Leptospirosis

Robert J. CaUan

Leptospirosis is a complex disease of both animals and humans caused by pathogenic species of Leptospira.¹⁹ Pathogenic Leptospira species persist as chronic infections of the renal tubules of the maintenance host species, often causing little or no disease.

Leptospira is a diverse genus of motile, gram-negative, obligate aerobic, tightly coiled spirochetes approximately 0.1 to 0.3 μm in diameter and 6 to 20 μm in length.^1,2,5,8,9 The bacteria can survive in the environment for up to 6 months.^2,5,10 Leptospira spp. prefer a warm, moist environment with a pH of 7.2 to 8.0. Survival is short under dry conditions or at temperatures below 10° C.² Leptospira does not survive freezing in the environment.¹¹

Before 1989, Leptospira was divided into the pathogenic species L. interrogans and nonpathogenic saprophytic species Leptospira biflexa. The current taxonomy and classification of Leptospira use a complex system of both genetic and serologic characteristics.^1,5,9,12 The Leptospira species is determined by DNA sequence similarity.^1,5,9 Twenty-two genomospecies have been identified on the basis of DNA sequence heterogeneity (e.g., Leptospira borgpetersenii).^9,12 Further classification by serovar is based on antigenic grouping of the lipopolysac­charide (LPS) and other outer surface antigens using the cross-agglutinin adsorption test (CAAT).¹² More than 250 serovars are characterized for pathogenic strains of Leptospira (e.g., L.

Epidemiology

Leptospirosis is predominantly observed under conditions where livestock come in direct or indirect contact with urine from an infected maintenance host (Table 34.4).^1,2,5,10 The prevalence of infection within a maintenance host population tends to be high (30% to 50%); in such populations, infection is often spread between animals by direct contact.² Transmission to incidental hosts is generally by contact with the environment, feed, or water that is contaminated with urine from an infected maintenance host. Transmission can also occur from contact with an infected fetus or uterine discharge. Survival of the bacteria in the environment and the incidence of infection in animals are increased in warm, humid climates. The seasonal incidence is higher during the summer or fall in temperate regions and during the rainy season in warm-climate regions.⁵ Environmental conditions that contribute to moist surroundings and foot abrasions may contribute to Leptospira transmission, particularly in housed dairy cattle.

Cattle are the primary maintenance host reservoir of L. interrogans serovar hardjo type hardjopragitno and L. borgpeterse­nii serovar hardjo type hardjobovis. L. interrogans serovar hardjo type hardjopragitno is isolated primarily from cattle in the United Kingdom, and L. borgpetersenii serovar hardjo type hardjobovis is observed worldwide. Sheep and goats may also serve as maintenance hosts for L. borgpetersenii serovar hardjo

B..- OL J

15

type haidjoυovis.

■ TABLE 34.4

Current Nomenclature, Maintenance Host, and Incidental Hosts for Common Leptospira Isolates in Ruminants

Serovars hardjo, pomona, and grippotyphosa are most often implicated in renal infection of cattle.¹⁶ Although data are scarce, renal disease caused by leptospirosis in small ruminants appears to be uncommon.^11,17 Detection of Leptospira spp. from vaginal fluid and semen of sheep and goats has been reported and suggests the possibility of venereal transmission in those 13 18

species.¹³,¹⁸ Sheep may serve as subclinical carriers of serovars hardjo, hardjobovιs, and pomona.^3,13,19,20 Some serovars of Leptospira have zoonotic potential, and humans are always considered an incidental host.^1,2,4,5

An abattoir study of more than 5000 cattle in the United States identified approximately 2% renal carriers of L. inter­rogans.¹⁶ Serovar hardjo was the most common renal isolate, followed by serovars pomona and grippotyphosa.

In general, infection of cattle with the host-adapted serovar hardjo rarely results in acute, severe disease. If present, signs of disease are usually mild in acutely infected cattle and may simply present as cases of undifferentiated fever. Persistent, latent urogenital infection usually follows acute infection of host-adapted serovars in cattle, with most overt losses attribut­able to adverse effects on reproduction.^4,23-25

Acute, severe renal disease is more characteristic of incidental (also termed accidental) infection of cattle, particularly calves, with a non-host-adapted serovar of Leptospira. However, exceptions to this generalization do occur because host immunity and virulence of the organism are variable.^3,4

Clinical Findings

Serovar hardjo is host adapted to cattle, and many infections are asymptomatic or result in nonspecific reproductive failure or abortion. Leptospira serovar hardjo infection of cattle may produce chronic interstitial nephritis of variable severity, but overt renal dysfunction is rarely observed.²⁶ Chronic infection of the genital tract of cows and bulls is common.^7,27 Protracted shedding of the organism in the urine often occurs, possibly lasting for the life of the animal.^26,27 Infertility, stillbirth, abor­tion, and birth of weak calves are typical clinical manifestations of infection with serovar hardjo in cows.^7,24,28,29 The fetus can be infected in utero, and if it survives the acute infection, it may be born persistently infected.² Fever, agalactia, and mastitis may occasionally occur, and the resulting syndrome has been termed the milk-drop syndrome³ or flabby udder³ in lactating cows.

In contrast, infection with non-host-adapted serovars can result in severe systemic disease, hemolytic anemia, hepatitis, interstitial nephritis, and tubular nephrosis in calves and less often in adult cattle.^7,26 Meningitis is a rare manifestation.¹⁷ Agalactia and mastitis often occur in lactating cows, and pregnant cows may abort. Urine shedding of non-host-adapted serovars by infected cattle can persist for weeks to months.^4,7,29 Renal lesions result from direct damage to the vascular endo­thelium during leptospiremia, hypoxia from endothelial damage and hemolysis, tubular epithelial damage from hemoglobin, and interstitial nephritis.^2,5,26 Some Leptospira serovars, par­ticularly pomona, produce hemolysins that can cause acute intravascular hemolysis and anemia in cattle.

Clinical signs associated with acute infection may include fever, anorexia, lethargy, decreased milk production, petechia- tion, hemolytic anemia, icterus, and hemoglobinuria. Oliguria may be seen with interstitial nephritis or hemoglobinuric nephrosis. Elevated creatinine concentration caused by prerenal or renal causes may be observed on serum chemistry analysis. Examination of the urine may show proteinuria, pyuria, and cellular or granular casts in cases of nephritis. Hemoglobinemia and hemoglobinuria may be observed in patients with leptospire- induced hemolysis and can occasionally result in hemoglobinuric nephrosis (pigment nephropathy).

Pathophysiology

Contaminated feed and surface water, wildlife, rodents, and domestic animals are potential sources of pathogenic serovars for cattle.^2-5 Leptospires penetrate external mucosal surfaces and scarified or macerated skin. The bacteria multiply locally during an incubation period that can last 2 to 20 days.^2,5 After the incubation phase, the organism enters the bloodstream through the lymphatics or by direct penetration into the blood vessel.

During the convalescent phase, leptospires may become localized in the mammary gland, kidney, or genital tract, where they appear to be protected from the immune response.^2,30 Depending on the virulence of the serovar involved, chronic renal infection may create few histologic changes, mild inter­stitial nephritis, or diffuse, severe, lymphocytic interstitial nephritis with fibrosis.¹⁷ Nephritis may persist long after the host immune response has cleared the organism. Chronic infection of the kidney or reproductive tract allows for transmission of the organism in urine, uterine and vaginal secretions, placenta, fetal tissues, and semen.^3,'^,∙ Shedding in the urine may last for weeks to months with non-host-adapted infections. Renal shedding of host-adapted serovars can persist for months to years. The bacteria reside in the lumen of the renal proximal tubules, where they are protected from phago­cytes and humoral antibodies. The bacteria do not stimulate a systemic immune response while localized in the proximal tubule lumen, so serum antibody titers can decline and become negative even though the kidney is infected and urinary shedding of bacteria continues.

Multiple potential virulence factors may contribute to leptospirosis.^1,5,8,32 Virulence mechanisms include the toxin effect of lipopolysaccharide, adhesion to cells and extracellular matrix, bacterial motility, hemolysins, and iron sequestration. Leptospiral LPS and outer membrane proteins are believed to contribute to the development of interstitial nephritis. Motil­ity is an important pathogenic mechanism and contributes to invasion and dissemination of the bacteria. As many as 50 genes are related to leptospiral motility.¹ Adherence to cells is in part conferred by fibronectin-binding protein present on the surface of pathogenic strains but not on nonpathogenic strains. Leptospiral immunoglobulin-like protein A, B, and C (LigA, LigB, LigC) may also be involved in attachment and invasion. Additional proteins that may contribute to virulence include hemolysins, sphingomyelinase C, sphingomyelinase H, and hemolysis-associated protein 1 (HAP-1).^1,5,8,32 There appears to be significant redundancy in proteins associated with virulence mechanisms, making it difficult to isolate individual virulence factors using single gene knockout mutations.

Diagnosis

Diagnosis of Leptospira spp. infections is challenging due to the imperfect nature of currently available tests.³³ The micro­scopic agglutination test (MAT) is the most widely used serologic test for the diagnosis of leptospirosis in cattle. The MAT detects antibodies to specific serovars, but cross-reactivity occurs between related serovars, particularly within the same serogroup. Thus, an infection with one strain may result in increased MAT titer to multiple serovars. Serum antibody enzyme-linked immunosorbent assay (ELISA) tests have been developed for research but have not yet been adopted in the routine clinical diagnostic setting for animals.^34-37

An elevated serum antibody titer is observed after the bacteremic phase and is suggestive of Leptospira infection when associated with concurrent clinical signs, but interpretation of a single titer is problematic in vaccinated animals or when endemic exposure is suspected.^7,8,30 A fourfold increase in MAT titer between acute and convalescent serum samples, or conver­sion from a negative titer to a titer of 100 or greater, supports a diagnosis for both host-adapted and non-host-adapted serovars. However, vaccinated animals may have a diminished serologic response after challenge and renal colonization with L. borgpetersenii serovar hardjo. Serologic detection of persistent infection with serovar hardjo can be difficult because paired serum titers may be increasing, static, decreasing, or undetect­able at the time of clinical examination (e.g., at abortion).^7,30 Serology is unsuitable for screening individual animals for carrier status because many animals that are chronically infected and shedding may have negative serologic status.^39,40 Considering the serovar with the highest titer as being the infecting strain is generally recommended. However, serologic antibody titers may not accurately predict the infecting strain.^40,41

Leptospira shedding in urine and semen can be detected by multiple tests, including urine culture, phase-contrast microscopy, darkfield microscopy, fluorescent antibody (FA), PCR, nucleic acid hybridization, and immunoblot. ^{8,16,30,31,42 44} Urine cultures are often unrewarding because of the fastidious nature of the organism, and conclusive results may not be obtained for up to 6 months.^8,31 Darkfield microscopy, FA, and PCR assays are typically used by veterinary diagnostic labo­ratories to identify Leptospira species from urine samples. None of these tests will determine the infecting serovar. The sensitivity of detecting Leptospira shedding can be improved by performing two tests on a single sample.^31,44 Evaluation of serovar-specific antibody titers from positive animals may aid in identification of the infecting serovar.

Second-voiding urine samples collected after administration of IV furosemide are recommended for urine testing.^11,45 Furosemide is administered at 0.5 to 1.0 mg/kg IV or IM, and the first-voided urine is discarded. A second urine sample is then collected after cleaning the vulva of gross debris. Approxi­mately 10 mL of urine should be collected and stored on ice (not frozen) for transport to the diagnostic laboratory. Urine from 10 to 15 adult animals should be tested when evaluating a herd for endemic Leptospira serovar hardjo infection.

Biopsy or necropsy samples of renal tissue may be treated with Warthin-Starry or Levaditi silver stains for microscopic examination.¹⁷ Immunoperoxidase staining was shown to be more sensitive for identifying Leptospira species in kidney and liver of naturally infected cattle than Levaditi silver stain.⁴⁶

Treatment and Prognosis treatments has not been demonstrated in acute infections with other species and serovars of Leptospira, but their use could be considered relative to the lack of clinical data showing specific antimicrobial efficacy for treating acute leptospirosis in ruminants and swine.

Prevention

a more potent opsonin than IgG1.⁵⁴ A multivalent vaccine containing L. borgpetersenii serovar hardjo (Vira Shield 6+VL5HB, Novartis Animal Health, Larchwood, Iowa) has also been shown to prevent renal and reproductive system colonization and decrease shedding in cattle.⁵⁷

Because Leptospira species are largely extracellular patho­gens, the relevant protective immune response appears to be antibody-mediated neutralization and opsonization during leptospiremia.^1,5 There is no indication that cytotoxic immunity is a relevant immune response for protection or resolution of Leptospira infection. However, the exact immunologic mechanism responsible for protection from colonization and shedding of L. borgpetersenii serovar hardjo type hardjobovis in cattle is not fully understood. Although vaccines help prevent colonization and shedding of Leptospira serovar hardjo from the kidney, they are not effective in resolving current renal infection and elimination of the carrier state. Thus, vaccination at an early age followed by annual boosters is recommended to prevent initial renal colonization. Renal shedding in previously infected animals can be resolved by antibiotic treatment, and vaccination can be used to prevent future colonization. The efficacy of combined antibiotic and vaccination programs in controlling L. borgpetersenii serovar hardjo type hardjobovis infection and reproductive losses at the herd level is not fully established. Combination treatment with oxytetracycline and vaccination with a monovalent L. borgpetersenii serovar hardjo type hardjobovis vaccine (Spirovac, Pfizer Animal Health, New York, N.Y.) did result in decreased detection of leptospires in urine and kidney samples from dairy cattle.⁵⁸ However, one study in beef cattle and one in dairy cattle did not show improvement in herd reproductive performance following similar intervention.⁵⁹,⁶⁰