Diagnosis of Malarial infection on Mongolian Troops who working UN mission in Africa / Эрүүл мэндийн лаборатори

Abstract@#Malaria is an ancient disease caused by parasites of the genus <i>Plasmodium</i> and transmitted by several species of female anopheline mosquitoes. The term „malaria‟ originates from <i>mal’aria</i> (Italian) –signifying „bad air‟ or miasmas arising from marshes. </br> Cognizant of the burden of the disease in antiquity, several efforts have been made to understand the disease notably, the detection of the <i>Plasmodium</i> parasite in the blood of infected humans in 1880, as well as proof of the complete life cycle of malaria parasites in mosquitoes in 1897. </br> Among 200 <i>Plasmodium</i> species identified <i>P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi </i>are known to be responsible for human malaria, while mortality due to malaria is mostly attributed to infections with <i>P. falciparum.</i></br> The <i>Anopheles</i> mosquito bites a human and injects sporozoite forms. These move to the liver and invade hepatocytes, in which they develop to produce exoerythrocytic merozoite forms that are released into the blood stream. Merozoites invade erythrocytes and grow into trophozoites and mature schizonts. Merozoites are released that reinvade new erythrocytes.</br> Gametocytes, formed from the asexual blood stage, are taken up by a feeding mosquito into the gut where they mature to form male and female gametes. The fertilized zygote develops to an ookinete and an oocyst and finally sporozoites that migrate to the salivary glands.</br> Malaria transmission exits in 99 countries throughout world, and the greater burden of the disease is carried by African countries. According to the World Health Organization (WHO), the estimated cases of and deaths due to malaria in 2016 were 219 million and 660,000, respectively with malaria deaths steadily decreasing since 1980. Despite the decline in the burden of malaria with the scaling-up of interventions the fact that the estimated (uncertainty exists) number of malaria deaths in 2016 exceeded that of 1980 calls for more efforts in the prevention and control of the disease.</br> Mongolian troops have been participating at UN mission since 2003. They work very complicated condition. One of the simple risks is Malaria. We had approximately 80 cases who had been infected by malaria at the mission area. </br> Mongolia is land without malaria infection. But our tourists can visit all of the world and troops works on mission area in Africa. They have a risk of malaria and our doctors have to be diagnosis and treat to malaria cases. This article provides an overview of malaria laboratory diagnosis and epidemiological data that will lead to the development of strategies to diagnose and reduce infection.

Potential Vaccine Targets against Rabbit Coccidiosis by Immunoproteomic Analysis

The aim of this study was to identify antigens for a vaccine or drug target to control rabbit coccidiosis. A combination of 2-dimensional electrophoresis, immunoblotting, and mass spectrometric analysis were used to identify novel antigens from the sporozoites of Eimeria stiedae. Protein spots were recognized by the sera of New Zealand rabbits infected artificially with E. stiedae. The proteins were characterized by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) analysis in combination with bioinformatics. Approximately 868 protein spots were detected by silver-staining, and a total of 41 immunoreactive protein spots were recognized by anti-E. stiedae sera. Finally, 23 protein spots were successfully identified. The proteins such as heat shock protein 70 and aspartyl protease may have potential as immunodiagnostic or vaccine antigens. The immunoreactive proteins were found to possess a wide range of biological functions. This study is the first to report the proteins recognized by sera of infected rabbits with E. stiedae, which might be helpful in identifying potential targets for vaccine development to control rabbit coccidiosis.

Visualization of Malaria Parasites in the Skin Using the Luciferase Transgenic Parasite,<i> Plasmodium berghei</i>

We produced a transgenic rodent malaria parasite (<i>Plasmodium berghei</i>) that contained the luciferase gene under a promoter region of elongation factor-1α. These transgenic (TG) parasites expressed luciferase in all stages of their life cycle, as previously reported. However, we were the first to succeed in observing sporozoites as a mass in mouse skin following their deposition by the probing of infective mosquitoes. Our transgenic parasites may have emitted stronger bioluminescence than previous TG parasites. The estimated number of injected sporozoites by mosquitoes was between 34 and 775 (median 80). Since luciferase activity diminished immediately after the death of the parasites, luciferase activity could be an indicator of the existence of live parasites. Our results indicated that sporozoites survived at the probed site for more than 42 hours. We also detected sporozoites in the liver within 15 min of the intravenous injection. Bioluminescence was not observed in the lung, kidney or spleen. We confirmed the observation that the liver was the first organ in which malaria parasites entered and increased in number.

Visualization of malaria parasites in the skin using the luciferase transgenic parasite, <i>Plasmodium berghei</i>

Weproduced a transgenic rodent malaria parasite (<i>Plasmodium berghei</i>) that contained the luciferase gene under apromoter region of elongation factor-1α. These transgenic (TG) parasites expressed luciferase inall stages of their life cycle, as previously reported. However, we were the firstto succeed in observing sporozoites as a mass in mouse skin following theirdeposition by the probing of infective mosquitoes. Our transgenic parasites mayhave emitted stronger bioluminescence than previous TG parasites. The estimatednumbers of injected sporozoites by mosquitoes were between 34 and 775 (median 80). Since luciferase activity diminished immediately after the death of theparasites, luciferase activity could be an indicator of the existence of liveparasites. Our results indicated that sporozoites survived at the probed sitefor more than 42 hours. We also detected sporozoites in the liver within 15 minof the intravenous injection. Apart from the liver, bioluminescence was notobserved in the lung, kidney, or spleen. We reconfirmed that the liver was thefirst organ for malaria parasites to enter and increase in number.

Vectorial capacity, basic reproduction number, force of infection and all that: formal notation to complete and adjust their classical concepts and equations

A dimensional analysis of the classical equations related to the dynamics of vector-borne infections is presented. It is provided a formal notation to complete the expressions for the Ross' Threshold Theorem, the Macdonald's basic reproduction "rate" and sporozoite "rate", Garret-Jones' vectorial capacity and Dietz-Molineaux-Thomas' force of infection. The analysis was intended to provide a formal notation that complete the classical equations proposed by these authors.

Mosquito Species Composition and Plasmodium vivax Infection Rates on Baengnyeong-do (Island), Republic of Korea

Vivax malaria is a significant military and civilian health threat in the north of the Republic of Korea (ROK). The island of Baengnyeong-do is the westernmost point of the ROK and is located close to the southwestern coast of the Democratic People's Republic of Korea (DPRK). Mosquitoes were collected using a black light trap on Baengnyeong-do, and Anopheles spp. were assayed by PCR, to identify the species, and screened for sporozoites of Plasmodium vivax. Of a subsample of 257 mosquitoes, Anopheles lesteri was the most frequently collected (49.8%), followed by Anopheles sinensis (22.6%), Anopheles pullus (18.7%), Anopheles kleini (7.8%), and Anopheles belenrae (1.2%). The overall sporozoite rate was 3.1%, with the highest rates observed in An. kleini (15.0%), An. sinensis (5.2%), and An. lesteri (1.6%). No sporozoite positive An. pullus or An. belenrae were observed. The results extend our knowledge of the distribution and potential role in malaria transmission of An. kleini, An. lesteri, and An. sinensis, for an area previously considered to be at a low risk for contracting vivax malaria.

Distribution of sibling species of Anopheles culicifacies s.l. and Anopheles fluviatilis s.l. and their vectorial capacity in eight different malaria endemic districts of Orissa, India

The study was undertaken in eight endemic districts of Orissa, India, to find the members of the species complexes of Anopheles culicifacies and Anopheles fluviatilis and their distribution patterns. The study area included six forested districts (Keonjhar, Angul, Dhenkanal, Ganjam, Nayagarh and Khurda) and two non-forested coastal districts (Puri and Jagatsingpur) studied over a period of two years (June 2007-May 2009). An. culicifacies A, B, C and D and An. fluviatilis S and T sibling species were reported. The prevalence of An. culicifacies A ranged from 4.2-8.41 percent, B from 54.96-76.92 percent, C from 23.08-33.62 percent and D from 1.85-5.94 percent (D was reported for the first time in Orissa, except for occurrences in the Khurda and Nayagarh districts). The anthropophilic indices (AI) were 3.2-4.8 percent, 0.5-1.7 percent, 0.7-1.37 percent and 0.91-1.35 percent for A, B, C and D, respectively, whereas the sporozoite rates (SR) were 0.49-0.54 percent, 0 percent, 0.28-0.37 percent and 0.41-0.46 percent for A, B, C and D, respectively. An. fluviatilis showed a similarly varied distribution pattern in which S was predominant (84.3 percent overall); its AI and SR values ranged from 60.7-90.4 percent and 1.2-2.32 percent, respectively. The study observed that the co-existence of potential vector sibling species of An. culicifacies (A, C and D) and An. fluviatilis S (> 50 percent) was responsible for the high endemicity of malaria in forested districts such as Dhenkanal, Keonjhar, Angul, Ganjam, Nayagarh and Khurda (> 5 percent slide positivity rate). Thus, the epidemiological scenario for malaria is dependent on the distribution of the vector sibling species and their vectorial capacity.

Post-challenge hematological evaluation with virulent strain of Eimeria tenella in broilers immunized with attenuated strain or sporozoite proteins from homologous strain / Avaliação hematológica pós-desafio com cepa virulenta de Eimeria tenella em frangos de corte imunizados com cepa atenuada ou proteínas de esporozoítos de cepa homóloga

Hematological parameters were evaluated in broilers immunized and challenged with Eimeria tenella. Broiler chickens of Hubbard strain, females, coccidian-free, were kept in wire cages and inoculated on the third day. The experiment was designed to include five sorts of treatment with three replicates each. T1 was the negative control group, T2 received 500 attenuated sporulated oocysts by gavage, T3 was the positive control, T4 received 50 µg of sporozoite protein + Quil A vaccine, and T5 received Quil A without sporozoite protein + PBS, the last two through nasal route on days 0, 7, and 21. On the 31st day, all treatments were challenged with homologous virulent strain of E. tenella in the dose of 8.0 × 10(4) oocysts, with the exception of T1. One week later, blood sampling, lesion scores, and cecal oocyst count were carried out. The parasitological parameters showed statistical significance (p < 0.05) and there was no damage to the hematological parameters of birds (p > 0.05) by ANOVA test. The correlations suggest that the blood parameters were impaired by effects of the parasite on tissue, showing levels of hemorrhage and/or hydration.

Foram avaliados os parâmetros hematológicos em frangos de corte imunizados e desafiados com Eimeria tenella. Pintos de corte fêmeas da linhagem Hubbard, livres de coccídios, foram mantidos em baterias metálicas e inoculados no terceiro dia. O experimento foi delineado por cinco tratamentos com três repetições cada, sendo: T1 controle negativo, T2 recebeu 500 oocistos esporulados atenuados via oral, T3 controle positivo, T4 recebeu vacina contendo 50 µg de proteínas de esporozoítos + Quil A e T5 recebeu Quil A + PBS, sendo os dois últimos por via nasal nos dias 0, 7 e 21. No dia 31, todos os tratamentos foram desafiados com cepa virulenta homóloga de E. tenella na dose de 8,0 × 10(4) oocistos, exceto T1. Uma semana depois, foi realizada amostragem de sangue, escore de lesão e contagem de oocistos cecais. Os parâmetros parasitológicos apresentaram significância estatística (p < 0,05), sem que causassem prejuízos aos parâmetros hematológicos das aves (p > 0,05), pelo teste ANOVA. As correlações sugerem que os parâmetros sanguíneos foram afetados pelos efeitos do parasita no tecido, apresentando níveis de hemorragia e/ou hidratação.

Hypothesis testing of critical infection rates for early warning of malaria epidemics: A study using pooled sampling method and sporozoite rate / 第二军医大学学报

Objective: To provide a minimum sample size approach and a sequential sampling approach for testing whether the sporozoite rate has exceeded the critical level of malaria epidemics using the pool sampling method. Methods: Formulas of the expected pooled sample size and the power of tests were deduced while controlling the probability of type I and type II errors. The optimal pool sizes of the 2 approaches were given by minimizing the expected pooled sample size; computer simulation was used to verify the outcomes. Results: The optimal pool size, programming of MATLAB, and the steps of trials of the 2 approaches were given. The minimum sample size approach could be used for routine surveillance and sequential sampling approach could be used for early warning. Conclusion: The optimal pool size in the present study can obtain satisfactory testing power (type I and type II errors are both lower than 5%) and can effectively decrease the pooled sample size.

Malaria Vector Surveillance in Ganghwa-do, a Malaria-Endemic Area in the Republic of Korea

We investigated the seasonality of Anopheles mosquitoes, including its species composition, density, parity, and population densities of mosquitoes infected with the parasite in Ganghwa-do (Island), a vivax malaria endemic area in the Republic of Korea. Mosquitoes were collected periodically with a dry-ice-tent trap and a blacklight trap during the mosquito season (April-October) in 2008. Anopheles sinensis (94.9%) was the most abundant species collected, followed by Anopheles belenrae (3.8%), Anopheles pullus (1.2%), and Anopheles lesteri (0.1%). Hibernating Anopheles mosquitoes were also collected from December 2007 to March 2008. An. pullus (72.1%) was the most frequently collected, followed by An. sinensis (18.4%) and An. belenrae (9.5%). The composition of Anopheles species differed between the mosquito season and hibernation seasons. The parous rate fluctuated from 0% to 92.9%, and the highest rate was recorded on 10 September 2008. Sporozoite infections were detected by PCR in the head and thorax of female Anopheles mosquitoes. The annual sporozoite rate of mosquitoes was 0.11% (2 of 1,845 mosquitoes). The 2 mosquitoes that tested positive for sporozoites were An. sinensis. Malarial infections in anopheline mosquitoes from a population pool were also tried irrespective of the mosquito species. Nine of 2,331 pools of Anopheles mosquitoes were positive. From our study, it can be concluded that An. sinensis, which was the predominant vector species and confirmed as sporozoite-infected, plays an important role in malaria transmission in Ganghwa-do.

Malaria entomological inoculation rates in gold mining areas of Southern Venezuela

A longitudinal study of malaria vectors aiming to describe the intensity of transmission was carried out in five villages of Southern Venezuela between January 1999-April 2000. The man-biting, sporozoite and entomological inoculation rates (EIR) were calculated based on 121 all-night collections of anophelines landing on humans, CDC light traps and ultra violet up-draft traps. A total of 6,027 female mosquitoes representing seven species were collected. The most abundant species were Anopheles marajoara Galvão & Damasceno (56.7 percent) and Anopheles darlingi Root (33 percent), which together accounted for 89.7 percent of the total anophelines collected. The mean biting rate for An. marajoara was 1.27 (SD + 0.81); it was 0.74 (SD + 0.91) for An. darlingand 0.11 (SD + 0.10) for Anopheles neomaculipalpus Curry and the overall biting rate was 2.29 (SD + 1.06). A total of 5,886 mosquitoes collected by all three methods were assayed by ELISA and 28 pools, equivalent to 28 mosquitoes, yielded positive results for Plasmodium spp. CS protein. An. neomaculipalpus had the highest sporozoite rate 0.84 percent (3/356), followed by An. darlingi 0.82 percent (16/1,948) and An. marajoara 0.27 percent (9/3,332). The overall sporozoite rate was 0.48 percent (28/5,886). The rates of infection by Plasmodium species in mosquitoes were 0.37 percent (22/5,886) for Plasmodium vivax(Grassi & Feletti) and 0.10 percent (6/5,886) for Plasmodium falciparum (Welch). The estimated overall EIR for An. darling was 2.21 infective bites/person/year, 1.25 for An. marajoara and 0.34 for An. neomaculipalpus. The overall EIR was four infective bites/person/year. The biting rate, the sporozoite rate and the EIR are too low to be indicators of the efficacy of control campaigns in this area.

Progress of research on detection of Plasmodium sporozoites-carrying of mosquitoes / 中国血吸虫病防治杂志

Detection of Plasmodium sporozoites-carrying mosquitoes is an important indicator in monitoring mosquitoes, evaluating the control of malaria and forecasting the incidence of malaria. Here, we review the detection techniques in Plasmodium sporozoites-carrying mosquitoes and the progress of their applied research, and then briefly discuss the principles of the detection and the prospects for the field.

Vector bionomics and malaria transmission in the Upper Orinoco River, Southern Venezuela

A longitudinal epidemiological and entomological study was carried out in Ocamo, Upper Orinoco River, between January 1994 and February 1995 to understand the dynamics of malaria transmission in this area. Malaria transmission occurs throughout the year with a peak in June at the beginning of the rainy season. The Annual Parasite Index was 1,279 per 1,000 populations at risk. Plasmodium falciparum infections accounted for 64 percent of all infections, P. vivax for 28 percent, and P. malariae for 4 percent. Mixed P. falciparum/P. vivax infections were diagnosed in 15 people representing 4 percent of total cases. Children under 10 years accounted for 58 percent of the cases; the risk for malaria in this age group was 77 percent higher than for those in the greater than 50 years age group. Anopheles darlingi was the predominant anopheline species landing on humans indoors with a biting peak between midnight and dawn. A significant positive correlation was found between malaria monthly incidence and mean number of An. darlingi caught. There was not a significant relationship between mean number of An. darlingi and rainfall or between incidence and rainfall. A total of 7295 anophelines were assayed by ELISA for detection of Plasmodium circumsporozoite (CS) protein. Only An. darlingi (55) was positive for CS proteins of P. falciparum (0.42 percent), P. malariae (0.25 percent), and P. vivax-247 (0.1 percent). The overall estimated entomological inoculation rate was 129 positive bites/person/year. The present study was the first longitudinal entomological and epidemiological study conducted in this area and set up the basic ground for subsequent intervention with insecticide-treated nets.

Detection of vivax sporozoites naturally infected in Anopheline mosquitoes from endemic areas of northern parts of Gyeonggi-do (province) in Korea

We investigated population densities of mosquitoes infected with sporozoites in three highly epidemic areas of Josan-ri and Jangpa-ri (Paju City) and Dongjung-ri (Yeoncheon County) in Korea. Anopheline mosquitoes were collected from both indoors and outdoors by human baiting collection method during the period of the first week of June to the second week of September 1999. Total 13,296 female mosquitoes were collected and 8,650 (65.1%) were Anophelines. Thirty seven percent (3,199) of the Anopheline mosquitoes were captured outdoors and 63.9% (5,531) indoors. Employing a sandwich enzyme-linked immunosorbent assay (ELISA), we analyzed a total of 7,820 Anopheline mosquitoes and found that 7 Anopheline mosquitoes were infected with sporozoites. The positive rate in Josan-ri was 0.14% (5/3,500) and 0.15% (2/1,370) in Jangpa-ri. The total positive rate in all three surveyed areas was 0.09% (7/7,820). The mosquitoes infected with the sporozoites were detected on June 28th (n=2), July 5th (n=1), July 19th (n=1), August 9th (n=1), September 6th (n=1), and the last one on September 13th (n=1). They were all classified as Anopheles sinensis, which showed positive reaction in ELISA test. Therefore it might be concluded that A. sinensis plays an important role in re-emerging malaria transmission in Korea.

Mechanism of Protective Immunity Induced by Irradiation-Attenuated Sporozoites and its Implication for Pre-Erythrocytic Malaria Vaccine Research / 中国寄生虫学与寄生虫病杂志

Irradiation-attenuated sporozoites are still the most effective pre-erythrocytic malaria vaccine.However,limitation of purified sporozoites and difficulty in attenuation controlling of sporozoites hamper its use in practice.Understanding the mechanism of protective immunity induced by irradiation-attenuated sporozoites will be helpful for the design of the efficient pre-erythrocytic malaria vaccine.This is a review on research progress in the mechanism of protective immunity induced by irradiation-attenuated sporozoites and current status of pre-erythrocytic malaria vaccine development.

Evaluation of the Enzyme-linked Immunosorbant Assay in Detecting Circumsporozoite Protein of Anopheline Vectors in Yunnan / 中国寄生虫学与寄生虫病杂志

Objective To detect circumsporozoite protein (CSP) in anopheline vectors from south Yunnan and to evaluate ELISA in the detection. \ Methods\ Salivary glands of the anopheline mosquitoes were taken for finding sporozoites by microscopy and part of the glands was used for detecting CSP by ELISA. An. minimus was experimentally infected by blood from vivax malaria patient (with Plasmodium vivax) and examined for sporozoites and CSP. Eight species of anopheline mosquitoes were caught in the field and examined. Monoclonal antibodies to P.falciparum (Pf2A10) and P.vivax (Pv210, Pv247) were used in ELISA for detecting CSP. \ Results\ Sporozoites were found in the salivary glands of 27 out of 36 An. minimus experimentally infected (75^0%), 29 were ELISA CSP positives (80^6%), and 26 of the 27 mosquitoes showed Pv210 CSP positive. Among \{1 010\} parous anopheline mosquitoes from the field, 7 were found sporozoite positive (0^69%), 8 were ELISA CSP positive (0^79%), and 6 of the 7 mosquitoes showed CSP positive. Of \{4 675\} wild mosquitoes in 8 anopheline species with different ages, 11 were found CSP positive (0^24%) including An.minimus, An.sinensis and An.maculatus with a positive rate of 0^20%, 0^24% and 0^39% respectively.Among the 11 mosquitoes, 9 were Pv210 positive and 2 were Pf2A10 positive. Conclusion CSP detection by ELISA is a useful method to monitor the malaria transmission capacity of anopheline vectors.

Effects of BCG, Dexamethasone and Carbon Particles on Invasion of Sporozoites and Development of Exoerythrocytic Forms of Plasmodium yoelii in Rat liver / 第三军医大学学报

Wistar rats pretreated with BCG, dexamethasone or carbon particles respectively were inoculated with P. yoelii sporozoites via caudal vein. 42 hours after inoculation, the animals were killed to examine the effects of these 3 agents on the invasion of sporozoites and the development of exoerythrocytic forms (EEF) .It was found that in the carbon group about 30% of the kupffer cells contained the particles, and the density of EEF apparently decreased as compared with that of the control. In the BCG group, a dosage of 6 ? 107 units also caused a marked decrease of EEF density, an,d the parasites were smaller with a higher rate of degenerated forms as compared with those of the control, In the dexamethasone group, however, the EEF density increased markedly up to the level 1.5-2 times higher than that of the control. In addition the EEF were larger and more numerous with vacuoles and some lobulated. The mechanism of these facts were discussed.