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Article

Isolation and Genetic Characterization of Toxoplasma gondii from a Patas Monkey (Erythrocebus patas) in China

by
Liulu Yang
1,
Hongjie Ren
1,
Niuping Zhu
1,
Shilin Xin
1,
Gaohui Mao
1,
Yiheng Ma
1,
Junbao Li
2,
Qunchao Liang
3 and
Yurong Yang
1,*
1
Veterinary Pathology, Henan Agricultural University, Zhengzhou 450000, China
2
Zhengzhou Zoo, Zhengzhou 450000, China
3
Henan Yinji Jiabao Amusement Park Management Co., Ltd., Xinmi 452300, China
*
Author to whom correspondence should be addressed.
Genes 2023, 14(8), 1606; https://doi.org/10.3390/genes14081606
Submission received: 30 June 2023 / Revised: 7 August 2023 / Accepted: 8 August 2023 / Published: 10 August 2023
(This article belongs to the Special Issue Genetic and Immunological Studies of Parasite–Host Interactions)
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Abstract

:
Many cases of Toxoplasma gondii infection have been reported worldwide in non-human primates (NHPs), especially in captive New World monkeys. However, few studies on toxoplasmosis in Old World monkeys have been conducted. In this study, serological and molecular biological analyses were carried out to look for T. gondii antibodies and T. gondii infection in 13 NHPs from China. T. gondii infection was confirmed in 8 NHP cases. T. gondii antibodies were detected in 1/5 New World monkeys and in 4/7 Old World monkeys. T. gondii DNA was detected in 3/5 New World monkeys and 5/7 Old World monkeys. The one ring-tailed lemur was negative for both antibodies and DNA of T. gondii. The most common clinical manifestations of T. gondii infection were malaise, poor appetite, emaciation, and foamy nasal discharge. The most common histopathological findings were interstitial pneumonia, necrotic hepatitis, necrotizing myocarditis, lymphadenitis, and necrotic splenitis. One viable T. gondii strain was successfully isolated from the myocardium of a patas monkey (Erythrocebus patas) by bioassay in mice. T. gondii tachyzoites were obtained from cell cultures and were designated as TgMonkeyCHn2. The genotype of this strain belongs to ToxoDB genotype #9, and the allele of ROP18/ROP5 gene was 3/6. TgMonkeyCHn2 tachyzoites were avirulent in Swiss mice. To our knowledge, this is the first report of fatal toxoplasmosis in a patas monkey. T. gondii infection in patas monkeys may indicate environmental contamination by oocysts. The patas monkey is a new host record for T. gondii.

1. Introduction

Toxoplasma gondii is a zoonotic intracellular apicomplexan protozoan parasite that infects most warm-blooded animals, including non-human primates (NHPs) and humans [1,2]. T. gondii infection in captive animals is of interest because many species, especially NHPs and some Australian marsupials, often die due to severe toxoplasmosis [3,4,5,6,7]. New World monkeys, such as squirrel monkeys, saki monkeys, howler monkeys, and ring-tailed lemurs, are more susceptible to T. gondii than Old World monkeys and often develop fatal disease [6,8,9,10]. To date, several T. gondii genotypes from viable strains (n = 7) in NHPs have been identified worldwide, namely ToxDB #163, ToxDB #4, ToxDB #206, and four mixed genotypes [11,12,13,14]. The objective of the present investigation was to estimate the T. gondii infection status of captive NHPs from China by testing IgG antibodies, isolation, and genetic characterization of T. gondii. Additionally, possible sources of T. gondii infection in the zoos were evaluated to prevent future occurrences of toxoplasmosis.

2. Materials and Methods

2.1. Sample Collection and Clinical Case Report

Between 2020 and 2021, 13 NHPs died in zoos in Henan province, China (Table 1). Whole or partial tissue samples, including brain, heart, liver, spleen, lung, kidney, tongue, leg muscles, diaphragm, and intestines, were collected from 13 monkeys and transported to the veterinary pathology laboratory, Henan Agricultural University (Zhengzhou, Henan, China) for pathological diagnosis. Details of these cases are provided in Table 1.

2.2. T. gondii Antibody and T. gondii DNA Were Detected in Monkey Tissues

The modified agglutination test (MAT) was used to detect the anti-T. gondii antibody in the heart fluid of the monkeys [15]. Formalin-treated tachyzoites of T. gondii were obtained from the University of Tennessee Research Foundation (Knoxville, TN, USA; https://utrf.tennessee.edu/, accessed on 1 February 2020). T. gondii-positive mouse sera were provided by Dr. J. P. Dubey (Beltsville, MD, USA) and were used as the reference sera. The heart juice samples were tested at a ratio of 1:2, and the dilution was then doubled to a titer of 1: 256. Positive samples were further diluted from 1:200 to the final titer. Negative and positive controls were included in each plate. DNA was extracted from monkey tissues and pepsin-digested tissues using a DNA extraction kit (DP304; Tiangen Biotec Co., Tiangen, China). T. gondii DNA was checked using the specific primer pair TOX5/TOX8 (5′-CGCTGCAGACACAGTGCATCTGGATT-3′ and 5′-CCCA GCTGCGTCTGTCGGGAT-3′) in PCR assays [16]. PCR reaction process: initial denaturation at 94 °C for 2 min; 35 cycles of amplification (94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min) and final extension at 72 °C for 10 min, 6 °C forever. The PCR product length was estimated to be 450 bp, and both negative and positive controls were included.

2.3. Histopathological Analysis

Tissue samples from all monkeys were fixed in 10% (v/v) neutral-buffered formalin. The tissues were processed using conventional histological techniques and embedded in paraffin. Paraffin sections (5 μm in thickness) of the samples were prepared and stained with hematoxylin and eosin (HE). Immunohistochemical (IHC) staining was performed on monkeys suspected to be infected with T. gondii [17,18]. The rabbit anti-T. gondii polyclonal antibody was provided by Dr. Dubey (Beltsville, MD, USA Agricultural Research Service, USDA). The rabbit-specific HRP/DAB immunohistochemical assay kit was purchased from Abcam (ab64264). Brain tissue sections from mice infected with T. gondii (VEG strain) were used as positive controls for IHC staining (provided by Dr. Dubey, ARS, USDA).

2.4. Isolation of Viable T. gondii from Monkey Tissues Using Mice Bioassay

Sample tissues from nine NHPs were bioassayed in mice as per previously described methods [1,17] (Table 1). Briefly, tissue samples (50 g, including 15 g brain, 15 g heart, 10 g leg muscle, 5 g diaphragm, and 5 g tongue) from monkeys were digested in a pepsin solution and inoculated into Swiss mice (n = 2–5) or gamma interferon (IFN-γ−/−) knockout mice (n = 1–2). The survival time of IFN-γ−/− mice infected with T. gondii is short, which is an ideal animal model of acute toxoplasmosis [19,20,21,22]. In addition, the homogenized tissues of case#16 (red howler monkey: myocardium, diaphragm, and skeletal muscles), case#26 (white-cheeked gibbon: pleural and peritoneal effusion) were inoculated directly into Swiss mice. Tissue samples (brain, heart, spleen, lung, liver, kidney, skeletal muscle, lymph nodes) from case#18 (squirrel monkey), case#24 (mona monkey), case#25 (mona monkey), and case#28 (ring-tailed lemur) were homogenized and inoculated into gamma interferon (IFN-γ−/−) knockout mice (n = 1) or Swiss mice (n = 3–5), respectively. Clinical signs were recorded daily. At 30 days after inoculation (dpi), the serum anti-T. gondii antibodies were detected by MAT at 1:25 and 1:200 dilutions. Tachyzoites or cysts were examined in the lungs or brains of dead and euthanized mice. If no cysts or tachyzoites were found in mouse tissues, then mouse lung, brain, heart, and tongue homogenates were subpassaged into a new group of mice.

2.5. In Vitro Cultivation and Genotyping

Tissues (brain, lung, or mesenteric lymph nodes) from T. gondii-positive mice were seeded in Vero cell culture flasks (RPMI 1640, 3% fetal bovine serum, 37 °C, and 5% CO2) [1]. DNA was extracted from the cell culture-derived tachyzoites. T. gondii strain genotyping was performed using 10 PCR-RFLP genetic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) as previously described by Su et al. [23]. Genotyping of the virulence genes ROP18 and ROP5 was performed as previously described [24,25,26]. All batches contained T. gondii reference DNA.

2.6. Evaluation of the Virulence of the T. gondii Strain Isolated from Monkeys

We evaluated the virulence of T. gondii isolated from monkeys in Swiss mice [27]. T. gondii tachyzoites were collected from cell cultures. They were counted in a disposable hemocytometer and diluted 10-fold from 10−1 to 10−4 to reach an endpoint of <1 tachyzoite. Then, <1, 100, 101, 102, and 103 tachyzoites were intraperitoneally inoculated into four Swiss mice at each dilution. Clinical signs were recorded, and the mice were monitored daily. After 30 days, all surviving mice were bled and tested for anti-T. gondii IgG antibodies by MAT with titers between 1:25 and 1:200. Mice were euthanized at 60 dpi, after which their brains were examined and tissue cysts enumerated. All tissues were fixed in 10% (v/v) neutral-buffered formalin. Virulence was evaluated according to the percentage of dead T. gondii-positive mice.

2.7. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). Data were analyzed using the Chi-squared test or Fisher’s exact test. Statistical significance was set at p < 0.05.

3. Results

3.1. Clinical Signs, Pathological Evaluation, and Immunohistochemistry

Samples were collected from 13 NHPs between 2020 and 2021 (Table 1). T. gondii parasites were not found in IHC- and HE-stained tissue sections of these monkeys. The most common clinical manifestations were emaciation (46%, 6/13), malaise (31%, 4/13), and poor appetite (31%, 4/13). Moreover, renal insufficiency (69%, 9/13), age-related atrophy (54%, 7/13), necrotic myocarditis (39%, 5/13), interstitial pneumonia (31%, 2/13), necrotic enteritis (31%, 2/13), necrotic metritis (8%, 1/13), suppurative cystitis (8%, 1/13), lymphadenitis (8%, 1/13), and necrotic splenitis (8%, 1/13) were observed. Some histopathological lesions are shown in Figure 1.

3.2. T. gondii Examination by MAT and PCR

Heart fluid and tissue samples from 13 NHPs were tested for T. gondii serology and molecular biology; all monkeys were adults. PCR confirmed that eight monkeys were infected with T. gondii.
T. gondii antibodies were detected in 38% (5/13) of the monkeys, with a titer of 1:4 in one case, 1:8 in one case, 1:64 in two cases, and 1:3200 in one case. The MAT showed that 57% (4/7) of Old World monkeys had T. gondii antibodies, while 20% (1/5) of New World monkeys had T. gondii antibodies. However, the difference in T. gondii antibodies between Old World and New World monkeys was not significant (p = 0.2929). The proportion of female monkeys with T. gondii antibodies was more (57%, 4/7) than the proportion of male monkeys with T. gondii antibodies (17%, 1/6), although this difference was not significant (p = 0.2657).
T. gondii DNA was detected in 8 of the 13 NHPs. The proportion of Old World monkeys infected with the T. gondii was more (71%, n = 5/7) than that of New World monkeys (60%, n = 3/5), and one ring-tailed lemur was negative for T. gondii DNA. Parasite DNA was mainly observed in the spleen, tongue, lymph nodes, lungs, and kidneys but less frequently in the heart, skeletal muscles, intestines, and other organs (Table 1). In addition, only four PCR-positive cases showed T. gondii antibody transformation (≥1:4) (Table 1).

3.3. Viable T. gondii Was Isolated from Monkey Tissue Samples Using Mouse Bioassays and Genetic Characterization

Tissues from 9 out of 13 NHPs were individually tested via bioassay in mice (Table 1). In the Tox#20-31 group, four mice were inoculated with digestive fluid from the myocardial and skeletal muscles of case#21 (patas monkey); two IFN-γ−/− mice (M#511, M#586) died after showing signs of toxoplasmosis at 18–19 dpi and T. gondii tachyzoites were observed in the lungs (Figure 2). The remaining two Swiss mice had seroconverted antibodies for T. gondii at 30 dpi, and cysts (30 cysts from M#512 and 110 cysts from M#458) were observed in the brain at 317 dpi. The T. gondii strain from the lung of M#511 was successfully propagated in cell cultures (17 DPI) and was designated as TgMonkeyCHn2. T. gondii tachyzoites were found and confirmed in the mouse lungs by IHC staining (Figure 2). Genotyping the isolate of TgMonkeyCHn2 indicated that it was ToxoDB#9 using 10 PCR-RFLP markers (Table 2). The ROP18/ROP5 allele of TgMonkeyCHn2 isolates was 3/6.
In the other eight cases, none of the mice (n = 2–5) had antibodies against T. gondii, and no parasite was observed in mice tissues at 30–393 dpi.

3.4. Virulence Evaluation of TgMonkeyCHn2 by Mice

As shown in Table 3, 103 TgMonkeyCHn2 tachyzoites infected all Swiss mice, as confirmed by MAT at 30 dpi. Most mice were asymptomatic within 60 dpi after intraperitoneal inoculation with tachyzoites. However, two mice inoculated with 103 TgMonkeyCHn2 tachyzoites died of toxoplasmosis at 46 and 54 dpi, respectively, and T. gondii tachyzoites were detected in lung tissue. T. gondii cysts (0–60 cysts) were detected in mice brains when euthanized at 67 dpi.

4. Discussion

This study investigated T. gondii infection in 13 captive monkeys who died of suspected toxoplasmosis or other diseases in China zoos. Eight monkeys were confirmed to be infected with T. gondii by PCR. Four of the eight monkeys in which T. gondii DNA was detected were serologically negative (titer < 1:4). This may be because of acute infection and infected monkeys may die of the disease before they can produce detectable IgG titers. Most monkey toxoplasmosis cases result in widespread histopathological lesions and intralesional T. gondii organisms [28,29,30,31,32]. Unfortunately, neither immunohistochemical staining nor HE staining of 1 cm2 tissue sections from 13 monkeys revealed T. gondii cysts or tachyzoites. Moreira et al. also confirmed a case of toxoplasmosis in a black-and-gold howler monkey by increasing anti-T. gondii antibody titers (IFAT, 1:16–1:256 for 36 days); blood was tested for T. gondii nucleic acid, and T. gondii was isolated from the liver and heart, but no cysts or tachyzoites in the lung and liver were detected by HE and IHC staining [33]. Here, PCR (8/13) was more sensitive than histopathology (0/13) and serology (5/13), especially for acute T. gondii infection or parasitemia. We also conclude that serology is still an important method for diagnosing T. gondii infection in NHPs in addition to molecular methods; blood cell samples should be taken to allow for nucleic acid detection.
MAT has been widely used to detect anti-T. gondii IgG antibodies in serum or body fluids of animals and its effectiveness has been demonstrated with T. gondii isolated from pigs, lambs, chickens, and monkeys [34,35,36,37]. However, the validity of MAT in NHPs remains unclear. Previous studies using MAT at 1:16 and 1:20 dilutions have detected toxoplasmosis or isolated viable T. gondii strains in monkeys [31,32,37]. Here, a viable T. gondii strain was isolated from the striated muscles of case#21 (patas monkey), with a T. gondii antibody titer of 1:3200, and T. gondii infection was also confirmed by PCR detection in tissues (spleen, lung, kidney, and mesenteric lymph nodes). Previous studies have also detected high T. gondii antibody titers (1:1024, 1:10,240, 1:3200, and ≥1:500) in four other patas monkeys by MAT [11,38,39,40,41]. This may be related to their ground-dwelling habits, as they have more exposure to T. gondii oocysts than monkeys with tree-dwelling habits.
In this study, from the eight infected T. gondii NHPs, the parasite DNA was mainly observed in the spleen, lungs, tongue, lymph nodes, kidney, heart, skeletal muscles, and pancreas. Viable TgMonkeyCHn2 isolates were successfully obtained from the myocardial and skeletal muscles of a patas monkey at a titer of 1:3200 via a mouse bioassay. However, T. gondii DNA was not detected in the myocardial or skeletal muscles in this case. This result suggests that the density of T. gondii parasites in the striated muscle of this patas monkey was low. In a previous report, T. gondii DNA was detected in the lungs, liver, heart, and brain of squirrel monkeys that died of toxoplasmosis [31]. In addition, T. gondii DNA has been isolated from the liver and brain of a captive ring-tailed lemur with toxoplasmosis [32]. These results indicate the relatively low density of T. gondii parasites in the organs of some monkeys diagnosed with toxoplasmosis.
Although the higher susceptibility of New World monkeys to T. gondii has not been fully elucidated, their specific immune response to the parasite may be due to the lack of feline contact for more than 20 million years during the evolution of New World monkeys. T. gondii can infect Old World monkeys, and they can survive, and there have been no reports of clinical natural toxoplasmosis in Old World NHPs [11].
According to the summary of T. gondii viable strains isolated from humans and animals in China (Table S1), at least more than 16 ToxoDB RFLP-genotypes were found. These include ToxoDB RFLP-genotype #9, #1–#6, #10, #17–#18, #20, #204–#205, #292, #319, and mix genotypes. Among these viable T. gondii strains isolated, 65% were ToxoDB #9 (Chinese 1); 7% were ToxoDB #2; and 4% were ToxoDB #3, #10, and #205. T. gondii ToxoDB #9 was in clade D and haplogroup 13; beyond China, it is also distributed in Mexico [42,43], Colombia, Vietnam, Sri Lanka, West Indies, and Brazil [44,45,46,47,48,49]. Most of ToxoDB #9 strains were avirulent [50]; however, they have also been confirmed to be associated with clinical toxoplasmosis in humans [51,52], pigs [48], and mice [53]. The natural host range of ToxoDB #9 involves cats, humans, pigs, sheep, tigers, and cheetahs [50,51,53,54,55,56] (Table S1), and patas monkeys were also demonstrated to be natural intermediate hosts of T. gondii ToxoDB #9 in the present study.
Dubey et al. reported a high success rate of genotyping of T. gondii (ToxoDB#3, ToxoDB#9, ToxoDB#11, ToxoDB#21, ToxoDB#36) from tissues of NHPs that died of acute toxoplasmosis [11], indicating that we could genotype the samples directly. However, none of these samples were successfully genotyped in this study because of their low DNA concentration.
TgMonkeyCHn2 was non-lethal, and the number of brain cysts was low in Swiss mice. Recent studies have shown that the genetic diversity and population structure of T. gondii can affect its virulence [57]. In this study, TgMonkeyCHn2 may be an avirulent phenotype, which was predicted by ROP18 (allele 3) and ROP5 (allele 6) [24,25]. The virulence phenotype was supported by mouse infection (low cyst formation rate and avirulence). The ROP18/ROP5 type of TgMonkeyCHn1 (3/6) has also been confirmed to be non-lethal in Swiss mice [37].
Most primates in zoos were not serologically tested for T. gondii at the time of introduction. Thus, it is impossible to determine the time and source of infection. Patas monkeys are omnivorous, and their diet consists of fruits, seeds, leaves, flowers, buds, bird eggs, insects, and arthropods. The source of T. gondii infection in the patas monkey is unclear. Vertical transmission of T. gondii could occur in ring-tailed lemurs via endogenous transplacental transmission [58]; however, this is unclear in other monkey species. The patas monkey may have acquired T. gondii by ingesting oocysts from the feces of felids or mechanical transporters (insects, arthropods, zookeepers, or cleaning tools). This indicated that T. gondii oocysts contaminate the habitat environment (water and soil) of monkeys. The high prevalence of antibodies against T. gondii in stray cats (50%), captive tigers (80%), servals (100%), caracals (67%), and cheetahs (100%) in central China supports this hypothesis [59,60,61].
The present study is the first to isolate T. gondii from a patas monkey and the first molecularly confirmed case of T. gondii infection in patas monkeys, providing direct evidence that patas monkeys are intermediate hosts of T. gondii. Patas monkeys could be used as good sentinel animals for monitoring environmental T. gondii contamination.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes14081606/s1, Table S1: Summary of viable T. gondii isolates (n = 169) from animals and humans in China [62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77].

Author Contributions

L.Y. performed the experiments and wrote the manuscript. H.R., N.Z., S.X., G.M., Y.M., J.L. and Q.L. participated in the collection of samples. Y.Y. designed the study, analyzed the results, and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financed by the Henan Province modern agricultural industrial technology system (mutton sheep: HARS-22-15-G1).

Institutional Review Board Statement

This study was approved by the Institutional Animal Use Protocol Committee of Henan Agricultural University, China. The protocol was approved by the Beijing Association for Science and Technology. All monkey tissues and serum and mice were handled in strict accordance with the good animal practices of the Animal Ethics Procedures, Recycle-Reduce-Reuse Principle, and Guidelines of the China (Approval Code SYXK [Beijing] 2007-0023; date of approval: 1 February 2020).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data supporting the findings of this study are available from the corresponding author upon request. The TgMonkeyCHn2 isolates were cryopreserved and available for further analysis.

Acknowledgments

We thank Wei Huang and Nan Jiang (Henan Agricultural University, Zhengzhou, China) for collecting the samples.

Conflicts of Interest

The authors declare no conflict of interest. None of the authors have any financial or personal relationships with other people or organizations that could inappropriately affect its content.

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Genes 14 01606 g001
Figure 1. Patas monkey (Case#21) and its microscopic appearance of major organs. (A) Patas monkey died on 25 December 2020; (B) moderate interstitial hemorrhage (arrowhead) with glomerular hemorrhage (arrow) and severe glomerulonephritis, kidney, HE; (C) focal myocardial fibrosis (arrow) and multifocal inflammatory cell infiltration (arrowhead) with myocardial necrosis, heart, HE; (D) multifocal inflammatory cell infiltration with hepatocyte necrosis, mild and diffuse hemosiderosis (arrow), mild steatosis (arrowhead), liver, HE; (E) moderate edema with fibrin (hyaline membrane) (arrow), mild hemorrhage; mild lymphocyte infiltration and interstitial pneumonia, lung, HE; (F) severe hemorrhage with gas gangrene (arrow); acute necrotizing splenitis and lymphoid depletion (arrowhead); spleen, HE; bar = 50 μm.
Figure 1. Patas monkey (Case#21) and its microscopic appearance of major organs. (A) Patas monkey died on 25 December 2020; (B) moderate interstitial hemorrhage (arrowhead) with glomerular hemorrhage (arrow) and severe glomerulonephritis, kidney, HE; (C) focal myocardial fibrosis (arrow) and multifocal inflammatory cell infiltration (arrowhead) with myocardial necrosis, heart, HE; (D) multifocal inflammatory cell infiltration with hepatocyte necrosis, mild and diffuse hemosiderosis (arrow), mild steatosis (arrowhead), liver, HE; (E) moderate edema with fibrin (hyaline membrane) (arrow), mild hemorrhage; mild lymphocyte infiltration and interstitial pneumonia, lung, HE; (F) severe hemorrhage with gas gangrene (arrow); acute necrotizing splenitis and lymphoid depletion (arrowhead); spleen, HE; bar = 50 μm.
Genes 14 01606 g001
Genes 14 01606 g002
Figure 2. (A) T. gondii tachyzoites (arrowhead) were found in the lungs of IFN-γ−/− mouse, 18 dpi, smear, unstained; (B) aggregation of tachyzoites with a parasitophorous vacuole (arrowhead) was detected in the lungs of IFN-γ−/− mouse, 24 dpi, IHC; bar = 50 μm.
Figure 2. (A) T. gondii tachyzoites (arrowhead) were found in the lungs of IFN-γ−/− mouse, 18 dpi, smear, unstained; (B) aggregation of tachyzoites with a parasitophorous vacuole (arrowhead) was detected in the lungs of IFN-γ−/− mouse, 24 dpi, IHC; bar = 50 μm.
Genes 14 01606 g002
Table 1. Background and isolation of Toxoplasma gondii from non-human primates in China.
Table 1. Background and isolation of Toxoplasma gondii from non-human primates in China.
Case No.Received
Date		SpeciesSex, AgePathology No.Clinical SignsPathological FindingsT. gondiiMice
Bioassay
MAT aPCR bSwissIFN-γ−/−
New World Monkeys
Case#1627
February 2020		Red howler monkey (Alouatta seniculus)F, adult3022AsymptomaticAge-related atrophy, fatty degeneration of the liver, lipofuscin deposition in multiple organs.1:64H, Sk, P, I c0/5 end
Case#1715 April 2020Red howler monkey (Alouatta seniculus)F, adult3024Weight loss, lost two teethRenal insufficiency, necrotic splenitis, hypoproteinemia.<1:2Spndnd
Case#189 Novemebr 2020Squirrel monkey
(Saimiri sciureus)		M, adult3080Malaise, poor appetite, emaciation. Foamy nasal and oral discharge, died 2 days after onset of symptomsNecrotic glomerulonephritis, necrotic enteritis, pulmonary congestion, interstitial pneumonia.<1:2- d0/40/1
Case#1913 March 2021Red howler monkey (Alouatta seniculus)F, adult3130Malaise, poor appetite, emaciationSuppurative pneumonia, glomerulonephritis, hypoproteinemia.<1:2Sp, Lu, T, Lyndnd
Case#2031 July 2021White-faced saki monkey (Pithecia pithecia)M, adult3203Malaise, poor appetite, emaciationCardiac insufficiency, hydropericardium, hydrothorax, ascites, glomerulonephritis, skeletal muscle atrophy.<1:2-ndnd
Old World Monkeys
Case#2125 December 2020Patas monkey (Erythrocebus patas)M, adult3108Tail gangreneMultiple organ gas gangrene, renal insufficiency, cardiac insufficiency, lipofuscin deposition in the liver, interstitial pneumonia.1:3200Sp, Lu, K, Ly2/22/2
Case#229 January 2020Hamadryas baboon (Papio hamadryas)F, adult3112One week after the delivery of the fetusNecrotizing metritis, necrotizing myocarditis.1:64-nd0/2
Case#2317 February 2021Hamadryas baboon (Papio hamadryas)M, 24 years3121Leader, asymptomaticParenchymatous myocarditis, renal insufficiency, fatty degeneration of the liver.<1:2-0/3nd
Case#2415 April 2021Mona monkey (Cercopithecus mona)F, adult3148EmaciationRenal insufficiency, skeletal muscle atrophy.1:8Sp0/5nd
Case#2515 April 2021Mona monkey (Cercopithecus mona)F, adult3149EmaciationRenal insufficiency, skeletal muscle atrophy, necrotizing hepatitis.1:4K, T, Ly0/3nd
Case#264 August 2021White-cheeked Gibbon
(Nomascus leucogenys)		M, adult3205Malaise, poor appetite, depression, fever, four limbs, neck and abdomen edema; died 35 days after treatmentCachexia, age-related atrophy, necrotic enteritis, renal insufficiency, and acute hemorrhagic lymphadenitis. <1:2H, Sp, K, T, Ly, Sk, D0/2nd
Case#2710 August 2021Black-and-white colobus (Colobus polykomos)M, adult3208EmaciationNecrotic myocarditis, multiple organ atrophy.<1:2T, Pndnd
Lemuriformes
Case#2822 September 2021Ring-tailed lemur
(Lemur catta)		F, adult3218White fluid comes out of the vulvaSuppurative hemorrhagic cystitis, urethral calculus.<1:2-0/3nd
nd: Experiment not done. a: Modified agglutination test, titer 1:2–1:12,800; b: Polymerase chain reaction; c: T. gondii nucleic acid was detected in tissue; H: Heart; Sp: Spleen; Lu: Lung; K: Kidney; T: Tongue; Ly: Mesenteric lymph nodes; Sk: Skeletal muscle; D: Diaphragm; P: Pancreas; I: Intestines; d: Negative result; e: No. of mice infected/No. of mice inoculated.
Table 2. Genotypes of Toxoplasma gondii isolates from patas monkey in China according to PCR-RFLP of 10 markers and virulence proteins.
Table 2. Genotypes of Toxoplasma gondii isolates from patas monkey in China according to PCR-RFLP of 10 markers and virulence proteins.
Isolated IDSAG1(3′ + 5′) SAG2Alt SAG2SAG3BTUBGRA6C22-8C29-2L358PK1ApicoROP18ROP5ToxoDB Genotype
GT1, referenceIIIIIIIIIII11#10
PTG, referenceII/IIIIIIIIIIIIIIIIIIIIIII22#1
CTG, referenceII/IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII33#2
TgCgCa1, referenceIIIIIIIIIIIIIIu-1Iu-2I25#66
MAS, referenceu-1IIIIIIIIIIIIu-1IIIIII44#17
TgCatBr5, referenceIIIIIIIIIIIIIIIIIIIu-1I44#19
TgCatBr64, referenceIIu-1IIIIIIIIIu-1IIIIIIII33#111
TgRsCr1, referenceu-1IIIIIIIIIIu-2IIIIII33#52
TgMonkeyCHn2u-1IIIIIIIIIIIIIIIIIIIIIII36#9, this study
Table 3. Evaluation of the virulence of Toxoplasma gondii TgMonkeyCHn2 strain in Swiss mice.
Table 3. Evaluation of the virulence of Toxoplasma gondii TgMonkeyCHn2 strain in Swiss mice.
No. of TachyzoitesNo. of Mice Infection/
No. of Mice Inoculation (%)		Days of Survival/Number of MiceNo. of Brain Cysts
1034/4 (100%)≥60 dpi/2, 46 dpi/1, 54 dpi/12.5 ± 2.5
1022/4 (50%)≥60 dpi/4Not found
1012/4 (50%)≥60 dpi/435.0 ± 25.0
12/4 (50%)≥60 dpi/4Not found
<10/4 (-)≥60 dpi/4Not found
Blank control0≥60 dpi/4Not found
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Yang, L.; Ren, H.; Zhu, N.; Xin, S.; Mao, G.; Ma, Y.; Li, J.; Liang, Q.; Yang, Y. Isolation and Genetic Characterization of Toxoplasma gondii from a Patas Monkey (Erythrocebus patas) in China. Genes 2023, 14, 1606. https://doi.org/10.3390/genes14081606

AMA Style

Yang L, Ren H, Zhu N, Xin S, Mao G, Ma Y, Li J, Liang Q, Yang Y. Isolation and Genetic Characterization of Toxoplasma gondii from a Patas Monkey (Erythrocebus patas) in China. Genes. 2023; 14(8):1606. https://doi.org/10.3390/genes14081606

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Yang, Liulu, Hongjie Ren, Niuping Zhu, Shilin Xin, Gaohui Mao, Yiheng Ma, Junbao Li, Qunchao Liang, and Yurong Yang. 2023. "Isolation and Genetic Characterization of Toxoplasma gondii from a Patas Monkey (Erythrocebus patas) in China" Genes 14, no. 8: 1606. https://doi.org/10.3390/genes14081606

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