Small wild mammals as a source of leptospirosis

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1 Rev. sci. tech. Off. int. Epiz., 1988, 7 (4), Small wild mammals as a source of leptospirosis D.TRAP * Summary: Microagglutination tests on 95 serum samples from coypu (Myocastor coypus), using 13 serovars of Leptospira interrogans revealed antibodies in 45 animals (47.3%), 41 of which (43.1%) were against serovar icterohaemorrhagiae, suggesting the role of the coypu as a vector of leptospirosis. Tests on serum from horses and cattle for antileptospiral agglutinins showed that infection of domestic animals by small wild mammals was related to the abundance and pattern of life of the latter. KEYWORDS: Domestic animals France Leptospirosis Myocastor Rodentia Vectors Wild animals. Leptospirosis is a bacterial zoonosis of worldwide distribution which may take on a variety of forms and degrees of intensity. It affects domestic and wild animals, as well as human beings, and cases of human infection often reveal the existence of inapparent infection among animals. The two chief factors involved in the epidemiology of leptospirosis are water and the fauna of small, wild mammals, principally rodents and hedgehogs. These animals excrete leptospires in their urine, thereby contaminating the environment. Survival and dissemination of leptospires is facilitated by water, when conditions are favourable. This report provides the results of a survey of coypu (nutria) and of tests for antileptospiral agglutinins in horses and cattle, with the purpose of demonstrating the role of small wild mammals in transmitting the disease to domestic animals. Materials Antigens MATERIALS AND METHODS These were prepared at the Leptospirosis Department of the Paris Pasteur Institute, a WHO Reference Centre. The antigens were prepared from the following serogroups of pathogenic leptospires: * Laboratoire Central de Recherches Vétérinaires, 22, rue PierreCurie, B.P. 67, Maisons Alfort Cedex, France.

2 894 Serogroup Icterohaemorrhagiae Grippotyphosa Australis Pomona Tarassovi Canicola Sejroe Ballum Panama Hebdomadis Autumnalis Pyrogenes Serovar (sv) icterohaemorrhagiae (ih) and copenhageni (copen) grippotyphosa (gt) australis (Aa) pomona (pom) tarassovi (tar) canicola (can) sejroe (sej) and hardjo (hard) ballum (bal) panama (pan) hebdomadis (heb) autumnalis (aut) pyrogenes (pyr) The "BuenosAires" (BA) strain of saprophytic leptospire was also used. The number of serovars tested varied with the type of investigation. Each antigen was a culture of the given serovar in serumfree medium. Sera Serum samples from 95 coypu (Myocastor coypus) were tested against 12 pathogenic serovars (ih, copen, Aa, bal, sej, pan, heb, can, gt, hard, aut, pyr) and the BA saprophytic strain. They were obtained from animals captured in the Vendée Department between December 1986 and May The animals showed no sign of illness; 98% of them were adults. There were rather fewer males than females, and onethird of the latter were pregnant. They weighed between 1 and 9 kg. Serum samples from 561 horses were tested against 7 serovars (ih, gt, Aa, pom, tar, can, sej). They were obtained from 134 horses with clinical signs attributable to leptospirosis (jaundice and eye disease) and 427 clinically healthy horses. The samples came from 25 different Departments, but mainly from three of them: Yvelines, Calvados and the Lyons region. Serum samples from 709 healthy Charollais cattle destined for export were tested against 8 serovars (ih, gt, Aa, pom, tar, can, sej, bal). The animals came from a region roughly covering the three Departments of Charollais. Samples for bacteriological examination Samples of urine and organs (kidneys plus uterus in females) were obtained from the 95 captured coypus, sent alive to the Vendée Laboratory of the Veterinary Services (Director: Dr Ramisse). Methods Serological testing All sera were tested by the microagglutination test (MAT), the reference method for leptospirosis. They were diluted 1/100 and interpreted as positive, according to

3 895 the recommendations of the 1982 edition of the OIE International Animal Health (ZooSanitary) Code, if 50% of leptospires ( + + reaction) were agglutinated at this dilution. Positive samples were diluted further to determine the final titre of the test. Notation of the results has been described previously (6). Bacteriological examination Samples of urine and organ homogenates were promptly inoculated into 'EMJH' culture medium* of Ellinghausen and McCullough (2) as modified by Johnson and Harris (4), together with enrichment medium. 1/10 dilutions of urine were inoculated immediately. Cultures incubated at 30 C were examined by darkground microscopy once a week for three months. Suspect cultures were reinoculated into EMJH enrichment medium in dilutions of 10 1 to RESULTS Tests on coypu The epidemiological investigation showed that antibodies were present in 45 of 95 coypu (47.3%). Table I shows the number and percentage of MATpositive sera for each serovar tested. Thus 40 coypu were positive to serovar icterohaemorrhagiae, and 15 to copenhageni. Of course, these two serovars belong to the same serogroup, and coagglutinins were present in some sera, so that 13 coypu were positive to both serovars, 26 were positive to ih only, and 2 were positive to copen only. Thus 41 of the 45 positive coypu (91%) possessed icterohaemorrhagiae antibodies. The incidence of other serovars was low or nil. TABLE I Number of coypu sera positive to the microagglutination test (MA T) for leptospirosis, using 12 pathogenic serovars and one saprophytic serovar Pathogenic leptospires MATpositive sera Serogroup Serovar Strain No. Percentage Icterohaemorrhagiae i icterohaemorrhagiae verdun j copenhageni M Australis australis ballico Ballum ballum Mus Sejroe sejroe M Panama panama CZ 214 K Hebdomadis hebdomadis hebdomadis Canicola canicola Utrecht IV 1 1 Grippotyphosa grippotyphosa Moscow V 0 0 Sejroe hardjo Hardjoprajitno 0 0 Autumnalis autumnalis Akiyami A 0 0 Pyrogenes pyrogenes Salinem 0 0 Saprophytic leptospire Buenos Aires * Bacto Leptospira medium base EMJH Difco code Bacto Leptospira enrichment medium EMJH Difco code

4 896 The number of animals positive to MAT at different dilutions of serum against each serovar is shown in Table II. Titres of 22 sera were equal to or greater than 1 /400 in the case of Icterohaemorrhagiae, while 9 were positive at 1/1000 or more. Although the precise significance of such antibody titres has not been established clearly in the case of wild mammals, they nevertheless seem to indicate an active infection. TABLE II Number of coypu sera positive at different MAT dilutions against each serovar Serovar Number of MATpositive sera at different dilutions 1/100 1/200 1/400 1/800 ^ 1/1000 icterohaemorrhagiae copenhageni 14 1 australis 4 ballum 3 1 sejroe 2 1 panama 3 hebdomadis 2 canicola 1 Table III shows the number of coypu possessing antibodies to one or more serovars. This phenomenon is usually due to the common occurrence of coagglutinins in serum, but the possibility cannot be ruled out that the animal was infected simultaneously with many strains, or that antibodies persist from earlier infections. TABLE III Number of coypu sera positive to one or more serovars Number of serovars Number of positive sera Unfortunately all attempts to isolate leptospires at the Vendée Laboratory of the Veterinary Services were negative. Tests on horses The testing of 561 serum samples from horses (6) revealed a clear increase in 1978 in the number of reactions to sejroe antigen: 17% were positive, compared with 14% to gt and 11.7% to Aa. A similar increase has been noted in other species of animals

5 897 at this time. Among the 134 horses showing clinical signs, 114 came from the Lyons region, where numerous cases of periodic ophthalmia (recurrent uveitis) have been recorded. 85 positive horses (74.5%) had been in contact with affected horses, 31 (36.4%) of them being positive to serovar sejroe at titres which were high in some cases (1/3000 or more). Enquiry at the premises concerned revealed that they were all located in damp areas, and that rodents had been unusually prevalent that year. This was particularly the case at an equestrian centre on an island in the River Rhone, where 20 horses with recurrent uveitis were positive to sejroe antigen. Tests on cattle Tests on serum samples from 709 clinically healthy Charollais cattle (1) showed that 28% of them were positive, with inapparent infection present in about onethird of the herds. The authors demonstrated a relationship between the presence of serum antibodies against certain serovars, husbandry conditions (which varied according to season) and the rodent populations to which the cattle were exposed. Table IV shows that cattle born during the first few months of the year and kept housed possessed antibodies against serovars ih and bal, while those born in spring and reared on pasture were positive to serovars gt, Aa and sej, which corresponded to wildlife populations having different living patterns. TABLE IV Relationship between the percentage of cattle positive to each serovar and their age (from ref. 1) Sérovar Jan. 8 Month of birth Feb. March April May Age at time of testing (months) Percentage positive icterohaemorrhagiae grippotyphosa australis sejroe ballum DISCUSSION Within the body, leptospires are found to persist in the kidneys. They are eliminated intermittently in the urine, thereby contaminating the environment. The duration of this leptospiruria is variable among domestic animals, but in small wild mammals which are healthy carriers and excretors, it can persist for life. The bacteria are very sensitive to drying and ultraviolet rays, but are very resistant in water which has a

6 898 slightly alkaline ph. Hence all damp places are suitable for the survival and dissemination of leptospires. Direct infection through bite wounds is rare. Transmission is essentially indirect through urine and contaminated water in contact with feed (which often happens in maize silage), bedding, pasture and drinking troughs. While the sources of infection are mainly provided by the inexhaustible reservoir of small wild mammals, it must not be overlooked that domestic animals can also act as sources of infection. There is no unique reservoir host for a given serovar. A rodent may carry several serovars just as one serovar may be present in many rodent species. However, it is recognised that the black rat {Rattus rattus), Norway or grey rat {Rattus norvegicus) and muskrat (Ondatra zibethica or Fiber zibethicus) are frequently responsible for icterohaemorrhagiae infections. The survey of coypu has shown that they can also carry this serovar, for 41 of 95 (43.1%) were positive, half of them having high titres which probably denoted a current infection. Although it proved impossible to isolate a strain, these animals probably play a role in the spread of icterohaemorrhagiae leptospirosis. Moreover it is obvious that the aquatic mode of life of coypu favours their role as vectors of the disease. However, there has been no abnormal increase in the coypu population in recent years. The incidence of leptospirosis among local livestock has been stationary for some years, and is no different from that observed elsewhere in France. The cattle survey shows that, at least during 1965, animals kept housed were more likely to become infected by serovars carried by rodents which live in buildings, such as ih carried by Norway (grey) rats and bal carried by house mice (Mus musculus). Cattle at pasture were liable to become infected with the serovars carried by hosts living outside buildings, such as gt carried by the common vole (Microtus arvalis), sej carried by the steppe mouse (Mus spicilegus) and the wood mouse (Apodemus sylvaticus), and Aa by the hedgehog (Erinaceus europaeus). The multiplication of vector hosts appears to be related directly to the importance of the infection in domestic animals. The survey of horses disclosed abnormally rapid multiplication of rodents in some areas. At the same time an increasing proportion of positive reactions to the sejroe antigen was noted. It was impossible, however, to establish whether this phenomenon was linked to an increase in population density of the hosts considered to be preferential reservoirs of this serovar, Mus spicilegus and Apodemus sylvaticus. Similarly, a survey of cattle (3) revealed a clear dominance of reactions to grippotyphosa in many regions. The common vole, often a carrier of this serovar, undergoes periodic fluctuations in a threeyear cycle. The survey showed that these rodents were particularly abundant in the regions from which the infected cattle came. Finally, little is known of the role in France of hedgehogs, although some 40% of them act as bacteriological carriers of serovar muenchen (of serogroup Australis) in certain regions (5). Thus, Arnault (personal communication) observed few cases of bovine leptospirosis in Creuse Department in 1987, which may be at least partly due to extensive destruction of these animals during the preceding year.

7 899 CONCLUSION Small wild mammals (principally rodents and hedgehogs) are the major vectors for infecting domestic animals with leptospires. These animals are healthy carriers which excrete leptospires in urine throughout their life. The urine contaminates the environment (animal houses, bedding, drinking water, feed, pasture, ponds, lakes, watercourses), facilitated by water. The frequency of leptospirosis in domestic animals is related directly to the abundance of the wildlife reservoir, its mode of life and the conditions required for dissemination of the infectious agent. * * * REFERENCES 1. CHEVRIER L. & GAUMONT R. (1974). Contribution à l'étude des leptospiroses bovines inapparentes en Charollais. Bull. Acad. vét. Fr., 47, ELLINGHAUSEN H.C. & MCCULLOUGH W.G. (1965). Nutrition of Leptospira pomona and growth of 13 other serotypes: fractionation of oleic albumin complex and a medium of bovine albumin and polysorbate 80. Am. J. vet. Res., 26, GAUMONT R. (1966). Enquête sur la présence d'agglutinines antileptospires chez les animaux domestiques en France. Bull. Off. int. Epiz., 66, JOHNSON R.C. & HARRIS V.C. (1967). Differentiation of pathogenic and saprophytic leptospires. 1. Growth at low temperatures. J. Bact., 94, MAILLOUX M. (1973). Le hérisson vecteur de leptospiroses dans l'ouest de la France. Méd. Mal. Inf., 34, TRAP D., GAUMONT R. & CHEVRIER L. (1979). Leptospirose équine : enquête sur la présence d'agglutinines antileptospires chez les chevaux en France. Prat. vét. équine, 11 (3),