Korean Journal of
Veterinary Service

Leptospirosis is a subacute or chronic zoonotic infectious disease affecting both humans and animals, caused by the bacterium Leptospira, which is prevalent globally in tropical regions and warm, humid climates. These environments facilitate close interactions among livestock, wildlife, and humans, influencing both cultural practices and environmental exposures (Levett, 2001; Ministry of Agriculture and NVRQS, 2003). The primary causative agent, Leptospira interrogans, is a long, thin, spiral-shaped bacterium, classified as a Category 3 infectious agent in humans in Korea (Ministry of Agriculture and NVRQS, 2003). This bacterium demonstrates significant heat sensitivity, being inactivated within 10 minutes at 50℃ and within 1 minute at 60℃. However, it can be persist in the kidneys of deceased animals and in the tissues of aborted fetuses for approximately three months at −20℃ and one month at 3∼5℃. Moreover, Leptospira shed in the urine of infected animals can survive for weeks in water or mud, although they are less resilient and perish swiftly in dry conditions.

Leptospira bacteria are excreted via kidneys of infected animals over prolonged periods, with urine acting as the primary transmission vector. The duration of bacterial shedding varies among species, with cattle typically shedding bacteria for about three months and rodents potentially for their lifespan, underscoring the importance of rodent control (Ministry of Agriculture and NVRQS, 2003). Leptospirosis poses significant public health challenges globally, with sporadic cases occurring throughout the year but predominantly in the fall (Kim, 2013). Annually, it is estimated that 1.03 million people contract leptospirosis, resulting in approximately 60,000 deaths, mostly in low-income, tropical countries (Thibeaux et al., 2020).

In Korea, leptospirosis predominantly affects farmers and fishermen, particularly from September to November, with higher incidence rates in Jellanam-do, Jeonbuk-do, and Chungnam-do (Joshi et al., 2017). From 2001 to 2023, there was an average of about 180 officially reported outbreaks per year (KDCA Infectious Disease Center, 2024). Symptoms in humans range from mild cold-like symptoms to severe manifestations like septicemia, characterized by sudden onset of fever, chills, conjunctival edema, headaches, muscle aches, nausea, vomiting, and in some cases hemoptysis (Infectious Disease Portal, 2024). Over 250 leptospira serotypes have been identified (Helman et al., 2023), with serotypes such as Pomona, Canicola, Icterohemorrhagiae, Hardjo, Braislava, and Grippotyphosa being particularly prevalent among various animal species including cattle, sheep, pigs, dogs, and horses (Ministry of Agriculture and NVRQS, 2003). Notably, the serotype Canicola is predominant among dogs, although other serotypes such as Icterohemorrhagiae and Grippotyphosa are also significant in clinically symptomatic canines.

Leptospirosis in dogs typically manifests as hepatic disease accompanied by renal failure and jaundice. The acute and chronic serotypes of the disease have been well-documented, with Icterohaemorrhagiae-type leptospirosis frequently resulting in hyperthermia, abortion, and death, and occasionally severe liver disease characterized by jaundice, depression, fever, and hemorrhage. Conversely, Canicola-type leptospirosis in dogs primarily causes acute renal interstitial inflammation and liver damage, which may be followed by uremia, enteritis, and death. Chronic hepatitis is commonly associated with the Grippotyphosa serotype (Rentko et al., 1992).

Despite the challenges in diagnosing leptospirosis, the World Organization for Animal Health (WOAH) prescribes the Microscopic Agglutination Test (MAT) as the standard diagnostic method. However, this method is problematic due to the requirement for live bacteria and complex antigen management, which complicates its use in livestock control institutions and veterinary hospitals. Therefore, there is a pressing need to develop alternative diagnostic methods that can replace the MAT. While diagnostic kits for humans are available, the survey and validation of leptospira antibodies in livestock, particularly through the IgM test in cattle, necessitate further research.

Given the widespread occurrence of canine leptospirosis and its implications as the primary source of infection for Canicola serotypes—along with incidental occurrences of other serotypes of L. interrogans in dogs—there is a significant risk of intra- and interspecies transmission between humans and dogs. This highlights the need for effective zoonotic disease management strategies (Perez-Garcia et al., 2022). In response, the Seoul Metropolitan Government, in collaboration with the Animal and Plant Quarantine Agency (APQA), conducted this survey to assess the status of leptospirosis infections in stray dogs in Seoul, aiming to develop disease prevention strategies and enhance antibody diagnostic methods by obtaining positive serum samples for canine leptospirosis.

Ethics statements

The study received funding through the utilization of blood samples collected from stray dogs housed in animal shelters during routine medical examinations. As the research did not directly involve any experimental procedures on the animals, it was experimental procedures on the animals, it was exempt from requiring approval from an animal care and use committee.

Experimental materials

The experimental materials consisted of serum samples from 330 stray dogs, phosphate-buffered saline (PBS) for dilution, a centrifuge for serum separation from blood, standard antisera, 15 reference leptospiral strains, an incubator for antigen sensitization, and a dark field microscope for observing bacterial motility and viability.

Research design

This study utilized blood samples collected from 330 animals housed in 25 local animal shelters across Seoul from 2020 to 2021. The seasonal and age distribution of the collected serum samples is summarized in Table 1, 2. After serum separation via centrifugation, the samples were submitted to APQA for analysis using the MAT, the standard diagnostic method for Leptospira as endorsed by the WOAH. This research investigated the distribution of antibody titers according to age, season, and regional characteristics.

Table 1 . Seasonal distribution of the collected serum samples.

	Season			Total (n=330)
	Spring (March∼May) (n=120)	Summer (June∼September) (n=120)	Fall (October∼November) (n=80)
No	40	214	76	330

Table 2 . Age distribution of the collected serum samples.

	Age				Total
	Juvenile (0∼6m)	Adolescent (∼2y)	Mature (∼7y)	Senior (>7y)
No	78	122	87	59	330

Standard antisera and leptospiral strains used were sourced from the Korea Agriculture and Livestock Quarantine Center prior to the commencement of the study. The strains included Australis, Autumnalis, Ballum, Bataviae, Bratislava, Canicola, Copenhageni, Grippotyphosa, Hardjo, Hebdomadis, Pomona, Pyrogenes, Sejroe, Szwajizak, and Tarassovi, classified as USDA Leptospira spp., serotype.

Microscopic Agglutination Test (MAT) protocol

Blood samples were centrifuged at 3,000 rpm for 10 minutes to separate the serum. The obtained serum was then diluted 12.5 times with PBS. For the assay, 25 μL of PBS was dispensed into all wells of a 96-well plate except for well 1, followed by the addition of 25 μL of the 12.5-fold diluted serum into wells 1 and 2. Serial dilutions were prepared starting from well 2.

Antigen preparation involved inoculating 25 μL of each standard strain (2×10⁸ cells/mL) into the corresponding wells following the dilution process. The activity and density of the antigen were verified under dark field conditions prior to inoculation.

Following inoculation, the mixture was incubated at 28℃ for 2 hours to allow for antigen sensitization. Post-incubation, the presence and motility of Leptospira were evaluated using dark field microscopy. Serum samples were classified based on their reactivity compared to the negative control: no difference was recorded as negative; 75% bacterial survival as +; 50% as ++; 25% as +++; and identical to positive serum as ++++.

Sera from 330 stray dogs were collected from animal shelters in the Seoul area and shelter were collected and tested for MAT against 15 standard strains of Leptospira, and all sera tested were negative.

No positive individuals were found when the WOAH standard MAT test was applied to 15 standard strains (Australis, Autumnalis, Ballum, Bataviae, Bratislava, Canicola, Copenhageni, Grippotyphosa, Hardjo, Hebdomadis, Pomona, Pyrogenes, Sejroe, Szwajizak, and Tarassovi) as summarized in Table 3.

Table 3 . Antibody positivity by age for 15 WOAH standard strains.

Leptospira serotype	Age				Total (n=330)
	Juvenile (0∼6m) (n=78)	Adolescent (∼2y) (n=120)	Mature (∼7y) (n=80)	Senior (>7y) (n=52)
Australis	0	0	0	0	0
Autumnalis	0	0	0	0	0
Ballum	0	0	0	0	0
Bataviae	0	0	0	0	0
Bratislava	0	0	0	0	0
Canicola	0	0	0	0	0
Copenhageni	0	0	0	0	0
Grippotyphosa	0	0	0	0	0
Hardjo	0	0	0	0	0
Hebdomadis	0	0	0	0	0
Pomona	0	0	0	0	0
Pyrogenes	0	0	0	0	0
Sejroe	0	0	0	0	0
Szwajizak	0	0	0	0	0
Tarassovi	0	0	0	0	0

Dogs represent a significant reservoir for the Canicola serotype of Leptospira interrogans and have been documented to disseminate this pathogen into the environment via the excretion of infected urine (Perez-Garcia et al., 2022). It is postulated that stray dogs, particularly those that roam freely in urban settings and public parks, may serve as vectors for the propagation of Leptospira, thereby contributing to environmental contamination. However, empirical support for this assertion remains limited (Goh et al., 2021). Epidemiological studies have established a correlation between the urban prevalence of leptospirosis and the presence of asymptomatic rodent carriers. Furthermore, the incidence and spread of leptospirosis outbreaks are acknowledged to be modulated by a confluence of geographical, environmental, and climatic variables (Zakharova et al., 2020).

In the context of this study, a cohort of 330 stray dogs in Seoul was evaluated for the presence of Leptospira antibodies during the period 2020∼2021, with all tests yielding negative results. Nonetheless, these findings should not be constructed to diminish the importance of ongoing management practices aimed at mitigating the risk of Leptospira transmission from stray dogs, given the potential public health implications. It remains imperative to assess the geographical, environmental, and climatic conditions prevailing during the sample collection interval, as well as to evaluate the efficacy of current urban rodent control measures implemented within Seoul.

In light of the negative results for Leptospira in the studied cohort of stray dogs, it is crucial to consider alternative vectors and transmission pathways that might contribute to the persistence and spread of leptospirosis in urban environments. While our findings indicate a low prevalence of Leptospira among stray dogs in Seoul, it is essential to remain vigilant regarding other potential reservoirs and carriers. Notably domestic dogs, which interact closely with human populations, have historically been considered at lower risk in regions with no recent reported cases of leptospirosis. However, the absence of infection in stray dogs does not preclude the potential for domestic dogs to act as carriers, particularly in scenarios where they might come into contact with infected wildlife or contaminated environments. Historical data from a 2007 investigation by the APQA revealed that among 64 stray dogs tested, 5 were found to be positive for various serotypes of Lai, two of Pyrogenes, and one of Sejroe (Miotto et al., 2018). This evidence, in conjunction with international studies on Leptospira in stray dogs from urban areas and the potential for zoonotic transmission through the relocation of these animals to shelter or through adoption processes, underscores the necessity for meticulous oversight and surveillance of stray dogs. Such measures are crucial to preemptively curtail the propagation of leptospirosis and other zoonotic diseases through indirect contact pathways facilitated by animal shelters and adoption services.

Further investigation into environmental and wildlife vectors is warranted. Studies have shown that urban wildlife, such as rodents and small mammals, can harbor and transmit Leptospira spp., potentially influencing the epidemiology of the disease without direct involvement from stray or domestic dogs (Smith et al., 2019). Additionally, water sources and soil contaminated with Leptospira from infected wildlife pose a risk to both animals and humans, suggesting that environmental management and monitoring should be integrated components of leptospirosis control strategies.

Given these complexities, a multifaceted approach to surveillance that includes assessment of various animal populations and environmental sampling is recommended. This strategy would not only enhance our understanding of the epidemiological dynamics of leptospirosis but also inform targeted interventions. Moreover, while the current evidence may not support widespread vaccination of domestic dogs in Korea, continuous re-evaluation of this stance is advisable as urban development ecological changes could alter exposure risks.

This study was supported by ‘The Government-wide R&D to Advance Infectious Disease Prevention and Control’, Republic of Korea (grant number: RS-2023-KH140418) and Animal and Plant Quarantine Agency, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea (B-1543069-2021-22-03).

No potential conflict of interest relevant to this article was reported.