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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., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

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., (© 2024. The Author(s).)

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.

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. We also confirm that our work is original and has not been submitted elsewhere. We, (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

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., (© 2023. The Author(s).)

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., (© 2022. The Author(s).)

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., (© N.M. Yusuf et al., published by EDP Sciences, 2022.)

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.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors have declared that no competing interests exist

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.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Land-use changes can impact infectious disease transmission by increasing spatial overlap between people and wildlife disease reservoirs. In Malaysian Borneo, increases in human infections by the zoonotic malaria Plasmodium knowlesi are hypothesised to be due to increasing contact between people and macaques due to deforestation. To explore how macaque responses to environmental change impact disease risks, we analysed movement of a GPS-collared long-tailed macaque in a knowlesi-endemic area in Sabah, Malaysia, during a deforestation event. Land-cover maps were derived from satellite-based and aerial remote sensing data and models of macaque occurrence were developed to evaluate how macaque habitat use was influenced by land-use change. During deforestation, changes were observed in macaque troop home range size, movement speeds and use of different habitat types. Results of models were consistent with the hypothesis that macaque ranging behaviour is disturbed by deforestation events but begins to equilibrate after seeking and occupying a new habitat, potentially impacting human disease risks. Further research is required to explore how these changes in macaque movement affect knowlesi epidemiology on a wider spatial scale.

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., (Copyright © 2019. Published by Elsevier B.V.)

Strongyloides fuelleborni is a soil-transmitted nematode parasite of non-human primates. The worm is prevalent also in human populations in Africa and South-East Asia. In this study, we amplified and sequenced a portion of the 18S ribosomal RNA gene (rRNA) and of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of Strongyloides adult males recovered from faecal samples from long-tailed macaques (Macaca fascicularis) in Thailand and Lao PDR. The prevalence in Thailand was 31.1% (55/177) and in Lao PDR it was 62.1% (41/66), with an overall prevalence of 39.5% (96/243). All 18S rRNA sequences that we obtained (n = 96) showed 100% identity with published S. fuelleborni sequences. The 96 cox1 sequences that we obtained represented 32 new haplotypes. When included with the 17 previously known haplotypes from S. fuelleborni, the cox1 sequences fell into four clusters, which had clear geographical structure. This is the first molecular confirmation of S. fuelleborni in long-tailed macaques in Thailand and Lao PDR. Clearly, awareness needs to be raised of the zoonotic potential of S. fuelleborni. A monitoring programme should be organized, taking into account the role of reservoir hosts (i.e. monkeys) in the natural background of human strongyloidiasis caused by S. fuelleborni.

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.

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 DBPβ 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.

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; χ 2  = 14.391, P = 0.0001), in animals with loose stools (41.4%, 151/365) than those with normal stool (28.5%, 310/1087; χ 2  = 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; χ 2  = 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; χ 2  = 1.54, P = 0.2152), in monkeys with loose stools (41.1%, 150/365) than those with normal stools (29.3%, 319/1087; χ 2  = 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; χ 2  = 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.

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.

Background: Macaca fascicularis (long-tailed macaque) is the most widespread species of macaque in Southeast Asia and the only species of monkey found naturally in the Philippines. The species is the natural host for the zoonotic malaria species, Plasmodium knowlesi and Plasmodium cynomolgi and for the potentially zoonotic species, Plasmodium inui. Moreover, other Plasmodium species such as Plasmodium coatneyi and Plasmodium fieldi are also natural parasites of M. fascicularis. The aims of this study were to identify and determine the prevalence of Plasmodium species infecting wild and captive long-tailed macaques from the Philippines., Methods: A total of 95 blood samples from long-tailed macaques in the Philippines were collected from three locations; 30 were from captive macaques at the National Wildlife Rescue and Rehabilitation Center (NWRRC) in Luzon, 25 were from captive macaques at the Palawan Wildlife Rescue and Conservation Center (PWRCC) in Palawan and 40 were from wild macaques from Puerto Princesa Subterranean River National Park (PPSRNP) in Palawan. The Plasmodium spp. infecting the macaques were identified using nested PCR assays on DNA extracted from these blood samples., Results: All 40 of the wild macaques from PPSRNP in Palawan and 5 of 25 captive macaques from PWRCC in Palawan were Plasmodium-positive; while none of the 30 captive macaques from the NWRRC in Luzon had any malaria parasites. Overall, P. inui was the most prevalent malaria parasite (44.2%), followed by P. fieldi (41.1%), P. cynomolgi (23.2%), P. coatneyi (21.1%), and P. knowlesi (19%). Mixed species infections were also observed in 39 of the 45 Plasmodium-positive macaques. There was a significant difference in the prevalence of P. knowlesi among the troops of wild macaques from PPSRNP., Conclusion: Wild long-tailed macaques from the island of Palawan, the Philippines are infected with P. knowlesi, P. inui, P. coatneyi, P. fieldi and P. cynomolgi. The prevalence of these Plasmodium spp. varied among the sites of collection and among troops of wild macaques at one site. The presence of these simian Plasmodium parasites, especially P. knowlesi and P. cynomolgi in the long-tailed macaques in Palawan presents risks for zoonotic transmission in the area.

Gastrointestinal parasites have diverse life cycles that can involve people, animals, and the environment (e.g., water and soil), demonstrating the utility of One Health frameworks in characterizing infection risk. Kosumpee Forest Park (Thailand) is home to a dense population of long-tailed macaques ( Macaca fascicularis ) that frequently interact with tourists and local residents. Our study investigated the presence of zoonotic parasites, and barriers to healthy coexistence by conducting stool analysis on macaques ( N = 102) and people ( N = 115), and by examining risk factors for infection with a household questionnaire ( N = 95). Overall, 44% of macaques and 12% of people were infected with one or more gastrointestinal helminths, including Strongyloides spp., Ascaris spp., and Trichuris sp. An adults-only generalized linear mixed model identified three factors significantly associated with human infection: household size, occupational exposure, and contact with macaque feces at home. Participants identified both advantages and disadvantages to living in close contact with macaques, suggesting that interventions to improve human and animal health in Kosumpee Forest Park would be welcome.

Research on host-associated microbiomes has highlighted major divisions between the role of eukaryotes in free-living and symbiont systems. These trends call into question the relevance of macroecological processes to host-associated systems and the relative importance of parasitism, commensalism, and mutualism as evolutionary patterns across the domains of life. However, it is unclear as to whether these apparent differences reflect biological realities or methodologies in community characterization: free-living eukaryotes tend to be characterized using metabarcoding whereas symbiont eukaryotes are typically characterized with microscopy. Here, we utilize an Illumina high-throughput metabarcoding approach to characterize the diversity and dynamics of eukaryotic symbiont communities in the feces of a wild non-human primate, Macaca fascicularis, revealing functionally and taxonomically diverse communities of eukaryotes hitherto unreported from any vertebrate. Importantly, community assembly was consistent with top-down and bottom-up trophic food web dynamics, highlighting the applicability of macroecological principles to these communities. Ultimately, our findings highlight vertebrate-associated symbiont communities of the gut that are much more similar to free-living systems than previously realized. Additionally, our results support a role for symbiosis as a major recurrent life strategy among eukaryotes and highlight the potential for vertebrates to host vast reservoirs of unexplored eukaryotic diversity., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The simian parasite Plasmodium knowlesi can cause severe and fatal human malaria. However, little is known about the pathogenesis of this disease. In falciparum malaria, reduced red blood cell deformability (RBC-D) contributes to microvascular obstruction and impaired organ perfusion. In P knowlesi infection, impaired microcirculatory flow has been observed in Macaca mulatta (rhesus macaques), unnatural hosts who develop severe and fatal disease. However, RBC-D has not been measured in human infection or in the natural host M fascicularis (long-tailed macaques). Using ektacytometry, we measured RBC-D in adults with severe and non-severe knowlesi and falciparum malaria and in healthy controls. In addition, we used micropipette aspiration to determine the relative stiffness of infected RBCs (iRBCs) and uninfected RBCs (uRBCs) in P knowlesi -infected humans and M fascicularis Ektacytometry demonstrated that RBC-D overall was reduced in human knowlesi malaria in proportion to disease severity, and in severe knowlesi malaria, it was comparable to that of severe falciparum malaria. RBC-D correlated inversely with parasitemia and lactate in knowlesi malaria and HRP2 in falciparum malaria, and it correlated with hemoglobin nadir in knowlesi malaria. Micropipette aspiration confirmed that in humans, P knowlesi infection increased stiffness of both iRBCs and uRBCs, with the latter mostly the result of echinocytosis. In contrast, in the natural host M fascicularis , echinocyte formation was not observed, and the RBC-D of uRBCs was unaffected. In unnatural primate hosts of P knowlesi , including humans, reduced deformability of iRBCs and uRBCs may represent a key pathogenic mechanism leading to microvascular accumulation, impaired organ perfusion, and anemia., (© 2018 by The American Society of Hematology.)

Ciliate protozoa of the genus Balantioides can parasitize a variety of animals. The morphology of the evolutionary forms of the parasite and the host species affected have long been the only characteristics used to taxonomically identify the species of these protozoa, but these variables are not very precise. To confirm species identity, molecular biology tools are currently used. In this context, this study aimed to analyze protozoan isolates maintained in culture medium and from fecal samples from captive animals in Rio de Janeiro, Brazil, by means of molecular tools. Forty isolates maintained in Pavlova modified medium (30 were isolated from feces of pigs and 10 from feces of cynomolgus macaques) were analyzed. In addition, 34 fecal samples (8 from pigs, 8 from cynomolgus macaques and 18 from rhesus macaques) containing Balantioides coli-like cysts were analyzed. All samples were subjected to DNA extraction and the polymerase chain reaction (PCR) to amplify the fragment ITS1 - 5.8s rRNA - ITS2, and the PCR products were purified and sequenced. All samples (100%) presented sequences that were grouped in the Balantioides coli cluster. The type A0 variant predominated. These sequences were 96% to 99% identical to those deposited in GenBank, including a B. coli sequence that had been obtained from human fecal material in Bolivia. It seems that the culturing system did not select variants, because this variant was also seen in the amplified sequences of fecal samples containing cysts. The isolate sequences in the cultures showed few ambiguities and substitutions, thus generating reliable chromatograms. This was the first study to identify B. coli in captive animals in Brazil, through molecular biology. In addition, it was the first to evaluate a large panel of isolates of the parasite through culturing., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Anopheles balabacensis of the Leucospyrus group has been confirmed as the primary knowlesi malaria vector in Sabah, Malaysian Borneo for some time now. Presently, knowlesi malaria is the only zoonotic simian malaria in Malaysia with a high prevalence recorded in the states of Sabah and Sarawak., Methodology/principal Findings: Anopheles spp. were sampled using human landing catch (HLC) method at Paradason village in Kudat district of Sabah. The collected Anopheles were identified morphologically and then subjected to total DNA extraction and polymerase chain reaction (PCR) to detect Plasmodium parasites in the mosquitoes. Identification of Plasmodium spp. was confirmed by sequencing the SSU rRNA gene with species specific primers. MEGA4 software was then used to analyse the SSU rRNA sequences and bulid the phylogenetic tree for inferring the relationship between simian malaria parasites in Sabah. PCR results showed that only 1.61% (23/1,425) of the screened An. balabacensis were infected with one or two of the five simian Plasmodium spp. found in Sabah, viz. Plasmodium coatneyi, P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Sequence analysis of SSU rRNA of Plasmodium isolates showed high percentage of identity within the same Plasmodium sp. group. The phylogenetic tree based on the consensus sequences of P. knowlesi showed 99.7%-100.0% nucleotide identity among the isolates from An. balabacensis, human patients and a long-tailed macaque from the same locality., Conclusions/significance: This is the first study showing high molecular identity between the P. knowlesi isolates from An. balabacensis, human patients and a long-tailed macaque in Sabah. The other common simian Plasmodium spp. found in long-tailed macaques and also detected in An. balabacensis were P. coatneyi, P. inui, P. fieldi and P. cynomolgi. The high percentage identity of nucleotide sequences between the P. knowlesi isolates from the long-tailed macaque, An. balabacensis and human patients suggests a close genetic relationship between the parasites from these hosts.

The macaque parasite Plasmodium knowlesi is a significant concern in Malaysia where cases of human infection are increasing. Parasites infecting humans originate from genetically distinct subpopulations associated with the long-tailed (Macaca fascicularis (Mf)) or pig-tailed macaques (Macaca nemestrina (Mn)). We used a new high-quality reference genome to re-evaluate previously described subpopulations among human and macaque isolates from Malaysian-Borneo and Peninsular-Malaysia. Nuclear genomes were dimorphic, as expected, but new evidence of chromosomal-segment exchanges between subpopulations was found. A large segment on chromosome 8 originating from the Mn subpopulation and containing genes encoding proteins expressed in mosquito-borne parasite stages, was found in Mf genotypes. By contrast, non-recombining organelle genomes partitioned into 3 deeply branched lineages, unlinked with nuclear genomic dimorphism. Subpopulations which diverged in isolation have re-connected, possibly due to deforestation and disruption of wild macaque habitats. The resulting genomic mosaics reveal traits selected by host-vector-parasite interactions in a setting of ecological transition.

Background: The zoonotic Plasmodium knowlesi is a major cause of human malaria in Malaysia. This parasite uses the Duffy binding protein (PkDBPαII) to interact with the Duffy antigen receptor for chemokines (DARC) receptor on human and macaque erythrocytes to initiate invasion. Previous studies on P. knowlesi have reported distinct Peninsular Malaysia and Malaysian Borneo PkDBPαII haplotypes. In the present study, the differential binding activity of these haplotypes with human and macaque (Macaca fascicularis) erythrocytes was investigated., Methods: The PkDBPαII of Peninsular Malaysia and Malaysian Borneo were expressed on the surface of COS-7 cells and tested with human and monkey erythrocytes, with and without anti-Fy6 (anti-Duffy) monoclonal antibody treatment. Binding activity level was determined by counting the number of rosettes formed between the transfected COS-7 cells and the erythrocytes., Results: Anti-Fy6 treatment was shown to completely block the binding of human erythrocytes with the transfected COS-7 cells, thus verifying the specific binding of human DARC with PkDBPαII. Interestingly, the PkDBPαII of Peninsular Malaysia displayed a higher binding activity with human erythrocytes when compared with the Malaysian Borneo PkDBPαII haplotype (mean number of rosettes formed = 156.89 ± 6.62 and 46.00 ± 3.57, respectively; P < 0.0001). However, no difference in binding activity level was seen in the binding assay using M. fascicularis erythrocytes., Conclusion: This study is the first report of phenotypic difference between PkDBPαII haplotypes. The biological implication of this finding is yet to be determined. Therefore, further studies need to be carried out to determine whether this differential binding level can be associated with severity of knowlesi malaria in human.

Objective: To investigate the infection situation of blood parasitic protozoa in farmed Macaca fascicularis in an animal breeding ground in Nanning, Guangxi Zhuang Autonomous Region, so as to provide the evidence for the prevention and control of human blood parasitic protozoa., Methods: A total of 993 blood samples from farmed M. fascicularis were collected and stored on FTA cards. Among them, 550 thin blood smears were made. Each 10 samples were mixed in groups, and then the Babesia spp . and Plasmodium spp. in the blood of M. fascicularis were detected by Nest-PCR and PCR, respectively. The positive groups were tested individually. The thin blood smears stained with Giemsa were examined microscopically when PCR reported the samples were positive., Results: When detected by Nest-PCR, the positive rate of Babesia. microti was 6.95% (69/993); only 1 positive sample with Plasmodium inui was detected by PCR. Among the 22 positive thin blood smears detected by PCR, 16 were determined with B. microti by microscopic examinations, on which the ring forms could be observed in the erythrocytes, but no hemozoin., Conclusions: The positive rate of B. microti in M. fascicularis in Guangxi Zhuang Autonomous Region is high, and the animal may play a role as a reservoir host in the transmission of B. microti . In the screening of B. microti with low infection density, Nest-PCR has a higher sensitivity.

Background: Cynomolgus macaques (Macaca fascicularis) represent a feasible model for research on Chagas disease since natural T. cruzi infection in these primates leads to clinical outcomes similar to those observed in humans. However, it is still unknown whether these clinical similarities are accompanied by equivalent immunological characteristics in the two species. We have performed a detailed immunophenotypic analysis of circulating leukocytes together with systems biology approaches from 15 cynomolgus macaques naturally infected with T. cruzi (CH) presenting the chronic phase of Chagas disease to identify biomarkers that might be useful for clinical investigations., Methods and Findings: Our data established that CH displayed increased expression of CD32+ and CD56+ in monocytes and enhanced frequency of NK Granzyme A+ cells as compared to non-infected controls (NI). Moreover, higher expression of CD54 and HLA-DR by T-cells, especially within the CD8+ subset, was the hallmark of CH. A high level of expression of Granzyme A and Perforin underscored the enhanced cytotoxicity-linked pattern of CD8+ T-lymphocytes from CH. Increased frequency of B-cells with up-regulated expression of Fc-γRII was also observed in CH. Complex and imbricate biomarker networks demonstrated that CH showed a shift towards cross-talk among cells of the adaptive immune system. Systems biology analysis further established monocytes and NK-cell phenotypes and the T-cell activation status, along with the Granzyme A expression by CD8+ T-cells, as the most reliable biomarkers of potential use for clinical applications., Conclusions: Altogether, these findings demonstrated that the similarities in phenotypic features of circulating leukocytes observed in cynomolgus macaques and humans infected with T. cruzi further supports the use of these monkeys in preclinical toxicology and pharmacology studies applied to development and testing of new drugs for Chagas disease.

The aim of this study was to examine helminths and protozoans in cynomolgus macaques (Macaca fascicularis) imported from registered breeding facilities in China and their relation to health risks for non-human primate handlers in biomedical research centers and in breeding facilities. Fresh fecal samples were collected from a total of 443 M. fascicularis and analyzed by copromicroscopical analysis, immunoenzymatic, or molecular assays. As to helminths, whose eggs were shed in 2.03% of the samples, Trichuris and Oesophagostomum were the only two taxa found, with low prevalence and low eggs per gram (EPG) values. Protozoans were more frequently detected (87.40%), with Entamoeba coli (85.19%) and Endolimax nana (79.26%) as the most prevalent species shed. Other parasites found by fecal smear examination were uninucleated-cyst-producing Entamoebas (78.52%), Iodamoeba bütschlii (42.96%), and Chilomastix mesnili (24.44%), while cysts of Balantidium coli (22.2%) were only observed by sedimentation. No coproantigens of Giardia duodenalis, Cryptosporidium spp., and Entamoeba histolytica complex were detected. Blastocystis sp. infection was noticed in 87.63% of macaques by PCR. These cynomolgus monkeys were infected with many subtypes (ST1, ST2, ST3, ST5, and ST7), where the predominant Blastocystis sp. subtypes were ST2 (77.5%), followed by ST1 (63.5%). Data collected confirmed the presence of potentially zoonotic parasites and a high parasite diversity, suggesting the need for appropriate and sensitive techniques to adequately control them and related health risks for handlers of non-human primates in biomedical research centers and in breeding facilities.

Background: Primates are important reservoirs for human diseases, but their infection status and disease dynamics are difficult to track in the wild. Within the last decade, a macaque malaria, Plasmodium knowlesi, has caused disease in hundreds of humans in Southeast Asia. In order to track cases and understand zoonotic risk, it is imperative to be able to quantify infection status in reservoir macaque species. In this study, protocols for the collection of non-invasive samples and isolation of malaria parasites from naturally infected macaques are optimized., Methods: Paired faecal and blood samples from 60 Macaca fascicularis and four Macaca nemestrina were collected. All animals came from Sumatra or Java and were housed in semi-captive breeding colonies around West Java. DNA was extracted from samples using a modified protocol. Nested polymerase chain reactions (PCR) were run to detect Plasmodium using primers targeting mitochondrial DNA. Sensitivity of screening faecal samples for Plasmodium was compared to other studies using Kruskal Wallis tests and logistic regression models., Results: The best primer set was 96.7 % (95 % confidence intervals (CI): 83.3-99.4 %) sensitive for detecting Plasmodium in faecal samples of naturally infected macaques (n = 30). This is the first study to produce definitive estimates of Plasmodium sensitivity and specificity in faecal samples from naturally infected hosts. The sensitivity was significantly higher than some other studies involving wild primates., Conclusions: Faecal samples can be used for detection of malaria infection in field surveys of macaques, even when there are no parasites visible in thin blood smears. Repeating samples from individuals will improve inferences of the epidemiology of malaria in wild primates.

Human malaria parasite species were originally acquired from other primate hosts and subsequently became endemic, then spread throughout large parts of the world. A major zoonosis is now occurring with Plasmodium knowlesi from macaques in Southeast Asia, with a recent acceleration in numbers of reported cases particularly in Malaysia. To investigate the parasite population genetics, we developed sensitive and species-specific microsatellite genotyping protocols and applied these to analysis of samples from 10 sites covering a range of >1,600 km within which most cases have occurred. Genotypic analyses of 599 P. knowlesi infections (552 in humans and 47 in wild macaques) at 10 highly polymorphic loci provide radical new insights on the emergence. Parasites from sympatric long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) were very highly differentiated (FST = 0.22, and K-means clustering confirmed two host-associated subpopulations). Approximately two thirds of human P. knowlesi infections were of the long-tailed macaque type (Cluster 1), and one third were of the pig-tailed-macaque type (Cluster 2), with relative proportions varying across the different sites. Among the samples from humans, there was significant indication of genetic isolation by geographical distance overall and within Cluster 1 alone. Across the different sites, the level of multi-locus linkage disequilibrium correlated with the degree of local admixture of the two different clusters. The widespread occurrence of both types of P. knowlesi in humans enhances the potential for parasite adaptation in this zoonotic system.

Helminthes have the capacity to modulate host immunity, leading to positive interactions with coinfecting microparasites. This phenomenon has been primarily studied during coinfections with a narrow range of geo-helminthes and intracellular microparasites in human populations or under laboratory conditions. Far less is known regarding differences in coinfection dynamics between helminth types, the range of microparasites that might be affected or the overall community-level effects of helminth infections on microparasites in wild systems. Here, we analysed the presence/absence and abundance patterns of enteric parasites in long-tailed macaques (Macaca fascicularis) on the island of Bali, Indonesia, to assess whether naturally occurring helminth infections were associated with increased shedding of the most common intracellular (Cryptosporidium spp., Isospora spp.) and extracellular (Entamoeba spp., Giardia spp.) microparasites. We also comparatively assessed the statistical correlations of different helminth taxa with microparasite shedding to determine if there were consistent relationships between the specific helminth taxa and microparasites. Helminth infections were associated with increased shedding of both intracellular and extracellular microparasites. Platyhelminthes repeatedly displayed strong positive correlations with several microparasites; while nematodes did not. Our results indicate that helminthes can influence microparasite community shedding dynamics under wild conditions, but that trends may be driven by a narrow range of helminthes.

Trypanosoma cruzi, the causative agent of Chagas disease, is endemic in south Texas due to the abundant vector and wild small mammalian reservoir populations. This situation predisposes nonhuman primate colonies exposed to outdoor housing to infection from ingestion or bite of triatomid insects. Using a T. cruzi-specific real-time PCR and Trypanosome spp.-specific ELISA, we revealed a prevalence rate of 8.5% in a colony of outdoor-housed cynomolgus macaques. By using a discriminating kinetoplastid minicircle PCR, we eliminated the possibility of mixed prevalence with nonpathogenic trypanosomes and showed the ELISA results were specific for T. cruzi. In this study, we found an inverse relationship between antibody titers and circulating parasite load. Also, 23% of T. cruzi IgG ELISA-positive macaques were negative by real-time PCR. Furthermore, in a subset of infected macaques, cardiac tissue was infiltrated by inflammatory mononuclear cells and contained T. cruzi genomic and kinetoplast DNA despite lacking microscopic evidence of discrete parasite stages. In addition, 19% of the infected macaques had titers for cardiac troponin I autoantibody, which could contribute to autoimmune myocarditis or interfere with circulating troponin I measurements. These findings indicate the possibility of T. cruzi to interfere with the assessment of cardiac safety signals in preclinical toxicology and safety pharmacology studies and the necessity for prestudy screening for T. cruzi in outdoor-housed nonhuman primates from endemic areas.

An adult male cynomolgus macaque (Macaca fascicularis) from Mauritius arrived at our facility in France after a 1-year period of quarantine in Spain. Clinical examination soon after arrival revealed the presence of numerous firm cutaneous and subcutaneous nodules (0.1-0.5 cm diameter) in the scrotal and inguinal areas, and persistent mild eosinophilia. On necropsy examination additional similar nodules were found in the peritoneum and abdominal wall, omentum and mesentery. Microscopical examination revealed disseminated eosinophilic granulomas containing tapeworm larvae identified as Spirometra erinaceieuropaei by direct sequencing of the cox1 gene., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human–wildlife interactions have reached unprecedented levels, and humans are influencing the earth’s ecosystems more rapidly and extensively than ever before. This situation is cause for serious concern, especially since disease interactions between wildlife and humans have been recognized as major conservation threats. In this study, long-tailed macaques, Macaca fascicularis, from 2 forest parks located in north-eastern Thailand were investigated to determine the influence of habitat modification by humans on helminth parasite associations in non-human primates. Macaque populations with contact to anthropogenically modified environments were compared with sylvatic groups in nearby natural environments. In order to test for human–non-human primate transmission of parasites, the local human populations were also examined. Humans were infected with a number of potentially pathogenic parasites, including Opisthorchis viverrini and Strongyloides stercoralis. However, eggs of these helminths were not detected in macaque feces. Thus, no direct parasite transfer from humans to non-human primates could be confirmed. However, macaque groups with more frequent contact with human modified habitats, and a higher portion of human-provided food in their diet, had significantly higher prevalences and intensities of Strongyloides fuelleborni and of an intestinal fluke (probably Haplorchis sp.) than sylvatic groups. Positive correlations were found between the time foraging on the ground and infection with S. fuelleborni, and the amount of human-provided food and intestinal fluke infection. Human alteration of habitat and associated modifications in nonhuman primate behaviour are likely to play a role in determining the occurrence, prevalence and intensity of zoonotic helminth infection of wild non-human primates.

This paper discusses the colonization of the stratiomyid species Ptecticus melanurus (Walker) (Diptera: Stratiomyidae) in monkey carrion and its potential for the determination of the minimum time since death (PMI). A study was conducted in a tropical forest at Bangi, Malaysia from 13 November 2009 to 8 June 2011. Twelve monkey carcasses (Macaca fascicularis Raffles) were used and divided in equal number into three different field trials. Adults of P. melanurus were first observed on monkey carrions on the second day the carcasses were placed in the field while their penultimate instar larvae were found in the wet soil under and beside carcass from day 8 to 31 days postmortem.

Background: An unusual case of visceral pentastomiasis in a male adult long-tailed macaque imported from China is reported., Methods: The monkey was part of a toxicologic study. A massive accumulation of C-shaped parasites in various visceral organs was found post-mortem., Results: Based on the morphology of the nymphs, pentastomiasis was diagnosed etiopathologically. The pentastome genus and species was identified as Armillifer agkistrodontis by PCR and respective sequencing., Conclusion: Molecular diagnostic methods are necessary tools to determine the exact species involved., (© 2012 John Wiley & Sons A/S.)

Objective: To observe the ultrastructure of nymphal Armillifer sp. isolated from Macaca fascicularis by using scanning electron microscope (SEM), and analyze the phylogenetic relationships based on 18S rRNA gene sequences., Methods: The parasite samples stored in 70% alcohol were fixed by glutaraldehyde and osmium peroxide. Ultrastructural characters of those samples were observed under SEM. Amplification and sequencing of the 18S rRNA gene were performed following the extraction of total genome DNA. Sequence analysis was performed based on multiple alignment using ClustalX1.83, while phylogenetic analysis was made by Neighbor-Joining method using MEGA4.0., Results: The nymphs were in cylindrical shape, the body slightly claviform tapering to posterior end. Abdominal annuli were gradually widened from anterior to posterior parts, the 12th-13th abdominal annuli of which were similar in width. The annuli ranged closer in the front half body, whereas in the latter part there were certain gaps between them. The circular-shaped mouth located in the middle of head ventrally. Folds were seen in inner margin of the mouth with a pair of curved hooks on both sides above it which practically disposed in a straight line. Two pairs of large sensory papillae were observed symmetrically over the last thoracic annulus of cephalothoraxs lying below the outer hook, and the first abdominal annulus was near the median ventral line. The number of abdominal annuli was 29, not including 2 incomplete terminal annuli. Rounded sensory papillae were fully distributed on the body surface, except the dorsal side of head and the ventral part of the terminal annulus. Agglomerate-like anus opening was observed at the end of ventral abdominal annuli and distinctly sub-terminal. These morphological features demonstrated that the nymphs were highly similar with that of Armillifer moniliformis Diesing, 1835. A fragment of 18SrRNA gene (1 836 bp) sequences was obtained by PCR combined with sequencing, and was registered to the GeneBank database with an accession number HM048870. The phylogenetic tree indicated that A. moniliformis, A.agkistrodon and A.armillatus were at the same clade with a bootstrap value at 95%, and A. moniliformis and A. agkistrodon were solo at a clade with a bootstrap value of 75%., Conclusion: The nymphs isolated from Macaca fascicularis are identified as A. moniliformis temporarily.

Long-tailed macaques (Macaca fascicularis) are bred in China for export and for use in experiments. Entamoeba infections in captive long-tailed macaques were surveyed in one of the biggest colonies located in Guangxi Province, China. One stool sample was obtained from each of the 152 different cages representing >3,000 macaques in the colony. The samples were examined by PCR for five Entamoeba species. The number of detected Entamoeba coli infections comprised 94% of the samples, 93% for Entamoeba chattoni, and 83% for Entamoeba dispar. In contrast, Entamoeba histolytica and Entamoeba nuttalli were not detected. Six isolates of E. dispar were obtained by culture in Tanabe-Chiba medium. Analysis of serine-rich protein genes in these isolates showed two genotypes, one of which is identical to that of the E. dispar SAW760 strain in humans. This suggests transmission of E. dispar between humans and nonhuman primates. These results demonstrate that Entamoeba infections are common, but virulent Entamoeba species are absent in this colony. This work also confirms the need for monitoring with PCR-based identification of Entamoeba species for captive macaques in breeding colonies to ensure animal health and protection of humans from zoonotic hazards.

The distribution of wildlife parasites in a landscape is intimately tied to the spatial distribution of hosts. In parasite species, including many gastrointestinal parasites, with obligate or common environmental life stages, the dynamics of the parasite can also be strongly affected by geophysical components of the environment. This is especially salient in host species, for example humans and macaques, which thrive across a wide variety of habitat types and quality and so are exposed to a wealth of environmentally resilient parasites. Here, we examine the effect of environmental and anthropogenic components of the landscape on the prevalence, intensity, and species diversity of gastrointestinal parasites across a metapopulation of long-tailed macaques on the island of Bali, Indonesia. Using principal-components analysis, we identified significant interaction effects between specific environmental and anthropogenic components of the landscape, parsing the Balinese landscape into anthropogenic (PC1), mixed environment (PC2), and non-anthropogenic (PC3) components. Further, we determined that the anthropogenic environment can mitigate the prevalence and intensity of specific gut parasites and the intensity of the overall community of gut parasites, but that non-anthropogenically driven landscape components have no significant effect in increasing or reducing the intensity or prevalence of the community of gut parasites in Balinese macaques.

Little is known of the seroprevalence of Toxoplasma gondii in bred cynomolgus monkeys ( Macaca fascicularis ) in China. In the present survey, 240 serum samples of cynomolgus monkeys from 4 nonhuman primate centers in Guangxi Zhuang Nationality Autonomous Region (GX), and 120 serum samples from 2 nonhuman primate centers in Guangdong Province (GD) were obtained, and the modified agglutination test (MAT) was performed to assay for anti- T. gondii antibodies. Antibodies to T. gondii (MAT titer of 1:16 or higher) were found in 0 of 120 (0/120) samples in GD, and 5 (2.1%) of 240 sera with titers of 1:16 in 1, 1:64 or higher in 4 in primate centers from GX. The total prevalence of T. gondii in bred cynomolgus monkeys was 1.4% (5/360, MAT titer > or = 1:16) in southern China according to our investigation. To our knowledge, this is the first seropositive report in China on the prevalence of anti- T. gondii antibodies in bred cynomolgus monkeys using MAT.