Experimental reproduction of canine interstitial nephritis

J. COMP. PATH. 1967.

77.

VOL.

413

EXPERIMENTAL

REPRODUCTION

INTERSTITIAL

OF CANINE

NEPHRITIS BY

LINDSAY Department

of Experimental

J. Veterinary

ANDERSON Medicine,

University

of Glasgow

INTRODUCTION

The importance of insterstitial nephritis in canine medicine has been recognised for many years (Bloom, 1937; Dahme, 1957; McIntyre and Stuart, 1949). In Britain, the condition is usually associated with infection by Leptospira canicola (McIntyre and Montgomery, 1952; Wettimuny, 1963). Although the clinical syndrome and the main pathological features have been described, many aspects of the pathogenesis are not understood, largely because interstitial nephritis has never been reproduced experimentally in a severe and progressive form. A few attempts have been made to reproduce interstitial nephritis by infecting dogs with L. c&cola (Monlux, 1948; McIntyre, 1954; Jull and Heath, 1960; Wettimuny, 1963), but none of the dogs developed any sign of renal dysfunction and the renal lesionsproduced were limited to a few small isolated foci of plasma cell and lymphocyte reaction. Suggested reasons for the lack of successinclude a possible lossof virulence in cultured spirochaetes, or the absenceof some essential predisposing factor. This paper reports the first successful reproduction of severe interstitial nephritis, with uraemia and progression to the sub-acute phase. Complete descriptions of the individual experiments in the series have been recorded elsewhere* (Mackey, 1965). MATERIALS

AND

METHODS

Five mongrel dogs, purchased from a city pet shop at the age of 6 weeks were used in the experiment. They were in good health and had negative serum tines to L. canicola and L. icterohaemorrhagiae. The age at the time of infection was 10 weeks. The dogs were kept in a warm dry pen, out of any contact with other animals. The strain of L. canicola used to infect the dogs had been isolated from the urine of a dog admitted to the hospital with acute nephritis. This strain was passaged once through dogs in a preceding experiment and re-isolated from kidney tissue. The first sub-culture grown in Stuart’s modification of Schuffner’s medium (Stuart, 1946) was used in the present experiment. Four of the five pups (24478/11 to 14) were infected by intraperitoneal injection of 1 ml. of culture. The fifth (24478/15) was not infected, but kept in the same pen to find whether transfer of infection would occur. The dogs were examined clinically each day for 12 weeks after infection. Rectal temperatures were recorded before infection and daily for the following 10 days. Weekly blood samples were taken and the serum titres to L. canicola and L. icterohaemorrhagiae were estimated. Full post-mortem and histological examinations were carried out at the end of the experiment. * Now

Anderson,

L.J.

414

REPRODUCTION

OF

CANINE

INTERSTITIAL

NEPHRITIS

RESULTS

Attempts

to Reproduce

Interstitial

Nephritis

(Summary

of Earlier

Experiments)

A series of 12 experiments was carried out, using a variety of methods designed to overcome the apparent natural resistance of healthy dogs to the potential effects of L. canicola infection and the possible loss of virulence in organisms maintained in prolonged culture. Full details are described elsewhere (Mackey, 1965) and this brief outline is intended merely to indicate in general terms the methods tested and the results obtained. The dogs used in the series were aged approximately 3 months when infected. Most were purchased at the age of 6 weeks from city pet shops and for one experiment, farm-bred collies were obtained. They were kept in isolation from other animals and were examined serologically to ensure the absence of antibodies to L. canicola and L. icterohaemorrhagiae before infection. The organisms used in some experiments had been maintained in prolonged culture. Later, a fresh strain of L. canicola was isolated from a dog suffering from acute nephritis. During the series, infection was introduced by the intravenous, intraperitoneal, intrarenal arterial, intranasal and oral routes. With each method of infection bacteraemia became established and was followed by localisation of the organisms in the kidneys and serological response. Thus the route of inoculation did not significantly affect the pathogenesis. To overcome host resistance, attempts were made to depress the immunological response before infection by administration of methotrexate (4 ammo N1’-methyl pteroylglutamic acid) in two experiments and cortisone (Betamethazone) in another. Stressing the kidney itself was also attempted by administering mercuric chloride before infection. In these trials, a proportion of the dogs succumbed to a form of acute hepatic failure which closely resembled the syndrome usuaIly associated with L. icterohaemorrhagiae infection in the field, but in no case did severe interstitial nephritis develop, although small focal renal lesions were found in most dogs. L. canicola, from culture, was passaged during the bacteraemic phase in series through dogs in two experiments to determine whether an increase in virulence would result. Again, a proportion of the dogs developed hepatic dysfunction with clinical jaundice. Most recovered and nephritis was absent when subsequent post-mortem examinations were made. There was no serological or pathological evidence of virus hepatitis. A field strain of L. canicola, isolated from the urine of a dog suffering from acute nephritis, was used to infect dogs, which developed sub-clinical focal interstitial nephritis. The re-isolated organism from one of the dogs was used in the successful experiment described below. It was then isolated again from kidney tissue and used to infect a further group of dogs, but only small renal lesions resulted on this occasion. The animals in the latter experiment were farm-bred collies, selected to avoid any immunity which might exist in pups bred in the city, where leptospirosis due to L. canicola is endemic. In one experiment, dogs were given repeated weekly infections for 12 weeks. Severe nephritis did not result and a state of “immune paralysis” was demonstrated.

LINDSAY

Reproduction

of Severe Interstitial

J.

415

ANDERSON

Nephritis

The origin of the dogs used in the experiment, routes of infection etc. are given under “hilaterials and Methods”. The infected dogs developed mild malaise on the 3rd, 4th and 5th days after infection. This was most pronounced in 24478/ 12, which had a reduced appetite at this time. A pyrexic response occurred on day 3 and persisted for 3 days. The temperatures over this period are shown in Table 1. The dogs recovered and no further clinical abnormalities were detected during the next 2 months except in dog 24478/l 1. This animal failed to grow as well as the others and remained very thin. The serum titres to L. canicola during the experiment are shown in Table 2. The development of a positive titre in dog 24478/ 15 was of interest, indicating that cross-infection took place. TABLE RECTAL

Post-iqfection Day

TEMPERATURE

24478/l 1

OF THE

INFECTED

1 DOGS

(IN

DEGREES

CENTIGRADE)

24478112

24478113

24478114

2

38.9

38.9

38.6

38.9

3

39.1

40.0

38.9

39.4

4

39.1

39.7

38.9

38-8

5

39.3

39.6

38.7

38.6

6

38.6

38.9

38.7

38.7

7

38.6

38.8

38.6

38.6

During the 11th week, dog 24478/ 11 died suddenly. On post-mortem examination, the liver was found to be pale and slightly enlarged. The renal cortices contained many white foci, 2 to 3 mm. in diameter, concentrated mainly in the juxta-medullary region together with intervening narrow radial bands of fibrosis. Histologically, moderately severe sub-acute interstitial nephritis was present (Figs. 1 and 2). The renal cortices contained large areas of plasma cell and lymphocyte reaction and also many foci of well-developed fibrosis. A moderate degree of fatty change affected the liver; inclusion bodies were absent and serological examination for evidence of virus hepatitis proved negative. During the next month, no clinical abnormalities were apparent in the remaining dogs and they were destroyed three months after infection. At post-mortem examination, extremely pronounced lesions were found in the kidneys of dog 24478/12, although there had been no evident illness in life. The kidneys were markedly swollen and very pale, with irregularity of the cortical surfaces and focal adhesion of the capsules (Fig. 3). The kidney tissue was abnormally firm in consistency and throughout the cortices there were very many pale foci 2 to 3 mm. diameter and intervening fine radial bands of fibrous tissue (Fig. 4). Histologically, the renal changes were those of severe, sub-acute interstitial nephritis. Large foci of intense plasma cell and lymphocyte reaction were scattered throughout the interstitial tissue of the cortices and destruction of many

416

REPRODUCTION

OF

CANINE

INTERSTITLiL

TABLE SERUM

24478/11

Post-infection

week

TITRES

NEPHRITIS

2

T O L.CANICOLA

24478/l 2

24478113

24478114

2

1: 3,000

1:

10,000

-

-

3

1: 3,000

1: 30,000

-

-

4

1: 3,000

> I: 30,000

-

-

1:

10,000

> 1: 30,000

-

1:

30,000

> 1: 30,000

5 6 8 10 12

’ >l:

1: 10

24478115

1:

100

1:

1,000

-

1:

300

10

1: 30,000

> 1: 30,000

-

-

1:

100

1:

> 1: 30,000

-

-

1:

10

-

-

1,000

1:

1,000

-

convoluted tubules was apparent (Fig. 5). Smaller foci of mononuclear cell reaction were present in the medulla. Radial areas of fibrosis were numerous in the cortices (Fig. 6) and closely related glomeruli were affected by varying degrees of hyalinisation of the capillary tuft. The arteries and arterioles were normal. Facilities for routine biochemical examinations were not available during this experiment, but when the severe renal lesions were found post-mortem, heart blood was taken from dog 24478/ 12 and the serum urea estimated. This was found to be 150 mg./lOO ml. L. canicola was successfully isolated from kidney tissue. No macroscopic or histological lesions were detected in the kidneys of dogs 24478/13 and 14. Mild, focal acute interstitial nephritis was present in dog 24478/15, seen as small scattered foci of plasma cell and lymphocyte reaction in the juxta-medullary region of the cortices. DISCUSSION

A reliable method for reproducing canine interstitial nephritis would be valuable for several reasons. Many aspects of the pathogenesis of this common clinical condition are not understood. Two of the most important unresolved features are the significance of the plasma cell and lymphocyte reaction characteristic of the acute phase and the mechanism responsible for the progressive loss of nephrons and increasing renal fibrosis in chronic interstitial nephritis. Additional studies by the present author indicate that hypertension is a major factor in the sequence (Mackey, 1965 ; Anderson, 1967 ; Anderson and Fisher, 1967). As well as providing an opportunity to elucidate the diseaseprocessin all its aspects, the experimental reproduction of interstitial nephritis might be useful in the study of hypertension, by forming a useful model for comparative investigations. Hitherto, experiments in dogs have involved the constriction of renal arteries and

LINDSAY

J.

ANDERSON

417

other artificial manipulations. A practical benefit would also accrue, in that adequate testing of commercial leptospiral vaccines might be possible. At present, the protective efficiency of such vaccines is judged only on ability to stimulate a positive antibody titre to L. canicola. There is no direct evidence that vaccinated animals become resistant to natural infection. In these experiments, severe progressive interstitial nephritis with uraemia has been successfully reproduced for the first time by infecting dogs with L. canicola. This is of considerable significance, as it indicates that the disease may be regularly reproducible if a reliable method can be found. The main factors determining the outcome of infection appear to be related to individual variation in the susceptibility of dogs and differences in the virulence of cultured and freshly isolated strains of L. canicola respectively. In general, experimental infection leads to bacteraemia with transient pyrexia followed by localisation of leptospirae in the kidneys, and the development of a marked serological reaction and mild focal acute interstitial nephritis. Dogs infected with recently isolated organisms showed a more marked pyrexic response, higher serum titre levels and more extensive renal lesions than when leptospirae maintained in prolonged culture were used. The effect of stressing the dogs with methotrexate, cortisone or mercuric chloride before infection was to produce an acute syndrome resembling Weil’s disease. This response may reflect a particular susceptibility in the immature animal, since a similar illness occurred in a proportion of pups through which the organism was passaged. The route of infection did not influence the outcome and any predisposing or synergistic factors operating under field conditions to produce the severe disease remain unknown. It is not certain why severe progressive sub-acute nephritis with uraemia was produced in one experiment. However, since two of the five dogs developed extensive renal lesions with fibrosis, the result is encouraging. As the dogs were kept in isolation for 4 weeks before the start of the experiment and they had negative serum titres to leptospirae, it is highly unlikely that these could have been natural cases. In conclusion, the present investigations have shown that severe progressive interstitial nephritis can be reproduced experimentally in dogs by infection with L. canicola, but that a reliable method giving predictable results is not yet available. SUMMARY

This paper describes the first successful experimental attempt to reproduce canine interstitial nephritis in a severe and progressive form. A group of 5 dogs were infected intraperitoneally with a strain of Le~tospira canicola recently isolated from the urine of a dog suffering from acute nephritis. Infection was followed by transient malaise and pyrexia, and a marked serological response. After 3 months, renal lesions typical of severe subacute interstitial nephritis were present in 2 of the dogs and uraemia was demonstrated in one of them. The experiment was one of a series of attempts to reproduce the disease and the methods used in other trials are outlined briefly. The results indicate that variations in the susceptibility of individual dogs and in the virulence of different strains of L. canicoZa are important in determining the outcome of infection. F

418

REPRODUCTION

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CANINE

INTERSTITIAL

NEPHRITIS

ACKNOWLEDGMENT

During the time in which this work was carried out, I was in receipt of a Scholarship from the Horserace Betting Levy Board, London. I am indebted to Dr. E. W. Fisher for his advice and assistance in performing experiments. REFERENCES

Anderson, Lindsay J. (1967). 1. Path. Bact., In press. Anderson, Lindsay J., and Fisher, E. W. (1967). Res. vet. Sk., In press. Bloom, F. (1937). J. Amer. vet. med. Ass., 91, 679. Dahme, E. (1957). Arch. exp. Vet.-Med., 11, 611. Jull, D. J., and Heath, K. R. (1960). J. small aim. Pratt., t, 245. McIntyre, W. I. M. (1954). Ph.D. Thesis; University of Edmburgh. McIntyre, W. I. M., and Montgomery, G. L. (1952). 1. Path. Butt., 64, 145. McIntyre, W. I. M., and Stuart, R. D. (1949). Vet. Rec., 61,411. Mackey, Lindsay J. (1965). Ph.D. Thesis; University of Glasgow. Monlux, W. S. (1948). Cornelt Vet., 38, 199. Stuart, R. D. (1946). 1. Path. Butt., 58, 343. Wettimuny, S. G. de S. (1963). Ph.D. Thesis; University of Glasgow. [Received

for publication,

]unuary

18th, 19671

the

LINDSAY

Fig. Fig.

1. Kidney 2. Kidney fibrosis, tion. H.

J.

ANDERSON

of dog 24478/l 1, showing an area of interstitial nephritis. H. & E. x 150. of dog 24,478/l 1, showing an area of chronic reaction with well developed interstitial protein casts within tubules, hyalinisation of glomeruli and minimal cellular infiltra& E. x 150. To face page 418

REPRODUCTION

OF

CANINE

INTERSTITIAL

NEPHRITIS

.

Fig. Fig.

3. Kidney of dog 24478/12, 4. Kidneys of dogs 24478/12 and the cellular infiltrate

x 1.5. showing the pale, swollen, nodular appearance of the cortex. (left) and 24478/15 (right), showing the enlargement of the former in the cortex. x 1.5.

LINDSAY

Fig. Fig.

J.

ANDERSON

3. Kidney of dog 24478/12, showing a large focus of intense plasma cell and lymphocyte reaction in the cortex. H. & E. x 100. 6. Kidney of dog 24478112, showing an area of well developed interstitial fibrosis in the cortex and a persisting focus of plasma cell and lymphocyte reaction. H. & E. x 150.