CHAPTER 33 LEPTOSPIRA INFECTIONS

RICHARD BIRTLES

School of Environment and Life Sciences, University of Salford, Greater Manchester, UK

Leptospirosis (also known as Weil’s disease, canicola fever, canefield fever, mud fever, nanukayami fever, rat catcher’s yellows, Fort Bragg fever and pretibial fever) is a disease of public health and veterinary importance that is encoun­tered throughout the world.

AETIOLOGY

Leptospira species are Gram-negative bacteria that exploit a wide range of mammals as reservoir hosts. Bacteria are shed from infected mammals in urine, and susceptible hosts usually acquire infection through contact with con­taminated watercourses. In reservoir hosts, infections are usually chronic and asymptomatic, with bacteria typically colonizing the renal tubules. Pathology can result from these chronic infections, but acute disease is more common in non- reservoir hosts; leptospirosis is one of the most common zoonoses on the planet and is recognized as a significant threat to livestock worldwide.

Members of the genus Leptospira are Gram-negative, spirochete bacteria. Before 1989, the genus comprised of

only two species: Leptospira interrogans, which embraced all disease-associated leptospires, and Leptospira biflexa, which contained saprophytic, environmental strains. Lept­ospira interrogans was subdivided into a large number of antigenically distinct serovars, many of which were identi­fied as being associated with particular mammalian reser­voir hosts, and therefore had ecological relevance. In 1989, taxonomic re-evaluation led to restructuring of the genus and the recognition of more species within it.

Infectious Diseases of Wild Mammals and Birds in Europe, First Edition. Edited by Dolores Gavier-Widen, J. Paul Duff, and Anna Meredith. © 2012 Blackwell Publishing Ltd. Published 2012 by Blackwell Publishing Ltd.

Leptospira species possess a characteristic long, fine, helical-shaped appearance. They are motile by means of flagellae that lie within the periplasmic space of the bacte­rium rather than extending from its outer membrane. The outer membrane contains lipopolysaccharide, and it is the structural heterogeneity in the carbohydrate component of this molecule that underlies leptospiral serovar diversity.

EPIDEMIOLOGY

Leptospira species are encountered more or less throughout the world.

Lagomorphs are also carriers of leptospires, with infec­tions reported in both rabbits ( Oryctolagus cuniculus) and hares (Lepus spp.). Evidence for the infections in mustelids has been published; leptospires, or anti- leptospiral anti­bodies, have been detected in various mustelid species, including European and American mink (Mustela lutreola and Mustela vison), western polecats (Mustela putorius), pine martins (Martes martes), stone martins (Martes foina) and badgers (Meles meles). Infections have also been recorded in other carnivores, including genets (Genetta genetta), mongooses (Herpestes ichneumon), red and arctic foxes (Vulpes vulpes and Alopex lagopus), wolves (Canis lupus), lynx (Lynx lynx) and brown bears (Ursus arctos). Leptospires have been detected in raccoons (Procyon lotor) in their native North America.

Leptospiral infections have been reported in both wild­living and domesticated ungulates; indeed, in the latter group of animals, leptospirosis is a well-recognized disease of veterinary and economic significance. Among wild­living ungulates, evidence for infection has been reported for wild boar (Sus scrofa), various deer species (including Cervus elaphus, Dama dama, Capreolus capreolus), mouflon (Ovis orientalis) and bison (Bison bonasus). Serological evidence of infections in moose (Alces alces) in Canada has been reported.

Marine mammals are also susceptible to leptospirosis. A catastrophic outbreak of acute disease in captive common seals (Phoca vitulina) was reported in 2006 in a zoo in the Netherlands, where, interestingly, the seals shared the same water system as a colony of coypu. Disease in captive and rehabilitated seals has also been reported in the USA, as have infections in wild-living pinnipeds.

As outlined above, it is now recognized that different Leptospira species and serovars have evolved to exploit dif­ferent mammals as reservoir hosts. For example, L. borg- petersenii serovar Hardjo and L. interrogans serovar Hardjo are most frequently associated with cattle, L. interrogans serovar Canicola with dogs, L. interrogans serovar Pomona with pigs, L. interrogans serovars Icterohaemorrhagiae and Copenhageni with rats, and L. interrogans serovar Bratis­lava with hedgehogs. The extent to which leptospiral species or serovars have adapted to specific hosts, and the mechanism that underlies such adaptation, are uncertain. For example, although rats are widely considered to be one of the most important reservoir hosts for zoonotic lept­ospiral strains, it remains uncertain as to whether this is primarily a reflection of the specific host adaptation of, say, L. interrogans serovar Icterohaemorrhagiae to rats or the result of other epidemiological factors, such as rat demo­graphics, population dynamics and/or habitat preferences (specifically their proximity to humans). Often, in envi­ronments where leptospires are thought to be maintained by rats, other mammals tend to harbour the same serovar; however, the relevant contribution of these mammals as hosts in leptospiral enzootic cycles is yet to be quantified. Thus, they may be acting merely as incidental hosts, or may be fulfilling a more important role in the natural maintenance of the bacteria.

The ecology of leptospires is not fully understood, but the current general paradigm is that they exploit mammal species as reservoir hosts by establishing chronic infections in the renal tubules of the kidneys (often referred to as the ‘carrier phase’) that can persist for months or longer.

Leptospiral infection does not necessarily provoke disease. Indeed, it is likely that, for those mammal species that serve as reservoir hosts, chronic, sub-clinical infection is the norm. However, in accidental hosts, or immunocompromised individuals within a reservoir host population, infection can result in systemic, potentially life-threatening disease.

PATHOGENESIS, PATHOLOGY

AND IMMUNITY

Acquisition of leptospiral infection occurs via mucosal surfaces and via cuts or trauma to the skin. The number of leptospires required to produce an infectious dose in nature is unknown, but estimates have been derived from experimental infections. These studies have shown that infectious dose is very much dependent on host, Leptospira species and serovar. For example, inoculation of hamsters with 10⁸ of L. interrogans serovar Icterohaemorrhagiae resulted in 50% mortality, whereas inoculation of the same number of L. interrogans serovar Copenhageni resulted in the death of all animals. In the absence of specific antibod­ies, leptospires are able to multiply rapidly on entering the vasculature, from where they disseminate and further rep­licate in numerous sites around the body. In immunocom­petent hosts, this systemic infection eventually provokes an antibody-mediated response that controls and clears the infection from most of the body, although bacteria colo­nizing the renal tubules often persist, perhaps as a result of somewhat immune-privileged nature of this site, and a chronic infection is established. In the absence of an effec­tive humoral response, systemic infection can be aug­mented, resulting in profound disease. The pathology of systemic leptospirosis is determined by a combination of direct effects of leptospires, and leptospiral products on cells and tissues, and damage resulting from immune response to infection. For example, leptospiral disruption of blood-vessel function can provoke ischaemia and thus precipitate damage to the parenchyma of various organs, and leptospires also produce toxins that may also have direct detrimental effects on infected tissues. However, in keeping with other Gram-negative bacteria, leptospiral lipopolysaccharide (endotoxin) is a potent activator of the innate immune system, and hence a potentially dam­aging inflammatory response. However, the progression of disease is not solely dependent on host susceptibility; dif­ferent serovars tend to be associated with different presen­tations. For example, in dogs, serovars Canicola and Bratislava are more often associated with renal dysfunc­tion, whereas serovars Icterohaemorrhagiae and Pomona provoke more hepatic disease. Although clinical disease is most frequently associated with acute-phase leptospiral infections, chronic infection is not without consequences. Interstitial nephritis and other renal lesions associated with chronic leptospirosis have been documented in a range of reservoir hosts, and it is thought these manifestations may progress to fibrosis and subsequent renal failure. Several studies have explored the means by which leptospires persist in renal tubules. This stage is mediated primarily by interaction between leptospires and tubule epithelium, with bacteria closely associated with epithelial cells and the extracellular matrix. Indeed, although the extent of infec­tion markedly varies, from one or two tubules in some animals, to nearly all tubules in others, the intensity of infection is usually low, with most bacteria lying in contact with the epithelium, forming only a thin lining to the lumen.

Although a handful of reports of leptospire-associated pathology have emerged from North America in wildlife species whose range extends into Europe (for example, a survey of red foxes ( Vulpes vulpes) in Canada revealed that severe haemorrhagic nephritis and interstitial nephritis were common), no European reports of leptospire-induced pathology in wildlife have appeared in the international literature. Thus, veterinary knowledge of the pathologic consequences of leptospiral infection is primarily based on observations of disease in dogs and livestock. In canine leptospirosis, external gross signs may include congested or icteric mucosa, with petechial or ecchymotic haemor­rhaging. Internally, kidneys become enlarged and take on a pale, yellowish appearance. In severe cases, the renal capsule may adhere to the kidney surface and subcapsular haemorrhages are common. In less severe cases, white spot­ting may be seen in the renal cortex, particularly along the corticomedullary junction. In chronically infected, or recovered, individuals, the kidneys may be scarred or shrunken. With hepatic leptospirosis, the liver becomes enlarged, takes on a yellow colour and develops interlobu­lar markings. Histological examination of infected kidneys reveals some variation, thought to be attributed to the specific behaviour of different serovars and individual immune responses. However, in general, renal lesions are relatively insubstantial, consisting of limited tubular necrosis and interstitial oedema. Chronic renal infections are histologically characterized by diffuse interstitial inflammation, particularly around the corticomedullary junction. This inflammation is primarily made up of plasma cells with fewer lymphocytes and macrophages.

This pathology can progress to diffuse interstitial fibrosis with some multifocal lymphoplasmacytic inflammation in kidneys that have been long infected. Leptospires can be observed in tissues using silver staining or immunohisto­chemistry. In early-stage infections, bacteria appear, adher­ing to the luminal surface of renal tubule epithelial cells, but they are less apparent in chronic leptospirosis.

CLINICAL SIGNS AND TREATMENT

No clinical case reports of acute leptospirosis in European wildlife have been published in the international literature; thus veterinary knowledge of the spectrum of clinical pres­entations associated with leptospires is drawn from obser­vations of infected dogs or livestock. The clinical signs of canine leptospirosis depend on the age and immune status of the animal and the identity of the infecting strain. Common signs associated with acute canine leptospirosis include pyrexia (39.5—40°C), shivers and myalgia, then subsequently vomiting, dehydration and peripheral vascu­lar collapse, although numerous other non-specific mani­festations have also been reported. Mucous membranes become congested and petechial and ecchymotic haemor­rhages can appear. Progressive deterioration in renal func­tion is reflected in oliguria or even anuria. Icterus is another common sign in canine leptospirosis, and has been observed in foxes as well as domesticated dogs. Intestinal and pul­monary manifestations can also develop as disease devel­ops. As mentioned above, renal dysfunction is also a consequence of chronic leptospiral carriage. Leptospiral infections in livestock (primarily cattle and pigs) are pri­marily recognized in the form of reproductive disorders, which are manifestations of the chronic form of infection. However, acute leptospirosis, albeit usually self-limiting, is observed in young animals, presenting as fever (40.5— 41°C), anorexia, dyspnoea from pulmonary congestion, icterus, haemoglobinuria and haemolytic anaemia. In older animals, reproductive disorders take the form of abortion, which is thought to occur several weeks or months after initial infection and is more common in animals nearing term. Alternatively, infected mothers may yield stillbirths, premature births or weakened infected offspring. An abor­tion storm in a breeding herd is often the first indication of leptospirosis infection in cattle, because the mild initial signs often pass unnoticed. Infertility may also be a pres­entation in endemically infected herds, possibly as a con­sequence of localization of infection in the uterus and oviducts. In dairy herds, acute leptospirosis may result in widespread pyrexia and a sudden drop in milk production of up to 75%. The remaining milk can be thick, yellow and blood-tinged, with thick clots and a high somatic cell count. The udder is typically soft and flabby, which is unique for leptospirosis. Milk production can return to normal within 2 weeks, even in the absence of treatment, but cows may not recover to full production during that lactation cycle.

DIAGNOSIS AND TREATMENT

Diagnosis of leptospirosis relies on either demonstration of bacterial presence in infected tissues or quantification of specific antibodies. Demonstration of the presence of bacteria can be achieved using a variety of approaches, including direct microscopy, immunodetection, culture and methods based on the polymerase chain reaction (PCR). The simplest or most ‘low-tech’ means of confirm­ing a leptospiral infection is by darkfield microscopic examination of clinical specimens, most commonly urine. Darkfield microscopy is employed as leptospires are not easily stained using standard bacterial stains. Wet-mount preparations allow the motility and flexing of leptospires to be observed, thereby facilitating discrimination of bac­teria from thread-like debris in the sample. However, this approach is not particularly sensitive, and it has been suggested that an infection intensity of as many as 10⁵ organisms/ml is required to be sure of observing bacteria. Thus, a negative result using darkfield microscopy should not curtail the use of other diagnostic approaches. Lept­ospira can also be observed histologically using Giemsa or silver stains. Again, the sensitivity of this approach is con­sidered to be limited. Immunodetection, most commonly in the form of direct fluorescent antibody tests, is also used to detect leptospires in tissue imprints or body fluids. However, owing to the extreme serological diversity of leptospires, tests are usually serovar- or serogroup-specific. Other forms of immunodetection have also been devel­oped — in particular, urinary antigen enzyme-linked immunosorbent assays (ELISA). The value of these assays appears to be good in terms of their specificity and sensi­tivity relative to other diagnostic approaches, ease of use and cost. However, again, the specificity of these ELISAs is limited to particular serovars or serogroups. PCR-based methods are also widely employed and can be designed to detect all members of the genus or to be specific to Lept­ospira species. DNA extracts for use in PCR can be pre­pared from fresh, frozen or fixed tissue suspected of being infected, and a range of PCR formats, including nested and real-time assays (the latter allowing quantification of infection intensity) have been described. Sequencing of PCR products can be used to facilitate identification of infecting Leptospira species, but not serogroup or serovar. Isolation of bacteria using specific culture techniques is also possible and in ideal circumstances is the preferred means of diagnosis. However, leptospires are notoriously fastidious organisms requiring specific media and pro­longed incubation times. Isolation attempts are usually made on suspected infected internal tissues or, more com­monly, on urine. Multiple urine samples should be tested because of the intermittent nature of leptospiral shedding. The most common medium used for isolation attempts is Ellinghausen-McCullough-Johnson-Harris (EMJH) medium. Leptospiral cultures are incubated under aerobic conditions at about 30°C for prolonged periods, typically at least 12 weeks, and often timescales of 30 weeks are advised. The presence of leptospires in these cultures is monitored by periodic darkfield observations.

Serological tests are perhaps the most widely used means ofdiagnosing leptospiral infections. The microscopic agglu­tination test (MAT) is considered the ‘gold standard’ of serological tests, and relies on the formation of bacterial aggregates when a dilution series of serum is mixed with suspensions of an appropriate serogroup. Although this approach has stood the test of time, it is considered labour­intensive, time-consuming and difficult to perform and control. Furthermore, its ability to accurately delineate serovars within serogroups has been frequently questioned. Diagnosis of infection may be based on a single titre, although the magnitude of this is dependent on circum­stance. Demonstration of a four-fold rise in MAT titre is a reliable means of serologically confirming acute disease. Other serological test formats have also been developed, including a latex agglutination test and ELISA. For the diagnosis of infections caused by serovars associated with human and companion animal leptospirosis, numerous commercial serological test formats are also available, including easy-to-use dipstick-style tests. Assays based on slide agglutination or haemagglutination that incorporate broadly reactive antigens are useful, as they allow the detec­tion ofspecific antibodies in a range ofdifferent host species.

Acute leptospirosis can be effectively treated with anti­biotics, and, by inhibiting the multiplication of bacteria in the blood system, early antibiotic intervention can have a significant impact on the progression of disease, curtail­ing the more profound complications of infection. It is therefore recommended that antibiotics be prescribed immediately on suspicion of leptospirosis. Penicillin and its derivatives are considered the drugs of choice for treat­ment of acute disease, although other classes of antibiotics have also proven to be effective in this role. Antibiotic prescription may also be successful in elimination of chronic infections.

MANAGEMENT, CONTROL AND REGULATIONS

Control of leptospirosis in livestock and companion animals is primarily achieved through vaccination. Vac­cines are most often inactivated suspensions of one or more leptospiral serovars, determined by the animal species being vaccinated and geographic location. Although numerous experimental vaccines based on cellular extracts have been tested, commercial vaccines are, with few exceptions, whole cell products combined with suitable adjuvants. Although vaccination is effective against acute, clinical disease, it does not necessarily prevent chronic leptospiral carriage; thus the usefulness of vaccination in eradicating leptospirosis from, for example, a herd, flock, kennel or farm, is limited. Ideally, infected animals should be isolated. Control of leptospirosis caused by rodent- associated serovars can be addressed by reducing rodent numbers in the vicinity of livestock or companion animals. Furthermore, as leptospire transmission involves an aquatic, environmental stage, effective removal of shed urine and maintenance of animals in clean, dry conditions will reduce transmission rates.

The veterinary and zoonotic importance of leptospirosis is reflected in its inclusion, for a long period of time, as a ‘listed’ (formerly list B) disease with The World Organisa­tion for Animal Health (OIE).

PUBLIC HEALTH CONCERN

In humans, the early phase of disease is typically character­ized by a flu-like illness. Progression to systemic disease occurs in 5 to 15% of cases, with pulmonary haemorrhage, myocarditis and kidney and liver failure among the more common complications. The International Leptospirosis Society estimates that up to 500,000 cases of human lept­ospirosis occur annually worldwide. However, this number is probably an underestimate, as surveillance is poor in many areas where leptospirosis is likely to be most common. Most cases occur in the tropics, where ‘warmth and wetness’ favour the environmental persistence of lepto- spires. Exposure to leptospirosis is associated with contact with water, for example as a result of rice farming, but also during periods of heavy rainfall. Outbreaks of leptospirosis are very common among residents of tropical and sub­tropical urban (rodent-infested) slums during these periods when infrastructural shortcomings facilitate transmission of the disease. Human leptospirosis is also of public health significance in temperate climes, and is often acquired by recreational exposure occurring in water sports. There have been several recent high-profile outbreaks of leptospirosis associated with competitive outdoor events, such as tria- thlons or adventure races.

SIGNIFICANCE AND IMPLICATIONS FOR ANIMAL HEALTH

Canine leptospirosis is widely recognized across Europe and in many countries and has led to widespread vaccination of dogs against serovars Icterohaemorrhagiae and Canicola. Not all canine leptospirosis is caused by these serovars, and, increasingly, leptospires belonging to serovars Australis, Bratislava, Grippotyphosa or Sejrc^ are being encountered. Cats are also susceptible to leptospirosis, but the disease is considered to be a far less important threat to the wellbeing of cats than dogs. Numerous cases of equine leptospirosis have been reported, in a variety of clinical presentations, ranging from common renal and hepatic dysfunction to rarer forms such as uveitis and reproductive disorders. Serological evidence of leptospiral infection is common in horses, and the predominant serovars reported are Pomona, Bratislava, Icterohaemorrhagiae and Grippoty- phosa. Among European production animals, leptospirosis is recognized most frequently in pigs and cattle. In pigs it is primarily a cause of reproductive disease, with strains belonging to serovars Bratislava and Pomona being most commonly associated with disease. In cattle, strains belong­ing to serovar Hardjo are commonly encountered, with infections being associated with a sudden drop in milk production, abortions, birth of weak calves or infertility.

Given the frequency of clinical leptospirosis in livestock and companion animals, it is reasonable to assume that cases do occur in wildlife but simply go undiagnosed. Our lack of awareness of such cases is probably associated with the usually non-fatal nature of infections and difficul­ties associated with diagnosis. Improved diagnostic tests, including serology and PCR, may be useful for identifying endemic leptospiral infections in wild species and indicat­ing their prevalences with a view to understanding epide­miology and natural cycles of infection.

Further reading about leptospirosis is recommended in the references given below^(1-6).

REFERENCES

1. Greene, C.E., Sykes, J.E., Brown, C.A. & Hartmann, K. Leptospirosis. In: Greene, C.E. (ed.). Infectious Diseases of the Dog and Cat. St Louis, MO: WB Saunders; 2006; pp. 402—17.

2. Evangelista, K.V. & Coburn, J. Leptospira as an emerging pathogen: a

review of its biology, pathogenesis and host immune responses. Future Microbiology. 2010;5:1413-25.

3. Fennestad, K.L. & Borg-Petersen, C. Leptospirosis in Danish mammals.

Journal of Wildlife Diseaset. 1972;8:343-51.

4. Twigg, G.I. & Cox, PJ. The distribution of leptospires in the kidney

tubules of some British wild mammals. Journal of Wildlife Diseases. 1976;12:318-21.

5. Adler, B. & de la Pena Moctezuma, A. Leptospira and leptospirosis.

Veterinary Microbiology. 2010;140:287-96.

6. Levett, PN. Leptospirosis. Clinical Microbiology Reviewt. 2001;14:

296-326.