Preferred citation: Anipedia, www.anipedia.org: JAW Coetzer and P Oberem (Directors) In: Infectious Diseases of Livestock, JAW Coetzer, GR Thomson,
NJ Maclachlan and M-L Penrith (Editors). MH Moseley, K Allan and P Oberem, Leptospirosis, 2018.
Leptospirosis

Leptospirosis

Previous authors: P HUNTER

Current authors:
M H MOSELEY - Researcher, BVSc Mphil, University of Aberdeen, School of Biological Sciences, Aberdeen, United Kingdom, AB24 2TZ
K ALLAN - Veterinary Training Fellow, BSc (Hons) BVM&S PhD MRCVS, Graham Kerr Building, University of Glasgow, University Avenue, Glasgow, Lanarkshire, United Kingdom, G12 8QQ
P OBEREM - Self Employed, BVsc Hons, 11 Bruce Avenue Mont Lorraine, Gauteng, South Africa

Introduction

Leptospirosis in livestock is caused by spirochaetes of the genus Leptospira and is characterised by fever, renal and hepatic failure and reproductive failure.2 In equids, leptospirosis may manifest as recurrent uveitis associated with an autoimmune response.122 Infection in livestock also may result in chronic renal infection and urinary shedding that persists for months to years following initial infection. Contact with livestock is an important risk factor for human leptospirosis,2, 144 a widespread but neglected zoonotic disease that is estimated to affect more than one million people and result in around 59 000 deaths worldwide each year.35

Leptospirosis was first recognized in humans in 1886 by Weil,194 who described it as an infectious disease characterized by icterus. The responsible organism was simultaneously isolated and reported in 1916 by two independent groups of researchers,105, 184 and shortly afterwards rats were shown to be a host of infection.104 Leptospira spp. are difficult organisms to culture, which presents challenges for the diagnosis of infection and has limited research into human and animal disease. However, the advent of molecular typing and, more recently, whole genome sequencing, has improved our knowledge of Leptospira taxonomy, virulence and transmission.

Aetiology

Leptospira spp. bacteria belong to the family Leptospiraceae. Leptospira spp. are thin (0.15µm), right-handed helical, motile organisms that are 10-20µm long with distinctive hooked ends.152 They are Gram-negative organisms with an inner membrane and peptidoglycan cell wall overlain by an outer membrane containing lipopolysaccharides (LPS).152 Leptospira LPS are structurally and immunologically similar to LPS from other Gram-negative organisms although they do not appear to exhibit the same endotoxicity.2, 3

Dark-field microscopy is used to visualise Leptospira, which aredifficult to visualise with conventional microscopy because they are so thin. Leptospira exhibit rapid darting movements in liquid media with flexing motions and rotation about their long axes.3 The characteristic motility of Leptospira is due to a periplasmic endoflagellum with polar insertions, which is unique to spirochaete bacteria.152 The motility of Leptospira allows them to move in viscous environments, which enables them to penetrate blood vessels and rapidly reach target organs through haematogenous dissemination.152

Based on genetic typing, 22 species of Leptospira have been identified, which are divided into three distinct phylogenetic clades (Figure 1).152 The genus comprises a clade of 10 pathogenic Leptospira species that have been associated with disease in humans and animals; a clade of five species with ‘intermediate’ pathogenicity traits that have been associated with mild clinical manifestations; and six saprophytic Leptospira species, which do not cause disease. Within the pathogenic Leptospira clade, four subgroups197 have been identified that are thought to correlate to the degree of virulence in humans.152 Subgroup I contains three species (L. interrogans, L. kirschneri, L. noguchii) that are most often associated with severe manifestations of leptospirosis. Subgroup II contains five species (L. borgpetersenii, L. mayottensis, L. santarosai, L. alexanderi, L. weilii) that are typically associated with milder disease and that show evidence of co-evolution with particular animal host species.152 Subgroups III and IV are represented by only one species each (L. alstoni and L. kmetyi respectively) and the virulence of these species remains unclear.

Figure 1 Maximum likelihood tree based on concatenated sequences of 491 core genes.152

The smallest taxonomic unit of Leptospira is the serovar and more than 250 distinct serovars have been described to date. Nomenclature for Leptospira serovars has changed over the years but now follows a format agreed by the Committee on the Taxonomy of Leptospira in 2002, where the genus and species are stated and italicised followed by the serovar name, which is capitalised but not italicised (for example; Leptospira interrogans serovar Icterohaemorrhagiae) (http://www.leptosociety.org/resources). This nomenclature is also useful as it reflects both the phylogenetic and serological classifications of each serovar. Although the serogroup is not strictly included in serovar nomenclature, it is commonly stated in parentheses after the serovar name (e.g. L. interrogans serovar Copenhagenii (serogroup Icterohaemorrhagiae)).

Identifying new Leptospira serovars is a complicated and time-consuming process requiring specialist facilities.152 Cross-agglutination absorption tests and monoclonal and polyclonal agglutination testing are used to characterise serovars based on their serological profile, determined by structural heterogeneity in the carbohydrate component of the LPS.21, 88, 122 Related serovars are grouped into serogroups based on their serological and antigenic characteristics. However, serogroups have no taxonomic standing per se. The serological classification of Leptospira is complicated by horizontal transfer of the LPS biosynthetic locus (rfb) between different Leptospira species, resulting in a single serogroup being represented by multiple Leptospira species. (Table 1)122 Despite this limitation, serogroups remain essential for leptospirosis diagnosis and have proved useful for understanding population-level trends in predominant serogroups.21

Table 1 The relationship between Leptospira genomospecies and Leptospira serogroup classifications122

Serogroup

Genomospecies

Andamana

L. biflexa

Australis

L. borgpetersenii, L. interrogans, L. kirschneri, L. noguchii

Autumnalis

L. borgpetersenii, L. interrogans, L. kirschneri, L. noguchii, L. santarosai

Ballum

L. borgpetersenii

Bataviae

L. borgpetersenii, L. interrogans, L. kirschneri, L. noguchii, L. santarosai

Canicola

L. inadai, L. interrogans, L. kirschneri

Celledoni

L. borgpetersenii, L. weilii

Codice

L. wolbachii

Cynopteri

L. kirschneri, L. santarosai

Djasiman

L. interrogans, L. kirschneri, L. noguchii

Grippotyphosa

L. interrogans, L. kirschneri, L. santarosai

Hebdomadis

L. alexanderi, L. borgpetersenii, L. interrogans, L. kirschneri, L. santarosai, L. weilii

Hurstbridge

L. fainei

Icterohaemorrhagiae

L. inadai, L. interrogans, L. kirschneri, L. weilii

Javanica

L. alexanderi, L. borgpetersenii, L. inadai, L. meyeri, L. santarosai, L. weilii

Louisiana

L. interrogans, L. noguchii

Lyme

L. inadai

Manhao

L. alexanderi, L. inadai, L. weilii

Mini

L. alexanderi, L. borgpetersenii, L. interrogans, L. meyeri, L. santarosai, L. weilii

Panama

L. inadai, L. noguchii

Pomona

L. interrogans, L. kirschneri, L. noguchii, L. santarosai

Pyrogenes

L. borgpetersenii, L. interrogans, L. noguchii, L. santarosai, L. weilii

Ranarum

L. interrogans, L. meyeri

Sarmin

L. interrogans, L. santarosai, L. weilii

Sejroe

L. borgpetersenii, L. interrogans, L. meyeri, L. santarosai, L. weilii

Semaranga

L. biflexa, L. meyeri

Shermani

L. inadai, L. noguchii, L. santarosai

Tarassovi

L. borgpetersenii, L. inadai, L. noguchii, L. santarosai, L. weilii

Leptospira are fastidious organisms and may take up to six months to grow in in vitro.122 Leptospires in culture grow at an optimal temperature of 29 to 30 °C. Cultivation requires the supplementation of growth media with long-chain fatty acids and with vitamins B1 and B12. Fatty acids are supplied by the addition of serum to growth media, or alternatively, an albumin-polysorbate 80 medium can be used. Commercially available culture media include Ellinghausen McCullough Johnson Harris (EMJH) medium45 and Fletcher’s medium, although some strains also require the addition of either pyruvate or rabbit serum.122 Protein-free media containing charcoal-detoxified polysorbates are also popular, especially for vaccine production.20, 39

Liquid, semi-solid or solid media can be used for cultivation. Most laboratories employ semi-solid media for initial isolation and maintenance of cultures and liquid media for antigen production. For isolation of leptospires from non-laboratory sources, it is essential to add 5-fluorouracil110 or other antibiotics4 in order to inhibit the growth of contaminating bacteria that may overgrow the target organism and hamper isolation. For clinical samples, it is essential to use fresh specimens of tissue or body fluids for isolation in order to lessen the detrimental effects of other bacteria and products of autolysis on leptospires. Tissue and urine samples may also have to be diluted to minimize these effects and increase the chance of successful isolation.

Epidemiology

Leptospira bacteria can infect all mammalian livestock species.  Leptospira infection occurs following contact with urine from an infected animal host or through sexual transmission. Contact with contaminated environmental sources such as soil, water or water-borne wastes such as slurry or run-off water from infected animal houses63 is epidemiologically important as some Leptospira species can survive for prolonged periods in the environment. Favourable conditions such as adequate moisture and a neutral to slightly alkaline pH promote environmental survival.71, 116 However, environmental persistence can vary amongst pathogenic Leptospira species. Certain species, such as Leptospira borgpetersenii, appear poorly adapted to survive in the environment29 while others, such as Leptospira interrogans, show prolonged environmental survival.154 A recently recognised factor that may influence the survival of Leptospira in the environment is their ability to form biofilms.158 This ability may be one of the main factors contributing to environmental persistence and disease transmission.

Leptospira infection in livestock can be broadly divided into two major epidemiological categories.123 Infection with host-adapted serovars, such as L. borgpetersenii serovar Hardjo in cattle, is characterised by subclinical infection with prolonged urinary shedding of bacteria. Animals that are chronically infected with host-adapted serovars are referred to as maintenance, reservoir or carrier hosts and are an important source of infection to other animals and humans.122 The second epidemiological category, described as incidental infections, includes infections of livestock with serovars that are adapted to and maintained by other animal species. Typically, these infections are associated with more severe clinical manifestations and may present as an outbreak of disease in affected herds. For example, L. interrogans serovar Icterohaemorrhagiae, a serovar that is carried and shed by brown rats (Rattus norvegicus) with little sign of disease, can result in severe disease in livestock infections and may be fatal.21, 52

A wide range of mammal species may act as carrier hosts forpathogenic Leptospira bacteria, with rodents considered a common source of Leptospira serovars associated with incidental infection,122 In Africa, Leptospira bacteria have been isolated from a range of rodent species8 including house mice (Mus musculus), brown rats, black rats (Rattus rattus), multimammate mice (Mastomys natalensis), African grass rats (Arvicanthus niloticus), giant pouched rats (Cricetomys gambianus) and a variety of other indigenous species.8 Bats and a variety of insectivores have been shown to be carriers of infection in Madagascar and the Indian Ocean Islands.43, 119 In Botswana, renal carriage of pathogenic Leptospira was identified by PCR in a wide range of mammalian, avian and reptilian wildlife species,107 including species that live in close proximity to humans, such as the banded mongoose (Mungos mungo).108

Leptospira infection has also been detected in a range of other wildlife species in Africa by serology. A serosurvey of free-living game in the northern KwaZulu-Natal game parks in South Africa detected antibodies against serogroup Mini in reedbuck (Redunca sp.), bushpig (Potamachoerus larvatus), and black wildebeest (Connochaetes gnu) and againstserogroup Tarassovi in a nyala (Tragelaphus angasii). Two black rhinoceroses (Diceros bicornis) had antibodies against multiple serogroups including Mini, Hardjo, Tarassovi, Copenhageni and Pomon.102 Serological reactivity against Leptospira has also been reported in a variety of wildlife species (buffalo [Syncerus caffer], zebra [Equus quagga] and lions [Panthera leo]) living alongside livestock in the Katavi-Rukwa Ecosystem of Tanzania.12 However, the role of mammalian wildlife species in the epidemiology of leptospirosis in livestock is not well understood and warrants further research, particularly in regions where contact between wildlife and livestock is common. Leptospira antibodies have been demonstrated in non-mammalian vertebrates (e.g. fish),137 reptiles and amphibians,185 and infection has been induced experimentally in chicken embryos.62 However, there is currently no evidence that non-mammalian hosts are susceptible to clinical disease or able to maintain and propagate Leptospira infection and therefore only mammalian hosts are thought to contribute to the epidemiology of the disease.

Host-serovar associations in livestock

Particular associations have been described between specific Leptospira serovars and certain host species. Classic examples include L. interrogans serovar Canicola in dogs, L. interrogans serovar Pomona in pigs, L. borgpetersenii serovar Hardjo in cattle and L. interrogans serovars Copenhagenii and Icterohaemorrhagiae in rats.2, 50, 152 However, these associations are not absolute and may vary in different geographic settings.

Cattle

Of all livestock species, cattle appear to be the most susceptible to leptospirosis and are frequently implicated as the species with the highest seroprevalence.171 Where culture has been attempted, a wide range of serogroups have been isolated from cattle in Africa although not all have been associated with clinical disease (Table 2).

Table 2 Serogroups of Leptospira isolated from cattle in Africa.8

Isolated serogroup

Country or Countries

Australis

Zimbabwe

Bataviae

Egypt

Grippotyphosa

Kenya, Zimbabwe

Hebdomadis

Zimbabwe

Icterohaemorrhagiae

Egypt, Tanzania and Zimbabwe

Pomona

Botswana, Egypt, South Africa and Zimbabwe

Pyrogenes

Nigeria, Zimbabwe

Sejroe

South Africa, Tanzania and Zimbabwe

Tarassovi

Zimbabwe

Leptospira borgpetersenii serovar Hardjo (serogroup Sejroe), a serovar that is associated with cattle worldwide,52 is known to have a reduced ability to survive in the environment.29 Infection with serovar Hardjo is therefore influenced more strongly by management systems that facilitate direct host-to-host transmission than environmental conditions, although environmental transmission may be possible in tropical conditions.130 For example, in Brazil, intensively managed dairy cattle were determined to be eight times more likely to be infected with leptospirosis than extensively managed beef cattle from the same farm.129 In Tanzania a serological survey of cattle at slaughter, where serogroup Sejroe was the predominant reactive serogroup, showed a high seroprevalence even in dry areas, supporting the disconnect between environmental conditions and transmission of this serovar.174

Leptospirosis in cattle in South Africa occurs mostly in relatively high rainfall areas —Mpumalanga, KwaZulu-Natal and the coastal area of the Eastern and Western Cape provinces (from Port Elizabeth in the east to the Western Cape Province area). A serological survey of cattle in KwaZulu-Natal identified a seroprevalence of 19.4 per cent with exposure to a wide diversity of serovars including Pomona, Tarrasovi, Bratislava, Canicola, Hardjo, Icterohaemorrhagiae, Szwajizak  and Grippotyphosa.96 However, leptospirosis has also been diagnosed in the dry Limpopo and North West provinces.92 Serovars Pomona, Mini, Canicola and Hardjo have been isolated from cattle in South Africa.95, 179 During 1986 to 1987, 28 per cent of bovine serum samples tested at the Onderstepoort Veterinary Institute were positive for serogroup Tarassovi, 25.5 per cent for serogroup Mini, and 20.8 per cent for serogroup Sejroe (serovar Hardjo).11 As none of the multivalent Leptospira vaccines used in South Africa contain either serovars Tarassovi or Mini, significant antibody levels against these serovars as a consequence of immunization are not possible and cattle are probably exposed to these strains from as yet unidentified sources. Serovar Mini has been reported as a cause of abortion in cattle70 but, at present, the role of serovar Tarassovi as a cause of bovine infertility is unknown.121 In Zimbabwe positive titres in 27 per cent of cattle have been recorded,66 the most common reactions being against serogroup Sejroe (serovar Hardjo) and serogroup Tarassovi. In Botswana serovar Pomona has been isolated from cattle with a history of abortions.93 The diversity of Leptospira identified in cattle in Africa suggests that the epidemiology and control of bovine leptospirosis is complex and requires further research.

Pigs

The serovars most commonly associated with pigs are L. interrogans serovar Pomona (serogroup Pomona) and L. interrogans serovar Bratislava (serogroup Australis).6, 52 Venereal transmission is thought to play an important role in the transmission of Bratislava amongst pig herds, which has been isolated from the genital tracts of sows and boars.57 Lack of rodent control and the use of artificial insemination have also been implicated as risk factors for leptospirosis in pigs.24 In temperate climates the risk of infection in pigs has been shown to be related to rainfall suggesting a potential environmental component to transmission.25

Evidence that pigs may act as reservoirs for leptospirosis appears to vary. A number of surveys have demonstrated a relatively low seroprevalence amongst pigs compared to other livestock species.171, 173  In Hawaii, however, an increase in the incidence of disease caused by serovar Australis has been postulated to be due to increasing populations of feral pigs and their movement into urban areas196 and, in Australia, feral pigs have also been implicated in leptospirosis transmission.91

Leptospirosis in pigs in South Africa is widespread, with serovars Canicola, Icterohaemorrhagiae and Pomona isolated from pigs in infected herds.103, 189 In Africa, novel serogroups have been detected in pigs. For example, in Tanzania, exposure to an as yet undescribed Leptospira serogroup has recently been identified,136 and in North Africa, a novel serovar of the Tarassovi group (L. borgpetersenii serovar Tunis) was first isolated in the 1960s,13 although there have been few reports of Leptospira infection in pigs in the region since then.

Sheep and goats

Sheep and goats have typically been thought to be incidental hosts of Leptospira and not considered important reservoirs of infection,38 but recent studies have demonstrated that both species may act as carriers of pathogenic Leptospira bacteria.40, 124 Urinary shedding in sheep has been demonstrated in an abattoir study in Morocco17 and sheep have, along with cattle, been implicated as maintenance hosts of serovar Hardjo.86, 128 Infection in goats has also been reported in association with chronic renal and genital tract carriage, reproductive problems and abortion outbreaks.128

Although sheep and goats have been thought of as the least susceptible of domestic animals to leptospirosis, severe outbreaks may occur.38 In South Africa, L. interrogans serovar Pomona infection caused an outbreak of leptospirosis in sheep where the source was an infected piggery.42 Studies of leptospirosis and the impacts it may have on small ruminant production are scarce, although recent studies have implicated leptospirosis as a major cause of poor productivity.128

Horses

Horses are usually implicated as incidental hosts of Leptospira, although leptospiral DNA has been detected in the urine of horses following infection, suggesting a possible role as a source of infection for humans or other animals.76 Moreover, demonstration of Leptospira DNA in equine semen suggests a potential venereal route of transmission in horses.76, 85 Infection of horses with leptospirosis has been reported from Australia,176 North America,85 Egypt,15 and the UK.53 In South Africa, a serosurvey of horses from three provinces, namely Gauteng, KwaZulu-Natal and the Western Cape, has demonstrated a high level of exposure with seroprevalence of 49 per cent, 37 per cent and 32 per cent respectively.164 Bratislava, Djasiman, Arborea and Tarrasovi were the serovars most frequently detected, although a serological response to 21 different serovars from 17 serogroups was noted. Historic evidence suggests that horses are have been exposed to other circulating serovars with 27 per cent of sera from horses tested in South Africa in 1986 to 1987 positive for L. interrogans serovar Pomona.11 However, no clinical disease in this species has as yet been reported in Africa.

Pathogenesis

Leptospira infection is acquired through direct contact with urine or indirectly by water or mud contaminated with infected urine. Leptospira bacteria penetrate the mucous membranes or abraded skin and enter the blood stream of the host where they multiply and establish a leptospiraemia.2 Thereafter organisms localize to target organs such as the kidneys, lungs, brain, eyes and genital tract of both male and female animals.132 Leptospires have also been isolated from the cerebrospinal fluid of infected animals.52

Dissemination of leptospires is facilitated by haemotogenous spread to tissue sites and is aided by the ability of pathogenic Leptospira to cross tissue barriers, migrate through the extracellular matrix and adhere to host cells. Haemorrhage and disruption of haemostasis are also common manifestations of acute leptospirosis. The pathogenesis is still not fully understood but includes endothelial damage, disruption of tissue integrity through the production of bacterial toxins or enzymes such as sphingomyelinase.33 Leptospira has been shown to bind fibrinogen, plasminogen and partially inhibit fibrin formation, all of which may also contribute to reduced clotting ability.148 Furthermore, some Leptospira serovars produce a haemolysin that causes haemolytic anaemia and subsequent haemoglobinuria and icterus. Intravascular haemolysis can also contribute to liver damage and jaundice through ischaemic injury to hepatic centrilobular areas.132

Secondary clinical signs, seen after an incubation period of 2 to 16 days, are dependent on the site of leptospiral localization. Localization in the proximal tubules of the kidneys in maintenance hosts causes mild chronic interstitial nephritis and long-term leptospiruria.182 Endothelial damage and subsequent ischaemia are thought to be important mechanisms in tissue damage associated with Leptospira infection. Enzymatic damage (e.g. from sphingomyelinases) to tissues may also contribute to the pathology associated with acute disease.2 Acute renal dysfunction is thought to be a combined result of reduced renal perfusion and ischaemia as a consequence of intravascular haemolysis, combined with direct tissue damage and inflammation following leptospiral renal invasion. Chronic renal injury is associated with an inflammatory response to invading Leptospira organisms resulting in a lymphoplasmacytic infiltrate in the renal interstitium.132

Foetal infection, caused by leptospires crossing the placenta, varies in outcome depending on the age of the foetus at the time of infection. Possible outcomes include abortion or stillbirth, foetal antibody production and clearance of infection, or poor neonatal viability associated with latent infections.64, 65 Leptospires that localize in the foetal brain may persist for long periods, giving rise to neurological signs in some cases.63

The pathogenesis of uveitis in horses results from intraocular leptospiral infection and an associated immune-mediated response. There is a close antigenic relationship between certain leptospiral antigens and lens and retinal proteins of horses that appears to aid in the development of chronic inflammation in infected horses.191 Chronic lymphoplasmacytic inflammation of the uvea can lead to irreversible ocular damage including the formation of posterior synechiae, increased intra-ocular pressure and retinal detachment.132

Clinical signs

The clinical signs of leptospirosis in livestock vary with the affected species and the infecting serovar. In general, the clinical signs seen in association with Leptospira infection can be grouped into four main syndromes: 1) severe, peracute disease with a high fatality rate, most commonly seen in susceptible young stock; 2) acute febrile disease in adult animals; 3) reproductive disease characterised by abortion, stillbirth and reduced fertility; and 4) subclinical, chronic renal infection. Other disease manifestations may also be seen such as recurrent uveitis in horses and, occasionally, meningitis in young stock. Although the clinical signs may vary by the organ system affected, in general there are a number of similarities between different livestock species.

Pyrexia, acute renal failure, jaundice and haemorrhage are typical manifestations of acute and peracute leptospirosis.132 The development of an acute febrile reaction coincides with the leptospiraemic phase of infection and is one of the earliest clinical signs of Leptospira infection. Young stock are particularly susceptible to severe, acute infection. Reproductive disease characterised by abortion, stillbirths and neonatal deaths is common amongst livestock species.52 Abortion storms may be seen in groups of naïve, reproductively active animals. However, subclinical chronic infection is also common in livestock and may only be detected in routine screening or investigation of poor reproductive performance and infertility. Leptospiruria occurs in acute, subclinical and chronic infections but the duration of shedding may vary. Acute infection with a non-host-adapted serovar may persist for 2-4 weeks post-infection whereas shedding can continue for more than two years in chronic, subclinical infections with adapted serovars.52

Cattle

Amongst livestock species, cattle appear relatively susceptible to acute disease following Leptospira infection. Globally, serovars from a wide variety of serogroups have been reported in association with clinical infection in cattle.52 Serogroups isolated from cattle in Africa are shown in Table 2. Clinical disease in sub-Saharan Africa has primarily been reported in association with L. interrogans serovar Pomona93 and serovars of the Grippotyphosa serogroup. Acute leptospirosis is characterized by fever, anorexia, malaise, haemoglobinuria, icterus and anaemia in cattle,132 affected calves may also have diarrhoea.147 Fever may last from one to five days and in experimental studies it has been reported that relapses recur for up to 53 days in 80 per cent of infected animals.49 Severely affected calves die within one to seven days of developing clinical signs.132 In adult animals, Leptospira infection is not usually fatal but may result in chronic renal infection, which is associated with mild, focal, interstitial nephritis.181

Agalactia (also referred to as ‘milk-drop’ or ‘flabby bag’ syndrome is a feature of acute leptospirosis in adult lactating animals and has been reported in association with a variety of serogroups including Pomona and Hardjo infection.52 Infection may result in a precipitous drop in milk production. All quarters of the udder may become flaccid, and milk may vary from thick, pasty and cream-coloured to bloody with a normal consistency. These cases may test positive on the California milk cell test.151 This agalactia resolves within a week and its course is not affected by treatment. It appears not to be a true mastitis as the affected udder lacks an inflammatory response, though leptospires can be isolated from the milk and udder tissue.80, 98, 126, 151, 172

Abortions and reproductive failure are also common presentations of Leptospira infection in cattle. In pregnant females, abortions may occur in the last trimester and occur from 1-12 weeks following acute infection. Other reproductive problems such as stillbirths or the birth of weak or premature calves may also occur in association with Leptospira infection, depending on the stage of gestation when infection occurred.58, 93, 151, 181

Although numerous serovars have been identified as causes of leptospirosis in cattle, serovars of the Sejroe serogroup, in particular the Hardjo serovars, have been recognized worldwide as being an important cause of bovine abortion. Infertility is also a feature of infection with Hardjo in cattle herds and low conception rates are reported inHardjo-infected herds.80 Hardjo infection has also been associated with early foetal loss so may present as an early return to oestrus in breeding animals, with no visible signs of embryo loss.132  Although some studies have demonstrated an association between Hardjo seroprevalence and early foetal loss or abortion in cattle herds,160  the use of serology to diagnose serovar Hardjo as the cause of reproductive failure remains fairly controversial, with some studies failing to demonstrate any association between seroprevalence and foetal loss, e.g34 It is also worth noting that there is a relatively poor correlation between serological status and chronic renal infection and shedding in cattle, particularly with serovar Hardjo.52 Although experimental studies have demonstrated that animals with Hardjo infection may have leptospiruria that can persist for up to 450 days or longer,44, 181 the duration of antibody response is variable and may decline to low or undetectable levels before infection is cleared from kidney or genital tract.

Pigs

Leptospirosis in pigs is an important cause of productivity loss in farming systems worldwide. Infection manifests most commonly as a reproductive syndrome characterized by abortions, infertility and the birth of premature and weak piglets. Abortion may occur from as early as six weeks’ gestation, although it mostly occurs towards term; many litters reach term and are either all stillborn or are a mixture of stillborn and live, weak piglets.56 The most common serogroups associated with reproductive failure in pigs are Pomona (especially Leptospira interrogans serovars Pomona and Kennewicki), Australis (L. interrogans serovar Bratislava) and serogroup Tarassovi.52  Acute systemic disease characterised by anorexia, jaundice and renal failure can also be seen, especially in young stock, and is typically caused by incidental infection with a variety of serogroups including Canicola, Hebdomadis, Icterohaemorrhagiae and Autumnalis.5

In Africa, data on clinical disease in pigs in Africa are limited. The majority of data comes from South Africa, where L. interrogans serovar Pomonahas been identified as a cause of abortions, stillbirths, perinatal deaths, and chronic interstitial nephritis in pigs.103 Asymptomatic chronic interstitial nephritis may be identified at slaughter in Pomona infections (‘white-spot’ kidneys).103 L. interrogans serovar Canicola has also been isolated from breeding sows in South Africa affected by abortion, poor neonatal survival and infertility.189

Sheep and goats

In general, sheep are thought to be relatively resistant to acute clinical leptospirosis. Acute systemic disease, characterized by icterus, haemolytica anaemia, haemoglobinuria, fever and death, has been reported in lambs in association with infections by the Pomona, Hebdomadis, Ballum and Grippotyphosa serogroups.53 This fatal haemolytic disease may kill up to 5 per cent of the lamb crop,161, 168 losses being experienced in two- to four-month-old lambs.161 Fatal haemolytic disease accompanied by icterus and haemoglobinuria has been induced in experimental infection of sheep with L. interrogans serovar Pomona.100 Abortion, stillbirths, and the birth of weak lambs have all been reported in associated with Hardjo infection.53 Infection with other serogroups such as Hebdomadis, Australis and Pomona are also thought to cause abortion with rates of up to 20 per cent being recorded.10, 53 Ewes can also show total or only partial agalactia, which resolves without treatment within three to four days. This syndrome may only be detected when lambs with a normal birth weight die of malnutrition.134

Goats are also susceptible to infection with Leptospira. Severe disease is most commonly seen in young kids and is associated with incidental infection. In Brazil, outbreaks of abortion and neonatal death associated with Leptospira infection have been reported.131  Infection with serovars from the Grippotyphosa serogroup have been experimentally shown to induce an acute disease characterized by haemoglobinuria, anaemia and icterus, and death in severe cases,52 whereas experimental infection with serovars Hardjo and Pomona caused only mild clinical signs including mild fever, and a drop in milk production in some animals.183

There is very little information available on clinical Leptospira infection in sheep and goats in sub-Saharan Africa. Serosurveys suggest that exposure is high in some settings,128 but there are no studies to indicate clinical disease or reproductive losses in association with infection in small ruminants. It is possible, however, given reports from other geographic regions, that leptospirosis remains an undiagnosed cause of production losses in small ruminant rearing systems.

Horses

Most cases of leptospirosis in horses are subclinical although,as with young of other species, foals are more susceptible to acute infections, which may be fatal.132 More common clinical syndromes include abortions, stillbirth, premature live birth54 and recurrent uveitis.132 Serogroups including Australis, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae and Pomona have been isolated from aborted equine foetuses.54, 75 Chronic leptospiral infections in horses are more commonly associated with the development of recurrent uveitis also referred to as periodic ophthalmia or moon blindness.191 Signs of uveitis develop some time (sometimes years 41) after the initial infection, and manifest as painful keratitis or ophthalmitis.162 It is recurrent, uni- or bilateral and characterized clinically by miosis, congestion of the sclera, photophobia, severe lachrymation and sometimes blindness.41, 167 Although recurrent uveitis is an important cause of equine vision loss around the world, there is limited evidence to date from Africa.164

Pathology

The gross presentation of Leptospira infection varies with the respective syndromes in affected animals. However, many features are consistent among the major livestock species.

In acute systemic disease, the macroscopic lesions are characteristic of a haemolytic crisis and septicaemia, and include icterus, anaemia, splenomegaly and subcutaneous and mucosal haemorrhages.132, 147 Pulmonary oedema, hepatomegaly and nephritis are also typical of acute cases of leptospirosis. Localization of leptospires to the kidney is associated with interstitial nephritis and tubular necrosis, which may result in the kidneys macroscopically appearing swollen and haemorrhagic.132

In subacute or chronic renal infection, multifocal white spots with or without a hyperaemic border may be seen, which may be as small as 1 to 2mm in diameter, and may be restricted to the cortex or also extend into the medulla. These lesions are sometimes accompanied by a few haemorrhages and small pitted areas. The renal capsule is adherent to the cortex over the affected areas.181 Histologically there is mild, focally disseminated, interstitial, lymphoplasmacytic nephritis. The infiltrate in the affected kidneys occurs primarily in the interstitial tissue and around the glomeruli 147 and while it consists mostly of lymphocytes and plasma cells, a few neutrophils and eosinophils may also be seen.115 Other lesions in the kidneys include degeneration, necrosis and atrophy of the tubular epithelium, few cellular and hyaline casts, and the development of fibrosis. Glomeruli may show swelling of the tuft, an accumulation of protein in the urinary space, and fibrosis of Bowman’s capsule. Focal areas of calcification may occur in the cortex and in the medulla.115 However, leptospiral organisms are not necessarily associated with lesions in the kidneys.49 The presence of leptospires in the kidneys does not necessarily induce nephritis165 and there is often no correlation between the severity of the renal lesions and the isolation of leptospires from the respective tissues, especially with host-adapted serovars.

Few specific lesions occur in the placenta of animals that have aborted. The intercotyledonary tissue may be oedematous while the cotyledons may be fawn-coloured and atonic. Hydropic degeneration and focal necrosis of the cotyledons occur in the affected placentas. The degree of change varies amongst animals and also between the cotyledons of the same animal.49 Leptospires may be detected histologically in the tissues of affected animals by the application of immunofluorescence techniques. Traditionally, silver stains and modified Warthin-Starry stains have been used in histopathological diagnosis; however, these stains are insensitive and have also been associated with false-positive results.132 In systemic Leptospira infection in juvenile livestock the organisms may be demonstrated in various tissues including the kidneys, liver and lungs, and organisms can also be seen in the kidneys, placenta, cotyledons, and the liver of animals with subclinical infections.181

Figure 2 Numerous cutaneous haemorrhages in a pig foetus aborted as a result of leptospirosis

Cattle

The pathology of leptospirosis in cattle varies according to the respective syndrome. In acute cases of systemic infection with an incidental serovar, pathology is consistent with that seen in other species as described above. In abortions, aborted foetuses may be quite fresh,49 oedematous or autolysed at the time of abortion. Leptospira organisms may be detected in the kidneys and focal tubular necrosis, sometimes associated with the early stages of interstitial nephritis may also occur.38, 132 Few lesions are seen in weak, live-born calves but kidneys may be oedematous and haemorrhages may occasionally occur in the urinary bladder, lungs and kidneys.181

Pigs

Abortions, which occur late in gestation, are often the most visible sign of Leptospira infection in pig herds. In aborted foetuses, multifocal haemorrhages are the most common macroscopic lesions (Figure 2). In infected neonates, icterus is also a common gross finding.132 Histologically, acute hepatitis, lymphocytic myocarditis and interstitial nephritis are seen in these cases. Chronic interstitial nephritis is frequently found in slaughter pigs infected with leptospires.99, 103 These lesions are seen as greyish foci, a few millimetres to a centimetre in diameter, in the cortex and medulla.81 Advanced lesions become fibrosed.82 Histologically, the lesions in the kidneys vary according to the stage of their development. Acute lesions are characterized by hyperaemia, oedema and degeneration of the tubular epithelium, but when infection becomes subacute, histology is characterized by the presence of monocytes and macrophages that are replaced at a later stage by plasma cells and lymphocytes. Even serovars of low virulence cause lesions in the kidneys. Leptospires occur in affected kidneys at all the stages of the development of the lesions. Initially they are present in all the structures, including blood vessels, but as the lesions progress in age they are restricted to the microvilli of the renal epithelium (Figures 3 and 4).36

Sheep and goats

Few lesions are seen in adult sheep with leptospirosis. Typically the only macroscopic lesions are acute nephritis and concurrent haemorrhage, although hepatitis has also been reported.72, 142, 168 Abortion may occur in some cases and leptospires have been detected by immunoperoxidase in the tissues of aborted or stillborn foetuses.53 In lambs and kids, systemic infection can cause a fatal haemolytic syndrome characterized by severe icterus, anaemia, haemoglobinuria, and pale brown168 to dark red or blackish kidneys.100, 161 On cut surface the kidneys manifest dark brown streaks and the focal white spots indicating interstitial nephritis. Cell swelling and necrosis of few hepatocytes are seen histologically in these animals, while haemosiderin pigmentation of the tubular epithelium and clumps of leptospires, hyaline casts, haemoglobin and cellular debris also occur in the kidneys.168

Horses

The sequestration of leptospires in the eyes causes progressive uveitis, thought to be the consequence of an immune-mediated hypersensitivity reaction. The anterior uveitis that is seen initially progresses during repeated attacks to involve the posterior uvea, cornea, retina and optic nerve, with resulting blindness.

Lesions are rarely encountered in aborted foetuses, even though the infection may be diagnosed by immunofluorescence, which is most successful in the kidneys and the liver, or by isolation of the organism.54 Few lesions are seen on histology of aborted foetuses. In rare cases dissociation of the hepatocytes, necrosis of individual hepatocytes, and confluent and multiple areas of necrosis can be seen. Non-specific lesions include degeneration of the renal tubules and perivascular haemorrhages in the brain.54 Concurrent infections such as equid herpesvirus 1 cause more severe lesions in the affected organs. In these cases viral inclusions are present in the affected cells and serve to confirm the diagnosis.51

Figure 3 Histological sections showing numerous leptospires entwined in microvilli and free in the lumen of a renal tubule (Warthin-Starry stain)

Figures 4 Histological sections showing numerous leptospires entwined in microvilli and free in the lumen of a renal tubule (Warthin-Starry stain)

Diagnosis

The history and clinical signs in affected animals may indicate leptospirosis. However, due to the variable nature of the clinical signs the diagnosis should be confirmed by laboratory tests. The slow growing and fastidious nature of Leptospira makes isolation of little use as a diagnostic test and therefore definitive diagnosis is achieved through serological or molecular detection assays. Typically the incubation period for leptospirosis is 7-12 days although it may range from 2 to 30 days. The first phase of leptospirosis is the leptospiraemic phase during which leptospires can be detected in the blood for up to 7 days.21 The second phase of the disease is characterised by an immune response during which IgM antibodies increase from the second week after infection, which coincides with tissue localisation of the leptospires and the onset of leptospiruria.122

Serological diagnostics

The two most commonly used serological tests for leptospirosis are commercially available enzyme-linked immunosorbent assays (ELISA) or the microscopic agglutination test (MAT).153 Although ELISA has been used with success in the diagnosis of leptospirosis in some species,5, 39 particularly in differentiating between acute and chronic infections,82 it cannot be used for conclusive serological diagnosis due to its poor sensitivity and specificity and requires confirmation with MAT or PCR.153

The microscopic agglutination test (MAT) is the gold standard serological test and can be used in any livestock species. The MAT requires maintenance of a panel of live Leptospira isolatesand the inclusion of local isolates in MAT antigen panels is critical and can help improve the sensitivity of Leptospira detection.136 A major limitation of the MAT is that a single MAT test performed on an acute blood sample from an animal may be insufficient for the routine diagnosis of leptospirosis in individual animals. A four-fold rise in antibody titre between paired acute and convalescent serum samples is the diagnostic standard although will only provide a retrospective diagnosis, which has limited value for the clinician.122 For practical purposes, a single MAT titre of ≥1:400 in the presence of clinical signs and an appropriate history is usually indicative of clinical infection.2 MAT testing is therefore often considered more useful for determining the presence of infection within a herd, which can be valuable for herd health strategies.94 Factors that limit the usefulness of serology in individual animals are the administration of antibiotics that suppress the development of antibodies, previous vaccination that hampers the interpretation of serological results, and the prevalence of significant antibody levels in endemic areas.

Serology is a poor diagnostic tool for confirming the role of leptospires in specific cases of recurrent uveitis in horses. Positive serological titres are more common than the occurrence of uveitis, and many serologically positive horses are clinically normal. Antibodies, however, appear also to be synthesized intra-ocularly, particularly during the active stages of recurrent uveitis, as their levels in the aqueous humour may exceed those in the serum.195 Antibody levels in aqueous humour obtained by paracentesis that are in excess of those in the blood,41 or the detection of Leptospira antigen using PCR61 are considered to be diagnostic for recurrent uveitis.

Molecular diagnostics

Polymerase chain reactions can be used for detection of Leptospira infections during the leptospiraemic phase of the disease 153 or to demonstrate urinary shedding in chronic infections.78 PCR is a useful diagnostic tool in outbreak situations where a rapid diagnosis is needed.129 Moreover, sequencing of PCR products may provide valuable phylogenetic data. For example, sequencing of PCR products obtained directly from urine has demonstrated the presence in cattle of Leptospira that have not yet been cultured.79

Although PCR is currently the best diagnostic technique available for a rapid diagnosis of leptospirosis26 it does have limitations. Some PCR techniques detect saprophytic Leptospira in environmental contamination, which would lead to false positives. Moreover, there is a lower limit (usually between 102 and 103 Leptospira/ml of clinical sample) below which PCR assays will not detect Leptospira DNA; sequence polymorphisms in the target genes may affect PCR performance and PCR assays may not be suitable for screening for undescribed pathogenic strains.26

Various PCR assays for leptospirosis have been described. In a recent study the SYBR green and TaqMan qPCR assays, targeting the lfb1and lipL32 genes respectively, were determined to be the most sensitive and specific assays for the detection of pathogenic Leptospira species.26 The detection of previously undescribed or non-pathogenic Leptospira can be achieved by using PCR assays targeting the 16s rRNA genes.26, 69 It has been suggested that more than one technique should be used to increase diagnostic sensitivity and specificity.26 Recently isothermal methods, which do not require a thermal cycler or gel electrophoresis, have also been used to detect infection, which may provide a less technically demanding means of diagnosing leptospirosis for use in less sophisticated laboratories.153

Differential diagnosis

In cattle the haemolytic syndrome caused by leptospirosis should be differentiated from other causes of a haemolytic anaemia such as babesiosis and anaplasmosis. Other causes of abortion, stillbirths, neonatal deaths and/or infertility in cattle include brucellosis, campylobacteriosis, chlamydiosis, coxiellosis(Q fever), Neospora infection and trichomonosis as well as a variety of viral infections such as bovine viral diarrhoea (BVD), Rift Valley fever (RVF), bluetongue or infectious bovine rhinotracheitis (IBR).

In pigs, the abortions, stillbirths and mummification caused by porcine parvovirus and other viral infections can be confused with leptospirosis. The multifocal, subacute to chronic nephritis in cases of leptospiral infection in pigs is similar to that caused by other bacteria such as Escherichia coli, Streptococcus spp. and Arcanobacterium pyogenes and by the mycotoxins (ochratoxin A, citrinin and others) produced by Aspergillus and Penicillium spp.113

Control

The complex multi-host epidemiology of leptospirosis can make control of the disease challenging. In animals a variety of antibiotics have been used to treat acute leptospirosis. Currently, a combination of penicillin and streptomycin is the treatment of choice. However, ampicillin, amoxicillin, tetracyclines, tulathromycin and third generation cephalosporins have also been used.52 Antibiotics may differ in their ability to clear renal carriage of Leptospira infection in chronically infected animals. For example, in hamster models, ampicillin (100mg/kg) was unable to clear the infection from the kidneys and heart while doxycyline (10mg/kg) cleared the infection from all organs in two days.21 In dogs, treatment with doxycycline (5mg/kg PO or IV q12h for 2 weeks) has been suggested for clearance of the leptospires from renal tubules.177 Antibiotic treatment may also play a role in the control of leptospirosis. For example, systemic treatment of herds may be considered in the case of abortion storms, new introductions to a herd should be treated to prevent introduction of the disease and semen diluent should be treated with a combination of penicillin and streptomycin to kill any leptospires.52  The most appropriate antibiotic treatment or control regime will vary depending on the livestock species involved, management system and other considerations such as the withdrawal period for meat or milk products.

Vaccination is a practical means of preventing clinical leptospirosis in many livestock species but may only be partially effective due to the serovar-specific nature of vaccine-induced immunity and the presence of local serovars not included in the vaccine.2 A wide range of vaccines are available for use in farm animals including several multivalent preparations.7 However, vaccination does not necessarily prevent renal colonization and leptospiruria despite conferring protection against clinical disease and abortion.31, 115, 143

The recommendations of the vaccine manufacturer should be followed as they differ for the different vaccines, but some general guidelines for effective vaccination can be given. A critical aspect of any vaccination program should be the establishment of protective immunity prior to breeding, as vaccination is unlikely to be effective if placental infection has already occurred.52 Pigs inoculated for the first time should receive two injections four to six weeks apart. Breeding sows should be vaccinated six weeks before breeding and revaccinated four to six weeks later. A booster should be given four weeks before each subsequent farrowing. Boars should be vaccinated every six months. Colostral antibodies from vaccinated sows protect piglets from leptospiral infection for approximately three months.9 Piglets of vaccinated sows should therefore be vaccinated at the age of three months, when passive immunity is waning. Inoculation of piglets less than one month old has been shown to be ineffective.30 Previously unvaccinated cattle should receive two inoculations one month apart. Testing for urinary shedding with subsequent antibiotic treatment of shedders may also be indicated prior to vaccination in herds that have been diagnosed with Leptospira infection. An annual booster is recommended by most manufacturers although it has been recommended that cattle should be vaccinated up to three times annually in order to prevent abortion and infertility.20 Due to a lack of cross protective immunity between Leptospira serovars, vaccinated livestock may still be susceptible to Leptospira serovars not included in vaccines.

Biosecurity to prevent new introductions of Leptospira into the herd is also an important component of leptospirosis control. Many wild animal species can carry Leptospira infection and act as a source of infection for livestock. In particular, rodents are an important source of a wide diversity of Leptospira serovars and incidental infection of livestock with rodent-associated Leptospira serovars may cause outbreaks of acute disease. Rodent control appears to be of particular importance to the control of leptospirosis in piggeries.52 Rodent control should, therefore, form an important part of leptospirosis control strategies.

References

  1. ABREY, A., 1981. Abortions in the Natal Midlands due to leptospirosis. Veterinary Clinician, 7–9.
  2. ADLER, B. & DE LA PENA MOCTEZUMA, A., 2010. Leptospira and leptospirosis. Veterinary Microbiology, 140(3–4), 287–296.
  3. ADLER, B., FAINE, S. & CHAPPEL, R.J., 1986. Development of improved selective medium for isolation of leptospires from clinical material. Veterinary Microbiology, 12, 377–381.
  4. ADLER, B., FAINE, S. & GORDON, L.M., 1981. The enzyme-linked immunosorbent assay (ELISA) as a serological test for detecting antibodies against Leptospira interrogans serovar hardjo in sheep. Australian Veterinary Journal, 57, 414–417.
  5. ADLER, B., LO, M., SEEMANN, T. & MURRAY, G.L.G., 2011. Pathogenesis of leptospirosis: the influence of genomics. Veterinary Microbiology, 153(1–2), 73–81.
  6. ALEXANDER, A.D., 1976. Immunity in leptospirosis. In: JOHNSON, R.C., (ed.). The Biology of Parasitic Spirochaetes. New York, San Francisco, London: Academic Press.
  7. ALLAN, K.J., BIGGS, H.M., HALLIDAY, J.E.B., KAZWALA, R.R., MARO, V.P., CLEAVELAND, S. & CRUMP, J.A., 2015. Epidemiology of leptospirosis in Africa: a systematic review of a neglected zoonosis and a paradigm for ‘One Health’ in Africa. PLoS Neglected Tropical Diseases, 9(9), e0003899. doi: 10.1371/journal.pntd.0003899.
  8. AMATREDJO, A. & CAMPBELL, R.S.F., 1975. Bovine leptospirosis. Veterinary Bulletin, 43, 875–891.
  9. ANDREANI, E., SANTARELLI, E. & DILIGENTI, R., 1974. Leptospirosi delgi ovi. Infezione naturale de serotype hardjo. Annali della Facolta di Medicina Veterinaria Universita di Pisa, 27, 33.
  10. ANON., 1986–1987. Bacteriology & Reproduction. Annual Report of the Veterinary Research Institute, Onderstepoort.
  11. BALL, M.G., 1966. Animal hosts of leptospires in Kenya and Uganda. American Journal of Tropical Medicine and Hygiene, 15, 523–530.
  12. BARSOUM, I.S., BOTROS, B.A.B. & MORCOS, M.B., 1978. Equine leptospirosis with some clinical observations. Annales de Recherche Veterinaire, 9, 115–118.
  13. BEAMER, P.D., HARDENBROOK, H. & MORRILL, C.C., 1953. Studies on leptospirosis in domestic animals. I. Leptospirosis in sheep. Veterinary Medicine, 48, 365–366.
  14. BERCOVICH, Z. TAAIJKE, R. & BOKHOUT, B.A., 1990. Evaluation of an ELISA for the diagnosis of experimentally induced and naturally occurring Leptospira infections in cattle. Veterinary Microbiology, 21, 255–262.
  15. BERNKOPF, H., 1946. Experimental work on leptospirosis in man and cattle in Palestine. Refuah Veterinarith, 3, 49.
  16. BEY, R.F. & JOHNSON, R.C., 1986. Current status of lepto vaccines. In: PANDY, R., (ed.). Progressive Veterinary Microbiology and Immunology. Basel: S. Karger.
  17. BHARTI, A.R, NALLY, J.E., RICALDI, J.N., MATTHIAS, M.A., DIAZ, M.M., LOVETT, M.A., LEVETT, P.N., GILMAN, R.H., WILLIG, M.R., GOTUZZO, E. & VINETZ, J.M., 2003. Leptospirosis: A zoonotic disease of global importance. Lancet Infectious Diseases, 3(12), 757–771.
  18. BLACKMORE, D.K. & HATHAWAY, S.C., 1979. The nodality of zoonoses. Proceedings of the Second International Symposium on Veterinary Epidemiology and Economics, Canberra.
  19. BOLIN, C.A., ZUERNER, R.L. & TRUEBA, G., 1989. Comparison of three techniques to detect Leptospira interrogans serotype hardjo var. hardjo-bovis in bovine urine. American Journal of Veterinary Research, 50, 1001–1003.
  20. BOQVIST, S., CHAU, B.L., GUNNARSSON, A., OLSSON ENGVALL, E., VÅGSHOLM, I. & MAGNUSSON, U., 2002. Animal- and herd-level risk factors for leptospiral seropositivity among sows in the Mekong delta, Vietnam. Preventive Veterinary Medicine, 53(3), 233–245.
  21. BOQVIST, S., ELIASSON-SELLING, L., BERGSTRÖM, K. & MAGNUSSON, U., 2012. The association between rainfall and seropositivity to Leptospira in outdoor reared pigs. Veterinary Journal, 193(1), 135–139.
  22. BOURHY, P., BREMONT, S., ZININI, F., GIRY, C. & PICARDEAU, M., 2011. Comparison of real-time PCR assays for detection of pathogenic Leptospira spp. in blood and identification of variations in target sequences. Journal of Clinical Microbiology, 49(6), 2154–2160.
  23. BRENDLE, J.J., ROGUL, M. & ALEXANDER, A.D., 1974. Deoxyribonucleic acid hybridization among selected leptospiral serotypes. International Journal of Systematic Bacteriology, 24, 205–212.
  24. BRENNER, D.G., KAUFFMANN, A.F., SULZER, K.R., STEIGERWALT, A.G., ROGER, F.C. & WEYANT, R.S., 1999. Further determination of DNA relatedness between serogroups and serovars in the family Leptospiraceae with proposal of Leptospira alexanderi sp. nov. and four new Leptospira genomospecies. International Journal of Systemic Bacteriology, 49, 839–858.
  25. BULACH, D.M., ZUERNER, R.L., WILSON, P., SEEMANN, T., MCGRATH, A., CULLEN, P.A., DAVIS, J., JOHNSON, M., KUCZEK, E., ALT, D.P., PETERSON-BURCH, B., COPPEL, R.L., ROOD, J.I., DAVIES, J.K. & ANDADLER, B., 2006. Genome reduction in Leptospira borgpetersenii reflects limited transmission potential. Proceedings of the National Academy of Sciences of the United States of America, 103(39), 14560–14565.
  26. CACCHIONE, R.A., CASCELLI, E.S., SARAVI, M.A. & MARTINEZ, E.S., 1981. Evaluacion de una vacuna trivalente antileptospira en bovina y porcinos de la Argentina. Revista de Medicina Veterinaria Argentina, 62, 24–31.
  27. CARGILL, C.F. & DAVOS, D.E., 1981. Renal leptospirosis in vaccinated pigs. Australian Veterinary Journal, 57, 236–238.
  28. CARLETON, H.M. & DRURY, R.A.B., 1957.Histological Technique. 3rd Edition. London: Oxford University Press.
  29. CHAPERON, K.M., DAVIS, R.B. & MILLER, N.G., 1979. Effect of a plasma factor in leptospirosis on platelets in vivo. Thrombosis Research, 14, 975–980.
  30. CHAPPEL, R.J., MILLAR, B.D., ADLER, B., HILL, J., JEFFERS, M.J., JONES, R.T., MCCAUGHAN, C.J., MEAD, L.J. & SKILBECK, N.W., 1989. Leptospira interrogans serovar Hardjo is not a major cause of bovine abortion in Victoria. Australian Veterinary Journal. 66, 330–333.
  31. CHERHUKCHA, Y.G., SHISHKINA, Z.S., BARYSHEV, P.M. & KOKOVIN, I.L., 1976. Zentralblatt für Bakteriologie, Abteilung, 1, 236-336
  32. CHEVILLE, N.F., HUHN, R. & CUTLIP, R.C., 1980. Ultrastructure of renal lesions in pigs with acute leptospirosis caused by Leptospira pomona. Veterinary Pathology, 17, 338–351.
  33. CHRISTOPHER, W., ADLER, B. & FAINE, S., 1982. Immunogenicity of leptospiral vaccines grown in protein-free medium. Journal of Medical Microbiology, 15, 493–501.
  34. CICERONI, L., LOMBARDO, D., PINTO, A., CIARROCCHI, S. & SIMEONI, J., 2000. Prevalence of antibodies to Leptospira serovars in sheep and goats in Alto Adige-South Tyrol. Journal of Veterinary Medicine. B, Infectious Diseases and Veterinary Public Health, 47(3), 217–223.
  35. COSTA, F., HAGAN, J.E., CALCAGNO, J., KANE, M., TORGERSON, P., MARTINEZ-SILVEIRA, M.S., STEIN, C., ABELA-RIDDER, B. & KO, A.I., 2015. Global Morbidity and Mortality of Leptospirosis : A Systematic Review. PLoS Neglected Tropical Diseases, 0–1.
  36. COUSINS, D.V. & ROBERTSON, G.M., 1986. Use of enzyme immuno assay in a serological survey of leptospirosis in sheep. Australian Veterinary Journal, 63, 36–39.
  37. DA SILVA, R., COSTA, V.M., SHIMABUKURO, F.H., RICHINI-PEREIRA, V.B., MENOZZI, B.D. & LANGONI, H., 2012. Frequency of Leptospira spp. in sheep from Brazilian slaughterhouses and its association with epidemiological variables. Pesquisa Veterinária Brasileira, 32(3), 194–198.
  38. DAVIDSON, M.G., NASISSE, M.P. & ROBERTS, S.M., 1987. Immunodiagnosis of leptospiral uveitis in two horses. Equine Veterinary Journal, 19, 155–157.
  39. DE LANGE, J.F., GUMMOW, B., TURNER, G.V. & REDMAN, A.R., 1987. The isolation of Leptospira interrogans serovar pomonaand related serological findings associated with a mixed farming unit in the Transvaal. Onderstepoort Journal of Veterinary Research, 54, 119–121.
  40. DOHERTY, P.C., 1967. Bovine Leptospira pomona infection: The disease in cattle infected during an experimental outbreak. Queensland Journal of Agriculture and Animal Science, 24, 351–364.
  41. ELLINGHAUSEN, H.C. & MCCULLOUGH, W.C., 1965. Nutrition of Leptospira Pomona. American Journal of Veterinary Research, 26, 45–51.
  42. ELLIS, R.M., ROBERTSON, G.M., HUSTAS, L. & KIRBY, M., 1983. Detection of leptospires in tissue using an immunoperoxidase staining procedure. Australian Veterinary Journal, 60, 364–367.
  43. ELLIS, T.M., BRYSON, D.G. & MCFERRAN, J.B., 1983. Abortion associated with mixed leptospira/equid herpesvirus 1 infection. The Veterinary Record, 98, 218–219.
  44. ELLIS, W.A. & MICHNA, S.W., 1976. Bovine leptospirosis: A serological and clinical study. The Veterinary Record, 99, 387–391.
  45. ELLIS, W.A. & MICHNA, S.W., 1977. Bovine leptospirosis: Experimental infection of pregnant heifers with a strain belonging to the Hebdomadis serogroup. Research in Veterinary Science, 22, 229–236.
  46. Ellis, W.A., 2015. Animal Leptospirosis. In: Leptospira and leptospirosis, ADLER, B (ed.), Springer.
  47. ELLIS, W.A., BRYSON, D.G., NEILL, S.D., MCPARLAND, P.J. & MALONE, F.E., 1983. Possible involvement of leptospires in abortion, stillbirths and neonatal deaths in sheep. The Veterinary Record, 112, 291–293.
  48. ELLIS, W.A., BRYSON, D.G., O’BRIEN, J.J. & NEILL, S.D., 1983. Leptospiral infection in aborted equine foetuses. Equine Veterinary Journal, 15, 321–324.
  49. ELLIS, W.A., HUSTAS, L., ROBERTSON, G.M. & MAYBERRY, C., 1984. Kidney disease of sheep infected by leptospires of the Serjoe serogroup. Australian Veterinary Journal, 61, 304–305.
  50. ELLIS, W.A., MCPARLAND, P.J., BRYSON, D.G. & CASSELLS, J.A., 1986. Prevalence of Leptospira infection in aborted pigs in Northern Ireland. The Veterinary Record, 118, 63–65.
  51. ELLIS, W.A., MCPARLAND, P.J., BRYSON, D.G. & MCNULTY, M.S., 1985. Leptospires in pig urogenital tracts and foetuses. The Veterinary Record, 117, 66–67.
  52. ELLIS, W.A., O’BREIN, J.J., PEARSON, J.K.L. & COLLINS, D.S., 1976. Bovine leptospirosis: Infection by the Hebdomadis serogroup and mastitis. The Veterinary Record, 99, 368–370.
  53. ELLIS, W.A., O’BRIEN, J.J., NEILL, S.D., FURGUSON, H.W. & HANNA, J., 1982. Bovine leptospirosis: Microbiological and serological findings in aborted foetuses. The Veterinary Record, 110, 147–150.
  54. ELLIS, W.A., SONGER, J.G., MONTGOMERY, J. & CASSELLS, J.A., 1986. Prevalence of Leptospira interrogans serovar hardjo in the genital and urinary tracts of non-pregnant cattle. The Veterinary Record, 118, 11–13.
  55. ELLIS, W.A., 1999. Equine leptospirosis. Proceedings of the Eighth International Conference on Equine Infectious Diseases, Dubai, 23–26 March 1998, 155–158.
  56. FABER, N., CRAWFORD, M., LEFEBVRE, R. B., BUYUKMIHCI, N. C., MADIGAN, J. E., & WILLITS, N. H., 2000. Detection of Leptospira spp.in the aqueous humor of horses with naturally acquired recurrent uveitis. Journal of Clinical Microbiology, 38(7), 2731–2733.
  57. FAINE, S., ADLER, B., BOLIN, C. & PEROLAT, P., 1999. Leptospira and Leptospirosis. Medisci, Melbourne.
  58. FAINE, S., 1982. Guidelines for the Control of Leptospirosis. WHO offset publication No. 6. Geneva: World Health Organization.
  59. FENNESTAD, K.L. & BORG-PETERSEN, C., 1958. Fetal leptospirosis and abortion in cattle. Journal of Infectious Diseases, 102, 227–236.
  60. FENNESTAD, K.L. & BORG-PETERSEN, C., 1962. Antibody and plasma cells in bovine fetuses infected with Leptospira saxkoebing. Journal of Infectious Diseases, 110, 63–69.
  61. FERESU, S.B., 1987. Serological survey of leptospiral antibodies in cattle in Zimbabwe. Tropical Animal Health and Production, 19, 209–214.
  62. FERGUSON, L.C., LOCOCO, S., SMITH, H.R. & HAMDY, A.H., 1956. The control and treatment of swine leptospirosis during a naturally occurring outbreak. Journal of the American Veterinary Medical Association, 129, 263.
  63. GANOZA, C. A., MATTHIAS, M. A., SAITO, M., CESPEDES, M., GOTUZZO, E., & VINETZ, J. M., 2010. Asymptomatic renal colonization of humans in the peruvian Amazon by Leptospira. PLoS Neglected Tropical Diseases, 4(2), e612.
  64. GLOSSER, J.W., SULZER, C.R., REYNOLDS, G.C. & WHITTSETT, D.K., 1975. Isolation of leptospiral serotype szwazizak from dairy cattle in Oregon. Proceedings of the United States 78th Annual Meeting Animal Health Association, 1974, 78, 119–125.
  65. GORDON SMITH, C.E. & TURNER, L.H., 1961. The effect of pH on the survival of leptospires in water. Bulletin of the World Health Organisation, 24, 35–43.
  66. GORDON, L.M., 1980. Isolation of Leptospira interrogans hardjo from sheep. Australian Veterinary Journal, 56, 348–349.
  67. GRÉGOIRE, N., HIGGINS, R. & ROBINSON, Y., 1987. Isolation of leptospires from nephritic kidneys of beef cattle at slaughter. American Journal of Veterinary Research, 48, 370–371.
  68. GSELL, O., 1944. The history of leptospirosis: 100 years. Zentralblatt für Bakteriologische und Mikrobiologische Hygiene, 257, 473–478.
  69. HAMOND, C., MARTINS, G., LAWSON-FERREIRA, R., MEDEIROS, M., & LILENBAUM, W., 2013. The role of horses in the transmission of leptospirosis in an urban tropical area. Epidemiology and Infection, 141(1), 33–35.
  70. HAMOND, C., MARTINS, G., LOUREIRO, A.P., PESTANA, C., LAWSON-FERREIRA, R., MEDEIROS, M. A, & LILENBAUM, W., 2013. Urinary PCR as an increasingly useful tool for an accurate diagnosis of leptospirosis in livestock. Veterinary Research Communications.
  71. HAMOND, C., MARTINS, G., MEDEIROS, M.A. & LILENBAUM, W., 2013. Presence of Leptospiral DNA in Semen Suggests Venereal Transmission in Horses. Journal of Equine Veterinary Science, 33(12), 1157–1159.
  72. HAMOND, C., PESTANA, C. P., MEDEIROS, M.A. & LILENBAUM, W., 2015. Genotyping of Leptospira directly in urine samples of cattle demonstrates a diversity of species and strains in Brazil. Epidemiology and Infection, 1–4.
  73. HANSON, L.E. & BRODIE, B.O., 1967. Leptospira hardjo infections in cattle. Proceedings of the 71st Annual Meeting of the United States Livestock Sanitary Association, 71, 210–215.
  74. HANSON, L.E. & FERGUSON, L.C., 1975. Leptospirosis. In: DUNNE, H.W. & LEMAN, A.D.,(eds). Diseases of Swine. Ames, Iowa: Iowa State University Press.
  75. HANSON, L.E. & TRIPATHY, D.N., 1981. In: LEMAN, A.D., GLOCK, R.D., MENGELING, W.L., PENNEY, R.H.C., SCHOLL, E. & STRAW, B., (eds.). Diseases of Swine. 5th Edition. Ames, Iowa: Iowa State University Press.
  76. HANSON, L.E., 1976. Pathogenesis of leptospirosis. In: JOHNSON, R.C., (ed). The Biology of Parasitic Spirochaetes. New York: Academic Press, Inc.
  77. HANSON, L.E., MARTIN, R.J., GIBBONS, R.N. & SCHAURRENBERGER, P.R., 1969. Equine leptospirosis. Proceedings of the 79th Annual Meeting, United States Animal Health Association.
  78. HARRINGTON, R., 1975. Leptospiral antibodies in serum from cattle, swine, horses, deer, sheep and goats: 1973–1974. American Journal of Veterinary Research, 36, 1367–1370.
  79. HARSTKEERL, R.A., COLLARES-PEREIRA, M., & ELLIS, W.A., 2011. Emergence, control and re-emerging leptospirosis: dynamics of infection in the changing world. Clinical Microbiology and Infection, 17(4), 494–501.
  80. HARTMAN, E.G., 1986. Detection of leptospira antibodies by the solid phase ELISA. In: ELLIS, W.A. & LITTLE, T.W.A., (eds.). The Present State of Leptospirosis Diagnosis and Control. Netherlands: Martinus Nijhoff Publishers.
  81. HATHAWAY, S.C. & LITTLE, T.W.A., 1981. Prevalence and clinical significance of leptospiral antibodies in pigs in England. The Veterinary Record, 108, 224–228.
  82. HATHAWAY, S.C., 1981. Leptospirosis in New Zealand: An ecological view. New Zealand Veterinary Journal, 27, 109–112.
  83. HEISE-PAVLOV, P.M. & HEISE-PAVLOV, S.R., 2003. Feral pigs in tropical lowland rainforest of northeastern Australia : ecology, zoonoses and management. Wildlife Biology, 9(Suppl. 1), 21–27.
  84. HELLSTROM, J.B. & MARSHALL, R.B., 1978. Survival of Leptospira interrogans serovar pomona in an acidic soil under simulated New Zealand field conditions. Research Veterinary Science, 25, 29–33.
  85. HERR, S. & WINNEN, G.M., 1983. The first isolation of Leptospira interrogans serovar pomona from cattle in Botswana. Journal of the South African Veterinary Association, 54, 83–84.
  86. HERR, S., RILEY, A.E., NESER, J.A., ROUX, D. & DE LANGE, J.F., 1982. Leptospira pomona associated with abortion in cattle. Onderstepoort Journal of Veterinary Research, 49, 57–62.
  87. HERR, S., 1987. Onderstepoort Veterinary Institute, South Africa. Unpublished data.
  88. HESTERBERG, U.W., BAGNALL, R., PERRETT, K., BOSCH, B., HORNER, R. & GUMMOW, B., 2008. A serological prevalence survey of leptospirosis in cattle of rural communities in the province of KwaZulu-Natal, South Africa. Journal of the South African Veterinary Association, 79(1), 15–18.
  89. HIGGINS, R.J., HARBOURNE, J.F., LITTLE, T.W.A. & STEVENS, A.E., 1980. Mastitis and abortion in dairy cattle associated with Leptospira of the serotype hardjo. The Veterinary Record, 107, 307–310.
  90. HO, H.F. & BLACKMORE, D.K., 1979. Effects of chilling and freezing on Leptospira interrogans serovar pomona in naturally infected pig kidneys. New Zealand Veterinary Journal, 27, 121–123.
  91. HODGES, R.T., 1974. Some observations on experimental Leptospira serotype pomonoa infection in sheep. New Zealand Veterinary Journal, 22, 151–154.
  92. HOUVIND-HOUGEN, K., 1986. Morphology of leptospires: Electron microscopic studies in relation to the classification of Leptospira. In: ELLIS, W.A. & LITTLE, T.W.A., (eds.). Present State of Leptospirosis Diagnosis and Control. Netherlands: Martinus Nijhoff Publishers.
  93. HUNTER, P., FLAMAND, J.R.B., MYBURGH, J. & VAN DER MERWE, S.M., 1988. Serological reactions to Leptospira species in game animals of northern Natal. Onderstepoort Journal of Veterinary Research, 55, 191–192.
  94. HUNTER, P., VAN DER VYVER, F.H., SELMER-OLSEN, A., HENTON, M.M., HERR, S. & DE LANGE, J.F., 1987. Leptospirosis as a cause of ‘white spot’ kidneys in South African pig abattoirs. Onderstepoort Journal of Veterinary Research, 54, 59–62.
  95. IDO, Y., HOKI, R., ITO, H. & WANI, H., 1917. The rat as a carrier of spirochaeta icterohaemorrhagiae. The causative agent of Weil’s disease. Journal of Experimental Medicine, 26, 341–353.
  96. INADA, R., IDO, Y., HOKI, R., KANEKO, R. & ITO, H., 1916. The aetiology, mode of infection and specific therapy of Weil’s disease (Spirochaetosis icterohaemorrhagica). Journal of Experimental Medicine, 23, 377–401.
  97. INZANA, T.J. & DAWE, D.L., 1979. Experimentally induced Leptospira interrogans autumnalis infections in young swine. American Journal of Veterinary Research, 40, 1355–1358.
  98. JOBBINS, S.E. & ALEXANDER, K.A., 2015. Evidence of Leptospira sp. infection among a diversity of African wildlife species: beyond the usual suspects. Transactions of the Royal Society of Tropical Medicine and Hygiene, 109(5), 349–351.
  99. JOBBINS, S.E. SANDERSON, C.E., & ALEXANDER, K.A., 2013. Leptospira interrogans at the human-wildlife interface in northern Botswana: A newly identified public health threat. Zoonoses and Public Health.
  100. JOHNSON, R.C. & FAINE, S., 1986.Bergey’s Manual of Systematic Bacteriology. Vol. I. Baltimore, London: Krieg, Holt, Williams & Wilkins.
  101. JOHNSON, R.C. & ROGERS, P., 1964. 5-Fluorouracil as a selective agent for growth of leptospirae. Journal of Bacteriology, 87, 422–426.
  102. JOHNSON, R.C., 1976. Comparative spirochaete physiology and cellular composition. In: JOHNSON, R.C., (ed.). Biology of Parasitic Spirochaetes. New York, San Francisco, London: Academic Press, Inc.
  103. JUBB, K.V.F., KENNEDY, P.C. & PALMER, N., 1985. Pathology of Domestic Animals. 3rd edition. New York: Academic Press.
  104. KELLERMAN, T.S., COETZER, J.A.W. & NAUDÉ, T.W., 1988.Plant Poisonings and Mycotoxicoses of Livestock in Southern Africa. Cape Town: Oxford University Press Southern Africa.
  105. KIKTENKO, V.S., BALASHOV, N.G. & RODINA, V.N., 1976. Leptospirosis infection through insemination of animals. Journal of Hygiene, Epidemiology, Microbiology and Immunology, 20, 207–213.
  106. KILLINGER, A.H., TAYLOR, P.L., HUHN, R.G., HANSON, L.E. & MANSFIELD, M.E., 1976. Immunity to leptospirosis: Renal changes in vaccinated cattle given challenge inoculum. American Journal of Veterinary Research, 37, 93–94.
  107. KIRSCHNER, L. & MAGUIRE, T., 1957. Survival of Leptospira outside their hosts. New Zealand Medical Journal, 56, 385–391.
  108. KITSON-PIGGOT, A.W. & PRESCOTT, J.F., 1987. Leptospirosis in horses in Ontario. Canadian Journal of Veterinary Research, 51, 44–451.
  109. KLARENBEEK, A. & SCHUFFNER, W., 1933. Het voorkomen van een afwykend Leptospiras in Nederland. Geneeskunde, Nederlandse Tijdschriften, 77, 4271–4276.
  110. LAUERMAN, L.H., KISER, D.D. & LONG, I.R., 1983. Leptospira tarassovi in Alabama cattle: Serologic evidence. Proceedings of the 87th Annual Meeting of the United States Animal Health Association, 87, 193–198.
  111. LEVETT, P.N., 2001. Leptospirosis. Clinical Microbiology Reviews, 14(2), 296–326.
  112. LILENBAUM, W. & MARTINS, G., 2014. Leptospirosis in cattle: a challenging scenario for the understanding of the epidemiology. Transboundary and Emerging Diseases, 61 Suppl 1, 63–68.
  113. LILENBAUM, W., VARGES, R., RISTOW, P., CORTEZ, A., SOUZA, S.O., RICHTZENHAIN, L.J. & VASCONCELLOS, S.A., 2009. Identification of Leptospira spp. carriers among seroreactive goats and sheep by polymerase chain reaction. Research in Veterinary Science, 87(1), 16–19.
  114. LITTLE, T.W.A. & SALT, G.F.H., 1976. Isolation of a leptospira of the Pomona serogroup from a field vole. The Veterinary Record, 97, 56.
  115. LITTLE, T.W.A., RICHARDS, M.S., HUSSAINI, S.N. & JONES, T.D., 1980. The significance of leptospiral antibodies in calving and aborting cattle in the south west England. The Veterinary Record, 106, 221–224.
  116. LOCOCO, S., BOHL, E.H. & SMITH, H.R., 1958. Treatment of porcine leptospiruria. Journal of the American Veterinary Medical Association, 132, 251–253.
  117. MARTINS, G. & LILENBAUM, W., 2014. Leptospirosis in sheep and goats under tropical conditions. Tropical Animal Health and Production, 46(1), 11–17.
  118. MARTINS, G., BRANDÃO, F.Z., HAMOND, C., MEDEIROS, M. & LILENBAUM, W., 2012. Diagnosis and control of an outbreak of leptospirosis in goats with reproductive failure. Veterinary Journal (London, England : 1997), 193(2), 600–601.
  119. MARTINS, G., PENNA, B. & LILENBAUM, W., 2010. Maintenance of Leptospira infection in cattle under tropical conditions. The Veterinary Record, 167(16), 629–630.
  120. MARTINS, G., PENNA, B. & LILENBAUM, W., 2012. Differences between seroreactivity to leptospirosis in dairy and beef cattle from the same herd in Rio de Janeiro, Brazil. Tropical Animal Health and Production, 44(3), 377–378.
  121. MAXIE, G., 2015. Jubb, Kennedy & Palmer's Pathology of Domestic Animals, 6th Edition. Elsevier
  122. MCGUIRE, C.D. & MEYERS, D.M., 1957. Leptospirosis in Egypt. American Journal of Tropical Medicine and Hygiene, 6, 532–539.
  123. MCKEOWN, J.D. & ELLIS, W.A., 1986. Leptospira hardjo agalactia in sheep. The Veterinary Record, 118, 482.
  124. MERCY, A.R., PEET, R.L. & HUSTAS, L., 1988. Macroscopic kidney lesions in slaughtered pigs as an indication of current leptospiral infections. Australian Veterinary Journal, 65, 35–36.
  125. MGODE, G.F., MHAMPHI, G.G., KATAKWEBA, A.S. & THOMAS, M., 2014. Leptospira infections in freshwater fish in Morogoro, Tanzania: A hidden public health threat. Tanzania Journal of Health Research, 16(2).
  126. MGODE, G.F., MACHANG’U, R.S., MHAMPHI, G.G., KATAKWEBA, A., MULUNGU, L.S., DURNEZ, L., LEIRS, H., HARTSKEERL, R.A. & BELMAIN, S.R., 2015. Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts. PLoS Neglected Tropical Diseases, 9(12).
  127. MICHNA, S.W., 1965. Further observations on abortion of the sow due to infections by Leptospira canicola. The Veterinary Record, 77, 802–810.
  128. MILLAR, K.R., HODGES, R.T., SHEPPARD, A.D. & HAMMINGTON, M.W., 1977. Clinical and biochemical changes in sheep inoculated with Leptospira interrogans serotype pomona. New Zealand Veterinary Journal, 25, 203–207.
  129. MINETTE, H.P., 1983. Leptospirosis in poikilothermic vertebrates: A review. International Journal of Zoonoses, 10, 111–121.
  130. MOCH, R.W., EBNER, E.E., BARSOUM, I.S. & BOTROS, A.M., 1975. Leptospirosis in Ethiopia: A serological survey in domestic and wild animals. Journal of Tropical Medicine and Hygiene, 78, 38–42.
  131. MORSE, E.V., MORTER, R.L., LANGHAM, R.F., LUNDBERG, A. & ULLREY, E., 1957. Experimental ovine leptospirosis: Leptospira pomona infection. Journal of Infectious Diseases, 101, 129–136.
  132. MORSI, H.M., SHIBLEY, G.F. & STROTHER, H.L., 1973. Renal leptospirosis: Challenge exposure of vaccinated and non-vaccinated cattle to Leptospira icterohaemorrhagiae and Leptospira canicola. American Journal of Veterinary Research, 34, 175–179.
  133. NEIL, S.D., REID, R.L., WEATHERUP, S.T.C. & ELLIS, W.A., 1986. The use of aminopeptidase activity in leptospira identification. In: ELLIS, W.A. & LITTLE, T.W.A.,(eds.). The Present State of Leptospirosis Diagnosis and Control. Netherlands: Martinus Nijhoff Publishers.
  134. NERVIG, R.M. & GARRETT, L.A., 1979. Use of furosemide to obtain bovine urine samples for leptospiral isolation. American Journal of Veterinary Research, 40, 1197–1200.
  135. NERVIG, R.M., CHEVILLE, N.F. & BAETZ, A.L., 1978. Experimental infection of calves with Leptospira interrogans serotype szwajizak. American Journal of Veterinary Research, 39, 523–525.
  136. ORR, H.S. & LITTLE, T.W.S., 1979. Isolation of leptospira of the serotype hardjo from bovine kidneys. Research in Veterinary Science, 27, 343–346.
  137. PARMA, A.E., SANTISTEBAN, C.G., VILLALBA, J.S. & BOWDEN, R.A., 1985. Experimental demonstration of an antigenic relationship between Leptospira and equine cornea. Veterinary Immunology and Immunopathology, 10, 215–224.
  138. PEARSON, K.L., MACKIE, D.P. & ELLIS, W.A., 1980. Milk drop syndrome resulting from Leptospira hardjo. The Veterinary Record, 106, 135–136.
  139. PICARDEAU, M., 2017. Virulence of the zoonotic agent of leptospirosis: still terra incognita? Nature Reviews Microbiology, 15(5), 297–307.
  140. PICARDEAU, M., BERTHERAT, E., JANCLOES, M., SKOULOUDIS, A.N., DURSKI, K. & HARTSKEERL, R.A., 2014. Rapid tests for diagnosis of leptospirosis: Current tools and emerging technologies. Diagnostic Microbiology and Infectious Disease, 78(1), 1–8.
  141. PICARDEAU, M., BULACH, D.M., BOUCHIER, C., ZUERNER, R.L., ZIDANE, N., WILSON, P.J., CRENO, S., KUCZEK, E.S., BOMMEZZADRI, S., DAVIS, J.C., MCGRATH, A., JOHNSON, M.J., BOURSAUX-EUDE, C., SEEMANN, T., ROUY, Z., COPPEL, R.L., ROOD, J.I., LAJUS, A., DAVIES, J.K., MÉDIGUE, C. & ADLER, B., 2008. Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis. PLoS ONE, 3(2), e1607.
  142. REILLY, J.R., HANSON, L.E. & SIMON, J., 1970. Experimentally induced predator chain transmission of Leptospira grippotyphosa from rodents to wild marsupialia and carnivora. American Journal of Veterinary Research, 31, 1443–1448.
  143. RINGEN, L.M. & BRACKEN, F.K., 1956. Studies on bovine leptospirosis. Journal of the American Veterinary Medical Association, 129, 266–271.
  144. RIS, D.R. & HAMEL, K.L., 1978. The detection of leptospirae in cattle urine. New Zealand Veterinary Journal, 26, 246, 255–256.
  145. RISTOW, P., BOURHY, P., KERNEIS, S., SCHMITT, C., PREVOST, M.C.C., LILENBAUM, W. & PICARDEAU, M., 2008. Biofilm formation by saprophytic and pathogenic leptospires. Microbiology, 154(5), 1309–1317.
  146. ROBERTS, S.J., 1958. A study of leptospirosis in a large artificial insemination stud. Cornell Veterinarian, 48, 363–371.
  147. SANHUEZA, J.M., HEUER, C. & WEST, D., 2013. Contribution of Leptospira, Neospora caninum and bovine viral diarrhea virus to fetal loss of beef cattle in New Zealand. Preventive Veterinary Medicine, 112(1–2), 90–98.
  148. SCHMITZ, J.A., COLES, B.M. & SHIRES, G.M., 1981. Fatal hemolytic disease in sheep attributed to Leptospira interrogans serotype hardjo infection. Cornell Veterinarian, 71, 175–182.
  149. SCHONBERG, A., 1986. Application of skin test and other diagnostic tests. In: ELLIS, W.A. & LITTLE, T.W.A.,(eds). Present State of Leptospirosis Diagnosis and Control. Netherlands: Martinus Nijhoff Publishers.
  150. SHEPHERD, A.J. & LEMAN, P.A., 1985. Bacterial surveillance of South African rodents. South African Journal of Science, 81, 302–308.
  151. SIMBIZI, V., SAULEZ, M.N., POTTS, A., LÖTTER, C. & GUMMOW, B., 2016. A study of leptospirosis in South African horses and associated risk factors. Preventive Veterinary Medicine, 134, 6–15.
  152. SKILBECK, N.W., FORSYTH, W.M. & DOHNT, M., 1988. Bovine leptospirosis: Microbiological and histological findings in cattle at slaughter. Australian Veterinary Journal, 65, 73–75.
  153. SLEIGHT, S.D. & WILLIAMS, J.A., 1961. Transmission of bovine leptospirosis by coition and artificial insemination. Journal of the American Veterinary Medical Association, 138, 151–152.
  154. SMITH, A.S., JONES, T.C. & HUNT, R.D., 1972. Veterinary Pathology. Philadelphia: Lea and Febiger.
  155. SMITH, B.P. & ARMSTRONG, J.M., 1975. Fatal haemolytic anemia attributed to leptospirosis in lambs. Journal of the American Veterinary Medical Association, 167, 739–741.
  156. SOUTH, P.J. & STOERNER, H.G., 1974. The control of outbreaks of leptospirosis in beef cattle by simultaneous vaccination and treatment with dihydrostreptomycin. Proceedings of the Annual Meeting of the United States Animal Health Association, 78, 126–130.
  157. STALHEIM, O.H.V., 1972. Swine leptospirosis certification: A proposal. Journal of the American Veterinary Medical Association, 161, 1244–1247.
  158. SUEPAUL, S.M., CARRINGTON, C.V, CAMPBELL, M., BORDE, G. & ADESIYUN, A.A., 2011. Seroepidemiology of leptospirosis in livestock in Trinidad. Tropical Animal Health and Production, 43(2), 367–375.
  159. SULLIVAN, N.D. & CALLAN, D.P., 1970. Isolation of Leptospira hardjo from cows with mastitis. Australian Veterinary Journal, 46, 123–125.
  160. SUWANCHAROEN, D., CHAISAKDANUGULL, Y., THANAPONGTHARM, W. & YOSHIDA, S., 2013. Serological survey of leptospirosis in livestock in Thailand. Epidemiology and Infection, 141(11), 2269–2277.
  161. SWAI, E. & SCHOONMAN, L., 2012. A survey of zoonotic diseases in trade cattle slaughtered at Tanga city abattoir: a cause of public health concern. Asian Pacific Journal of Tropical Biomedicine, 2(1), 55–60.
  162. SWAN, R.A., WILLIAMS, E.S. & TAYLOR, E.G., 1981. Clinical and serological observations on horses with suspected leptospirosis. Australian Veterinary Journal, 57, 528–529.
  163. SWART, K.S., CALVERT, K. & MENERY, C., 1982. The prevalence of antibodies to serovars of Leptospira interrogans in horses. Australian Veterinary Journal, 59, 25–27.
  164. SYKES, J.E., HARTMANN, K., LUNN, K.F., MOORE, G.E., STODDARD, R.A. & GOLDSTEIN, R.E., 2011. ACVIM Consensus Statement. Journal of Veterinary Internal Medicine, 25, 1–13.
  165. TARASSOFF, S., 1928. Trais éclosions d’épedemie de fiévre aquatique du Leptospirosis grippotyphosa aquatilis en URSS.Bulletin Office International Hygiene, 27, 928.
  166. TE BRUGGE, L.A. & DREYER, T., 1985. Leptospira interrogans serovar hardjo associated with bovine abortion in South Africa. Onderstepoort Journal of Veterinary Research, 52, 51–52.
  167. THIERMANN, A.B. & ELLIS, W.A., 1986. Identification of leptospires of veterinary importance by restriction endonuclease analysis. In: ELLIS, W.A. & LITTLE, T.W.A., (eds). The Present State of Leptospirosis Diagnosis and Control. Netherlands: Martinus Nijhoff Publishers.
  168. THIERMANN, A.B., 1984. Leptospirosis: Current development and trends. Journal of the American Veterinary Medical Association, 184, 722–725.
  169. THIERMANN, A.B., 1982. Experimental leptospiral infections in pregnant cattle with organisms of the Hebdomadis group. American Journal of Veterinary Research, 43, 780–784.
  170. TRIPATHY, D.N., HANSON, L.E., MANSFIELD, M.E. & THILSTED, J.P., 1985. Experimental infection of lactating goats with Leptospira interrogans serovars pomona and hardjo. American Journal of Veterinary Research, 46, 2512–2514.
  171. UHLENHUTH, P. & FROMME, W., 1916. Untersuchungen über die Aetiologie, Immunität und specifische Behandlung der Weilsche Krankheit (Icterus Infectiousus). Zeitschrift für Immunitätsforschung, 25, 317.
  172. VAN DER HOEDEN, J., 1966. Leptospiral antibodies in cold blooded animals. Annales de la Société Belge de Médicine Tropicale (Antwerpen), 46, 171-172.
  173. VAN DER HOEDEN, J., 1958. Leptospira canicola in cattle. Journal of Comparative Pathology, 65, 278–283.
  174. VAN DER HOEDEN, J., 1964. Zoonoses. Elsevier Publishing Company.
  175. VAN EYS, G.J., GRAVENKAMP, C., GERRITSEN, M.J., QUINT, W., CORNELISSEN, M.T., SHEGGET, J.T. & TERPSTRA, W.J., 1989. Detection of leptospires in urine by polymerase chain reaction. Journal of Clinical Microbiology, 27, 2258–2262.
  176. VAN RENSBURG, W.J.J., 1973. Isolasie van Leptospira canicola by varke en honde in Suid-Afrika. Journal of the South African Veterinary Association, 44, 435–436.
  177. VERMA, A., STEVENSON, B. & ADLER, B., 2013. Leptospirosis in horses. Veterinary Microbiology, 167(1-2), 61-66
  178. WANYANGU, S.W., PALMER, M.F., ZOCHOVSKI, W.J. & WAITKENS, S.A., 1987. Comparison of the DIFCO and Patoc 1 slide antigens in the screening of leptospirosis. Comparative Immunology and Microbial Infectious Diseases, 10, 155–161.
  179. WEIL, A., 1886. Über eine eigentumliche mit Miltztumor, Ikterus und Nephritis einhergehende akute Infektionskrankheit. Deutches Archiv für Klinische Medicin, 39, 209.
  180. WITNER, R.H., 1954. Periodic ophthalmia in horses. American Journal of Ophthalmology, 37, 961–967.
  181. WONG, M., KATZ, A.R., LI, D. & WILCOX, B.A., 2012. Leptospira infection prevalence in small mammal host populations on three Hawaiian islands. The American Journal of Tropical Medicine and Hygiene, 87(2), 337–341.
  182. XU, Y., ZHU, Y., WANG, Y., CHANG, Y.F., ZHANG, Y., JIANG, X., ZHUANG, X., ZHU, Y., ZHANG, J., ZENG, L., YANG, M., LI, S., WANG, S., YE, Q., XIN, X., ZHAO, G., ZHENG, H., GUO, X. & WANG, J., 2016. Whole genome sequencing revealed host adaptation-focused genomic plasticity of pathogenic Leptospira. Scientific Reports, 6, 20020.
  183. YOUNG, B.J., 1969. A reliable method for demonstrating spirochaetes in tissue sections. Journal of Medical Laboratory Technology, 26, 248–252.