Abstract
Leptospira kirschneri is an emerging human and animal pathogen. Here we describe 2 unrelated outbreaks of acute fatal leptospirosis caused by L. kirschneri in suckling lambs and calves diagnosed following a flooding event in the spring of 2023 in northern Uruguay. In outbreak 1, the sheep flock was raised in a low-lying area previously used for rice cultivation; 20 lambs died after developing apathy, jaundice, and hemoglobinuria. In outbreak 2, 2 calves were found dead. At autopsy, 4 lambs and 2 calves had marked jaundice, hemoglobinuria, dark-red kidneys, and hepatomegaly. Microscopically, we found marked hemoglobinuric nephrosis, lymphohistiocytic tubulointerstitial nephritis, hepatocellular dissociation, and random hepatocellular necrosis with periportal lymphohistiocytic hepatitis and canalicular cholestasis. Positive immunostaining for Leptospira sp. was found in the liver (lamb C), both the liver and kidney (calf B), and the liver (calf A). Leptospira kirschneri was PCR-confirmed using kidney and liver samples from the autopsied animals. Acute leptospirosis caused by L. kirschneri has not been reported previously in sheep and cattle, to our knowledge. Our findings highlight that L. kirschneri infection can cause acute leptospirosis in unvaccinated lambs and calves.
Leptospirosis is an important zoonotic disease in tropical and subtropical regions. Leptospira interrogans, L. noguchi, and L. borgpetersenii are pathogenic species most frequently associated with acute and chronic disease. 8 Several studies have identified Leptospira kirschneri as an emerging pathogenic species. L. kirschneri infections have been reported in humans in Malaysia, 13 Tanzania, 1 Germany, 5 Brazil,2,3 and Uruguay. 10
In addition to humans, infection by L. kirschneri has been documented in common dolphins (Delphinus delphis delphis), 15 raccoon dogs (Nyctereutes procyonoides), 9 aborted mares, 21 a prematurely born foal, 18 and cats. 12 In France, water voles (Arvicola terrestris) were identified as carriers of L. kirschneri serogroup Grippotyphosa. 7 In Brazil, serologic evidence indicates exposure to L. kirschneri serogroup Cynopteri in humans 13 and L. kirschneri serogroup Grippotyphosa in slaughtered cattle. 16 We retrieved no cases of acute leptospirosis caused by L. kirschneri infection in ruminants in a search of Google, PubMed, CAB Direct, Web of Science, and Scopus, using the search terms “Leptospira kirschneri”, “ruminants”, “sheep”, and “cattle”, suggesting that this condition has not been reported previously in sheep and cattle. Here we describe the epidemiologic, clinicopathologic, immunohistochemical, and molecular confirmation of 2 fatal outbreaks of L. kirschneri infection in lambs and calves in Uruguay.
Outbreak 1 occurred in the Department of Rivera in October 2023 (spring season in the Southern Hemisphere). Following a severe flooding event, 20 of 450 (4.4%) Texel lambs <30-d-old died. The flock was grazing natural grassland in a low-lying area used previously for rice cultivation. The affected lambs had severe apathy, jaundice, and hemoglobinuria, progressing to death within 24–48 h. Outbreak 2 took place 1 wk later on an unrelated farm, located in the Department of Tacuarembó, 158 km away from the site of outbreak 1. Two of 41 (4.8%) Angus calves <30-d-old were found dead. In both outbreaks, none of the animals had been vaccinated against Leptospira.
At autopsy, 4 lambs (lambs A–D) and 2 calves (calves A, B) had severe jaundice (Fig. 1A), hepatomegaly, diffusely dark-red kidneys, hemoglobinuria (Fig. 1A), and multifocal pulmonary hemorrhages. Histologic examination revealed lymphohistiocytic tubulointerstitial nephritis (Fig. 1B), hemoglobinuric nephrosis, hepatocellular dissociation, random hepatocellular necrosis with periportal lymphohistiocytic hepatitis (Fig. 1C), and canalicular cholestasis.
Gross, histologic, and immunohistochemical findings in lambs and calves infected spontaneously by Leptospira kirschneri.
Formalin-fixed paraffin-embedded sections of the liver and kidney (lambs C, D; calves A, B) were processed for immunohistochemistry (IHC) for Leptospira spp. antigen detection at the INIA La Estanzuela histology laboratory (Colonia, Uruguay). Tissue sections were deparaffinized and rehydrated, and the endogenous peroxidase was blocked with 3% hydrogen peroxide (Sigma Aldrich) for 10 min. No antigenic recovery method was applied. A commercial rabbit serum (Leptospira multivalent fluorescent antibody conjugate, LEP-FAC; USDA National Veterinary Services Laboratories) was used as the primary antibody at a 1:1,000 dilution. The secondary antibody was a goat anti-rabbit serum conjugated with a polymer labeled with horseradish peroxidase (EnVision+ System HRP, K4003; Dako); 3-amino-9-ethylcarbazole (AEC, K3464; Dako) was used as the chromogen/substrate solution. Kidney sections from a mouse experimentally inoculated with L. interrogans (kindly provided by Dr. Leticia Zarantonelli from the Institut Pasteur de Montevideo) were used as a positive control. Negative controls consisted of serial sections from all analyzed tissues, including those from the positive control, processed as described above except that the primary antibody was replaced by normal rabbit serum (NC499H; Biocare Medical). The slides were counterstained with hematoxylin and coverslipped with aqueous mounting medium. A run was considered valid when strong immunoreactivity was observed in the lumen of the cortical renal tubules in the positive control and no immunoreactivity in the negative controls. Focal and multifocal immunoreactivity was detected in the hepatic sinusoids (Fig. 1D) of lamb C, and calves A and B, as well as in the lumen of renal cortical tubules of calf A (Table 1; Fig. 1E).
Results of immunohistochemistry (IHC) for Leptospira spp. antigen detection and detection of DNA and molecular typing in tissue samples.
NA = not applicable; ND = not detected; + = positive, – = negative.
To detect pathogenic Leptospira, total DNA extraction was performed on frozen liver and kidney tissue samples, which were subsequently analyzed using a real-time PCR targeting the lipL32 gene. Samples were collected during autopsy and stored at −20°C until shipment to the Institut Pasteur (Montevideo, Uruguay) for analysis. Total DNA extraction was performed on 30-mg frozen tissue samples (PureLink genomic DNA minikit; Invitrogen) following the manufacturer’s instructions. The lipL32 gene amplification was performed with oligonucleotide primers and probes as described elsewhere 17 (Table 2). The PCR was performed (SensiFAST Probe No-ROX kit, Meridian Bioscience; QuantStudio 3 real-time PCR instrument, Applied Biosystems) using 2.5 μL of template DNA. As a control for DNA extraction efficiency and for the presence of potential inhibitors of the amplification reaction, all samples were tested for the reference gene, β-actin (Table 2).
Oligonucleotide primers and probes used in our study of Leptospira kirschneri infection.
All DNA samples that tested positive by lipL32 qPCR underwent further analysis by conventional PCR amplification followed by sequencing of the rrs gene as described previously.14,20 The resulting 331-bp amplicons were sequenced in both directions using internal oligonucleotide primers (Table 2). Sequence quality was verified (Chromas software v.2.6.5; Technelysium), and consensus sequences were defined (BioEdit v.7.05; Ibis Therapeutics). The consensus sequences were then compared to available sequences in GenBank using BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Pathogenic Leptospira spp. DNA was confirmed in 4 lamb cases, with positive amplification detected in 4 kidney samples and 3 liver samples from autopsied lambs. Additionally, the kidney of one calf tested positive. Purified DNA from lipL32 qPCR–positive cases underwent partial rrs gene amplification and sequencing, revealing 100% identity with L. kirschneri in all detected cases (Table 1).
Our study provides laboratory-confirmed evidence of fatal L. kirschneri infection in unvaccinated lambs and calves following the rainfall season. L. borgpetersenii, L. interrogans, and L. noguchii have been reported previously in Uruguay,6,10,20 demonstrating that the 4 pathogenic species of leptospires can cause disease in this country.
The hallmarks of acute leptospirosis include severe acute intravascular hemolysis, jaundice, hemoglobinuria, and rapid progression to death. In our case, lambs and calves had clinical signs, as well as gross and histologic lesions, similar to those described for other Leptospira species. 8 In both outbreaks, the etiologic diagnosis was established through IHC and molecular detection in kidney and/or liver samples. Although DNA of L. kirschneri was confirmed in the kidney tissue in only 1 of the 2 calves, bacterial antigens were detected by IHC in both cases. Our findings highlight the importance of employing complementary laboratory methods alongside clinical and pathology evaluations in a comprehensive diagnostic investigation.
Reports of leptospiral species confirmed to cause acute leptospirosis in ruminants remain scarce in South America.4,6 In Uruguay, 2 sheep flocks from the departments of Paysandú and Canelones were affected by L. interrogans serogroup Pomona serovar Kennewicki in September and October 2017. 6 Interestingly, the cases described in our study occurred in the departments of Rivera and Tacuarembó, during the same season and under similar epidemiologic conditions. Pathogenic Leptospira species are endemic in these areas of Uruguay, and they can and do cause acute leptospirosis in livestock.
Although leptospirosis is a well-known disease worldwide, data on L. kirschneri infection in livestock are limited. In Brazil, L. kirschneri serogroup Grippotyphosa has been detected in slaughtered cattle, 16 demonstrating the potential for host–pathogen interactions. L. kirschneri serogroup Pomona serovar Mozdok has been identified as a cause of abortion in mares in Croatia 21 and the United States. 18 Our investigation confirms that suckling lambs and calves are susceptible to L. kirschneri infection, resulting in typical acute leptospirosis. The species responsible for the outbreaks were successfully identified through molecular analysis of autopsied tissue samples that had been preserved at low temperatures. However, a limitation of our study was the absence of bacterial culture for Leptospira isolation, preventing the determination of the serogroup and/or serovar involved.
Although L. kirschneri had not been identified in naturally infected cattle, 20 it is well documented as a pathogenic Leptospira species affecting rural workers in Uruguay. 10 The detection of L. kirschneri increases our knowledge of the diversity of Leptospira species circulating in cattle and sheep within the country, reinforcing the importance of diagnosing this zoonotic disease in animals from a One Health perspective. Furthermore, this diversity of hosts affected by L. kirschneri underscores the possible wide-reaching species impact.
Footnotes
Acknowledgements
We thank Alejandra Custodio for technical assistance with the histologic techniques, Sabrina Pimentel and Eleana Luque for their support, and Karina Cresci for the clinical data provided.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The research was funded by grants from the Unidad Mixta Pasteur + INIA, INIA (PL_27), and from FOCEM (Fondo para la Convergencia Estructural del Mercosur; COF 03/11).
