seen in the low melting domain and not in the GC-clamp (Fig. 4). The side is selected in such a manner that the melting temperature profile increases towards the GC-clamp. This is to avoid “dips” in the low temperature melting domain [16, 32], which would give peak broadening and loss of resolution. However, the tool also allows the user to determine the specific placement of the GC-clamp. As seen in Fig. 1, the drop-down menu for ‘GC clamp location’ has several options for placement of the GC-clamp: “none”, “automatic” (i.e. selecting the side with highest average melting temperature), “5-prime end” or “3-prime end”. This functionality will allow the user to explore the effect of the GC-clamp in relation to the melting behavior of the fragments. The result column “Avg Melt Temp” calculates the average melting temperature of the target sequence between the primers. When corrected for urea concentration in the polymer (−3 °C/1 M urea) [33, 34], this would yield the analysis temperature used in a capillary electrophoresis instrument. An example of the melting profile is shown in Fig. 4 and part of Fig. 5. The vertical line in the melting graph indicates the variant position (Figs. 4, 5, 6). The Google chart feature has sliders that allows the user to focus the graph at the X-axis (Fig. 5). A core feature of the Galaxy framework is that the results of each analysis are stored in a history. It is optional for the user to register an account that allows for storage of the amplicon design history (Fig. 1). An arbitrary variant (rs2252586) was used as a test on the amplicon design, subsequently followed by ordering of the suggested primers and analysis of the amplified amplicon by cycling temperature capillary electrophoresis and high resolution melting (HRM). The PCR reaction was optimized in a temperature gradient thermo cycler. The gradient used was from 52.2–60.2 °C. The optimal annealing temperature observed for this amplicon was approximately 4 °C lower than the theoretical value given in the results list, which is in accordance to general PCR protocols . Figure 5 depicts the clear baseline separation of the alleles in the amplicon. The single strand (X) is recognized as a distribution, in contrast to the double stranded target with appropriate melting properties. As previously demonstrated, single stranded DNA will not significantly alter its position in the electropherogram with changes in the denaturing conditions . To test the workflow further, primer sets for 2500 SNP reference numbers were also successfully designed in one run.
Figure 6 illustrates the melting profile of the amplicon (rs2252586) without the GC-clamp. The difference in melting temperature of the alleles is exploited in the HRM assay. By slowly heating the PCR product and recording the fluorescence for each temperature increment, a temperature dependent decrease in the signal occurs due to strand dissociation [26–30]. After normalization of the data and subtraction of the values of a known reference, genotypes can be visualized as in Fig. 6, lower part. To further explore the HRM assay, a set of twelve rs numbers (Table 1) previously analyzed by CTCE (manuscript in preparation) were subjected to analysis with Variant Melting Profile (batch mode) with default settings and no GC-clamp. For each amplicon, eight samples were subjected to real time-PCR following HRM. Figure 7 displays the analysis of the melting curve data, normalized and plotted as differences to a known heterozygote sample. The data suggest that HRM could be used in 8 or 9 amplicons as a genotyping tool. It is noteworthy that one amplicon (rs1011970) failed due to a second SNP (rs188512825) in the fragment. However, with better temperature control, HRM could in theory resolve the allele combinations of two neighboring SNPs (micro-haplotypes), given that the variants changes the DNA melting properties sufficiently.
This is the first web application, to our knowledge, that combines the three features of primer selection around a DNA variant, controlling primer pair for specificity, and of computing the melting properties of the amplicon with or without a GC-clamp. Hence, this tool is novel with respect to covering the whole workflow necessary for groups that currently use melting equilibrium techniques to separate DNA variants.
Variant melting profile
The Variant Melting Profile is served as a tool within the Galaxy framework, which is an open source, web-based platform for data intensive biomedical research [23–25]. Galaxy is here used in combination with components from the Genomic Hyperbrowser [36, 37] as well as Google Charts for visualization (https://developers.google.com/chart/).
DNA variants (single nucleotide variants) can be given either as genomic coordinates (chromosome:position:reference_allele:alternative_allele), or as dbSNP reference IDs (e.g. rs9648696). User-specified parameters for selection of PCR primers (e.g. optimum primer sizes and primer melting temperatures etc.), as given by the Primer3 program  is used for finding suitable primers. The top candidate amplicon within the list of candidate primers is checked for specificity in the human genome using UCSC In-Silico PCR . If the amplicon maps uniquely, a GC clamp can be added to the PCR amplicon, and the DNA melting profiles for the nucleotide variants are visualized in an interactive fashion. The melting profiles are generated using the thermodynamic model provided by Blossey and Carlon .
DNA extraction, amplification and CTCE
Genomic DNA was extracted from anonymous blood donor samples by use of GenoVision M48 extraction robot (Biorobot M48 station, Qiagen, Norway), following standard procedures as given by the instrument manufacture.
A 42 base pair sequence of dGTP and dCTP, also known as a GC-clamp, labeled with 6-FAM, was incorporated at one end of the amplified target using a set of three primers in the PCR setup. An amplicon was designed for analysis of DNA variation identified by NCBI SNP references number, rs2252586.
Reverse ½ GC-lamp tailed
The PCR reaction mixture contained approximately 5 ng/µl genomic DNA, 0.4 mM dNTPs (0.1 mM of each dNTP) (VWR, Oslo, Norway), 1X Taq buffer, 0.075unit Taq/µl, 0.15 µM each of labeled GC clamp and the 1/2 GC-clamp tailed primer, while 0.3 µM of the “forward” primer (Integrated DNA Technologies, Leuven, Belgium) and 3 mM MgCl in a total reaction volume of 10 µl. Amplification was performed in a Eppendorf Mastercycler ep gradient S (Eppendorf, Hamburg, Germany) cycling 35 time between the temperatures of; cycles of denaturation for 10 s at 94 °C, annealing at 55.7 °C for 20 s and elongation at 72 °C for 30 s.
Six-fam labeled PCR products were analyzed with a 96-capillary DNA analyzer, i.e. the MegaBACE™ 1000 DNA Analysis System (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The instrument was modified to allow for elevated temperatures up to 65 °C. For detailed information about the modification, please contact the author P.O. Ekstrøm. Standard sequencing polyacrylamide (MegaBACE LPA) containing urea was replaced in the capillaries prior to each run. Samples were loaded into the capillaries from 96-well plates by electrokinetic injection at 161 V/cm for 15 s. The electrophoresis was carried out at a constant field of 145 V/cm. Laser-induced fluorescence was used with excitation at 488 nm (blue laser) and detection of emission at 520 nm (FAM channel). The scan rate was 1.75 Hz. The optimal separation temperature proposed by these programs was adjusted based on the urea concentration in the matrix. For each molar increment of urea, the temperature was lowered approximately 3 K (Kelvin) [40, 41].
The denaturing temperature in the capillary chamber, the cycling temperature, was programmed in the macro.ini file used by the Instrument Control Manager (ICM) software package (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Data were converted to text files by MegaBACE Sequence Analyzer View and Edit software.
Real time-PCR and high resolution melting (HRM)
A 25 µl Real time-PCR reaction was made up of 12.5 µl 2xMIX (PerfeCTa® SYBR® Green SuperMix, Quanta Biosciences, Gaithersburg, USA) 0.75 µl forward and 0.75 µl reveres primer with a concentration of 0.3 µM each. 2 µl DNA template and 9 µl H2O. The mixture was cycled 50 times with the following temperature, 95 °C for 15 s and 60 °C for 30 s. The fluorescence was read for each cycle, in a CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories AB, Oslo, Norway).
High resolution melting was performed after the temperature cycling by slowly increasing the temperature from 65–95 °C in increments of 0.1 °C. Fluorescent signal was recorded for 5 s after each 0.1 °C increment.
Primers used for the real-time PCR and HRM were selected by the Variant Melting Profile tool, checked for specificity by ePCR ordered from IDTDNA, with the following base composition: “Reverse” primer 5′CAACACGTTTCACCAGTGCA3′ and “forward” primer 5′TGCAAGATTGTACCTTCCTTGGT3′.
HRM curve data were normalized to the pre-melt (initial fluorescence) and post-melt (final fluorescence) signals. Thus, all samples were set to uniform, relative values from 100–0 %. The temperature axis of the normalized melting curves was shifted to the point where the entire double-stranded DNA was completely denatured. Differences in melting curve shape were further analyzed by subtracting the curves from a reference curve. We used a heterozygote sample as reference.
Following the initial test of HRM, 12 fragments were automatically designed on SNPs with references number given in Table 1. The fragments were subjected to Real time-PCR and HRM as described above, save for the annealing temperature which was set to 57 °C.
Availability and requirements
Project name: MeltPrimer
Project home page: http://meltprimer.ous-research.no/
Operating system(s): Linux
Programming language: HyperBrowser/Galaxy framework, SQLite, Blat/gfServer/gfPcr, Primer3
Any restrictions to use by non-academics: no licenses needed.
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POE specified the underlying software components and was responsible for testing of the Variant Melting Profile tool. POE amplified DNA by PCR and separated variants. SN and MJ implemented the computational workflow underlying Variant Melting Profiles. EH participated in the design of the study. All authors contributed equally in the writing of the manuscript and have read and approved the final version. All authors read and approved the final manuscript.
This study was supported by Research Council of Norway Grants 221580 and 218241, and Grant 71220 -PR-2006-0433 from The Norwegian Cancer Society.
The authors declare that they have no competing interests.
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Cite this article
Ekstrøm, P.O., Nakken, S., Johansen, M. et al. Automated amplicon design suitable for analysis of DNA variants by melting techniques. BMC Res Notes 8, 667 (2015). https://doi.org/10.1186/s13104-015-1624-8


REVIEW 3: NUTRITION, RESPIRATION & PHOTOSY/NTHESIS
NUTRITION AND METABOLISM
All living organisms need energy and nutrients. The energy is used for many purposes, such as
synthesis → building more molecules
growth → making new cells to grow larger
repair → making new cells to mend injuries
locomotion → moving around in the environment
The nutrients are also used for many purposes, such as
raw materials → the building blocks that new molecules and cells are built from
fuel → used to make energy in cell respiration
Animals have to eat food to both make energy and get nutrients. When animals take in food, it’s called ingestion. When they breakdown the food, it’s called digestion. When they take the food into their cells, it’s called absorption. Since they have to eat other organisms, animals are called heterotrophs. Hetero means “others”, troph means “feeding”, so heterotroph means
“feeding on others”.
Some organisms can harvest the energy from the sun and use it to synthesize the molecules and cells of their bodies. These organisms are plants. The process that allow plants to capture the sun and use it for synthesis is photosynthesis. Because, in this way, plants make their own food, they are also called autotrophs. Auto means “self”, troph means “feeding”, so autotroph means “self-feeding”. In photosynthesis, plants take in simple inorganic compounds (CO2 &
H2O) and build organic nutrients such as sugars (C6H12O6)
1. Nutrition: Organisms take in nutrients (food) for various activities including:
a. Ingestion: ______________________________________________________________
b. Digestion: ______________________________________________________________
Nutrients must be broken down into smaller parts so that they can be
________________________ into the blood and cells of organisms.
Starches are digested into ______________________________________________
Proteins are digested into _______________________________________________
1 of 6
2. Autotrophic Nutrition:
a. Organisms take inorganic materials ( ______ & ______ ) and convert them into organic nutrients (__________________).
b. Auto = ___________; troph = ___________ so Autotroph = _______________________
c. What process do autotrophs use to do this? ____________________________________
d. Examples of organisms that do this: __________________________________________
3. Heterotrophic Nutrition:
a. Organisms must __________________ nutrients made by other organisms.
b. Hetero = ___________; troph = ___________ so Heterotroph = ____________________
c. Examples of organisms that do this: __________________________________________
d. These organisms include:
4. Write the formula for photosynthesis: chemical formulas
5. Photosynthesis is the process in which sun’s energy is trapped in the chemical bonds of sugar. a. Requires _________________, _________________, and _________________.
b. Makes _________________ (______________) which is used as food in the plant.
c. Waste product produced is _________________.
Provides food for all plants and animals → the whole food chain.
Provides _________________ to breathe.


Name: Common Darter
Scientific name: Sympetrum striolatum
Nature Stars: 30
About: A small, narrow bodied dragonfly, up to 6cm long. The male is bright red, while females and immatures are golden brown. On the wing from the end of June right through to October or even November in a warm autumn. This is a very common dragonfly, breeding in all sorts of water bodies, from ponds and ditches to rivers and lakes.
How to identify: There are several similar red darters, which can be very difficult to tell apart.
Where: Found throughout the country
Fantastic fact: In the late summer, Common Darters can be found almost everywhere, sat on twigs and fences angling their wings towards the sun to warm up.
Photograph credit: Robbie G1


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- Hello. I had a dream of a rather fat, yellow jacket wasp, with intention to sting. It broke into my snakes cage and stung it several times. The wasp finally escaped leaving my snake in critical condition. Through out my dream, we’re doing everything we can to save it, in the end, she surivived.
- Aug 18, 2020 · The short answer is yes, but only mildly. The venom is similar to that of a black widow spider, but nowhere near as strong and not fatal under normal circumstances. A bite will typically cause symptoms such as redness, blisters and pain around the bite. These symptoms will normally go away after a day or so.
Giant Bee Can Kill up to 24 Humans, attacks small children Giant Bee Can Kill up to 24 Humans, attacks small children. As you may have guessed by the name, the Asian Giant Hornet is actually the largest hornet species in the world. I am curious to see if you can guess where you find these massive hornets. Wow! You are good.
- *Minor Edit* This answer was merged at some point to include “dangerous to humans”. To put it bluntly, you'd need to be actively trying to get killed by tarantulas before you suffered any permanent damage from their venom.
Wasp Venom - Wasp venom assaults the nervous system the moment wasp venom is injected. Learn how a small dose of wasp venom can deter even the After all, why does a wasp sting hurt so much? Wasp venom is far more than just stuff that hurts; it's a multi-step micro-assault with a twofold aim
- Dec 09, 2020 · Snake venom affects the human body in a number of ways, depending on the snake, the type of venom, and how much venom is released. Different snakes produce different types of venom, and even within a snake species, the components of venom appear to vary, depending on geographic location. This is why ...
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- For example, the ovaries of the parasitoid wasp, Asobara, undergo apoptosis if signals from their Wolbachia symbionts are lacking (Pannebakker et al. 2007). And the newborn of the squid Euprymna scolopes lacks a light organ, which is developed in cooperation between the squid and the luminescent bacteria ( Vibrio fisheri) absorbed by its ...
Share — copy and redistribute the material in any medium or format Adapt — remix, transform, and build upon the material for any purpose, even commercially. The licensor cannot revoke these freedoms as long as you follow the license terms. ShareAlike — If you remix, transform, or build upon ...
- Jun 19, 2014 · The Horse’s experts answer your questions during a monthly ... and African bees that were inadvertently released in Brazil in the 1950s. ... to large amounts of bee venom are susceptible to ...
Venom belonging to the Brazilian social wasp Polybia paulista contains the antimicrobial peptide Polybia-MP1 (MP1), which has been demonstrated to inhibit multiple forms of cancerous cells such as prostate cancer, bladder cancer and multidrug-resistant leukemic cells.
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Jul 12, 2014 · The Type 26 frigate will be a powerful addition to the surface fleet. The remaining fleet of six destroyers, thirteen frigates, four patrol ships and seven attack submarines is sufficient to provide escort for two carrier groups plus one amphibious group and patrol the routes to the Faslane Trident SSBN base but they can only provide a limited anti-submarine capability.


Mango leaves tea is a specific component of mango leaves that has medicinal properties. It has a wide spectrum of medicinal and restorative characteristics that can help with everything from minor to severe health problems. Even if you have diabetes or varicose veins, it will be beneficial to you.
The amazing health benefits of drinking mango leaves tea on an empty stomach first thing in the morning:-
Blood sugar management
Mango leaves, according to WebMD, are highly effective in the treatment of diabetes. They have the capacity to boost insulin production, glucose distribution, and blood sugar control. Soak the leaves in warm water overnight. Then, to aid with diabetic symptoms, filter the water and consume it.
Controlling high blood pressure
Mango leaves have hypotensive properties and are abundant in fiber and pectin, which can help decrease bad cholesterol, manage blood pressure, and strengthen blood vessels.
Relief from a slight fever for a short period of time
Drinking a decoction made from mango leaves can help decrease a fever-induced elevated body temperature. Fever may be effectively treated by boiling fresh mango leaves with other plant leaves such as paw-paw, neem, cashew, bitter leaf and others.
Mango leaves; according to healthline contain antioxidants that can help fight inflammation and prevent damage produced by free radicals in the environment; which can harm cells and lead to diseases such as cancer and other ailments.
Respiratory problems are resolved
When you have a cold, bronchitis, or asthma, mango leaves are very useful. Drinking a decoction made by cooking mango leaves in water with a little honey can effectively relieve cough. It also helps in the treatment of throat tension-related voice loss.
Get rid of stomach aches and pains
Boiling mango leaves in the morning creates a stomach tonic that relieves stomach discomfort, promotes bowel movement, flushes toxins from the body and keeps the stomach clean.
Keeping your weight in check
Mango leaves help the body store fat by increasing the manufacture of the hormone leptin; which is vital for fat management


Ayur Vera Juice: Ayur Vera Juice is a very good detoxifier.It Cleans all the toxins from the body and very effective in constipation. It helps to cure stomach ulcers, it gives rashes free and smooth skin. It gives strength to the muscles and enhances the functions of all the endocrine organs. Ayur Vera help to prevent insomnia.
AyuRakshak Juice: AyuRakshak Juice is the mixture of Giloy, Papaya and Wheatgrass leaves juices. It is a very good immunity booster and gives the power to fight all types of disease especially all the infectious diseases. It is helpful to produce all the particles of blood like RBC, WBC and Platelets. It is very helpful to control viral fever, influenza, Dengue Fever, Malaria, Chickengunia etc.
Livon DS: Livon-DS is very effective liver syrup. This is the complete rejuvenator of human liver. It helps to enhance the functionality of liver. It prevents liver cirrhosis. It controls liver enlargement due to alcohol consumption. It is effective to use this product in Hepatitis.
Arshcure: Arshcure capsules herbal remedy of piles and fistula. It helps to controls bleeding and pain. Please don’t eat alcohol, ginger, meat etc. While treatment, and use it with buttermilk.
Triphala Kadha: Triphala Kadha is a combination of three essential herbs Hard, Baheda and Amla. It is an ancient ayurvedic supplement and just like an elixir for our gut. It reduces constipation, controls indigestion, removes gas and acidity. It is also helpful to detoxify our digestion system. It improves eyesight and helps to repair hair problems
Kabzgo Churna: Kabzgo Churna is an effective combination of herbs to remove constipation by its root. It is an advanced remedy to strengthen the digestive system. It also cures making extra gases.


Diet and lifestyle changes may control symptoms of crohn’s disease and lengthen the time between flare-ups.
An inflammatory bowel disease, crohn’s disease is marked by symptoms of abdominal pain, severe diarrhea, fatigue, weight loss and malnutrition. Crohn’s disease may affect different areas of digestive tract, leading to debilitating and life-threatening complications. Crohn’s disease may be effectively treated by following certain diet and lifestyle changes.
- Limit dairy products: By limiting and eliminating dairy products the fall-out of inflammatory bowel disease evident as diarrhea, abdominal pain and gas can be controlled effectively.
- Having low-fat foods: If you are diagnosed for crohn’s disease, it is better to avoid low-fat foods such as butter, margarine, cream sauces and fried foods. People with crohn’s disease of small intestine may not be able to digest fat normally.
- Limit fiber: In some people with crohn’s disease, high fiber foods may worsen the symptoms. Instead of taking raw fruits and vegetables, it is better to take them in steamed or baked form.
- Avoid problems foods: It is always better to avoid spicy foods, alcohol and caffeine as they may worsen the symptoms of crohn’s disease.
- Eat small meals: Those with the condition of crohn’s disease should break from the routine of tiffin, lunch and dinner. You may eat five to six small meals in a day that may help to reduce the occurrence of crohn’s disease.
- Drink plenty of liquids: Water is the best drink for those affected by crohn’s disease. Alcohol, caffeine and carbonated drinks should be avoided as they produce gas and may aggravate diarrhea.
- Take multivitamins and mineral supplements: Crohn’s disease inhibits the body’s ability to absorb nutrients. Taking adequate amount of multivitamin and mineral supplements ensures proper supply of nutrients to the body.
- Overcome stress: Stress may not cause crohn’s disease, but it may aggravate its symptoms. You can manage stress effectively by practicing relaxation and breathing exercises.


Typically, people breathe through their nose when they sleep. If you snore, it means you’re breathing through your mouth. It not only can affect your sleep, but it can cause partners to sleep in separate rooms. Recent evidence suggest snoring may cause thickening of the carotid arteries over time and potentially increase the risk of stroke. Snoring may be a sign of obstructive sleep apnea. Lifestyle changes, such as losing weight, avoiding alcohol close to bedtime or sleeping on your side, can help stop storing. If these changes do not help, your doctor can help identify if you have obstructive sleep apnea.


A banana is the common name for a type of fruit and also the name for the herbaceous plants that grow it. These plants belong to the genus Musa.They are native to the tropical region of southeast Asia. It is thought that bananas were grown for food for the first time in Papua New Guinea. Today, they are cultivated in tropical regions around the world. Most banana plants are grown for their.
Here you can read about 10 uses of banana plants. Uses of Banana Trees. You can eat edible sweet bananas. You can eat edible banana fruit peels. You can even eat the stem. You can use stem fibres as natural craft materials. You can use banana plant fibres to make garments. Banana leaves can be used as natural leaf platters.
The banana is an edible fruit, botanically a berry, produced by several kinds of large herbaceous flowering plants in the genus Musa.In some countries, bananas used for cooking may be called plantains. The fruit is variable in size, color and firmness, but is usually elongated and curved, with soft flesh rich in starch covered with a rind which may be green, yellow, red, purple, or brown when.
Essay on banana tree in telugu language for help me write best critical thinking. 17 think critically about illustrations because illustrations always select some examples of the earth below: Large buildings smaller than xxxl. Now all she wants to do. But divesting myself of the collective imagination.
Short essay on banana tree Ask for details; Follow Report by Sidpower3774 18.12.2017 Log in to add a comment Answers asam Ambitious; Banana is an indigenous plant of South-East Asia. It was grown in the country since ancient times. There is reference of its cultivation in the Pali literature (500-600 BC).
Here is a list of banana tree uses. According to Ayurveda, all the parts of the Banana tree have healing properties. The banana tree uses are listed as follows: juice of banana flowers helps during painful menstruation, juice of banana stem helps in flushing out kidney stones, banana leaves alleviate stomach disorders, banana corm reduces acidity and burning sensation.
Find an answer to your question 5 lines essay on banana Banana is a fruit which is yellow .This plant belong to the Guinness Moosa they are native to the tropical region of south east asia there are about 110 different spices of Banana it is thought that banana we are grown for food for the first time in Papua New Guinea today they are cultivated in tropical regions around the world most.
James began to publish short stories and writing plays. In 1976 he published Bluefoot Traveller, a book of black British poetry. Since his early days in this country, James has remained an active campaigner on the part of black people and has helped to promote young black writers in particular.
Adopting a brand extension strategy to bring the two under the umbrella “Banyan Tree” brand could have had a positive halo effect on the perceived quality of Angsana Spa and Colours of Angsana on one hand but on the other, it could have led to “Banyan Tree” brand dilution and dented the customer perceived value because the customers no longer would be able to identify with BTHL as a.
Banana trees are one of the common trees that come to mind when dreaming of the tropics, but did you know that it is not really a tree? It is actually the world's largest herb. On banana plantations in the tropic the plants must be pruned to the ground after fruiting. The trunk is composed of the main fruiting stem enrobed by leaves.
Essay on trees are able to be used in sanskrit tree in sanskrit, sanskrit. Essay banana tree in sanskrit get help with your writing 1 through 30. 10 lines on banana tree in sanskrit language in the banana tree. Jpg. Free essays on the air and doing your writing 1 through 30. Title: the back of a banana tree, sanskrit, the tree in sanskrit search.
Essay text: That same large banana even has 2 grams of protein and 4 grams of fiber. No wonder the banana was considered an important food to boost the health of malnourished children, those reducing sodium in their diets cannot go wrong with a banana with its mere 2 mgs of sodium.
Banana tree essay.. .. Write a narrative essay on a lost key. Menu. Banana tree essay.
This is what was so special to him. Moral Genre of the story. The Banana Tree The genre of this story is, an exciting story in as much as it keeps you on your toes, because you don't know what will happen to the boy. It is also quite a sad story because you do feel for the.
Banana plant is not a tree,it is the largest herb in the world. It can grow upto 20 to 25 feet high. The functional trunk formed by upright concentric sheathing base of long broad leafs. It is a perennial herb. Bananas propagate through rootstalk rhizome. Banana flower is large, long, oval, tapering and purple color.It looks like a large bud.Translation for 'banana' in the free English-Hindi dictionary and many other Hindi translations.Bananas grow on plants called banana palms.While many people think that banana palms are trees, they are actually considered to be an herb. In fact, the banana palm is the largest herb in the world.


The rising global demand for animal products and the growing public concerns about the environment and animal welfare require dairy farms to improve their efficiency and apply more sustainable farming systems. Precision Livestock Farming (PLF) could represent a valuable support in addressing these challenges. In recent years, dairy farms have been modernising and introducing new sensors and automatic systems for managing the herd. However, the diffusion of new technologies in Italian dairy farms is still limited and farmers are reluctant to invest in precision systems. The aim of the study was to investigate the presence of PLF tools in Italian dairy farms, the motivations, benefits and limits of technological investments from the farmers' point of view and the factors affecting the diffusion of technology. From November 2020 to June 2021, an online questionnaire was distributed and 52 responses were obtained. About 79% of the farms were located in Lombardy. The...
Diffusion of precision livestock farming technologies in dairy cattle farms.
A. Sandrucci, Department of Agricultural and Environmental Sciences, University of Milan, 20122 Milan, Italy. E-mail firstname.lastname@example.org
Bianchi, M. C., Bava, L., Sandrucci, A., Tangorra, F. M., Tamburini, A., Gislon, G., Zucali, M.; Diffusion of precision livestock farming technologies in dairy cattle farms.. IFIS Food and Health Sciences Database 2023; doi:
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by Republic of Kenya, National Council for Science and Technology in [Nairobi] .
Written in English
|Series||NCST ;, no. 40, NCST (Series) ;, no. 40.|
|Contributions||National Council for Science and Technology (Kenya)|
|LC Classifications||SB295.Q38 M35 1996|
|The Physical Object|
|Pagination||ii, 52 p. :|
|Number of Pages||52|
|LC Control Number||99890362|
Catha edulis (khat) is a plant grown commonly in the horn of Africa. The leaves of khat are chewed by the people for its stimulant action. Its young buds and tender leaves are chewed to attain a state of euphoria and stimulation. Khat is an evergreen shrub, which is cultivated as a bush or small tree. The leaves have an aromatic odor. Alsalahi A, Alshawsh MA, Mohamed R, et al. Conflicting reports on the role of the glycemic effect of Catha edulis (Khat): A systematic review and meta-analysis. J Ethnopharmacol. ; Khat is a stimulant made up of fresh leaves of the Catha edulis shrub found in East Africa and southern Arabia. It contains a variety of chemicals. Abstract. The khat plant (Catha edulis) is produced in certain areas of East Africa and the Arabian peninsula, and the leaves are chewed for their stimulating effect. Fresh material, which is preferred by users, contains a substance that is rapidly converted to (+)-norpseudoephedrine. As a consequence of the traditional means of consumption, intoxication with khat is self-limiting but chronic.
Catha edulis Forsk., its preparations, derivatives, alkaloids and salts, including: (1) Cathine (d–threo–2–amino–1–hydroxy–1–phenylpropane). Flanman31 , 16 September (UTC) While there are numerous spellings, and all of them are acceptable, there is always a preferred spelling. Catha Edulis is a psychoactive plant found in East Africa and Western Arabia. Depending on who you ask, Khat may be considered a deadly drug or a cultural tradition. This book goes into detail on the psychoactive effects whether Instantaneous, Long Term Usage, or : $ Jeffrey K. Aronson, in Manson's Tropical Infectious Diseases (Twenty-third Edition), Khat. Khat or qat (cafta, miraa, muiragi) is a stimulant commonly used in East Africa, Yemen, and southern Saudi Arabia and is derived from a small tree, Catha edulis. The leaves and twigs are chewed while fresh, but can also be smoked, infused in tea, or sprinkled on food. Catha edulis (khat, qat frae Arabic: القات ) is a flouerin plant native tae the Horn o Africae an the Arabian commonties frae these areas, khat chowin has a history as a social custom datin back thoosands o years. References.
Catha edulis will grow well in full sun or part shade. It’s ideal is 80 degrees (f) at about the ′ elevation in the tropics. This translates well to part shade, 80 degrees (f) at lower elevations. Generally, the more red on the stems and veins of the leaves, the stronger the Khat is considered to be. Khat (catha edulis) is a psychoactive shrub whose tender leaves and twigs are chewed in the Horn of Africa and the Arabian Peninsula for their euphonizing effects for a millennium. A stimulant grown in small gardens for consumption on cultural and religious occasions around the turn of the last century, khat has now become the preferred and Author: Ezekiel Gebissa. Simon Gibbons, Warunya Arunotayanun, in Novel Psychoactive Substances, Catha edulis (Khat). Khat (Catha edulis Forsk. ex Endl.) is a small tree from the Celastraceae plant family that grows in East Africa and the Southern Arabian crude drug material has a number of names in various locations such as ‘qat’ (Yemen), ‘tchat’ (Ethiopia), ‘qaad or jaad’ (Somalia) or. Catha forskalii . Catha inermis . Celastrus edulis Vahl. Dillonia abyssinica Sacleux.. Methyscophyllum glaucum Eckl. & Zeyh. Trigonotheca serrata Hochst.


Your liver is an amazing organ that can regenerate very quickly given the proper rest, lifestyle habits, and nutrition. If it becomes tired, sick, or injured you will notice specific symptom patterns and body changes that get worse over time. Use this article to educate yourself and your family on liver function.
Learn about the symptom pattern and body type of the ovaries. The life-giving ovaries produce hormones that are important to the emotional and physical wellbeing of women of any age. Use the information to understand if your symptoms are directly tied to your ovaries and what to do about it.
The thyroid, located in the middle of the neck sends signals (hormones) to the liver that in turn regulates our energy production. Learn how the thyroid impacts metabolism and how this is experienced through symptom patterns and body composition changes.
The pituitary gland is only the size of a pea, yet it regulates our entire hormonal system. This gland is very sensitive as can be malnourished or overstimulated causing loads of problems into adulthood including extreme under and overdevelopment, infertility problems, and tumor susceptibility.
The gonads are a combination of glands that include the testes, prostate, and cowper's. These glands work together to produce hormones and function to the male reproductive system. If not properly nourished and rested, these glands cannot produce enough hormones and thus unfavorable physical results.
The adrenal glands do so much considering they are about the size of a walnut. They help balance blood sugar, they help make energy, and they even help make a fertility hormone. Learn about the symptoms and body composition patterns of these remarkable glands.
You can benefit from learning how to balance your hormones naturally. Use the information and quiz in this article to reduce food cravings, lose weight-gain in specific areas, and reduce common symptoms.


You wouldn’t apply a tourniquet to a bruise. Or put an ice pack on an ulcer. Those would be the wrong treatment protocols for those particular conditions.
Yet, when it comes to care for chronically infected gums and loss of bone—the markers of periodontitis—dental professionals are trained to provide acute wound treatment protocols.
It may not seem like a huge difference. But let’s consider the fundamental differences between treating acute wounds and treating chronic wounds. And let’s take the tourniquet off the bruise.
Traditionally, gum disease (periodontitis) has been addressed the same way you might approach a broken bone. Namely, it has been treated as a trauma with acute care, including scaling, root planing and even surgery.
And it’s hard to argue with this “head-on” approach to addressing this troubling issue.
Gum disease is a disruptive and destructive problem, and no one wants to withstand it any longer than absolutely necessary. Oddly enough, an acute response alone can actually prolong the disease if it is not matched with chronic wound care.
Studies indicate that scaling and root planing, the most common treatments of periodontitis that physically remove bacterial colonies called biofilms, leave behind the bacteria that caused the infection in the first place. And worse yet, the pathogens repopulate because it is impossible for the instruments to remove all of the biofilm.
So, what’s the answer?
If you ask the American Medical Association (AMA), chronic wound management is it. And gum disease, specifically periodontitis, fits the chronic wound model.
Now the questions become, “how is chronic care different, and how do you deliver it to the gums?”
Chronic care protocols address the infectious agents first. Then, they debride the wound and encourage it to heal from within. In order to do that for the gums, we prescribe a customized sealed Perio Tray to deliver a low concentration of hydrogen peroxide gel deep below to gums to establish an oxygen rich environment that simultaneously debrides pathogens, and encourages wound healing.
The Perio Tray® does more than lock in the treatment. It compresses the wound and increases the oxygen levels below the gums. With the sealed tray, the hydrogen peroxide release of oxygen takes place at the site of the infection. Peroxide performs as an oral debriding agent and oral wound cleanser. The Perio Tray is important because it delivers the medication deep below the gums where these chronic oral wounds hide from toothbrushes, rinse and floss.
If this all seems revolutionary, it’s not.
The Perio Protect Method, combining acute and chronic care, simply follows the AMA guidelines for treating chronic wounds, and the results speak for themselves.
Research and case studies have shown treatment of chronic periodontal disease with a direct medication delivery method significantly decreases bleeding upon probing and shrinks the depth of periodontal pockets1.
By creating the right environment for treatment, this approach compresses the wound and addresses the bacterial cause, allowing the wound to heal from the tissue level. The treatment suppresses damaging bacteria and encourages healthy bacteria for healthy gums.
Where acute care addresses the immediate needs, chronic care—like daily Perio Tray delivery—addresses the cause.
1 Cochrane RB and Sindelar B. Case Series Report of 66 Refractory Maintenance Patients Evaluating the Effectiveness of Topical Oxidizing Agents. The Journal of Clinical Dentistry 2015;26:109-114. Putt M, Mallatt M, Messmann L, and Proskin H. A 6-month clinical investigation of custom tray application of peroxide gel with or without doxycycline as adjuncts to scaling and root planning for treatment of periodontitis. American Journal of Dentistry. 2014;27:273-284. Putt M and Proskin H. Custom Tray Application of Peroxide Gel as an Adjunct to Scaling and Root Planing in the Treatment of Periodontitis: Results of a Randomized, Controlled Trial after 6 Months. The Journal of Clinical Dentistry. 2013;24:100-107.


My research focuses on the behaviour and wellbeing of companion animals, and their interactions with humans. I am particularly interested in play behaviour as shown below, and in developing ways ...
Nicola graduated from Leicester University (Hons, 1st class), before completing a PGCE. After studying mink anatomy and diet, (WildCRU, Oxford University), she moved to Southampton University to complete a ...
Research: Animal Welfare and Behaviour
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Statewide Wild Turkey Studies Underway – Laker Country 104.9 FM WJRS
Two ongoing research projects should shed light on why Kentucky’s turkey crop has fallen since its 2010 record.
Hunters reported harvesting 26,836 turkeys during the state’s 2022 spring seasons. While this level is comparable to the peak of the turkey population boom in the early 2000s, it is well below Kentucky’s record spring harvest of more than 36,000 turkeys 12 years ago, and the average of the last 10 years of 30,822 harvested.
Kentucky is not alone in suffering a decline in its turkey crop.
“This is a range-wide phenomenon – it’s happening in other states as well,” said Zak Danks, turkey program coordinator for the Department of Fish and Wildlife Resources of the United States. Kentucky. “Researchers are talking and collaborating with each other, trying to figure out what’s going on in the wild turkey’s range.”
Kentucky is currently working with Tennessee Tech University and the University of Georgia on a turkey breeding study. Researchers are studying the nesting success and survival rates of young birds. Biologists will study how predators, weather, habitat and gobage affect the number of turkeys that reach adulthood.
Another ongoing study examines the impact of hunting on the herd. For this research, conducted in partnership with the Tennessee Wildlife Resources Agency, biologists began banding male turkeys earlier this year. Researchers will continue to band birds until 2025 to better understand the number of birds captured by hunters compared to the number of birds that die from other causes, such as predators.
Ben Robinson, acting director of the department’s Wildlife Division, warned that researchers need to complete the entire project before beginning to interpret the results.
“Drawing conclusions now would be like calling the Kentucky Derby halfway through the race,” he said.
Several factors impacted the turkey season this spring, including spells of cold and wet weather. The holidays may also have affected the number of hunters: Easter fell during the opening weekend while the spring season ended on Mother’s Day. Other factors during the spring season, such as the Kentucky Derby, youth sports or other group activities, once again competed for people’s time.
License sales figures this year indicate that the uptick in hunting and fishing participation during the pandemic has declined as COVID restrictions have eased.
Sales of licenses and permits authorizing the hunting of turkeys fell 6.6% in 2022 compared to the previous year. Still, an estimated 75,000 to 85,000 people hunted in Kentucky during the spring 2022 turkey season.
In preliminary results of a currently ongoing survey of 2,022 turkey hunters, a significant percentage reported that they stopped hunting after harvesting a turkey due to concerns about declining turkey numbers. Final results will be available upon completion of the survey later this month. This factor and other factors related to participation may explain the drop in the 2022 harvest.
Less gobbling observed by turkey hunters this spring – as indicated in the current post-season survey – is likely correlated with below average poult recruitment in 2020, reflected in the results of the annual brood survey statewide summer. However, last year’s brood survey indicates above average recruitment of young turkeys.
“We’ve heard from many hunters who have seen more jakes this spring, raising expectations for a good crop of 2-year-old gobblers to hunt next year,” Danks said.
As researchers and wildlife managers seek to better understand what might be happening with turkeys in their range, the Kentucky Fish and Wildlife Commission offered several recommended changes at its meeting last December to combat proactive against perceived decline.
The committee recommended:
A bag limit of one turkey per hunter per Wildlife Management Zone per season.
Eliminate the harvest of beardless turkeys during fall hunting seasons.
Extend wildlife baiting ban from May 31 to July 31. Bait piles promote artificially high concentrations of predators and turkeys at bait sites, likely increasing predation and health risks to turkeys.
Extend the night coyote hunting season with rifles until June 30, except that the season would be closed from April 1 to May 15. Hunters on private land could use any legal modern firearm, centerfire and rimfire.
The recommended changes to the regulations are not yet in effect, as they must receive legislative approval before becoming law. Before leaving, hunters should always consult the hunting guide in force, published by the department to summarize the regulations in force. Hunting guide booklets are available at fw.ky.gov and from licensing agents.
Danks said crop fluctuations could be part of the normal cycles of a turkey population that has already peaked. In 1978, Kentucky’s turkey population was estimated at only 2,000 turkeys. It took almost two decades before the flock exceeded 100,000 birds. Flock estimates have stabilized at around 200,000 to 250,000 since 2002, indicating that Kentucky could have as many turkeys as the landscape will support.
“When populations increase, they generally have a higher reproductive rate,” Danks said. “When a population reaches the carrying capacity of the environment, it exceeds that capacity for a short time and then declines, followed by fluctuations.”
The 2010 season is an example of overflow. A large outbreak of 17-year-old cicadas in 2008 provided poults with a substantial food source that also distracted predators from their focus on turkeys and other prey. Survival rates skyrocketed, leading to the record harvest two years later.
This is why studies will focus on the survival of turkeys. Have all the best nesting sites and habitats been captured due to the number of birds in the flock?
“There is only a limited amount of habitat available for turkeys,” Robinson said. “Once we hit the carrying capacity of the turkey flock, we start to see fluctuations.”
Hunters and other wildlife enthusiasts can help monitor the wild turkey population by submitting sightings of turkeys in July and August each year. Visit fw.ky.gov and search the keywords “turkey survey” and select the preferred method for completing the summer brood survey.


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