Study of Seroprevalence and Seroconversion to A Novel Bacterial Pathogen in Thoroughbred Horses

Introduction

Kingella kingae is a gram-negative aerobic coccobacillus, first isolated in 1960 from children, and originally classified as Moraxella but subsequently transferred to the genus Kingella [1]. It was not until 1990 that culture and other techniques indicated that it was a significant cause of infection in young children [2] We have recently detected a novel bacterial species, identified by MALDI-TOF as Kingella kingae, in synovial and bone tissues surgically debrided from thoroughbred horses, aged between 3 and 15 months, with osteoarticular infection (OAI). Molecular studies (16s rRNA) indicated the organism was not Kingella kingae, but is likely a related but novel species (see GenBank Accession number KR494280). There are no data on Kingella infection in horses and there is only one other animal isolate of Kingella (Kingella potus) - isolated from the wound of a zookeeper bitten by a kinkajou (Potus flavus) [3]. The latest publication of Equine Medicine [4] does not mention any potential infection with Kingella species, nor is there any literature found on internet search, using the words “Kingella”, “horses”, “equine”. K. kingae infection in humans is an emerging infection with clinical manifestations including OAI such as osteomyelitis, septic arthritis, vertebral osteomyelitis. K. kingae is part of the normal flora of the upper respiratory tract and oral cavity in young children and is likely transmitted via respiratory droplets or direct contact [5]. Yagupsky reported high incidence of K. kingae among children, and high rates of transmission, in day care centers (DCC) in the United States, France, Israel and other countries [5]. K. kingae is reported as the commonest cause of OAI in young children, between 6 months and 5 years of age in France and Italy, and likely most developed countries [5, 6, 7]. When it causes disease, clinical presentation is often subtle and preceded by a recent history of stomatitis or upper respiratory infection [5]. As our recently discovered isolate from a bone specimen taken from a young equine with OAI appears to be related to K. kingae (based on 16s rRNA sequence deposited in GenBank - Accession number KR494280), there may be similarities between human and equine infection. A sporadic case report is analogous to the situation in humans that led to the discovery of widespread infection and invasive disease due to K. kingae in humans (children) [2] [5]. We have investigated the possibility of developing a reliable serology assay to detect sero- conversion as this should assist in epidemiological investigation, prevention and control of the infection. A serum–based IFAT (LED-immunofluorescent antibody test) was investigated. LED-IFAT has often been used as a gold standard test for the diagnosis of several infections [8, 9, 10, 11] as such test could be used as a first step in developing an automated assay that facilitates testing large numbers of specimens.

Results

Serum Samples from Mares and their Respective Foals (now Weanlings or Yearlings) on Farm A were Tested for any Evidence of Antibodies by LED-IFAT

Results are shown in Table 1 below. As the LED-IFAT test was negative for all 8 samples derived from Farm A, (denoting no evidence of exposure to Kingella bacteria in this herd), residents of Farm A (and their serum samples) served as negative controls.

Rajasekariah GH, et al. Study of Seroprevalence and Seroconversion to A Novel Bacterial Pathogen in Thoroughbred Horses. Virol Immunol J 2019, 3(1): 000203.

Discussion

Invasive infection with K. kingae is thought to begin with the bacterium colonizing the pharynx, crossing the mucosa, thence entering the circulation and reaching deeper tissues, such as bones and joints. Poor oral hygiene, pharyngitis, mucosal ulceration and recent virus infection, including herpes group viruses, may be predisposing factors for invasive infection [8]. In humans, infection has been shown to have higher prevalence in the autumn and winter months. As an oro-pharyngeal colonizer, K. kingae is transmitted via respiratory secretions, saliva, direct contact and, potentially, oral contact with contaminated objects (e.g. fomite spread). Recent studies suggest K. kingae strains demonstrate varying degrees of pathogenicity [8]. These points may also be relevant to equine infection with the novel bacterium, referred to as K. equi for now. with respect to acquisition, transmission and invasive disease such as OAI. Development of a reliable LED-IFAT depends upon careful selection of the optimal serum dilution to be used for the given test. Unless this is investigated thoroughly, non-specific binding of antibodies may be difficult to overcome. LED-IFAT as “the test of choice” for detection of antibodies has been applied to the investigation of several types of infection in horses: (a) equine protozoal myeloencephalitis [9], (b) Potomac horse fever due to Ehrlichia [10, 11], and (c) equine piroplasmosis [12, 13, 14]. These reports adjudge IFAT to be both reliable and the test of choice for detection of seroconversion. LED- IFAT is being extensively used for investigating the Sarcocystis neurona [15] which is showing 83% sensitivity and 97% specificity whereas Duarte et al. [16] compared IFAT and western blots for diagnosis of equine protozoal myeloencephalitis and recorded very higher (91 to 100%). In Korea seroprevalence of foals is investigated in Jeju Island, South Korea against Neospora, Toxoplasma and Sarcocystis and recorded very low seroconversion (2% and 2.6% seroconversion) by IFAT for Neospora and Toxoplasma respectively whereas horses do not show response against Sarcocystis [17]. The main intention of this pilot serology survey was to examine whether yearlings have antibodies to Kingella and, thereafter, to detect seroconversion, as the latter can assist with further investigation of events surrounding such seroconversion. Apart from aiding with such investigation, it can also reinforce the basic principle of clinical and subclinical illness. Some horses may seroconvert with no evidence of infection while others may seroconvert and show clinical illness e.g. OAI. It is hypothesized that the immune system of the horses must have responded to bacterial invasion (by Kingella) and thereby generated specific antibodies. Those yearlings, which were found to be positive for antibodies, have been designated as “seropositives”. We found 5 seropositives out of 10 foals tested from Farm B whereas there was only 1 seropositive out of 9 foals tested from Farm C. This preliminary investigation supports the hypothesis that invasive Kingella infection occurs in yearlings and that some of the yearlings affected by invasive infection develop OAI. When a more virulent strain is introduced into the herd (or develops by mutation of a low virulence strain that is already in the herd), more cases of OAI will likely be detected. Further testing on a larger number of sera was advised to confirm this finding. The hypothesis is therefore that a positive LED-IFAT result likely indicates exposure to Kingella, and infection monitor is suggested (Table 3). A larger serosurvey, supported by molecular assays, will enable determination of prevalence of horses in the herd with antibodies (seroprevalence) as well as the percentage of horses that are seroconverters. Additionally, closer assessment of the time at which seroconversion has occurred should enable clarification of the epidemiology of the infection. Specifically, horse movements and exposures around the time of seroconversion could be pinpointed with a view to implementing control measures. For example, one horse (B-8) seroconverted between 28 August 2012 and 15 October 2012. Exposure to K. equi occurred more likely sometime between 18 August 2012 and early October 2012. By checking movements and exposures of this horse one may gain insight into why exposure occurred and how it could be prevented. Likewise, for any other yearlings who have an initial negative Kingella antibody test result but then become Kingella antibody positive, it may be possible to glean valuable information on exposure. This could lead to implementation of measures that may facilitate prevention of invasive disease including OAI. In conclusion, this report is the first recorded serosurvey for detection of equine antibodies to a novel bacterial pathogen, resembling K. kingae. The method used (LED-IF) is robust and easy to perform and could potentially be used to screen equine populations on a wider basis, for individual testing (infection status – exposed, unexposed, seroconverted) as well as herd testing to pre-emptively identify introduction of the novel Rajasekariah GH, et al. Study of Seroprevalence and Seroconversion to A Novel Bacterial Pathogen in Thoroughbred Horses. Virol Immunol J 2019, 3(1): 000203.

bacterial pathogen into the herd and identify the need for further epidemiological investigations.