A review on Leptospirosis
G Abdollahpour; DVM, PhD.
University of Tehran
Postal address: G. Abdollahpour, Leptospira Research Laboratory, University of Tehran,
P. O. Box: 14155-6453 Tehran, Iran.
Fax: 0098 26 36204023 and 0098 21 66933222
Table of Contents
....................... A. Source of infection
........... B. Modes of transmission
3. HUMAN LEPTOSPIROSIS
6. CLINICAL FINDINGS
7. CLINICAL LABORATORY DIAGNOSIS
.......... A. Culture of the organism
...................... B. Dark-field Microscopy
.......... C. Serological tests
....................... D. Restriction Endonuclease Analysis (REA)
.......... E. DNA hybridisation
................ F. Polymerase chain reaction (PCR)
8. NECROPSY FINDINGS
11. ECONOMIC IMPORTANCE OF LEPTOSPIROSIS
12. MANAGEMENT AND CONTROL
........... A. Eradication
........... B. Vaccination
The earliest recognised accounts of leptospirosis were descriptions of severe illness with jaundice and renal involvement in man published by Landouzy (1883), Weil (1886), and Vasiliev (1888): these were clinically distinct from other icteric and nephritic illnesses, but the cause was not determined (Turner, 1966).
The first report of bovine leptospirosis was published in the former USSR in 1935 where Leptospira interrogans. Serovar Grippotyphosa was isolated from calves with acute infection (Amatredjo, Campbell, 1975). The first isolation of L. hardjo from cattle was made by Roth and Galton (1960) in America, later in Canada by Robertson et al. (1964). In Australia, the first isolation of L. hardjo was made by Sullivan and Stallman, (1969). Since then, further serovars were discovered throughout the world and now all pathogenic leptospires are classified into one species Leptospira interrogans containing 223 serovars arranged into 23 serogroup (Ramadass et al., 1992; Woodward and Redstone, 1994).
In I. R. of Iran, the earliest recognised report of leptospirosis is published by Rafyi and Magami in 1968. Since then the most prevalent leptospires reported in Iran includes Leptospira interrogans serovars Hardjo, Pomona, Grippotyphosa, Canicola and Icterohaemorrhagia (Abdollahpour, et al. 2005; Hooshmand Rad P; Magami GH 1979, Rafyi A; Magami GH 1968, Magami GH, et al, 1977, Haji Hajikolaei et al 2005, Abdollahpour, et al. 2009, Sakhaee et al 2010). In a more recent serological study In 2012, two new serovars of Leptospira interrogans serovars Australis and Tarassovi have been reported in Iran (Abdollahpour 2012).
In a serological study in cattle in Tehran province in 2001, it was shown that 46.8% samples belonging to 39 herds had a positive reaction against one or more serogroups. The most prevalent Leptospira serogroup was Canicola (59 samples) and then Ballum (37 samples), Grippotyphosa (36 samples). The less prevalent Leptospira serogroup was Hardjo (7 samples). None of the samples had seropositive reaction against serogroup Icterrohamoragia (Abdollahpour et al, 2004). Another serological study in Gilan province of Iran in 2004 in 205 bovine serum samples showed that 53 (25.8%) samples had a positive reaction against one or more serovars. The most prevalent Leptospira serovar was Canicola 24 (11.7%) samples, and the less prevalent Leptospira serovars were Icterohaemorrhagiae 1 (0.5%) sample and Hardjo 2 (1%) samples (Abdollapour et al, 2009). Another study was conducted on 95 horses in Tabriz area in Iran in order to seroprevalence of leptospiral infection in this species. The prevalence of leptospiral infection was 41.05% in horses. 42.68% of male horses and 30.77% of female horses were positive. There was significant difference between males and females (P < 0.05). There was no significant relationship between aging and the incidence of leptospiral infection and between breed of the horses. The highest number of reactors in horses (46.15%) was due to serovar Pomona, followed in descending order by Grippothyphosa (41.03%), Icterohaemorrhagiae (17.95%), Canicola (12.82%) and Hardjo (2.56%). These results confirm that the majority of leptospiral infections is asymptomatic and the presence of antibodies in the absence of infection indicates exposure to the organism in studied horses (Hassanpour et als, 2009). However, a serological study of leptospirosis in dairy farms in semidry area of Iran, showed that 10.6% of the studied animals had a positive reaction against one or more sarovars of leptospira (Abdollahpour, et al., 2010).
Leptospirosis occurs worldwide wherever
there is a risk of direct or indirect contact with the urine of infected
animals. Theoretically, any mammal is
capable of being infected by any serovar of Leptospira interrogans. However, only a few serovars are enzootic in
any particular region. Optimal
conditions for survival are a warm and wet environment, with neutral or
slightly alkaline water; however, leptospires are
known to survive acidity of Ph 5 to 6.2 for limited periods. They can survive in cold water, provided that
it does not freeze (Thiermann, 1984). Because of the importance of water as a means
of spreading infection, new cases are most likely to occur in wet seasons and low lying areas, especially when contamination and
susceptibility are high (Nervig et al.,
1978). A differential distribution has
been observed in the prevalence of seropositives in
Rodents are considered to be the
most important carrier hosts for most serovars of leptospira
(Magami et al., 1977), but serovars
Two serovars of Leptospira interrogans namely L. hardjo
Domestic animals in contact with reservoir rodents or the contaminated urine of infected animals may acquire the infection, which may then be maintained as an enzootic disease within the herd or flock. The rate of transmission between mammals, by indirect contact largely depends upon those environmental conditions that favour the survival of leptospires (Hookey, 1991). In general terms the disease is most common in areas or seasons when the climate is warm and humid, soils are alkaline and there is an abundance of surface water (Faine, 1994).
In 1990, Ramadass et al. proposed to remove the prefix hardjo from the strain name hardjobovis and call it L. borgpetersoni serovar hardjo strain Bovis because they did not find any genetic relatedness between strains hardjobovis and hardjoprajitno using DNA hybridisation.
The urine of infected animals or healthy carriers, which may contaminate soil, pasture, drinking water and feed is the main source of infection. In the case of leptospiral abortion, infection can be spread by the aborted foetus and uterine discharges (Michna, 1970; Pritchard et al., 1989; Faine, 1994). An infected foetus can carry the infection for up to 7 weeks after birth (Giles et al., 1983). The semen of an infected bull may carry leptospires and transmission from such a bull to heifers by coitus and artificial insemination has been observed (Thiermann, 1984). Field observations of herd outbreaks of L. hardjo infection have frequently implicated a bull as the source of introduced infection (Ellis et al., 1985b).
In cattle, excretion of leptospires in the urine may take up to 542 days (Thiermann, 1982). L.
hardjo is excreted from the genital tract of
aborting cows for as long as 8 days after abortion or calving and is detectable
in the oviducts and uterus for up to 90 days after experimental infection and
in naturally infected cows (Ellis et al., 1985a; Ellis et al.,
1986b). Ellis et al. (1986b)
concluded that the genital tract appeared to be as important a site of L. hardjo localisation as the urinary tract. Infection may also persist in the mammary
gland, as organisms have been isolated from milk of an experimentally infected
cow for as long as 91 days after infection (Thiermann,
1982). Leptospiral infection has been
known to persist for up to 142 days in the pregnant bovine uterus and for 96
days in the nonpregnant uterus (Thiermann,
1984). Experimental infection indicated
that sheep did not shed the organisms in significant numbers or for long enough
Leptospiral organisms enter into the body most often through cutaneous or mucosal abrasions. Oral transmission may occur when animals are feeding on contaminated pasture or feedstuffs, or drinking or standing in contaminated water. However, oral dosing is not a satisfactory method for experimental transmission as compared to injection and instillation into the nasal cavities, conjunctival sac and vagina (Amatredjo and Campbell, 1975).
The finding of L. hardjo in the genital tract, specially the vagina of
non-pregnant cattle, indicates that venereal transmission may also play a part
in the epidemiology of bovine leptospirosis (Ellis et al., 1986b). Transplacental transmission is not common but
neonatal infection, probably contracted in the uterus, has been recorded (Michna, 1970). It is
also commonly found in the genital tract of bulls and venereal spread of the
infection is thought to occur in cattle (Ellis et al., 1986b). Leptospires have
been isolated from bull semen for 18 to 38 days after infection, and
leptospiral antibodies have been detected in the semen of 54% of 71 bulls
Leptospirosis is a classical example of a disease that will require close
collaboration between human medical and veterinary medical communities, if
public health is to be maintained.
Veterinarians can best participate in this process by encouraging
farmers to prevent this disease (Songer and Thiermann, 1988).
The incidence of positive agglutination tests in humans in contact with
cattle is surprisingly low and clinical cases in man, in which the infection is
acquired from animals, are not common.
Human infection is most likely to occur by exposure to infected urine,
uterine contents or contaminated water (Chen et al., 1990). Dairy cattle farmers are recognised as the
group most at risk, particularly from L. hardjo
infections. This Leptospira serovar is
maintained within the herd and transmitted to people by direct contact with
contaminated urine during milking (Ellis, 1986a). Although leptospires
may be present in milk for a few days at the peak of fever in an acute case,
the bacteria do not survive for long in the milk and do not withstand pasteurisation. However, farm workers who actually milk cows
are highly susceptible to L. hardjo infection,
The results of Swart et al.
There is a case report of L. hardjo infection in a woman who suffered mild fever and malaise of 3 days' duration, followed in 10 days by higher fever, severe headaches and myalgia and joint pain. Since the patient continued to breast-feed her 4-month-old infant throughout the illness, 21 days after the initial appearance of other clinical signs, the infant became acutely ill and experienced fever, anorexia, irritability, and lethargy (Songer and Thiermann, 1988).
The genus Leptospira is represented by Gram-negative flexuous, helical organisms measuring 0.1 μm x 150-600 μm. Strains are obligate aerobes growing at optimum temperatures of 28-30°C in media containing vitamins, bovine serum albumin and rabbit serum. Some are pathogenic for animals and humans, while others are non-pathogenic and are present in soil, fresh water or marine environments (Hookey, 1991).
The classification of leptospires has been confusing and complicated. Two methods that can be used for classification of the genus Leptospira, include serology and molecular biology based methods. However, there is a general move in bacteriological taxonomy towards classification based on genetic relatedness. The work of Yasuda et al. (1987) is a major contribution on genetic taxonomy.
In 2002, the Committee on the Taxonomy of Leptospira of the International Union of Microbiological Societies approved the following nomenclature for serovars of Leptospira. Genus and species must of course be italicized, with the serovar name not italicized and with an upper case first letter. Members are requested to adhere to this official nomenclature and to insist upon it in all papers they review. For example: Leptospira interrogans serovar Icterohaemorrhagiae or L. borgpetersenii serovar Hardjo.
A. Serological taxonomy
The genus Leptospira belongs to the family Leptospiracea, of the order Spirochaetales, comprising two species, L. interrogans (pathogenic) and L. biflexa (non-pathogenic). Each species is divided into serogroups on the basis of serological cross-reactivity and these are subdivided into serovars. On the other hand, the term serovar is a description of a leptospira whose homologous rabbit antiserum agglutinates it, but not other strains of leptospires. Serovars that show 10% cross-agglutination are collected into a serogroup (Faine, 1994). According to this classification, Leptospira interrogans contains over 223 serovars arranged into 23 serogroup (Ramadass et al., 1992; Woodward and Redstone, 1994).
B. Genetic taxonomy
Classification and identification of leptospires using serology is a difficult and long procedure. Recently molecular biology techniques have been introduced into the field of leptospirosis. These techniques represent the first available to facilitate the study of the epidemiology of leptospirosis (Herrmann, 1993). Genetic taxonomy involves DNA/DNA hybridisation and the guanine-plus-cytosine mol percentages (G+C mol %) content of DNA. Yasuda et al. (1987) proposed a new classification of the genus Leptospira based on a DNA homology study on 46 pathogenic and nonpathogenic serovars. These authors proposed seven new genospecies named L. borgpetersoni, L. inadai, L. noguchii, L. santarosai, L. weilii, L. meyeri, and L. wolbachii. Ramadass et al. (1992) analysed 66 serovars of potentially pathogenic leptospires by DNA hybridisation, and obtained data in close agreement with that of Yasuda et al. (1987); L. Kirschneri was proposed as a new genospecies by these authors.
Woodward and Redstone, (1994) developed a polymerase chain reaction (PCR) combined with restriction fragment length polymorphism which can be used for differentiation of leptospira serovars. This PCR assay can amplify the flaB gene of 23 serovars of the genus Leptospira. The PCR products are then digested to completion with different restriction enzymes. Each enzyme gave different restriction fragments which can be used for classifying the leptospiral isolates. These authors suggested that the PCR combined with restriction fragment length polymorphism as a useful tool for rapid detection and preliminary differentiation of leptospires.
However, according to the Taxonomic Subcommittee on Leptospira (TSL) of the International Committee on Systematic Bacteriology (ICSB), the "official" Taxonomic scheme which is accepted for leptospires is based on serology (Faine, 1994).
Leptospires enter into the body of a susceptible host through mucous membrane or abraded skin. After 4 to 10 days, the host becomes bacteraemic, this period lasting from hours to 7 days, and may be characterised by pyrexia, leptospires in the milk and anorexia. However, clinical symptoms will depend on the susceptibility of the host, the serotype of leptospira and the virulence of the organisms (Thiermann, 1984).
After the number of the leptospires in the blood and tissues reach a critical level, lesions due to the action of leptospiral toxin and consequent symptoms appear (Faine, 1994). Capillary damage is common to all serotypes and during the septicaemic phase, petechial haemorrhages in mucosae are a common expression of this (Radostits et al., 1994). Immunity appears to be solely humoural with circulating antibodies opsonising leptospires, causing cessation of bacteraemia. With the appearance of circulating antibodies, leptospires localise and persist in a number of organs, especially in the proximal renal tubes and in the female genital tract. The duration of such localisation, and urinary shedding will depend on host-serovar adaptation. When leptospires are infecting a definite host, infection of these organs will be long-term, perhaps even for life. In the case of accidental host infections, colonisation is short-term and urinary shedding may not occur (Thiermann, 1984; Ellis, 1984).
In the case of L.
A recent study on physiopathology of leptospirosis (Younesibrahim et al., 1995) indicated that a glycolipoprotein (GLP) fraction which was extracted from Leptospira interrogans inhibited Na,K-ATPase activity in rabbit kidney epithelial and medulla cells, whereas it had no effect on other enzymes. These authors concluded that this GLP which is present in the diseased tissues, might cause cellular dysfunctions, in particular electrolytical disorders in infected animals.
In the field, leptospirosis manifests itself as a disease with different forms. There are acute and subacute forms, a so-called chronic or abortion form and an occult form in which there is no clinical illness. Which form of the disease occurs, depends largely on the species of the host. Variations between serotypes of L. interrogans in their pathogenicity also affects the nature of the signs which appear.
Leptospirosis in cattle may appear as acute, subacute or chronic forms. The number of animals clinically affected in a cattle herd depends on the host-serovar adaptation, and the susceptibility of the herd to the infecting serovar. In all animals the incubation period is from 3 to 7 days (Thiermann, 1984).
A. Acute leptospirosis
Acute leptospirosis in cattle is characterised by one or more symptoms of: fever, haemolytic anaemia, haemoglobinuria, hepatitis, jaundice, interstitial nephritis, meningitis, and agalactia. Ellis, (1986a) enumerated the following features for diagnosis of acute leptospirosis; a) sudden onset of agalactia in adult cattle, b) icterus in young animals; and c) meningitis. For diagnosis of chronic leptospirosis the following signs should be present; a) abortion or premature calving b) infertility c) periodic ophthalmia in horses. L. hardjo causes a milk drop syndrome associated with "flabby udder mastitis" in the absence of other symptoms. Agalactia has also been observed in L. hardjo infected sheep and is characterised by starving lambs in the first week of life (Ellis et al., 1986a).
B. Subacute leptospirosis
This form of the disease differs from the acute form only in degree, approximately the same signs being observed in a number of affected animals but not all of the signs necessarily being present in the one animal.
C. Chronic leptospirosis
Animals which have recovered from acute leptospirosis may develop a carrier condition in which leptospires grow and remain in the renal tubes for periods of days to years. These excretory animals are the central points of distribution of leptospires to other animals or people (Faine, 1994). However, L. hardjo can also persist in other organs, notably the genital tract of cattle (Ellis et al., 1986b). There is strong presumptive evidence that the chronic form of leptospirosis in horses causes periodic ophthalmia (Hathaway et al., 1981; Radostits et al., 1994).
In L. hardjo infection there is a sudden onset of fever, anorexia, immobility and agalactia. The milk is yellow to orange and may contain clots. The udder is flabby, has no heat or pain, and all four quarters are equally affected (Durfee and Allen, 1980; Tripathy et al., 1985a and 1985b). This may affect up to 50% of cows at one time, causing a precipitate fall in the herd's milk yield (Higgins et al., 1980). Abortion may occur several weeks later, but may also occur as the only evidence of the disease (Ellis et al., 1985c). In some areas or circumstances it is the principal clinical manifestation of leptospirosis due to L. hardjo, and the principal cause of abortion in cattle (Sullivan, 1970a; Sullivan, 1970b). It is also possible that many cows in a herd show subclinical infections with L. hardjo in which only a fall in milk yield may be detectable (Radostits et al., 1994).
Clinical symptoms of leptospirosis are not so highly specific that a conclusive diagnosis can be made without laboratory confirmation. Therefore, laboratory procedures are very important tools in the diagnosis of leptospirosis. Depending on the aims and needs, different techniques can be used to confirm the diagnosis. However, the faster and more reliable diagnostic methods would allow the clinician and the farmer to commence appropriate treatment and vaccination regimens with minimal delay.
The common techniques that are used for the diagnosis of leptospirosis include cultural isolation of the organism, serological tests, microscopic examination of urine, fluorescent staining, the hamster inoculation test, the growth inhibition test and molecular biology techniques. During the septicaemic stage, leptospires are present only in the blood. There is laboratory evidence of acute haemolytic anaemia and increased erythrocyte fragility and often haemoglobinuria. A leucopoenia has been observed in cattle while in other species there is a mild leucocytosis. However, the only positive diagnostic measure at this stage of the disease is culture of the blood. If abortion occurs, the kidney, lung and pleural fluid of the aborted foetus should also be examined for the presence of the organism. Serological testing at the time of abortion may be seriously inaccurate (Ellis et al., 1982a). In the stage immediately after the subsidence of the fever, antibodies begin to develop and the leptospires disappear from the blood and appear in the urine. The leptospiruria is accompanied by albuminuria of varying degrees and persists for varying lengths of time in different species (Radostits et al., 1994).
Definitive diagnosis of leptospirosis is usually provided by bacteriologic culture of the infecting organism (Bolin et al., 1989b). In addition, the organisms are needed for typing, as specific antigens in serological tests and for determination of pathogenicity (Thiermann, 1984). Leptospires are commonly isolated from urine or kidney of infected animals, but one should also consider other tissues as possible sources for isolation of the organism (Ellis et al., 1986d; Anderson et al., 1993).
The major advantage of bacteriologic culture is that leptospires of any serovar can be detected and subsequently can be identified. However, bacteriologic culture procedures are too expensive and too slow for routine use, because fresh samples are necessary and 4 to 6 months may be required for conclusive results (Bolin et al., 1989b). Ellis et al. (1986d) reported that even in a fresh foetus the positive identification of leptospirae in lesions is very difficult, especially with L. hardjo which is very fastidious in its cultural requirements. These authors also stated that genotype hardjoprajitno strains are much more difficult to isolate than are genotype hardjobovis strains.
Isolation of leptospires from the urine of vaccinated cows is usually unsuccessful. This difficulty may be because of the presence of antibodies or other substances in the urine of vaccinated cows which interfere with the growth of leptospires (Bolin et al., 1989b).
For selecting proper tissues or samples for isolation purposes, it is important to first determine the stage of the disease. In cases of acute disease, isolation should focused on blood samples. In the chronic form after the development of serological responses, isolation should be attempted from the urine. In the case of clinical disease or with aborted foetus, isolation should be attempted from kidney, liver and the aqueous humour (Thiermann, 1984; Faine, 1994).
Urine samples should be obtained from as many affected and non-affected (in-contact) animals as possible. For maximum efficiency, one-half of each urine sample should be submitted with added formalin (1 drop to 20-30 mL of urine) and the other half submitted in the fresh state. The formalin prevents bacterial overgrowth and the fresh urine sample may be used for culture. Culture of the organism from blood, urine and milk may be attempted by injection of fresh samples directly into hamsters or guinea-pigs on the farm or using special media (Radostits et al., 1994). The best laboratory animals for this purpose are 3 to 4 week old hamster. The animals should be injected via IP or SC with 0.5 Ml of the material and observed closely for signs of infection (Torten, 1979).
The quality of the medium is an important factor in isolating leptospires successfully. Media based on polysorbate-80 and bovine serum albumin (BSA), like EMJH (Johnson et al., 1973), T80-40LH medium (Ellis et al., 1985a) are the most useful for the isolation of leptospires from infected animals (Thiermann, 1984; Ellis, 1986a). Supplements have been recommended by many workers to increase the rate of isolation (Adler et al., 1986). The addition of fresh rabbit serum and 5-fluorouracil (Ris and Hamel, 1978; Ellis et al., 1982c; Oie et al., 1986), as well as the extensive dilution of the sample in 1% BSA solution are highly recommended (Thiermann et al., 1985). For isolation purpose these media are usually used in a semisolid form obtained by adding purified agar to a final concentration of 0.1 to 0.2 percent agar (Ellis, 1986a). Culture media are incubated at 28°C to 30°C and examined by dark-field microscope at least fortnightly for 3-6 months (Ellis, 1986a; Faine, 1994).
Microscopic examination of the centrifuged urine using dark-field illumination is considered to be a convenient and rapid diagnostic test. However, dark-field microscopic (DFM) examination is insensitive and requires a skilled observer to differentiate leptospires from artefacts (Bolin et al., 1989b).
The diagnosis of bovine leptospirosis is based primarily on serological tests because isolation of leptospires is difficult and time consuming (Thiermann and Garrett, 1983). It would appear that the bacterin induces an anamnestic response in cattle with prior natural exposure, and the resulting titres persist for such a length of time that they might be confused with titres induced by active infection (Stringfellow et al., 1983). However, serology is known to give only a limited information on the prevalence of leptospirosis because bovine leptospirosis often occur in the absence of detectable serologic titres (Mackintosh et al., 1980; Ellis et al., 1982a; Thiermann, 1983).
The common serological tests used are the enzyme-linked immunosorbent assay (ELISA) test and microscopic agglutination test (MAT), formerly known as the agglutination-lysis test.
a. Microscopic Agglutination Test (MAT)
The MAT which was originally described by Galton et al. (1965) and modified by Cole et al. (1973), is the most widely used serological test for leptospirosis (Thiermann and Garrett, 1983; Ellis, 1986a). The MAT is best used as a screening test when investigating the possibility of L. hardjo infection in groups or herds of cattle. At least 30 animals (or 10% of large groups) should be bled and animals of various ages should be included (Hathaway et al., 1986). The MAT is particularly useful in the diagnosis of disease caused by incidental, non-host-adapted serovars or acute disease caused by host-adapted serovars. However, because of the frequent low or possibly negative MAT titres in animals recently infected with L. hardjo, making a diagnosis on the basis of a serological result from one animal is extremely difficult (Elder et al., 1985). Ellis et al. (1982a) reported that there was no value in examining paired serum samples from individual cows after abortion because titres are either falling or static at the time of abortion.
For a diagnosis of leptospiral
abortion in cattle, a reciprocal titre of 3000 is proposed by Elder et al.
(1985) as the threshold for L.
Cross-reactions caused by exposure to leptospires of the same serogroup can occur, for example, infection by L. balcanica (Mackintosh et al., 1981) and L. medanensis can produce false positive L. hardjo reactions. The MAT has the disadvantages that it is tedious and time consuming (Cousins et al., 1985), and the use of live culture imposes a risk of human infection. Another disadvantage is the failure of the MAT to differentiate between titres after vaccination and those after natural infection, since the titres may be of similar magnitude (Adler et al., 1982; Hodges and Day, 1987).
b. One-point Microcapsule Agglutination Test; (MCAT)
The MCAT is claimed to be more reactive to the IgM antibody than MAT, and is superior in detecting antibody in the early stages of the disease (Arimitsu et al., 1987; Seki et al., 1987; Cui et al., (1991),.
c. Complement fixation test (CFT)
The CFT as described by Hodges et al. (1979) can be a useful test in detecting current infection. This test can be used to screen sera before testing them by the MAT, so it can eliminate the need for a lot of potentially unnecessary MAT. It does, however, have the disadvantages that it is even less sensitive than MAT and fails to detect low levels of antibody (Ellis et al., 1982a; Ellis, 1986a). False-positive results may occur with serum samples from patients with other infections, such as hepatitis A, cytomegalovirus, syphilis, or mycoplasma (Hookey, 1991).
d. Enzyme-Linked Immunosorbent Assay; (ELISA)
The ELISA test is potentially the most useful serological test (Ellis, 1986a). This test is highly sensitive, easy to perform and can measure IgM and IgG antibody levels in serum without prior fractionation, by using specific anti-IgM and anti-IgG enzyme conjugates (Adler et al., 1981; Cousins et al., 1985; Thiermann and Handsaker, 1985).
It has a number of advantages over the MAT. It uses a killed antigen; results can be read objectively rather than subjectively, and it can measure different immunoglobulin classes without prior fractionation of sera (Cousins et al., 1985). The ELISA test is much more accurate than the other serological tests and has many advantages from the point of view of laboratory practice (Thiermann and Garrett, 1983; Cho et al., 1989). Some difficulty is encountered in interpreting the significance of titres of antibody in serum. Although there is no marked difference between vaccinal and infection titres (Trubea et al., 1990), it is apparent from a heifer trial that infection titres generally persist much longer (Broughton et al., 1984).
In an experimental study, all 3 groups of animals responded to inoculation with live leptospires with a classical immunological response. IgM antibody levels increased in the first week, but became negative within 3-5 weeks in most animals. The IgG antibody was detected at about the same time as IgM, but presented for much longer. Because IgM is positive for only a short time, the IgM ELISA appears to be a suitable method of detecting recent exposure of leptospires in cattle (Cousins et al., 1985).
Because the ELISA technique can be readily scaled up to test many samples, it would be particularly useful for epidemiological studies. The high level of sensitivity, and specificity obtained for the ELISA-IgG, indicated that this test would be a good alternative to the MAT for detection of leptospirosis (Cousins et al., 1986).
In a comparative study, sera collected from calves experimentally inoculated with L. hardjo showed positive reaction to the MAT as early as 10 days after inoculation; these sera did not react positively in the ELISA until 25 days after the first inoculation. Field sera from 704 adult cattle on 90 farms were also examined by the MAT and the ELISA. The results showed that there was 90% correlation between the two tests. Sera from 86 calves inoculated with other serovars of Leptospira, and 227 field sera from adult cattle naturally infected with leptospirosis other than hardjo were examined by the ELISA. Less than 1% of these heterologous sera reacted with L. hardjo antigen in the ELISA. The results of field sera showed that 2.4% of the sera that reacted positive in the MAT were found negative in the ELISA. Positive MAT and negative ELISA results were also found in the acute phase of the experimentally infected calves. In contrast, 8.1% of the MAT-negative sera were positive in the ELISA, which was probably caused by non-agglutinating antibodies detected by the ELISA only. In this study the authors concluded that the ELISA is an advantageous alternative to the MAT for diagnosing leptospirosis (Bercovich et al., 1990). In a more recent comparative study, two serological techniques (MAT and ELISA), were used for detection of leptospiral antibodies in cattle. The results of this study showed that 2.25% of the sera that reacted positive in the MAT were negative by the ELISA, and 8.45% of the MAT negative sera were positive by the ELISA. Comparison between two techniques showed that there was a high correlation (0.61 < kappa= 0.649 < 0.8) between the ELISA and MAT for detection of leptospiral antibodies in cattle (Sakhaee et al, 2010).
A serological study of experimentally L. hardjo infection in calves conducted by Goddard et al. (1991) indicated that the rapid and high rise in IgM levels following challenge made the anti-IgM ELISA a potentially good indicator of recently established infection, although some transitory high levels were seen where infection did not become established. The low IgG response to infection made the anti-IgG ELISA of limited diagnostic use (Goddard et al., 1991).
e. Fluorescent antibody test (FAT)
The FAT is a very useful technique for demonstrating leptospires in tissues from animals (including foetuses) which have died of leptospirosis (Cook et al., 1972; Kirkbride and Halley, 1982). Studies of Smith et al. (1966) showed that the FAT was superior to culture and histopathological methods in demonstrating the presence of leptospires in autolysed materials. In another study, Smith et al. (1967) reported that in fresh material homogenates with live leptospires the supernatant fluid contained more organisms, whereas in autolysed materials with dead organisms, the sediment was more likely to be positive.
Fluorescent staining of antibody in urine or cultures is a fast and accurate diagnostic method for detecting the presence of leptospirae and for identifying serotypes (Hodges and Ekdahl, 1973). Antibodies also appear in urine and milk and their measurement may have some significance in special circumstances. This test may be used with fresh or frozen tissues and urine and aids in the discrimination of leptospires from artefacts (Bolin et al., 1989c).
Ellis et al. (1982c) enumerated the following features for using the FAT to demonstrate leptospires in aborted foetuses: using incident-light illumination with high magnification oil immersion objectives and low power eye pieces gave the best results. Examination of kidney and lung homogenates was very useful for demonstrating of the organism. The presence of foetal serum antibodies which might block fluorescent staining of leptospires in homogenate smears had minimum effect in cryostat sections.
The FA-positive, culture-negative cases are usually the result of the overgrowth of cultures by contaminating microorganisms (Ellis et al., 1982c). In a recent study conducted by Miller et al. (1991b), it was concluded that the FA test utilising multivalent conjugates could be used successfully as an additional method for the diagnosis of leptospirosis (Miller et al., 1989).
The traditional methods of
classifying genus Leptospira which are based on serology, are subjective
and time consuming, and have caused major problems in studying the epidemiology
of closely related strains found in different animal species and
populations. These difficulties have
been particularly obvious with those strains belonging to the Sejroe serogroup found in cattle and
In recent years, scientific developments have opened the way for new methods to be utilised in the classification of leptospires. REA has been employed successfully to type leptospires, including strains that are serologically indistinguishable (Gerritsen et al., 1991; Zuerner and Bolin, 1990; Silbreck and Davis, 1989).
The identification of leptospira interrogans serovars by REA was first described by Marshall et al. (1981). Subsequent studies have confirmed the usefulness of REA as a means of typing leptospira serovars and have revealed genotype differences between field isolates of some serovars and their corresponding reference strains which are not revealed by traditional cross absorption typing (Silbreck and Davies, 1989).
The REA technique has allowed differentiation between closely related serovars. In this technique, a large number of fragments are generated after restriction with an endonuclease enzyme such as EcoRI, HhaI, or HindIII (Herrmann et al., 1992; Thiermann et al., 1985). Djordjevic et al. (1993) reported that silver staining of polyacrylamide gel (PAG) gave enhanced resolution of REA fragments compared with the ethidium bromide staining of agarose gels.
Recently, pulsed-field gel electrophoresis (PFGE) has also been introduced for identification and epidemiological studies of leptospiral isolates (Herrmann et al., 1992). PFGE of large DNA fragments produced by rare-cutting restriction enzymes offers the advantage of a simple interpretation combined with a rapid result (Herrmann et al., 1991).
DNA hybridisation with genomic probes are widely used for rapid, specific and sensitive diagnosis of many infectious diseases. The use of such probes for diagnosis of leptospirosis has been put forward by different authors (Terpstra et al., 1987; Millar et al., 1987; Fach et al., 1991). Detection of leptospires in clinical samples using a DNA probe was first described by Terpstra et al. (1986). LeFebvre, (1987) described a DNA probe that could identify L. hardjobovis in cattle. Later on, Zuerner and Bolin, (1988) cloned a repetitive DNA sequence from L. hardjobovis, and found it to be a sensitive and specific probe for the diagnosis of L. hardjobovis in cattle.
In a comparative study, Bolin et al. (1989b) showed that DNA hybridisation was more sensitive than either FAT or culture techniques for the diagnosis of bovine leptospirosis.
The PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridise to opposite strands and flank the region of interest in the target DNA (Erlich, 1989). It has been used to diagnose infectious diseases caused by fastidious bacteria (Bej et al., 1991; Merien et al., 1992). It is a rapid, reliable and sensitive test for the diagnosis of leptospirosis (Van Eys et al., 1989; Woodward et al., 1991). The application of the PCR for the diagnosis of leptospirosis has been reported by different workers.
In 1989, Van Eys et al. developed a PCR assay that could detect less than 10 L. hardjobovis added into the urine samples. Woodward et al. (1991) also developed a PCR which was specific for L. hardjobovis. Gerritsen et al. (1991) described a protocol to prepare a bovine urine sample for PCR assay, which improved the sensitivity of the assay.
Merien et al. (1992) described a PCR assay which could detect all pathogenic and non-pathogenic leptospires in clinical samples. Gravekamp et al. (1993) also developed a PCR assay using two sets of primers derived from genomic DNA libraries of L. icterohaemorrhagiae and bim which could detect all pathogenic leptospires in serum samples.
In 1994, Woodward and Redstone developed a PCR to amplify the flaB gene of 23 serovars of the genus Leptospira. The PCR products were subjected to REA and the profiles correlated well with phylogenetic relationships between these serovars. These data suggest that the PCR combined with REA could be a useful tool for rapid detection and preliminary differentiation of leptospires.
In a comparative study (Merien et al., 1995), PCR was found to be more sensitive than either MAT or culture methods for the diagnosis of human leptospirosis. This study also showed that PCR can be an efficient tool for early diagnosis of the disease, especially when the clinical expression of the disease is confusing.
The main concern with PCR assay is the false-positive results which are caused by contamination with previously amplified DNA or target DNA. Chemical or biological reagents are important factors which may reduce the sensitivity of the PCR assay. There is a need to investigate and determine these inhibitors and eliminate the effects of these reagents on PCR assay.
In spite of the large volume of literature on post-mortem pathology of leptospirosis in cattle, no definite pathogonomic lesions have been described (Van der Hoeden, 1964; Alston and Broom, 1958; Murphy and Jensen, 1969; Amatredjo and Campbell, 1975). Descriptions of the pathology of classical leptospirosis were recorded in some publications, but classical cases are the exception to the rule. The commonest lesions are seen in the kidneys, where cortical cellular necrosis, petechiae and ecchymotic haemorrhages occur, especially in the glomeruli and the proximal convoluted tubules (Faine, 1994). However, the extent and severity of lesions depend on the infecting serovar, age of animal and the stage of the disease.
The main pathological signs which may be observed in the acute form are; anaemia, jaundice, haemoglobinuria and subserous and submucosal haemorrhages. There may be ulcers and haemorrhages in the abomasal mucosa in cattle, and if haemoglobinuria has been severe there may be associated pulmonary oedema and emphysema (Radostits et al., 1994).
As with all leptospiral infections, the disease and lesions in calves are much more severe than in adult cattle (Baskerville, 1986). Although there is little detailed histopathology in the literature, it appears that some of the lesions are common in several infecting serovars. The recent increase in awareness of the prevalence and significance of L. hardjo in reproductive disease is reflected in studies on pregnant cattle (Ellis et al., 1982a; Ellis et al., 1982b; Ellis et al., 1982c; Thiermann, 1982; Ellis et al., 1985b; Ellis et al., 1985c).
In animals recovered from the
acute form of the disease the characteristic finding is a progressive
interstitial nephritis manifested by small, white, raised areas in the renal
cortex. Many clinically normal cattle
presented to abattoirs have these lesions of interstitial nephritis. Aborted bovine foetuses are usually autolysed
to the point where no lesions or bacteria can be demonstrated (Radostits et al., 1994). However, there are no lesions which are
consistently found in aborted foetuses or their membranes. Placental oedema, placentitis
and nephritis occur inconsistently. The
placental lesions are more likely to have caused foetal death but they are not
unique to leptospirosis and may be masked by the decomposition which usually
affects aborted bovine placentas (Boulton, 1993). Liver lesions differ markedly in their
severity between calves and adult animals and are particularly a feature of L.
In sheep, the main pathological observations in the acute leptospirosis are a variable extent of jaundice, and widespread haemorrhage and anaemia, with bloodstained exudates and urine. Foetal deaths and congenital infection of survivors can occur (Hartley, 1952; Clark, 1994; Faine, 1994).
Positive diagnosis of leptospirosis can be made by demonstrating of leptospires organisms in clinical samples, isolation of the organism in the culture medium, serological responses of the host, and recently by demonstrating the bacterial DNA in clinical samples. Different diagnostic tests are discussed in this chapter.
Since leptospires are fastidious organisms, most practical diagnostic attempts concentrate on serological methods and recently on molecular biology techniques such as PCR. Nevertheless, isolation of the leptospires and histologic examination of tissues are of major importance (Thiermann, 1984).
Since the clinical symptoms of leptospirosis are extremely variable, pathogonomic lesions are not present in most cases, the diagnosis of leptospirosis is much easier on a herd basis than in a single animal. In an infected herd some animals are certain to have high titres and the chances of demonstrating or isolating the organism in urine or milk are increased with samples being taken from many animals; whereas in a single animal, depending on when the infection occurred, the titre may have declined to a low level and be difficult to interpret. This becomes particularly important for the clinician confronted with a diagnosis of abortion due to leptospirosis in which the infection may have occurred several weeks previously and the serum may be negative or the titres too low for an accurate interpretation. Examination of the urine may be useful in these cases. If possible wildlife or rodents which are known to inhabit the farm and use nearby water supplies should be captured and laboratory examinations of their tissues and blood carried out and the results compared with those obtained in the farm animals (Radostits et al., 1994).
When attempting to isolate leptospires from tissues including blood, kidney, brain or cerebrospinal fluid it should always be remembered that excess tissue in the test tube is harmful to the microorganisms and will usually result in isolation failure. It has been found that the best procedure for isolation of leptospires from tissue material is either squeezing the liquid from a small tissue sample into the test tube and removing the remaining solids or preparing serial dilutions from homogenised or minced tissue (Torten, 1979).
A. Differential clinical diagnosis
The acute and subacute forms of leptospirosis in cattle need to be differentiated from babesiosis, anaplasmosis, rape and kale poisoning, postparturient haemoglobinuria, bacillary haemoglobinuria, and the acute haemolytic anaemia which occurs in calves after drinking large quantities of water (Radostits et al., 1994). Leptospiral agalactia needs to be differentiated from mycoplasma bovis mastitis, tick-born fever, sudden water deprivation, acute infectious bovine rhinotracheitis (IBR) and cold cow syndrome (Ellis, 1986a).
Considerable effort was made in
the 1950s and early 1960s to investigate
optimal antimicrobial treatment for leptospirosis in animals and humans (
Recommended treatments for acute
leptospirosis in cattle are the use of intramuscular injections of
dihydrostreptomycin (12.5mg/kg, twice daily) or tetracycline (10-15mg/kg, twice
daily). In the case of chronic
leptospirosis due to L.
Recently, Gerritsen et al. (1993) and Gerritsen et al. (1994b) studied the effect of DHS in the treatment of experimentally and naturally L. hardjo infected cows. On both occasions, these authors reported that all infected cows were treated successfully with a single intramuscular injection of 25mg/kg body weight.
The economic importance of bovine leptospirosis includes direct or indirect costs of abortion, loss of milk production and related veterinary costs and human infection. There are also costs associated with vaccination and surveillance, including testing of serum samples before international transport, and in the case of human infection, medical treatment, loss of productive working time and capacity (Faine, 1994). The economic importance of L. hardjo abortion, mastitis and human infection has recently been recognised in many countries (Broughton et al., 1984). Although it is difficult to determine the actual rate of economic loss due to leptospirosis, there are some reports that show the importance of these losses.
Economic losses due to
leptospirosis can be caused by any of the acute, subacute or chronic forms of
the disease. It is impossible to assess
what the total cumulative losses are in an individual herd (Ellis, 1984). There is a report which indicated that in a 6
years study with a split herd, vaccination experiments with a L. hardjo bacterin, conducted in the dry tropics of
Control of bovine leptospirosis
is based on measures which minimise the risk of accidental infection of cattle
by leptospires maintained by other hosts, and
minimise spread of infection within a herd by vaccination or combined
vaccination and DHS therapy (Ellis, 1986b).
To minimise infection of cattle by leptospires
maintained by other species of animals, cattle should be separated from pigs
and sheep, and with fencing off cattle pasture from streams, ponds and marshes
to reduce contact with potentially contaminated water. It has been recommended that replacement
animals should be given a single dose of DHS and vaccinated immediately on
arrival on the farm (Ellis, 1986b; Bennet, 1991). Little et al. (1992a and 1992b)
reported that L. hardjo infection in a closed
herd of 800 beef cattle on the
Programmes to control bovine leptospirosis are designed to prevent reproductive losses, renal colonisation and urinary shedding. Within the herd, control can most economically be achieved by vaccinating all members of the herd. In closed herds vaccination should take place annually, while in open herds vaccination should be repeated every 6 months (Ellis, 1986b).
Although absolute eradication of
leptospirosis is a very difficult task, proper prevention and control methods
could greatly reduce the incidence of this disease in both man and domestic
animals (Torten, 1979). However, because of the development of
diagnostic methods, and pharmaceutical elimination of the carrier state, it is
now reasonable to attempt eradication of the disease from individual herds, and
possibly from areas. The principal
hazard in such a scheme is the introduction of carrier animals of any species,
or by reintroduction of the infection by rodents or other wildlife. In a serological investigation of 25 species
of wildlife in south-eastern
In cattle herds the eradication might seem simpler because of the easier identification of carriers. However, an eradication programme is rarely if ever adopted. Some consideration must be given to the bulls because if they are infected they should not be used naturally or for artificial insemination, even though the standard concentration of penicillin and streptomycin in the semen diluent is sufficient to ensure that no spread occurs (Radostits et al.,1994).
If eradication is attempted and completed, then introduced animals should be required to pass a serological test on two occasions at least 2 weeks apart before allowing them to enter the herd. Urine examination for leptospirae should be carried out if practicable (Radostits et al., 1994). Control of shipment of domestic animals across country and international is also an important key to the eradication of leptospirosis. It is strongly recommended that each country or region planing to import domestic animals demand certificates of leptospiral free herds with each shipment (Dobson, 1971)
Hygienic methods for prevention of direct and indirect human contact with animal urine have often been recommended (Torten, 1979). If the immediate source of infection is identifiable, in the form of yards, marshes and damp calf pens, every attempt must be made to avoid contact between animals and infected surroundings. Damp areas should be drained or fenced and pens disinfected after use by infected animals. The possibility that rats and other wild animals may act as a source of infection suggests that contact between them and farm animals should be controlled (Radostits et al., 1994).
An ideal leptospiral vaccine would prevent or minimise bacteraemia and consequently would prevent renal and foetal infection (Bolin et al., 1989c; Bolin, 1990). The majority of vaccines are formalin-inactivated bacterins which contain one or more serotypes (Thiermann, 1984; Marshall, 1991) and also aluminium hydroxide. Vaccines containing Freund's complete adjuvant stimulate much higher serological responses, but do not provide additional protection. The immune response provided by the bacterins is serotype-specific, and protection is dependent on the use of bacterins containing serotypes prevalent in the area. The bacterins stimulate the production of a low titre to the MAT which appears early and declines after several weeks, although protective immunity against the disease and renal infection has been demonstrated to last at least 12 months in cattle (Radostits et al., 1994). Regular serological testing in herds which are being vaccinated annually can be used successfully to monitor new infections, since these stimulate a titre to the MAT. Vaccination as part of a herd health programme should start with calves at 4-6 months of age (Schollum and marshall, 1985), followed by revaccination annually. These programmes should provide significant rises in calving rates, but have little or no effect on perinatal or postnatal losses.
There is no cross-immunity
Vaccination of animals less than 3 months of age is unlikely to be effective and is not recommended (Schollum and marshall, 1985), but vaccination of cows in late pregnancy gives effective immunity to their calves. Palit et al. (1991) findings indicated that the pre-vaccination titre determines the response to vaccination. The post-vaccination rise in titre is inversely proportional to the pre-vaccination titre. Vaccination in the presence of circulating leptospiral antibody derived from colostrum, retards the post-vaccination serological response. These results suggest that calves as young as 4 weeks of age may be effectively vaccinated against L. hardjo in the presence of circulating maternally-derived antibody. It is likely that calves from vaccinated dams would be protected against leptospirosis by maternal antibody for the first few weeks. It has been shown that relatively early vaccination with a potent L. hardjo vaccine is effective, and would reduce the chances of calfhood infection. A booster vaccination at 6-8 months of age should ensure a continued high level of protection (Palit and Middleton, 1990).
Vaccines for animal use should be potency-tested by challenge the vaccinated animals with virulent serovars isolated from the region where they are to be used. The best vaccines are those which include only locally known serovars proved to cause leptospirosis in either man or domestic animals (Torten, 1979).
If the disease is spreading rapidly, as evidenced by frequent appearance of clinical cases, and a high range of titres are seen in a number of animals, all clinical cases and sero-positive animals should be treated. Negative animals should be vaccinated, and moved on the first day of treatment to a clean field. Retesting a group to determine the rate of spread would be an informative procedure but active measures must usually commence before this information is available. During an outbreak of leptospiral abortion, vaccination and DHS (25 mg/kg) can administrated simultaneously, whereas in agalactia outbreaks vaccination alone is more cost-effective (Ellis, 1986b). In beef herds and dairy herds which calve seasonally, this means treatment of the entire herd. The objective of administration of streptomycin is to eliminate carriers of leptospira. However, the effectiveness of antibiotic in treatment and control of leptospirosis is quite controversial (Bernard et al., 1993; Gerritsen et al., 1994b). The results of study conducted by Ellis et al. (1985a) showed that DHS failed to removed established genital or renal infection of L. hardjo in cattle.
Vaccination of the animals already shedding leptospires in their urine had no significant effect in reducing the prevalence of leptospiruria in infected cattle. However, in endemic areas regular calfhood vaccination of all replacement cattle should feasibly reduce the incidence of the disease in dairy farmers (Thiermann, 1984).
One of the theoretical disadvantages of vaccination against leptospirosis is the possible development of renal carrier animals which are sufficiently immune to resist systemic invasion but not the colonisation of the kidney, leading to the development of a carrier animal showing transient leptospiruria. An experimental study conducted in cattle with hardjoprajitno vaccine indicated that a single genotype vaccine completely protected all 12 animals from renal infection by homologous and heterologous L. hardjo strains (Ellis and Zygraich, 1986).
In contrast to the reports which indicate the effectiveness of vaccination (Marshall et al., 1979; Allen et al., 1982; Hancock et al., 1984), recent experimental and field evidence in cattle vaccinated against L. hardjo has shown different results in preventing L. hardjo infection. In an experimental study, vaccination with pentavalent leptospiral vaccine containing type hardjoprajitno did not protect cows from infection with L. hardjobovis 6 months after the last of 2 vaccinations, and the cows became infected and developed renal and foetal infection (Bolin et al., 1989c).
In a comparative study conducted by Bobin et al. (1989a) vaccination of cattle with pentavalent leptospiral vaccines containing type hardjoprajitno, with one containing type hardjobovis, failed to protect steers from infection with hardjobovis 6 months after vaccination. Modification of the vaccination regimen to include 2 doses of vaccine 3 weeks apart also failed to protect steers. Although it was not protective, the hardjobovis vaccine was more antigenic than the hardjoprajitno vaccine, as measured by higher antibody titres in vaccinated steers. These authors also concluded that the presence of high concentrations of anti-hardjo IgG antibody was not protective. This differs from other reports, (Cousins et al., 1985; Elder and Ward, 1978; Goddard et al., 1991; Turner, 1966) which indicate that the presence of specific anti-leptospira IgG antibodies, as measured by hamster protection tests and in vitro growth inhibition, is correlated with protection (Bolin et al., 1989a).
Bolin et al. (1991) reported that, cattle vaccinated with monovalent vaccine containing L. hardjo genotype hardjobovis were not protected from challenge 2 months after completion of vaccination, despite the presence of high titres of anti-hardjo antibodies. Quinland and McNicholl, (1993) also reported L. hardjo infection in a 100-cow dairy herd 7 months after vaccination. This report indicated that 29% of the cows were shedding leptospires in the urine. These findings are very disturbing, since vaccination has became the backbone of leptospiral control programme. It is now clear that there is a need for further investigation into L. hardjo infection in cattle.
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