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

Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease.

BACKGROUND:Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. METHODS:In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities. RESULTS:The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test). CONCLUSIONS:These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica

Determination of the specificity of MoAb 4E3 with CE from <i>F</i>. <i>gigantica</i>, other trematode, cestode and nematode parasites by using indirect ELISA and immunoblot analysis.

<p>(A) ELISA OD values of cross-reactivities of MoAb 4E3 and antigens from various trematode, cestode and nematode species. Fg = <i>F</i>. <i>gigantica</i>, Fh = <i>F</i>. <i>hepatica</i>, Ge = <i>G</i>. <i>explanatum</i>, Ep = <i>E</i>. <i>pancreaticum</i>, Pc = <i>P</i>. <i>cervi</i>, Pg = <i>P</i>. <i>gracile</i>, Cc = <i>C</i>. <i>cotylophorum</i>, Fc = <i>F</i>. <i>cobboldi</i>, Gc = <i>G</i>. <i>crumenifer</i>, Ss = <i>S</i>. <i>spindale</i>, Mb = <i>M</i>. <i>benedeni</i>, Ac = <i>A</i>. <i>centripunctata</i>, Ts = <i>Trichuris</i> sp., Hp = <i>H</i>. <i>placei</i> and Sp = <i>S</i>. <i>labiato-papillosa</i>, Sm = <i>S</i>. <i>mansoni</i>, Sme = <i>S</i>. <i>mekongi</i>, Sj = <i>S</i>. <i>japonicum</i>, and Ov = <i>O</i>. <i>viverrini</i>. Undil = undiluted MoAb hybridoma fluid, MoAb dilution = MoAb hybridoma fluid diluted at 1:10, 1: 100, 1: 500 and 1: 1,000. The cut-off value is indicated by a horizontal dashed line and calculated as the mean OD of negative controls plus 3 SD. The OD values greater than this cut-off value are considered to be positive. (B) Immunoblotting detection of the cross-reactivities of MoAb 4E3 with CE from <i>F</i>. <i>gigantica</i> and other trematode parasites. Fg = <i>F</i>. <i>gigantica</i> (lane 2), Fh = <i>F</i>. <i>hepatica</i> (lane 3), Ge = <i>G</i>. <i>explanatum</i> (lane 4), Ep = <i>E</i>. <i>pancreaticum</i> (lane 5), Pc = <i>P</i>. <i>cervi</i> (lane 6), Pg = <i>P</i>. <i>gracile</i> (lane 7), Cc = <i>C</i>. <i>cotylophorum</i> (lane 8), Fc = <i>F</i>. <i>cobboldi</i> (lane 9) and Gc = <i>G</i>. <i>crumenifer</i> (lane 10) and Ss = <i>S</i>. <i>spindale</i> (lane 11), (C) Immunoblotting detection of the cross-reactivities of MoAb 4E3 with CE from <i>F</i>. <i>gigantica</i>, other trematode, cestode and nematode parasites. Mb = <i>M</i>. <i>benedeni</i> (lane 3), Ac = <i>A</i>. <i>centripunctata</i> (lane 4), Ts = <i>Trichuris</i> sp. (lane 5), Hp = <i>H</i>. <i>placei</i> (lane 6), Sp = <i>S</i>. <i>labiato-papillosa</i> (lane 7), Sm = <i>S</i>. <i>mansoni</i> (lane 8), Sme = <i>S</i>. <i>mekongi</i> (lane 9), Sj = <i>S</i>. <i>japonicum</i> (lane 10) and Ov = <i>O</i>. <i>viverrini</i> (lane 11). Lane 1 of B and C is CE from <i>F</i>. <i>gigantica</i> blotted with the myeloma culture fluid (MCF), which is used as the negative control. STD is the lane containing standard protein molecular weight markers indicated on the left side.</p

An immunochromatographic (IC) strip test is developed for diagnosis of fasciolosis by <i>F</i>. <i>gigantica</i>: Experiment trial.

<p>(A) A schematic diagram of the immunochromatographic (IC) strip test showing several components: a sample pad, a conjugate pad, an immobilized nitrocellulose membrane (control and test antibody) and an absorbent pad. (B) The samples of the IC strip test for deciding the results: a positive result shows two red dots at the test and control regions, while a negative result exhibits only one red dot in the control region. The strip tests are invalid when there is no red dot at the control region. (C-E) Sensitivity testing of the IC strip was studied using a series of dilutions in a buffer (0.0512–20,000 ng/ml) of adult <i>F</i>. <i>gigantica</i> recombinant cathepsin L1 (rCatL1) (C), crude extract (CE) (D), and excretory-secretory (ES) antigens (E). 1 = 20,000 ng/ml, 2 = 4,000 ng/ml, 3 = 800 ng/ml, 4 = 160 ng/ml, 5 = 32 ng/ml, 6 = 6.4 ng/ml, 7 = 1.2 ng/ml, 8 = 0.256 ng/ml, 9 = 0.0512 ng/ml, and 10 = buffer control. (F) The specificity of the IC strips were tested against CE from <i>F</i>. <i>gigantica</i>, other trematode, cestode and nematode parasites. All antigens were tested at 1,000 μg/ml. Fg = <i>F</i>. <i>gigantica</i>, Bf = buffer control, Fh = <i>F</i>. <i>hepatica</i>, Ge = <i>G</i>. <i>explanatum</i>, Ep = <i>E</i>. <i>pancreaticum</i>, Pc = <i>P</i>. <i>cervi</i>, Pg = <i>P</i>. <i>gracile</i>, Cc = <i>C</i>. <i>cotylophorum</i>, Fc = <i>F</i>. <i>cobboldi</i>, Gc = <i>G</i>. <i>crumenifer</i>, Ss = <i>S</i>. <i>spindale</i>, Mb = <i>M</i>. <i>benedeni</i>, Ac = <i>A</i>. <i>centripunctata</i>, Ts = <i>Trichuris</i> sp., Hp = <i>H</i>. <i>placei</i>, Sp = <i>S</i>. <i>labiato-papillosa</i>, Sm = <i>S</i>. <i>mansoni</i>, Sme = <i>S</i>. <i>mekongi</i>, Sj = <i>S</i>. <i>japonicum</i> and Ov = <i>O</i>. <i>viverrini</i>.</p

A field trial of the immunochromatographic (IC) strip test for diagnosis of fasciolosis by <i>F</i>. <i>gigantica</i>.

<p>(A) Detection of circulating CatL1 antigens in the serum samples of mice experimentally infected with <i>F</i>. <i>gigantica</i> as compared with non-infected mice. D1 = day 1 post infection, D4 = day 4 post infection, D7 = day 7 post infection, D = day 21 post infection, D35 = day 35 post infection, IS = infected serum, and NS = normal serum. (B) Detection of circulating CatL1 antigens in the serum samples of cattle naturally infected with <i>F</i>. <i>gigantica</i> (strip 1), <i>P</i>. <i>cervi</i> (strip 2), <i>M</i>. <i>benedeni</i> (strip 3), Strongylids (strip 4), <i>Trichuris</i> sp. (strip 5), and <i>Strongyloides</i> sp. (strip 6). Serum sample from non-infected cattle was used as the negative control (strip 7). (C) Detection of circulating CatL1 antigens in the serum samples of mice experimentally infected with <i>F</i>. <i>gigantica</i> as compared with non-infected mice, showing results of positivity (+VE) and negativity (-VE) as measured by IC test. (D and E) Comparison between positivity (+VE) and negativity (-VE) of different parasite species as measured by IC test. (D) Sera from mice experimentally infected with fasciolosis (1), opisthorchiasis (2), schistosomiasis (3), non-infected mice (4), and non-infected hamsters (5). (E) Sera from cattle naturally infected with fasciolosis (1), paramphistomosis (2), monieziasis (3), strongylid infection (4), trichuriasis (5), strongyloidiasis (6) and non-infected cattle (7).</p

Immunofluorescence staining of CatL1 proteases of adult <i>F</i>. <i>gigantica</i> using the specific MoAb 4E3 as a probe.

<p>(A) The negative control of a cross section stained with myeloma culture fluid, showing tegument (Te), spine (Sp), muscle (Mu), caecum (Ca), and parenchymal cells (Pc), while vitelline glands (Vi) appear only nonspecific orange autofluorescence. (B) A low magnification micrograph showing intense fluorescence in both caecal epithelium and in the lumen of the caecum, while the tegument (Te), spine (Sp), muscle (Mu) and parenchymal cells (Pc) are not stained. (C and D) Medium and high magnification micrographs showing intense fluorescence in both caecal epithelium and in the lumen of the caecum, while parenchymal cells (Pc) are not stained.</p

A reliable monoclonal antibody (MoAb)-based sandwich enzyme-linked immunosorbent assay (sandwich ELISA) is developed for diagnosis of fasciolosis by <i>F</i>. <i>gigantica</i>.

<p>(A and B) The lowest concentrations of rCatL1, CatL1 in CE of variuos stages of <i>F</i>. <i>gigantica</i>, and in ES antigens of adult <i>F</i>. <i>gigantica</i> as detected by the sandwich ELISA. (A) Lines with black circle, white triangle and black square denote the concentration levels of rCatL1 and CatL1 in CE and ES antigens of adult <i>F</i>. <i>gigantica</i>. The arrows indicate the lowest concentrations of CatL1 that could still be detected. (B) Lines with black circle, white circle, black triangle, white triangle, black square and white square show the concentration levels of CatL1 in CE of adult, 5-, 3-, 1-week-old juveniles, NEJ and Met of <i>F</i>. <i>gigantica</i>, respectively. The arrows indicate the lowest concentrations that CatL1 could still be detected. (C) Detection of circulating CatL1 antigens in the serum samples of mice experimentally infected with <i>F</i>. <i>gigantica</i> as compared with non-infected mice by sandwich ELISA. Black circles denote OD values of individual mouse serum; white squares represent mean OD values of all mice in each experimental group. The horizontal dotted line represents the cut-off value for a positive detection. (D) The relative levels of circulating CatL1 antigens in the serum samples from mice infected with <i>F</i>. <i>gigantica</i> and <i>S</i>. <i>mansoni</i> as well as hamster infected with <i>O</i>. <i>viverrini</i> as examined by sandwich ELISA (OD values at 450 nm). The serum samples from non-infected mice and hamsters are used as negative controls. The horizontal dotted line is the cut-off value for a positive detection. (E) The relative levels of circulating CatL1 antigens in the serum samples from cattle naturally infected with <i>F</i>. <i>gigantica</i> (fasciolosis), <i>P</i>. <i>cervi</i> (paramphistomosis), <i>M</i>. <i>benedeni</i> (Monieziasis), Strongylids (strongylid infection), <i>Trichuris</i> sp. (Trichuriasis), and <i>Strongyloides</i> sp.(strongyloidiasis) as measured by sandwich ELISA (OD values at 450 nm). Serum samples from non-infected cattle are used as negative controls. The horizontal dotted line represents the cut-off value for a positive detection.</p

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

Abstract 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 occurrence and genetic diversity of Ehrlichia canis in naturally infected dogs from Thailand

Abstract Canine monocytic ehrlichiosis is cause by Ehrlichia canis resulting in hematologic disorders and severe clinical signs. The aim of this study was to scrutinize the molecular detection and genetic diversity of E. canis based on the trp36 gene in dogs from Thailand’s northern and central regions. A total of 120 dogs blood samples were amplified for trp36 gene of E. canis using the polymerase chain reaction (PCR). Forty-seven out of 120 dog blood samples (39.16%, 47/120) were positive for E. canis the trp36 DNA with 790 bp of PCR amplicon size. The factor significantly associated with E. canis infection is animal housing status (p < 0.05). Sequence and phylogenetic analysis showed that E. canis trp36 gene of Thailand isolates was clustered into 1st clade with similarity ranging from 95.65 to 100% together with the US genogroup. The 14 haplotypes of the trp36 gene shown in TCS network exhibited that haplotype #1–4 was found in Thailand. The entropy analysis of the trp36 gene illustrated 751 polymorphic sites and 271 entropy peaks of nucleic and amino acid sequences, respectively. Hence, these findings are crucial for better understanding the epidemiology of Ehrlichia infection and could be helpful for implementing control measures in Thailand