Validation of a point-of-care polymerase chain reaction assay for detection of Streptococcus equi subspecies equi in rostral nasal swabs from horses with suspected strangles

This study aimed to validate a point-of-care polymerase chain reaction (PCR) assay for detection of Streptococcus equi subsp. equi (S. equi) in rostral nasal swabs from horses with suspected acute strangles and to compare the results against the molecular gold standard of quantitative polymerase chain reaction (qPCR). Two hundred thirty-two individual swabs of rostral nasal passages were characterized by qPCR as S. equi positive, S. equi subsp. zooepidemicus (S. zooepidemicus) positive, or S. equi and S. zooepidemicus negative. The specificity and sensitivity of the point-of-care PCR assay were 89% and 84%, respectively. The limits of detection of the qPCR assay and the point-of-care PCR analyzer were 3 and 277 eqbE target genes of S. equi, respectively. Overall agreement and short turnaround time make the point-of-care PCR assay a potential molecular diagnostic platform that will enhance the capability of equine veterinarians to timely support a diagnosis of strangles and institute proper biosecurity protocols.     

Bacteriological and Molecular Detection of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in Equines of Northern India

Present study was undertaken to study the prevalence of β-haemolytic streptococci in equine of northern temperate region of Jammu and Kashmir, India. One hundred and forty one samples were collected in duplicate from nasopharyngeal tract of diseased (53) and apparently healthy equine (88) for isolation and direct PCR. A total of 77 isolates of streptococci were recovered from 141 samples with an overall prevalence rate of 54.60%. Out of these 77 isolates, 52 were from diseased and 25 from apparently healthy animals. Of the 77 isolates, 4 were identified as Streptococcus equi subsp. equi, 56 as S. equi subsp. zooepidemicus and 17 as S. dysgalactiae subsp. equisimilis. Thus the overall prevalence of S. equi subsp. equi, S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis was 2.83, 39.71 and 12.05% respectively. The sensitivity of the PCR for the detection of S. equi species was found higher when attempted from direct swab samples.     

Cytokine expression by neutrophils of adult horses stimulated with virulent and avirulent Rhodococcus equi in vitro

Rhodococcus equi is an intracellular pathogen of macrophages that causes rhodococcal pneumonia in foals and immunocompromised people. Evidence exists that neutrophils play a vital role in resistance to infection with R. equi; however, the means by which neutrophils exert their effects have not been clearly defined. In addition to directly killing bacteria, neutrophils also may exert a protective effect by linking innate and adaptive immune responses. In the present study we evaluated the cytokine expression profiles of adult equine neutrophils in response to stimulation with isogenic strains of virulent and avirulent R. equi in vitro. After 2 and 4 h incubation with virulent or avirulent R. equi, adult equine neutrophils expressed significantly (P < 0.05) greater tumor necrosis factor alpha (TNFα), interleukin (IL)-12p40, IL-6, IL-8 and IL-23p19 mRNA, but not interferon gamma (IFNγ) or IL-12p35 mRNA than unstimulated neutrophils. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA than avirulent R. equi. These results demonstrate that R. equi-stimulated neutrophils are a source of many proinflammatory cytokines. Furthermore, these results suggest that IL-23 may be preferentially expressed over IL-12 in response to exposure with R. equi, and that this response may be more strongly induced by virulent R. equi than avirulent R. equi. Collectively, the data presented herein suggest a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.     

Effects of inoculum size on cell-mediated and humoral immune responses of foals experimentally infected with Rhodococcus equi: A pilot study

The objective of this pilot study was to compare the cytokine profile as well as cell-mediated and antibody responses of foals infected with a low inoculum of virulent Rhodococcus equi resulting in subclinical pneumonia to that of foals infected with a high inoculum resulting in severe clinical pneumonia. The mean (±SD) ratio of post-infection to pre-infection anti-R. equi IgG(T) concentration was significantly (P = 0.002) higher in foals infected with the high inoculum (195 ± 145; range 62–328) compared to foals infected with the low inoculum (3.9 ± 4.5; range 0.5–11). Similarly, mean (±SD) ratio of post-infection to pre-infection IgM concentration was significantly (P = 0.002) higher in foals infected with the high inoculum (12 ± 4.0; range 7.4–14) compared to foals infected with the low inoculum (2.5 ± 1.5; range 1.2–4.7). Proliferative responses to R. equi antigens as well as expression of mRNA for IL-2, IL-4, IL-10, and IFN-γ in BLN were not significantly different between the two groups. There was a tendency (P = 0.073) towards a higher IFN-γ/IL-4 ratio in the low inoculum group. This study demonstrates that the size of inoculum modulates the IgG subisotype response and possibly the cytokine profile of foals.     

Genotypic characterization of VapA positive Rhodococcus equi in foals with pulmonary affection and their soil environment on a warmblood horse breeding farm in Germany

Pulsotypes of VapA positive Rhodococcus equi isolated from foals and soil on a farm in Germany were characterized on the basis of nasal and tracheal samples simultaneously collected in 2003 from 217 foals with sonographic evidence of pneumonia or pulmonary abscesses. Of the 217 double samples, R. equi was isolated in 118 (54%) of the tracheal samples and in 52 of the nasal swab samples (24%) (P < 0.001). Furthermore, 37 and 55 isolates were also randomly selected from nasal swabs and the tracheal samples, respectively, and further processed to determine the presence of VapA by colony blot enzyme-linked immunosorbent assay. This method showed that 26 (68%) of the nasal swabs and 40 (73%) of the tracheal samples were VapA-positive.R. equi was isolated from 56 (87%) of the 64 soil samples taken from the paddocks and stables in March and from 17 (68%) of the 25 samples taken in July of 2003. Three (21%) of these randomly selected 14 isolates from March and 13 (81%) of the 16 from July were VapA-positive.The VapA positive isolates from foals and soil were genotyped by plasmid profiling, restriction fragment length polymorphism analysis and pulsed-field gel electrophoresis. Of the 83 isolates, 80 contained an 85-kb type I plasmid and three contained an 87-kb type I plasmid. Pulsed-field gel electrophoresis yielded four distinct VspI profiles dividing 83 isolates into three major (A1, 51; D, 14; and 11 isolates) and three minor (C, 4; A3, 2; and A2, 1 isolates) groups. These results suggest that the majority of foals were exposed to and infected with three pulsotypes of VapA positive R. equi containing an 85-kb type I plasmid.     

An outbreak of fatal hemorrhagic pneumonia caused by Streptococcus equi subsp. zooepidemicus in shelter dogs

An outbreak of fatal hemorrhagic pneumonia with 70~90% morbidity and 50% mortality occurred in an animal shelter in Yangju, Gyeonggi Province, Korea. Clinically, the affected dogs showed severe respiratory distress within 48 h after arriving in the shelter. The dead were found mainly with nasal bleeding and hematemesis. At necropsy, hemothorax and hemorrhagic pneumonia along with severe pulmonary consolidation was observed, though histopathological analysis showed mainly hemorrhagic bronchopneumonia. Lymphoid depletion was inconsistently seen in the spleen, tonsil and bronchial lymph node. Gram-positive colonies were shown in blood vessels or parenchyma of cerebrum, lung, liver, spleen, and kidney. Also, Streptococcus (S.) equi subsp. zooepidemicus was isolated from the various organs in which the bacterium was microscopically and histologically detected. In addition, approximately 0.9 Kb specific amplicon, antiphagocytic factor H binding protein, was amplified in the bacterial isolates. In this study, we reported an outbreak of canine hemorrhagic bronchopneumonia caused by S. equi subsp. zooepidemicus in an animal shelter in Yangju, Korea.     

Seroprevalence and risk factors associated with Babesia caballi and Theileria equi infection in equids

A cross-sectional study was carried out on equids (horses, mules and donkeys) in Andalusia, Southern Spain, to assess the level of exposure to equine piroplasmosis and to investigate risk factors associated with these infections. At least one animal seropositive for Theileria equi and/or Babesia caballi was detected in 222/380 (58.4%) herds sampled by competitive inhibition ELISAs. The seroprevalences for B. caballi and T. equi were 13.2% and 56.1%, respectively; there was serological evidence of co-circulation of both piroplasms in 10.8% of herds. Antibodies against equine piroplasms were detected in 286/537 (53.3%) animals; 61 (11.4%) were seropositive for B. caballi, 270 (50.3%) were seropositive for T. equi and 24 (8.4%) were seropositive for both T. equi and B. caballi.There was a significantly higher seroprevalence of B. caballi in mules (32.1%) compared with donkeys (17.0%) and horses (7.9%), and a significantly higher seroprevalence of T. equi in mules (66.1%) in comparison with horses (48.6%), but not donkeys (47.2%). There were significant differences in prevalence of both piroplasms among locations; the seroprevalence of B. caballi ranged from 0 to 22.5%, while the seropositivity to T. equi ranged from 26.7 to 63.3%. A multiple logistic regression model indicated that the risk factors associated with a higher T. equi seroprevalence were increased age, presence of ticks and vaccination against other diseases. Risk factors associated with a higher seroprevalence of B. caballi were species (mules compared to horses), entry of horses in the last 6 months, presence of ticks and presence of shelter. The findings indicate widespread exposure to equine piroplasmosis in Southern Spain.     

Adherence of Streptococcus equi on tongue,cheek and nasal epithelial cells of ponies

Streptococcus equi was found to adhere to tongue, cheek and nasal epithelial cells of ponies, in vitro. Maximum adherence was observed at pH 7.5 after one hour of incubation of bacteria with epithelial cells. This adherence was more on epithelial cells from adult animals than foals. Streptococci exposed to heat (60°C for 10 min) or treated with pepsin or trypsin showed a reduced adherence, whereas an increase occurred on treatment with hyaluronidase. Antibodies against whole S. equi cells or M-like protein blocked the adherence, whereas antibodies against group-specific carbohydrate or lipoteichoic acids did not. Pretreatment of epithelial cells with either the M-like protein or crude extract of S. equi lowered the adherence, whereas an extract of S. zooepidemicus did not. Adherence of S. equi to the epithelial cells was considered to be mediated by structures specific to S. equi.     

Occurrence of canine hemotropic mycoplasmas in domestic dogs from urban and rural areas of the Valdivia Province,southern Chile

This is the first study to investigate the occurrence, risk factors and hematological findings of hemoplasmas in dogs from Chile. Complete blood count and 16S rRNA conventional PCR for Mycoplasma spp. were performed in 278 blood samples from rural (n = 139) and urban (n = 139) dogs in Valdivia. Real time 16S rRNA PCR (qPCR) allowed species identification. Mycoplasma spp. occurrence was 24.8%. ‘Candidatus M. haematoparvum’ (CMhp) was identified in 12.2% and Mycoplasma haemocanis (Mhc) in 11.9% dogs. It was not possible to identify species in two Mycoplasma spp. samples by qPCR. Sequencing allowed identifying one of them as ‘Candidatus M. turicensis’ (CMt). Frequency in rural localities was higher (41.7%) than in urban (7.9%). Rural locality, maleness and older age were risk factors for hemoplasmosis. Hemoplasma-positive dogs had a higher total protein. This is the first report of Mhc, CMhp and CMt in dogs from Chile, with a high occurrence in rural localities.     

Streptococcus equi subsp. zooepidemicus isolates from equine infectious endometritis belong to a distinct genetic group

Streptococcus equi subsp. zooepidemicus is the pathogen most commonly isolated from the uterus of mares. S. zooepidemicus is an opportunistic pathogen and part of the resident flora in the caudal reproductive tract. The aim of this study was to investigate whether a genotypically distinct subpopulation of S. zooepidemicus is associated with endometritis in the mare, by genotyping and comparing uterine S. zooepidemicus strains with isolates from the vagina and clitoral fossa. Mares with (n = 18) or without (n = 11) clinical symptoms of endometritis were included. Uterine samples were obtained using a guarded endometrial biopsy punch, whereas a swab was used to recover samples from the cranial vagina and the clitoral fossa. If S. zooepidemicus was present, up to three colonies were selected from each anatomical location (max. 9 isolates per mare). Bacterial isolates were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). S. zooepidemicus was isolated from the endometrium of 12 mares. A total of 88 isolates were analyzed by PFGE: 31 from the endometrium, 26 from the cranial vagina and 31 isolates from the clitoral fossa. For MLST 21 isolates were chosen. Results demonstrated a higher genetic similarity of the isolates obtained from infectious endometritis compared to isolates obtained from the caudal reproductive tract. In conclusion, we demonstrate for the first time that a genetically distinct group of S. zooepidemicus is associated with infectious endometritis in the mare.     

Multiplex PCR-based identification of Streptococcus canis,Streptococcus zooepidemicus and Streptococcus dysgalactiae subspecies from dogs

Streptococcus canis (S. canis), Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) and Streptococcus dysgalactiae subspecies (S. dysgalactiae subspecies) are β-haemolytic Gram positive bacteria infecting animals and humans. S. canis and S. zooepidemicus are considered as two of the major zoonotic species of Streptococcus, while more research is needed on S. dysgalactiae subspecies bacteria. In this work, a multiplex-PCR protocol was tested on strains and clinical samples to detect S. canis, S. dysgalactiae subspecies and S. equi subspecies bacteria in dogs. All strains were correctly identified as S. canis, S. equi subspecies or S. dysgalactiae subspecies by the multiplex-PCR. The main Streptococcus species isolated from symptomatic dogs were confirmed S. canis. The multiplex-PCR protocol described is a rapid, accurate and efficient method for identifying S. canis, S. equi subspecies and S. dysgalactiae subspecies in dogs and could be used for diagnostic purposes and for epidemiological studies.     

Comparison of three feline leukaemia virus (FeLV) point-of-care antigen test kits using blood and saliva

Feline leukaemia virus (FeLV) can be a challenging infection to diagnose due to a complex feline host-pathogen relationship and occasionally unreliable test results. This study compared the accuracy of three point-of-care (PoC) FeLV p27 antigen test kits commonly used in Australia and available commercially worldwide (SNAP FIV/FeLV Combo, Witness FeLV/FIV and Anigen Rapid FIV/FeLV), using detection of FeLV provirus by an in-house real-time polymerase chain reaction (qPCR) assay as the diagnostic gold standard. Blood (n = 563) and saliva (n = 419) specimens were collected from a population of cats determined to include 491 FeLV-uninfected and 72 FeLV-infected individuals (45 progressive infections [p27 and qPCR positive], 27 regressive infections [p27 negative, qPCR positive]). Sensitivity and specificity using whole blood was 63% and 94% for SNAP Combo, 57% and 98% for Witness, and 57% and 98% for Anigen Rapid, respectively. SNAP Combo had a significantly lower specificity using blood compared to the other two kits (P = 0.004 compared to Witness, P = 0.007 compared to Anigen Rapid). False-positive test results occurred with all three kits using blood, and although using any two kits in parallel increased specificity, no combination of kits completely eliminated the occurrence of false-positive results. We therefore recommend FeLV proviral PCR testing for any cat that tests positive with a PoC FeLV antigen kit, as well as for any cat that has been potentially exposed to FeLV but tests negative with a FeLV antigen kit, before final assignment of FeLV status can be made with confidence. For saliva testing, sensitivity and specificity was 54% and 100%, respectively, for all three test kits. The reduced sensitivity of saliva testing compared to blood testing, although not statistically significant, suggests saliva testing with the current generation of PoC FeLV antigen kits is unsuitable for screening large populations of cats, such as in shelters.     

Genetic diversity of Streptococcus equi subsp. zooepidemicus isolated from horses

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic and zoonotic pathogen of horses. In this study, genetic intraspecies variability of SEZ obtained mainly from respiratory and genital samples of horses was investigated by analysis of the 16S–23S rRNA intergenic spacer region (ISR) and of the 16S rRNA gene. 16S–23S ISR rRNA type A1 was predominant, although a high rate of multiple products (30.5%) was obtained. Phylogenetic analysis of the 16S rRNA gene detected three genogroups (I, II and III). 16S rRNA variable regions V1 and V2 are the most important regions for evaluating SEZ intraspecies variability, but at least V1-V5 regions should be considered to avoid mistakes. Analysis of all 16S rRNA sequences available in databases assigned human SEZ to groups I and III but not to group II. These results show a high genetic variability in SEZ collected from different specimens of horses from various regions of Italy.     

Diagnostic testing patterns for Streptococcus equi subsp. equi in Ontario horses during the years 2008 to 2018

This retrospective study describes testing patterns and the incidence of Streptococcus equi subsp. equi in Ontario to assess the utility of laboratory data for surveillance purposes. Laboratory records for equine infectious disease test submissions were extracted from the Animal Health Laboratory (AHL) at the University of Guelph for the years 2008 to 2018. Yearly and seasonal trends in S. equi testing and the proportion of tests that returned positive results were assessed. The number of samples submitted for S. equi testing decreased over the 11-year period (odds ratio = 0.96, 95% confidence interval: 0.92 to 0.999; P = 0.04). A generalized linear model identified a significant seasonal effect for animals recognized as clinically ill, with the highest test positivity noted in the winter. Although this study identified important trends in the incidence of S. equi in Ontario, the variability in information accompanying test submissions made the data challenging to interpret, highlighting the need for more complete diagnostic submission data for S. equi.     

Development and use of real-time PCR to detect and quantify Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum” in dogs

Two canine haemoplasma species have been recognised to date; Mycoplasma haemocanis (Mhc), which has been associated with anaemia in splenectomised or immunocompromised dogs, and “Candidatus Mycoplasma haematoparvum” (CMhp), recently described in an anaemic splenectomised dog undergoing chemotherapy. The study aim was to develop quantitative real-time PCR assays (qPCRs) incorporating an endogenous internal control to detect Mhc and CMhp and to apply these assays to DNA samples extracted from canine blood collected in Northern Tanzania (n = 100) and from dogs presented to a Trinidadian veterinary hospital (n = 185).QPCRs specific for Mhc and CMhp were designed using 16S rRNA gene sequence data, and each was duplexed with an assay specific for canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The assays detected ≤10 copies of a sequence-specific haemoplasma plasmid per reaction and neither assay showed cross-reactivity with 10⁶ copies of the sequence-specific plasmid from the non-target canine haemoplasma species.Nineteen of the 100 Tanzanian samples (19%) were positive for Mhc alone and one (1%) was dually infected. One Trinidadian sample was negative for canine GAPDH DNA and was excluded from the study. Of the 184 remaining Trinidadian samples, nine (4.9%) were positive for Mhc alone, five (2.7%) for CMhp alone, and two (1.1%) dually infected.This is the first report of canine haemoplasma qPCR assays that use an internal control to confirm the presence of amplifiable sample DNA, and their application to prevalence studies. Mhc was the most commonly detected canine haemoplasma species.     

Characterization of a Streptococcus equi ssp. equi Isolate From a Strangles Outbreak in Thailand

Strangles, which is caused by Streptococcus equi ssp. equi, is one of the major infectious respiratory diseases in horses. Knowledge of isolates from different areas of the world is important for investigating the different strains of the disease. In contrast to many other countries, currently little is known about S. equi ssp. equi isolates in Thailand. In 2014, a farm in Thailand imported 20 horses from Europe. Approximately 1 month after arrival, 50% of the horses had developed pyrexia, mucopurulent nasal discharge, and abscesses of the mandibular lymph nodes. Nasal swabs of mucopurulent discharge were sent to a diagnostic laboratory, and two isolates of S. equi ssp. equi were identified. One of the isolates was further characterized using seM gene polymerase chain reaction and sequence analysis. The seM sequence was then compared to the database of PubMLST-seM. It was found to contain SeM allele 48, an allele isolated from horses in the United Kingdom in 2006 and 2010. This result demonstrates the usefulness of SeM allele identification as a tool for investigating the source of related strains and for the epidemiologic study of strangles. To the best of the authors' knowledge, this is the first report of the identification of an SeM allele of the S. equi ssp. equi isolate in Thailand.     

The critical threshold of Lawsonia intracellularis in pig faeces that causes reduced average daily weight gains in experimentally challenged pigs

Serology indicates that Lawsonia intracellularis infection is widespread in many countries, with most pigs seroconverting before 22 weeks of age. However, the majority of animals appear to be sub-clinically affected, demonstrated by the low reported prevalence of diarrhoea. Production losses caused by sub-clinical proliferative enteropathy (PE) are more difficult to diagnose, indicating the need for a quantitative L. intracellularis assay that correlates well with disease severity. In previous studies, increasing numbers of L. intracellularis in pig faeces, quantified with a real time polymerase chain reaction (qPCR), showed a strong negative correlation with average daily gain (ADG).In this study, the association between faecal L. intracellularis numbers and PE severity was examined in two L. intracellularis experimental challenge trials (n₁ = 32 and n₂ = 95). The number of L. intracellularis shed in individual faeces was determined by qPCR on days 0, 7, 14, 17 and 21 days post challenge, and average daily gain was recorded over the same period. The severity of histopathological lesions of PE was scored at 21 days post challenge. L. intracellularis numbers correlated well with histopathology severity and faecal consistency scores (r = 0.72 and 0.68, respectively), and negatively with ADG (r = ?0.44). Large reductions in ADG (131 g/day) occurred when the number of L. intracellularis shed by experimentally challenged pigs increased from 10⁷ to 10⁸ L. intracellularis, although smaller ADG reductions were also observed (15 g/day) when the number of L. intracellularis increased from 10⁶ to 10⁷ L. intracellularis.     

Haemorrhagic pneumonia in sled dogs caused by Streptococcus equi subsp. zooepidemicus - one fatality and two full recoveries: a case report

In spite of yearly vaccination, outbreaks of canine infectious respiratory disease are periodically seen amongst domestic dogs. These infections compromise host defense mechanisms, and, when combined with other stressful events, allow opportunistic pathogens like Streptococcus equi subsp. zooepidemicus to create serious disease. Early recognition and treatment are tremendously important for a successful outcome in these cases. A polyvalent vaccine was given to 22 racing dogs three days after a competition, followed by two days of rest, and then the dogs were returned to regular training. Coughing was noticed among the dogs four days after immunisation. Three days after this outbreak one of the dogs was unusually silent and was found dead the next morning. Simultaneously two other dogs developed haemorrhagic expectorate, depression and dyspnea and were brought in to the veterinary hospital. Streptococcus equi subsp. zooepidemicus was isolated in pure culture from all three cases. They were treated and rehabilitated successfully, and won a sledge race three months later. This paper discusses the necropsy results, treatment regime, rehabilitation and the chronology of vaccination, stressful events and disease.     

First Report of Molecular Characterization of Argentine Isolates of Streptococcus equi subsp. equi by Pulsed-Field Gel Electrophoresis

Strangles is one of the most frequently diagnosed equine respiratory infectious diseases in the world. It is caused by Streptococcus equi subsp. equi (S. equi), and it is an acute infection characterized by pyrexia, nasal discharge, pharyngitis, and abscessation of lymph nodes. Frequently, healthy horses might continue to harbor S. equi after clinical recovery. Although the genetic distance between S. equi isolates is short, strains can be differentiated by pulsed-field gel electrophoresis (PFGE) and single locus sequence typing for epidemiological studies. The aim of this study was to characterize by PFGE Argentine isolates of S. equi obtained from horses with acute strangles and those that had recovered. Bacterial isolation and identification of 80 S. equi isolates by phenotypic and genotypic tests were performed using samples from 29 horses with acute strangles and 95 from healthy animals. Also, the isolates were characterized by PFGE using Bsp120I and SmaI. Visual comparison of macrorestriction patterns generated with both enzymes revealed three different DNA fragment profiles with variations of one or two bands. Interestingly, an identical profile was found in isolates from the same horse and from horses that were infected at the same time, and the horses recovered from strangles continue to carry the same strain. Some vaccinated horses have been mild infected for a different strain from that of carriers suggesting other source of infection. This is the first molecular characterization of Argentine isolates of S. equi, which shows the presence of three strains between 2010 and 2013 in Buenos Aires.     

Rapid identification and differentiation of Theileria sergenti and Theileria sinensis using a loop-mediated isothermal amplification (LAMP) assay

The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of two benign bovine Theileria species – T. sergenti and T. sinensis. The LAMP assay is inexpensive and easy to perform and involves a rapid reaction-the amplification can be performed in 55 min or 50 min under isothermal conditions of 61 °C or 63 °C, respectively, by employing a set of four species-specific primer mixtures. The results can be checked using agarose gels. The optimal assay conditions, under which the assay exhibited with no cross-reaction with other closely related tick-borne parasites (T. annulata, Babesia bovis, B. bigemina, B. major, B. ovata, B. U. sp., Anaplasma marginale) or between the two Theileria species of interest, was established. The assay is approximately 10-fold more sensitive than the conventional specific PCR assay. The LAMP assay was validated using DNA from 6 standard stocks in the laboratory and was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infection cattle or yaks in China. These findings indicate that this Theileria species-specific LAMP assay may have potential clinical applications for the detection and differentiation of two benign bovine Theileria species – T. sergenti and T. sinensis, especially in endemic countries.