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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. Prescription Animal Remedy (P.A.R) Class I. For use only under the authority or prescription of a veterinarian. Registered pursuant to the ACVM Act 1997, No’s A1948 and A3876. ®Registered trademark. Schering-Plough Animal Health Limited, 33 Whakatiki Street, Upper Hutt. 0800 800 543. LEP-511-2009.