- Email: [email protected]
Leptospirosis as a Cause of Reproductive Failure
DIAGNOSIS OF ABORTION
0749-0720/94 $0.00 + .20
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTIVE FAILURE William A. Ellis, PhD, BVMS, FRCVS
Leptospirosis is a zoonotic disease caused by members of the genus
Leptospira. Veterinarians' perception of leptospirosis as a disease of domestic animals has undergone considerable modification in the past decade or so as they have increasingly appreciated the role of the hostmaintained leptospires as causes of reproductive wastage in their respective host species kept under modem intensive management systems. Large discrepancies exist in our understanding of leptospirosis among different domestic ruminants. A considerable amount of data has been published on the condition in cattle, whereas much less is known about leptospirosis in sheep, and even less in goats. This imposes a bias toward discussion of cattle in this review. Even with cattle, many aspects remain poorly understood-e.g., variations in disease pattern and disease impact associated with different strains of the same host-maintained serovar in different management systems and different parts of the world. The paucity of good data on leptospirosis in sheep and goats could be due to one or more of the following factors: Sheep and goats may be much less susceptible to leptospiral infection; developed economies place much lower value on those species, especially goats, so less research has been done; and they rarely are kept in intensive management systems. The organisms of the genus Leptospira are small aerobic spirochetes that can be found in a wide variety of animal species (the parasitic strains) and in water (the saprophytic strains). These two groupings,
From the Veterinary Sciences Division, Department of Agriculture, Northern Ireland, and Queen's University, Belfast, Northern Ireland
VETERINARY CLINICS OF NORTH AMERICA: FOOD ANIMAL PRACTICE VOLUME 10 • NUMBER 3 • NOVEMBER 1994
463
464
ELLIS
until recently, were referred to as the "interrogans" and "biflexa" complexes, and can be differentiated by their growth requirements and biochemical reactions. For taxonomic purposes and as an aid to epidemiologic studies, the parasitic leptospires were subdivided into serogroups on the basis of antigenic relationships as determined by cross agglutination reactions and further subdivided into serovars by agglutinationabsorption patterns. Twenty-three serogroups are recognized, containing approximately 212 serovars. 37 Alternative serologic typing systems based on either monoclonal antibodies or factor analysis give results comparable to conventional serotyping methods. 12,37 The advent of genetic typing methods has thrown the taxonomy of the genus into some confusion, but has provided rapid, reproducible typing protocols. Seven genospecies of parasitic leptospires have been described based on DNA relatedness and guanine plus cytosine content studies. 56, 68 The value of this system is limited with respect to epidemiology. Other genetic typing methods have proved of value and increasingly are being used as supplementary or alternative typing systems, particularly in epidemiologic studies, because they identify strain differences at the subserovar level. These systems include restriction endonuclease analysis of chromosomal DNA by fixed field 24, 45, 62,63 or pulsed field gel electrophoresis,34, 35 recombinant DNA probes,39, 58, 66, 76 and ribotyping. 52,53 EPIDEMIOLOGY Distribution
The epidemiology of leptospirosis in domestic animals is potentially very complicated because animals can be infected by any of the pathogenic serovars. Fortunately, only a small number of serovars are endemic in any particular region or country. Furthermore, leptospirosis is a disease that shows a natural nidality and each serovar tends to be maintained in specific maintenance hosts. In any region, therefore, an animal species may be infected by serovars maintained by its own species or serovars maintained by other animal species present in the area. The relative importance of these incidental infections is determined by the opportunities for contact and transmission of leptospires from other species to the target host species provided by prevailing social, management, and environmental factors. 31 In the developed, intensive-agricultural economies, incidental infections playa much less significant role than do host-maintained infections. The number of Leptospira serovars maintained by domestic animals is small and, although regional differences occur, local geographic differences are not as important as with incidental infections.
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTIVE FAILURE
465
Hardjo is the serovar maintained by cattle. It has a worldwide distribution, with high serologic and microbiologic prevalences identified in most countries that have carried out appropriate investigations. Although differences in cattle management practices and resulting herd immunity patterns probably contribute to the differing clinical situations observed in different countries, it also is probable that strain differences highlighted by a variety of DNA technologies, make a contribution. Two major genotypes of hardjo are found in cattle and sheepHardjobovis and Hardjoprajitno. Hardjobovis appears to be a betteradapted parasite than Hardjoprajitno. It is excreted in much larger numbers in cattle urine and is the strain found in most countries. Hardjoprajitno so far has been recovered only in the United Kingdom, Nigeria, India, Malaysia, Brazil, Mexico, and the United States. Serovar pomona, maintained by swine and a variety of free living animals, is the most important source of incidental infection of cattle in North America, Australia, and New Zealand. In parts of Africa, Russia, and Israel, grippotyphosa constitute an important incidental infective agent of cattle. In parts of the Caribbean, this role is filled by Autumnalis strains. Strains belonging to the Pyrogenes serogroup are found in cattle in parts of Africa, and serovar zanoni recently was shown to be common in Queensland. Serologic prevalences tend to be much lower in sheep than in cattle, perhaps in part because of complement interfering with the microscopic agglutination testt3 and the fact that sheep are kept in extensive management systems with less opportunity for transmission. There is consistent serologic evidence that hardjo infection is the major one found in sheep worldwide, however, in some countries there even is evidence that sheep may maintain the serovar without the presence of cattle.9 Seroprevalence data indicate that other leptospiral infections in sheep are uncommon and are incidental, and the limited culture surveys reported support that view. I , 2 The serovars involved in such incidental infections vary by region and reflect the serovars commonly found in other domestic and wild animals in a region. No cultural or serologic evidence exists to suggest that goats act as maintenance hosts for leptospires. l , 26, 57 High seroprevalences have been reported in some countries, but the predominant infecting serovar varies, suggesting an incidental pattern of infection.
Transmission
Direct transmission can occur among animals via infected urine, postabortion uterine discharge, infected placenta, sexual contact, or in utero infection. It is probably of greatest importance with serovar hardjo,
466
ELLIS
with which infection appears to be largely independent of rainfall and cattle and sheep management systems. Indirect transmission plays a much greater role in the transmission of incidental infections. It occurs through exposure to an environment contaminated with infective material-usually one that permits the maximum survival of leptospires outside the host-and a management system that facilitates close contact between carrier and susceptible animals. The optimum conditions for survival outside the host are warm, moist conditions, with a pH close to neutral. Where such conditions are found, the prevalence of incidental infections is likely to be highest. Areas such as waterholes, where animals congregate, frequently are incriminated in outbreaks of leptospirosis. Environments favorable to the survival of leptospires are much less important in the epidemiology of host-maintained leptospires. The major risk factors in introducing infection into a clean herd are: 1. An open herd policy, with the consequent risk of purchasing
carrier animals; 2. Close proximity of another domestic host species-e.g., sheep in the case of hardjo infection in cattle; 3. Access to other animals of the same species-in particular, the use of a shared bull; 4. Access to a water course exposed to other animal hosts upstream; The major factors in maintaining infection in a herd are: 1. The persistently infected carrier: Some animals are carriers for
life, but it remains uncertain whether they remain infective throughout that period. 2. A regular supply of susceptible animals: The standard practice in dairy herds of removing replacement heifers from their dams shortly after birth and keeping them separated from the main herd until shortly before or after calving ensures such a regular supply of nonimmune animals.
PATHOGENESIS
Infection of susceptible animals occurs through the mucous membranes of the eyes, mouth, nose, vagina, and penis and through abraded or water-softened skin and is followed, after a 4- to lO-day incubation period, by a bacteremic phase that may last from a few hours to 7 days. That phase may be subclinical but can be characterized by pyrexia, excretion of leptospires in milk, and, with some serovars, functional damage to the internal organs, especially in younger animals. During this period, leptospires can be isolated from most organs of the body and from
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTIVE FAILURE
467
cerebrospinal fluid. Acute clinical disease coincides with this bacteremic phase. The primary bacteremic phase ends with the appearance of circulating antibodies, usually detectable by 10 days after infection. Peak titers and the time for which they persist vary considerably, depending on the animal species, the infecting serovar, and the route of infection. Serovar hardjo titers (as determined by the microscopic agglutination test, MAT) in cattle infected with some genotype Hardjobovis strains, can reach levels of greater than 1:10,000 by 4 to 6 weeks postinfection and can remain detectable for more than 1 year. 41 The initial response is an IgM one that peaks 2 to 3 weeks postinfection. The IgG response is much slower, with peak levels occurring between 12 and 30 weeks after infectionY In contrast, serovar hardjo titers (as determined by the MAT) in sheep are low and transient. Following the period of leptospiremia, the leptospires localize and persist in a number of organs, especially the proximal renal tubules (all ages) and the genital tracts of sexually mature females and entire males. Relapses of pyrexia may occur; whether they are associated with persistent foci of infection giving rise to recurring bacteremia or, possibly, toxemia is unknown. Differences have been found in tissue tropisms exhibited by different strains of hardjo.4 Some strains of Hardjobovis have been shown to persist primarily in the kidney, others have a predilection for the genital tract, and still others persist in both organ systems. Leptospires localized in the proximal renal tubules multiply and are voided in the urine. The duration and intensity of urinary shedding varies from animal to animal and with the infecting serovar. In the case of host-maintained infections such as hardjo infection in cattle, the intensity of excretion is greatest during the first 4 to 6 weeks of shedding. Leptospiruria is constant during that period. A variable period of intermittent, low-intensity leptospiruria then ensues, frequently lasting for 6 to 12 months in cattle but sometimes persisting for life. Excretion of hardjo for prolonged periods also has been observed in sheep. In contrast, urinary shedding is of very low intensity in other host-maintained infections in which the venereal route probably is more important, such as Hardjoprajitno infection in cattle and sheep. The factors involved in the cessation of urinary excretion are poorly understood, but one recent study showed it invariably is associated with a sharp increase in urinary antileptospiral IgG and IgA antibody levels.41 Localization of leptospires in the pregnant and nonpregnant uterus of cows has been shown to persist for up to 142 days and 97 days postinfection, respectively. Localization in the pregnant uterus, in turn, may be followed by fetal infection, with subsequent chronic reproductive wastage and excretion of the leptospires in the postcalving uterine dis-
468
ELLIS
charge. Bovine and ovine fetuses infected during the latter stages of gestation may develop detectable antibody titers. Localization of serovar hardjo has been reported in the testes and accessory glands of bulls,17 and leptospires of the pomona and Hebdomadis serogroups have been demonstrated in bull semen. Leptospiral antibodies have been detected in seminal plasma, suggesting local antibody production. Persistence of serovar hardjo in the mammary glands of cattle61 and goats64 also has been reported.
CLINICAL CONCERNS
The vast majority of leptospiral infections are subclinical. Two groups of animals are most likely to experience clinical infections: (1) Very young animals that contract an incidental infection may experience severe illness characterized by jaundice, hematuria, hemaglobinuria, evidence of renal damage, meningitis, and, in some cases, death. Such acute clinical events very occasionally are seen in older animals. (2) Sexually mature lactating or pregnant females infected by either incidental or host-maintained leptosires may demonstrate agalactia (in cattleS1 and sheep46) or reproductive wastage.
Reproductive Wastage in Cattle
This feature is the most important aspect of leptospirosis in terms of its effects on farm economics. It is a chronic sequel to leptospirosis in breeding cows. Fetal infection with resulting stillbirths, abortions, and the birth of weak offspring of reduced viability may occur. Although such reproductive wastage can occur in cattle infected by many parasitic leptospires, it is particularly a feature of pomona and hardjo infections. Abortion and the other effects usually occur 1 to 6 weeks (pomona infection) or 4 to 12 weeks (hardjo infection) after the acute phase of infection, but such animals frequently show no clinical evidence of acute infection. With pomona infections, the same events usually occur in the last 3 months of gestation. Abortion has been observed in the second trimester in cases of autumnalis infection.lO With hardjo infection, abortion has been diagnosed at all stages, from the fourth month through term, and circumstantial evidence indicates it also may cause early embryonic death. 13 It has been difficult to define the importance of leptospirosis relative to other factors in the etiology of bovine abortion, stillbirth, and premature live birth because of difficulties in diagnosis and regional differences
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTIVE FAILURE
469
in the distribution of genetic subtypes. In a study in Northern Ireland, 42% of aborted bovine fetuses had been infected by leptospires (almost all hardjo),21 whereas evidence of leptospiral infection was found in 10%
of aborted fetuses examined in South Dakota. 36 The incidence of abortion on individual farms can be very high following an epizootic of pomona infection; incidences of up to 50% have been reported. 38 Abortion rates tend to be much lower (3%-10%) with hardjo infection, but rates of more than 30% occasionally occur.13 Hardjo represents the most important leptospiral infection of cattle, however, because it is associated with long-term herd infections on farms, with the potential for ongoing losses year after year. 28 This recurring loss is most noticeable where management practices ensure the regular introduction of susceptible animals into an infected herd. IS, 32 No association between pomona infection and infertility has been shown,42 but infertility has been a common field observation in hardjoinfected herds29,s4 and improvements in breeding efficiency have been noted in herds after hardjo vaccination. 3D Attempts to establish herd data to support those observations and preliminary supportive findings have been reported. 33, 49
Reproductive Wastage in Sheep
Relative to cattle and pigs, sheep have always been considered resistant to leptospiral infections. Although that view still generally is true in many parts of the world, it is becoming apparent that, under certain intensive management systems now used in countries such as the United Kingdom and Ireland, leptospirosis can cause overt clinical disease in the last 2 weeks of gestation and in the immediate postpartum period, when sheep are immunologically compromised. This can cause considerable loss to individual farmers using such management regimens. Reproductive wastage in sheep is seen as late-term abortion, stillbirth, and the birth of weak lambs. In a study in Northern Ireland, approximately 17% of 872 aborted lambs examined between 1981 and 1987 were positive for leptospires on fluorescent antibody examination. Culture data indicated that the infecting strain in the majority of cases was serovar hardjo, with a small number caused by pomona, ballum, or bratislava. 14 In a South Dakota study,36 8% of aborted fetuses had antibodies to leptospires. A study in Spain40 estimated that 1.7% of abortions were due to leptospires (mainly pomona). Serovar pomona also has been implicated in abortions in Albania,36 whereas grippotyphosa was deemed to be the cause of abortions in Hungary.27 Ellis I4 observed that hardjo abortion rarely was found in extensively managed flocks. It was confined primarily to ewes bred in an extensive
470
ELLIS
system and subsequently bought as replacements for intensively managed flocks with high stocking rates and indoor housing for ewes. Reproductive wastage and agalactia were seen in such animals in their first lambing season following introduction, but not in subsequent years.
Reproductive Wastage in Goats
There are few reports of leptospirosis causing reproductive wastage in goats. A report from Spain40 attributed 2.7% of goat abortions to leptospirosis (mainly pomona). In Israel, van der Hoeden65 described an outbreak of acute grippotyphosa infection in goats, a sequel to which was a high incidence of abortion. Abortion also has been reported in goats in Guyana47 and India.5o
FETAL AND UTERINE PATHOLOGY
Postmortem examination of fetuses aborted because of leptospirosis usually reveals only nonspecific findings that either result from autolysis or cannot be differentiated satisfactorily from autolytic changes. Autolysis is a particular feature of fetuses aborted before 6 months. Jaundice occasionally may be observed in the subcutaneous tissues of infected fetuses aborted in the last stages of gestation, whereas stillborn fetuses frequently exhibit lesions similar to those produced by anoxia-i.e., petechial hemorrhages on the surface of the thymus, thyroid, lungs, and heart and in the parietal pluera, peritoneum, and mesentery. The only consistent histologic changes observed in experimental leptospiral abortion have been in the fetal kidney. They have consisted of foci of tubular necrosis with interstitial and perivascular infiltration of lymphocytes, along with a few plasma cells and polymorphonuclear leukocytes.16, 25, 48 Severe vascular lesions were a feature of the histopathologic findings in two reports of hardjo and icterohaemorrhagiae infection in aborted fetuses. 19,2o The lesions were particularly severe in the liver and appeared to a lesser extent in the thymus, cerebral meninges, and interlobular septa of the lungs. The vessels often were congested and exhibited mural edema and necrosis. Perivascular hemorrhage was noted. Polyarthritis was reported in a live calf born to a cow experimentally infected with hardjo.61 In cases of pomona abortion, the cotyledons appear light tan to yellow in color and avascular and are affected uniformly,u A controlled serial kill experiment,48 however, failed to demonstrate any histologic changes in the pregnant uterus or fetal membranes of infected heifers
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTNE FAILURE
471
that were not present in control heifers and that have not been reported in so-called normal placentae. 3
DIAGNOSIS
The diagnosis of reproductive wastage due to leptospirosis often is unsatisfactory. Unfortunately, reproductive wastage often occurs without any previous clinical evidence of leptospirosis having been observed in a herd18 or flock, and the veterinarian has to base his or her diagnosis on laboratory findings. Laboratory diagnostic procedures for leptospirosis fall into two groups: (1) tests for the demonstration of leptospires in fetal tissues and (2) tests for antibody detection. The selection of tests to be carried out depends on the resources available. The diagnosis of fetal infection ideally should be based on microbiologic investigation, but microbiologic techniques are not as widely available as serologic methods, so many veterinarians have to rely on serologic data.
Microbiologic Investigation Versus Immunochemical Methods
Microbiologic investigation should be based on the demonstration of leptospires in the internal organs (liver, lung, brain, kidney, and adrenal glands) and body fluids (blood, cerebrospinal, thoracic, and peritoneal) of aborted or stillborn calves. Isolation is difficult and time consuming and is a job for specialist laboratories, as is the identification of isolates. The demonstration of leptospires by immunochemical staining methods is more suited to diagnostic laboratories, but success or failure of the tests depends on the numbers of organisms and the tests lack the sensitivity of culture. The immunochemical methods that have been used for diagnosis include immunofluorescence, peroxidase-antiperoxidase, avidin-biotin, and immunogold techniques. Although these methods are not as sensitive as culture, one of them-immunofluorescence 21 -has been used widely in the diagnosis of fetal leptospirosis. It has the advantage of providing better contrast between the leptospires and the tissue background than other methods. That is particularly important because leptospires are very small and filamentous, making them difficult to differentiate from some connective tissue elements and cilia. Immunofluorescence has the disadvantage that the production of good quality polyclonal antisera requires long innoculation regimens in rabbits. Single monoclonal conjugates have not proved a viable alternative to good
472
ELLIS
polyclonal conjugates because good fluorescent antibody sera must be able to recognize a variety of epitopes that will be exposed as leptospires are degraded by autolytic change. The use of suitable pooled monoclones may overcome this problem. A variety of antigen capture methods have been described, including radioimmunoassay, enzyme linked immunosorbent assays, latex agglutination, chemiluminescence, and time resolved fluorimetry, but they have not been evaluated by diagnostic laboratories. Dark-ground microscopy of fetal fluids has been used widely and can be a useful tool in the hands of an experienced diagnostician, but many tissue artifacts can be mistaken for leptospires. Leptospires do not stain satisfactorily with the aniline dyes, and silver staining techniques lack sensitivity and specificity. DNA Hybridization Techniques
There have been a number of reports of blot and in situ hybridization, with both radioactive and nonradioactive, genomic and specific probes being examined as diagnostic tools for leptospirosis. In general terms, the radioactive probes have proved to be more sensitive than nonradioactive probes. 46, 59, 60 A repetitive sequence probe has proved to be about four times more sensitive than a genomic probe in detecting hardjo in cattle urine. As is the case with immunologic detection methods, the limiting factor in these methods is the number of leptospires present: The lower detection limit for DNA probes is about 103 organisms?' 69 Diagnostic laboratories have not published evaluations of such methods with respect to diagnosing fetal leptospirosis. Considerable interest is being shown in the use of the polymerase chain reaction (PCR) as a method of amplifying the amount of leptospiral DNA present before hybridization, and its use in detecting leptospires in bovine urine has been reported. 67 Its use on fetal material has not been reported. Serologic Tests
Serologic testing using the MAT is the laboratory procedure most widely used for the diagnosis of leptospiral abortion. The minimum antigen requirement is that the test should employ representative strains of all the serogroups known to exist in the country plus those known to be maintained by the particular animal elsewhere. The MAT is used best as a herd or flock test, rather than for an individual animal, although it still may play a limited role when only individual animal samples are available.
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTNE FAILURE
473
Because of individual variations in antibody response (many have declining or very low antibody titers at the time of abortion) it is necessary to test at least 10 animals or 10% of the herd, whichever is the greater, to obtain useful information. A retrospective diagnosis of pomona abortion may be made when the majority of affected animals have titers of 1:1000 or more. Increasing the sample size and sampling a number of different cohorts markedly improves disease investigation and assessments of vaccination needs. The herd test approach is not very useful for the diagnosis of hardjo-associated abortion in endemically infected herds because of the insidious and chronic nature of herd infections and the very low levels of antibody that may be found in postabortion cow sera. The limited reliable information available suggests that if a high MAT titer (1:1000 or more) is found in an individual serum sample taken from a hardjo-infected cow shortly after abortion then there is a strong probability (approximately 80%) that the fetus was infected. 22 Unfortunately, the converse is not true; approximately one third of aborting cows with low levels of antibody « 1:100) in one study were shown to have had leptospiral-infected fetuses. 22 Low titers at the time of abortion are the norm in hardjo-associated sheep abortions. There is no value in examining postabortion paired serum samples from individual cows or sheep because titers are either static or falling. 22 Examination of fetal sera by the MAT always should be carried out when possible because up to one in six infected bovine fetuses and a much lower proportion of sheep fetuses may have developed MAT titers, which will range from 1:10 upward. The plate test is not sufficiently sensitive for examining fetal sera.
TREATMENT AND CONTROL
Incidental and host-maintained infections present as different control problems. With incidental infections, it is desirable to reduce contact with wildlife and to use vaccination. Vaccination considerations are the same as those outlined subsequently for host-maintained infections. Fundamental to developing strategies for the control of infection with host-maintained leptospires is an understanding of the epidemiology of infection and methods of transmission, and the identification of major risk factors such as those described in the epidemiology section. In devising a control strategy, it also is important to consider the reasons why you want to control leptospirosis. The arguments for control go beyond the obvious need to prevent clinical disease and economic loss: There also is the need to minimize the risk of human infection by controlling exposure of domestic animals. For some farmers, it may be
474
ELLIS
necessary to take a control program to the point at which eradication is a feasible option. Although eradication may be possible, it may not always be desirable. Cattle free from hardjo infection are not an attractive proposition to commercial dairy farmers, for example; they require immune cattle, not totally susceptible cattle. The exceptions to this argument are farmers selling valuable sires to artificial insemination centers, or artificial insemination centers wishing to sell semen abroad; freedom from infection is an important requirement for that relatively small group of managers.
Tools for Control
The next step in determining a control strategy is a consideration of tools available to support such a program. Four or five approaches are available. Antibiotic Therapy
Streptomycin (and its dihydro analogue), given at a dose of 25 mg/ kg, will markedly reduce the number of organisms an infected animal excretes but will not give a microbiologic cure?3 It will not prevent reinfection, so when it is used as part of a control program, it is best used either on a whole herd at the same time and given to added animals held in isolation. There are problems with this approach in the United States because streptomycin is being withdrawn from veterinary use and no antibiotic of comparable efficacy is available. Vaccination
Vaccines are the best tool available, but they have limitations. They may not protect adequately against all genotypes of a serovar such as hardjo. Almost all the hardjo products available on the world market are produced using genotype Hardjoprajitno strains, whereas the predominant strain found in cattle is genotype Hardjobovis. Major doubts have been cast on the efficacy of some leptospiral vaccines.s, 6, 8 It always has been recognized that immunity to infection may not be as good as immunity to clinical disease. Vaccines alone will not eradicate infection from an endemically infected herd; they will not stop excretion in the animal that already is leptospiruric; and they will not always prevent abortion in animals in which placental localization has already occurred. National Animal Disease Centers, 6, 8 work suggested that the products tested by them did not even give clinical protection.
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTIVE FAILURE
475
Identification of Infected Animals
This is an important tool in other disease control programs, either as a means of preventing introduction of infection to a herd or to remove carrier animals already in a herd. No worthwhile methods are available. Management
Management has an important part to play in the control of risk factors. By suitable planning of land and buildings, access to other animals and contaminated water supplies can be minimized. The management of added animals is important in herds that have a control program. New animals should be isolated, treated with streptomycin, and vaccinated before being added to the herd. Permutations of the Approaches Already Mentioned
Any control strategy must employ a mixture of the aforementioned methods. Management methods run across all the control strategy options.
Disease Outbreak
In the face of a reproductive wastage problem, it is essential to vaccinate and treat those animals still pregnant, then move into a control program as described for an endemic herd. Endemically Infected Herd
A control program ideally should start with treating all adult animals at a time when the cost of lost product (such as milk) can be minimized. That may mean deferring treatment until animals go dry. Vaccinate the whole herd (all females destined for breeding and all intact males) annually in the case of cattle or before to service in the case of sheep. When doubts exist as to the efficacy of the vaccine reduce cattle vaccination intervals to 6 months or less, and when infertility is a feature, vaccination also initially should take place at intervals of 6 month or less. Farmers should be warned that they should stay with a control program once it is started. Stopping vaccination to save money after a few years will leave them with a totally susceptible herd and potentially worse off than if they had never started the program. Current leptospiral vaccines are not licenced for use in sheep, but experience has shown that they can be used at a quarter of the cattle dosage.
476
ELLIS
References 1. Adinarayanan N, James PC: Studies on leptospirosis in farm stock and wildlife in Kerala. Indian J Anim Health 13:93, 1980 2. Blackmore DK, Bahaman AR, Marshall RB: The epidemiological interpretation of serological responses to leptospiral serovars in sheep. NZ Vet J 30:38, 1982 3. Bjorkman N, Sollen P: Morphology of the bovine placenta at normal delivery. Acta Vet Scand 1:347, 1960 4. Bolin CA: Oral presentation to the 36th Annual Meeting, American Leptospirosis Research Conference, Chicago, 1993 5. Bolin CA, Cassells JA, Zuemer RL, et al: Effect of vaccination with a monovalent Leptospira interrogans serovar hardjo type Hardjobovis vaccine on type Hardjobovis infection of cattle. Am J Vet Res 52:1639, 1991 6. Bolin CA, Thiermann AB, Handsaker A, et al: Effect of vaccination with a pentavalent leptospiral vaccine on Leptospira interrogans serovar hardjo type Hardjobovis infection in pregnant cattle. Am J Vet Res 50:161, 1989 7. Bolin CA, Zuemer RL, Trueba G: Comparison of three techniques to detect Leptospira interrogans serovar hardjo type Hardjobovis in bovine urine. Am J Vet Res 50:1001,1989 8. Bolin CA, Zuemer RL, Trueba G: Effect of vaccination with a pentavalent leptospiral vaccine containing serovar hardjo type Hardjobovis on type Hardjobovis infection in cattle. Am J Vet Res 50:2004, 1989 9. Cousins DW, Ellis TM, Parkinson J, et al: Evidence for sheep as a maintenance host for Leptospira interrogans serovar hardjo. Vet Rec 124:123, 1989 10. Damude DF, Jones CJ, Myers DM: A study of leptospirosis among animals in Barbados. Trans R Soc Trop Med Hyg 73:161, 1979 11. Dennis SM: Diagnosis of infectious abortion in cattle. Vet Med Small Anim Clin 64:423, 1969 12. Dikken H, Kmety E: Serological typing methods of leptospires. Methods in Microbiology 11 :259, 1978 13. Ellis WA: Recent developments in bovine leptospirosis. In Grunsell CSG, Hill FWG (eds): The Veterinary Annual, 23rd issue. Bristol, Scientechnica, 1983, pp 91-95 14. Ellis WA: Leptospirosis. In Martin WB, Aitkin ID (eds): Diseases of Sheep, ed 2. Oxford, Blackwell Scientific Publications, 1991, pp 78-80 15. Ellis WA, Michna SW: Bovine leptospirosis: Infection by the Hebdomadis serogroup and abortion-a herd study. Vet Rec 99:409, 1977 16. Ellis WA, Michna SW: Bovine leptospirosis: Experimental infection of pregnant heifers with a strain belonging to the Hebdomadis serogroup. Res Vet Sci 22:229, 1977 17. Ellis WA, Cassells JA, Doyle J: Genital leptospirosis in bulls. Vet Rec 118:333, 1986 18. Ellis WA, O'Brien H, Bryson DG, et al: Bovine leptospirosis: Some clinical features. Vet Rec 117:101, 1985 19. Ellis WA, O'Brien H, Neill S, et al: The isolation of a leptospire from an aborted bovine fetus. Vet Rec 99:458, 1976 20. Ellis WA, O'Brien H, Neill S, et al: The isolation of a strain of Leptospira serogroup icterohaemorrhagiae from an aborted bovine fetus. Br Vet J 133:108, 1977 21. Ellis WA, O'Brien H, Neill SD, et al: Bovine leptospirosis: Microbiological and serological findings in aborted fetuses. Vet Rec 110:147, 1982 22. Ellis WA, O'Brien H, Neill SD, et al: Bovine leptospirosis: Serological findings in aborting cows. Vet Rec 110:178, 1982 23. Ellis WA, Montgomery J, Cassells JA: Dihydrostreptomycin treatment of bovine carriers of Leptospira interrogans serovar hardjo. Res Vet Sci 39:292, 1985 24. Ellis WA, Thiermann AB, Montgomery J, et al: Restriction endonuclease analysis of Leptospira interrogans serovar hardjo isolates from cattle. Res Vet Sci 44:375, 1988 25. Fennestad KL, Borg-Petersen C: Foetal leptospirosis and abortion in cattle. J Infect Dis 102:227, 1958 26. Flint SH, Comer RJ, Marshall RB: Leptospirosis in farmed goats. N Z Vet J 36:156, 1988 27. Hajtos I, Malik G, Banfalvi E, et al: Abortion in sheep caused by Leptospirae in Hungary. Magyar Allat Lapja 38:721, 1983 28. Hanson LE: Control of bovine leptospirosis. Bovine Pract 7:17, 1972
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTIVE FAILURE
477
29. Hanson LE, Brodie BO: Leptospira hardjo infections in cattle. In Proceedings of the 71st Annual Meeting of the United States. Livestock Sanitary Association, Phoenix, AZ, 1967, P 210 30. Hanson LE, Tripathy DN, Killinger AH: Current status of leptospirosis immunization in swine and cattle. J Am Vet Med Assoc 161:1235, 1972 31. Hathaway SC: Leptospirosis in New Zealand: An ecological view. NZ Vet J 29:109, 1981 32. Hathaway SC, Little TWA: Epidemiological study of Leptospira hardjo infection in second calf dairy cows. Vet Rec 112:215, 1983 33. Heath S: Oral presentation to the 36th Annual Meeting, American Leptospirosis Research Conference, Chicago, 1993 34. Hermann JL, Baril C, Bellenger E, et al: Conservation of the genome among isolates of Leptospira interrogans. J BacterioI173:7582, 1991 35. Hermann JL, Bellenger E, Perolat P, et al: Pulse-field gel electrophoresis of Non digests of leptospiral DNA: A new rapid method of serovar identification. J Clin Microbiol 30:1696, 1992 36. Kirkbride CA, Johnson MW: Serological examination of aborted ovine and bovine fetal fluids for the diagnosis of border disease, bluetongue, bovine viral diarrhea, and leptospiral infections. J Vet Diagn Invest 1:132, 1989 37. Kmety E, Dikken H: Revised list of Leptospira serovars. Groningen, University Press, 1988 38. Knott SG, Dadswell LP: An outbreak of bovine abortions associated with leptospirosis. Aust Vet J 46:385, 1970 39. LeFebvre RB: DNA probe for detection of the Leptospira interrogans serovar hardjo genotype Hardjobovis. J Clin MicrobioI25:2094, 1987 40. Leon - Vizcaino L, Hermoso de Mendoza M, Garrido F: Incidence of abortions caused by leptospirosis in sheep and goats in Spain. Comp Immun Microbiol Infect Dis 10:147, 1987 41. Leonard FC, Quinn pJ, Ellis WA, et al: Association between cessation of leptospiruria in cattle and urinary antibody levels. Res Vet Sci 55:195, 1993 42. Lingard ER, Hanson LE: Effect of Leptospira pomona on the reproductive efficiency of cattle. J Am Vet Med Assoc 139:449, 1961 43. Malkin K: Enhancement of Leptospira hardjo agglutination titers in sheep and goat serum by heat inactivation. Can J Comp Med 48:208, 1984 44. McKeown JD, Ellis WA: Leptospira hardjo agalactia in sheep. Vet Rec 118:482, 1986 45. Marshall RB, Wilton BE, Robinson AJ: Identification of Leptospira serovars by restriction endonuclease analysis. J Med MicrobioI14:163, 1981 46. Miller BD, Chappel RJ, Adler B: Detection of leptospires in biological fluids using DNA hybridization. Vet MicrobioI15:71, 1987 47. Motie A, Meyers DM: Leptospirosis in sheep and goats in Guyana. Trop Anim Health Prod 18:113, 1986 48. Murphy JC, Jensen R: Experimental pathogenesis of leptopiral abortion in cattle. Am J Vet Res 30:703, 1969 49. Murray RD, Dhaliwal GS, Downham DY, et al: Effects of leptospirosis on fertility of dairy cattle. Cattle Practice 1:99-105, 1993 50. Nigam SK, Shukla V, Ranat R: Abortion in goats probably due to Leptospira infection in a sheep farm in Madhya Pradesh. Indian Journal of Animal Health 13:93, 1974 51. Pearson JKL, Mackie DP, Ellis WA: Milk drop syndrome resulting from Leptospira hardjo. Vet Rec 106:135, 1980 52. Perolat P, Grimont F, Regnault B, et al: rRNA gene restriction patterns of Leptospira: A molecular typing system. Res MicrobioI141:159, 1990 53. Perolat P, Lecuyer I, Postic D, et al: Diversity of ribosomal DNA fingerprints of Leptospira serovar provides a database for subtyping and species assignation. Res Microbiol 144:5, 1993 54. Quinlan JF, Nicholl VJ: Agalactia and infertility due to Leptospira interrogans serovar hardjo infection in a vaccinated dairy herd. Irish Vet J 46:97, 1993 55. Rako A: Leptospirosis in sheep. Bul Shk Zootek Vet 4:71, 1986 56. Ramadass P, Jarvis BDW, Comer RJ: Genetic characterisation of pathogenic Leptospira species by DNA hybridization. Int J Syst Bact 42:215, 1992 57. Schollum LM, Blackmore DK: The serological and cultural prevalence of leptospirosis in a sample of feral goats. NZ Vet J 29:104, 1981
478
ELLIS
58. Segers RPAM, Van Gestel JA, Van Eys GJJM, et al: Presence of putative spingomyelinase genes among members of the family Leptospiraceae. Infect Immun 60:1707, 1992 59. Terpstra WJ, Schoone GJ, Schegget JT: Detection of leptospiral DNA by nucleic acid hybridization with 32-P and biotin-labelled probes. J Med Microbio122:23, 1986 60. Terpstra WJ, Schoone GJ, Lighart GS, et al: Detection of Leptospira interrogans in clinical specimens by in situ hybridization using biotin-labelled DNA probes. J Gen Microbiol 133:911, 1987 61. Thiermann AB: Experimental leptospiral infections in pregnant cattle with organisms of the Hebdomadis serogroup. Am J Vet Res 43:780, 1982 62. Thierman AB, LeFebvre RB: Restriction endonuclease analysis and other molecular techniques in the identification of Leptospira and other pathogens of veterinary importance. In Swaminathan B, Prakash G (eds): Nucleic Acid and Monoclonal Antibody Probes. Applications in Diagnostic Microbiology. New York, Marcel Dekker, 1989, pp 145-183 63. Thiermann AB, Handsaker AL, Foley JW, et al: Reclassification of North American isolates belonging to serogroups Mini and Sejroe by restriction endonuclease analysis. Am J Vet Res 47:61, 1986 64. Tripathy DN, Hanson LE, Bedoya M, et al: Experimental infection of pregnant and lactating goats with Leptospira interrogans serovars hardjo and szwajizak. Am J Vet Res 46:2515, 1985 65. Van der Hoeden J: Leptospirosis among goats in Israel. J Comp Patho163:101, 1953 66. Van Eys GJJM, Gerritsen MJ, Korver H, et al: Characterization of serovars of the genus Leptospira with hardjobovis and icteroheamorrhagiae recombinant probes with special attention to serogroup Sejroe. J Clin Microbio129:1042, 1991 67. Van Eys GJJM, Gravekamp C, Gerritsen MJ, et al: Detection of leptospirosis in urine by polymerase chain reaction. J Clin Microbiol 27:2258, 1989 68. Yasuda PH, Steigerwalt AG, Sulzer KR, et al: Deoxyribonucleic acid relatedness between serogroups and serovars in the family Leptospiraceae with proposals for seven new Leptospira species. Int J Syst Bacterio137:407, 1987 69. Zuerner RL, Bolin CA: Repetitive sequence element cloned from Leptospira interrogans serovar hardjo type Hardjobovis provides a sensitive diagnostic probe for bovine leptospirosis. J Clin Microbio126:2495, 1988 70. Zuerner RL, Bolin CA: Nucleic acid probe characterises Leptospira interrogans serovars by restriction fragment length polymorphisms. Vet Microbio124:355, 1990
Address reprint requests to W.A. Ellis, PhD, BVMS, FRCVS Veterinary Sciences Division Department of Agriculture Stormont, Belfast Northern Ireland BT4 35D
0749-0720/94 $0.00 + .20
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTIVE FAILURE William A. Ellis, PhD, BVMS, FRCVS
Leptospirosis is a zoonotic disease caused by members of the genus
Leptospira. Veterinarians' perception of leptospirosis as a disease of domestic animals has undergone considerable modification in the past decade or so as they have increasingly appreciated the role of the hostmaintained leptospires as causes of reproductive wastage in their respective host species kept under modem intensive management systems. Large discrepancies exist in our understanding of leptospirosis among different domestic ruminants. A considerable amount of data has been published on the condition in cattle, whereas much less is known about leptospirosis in sheep, and even less in goats. This imposes a bias toward discussion of cattle in this review. Even with cattle, many aspects remain poorly understood-e.g., variations in disease pattern and disease impact associated with different strains of the same host-maintained serovar in different management systems and different parts of the world. The paucity of good data on leptospirosis in sheep and goats could be due to one or more of the following factors: Sheep and goats may be much less susceptible to leptospiral infection; developed economies place much lower value on those species, especially goats, so less research has been done; and they rarely are kept in intensive management systems. The organisms of the genus Leptospira are small aerobic spirochetes that can be found in a wide variety of animal species (the parasitic strains) and in water (the saprophytic strains). These two groupings,
From the Veterinary Sciences Division, Department of Agriculture, Northern Ireland, and Queen's University, Belfast, Northern Ireland
VETERINARY CLINICS OF NORTH AMERICA: FOOD ANIMAL PRACTICE VOLUME 10 • NUMBER 3 • NOVEMBER 1994
463
464
ELLIS
until recently, were referred to as the "interrogans" and "biflexa" complexes, and can be differentiated by their growth requirements and biochemical reactions. For taxonomic purposes and as an aid to epidemiologic studies, the parasitic leptospires were subdivided into serogroups on the basis of antigenic relationships as determined by cross agglutination reactions and further subdivided into serovars by agglutinationabsorption patterns. Twenty-three serogroups are recognized, containing approximately 212 serovars. 37 Alternative serologic typing systems based on either monoclonal antibodies or factor analysis give results comparable to conventional serotyping methods. 12,37 The advent of genetic typing methods has thrown the taxonomy of the genus into some confusion, but has provided rapid, reproducible typing protocols. Seven genospecies of parasitic leptospires have been described based on DNA relatedness and guanine plus cytosine content studies. 56, 68 The value of this system is limited with respect to epidemiology. Other genetic typing methods have proved of value and increasingly are being used as supplementary or alternative typing systems, particularly in epidemiologic studies, because they identify strain differences at the subserovar level. These systems include restriction endonuclease analysis of chromosomal DNA by fixed field 24, 45, 62,63 or pulsed field gel electrophoresis,34, 35 recombinant DNA probes,39, 58, 66, 76 and ribotyping. 52,53 EPIDEMIOLOGY Distribution
The epidemiology of leptospirosis in domestic animals is potentially very complicated because animals can be infected by any of the pathogenic serovars. Fortunately, only a small number of serovars are endemic in any particular region or country. Furthermore, leptospirosis is a disease that shows a natural nidality and each serovar tends to be maintained in specific maintenance hosts. In any region, therefore, an animal species may be infected by serovars maintained by its own species or serovars maintained by other animal species present in the area. The relative importance of these incidental infections is determined by the opportunities for contact and transmission of leptospires from other species to the target host species provided by prevailing social, management, and environmental factors. 31 In the developed, intensive-agricultural economies, incidental infections playa much less significant role than do host-maintained infections. The number of Leptospira serovars maintained by domestic animals is small and, although regional differences occur, local geographic differences are not as important as with incidental infections.
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTIVE FAILURE
465
Hardjo is the serovar maintained by cattle. It has a worldwide distribution, with high serologic and microbiologic prevalences identified in most countries that have carried out appropriate investigations. Although differences in cattle management practices and resulting herd immunity patterns probably contribute to the differing clinical situations observed in different countries, it also is probable that strain differences highlighted by a variety of DNA technologies, make a contribution. Two major genotypes of hardjo are found in cattle and sheepHardjobovis and Hardjoprajitno. Hardjobovis appears to be a betteradapted parasite than Hardjoprajitno. It is excreted in much larger numbers in cattle urine and is the strain found in most countries. Hardjoprajitno so far has been recovered only in the United Kingdom, Nigeria, India, Malaysia, Brazil, Mexico, and the United States. Serovar pomona, maintained by swine and a variety of free living animals, is the most important source of incidental infection of cattle in North America, Australia, and New Zealand. In parts of Africa, Russia, and Israel, grippotyphosa constitute an important incidental infective agent of cattle. In parts of the Caribbean, this role is filled by Autumnalis strains. Strains belonging to the Pyrogenes serogroup are found in cattle in parts of Africa, and serovar zanoni recently was shown to be common in Queensland. Serologic prevalences tend to be much lower in sheep than in cattle, perhaps in part because of complement interfering with the microscopic agglutination testt3 and the fact that sheep are kept in extensive management systems with less opportunity for transmission. There is consistent serologic evidence that hardjo infection is the major one found in sheep worldwide, however, in some countries there even is evidence that sheep may maintain the serovar without the presence of cattle.9 Seroprevalence data indicate that other leptospiral infections in sheep are uncommon and are incidental, and the limited culture surveys reported support that view. I , 2 The serovars involved in such incidental infections vary by region and reflect the serovars commonly found in other domestic and wild animals in a region. No cultural or serologic evidence exists to suggest that goats act as maintenance hosts for leptospires. l , 26, 57 High seroprevalences have been reported in some countries, but the predominant infecting serovar varies, suggesting an incidental pattern of infection.
Transmission
Direct transmission can occur among animals via infected urine, postabortion uterine discharge, infected placenta, sexual contact, or in utero infection. It is probably of greatest importance with serovar hardjo,
466
ELLIS
with which infection appears to be largely independent of rainfall and cattle and sheep management systems. Indirect transmission plays a much greater role in the transmission of incidental infections. It occurs through exposure to an environment contaminated with infective material-usually one that permits the maximum survival of leptospires outside the host-and a management system that facilitates close contact between carrier and susceptible animals. The optimum conditions for survival outside the host are warm, moist conditions, with a pH close to neutral. Where such conditions are found, the prevalence of incidental infections is likely to be highest. Areas such as waterholes, where animals congregate, frequently are incriminated in outbreaks of leptospirosis. Environments favorable to the survival of leptospires are much less important in the epidemiology of host-maintained leptospires. The major risk factors in introducing infection into a clean herd are: 1. An open herd policy, with the consequent risk of purchasing
carrier animals; 2. Close proximity of another domestic host species-e.g., sheep in the case of hardjo infection in cattle; 3. Access to other animals of the same species-in particular, the use of a shared bull; 4. Access to a water course exposed to other animal hosts upstream; The major factors in maintaining infection in a herd are: 1. The persistently infected carrier: Some animals are carriers for
life, but it remains uncertain whether they remain infective throughout that period. 2. A regular supply of susceptible animals: The standard practice in dairy herds of removing replacement heifers from their dams shortly after birth and keeping them separated from the main herd until shortly before or after calving ensures such a regular supply of nonimmune animals.
PATHOGENESIS
Infection of susceptible animals occurs through the mucous membranes of the eyes, mouth, nose, vagina, and penis and through abraded or water-softened skin and is followed, after a 4- to lO-day incubation period, by a bacteremic phase that may last from a few hours to 7 days. That phase may be subclinical but can be characterized by pyrexia, excretion of leptospires in milk, and, with some serovars, functional damage to the internal organs, especially in younger animals. During this period, leptospires can be isolated from most organs of the body and from
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTIVE FAILURE
467
cerebrospinal fluid. Acute clinical disease coincides with this bacteremic phase. The primary bacteremic phase ends with the appearance of circulating antibodies, usually detectable by 10 days after infection. Peak titers and the time for which they persist vary considerably, depending on the animal species, the infecting serovar, and the route of infection. Serovar hardjo titers (as determined by the microscopic agglutination test, MAT) in cattle infected with some genotype Hardjobovis strains, can reach levels of greater than 1:10,000 by 4 to 6 weeks postinfection and can remain detectable for more than 1 year. 41 The initial response is an IgM one that peaks 2 to 3 weeks postinfection. The IgG response is much slower, with peak levels occurring between 12 and 30 weeks after infectionY In contrast, serovar hardjo titers (as determined by the MAT) in sheep are low and transient. Following the period of leptospiremia, the leptospires localize and persist in a number of organs, especially the proximal renal tubules (all ages) and the genital tracts of sexually mature females and entire males. Relapses of pyrexia may occur; whether they are associated with persistent foci of infection giving rise to recurring bacteremia or, possibly, toxemia is unknown. Differences have been found in tissue tropisms exhibited by different strains of hardjo.4 Some strains of Hardjobovis have been shown to persist primarily in the kidney, others have a predilection for the genital tract, and still others persist in both organ systems. Leptospires localized in the proximal renal tubules multiply and are voided in the urine. The duration and intensity of urinary shedding varies from animal to animal and with the infecting serovar. In the case of host-maintained infections such as hardjo infection in cattle, the intensity of excretion is greatest during the first 4 to 6 weeks of shedding. Leptospiruria is constant during that period. A variable period of intermittent, low-intensity leptospiruria then ensues, frequently lasting for 6 to 12 months in cattle but sometimes persisting for life. Excretion of hardjo for prolonged periods also has been observed in sheep. In contrast, urinary shedding is of very low intensity in other host-maintained infections in which the venereal route probably is more important, such as Hardjoprajitno infection in cattle and sheep. The factors involved in the cessation of urinary excretion are poorly understood, but one recent study showed it invariably is associated with a sharp increase in urinary antileptospiral IgG and IgA antibody levels.41 Localization of leptospires in the pregnant and nonpregnant uterus of cows has been shown to persist for up to 142 days and 97 days postinfection, respectively. Localization in the pregnant uterus, in turn, may be followed by fetal infection, with subsequent chronic reproductive wastage and excretion of the leptospires in the postcalving uterine dis-
468
ELLIS
charge. Bovine and ovine fetuses infected during the latter stages of gestation may develop detectable antibody titers. Localization of serovar hardjo has been reported in the testes and accessory glands of bulls,17 and leptospires of the pomona and Hebdomadis serogroups have been demonstrated in bull semen. Leptospiral antibodies have been detected in seminal plasma, suggesting local antibody production. Persistence of serovar hardjo in the mammary glands of cattle61 and goats64 also has been reported.
CLINICAL CONCERNS
The vast majority of leptospiral infections are subclinical. Two groups of animals are most likely to experience clinical infections: (1) Very young animals that contract an incidental infection may experience severe illness characterized by jaundice, hematuria, hemaglobinuria, evidence of renal damage, meningitis, and, in some cases, death. Such acute clinical events very occasionally are seen in older animals. (2) Sexually mature lactating or pregnant females infected by either incidental or host-maintained leptosires may demonstrate agalactia (in cattleS1 and sheep46) or reproductive wastage.
Reproductive Wastage in Cattle
This feature is the most important aspect of leptospirosis in terms of its effects on farm economics. It is a chronic sequel to leptospirosis in breeding cows. Fetal infection with resulting stillbirths, abortions, and the birth of weak offspring of reduced viability may occur. Although such reproductive wastage can occur in cattle infected by many parasitic leptospires, it is particularly a feature of pomona and hardjo infections. Abortion and the other effects usually occur 1 to 6 weeks (pomona infection) or 4 to 12 weeks (hardjo infection) after the acute phase of infection, but such animals frequently show no clinical evidence of acute infection. With pomona infections, the same events usually occur in the last 3 months of gestation. Abortion has been observed in the second trimester in cases of autumnalis infection.lO With hardjo infection, abortion has been diagnosed at all stages, from the fourth month through term, and circumstantial evidence indicates it also may cause early embryonic death. 13 It has been difficult to define the importance of leptospirosis relative to other factors in the etiology of bovine abortion, stillbirth, and premature live birth because of difficulties in diagnosis and regional differences
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTIVE FAILURE
469
in the distribution of genetic subtypes. In a study in Northern Ireland, 42% of aborted bovine fetuses had been infected by leptospires (almost all hardjo),21 whereas evidence of leptospiral infection was found in 10%
of aborted fetuses examined in South Dakota. 36 The incidence of abortion on individual farms can be very high following an epizootic of pomona infection; incidences of up to 50% have been reported. 38 Abortion rates tend to be much lower (3%-10%) with hardjo infection, but rates of more than 30% occasionally occur.13 Hardjo represents the most important leptospiral infection of cattle, however, because it is associated with long-term herd infections on farms, with the potential for ongoing losses year after year. 28 This recurring loss is most noticeable where management practices ensure the regular introduction of susceptible animals into an infected herd. IS, 32 No association between pomona infection and infertility has been shown,42 but infertility has been a common field observation in hardjoinfected herds29,s4 and improvements in breeding efficiency have been noted in herds after hardjo vaccination. 3D Attempts to establish herd data to support those observations and preliminary supportive findings have been reported. 33, 49
Reproductive Wastage in Sheep
Relative to cattle and pigs, sheep have always been considered resistant to leptospiral infections. Although that view still generally is true in many parts of the world, it is becoming apparent that, under certain intensive management systems now used in countries such as the United Kingdom and Ireland, leptospirosis can cause overt clinical disease in the last 2 weeks of gestation and in the immediate postpartum period, when sheep are immunologically compromised. This can cause considerable loss to individual farmers using such management regimens. Reproductive wastage in sheep is seen as late-term abortion, stillbirth, and the birth of weak lambs. In a study in Northern Ireland, approximately 17% of 872 aborted lambs examined between 1981 and 1987 were positive for leptospires on fluorescent antibody examination. Culture data indicated that the infecting strain in the majority of cases was serovar hardjo, with a small number caused by pomona, ballum, or bratislava. 14 In a South Dakota study,36 8% of aborted fetuses had antibodies to leptospires. A study in Spain40 estimated that 1.7% of abortions were due to leptospires (mainly pomona). Serovar pomona also has been implicated in abortions in Albania,36 whereas grippotyphosa was deemed to be the cause of abortions in Hungary.27 Ellis I4 observed that hardjo abortion rarely was found in extensively managed flocks. It was confined primarily to ewes bred in an extensive
470
ELLIS
system and subsequently bought as replacements for intensively managed flocks with high stocking rates and indoor housing for ewes. Reproductive wastage and agalactia were seen in such animals in their first lambing season following introduction, but not in subsequent years.
Reproductive Wastage in Goats
There are few reports of leptospirosis causing reproductive wastage in goats. A report from Spain40 attributed 2.7% of goat abortions to leptospirosis (mainly pomona). In Israel, van der Hoeden65 described an outbreak of acute grippotyphosa infection in goats, a sequel to which was a high incidence of abortion. Abortion also has been reported in goats in Guyana47 and India.5o
FETAL AND UTERINE PATHOLOGY
Postmortem examination of fetuses aborted because of leptospirosis usually reveals only nonspecific findings that either result from autolysis or cannot be differentiated satisfactorily from autolytic changes. Autolysis is a particular feature of fetuses aborted before 6 months. Jaundice occasionally may be observed in the subcutaneous tissues of infected fetuses aborted in the last stages of gestation, whereas stillborn fetuses frequently exhibit lesions similar to those produced by anoxia-i.e., petechial hemorrhages on the surface of the thymus, thyroid, lungs, and heart and in the parietal pluera, peritoneum, and mesentery. The only consistent histologic changes observed in experimental leptospiral abortion have been in the fetal kidney. They have consisted of foci of tubular necrosis with interstitial and perivascular infiltration of lymphocytes, along with a few plasma cells and polymorphonuclear leukocytes.16, 25, 48 Severe vascular lesions were a feature of the histopathologic findings in two reports of hardjo and icterohaemorrhagiae infection in aborted fetuses. 19,2o The lesions were particularly severe in the liver and appeared to a lesser extent in the thymus, cerebral meninges, and interlobular septa of the lungs. The vessels often were congested and exhibited mural edema and necrosis. Perivascular hemorrhage was noted. Polyarthritis was reported in a live calf born to a cow experimentally infected with hardjo.61 In cases of pomona abortion, the cotyledons appear light tan to yellow in color and avascular and are affected uniformly,u A controlled serial kill experiment,48 however, failed to demonstrate any histologic changes in the pregnant uterus or fetal membranes of infected heifers
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTNE FAILURE
471
that were not present in control heifers and that have not been reported in so-called normal placentae. 3
DIAGNOSIS
The diagnosis of reproductive wastage due to leptospirosis often is unsatisfactory. Unfortunately, reproductive wastage often occurs without any previous clinical evidence of leptospirosis having been observed in a herd18 or flock, and the veterinarian has to base his or her diagnosis on laboratory findings. Laboratory diagnostic procedures for leptospirosis fall into two groups: (1) tests for the demonstration of leptospires in fetal tissues and (2) tests for antibody detection. The selection of tests to be carried out depends on the resources available. The diagnosis of fetal infection ideally should be based on microbiologic investigation, but microbiologic techniques are not as widely available as serologic methods, so many veterinarians have to rely on serologic data.
Microbiologic Investigation Versus Immunochemical Methods
Microbiologic investigation should be based on the demonstration of leptospires in the internal organs (liver, lung, brain, kidney, and adrenal glands) and body fluids (blood, cerebrospinal, thoracic, and peritoneal) of aborted or stillborn calves. Isolation is difficult and time consuming and is a job for specialist laboratories, as is the identification of isolates. The demonstration of leptospires by immunochemical staining methods is more suited to diagnostic laboratories, but success or failure of the tests depends on the numbers of organisms and the tests lack the sensitivity of culture. The immunochemical methods that have been used for diagnosis include immunofluorescence, peroxidase-antiperoxidase, avidin-biotin, and immunogold techniques. Although these methods are not as sensitive as culture, one of them-immunofluorescence 21 -has been used widely in the diagnosis of fetal leptospirosis. It has the advantage of providing better contrast between the leptospires and the tissue background than other methods. That is particularly important because leptospires are very small and filamentous, making them difficult to differentiate from some connective tissue elements and cilia. Immunofluorescence has the disadvantage that the production of good quality polyclonal antisera requires long innoculation regimens in rabbits. Single monoclonal conjugates have not proved a viable alternative to good
472
ELLIS
polyclonal conjugates because good fluorescent antibody sera must be able to recognize a variety of epitopes that will be exposed as leptospires are degraded by autolytic change. The use of suitable pooled monoclones may overcome this problem. A variety of antigen capture methods have been described, including radioimmunoassay, enzyme linked immunosorbent assays, latex agglutination, chemiluminescence, and time resolved fluorimetry, but they have not been evaluated by diagnostic laboratories. Dark-ground microscopy of fetal fluids has been used widely and can be a useful tool in the hands of an experienced diagnostician, but many tissue artifacts can be mistaken for leptospires. Leptospires do not stain satisfactorily with the aniline dyes, and silver staining techniques lack sensitivity and specificity. DNA Hybridization Techniques
There have been a number of reports of blot and in situ hybridization, with both radioactive and nonradioactive, genomic and specific probes being examined as diagnostic tools for leptospirosis. In general terms, the radioactive probes have proved to be more sensitive than nonradioactive probes. 46, 59, 60 A repetitive sequence probe has proved to be about four times more sensitive than a genomic probe in detecting hardjo in cattle urine. As is the case with immunologic detection methods, the limiting factor in these methods is the number of leptospires present: The lower detection limit for DNA probes is about 103 organisms?' 69 Diagnostic laboratories have not published evaluations of such methods with respect to diagnosing fetal leptospirosis. Considerable interest is being shown in the use of the polymerase chain reaction (PCR) as a method of amplifying the amount of leptospiral DNA present before hybridization, and its use in detecting leptospires in bovine urine has been reported. 67 Its use on fetal material has not been reported. Serologic Tests
Serologic testing using the MAT is the laboratory procedure most widely used for the diagnosis of leptospiral abortion. The minimum antigen requirement is that the test should employ representative strains of all the serogroups known to exist in the country plus those known to be maintained by the particular animal elsewhere. The MAT is used best as a herd or flock test, rather than for an individual animal, although it still may play a limited role when only individual animal samples are available.
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTNE FAILURE
473
Because of individual variations in antibody response (many have declining or very low antibody titers at the time of abortion) it is necessary to test at least 10 animals or 10% of the herd, whichever is the greater, to obtain useful information. A retrospective diagnosis of pomona abortion may be made when the majority of affected animals have titers of 1:1000 or more. Increasing the sample size and sampling a number of different cohorts markedly improves disease investigation and assessments of vaccination needs. The herd test approach is not very useful for the diagnosis of hardjo-associated abortion in endemically infected herds because of the insidious and chronic nature of herd infections and the very low levels of antibody that may be found in postabortion cow sera. The limited reliable information available suggests that if a high MAT titer (1:1000 or more) is found in an individual serum sample taken from a hardjo-infected cow shortly after abortion then there is a strong probability (approximately 80%) that the fetus was infected. 22 Unfortunately, the converse is not true; approximately one third of aborting cows with low levels of antibody « 1:100) in one study were shown to have had leptospiral-infected fetuses. 22 Low titers at the time of abortion are the norm in hardjo-associated sheep abortions. There is no value in examining postabortion paired serum samples from individual cows or sheep because titers are either static or falling. 22 Examination of fetal sera by the MAT always should be carried out when possible because up to one in six infected bovine fetuses and a much lower proportion of sheep fetuses may have developed MAT titers, which will range from 1:10 upward. The plate test is not sufficiently sensitive for examining fetal sera.
TREATMENT AND CONTROL
Incidental and host-maintained infections present as different control problems. With incidental infections, it is desirable to reduce contact with wildlife and to use vaccination. Vaccination considerations are the same as those outlined subsequently for host-maintained infections. Fundamental to developing strategies for the control of infection with host-maintained leptospires is an understanding of the epidemiology of infection and methods of transmission, and the identification of major risk factors such as those described in the epidemiology section. In devising a control strategy, it also is important to consider the reasons why you want to control leptospirosis. The arguments for control go beyond the obvious need to prevent clinical disease and economic loss: There also is the need to minimize the risk of human infection by controlling exposure of domestic animals. For some farmers, it may be
474
ELLIS
necessary to take a control program to the point at which eradication is a feasible option. Although eradication may be possible, it may not always be desirable. Cattle free from hardjo infection are not an attractive proposition to commercial dairy farmers, for example; they require immune cattle, not totally susceptible cattle. The exceptions to this argument are farmers selling valuable sires to artificial insemination centers, or artificial insemination centers wishing to sell semen abroad; freedom from infection is an important requirement for that relatively small group of managers.
Tools for Control
The next step in determining a control strategy is a consideration of tools available to support such a program. Four or five approaches are available. Antibiotic Therapy
Streptomycin (and its dihydro analogue), given at a dose of 25 mg/ kg, will markedly reduce the number of organisms an infected animal excretes but will not give a microbiologic cure?3 It will not prevent reinfection, so when it is used as part of a control program, it is best used either on a whole herd at the same time and given to added animals held in isolation. There are problems with this approach in the United States because streptomycin is being withdrawn from veterinary use and no antibiotic of comparable efficacy is available. Vaccination
Vaccines are the best tool available, but they have limitations. They may not protect adequately against all genotypes of a serovar such as hardjo. Almost all the hardjo products available on the world market are produced using genotype Hardjoprajitno strains, whereas the predominant strain found in cattle is genotype Hardjobovis. Major doubts have been cast on the efficacy of some leptospiral vaccines.s, 6, 8 It always has been recognized that immunity to infection may not be as good as immunity to clinical disease. Vaccines alone will not eradicate infection from an endemically infected herd; they will not stop excretion in the animal that already is leptospiruric; and they will not always prevent abortion in animals in which placental localization has already occurred. National Animal Disease Centers, 6, 8 work suggested that the products tested by them did not even give clinical protection.
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTIVE FAILURE
475
Identification of Infected Animals
This is an important tool in other disease control programs, either as a means of preventing introduction of infection to a herd or to remove carrier animals already in a herd. No worthwhile methods are available. Management
Management has an important part to play in the control of risk factors. By suitable planning of land and buildings, access to other animals and contaminated water supplies can be minimized. The management of added animals is important in herds that have a control program. New animals should be isolated, treated with streptomycin, and vaccinated before being added to the herd. Permutations of the Approaches Already Mentioned
Any control strategy must employ a mixture of the aforementioned methods. Management methods run across all the control strategy options.
Disease Outbreak
In the face of a reproductive wastage problem, it is essential to vaccinate and treat those animals still pregnant, then move into a control program as described for an endemic herd. Endemically Infected Herd
A control program ideally should start with treating all adult animals at a time when the cost of lost product (such as milk) can be minimized. That may mean deferring treatment until animals go dry. Vaccinate the whole herd (all females destined for breeding and all intact males) annually in the case of cattle or before to service in the case of sheep. When doubts exist as to the efficacy of the vaccine reduce cattle vaccination intervals to 6 months or less, and when infertility is a feature, vaccination also initially should take place at intervals of 6 month or less. Farmers should be warned that they should stay with a control program once it is started. Stopping vaccination to save money after a few years will leave them with a totally susceptible herd and potentially worse off than if they had never started the program. Current leptospiral vaccines are not licenced for use in sheep, but experience has shown that they can be used at a quarter of the cattle dosage.
476
ELLIS
References 1. Adinarayanan N, James PC: Studies on leptospirosis in farm stock and wildlife in Kerala. Indian J Anim Health 13:93, 1980 2. Blackmore DK, Bahaman AR, Marshall RB: The epidemiological interpretation of serological responses to leptospiral serovars in sheep. NZ Vet J 30:38, 1982 3. Bjorkman N, Sollen P: Morphology of the bovine placenta at normal delivery. Acta Vet Scand 1:347, 1960 4. Bolin CA: Oral presentation to the 36th Annual Meeting, American Leptospirosis Research Conference, Chicago, 1993 5. Bolin CA, Cassells JA, Zuemer RL, et al: Effect of vaccination with a monovalent Leptospira interrogans serovar hardjo type Hardjobovis vaccine on type Hardjobovis infection of cattle. Am J Vet Res 52:1639, 1991 6. Bolin CA, Thiermann AB, Handsaker A, et al: Effect of vaccination with a pentavalent leptospiral vaccine on Leptospira interrogans serovar hardjo type Hardjobovis infection in pregnant cattle. Am J Vet Res 50:161, 1989 7. Bolin CA, Zuemer RL, Trueba G: Comparison of three techniques to detect Leptospira interrogans serovar hardjo type Hardjobovis in bovine urine. Am J Vet Res 50:1001,1989 8. Bolin CA, Zuemer RL, Trueba G: Effect of vaccination with a pentavalent leptospiral vaccine containing serovar hardjo type Hardjobovis on type Hardjobovis infection in cattle. Am J Vet Res 50:2004, 1989 9. Cousins DW, Ellis TM, Parkinson J, et al: Evidence for sheep as a maintenance host for Leptospira interrogans serovar hardjo. Vet Rec 124:123, 1989 10. Damude DF, Jones CJ, Myers DM: A study of leptospirosis among animals in Barbados. Trans R Soc Trop Med Hyg 73:161, 1979 11. Dennis SM: Diagnosis of infectious abortion in cattle. Vet Med Small Anim Clin 64:423, 1969 12. Dikken H, Kmety E: Serological typing methods of leptospires. Methods in Microbiology 11 :259, 1978 13. Ellis WA: Recent developments in bovine leptospirosis. In Grunsell CSG, Hill FWG (eds): The Veterinary Annual, 23rd issue. Bristol, Scientechnica, 1983, pp 91-95 14. Ellis WA: Leptospirosis. In Martin WB, Aitkin ID (eds): Diseases of Sheep, ed 2. Oxford, Blackwell Scientific Publications, 1991, pp 78-80 15. Ellis WA, Michna SW: Bovine leptospirosis: Infection by the Hebdomadis serogroup and abortion-a herd study. Vet Rec 99:409, 1977 16. Ellis WA, Michna SW: Bovine leptospirosis: Experimental infection of pregnant heifers with a strain belonging to the Hebdomadis serogroup. Res Vet Sci 22:229, 1977 17. Ellis WA, Cassells JA, Doyle J: Genital leptospirosis in bulls. Vet Rec 118:333, 1986 18. Ellis WA, O'Brien H, Bryson DG, et al: Bovine leptospirosis: Some clinical features. Vet Rec 117:101, 1985 19. Ellis WA, O'Brien H, Neill S, et al: The isolation of a leptospire from an aborted bovine fetus. Vet Rec 99:458, 1976 20. Ellis WA, O'Brien H, Neill S, et al: The isolation of a strain of Leptospira serogroup icterohaemorrhagiae from an aborted bovine fetus. Br Vet J 133:108, 1977 21. Ellis WA, O'Brien H, Neill SD, et al: Bovine leptospirosis: Microbiological and serological findings in aborted fetuses. Vet Rec 110:147, 1982 22. Ellis WA, O'Brien H, Neill SD, et al: Bovine leptospirosis: Serological findings in aborting cows. Vet Rec 110:178, 1982 23. Ellis WA, Montgomery J, Cassells JA: Dihydrostreptomycin treatment of bovine carriers of Leptospira interrogans serovar hardjo. Res Vet Sci 39:292, 1985 24. Ellis WA, Thiermann AB, Montgomery J, et al: Restriction endonuclease analysis of Leptospira interrogans serovar hardjo isolates from cattle. Res Vet Sci 44:375, 1988 25. Fennestad KL, Borg-Petersen C: Foetal leptospirosis and abortion in cattle. J Infect Dis 102:227, 1958 26. Flint SH, Comer RJ, Marshall RB: Leptospirosis in farmed goats. N Z Vet J 36:156, 1988 27. Hajtos I, Malik G, Banfalvi E, et al: Abortion in sheep caused by Leptospirae in Hungary. Magyar Allat Lapja 38:721, 1983 28. Hanson LE: Control of bovine leptospirosis. Bovine Pract 7:17, 1972
LEPTOSPIROSIS AS A CAUSE OF REPRODUCTIVE FAILURE
477
29. Hanson LE, Brodie BO: Leptospira hardjo infections in cattle. In Proceedings of the 71st Annual Meeting of the United States. Livestock Sanitary Association, Phoenix, AZ, 1967, P 210 30. Hanson LE, Tripathy DN, Killinger AH: Current status of leptospirosis immunization in swine and cattle. J Am Vet Med Assoc 161:1235, 1972 31. Hathaway SC: Leptospirosis in New Zealand: An ecological view. NZ Vet J 29:109, 1981 32. Hathaway SC, Little TWA: Epidemiological study of Leptospira hardjo infection in second calf dairy cows. Vet Rec 112:215, 1983 33. Heath S: Oral presentation to the 36th Annual Meeting, American Leptospirosis Research Conference, Chicago, 1993 34. Hermann JL, Baril C, Bellenger E, et al: Conservation of the genome among isolates of Leptospira interrogans. J BacterioI173:7582, 1991 35. Hermann JL, Bellenger E, Perolat P, et al: Pulse-field gel electrophoresis of Non digests of leptospiral DNA: A new rapid method of serovar identification. J Clin Microbiol 30:1696, 1992 36. Kirkbride CA, Johnson MW: Serological examination of aborted ovine and bovine fetal fluids for the diagnosis of border disease, bluetongue, bovine viral diarrhea, and leptospiral infections. J Vet Diagn Invest 1:132, 1989 37. Kmety E, Dikken H: Revised list of Leptospira serovars. Groningen, University Press, 1988 38. Knott SG, Dadswell LP: An outbreak of bovine abortions associated with leptospirosis. Aust Vet J 46:385, 1970 39. LeFebvre RB: DNA probe for detection of the Leptospira interrogans serovar hardjo genotype Hardjobovis. J Clin MicrobioI25:2094, 1987 40. Leon - Vizcaino L, Hermoso de Mendoza M, Garrido F: Incidence of abortions caused by leptospirosis in sheep and goats in Spain. Comp Immun Microbiol Infect Dis 10:147, 1987 41. Leonard FC, Quinn pJ, Ellis WA, et al: Association between cessation of leptospiruria in cattle and urinary antibody levels. Res Vet Sci 55:195, 1993 42. Lingard ER, Hanson LE: Effect of Leptospira pomona on the reproductive efficiency of cattle. J Am Vet Med Assoc 139:449, 1961 43. Malkin K: Enhancement of Leptospira hardjo agglutination titers in sheep and goat serum by heat inactivation. Can J Comp Med 48:208, 1984 44. McKeown JD, Ellis WA: Leptospira hardjo agalactia in sheep. Vet Rec 118:482, 1986 45. Marshall RB, Wilton BE, Robinson AJ: Identification of Leptospira serovars by restriction endonuclease analysis. J Med MicrobioI14:163, 1981 46. Miller BD, Chappel RJ, Adler B: Detection of leptospires in biological fluids using DNA hybridization. Vet MicrobioI15:71, 1987 47. Motie A, Meyers DM: Leptospirosis in sheep and goats in Guyana. Trop Anim Health Prod 18:113, 1986 48. Murphy JC, Jensen R: Experimental pathogenesis of leptopiral abortion in cattle. Am J Vet Res 30:703, 1969 49. Murray RD, Dhaliwal GS, Downham DY, et al: Effects of leptospirosis on fertility of dairy cattle. Cattle Practice 1:99-105, 1993 50. Nigam SK, Shukla V, Ranat R: Abortion in goats probably due to Leptospira infection in a sheep farm in Madhya Pradesh. Indian Journal of Animal Health 13:93, 1974 51. Pearson JKL, Mackie DP, Ellis WA: Milk drop syndrome resulting from Leptospira hardjo. Vet Rec 106:135, 1980 52. Perolat P, Grimont F, Regnault B, et al: rRNA gene restriction patterns of Leptospira: A molecular typing system. Res MicrobioI141:159, 1990 53. Perolat P, Lecuyer I, Postic D, et al: Diversity of ribosomal DNA fingerprints of Leptospira serovar provides a database for subtyping and species assignation. Res Microbiol 144:5, 1993 54. Quinlan JF, Nicholl VJ: Agalactia and infertility due to Leptospira interrogans serovar hardjo infection in a vaccinated dairy herd. Irish Vet J 46:97, 1993 55. Rako A: Leptospirosis in sheep. Bul Shk Zootek Vet 4:71, 1986 56. Ramadass P, Jarvis BDW, Comer RJ: Genetic characterisation of pathogenic Leptospira species by DNA hybridization. Int J Syst Bact 42:215, 1992 57. Schollum LM, Blackmore DK: The serological and cultural prevalence of leptospirosis in a sample of feral goats. NZ Vet J 29:104, 1981
478
ELLIS
58. Segers RPAM, Van Gestel JA, Van Eys GJJM, et al: Presence of putative spingomyelinase genes among members of the family Leptospiraceae. Infect Immun 60:1707, 1992 59. Terpstra WJ, Schoone GJ, Schegget JT: Detection of leptospiral DNA by nucleic acid hybridization with 32-P and biotin-labelled probes. J Med Microbio122:23, 1986 60. Terpstra WJ, Schoone GJ, Lighart GS, et al: Detection of Leptospira interrogans in clinical specimens by in situ hybridization using biotin-labelled DNA probes. J Gen Microbiol 133:911, 1987 61. Thiermann AB: Experimental leptospiral infections in pregnant cattle with organisms of the Hebdomadis serogroup. Am J Vet Res 43:780, 1982 62. Thierman AB, LeFebvre RB: Restriction endonuclease analysis and other molecular techniques in the identification of Leptospira and other pathogens of veterinary importance. In Swaminathan B, Prakash G (eds): Nucleic Acid and Monoclonal Antibody Probes. Applications in Diagnostic Microbiology. New York, Marcel Dekker, 1989, pp 145-183 63. Thiermann AB, Handsaker AL, Foley JW, et al: Reclassification of North American isolates belonging to serogroups Mini and Sejroe by restriction endonuclease analysis. Am J Vet Res 47:61, 1986 64. Tripathy DN, Hanson LE, Bedoya M, et al: Experimental infection of pregnant and lactating goats with Leptospira interrogans serovars hardjo and szwajizak. Am J Vet Res 46:2515, 1985 65. Van der Hoeden J: Leptospirosis among goats in Israel. J Comp Patho163:101, 1953 66. Van Eys GJJM, Gerritsen MJ, Korver H, et al: Characterization of serovars of the genus Leptospira with hardjobovis and icteroheamorrhagiae recombinant probes with special attention to serogroup Sejroe. J Clin Microbio129:1042, 1991 67. Van Eys GJJM, Gravekamp C, Gerritsen MJ, et al: Detection of leptospirosis in urine by polymerase chain reaction. J Clin Microbiol 27:2258, 1989 68. Yasuda PH, Steigerwalt AG, Sulzer KR, et al: Deoxyribonucleic acid relatedness between serogroups and serovars in the family Leptospiraceae with proposals for seven new Leptospira species. Int J Syst Bacterio137:407, 1987 69. Zuerner RL, Bolin CA: Repetitive sequence element cloned from Leptospira interrogans serovar hardjo type Hardjobovis provides a sensitive diagnostic probe for bovine leptospirosis. J Clin Microbio126:2495, 1988 70. Zuerner RL, Bolin CA: Nucleic acid probe characterises Leptospira interrogans serovars by restriction fragment length polymorphisms. Vet Microbio124:355, 1990
Address reprint requests to W.A. Ellis, PhD, BVMS, FRCVS Veterinary Sciences Division Department of Agriculture Stormont, Belfast Northern Ireland BT4 35D