Of 300 animal blood samples provided by the diagnostic lab, two samples were successfully amplified based on the gltA gene of Rickettsia with an overall infection rate of 0.67% (2/300). The positive samples were derived from a goat with an infection rate of 5.56% (1/18) and a sheep with an infection rate of 2.22% (1/45). None of the blood samples of wildlife animals reported rickettsial infections. The goat and sheep sample originated from a private and government farm, respectively. Our records state that the goat sample was sent for a diagnostic test as it presented symptoms such as lack of appetite, paleness, and weight loss while the sheep sample was sent for routine screening.
Through BLAST analysis, the sequence obtained from the goat (G325) and sheep (S102) both demonstrated 99.64% sequence similarity to Rickettsia asembonensis (MK923743), a recently discovered isolate from the ectoparasites of domestic animals in Peru, distinct from the original Kenya strain (Maina et al., 2016). The neighbour joining dendrogram constructed based on the gltA gene sequence in Fig. 1 revealed that both sequences G325 and S102, clustered with the respective reference R. asembonensis sequences (KY445724, KY445725, KX196267, AF516333) retrieved from the NCBI GenBank and showed a close relationship with R. senegelensis thus, confirming the findings obtained from the BLAST analysis. We reported the first detection of a genotype of Rickettsia asembonensis in sheep and goat in Malaysian farms.
Rickettsia asembonensis is a newly identified species (Maina et al., 2016), closely related to R. felis, and together with Ca. R. senegalensis and other genetically related species grouped as R. felis-like organisms (RFLO). There have been several reports of partially characterized agents that were closely related to R. asembonensis prior to its first complete characterization including the Rickettsia sp. RF2125, which was first identified in the flea specimens along the Thai-Myanmar border (Parola et al., 2003). Reports of Rickettsia asembonensis and those genetically related, have been identified in various flea species (predominantly Ctenocephalides felis) collected from domestic animals across the world (Maina, et al., 2016). In Malaysia, Rickettsia sp. Rf31, a strain of R. senegalensis and Rickettsia sp. RF2125, a strain of R. asembonensis have been reported in fleas (Ctenocephalides felis and Ctenocephalides orientis) and tick (Rhipicephalus sanguineus) parasitizing dogs and cats in several studies (Low et al., 2017; Mokhtar & Tay, 2011; Tay et al., 2014). In a vector surveillance study among farm animals and peri-domesticated animals in rural villages of Peninsular Malaysia, Rickettsia sp. RF2125 was detected in ticks (predominantly Haemaphysalis spp.) parasitizing cattle and the peri-domesticated animals (Kho et al., 2017). As a result, the potential of ectoparasites of domestic animals as vectors of this emerging pathogen should not be disregarded.
Despite the extensive research on the ectoparasite vectors of R. asembonensis, there is limited study on its animal reservoirs. Thusfar, it has been detected in the blood sample of healthy cynomolgus monkeys in Malaysia (Tay et al., 2015) and in blood samples of cats in Thailand (Phoosangwalthong et al., 2018). The identification of R. asembonensis in a goat and a sheep in this study therefore suggests that the infection caused by this species may be present in a wider range of animals, specifically farm animals.
The absence of rickettsial infections in other domestic animals such as cattle, may be due to better tick control or compliance to the Herd Health Program in the farms (Rohaya et al., 2017; Tay et al., 2014). Their compliance to these programmes encourages the implementation of additional control measures to avoid any disease-causing factors, such as eliminating potential vectors. Admittedly, these samples were sent to the diagnostic lab on account of suspicion of a parasitical infection, implying that these samples may not be a good representation of the population for a bacterial study. The PCR assays in this study were based on the gltA gene fragment of Rickettsia spp. as it is well-conserved, and a more extensive reference sequences are available in the NCBI GenBank for phylogenetic analysis. A gltA gene amplicon provides conclusive evidence that the agent belongs to the genus, Rickettsia (Parola et al., 2005). However, previous studies have used gltA PCR assay for initial screening of Rickettsia DNA before amplifying other Rickettsia specific genes such as ompA and ompB gene fragment (Kho et al., 2017). Since the detection was solely by gltA gene amplification, it is possible that it has remained undetected in most of the blood samples.
Our findings pinpoint the need of continuous surveillance of emerging and reemerging pathogens in animal reservoirs. Although low infection rate of R. asembonensis was detected in sheep and goat samples in this study, its zoonotic potential should not be disregarded in view of the close contact of animal farm workers or handlers with the animals. However, human pathogenesis of R. asembonensis remains unclear. There is an urgency to identify the true hosts responsible for transmission since human infections caused by this species of Rickettsia have been reported. For instance, R. asembonensis was identified in four patients displaying common febrile symptoms in Peru (Palacios-Salvatierra et al., 2018) and two human blood samples in Zambia (Moonga et al., 2021). Similarly, in Malaysia, the first report of this infection was in a 15-year-old boy presenting typical febrile symptoms however, with no signs of a tick bite (Kho et al., 2016). Interestingly, another case presented a co-infection with malaria in Sabah (Tay et al., 2019).
Our study has revealed the possibility of domestic animals (sheep and goat) as a potential host of rickettsial infections in Malaysia. The tropical climate of Malaysia creates a favourable environment for the proliferation of Rickettsia-infected ticks and fleas. An increased exposure to these vectors and its reservoirs, therefore, pose a zoonotic threat to the local population, especially animal handlers, and farm workers. This preliminary study has yet to establish the competency of the goat and sheep as reservoirs of this emerging pathogen however, the molecular evidence of rickettsioses in these samples introduces concern over the complexity of the host-reservoir system of this bacterium.