Dataset Information

Validation of a multiplex PCR assay to detect Babesia spp. and Anaplasma marginale in cattle in Uruguay in the absence of a gold standard test.

ABSTRACT: Detection of bovine Babesia spp. and Anaplasma marginale is based on the reading of Giemsa-stained blood or organ smears, which can have low sensitivity. Our aim was to improve the detection of bovine Babesia spp. and A. marginale by validating a multiplex PCR (mPCR). We used 466 samples of blood and/or organs of animals with signs and presumptive autopsy findings of babesiosis or anaplasmosis. The primers in our mPCR amplified the rap-1a gene region of Babesia bovis and B. bigemina, and the msp-5 region of A. marginale. We used a Bayesian model with a non-informative priori distribution for the prevalence estimate and informative priori distribution for estimation of sensitivity and specificity. The sensitivity and specificity for smear detection of Babesia spp. were 68.6% and 99.1%, and for A. marginale 85.6% and 98.8%, respectively. Sensitivity and specificity for mPCR detection for Babesia spp. were 94.2% and 97.1%, and for A. marginale 95.2% and 92.7%, respectively. Our mPCR had good accuracy in detecting Babesia spp. and A. marginale, and would be a reliable test for veterinarians to choose the correct treatment for each agent.

SUBMITTER: Parodi P

PROVIDER: S-EPMC7758682 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Validation of a multiplex PCR assay to detect Babesia spp. and Anaplasma marginale in cattle in Uruguay in the absence of a gold standard test.

Parodi Pablo P Corbellini Luis G LG Leotti Vanessa B VB Rivero Rodolfo R Miraballes Cecilia C Riet-Correa Franklin F Venzal José M JM Armúa-Fernández María T MT

Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 20201130 1

Detection of bovine Babesia spp. and Anaplasma marginale is based on the reading of Giemsa-stained blood or organ smears, which can have low sensitivity. Our aim was to improve the detection of bovine Babesia spp. and A. marginale by validating a multiplex PCR (mPCR). We used 466 samples of blood and/or organs of animals with signs and presumptive autopsy findings of babesiosis or anaplasmosis. The primers in our mPCR amplified the rap-1a gene region of Babe ...[more]

PMID: 33252025

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Project description:Tropical theileriosis, bovine babesiosis and anaplasmosis are tick-borne protozoan diseases that impose serious constraints on the health and productivity of domestic cattle in tropical and sub-tropical regions of the world. A common feature of these diseases is that, following recovery from primary infection, animals become persistent carriers of the pathogen and continue to play a critical role in disease epidemiology, acting as reservoirs of infection. This study describes development and evaluation of multiplex and single PCR assays for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Following in silico screening for candidate target genes representing each of the pathogens, an optimised multiplex PCR assay was established using three primer sets, cytob1, MAR1bB2 and bovar2A, for amplification of genomic DNA of T. annulata, A. marginale and B. bovis respectively. The designed primer sets were found to be species-specific, generating amplicons of 312, 265 and 166 base pairs, respectively and were deemed suitable for the development of a multiplex assay. The sensitivity of each primer pair was evaluated using serial dilutions of parasite DNA, while specificity was confirmed by testing for amplification from DNA of different stocks of each pathogen and other Theileria, Babesia and Anaplasma species. Additionally, DNA preparations derived from field samples were used to evaluate the utility of the single and multiplex PCRs for determination of infection status. The multiplex PCR was found to detect each pathogen species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA representing the other pathogens. Moreover, single and multiplex PCRs were able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of T. annulata, B. bovis and A. marginale, and no evidence of non-specific amplification from non-target species was observed. Validation that the multiplex PCR efficiently detects single and mixed infections from field samples was demonstrated. The developed assay represents a simple and efficient diagnostic for co-detection of tropical theileriosis, bovine babesiosis and anaplasmosis, and may be a valuable tool for epidemiological studies aimed at assessing the burden of multiple infection with tick-borne pathogens and improving control of the associated diseases in endemic regions.

Project description:Tick-borne pathogens (TBPs) seriously affect cattle production and can be economically damaging. The epidemiology of these organisms in the Chongqing municipality of China is not well described. This study aimed to investigate the prevalence and risk factors of TBPs including Anaplasma spp., Babesia spp. and Theileria spp. in cattle in Chongqing municipality. The results showed that 43.48% (150/345) of cattle were infected with at least one TBP, of which single infections were detected in 104 (30.14%), double infections in 34 cattle (9.86%) and triple infections in 12 (3.48%) of the cattle. The overall prevalence of Anaplasma spp., Theileria spp. and B. bigemina were 22.32%, 23.19% and 7.24%, respectively. Among these, the prevalence of A. bovis, A. central, A. phagocytophilum, A. platys, A. marginale, T. sinensisi and T. orientalis were 8.41%, 7.83%, 4.93%, 4.35%, 2.61%, 22.32% and 2.60%, respectively. We could not detect B. bovis, T. annulata, T. luwenshuni or T. uilenbergi in cattle. Cattle ≥1-year-old were more likely to be infected with Theileria spp. [adjusted odd ratio (AOR) = 2.70, 95% CI = 1.12-6.56)] compared with younger cattle, while cattle ≥1-year-old had reduced susceptibility to B. bigemina (AOR = 0.14, 95% CI = 0.03-0.60). Cattle living at higher altitude (≥500 m) were more susceptible to B. bigemina (AOR = 6.97, 95% CI = 2.08-23.35) and Theileria spp. infection (AOR = 1.87, 95% CI = 1.06-3.32). The prevalence of Theileria spp. on farms with cats was significantly higher than that without cats (AOR = 2.56, 95% CI = 1.12-5.88). Infection with A. bovis and A. central were significantly associated with A. phagocytophilum infection. Furthermore, there were significant associations between A. bovis and A. central infection, T. sinensisi and A. marginale infection, and B. bigemina and T. orientalis infection. This study provides new data on the prevalence of Anaplasma spp., Babesia spp. and Theileria spp. in cattle in Chongqing, and for the first time we reveal a possible relationship between the afore-mentioned pathogens, which will help in formulating appropriate control strategies for these pathogens in this area.

Project description:BackgroundTick-borne diseases (TBDs) constitute a major constraint for livestock development in sub-Saharan Africa, with East Coast fever (ECF) being the most devastating TBD of cattle. However, in Burundi, detailed information is lacking on the current prevalence of TBDs and on the associated economic losses from mortality and morbidity in cattle as well as the costs associated with TBD control and treatment. The aim of this study was, therefore, to assess the prevalence and spatial distribution of tick-borne pathogens (TBPs) in cattle across the major agro-ecological zones (AEZs) in Burundi.MethodsIn a cross-sectional study conducted in ten communes spanning the five main AEZs in Burundi, blood samples were taken from 828 cattle from 305 farms between October and December 2017. Evidence of Theileria parva infection was assessed by antibody level, measured using a polymorphic immunodominant molecule (PIM) antigen-based enzyme-linked immunosorbent assay (ELISA) and by a T. parva-specific p104 gene-based nested PCR. Antibodies against Theileria mutans infection were detected using the 32-kDa antigen-based indirect ELISA, while the 200-kDa antigen and the major surface protein 5 (MSP5)-based indirect ELISA were used to detect antibodies against Babesia bigemina and Anaplasma marginale, respectively.ResultsThe prevalence of T. parva across the ten communes sampled ranged from 77.5 to 93.1% and from 67.8 to 90.0% based on the ELISA and PCR analysis, respectively. A statistically significant difference in infection was observed between calves and adult cattle; however, T. parva infection levels were not significantly associated with sex and breed. The seroprevalence indicating exposure to T. mutans, B. bigemina and A. marginale ranged from 30 to 92.1%, 33.7 to 90% and 50 to 96.2%, respectively. Mixed infections of TBPs were detected in 82.91% of cattle sampled, with 11 different combinations of pathogen species detected .ConclusionsThe findings indicate that T. parva, A. marginale and B. bigemina infections are endemic in Burundi. Knowledge of the spatial distribution of TBPs will facilitate the design of effective targeted strategies to control these diseases. There is a need for further investigations of the distribution of tick vectors and the population structure of TBPs in order to identify the key epidemiological factors contributing to TBD outbreaks in Burundi.

Dataset Information

Validation of a multiplex PCR assay to detect Babesia spp. and Anaplasma marginale in cattle in Uruguay in the absence of a gold standard test.

Publications

Validation of a multiplex PCR assay to detect <i>Babesia</i> spp. and <i>Anaplasma marginale</i> in cattle in Uruguay in the absence of a gold standard test.

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