Download Leptospirosis Diagnosis - Part 1: The Individual Animal

LEPTOSPIROSIS: THE HARD FACTS
Leptospirosis Update #4.1
Leptospirosis Diagnosis - Part 1:
The Individual Animal
Of the 6 endemic serovars in NZ*, only Pomona, Hardjo, Ballum and Copenhageni are associated with disease in cattle,
sheep and deer.
- Pomona causes abortion and milk drop in cows, and outbreaks of haemoglobinuria and death in young calves 1-3
and lambs4.
- Copenhageni and Ballum causes acute nephritis and death in calves5.
- Hardjo while a common cause of abortion, milk drop, still or weak births, and recently, infertility and early embryonic
death globally6,7 has not been shown to be a pathogen in sheep or cattle in NZ. In deer it is associated with reduced
reproduction and weight gain in deer8.
Part 1 of this Hardfacts discusses individual animal diagnosis due to these serovars.
Part 2 discuses herd level diagnosis.
A diagnosis of Leptospirosis depends on good clinical and vaccination history and the thoughtful use of diagnostic
tests. The ease of diagnosis often depends on the serovar involved.
All current diagnostic tests have their strengths and weaknesses (See Table 1) and generally no single test can lead to
a definitive serovar specific diagnosis. To maximize the likelihood of reaching a diagnosis contact the laboratory prior
to submission to ensure suitable samples are appropriately collected.
Haemoglobinuria, Nephritis or death
in young animals due to Pomona
or Copenhageni.
High antibody titers (≥ 1:1600) in serum or fluids
can be considered confirmatory. Lower titres are
suggestive; PCR of urine, kidney or pleural fluid can be
used for confirmation. Note asymptomatic infection
with either Pomona or Copenhageni is reported9.
Antibody determination of dams, recent arrivals and other
in-contact animals may be used to shed some light on the
source of the infection.
History, presentation, gross post-mortem and histological
findings are often strongly suggestive of Leptospirosis.
The history usually includes a lack of vaccination, recent
introduction of stock and exposure to surface water.
Post-mortem may reveal jaundice, dark urine (NB can
confirm as haemoglobinuria by dipstick), swollen
dark kidneys with diffuse haemorrhagic and pale areas.
The liver is often swollen and yellowish.
Note if MAT titres are determined early in the serological
response, cross reaction between serovars is common,
and the highest titer may not be the infecting serovar.
It is best to repeat testing in samples taken 2-3 weeks
later and sample in contact animals to confirm the
serovar involved.
See Leptospirosis in NZ Dairy Herds Technical Bulletin for details.
*
Table 1: Summary of leptospiral diagnostic methods
Method
Samples
Material
detected
ID
serovar
Comments
Dark-field
microscopic
Blood and urine
Live leptospires
no
Rapid, but low sensitivity, and requires a high
degree of skill.
Culture
Blood, urine, csf, kidney
or other tissues
Live leptospires
yes
Slow (weeks to months).
Polymerase
chain reaction
(PCR) assay
Urine, tissues e.g. kidney
placenta, foetal fluids
Leptospiral
DNA
no
Very specific and sensitive. False-negatives due to inhibitors
in tissues preventing DNA amplification. False-positives as
exquisitely sensitive to contamination with leptospiral DNA.
Microscopic
agglutination
test (MAT)
Serum, fluid from tissues
or body cavities
Agglutinated
antibodies
yes
Highly specific. In acute cases animals may
die before they seroconvert.
Histology +/Silver staining
Any body organ
e.g. kidney, placenta.
Intact
leptospires
no
Non-specific and silver staining will identify
intact organisms only. Poor sensitivity.
Leptospirosis Update #4.1
Stillbirth or Abortion
Pomona and Hardjo (although not confirmed in NZ)
can cause abortion at any stage of pregnancy, however
usually they induce abortion in the last ½ of gestation.
Aborting dams are often asymptomatic, however fever,
anorexia, agalactia, malaise, dark urine, jaundice and
anaemia are reported10. The interval from infection
to abortion varies with serovar, with Pomona induced
abortion occurring within 1-2 weeks; whereas Hardjo
infected animals may abort several months after
infection. MAT titres are very variable. A Pomona titre
≥1:1600 or a Hardjo titre ≥1:200, along with typical
signs and history are considered strongly suggestive;
however, Hardjo titres are often lower or negative
(i.e. <1:50) as infection can occur months before
abortion. A NZ researcher states the arbitrary cutoff
of 1:50 used by labs is too high for Hardjo and
suggested a dilution of 1:25 should be used11.
Paired sera are also often not diagnostic as the
serological peak has occurred before the animal aborts.
Furthermore, as many animals in the same mob may
have similar titres without evidence of abortion and
titres due to natural infection can last for up to two
years, confirmation of diagnosis should be made by PCR
or culture of urine, or if available renal tissue collected
at slaughter. Ideally, urine should be collected after
the injection of furosemide, as the increased glomerular
filtration flushes more leptospires from the kidneys
and the dilute urine enhances their survival12.
In interpreting a positive urine sample, note that
some cattle have been shown to excrete Hardjo
for 542 days and it has been speculated for life13.
Postmortem examination of foetuses usually
reveals only non-specific autolytic findings, although
subcutaneous jaundice, foci of renal tubular necrosis
and vascular lesions in many organs are observed
occasionally14. Placenta, if available, is histologically
non-diagnostic, however some researchers have found
the placenta the most likely organ to be MAT or PCR
positive13. Identification of leptospires by silver staining
in aborted tissues is diagnostic, but not serovar specific.
Pooled foetal fluid from the pericardial, thoracic and
abdominal cavities, or pooled foetal kidney, adrenal
gland, lung and placenta are useful tissues to examine
for leptospiral antigen by culture or PCR. For Hardjo, a
prominent researcher states for MAT on foetal fluids
dilutions should be started a 1:10, in contrast to the usual
starting dilution of 1:5012.
Reproductive failure and other production
related diseases associated with Hardjo
As reproductive or production loss associated with
Hardjo generally manifests subclinically, and in most
unvaccinated herds the within herd seroprevalence
is high, it is difficult to diagnose Leptospirosis as
the cause. Conformation of endemic Hardjo within a
herd is the first step (See Part 2). Secondly, showing
evidence of seroconversion during mating or lactation
is useful. Lastly, response to Leptavoid® vaccination has
been used in NZ deer8 and lactating cattle in the UK15
as a means of gauging the impact of infection.
Author
Dr. John Moffat
References
1. Carter, M.E., et al., Leptospirosis: II. Investigation of clinical disease in dairy cattle in the Waikato district of NZ. NZVJ, 1982. 30(9): 136-40.
2. Cordes, D.O., et al., Leptospirosis: I. Clinical investigation of the infection in dairy cattle in the Waikato district of NZ. NZVJ, 1982. 30(8): 122-4.
3. Hathaway, S.C. & D.K. Blackmore, Ecological aspects of the epidemiology of infection with leptospires of the Ballum serogroup in the black rat (Rattus rattus)
and the brown rat (Rattus norvegicus) in NZ. Jou of Hygiene, 1981. 87(3): 427-36.
4. Vermunt, J., et al., Observations on three outbreaks of Leptospira interrogans serovar pomona infection in lambs. NZVJ, 1994. 42(4): 133-136.
5. Dodd, D. & D. Brakenridge, Leptospira icterohaemorrhagiae infection in calves. NZVJ, 1960. 8(4): 71-76.
6. Dhaliwal, G.S., et al., Reduced conception rates in dairy cattle associated with serological evidence of Leptospira interrogans serovar hardjo infection. Vet Rec, 1996. 139(5): 110-4.
7. Dhaliwal, G.S., R.D. Murray, & W.A. Ellis, Reproductive performance of dairy herds infected with Leptospira interrogans serovar hardjo relative to the year of diagnosis. Vet Rec, 1996. 138(12): 272-6.
8. Ayanegui-Alcerreca, M., et al., Leptospirosis in farmed deer in New Zealand: A review. NZVJ, 2007. 55(3): 102-108.
9. Ris, D.R., D.E. Lake, & J.T. Holland, The isolation of Leptospira serotypes Copenhageni and Ballum from healthy calves. NZVJ, 1973. 21: 218-220.
10.Radostitis, O.M., et al., Veterinary Medicine. 9 ed. 2000, London: WB Saunders.
11.Blackmore, D.K. Dairy Cattle Production, Proceedings No 78. 1985. University of Sydney.
12.Grooms, D.L. & C.A. Bolin, Diagnosis of fetal loss caused by bovine viral diarrhea virus and Leptospira spp. Vet.Clin. Nth Am-Food Anim Pract, 2005. 21(2): 463-472.
13.Ellis, W.A., Bovine Leptospirosis in the tropics. PVM, 1984. 2: 411-421.
14.Ellis, W.A., Leptospirosis as a cause of reproductive failure. Vet Clin Nth Am Food Anim Pract, 1994. 10(3): 463-78.
15.Dhaliwal, G.S., et al., Effect of vaccination against Leptospira interrogans serovar hardjo on milk production and fertility in dairy cattle. Vet Rec, 1996. 138(14): 334-5.
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