Clinical and molecular characterization of an outbreak of leptospirosis in dogs from Los Angeles County, California, USA, 2021

In 2021, an outbreak of *Leptospira interrogans* serovar Canicola affected over 200 unvaccinated dogs in Los Angeles County, with whole genome sequencing confirming distinct strains, urine PCR proving the most sensitive diagnostic tool, and indoor congregate facilities identified as a significant risk factor.

The Gist

Imagine a city where dogs are the main residents, and suddenly, a silent, invisible enemy starts making them very sick. This is the story of a 2021 outbreak in Los Angeles County, California, where over 200 dogs fell ill with leptospirosis, a nasty bacterial infection.

Here is the story of what happened, broken down into simple terms with some helpful analogies.

The Invisible Invader

Think of Leptospira bacteria as tiny, microscopic "ghosts" that live in the urine of animals like rats and, in this case, other dogs. These ghosts don't just sit there; they can swim through water or soil and jump onto a dog's skin or into their mouth if the dog drinks from a puddle or sniffs the ground.

Once inside, these ghosts attack the dog's kidneys and liver, causing fever, vomiting, and extreme thirst. It's like a plumbing disaster happening inside the dog's body.

The Mystery of the Outbreak

In 2021, veterinarians in West Los Angeles noticed something strange. Dogs were getting sick at the same time and in the same area. It wasn't just one or two dogs; it was a wave.

The researchers (the detectives of this story) wanted to know three things:

1. Who is the bad guy? (Which specific bacteria is causing this?)
2. How did they catch it? (Where did the infection come from?)
3. How do we catch the bad guy in the act? (Which tests work best?)

The Detective Work: DNA and Fingerprints

The team took samples from the sick dogs and used a high-tech tool called Whole Genome Sequencing. Imagine this as taking a DNA fingerprint of the bacteria.

• The Verdict: The bad guy was identified as Leptospira interrogans serovar Canicola. This is a specific "brand" of bacteria that dogs are known to carry.
• The Twist: Even though it was the same "brand," the DNA fingerprints were slightly different for each dog. This is like finding five different people wearing the same red hoodie. It means the dogs didn't all catch it from one single "patient zero." Instead, there were multiple sources of infection happening at the same time.

The Suspects: Where Did It Come From?

The researchers looked for clues on where the dogs had been. They found a strong link to Indoor Congregate Facilities (ICFs).

• The Analogy: Think of a doggy daycare or a boarding kennel like a crowded school cafeteria. If one kid brings a cold, it spreads fast.
• The Finding: Many of the sick dogs had recently been to a doggy daycare or boarding facility. The bacteria likely spread through urine in these crowded places.
• The "Unvaccinated" Factor: Almost all the sick dogs were unvaccinated. It's like a school where no one got the flu shot; when the virus arrives, everyone gets sick. The few dogs that were vaccinated had either just started their shots (and weren't fully protected yet) or were unlucky enough to get sick right after.

The Tools: How to Spot the Ghost

Diagnosing this disease is tricky because the bacteria hide. The researchers tested different methods to see which one was the best "flashlight" to find the infection.

1. Blood Test (PCR): This looks for the bacteria in the blood.
   • Result: It only worked 27% of the time.
   • Why? The bacteria are like a flash mob in the blood; they are there for a very short time at the beginning of the illness and then vanish.
2. Urine Test (PCR): This looks for the bacteria in the pee.
   • Result: It worked 91% of the time!
   • Why? Once the bacteria move from the blood to the kidneys, they start "showering" the dog with bacteria in their urine. This is the best place to look.
3. Antibody Tests (The "Wanted" Posters): These tests check if the dog's immune system has built a "wanted poster" (antibodies) for the bacteria.
   • Result: These worked well (about 75-89%), but only if the dog had been sick for a few days. If you test a dog too early (within the first 24-48 hours), the immune system hasn't built the poster yet, so the test says "negative" even if the dog is sick.

The Lesson: To catch the ghost, you need to look in the urine and wait a few days for the immune system to react.

The Outcome: A Happy Ending for Most

The good news is that 92% of the dogs survived.

• The doctors treated them with fluids (to fix the plumbing) and antibiotics (to kill the bacteria).
• Some dogs needed dialysis (a machine to clean their blood) because their kidneys were in bad shape, but even they recovered.
• Sadly, a few dogs were too sick to save, mostly because their kidneys failed completely or they had bleeding in their lungs.

The Big Takeaway

This outbreak was a wake-up call for Los Angeles.

1. Vaccinate: The best defense is a good offense. Getting dogs vaccinated against this specific bacteria prevents the "flu shot" gap.
2. Watch the Daycares: Crowded places need to be extra clean to stop the spread of urine-borne germs.
3. Test Smart: If a dog is sick, don't just do one test. Check the urine and wait a few days for antibody tests to be accurate.

In short: A group of unvaccinated dogs caught a bacterial infection from a few different sources, likely spreading through crowded doggy daycares. By using the right tests (urine PCR) and treating them quickly, most dogs made a full recovery. The lesson for all dog owners? Get the shots, and keep an eye on your pup if they start drinking and peeing like crazy!

Technical Summary

1. Problem Statement

In 2021, the Los Angeles County (LAC) Department of Public Health identified a suspected outbreak of leptospirosis involving over 200 client-owned dogs in West Los Angeles. While leptospirosis is a known zoonotic disease, outbreaks in urban settings are rare, and the specific epidemiological drivers, causative serovars, and diagnostic performance in this context were unclear. The outbreak was initially suspected to be driven by dog-to-dog transmission in indoor congregate facilities (ICFs) following high MAT titers to Leptospira interrogans serovar Canicola. The study aimed to:

• Accurately identify the causative agent and its genetic diversity.
• Characterize clinical risk factors, diagnostic test performance, and outcomes.
• Determine if the outbreak represented a single source or multiple independent infections.
• Assess the public health implications of the outbreak.

2. Methodology

The study employed a mixed-methods approach combining molecular epidemiology, retrospective medical record review, and spatial analysis.

• Molecular Characterization:
  • Isolation: Blood and urine samples from four highly suspected dogs (LAD1, LAD3-LAD5) were cultured using HAN and T80/40/LH media.
  • Genome Enrichment: For a fifth dog (LAD2) where culture failed but urine PCR was positive, DNA capture and enrichment were performed to obtain a genome.
  • Sequencing & Analysis: Isolates underwent Whole Genome Sequencing (WGS) via Illumina. Phylogenomic analysis was conducted using Single Nucleotide Polymorphisms (SNPs) to compare the five dog genomes against 13 publicly available L. interrogans serovar Canicola genomes. Serotyping was confirmed via Microscopic Agglutination Test (MAT) with monoclonal antibodies.
• Epidemiological & Clinical Review:
  • Cohort: 59 confirmed cases (diagnosed via PCR, MAT titer $\ge$ 1:6400, or 4-fold rise) from two specialty hospitals (ASEC and WLA) between April and December 2021.
  • Controls: 15,536 dogs from the same hospitals during the same period.
  • Data Collection: Extracted data included vaccination history, demographics, clinical signs, laboratory values (hematology, biochemistry, urinalysis), diagnostic test results (PCR, MAT, Point-of-Care [POC] tests), and outcomes.
  • Statistical Analysis: Univariate analysis (Fisher's exact test, Mann-Whitney U) compared cases to controls. Spatial analysis (ArcGIS Pro) utilized Global Moran's I and Getis-Ord Gi* statistics to identify clustering and hotspots.
• Diagnostic Performance: Sensitivity of blood PCR, urine PCR, MAT, and POC assays (WITNESS Lepto and SNAP Lepto) was calculated against the study's case definition.

3. Key Contributions

• First US Isolation in Decades: This is the first study to isolate and characterize pathogenic Leptospira from dogs in the US since the early 1990s.
• Genomic Resolution: It utilized WGS to demonstrate that while all isolates were L. interrogans serovar Canicola ST34, they were genetically distinct, suggesting multiple infection sources rather than a single point-source outbreak.
• Risk Factor Identification: The study identified a strong association between the outbreak peak and exposure to Indoor Congregate Facilities (ICFs), specifically boarding and daycare.
• Diagnostic Benchmarking: It provided a comparative analysis of diagnostic modalities in an outbreak setting, highlighting the superior sensitivity of urine PCR over blood PCR and the limitations of serology in the acute phase.

4. Key Results

• Microbiology & Genomics:
  • All five sequenced genomes were L. interrogans serovar Canicola ST34.
  • The isolates were distinct, separated by 12 to 224 SNPs, indicating multiple independent infection sources rather than direct dog-to-dog transmission of a single strain.
  • Two genomes (LAD1 and LAD2) were the most similar (12 SNPs apart) and originated from contiguous zip codes.
• Epidemiology & Risk Factors:
  • Demographics: Cases were significantly more likely to be <6 years old, $\ge$15 kg, and male. Among females, intact status was a risk factor.
  • Breed: Golden Retrievers and Siberian Huskies were overrepresented.
  • Exposure: 53% of cases had exposure to ICFs. During the peak period, dogs had 5.7 times higher odds of ICF exposure compared to non-peak cases.
  • Vaccination: Of 47 dogs with known history, 87% were unvaccinated. The few vaccinated dogs were either in the middle of a primary series or had received the vaccine very recently (within days of onset).
  • Spatial/Temporal: Cases clustered temporally (July–August 2021) and spatially in zip codes west and south of the clinics.
• Clinical Findings:
  • Signs: Lethargy (76%), hyporexia (54%), and vomiting (47%) were most common.
  • Pathology: 73% of dogs with available data had renal impairment; 15% had concurrent renal and hepatic impairment.
  • Outcome: 92% (54/59) survived to discharge. Four dogs were euthanized due to severe azotemia or pulmonary hemorrhage syndrome.
• Diagnostic Test Performance:
  • Urine PCR: 91% sensitivity (49/54 positive).
  • Blood PCR: 27% sensitivity (15/56 positive).
  • MAT: 76% sensitivity (22/29 positive); many acute samples were negative, but titers were high in positive cases.
  • POC Tests: WITNESS Lepto (74% sensitivity) and SNAP Lepto (89% sensitivity).
  • Timing: Negative serologic results were strongly associated with illness duration of $\le$7 days.

5. Significance and Clinical Implications

• Public Health: The outbreak highlights that L. interrogans serovar Canicola can cause significant disease in urban dogs and poses a zoonotic risk, even in the absence of identified human cases. The high rate of leptospiruria (91% urine PCR positivity) suggests a potential for dog-to-dog and dog-to-human transmission.
• Vaccination Policy: The findings challenge the notion that leptospirosis is only a risk for rural dogs. The study supports the recommendation for widespread annual vaccination of all dogs, particularly in urban areas with high dog ownership and density.
• Diagnostic Strategy: Clinicians should prioritize urine PCR for early diagnosis, as blood PCR has low sensitivity after the first week. Serology (MAT) is less sensitive in the acute phase (<7 days) but essential for confirmation. A combination of PCR and serology is recommended for optimal diagnosis.
• Epidemiological Insight: The outbreak was likely fueled by a combination of factors: increased pet ownership during the pandemic leading to overcrowding in ICFs, potential rodent exposure, and a lack of vaccination. The genetic diversity of the isolates suggests the environment (e.g., contaminated water/soil in ICFs or rodent reservoirs) acted as a common source for multiple distinct strains.