Fatal coinfection of blastocystosis and intestinal trichomoniasis in a rhesus macaque (Macaca mulatta)

Abstract

A 3-year-old male rhesus macaque was presented at Referral Veterinary Polyclinic-Teaching Veterinary Clinical Complex, with a chief complaint of chronic diarrhoea and swelling of dependent body parts. The patient's history indicates that the monkey had been experiencing diarrhoea for the past month, with 2–3 episodes of vomiting in the last 2 days. Additionally, oedema has developed within the last 2 weeks. The clinical examination findings revealed dullness and depression, the mucus membrane appeared pale, with a temperature-102.1 °F, a respiration rate-28/min, and a heart rate-92/min. The capillary refill time was 4 s. During the physical examination, the animal exhibited oedema on the dependent part of the body and faecal staining around the perineum along with loose yellow stool. Direct saline and iodine mount faecal smear examination revealed the presence of many motile pear-shaped flagellated protozoa and round vacuolated Blastocystis organisms. Giemsa-stained faecal smear cytology confirmed the presence of Pentatrichomonas sp. and Blastocystis sp. along with many microbes. The faecal culture was negative for all pathogenic microbes. The case was diagnosed as co-infection Blastocystosis and intestinal trichomoniasis. The treatment was initiated with a combination of sulfamethoxazole + trimethoprim @ 35 mg/kg body weight and metronidazole @25 mg/kg administered orally once daily for 7 days. Supportive therapy includes hematinic injection (iron sorbitol, folic acid and vitamin B12) @ 1 ml total dose, administered intramuscularly on alternate days for four occasions as well as intravenous infusion of crystalline amino acid @ 5 ml total dose on alternate days for four occasions. To manage vomition, injection ondansetron was administered@0.5 mg/kg intramuscularly, twice daily for 3 days and H2 blockers, including injection ranitidine@2 mg/kg intramuscularly twice daily for 3 days. Electrolyte and probiotic supplementation were administered orally. After 7 days of therapy, the oedema had significantly improved and episodes of vomition were stopped but there was no significant improvement in the episode of diarrhoea and consistency of faeces. Unfortunately, on the 10th day of therapy, the animal suddenly collapsed. Understanding the virulence pattern of opportunistic protozoa in primates is crucial, and identifying suitable therapeutic candidates to prevent fatal outcomes is the need of the hour, especially considering protozoal infections as an important differential diagnosis in gastrointestinal tract-related ailments. Our study successfully demonstrated the co-occurrence of blastocystosis and intestinal trichomoniasis, both uncommon infections with potential zoonotic implications.

Introduction

Rhesus macaques (Macaca mulatta) kept in captivity frequently suffer from chronic diarrhoea (Kanthaswamy et al. 2014; Laing et al. 2018). Chronic diarrhoea in rhesus macaques can result in dehydration, malnutrition, stunted development, weight loss, a weakened immune system, and even death. This has a significant negative impact on the ability of captive rhesus macaques to reproduce (Kanthaswamy et al. 2014; Prongay et al. 2013). The inefficiency of antibiotic therapies is one of the main features of chronic diarrhoea in rhesus macaques (Westreich et al. 2019). A variety of gastrointestinal parasites are present in several species of monkeys (Petrášová et al. 2010). However, a number of intestinal parasites are zoonotic, which makes them a potential source of infection transmitted from animals to humans. There have been several reports of gastrointestinal protozoa and helminths in various species of monkeys (Parr et al. 2013). Certain gastrointestinal protozoa and helminths are typically non-pathogenic, while several of them can be zoonotic pathogens, including Entamoeba histolytica, Giardia lamblia, and Blastocystis hominis (B. hominis) (Dalimi et al. 2016). The eukaryotic parasite Blastocystis is a unicellular, anaerobic organism that inhabits the intestinal tract of numerous animals, including humans. The pathogenicity of Blastocystis remains uncertain, and there is an ongoing debate regarding whether it should be classified as a pathogen or a commensal organism. This is because Blastocystis can be found in the intestines of both asymptomatic individuals and those who are experiencing symptoms of illness (Mutlag et al. 2019). Diarrhoea is indeed a common gastrointestinal symptom associated with Blastocystis infection (Laodim et al. 2013). Furthermore, there is evidence suggesting a potential link between this organism and irritable bowel syndrome (IBS) (Poirier et al. 2012). Blastocystis sp. has also been discovered to have a connection with inflammatory bowel disease (IBD) (Dogruman-Al et al. 2009). However, some colonies of monkeys experienced deadly diseases associated with blastocystosis. B. hominis infections in zoo-housed apes were effectively treated with trimethoprim-sulfamethoxazole, while a pig-tailed Macaque's infection was successfully cured using diiodohydroxyquin (McClure et al. 1980).

Pentatrichomonas hominis (P. hominis) is thought to be a commensal organism that mostly lives in the large intestines of nonhuman primates (Kondova et al. 2005; Tolbert et al. 2012); Disease can result from opportunistic overgrowth of P. hominis, often characterized by diarrhoea (Jergens and Willard 2000). Nonetheless, there is a scarcity of data regarding the pathogenicity of these trichomonads. Numerous investigations have discovered a link between certain trichomonad species and digestive problems. As per certain research findings, P. hominis infection in dogs and cats leads to the development of diarrhoea (Kim et al. 2010; Gookin et al. 2007). Gastrointestinal problems in children were determined to be most likely caused by P. hominis (Meloni et al. 2011). While there have been limited accounts of trichomonad-induced lesions in nonhuman primates, there have been documented instances of invasive trichomoniasis affecting the stomach and colonic mucosa. These cases were caused by Tritrichomonas mobilensis and were observed in simian immunodeficiency virus-infected rhesus macaques (Kondova et al. 2005) and titi monkeys (Bunton et al. 1983), respectively.

History and case description

A 3-year-old male rhesus macaque weighing 3 kg was brought to the Referral Veterinary Polyclinic-Teaching Veterinary Clinical Complex (RVP-TVCC), ICAR-Indian Veterinary Research Institute, with the following concern: diarrhoea persisting for one month and 2–3 episodes of vomiting over the past two days, along with recent development of oedema during the last two weeks. On clinical examination, the mucus membrane appeared pale, the temperature was 102.1 °F, the respiration rate was 28/ min, the heart rate was 92/ min, and the general status was dull and depressed. The capillary refill time was 4 s, and the skin tenting time was 6 s indicating moderate dehydration. Physical examination revealed oedema on the dependent portions of the body (thigh, leg and scrotum), faecal staining around the perineum with watery yellow faeces, and a dilated anus (Fig. 1). Auscultation of the stomach revealed a gurgling sound.

Fig. 1.

Rhesus macaque suffering from chronic diarrhoea. a Faecal staining around the perineum along with yellow diarrheic faeces, b Oedema of leg and ventral part of the body, c Pale conjunctival mucus membrane, d Pale oral mucus membrane

Laboratory investigation

A freshly passed loose stool sample from the patient was collected and routine stool microscopy was performed immediately after receiving the sample. The faecal sample was suspended in an equal volume of normal saline and microscopically examined under a cover slip using magnification of 100X, 400X and 1000X. Direct examination revealed the presence of Pentatrichomonas sp. and Blastocystis sp. The Pentatrichomonas sp. trophozoites featuring a pyriform body and exhibiting jerky, progressive forward motion, were present in large numbers. Pentatrichomonas sp. displays distinct motility traits compared to Giardia sp., as it exhibits a "falling leaf" type of motility (Yao and Köster 2015). The Balstocystis sp. exhibited a large central vacuole occupying the central part of the organism, with a thin layer of cytoplasm pushed to the surface of the cell by the central vacuole. Usually, one or two nuclei were found in the narrow rim of the cytoplasm, although sometimes as many as four or more nuclei (Fig. 2). The diameters of these organisms ranged from 8 to 18 μm. Iodine mount examination revealed round to oval Blastocystis sp. and pear-shaped five flagellates’ protozoa (Fig. 2). Further confirmation was achieved through Giemsa staining of faecal smear, which confirms the presence of round to oval Blastocystis sp. and Pentatrichomonas sp. with five anterior flagella with an undulating membrane (Fig. 3). Importantly, no pathogenic bacteria were detected in faecal cultures.

Fig. 2.

Lugol’s iodine wet mount microscopy, a Pear-shaped flagellated trophozoites of Pentatrichomonas sp. (arrow marked), b Round to oval Blastocystis sp. (arrow marked), c Single Blastocystis sp. with two nuclei on the periphery (arrow marked), d Both Blastocystis sp. and Pentatrichomonas sp. are in one field (arrow marked)

Fig. 3.

Giemsa-stained faecal cytology, a Pentatrichomonas sp. (arrow marked) one in elongated shaped and one in pyriform shaped, b Pear-shaped Pentatrichomonas sp. with clearly visible five anterior flagella and one posterior flagella run along with undulating membrane, c Large number of Blastocystis sp. (arrow marked) along with faecal microbes, d Oval shaped Blastocystis sp. with clearly visible two nuclei in the periphery

Haemato-biochemical alteration

The haematological examination results indicated severe anaemia, characterized by lower haemoglobin levels, a reduced red blood cell count, and a decreased packed cell volume. Additionally, there was an increase in the total white blood cell (WBC) count and neutrophil percentage, coupled with a decrease in lymphocyte count, suggesting a chronic infection. However, it's important to note that all other haematological parameters were within the normal range. In the serum biochemical examination, there was a severe deficiency of proteins, specifically hypoalbuminemia, hypoproteinemia, and hypoglobulinemia. On a positive note, the hepatic and renal function parameters were found to be within normal ranges (Table 1).

Table 1.

Haemato-biochemical changes in Rhesus Macaque suffering from blastocystosis and intestinal trichomoniasis

Parameters	Value	Reference ranges
Heamoglobin(g/dl)	6.1	11.85–15.39*
RBC(× 106µL)	2.81	5.00–6.39*
PCV (%)	21.5	36.17–50.29*
MCV (fL)	76.8	68.02–83.88*
MCH (pg)	21.7	21.91–26.04*
MCHC (g/L)	29	28.97–34.22*
WBC (× 103µL)	19.5	4.42–14.38*
Neutrophils (%)	72	11.58–63.62*
Lymphocyte (%)	18	30.24–77.68*
Monocyte (%)	10	1.83–11.70*
Eosinophils (%)	0	0.30–5.95*
Basophils (%)	0	0.00–2.33*
Platelets (× 103µL)	532	203.64–517.69*
SGPT (IU/L)	31.82	20.22–70.40*
SGOT(IU/L)	28.54	9.51–74.07*
BUN (mg/dL)	20.68	10.2–22.6#
Creatinine (mg/dL)	0.53	0.62–1.41*
Total protein (mg/dL)	1.35	7.03–8.78*
Albumin (mg/dL)	0.20	2.50–4.30*
Globulin (mg/dL)	1.15	2.50–4.30*

^#Koo et al. (2019)

*Shah et al. (2022)

Treatment

The treatment began with metronidazole @25 mg/kg body weight (BW) and sulfamethoxazole + trimethoprim combination @35 mg/kg BW, administered orally once daily for 7 days. This was followed by supportive therapy, including crystalline amino acid infusion (Astymin 3) @5 ml total dose, administered intravenously on an alternate day for four occasions. Additionally, haematinic injection Feritas (Iron sorbitol, folic acid, and vitamin B12) @1 ml was administered intramuscularly, with a 48 h interval, for four occasions. To manage vomition, injection of ondansetron was administered @ 0.5 mg/kg intramuscularly, twice daily for 3 days and H2 blockers, including injection of ranitidine @ 2 mg/kg intramuscularly twice daily for 3 days. Electrolyte and probiotic supplementation were administered orally. After 7 days of treatment, the animal condition significantly improved with a notable reduction in oedema and cessation of vomition episodes, however, the diarrhoea episode did not improve. Unfortunately, on the 10th day following the initiation of therapy, the animal passed away and the animal's necropsy was not permitted by the owner.

Discussion

Primate species are often kept in close proximity to humans as pets, despite the practice being illegal but common (Inoue et al. 2015). Nonhuman primates including chimpanzees, mandrills, macaques, and baboons have been documented to contract Blastocystis sp. infection, which is crucial from the standpoint of public health (Ma et al. 2020). Blastocystis sp. is an intestinal parasite that thrives in anaerobic conditions and can infect both humans and a broad spectrum of animal species (Tan 2008; Wawrzyniak et al. 2013). The primary methods employed for diagnosing Blastocystis sp. typically involve direct examination of faecal smears using physiological saline and iodine staining under a light microscope, or cultivating the parasite in xenic in-vitro cultures (Tan 2008). Three different types of B. hominis have been seen under a light microscope: vacuolated, granular, and amoeboid (Zierdt and Tan 1976; Wawrzyniak et al. 2013). In our case, we observed a large number of Blastocystis sp. and Pentatrichomonas sp. in monkey faeces with chronic diarrhoea. Similar to this, research by Bunton et al. (1983) found Blastocystis sp., Entamoeba coli, Chilomastix sp., and Trichomonas sp. in the excrement of Callicebus moloch that had diarrhoea. Additionally, it has been reported in earlier investigations that B. hominis is implicated as a cause of gastrointestinal illness and severe diarrhoea in people (Zierdt and Tan 1976). In a study by McClure et al. (1980), a case was documented in which a significant quantity of B. hominis was found in the faeces of a pig-tailed macaque (Macaca nemestrina). This finding suggests its potential role as an enteric pathogen in nonhuman primates with chronic diarrhoea. Reports of Blastocystis sp. infections have emerged in pigs experiencing diarrhoea or dysentery in England. Nevertheless, there is currently no conclusive evidence to indicate that Blastocystis plays a pathogenic role in these cases (Burden et al. 1978/1979). According to Mansfield (2003), many colonies of some callitrichid species have trichomonad protozoa in their large intestines. In relation to digestive problems, little research has been done on alterations in the commensal eukaryotic population of the colon. The prevalence of trichomonad parasites, especially P. hominis, has been found to be higher in animals with idiopathic chronic diarrhoea (ICD). However, P. hominis is commonly recognized as non-pathogenic in humans and macaques (Li et al. 2016). Conversely, certain studies propose that an overgrowth of P. hominis may be associated with the occurrence of diarrhoea (Kim et al. 2010). The results reported by Inoue et al. (2015) indicate that P. hominis infection may not have been the primary cause of diarrhoea or colitis in marmosets, as they found similar positive rates for the presence of trophozoites in both normal and diarrheal faeces. The transmission of Pentatrichomonas sp. occurs through the oral ingestion of trophozoites or pseudocysts excreted in faeces, emphasizing the importance of good hygiene practices to prevent its spread (Inoue et al. 2015). Nonetheless, there have been limited accounts of trichomonads causing gastrointestinal illness in immune-compromised rhesus macaques and various other species (Bunton et al. 1983; Tolbert and Gookin 2016). Although an increase in trichomonads could just be a symptom of the dysbiosis linked to ICD, more investigation into P. hominis as a potential pathogen is required.

The intestinal parasite B. hominis has been associated with gastrointestinal illnesses such as IBS, persistent diarrhoea, and IBD. IBD is characterized by mucosal inflammation, which can lead to mucosal malabsorption and anaemia. In cases of IBD, anaemia may also be worsened by continuous blood loss (Gasche et al. 2004). In our investigation, there was lower haemoglobin, red cell count, packed cell volume, higher white blood cell count, and neutrophil percent. In Mutlag and coworkers (2019) study, individuals with Blastocystis infection exhibited slightly lower levels of haemoglobin, haematocrit, and erythrocyte counts compared to the control group. Additionally, as reported by Yavasoglu et al. (2008), either on its own or in conjunction with other factors, B. hominis might play a role in the development of iron deficiency anaemia. The observations made align with the discoveries of Andiran et al. (2006) and Mutlag et al. (2019), both of whom reported a significant increase in white blood cell counts in the patient group when compared to the control group. Leukocytes possess the ability to stimulate and modulate the production of proinflammatory cytokines within intestinal epithelial cells, which is why their levels tend to rise in cases of Blastocystis infection, especially when digestive system symptoms are present (Cheng et al. 2003). Remarkably, this study reveals a notable rise in the neutrophil percentage accompanied by a decline in the lymphocyte percentage. This observation aligns with the results reported in many earlier studies. Laodim et al. (2013) documented a substantial increase in neutrophils and monocytes among Thai individuals infected with Blastocystis compared to the healthy subjects in the control group. Similarly, the investigation by Al-Mozan et al. (2017) in Dhi Qar, Iraq, revealed a proportional reduction in lymphocyte levels in cases of intestinal parasitic infection. More recently, Ismail et al. (2022) reported a significant decrease in total lymphocyte and platelet counts, accompanied by a noteworthy increase in monocytes and neutrophils in the blood of both IBS patients and non-IBS individuals infected with Blastocystis parasites, when compared to the healthy control group. Furthermore, the notable increase in the neutrophil count in macaque infected with Blastocystis and intestinal trichomoniasis could be attributed to the role of neutrophils as the primary line of defence against pathogenic agents. Neutrophils play a crucial role in recognizing and engulfing invading pathogens (Ismail et al. 2022). The decrease in lymphocyte numbers may be attributed to the colonization of Blastocystis parasites in the gut, which could trigger the activation or influx of T cells, monocytes/macrophages, and/or natural killer cells in local tissues (Iguchi et al. 2009).

A number of disorders have been linked to hypoalbuminemia as a predictor. In children with chronic diarrhoea, it represents a significant risk factor for mortality (Umamaheswari et al. 2010). Serum albumin is a crucial protein that plays several essential roles in the body. It has osmotic effects, anti-inflammatory properties, and the ability to bind various molecules and drugs (Arques and Ambrosi 2011. In our investigation, we documented a substantial presence of hypoproteinemia, hypoalbuminemia, and hypoglobulinemia, culminating in debilitation and the onset of edema in anatomically dependent regions. Similarly, hypoalbuminemia linked to B. hominis infection has been documented in two individuals (Levy et al. 1996; Nassir et al. 2004). According to reports, persons with Blastocystis infection, who are underweight are more likely to get diarrhoea. B. hominis itself has the potential to harm the intestinal mucosa and exacerbate hypoalbuminemia due to poor mucosal absorption. Hypoalbuminemia may also be brought on by prolonged diarrhoea caused by Blastocystis sp. Additionally, it was said to affect intestinal barrier functions and damage gut permeability (Tan 2008; Dagci et al. 2002).

Metronidazole is the drug of choice for treating blastocystosis and enteric trichomoniasis, albeit it has limited efficacy in some cases. The other drugs used to treat these infections include trimethoprim-sulfamethoxazole, paromomycin, and furazolidone (Kurt and Tanyüksel 2016). In this investigation, the monkey received the standard treatment— which is widely regarded as the best drug for treating blastocystosis and intestinal trichomoniasis caused by Blastocystis sp. and Pentatrichomonas sp.—did not respond to it which raises the possibility that the parasite has become resistant to it or that the owner did not give it properly. Further research is necessary to demonstrate the pathogenicity of Blastocystis sp. and Pentatrichomonas sp. as well as their resistance to standard drugs (Sekar and Shanthi 2013; Bastos et al. 2018).

Conclusion

The case involved a rhesus macaque with chronic diarrhoea, vomiting, and oedema, found to have Pentatrichomonas and Blastocystis in its faeces. The macaque exhibited severe anaemia and protein deficiencies. Treatment with standard drugs was ineffective, and the animal ultimately died. Though protozoa species like Pentatrichomonas and Blastocystis are classified as commensal organisms in primates they pose a significant risk of becoming opportunistically pathogenic and even fatal. The underlying mechanisms that are evolving virulence and pathogenicity of these organisms must be considered in future research and pharmaceutical candidates should be identified that possess significant therapeutic efficacy in such infections. As rhesus macaques share a close relationship in physiology to humans the risk of spillover of these pathogenic agents must always be considered and tackled accordingly. This case underscores the potential pathogenicity of these parasites in nonhuman primates, their zoonotic potential, and the challenge of treatment resistance. Additionally, it raises concerns about the enhanced pathogenicity of parasites during concurrent infections. Further research is needed to better understand and treat such infections.

Authors’ contributions

This is a unique clinical case of coinfection with Blastocystis sp. and Pentatrichononas sp. infection in a rhesus macaque, being reported by Varun Kumar Sarkar. Ujjwal Kumar De guided to building the manuscript. Devendra Prasad Pateer helped in the microscopic examination of Blastocystis sp. and Pentatrichomonas sp. whereas, Sudhir Kumar Prajapati helped in the faecal culture for pathogenic microbes. Harshit Saxena, Shivansh Mehra and Pooja Solanki helped in the correction of the manuscript.

Fundig

The authors have not disclosed any funding.

Declarations

Conflict of interest

None of the authors have any financial or personal affiliations with individuals or organizations that might unduly influence or prejudice the content of the paper.

Ethical approval

This study did not necessitate official or institutional ethical approval as it did not involve experimental procedures. The examination of the macaque in this case was conducted with the written consent of its owner.