Prevalence and genetic diversity of simian malaria in wild macaque populations across Thailand: Implications for human health.

Over the past year, P. falciparum infections have declined in Thailand, yet nonhuman primate malaria infections have correspondingly increased, including Plasmodium knowlesi and P. cynomolgi. Nevertheless, little is known about simian malaria in its natural macaque hosts, Macaca mulatta and Macaca fascicularis. This study aims to address several research questions, including the prevalence and distribution of simian malaria in these two Thai wild macaque species, variations in infection between different macaque species and between M. fascicularis subspecies, and the genetic composition of these pathogens. Blood samples were collected from 82 M. mulatta and 690 M. fascicularis across 15 locations in Thailand, as well as two locations in Vietnam and Myanmar. We employed quantitative real-time PCR targeting the Plasmodium genus-specific 18S ribosomal RNA (rRNA) gene to detect malaria infection, with a limit of detection set at 1,215.98 parasites per mL. We genotyped eight microsatellite markers, and the P. cynomolgi dihydrofolate reductase gene (DHFR) was sequenced (N = 29). In total, 100 of 772 samples (13 %) tested positive for malaria, including 45 (13 %) for P. cynomolgi, 37 (13 %) for P. inui, 16 (5 %) for P. coatneyi, and 2 (0.25 %) for Hepatocystis sp. in Saraburi, central and Ranong, southern Thailand. Notably, simian malaria infection was observed exclusively in M. fascicularis and not in M. mulatta (P = 0.0002). Particularly, P. cynomolgi was detected in 21.7 % (45/207) of M. f. fascicularis living in Wat Tham Phrapothisat, Saraburi Province. The infection with simian malaria was statistically different between M. fascicularis and M. mulatta (P = 0.0002) but not within M. fascicularis subspecies (P = 0.78). A haplotype network analysis revealed that P. cynomolgi shares a lineage with reference strains obtained from macaques. No mutation in the predicted binding pocket of PcyDHFR to pyrimethamine was observed. This study reveals a significant prevalence of simian malaria infection in M. fascicularis. The clonal genotypes of P. cynomolgi suggest in-reservoir breeding. These findings raise concerns about the potential spread of nonhuman primate malaria to humans and underscore the need for preventive measures.

Ultrasensitive malaria detection system for Anopheles mosquito field surveillance using droplet digital PCR.

Malaria remains a significant global public health concern, with a recent increase in the number of zoonotic malaria cases in Southeast Asian countries. However, limited reports on the vector for zoonotic malaria exist owing to difficulties in detecting parasite DNA in Anopheles mosquito vectors. Herein, we demonstrate for the first time that several Anopheles mosquitoes contain simian malaria parasite DNA using droplet digital PCR (ddPCR), a highly sensitive PCR method. An entomological survey was conducted to identify simian malaria vector species at Phra Phothisat Temple (PPT), central Thailand, recognized for a high prevalence of simian malaria in wild cynomolgus macaques. A total of 152 mosquitoes from six anopheline species were collected and first analyzed by a standard 18S rRNA nested-PCR analysis for malaria parasite which yielded negative results in all collected mosquitoes. Later, ddPCR was used and could detect simian malaria parasite DNA, i.e. Plasmodium cynomolgi, in 25 collected mosquitoes. And this is the first report of simian malaria parasite DNA detection in Anopheles sawadwongporni. This finding proves that ddPCR is a powerful tool for detecting simian malarial parasite DNA in Anopheles mosquitoes and can expand our understanding of the zoonotic potential of malaria transmission between monkeys and humans.

The prevalence of simian malaria in wild long-tailed macaques throughout Peninsular Malaysia.

The parasite Plasmodium knowlesi has been the sole cause of malaria in Malaysia from 2018 to 2022. The persistence of this zoonotic species has hampered Malaysia's progress towards achieving the malaria-free status awarded by the World Health Organisation (WHO). Due to the zoonotic nature of P. knowlesi infections, it is important to study the prevalence of the parasite in the macaque host, the long-tailed macaque (Macaca fascicularis). Apart from P. knowlesi, the long-tailed macaque is also able to harbour Plasmodium cynomolgi, Plasmodium inui, Plasmodium caotneyi and Plasmodium fieldi. Here we report the prevalence of the 5 simian malaria parasites in the wild long-tailed macaque population in 12 out of the 13 states in Peninsular Malaysia using a nested PCR approach targeting the 18s ribosomal RNA (18s rRNA) gene. It was found that all five Plasmodium species were widely distributed throughout Peninsular Malaysia except for states with major cities such as Kuala Lumpur and Putrajaya. Of note, Pahang reported a malaria prevalence of 100% in the long-tailed macaque population, identifying it as a potential hotspot for zoonotic transmission. Overall, this study shows the distribution of the 5 simian malaria parasite species throughout Peninsular Malaysia, the data of which could be used to guide future malaria control interventions to target zoonotic malaria.

The postmortem diagnosis of an Armillifer infection case in a cynomolgus macaque (Macaca fascicularis).

Armillifer moniliformis belongs to the order Porocephalida and family Porocephalidae, and it can cause zoonotic pentastomiasis. A suspected parasitic infection was incidentally discovered in the abdominal cavity of a cynomolgus macaque that died of persistent diarrhea. 18S rDNA amplification and sequencing revealed a high similarity (99.83%) to the Armillifer moniliformis Guangxi isolate. The isolated parasite was named the Armillifer moniliformis Yunnan isolate (GenBank accession no. HM048870). Our report presents a case of Armillifer moniliformis infection in macaques. The results indicated that early quarantine and diagnosis should be employed for animal health.

Zoonotic simian malaria parasites in free-ranging Macaca fascicularis macaques and human malaria patients in Thailand, with a note on genetic characterization of recent isolates.

Despite the natural occurrences of human infections by Plasmodium knowlesi, P. cynomolgi, P. inui, and P. fieldi in Thailand, investigating the prevalence and genetic diversity of the zoonotic simian malaria parasites in macaque populations has been limited to certain areas. To address this gap, a total of 560 long-tailed macaques (Macaca fascicularis) and 20 southern pig-tailed macaques (M. nemestrina) were captured from 15 locations across 10 provinces throughout Thailand between 2018 and 2021 for investigation of malaria, as were 15 human samples residing in two simian-malaria endemic provinces, namely Songkhla and Satun, who exhibited malaria-like symptoms. Using PCR techniques targeting the mitochondrial cytb and cox1 genes coupled with DNA sequencing, 40 long-tailed macaques inhabiting five locations had mono-infections with one of the three simian malaria species. Most of the positive cases of macaque were infected with P. inui (32/40), while infections with P. cynomolgi (6/40) and P. knowlesi (2/40) were less common and confined to specific macaque populations. Interestingly, all 15 human cases were mono-infected with P. knowlesi, with one of them residing in an area with two P. knowlesi-infected macaques. Nucleotide sequence analysis showed a high level of genetic diversity in P. inui, while P. cynomolgi and P. knowlesi displayed limited genetic diversity. Phylogenetic and haplotype network analyses revealed that P. inui in this study was closely related to simian and Anopheles isolates from Peninsular Malaysia, while P. cynomolgi clustered with simian and human isolates from Asian countries. P. knowlesi, which was found in both macaques and humans in this study, was closely related to isolates from macaques, humans, and An. hackeri in Peninsular Malaysia, suggesting a sylvatic transmission cycle extending across these endemic regions. This study highlights the current hotspots for zoonotic simian malaria and sheds light on the genetic characteristics of recent isolates in both macaques and human residents in Thailand.

Subtyping of Nonhuman Primate-Adapted Cryptosporidium hominis in Macaca Fascicularis and Macaca mulatta in Yunnan Province, Southwestern China.

Background: Cryptosporidium spp. are a type of protozoan parasite responsible for causing diarrheal illness worldwide. They infect a broad range of vertebrate hosts, including both non-human primates (NHPs) and humans. In fact, zoonotic transmission of cryptosporidiosis from NHPs to humans is frequently facilitated by direct contact between the two groups. However, there is a need to enhance the information available on the subtyping of Cryptosporidium spp. in NHPs in the Yunnan province of China. Materials and Methods: Thus, the study investigated the molecular prevalence and species of Cryptosporidium spp. from 392 stool samples of Macaca fascicularis (n = 335) and Macaca mulatta (n = 57) by using nested PCR targeting the large subunit of nuclear ribosomal RNA (LSU) gene. Of the 392 samples, 42 (10.71%) were tested Cryptosporidium-positive. Results: All the samples were identified as Cryptosporidium hominis. Further, the statistical analysis revealed that age is a risk factor for the infection of C. hominis. The probability of detecting C. hominis was found to be higher (odds ratio = 6.23, 95% confidence interval 1.73-22.38) in NHPs aged between 2 and 3 years, as compared with those younger than 2 years. Sequence analysis of the 60 kDa glycoprotein (gp60) identified six (IbA9 n = 4, IiA17 n = 5, InA23 n = 1, InA24 n = 2, InA25 n = 3, and InA26 n = 18) C. hominis subtypes with "TCA" repeats. Among these subtypes, it has been previously reported that the Ib family subtypes are also capable of infecting humans. Conclusion: The findings of this study highlight the genetic diversity of C. hominis infection among M. fascicularis and M. mulatta in Yunnan province. Further, the results confirm that both these NHPs are susceptible to C. hominis infection, posing a potential threat to humans.

Population genetic analysis of Plasmodium knowlesi reveals differential selection and exchange events between Borneo and Peninsular sub-populations.

The zoonotic Plasmodium knowlesi parasite is a growing public health concern in Southeast Asia, especially in Malaysia, where elimination of P. falciparum and P. vivax malaria has been the focus of control efforts. Understanding of the genetic diversity of P. knowlesi parasites can provide insights into its evolution, population structure, diagnostics, transmission dynamics, and the emergence of drug resistance. Previous work has revealed that P. knowlesi fall into three main sub-populations distinguished by a combination of geographical location and macaque host (Macaca fascicularis and M. nemestrina). It has been shown that Malaysian Borneo groups display profound heterogeneity with long regions of high or low divergence resulting in mosaic patterns between sub-populations, with some evidence of chromosomal-segment exchanges. However, the genetic structure of non-Borneo sub-populations is less clear. By gathering one of the largest collections of P. knowlesi whole-genome sequencing data, we studied structural genomic changes across sub-populations, with the analysis revealing differences in Borneo clusters linked to mosquito-related stages of the parasite cycle, in contrast to differences in host-related stages for the Peninsular group. Our work identifies new genetic exchange events, including introgressions between Malaysian Peninsular and M. nemestrina-associated clusters on various chromosomes, including in parasite invasion genes (DBP[Formula: see text], NBPX[Formula: see text] and NBPX[Formula: see text]), and important proteins expressed in the vertebrate parasite stages. Recombination events appear to have occurred between the Peninsular and M. fascicularis-associated groups, including in the DBP[Formula: see text] and DBP[Formula: see text] invasion associated genes. Overall, our work finds that genetic exchange events have occurred among the recognised contemporary groups of P. knowlesi parasites during their evolutionary history, leading to apparent mosaicism between these sub-populations. These findings generate new hypotheses relevant to parasite evolutionary biology and P. knowlesi epidemiology, which can inform malaria control approaches to containing the impact of zoonotic malaria on human communities.

Plasmodium spp. in macaques, Macaca fascicularis, in Malaysia, and their potential role in zoonotic malaria transmission.

Macaques, Macaca fascicularis, are a known reservoir of Plasmodium knowlesi, the agent of simian malaria which is the predominant zoonotic species affecting humans in Malaysia and other Southeast Asian countries. Recently, a naturally acquired human infection of another simian malaria parasite, P. cynomolgi has been reported. Thus, it is crucial to study the distribution of simian Plasmodium infections with particular attention to the macaques. Four hundred and nineteen (419) long-tailed macaques (Macaca fascicularis) were trapped in selected areas where human cases of P. knowlesi and P. cynomolgi have been reported. Nested polymerase chain reaction (PCR) was conducted to identify the Plasmodium spp., and circumsporozoite protein (CSP) genes of P. knowlesi samples were sequenced. Plasmodium cynomolgi infection was shown to be the most prevalent among the macaque population (68.4%). Although 50.6% of analyzed samples contained single infections either with P. knowlesi, P. cynomolgi, P. inui, P. coatneyi, or P. fieldi, mixed infections with double, triple, quadruple, and all 5 species were also detected. Infection with P. cynomolgi and P. knowlesi were the highest among Malaysian macaques in areas where humans and macaques are in close contact. The risk of zoonotic infection in these areas needs to be addressed since the number of zoonotic malaria cases is on the rise. With the elimination of human malaria, the risk of humans being infected with simian malaria is very high and steps should be taken to mitigate this issue.

Title: Plasmodium spp. chez les macaques, Macaca fascicularis, en Malaisie, et leur rôle potentiel dans la transmission zoonotique du paludisme. Abstract: Les macaques, Macaca fascicularis, sont un réservoir connu de Plasmodium knowlesi, l'agent du paludisme simien qui est l'espèce zoonotique prédominante affectant les humains en Malaisie et dans d'autres pays d'Asie du Sud-Est. Récemment, une infection humaine acquise naturellement par un autre parasite du paludisme simien, P. cynomolgi, a été signalée. Ainsi, il est crucial d'étudier la distribution des infections simiennes à Plasmodium avec une attention particulière pour les macaques. Quatre cent dix-neuf (419) macaques à longue queue (Macaca fascicularis) ont été piégés dans des zones sélectionnées où des cas humains de P. knowlesi et P. cynomolgi avaient été signalés. La réaction en chaîne par polymérase (PCR) nichée a été menée pour identifier les Plasmodium spp. et les gènes de la protéine circumsporozoïte (CSP) des échantillons de P. knowlesi ont été séquencés. L'infection à P. cynomolgi s'est avérée la plus répandue parmi la population de macaques (68,4 %). Bien que 50,6 % des échantillons analysés montraient des infections simples avec soit P. knowlesi, P. cynomolgi, P. inui, P. coatneyi ou P. fieldi, des infections mixtes avec deux, trois, quatre ou même les cinq espèces ont également été détectées. L'infection par P. cynomolgi et P. knowlesi était la plus élevée parmi les macaques malais dans les zones où les humains et les macaques sont en contact étroit. Le risque d'infection zoonotique dans ces zones doit être pris en compte car le nombre de cas de paludisme zoonotique est en augmentation. Avec l'élimination du paludisme humain, le risque d'être infecté par le paludisme simien est très élevé et des mesures doivent être prises pour atténuer ce problème.

Macaca fascicularis and Macaca nemestrina infected with zoonotic malaria parasites are widely distributed in Sarawak, Malaysian Borneo.

Human infections with Plasmodium knowlesi, a malaria parasite of Macaca fascicularis and Macaca nemestrina (long-tailed and pig-tailed macaques respectively), occur throughout Southeast Asia, especially Malaysian Borneo. Other naturally-acquired human infections with malaria parasites from macaques in Southeast Asia are P. cynomolgi, P. inui-like, P. coatneyi and P. simiovale. In Sarawak, Malaysian Borneo, M. fascicularis and M. nemestrina from only the Kapit Division have been examined previously for malaria parasites. In order to determine the distribution of P. knowlesi and other zoonotic malaria parasites, 73 macaque blood samples derived from 7 other administrative divisions in Sarawak were studied. Of 45 blood samples from M. fascicularis and 28 from M. nemestrina tested by nested PCR assays, 23 (51.1%) M. fascicularis and 15 (53.6%) M. nemestrina samples were positive for Plasmodium DNA. Thirty-two of these macaques from 7 divisions sampled, harboured either single (n = 12), double (n = 9), triple (n = 7) or quadruple (n = 4) infections of P. knowlesi, P. inui, P. cynomolgi and P. coatneyi, while the infecting species of Plasmodium could not be identified for 6 samples. P. knowlesi was detected in 15.5% (7/45) M. fascicularis and in 7.1% (2/28) M. nemestrina sampled. Despite the small number of samples analysed from each administrative division, the current study indicates that macaques infected with the zoonotic malaria parasites P. knowlesi, P. cynomolgi, P. inui and P. coatneyi are widely distributed throughout Sarawak, Malaysian Borneo. Travelers to forested areas in Sarawak should be made aware of the potential risk of acquiring zoonotic malaria.

Evaluation of Mosquito Magnet and other collection tools for Anopheles mosquito vectors of simian malaria.

BACKGROUND: Vector surveillance is essential in determining the geographical distribution of mosquito vectors and understanding the dynamics of malaria transmission. With the elimination of human malaria cases, knowlesi malaria cases in humans are increasing in Malaysia. This necessitates intensive vector studies using safer trapping methods which are both field efficient and able to attract the local vector populations. Thus, this study evaluated the potential of Mosquito Magnet as a collection tool for Anopheles mosquito vectors of simian malaria along with other known collection methods. METHODS: A randomized 4 × 4 Latin square designed experiment was conducted to compare the efficiency of the Mosquito Magnet against three other common trapping methods: human landing catch (HLC), CDC light trap and human baited trap (HBT). The experiment was conducted over six replicates where sampling within each replicate was carried out for 4 consecutive nights. An additional 4 nights of sampling was used to further evaluate the Mosquito Magnet against the "gold standard" HLC. The abundance of Anopheles sampled by different methods was compared and evaluated with focus on the Anopheles from the Leucosphyrus group, the vectors of knowlesi malaria. RESULTS: The Latin square designed experiment showed HLC caught the greatest number of Anopheles mosquitoes (n = 321) compared to the HBT (n = 87), Mosquito Magnet (n = 58) and CDC light trap (n = 13). The GLMM analysis showed that the HLC method caught significantly more Anopheles mosquitoes compared to Mosquito Magnet (P = 0.049). However, there was no significant difference in mean nightly catch of Anopheles mosquitoes between Mosquito Magnet and the other two trapping methods, HBT (P = 0.646) and CDC light traps (P = 0.197). The mean nightly catch for both An. introlatus (9.33 ± 4.341) and An. cracens (4.00 ± 2.273) caught using HLC was higher than that of Mosquito Magnet, though the differences were not statistically significant (P > 0.05). This is in contrast to the mean nightly catch of An. sinensis (15.75 ± 5.640) and An. maculatus (15.78 ± 3.479) where HLC showed significantly more mosquito catches compared to Mosquito Magnet (P < 0.05). CONCLUSIONS: Mosquito Magnet has a promising ability to catch An. introlatus and An. cracens, the important vectors of knowlesi and other simian malarias in Peninsular Malaysia. The ability of Mosquito Magnet to catch some of the Anopheles mosquito species is comparable to HLC and makes it an ethical and safer alternative.

High variation in immune responses and parasite phenotypes in naturally acquired Trypanosoma cruzi infection in a captive non-human primate breeding colony in Texas, USA.

Trypanosoma cruzi, the causative agent of human Chagas disease, is endemic to the southern region of the United States where it routinely infects many host species. The indoor/outdoor housing configuration used in many non-human primate research and breeding facilities in the southern of the USA provides the opportunity for infection by T. cruzi and thus provides source material for in-depth investigation of host and parasite dynamics in a natural host species under highly controlled and restricted conditions. For cynomolgus macaques housed at such a facility, we used a combination of serial blood quantitative PCR (qPCR) and hemoculture to confirm infection in >92% of seropositive animals, although each method alone failed to detect infection in >20% of cases. Parasite isolates obtained from 43 of the 64 seropositive macaques were of 2 broad genetic types (discrete typing units, (DTU's) I and IV); both within and between these DTU groupings, isolates displayed a wide variation in growth characteristics and virulence, elicited host immune responses, and susceptibility to drug treatment in a mouse model. Likewise, the macaques displayed a diversity in T cell and antibody response profiles that rarely correlated with parasite DTU type, minimum length of infection, or age of the primate. This study reveals the complexity of infection dynamics, parasite phenotypes, and immune response patterns that can occur in a primate group, despite being housed in a uniform environment at a single location, and the limited time period over which the T. cruzi infections were established.

Historical contingency, geography and anthropogenic patterns of exposure drive the evolution of host switching in the Blastocystis species-complex.

Parasites have the power to impose significant regulatory pressures on host populations, making evolutionary patterns of host switching by parasites salient to a range of contemporary ecological issues. However, relatively little is known about the colonization of new hosts by parasitic, commensal and mutualistic eukaryotes of metazoans. As ubiquitous symbionts of coelomate animals, Blastocystis spp. represent excellent candidate organisms for the study of evolutionary patterns of host switching by protists. Here, we apply a big-data phylogenetic approach using archival sequence data to assess the relative roles of several host-associated traits in shaping the evolutionary history of the Blastocystis species-complex within an ecological framework. Patterns of host usage were principally determined by geographic location and shared environments of hosts, suggesting that weight of exposure (i.e. propagule pressure) represents the primary force for colonization of new hosts within the Blastocystis species-complex. While Blastocystis lineages showed a propensity to recolonize the same host taxa, these taxa were often evolutionarily unrelated, suggesting that historical contingency and retention of previous adaptions by the parasite were more important to host switching than host phylogeny. Ultimately, our findings highlight the ability of ecological theory (i.e. 'ecological fitting') to explain host switching and host specificity within the Blastocystis species-complex.

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.

Multilocus sequence typing of Enterocytozoon bieneusi in crab-eating macaques (Macaca fascicularis) in Hainan, China.

BACKGROUND: Enterocytozoon bieneusi is one of common intestinal pathogens in humans and animals including non-human primates (NHPs). Many zoonotic pathogens including E. bieneusi have been found in these animals. However, there are few studies on the population structure of E. bieneusi in NHPs. To infer the gene diversity and population genetics of E. bieneusi, we selected 88 E. bieneusi-positive samples from crab-eating macaques for multilocus characterizations in this study. METHODS: The E. bieneusi isolates examined belonged to three common genotypes with different host ranges by sequence analysis of the ribosomal internal transcribed spacer (ITS): Type IV (n = 44), Macaque3 (n = 24) and Peru8 (n = 20). They were further characterized by sequence analysis at four microsatellite and minisatellite loci (MS1, MS3, MS4 and MS7). DnaSP, Arlequin and LIAN were used to analyze the sequence data together with those from the ITS locus to infer the population genetics. Subpopulation structure was inferred using phylogenetic and STRUCTURE analyses. RESULTS: Seventy-two (81.8%), 71 (80.7%), 76 (86.4%) and 79 (89.8%) samples were amplified and sequenced successfully at the MS1, MS3, MS4 and MS7 loci, respectively, with 53 having sequence data at all five MLST loci including ITS. Altogether, 33 multilocus genotypes (MLGs) were produced based on concatenated sequences from the 53 samples. In phylogenetic analyses of sequences and allelic data, four major subpopulations (SPs) were observed with different ITS genotypes in each of them: Type IV and Peru8 in SP1 and SP2; Type IV, Macaque3 and Peru8 in SP3; and Type IV and Macaque3 in SP4. SP3 and SP4 were phylogenetically related and might be NHP-specific based on the fact that Macaque3 is mostly found in NHPs. A strong linkage disequilibrium (LD) was observed among the multilocus sequences and allelic data. CONCLUSIONS: The significant LD in the multilocus sequence analysis indicated the presence of an overall clonal population structure of E. bieneusi in crab-eating macaques. The inconsistent segregation of MLGs among ITS genotypes suggested some occurrence of genetic recombination. These observations should improve our understanding of the population genetics of E. bieneusi in NHPs.

Genotyping of Enterocytozoon bieneusi among captive long-tailed macaques (Macaca fascicularis) in Hainan Province: High genetic diversity and zoonotic potential.

Enterocytozoon bieneusi is a potentially important zoonotic pathogen. However, there is no information on E. bieneusi infection of captive long-tailed macaques (Macaca fascicularis) in Hainan Province, China. Here 193 fecal specimens of M. fascicularis were collected from a breeding base in Hainan Province, China, housing non-human primates for experimental use. E. bieneusi was identified and genotyped by nested PCR analysis of the internal transcribed spacer (ITS) region of the rRNA gene. A total of 59 (30.6%) specimens were PCR-positive for E. bieneusi and 16 ITS genotypes were identified including nine known genotypes: Type IV (n = 19), D (n = 11), CM1 (n = 8), PigEBITS7 (n = 4), Pongo2 (n = 4), Peru8 (n = 3), Peru11 (n = 1), WL21 (n = 1) and CM2 (n = 1) and seven novel genotypes HNM-I to HNM-VII (one each). Importantly, genotypes D, Type IV, Peru8, PigEBITS7, and Peru11, which were the predominant (38/59, 64.4%) genotypes identified among captive M. fascicularis in this study, are also well-known human-pathogenic genotypes. All the genotypes of E. bieneusi identified here, including the seven novel ones, belonged to zoonotic Group 1. This is the first report of the identification of E. bieneusi in M. fascicularis in Hainan Province, China. The finding that the numerous known human-pathogenic types and seven novel genotypes of E. bieneusi all belong to zoonotic Group 1 indicates the possibility of transmission of this important pathogenic parasite between M. fascicularis and humans.

Human Plasmodium knowlesi infections in South-East Asian countries.

Plasmodium knowlesi is now regarded as the fifth malaria parasite causing human malaria as it is widely distributed in South-East Asian countries especially east Malaysia where two Malaysian states namely Sabah and Sarawak are situated. In 2004, Polymerase Chain Reaction (PCR) was applied for diagnosing knowlesi malaria in the Kapit Division of Sarawak, Malaysia, so that human P. knowlesi infections could be detected correctly while blood film microscopy diagnosed incorrectly as Plasmodium malariae. This parasite is transmitted from simian hosts to humans via Anopheles vectors. Indonesia is the another country in South East Asia where knowlesi malaria is moderately prevalent. In the last decade, Sarawak and Sabah, the two states of east Malaysia became the target of P. knowlesi research due to prevalence of cases with occasional fatal infections. The host species of P. knowlesi are three macaque species namely Macaca fascicularis, Macaca nemestrina and Macaca leonina while the vector species are the Leucosphyrus Complex and the Dirus Complex of the Leucophyrus Group of Anopheles mosquitoes. Rapid diagnostic tests (RDT) are non-existent for knowlesi malaria although timely treatment is necessary for preventing complications, fatality and drug resistance. Development of RDT is essential in dealing with P. knowlesi infections in poor rural healthcare services. Genetic studies of the parasite on possibility of human-to-human transmission of P. knowlesi were recommended for further studies.

Cryptosporidium parvum and Cryptosporidium hominis subtypes in crab-eating macaques.

BACKGROUND: Non-human primates are often infected with human-pathogenic Cryptosporidium hominis subtypes, but rarely with Cryptosporidium parvum. In this study, 1452 fecal specimens were collected from farmed crab-eating macaques (Macaca fascicularis) in Hainan, China during the period April 2016 to January 2018. These specimens were analyzed for Cryptosporidium species and subtypes by using PCR and sequence analysis of the 18S rRNA and 60 kDa glycoprotein (gp60) genes, respectively. RESULTS: Altogether, Cryptosporidium was detected using 18S rRNA-based PCR in 132 (9.1%) sampled animals, with significantly higher prevalence in females (12.5% or 75/599 versus 6.1% or 43/706), younger animals (10.7% or 118/1102 in monkeys 1-3-years-old versus 4.0% or 14/350 in those over 3-years-old) and animals with diarrhea (12.6% or 46/365 versus 7.9% or 86/1087). Four Cryptosporidium species were identified, namely C. hominis, C. parvum, Cryptosporidium muris and Cryptosporidium ubiquitum in 86, 30, 15 and 1 animal, respectively. The identified C. parvum, C. hominis and C. ubiquitum were further subtyped by using gp60 PCR. Among them, C. parvum belonged to subtypes in two known subtype families, namely IIoA14G1 (in 18 animals) and IIdA19G1 (in 2 animals). In contrast, C. hominis mostly belonged to two new subtype families Im and In, which are genetically related to Ia and Id, respectively. The C. hominis subtypes identified included ImA18 (in 38 animals), InA14 (in six animals), InA26 (in six animals), InA17 (in one animal) and IiA17 (in three animals). The C. ubiquitum isolates belonged to subtype family XIId. By subtype, ImA18 and IIoA14G1 were detected in animals with diarrhea whereas the remaining ones were mostly found in asymptomatic animals. Compared with C. parvum and C. muris, higher oocyst shedding intensity was observed in animals infected with C. hominis, especially those infected with the Im subtype family. CONCLUSIONS: Data from the study suggest that crab-eating macaques are infected with diverse C. parvum and C. hominis subtypes. The C. parvum IIo subtype family previously seen in rodents in China has apparently expanded its host range.

Whole genome sequencing of amplified Plasmodium knowlesi DNA from unprocessed blood reveals genetic exchange events between Malaysian Peninsular and Borneo subpopulations.

The zoonotic Plasmodium knowlesi parasite is the most common cause of human malaria in Malaysia. Genetic analysis has shown that the parasites are divided into three subpopulations according to their geographic origin (Peninsular or Borneo) and, in Borneo, their macaque host (Macaca fascicularis or M. nemestrina). Whilst evidence suggests that genetic exchange events have occurred between the two Borneo subpopulations, the picture is unclear in less studied Peninsular strains. One difficulty is that P. knowlesi infected individuals tend to present with low parasitaemia leading to samples with insufficient DNA for whole genome sequencing. Here, using a parasite selective whole genome amplification approach on unprocessed blood samples, we were able to analyse recent genomes sourced from both Peninsular Malaysia and Borneo. The analysis provides evidence that recombination events are present in the Peninsular Malaysia parasite subpopulation, which have acquired fragments of the M. nemestrina associated subpopulation genotype, including the DBPß and NBPXa erythrocyte invasion genes. The NBPXb invasion gene has also been exchanged within the macaque host-associated subpopulations of Malaysian Borneo. Our work provides strong evidence that exchange events are far more ubiquitous than expected and should be taken into consideration when studying the highly complex P. knowlesi population structure.

Molecular prevalence and subtyping of Cryptosporidium hominis among captive long-tailed macaques (Macaca fascicularis) and rhesus macaques (Macaca mulatta) from Hainan Island, southern China.

BACKGROUND: Cryptosporidium is an important zoonotic parasite that is commonly found in non-human primates (NHPs). Consequently, there is the potential for transmission of this pathogen from NHPs to humans. However, molecular characterization of the isolates of Cryptosporidium from NHPs remains relatively poor. The aim of the present work was to (i) determine the prevalence; and (ii) perform a genetic characterization of the Cryptosporidium isolated from captive Macaca fascicularis and M. mulatta on Hainan Island in southern China. METHODS: A total of 223 fresh fecal samples were collected from captive M. fascicularis (n = 193) and M. mulatta (n = 30). The fecal specimens were examined for the presence of Cryptosporidium spp. by polymerase chain reaction (PCR) and sequencing of the partial small subunit (SSU) rRNA gene. The Cryptosporidium-positive specimens were subtyped by analyzing the 60-kDa glycoprotein (gp60) gene sequence. RESULTS: Cryptosporidium spp. were detected in 5.7% (11/193) of M. fascicularis. All of the 11 Cryptosporidium isolates were identified as C. hominis. Subtyping of nine of these isolates identified four unique gp60 subtypes of C. hominis. These included IaA20R3a (n = 1), IoA17a (n = 1), IoA17b (n = 1), and IiA17 (n = 6). Notably, subtypes IaA20R3a, IoA17a, and IoA17b were novel subtypes which have not been reported previously. CONCLUSIONS: To our knowledge, this is the first reported detection of Cryptosporidium in captive M. fascicularis from Hainan Island. The molecular characteristics and subtypes of the isolates here provide novel insights into the genotypic variation in C. hominis.

Genotypes and public health potential of Enterocytozoon bieneusi and Giardia duodenalis in crab-eating macaques.

BACKGROUND: Enterocytozoon bieneusi and Giardia duodenalis are common human and animal pathogens. Studies have increasingly shown that non-human primates (NHPs) are common hosts of these two zoonotic parasites. However, few studies have explored the genetic diversity and public health potential of these pathogens in laboratory monkeys. In this study, we examined the genetic diversity of the two pathogens in crab-eating macaques (Macaca fascicularis) in a commercial facility in Hainan, China. RESULTS: Enterocytozoon bieneusi and G. duodenalis were detected by PCR analysis in 461/1452 (31.7%) and 469/1452 (32.3%) fecal specimens from the animals, respectively. Significantly higher detection rates of E. bieneusi were detected in males (36.5%, 258/706) than in females (26.7%, 160/599; χ2 = 14.391, P = 0.0001), in animals with loose stools (41.4%, 151/365) than those with normal stool (28.5%, 310/1087; χ2 = 20.83, P < 0.0001), and in animals of over 3 years of age (38.6%, 135/350) than those of 1-3 years (29.6%, 326/1,102; χ2 = 9.90, P = 0.0016). For G. duodenalis, the detection rate in males (33.4%, 236/706) was higher than in females but not statistically significant (30.2%, 181/599; χ2 = 1.54, P = 0.2152), in monkeys with loose stools (41.1%, 150/365) than those with normal stools (29.3%, 319/1087; χ2 = 17.25, P < 0.0001), and in monkeys of 1-3 years of age (36.6%, 403/1102) than those over 3 years (18.9%, 66/350; χ2 = 38.11, P < 0.0001). Nine E. bieneusi genotypes were detected in this study by DNA sequence analysis of the internal transcribed spacer of the rRNA gene, namely Type IV (236/461), Peru8 (42/461), Pongo2 (27/461), Peru11 (12/461), D (4/461) and PigEbITS7 (1/461) previously seen in NHPs as well as humans, and CM1 (119/461), CM2 (17/461) and CM3 (3/461) that had been only detected in NHPs. DNA sequence analyses of the tpi, gdh and bg loci identified all G. duodenalis specimens as having assemblage B. Altogether, eight (4 known and 4 new), seven (6 known and 1 new) and seven (4 known and 3 new) subtypes were seen at the tpi, gdh and bg loci, leading to the detection of 53 multi-locus genotypes (MLG-B-hn01 to MLG-B-hn53). Most of them were genetically related to those previously seen in common Old-World monkeys. CONCLUSIONS: Data from this study indicate a common occurrence of zoonotic genotypes of E. bieneusi and assemblage B of G. duodenalis in farmed crab-eating macaques in Hainan, China.