Paper
Paper
Potential differences between Leptospira
serovars, host-adapted (Bratislava) and
incidental (Copenhageni), in determining
reproductive disorders in embryo transfer
recipient mares in Brazil
A. Pinna, G. Martins, C. Hamond, M. A. Medeiros, G. N. de Souza, W. Lilenbaum
The objective was to investigate potential differences between two Leptospira serovars,
host-adapted (Bratislava) and incidental (Copenhageni), in causing reproductive disorders in
mares. From August 2009 to March 2011, 608 adult recipient mares from eight studs located
in the state of Rio de Janeiro, Brazil, were screened for leptospirosis. These mares were
3–8 years of age, of various breeds, and were managed in a semiextensive system (embryo
transfer centres). According to the reproductive history of these mares, the studs were
categorised as Group A (357 mares: high prevalence of reproductive problems), and Group B
(251 mares: all pregnant). Of the 608 samples tested serologically, 273 (44.9 per cent) were
reactive (titres ≥200), predominantly against Bratislava (62.3 per cent of reactive mares).
In Group A, 247/357 sera (69.2 per cent) were reactive, with reactivity against Bratislava
(64.8 per cent of reactive). By contrast, in Group B, only 26/251 sera (10.4 per cent) were
reactive, the majority against Copenhageni (61.5 per cent of reactive). Seroreactivity against
Bratislava was more likely to be associated (P<0.001) with reproductive problems than
seroreactivity against Copenhageni; this association was true for early embryonic death
(P<0.001), perinatal death (P<0.01), and abortion (P<0.001). Additionally, 29 urine samples
were collected (from Group A mares) for Leptospira species PCR, of which 16 were positive
(55.2 per cent). We concluded that leptospirosis was associated with poor reproductive
performance in embryo recipients, with the majority due to host-adapted Bratislava.
Introduction
Leptospirosis in animals may present in various forms, depending
on host species, environmental conditions and the infecting serovar.
Whereas incidental serovars usually determine acute clinical syndromes including haematuria, fever, jaundice, anorexia and respiratory
distress, host-adapted serovars typically cause chronic or subclinical
infections (Yan and others 2010).
In horses, in addition to well-known ophthalmic alterations (eg, uveitis, which is also termed moon blindness), leptospirosis may lead to
serious reproductive disorders, including abortion, perinatal mortality,
Veterinary Record (2014)
A. Pinna, DVM, PhD,
G. Martins, DVM, MSc,
C. Hamond, DVM, MSc,
W. Lilenbaum, DVM, PhD,
Laboratory of Veterinary Bacteriology,
Universidade Federal Fluminense,
Niterói, RJ 24210-130, Brazil
M. A. Medeiros, DBSc, PhD,
Bio-Manguinhos, Oswaldo Cruz
Foundation, Brazilian Ministry of Health,
Rio de Janeiro, RJ 21040-360, Brazil
doi: 10.1136/vr.101444
G. N. de Souza, DVM, PhD,
Embrapa Dairy Cattle,
Juiz de Fora, MG 36038-330,
Brazil
E-mail for correspondence:
[email protected]
Provenance: not commissioned;
externally peer reviewed
Accepted 18 March 2014
placentitis and premature foaling, with substantial economic impacts
(Dixon and Coppack 2002, Pinna and others 2007). Abortion due
to leptospirosis is a consequence of fetal leptospiremia, since these
bacteria may cross the placental barrier (Sebastian and others 2005).
Nevertheless, not all infected horses have clinical signs of the disease;
subclinical forms are very common, particularly in endemic regions.
In this latter form of the infection, animals can carry leptospires and
spread the organism even in the absence of clinical signs (Houwers
and others 2011).
Regarding incidental infections, in North America (USA and
Canada), serovar Pomona seems to be predominant (Timoney and
others 2011), whereas in tropical countries, mainly Brazil, India,
Thailand, Vietnam, Australia and Barbados, serovars belonging to the
Icterohaemorrhagiae serogroup, for example, Copenhageni, are generally the most prevalent (Båverud and others 2009, Hamond and others
2011). Members of the Icterohaemorrhagiae serogroup are maintained
and transmitted by rodents, particularly Rattus norvegicus. By contrast,
Bratislava (Australis serogroup) is considered a host-adapted serovar in
horses and may be transmitted from one animal to another by direct or
indirect contact with contaminated urine (Ellis and others 1983).
Although serological evidence of this serovar has been reported
worldwide (Odontsetseg and others 2005, Pinna and others 2007,
Båverud and others 2009), there is a paucity of studies regarding
the real role of Bratislava in reproductive problems caused by to
May 24, 2014 | Veterinary Record
Paper
leptospirosis in horses. Probably due to the reported extreme difficulty in cultivating this bacterium (Bolin and Cassells 1992), this
serovar is very seldom isolated worldwide and has apparently never
been reported to have been isolated in Brazil. Consequently, nearly
all studies regarding the epidemiology of that serovar are based on
serological evidence, and therefore subject to inherent limitations,
mainly lack of sensitivity and specificity. Although reliable to differentiate among serogroups, microscopic agglutination test (MAT)
cannot distinguish serovars (OIE 2008). Additionally, seroreactivity
is not a guarantee that the agent is actively circulating on the farm.
In the current study, despite that limitation and in order to facilitate
interpretation, seroreactivity against serogroup Australis (Bratislava)
and Icterohaemorrhagiae (Copenhageni) were referred to as Bratislava
and Copenhageni, respectively.
Although not frequently described in horses, the role of Bratilsava
in reproductive disorders has been clearly demonstrated in pigs; it has
been isolated from the kidney and uterus in repeat-breeding sows (Ellis
and Thiermann 1986), from placentae and from weak newborn piglets (Bolin and Cassells 1992) and was associated with a prolonged
interval between farrowings and infertility (Ramos and others 2006).
Differences between leptospirosis determined by incidental or adapted
serovars, as well as the association of each serovar with specific reproductive disorders, have apparently never been described in horses.
The objective of the present study was to investigate potential differences between host-adapted (Bratislava) and incidental
(Copenhageni) Leptospira-serovars as the cause of reproductive disorders in recipient mares.
Materials and methods
Study population
From August 2009 to March 2011, 608 recipient mares from eight
studs located in four cities around Rio de Janeiro, Brazil, were screened
(serologically) for leptospirosis. Samples were collected during the rainfall season (October to March). The studied mares were 67±29 months
(range, 38–96 months), of various breeds, and were included in a semiextensive breed system, fed once a day in barns, and ad libitum at field.
It was noteworthy that all these mares had access to flooded areas of
the farm where they probably drank water. Additionally, as inclusion
criteria, those studs provided daily assistance by the resident veterinarian, including reproductive management based on ultrasonography
(US), adequate nutritional support; vaccination against herpesvirus
(seronegativity of all mares); absence of Streptococcus zooepidemicus (confirmed by bacteriological culture); and absence of a specific programme
of vaccination against leptospirosis (none of the mares had a history of
vaccination against leptospires). Since all mares from Groups A and B
were involved in an embryo transfer programme, hygienic and management conditions were similar. During this study, no specific control
or treatment measures for leptospirosis were conducted.
Transrectal ultrasongraphic assessment of the uterus and conceptus
was done at approximately 15, 45, 90, 180, and 270 days of pregnancy. To minimise perinatal deaths, mares were closely monitored
and managed during the foaling process. To identify mares with
endometritis, ultrasound and uterine cytology were conducted before
embryo transfer. According to the reproductive history of the mares
(data from the clinical veterinarian), the studs were categorised into
Group A (5 studs, 357 mares), with a high prevalence of reproductive
problems (embryonic recovery <50 per cent, embryonic death >10 per
cent, abortions >5 per cent, and frequent perinatal death), and Group
B (3 studs, 251 mares), previously selected based on all mares being
pregnant (confirmed by ultrasound).
In the second part of the study, from June 2011 to August 2011,
two studs from Group A with a high prevalence of seroreactive mares
(>40 per cent) were selected, and urine samples from 29 mares with
high titres (≥800) at serology were collected for bacteriological and
molecular assessments.
Sampling
Blood samples were collected (into evacuated tubes) by jugular
venipuncture. The vulva was carefully cleaned and urine samples (second voiding of urine) were collected in sterile vials. These
samples were immediately used to inoculate 10 per cent liquid
Veterinary Record | May 24, 2014
Ellinghausen-McCullough-Johnson-Harris medium (EMJH; Difco,
Detroit, Michigan, USA)/saline and transported to the laboratory at
room temperature. Urine was also chilled and transported to the laboratory for PCR.
Laboratory procedures
Serology
In the laboratory, blood samples were centrifuged (1000×g for 10 minutes) and examined for anti-Leptospira antibodies by MAT, as described
(OIE 2008). Briefly, the antigens were a panel of 24 strains (representing all described serogroups) of live Leptospira grown in liquid medium
EMJH (Difco), and free of contamination or self-agglutination. Titres
≥200 were considered reactive (Hamond and others 2013). The highest titre was designated the infective serogroup.
Bacteriology
For bacteriological culturing, samples were inoculated using a serial
dilution technique (to 10−2 and 10−3 dilutions), into Fletcher semisolid medium, which contained 300 mg/l 5-fluorouracil (Pharmacia,
Kalamazoo, Michigan, USA) and 20 mg/l nalidixic acid (Oxoid,
Basingstoke, UK), and incubated for 24 hours at 28°C. After 24 hours
of incubation, tubes were seeded into Fletcher semisolid medium
(Difco, Detroit, Michigan, USA) without antibiotics, incubated at
28–30°C, and examined by darkfield microscopy once weekly for
20 weeks (Lilenbaum and others 2007).
PCR
Prior to PCR, DNA was extracted with the Promega Wizard SV kit
genomic DNA Purification System (Madison, Wisconsin, USA).
The primers (Stoddard and others 2009) were LipL32-45F (5′-AAG
CAT TAC CGC TTG TGG TG-3′) and LipL32-286R (5′-GAA
CTC CCA TTT CAG CGA TT- 3′), with a referred specificity and
sensitivity of 100 per cent. For amplification reactions, 0.6 µM of
forward and reverse primers were used, as well as 1.0 U Taq polymerase, 10× reaction buffer (50 mMKCl, 75 mMTrisHCl - pH
9.0, (NH4) 2SO4 20 mM), 2.4 µM MgCl2, 0.3 mM deoxyribonucleotide triphosphates, and 3.5 µl of DNA (final volume, 25 µl).
The reaction was incubated at 94°C for 5 minutes for DNA denaturation, followed by 35 cycles of: denaturation at 94°C for 30 seconds,
annealing at 53°C for 30 seconds, extension at 72°C for 1 minute, and
a final extension at 72°C for 5 minutes. The products were separated on
2 per cent agarose gels and stained with ethidium bromide. A molecular
weight marker was included for assessing amplicon sizes, and positive
(DNA of Leptospira interrogans serovar Copenhagen strain L1-130) and
negative (ultrapure water) controls were applied. After electrophoresis,
gels were examined on an UV light transiluminator and photographed.
Reproductive parameters
Reproductive parameters were defined according to the history of each
mare prior to sample collections. Early embryonic death (≤45 days
of pregnancy), abortions (from 45 days to term) and perinatal death
(≤3 days after birth) were considered. Reproductive tract examinations
were done with transrectal palpation and ultrasound (Aloka SSD-500,
Aloka, Wallingford, Connecticut, USA). Mares were examined as necessary by ultrasound during the postbreeding period, and at approximately 15, 45, 90, 180, and 270 days of pregnancy.
Statistics
The results of overall seroreactivity, as well as the predominance of
serovars and associations with reproductive failures, were analysed
with Fisher’s Exact test, using GraphPad InStat V,3.05 (GraphPad
Software, San Diego, California, USA). Additionally, ORs were calculated. Analyses with a CI exceeding 95 per cent (P<0.05) were considered significant.
Results
Reproductive parameters
None of the mares had clinical systemic signs compatible with acute
leptospirosis. Regarding reproductive problems, 74 of 357 mares from
Group A had early embryonic death (20.7 per cent), whereas 78
(21.8 per cent) aborted (confirmed by veterinary observation and/or
Paper
ultrasonography) during the study period and 18 (5.0 per cent) had
perinatal death of the foals (direct veterinary observation; Table 1).
Overall, 170 (47.6 per cent) of mares in Group A had reproductive
problems, whereas there were no reproductive problems in any of the
mares in Group B.
Other possible causes of pregnancy failure, for example, twin pregnancies, abnormal implantation area or abnormal embryonic development were not identified. No postmortem examinations were done
on aborted foals.
100
90
Frequency of seroreacitive mares (%)
80
Serology
Of the 608 serum samples tested by MAT, 273 (44.9 per cent) were reactive, predominantly against Bratislava (170 sera, 62.3 per cent of reactive), but also against Copenhageni (103 sera, 37.7 per cent of reactive).
Cross-reactions occurred at low titres among various serogroups (from
the 24 tested), but were not sustained at higher dilutions. Cross-reactions
between Australis and Icterohaemorrhagiae serogroups were not noticed.
In Group A, 247 of 357 sera (69.2 per cent) were reactive, distributed
between reactivity against Bratislava (160 sera, 64.8 per cent of reactive) and Copenhageni (87 sera, 35.2 per cent of reactive). Regarding
the titres, 103 (41.7 per cent) sera had titres of 200, whereas 101 (40.9
per cent) had titres of 400, and 43 (17.4 per cent) sera had high titres
(≥800), combined for all studs tested.
In Group B, 26 of 251 sera (10.4 per cent) were reactive, against
Bratislava (10 sera, 38.5 per cent of reactive) or Copenhageni (16 sera, 61.5
per cent of reactive; Fig 1). In that group, three samples (11.5 per cent) had
titres of 400, whereas the remaining 23 (88.5 per cent) had titres of 200.
69.2%
Serovars
70
Bratislava
60
Copenhageni
24.4%
50
40
30
44.8%
20
10.4%
10
6.4%
0
4.0%
Group A
Studied groups
Group B
Bacteriology
FIG 1: Distribution of serovars in seroreactive mares from Rio de
Janeiro, Brazil with (Group A) or without (Group B) reproductive
problems
PCR
as well as other tropical (Donahue and others 1995, Léon and others 2006), and non-tropical countries (Odontsetseg and others 2005),
as well as the predominance of Bratislava. This serovar is considered
to be adapted to horses (Ellis and others 1983), and has already been
reported (serology) in the studied region (Pinna and others 2007).
Although further investigations are required, particularly those
concerning recovery of the agent from clinical cases, seroreactivity against this serovar has been broadly associated with reproductive failure in horses, often without other clinical signs (Pinna and
others 2010). Furthermore, the high prevalence of seroreactivity
for serovar Copenhageni was not surprising, since members of
Icterohaemorrhagiae serogroup represent the major agent of leptospirosis in many species, including horses in the tropics, particularly in
Brazil (Hamond and others 2011).
Apart from reproductive problems, it was noteworthy that mares
lacked other clinical signs of leptospirosis, including jaundice, fever or
ocular manifestations. Leptospirosis in horses is frequently subclinical
and commonly manifested only by reproductive failure (Houwers and
others 2011), although the causative organism is often not identified.
In that regard, culture requires a prolonged interval, is difficult, and has
a high incidence of false negatives (Pinna and others 2011); therefore,
serology is still widely used and represents the majority of reports.
Therefore, despite representing a major drawback of this study, the
absence of leptospiral recovery by culture is common.
The limitations of serology and bacteriology for detection of leptospirosis have been the greatest impetus for application of molecular tools,
None of the 29 urine samples were positive on darkfield examination
and no pure isolates were obtained.
There were 29 urine samples collected from two studs (14 and 15
samples, respectively). In these two studs, 7 of 14 (50 per cent) and
9 of 15 (60 per cent) of the urine samples were positive (pathogenic
Leptospira species-specific).
Association between serology and reproductive
parameters
Seroreactivity for leptospirosis was much more common (P<0.001)
in studs with reproductive problems (Group A) than those without
reproductive problems (Group B). In that regards, the probability of
a seroreactive mare having reproductive problems was 19.4 times
greater (OR) than a seronegative one (95% CI 12.2 to 30.9).
Reactivity against leptospirosis (independent of the causative serovar)
was associated with early embryonic death (P<0.001; OR 6.8; 95% CI
3.9 to 11.7), perinatal death (P<0.001; OR 13.2; 95% CI 3.7 to 46.4),
and abortion (P<0.001; OR 203.8; 95% CI 28.1 to 1479.9). Interestingly,
seroreactivity against Bratislava was more often associated with reproductive problems than seroreactivity against Copenhageni (P<0.001;
OR 3.6; 95% CI 2.5 to 5.3). This was true for early embryonic death
(P<0.001; OR 5.3; 95% CI 3.1 to 8.9), perinatal death (P<0.01; OR 4.5;
95% CI 1.6 to 12.5), and abortions (P<0.001; OR 2.3; 95% CI 1.9 to 3.0).
Discussion
The high rate of seroreactive mares was consistent with previous
reports in Brazil (Pinna and others 2007, Hamond and others 2011)
TABLE 1: Reproductive failures in 357 mares from five studs (Group A) with a history of reproductive problems in Rio de Janeiro, Brazil
Reproductive failures (%)
Stud
N° mares
EED
Abortion*
1
2
3
4
5
TOTAL
53
79
70
93
62
357
5 (9.4)
11 (13.9)
10 (14.3)
17 (18.3)
31 (50.0)
74 (20.7)
16 (30.2)
37 (46.8)
11 (15.7)
14 (15.1)
0
78 (21.8)
PD
7 (13.2)
5 (6.3)
6 (8.6)
0
0
18 (5.0)
Total RF
Pregnancy rate
28 (52.8)
53 (67.1)
27 (38.6)
31 (33.3)
31 (50.0)
170 (47.6)
47.2
32.9
61.4
66.7
50.0
*Occurred in all stages of pregnancy
EED, early embryonic death; PD, perinatal death; RF, reproductive failures
May 24, 2014 | Veterinary Record
Paper
namely PCR (Hamond and others 2012). In the current study, although
necropsy and histopathology of aborted foals was not done, and culturing of leptospires was unrewarding, PCR was valuable for confirming
the presence of the agent circulating among mares. We concluded that
PCR should be used more often to diagnose leptospirosis in horses and
to detect carriers of the bacterium; in that regard, PCR was recently
used for diagnosis of equine pregnancy losses caused by Leptospira species (Whitwell and others 2009, Timoney and others 2011) and, more
recently, our group used PCR to diagnose leptospirosis in an aborted foal
(Pinna and others 2011). Furthermore, PCR was used to determine the
role of Leptospira in the pathogenesis of recurrent uveitis in horses (Pearce
and others 2007, Gilger and others 2008). Nevertheless, we acknowledge
that the absence of a control group (Group B) for PCR was a drawback of
our study. Unfortunately, despite our repeated requests, owners did not
authorise further studies on seronegative-pregnant mares.
The association between seroreactivity and leptospirosis with
reproductive problems is clear and has been extensively reported, albeit
usually in outbreaks and often without a clear statistical significance
(Donahue and others 1995, Sebastian and others 2005, Szeredi and
Haake 2006, Pinna and others 2007, Pinna and others 2011). In the current study, mares with reproductive problems were significantly more
frequently seroreactive than those without reproductive problems.
Abortion, the most frequent reproductive problem associated with seroreactivity for leptospirosis, may be associated with placentitis (Szeredi
and Haake 2006), whereas early embryonic and perinatal death, even
when not generally perceived as being caused by leptospirosis, were also
more common in seroreactive mares, consistent with the few reports
regarding neonatal (Pinna and others 2007) or perinatal (Donahue and
others 1995) deaths. Some similar findings have been reported in horses
(Pinna and others 2010, Pinna and others 2012), deer (Subharat and others 2010), and cattle (Guitian and others 1999). Nevertheless, there are
other reasons related to reproductive failures in horses, for example, age
of mares; other infectious and non-infectious process; and inadequate
nutrition. These factors should not be neglected during the process of
diagnosis and reduction of reproductive failures in equine studs.
In our opinion, the most important outcome of the present study
was that association of seroreactivity to leptospirosis with reproductive problems was not the same for host-adapted (Bratislava) and incidental (Copenhageni) leptospiral serovars, since seroreactivity against
Bratislava had a much stronger association with reproductive problems than that against Copenhageni. In that regard, a mare seroreactive to Bratislava was 3.6 times more likely to have reproductive
disorders than a mare seroreactive to Copenhageni, although the latter were still more likely to have reproductive problems (all categories
studied) than seronegative mares. We consider that these results represent new knowledge regarding the association between leptospirosis
and reproductive problems in domestic animals, particularly in mares.
Additionally, although the relationship between host-adapted serovars
and reproductive problems has been highlighted since the 1980s, possible differences between host-adapted and incidental leptospirosis in
animal reproduction has been largely neglected in horses, although
reported in sows (Ramos and others 2006).
In conclusion, the present study provided evidence that seroreactivity
for leptospirosis, among other causes, may be associated with reproductive failures in horses, including, but not limited to, abortion. Furthermore,
it was noteworthy that mares reactive against Bratislava, a host-adapted
serovar, were more likely to have reproductive problems than those reactive against incidental serovars, for example, Copenhageni.
Acknowledgments
The authors express their gratitude to Professor J. Kastelic, L. A.
Nogueira, S. Vasconcellos, M. Alvarenga, and J. Jacob, for critical
reviews of the manuscript.
References
BÅVERUD, V., GUNNARSSON, A., ENGVALL, E. O., FRANZÉN, P. & EGENVALL, A.
(2009) Leptospira seroprevalence and associations between seropositivity, clinical disease
and host factors in horses. Acta Veterinaria Scandinavica 51, 1–10
BOLIN, C. A. & CASSELLS, J. A. (1992) Isolation of Leptospira interrogans serovars bratislava and hardjo from swine at slaughter. Journal of Veterinary Diagnostic Investigation 4,
87–89
DIXON, P. & COPPACK, R. (2002) Equine recurrent uveitis. Veterinary Record 150, 556
Veterinary Record | May 24, 2014
DONAHUE, J. M., SMITH, B. J., POONACHA, K. B., DONAHUE, J. K. & RIGSBY,
C. L. (1995) Prevalence and serovars of leptospira involved in equine abortions in
central Kentucky during the 1991–1993 foaling seasons. Journal of Veterinary Diagnostic
Investigation 7, 87–91
ELLIS, W. A., O’BRIEN, J. J., CASSELLS, J. A. & MONTGOMERY, J. (1983) Leptospiral
infection in horses in Northern Ireland: serological and microbiological findings. Equine
Veterinary Journal 15, 317–320
ELLIS, W. A. & THIERMANN, A. B. (1986) Isolation of Leptospira interrogans serovar
bratislava from sows in Iowa. American Journal of Veterinary Research 47, 1458–1460
GILGER, B. C., SALMON, J. H., YI, N. Y., BARDEN, C. A., CHANDLER, H. L.,
WENDT, J. A. & COLITZ, C. M. (2008) Role of bacteria in the pathogenesis of recurrent uveitis in horses from the southeastern United States. American Journal of Veterinary
Research 69, 1329–1335
GUITIAN, J., THURMOND, M. C., HIETALA, S. K. (1999) Infertility and abortion
among first-lactation dairy cows seropositive or seronegative for Leptospira interrogans
serovar hardjo. Journal of the American Veterinary Medical Association 215, 515–518
HAMOND, C., MARTINS, G., LAWSON-FERREIRA, R., MEDEIROS, M. A. &
LILENBAUM, W. (2013) The role of horses in the transmission of leptospirosis in an
urban tropical area. Epidemiology and Infection 141, 33–35
HAMOND, C., MARTINS, G., LILENBAUM, W. & MEDEIROS, M. A. (2012) PCR
detection of leptospiral carriers among seronegative horses. Veterinary Record 171,
105–106
HAMOND, C., MARTINS, G., REIS, J., KRAUS, E., PINNA, A. & LILENBAUM, W.
(2011) Pulmonary hemorrhage in horses seropositive to leptospirosis. Pesquisa Veterinária
Brasileira 31, 413–415
HOUWERS, D. J., GORIS, M. G., ABDOEL, T., KAS, J. A., KNOBBE, S. S., VAN
DONGEN, A. M., WESTERDUIN, F. E., KLEIN, W. R. & HARTSKEERL, R. A.
(2011) Agglutinating antibodies against pathogenic Leptospira in healthy dogs and horses
indicate common exposure and regular occurrence of subclinical infections. Veterinary
Microbiology 148, 449–451
LÉON, A., PRONOST, S., TAPPREST, J., FOUCHER, N., BLANCHARD, B., ANDRÉFONTAINE, G., LAUGIER, C., FORTIER, G. & LECLERCQ, R. (2006) Identification
of pathogenic Leptospira strains in tissues of a premature foal by use of polymerase chain
reaction analysis. Journal of Veterinary Diagnostic Investigation 18, 218–221
LILENBAUM, W., MORAIS, Z. M., GONÇALES, A. P., SOUZA, G. O.,
RICHTZENHAIN, L. & VASCONCELLOS, S. A. (2007) First isolation of leptospires
from dairy goats in Brazil. Brazilian Journal of Microbiology 38, 507–510
ODONTSETSEG, N., BOLDBAATAR, D., MWEENE, A. S. & KIDA, H. (2005)
Serological prevalence of Leptospira interrogans serovar Bratislava in horses in Mongolia.
Veterinary Record 157, 518–519
OIE (2008) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Paris: World
Organisation for Animal Health. pp. 254–255
PEARCE, J. W., GALLE, L. E., KLEIBOEKER, S. B., TURK, J. R., SCHOMMER, S. K.,
DUBIELIZIG, R. R., MITCHELL, W. J., MOORE, C. P. & GIULIANO, E. A. (2007)
Detection of Leptospira interrogans DNA and antigen in fixed equine eyes affected with
end-stage equine recurrent uveitis. Journal of Veterinary Diagnostic Investigation 19, 686–690
PINNA, A., MARTINS, G. & LILENBAUM, W. (2012) Leptospirosis and embryo recovery rate in mares. Veterinary Record 170, 60
PINNA, A. E., MARTINS, G., HAMOND, C., LILENBAUM, W. & MEDEIROS, M.
A. (2011) Molecular diagnostics of leptospirosis in horses is becoming increasingly
important. Veterinary Microbiology 153, 413
PINNA, M., MARTINS, G., FREIRE, I. & LILENBAUM, W. (2010) Seropositivity
to Leptospira interrogans serovar Bratislava associated to reproductive problems without significant biochemical or hematological alterations in horses. Ciência Rural 40,
2214–2217
PINNA, M. H., VARGES, R., ABREU, R. & LILENBAUM, W. (2007) Outbreak of
equine leptospirosis by s. Bratislava. Online Journal of Veterinary Research 11, 1–4
RAMOS, A. C., SOUZA, G. N. & LILENBAUM, W. (2006) Influence of leptospirosis on
reproductive performance of sows in Brazil. Theriogenology 66, 1021–1025
SEBASTIAN, M., GILES, R., ROBERTS, J., POONACHA, K., HARRISON, L.,
DONAHUE, J. & BENIRSCHKE, K. (2005) Funisitis associated with leptospiral abortion in an equine placenta. Veterinary Pathology 42, 659–662
STODDARD, R. A., GEE, J. E., WILKINS, P. P., MCCAUSTLAND, K. &
HOFFMASTER, A. R. (2009) Detection of pathogenic Leptospira spp. through TaqMan
polymerase chain reaction targeting the LipL32 gene. Diagnostic Microbiology and Infectious
Disease 64, 247–255
SUBHARAT, S., WILSON, P. R., HEUER, C. & COLLINS-EMERSON, J. M. (2010)
Investigation of localisation of Leptospira spp. in uterine and fetal tissues of non-pregnant and pregnant farmed deer. New Zealand Veterinary Journal 58, 281–288
SZEREDI, L. & HAAKE, D. A. (2006) Immunohistochemical identification and pathologic findings in natural cases of equine abortion caused by leptospiral infection.
Veterinary Pathology 43, 755–761
TIMONEY, J. F., KALIMUTHUSAMY, N., VELINENI, S., DONAHUE, J. M.,
ARTIUSHIN, S. C. & FETTINGER, M. (2011) A unique genotype of Leptospira interrogans serovar Pomona type kennewicki is associated with equine abortion. Veterinary
Microbiology 150, 349–353
WHITWELL, K. E., BLUNDEN, A. S., MILLER, J. & ERRINGTON, J. (2009) Two
cases of equine pregnancy loss associated with Leptospira infection in England. Veterinary
Record 165, 377–378
YAN, W., FAISAL, S. M., DIVERS, T., MCDONOUGH, S. P., AKEY, B. & CHANG,
Y. F. (2010) Experimental Leptospira interrogans serovar Kennewicki infection of horses.
Journal of Veterinary Internal Medicine 24, 912–917