778

TWSACTIONS OF THE Roulu. SWIE~ OF TROPICAL MEDICINE AND HYGIENE, VOL. 76, No. 6, 1982

S-urvival of Loa loa following transplantation from drills (Mandrillus leucophaeus) into jirds (Meriones unguiculatus): parasitology and

pathology

C. D. MACKENZIE, R. R. SUSWLLO AND D. A. DENHAM Dept. of Medical Helminthology, London School of Hygiene and Tropical Medicine, Keppel Street, London

WClE 7HT

Summary Two drills infected with Loa loa maintained a

microflaraemia for four and a half years ranging from less than 1 mf/lOO ~1 to 1150 mf/lOO ul. No signi- ficant tissue reactions to the adult worms were seen at autopsy. Adult worms were transplanted into the peritoneal cavities of naive jirds when, a persistent microfilaraemia lirst developed by 17 days. Retrans- nlantation of adult worms into naive jirds produced a microtilaraemia and microfilariae in the peritoneal cavities of three out of five animals. These three animals were all negative for circulating parasites by eight and a half months. The tissue reactions to the worms in the jirds are described, including a granulo- matous response surrounding adults and a myositis involving microIilariae.

Introduction Loa loa has been maintained and studied in various

primates including drills (Mandrillus leucophaeus), baboons (Papio anubis) and patas monkeys (Erythrocebus patas) (see DUKE, 1972; ORIHEL & MOORE, 1975; EBEHARD & ORIHEL, 1981). However, successful infection of small laboratory animals has not as yet been described.

SUSWILLO et al. (1977) attempted unsuccessfully to infect jirds with Loa Zoa by injecting third stage larvae from Chtysops silacea into subcutaneous tissues and into the peritoneal cavity. In this communication we describe the parasitological and pathological events following the transplantation of primate-derived adult L. loa into jirds.

Materials and Methods Two drills (M. leucophaeus) were each infected in

Cameroon with infective larvae on two occasions six days apart, 40 larvae initially and 200 or 500 on the second occasion. These larvae were obtained from wild C. silacea that had been captured and fed on Loa Zoa infected human volunteers. The infected drills were transported to the UK where they were bled, either bv finger-Ian&a (nerinheral samples) or by venepun&ure~ and the& r%cr&Iariae enumerated at various intervals by the filter technique (DENNIS & KEAN, 1971). Four and a half years after infection they were killed and autopsies performed, parasites sought and collected and tissues taken for histological examination. Isolated adult L. Zoa were implanted into the peritoneal cavities of jirds (M&s unguicu- latus) usine the techniaue of SUSWLLO & DENHAM (1977). T&sue for hi&pathological examination was taken from jirds 15 days after retransplantation of

adult worms, fixed in form01 saline and processed by standard methods. Haematoxylin and eosin, chroma- trope R and Giemsa stains were used.

Results The infection in drills

Parasitological jindings: Neither drill had a high circulating microfilarial load at any time during this study. During the Iirst eight months of infection one animal had counts ranging between 10 and 12 microfilariae (mf) per 100 ul of peripheral blood. The number of circulating parasites in this animal then appeared to drop and microfilariae were only occa- sionally detected when small samples (100 fl) of blood were tested. However, whenever 2 ml of blood were taken from the animal and filtered (DENNIS & KEAN, 1971) microfilariae were always found. At 20 months after infection this animal had a circulating parasitaemia of 1150 mf/lOO u.l and at two and three-quarter years 40 mf/lOO l.rl (venous samples).

In the first phase of infection the second drill had circulating microfilarial counts of up to 45 mf/lOO ul, these then again dropped to 0 to 11100 yl (peripheral samples). At 20 months post infection a count of 110 mf/lOO ~1 blood was obtained using the Nucle- pore filter method.

Autopsy findings: The animals were in good condition at the time of death and no general signs of debility were seen, although one animal appeared to have a low grade pulmonary infection. In sharp contrast to the observations by DUKE (1972) there were no gross changes detected in the spleen.

22 females and 20 males L. Zoa adults were found in one drill and four females and one male in the other animal. Microfilariae were present in the heart blood of these animals at levels of 10 and 9 mf/lOO ul blood respectively. The adult worms were mostly found lying between muscle blocks in the fascia surrounding these muscles, male and female worms being seen in close proximity to each other. Single worms were also found on each kidney and in the peritoneal cavity. There was rarely any macroscopic evidence of changes in host tissues due to the presence of these worms.

Transplantation experiments in jirds Parasitological findings: Two female and one male

L. Zoa adults taken from the first drill at autopsy were maintained in sterile RPMI-1640 tissue culture medium (Gibco Biocult Ltd.) and within four hours were transplanted into the abdominal cavity of a jird.

C. D. MACKENZIE et d. 779

Fig. 1. Gramdomatous reactions surrounding an adult Lou Zoa I5 days after transplantation into jirds. A. Reactive tissue, consisting mainly of fibrous connective tissue and accumulations of eosinophils. (X 95) B. High power of area of eosinophil inlilaation showing character-

istic nuclear structure of eosinophils (arrow). (X 950) C. An area close to parasite (P) showing eosinophils (arrow) in close

apposition to its surface. (X 950)

780 Loa loa IN JIRDS

Fig. 2. Lumbar muscle in the proximity of adult Loa loa. Accumulations of inflammatory cells can be seen in between the muscle-bundles. An area of muscle degeneration is shown (D). (X 95)

Fig. 3. A bigh photomicmgraph of au inknmatory cell accumulation in muscle showing presence of microlilariae (arrow). Eosinophil leucocym

with typical nuclear suucture are also present. (X 950)

c. D. MACKENZIE et al. 781

Within a week, aspirates of peritoneal fluid contained microfilariae and after 17 days the jird developed a microfilaraemia of 1 mf/lOO ~1 of tail blood which persisted until autopsy six weeks later. Two apparent- ly healthy female L. Zoa were recovered from the abdominal cavity and re-implanted into a second jird. No microfilariae were ever found in this second recipient, either in the blood or the peritoneal fluid, and at autopsy three months later no worms or worm debris were recovered.

A total of 19 worms, including five males, were isolated from the second drill at autopsy. These were implanted into the peritoneal cavities of five jirds, each animal receiving from two to nine worms including a single male worm. Microfrlariae were detected in the peritoneal aspirates of three of these animals within a week of transplantation. The other two jirds remained microfilaria-negative from the time of transplantation to the time of autopsy at three and a half months when there were no visible signs of any worms. The three jirds that successfully carried the parasites in their peritoneal cavities also had circulat- ing microfilariae, their microfilaraemias ranging from 15 to 900 parasites per 100 ~1 of tail vein blood. 10% of the peritoneal microfilariae recovered by aspiration from these animals at two and a half to three months appeared dead. After three and a half months of infection up to 60% of the peritoneal microfilariae were found to be immotile, had a straight rigid conformation and appeared to be disintegrating with- out provoking any local host inflammatory response. At five and a half months one jird still carried living rnicrofilariae in its abdominal cavity, another con- tained microfilariae that were all dead and the third animal had no microfilariae at all present in its peritoneal cavity. The levels of microfllaraemia in these three animals also began to decrease slowly after three months. One animal was negative for circulating parasites at five and a half months and all these jirds were negative by eight and a half months, at which time they were all autopsied. Nine female worms were found but no males seen.

The female worms which appeared alive and healthy were again implanted, four worms each being placed in the -peritoneal cavities of two naive jirds. One of these animals develoned a low microlihuaemia (5 mf/lOO lrl 1 blood) and had microfilariae present in its abdominal cavity. Both these animals were autop- sied at 15 days when half the re-implanted worms were found to have migrated out of the peritoneal cavity and were lying ei&er over the muscles of the seine in the hiah lumbar (Ll to L3) reaion of the back? or were “unongst the adipose’ tis&es of the ingumal region. In one jird, two worms were found embedded in the adipose tissues close to the right kidney.

Histopathological findings in jirds: Histopathological examination of samples from jirds 15 days after retransplantation of adult L. ba showed a number of significant changes in tissues adjacent to the worms that were lying on the dorsal muscles and in the renal area. Worms lying in the adipose connective tissue on the dorsal muscles were usually viable and fecund, and were surrounded by a granulomatous reaction (Fig. 1). This host reaction comprised of a loosely packed fibrous tissue with a considerable number of

eosinophils scattered amongst the fibrocytes and fibres (Fig. 1). EOS~IIOD~~~S were more common in the tissues lymg’nearer to the worm and to the nearby blood vessels. Cells, often clearly discernible as eosinophils (Fig. lc), were seen attached to the outer surface of adult parasites in sections where the granulomatous response occurring around these par- ticular worms commonly contained foci of eosin- ophils. Where the cellular reaction around the adult worm was solely fibrocytic no adherence of eosin- ophils or other cell types to its surface was seen. Macrophages and a few giant cells (polykaqons) were present in the granulomatous tissue and there was __~~. ~~ the o&a&~ focus ofplasmacytes evident. Pocketsof inflammatory cells were common in the surrounding adipose tissue itself, and here eosinophils and mono- nuclear cells were the predominant cell types; a few of these foci had microfilariae in the midst of the cells.

Changes were also seen in the muscle tissues adjacent to the adult parasites. The capsule surround- ing the muscle bundles appeared thickened due to a proliferation of connective tissue and an invasion of i&unmatory cells that were almost exclusively eosi- nophils, a few foamy-type macrophages were also seen. Microfilariae were present in the capsular area, either amongst the proliferating connective tissue cells or actually outside of the capsule. Foci containing many eosinophils, a few mononuclear cells., and sometimes neutrophils, were seen in the petvnysia separating the fasciculi of muscle fibres (Fig. 2). Microfilariae were commonly found in these cellular foci (Fig. 3). In areas where there was an intense infiltration of cells and parasites the adjacent muscle fibres were sometimes undergoing ischaemic degen- eration, with no apparent regenerative process (Fig. 2). Blood vessels in the wrimvsia were usuallv cuffed with inflammatory cells*parti&arly eosinoptis, with neutrophils sometimes being present.

The tissue taken from the perirenal area containing calcified worms was essentially comprised of a mass of fibrous tissue that tightly surrounded the shells of adult worms. Only part of the cuticles of these worms remained intact and their centres had become cal- cified. Eosinophils were scattered amongst the fibrous tissue.

All the lymph nodes ezamined had moderately active germinal centres, paracortical zones and medul- lae; eosinophils were seen in the medullary zones of these nodes. Minor interstitial foci of in&mmation were present in the kidneys of most animals, although the significance of these particular findings is un- known.

It is apparent from this current study that L. loa adults can be successfully maintained in the peritoneal cavity of jirds for three to four months, thus providing a laboratory model for the study of loiasis. Various aspects of the chemotherapy of this disease and the infection of vectors could also be studied with this model. If a good supply of adult Loa could be obtained it would be possible to use this model to screen drugs for filaricidal activity, as is done with &ugia pahygi (SE Suswrq~o 8z DENHAM, 1977).

The granulomatous changes in the tissues adjacent to the worms are consistent with other filarial diseases and reflect an active cell-mediated response to the

782 Loa loa IN JIRDS

presence of the adult parasites. The predominance of eosinopbil leucocytes in these reactions is also consis- tent with findings in other filarial infections. The presence of inflammatory foci in the muscle tissues and the damage to muscle fibres is noteworthy. The presence of microfilariae in these foci suggests tbat tbis particular parasitic stage may be contributing significantly to tbe pathology occurring in these particular tissues. The common occurrence of eosin- opbils around these parasites may indicate that there is an immunologically-mediated destruction of micro- filariae taking place at these foci. Whether or not there is any connection between these observations and clinical symptoms such as muscle pain and cellulitis sometimes seen in humans infected with Loa loa is unknown but such reactions in muscle tissues may be contributory.

Acknowledgements The drills used in this study were infected by Dr. B. 0. L.

Duke, MRC Helminthiasis Unit, Kumba, United Republic of Cameroon, and his efforts in supplying these animals are gratefully acknowledged. Dr. A. E. Blanc0 is also thanked for his collaboration as is H. A. Furse for his expertise in preparing tissues for histological examination. D.A.D. is an external staff member of the Medical Research Council, UK.

;fthi~rp supported by the Tropical Medicine Research 3 .

References Dennis, D. T. 81 Kesn, B. H. 11971). Isolation of

microhlariae: report of a new method. Journal of Parasitology, 57, 1146.

Duke, B. 0. L. (1972). Behavioral aspects of the life cycle of Loa. In: Behavioral aspects of parasitic transmission. Canning, E. V. & Wright, C. A. (Editors). London: Academic Press.

Ehehard, M. L. & Orihel, T. C. (1981). Development and larval morphology of Loa loa in experimental primate hosts. Journal of Parasitology, 67, 556-564.

Orlhel, T. C. & Moore, P. J. (1975). Loa loa experimental infection in two species of African primates; American Journal of Tropical Medicine and Hygiene, 24, 606-609.

Suswillo, R. R., Nelson, G. S., Muller, R., McGreevy, P. D., Duke, B. 0. L. & Denham, D. A. (1977). Attempts to infect jirds (Merianes unguiculatus) with Wuchereria bancroft, Onchocerca volvultts, Loa loa loa and Mansonella ozzardi. 3ournal of Helminthologv, 51, 132-134.

Suswillo, R. R. & Denham, D. A. (1977). A new system of testing for hlaricidal activity using transplanted adult Brugia in the jlrd. 3ownal of Parasitology, 63, 591-592.

Accepted for publication 30th March, 1982.