Evaluation of the Leptospira species microscopic agglutination test in experimentally vaccinated cats and Leptospira species seropositivity in aged azotemic client-owned cats

Abstract

Objectives

The objectives of this study were to validate the microscopic agglutination test (MAT) using feline sera, determine cross-reactivity of Borrelia burgdorferi antibodies in the MAT, and evaluate if there is an association between Leptospira species seropositivity in aged (⩾10 years) client-owned cats with and without azotemia (creatinine >2 g/dl).

Methods

A four-serovar canine leptospiral vaccine was administered to two specific pathogen-free (SPF) cats on days 0 and 14. The MAT was performed intermittently until day 42 for the serovars Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, Pomona and Bratislava, with a cut-off value of ⩾1:100. Five purpose-bred cats were infested with wild-caught Ixodes scapularis adults with an average B burgdorferi infection rate of 50%, and tested for antibodies against B burgdorferi C6 peptide and DNA in skin biopsies, as well as by MAT. Sera from 66 azotemic and 75 non-azotemic cats ⩾10 years of age were tested for Leptospira species antibodies using the MAT and results were compared by the χ² test.

Results

Both SPF cats seroconverted by week 3 and formed antibodies against at least one serovar. There was no cross-reactivity in the MAT using samples from cats with antibodies to B burgdorferi. MAT results were positive for 4/66 azotemic cats and 8/75 non-azotemic cats; these results were not statistically different.

Conclusions and relevance

The MAT can be interpreted using feline serum and does not appear to cross-react in cats with B burgdorferi antibodies. There was no association between Leptospira species MAT results and azotemia in this group of aged client-owned cats but further studies are needed to determine if leptospirosis contributes to feline chronic kidney disease.

Introduction

Leptospira species are commonly associated with acute renal failure in dogs, and residual damage has also been theorized to result in canine chronic kidney disease (CKD). ¹ CKD is a common cause of morbidity and mortality in cats worldwide, and has been suggested to be the number one cause of death in aged cats.^2,3 Although cats have historically been considered to be naturally resistant to developing leptospirosis, results of several studies have called this assumption into question. For example, experimental inoculation of cats with Leptospira species can result in nephritis, and Leptospira species seropositivity has been associated with clinical signs such as polydipsia and polyuria,^4–9 thus making it plausible that these organisms could be associated with CKD in this species. Additionally, the potential known exposure of cats (particularly outdoor cats) to reservoir hosts and the documentation that cats can shed leptospires in their urine raises additional concern for transmission and disease manifestations in humans.^4,10–12 These factors make investigation of leptospirosis of particular interest to practitioners invested in improving feline medicine and preventing potential zoonotic disease.

Currently, there are several ways to detect a leptospirosis infection in cats, including culture to help document an active infection, but this is rarely performed and not readily available. ¹³ Recently, however, the use of PCR assays to amplify Leptospira species DNA from feline urine has been used to suggest current infection in several studies.^10–12 Additionally, antibodies against Leptospira species in cat serum are detected by the modified agglutination test (MAT) and suggest either current or previous infection.^{1,8–13,14–27} Leptospira species antibody prevalence rates in feline sera have varied between 4.8% and 48.0%, depending on the geographic location and sampling method.^{8,10,11,14–27} A recent study utilizing both the MAT and PCR demonstrated a higher prevalence of antibodies to Leptospira species in cats with kidney disease than in healthy cats, ¹⁰ suggesting that Leptospira species infections could be associated with CKD in naturally exposed cats. However, while the MAT is thought to be accurate for use with serum from any mammal, information concerning validation of this assay in cats is minimal. For example, the magnitude and duration of Leptospira species antibodies as detected by the MAT in cat sera is largely unknown. While the MAT is believed to be specific for Leptospira species antibodies, it is unknown whether antibodies against other spirochetes in feline sera can lead to falsely positive results. When considering false-positive test results, it is known that naturally exposed and experimentally infected cats develop antibodies against Borrelia burgdorferi but there is no information available that evaluates whether B burgdorferi antibodies in feline sera are detected in the Leptospira species MAT. Furthermore, B burgdorferi is known to cause nephritis in dogs,^28,29 making the possibility of cross-reactivity a true concern when interpreting test results in cats.

Three experiments were designed to address these clinical questions. The objectives of the first two experiments were to gather and report new information validating the use of MAT for detection of Leptospira species antibodies in cats. We hypothesized that cats would have variable Leptospira species antibody responses when administered a four-serovar vaccine marketed for dogs and that serum antibodies from cats experimentally infected with B burgdorferi would not be detected in the Leptospira species MAT. The objective of the third experiment was to evaluate if there is an association between antibodies to Leptospira species and azotemia in aged client-owned cats. We hypothesized that cats ⩾10 years of age with azotemia would be more likely to be positive for Leptospira species antibodies than cats ⩾10 years of age without azotemia.

Materials and methods

Detection of Leptospira species antibodies in vaccinated cats

Adult (n = 2) purpose-bred laboratory-housed cats with no potential exposure to Leptospira species reservoir hosts were transferred from another study to the current study with approval from the Institutional Animal Care and Use Committee. Both cats were administered a subcutaneous dose of a commercially available canine vaccine (Vanguard L4; Zoetis Animal Health), which contains the Leptospira serovars Canicola, Grippotyphosa, Icterohaemorrhagiae and Pomona, on days 0 and 14. Sera were collected from the cats prior to inoculation and intermittently until day 42. Each serum sample was evaluated for Leptospira species antibodies (serovars Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, Pomona and Bratislava) by MAT performed by standard operating procedures at an accredited laboratory (Veterinary Diagnostic Laboratory, Colorado State University, Fort Collins, CO, USA). Samples with agglutination at ⩾1:100 were considered positive for the respective serovar.

Evaluation for B burgdorferi antibody cross-reactivity with Leptospira species using MAT

Laboratory-reared young adult specific pathogen-free cats (n = 5) were infested with wild-caught Ixodes scapularis adults (from Rhode Island) with an average B burgdorferi infection rate of 50% in an unrelated experiment. ²⁹ All of the cats developed antibodies against B burgdorferi C6 peptide and were positive for B burgdorferi DNA in skin biopsies taken at the sites of I scapularis attachment. The sera had been stored at −80°C until assayed for Leptospira species antibodies by MAT, as previously described.

Leptospira species antibodies in serum of aged client-owned cats with and without azotemia

In a separate study on vaccine-associated renal antibodies, clinicians at Colorado State University and a select group of feline practitioners in the USA were contacted and asked to submit serum from cats ⩾10 years of age with serum creatinine concentrations >2 mg/dl that were believed to have stable CKD. The veterinarians were also asked to send serum from cats ⩾10 years of age evaluated in the clinic during the same time as the azotemic cats but with serum creatinine concentrations ⩽2 mg/dl. On arrival, the serum creatinine concentration was determined and then the samples were stored at −80°C until assayed in the Leptospira species MAT. Positive MAT prevalence rates were compared between groups by the χ² test with a P value <0.05 considered significant.

Results

Detection of Leptospira species antibodies in vaccinated cats

Antibodies against at least one Leptospira species serovar were detected from both vaccinated cats, as reported in Table 1. One cat was positive for antibodies against serovar Hardjo by day 12 and ultimately developed serum antibodies against serovars Hardjo, Grippotyphosa and Bratislava. The other cat was positive for antibodies against serovar Hardjo on postinoculation days 19 and 36 (1:100) but was negative by day 42.

Table 1.

Leptospira species modified agglutination test antibody titers from two adult specific pathogen-free cats administered two doses of a commercially available canine vaccine

	Day 0		Day 12		Day 19		Day 36		Day 42
Serovar	Cat 1	Cat 2	Cat 1	Cat 2	Cat 1	Cat 2	Cat 1	Cat 2	Cat 1	Cat 2
Canicola	0	0	0	0	0	0	0	0	0	0
Grippotyphosa	0	0	0	0	0	0	800	0	800	0
Hardjo	0	0	200	0	100	100	400	100	400	0
Icterohaemorrhagiae	0	0	0	0	0	0	0	0	0	0
Pomona	0	0	0	0	0	0	0	0	0	0
Bratislava	0	0	0	0	0	0	100	0	200	0

Evaluation for B burgdorferi antibody cross-reactivity with Leptospira species using MAT

None of the five cats with antibodies against B burgdorferi C6 peptide were positive for antibodies against any Leptospira species by MAT.

Leptospira species antibodies in serum of aged client-owned cats with and without azotemia

Of the 66 cats with serum creatinine concentrations >2 mg/dl, 4/66 (6%) were positive for antibodies against one Leptospira serovar. None of the cats had antibodies to more than one serovar. Antibodies against serovar Icterohaemorrhagiae were detected in the serum of two cats from Ohio (1:100 titers), and antibodies against serovar Bratislava were detected in one cat from Ohio (1:200 titer) and one cat from West Virginia (1:100 titer). Of the 75 cats with serum creatinine concentrations ⩽2 mg/dl, 8/75 (11%) were positive for antibodies against one Leptospira serovar. None of the cats had antibodies to more than one serovar. Antibodies against serovar Canicola were detected in the serum of one cat from Ohio (1:100), one cat from New York (1:100) and one cat from Oklahoma (1:200). Antibodies against serovar Icterohaemorrhagiae were detected in the serum of one cat from Ohio (1:100 titer). Antibodies against serovar Bratislava were detected in the serum of one cat from Colorado (1:100), one cat from South Dakota (1:100 titer) and two cats from Ohio (1:100 titers). The difference in Leptospira species antibody prevalence rates between the two groups of cats was not statistically different (P = 0.3281).

Discussion

In this study, it was documented that cats can produce Leptospira species agglutinating antibodies when exposed to the inactivated organisms within a commercially available four-serovar Leptospira-containing canine vaccine. However, only one of the cats maintained a positive titer to one serovar over the 42 day study period, and the highest titer documented was 1:800. The magnitude of the titers observed in this study was drastically lower than in a recent study performed in presumably naturally exposed healthy cats and cats with kidney disease that documented a maximal titer of ⩾1:12,800 depending on the serovar. ¹⁰ A previous study in Leptospira serovar-vaccinated dogs also documented higher titer magnitudes where maximal titers of 1:6400 were detected transiently, depending on the serovar. ³⁰ In addition, antibodies against three of the serovars contained within the vaccine were not detected, whereas antibodies against two serovars (Bratislava and Hardjo), which are not contained within the vaccine, were detected. The same problems also occur when interpreting canine Leptospira species MAT results as variable titer magnitudes are observed and cross-reactivity to serogroups not within the vaccine can occur. Results using cat sera should also be interpreted carefully as the Leptospira species MAT detection assay is optimized for use in dogs not cats. Vaccinated cat sera were used in this study exclusively to help assess the validity of the MAT by attempting to induce Leptospira species antibodies in research cats. Thus, this information should not be overinterpreted and should not be used as a recommendation to administer this canine vaccine to cats. As this vaccine was not titrated for use in cats, these serological test results should not be interpreted as a lack of response of cats to optimized vaccines but could help to explain the observed differences in titer magnitude. If a feline Leptospira species vaccine is to be developed, further research will be needed to determine initial and booster dosing recommendations, effect of adjuvants, duration of immunity characteristics, and degree of cell-mediated or humoral immunity response. Along this line, the immune system status could also have influenced the titer magnitude depending on whether a weak or strong cell-mediated immunity response occurred following vaccination. All of these factors may help to explain the differences in titer magnitudes seen in cats and underscore the need for further studies to determine what factors affect Leptospira serovar titer magnitude and duration in cats.

B burgdorferi has been documented to cause nephritis in some dogs, and it is now known that it can induce antibody production against the C6 peptide in cats, but whether this organism causes kidney disease in cats is unknown.^28,29,31 B burgdorferi-endemic areas are also endemic for leptospirosis, making distinguishing these infections from each other of clinical importance in both dogs and cats. ³² We hypothesized that it would be unlikely for B burgdorferi antibodies to cross-react with Leptospira species antibodies, but it has not been extensively investigated in dogs and has never been evaluated in cats. The results of our study show that cats experimentally infested with wild-caught I scapularis with an average B burgdorferi infection rate of 50% in the absence of Leptospira species reservoir exposure produce antibodies against B burgdorferi but not Leptospira species. Thus, positive Leptospira species MAT results from cats in the field likely reflect antibodies against leptospires not B burgdorferi.

In the experiment studying aged client-owned cats with and without azotemia, the non-azotemic cats were more likely to have antibodies against Leptospira species than cats with azotemia. This is in contrast to a recent study that showed that cats with kidney disease were statistically more likely to be seropositive than healthy cats. This study included cats with CKD, as well as cats with acute kidney injury (AKI), ¹⁰ whereas the azotemic cats in our study were only considered to have stable CKD. The geographic location was also different between these two studies; Rodriguez et al sampled cats from various areas in Quebec, Canada, ¹⁰ and the present study sampled cats from various regions within the USA. The previous study also had a much larger sample set than the present one. The disease classification (CKD vs AKI), geographic location and number of cats all could explain the different results observed in these two studies.

Several papers have shown that cats with antibodies to Leptospira species have either signs associated with renal disease or histopathologic evidence of renal inflammation.^4–9,10 It is unknown, however, if leptospirosis causes an AKI that then leads to the development of CKD in cats. Therefore, determining if an active infection is present or not would help to better characterize the clinical disease course in cats. Similar to the recommendations for dogs and based on recent studies in cats, additional testing other than antibody testing is likely needed to determine if an active infection is present.^1,10 In dogs, PCR testing for leptospires in the urine can help to document an active infection but false-negatives do occur. ¹ Combining the Leptospira species MAT and urine PCR assays could help increase the likelihood of detecting active leptospirosis in cats. This is also of relevance to human health owing to the known zoonotic risk of pathogenic leptospires in humans. ¹ As a result, additional studies for accurate detection of leptospirosis in cats utilizing MAT and urine PCR are needed to help determine if feline leptospirosis plays a role in the development of feline CKD.

There were several limitations of this study. This study was partially retrospective rather than a double-blinded controlled prospective study. Additionally, the housing status and hunting tendencies of the cats in our study were not known, which could certainly have affected the results as Rodriguez et al showed that cats which were outdoors or which had hunting tendencies were considered to be at risk for Leptospira species seropositivity. ¹⁰ The sample set of cats was also not large, which would likely affect the ability to draw conclusions from the statistics performed. It is also unknown for the azotemic cats how their CKD was classified as stable disease. Additionally, for the aged client-owned non-azotemic cats, it was unknown why sera were collected and if they were clinically ill from something other than renal disease. This information could have aided in the interpretation of the prevalence rates observed in cats with and without azotemia.

Conclusions

This study showed that cats given a commercially available canine Leptospira species vaccine form antibodies that are detectable via a MAT and that variable titer magnitudes are observed against different serovars similar to what is observed in dogs.

To our knowledge, this is the first time that it has been documented that cats experimentally infected with B burgdorferi do not form antibodies that cross-react with the Leptospira species MAT.

There was no difference between Leptospira species seropositivity in aged client-owned cats with and without azotemia.