Updating estimates of Plasmodium knowlesi malaria risk in response to changing land use patterns across Southeast Asia.

BACKGROUND: Plasmodium knowlesi is a zoonotic parasite that causes malaria in humans. The pathogen has a natural host reservoir in certain macaque species and is transmitted to humans via mosquitoes of the Anopheles Leucosphyrus Group. The risk of human P. knowlesi infection varies across Southeast Asia and is dependent upon environmental factors. Understanding this geographic variation in risk is important both for enabling appropriate diagnosis and treatment of the disease and for improving the planning and evaluation of malaria elimination. However, the data available on P. knowlesi occurrence are biased towards regions with greater surveillance and sampling effort. Predicting the spatial variation in risk of P. knowlesi malaria requires methods that can both incorporate environmental risk factors and account for spatial bias in detection. METHODS & RESULTS: We extend and apply an environmental niche modelling framework as implemented by a previous mapping study of P. knowlesi transmission risk which included data up to 2015. We reviewed the literature from October 2015 through to March 2020 and identified 264 new records of P. knowlesi, with a total of 524 occurrences included in the current study following consolidation with the 2015 study. The modelling framework used in the 2015 study was extended, with changes including the addition of new covariates to capture the effect of deforestation and urbanisation on P. knowlesi transmission. DISCUSSION: Our map of P. knowlesi relative transmission suitability estimates that the risk posed by the pathogen is highest in Malaysia and Indonesia, with localised areas of high risk also predicted in the Greater Mekong Subregion, The Philippines and Northeast India. These results highlight areas of priority for P. knowlesi surveillance and prospective sampling to address the challenge the disease poses to malaria elimination planning.

Improving in vitro continuous cultivation of Plasmodium cynomolgi, a model for P. vivax.

The absence of a routine continuous in vitro cultivation method for Plasmodium vivax, an important globally distributed parasite species causing malaria in humans, has restricted investigations to field and clinical sampling. Such a method has recently been developed for the Berok strain of P. cynomolgi, a parasite of macaques that has long been used as a model for P. vivax, as these two parasites are nearly indistinguishable biologically and are genetically closely related. The availability of the P. cynomolgi Berok in routine continuous culture provides for the first time an opportunity to conduct a plethora of functional studies. However, the initial cultivation protocol proved unsuited for investigations requiring extended cultivation times, such as reverse genetics and drug resistance. Here we have addressed some of the critical obstacles to this, and we propose a set of modifications that help overcome them.

Gastro-intestinal parasites in two subspecies of toque macaque (Macaca sinica) in Sri Lanka and their zoonotic potential.

Gastro-intestinal (GI) parasites of primates have a greater potential of becoming zoonotic. This potential may vary in different primates based on multiple factors such as proximity to human settlements and the climate of their habitat. We examined the GI parasites in two subspecies of toque macaque: Macaca sinica sinica (confined to the dry zone) and Macaca sinica aurifrons (confined to the wet zone) of Sri Lanka. Fresh faecal samples were collected and analysed following a modified Sheather's sucrose floatation method. A total of 90.8% (89/98) macaques were infected with one or more parasite species. There was no difference in the overall prevalence of GI parasites between the two subspecies, M. s. aurifrons (95.9%) and M. s. sinica (85.7%; χ2;χ = 3.059, p = 0.080). Sixteen parasite species were recorded including, 15 species in the M. s. sinica and 12 species in the M. s. aurifrons. Among the helminths identified, Anatrichosoma sp., Ancylostoma spp., Capillaria spp., Oesophagostomum /Bunostomum spp. and Physaloptera spp. are known to be zoonotic while Ascaris spp., Enterobius sp., Strongyloides spp. and Trichuris spp. have both zoonotic and anthroponotic potential. Among the protozoans, Balantidium coli and Buxtonella sp. are known to be zoonotic, while Entamoeba spp. and Cryptosporidium spp. have both anthroponotic and zoonotic potential. This study provides the first record of Anatrichosoma sp. and Buxtonella sp. in Sri Lanka and the first record of Cryptosporidium spp. in M. s. aurifrons. The molecular data allowed further identification and differentiation of Entamoeba nuttalli and E. coli that are known to be zoonotic and anthroponotic, respectively. The two subspecies of macaques have close interactions with humans; hence, in-depth epidemiological studies are required to understand the potential public-health risks to humans and conservation implications for macaque populations.

Preliminary review on the prevalence, proportion, geographical distribution, and characteristics of naturally acquired Plasmodium cynomolgi infection in mosquitoes, macaques, and humans: a systematic review and meta-analysis.

BACKGROUND: Plasmodium cynomolgi is a simian malaria parasite that has been reported as a naturally acquired human infection. The present study aims to systematically review reports on naturally acquired P. cynomolgi in humans, mosquitoes, and macaques to provide relevant data for pre-emptive surveillance and preparation in the event of an outbreak of zoonotic malaria in Southeast Asia. METHODS: The protocol of the systematic review was registered at PROSPERO with approval ID CRD42020203046. Three databases (Web of Science, Scopus, and MEDLINE) were searched for studies reporting the prevalence of P. cynomolgi infections in Southeast Asian countries between 1946 and 2020. The pooled prevalence or pooled proportion of P. cynomolgi parasitemia in humans, mosquitoes, and macaques was estimated using a random-effects model. Differences in the clinical characteristics of P. cynomolgi infections were also estimated using a random-effects model and presented as pooled odds ratios (ORs) or mean differences (MDs) with 95% confidence intervals (CIs). RESULTS: Thirteen studies reporting on the prevalence of naturally acquired P. cynomolgi in humans (3 studies, 21 cases), mosquitoes (3 studies, 28 cases), and macaques (7 studies, 334 cases) were included. The results demonstrated that the pooled proportion of naturally acquired P. cynomolgi in humans was 1% (95% CI, 0.1%, I2, 0%), while the pooled proportion of P. cynomolgi infecting mosquitoes was 18% (95% CI, 10-26%, I2, 32.7%). The pooled prevalence of naturally acquired P. cynomolgi in macaques was 47% (95% CI, 27-67%, I2, 98.3%). Most of the cases of naturally acquired P. cynomolgi in humans were reported in Cambodia (62%) and Malaysia (38%), while cases of P. cynomolgi in macaques were reported in Malaysia (35.4%), Singapore (23.2%), Indonesia (17.3%), Philippines (8.5%), Laos (7.93%), and Cambodia (7.65%). Cases of P. cynomolgi in mosquitoes were reported in Vietnam (76.9%) and Malaysia (23.1%). CONCLUSIONS: This study demonstrated the occurrence of naturally acquired P. cynomolgi infection in humans, mosquitoes, and macaques. Further studies of P. cynomolgi in asymptomatic human cases in areas where vectors and natural hosts are endemic are extensively needed if human infections with P. cynomolgi do become public health problems.

Investigation of possible alternate animal reservoir hosts of Opisthorchis viverrini.

Chronic opisthorchiasis caused by Opisthorchis viverrini (O. viverrini) adversely affects human health and is associated with a fatal bile duct cancer (cholangiocarcinoma). Although cats and dogs are known animal reservoir hosts of opisthorchiasis, there is limited information about whether other fish-eating animals are fluke reservoirs. Wetlands along Chi River of Thailand have abundant intermediate host snails and fish for O. viverrini and diverse other animal species. This led to our investigation into whether other fish-eating animals can also become infected and be alternate reservoir hosts for human opisthorchiasis. Our preliminary study focused on the O. viverrini infection status of crab-eating or long-tailed macaques roaming in Kosumpi National Forest Park in Chi River Basin, Kosumpisai District of Mahasarakam Province, and rodents, small residential mammals and fish-eating birds living in Lawa wetland complex of Khon Kaen Province. Fecal samples of each animal were collected and modified formalin ether concentration technique was applied to identify infections. Additionally, participatory epidemiology was used to retrieve information from local communities on opisthorchiasis transmission in these animals. No O. viverrini infection was found in any fecal samples suggesting that monkeys, rodents, small residential mammals and birds in these two wetlands probably do not serve as alternate reservoir hosts of O. viverrini.

A chemokine-fusion vaccine targeting immature dendritic cells elicits elevated antibody responses to malaria sporozoites in infant macaques.

Infants and young children are the groups at greatest risk for severe disease resulting from Plasmodium falciparum infection. We previously demonstrated in mice that a protein vaccine composed of the chemokine macrophage inflammatory protein 3α genetically fused to the minimally truncated circumsporozoite protein of P. falciparum (MCSP) elicits high concentrations of specific antibody and significant reduction of liver sporozoite load in a mouse model system. In the current study, a squalene based adjuvant (AddaVax, InvivoGen, San Diego, Ca) equivalent to the clinically approved MF59 (Seqiris, Maidenhead, UK) elicited greater antibody responses in mice than the previously employed adjuvant polyinosinic:polycytidylic acid, ((poly(I:C), InvivoGen, San Diego, Ca) and the clinically approved Aluminum hydroxide gel (Alum, Invivogen, San Diego, Ca) adjuvant. Use of the AddaVax adjuvant also expanded the range of IgG subtypes elicited by mouse vaccination. Sera passively transferred into mice from MCSP/AddaVax immunized 1 and 6 month old macaques significantly reduced liver sporozoite load upon sporozoite challenge. Protective antibody concentrations attained by passive transfer in the mice were equivalent to those observed in infant macaques 18 weeks after the final immunization. The efficacy of this vaccine in a relevant non-human primate model indicates its potential usefulness for the analogous high risk human population.

Prevalence of simian malaria parasites in macaques of Singapore.

Plasmodium knowlesi is a simian malaria parasite currently recognized as the fifth causative agent of human malaria. Recently, naturally acquired P. cynomolgi infection in humans was also detected in Southeast Asia. The main reservoir of both parasites is the long-tailed and pig-tailed macaques, which are indigenous in this region. Due to increased urbanization and changes in land use, there has been greater proximity and interaction between the long-tailed macaques and the general population in Singapore. As such, this study aims to determine the prevalence of simian malaria parasites in local macaques to assess the risk of zoonosis to the general human population. Screening for the presence of malaria parasites was conducted on blood samples from 660 peridomestic macaques collected between Jan 2008 and Mar 2017, and 379 wild macaques collected between Mar 2009 and Mar 2017, using a Pan-Plasmodium-genus specific PCR. Positive samples were then screened using a simian Plasmodium species-specific nested PCR assay to identify the species of parasites (P. knowlesi, P. coatneyi, P. fieldi, P. cynomolgi, and P. inui) present. All the peridomestic macaques sampled were tested negative for malaria, while 80.5% of the 379 wild macaques were infected. All five simian Plasmodium species were detected; P. cynomolgi being the most prevalent (71.5%), followed by P. knowlesi (47.5%), P. inui (42.0%), P. fieldi (32.5%), and P. coatneyi (28.5%). Co-infection with multiple species of Plasmodium parasites was also observed. The study revealed that Singapore's wild long-tailed macaques are natural hosts of the five simian malaria parasite species, while no malaria was detected in all peridomestic macaques tested. Therefore, the risk of simian malaria transmission to the general human population is concluded to be low. However, this can be better demonstrated with the incrimination of the vectors of simian malaria parasites in Singapore.

Prevalence, molecular epidemiology and zoonotic risk of Entamoeba spp. from experimental macaques in Yunnan Province, southwestern China.

Amebiasis is a worldwide parasitic zoonosis, with symptoms of abdominal discomfort, indigestion, diarrhea, and even death. However, limited information about the prevalence of Entamoeba spp. in experimental nonhuman primates (NHPs) in southwestern China is available. The objective of the current study was to investigate the frequency and species identity of Entamoeba to evaluate potential zoonotic risk factors for Entamoeba spp. infection in experimental NHPs. A total of 505 fecal samples were collected from NHPs (macaques) and analyzed by PCR analysis the small subunit rRNA (SSU rRNA) gene of Entamoeba spp. Forty-seven specimens were positive for Entamoeba spp., and the prevalence of Entamoeba spp. was 9.31% (47/505). Significant differences in the prevalence rates among the three breeds (P = 0.002 < 0.01, df = 2, χ2 = 12.33) and feed types (P = 0.001 < 0.01, df = 1, χ2 = 10.12) were observed. Altogether, four Entamoeba species, including E. dispar (57.44%), E. chattoni (29.78%), E. histolytica (6.38%), and E. coli (6.38%), were identified by DNA sequence analysis. The results suggested a low prevalence but high diversity of Entamoeba species in experimental NHPs in Yunnan Province, southwestern China. Results of this study contribute to the knowledge of the genetic characteristics of Entamoeba spp. in NHPs.

The Entamoeba lysine and glutamic acid rich protein (KERP1) virulence factor gene is present in the genomes of Entamoeba nuttalli, Entamoeba dispar and Entamoeba moshkovskii.

The lysine and glutamic acid rich protein KERP1 is a cell surface-expressed virulence factor in the human pathogen Entamoeba histolytica. It was originally suggested that the gene was absent from the related, avirulent human commensal Entamoeba dispar, an absence which would be relevant to the differential virulence of these species. Here, the gene is shown to be present in E. dispar, and its sequence is presented, as well as in a virulent parasite of macaques, Entamoeba nuttalli, and the primarily free living, opportunistically parasitic Entamoeba moshkovskii.

Natural Plasmodium infection in wild macaques of three states in peninsular Malaysia.

Zoonotic cases of Plasmodium knowlesi account for most malaria cases in Malaysia, and humans infected with P. cynomolgi, another parasite of macaques have recently been reported in Sarawak. To date the epidemiology of malaria in its natural Macaca reservoir hosts remains little investigated. In this study we surveyed the prevalence of simian malaria in wild macaques of three states in Peninsular Malaysia, namely Pahang, Perak and Johor using blood samples from 103 wild macaques (collected by the Department of Wildlife and National Parks Peninsular Malaysia) subjected to microscopic examination and nested PCR targeting the Plasmodium small subunit ribosomal RNA gene. As expected, PCR analysis yielded significantly higher prevalence (64/103) as compared to microscopic examination (27/103). No relationship between the age and/or sex of the macaques with the parasitaemia and the Plasmodium species infecting the macaques could be identified. Wild macaques in Pahang had the highest prevalence of Plasmodium parasites (89.7%), followed by those of Perak (69.2%) and Johor (28.9%). Plasmodium inui and P. cynomolgi were the two most prevalent species infecting the macaques from all three states. Half of the macaques (33/64) harboured two or more Plasmodium species. These data provide a baseline survey, which should be extended by further longitudinal investigations that should be associated with studies on the bionomics of the anopheline vectors. This information will allow an accurate evaluation of the risk of zoonotic transmission to humans, and to elaborate effective strategies to control simian malaria.

Three-dimensional electron microscopy analysis reveals endopolygeny-like nuclear architecture segregation in Plasmodium oocyst development.

The genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the production of haploid parasites in the Plasmodium life cycle: the oocyst stage in mosquitoes and the liver and blood stages in mammalian hosts. The Plasmodium oocyst stage plays an important role in the production of the first generation of haploid parasites. Nuclear division is the most important event that occurs during the proliferation of all eukaryotes. However, obtaining the details of nuclear division at the oocyst stage is challenging owing to difficulties in preparation. In this study, we used focused-ion-beam-milling combined with scanning-electron-microscopy to report the 3D architecture during nuclear segregations in oocyst stage. This advanced technology allowed us to analyse the 3D details of organelle segregation inside the oocyst during sporogony formation. It was revealed that multiple nuclei were involved with several centrosomes in one germ nucleus during sporozoite budding (endopolygeny). Our high-resolution 3D analysis uncovered the endopolygeny-like nuclear architecture of Plasmodium in the definitive host. This nuclear segregation was different from that in the blood stage, and its similarity to other apicomplexan parasite nuclear divisions such as Sarcocystis is discussed.

Robust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages.

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most widely distributed and difficult to treat malaria parasite, a strict preference for reticulocytes thwarts efforts to maintain it in vitro. Cultivation of P. cynomolgi, a macaque-infecting species phylogenetically close to P. vivax, was briefly reported in the early 1980s, but not pursued further. Here, we define the conditions under which P. cynomolgi can be adapted to long term in vitro culture to yield parasites that share many of the morphological and phenotypic features of P. vivax. We further validate the potential of this culture system for high-throughput screening to prime and accelerate anti-P. vivax drug discovery efforts.

Genetic diversity of Plasmodium knowlesi among human and long-tailed macaque populations in Peninsular Malaysia: The utility of microsatellite markers.

It has been discovered that Plasmodium knowlesi (P. knowlesi) is transmitted from macaque to man. Thus, the aim of the present study was to determine P. knowlesi genetic diversity in both human (n = 147) and long-tailed macaque (n = 26) samples from high- and low-endemicity localities. Genotyping was performed using seven neutral microsatellite loci markers. The size of the alleles, multiplicity of infection (MOI), mean number of alleles (Na), expected heterozygosity (HE), linkage disequilibrium (LD), and genetic differentiation (FST) were determined. In highly endemic P. knowlesi localities, the MOI for human and long-tailed macaque isolates was 1.04 and 1.15, respectively, while the Na was 11.14 and 7.86, respectively. Based on the allele frequency distribution for all loci, and with FST < 0.1, no genetic differentiation was seen between human and long-tailed macaque. In localities characterised by lower P. knowlesi endemicity, the MOI for human and long-tailed macaque isolates was 1.05 and 1.11, respectively, while the Na was 6.14 and 2.71, respectively. Further molecular analysis of the allele frequencies indicated that there was a significant genetic differentiation in human P. knowlesi isolates as compared to long-tailed macaque isolates, with a very low fixation index (FST = 0.016, p < .05) based on multiple loci analysis. Our results further indicate that, in Peninsular Malaysia, humans are mostly affected by P. knowlesi of a single genotype, while long-tailed macaque tend to acquire polyclonal infections, which supports the assumption that there is a higher rate of transmission among long-tailed macaque. Understanding the genetic diversity of P. knowlesi isolates can provide invaluable information for characterising patterns of the population structure and the migration rate of P. knowlesi in peninsular Malaysia.

ENTAMOEBA SPP. IN WILD FORMOSAN ROCK MACAQUES (MACACA CYCLOPIS) IN AN AREA WITH FREQUENT HUMAN-MACAQUE CONTACT.

Entamoeba is a genus of gastrointestinal protozoon that is transmitted through contaminated food and water. This protozoon is commonly found in human and nonhuman primates. Contact between humans and Formosan rock macaques (Macaca cyclopis) has become more frequent due to food provisioning; accordingly, concerns regarding zoonotic pathogen transmission through the fecal-oral route have increased. For example, surveillance of intestinal parasites in wild Formosan rock macaques indicated that Entamoeba infection was the most prevalent type of intestinal parasite infection. The morphologies of pathogenic and nonpathogenic species are difficult to distinguish. In this study, we collected fecal samples from wild Formosan rock macaques in the Shoushan National Nature Park (Kaohsiung, Taiwan) and adopted both morphologic and molecular methods for Entamoeba species identification. In total, we collected 208 fecal samples with a 57.7% (120/208, 95% confidence interval: 50.9-60.4%) prevalence of Entamoeba infection. Four Entamoeba species were identified: three nonpathogenic species, Entamoeba coli (19%), Entamoeba chattoni (50%), and Entamoeba hartmanni (11%), and one potentially pathogenic species, Entamoeba nuttalli (20%). Our study revealed the risk of zoonotic transmission of these Entamoeba species to humans. To address relevant public health and wildlife conservation concerns, further research is required to fully understand the virulence of E. nuttalli isolated from Formosan rock macaques.

The Role of Ecological Linkage Mechanisms in Plasmodium knowlesi Transmission and Spread.

Defining the linkages between landscape change, disease ecology and human health is essential to explain and predict the emergence of Plasmodium knowlesi malaria, a zoonotic parasite residing in Southeast Asian macaques, and transmitted by species of Anopheles mosquitos. Changing patterns of land use throughout Southeast Asia, particularly deforestation, are suggested to be the primary drivers behind the recent spread of this zoonotic parasite in humans. Local ecological changes at the landscape scale appear to be increasing the risk of disease in humans by altering the dynamics of transmission between the parasite and its primary hosts. This paper will focus on the emergence of P. knowlesi in humans in Malaysian Borneo and the ecological linkage mechanisms suggested to be playing an important role.

Vigilancia molecular de la malaria en potenciales reservorios silvestres que viven en una región históricamente endémica de Paraguay / Molecular surveillance of malaria in potential wild reservoirs that live in a historically endemic region of Paraguay

En Paraguay, no se han registrado casos autóctonos de malaria desde el 2011. Se realizó un estudio descriptivo observacional transversal en 6 monos y 23 aves que vivían en una región históricamente endémica de Paraguay para buscar presencia de reservorios silvestres de parásitos plasmodios causantes de la malaria. El ADN se extrajo por el método de Chelex a partir de una gota de sangre en un papel de filtro, y la detección del parásito se realizó mediante la PCR múltiple semianidada. Por este método, no se detectaron parásitos en ninguna de las 29 muestras. Se evaluó el riesgo potencial de circulación selvática de los parásitos que causan la malaria. Teniendo en cuenta la presencia de mosquitos anofelinos vectores en la zona, el hecho de que no se hayan observado casos positivos es un buen indicador teniendo en cuenta que nuestro país fue declarado recientemente como país libre de malaria por la OMS(AU)

In Paraguay, autochthonous cases of malaria have not been recorded since 2011. A cross-sectional observational descriptive study was conducted in 6 monkeys and 23 birds living in a historically endemic region of Paraguay to identify wild reservoirs of plasmodium parasites that cause malaria. DNA was extracted by the Chelex method from a blood drop in a filter paper, and parasite detection was performed by the seminested multiplex PCR. By this method, parasites were not detected in any of the 29 samples. The risk of potential sylvatic circulation of the parasites causing malaria was evaluated. Considering the presence of anopheline mosquitoes in the area, the fact that we did not find any positive cases is a good indicator as our country was recently certified as a malaria-free country by the WHO(AU)

Comparative study of three xenic media culture for cultivation of Balantidium coli strains.

The aim of the present study was to evaluate the growth rate of Balantidium coli in three xenic media cultures. Between 2013 and 2015, 10 B. coli isolates obtained from feces of Cynomolgus macaques, and 30 isolates from feces of pigs were studied. An inoculum of 500 trophozoites was transferred to tubes containing LES, TYSGM-9 and Pavlova media. These cultures were evaluated at incubation times of 24, 48, 72 and 96 hours. In most of strains analyzed wasn't showed significant difference in the growth rate comparing TYSGM-9 and Pavlova media (Wilcoxon p>0.016). In Pavlova medium, the trophozoites showed a maximum growth at 72 hours with significant difference when compared with the times of 24 h and 96 h (Wilcoxon <0.008). In LES, viable trophozoites were observed until 24 hours, with a significant difference (Friedman p<0.05, Wilcoxon p<0.016) in the number of parasite cells compared with Pavlova and TYSGM-9 media cultures. Thus, LES medium seemed to be less adequate than the other media for maintenance of B. coli. Despite the satisfactory results in TYSGM-9, Pavlova medium was considered ideal for the maintenance of this protozoan strain, guaranteeing the viability of the parasite with subculture every three days, presenting lower costs.

Parasites may exit immunocompromised northern pig-tailed macaques (Macaca leonina) infected with SIVmac239.

Parasites can increase infection rates and pathogenicity in immunocompromised human immunodeficiency virus (HIV) patients. However, in vitro studies and epidemiological investigations also suggest that parasites might escape immunocompromised hosts during HIV infection. Due to the lack of direct evidence from animal experiments, the effects of parasitic infections on immunocompromised hosts remain unclear. Here, we detected 14 different parasites in six northern pig-tailed macaques (NPMs) before or at the 50th week of simian immunodeficiency virus (SIV) infection by ELISA. The NPMs all carried parasites before viral injection. At the 50th week after viral injection, the individuals with negative results in parasitic detection (i.e., 08247 and 08287) were characterized as the Parasites Exit (PE) group, with the other individuals (i.e., 09203, 09211, 10205, and 10225) characterized as the Parasites Remain (PR) group. Compared with the PR group, the NPMs in the PE group showed higher viral loads, lower CD4+ T cells counts, and lower CD4/CD8 rates. Additionally, the PE group had higher immune activation and immune exhaustion of both CD4+ and CD8+ T cells. Pathological observation showed greater injury to the liver, cecum, colon, spleen, and mesenteric lymph nodes in the PE group. This study showed more seriously compromised immunity in the PE group, strongly indicating that parasites might exit an immunocompromised host.

Comparative study of three xenic media culture for cultivation of Balantidium coli strains / Estudo comparativo entre três meios de cultivo xênicos para o cultivo de cepas de Balantidium coli

Abstract The aim of the present study was to evaluate the growth rate of Balantidium coli in three xenic media cultures. Between 2013 and 2015, 10 B. coli isolates obtained from feces of Cynomolgus macaques, and 30 isolates from feces of pigs were studied. An inoculum of 500 trophozoites was transferred to tubes containing LES, TYSGM-9 and Pavlova media. These cultures were evaluated at incubation times of 24, 48, 72 and 96 hours. In most of strains analyzed wasn't showed significant difference in the growth rate comparing TYSGM-9 and Pavlova media (Wilcoxon p>0.016). In Pavlova medium, the trophozoites showed a maximum growth at 72 hours with significant difference when compared with the times of 24 h and 96 h (Wilcoxon <0.008). In LES, viable trophozoites were observed until 24 hours, with a significant difference (Friedman p<0.05, Wilcoxon p<0.016) in the number of parasite cells compared with Pavlova and TYSGM-9 media cultures. Thus, LES medium seemed to be less adequate than the other media for maintenance of B. coli. Despite the satisfactory results in TYSGM-9, Pavlova medium was considered ideal for the maintenance of this protozoan strain, guaranteeing the viability of the parasite with subculture every three days, presenting lower costs.

Resumo O objetivo do presente estudo foi avaliar a taxa de crescimento de Balantidium coli em três meios de cultura xênicos. Entre 2013 e 2015 foram estudados 10 isolados de B. coli obtidos de Cynomolgus macaques e 30 isolados de suínos. Um inóculo contendo 500 trofozoítos foi transferido para tubos contendo os meios LES, TYSGM-9 e Pavlova. Os cultivos foram avaliados com tempos de incubação de 24, 48, 72 e 96 horas. Na maioria das cepas analisadas não foi observado diferença significativa na taxa de crescimento comparando TYSGM-9 e Pavlova (Wilcoxon p>0,016). Em Pavlova, os trofozoítos apresentaram máximo de crescimento a 72 h com diferença significativa quando se comparou com os tempos de 24 h e 96 h (Wilcoxon <0,008). Em LES observou-se trofozoítos viáveis até 24 horas com diferença significativa (Friedman p<0,05 e Wilcoxon p<0,016), na quantidade de células parasitárias, quando comparado com Pavlova e TYSGM-9. Dessa forma, o meio LES mostrou-se ser menos adequado do que os outros, para a manutenção de B. coli. Apesar do resultado satisfatório em TYSGM-9, Pavlova foi considerado ideal para manutenção do protozoário, por garantir a viabilidade do parasito com subcultivos a cada três dias, além de apresentar menor custo.