Loiasis, a filarial infection caused by Loa loa usually thought to cause relatively minor morbidity, can cause serious and often fatal reactions in patients carrying very high levels of circulating Loa loa microfilariae (mf) following administration of microfilaricidal drugs. An experimental model of this condition would greatly aid the definition of the optimal management of this important clinical presentation. Fifteen baboons (Papio anubis) were infected with 600 infective larvae (L3) isolated from Chrysops vector flies. Animals were observed for any clinical changes; blood samples were collected every 1–2 months for 22 months, and analysed for parasitological, hematological and biochemical profiles using standard techniques. All animals became patent but remained clinically normal throughout the study. The parasitological pre-patent period was between 4–8 months, with a majority (60%) of animals becoming patent by 5 months post infection (MPI); all animals were patent by 8 MPI. Microfilarial loads increased steadily in all animals and reached a peak at 18 MPI. By 10 MPI >70% of animals had mf >8,000 mf/mL, and at 18 MPI >70% of animals had mf >30,000mf/mL with 50% of these animals with mf >50,000mf/mL. Absolute eosinophil, creatinine, Ca2+ and K+ levels were generally above normal values (NV). Positive associations were seen between microfilariaemia and eosinophilia, Hb, Ca2+, and gamma-GT values, whilst significant negative associations were seen between microfilariaemia and potassium, glucose and mononuclear leukocyte levels. Infection of splenectomised baboons with L. loa can induce levels of circulating microfilariae, and corresponding haematological profiles, which parallel those seen in those humans in danger of the severe post-microfilariacide clinical responses. Utilization of this experimental model could contribute to the improved management of the loiasis related adverse responses in humans.
Loa loa is a parasitic filarial nematode of humans, and a member the super family Filariodea which includes infections that are targeted for elimination, such as lymphatic filariasis (Wuchereria bancrofti, Brugia sp), and onchocerciasis (Onchocerca volvulus). L. loa is found in the tropics [1] restricted to the rainforest and forest fringes of West and Central Africa [2] and causing a relatively well tolerated medical condition known as loiasis. The geographic distribution of this infection is limited by the presence of the two biting tabaniid vectors, Chrysops silicea and C. dimidiata, which generally prefer rainforest-like environment; however, recently the disease has been described in the Guinean savannah [3]. It is estimated that some 14. 4 million people live in high risk areas where the prevalence of loiasis (i. e. a history of “eye worm”) is greater than 40%, with 15. 2 million in intermediate risk areas where the estimated eye worm prevalence is between 20 and 40% [4]. This disease has been recognized as one of public health importance, not so much because of its own clinical manifestations, but because of its negative impact on the control of onchocerciasis and lymphatic filariasis in areas of co-endemicity. There have been increasing reports in the past decade of serious adverse events (SAE) following the administration of ivermectin for these two major filariae, onchocerciasis and lymphatic filariasis, in L. loa endemic areas. These SAE are characterized by a severely disabling and potentially fatal encephalopathy. Evidence exists that these SAE appear to correlate with high loads of L. loa microfilariaemia (>30,000 mf/mL) [5–8]. Despite advances in defining the epidemiological aspects of these L. loa associated SAE, their pathogenesis and treatment still remains obscure. It is not known if the genesis of the encephalopathy is associated with the increased presence of L. loa in the brain tissue, although a vasculopathy associated with the presence of microfilariae has been proposed as a possible aetiology [9,10]. To be able to properly manage these cases, it is necessary to better understand the mechanism of pathogenesis of the post-ivermectin events in these heavily infected individuals. Very limited progress has been made in research on the pathogenesis of encephalopathy, due in most part to the lack of material from human cases, as well as a lack of any useful animal models to investigate the etiology and test new potential clinical management procedures. Although the natural hosts of L. loa are humans, the entire life cycle of L. loa can be maintained experimentally. L. loa readily infects mandrills (Mandrillus leucophaeus) [11], baboons (Papio anubis) [12], and patas monkeys (Erythrocebus patas) [12]. Non-human primates (NHPs) are therefore considered the best models for the much needed investigation in human loiasis, especially as suitable in vitro models for loiasis have not yet been developed, and indeed such artificial models are not easily extrapolated to humans [13–15]. Although the mandrill is an excellent experimental host, there are ethical concerns with using this now protected animal for research, and it is no longer used in biomedicine. The use of Patas monkeys also is limited as the parasite does not behave in the same way like it does in the more human-like drill [12]. The baboon (P. anubis) however, has potential as an experimental model to study the mechanisms behind the SAEs that develop in L. loa infected people as in this animal the parasite here behaves essentially in the same way as it does in the drill [12], and therefore is comparable with the situation in humans. Secondly, the use of baboons in biomedical research is accepted by the International Union for Conservation of Nature-IUCN [16]. In the simian host, the spleen usually becomes enlarged and granulomatous in filarial infections as it is the site of destruction of a large proportion of circulating Loa microfilariae [17,18]. If the spleen is removed very high levels of circulating microfilariae develop in the blood (>50,000 mf/mL), levels that are similar to those found in patients developing the post-treatment Loa-associated encephalopathy. Thus the baboon, with its remarkable similarities to humans in many anatomical and physiological parameters [19,20,21], appears to likely be a suitable model for studying this important clinical phenomenon.
Ethical and administrative clearances for the use of baboons in this study were obtained from the Ministry of Scientific Research and Innovation of Cameroon (Research permit #028/MINRESI/B00/C00/C10/C12). The animal procedures were conducted in accordance with the guidelines with animal care and use committee at the National Institutes of Health (USA) and University of Georgia, Athens, USA. Ethical clearance for the involvement of human subjects in the production of infective larvae was obtained from the Institutional Review Board of the Medical Research Station of Kumba, Cameroon. All volunteers were handled according to the Helsinki declaration on the use of humans in biomedical research. The use of non-human primates for research was approved by the Committee on the Ethical Use of Animals in Research (CEUAR) within the Research Foundation for Tropical Diseases and Environment (REFOTDE), Cameroon. All relevant aspects of the International Primatological Society (IPS) 2007 guidelines on the acquisition, care and breeding of non-human primates for research were followed. Baboons of both sexes were trapped in different parts of Cameroon according to IPS standard accepted procedures These animals were transported to the animal facilities in the Tropical Medicine Research Station, Kumba, South West Region and quarantined for a period of two months during which they were pre-screened for a panel of natural infections (loiasis, other blood-borne parasites, and intestinal worms). Each animal was observed daily by the veterinary staff to ensure that they were healthy, and any animal found to be ill was immediately given appropriate treatment, both in the quarantine period and during the main study period. The animals were housed individually in large custom built cages that allowed the animals to move about freely and be allowed to display their normal repertoire of locomotor behavior (walking, climbing, running, jumping and swinging) by providing them with vertical climbing surfaces and perches. Horizontal surfaces were also provided to allow them to rest comfortably and perform their social interactions such as sprawling during grooming. The housing facility was well aerated and equipped with a system that provided water ad libitum for each animal. Each baboon’s behavior was regularly monitored to identify any indications of poor welfare. Baboons received a diet of food that mimicked their natural diet (leaves, grass, roots, bark, flowers, fruit, lichens, tubers, seeds, mushrooms, corms, and rhizomes). They were also fed a supplement of a nutritionally complete commercial-available diet. The health and well-being of the baboons were regularly assessed during the study by an animal welfare officer who advised on matters such as disease prophylaxis, zoonoses, anesthesia, and methods of humane euthanasia and provision of health certificates. All measures were taken to minimize suffering during capture, captivity and experimentation. Health screening of workers in contact with the baboons was performed regularly to prevent animal losses from diseases transmitted from humans to baboons as well as zoonotic transmission of disease from baboons to workers. A total of 15 animals (6 males, 9 females) were used in this study with each animal being given a project animal number (BAB-1 to BAB -15). Splenectomy was carried out by a licensed veterinarian following previously published procedures [17,18]. The animals were anaesthetized using an intra-peritoneal injection of 4 mg of betamethasone (Septon, Europe) and 5 mg ketamine (Imalgene, Merial, France); 2mg/kg morphine sulphate (Hamelin Pharmaceuticals Ltd, UK) as also added to the administration as an analgesic. Spleens were removed in approximately 25 minutes under aseptic conditions, the skin wound sutured and disinfected with an antibiotic spray (2. 0g Chlortetracycline, 0. 5g Gentian violet, 100 mL excipient), and the incision site bandaged. Dressing were changed daily and the animals given a daily 1 mL injection containing 1. 2 million units of penicillin and 5 mg of streptomycin with 1 mL of anticoagulation factor (Vitamin K) for a week post-surgery. The surgical wound was dressed daily using an antimicrobial and insect repellent (Veto Spray—Vétoquinol) to protect against flies. The sutures were removed 7 to 8 days after splenectomy. The general welfare of the animals was monitored is on a daily basis for a period of approximately 2 months before infecting the animals with L. loa. The normal ranges for various blood parameters in baboons used for comparison were those provided by the Association of Primate Veterinarians Primate Formulary (1999) as listed for baboons housed individually in large custom cages. The data were entered into Epi Info version 3. 5. 3 (C. D. C. Atlanta, GA, USA) and analysed using the Software Package SPSS version 20. Descriptive statistical analyses were performed to compute the mean, median and standard deviations of Loa microfilarial counts, different haematological and biochemical parameters in the general study group, and in both males and females. Graph PadPrism software was used to draw the scatter plots comparing microfilariaemia and the different haematological and biochemical parameters to test for any association. The Kruskal and Wallis test was used to test for significant differences in levels of microfilariaemia, haematological and biochemical parameters before inoculation and at different time points of observation in the general study population. The Mann-Whitney test was used to test for significant differences in the different haematological and biochemical parameters between males and females. The Jonchkeere-Terpstra (J-T) test was used to test for any trend of linearity in the different parameters at different months. All tests were performed to a 5% significance level.
All fifteen animals appeared well fed and remained healthy throughout the study. The surgical incision sites post-splenectomy all healed without any evidence of infection, and no animal showed fever nor any intestinal disturbances during the study. The pre-study screening did not detect the presence of malaria or any intestinal parasites, in the test animals. The pre-patent period of this infection in baboons ranged from 4–8 months, with a median of 5 months. 1 of 15 (6. 7%), 9 of 15 (60%) baboons, 4 of 15 (26. 7%), 1 of 15 (6. 7%) had pre-patent periods respectively of 4,5, 6, and 8 MPI (Figs 1 and 2). The pre-patent period for males ranged from 4–6 months (median 5 months) with 3 out of 6 males (50%) becoming patent at 5 months post infection (Fig 1). The pre-patent period of females ranged from 5–8 months (median 5 months) with 6 out of 9 females (66. 67%) becoming patent at month 5 post infection (Fig 1). There was no significant difference between the median pre-patent period of males and that of females (p = 0. 504). The month at which each baboon started having microfilariae in blood (pre-patency period) and the month at which each baboon developed its highest microfilariaemia is shown in Fig 1. Animals were followed up for 22 months by which time all animals had become patent (Fig 2). By month 4 post inoculation (MPI) about 7% of infected baboons had microfilariae present in their circulation, and by month 5 MPI >70% of infected animals had developed microfilariaemia. By month 8 MPI all animals were parasitologically positive (Fig 2B). Generally, the mf increased steadily in all animals from the onset of patency to reach a median of 48,790 mf/mL by month 18. The mf loads at different time points during the course of infection was highly variable (Fig 2A). Male baboons generally developed higher microfilariaemia than females (Fig 1), although this difference was not statistically significant (p = 0. 06). By 6 MPI about 7% of animals had developed mf loads >8,000 mf/mL; at 8 MPI 50% of them had developed mf loads >8,000 mf/mL, and at 10 MPI >70% of animals had developed mf loads >8,000 mf/mL (Figs 1 and 2A). With regards especially high blood microfilarial loads, about 20% of animals had developed mf loads >30,000 mf/mL by 10 MPI. At 14 MPI, 50% of infected animals had developed mf loads that were >30,000 mf/mL and at 18 MPI >70% of infected animals had developed mf loads >30,000 mf/mL (Fig 2B). By 10 MPI about 7% of infected animals had developed extremely high blood microfilarial loads of >50,000 mf/mL and by 18 MPI almost 50% of them had developed these very high microfilarial loads (Fig 2B). RBC counts ranged from 2. 65–4. 3 x106 cells/mm3 (median 3. 48 x106 cells/mm3; NV = 3. 76–5. 61 x106 cells/mm3). Again males differed significantly from females in RBC values (p<0. 001). Most RBC values recorded in all animals were below the NV at the different time points (Fig 3A). Hemoglobin (Hb) values ranged from 10–16. 2g/dl (median: 13. 90g/dl) essentially close to normal values (NV) of 9. 5–14. 5g/dl (Fig 3B), with a slight upward trend during the infection (see S1 File). There was significant difference between males and females: Hb values in males ranged from 10–16. 2g/dl (median: 14. 4g/dl) whilst for females the values were 11–15. 2g/dl (median 13. 6g/dl); these were significantly different (p<0. 01). The total white cell counts were generally all within the NV (Fig 4A). Absolute neutrophil counts also stayed within NV ranging from 1,000–19,500 cells/mm3 (Median: 2,250 cells/mm3) (Fig 4B). Absolute mononuclear (lymphocyte + monocyte) cell counts ranged from 2,400–34,760 cells/mm3 and were within the NV (see S1 File) although was a marked variation between different time points (Fig 4C). with a median of 3,796 cells/mm3 (NV: 810–19,728 cells/mm3). The values for males were 1,800–39,000 cells/mm3 (median: 3,788 cells/mm3) while in females these counts ranged from 1,848–6,030 cells/mm3 (median: 3,810 cells/mm3). These mononuclear cell counts did not vary significantly between males and females (p = 0. 904). Absolute counts at different time points however did vary significantly (p<0. 001) and showed a significant linear trend (p<0. 001). Mononuclear counts recorded were within the NV (Fig 4C). Absolute eosinophil counts ranged from 0–6,500 cells/mm3 (Median: 978 cells/mm3; NV: 0–822 cells/mm3). Absolute eosinophil values in males ranged from 0–6,500 cells/mm3 (Median: 1,013 cells/mm3) while in females absolute eosinophil levels values ranged from 0–2,640 cells/mm3 with a median of 972 cells/mm3; these were not significantly different (p = 0. 473). Eosinophil counts at different time points, however did vary significantly (p<0. 001) and showed a general increase over the 18 months studied. All animals recorded absolute eosinophil values out of the normal range at different time points (Fig 5A) The absolute eosinophil results for each animal are given in Fig 5B. Basophils were not identified in these study samples. There was no significant difference in SGPT values between males and females (p = 0. 342), nor did the SGPT values at different time points vary significantly (p = 0. 086) or show a significant linear trend (p = 0. 110) with the majority of SGPT values being within the NV (Fig 6A). The SGOT values at different time points varied significantly (p<0. 05) although there was no significant linear trend (p = 0. 356). The majority of SGOT values were within the NV, even though all baboons except BAB 04 showed SGOT values both below or above the NV at different time points (Fig 6B). The γ-GT values at different time points varied significantly (p<0. 001), and there was a significant linear relationship between microfilariaemia and the duration of infection (p<0. 05). However, most of the γ-GT values were within the NV (Fig 6C). The creatinine values at different time points varied significantly (p<0. 001), although the values did not show any significant linear relationship with the duration of infection (p = 0. 068). Most of the creatinine values were above the NV at the different time points (Fig 7A). Glucose values varied significantly (p<0. 001) at different time points, and there was significant linear relationship between blood glucose level and the duration of infection (p<0. 001). Majority of the glucose values were within the normal range, although all baboons showed glucose values out of the normal range at different time points (Fig 7B). Calcium values varied significantly at different time points (p<0. 001) and showed a significant linear relationship with the duration of infection (p<0. 001). Majority of the calcium values in all animals at different time points were below the NV (Fig 8A). The potassium values at different time points varied significantly (p<0. 001), and showed a significant negative linear relationship with the duration of infection (p<0. 001). The majority of the potassium values in all animals were out of the NV (Fig 8B). Hb values showed a slow increase from 12. 4 g/dL before inoculation to 14. 1 g/dL at 3 MPI after which the values dropped to 13. 1 g/dL at 6 MPI and increased steadily over time to 14. 1 g/dL at 16 MPI where a slight dropped to 13. 3 g/dL was noticed as mf culminated at 18 MPI. Overall, there was a positive significant association (r = 0. 180, p>0. 05) between mf and Hb values (Fig 9B). The eosinophil count increased sharply from 0 cells/mm3 before inoculation to 1,500 cells/mm3 at 18 MPI when the mf reached its highest level. Overall, there was a strong positive significant association (r = 0. 730, p<0. 001) between eosinophil and mf (Fig 10C). The mononuclear count decreased steadily from 4,800 cells/mm3 at 1 MPI to 3,800 cells/mm3 at 16 MPI from where its values dropped slightly to 3,200 cells/mm3 at 18 MPI when the mf peaked. Overall, there was a negative significant association (r = -0. 368, p<0. 001) between mf and mononuclear counts (Fig 10D). There was no significant association between mf and RBC, WBC and neutrophil (Figs 9A, 10A and 10B). The enzymes SGPT and SGOT did not show any significant association with mf (Fig 11A and 11B). Overall, there was a positive significant association (r = 0. 281, p<0. 001) between γ-GT and mf (Fig 11C). γ-GT values increased sharply from 0 IU/L before inoculation to 50 IU/L at 8 MPI, then decreased slightly over time to 29 at 14 MPI after which time its values decreased gradually over time to 42 IU/L at 18 MPI when mf peaked and then dropped again. There was a negative, non-significant, association between mf and creatinine (Fig 12A). Glucose values decreased gradually from 1. 5 g/L at 1 MPI to 0. 6 g/L at 6 MPI from where the values increased slightly to 1. 1 g/L at 10 MPI after which its values decreased to 0. 6 g/L at 18 MPI. Overall, there was a negative significant association (r = -0. 171, p<0. 05) between glucose and mf, (Fig 12B). Calcium values increased steadily over time from 9 mg/L before inoculation to 25 mg/L at 10 MPI at which point it increased sharply to 74 mg/L at 12 MPI after which its values decreased steeply over time to 15 mg/L at 20 MPI. Overall, there was a positive significant association (r = 0. 410, p<0. 001) between calcium and mf (Fig 13A). Potassium values plummeted from 170 mmol/L before inoculation to 5 mmol/L at 18 MPI when the mf peaked. Overall, there was a negative significant association (r = -0. 423, p<0. 001), between mf and potassium (Fig 13B).
This study is the first showing parasitological, haematological and biochemical characterization of hyper-microfilaraemic loiasis in the splenectomized baboon (P. anubis). Parasite pre-patency was between 4–8 months, with the majority (60%) of animals becoming patent 5 months post inoculation; all animals developed patency; Microfilariaemia rose steadily in all animals and culminated at a peak level by month 18 post infection with males showed higher microfilariaemia than females. By month 10 post inoculation >70% of infected animals developed microfilariaemia >8,000mf/mL; by month 18 post inoculation >70% of infected animals had developed microfilariaemia >30,000mf/mL, and 50% of them developed >50,000mf/mL a level where in humans that predisposed for severe adverse reactions post treatment. Significant positive associations were seen between microfilariaemia and eosinophil, haemoglobin, calcium and gamma-GT, whilst there was a negative significant correlation between microfilariaemia and mononuclear leucocytes, glucose and potassium. This model has the potential of helping to understand the mechanism (s) involved in the development of Loa-encephalopathy post-ivermectin treatment in heavily Loa microfilariaemic humans, and could help in designing improved management of such cases.
Loiasis is a filarial infection of humans that, in addition to causing severe direct clinical effects, is of concern to the global community’s efforts to eliminate the important filarial diseases, onchocerciasis and lymphatic filariasis, through causing interruption to mass drug distribution activities. Hyper-microfilariaemia has been seen to be the characteristic parameter in patients suffering from post ivermectin encephalopathy, a condition which sometimes leads to death. Understanding and developing appropriate approaches to the treatment and prevention of these severe adverse reactions has been difficult due to the lack of suitable models. As primates can be infected with human L. loa, and can develop hyper-microfilariaemia, it is likely that they therefore can serve as suitable models for the investigation of this syndrome in humans. This current study shows that following splenectomy the circulating microfilarial loads are similar to those seen in humans, and that the clinical pathology profile following infection also appears to be similar. The consistent ability to induce microfilariae levels of above 30,000 mf/ml in more than 70% of the tested animals suggests that this is indeed a practical model for investigating the adverse events occurring in hyper-loiasis.
Parasitological, Hematological and Biochemical Characteristics of a Model of Hyper-microfilariaemic Loiasis (Loa loa) in the Baboon (Papio anubis)