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

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.


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).

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, 10 0 , 10 1 , 10 2 , and 10 3 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.

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.

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).

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 3/6.57% (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).

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.

Virulence Evaluation of TgMonkeyCHn2 by Mice
As shown in Table 3, 10 3 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 10 3 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.

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 cm 2 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.
Genes 2023, 14, 1606 9 of 13 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].
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.
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.

Table 1 .
Background and isolation of Toxoplasma gondii from non-human primates in China.

Table 2 .
Genotypes of Toxoplasma gondii isolates from patas monkey in China according to PCR-RFLP of 10 markers and virulence proteins.

Table 3 .
Evaluation of the virulence of Toxoplasma gondii TgMonkeyCHn2 strain in Swiss mice.