Letter to the Editor Rapid and efficient diagnosis of leptospirosis in an aborted foal by PCR of gastric juice To the Editor, Our group has recently demonstrated that molecular diagnostics of leptospirosis in horses is becoming increas- ingly important (Pinna et al., 2011). At this moment, we would like to share our successful experience employing the molecular diagnostic (PCR) of leptospiral infection in the gastric juice of an aborted foal. Leptospirosis in horses is an important disease of the reproductive sphere, since it may lead to the birth of weak foals, stillbirth or neonatal mortality and abortion, usually around the six month of pregnancy (Timoney et al., 2011). Nevertheless, not all infected animals present with the acute disease, and subclinical forms are common in endemic regions (Houwers et al., 2011). Titers to several serovars have been reported in horses. While in North America and other temperate countries serovar Pomona seems to be predominant (Timoney et al., 2011), in tropical countries serovars belonging to the Icterohaemorrhagiae serogroup, such as Icterohaemorrha- giae or Copenhageni, tend to be most prevalent (Hamond et al., 2012). Although useful for herd diagnosis, serology not always is a reliable tool for detecting individual cases of leptospirosis, since not all animals produce detectable titres of specific agglutinins (Houwers et al., 2011). The isolation procedures are cumbersome, time consuming and require fresh samples with a significant concentration of leptospires (Hamond et al., 2012). Therefore, molecular tools such as polymerase chain reaction (PCR) have been increasingly been employed for diagnosis leptospirosis as a reproductive disease in horses. Leptospiral DNA has been detected in tissues (kidney and liver) (Whitwell et al., 2009) and in thoracic fluid of aborted fetuses (Pinna et al., 2011). A total of 16 Thoroughbred mares of the same flock were studied. The horses ranged from five to seven years old. Despite it is an endemic area for leptospirosis, none of these animals had been vaccinated for leptospirosis. Six out of the 16 mares were struggling to get pregnant and miscarried in the eighth month of pregnancy. In a routine checking for reproductive health of the mares, serology (microscopic agglutination test – MAT) was performed using 22 live serovars of live leptospires as antigens and cut-off point at 200. Of the 16 serum samples tested, 14 (87.5%) were reactive, and Copenha- geni was by far the most frequent serovar (12 out the 14 reactive sera). In this meantime, one mare at the eight month of pregnancy aborted. The abortion was necropsied and jaundice and petechiae were observed. Since liver and kidneys were friable and deteriorated, gastric juice was collected and sent to the laboratory for bacterial culturing and PCR. Additionally, urine samples were collected from all the mares for culturing and PCR. Bacterial culturing was performed by inoculation of the samples into tubes with Fletcher and EMJH media (BD Difco, Franklin Lakes, NJ, USA) for 20 weeks, while PCR was performed by extraction of DNA with the Promega Wizard SV kit genomic DNA Purification System1 and amplification targeting on primers LipL32_45F (50 AAG CAT TAC TTG CGC TGG TG 30) and LipL32_286R (50 TTT CAG CCA GAA CTC CGA TT 30) (Pinna et al., 2011). Serology of the mare that aborted was also performed (MAT). None leptospiral culture was obtained. Nevertheless, six (37.5%) urines and the gastric juice were positive by PCR, what confirms the infection. Importantly, although the presence of leptospiral DNA in aborted foals has already been demonstrated, in various tissues and thoracic fluid (Whitwell et al., 2009; Timoney et al., 2011; Pinna et al., 2011), this is the first report of detection of leptospiral DNA in gastric juice of an equine abortion. Leptospires have already been reported in the gastric juice of an equine abortion by darkfield microscopy (Santa Rosa, 1970), but, probably due to the acid pH of the gastric juice, it has never been cultured from that sample. Since DNA can withstand on acidic pH, PCR may be a valuable tool for diagnosing leptospires in gastric juice of aborted foals when tissues are not in good condition. Acknowledgements The aa. are thankful for the help of Dr. E. Kraus. This study was supported by CNPq and FAPERJ, Brazil. WL is a CNPq fellow. Veterinary Microbiology 160 (2012) 274–275 Contents lists available at SciVerse ScienceDirect Veterinary Microbiology jou r nal h o mep ag e: w ww .els evier .co m/lo c ate/vetm ic 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.05.030