Rethinking Coxiella infections in Australia

Coxiellaburnetii is the causative agent of coxiellosis in animals and Q fever in humans. Despite being a vaccine preventable disease, Q fever remains a frequently reported zoonotic infection in Australia. Recently, a Coxiella species was identified in brown dog ticks (Rhipicephalus sanguineus) in urban and rural regions of Australia. Further molecular characterisation revealed that it is genetically identical to ‘Candidatus Coxiella massiliensis’ (KM079627) described in R. sanguineus ticks removed from humans with eschars in France and serologic cross-reactivity among ‘Ca. Coxiella massiliensis’ and C.burnetii may occur. This report highlights the need for molecular testing of seropositive companion animals and humans to determine which species of Coxiella they are infected with, in order to further assess Coxiella species associated with Coxiella infections in Australia.

Coxiella burnetii is a small, obligate intracellular, Gram-negative coccobacillus found worldwide (except in New Zealand) and has a sylvatic lifecycle involving wildlife and domestic mammals, birds, and arthropods^1,2. Coxiella burnetii was first described in the 1930s as the causative agent of Q (query) fever in abattoir workers in Brisbane, Queensland, Australia³. Coxiella burnetii is also the known cause of coxiellosis in animals and is persistently shed by infected animals in secretions and parturient by-products. Transmission occurs predominantly through direct or indirect contact with infected tissues from domestic ruminants and companion animals, rather than as a consequence of tick bite⁴. Clinical presentations of Q fever range from acute to chronic, and can lead to post-Q fever fatigue syndrome, although asymptomatic Q fever represents >54–60% of infections³. High annual reports of human Q fever in Australia persist despite a readily available vaccine⁵; over 4800 cases were reported between 2007 and 2017, with 716 notifications of Q fever in the past 18 months⁶.

Australian serological surveys have reported the number of infected dogs with C. burnetii has increased over 26 years to nearly 22%⁷, with free-roaming dogs within Indigenous communities having the highest seroprevalence compared with breeding, pet, or shelter dogs, in a most recent study⁸. It has been proposed that dogs become infected with C. burnetii through consumption of infected raw meat, hunting, and scavenging wildlife, or due to heavy tick infestations⁸, most commonly with Rhipicephalus sanguineus ticks⁹. While our knowledge about the epidemiology of C. burnetii in companion animals continues to increase, it is unclear whether the high C. burnetii-seropositivity observed in these animals contributes to increasing reports of Q fever cases in humans.

In addition to C. burnetii, several other Coxiella species and subtypes of the genus have been identified in a range of different hosts, including C. cheraxi, the cause of mass mortalities in Australian redclaw crayfish, (Cherax quadricarinatus)¹⁰; Coxiella spp. endosymbionts of ticks¹¹; and more recently, ‘Candidatus Coxiella massiliensis’, associated with ticks removed from humans with eschars¹². Molecular evidence suggests that C. burnetii originated from an inherited symbiont in soft ticks and acquired virulence factors enabling it to infect vertebrate cells¹¹. To date, over 40 tick species have been associated with C. burnetii and Coxiella spp. Amblyomma, Dermacentor,Ixodes, and Rhipicephalus species are the most frequently implicated vectors^11,13.

Tick-associated Coxiella spp. have a role in maintaining tick health and influence the vertical transmission of other tick-borne pathogens¹⁴. Due to their symbiotic role in ticks, Coxiella spp. endosymbionts of ticks are considered non-pathogenic to vertebrates, however, the dogma of what is considered an endosymbiont versus a pathogen has been challenged recently though the observation of serological reactions to a number of tick-associated endosymbionts in people following a tick bite^14,15. Furthermore, a retrospective study identified Coxiella sp. (‘Ca. Coxiella massiliensis’) in several tick species, including R. sanguineus ticks removed from patients presenting with scalp eschars, cervical lymphadenopathy, fever, increased C-reactive protein and thrombocytopenia^11,12. Following the recent molecular characterisation of a Coxiella sp. in R. sanguineus ticks in Australia¹⁶, this present study screened 41 R. sanguineus ticks with a Coxiella-specific GroEL PCR assay to determine the genetic relatedness to ‘Ca. Coxiella massiliensis’.

A Coxiella-specific PCR assay, targeting a 659 bp region of the GroEL gene was performed using the primers Cox-660f (GGCGCICARATGGTTAARGA) and Cox-1320r (AACATCGCTTTACGACGA) according to Angelakis et al.¹², with the following modifications: each 25 μL PCR reaction contained 1× Perfect Taq buffer (5 Prime, Germany), 1 mg/mL BSA (Fisher Biotech, Australia), 2.5 mM MgCl₂, 1 mM dNTPs, 400 nM of each primer, 1.25 U Perfect Taq polymerase (5 Prime, Germany) and 2 μL of undiluted DNA. All samples were performed under the following thermal conditions: initial denaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 30s, annealing at 52°C for 30 s, extension at 72°C for 1 min, and a final extension at 72°C for 5 min. A phylogenetic tree was constructed with a 547 bp trimmed alignment of all known Coxiella GroEL sequences, including those obtained in this study, with MrBayes 3.2.6¹⁷.

DNA was successfully amplified in 80% (33/41) of the R. sanguineus ticks and Sanger sequencing was conducted on 10 positive samples according to Oskam et al.¹⁶. All 10 sequences were identical to each other (MK119208), and 100% similar to ‘Ca. Coxiella massiliensis’ isolated from R. sanguineus in France (KM079627). Phylogenetic analysis revealed the ‘Ca. Coxiella massiliensis’ identified in this study had high support (posterior probability 1.0) to ‘Ca. Coxiella massiliensis’ found within other R. sanguineus ticks (Figure 1)¹². The prevalence of ‘Ca. Coxiella massiliensis’ in this study was higher than the ‘Ca. Coxiella massiliensis’ prevalence of 35% (7/20) reported by Angelakis et al. in R. sanguineus¹².

Figure 1. Phylogenetic tree based on 547 bp GroEL gene sequences including Coxiella associated with ticks, C. burnetii reference strain and an outgroup, Rickettsiella gyrll (cropped). The proposed ‘Candidatus Coxiella Massiliensis’¹² is highlighted by the teal box. The Bayesian tree was constructed using MrBayes 3.2.6¹⁶ with posterior probabilities and the following parameters were used: substitution model GTR, gamma category 5, chain length 1,100,000, sampling every 200 trees and burn-in length 100,000. Bold type indicates the consensus sequence from this study. Abbreviations: A., Amblyomma; D., Dermacentor; I., Ixodes; O., Ornithodoros; R., Rhipicephalus.

It is still unknown whether ‘Ca. Coxiella massiliensis’ can be transmitted to humans via tick bite or aerosol inhalation in Australia, however it prompts further investigation to determine if cross-reactions can occur among other Coxiella sp. in Q fever serological tests. This study highlights the need for molecular testing of companion animals and humans that are seropositive for C. burnetii to determine which species of Coxiella they are infected with and to comprehensively assess all species of Coxiella in Australia for health risks.

The authors declare no conflicts of interest.