Theileriosis, a significant tick-borne hemoprotozoan disease affecting cattle in tropical and subtropical regions, is caused by intracellular protozoan parasites of the genus Theileria (d’Oliveira et al., 1995). Among these, Theileria orientalis was haemoprotozoanconsidered a benign parasite, typically associated with subclinical infections characterised by anaemia and nonspecific clinical signs (Izzo et al., 2010; Kamau et al., 2011). However, in recent years, T. orientalis has emerged as a growing threat to the global livestock industry, with increasing reports of clinical oriental theileriosis in the Asia-Pacific region, resulting in substantial economic losses (Sivakumar et al., 2013; Eamens et al., 2013; Perera et al., 2014). A 48.84 % in Assam (Brahma et al., 2018), 82.29 % in Odisha (Selim et al., 2020) and 46.4 % in Kerala (Priya et al., 2017) undoubtedly depicts a higher prevalence of this infection on blood smear examination in India. In Kerala, fatal cases of T. orientalis infection in crossbred cattle have highlighted the severity of the disease and its significance as a major threat (Aparna et al., 2011).

Epidemiological studies have highlighted the widespread prevalence and genetic diversity of T. orientalis worldwide. In Australia, Perera et al. (2013) reported a high prevalence rate of 70.8 % for T. orientalis using MPSP-PCR among cattle. Similarly, in Sri Lanka, Sivakumar et al. (2013) observed a 62 % prevalence in cattle. Wang et al. (2018) identified a 36.5 % infection rate in cattle from seven provinces in China, while Park et al. (2017) documented a 41.3 % prevalence in the Republic of Korea. Annual economic losses associated with theileriosis were reported to be $250–300 million annually (Kovalchuk, 2022). The global burden of T. orientalis, particularly the Ikeda genotype, has become a significant concern across the Asia-Pacific region. In Australia, the disease has resulted in considerable economic losses, estimated at over $20 million annually in the beef and dairy sectors since 2006 (Jenkins, 2018). In Japan, an epidemiological survey of grazing cattle in eastern Hokkaido revealed widespread distribution of T. orientalis, with periodic outbreaks contributing to serious economic consequences for the livestock industry (Ota et al., 2009).

Routine diagnostic methods, such as blood smear examination, lack sensitivity in cases of low parasitaemia and making it challenging to differentiate between Theileria species or detect intra-erythrocytic piroplasms of T. orientalis, especially in subclinical or latent infections (d’Oliveira et al., 1995; Bilgic et al., 2010). Serological techniques, including immunofluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA), detect antibodies against Theileria antigens; however, they cannot distinguish active infections from carriers or residual antibodies and long-term carriers may test negative while continuing to transmit the parasite to ticks (Bilgic et al., 2010; Santos et al., 2013). To address these limitations, molecular diagnostics such as PCR have proven effective for detecting T. orientalis infections at levels far below the sensitivity of traditional methods and facilitate species and genotype identification (d’Oliveira et al., 1995). These tests excel in diagnosing clinical, subclinical and carrier status, which is crucial for identifying carriers that play a key role in transmitting infection (d’Oliveira et al., 1995; Santos et al., 2013). However, PCR techniques require expensive equipment and trained personnel limiting its use in low resource settings. Therefore, a rapid, sensitive, highly specific and cost-effective field-oriented diagnostic method for detection of T. orientalis is the need of the hour.

In this context, a novel new generation DNA amplification method, Loop-mediated isothermal amplification (LAMP) assays, offers a promising alternative for the early diagnosis of infectious agents (Mandal et al., 2015). It is highly sensitive and specific, does not require sophisticated equipment and produces results visible to the naked eye when used with colorimetric dyes compared to PCR and real-time PCR (Notomi et al., 2000). Single-step LAMP assays further reduce contamination risks by eliminating the need for post-reaction tube opening, a significant advantage over two-step DNA-binding dye methods (Tomita et al., 2008). For animal parasites, LAMP has been developed successfully for the detection of Toxoplasma gondii (Krasteva et al., 2009), Trypanosoma evansi (Thekisoe et al., 2005), Babesia gibsoni (Mandal et al., 2015), Cryptosporidium parvum (Karanis et al., 2007), Tritrichomonosis foetus (Oyhenart et al., 2013) and T. annulata (Liu et al., 2012). The recent development of a dry LAMP assay for diagnosing tropical theileriosis (Theileria annulata) (Divakar et al., 2024) highlights its potential as a rapid, field-ready diagnostic tool for resource-limited settings.

This study aimed to develop a LAMP assay using specifically designed primers targeting the MPSP gene for the diagnosis of Theileria orientalis infection in cattle, marking its first report globally, to the best of our knowledge. While previous studies have developed LAMP assays for the detection of Theileria spp. Thekisoe et al. (2005) developed two LAMP assays for diagnosis of East Coast fever (ECF) caused by Theileria parva, which targeted polymorphic immunodominant molecule (PIM) and p150 genes in African cattle. Liu et al. (2012) developed two LAMP assays targeting 18S rRNA and ITS sequences for detecting T. annulata in China. Chaouch et al. (2018) developed a LAMP assay targeting the Cyt b gene for diagnosing tropical theileriosis. However, this study targeted the MPSP gene for the detection of T. orientalis using LAMP, as it is a well-known species-specific marker that offers greater specificity compared to more conserved genes such as 18S rRNA. This makes our assay a potentially more accurate diagnostic tool, particularly in distinguishing T. orientalis from other Theileria spp. or co-infecting hemoparasites. Its cross-reactivity was evaluated with other common haemoparasites of cattle viz., Theileria annulata, Babesia bigemina, Anaplasma marginale and Trypanosoma evansi. Furthermore, the sensitivity of the LAMP assay was assessed and compared with the diagnostic outcomes of microscopy and PCR, highlighting its potential as a precise and reliable tool for detecting bovine oriental theileriosis.