PCR Detection Research Articles

Identification and virulence test of a new pathogen that causes verticillium striping on rapeseed in northwestern China

Five stems of rapeseed with abundant black microsclerotia were collected from Huangyuan County of Qinghai Province, China, and fungal isolates were obtained from the stems. They were identified based on morphology, molecular features and specific PCR detection. The results showed that the 10 fungal isolates belonged to Verticillium longisporum lineage A1/D3. One of the 10 isolates (HW7-1) was tested for virulence on three species of rapeseed, including B. napus Zhongshuang 9, B. rapa Qingyou 9 and B. juncea Tayou 2 by conidia inoculation of HW7-1 on roots of young seedlings. Control seedlings were inoculated with V. dahliae conidia or water alone. The seedlings of these treatments were transplanted in culture mix and incubated in a growth chamber (20 ​°C). Results suggested that the control seedlings of three cultivars appeared quite healthy, while the seedlings inoculated with HW7-1 turned yellowing leaves, seedling stunting or even death after 22 days post-inoculation. V. longisporum was re-isolated from he yellow leaves, thus fulfilling Koch's postulates. Moreover, compared to the control treatments, inoculation with HW7-1 caused flowering delay and seed yield reduction on Tayou 2 with production of microsclerotia on the stems. To our knowledge, this is the first report of V. longisporum lineage A1/D3 on rapeseed in northwestern China.

Retroviral Insertion Polymorphism (RIP) of Porcine Endogenous Retroviruses (PERVs) in Pig Genomes.

Endogenous retroviruses (ERVs) are one of the superfamilies of long terminal repeat retrotransposons (LTRs) in mice and humans. Approximately 8% of the pig genome is composed of sequences derived from LTRs. While the majority of ERVs in pigs have decayed, a small number of full-length copies can still mobilize within the genome. This study investigated the unexplored retroviral insertion polymorphisms (RIPs) generated by the mobilization of full-length ERVs (Fl-ERVs), and evaluated their impact on phenotypic variation to gain insights into the biological role of Fl-ERVs in pigs. Overall, 39 RIPs (insertions or deletions relative to the pig reference genome) generated by Fl-ERVs were predicted by comparative genomic analysis, and 18 of them were confirmed by PCR detection. Four RIP sites (D5, D14, D15, and D18) were further evaluated by population analysis, and all of them displayed polymorphisms in multiple breeds. The RIP site of ERV-D14, which is a Fl-ERV inserted in the STAB2-like gene, was further confirmed by sequencing. Population analysis of the polymorphic site of ERV-D14 reveals that it presents moderate polymorphism information in the Large White pig breed, and the association analysis reveals that the RIP of ERV-D14 is associated with age variations at 30 kg body weight (p < 0.05) and 100 kg body weight (p < 0.01) in the population of Large White pigs (N = 480). Furthermore, the ERV-D14 RIP is associated with changes in the expression of the target gene STAB2-like in the liver, backfat, and leaf fat in Sushan pigs. These data suggest that some Fl-ERVs are still mobilizing in the pig's genome, and contribute to genomic and phenotypic variations.

Characterizing the detection of inactivated Mycoplasma hyopneumoniae DNA in the respiratory tract of pigs

A positive Mycoplasma hyopneumoniae PCR result in a clinical specimen may eventually represent the mere detection of non-viable bacteria, complicating the diagnostic interpretation. Thus, the objective of this study was to evaluate the PCR detection of non-viable M. hyopneumoniae and its residual cell-free DNA in live pigs. Pigs were inoculated with either active or inactivated M. hyopneumoniae and were sampled for up to 14 days. Mycoplasma hyopneumoniae was not detected by PCR at any timepoint in pigs inoculated with the inactivated bacterium, suggesting that in healthy pigs, the non-viable M. hyopneumoniae DNA was rapidly sensed and cleared.

Virus removal by high-efficiency air (HEPA) filters and filtration capacity enhancement by nanotextiles: a pilot study.

Portable household air purifiers are widely used devices designed to maintain a high-quality level of indoor air. Portable air purifiers equipped with the high-efficiency air (HEPA) filter served 100h in a household space occupied by two adults without any symptoms of respiratory tract infection. The main objective of the study was to determine microbial contamination on the HEPA filter and to investigate if the selected nanotextile monolayer made of polyamide 6 (PA6) nanofibers can capture potential microorganisms when installed downstream of the HEPA filter as the final filtration medium. Samples were taken from the inlet and outlet surfaces. Samples from the nanotextile were collected in the same manner as from the HEPA filter. QIAStat DX® 1.0 Analyzer using the Respiratory SARS CoV-2 Panel multiplex PCR detection system was selected for microorganism detection. Adenovirus was detected on the inlet surface of the HEPA filter. The outlet surface of the filter contained no viruses included in the Respiratory SARS CoV-2 Panel portfolio. The nanotextile monolayer was replaced twice during the 100h of operation, so three pieces were used and all contained coronavirus 229 E. Coronavirus 229 E was then detected in the nasopharynx of one of the members of the household as well. It may be assumed that the selected nanotextile is capable of capturing a virus of a small size.

Rapid and sensitive detection of large yellow croaker iridovirus by real-time RPA and RPA-LFD.

Large yellow croaker (Larimichthys crocea) is a vital marine-cultured species in China. Large yellow croaker iridovirus (LYCIV) can cause a high mortality rate in L. crocea. Rapid and convenient detection of LYCIV is an urgent demand for diagnosis. In this study, rapid and simple recombinase polymerase amplification (RPA), real-time RPA and RPA combined with lateral flow dipstick (RPA-LFD) methods were developed for the detection of LYCIV based on the conserved sequence of the LYCIV major capsid protein (MCP) gene. With these optimized RPA analyses, LYCIV detection could be completed within 20 min at 40°C. Both RPA and real-time RPA could detect viral DNA as low as 102 copies/μL, while the detection limit of RPA-LFD was 101 copies/μL, and there was no cross-reaction with other aquatic pathogens (KHV, CyHV-2, GCRV-JX01, SVCV, LCDV and LMBV). In practical evaluation of RPA, real-time RPA and RPA-LFD methods, the results showed consistency with the general PCR detection. In short, the developed RPA, real-time RPA and RPA-LFD analyses could be simple, rapid, sensitive and reliable methods for field diagnosis of LYCIV infection and have significant potential in the protection of LYCIV infection.

Development and application of targeted multiplex polymerase chain reaction to help diagnose infectious uveitis

Abstract&#x0D; Introduction &amp; Objectives : Clinical pattern recognition is paramount in uveitis diagnosis. Pathogen detection from ocular fluid samples is often necessary to support infectious uveitis diagnosis, particularly in cases presenting with atypical clinical appearance. This study aims to present the process of developing targeted multiplex PCR and its application in infectious uveitis.&#x0D; Methods : This was a cross sectional study to evaluate the diagnostic performance of targeted multiplex PCR in infectious uveitis. We obtained ocular fluid samples and reviewed medical records of uveitis patients who underwent ocular fluid analysis at Cipto Mangunkusumo Hospital from February 2022 to March 2023. PCR detection threshold values (DNA copies/mL) were 10.9 for Mycobacterium tuberculosis (Mtb), 672 for Epstein-Barr virus, 4.77 for Cytomegalovirus, 6.37 for Toxoplasma gondii, and 5.53 for Herpes simplex virus. With every two-fold increase of pathogen selection, this method requires a half volume of extracted DNA template from aqueous/vitreous samples than uniplex PCR. The ophthalmologist selected the pathogen combination to be detected, allowing for a tailored examination.&#x0D; Results : Forty-seven aqueous or vitreous samples were analyzed. The positivity rate was 23.4% (11/47) with Mtb yielded the highest positivity (7/34; 20.6%). With final diagnosis as a reference, targeted multiplex PCR resulted in 32.3% sensitivity, 93.8% specificity, 90.9% positive predictive value and 19.2% negative predictive value.&#x0D; Conclusion : With its high specificity, targeted multiplex PCR is useful as a confirmatory but not screening tool in uveitis diagnosis. Ocular fluid analysis is an important part of stepwise diagnostic approach in uveitis.

Comparison of the adherence of nontypeable haemophilus influenzae to lung epithelial cells

ObjectiveNontypeable Haemophilus influenzae (NTHi) plays an important role in respiratory tract infections, and adherence to lung epithelial cells is the first step in lung infections. To explore the role of NTHi in childhood lung infections, a comparative study was conducted on the adherence of strains isolated from sputum culture and bronchoalveolar lavage fluid to A549 lung epithelial cells.MethodsHaemophilus influenzae strains were obtained from the sample bank of Shenzhen Children’s Hospital, and identified as NTHi via PCR detection of the capsule gene bexA. NTHi obtained from healthy children’s nasopharyngeal swabs culture were selected as the control group, and a comparative study was conducted on the adherence of strains isolated from sputum culture or bronchoalveolar lavage fluid of patients to A549 cells.ResultsThe adherence bacterial counts of NTHi isolated from the nasopharyngeal cultures of healthy children to A549 cells was 58.2 CFU. In patients with lung diseases, NTHi isolated from bronchoalveolar lavage fluid was 104.3 CFU, and from sputum cultures was 115.1 CFU, both of which were significantly higher in their adherence to A549 cells compared to the strains isolated from the healthy control group. There was no significant difference in adherence between the strains isolated from sputum cultures and bronchoalveolar lavage fluid (t = 0.5217, p = 0.6033).ConclusionNTHi played an important role in childhood pulmonary infections by enhancing its adherence to lung epithelial cells.

Polluted surface water is a repository of antibiotic-resistant Escherichia coli harbouring Plasmid-Mediated Quinolone Resistance (PMQR) determinants

ABSTRACT We investigated the carriage of PMQR determinants by E. coli from surface water and their antibiotypes. Water samples were collected and analysed from two surface water: Agbowo (AG) and Arulogun (AR) in South-west Nigeria for E. coli, which were identified using PCR detection of uidA. Susceptibility to antibiotics was done using the disc-diffusion method. Detection of PMQR genes in isolates showing complete resistance or intermediate susceptibility to the fluoroquinolone antibiotics was done using primer-specific PCR. Thirty-six E. coli isolates were obtained [AG (20) and AR (16)]. The isolates showed different levels of resistance to the tested antibiotics, with 100% resistance to amoxicillin-clavulanate. None of the isolates was positive for extended spectrum β-lactamase (ESBL) production, but 25 showed complete resistance or intermediate susceptibility to the fluoroquinolone antibiotics. Two isolates each from AG and AR carried qnrA, one isolate carried qnrS, with one each from AG and AR carrying aac(6′)-lb-cr. Four isolates carried oqxAB, but, qnrB and qepA were not detected in any isolate. This study revealed that polluted surface water is a ‘hub’ of antibiotic-resistant E. coli harbouring PMQR genes.

Transmission risk evaluation of transfusion blood containing low-density Babesia microti.

Babesia is a unique apicomplexan parasite that specifically invades and proliferates in red blood cells and can be transmitted via blood transfusion, resulting in transfusion-transmitted babesiosis. However, detecting Babesia in blood before transfusion has not received enough attention, and the risk of transfusing blood containing a low density of Babesia microti (B. microti) is unclear, possibly threatening public health and wellness. This study aimed to determine the lower detection limit of B. microti in blood and to evaluate the transmission risk of blood transfusion containing low-density B. microti. Infected BALB/c mouse models were established by transfusing infected whole blood with different infection rates and densities of B. microti. Microscopic examination, nested Polymerase Chain Reaction (nested PCR), and an enzyme-linked immunosorbent assay (ELISA) were used to evaluate the infection status of the mouse models. Meanwhile, the nested PCR detection limit of B. microti was obtained using pure B. microti DNA samples with serial concentrations and whole blood samples with different densities of B. microti-infected red blood cells. Thereafter, whole mouse blood with a B. microti density lower than that of the nested PCR detection limit and human blood samples infected with B. microti were transfused into healthy mice to assess the transmission risk in mouse models. The infection status of these mice was evaluated through microscopic examination, nested PCR tests, and ELISA. The mice inoculated with different densities of B. microti reached the peak infection rate on different days. Overall, the higher the blood B. microti density was, the earlier the peak infection rate was reached. The levels of specific antibodies against B. microti in the blood of the infected mice increased sharply during the first 30 days of infection, reaching a peak level at 60 days post-infection, and maintaining a high level thereafter. The nested PCR detection limits of B. microti DNA and parasite density were 3 fg and 5.48 parasites/μL, respectively. The whole blood containing an extremely low density of B. microti and human blood samples infected with B. microti could infect mice, confirming the transmission risk of transfusing blood with low-density B. microti. Whole blood containing extremely low density of B. microti poses a high transmission risk when transfused between mice and mice or human and mice, suggesting that Babesia detection should be considered by governments, hospitals, and disease prevention and control centers as a mandatory test before blood donation or transfusion.

Bacillus thuringiensis translocation in citrus scion/rootstock combinations and binding of cry toxins with the Asian citrus psyllid gut receptors

Bacillus thuringiensis (Bt) can be effective in controlling insect pests either in spray products or by their toxins expressed in transgenic crops. The discovery of Bt as endophytic in citrus opened new perspectives for the control of Diaphorina citri Kuwayama, the vector of the bacteria Candidatus Liberibacter spp. associated with Huanglongbing. In this study, the endophytic translocation of Bt in citrus seedlings (small and large plants) and in different scion-rootstock combinations were investigated. We also assessed the lethal concentration of D. citri nymphs caused by systemic Bt in citrus and, moreover, the binding of Cry toxins with the Asian citrus psyllid (ACP) gut receptors. Endophytic translocation of Bt in citrus seedlings and nursery trees was confirmed by colony isolation, PCR detection, and Btk-GFP microscopy analyses, which demonstrated that, after Bt inoculation into the substrate, the bacteria enter through lateral root emergence sites and are localized in stem xylem vessel elements. Mean lethal concentrations for D. citri third instar nymphs were 4.92 × 104 and 2.19 × 104 spores mL−1 for S1450 and S1302 strains, respectively. Nymphal mortality induced by the bacteria was higher in citrus seedlings than in citrus nursery trees. All Cry-toxins tested were able to bind to D. citri brush border membrane vesicles, suggesting the potential pathogenicity of Bt strains against psyllid. This study clearly demonstrates the penetration and Bt translocation in the xylem of small and large citrus plants and the potential of the bacteria to cause ACP nymphs mortality that fed on shoots from Bt-treated citrus.

Multiplex Detection of RNA Viruses Based on Ligation Reaction and Universal PCR Amplification.

To detect several RNA viruses simultaneously, a method based on multiplex ligation reaction combined with multiplex qPCR or multiplex PCR+capillary electrophoresis was established to detect four RNA viruses: human immunodeficiency virus (HIV), hepatitis C (HCV), influenza A virus (IAV) H1N1 and H5N1. The experimental conditions including ligation probe concentration, annealing procedure, ligation temperature and ligase dosage were optimized intensively. We found that the specificity of the ligation reaction was affected by the probe concentration predominantly, high-probe concentration (100nM) resulted in splint-independent ligation with efficiency comparable to that with RNA splint. The sensitivity of the ligation reaction was affected by the annealing mode apparently as the sensitivity of the step-down annealing mode was 100 times higher than that of the isothermal annealing at 37°C. Under the optimized condition, this assay could detect virus RNA as low as 16 viral copies per reaction in doubleplex and triplex real-time quantitative PCR detection with satisfactory specificity and precision. By multiplex PCR+capillary electrophoresis, four RNA viruses could be detected in one tube with the sensitivity of 10 copies per reaction.

Non-lethal detection of Eubothrium crassum (Cestoda) in farmed Atlantic salmon, Salmo salar, using anal swabs and real-time PCR.

Detection of intestinal parasites in fish typically requires autopsy, resulting in the sacrifice of the fish. Here, we describe a non-lethal method for detecting the tapeworm Eubothrium crassum in fish using anal swabs and real-time PCR detection. Two assays were developed to detect cytochrome oxidase I (COI) mitochondrial DNA and 18S ribosomal DNA sequences of E. crassum, respectively. The assays were tested on swab samples from confirmed pathogen free Atlantic salmon (Salmo salar L.) and on samples from farmed Atlantic salmon, where the presence and intensity of parasites had been established through autopsy. The COI assay was shown to be specific to E. crassum, while the 18S assay also amplified the closely related E. salvelini, a species infecting Arctic charr (Salvelinus alpinus L.) in freshwater. The COI assay detected E. crassum in all field samples regardless of parasite load while the 18S assay failed to detect the parasite in two samples. The results thus demonstrates that this non-lethal approach can effectively detect E. crassum and can be a valuable tool in assessing the prevalence of infection in farmed salmon, aiding in treatment decisions and evaluating treatment effectiveness.

Minimum Minutes Machine-Learning Microfluidic Microbe Monitoring Method (M7).

Frequent outbreaks of viral diseases have brought substantial negative impacts on society and the economy, and they are very difficult to detect, as the concentration of viral aerosols in the air is low and the composition is complex. The traditional detection method is manually collection and re-detection, being cumbersome and time-consuming. Here we propose a virus aerosol detection method based on microfluidic inertial separation and spectroscopic analysis technology to rapidly and accurately detect aerosol particles in the air. The microfluidic chip is designed based on the principles of inertial separation and laminar flow characteristics, resulting in an average separation efficiency of 95.99% for 2 μm particles. We build a microfluidic chip composite spectrometer detection platform to capture the spectral information on aerosol particles dynamically. By employing machine-learning techniques, we can accurately classify different types of aerosol particles. The entire experiment took less than 30 min as compared with hours by PCR detection. Furthermore, our model achieves an accuracy of 97.87% in identifying virus aerosols, which is comparable to the results obtained from PCR detection.

Application of Silica Nanoparticles in Combination with Bacillus velezensis and Bacillus thuringiensis for Anthracnose Disease Control in Shallot.

&lt;b&gt;Background and Objective:&lt;/b&gt; Anthracnose in shallot contributes to significant losses. To solve this issue, silica nanoparticles, in combination with &lt;i&gt;Bacillus velezensis&lt;/i&gt; and &lt;i&gt;Bacillus thuringiensis&lt;/i&gt; were used together. &lt;b&gt;Materials and Methods:&lt;/b&gt; &lt;i&gt;In vitro&lt;/i&gt; antagonistic test of &lt;i&gt;Bacillus velezensis&lt;/i&gt; B-27 with &lt;i&gt;Colletotrichum gloeosporioides&lt;/i&gt; was carried out using dual culture and co-culture methods. Treatment in greenhouse experiments was carried out using single application of silica, &lt;i&gt;B. thuringiensis&lt;/i&gt;, &lt;i&gt;B. velezensis&lt;/i&gt;, a combination of &lt;i&gt;B. thuringiensis&lt;/i&gt; and &lt;i&gt;B. velezensis&lt;/i&gt; and a combination of &lt;i&gt;B. thuringiensis&lt;/i&gt;, &lt;i&gt;B. velezensis&lt;/i&gt; and silica. Detection of &lt;i&gt;B. velezensis&lt;/i&gt; in the roots of shallot plants was carried out by PCR using a pair of specific primers. &lt;b&gt;Results:&lt;/b&gt; &lt;i&gt;Bacillus velezensis&lt;/i&gt; was able to inhibit the growth of &lt;i&gt;C. gloeosporioides&lt;/i&gt; mycelium &lt;i&gt;in vitro&lt;/i&gt;, both in the dual culture and co-culture methods, by 62.8 and 77.17%, respectively. Treatment of &lt;i&gt;B. thuringiensis&lt;/i&gt; and &lt;i&gt;B. velezensis&lt;/i&gt;, either individually or in combination with silica, could reduce the intensity of anthracnose disease by 20% each and stimulate the growth of shallot plants. The PCR detection using specific primers on the roots of shallot plants showed that &lt;i&gt;B. velezensis&lt;/i&gt; was detected with a DNA band length of ±576 bp. &lt;b&gt;Conclusion:&lt;/b&gt; &lt;i&gt;Bacillus velezensis&lt;/i&gt; can inhibit the growth of &lt;i&gt;C. gloeosporioides&lt;/i&gt; mycelium &lt;i&gt;in vitro&lt;/i&gt;. Applying &lt;i&gt;B. velezensis&lt;/i&gt;, &lt;i&gt;B. thuringiensis&lt;/i&gt; and silica can reduce the intensity of anthracnose disease, promote plant growth and increase plant productivity. Furthermore, &lt;i&gt;B. velezensis&lt;/i&gt; was detected in the roots of shallot plants, revealing that the bacteria are well-established.

Relevance of Secondary Enrichment in the Detection of Salmonella spp. in Food Samples by qPCR According to DIN 10135.

When detecting Salmonella spp. in food samples, unlike with culture-based procedures where there are solid standards, PCR techniques are generally dominated by commercial solutions, often backed by the conformity of reference organizations, and based on rigorous validation studies. The few independent standards that exist are not subject to revision and improvement to the same extent as the manufacturer's methods. Moreover, since commercial networks do not promote them, they are implemented less in everyday practice. The German standard DIN 10135 is an example of this. In this method, before PCR detection, a primary enrichment (16-20 h) followed by a secondary selective enrichment of at least 6 h is needed. Nevertheless, it allows the possibility of only applying the first step if evidence of their correct operation is provided. To evaluate how necessary is the secondary enrichment for DIN 10135 standard. Short and complete enrichment steps were compared in the context of the evaluation of the limit of detection for 11 types of food. Additionally, a blind assay was performed with 75 food samples. The data show that a simple primary enrichment may be sufficient and that the second selective enrichment with the tested matrixes would not be strictly essential. The blind study obtained a 98.6% of trueness and precision of 100%. At least for the end consumer products, a secondary enrichment of 6 h is not necessary for all the products tested. In the context of the DIN 10135 standard, the primary enrichment (16-20 h, 37 ± 1°C) can be enough for detecting Salmonella spp.

Plasma-derived exosomal protein SHP2 deficiency induces neutrophil hyperactivation in Behcet's uveitis

To investigate the effect of plasma-derived exosomal proteins on neutrophil hyperactivation in Behcet's uveitis (BU), we treated neutrophils from healthy controls with plasma-derived exosomes from active BU patients, and determined the level of neutrophil activation by real-time quantitative PCR (RT-qPCR) and cytokine detection assay. The results revealed that exosomes from active BU patients could activate neutrophils as shown by increasing the expression levels of pro-inflammatory cytokines (IL-17 and IL-6), chemokines (IL-8 and MCP-1), and NETs (MPO and ELANE). Label-free quantitative proteomic analysis of plasma-derived exosomes from patients and healthy controls found a remarkably distinct protein profile and identified differentially expressed proteins (DEPs) between the two groups. The results of GO, KEGG, and GSEA enrichment analysis showed that DEPs were enriched in innate immune-mediated and neutrophil hyperactivation-related signaling pathways. The protein-protein interaction (PPI) analysis determined that SHP2 was a downregulated key hub protein in the exosomes of active BU patients. Knockdown of SHP2 in human neutrophil cell lines (NB4 cells) was shown to promote the secretion of pro-inflammatory cytokines, chemokines, and NETs. The converse effects were observed following SHP2 overexpression. In conclusion, we highlighted a pathogenic role of plasma-derived exosomal SHP2 deficiency in facilitating neutrophil activation and suggested that SHP2 might be an immunoprotective factor in BU pathologic process.

Parasitological, Serological and Molecular Prevalence of Trypanosoma evansi among Arabian Camels (Camelus dromedaries) with Evaluation of Antitrypanosomal Drugs.

This study was carried out to assess the prevalence of Trypanosoma evansi infection in naturally diseased Dromedary camels in Dammam, Eastern region of Saudi Arabia. The detection of Trypanosoma evansi was performed using the parasitological, serological, and molecular diagnosis and a comparison between such methods were analyzed. In addition, evaluation of therapeutic efficacy of selected antitrypanosomal drugs, cymelarsan and quinapyrmine (aquin-1.5), was trialed for treatment of diagnosed infected cases. A total 350 randomly selected camels were evaluated using thin blood smear (TBS), RoTat1.2 PCR and CATT/T. evansi techniques. The total prevalence was 6.9%, 7.7%, and 32.8% by TBS, RoTat1.2 PCR and CATT/T. evansi techniques, respectively. Although PCR detect T. evansi in more samples than TBS, the agreement was good (K = 0.9). Among the CATT/T. evansi results, PCR detect T. evansi in 12 and 15 CATT positive and negative camels, respectively, with low agreement (Kappa = 0.1). The use of cymelarsan and quinapyramine sulfate in the treatment of naturally infected cases demonstrated a very efficient therapeutic response. It was found that 1. Comparing the CATT/T. evansi and PCR results, the positivity of CATT was higher than PCR detection, while the agreement was poor (K = 0.1). 2. Cymelarsan and aquin-1.5 proved to be effective in the treatment of naturally infected camels, but cymelarsan presented with higher effectiveness (100%) than aquin-treated camels (83.3%). a 3. The use of cymelarsan and CATT is recommended for disease treatment and control.

SNP IFNG rs1861494 gene polymorphism in pediatric patients with gastroenteritis and its relation to co-infectious with Adenovirus and Cryptosporidium

Introduction and Aim: The most common cause of infantile gastroenteritis is enteric adenoviruses, while Cryptosporidiosis is caused by Cryptosporidium. The function of IFN-gamma (IFNG/IFN-y) in immunological responses, inflammation, and autoimmune disease is increasingly recognized. This study aimed to examine intestinal co-infections of Cryptosporidium and adenovirus in pediatrics and the role of IFNG rs1861494 polymorphism to gastroenteritis among Iraqi children. Materials and Methods: This case control study included 75 pediatric patients with severe gastroenteritis, aged 3-120 months, with a mean age of 30.64 months. The apparently healthy control (AHC) in this study included 25 pediatric individuals with a mean age of 27.64 months. Total genomic DNA extracted from stool specimens were subjected to PCR detection of human adenovirus virus. SNP IFNG rs1861494 polymorphism was detected using the ARMS-PCR technique. Results: The percentage of males to female ratio in this study was 54:46. The positive PCR result of HADV7 was 28% (14 of 50 cases) while the positive result of Cryptosporidium was 6% (6 of 75 cases).Genotyping studies showed the GG genotype increased at rate OR=2.67 as compared to AG and AA genotypes for the SNP IFNG rs1861494. The SNP IFNG rs1861494 results in gastroenteritis were highly correlated with HADV7 and Cryptosporidium (respectively: r = 0.968, P = 0.007 as well as r = 0.984, P = 0.008). Conclusions: Adenovirus diarrhea in Iraqi pediatric patients is associated with SNP rs1861494 polymorphism and could possibly be used as a biomarker in assessing the susceptibility of Iraqi children to the virus.

Simultaneous PCR detection of Paenibacillus larvae targeting insertion sequence IS256 and Melissococcus plutonius targeting pMP1 plasmid from hive specimens

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)—American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I–IV (strain DSM 7030—ERIC I, DSM 25430—ERIC II, LMG 16252—ERIC III, DSM 3615—ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.

Macrophthalmus (Macrophthalmus) abbreviatus Manning & Holthuis, 1981, a new natural host for Hematodinium perezi infection.

Recent reports have shown that wild crabs may be important hosts involved in the transmission and spread of the parasitic Hematodinium in cultured marine crustaceans. Therefore, monitoring the prevalence of Hematodinium infections in wild crabs is necessary to develop effective strategies for the prevention and control of Hematodinium disease. Here we report a wild crab species, Macrophthalmus (Macrophthalmus) abbreviatus Manning & Holthuis, 1981, as a new natural host for Hematodinium sp. infection. It is one of the common wild crab species dwelling in the ponds or waterways connected to the polyculture ponds located on the coast of Rizhao or Weifang, Shandong Peninsula, China. According to the results of PCR detection and phylogenetic analysis targeting the internal transcribed spacer 1 (ITS 1) region, these Hematodinium sp. isolates were identified as H. perezi and fell into the genotype II category within H. perezi. A high monthly prevalence of H. perezi infection was observed during the 2021-2022 field survey, ranging from 33.3% to 90.6% in M. abbreviatus originating from Weifang (n=304 wild crabs) and from 53.6% to 92.9% in those from Rizhao (n=42 wild crabs). Artificial inoculation infection experiments demonstrated that M. abbreviatus could be infected by H. perezi, and massive Hematodinium cells and typical histopathological changes were observed in the hepatopancreas and gill tissues of the infected crabs. To our knowledge, this is the first report of M. abbreviatus as a new natural host for H. perezi infection. Results in the present study extend the known host spectrum for this emerging parasite pathogen, and also provide valuable information for epidemic surveillance of the Hematodinium disease as well.