Abstract. The mouse/Necator americanus model was studied to assess the histopathological changes that oc- cur in the lungs following primary and secondary ...
Parasitol Res (1990) 76:386-392
Parasitology Research 9 Springer-Verlag 1990
Necator americanus in the mouse:
histopathological changes associated with the passage of larvae through the lungs of mice exposed to primary and secondary infection M.J. Wilkinson 1, C. Wells 2, and J.M. Behnke 2 1 Department of Pathology, University Hospital, Queen's Medical Centre, University of Nottingham, and 2 The MRC Experimental Parasitology Research Group, Department of Zoology, University of Nottingham, UniversityPark, Nottingham NG7 2RD, UK Accepted December 20, 1989 Abstract. The mouse/Necator americanus model was studied to assess the histopathological changes that occur in the lungs following primary and secondary exposure to infective larvae. Groups of BALB/c mice were infected percutaneously and killed on various days post infection. Parasite numbers were counted, the bronchoalveolar leukocyte response was quantified and histological sections of lung material were examined for evidence of host protective inflammatory reactions. An increase in the inflammatory infiltration was observed between days 5 and 9 in both primary and secondary infections but was considerably more intense in re-infected animals. This involved a marked change in the character of the infiltrate, particularly in the number of eosinophils that were recovered in lavage fluid. More worms were trapped in the lungs of challenged mice, as assessed through their inability to escape from lung material incubated in vitro. Overall, the results were found to be compatible with the development of acquired resistance to N. americanus and the expression of host protective immunity during the development of challengeqnfection larvae in the lungs.
Hookworms are endemic throughout most tropical and sub-tropical regions, with two species, Ancylostorna duodenale and Necator americanus, predominating as the major anthropophilic species, whereas a third, A. ceylanicum, causes local infections in parts of Asia. Collectively, hookworms are believed to affect some 900 million people worldwide (Banwell and Schad 1978; Crompton 1989; Keymer and Bundy 1989). Both A. duodenale and N. americanus can penetrate human skin, but the latter species is almost exclusively dependent on this route of infection, whereas the former establishes more successfully following oral ingestion of infective larvae (Hoagland and Schad 1978; Komiya and Yasuraoka 1966; Mizuno and Yanagisawa 1963). After Reprint requests to : J.M. Behnke
successful penetration of the host's skin, the L3 of N. americanus migrate to the lungs, where they undergo
a period of development usually lasting about 5 days and involving significant changes in larval morphology (Behnke et al. 1986a). A proportion of larvae may be delayed in the lungs for > 1 week. This period in the lungs appears to be obligatory in N. americanus, enabling further development and subsequent establishment in the intestine. The passage of hookworm larvae through human lungs is known to be associated with respiratory distress including bronchitis, transient bronchopneumonia (Kalmon 1954) and, occasionally, with death (Zimmerman 1946). Furthermore, in parts of Japan the onset of Wakana disease in late summer months, characterised by persistent asthmatic cough, is considered to be linked with seasonal exposure to hookworm larvae during the transmission season (Harada 1962). These studies indicate that the development as well as passage of hookworm larvae through the lungs is not without pathological consequences for the host, but overall, very little is known about the induction of pulmonary pathology during hookworm infection, principally because the parasites are so specific to man and manipulable laboratory models for human hookworm disease are scarce (Behnke 1989). During the last 20 years a strain of N. americanus has been adapted to passage through laboratory hamsters (Behnke et al. 1986a; Sen 1972), and larvae maintained in this host were found to follow the normal migration pathway when presented percutaneously to mice (Wells and Behnke 1988a). Subsequent studies demonstrated that mice acquired immunity to N. americanus and that this was reflected in the arrival of fewer larvae in the lungs following percutaneous infection (Wells and Behnke 1988 b). In the present paper we report a detailed study of the histopathological changes that accompany the expression of acquired resistance to N. americanus in mouse lungs, and we conclude that in addition to the skin, the lungs may also play a significant role in parasite attrition.
M.J. Wilkinson et al. : Pulmonary histopathology in N. americanus infection
387
Materials and methods
Statistical analysis of results
Animals
Groups were compared using the non-parametric Mann-Whitney U-test (Sokal and Rohlf 1969). The data are presented as mean values+standard deviation (SD) or as percentages, unless otherwise stated.
Male BALB/c mice were bred and maintained under conventional conditions. The original breeding stock was acquired from Harlam Olac Ltd., Bicester Oxon, in 1976. The animals were provided with food and water ad libitum and were generally infected when 6-8 weeks old.
Infections N. americanuswas maintained in hamsters as previously described (Behnke et al. 1986a). Mice were infected percutaneously after anaesthesia with Sagatal (M 7B Veterinary Products). The abdomen of each animal was shaved and cleaned and 350 infective larvae were applied on a gauze pad secured to the skin with adhesive tape; the pad was removed after 24 h. The full details of this technique have been described elsewhere (Behnke et al. 1986b). Animals were killed by inhalation of chloroform in groups at set inter'(als post infection, with five animals being used for broncho-alveoIar lavage and three, for histological assessment at each time point.
Histological assessment The lungs were fixed in distension using Bouin's fluid and subsequently embedded in paraffin wax, giving two blocks for each group of animals. Four sections were cut at 5-gm thickness from each block and stained, one each with haematoxylin and eosin (H&E), periodic acid-Shift (PAS), sirius red and carbol chromati'ope. The slides were examined qualitatively and a quantitative assessment was made by point counting. The H&E-stained sections Were projected onto a grid with points set at the apices of triangles with sides 5 cm long. The area of inflammatory infiltrate was secondarily counted on a grid with points 1 cm apart, giving a ratio Of 1 : 15 between the two grids. The final magnification after projection was 180. Worms were most easily detected on the PAS-stained tissue sections.
Results
Changes in total parasite numbers in the lungs following infection with N. a m e r i c a n u s A n u m b e r o f experiments have been carried o u t to q u a n tify the passage o f larvae t h r o u g h the lungs at different times post infection. Some o f the results have been p u b lished elsewhere (Wells a n d Behnke 1988a, b), b u t two representative d a t a sets are presented i n Fig. 1. Parasites were first detected in the lungs o n d a y 3, a n d in m o s t experiments the highest yields o f active larvae were rec o r d e d at this time, reflecting the s i m u l t a n e o u s arrival o f w o r m s f r o m the skin p e n e t r a t i o n site 2-3 days post infection (p.i.). T h e w o r m b u r d e n s fell slowly, a l t h o u g h n o t significantly w h e n c o m p a r e d statistically, over the following 2 days a n d then m o r e drastically by day 9, with o n l y a few larvae persisting for longer. The initial arrival o f larvae i n the lungs of mice t h a t h a d previous experience of i n f e c t i o n with N. americanus was significantly reduced, a n d a l t h o u g h w o r m b u r d e n s also declined m a r k e d l y by d a y 9 w h e n assessed by recovery o f active larvae, the m a j o r i t y o f persisting larvae (Ta-
80 §
9 17 [] []
LU Q:
Broncho-alveolar lavage The mice were killed and bled from the heart. The lungs were exposed, the trachea was ligated and 0,5 ml phosphate-buffered saline was injected into the trachea using a 21-gauge needle. The saline was aspirated and the process repeated, The total number of cells obtained from each animal was counted, the aspirates were pooled and centrifuged and cytopsin preparations were made. These were stained using Wright's stain and differential counts were completed on at least 500 cells/slide.
uJ >
60
o o
no
40
k~
o rw m co
Enumeration of larvae
EXPTI PRIMARY EXPTISECONDARY EXPT2PRIMARY EXPT2SECONDARY
20
I[ Z
The number of larvae in the lungs was determined using two approaches. The distribution of larvae between left and right lungs is always identical. The left lung was cut into 2-mm cubes and each section was squashed between 2 microscope slides. The total number of larvae could thus be accurately determined. Active larvae that were not trapped by the pulmonary reaction were counted by mincing the right lung and incubating the tissues in Hanks' saline for 24 h as previously described (Behnke et al. 1986a). Larvae migrating from the tissues were collected from the supernatant saline at 2, 6 and 24 h, then counted and removed. The number of trapped larvae was calculated from the difference in recovery between these two methods.
Z < m
I[
o 5 DAYS
6
7
POST
INFECTION
g
10
13
Fig. 1. Recovery of active larvae from the lungs of mice exposed to primary or secondary infection with N. americanus. The figure shows results from two experiments, one of which (Expt 1) has been published elsewhere (Wells and Behnke 1988b). Five mice were killed at each time point; in experiment 2, additional groups were also killed on days 16 and 2l but no worms were recovered
388
M.J. Wilkinson et al. : Pulmonary histopathology in N. amerieanus infection
Table 1. Evidence for a higher rate of larval entrapment in the lungs of immune mice challenged with N. arnericanus relative to controls Experiment
1 2 3 4 5 6 7
Treatment"
Primary Secondary Primary Secondary Primary Secondary Primary Primary Secondary Secondary
Mean number of worms recovered • SD Active migrating larvae (right lung) b
Total larvae (left lung) ~
15.5+_5.3 12.0_+5.8 6.2+-3.0 2.0+-1.6 9.3_+4.6 15.8+-9.0 8.2+_2.2 5.2+_1.3 4.8+_2.2 10.0+_4.6
18.7_+ 5.0 33.3+- 6.1 7.4+- 1.8 8.4+_ 3.6 19.0+- 3.5 42.3+-10.6 9.4_+ 3.4 7.2+_ 3.4 16.4+_ 3.9 34.7+_ 8.1
Index of lung trappingd
17.0 64.0 16.2 76.2 50.9 62.7 12.8 27.8 71.6 71.2
a Groups of mice were killed 9 days after primary or secondary infection. The first three experiments were direct comparisons between immune and control animals. Experiments 4-7 represent additional data collected from experiments in which immune and control animals were not directly compared b Larvae migrating from minced lungs during 24 h of incubation in Hanks' saline at 37~ C c Total larval burdens of the left lung were determined by counting worms in situ following meticulous examination of squashed sections of tissue d The index of lung trapping was calculated as: 100-(-active migratingt~talal la~vae i~eftlu~-glarvae in right lung x l00)
Table 2, The broncho-alveolar leukocyte response to N. amerieanus
in the lungs of mice exposed to primary and secondary infection Recovery of broncho-alveolar leukocytes
Day after infection
Treatmerita
Control 2 2 5 5 9 9
None 4.03_+1.16 98.7 Primary 3.58+0.99 94.4 Secondary 6.94___2.08 96.3 Primary 6.98-+1.78 97.4 Secondary 8.50+_1.39 60.7 Primary 7.68_+2.11 46.1 Secondary 16.80-t-_3.20 43.0
Total cells Macro- Lym- Neutro- Eomean_+ SDb phages pho- phils sino(%) cytes (%) phils (%) (%) 0.7 2.0 1.1 1.6 8.2 8.5 11.0
0.3 3.2 2.1 0.5 6.4 3.2 1.2
0.3 0.4 0.5 0.5 24.7 42.1 44.8
" Mice were killed following primary or secondary infection on the days shown and a group of naive control mice was included for comparison b This represents the mean number of cells recovered x 10s ml- 1 lavage fluid 2@
1
9 macroph~ges [] lymphocyles [] neulrophils
,
[]
o
eosimoDhils
X LU LU > 0 U LU
10
J Lu (3
ble 1, 6 2 % - 7 6 % ) appeared to be trapped and did not emerge on incubation in saline.
O
Changes in broncho-alveolar leukocytes during infection with N. americanus
GRoup Lung larvae of normal mice generally yielded about 4.03 x 105 cells m1-1, comprising 98.7% alveolar macrophages. During primary infection there was a steady increase in the total n u m b e r of cells collected, and this was accompanied by marked changes in the proportions of the various cell types (Table 2). A particularly prominent eosinophilia was evident by day 9 p.i. The secondary infection was accompanied by more m a r k e d changes initiated earlier in relation to the day of exposure to infective larvae. Eosinophilia was detectable as early as day 5 (Fig. 2). In b o t h the p r i m a r y and the secondary infection, considerable b a e m o r r h a g e was observed in the lavage fluid at 5 days p.i. At autopsy, the lung pleura typically showed localized petechial spots, ranging to ecchymotic areas, and often the entire extremities of the lung lobes seemed to be darkened by extensive haemorrhage. The magnitude of the h a e m o r r h a g e detected in broncho-alveolar leukocyte (BAL) lavage fluid and observed on the lung pleura (assessed subjectively) was reduced following secondary exposure.
D A Y P.i.
CO.TRO, DAY
0
1 DAY
2 2
1 DAY
2 5
1 DAY
2 9
Fig. 2. The number and class of broncho-alveolar leukocytes recovered following lung lavage on days 2, 5 and 9 post infection (p.i.) with N. americanus, l, primary infection; 2, secondary infection Histological assessment At 5 days following infection, mice exposed to both prim a r y and secondary infection showed intra-alveolar oedema (Fig. 3 b), but the p r o p o r t i o n of total alveolar volume affected was different (Table 3; 50% during primary infection vs 12.4% following challenge). An inflammatory infiltrate composed predominantly of acute inflamm a t o r y cells with occasional lymphocytes and scanty eosinophils was observed in a peri-bronchial location and was significantly more marked in the challenged animals (compare Figs. 3a, b and 4a). Sections of larvae were seen, consisting of a basophilic cuticle between 15 and 30 gm in diameter surrounding the empty digestive tract.
M.J. Wilkinson et al. : Pulmonary histopathology in N. americanus infection
389
Table 3. Quantification of the histopathological changes in the lungs of mice infected with N. americanus by point counting from histological sections Day
Treat-
after infection
ment a
Control 5 5 9 9
Alveolar parenchyma c Percentage of lung affected by Mean_+ SD (%)b
None Primary Secondary Primary Secondary
Total points counted
86.4+_1.0 84.6_+1.0 84.2_+1.1 73.7+-1.3 68.6+1.4
Affected by oedema
(%)
0 50.0 12.4 3.4 0
5-cm 1-cm grid grid
inflammation a Mean +- SD b Peribronchial
Parenchymal
1.9+_0.7 5.4+-0.1 12.3_+0.2 10.6+-0.2 17.0+-0.3
0 0 0 23.1 28.3
100.0 100.0 100.0 76.9 71.7
2658 2465 2295 2709 2856
2146 3888 4228 6895 794
a Mice were killed following primary or secondary infection as described in the text and a group of naive control mice was included for comparison b Percentage of total lung volume represented by the parenchyma or affected by inflammation was calculated in each case c The 5-cm grid was used to estimate the percentage of lung parenchyma on each section as well as to determine the proportion of alveolar space affected by oedema a The 1-cm grid was used to calculate the total percentage of inflammatory infiltrate on each section and to estimate the extent of peribronchial vs parenchymal infiltration
Fig. 3a-d. Histopathological changes in the lungs of mice 5 and 9 days after primary infection with iV. americanus, a Normal lung parenehyma ( x 101.8). b Lung parenchyma in infected mice on day 5 p.i,, showing extensive oedema (arro~4:s) and parasites (P) in the small airways ( x 101.8). c Higher magnification of part of
3b ( x 69.4), showing a close up of the larvae. There is no sign of a local response to the parasites, d Day 9 after primary infection, showing the more intense parenchymal infiltrate (arrows) detected in the later stages of infection and the absence of oedema ( x 39.8)
390
M.J. Wilkinson et al. : Pulmonary histopathology in N. americanus infection
Fig. 4a-d. Histopathological changes in the lungs of mice 5 and 9 days after secondary infection with N. arnericanus, a Extensive inflammation in the parenchyma 5 days after challenge infection ( x 25.9). b Inflamed lung parenchyma, peri-bronchiolar infiltration of leukocytes and a granulomatous reaction (arrow) surrounding a larva at 9 days p.i. (x 25.9). c Closeup of 4b, showing details of the cellular reaction around the parasite (x 2/2.9). d Section of lungs 9 days after challenge infection, showing parasites (P) free in the bronchioles ( x/06.4)
The larvae were mainly located in the small bronchi and alveolar ducts and did not appear to have excited any local cellular inflammatory response (Figs. 3 b, c). By day 9 the oedema was significantly reduced in the primary infection group and had disappeared completely from the mice exposed to the secondary infection (Fig. 4b). The intensity of the cellular inflammatory infiltrate had increased in both groups and, in addition to the polymorphonuclear leukocytes, lymphocytes and macrophages were evident with the formation of giant cells in some sites (Fig. 4c), particularly in challenged animals. In accordance with the lavage results, eosinophils were seen as a higher proportion of the infiltrate on tissue sections. Some larvae (or debris) in the lung parenchyma of challenged mice were surrounded by inflammatory cells (Fig. 4c), but in others, larvae devoid of any adherent cells were also clearly apparent (Fig. 4d). The inflammatory cells surrounding larvae
showed the same mixture of cell types as in the peribronchial infiltrate.
Discussion The passage of the larval stages of N. americanus through the mouse lungs was clearly associated with marked changes in the composition of the leukocytes located in the organ, with temporary haemorrhage, with oedema and, possibly, with the entrapment of developing parasites, particularly during secondary exposure. It is not possible to be certain on the basis of our results whether larvae were actually killed within the lungs or whether their development and onward migration were simply retarded. The greater numbers of apparently trapped parasites in secondarily exposed mice suggest that acquired immunity was involved and that the more rapid BAL response on re-exposure to infective larvae was capable of affecting a larger proportion of the developing parasites. Massive haemorrhage seen in the broncho-alveolar lavage fluid on day 5 appeared to be more severe in the primary infection than in the challenged animals but was not precisely quantified. Our observations on petechial and ecchymotic areas on the pleural surface as well as on the more extensive haemorrhage typically localized at the extremities of lung lobes were in accor-
M.J. Wilkinson et al. : Pulmonary histopathology in N. americanus infection dance with an earlier report on comparable pathology observed during the development of N. americanus in the lungs of guinea pigs (Schwartz and Alicata 1934). The disappearance of haemorrhage by the 9th day suggests that this occurred in the early stages of migration, possibly as the larvae left the arteries, the red cells following through the lesions created in the process. Schwartz and Alicata similarly reported that the haemorrhagic lung lesions that they had observed in guinea pigs in the early stages of infection had more or less resolved by the 2nd week. However, haemorrhage was not seen on histological sections, suggesting that it was not an effect solely attributable to damage to lung tissue sustained through the activities of migrating larvae. The passage of L3 through the arterial walls may have rendered the latter more fragile, enabling disruption by the trauma of broncho-alveolar lavage. Intra-pulmonary haemorrhage can be induced in rats infected with live Nippostrongylus brasiliensis larvae but not with inactive larvae (Salman and Brown 1980), supporting this suggestion. At 5 days after infection, the mice exposed to primary infection showed 50% of the total alveolar volume to be filled with oedematous fluid. In challenged mice only 12% of the alveolar volume was affected, but the percentage declined further in both groups by day 9, possibly implicating vessel leakage during the earlier stages of infection. The Iess severe haemorrhage arid oedema experienced by the challenged animals most likely reflected the arrival of fewer parasites from the infecting dose in the lungs as a result of the expression of pre-lung resistance (Fig. 1; also see Wells and Behnke 1988b); therefore, pulmonary pathology may be dose-related. In addition, it is possible that the normal damping of the acute inflammatory response in immune animals was involved. Our results demonstrate that in keeping with other models (Egwang et al. 1984), N. americanus induced marked inflammatory changes in the mouse lung, with the infiltrate initially being typically composed of acute inflammatory cells (day 5) and then progressing to a characteristic chronic inflammatory picture (day 9). Immune animals experienced more intense reactions, which were confirmed by examination of both broncho-alveolar lavage cells and histological sections of the affected lungs. At 5 days after infection the acute inflammatory infiltrate was entirely peribronchial, but by day 9, at the height of the response as judged by BALs in lavage fluid, it affected some 20%-30% of the lung parenchyma. Worms were observed mainly in the small bronchioles and on day 5 were not associated with any local inflammatory response. By day 9, both primarily and secondarily infected mice had observable worms in their alveoli; in some cases, particularly in immune animals, these were surrounded by collections of inflammatory cells with occasional giant cells (Fig. 4b). The composition of these accumulations of inflammatory cells was broadly the same as in the BAL population recovered through lavage. Similar features of the lung response have previously been reported for Strongyloides ratti (Dawkins
391
etal. 1981) and N. brasiliensis (Egwang etal. 1984, 1985). The use of the BAL lavage to characterise the inflammatory changes enabled a more precise quantitative determination of the proportions of different types of inflammatory cells in the lungs, but it is important to note that the technique is biased towards cells capable of migrating into the alveolar spaces. Tissue-bound cells, such as many lymphocytes, are not as easily detected. Nevertheless, changes in the composition of cells recovered by lavage are indicative of an ongoing inflammatory response in the lungs. Our results showed a disproportionate rise in eosinophils and a fall in the relative proportion of macrophages, although alveolar macrophages were numerically the dominant cell population throughout (Wells and Behnke 1988b). In challenged animals the sequence of events was more rapid, such that as early as day 5 the composition of the infiltrates was comparable with that of those seen on day 9 of a primary infection. Thus, whereas during the primary infection the peak response occurred after the majority of larvae had left the lungs, during the secondary infection the intense response evident as early as day 5 would have coincided with the simultaneous presence of a substantial proportion of the invading larvae in the lungs. It is therefore realistic to suggest that some parasites may have been impaired, and evidence to support such a conclusion is found in the increased percentage of trapped larvae as assessed through their inability to escape from lung material during in vitro incubation (Table I) and the histological picture of some larvae surrounded, possibly entrapped, with in granulomatous foci in the lungs (Fig. 4c). The lungs may thus play a role in protective immunity to N. americanus in this laboratory model, as has been proposed for A. caninum in dogs. Miller (1965, 1966, 1979) concluded that the lungs were a major site of antigenic stimulation as well as effective immunological reaction to subsequent invading larvae. Apart from the present work and our earlier paper (Wells and Behnke 1988 b), the only evidence to suggest that human hookworms may elicit protective immunity in the lungs comes from D'Abrera (1958), who identified macrophages and giant cells around fragments of N. americanus larvae in human lungs but provided no quantitative data on larval entrapment. Two studies have indicated that there is no host protective immunity in experimentally infected volunteers given repeated doses of N. americanus, despite enhanced immunological reactivity as measured by antibody responses (Ball and Bartlett 1969; Ogilvie et al. 1978). The difference in response of mice and humans may be doserelated: mice received larvae at a dose of about 15 000/kg and humans received doses of < 1/kg and it is possible that in human subjects the quantity of antigen was below the threshold required to elicit immunity. Alternatively, it is conceivable that hookworms use immunomodulatory strategies to avoid immunity in man and ensure their own survival. If so, the mechanisms involved are likely to be finely tuned to the normal human host of these worms, whereas in abnormal hosts such as mice,
392
M.J. Wilkinson et al. : Pulmonary histopathology in N. americanus infection
w h e r e r e l e v a n t strategies m a y n o t o p e r a t e o p t i m a l l y , acq u i r e d i m m u n i t y m a y be e x p r e s s e d effectively, to the d e t r i m e n t o f the p a r a s i t e ( B e h n k e 1987a, b). F i n a l l y , in this p a p e r we p r o v i d e the first d e t a i l e d d e s c r i p t i o n o f the h i s t o p a t h o l o g i c a l c h a n g e s t h a t a c c o m p a n y the dev e l o p m e n t o f N. americanus in the lungs o f mice, a n d o u r o b s e r v a t i o n s are c o m p a t i b l e w i t h a role for p u l m o n a r y i n f l a m m a t o r y r e a c t i o n s in h o s t p r o t e c t i v e i m m u n i t y to this h u m a n h o o k w o r m . Acknowledgements. We would like to express our deep appreciation to the trustees of the Sir Halley Stewart and the Sir Samuel Scott of Yews Trusts, who provided generous support for our hookworm researc1~ programme. We are aIso gra~eful for a contribution from the Daniel Falkner Charitable Trust. CW held an SERC studentship in the Department of Zoology. We acknowledge the technical assistance of Mr. K. Cosgrove, Mr. S. McKeag and Mr. K. Gordon, and we thank Profs. D. Wakelin and P.N.R. Usherwood for the facilities provided for our research.
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responses during primary and secondary Nippostrongylus brasiliensis infection in rats. Parasite Immunol 6:191-201 Egwang TG, Richards CD, Stadnyk AW, Gauldie J, Befus AD (1985) Multinucleate giant cells in routine and rat lungs during NippostrongyIus brasitiensis infections. A study of the kinetics of the response in vivo, cytochemistry IgG and C3 mediated functions. Parasite Immunol 7:11-18 Harada Y (1962) Wakana disease and hookworm allergy. Yonago Acta Med 6:109-118 Hoagland KE, Schad GA (1978) Neeator amerieanus and Ancylostoma duodenate: life history parameters and epidemiological implications of two sympatric hookworms of humans. Exp Parasitol 44:36-49 Kalmon EH (1954) Creeping eruptions associated with transient pulmonary infiltrations. Radiology 2:222-226 Keynler A, 8undy D (1989) Seventy-five years of solicitude (abstract). Nature 337:114 Komiya Y, Yasuraoka K (1966) The biology of hookworms. Prog Parasitol Jpn 3 : 1-114 Miller TA (1965) Effect of route of administration of vaccine and challenge on the immunogenic efficiency of double vaccination with irradiated Ancylostorna eaninum larvae. J Parasitol 51 : 200-206 Miller TA (1966) Comparison of the immunogenic efficiencies of normal and X-irradiated Ancylostoma caninum larvae in dogs. J Parasitol 52: 512-519 Miller TA (1979) Hookworm infection in man. Adv Parasitol 17:315-353 Mizuno T, Yanagisawa R (1963) Studies on the infection route of hookworms with reference to experimental infection in human hosts with larvae of Ancylostoma duodenale and Necator amerieanus. Jpn J Hyg 13:311-335 Ogilvie BM, Bartlett A, Godfrey FC, Turton JA, Worms MJ, Yeates RA (1978) Antibody responses in self infections with Necator americanus. Trans R Soc Trop Med Hyg 72:66-71 Salman SK, Brown PJ (1980) A study of the pathology of the lungs of rats after subcutaneous or intravenous injection of active or inactive larvae of Nippostrongylus braziIiensis. J Comp Pathol 90:447-455 Schwartz B, Alicata JE (1934) Development of the human hookworm, Necator arnericanus, in guinea pigs. Am J Hyg 20: 317328 Sen HG (1972) Necator americanus: behaviour in hamsters. Exp Parasitol 32: 26-32 Sokal RR, Rohlf FJ (1969) Biometry. Freeman, San Francisco Wells C, Behnke JM (1988a) The course of primary infection with Necator americanus in syngeneic mice. Int J Parasitol 18:47-51 Wells C, Behnke JM (1988b) Acquired resistance to the human hookworm Necator americanus. Parasite Immunol 10:493-505 Zimmerman HM (1946) Fatal hookworm disease in infancy and childhood on Guam. Am J Pathol 22:1081-1100