RPA-CRISPR/Cas12a assay for the diagnosis of bovine Anaplasma marginale infection.

Anaplasma marginale infection is one of the most common tick-borne diseases, causing a substantial loss in the beef and dairy production industries. Once infected, the pathogen remains in the cattle for life, allowing the parasites to spread to healthy animals. Since clinical manifestations of anaplasmosis occur late in the disease, a sensitive, accurate, and affordable pathogen identification is crucial in preventing and controlling the infection. To this end, we developed an RPA-CRISPR/Cas12a assay specific to A. marginale infection in bovines targeting the msp4 gene. Our assay is performed at one moderately high temperature, producing fluorescent signals or positive readout of a lateral flow dipstick, which is as sensitive as conventional PCR-based DNA amplification. This RPA-CRISPR/Cas12a assay can detect as few as 4 copies/µl of Anaplasma using msp4 marker without cross-reactivity to other common bovine pathogens. Lyophilized components of the assay can be stored at room temperature for an extended period, indicating its potential for field diagnosis and low-resource settings of anaplasmosis in bovines.

Molecular characterization and genetic diversity of Babesia bovis and Babesia bigemina of cattle in Thailand.

Babesia bovis and B. bigemina are the most common tick-borne parasites that cause bovine babesiosis which effects livestock production, leading to economic losses in tropical and subtropical areas of the world. The aims of this study were to determine the molecular detection, genetic diversity and antigenicity prediction of B. bovis based on spherical body protein 2 (sbp-2) gene and B. bigemina based on rhoptry-associated protein 1a (rap-1a) gene in cattle in Thailand. By PCR assay, the molecular detection of B. bovis and B. bigemina infection revealed levels of 2.58% (4/155) and 5.80% (9/155), respectively. The phylograms showed that B. bovis sbp-2 and B. bigemina rap-1a sequences displayed 5 and 3 clades with similarity ranging between 85.53 to 100% and 98.28 to 100%, respectively, when compared within Thailand strain. Diversity analysis of sbp-2 and rap-1a sequences showed 18 and 4 haplotypes, respectively. The entropy analysis illustrated 104 and 7 polymorphic sites of sbp-2 and rap-1a nucleic acid sequences, respectively, while those of sbp-2 and rap-1a amino acid sequences showed 46 and 4 high entropy peaks, respectively. Motifs analysis exhibited the distribution and conservation among sbp-2 and rap-1a sequences. The continuous and discontinuous B-cell epitopes have also been evaluated in this work. Therefore, our findings may be used to ameliorate the understanding inputs of molecular phylogeny, genetic diversity and antigenicity of B. bovis and B. bigemina Thailand stains.

Anaplasma marginale: Molecular discrimination, recombinant expression and characterization of major surface protein 2.

A. marginale's major surface protein 2 (MSP2) is an immunodominant protein that is encoded by a multigene family. Phylogenetic analysis revealed that the msp2 sequence Thailand strain was clustered in third clade, with similarity values between 90.4 and 100%. The haplotype diversity showed 10 haplotypes of the msp2 genes. The entropy analysis of the nucleic and amino sequences revealed 289 and 117 high entropy peaks, respectively. Interestingly, one predicted allele belonging to MHC-II represented the hypervariable region (HVR) of MSP2. A. marginale's recombinant MSP2 (rAmMSP2), which has a molecular weight of 42 kDa, was examined in SDS-PAGE. Antigenicity of rAmMSP2 (42 kDa) and AmMSP2 (36 kDa) showed the conserved epitopes. The distribution of AmMSP2 on infected erythrocytes' membrane and outside was demonstrated by immunofluorescence detection. Therefore, the rMSP2 could be utilized in the establishment of immunodiagnostic tools and vaccine approaches for the monitoring of anaplasmosis.

Antigenic components, identification, and characterization of whole worm extract of Platynosomum illiciens.

The antigenic components of adult Platynosomum illiciens were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cats naturally infected with P. illiciens, Dipylidium caninum, Toxocara cati and uninfected cat sera. The whole worm extract (WWE) of P. illiciens was fractionated by Sephadex G-200 gel filtration chromatography. The results showed that WWE fraction and F2 were highly antigenic as well as F1 and F3, which were moderately antigenic. For SDS-PAGE and immunoblotting, the antigenic molecules of WWE and all three fractions were mostly at molecular weights (MW) ranging from 11 to 150 kDa. Four antigenic proteins of 11, 18, 27 and 75 kDa detected in WWE and F1-F3 were found to give a reaction with sera from P. illiciens infected cats, and these proteins were also identified using liquid chromatography-mass spectrometry (LC-MS/MS). For immunolocalization observation, it was revealed that the P. illiciens antigen was present in high concentration in the cytoplasm of vitelline cells in the vitelline glands, the shell of the eggs and the eggs within the uterus, but not in other organs, i.e., tegument, muscle, parenchymal cells, testes and oral and ventral suckers of adult fluke. This finding indicates that these proteins may be potential antigen candidates for the immunodiagnosis of feline platynosomosis caused by P. illiciens.

Molecular genetic diversity and bioinformatic analysis of Leucocytozoon sabrazesi based on the mitochondrial genes cytb, coxI and coxIII and co-infection of Plasmodium spp.

Leucocytozoon sabrazesi is an intracellular haemoprotozoan parasite responsible for leucocytozoonosis, which is transmitted by insect vectors and affects chickens in tropical and subtropical areas in many countries. It causes huge economic losses due to decreased meat and egg production. In the present study, we used nested PCR to determine the genetic diversity of L. sabrazesi based on the cytb, coxI, coxIII and concatenated genes in chickens in Thailand. In addition, we found co-infections between L. sabrazesi and Plasmodium spp. (P. gallinaceum or P. juxtanucleare) in chickens that were not identified by microscopic examination of blood smears. The phylogenetic analysis indicated that L. sabrazesi cytb and coxIII genes were conserved with similarity ranging from 99.9 to 100% and 98 to 100%, respectively whereas the coxI gene was diverse, with similarities ranging from 97 to 100%. These findings ascertained the nucleotide analysis of the cytb, coxI, coxIII and concatenated sequences in which 4, 8, 10 and 9 haplotypes were found, respectively. In addition, it was found that the large number of synonymous substitutions and conservative amino acid replacements in these mitochondrial genes occurred by non-synonymous substitution. The evolutionary analysis of the Ka/Ks ratio supported purifying selection and the negative values of both Fu's Fs and Tajima's D indicate selective sweep especially for the coxI gene. The entropy and Simplot analysis showed that the genetic variation in populations of Plasmodium spp. was higher than in Leucocytozoon. Hence, the nucleotide sequences of three mitochondrial genes could reflect the evolutionary analysis and geographic distribution of this protozoan population that switches hosts during its life cycle.

Title: Diversité génétique moléculaire et analyse bioinformatique de Leucocytozoon sabrazesi basée sur les gènes mitochondriaux cytb, coxI et coxIII et la co-infection avec Plasmodium spp. Abstract: Leucocytozoon sabrazesi est le parasite hémoprotozoaire intracellulaire responsable de la leucocytozoonose, qui est transmise par des insectes vecteurs et affecte les poulets dans les zones tropicales et subtropicales de nombreux pays. Il provoque d'énormes pertes économiques en raison de la diminution de la production de viande et d'œufs. Dans la présente étude, nous avons utilisé la PCR nichée pour déterminer la diversité génétique de L. sabrazesi sur la base des gènes cytb, coxI, coxIII et concaténés chez des poulets en Thaïlande. De plus, nous avons trouvé des co-infections entre L. sabrazesi et Plasmodium spp. (P. gallinaceum ou P. juxtanucleare) chez des poulets, qui n'ont pas été identifiées par l'examen microscopique de frottis sanguins. L'analyse phylogénétique a indiqué que les gènes cytb et coxIII de L. sabrazesi étaient conservés avec une similarité allant respectivement de 99,9 à 100 % et de 98 à 100 %, alors que le gène coxI était diversifié, avec des similarités allant de 97 à 100 %. Ces découvertes ont confirmé l'analyse des nucléotides des séquences cytb, coxI, coxIII et concaténées dans lesquelles 4, 8, 10 et 9 haplotypes ont été trouvés, respectivement. De plus, il a été constaté que le grand nombre de substitutions synonymes et de remplacements conservateurs d'acides aminés dans ces gènes mitochondriaux se produisaient par substitution non synonyme. L'analyse évolutive du rapport Ka/Ks a soutenu la sélection purificatrice et les valeurs négatives des Fs de Fu et D de Tajima indiquent un balayage sélectif, en particulier pour le gène coxI. L'entropie et l'analyse Simplot ont montré que la variation génétique de la population de Plasmodium spp. était plus élevée que pour Leucocytozoon. Par conséquent, les séquences nucléotidiques de trois gènes mitochondriaux pourraient refléter l'analyse évolutive et la répartition géographique de cette population de protozoaires qui changent d'hôte au cours de leur cycle de vie.

Molecular discrimination and genetic diversity of three common tick-borne pathogens in dogs in Thailand.

There was little information regarding the occurrence of canine vector-borne disease (CVBDs) in shelter dogs in Thailand. This work is the first report regarding a molecular method used to determine the occurrence and genetic diversity of three canine tick-borne pathogens (TBPs) (Hepatozoon canis, Anaplasma platys and Ehrlichia canis) in blood samples from 275 shelter dogs in the north and central areas of Thailand. The PCR results based on the 18S rRNA and 16S rRNA genes showed that 71 (25.82%) dogs were positive for at least a TBP. The overall occurrence rates of H. canis, A. platys and E. canis infections were 1.81, 16.36 and 7.64%, respectively. For the phylogenetic analysis, A. platys 16S rRNA gene was genetically diverse, while H. canis 18S rRNA and E. canis 16S rRNA genes were conserved. The haplotype diversity exhibited 12 and 2 haplotypes as well as 78 and 178 polymorphic sites of A. platys and E. canis 16S rRNA genes, respectively. Our findings could be used to improve the understanding of phylogeny and genetic diversity of TBP rRNA genes and used to ameliorate the diagnosis and control programmes for the diseases in Thailand.

Molecular characterization of Anaplasma marginale based on the msp1a and msp1b genes.

Anaplasma marginale is an intracellular rickettsial bacterium causing anaplasmosis in ruminants. A. marginale is transmitted biologically by ticks and mechanically by blood-sucking vectors. Anaplasmosis occurs in tropical and subtropical areas of the world. This disease causes huge economic losses due to decreasing meat yield and milk production. The aims of this study were to determine the genetic diversity and antigenicity of A. marginale based on the msp1a and msp1b genes in cattle in Thailand. The A. marginale msp1a and msp1b genes were amplified by the polymerase chain reaction (PCR). There have been four copies of MSP1a tandem repeats among A. marginale Thailand strain, and thirteen different MSP1a tandem repeats were found including repeats B, 25, 27, M, 3, S, C, H, ß, 80, 4, TH1 and TH2. Notably, this study showed two copies of the novel conserved tandem sequences namely Thailand Type 1 (TH1) and Type 2 (TH2). The phylogenetic analysis revealed that A. marginale msp1a and msp1b genes were genetically diverse and showed 9 and 5 clades with similarity ranging from 98 to 100% and 79.5 to 100%, respectively, when compared within the isolates of this study. The results of diversity analysis showed 18 and 16 haplotypes of the msp1a and msp1b genes, respectively. The entropy analyses of msp1a and msp1b nucleic acid sequences showed 39 and 900 high entropy peaks with values ranging from 0.35 to 0.85 and from 0.41 to 1.48, respectively, while those of MSP1a and MSP1b amino acid sequences exhibited 75 and 72 high entropy peaks with values ranging from 0.35 to 1.06 and from 0.41 to 1.55, respectively. In addition, B-cell and T-cell epitopes have also been investigated in this study. Hence, our results could be employed to improve the insight input of molecular phylogenetics, genetic diversity and antigenicity of A. marginale Thailand strain.

Molecular detection and genetic diversity of Leucocytozoon sabrazesi in chickens in Thailand.

Leucocytozoon sabrazesi is the intracellular protozoa of leucocytozoonosis, which is transmitted by the insect vectors and affects chickens in most subtropical and tropical regions of the globe, except South America, and causing enormous economic losses due to decreasing meat yield and egg production. In this study, L. sabrazesi gametocytes have been observed in the blood smears, and molecular methods have been used to analyse the occurrence and genetic diversity of L. sabrazesi in blood samples from 313 chickens raised in northern, western and southern parts of Thailand. The nested polymerase chain reaction (nested PCR) assay based on the cytb gene revealed that 80.51% (252/313) chickens were positive of L. sabrazesi. The phylogenetic analysis indicated that L. sabrazesi cytb gene is conserved in Thailand, showed 2 clades and 2 subclades with similarity ranged from 89.5 to 100%. The diversity analysis showed 13 and 18 haplotypes of the sequences from Thailand and from other countries, respectively. The entropy analyses of nucleic acid sequences showed 26 high entropy peaks with values ranging from 0.24493 to 1.21056, while those of amino acid sequences exhibited 5 high entropy peaks with values ranging from 0.39267 to 0.97012. The results; therefore, indicate a high molecular occurrence of L. sabrazesi in chicken blood samples with the associated factors that is statistically significant (p < 0.05). Hence, our results could be used to improve the immunodiagnostic methods and to find appropriate preventive control strategies or vaccination programs against leucocytozoonosis in order to mitigate or eliminate the harmful impact of this infection on chicken industry.

Molecular and recombinant characterization of major surface protein 5 from Anaplasma marginale.

Anaplasmosis is a tick-borne disease caused by the intracellular rickettsia Anaplasma marginale, which affects cattle and other ruminants in both tropical and subtropical regions of the world, and also causing tremendous economic losses due to decreasing livestock production. The major surface protein 5 (MSP5) of A. marginale is an immunodominant and highly conserved protein encoding by a single gene. In the present study, the complete full-length of the msp5 coding sequence of A. marginale Thailand strain was cloned and determined at a size of 633 bp. Phylogenetic analysis based on neigh-joining (NJ) method showed that the msp5 sequence Thailand strains were clearly distributed in 3rd clade and conserved when compared with other strains. The results showed 9 haplotypes of the msp5 genes, and the entropy analysis of MSP5 amino acid sequences displayed 92 high entropy peaks with value ranging from 0.198 to 0.845 Additionally, a recombinant MSP5 of A. marginale (rAmMSP5) was over-expressed in the E. coli BL21 Star™ (DE3) host cell, affinity purified, and found in SDS-PAGE at a molecular weight of 26 kDa. The antigenicity of rAmMSP5 (26 kDa) and AmMSP5 (19 kDa) was recognized by rabbit anti-rAmMSP5 antisera and A. marginale-infected cattle sera. Both rAmMSP5 and AmMSP5 were perceived by these sera manifesting that recombinant and native AmMSP5 have conserved epitopes. Immunofluorescence technique using rabbit anti-rAmMSP5 antisera exhibited that the AmMSP5 is distributed on both the membrane and the outside of infected erythrocytes. Therefore, the recombinant MSP5 could be used for the development of immunodiagnostic assays and vaccine purposes for controlling anaplasmosis.

The anthelmintic potentials of medicinal plant extracts and an isolated compound (rutin, C27H30O16) from Terminalia catappa L. against Gastrothylax crumenifer.

Paramphistomosis is a pathogenic disease that occurs frequently in tropical and subtropical countries including Thailand. This disease is affected in the parasites causing severe gastrointestinal disorders and death in infected animals. In the present study, we examined the anthelmintic efficacy of albendazole (ABZ) and crude plant extracts from barks of Bombax ceiba L., Diospyros rhodocalyx Kurz. and Vitex glabrata R.Br., and leaves of Terminalia catappa L. and Cassia alata L. against Gastrothylax crumenifer. The hightest anthelmintic activity on the parasites after 24 h incubation was observed in the n-butanol extract of T. catappa leaf. In this study, fractionation bioassay of n-butanol extract of T. catappa leaf was conducted to both separation and discrimination of rutin served as a new efficient compound (LC50 = 28.96; LC90 = 88.75 µg/mL) against G. crumenifer. This compound was confirmed by 1H nuclear magnetic resonance (1H NMR), 13C NMR, infrared (IR) and ultraviolet (UV) spectra as well as mass spectra data. The rutin-treated parasites with all dosages showed swift decrease of the motility and the relative motility (RM) and survival index (SI) were decreased obviously from 3 h until flukes were killed after 12 h of incubation. When observed with light microscopy, the parasites showed the earliest change in a limited region of the tegument. When observed by scanning electron microscopy, the parasites' tegument exhibited similar sequences of surface changes after treatments with rutin and ABZ, but less severity in ABZ treatment. The sequences of changes comprised swelling of folds and ridges, formation of blebbing, rupturing of blebs, erosions, lesions and the tegument demolition. Hence, rutin could be considered as the potential anthelmintic agent for treatment of paramphistomosis.

Molecular detection and genetic diversity of Anaplasma marginale based on the major surface protein genes in Thailand.

Anaplasma marginale is the rickettsial agent of anaplasmosis, a tick-borne disease, which affects cattle and other ruminants in tropical and subtropical areas of the world, and causing huge economic losses because of decreasing meat and milk production. In the present study, molecular methods have been used to determine the occurrence and genetic diversity of A. marginale, based on the genes encoding the major surface proteins (msps) genes, in blood samples from 520 cattle and 121 buffaloes in the north and northeastern regions of Thailand. The polymerase chain reaction (PCR) results based on the msp4 gene indicated that 66 (10.30%) cattle were positive for A. marginale, whereas no positive result was obtained from buffaloes. The phylogenetic analysis based on the maximum likelihood method using 13, 29 and 27 nucleotide sequences from msp2, msp4, msp5 clones, respectively, revealed that the sequences detected in this study are obviously distributed in different clusters. The sequence analysis demonstrated that msp2 gene is genetically diverse, while msp4 and msp5 genes are conserved in Thailand. These findings corroborated the diversity analysis of the same sequences, which showed 13, 27 and 27 haplotypes of the msp2, msp4 and msp5 genes, respectively. In addition, the entropy analyses of amino acid sequences exhibited 127, 75 and 51 high entropy peaks with values ranging from 0.27119 to 2.45831, from 0.14999 to 2.17552 and from 0.15841 to 1.05453 for MSP2, MSP4 and MSP5, respectively. Therefore, the results indicate a low molecular occurrence of A. marginale in cattle blood samples in Thailand. From these results; however, a high degree of genetic diversity was observed in the analyzed A. marginale population. Hence, our finding could be used to improve the immunodiagnostics and vaccination programs for anaplasmosis.

Recombinant expression and characterization of major surface protein 4 from Anaplasma marginale.

Anaplasma marginale is the rickettsia which causes the bovine anaplasmosis. The distribution of A. marginale is both tropical and subtropical regions of the world. The major surface protein 4 (MSP4) of this parasite was identified as an immunodominant protein. In this study, the full length of DNA encoding A. marginale MSP4 (AmMSP4) was cloned from the parasites. The open reading frame of msp4 coding sequence of Thailand strain is 849 bp. Phylogenetic analysis revealed that the msp4 coding sequence of A. marginale was highly conserved when compared with Anaplasma phagocytophilum. The recombinant plasmid was further transformed into the BL21-CodonPlus (DE3)-RIPL competent cells for over-expression of the recombinant major surface protein 4 of A. marginale (rAmMSP4). Sera from rabbit immunized with rAmMSP4 and from cattle infected with A. marginale were used to study the antigenicity of rAmMSP4 (35 kDa) and AmMSP4 (31 kDa). Both rAmMSP4 and AmMSP4 were recognized by these sera showing that recombinant and native AmMSP4 have conserved epitopes. Localization of Anaplasma parasites by immunofluorescence showed these parasites are distributed on both the membrane and the outside of infected erythrocytes. Regarding antigenicity, recombinant MSP4 could be used for immunodiagnostic purposes and as a possible vaccine candidate against anaplasmosis.

Production and characterization of a monoclonal antibody specific to 16 kDa antigen of Paramphistomum gracile.

A number of monoclonal antibodies (MoAbs) against the 16 kDa antigen of Paramphistomum gracile (16 kDaAgPg) were produced in vitro by hybridoma technique. Reactivity and specificity of these MoAbs were evaluated by ELISA and immunoblotting assays. Seven MoAb clones were selected from the stable hybridoma clones, namely 1D10, 2D7, 3B10, 3D9, 4F1, 4G4, and 5G12. It was found to be IgM and kappa light chain isotypes. By immunoblotting and ELISA, all MoAbs reacted with purified 16 kDaAgPg at molecular weight (MW) of 16 kDa and with the native 16 kDa antigen at MW of 16 kDa in the whole body (WB) and excretory-secretory (ES) fractions, but not with tegumental antigens (TA) of adult fluke. All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants, including Eurytrema pancreaticum, Gigantocotyle explanatum, Schistosoma spindale, Moniezia benedeni, Avitellina centripunctata, Haemonchus placei, Trichuris sp., and Setaria labiato-papillosa. Localization and distribution of the native 16 kDaAg in adult P. gracile by immunohistochemistry, using MoAbs as probes, showed that the native 16 kDaAg was present in high concentration in the cytoplasm of vitelline cells, eggshell globules, and the shells of eggs, but not in the tegument, muscle, parenchymal cells, and cecum of adult fluke. This finding indicated that the 16 kDaAg is a copiously expressed parasite protein that is released into the ES; thus, 16 kDaAg and its MoAb could be a good candidate for immunodiagnosis of paramphistomosis in ruminants.