Anaplasma species are gram negative obligate intracellular bacteria of family Anaplasmataceae and order Rickettsiales. It is primarily transmitted by the tick vectors of genera Rhipicephalus, Ixodes, Dermacentor and Amblyomma. Anaplasma marginale appeared to be more pathogenic than A. centrale, A. bovis, A. ovis, A. capra, A. platys and A. phagocytophilum in the bovine. Bovine anaplasmosis, caused by A. marginale, is prevalent in tropical countries including India[3,4,5]. It causes huge economic losses to the bovine sector. The annual loss due to anaplasmosis in the USA amounts to USD 100 million. There is no breed-wise and age-wise variation in the level of susceptibility for anaplasmosis in the bovines[8,9]. Cattle are said to be more susceptible for anaplasmosis than the buffaloes.
Anaplasma marginale multiplies by forming membrane bound round basophilic inclusion bodies (0.5-1 mm) of 4–8 rickettsiae usually in the periphery of erythrocytes. Infected erythrocytes are phagocytosed by the reticuloendothelial cells, resulting in mild to severe degree of anemia and icterus. Along with progressive hemolytic anaemia the other common clinical signs in cattle are fever, icterus, anorexia, decreased milk yield, weight loss, weakness, sometime abortion in the pregnant animal and often death of the older animals. Following recovery, the animals can become carrier host and serve a source of infection to the vectors.
The tentative diagnosis of bovine anaplasmosis is mainly based on the clinical signs shown by the infected animals. Microscopic examination of stained blood smear is done for the confirmatory diagnosis. Although it is a gold standard detection technique but it frequently failed to do the same in the subclinical and chronic infection with low level of parasitaemia. Moreover, due to similarity in the morphology of Anaplasma species and other pathogens, it is difficult to distinguish these organisms microscopically. Therefore, several serological tests have been established. Unfortunately, because of cross reactivity, these tests failed to differentiate closely-related pathogens of genus Anaplasma and other rickettsia. Thus nucleic acid-based assays, notably polymerase chain reaction (PCR) and its types, with a high degree of sensitivity and specificity are widely used to identify Anaplasma species[5,16,17].
However, due to economic and/ or practical reasons these methods may not always be available in low structural facilities. These limitations of PCR have inspired the development of platforms for the isothermal nucleic acid amplification technique. Among various assays, the loop mediated isothermal amplification (LAMP) technique got the attention of the scientist all around the world for the diagnosis of diseases of animals and human[18,19]. The assay is inherited with the high degree of simplicity, as it can be performed at a particular temperature and final amplified product can be detected visually in the tube. Wen et al and Giglioti et al successfully developed a LAMP test for rapid and easy diagnosis of cattle anaplasmosis. The present study provides insights into the isothermal amplification of nucleic acid of A. marginale in cattle of South Gujarat, India.
MATERIAL AND METHODS
Study areas and animal under study
The focus of the study was on the cattle presented before the veterinarian for the diagnosis and treatment of the diseases in south Gujarat, India. The animals especially with the history of tick infestation, fever and anaemia were included in the present study. The animals were screened for Anaplasma infection using classical and molecular assays.
Collection of blood and cytological examination Approximately 2-3 milliliters of blood per cattle was collected aseptically by veni-puncture in sterile vacutainer with K3 EDTA and transported on ice to the departmental laboratory for DNA extraction. Ear tip was pricked aseptically to prepare a thin peripheral blood smear. A thin smear was prepared from 1 µl of 1: 100 diluted capillary blood with the RBC diluting fluid on clean grease free glass slide. After methanol fixation, the thin blood smear was stained with diluted Giemsa stain solution in PBS buffer in 1:9 ratio. Preliminary identification was carried out on the basis of morphological features of Anaplasma under the 100x oil immersion microscope. The infection intensity was estimated by multiplying the average number of organisms per microscopic field and approximate number of microscope fields in the prepared smear, and the value was further adjusted by the dilution factors.
Genomic DNA extraction
Genomic DNA was extracted from the anticoagulant treated whole blood of the animals using the DNeasy Blood & Tissue kit (Qiagen, Germany), according to the manufacturer’s instructions. The eluted whole DNA having an OD ratio at 260 and 280 nm of 1.7–1.9 in a NanoDrop™ 2000c spectrophotometer (Thermo Scientific, USA) was stored at -20°C for further use.
Non-isothermal nucleic acid amplification assay for Anaplasma DNA detection
For the detection of the rickettsial organism, two rounds of PCR were performed in a S1000™ thermal cycler (Bio-Rad, USA) under complete aseptic conditions. First round of PCR (Ana429 PCR) was performed to amplify the 429 bp of 16S rRNA gene of Anaplasma genus by 5’-tacctctgtgttgtagctaacgc-3’: 5’-cttgcgacattgcaacctattgt-3’ primer pair. Second round of PCR was performed to amplify 457 bp of msp5 gene of A. marginale by 5’-gcatagcctccgcgtctttc-3’: 5’-tcctcgccttggccctcaga-3’ (Anamar457 PCR) primer pair. Each 25 µL of PCR reaction was prepared by mixing 1 µL (25-50 ng) of DNA template, 1 µL (10-20 pM) of each primers, 12.5 µL of 2× Taq PCR master mix (2x buffer, 0.4 mM deoxynucleotide and 4 mM MgCl2) of Qiagen, Germany and 9.5 µL nuclease free water in sterile 0.2 mL PCR tube. Gradient PCR was done to standardize the amplification conditions for each primer set using reference/ non-template control. The standardized amplification condition Anamar457 PCR was initial denaturation at 94°C for 5 min followed by 35 cycles of 3 steps consisting of denaturation at 94°C for 1 min, annealing temperature 55°C for 1 min and elongation at 72°C for 1 min, final elongation at 72°C for 10 min, and storage at 4°C for an infinite period. The PCR products were visualized under UV light following 1% agarose gel electrophoresis with ethidium bromide stain.
Restriction digestion of PCR Products
The amplified products were made free from enzyme and salts using a PCR purification kit (Qiagen, Germany), following the manufacturer’s instructions. To further confirm the species specific amplification of the standardized PCR, 10 µL of purified PCR amplicon of msp5 DNA of A. marginale was digested with 1 µL of EcoRI restriction endonuclease (10 U/µL, Thermo Fisher Scientific, USA) in 2 µL of 10x buffer and 7 µL of nuclease-free water at 37°C for 4 h. The digested product was stained with ethidium bromide in 1% agarose gel, and visualized under UV light of gel documentation instrument (Bio-Rad, USA).
Gel extraction and sequencing of the PCR products
One hundred and fifty ml of PCR product was electrophoresed in 1% agarose gel. After visualizing under long range UV light, the desired band was excised out and transferred into a sterile 1.5 ml centrifuge tube. The DNA was eluted from the agarose gel slice using a gel extraction kit (Qiagen, Germany) and send for sequencing. The msp5 DNA fragments were sequenced in both directions, using the same primers used in the PCR reaction. The reaction was performed by Sanger’s di-deoxy chain termination method using Dye terminator Cycle Sequencing Kit (Applied Biosystems Inc., USA) and an ABIDNA sequencer at the Sequencing Department, Eurofins Genomics, Bengaluru, India.
Nucleotide sequence analysis of msp5 DNA of Anaplasma The newly generated raw sequence data were analyzed in the Bio Edit program (version 18.104.22.168) and chromatogram was also checked for their data quality. The 5' and 3' ends of the msp5 gene sequence of Anaplasma were initially determined by comparing them with previously published sequences. These raw sequences were analyzed in Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/BLAST), and results were complied for further analysis. The parameters observed were max score, total score, query coverage, percent identity and E-value (Expectation value). Identification to the species level was defined as msp5 gene sequence similarity of ≥99% with that of the sequence in GenBank. As per blast report the raw sequences of msp5 were trimmed from one or both ends, and used in the construction of the phylogenetic tree.
Phylogenetic analysis of msp5 DNA of Anaplasma
The phylogenetic relationship among Anaplasma species was reconstructed based on msp5 DNA sequences of NCBI GenBank from India and other countries of Class: Alphaproteobacteria, Order: Rickettsiales, Family: Anaplasmataceae in the Neighbor-Joining (NJ) method (maximum composite likelihood model). Multiple sequence alignments were performed using ClustalW algorithm. Evolutionary analyses were conducted in the Molecular Evolutionary Genetics Analysis (MEGA) 10 (version 10.1.7) with a gap opening penalty of 15 and gap extension penalty of 6.66 in both pairwise or multiple alignments, respectively. The tree stability was estimated by bootstrap analysis for 1000 replications. Anaplasma phagocytophilum msp5 DNA sequences was used as outgroup in the tree reconstructions. The evolutionary distances were in the units of the number of base substitutions per site, and computed using the Maximum Composite Likelihood method. This analysis involved 22 nucleotide sequences. Codon positions included were 1st +2nd +3rd+ noncoding. All ambiguous positions were removed for every sequence pair (pairwise deletion option). There was of 996 positions within the final dataset. The optimal tree with the sum of branch length = 2.43679930 was shown in the NJ method.
Isothermal nucleic acid amplification assay for Anaplasma DNA detection
The pathogen specific primers were used to amplify msp5 gene of A. marginale in the LAMP. A 10x primer concentration was prepared by mixing 6 LAMP primers [each outer forward (5’-accttctgctgttcgttg-3’) and backward (5’-gagaagccatgcctaactc-3’) primers of 2 µM (micro-mole), each inner forward (5’-gttgaaagacgcggaggctaaatcggcgagaggtttac-3’) and backward (5’-ccgtcagtagcggcgatttgtact-tacaggctgagaagc-3’) primers of 16 µM, each loop forward (5’-tgccctcacttacaacttcg-3’) and backward (5’-cggcaagcacatgttggta-3’) primers of 4 µM] in nuclease free water and stored in aliquots at -20°C for future use. The LAMP master mix was prepared immediately before the test at a designated workplace with a sterile plastic wares. The LAMP reaction was set in a 25 µL reaction mixture containing 12.5 µL of WarmStart Colorimetric LAMP 2x Master Mix (New England BioLabs Inc.), 2.5 µL of LAMP primer mix (10x), 1 µL of template DNA and 9 µL of nuclease free water in a sterile 0.2 mL PCR tube. The reaction mixture was mixed by pipetting, and the tube was instantly sealed with an optically clear adhesive seal. Each reaction solution was examined for its bright pink color before incubation. Incubation temperature and time were standardized using a range of temperature and time in an incubation chamber, respectively. Reaction tube was removed from the incubation chamber and allowed to cool at room temperature. Examined by naked eye and colorimetric change was photographed. Alternatively, LAMP products were resolved in 1% agarose gel electrophoresis.
Statistical analysis and reference control
Pathogen prevalence rate as per different assays was calculated. The P value <0.05 was considered significant, and was calculated using R software33. The DNA samples from previous study with and without A. marginale was used to standardize the assays. The DNA sample positive for Trypanosoma evansi, Babesia bigemina and Theileria annulata and A. centrale was used to evaluate the specificity of the tests. For non-template reaction equivalent volume of nuclease free water was added in place of template DNA. The sensitivity (detection limit) of the standardized assays was determined using 10-fold serial dilutions (100-10-8) of 0.1 µg total genomic DNA of A. marginale as templates. The standardized LAMP assay was evaluated in terms of diagnostic sensitivity, diagnostic specificity, positive predictive value and negative predictive value at 95% confidence intervals (CI).
As the current study was conducted with clinical cases, so ethical committee approval was not required.
Blood collection and its processing
A total of 280 blood samples of cattle was collected. In microscopic, A. marginale organism was mostly seen located near the margin of erythrocytes as blue to purple round or spherical basophilic inclusion body with a light halo around it. The average level of rickettsiaemia in the infected animals was 2003.82±25.998×106 infected erythrocytes/ml of blood.
PCR assay and EcoR1 digestion of msp5 DNA of Anaplasma
Ethidium bromide stained Ana429 PCR amplicon of 429 bp (Lane 1 of [Figure 1]) of 16S rRNA gene of Anaplasma was visualized under UV light following agarose gel electrophoresis in 58 cattle (20.71%). Similarly, the species specific Anamar457 PCR had amplified 457 bp (Lane 2 of [Figure 1]) of msp5 of A. marginale in 43 cattle (15.36%). The detection limit of species specific PCR (Anamar457 PCR) was up to 10-6 genomic DNA of A. marginale in the sensitivity experiment. There was no any cross amplification of the genomic DNA of T evansi, B. bigemina and T. annulata in the Ana429 PCR/ Anamar457 PCR, and A. centrale using species specific Anamar457 PCR, indicative of a high level of specificity of the assays against these pathogens. Further, no any amplification was observed in negative and non-template controls in the standardized PCR. The EcoRI digested the PCR products of msp5 gene of A. marginale into two fragments of 265 bp and 192 bp.
Sequence analysis and phylogenetic tree of msp5 DNA of Anaplasma
The NCBI-BLAST study confirmed that the obtained sequence was of msp5 DNA of A. marginale (MW538962 and MW538961). The current sequence showed 100% homology and 771 total score with published nucleotide sequences of accession no. MK188829, LC467711, LC467709, etc., in the NCBI-BLAST study. The current msp5 genotype formed a tight cluster with the A. marginale originated from different locations within the family Anaplasmataceae in the phylogram [Figure 2]. Monophyletic lineage relationships were observed with high bootstrap proportion for current and most of the published nucleotide sequences of A. marginale. Phylogram revealed major clades for A. marginale, A. centrale, A. ovis and A. phagocytophilum [Figure 2].
LAMP amplification assay based on msp5 DNA of Anaplasma
The optimum incubation temperature of LAMP with
A. marginale primers was established using a range of temperatures from 50°C, 55°C, 60°C, 65°C and 70°C, and 65°C was chosen as the reaction temperature for all applications. Time periods for the reactions ranging from 15 to 60 min were then tested in this reaction temperature. Analysis of the results indicated that incubation for 45 min was sufficient to allow the reaction to occur [Figure 3]. Positive reaction was turned in yellow colour while the negative control depicted original pink colour. The positive LAMP amplicons presented ladder-like DNA pattern in agarose gel electrophoresis. LAMP reaction at standardized condition with genomic DNA from T. evansi, B. bigemina, T. annulata and A. centrale as templates failed to get the amplicons, in contrast, the amplification was confirmed in LAMP with the genomic DNA of cattle A. marginale. This indicated the high level of specificity of the standardized LAMP assay against these pathogens. The sensitivity of the LAMP assay was up to 10-8 dilution of genomic DNA of A. marginale as a template for the detection of cattle anaplasmosis i.e., 100 times more sensitive than that of PCR assay.
Comparative evaluation of microscopy, PCR and LAMP assays for the detection of cattle anaplasmosis Microscopic examination of the 280 blood samples revealed that 17 (6.07%) cattle were infected with A. marginale. Anaplasma marginale msp5 DNA was detected in 43 (15.36%) and 62 (22.14%) samples by PCR and LAMP, respectively. The prevalence rate of A. marginale in the cattle was significantly higher (p<0.05) in the PCR (15.36%) and LAMP (22.14%) than the microscopy (6.07%).
Data presented in Table 1 were used to calculate the diagnostic sensitivity, specificity, positive and negative predictive values at 95% CI for LAMP assay with respect to PCR for the detection of A. marginale. The sensitivity and specificity at 95% CI for LAMP assay were 93.02% (80.94%-98.54%) and 90.72% (86.28%-94.09%), respectively. The positive and negative predictive values at 95% CI were 64.52% (54.77%-73.19%) and 98.62% (96.01%-99.53%), respectively.
Timely diagnosis and intervention is of paramount importance to minimize the economic losses in vector-borne bovine anaplasmosis. In the current study, detection of A. marginale infection was done by amplifying the nucleic acid of the pathogen in the variable temperature amplification assay, PCR and single temperature amplification assay, LAMP. Instead of having the most advanced assays, the importance of the gold standard microscopic detection technique cannot be ignored. The detection limit of the microscopic technique was in the multiple of 10 infected erythrocytes/ml of blood, according to the finding of Kumar et al, Palmer et al and Gale et al. The assay based on the detection of the nucleic acid of the pathogen showed its superiority over the classical or serological assays in the diagnosis of the diseases. Out of various targeted genes, msp5 gene of the msp gene family is one of the potential molecular targets for the diagnosis of A. marginale induced cattle anaplasmosis.
The 16S rRNA gene was amplified in the first round PCR for the identification of Anaplasma genus. The specificity of the A. marginale PCR targeting msp5 gene was confirmed by the amplification of an expected product size, restriction enzyme analysis in EcoR1[25,37] and sequencing of the PCR product. The present A. marginale isolates appeared phylogenetically closer to the isolates of Indian states, Philippines, Egypt, Kenya, China, Benin, Brazil and Cuba, as it occupies same major clade with closest relationship. A monophyletic lineage was observed in the msp5 gene of A. marginale isolated in the Philippines, and was phylogenetically associated with the strains isolated in Brazil or China.
The LAMP is based on strand displacement activity of the Bst DNA polymerase to amplify the nucleic acid in the isothermal condition. A set of primers (each of two outer, inner and loop primers) binds to different regions of the target gene. The loop primers further reduce the amplification time to one third of that of the original LAMP method. The assay generates an outsized amount of amplification products in the positive samples causing assembly of protons and subsequent drop in the pH of the reaction mixture, which may be detected visually within an hour by colour change, thus, thermal cyclers and electrophoresis chambers are not required[18,19,40]. In the current study an attempt was made to have a LAMP protocol for the detection of A. marginale infection in the cattle by amplifying the msp5 gene at 65°C for 45 min[20,21]. A colour change from pink to yellow colour was observed in the positive LAMP reaction. The positive LAMP amplicon presented ladder-like DNA pattern on agarose gel electrophoresis, the reason can be looked into the use of multiple primer sets which amplify the various region of the targeted gene[18,19,40]. In contrast, carryover contamination is the main limitation of LAMP, which leads to false positive results. To avoid contamination, in our LAMP reactions, all essential precaution was adopted, and just after making the reaction the tube was sealed properly. The sensitivity of standardized LAMP assay (10-8 dilution of genomic DNA of A. marginale) was 100 times more than that PCR assay for the detection of cattle anaplasmosis. Wen et al in their study too found LAMP targeting msp5 gene as 100 times more sensitive as of conventional PCR in the detection of A. marginale in the cattle. The sensitivity of the LAMP (5×10° copies/µL) by targeting 16S rRNA gene was 100 times more than that of conventional PCR (5×102 copies/µL) for the specific detection of A. bovis in sheep and goats. A high level of specificity of the LAMP assay was found against T. evansi, B. bigemina, T. annulata and A. centrale of cattle, as it failed to get the amplicons with the template DNA of these parasites. Wen et al following visual inspection or agarose gel electrophoresis confirmed the absence of LAMP amplicon of msp5 gene of A. marginale in Besnoitia besnoiti, Trypanosoma sp., Toxoplasma gondii and B. bigemina infected cattle. The LAMP based on the msp1b sequence as a specific assay for detecting A. marginale in cattle, and no any cross amplification were observed against the DNA template of Babesia bovis, B. bigemina and A. centrale infected cattle. Following the LAMP reaction at 62°C for 60 min in a water bath Wang et al noted no any amplification of A. ovis, A. phagocytophilum, Theileria luwenshuni, B. motasi and Schistosoma japonicum and used 16S rRNA gene based LAMP for the specific detection of A. bovis in sheep and goats. The sensitivity and specificity of the assay for the detection of the pathogen mainly depend upon the selection of the target gene and its corresponding primers.
The present study calculated 6.07% positivity of A. marginale in the animals using microscopic technique while it was significantly higher in PCR (15.36%) and LAMP (22.14%). The molecular assay, always noted a high level of prevalence of the pathogen than the conventional assay[5,14,15]. The geographical location, climatic condition and animal rearing condition, played a major role in determining the level of prevalence of the pathogen in the animals. In the msp5 based PCR, Singh et al calculated that 45.2% of carrier cattle of Punjab, India had the A. marginale infection while routine blood smear examination had revealed inclusion bodies in only 12.5% of samples. A positive LAMP result based on 16S rRNA gene for A. phagocytophilum was found in 56.4% sheep, whereas only 12.8% and 3.2% in the nested and conventional PCR, respectively.
The diagnostic sensitivity and specificity at 95% CI for LAMP assay with respect to PCR for the detection of A. marginale were 93.02% and 90.72%, respectively. The positive and negative predictive values at 95% CI were 64.52% and 98.62%, respectively. The same level of diagnostic sensitivity (95.45%) was calculated by Ma et al for LAMP for the specific detection of A. ovis in sheep using primers designed for msp4 gene. Therefore, current LAMP assay based on diagnostic test evaluation parameters defined its applicability for the specific detection of the A. marginale over A. centrale, T. evansi, B. bigemina and T. annulata.
It can be inferred from this study that nucleic acid detection assays, PCR (non-isothermal amplification assay) and LAMP (isothermal amplification assay) following amplification of msp5 gene noted the presence of A. marginale in the cattle of South Gujarat, India. Positive sample following PCR observed a band of 457 bp of msp5 DNA of A. marginale in the ethidium bromide stained 1% agarose gel under UV light. Positive LAMP reaction turned into yellow colour while the negative control depicted original pink colour in the visual detection. The positive LAMP amplicons presented ladder-like DNA pattern on agarose gel electrophoresis. The detection limit of PCRs was up to 10-6 of the original genomic DNA of A. marginale while it was 100 times more (10-8) for LAMP in the sensitivity experiment. The standardized PCR and LAMP observed a high level of specificity against T. evansi, B. bigemina and T. annulata and A. centrale. A single cut site of EcoRI was observed in the amplified PCR product of msp5 gene of A. marginale. The current sequences of msp5 DNA of A. marginale (MW538962 and MW538961) showed 100% homology and 771 total score with published nucleotide sequences in the NCBI BLAST study. The monophyletic lineage type relationship was observed with high bootstrap proportion for current and published nucleotide sequences of msp5 DNA of A. marginale in the phylogram. The prevalence rate of A. marginale in the cattle was significantly higher (p<0.05) in the PCR [43 out of 280 (15.36%)] and LAMP [62 out of 280 (22.14%)] than the microscopic technique [17 out of 280 (6.07%)]. The diagnostic sensitivity, specificity, positive and negative predictive values at 95% CI for LAMP assay with respect to PCR for the detection of A. marginale were 93.02%, 90.72%, 64.52% and 98.62%, respectively. Thus LAMP could provide an accurate, sensitive, easy-to-use method, and a practical alternative to PCR for the diagnosis of A. marginale infection in the cattle.
This study was summarized from the master thesis of the first author. The authors are thankful to the Vice-chancellor, Director of Research and Principal, College of Veterinary Science and Animal Husbandry, Navsari, Navsari Agricultural/ Kamdhenu University, and also investigator of NAHEP-CAAST for providing necessary facilities and funds to complete this research work.
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