Immunoproteomics to identify species-specific antigens in Neospora caninum recognised by infected bovine sera

Bovine neosporosis is a disease of concern due to its global distribution and significant economic impact through massive losses in the dairy and meat industries. To date, there is no effective chemotherapeutic drug or vaccine to prevent neosporosis. Control of this disease is therefore dependent on efficient detection tests that may affect treatment management strategies. This study was conducted to identify the specific immunoreactive proteins of Neospora caninum tachyzoites recognised by sera from cattle infected with N. caninum, Toxoplasma gondii, Cryptosporidium parvum, Babesia bovis and B. bigemina, and by sera from uninfected cattle using two-DE dimensional gel electrophoresis (2-DE) combined with immunoblot and mass spectrometry (LC-MS/MS). Among 70 protein spots that reacted with all infected sera, 20 specific antigenic spots corresponding to 14 different antigenic proteins were recognised by N. caninum-positive sera. Of these immunoreactive antigens, proteins involved in cell proliferation and invasion process were highly immunogenic, including HSP90-like protein, putative microneme 4 (MIC4), actin, elongation factor 1-alpha and armadillo/beta-catenin-like repeat-containing protein. Interestingly, we discovered an unnamed protein product, rhoptry protein (ROP1), possessing strong immunoreactivity against N. caninum but with no data on function available. Moreover, we identified cross-reactive antigens among these apicomplexan parasites, especially N. caninum, T. gondii and C. parvum. Neospora caninum-specific immunodominant proteins were identified for immunodiagnosis and vaccine development. The cross-reactive antigens could be evaluated as potential common vaccine candidates or drug targets to control the diseases caused by these apicomplexan protozoan parasites

Prevalence and Subtype Distribution of Blastocystis Infection in Patients with Diabetes Mellitus in Thailand

Diabetes mellitus (DM) is a major global public health problem with an increasing prevalence. DM increases the risk of infections caused by bacteria, fungi, viruses, and parasites. We examined the prevalence, subtypes, and risk factors of Blastocystis infection in patients with and without DM in central Thailand. Stool samples and questionnaires were obtained from 130 people in the DM group and 100 people in the non-DM group. Blastocystis infection was identified via a nested polymerase chain reaction and subtyped via sequencing of the partial small-subunit ribosomal RNA (SSU rRNA) gene. Analysis of potential risk factors was conducted via binary logistic regression. The overall prevalence of Blastocystis infection was 10.8%, including rates of 9% and 12.3% in the non-DM and DM groups, respectively. The most prevalent subtype was ST3, followed by ST1, and ST4. Factors that potentially increased the risk of Blastocystis infection include patients being >65 years old, the presence of DM, a DM duration of ≥10 years, a low level of education, and animal ownership. In conclusion, this is the first study of Blastocystis infection in DM, and a high prevalence was found among this population. Therefore, health education promoting sanitation and hygiene is necessary to reduce and prevent infection in the community

Prevalence and Subtype Distribution of Blastocystis Isolated from School-Aged Children in the Thai-Myanmar Border, Ratchaburi Province, Thailand

Blastocystis is one of the most common enteric protozoa that inhabits the intestinal tract of humans and different animals. Moreover, it has a worldwide geographic distribution. Its main mode of transmission is via the fecal-oral route. At present, 26 subtypes are widely distributed across both humans and animals. The current study aimed to determine the prevalence and subtype distribution of Blastocystis among school-aged children living on the Thai-Myanmar border, Ratchaburi province, Thailand. In total, 508 samples were collected from children at six schools. The prevalence of Blastocystis infection was amplified and sequenced in the 600 bp barcode region of the small-subunit ribosomal RNA (SSU rRNA). The overall prevalence of Blastocystis infection was 3.35% (17/508). ST3 (11/17) was the most predominant subtype, followed by ST1 (5/17) and ST2 (1/17). A phylogenetic tree was constructed based on the Tamura92+G+I model using the maximum-likelihood algorithm. Surprisingly, all sequences of the ST3-positive samples were closely correlated with the cattle-derived sequence. Meanwhile, all sequences of the Blastocystis ST1-positive samples were closely correlated with the human-derived sequence. Nevertheless, further studies should be conducted to validate the zoonotic transmission of Blastocystis. Based on our findings, personal hygiene and sanitation should be improved to promote better health in children in this area

Immunoproteomics to identify species-specific antigens in

Bovine neosporosis is a disease of concern due to its global distribution and significant economic impact through massive losses in the dairy and meat industries. To date, there is no effective chemotherapeutic drug or vaccine to prevent neosporosis. Control of this disease is therefore dependent on efficient detection tests that may affect treatment management strategies. This study was conducted to identify the specific immunoreactive proteins of Neospora caninum tachyzoites recognised by sera from cattle infected with N. caninum, Toxoplasma gondii, Cryptosporidium parvum, Babesia bovis and B. bigemina, and by sera from uninfected cattle using two-DE dimensional gel electrophoresis (2-DE) combined with immunoblot and mass spectrometry (LC-MS/MS). Among 70 protein spots that reacted with all infected sera, 20 specific antigenic spots corresponding to 14 different antigenic proteins were recognised by N. caninum-positive sera. Of these immunoreactive antigens, proteins involved in cell proliferation and invasion process were highly immunogenic, including HSP90-like protein, putative microneme 4 (MIC4), actin, elongation factor 1-alpha and armadillo/beta-catenin-like repeat-containing protein. Interestingly, we discovered an unnamed protein product, rhoptry protein (ROP1), possessing strong immunoreactivity against N. caninum but with no data on function available. Moreover, we identified cross-reactive antigens among these apicomplexan parasites, especially N. caninum, T. gondii and C. parvum. Neospora caninum-specific immunodominant proteins were identified for immunodiagnosis and vaccine development. The cross-reactive antigens could be evaluated as potential common vaccine candidates or drug targets to control the diseases caused by these apicomplexan protozoan parasites

Additional file 1: Figure S1. of Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis

Map showing the sampling areas and the types of samples collected in this study. (TIFF 2909 kb

Molecular identification of

Zoonotic Cryptosporidium spp., particularly C. meleagridis, C. canis, and C. felis, are enteric protozoa responsible for major public health concerns around the world. To determine the spread of this parasite in Thailand, we conducted molecular identification of Cryptosporidium spp. from animal samples around the country, by collecting and investigating the feces of seagulls (Chroicocephalus brunnicephalus and Chroicocephalus ridibundus), domestic pigeons (Columba livia domestica), dogs, and cats. Seagull and pigeon samples were collected at the seaside and on the riverside to evaluate their potential for waterborne transmission. Ten pigeon samples were combined into one set, and a total of seven sets were collected. Seventy seagull samples were combined into one set, and a total of 13 sets were collected. In addition, 111 dog samples were collected from cattle farms, and 95 dog and 80 cat samples were collected from a temple. We identified C. meleagridis in pigeons, Cryptosporidium avian genotype III in seagulls, C. canis in dogs, and C. felis in cats. In the temple, the prevalence was 2.1% (2/95) for dogs and 2.5% (2/80) for cats. No Cryptosporidium was found in dog samples from cattle farms. These are the first findings of C. meleagridis in domestic pigeons, and Cryptosporidium avian genotype III in seagulls. Our study invites further molecular epidemiological investigations of Cryptosporidium in these animals and their environment to evaluate the public health risk in Thailand

Agreement (Kappa) between the detection of <i>N</i>. <i>fowleri</i> by LAMP and PCR in water samples.

<p>*<i>P</i> <0.001</p><p>Agreement (Kappa) between the detection of <i>N</i>. <i>fowleri</i> by LAMP and PCR in water samples.</p

Categories of water samples tested in this study with number <i>N</i>. <i>fowleri</i>-positive by LAMP and PCR.

<p>*<i>P</i> value for McNemar's test = 0.25</p><p>Categories of water samples tested in this study with number <i>N</i>. <i>fowleri</i>-positive by LAMP and PCR.</p

Development of a Rapid, Simple Method for Detecting <i>Naegleria fowleri</i> Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP)

<div><p><i>Naegleria fowleri</i> is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of <i>N</i>. <i>fowleri</i> using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for <i>N</i>. <i>fowleri</i>. Time to results is about 90 min and amplification products were easily detected visually using <i>hydroxy naphthol blue</i>. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. <i>N</i>. <i>fowleri</i> was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for <i>N</i>. <i>fowleri</i>. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing <i>N</i>. <i>fowleri</i> in water and clinical samples, particularly in resource-poor settings.</p></div