Serological Detection of Anti-Leptospira Antibodies in Shelter Cats in Malaysia

Thesis Overview

Introduction

Leptospirosis is one of the most globally widespread zoonotic diseases, and is frequently reported in tropical regions.1 The causative agent for the infection is Leptospira, a bacterium belonging to the spirochetes phylum.2 Almost every mammalian species can become a carrier for Leptospira after exposure.3 The high mortality rate associated with leptospirosis in people exceeds other serious diseases such as Viral Hemorrhagic Fevers, with annual mortality of more than 1 million worldwide.4, 5 Transmission occurs in 2 ways; through direct contact with contaminated urine or indirectly through exposure to a contaminated environment.6Cats showed susceptibility to Leptospira infection, with seroprevalence varying between 4.8% and 48% in published studies.7, 8, 9, 10, 11 Several pathogenic serovars have been reported across feline species, namely L interrogans and L kirschneri.12, 13, 14 Infection with Pomona, Icterohaemorrhagiae, Canicola, Bataviae, and Ballum have also been reported. Most previously published anti-Leptospira antibodies detection studies in cats have been based on Microscopic Agglutination Test (MAT), and infective serogroups identified varied due to geographic location, endemicity of serovars, and also the preferred hosts.12According to a recent study in Greece, based on a 1:50 cut-off point, the seroprevalence in household cats (n = 99) was 33%, and most of them were positive for Autumnalis.9 Another study reported a 25% seropositivity in cats (n = 40) presented to a Canadian referral hospital.15 It is known that rodents are the main asymptomatic reservoir of Leptospira.16 Rats are likely to be the main source of infection for cats due to cats’ hunting instinct.12 Clinical signs of leptospirosis in cats are yet to be investigated; however, in previous studies, cats with polyuria and polydipsia were reported to be more likely to have anti-Leptospira antibodies.8, 17Leptospiruria has been observed in experimentally infected cats and both stray and household cats have been reported to shed the DNA from pathogenic Leptospiraspecies in urine.17, 18, 19, 20, 21 Therefore, cats might be reservoir or incidental host of Leptospira; however, the role of cats as potential source of the infection is unclear.12In Malaysia, cats are the most common companion animal and live in close vicinity people. There is a paucity of published data regarding leptospirosis in cats and their predominant serovars.22 This preliminary study aimed to investigate feline leptospirosis serologically among the shelter cat population in Malaysia and to determine the predominant serogroups.

Methods and Materials

Blood Sample Collection

This study was approved by the Institutional Animal Care and Use Committee(UPM/IACUC/AUP-R050/2017). Blood samples were obtained randomly from sheltered cats, between June 2017 and February 2018. Demographic details such as sex, as well as environmental/weather-related factors that could influence likelihood of exposure to Leptospira were recorded. A total of 3 mL of blood was collected from the jugular vein, using a 23-gauge needle and placed in plain tubes, which were centrifuged at 4000 rpm for 5 minutes. Serum samples were stored at −80°C for further investigation.

Leptospiral Antigens

Microscopic Agglutination Test

Serial dilutions of the sera samples, negative, and positive controls were prepared in the sterile microtiter plate wells. The serum was tested with a starting titer of 1:50-1:1600. A total of 96 µL Phosphate buffered saline (PBS) and 4 µL serum were distributed into the first wells and followed by a 2-fold dilution, then 50 µL antigen cultures at a density of 1 × 108 cell/mL were added equally into each well, then labeled. The flat-bottomed microtiter plates with the samples and negative controls were incubated for 2 hours at 30°C. Dark-field microscope examination was performed on the sera per each well. Samples were recorded as positive when agglutination of more than 50% compared to the negative control was detected, with a cut-off antibody titer of ≥1:100.

Results

A total of 110 feline blood samples were collected from 4 shelters located in different states in Malaysia (Johor and Selangor). Only adult cats were recruited in this study, but the exact age of the cats was unknown since all were previously stray. Of the 110 cats sampled, 38 were males (34.5%) and 72 were females (65.4%). Despite occasionally raining periods, no flooding incidence was reported in the shelters during period of time over which samples were collected. Generally, the cats selected for sampling appeared healthy, with minimal evidence of feline upper respiratory disease. Using the ≥1:100 titer cut-off point, anti-Leptospira antibodies were detected in 18.18% (n = 20/110) of the cats. The predominant serogroups found were Bataviae, Javanica, and Ballum.

Discussion

Leptospirosis is endemic in Malaysia, with reported human cases on the rise from 263 cases in 2004, to 7806 cases and 92 human deaths reported in 2014. The highest incidence was reported in Malacca in 2012, with a prevalence as high as 11.12 per 100,000 people.1, 23 High humidity and temperature in Malaysia provide an optimal atmosphere for the survival of the Leptospira species. Cats in Malaysia are the most common companion animal and live in close proximity to the human population. Most of the households keep more than 3 cats and cats are considered as semi-roamer. Due to the lack of proper population control and public awareness, the stray cat population has grown to become a major issue in recent years. The stray cats hunt rats and roam freely. In this study, we demonstrated that as high as 18.18% of the sheltered cat population was seropositive to Leptospira. The seroprevalence of Leptospira in the current study is similar to that reported in Germany, where 17.9% of 195 outdoors cats were seropositive.21 Another study evaluating 124 owned cats from urban and rural areas in Chile, reported a lower seropositivity of 8.1% against serogroups Canicola, Autumnalis, Grippotyphosa, and Bataviae by using the MAT.24 The variability of serodetection of Leptospira in cats in the published literature may be due to regional differences in the endemicity of the infection and MAT restrictions used for identifying “positive” cases (cut-off value and serovars panel). When a lower cut-off titer than 1:100 was utilized in previous study in Greece, it revealed seroprevalence of 33%.9In the present study, the predominant serogroups detected were Bataviae, Javanica, and Ballum. Serovars Javanica and Bataviae have been reported among the pathogenic serovars with the highest prevalence in Malaysia,1 and both were detected in 84 human cases of leptospiral infection in a Malaysian hospital.25Previous studies from other regions (Indonesia, Greece, Taiwan, and Chile) also reported Bataviae and Javanica among the serovars initially detected in cats.9, 20, 24, 26 It is noteworthy that both serovars were also detected in a recent rat study conducted in Malaysia, and were the most common serovars among the rats (Rattus rattus and Rattus norvegicus), with seroprevalence as high as 20.2% (n = 19/112) for Javanica and 16% (n = 15/112) for Bataviae.27 In another characterization study carried out in Malaysia, all 13 leptospiral isolates obtained from screening 357 rats were identified as either Bataviae or Javanica.28 The predator instinct of cats for rats is suggested as a major route of leptospirosis transmission between the 2 species. Additionally, studies performed on both sheltered and working dogs in Malaysia revealed that Javanica and Bataviae were the predominant infective serovars in this species as well.29, 30 Serovar Ballum has been previously detected in rodents from different geographic locations, but not in Malaysia.31, 32, 33 According to previous study on experimentally infected cats, cats infected with Ballum had longer carrier status and showed more severe nephritis as compared to cats infected with other serovars.34 Hence, more studies need to be performed in order to investigate this particular serovar.The cut-off point for seropositivity used in this study was 1:100 following the World Organization for Animal Health recommendations.35 Among the 20 seropositive cats identified in the present study, most showed positivity with a titer of ≥1:200 (n = 14/20). Previous study on leptospirosis in cats have suggested that a titer of ≥1:200 should be considered an active infection due to the absence of leptospiral vaccination in cats.15 Two positive sera samples of 20 showed agglutination at titers of 1:1600 and 1:800 to leptospiral serovars (Table 2). The recorded titers were among the highest ever reported in cat studies, which normally reported titers ranging from 1:100 to 1:400.15, 20, 24, 36, 37 According to the previous literature, in endemic countries, a single titer of ≥1:800 is an indication of infection.16 In Taiwan, 38% (8/21) of seropositive cats at titers of 1:100-1:400 were able to shed DNA from pathogenic Leptospira species in the urine.20 Thus, the role of the feline population in Malaysia as a carrier of disease is worthy of further investigations given the fact that urinary shedding in cats may last up to 8 months.21 Although the MAT is a gold standard test for the detection of Leptospira,38 it is suggested that MAT might provide false positive results in cats through cross-reactivity with other feline spirochetes. However, a recent study revealed no cross-reaction with anti-Leptospira antibodies using the MAT in cats experimentally infected with B burgdorferi.39 In our study, 15 of 110 cats had Leptospira titers of 1:50. These cats were considered seronegative in order to avoid over interpretation of the result, which might due to background noise or cross-reaction at low titres.20 Nevertheless, the possibility of cross-reactivity among the different serovars cannot be ruled out completely. In this study, 2 of 20 seropositive cats revealed a reaction to more than 1 serogroup, namely, Bataviae and Javanica. This multiple reaction could be interpreted as a concomitant or coinfection.40 Titers of 1:400, 1:800, and 1:1600 are presumptive serogroups detection for Ballum, Bataviae, and Javanica, respectively, since a cross-reaction of antibodies will usually appear at lower concentrations.41 In addition to that, 90% (n = 18/20) of the seropositive cats sera in this study were agglutinated only to a single serovar antigen. The results of the current study indicate that cats are potentially exposed to leptospiral infection, but it is speculated that cats may act as sources of infection during optimal environmental conditions.36MAT is the gold standard used to identify the presumptive Leptospira serogroups of the infection.38, 42 As a preliminary study for anti-Leptospira antibody detection in cats in Malaysia, MAT was used in this study. The antigens panel used in the MAT contained the common Leptospira serovars present in Malaysia. However, there were a few serovars, which have been reported in cats in other countries (such as Castellonis and Butembo) that were not included in the panel.40 Based on the detection of anti-Leptospira antibodies in cats in Malaysia, demonstrated by the MAT results, further studies are warranted for investigation of urine shedding in naturally infected cats with leptospirosis, using PCR and organism isolation followed by serovars identification.

Conclusion

Anti-Leptospira antibodies were detected in 20 of the 110 sheltered cats sampled, and the 3 predominant serovars found were Bataviae, Javanica, and Ballum. The high titers detected suggest the possibility of an active infection in cats. The cross-reaction to both Javanica and Bataviae serovars indicates that cats may harbor or are exposed to more than 1 serovar concurrently.