Lymphatic patterns of cats infected with Brugia malayi and streptococcus

TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICAL MEDICINE AND HYGIENE, VOL. 7 1, No. I, 1977.

Lymphatic

patterns of cats infected with Brugia malayi and streptococcus *

Department of Microbiology,

W. BOSWORTHAND A. EWERT University of’ Texas Medical Branch, Galveston, Texas 77550, U.S.A.

Summary

Approximately 50% of the hind legs of cats infected with Erugiu malayi and insulted with a beta haemolytic streptococcus became elephantoid in appearance after four to six weeks. This condition was found to be reversible. Collateral lymphatic vessels were seen by lymphography in most of the Brlrgiu-streptococcusinfected legs. The popliteal draining systems in two of three cats, which harboured no worms, appeared normal by lymphography at I8 weeks. Introduction

Lymphography and xerography have been used to visualize it? situ lymphatic pathology resulting from Brttgiu infections in the hind limbs of dogs and cats. The age, number and intensity of BrL/giu infections, as well as the sex of the vertebrate host, have been considered in interpreting attendant lymphatic lesions (EWERT et cd., 1972; GOONERATNE, 1973; SCHACHER et d., 1973; ROGERS~~ a/., 1975).Recently, DENHAM & ROGERS(~ 975)

treated Brttgia puhangi-infected cats with an active macrofilaricide and showed by xerography that the enlarged lymphatics soon returned to their pre-infection size. We have been able to amplify gross effects including lymphangitis, oedema and fibroplasia in the B. ttzalayiinfected hind limb of cats with a streptococcal insult and have noted at necropsy a decrease in the worm burdens of these cats (BOSWORTH et al., 1973; BOSWORTH & EWERT, 1975). The present study was intended to note the condition of the filaria-infected lymphatics at various intervals following bacterial infection and to correlate the lymphographic picture with the gross pathology of the affected limb. Materials

and methods

Thirteen cats were initially exposed on the volar surface of one hind foot to 100 B. ttdayi larvae and after three weeks re-exposed on the same foot to an additional 100 larvae. Four weeks after reinfection, a Group G beta haemolytic streptococcus was applied to the hind limbs of these cats in a manner similar to that described by BOSWORTH & EWERT (1975). Six control cats were concurrently exposed to the same number of Btwgiu larvae but not to streptococcus. Within the first two-week period following the streptococcal insult, cats were examined daily for possible sequelae, i e., erythema, oedema, lymphangitis, cellulitis or necrosis of the affected limb, and supportive therapy *This investigation was supported in part by a McLaughlin Fellowship Award from the University of Texas Medical Branch and in part by the United States-Japan Cooperative Medical Science Program administered by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, Department of Health, Education and Welfare.

was given as deemed necessary. Thereafter, cats were checked until lymphography and necropsy for changes in the gross appearance of the affected limb. Measurements of the hind limbs were made with Lange Skinfold calipers at the time of the streptococcal insult and at necropsy (BOSWORTH & EWERT, 1975). Cats were examined by lymphography four, IO or I8 weeks after streptococcal involvement. Two control cats, exposed only to Brugiu, were also examined by lymphography at these times. The lymphographic procedure employed in the present study is similar to that used by EWERT ef a/ (1972). Ethiodol was introduced into the afferent lymphatic vessels in the metatarsal region of the foot via a 27 or 30 gauge needle of a lymphangiography set”. A constant rate motorized injector was used to deliver the ethiodol. If the pressure became too great and the lymphatic vessel ballooned and opacified or ruptured, another site was cannulated. The actual amount of ethiodol injected into the infected leg varied while 0.5 ml of ethiodol adequately delineated the lymphatics of the uninfected, contralateral leg. Necropsy was performed the same day lymphograms were made or on the following morning. This allowed for comparing and contrasting the lymphograms with the condition of the lymphatics. The number and location of worms present were recorded. Results

Most of the Brlrgiu-infected legs were comparable in size and appearance with the uninfected leg at the time of the streptococcal insult (Table I). I2 of the 13 Bntgia plus streptococcus-infected legs displayed sequelae ranging from localized oedema about the afferent lymphatic vessels in the hock region to enlargement of the entire leg accompanied by abscess formation and sloughing of skin from necrotic areas. By four to six weeks after streptococcal infection, approximately 50 X, of the affected legs were elephantoid in appearance, but by the scheduled lymphography at 10 or 18 weeks, the elephantoid condition appeared to be waning or resolved in most of the legs. The Brrrgia-infected leg of one of the six control cats was lymphoedematous at the initial measurement. The Brligia-infected leg and contralateral leg of the two control cats examined at four or 10 weeks were comparable in size and appearance. The Brlrgia-infected leg which was lymphoedematous initially remained so until necropsy at 18 weeks, and the infected leg of the other control cat examined at 18 weeks became lymphoedematous in the hock region late in the study. The lymphographic picture of the Brugia plus streptococcus-infected legs varied with the individual cat. Collateral lymphatic networks were present at four, 10 or 18 weeks in most of the filaria-infected legs which RBecton, Dickinson and Company, Rutherford, N.J., U.S.A.

22

LYMPHATIC

PATTERNS

OF CATS INFECTED

WITH

BRUCIA

MALAY1

AND STREPTOCOCCUS

Table I - Size of the calcaneal tendon area (C.T.A.) of the Brugia-infected and uninfected leg at time of streptococcal insult and at lymphography Meusurement of’C.T.A. Cat No. 1

2 3 --4*

Isi measurement In/. leg Uninf. leg

3 4 2 2

3 2 1 2

in mm at insalt andat 4 weeks

2nd measurement Uninf: leg hf. leg 2 4 14 2

2 2 3 2

Measurement of C. T. A. in mm at insalt and at 10 weeks

5

6 I 8

1st measurement hf. leg Uninf. leg I 1

3 2 2 Measurement

1 2 2

4 2 2 2 15

6 8 6 IO

4 3 6 4

of C.T.A. in mm at insult andat 18 weeks

1st measurement Inf leg UninJ leg

9 10 11 12 13 -

2nd measurement Inf. leg UninJ leg

4 2 2 2 3

2nd measurement Inf. leg Uninf. leg

2 6 4 11 5

2 2 4 2 5

*No observable sequelae from streptococcal insult.

displayed sequelae to the bacterial infection. In most Brugia plus streptococcus-infected legs examined at four weeks, collateral networks were most prominent in the metatarsal, calcaneal tendon and hock regions of the elephantoid limbs (Fig. 1). At 10 weeks, elaborate collateral lymphatics were present in these same areas of legs which were still displaying some gross enlargement or had been elephantoid for some period following the bacterial infection (Fig. 2). By 18 weeks the collateral networks did not appear to be as prominent in the distal portion of the leg and there was more of a tendency to find the collateral vessels coursing the entire length of the leg (Fig. 3) and draining into the external iliac lymph node. Lymphographically, the popliteal lymph node was not readily detectable in legs examined four or 10 weeks after the Brugia-streptococcus interaction, but improved vessel-node function permitted visualization of some nodes by week 18 (Fig. 3). Affected vessels were usually dilated in the metatarsal region of the foot but, while the afferent vessels tapered in the hock region and were not identifiable thereafter in some legs, they were prominent until junction with the popliteal node in others. The afferent vessels which successfully conducted the contrast media into the popliteal lymph nodes at week 18 were comparable lymphographically with the vessels of the uninfected, contrafateral leg. In addition to the popliteal afferent lymphatics, lymphatic vessels alongside the saphenous vein were apparent in some of the legs at each of the lymphographic examining times. In the contralateral legs, the lymphograms showed that streptococcus alone had not produced any deleterious effects on the structure or function of the vessels or node

Fig. I. Lymphogram of elephantoid limb of Cat No. 3 at four weeks after streptococcal insult of Brugiu-infected leg. Collateral vessel networks are evident in the metatarsal, hock region and calcaneal tendon area of the leg. The main afferent vessels can be seen crossing the tibia (t).

of the popliteal draining system (Fig. 4). The lymphatic vessels alongside the great saphenous vein were also evident in severa of these legs examined at each interval. The legs of control cats exposed only to Bragia larvae before lymphography usually lacked collateral lymphatic networks, except in the two which were lymphoedematous (Fig. 5). Additionally, the conditions of the afferent vessels ranged from total occlusion, supplemented by dilated vessels alongside the saphenous vein, to being grossly dilated until joining the popliteal lymph node. However, the affected popliteal lymph nodes in these legs also failed to fill with ethiodol. The gross condition of the lymphatics at necropsy corresponded well with the lymphogram. It was difficult though to visualize collateralsper se, especially when they were in the collagenous beds in the distal regions of the leg. The failure of ethiodol to delineate completely afferents and lymph nodes could be associated in some

W. BOSWORTH

AND A. EWER-T

23

Fig. 2. Lymphogram of the Brugiu plus streptococcusinfected limb of Cat No. 6 at IO weeks after streptococcal insult, showing collateral vessels. A lymphatic vessel (t) alongside the great saphenous vein is evident and there is dermal backflow in the metatarsal region of the foot.

Fig. 3. Lateral aspect of the Brr/gia plus streptococcusinfected leg of Cat No. II at IX weeks after streptococcal insult. Improved vessel-node function permits visualization of the entire popliteal draining system. Collateral vessels and lymphatics (t) alongside the great saphenous vein are coursing the entire length of the leg.

cases with the presence of lymph thrombi, boluses of live and dead worms or apparent atrophy of lymphatic structures. The worms were usually more distally located in the lighter infections and more evenly dispersed in the afferent vessels in heavier infections. The worm recovery ranged from 0 to 45 with a mean of 13 in experimental cats and from 12 to 56 with a mean of 29 in control cats.

lymphatic pathology might produce a gross disease manifestation such as elephantiasis. Animal studies engaging B. pahangi and 8. malayi have permitted localization of worms, as well as known intensity and duration of infections, for lymphographic examinations. Attempts to alter any of these variables and produce lymphoedema or elephantiasis have met with limited success. With the development of an elephantoid condition of the hind legs of cats infected with both B. nzalayi and streptococcus (BOSWORTH & EWERT, 1975) the opportunity to unmask at least a portion of the pathogenesis of elephantiasis appeared to be available. We established that the excess tissue found four weeks after streptococcal infection was collagen and noted, in most instances, that the afferent lymphatic vessels and popliteal lymph node were severely affected. In the present study, we found that the experimentally

Discussion

The occurrence of lymphoedema and elephantiasis in humans infected with filariae has been investigated lymphographically (COHEN et a/. 1961; CAHILL & KAISER, 1964; CARAYON et al., 1968). In affected areas of the body so examined, dilated, tortuous vessels have been found frequently. However, it has been difficult to establish by what means the filarial infection and resultant

24

LYMPHATIC

PATTERNSOFCATSINFECTED

Fig. 4. Uninfected, contralateral leg of Cat No. showing lymphatic draining patterns.

WITHBRUGIAMALAYIANDSTREPTOCOCCUS

11

produced elephantoid condition was not irreversible. The Brlrgia-infected legs were at their maximum size by the fourth to sixth week after streptococcal involvement, but thereafter the affected limbs began to return to their normal size. Lymphographically, it was possible to correlate changes in the gross appearance of the Evugiu plus streptococcus-infected leg with the condition of the lymphatics. The vast collateral lymphatic networks found in the elephantoid limbs at four weeks were in regions where collagen was most prevalent. The production of collagen as scar tissue is accompanied by the formation of extensive blood and lymphatic vessel networks. Both of these systems are involved with bringing an area into equilibrium. The collateral lymphatics were also present in cats examined lymphographically at IO weeks although the elephantoid condition was waning. We speculate here that these collaterals are actively involved in the resolution ofthecollagen matrix. CLOIXUS et al. (1976) examined tissue tonicity in lymphoedema and reported that the rate of formation or destruction of existing fibrotic tissue is determined by the balance between collagen formation and lysis. Conservative treatment with Venalota was

Fig. 5. Lymphogram of Br@r-infected limb serving as control for Cat No. 11 (Figs. 3 and 4). Note dilated, tortuous afferent vessels. Some collateral vessels have formed in the calcaneal tendon area and in the mid-leg region.

found to restore tissue compressibility and increase lysis of collagen. The probable mode of action of this drug is to cause proteolysis in the tissues. Experimental lymphoedema has been produced by ligating lymphatics and successfully treated by using coumarin (CASLEY~MITH et al., 1974; FBLDI-B~RCSBK et al., 1972). Among the suggested reasons for the resolution of lymphoedema have been the uptake of protein molecules by blood capillaries, catabolism of plasma protein or more rapid opening of collateral lymphatic vessels. Filarial lymphoedema can also provoke collateral formation as was seen in our Brugia control cats. The rate of formation of RSchaper &Brtimmer,

Western Germany.

W.

BOSWORTH

these collateral vessels could not be determined, but one of the legs only became lymphoedematous late in the study and presumably the collaterals seen by lymphography in the affected areas were recently formed. We have no way of knowing if the collaterals would have contributed significantly to the resolution of the lymphoedema in this case. It would be interesting, though, to compare resolution rates of lymphoedema of cats treated with coumarin or Venalot with that of untreated, infected animals. The failure of ethiodol to enter the affected popliteal lymph node at four to IO weeks is also of particular interest, in that previous studies have usually reported contrast media in the nodes. The multiplicity of factors in the present study probably contributed to this finding. It is of interest that by I8 weeks the affected popliteal node of cats Nos. 9 and I I was evident by lymphcgraphy; neither of these cats harboured any worms at necropsy, they did however have some collateral lymphatic vessels coursing the entire leg. This correlates with the findings of DENHAM & RICERS (1975) who reported that after treatment of experimental filarial infections in cats, lymph vessels and nodes returned to normal. It would thus appear that elimination of the filarial infection either by a known, standard filaricide or by a secondary bacterial infection with the accompanying inflammatory reaction may permit the affected lymphatics to re-establish their normal structure and function. The formation of collateral lymphatic vessels also influences the ultimate pathological condition seen in filarial infections. References Bosworth, W. & Ewert, A. (1975). The effect of streptococcus on the persistence of Brtrgirt /rtu/u.r’i and on the production of elephantiasis in cats. Irr/er~ario~rrl Jourt~crlfkw Purasitology, 5, 583-589. Bosworth, W., Ewert. A. & Bray J. (1973). The interaction of Brtrgiu mzlqi and streptococcus in an animal model. Americurl Jomxctl of’ Tropical Medicitre md Hygiene, 22, 7 14-7 I 9. Cahill, K. M. & Kaiser, R. L. (1964). Lymphangiography in bancroftian filariasis. Trnrtstrctiom of the

AND

A. EWERT

2s

Royal Sorierv of’ Tropieul Medicitre mod Hj,giewe, 58, 356-362. Carayon, A., Coubril, L. J. & Colomar, R. (1968). Progrbs apportes par la lymphographie a la comprthension de la filariose de Bancroft. Med;citre D’Afiiqne Noire, 15, 2 I-24. Casley-Smith, J. R., Fbldi-Bdrcsok, E. 6i Foldi, M. (1974). Fine structural aspects of lymphoedema in various tissues and the effects of treatment with coumarin and troxerutin. Briti,sh Jormrtrl of E.yperimentul PuthOIOg~~,55, 8% 93. Clodius, L., Deak, L. & Piller, N. B. (1976). A new instrument for the evaluation of tissue tonicity in lymphoedema. L.r~?rplto/og>., 9, I 5. Cohen, L. B., Nelson, G., Wood, A. M., Manson-Bahr, P. E. C. & Bowen. R. (1961). Lymphangiography in filarial lymphoedema and elephantiasis. Americm Jo~rrrrul of Tropical Medicitw und H.vgietre, 10, 843--X48. Denham, D. A. & Rogers. R. (1975). Structural and functional studies on the lymphatics of cats infected with Brrlgirr puhutrgi. Trmscrctions of the Ro~nl Societ>j of Tropiccrl Medicine urtd Hygiette, 69, 173 176. Ewert, A., Balderach, R. & El Bihari, S. (1972). Lymphographic changes in regional lymphatics of cats infected with Brugirr mrrlu>.i. Atrrericutr Jo~rrrtrl of Tropicrd Medicine uttd H.vgiene, 21, 4074 14. Foldi-Borcsbk. E., Casley-Smith, J. R. & Foldi, M. (1972). The treatment of experimental lymphedema. Attgiologica, 9, 92-~98. Gooneratne, B. W. M. (1973). A chronological study of cats experimentally infected with Brulrgia filariasis from five days to tive years. L.vmphology, 6, l27- 149. Rogers, R., Davis R. & Denham D. A. (1975). A new technique for the study of changes in lymphatics caused by filarial worms. Jor~rtrrrl of’ Helttritrthology, 49, 31-32. Schacher, J. F., Edeson, J. F. B., Sulahian, A. & Rirk, G. (1973). An l&month longitudinal study of hlarial disease in dogs infected with Brugiu puhmgi (Buckley & Etleson, 1956). Awt7rrl.v of’ Tropicul Medicitte ctttd Ptrmcitolog)~, 67, 8 I-94. Acceptedfiw

prthlicutiotr

17th September IY7fi.