A
review on Leptospirosis
G Abdollahpour; DVM, PhD.
WHO/LERG Advisor
University of Tehran
Postal address: G. Abdollahpour, Leptospira Research Laboratory, University of
Tehran,
P. O.
Box: 14155-6453
Tehran, Iran.
Fax: 0098 261
6204023 and 0098
21 66933222
Email:
greza@ut.ac.ir, drgabdollahpour@gmail.com
April 2011
Table
of Contents
1. HISTORY
2. EPIDEMIOLOGY
....................... A. Source of infection
........... B. Modes of transmission
3. HUMAN
LEPTOSPIROSIS
4. CLASSIFICATION
5. PATHOGENESIS
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
9. DIAGNOSIS
10. TREATMENT
11. ECONOMIC
IMPORTANCE OF LEPTOSPIROSIS
12. MANAGEMENT AND
CONTROL
........... A. Eradication
........... B. Vaccination
13. References:
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 L. 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 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 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, 2001). 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, Kaykhosravi
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 cattle 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 and L.
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 under
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 examined in
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,
and one
The results of Swart et al.
(1983) in
In
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.
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).
7. CLINICAL LABORATORY DIAGNOSIS
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).
D. Restriction Endonuclease Analysis (REA)
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.
F. Polymerase chain reaction (PCR)
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.
11. ECONOMIC IMPORTANCE OF LEPTOSPIROSIS
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
In
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
between L.
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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