Positive Samples Research Articles

Next step towards point-of-care molecular diagnosis of female genital schistosomiasis (FGS): evaluation of an instrument-free LAMP procedure

Detection of Schistosoma spp. DNA in gynaecological samples by quantitative real-time polymerase chain reaction (qPCR) is considered to be the reference diagnostic test for female genital schistosomiasis (FGS). However, qPCR needs expensive laboratory procedures and highly trained technicians. Loop-mediated amplification (LAMP) is a more field-friendly isothermal procedure for the detection of parasite-specific DNA, but it still requires electrically powered equipment. Here, we validated a Schistosoma haematobium-specific Sh-LAMP procedure and tested a fully instrument-free isothermal amplification using a novel low-cost, and reusable Temperature-cup (T-cup) device. Specific primers were selected based on published assays, targeting the ribosomal intergenic spacer (IGS) region of S. haematobium. Technical validation of the IGS-Sh-LAMP was performed using 20 negative controls, including DNA extracts of soil-transmitted helminths and S. mansoni, and a 10-fold dilution series (100–10−3) of DNA extracted from a single S. haematobium egg (n=4). For clinical validation, the IGS-Sh-LAMP was tested on 125 DNA samples extracted from vaginal swabs of a previous FGS study in Madagascar. Results were compared with the quantification cycle value (Cq) of the standard ITS-2 targeting qPCR. Single S. haematobium egg DNA up to a 10–2 dilution and an ITS-2 Cq <35 tested positive in the IGS-Sh-LAMP. The specificity was found to be excellent (100%). In the clinical samples, IGS-Sh-LAMP showed comparable results with the qPCR, with 35.2% and 33.6% positives, respectively, and a concordance of 79.2% (99/125). Of the 12 false-negatives, 5 corresponded to the 7 qPCR positive samples with very low DNA levels (Cq ≥35). On the other hand, IGS-Sh-LAMP detected 14 additional cases that were not detected by qPCR. The T-cup IGS-Sh-LAMP performance was evaluated in a representative sub-selection (n=10) of IGS-Sh-LAMP positive clinical samples. The T-cup IGS-Sh-LAMP was found to be a very user-friendly method, but in different runs, it missed 1 to 4 of the 10 IGS-Sh-LAMP positive samples, specifically those with a low DNA load. Our results show that the IGS-Sh-LAMP is a suitable alternative to the ITS-2 qPCR for the diagnosis of FGS in gynaecological samples, with high potential for the T-cup as a fully instrument-free isothermal amplification device for point-of-care diagnosis in low-resource settings.

Molecular Detection and Characterization of Hemotropic Mycoplasma in Assamese Macaques (Macaca assamensis) of Northern Thailand

Hemotropic mycoplasmas, also known as hemoplasmas, are parasitic bacteria that infect red blood cells, potentially leading to varying degrees of anemia across numerous mammalian species, including nonhuman primates. The present study aims to investigate the prevalence of hemoplasma infection and identify the species involved among free-ranging Assamese macaques (Macaca assamensis) inhabiting northern Thailand. A total of 133 blood samples were collected from Assamese macaques in Chiang Rai province, Thailand, and subjected to screening for hemoplasma infection utilizing nested PCR amplification targeting the 16S rRNA gene. Positive samples were subsequently analyzed through nucleotide sequencing and phylogenetic analysis for putative species identification. Current study results revealed that 17.3% (23/133; 95% CI 11.29-24.81) of Assamese macaques tested positive for hemoplasma infection using the nested PCR assay. Partial 16S rRNA sequences derived from hemoplasma isolates in Assamese macaques exhibited 99% homology, forming a cluster within the same phylogenetic clade as “Candidatus Mycoplasma haematomacacae,” previously identified in long-tailed macaques, rhesus macaques, and Japanese macaques. These findings suggest the presence of “Ca. M. haematomacacae” not only in long-tailed macaques and rhesus macaques but also in Assamese macaques in Thailand. To our knowledge, this marks the first molecular detection of “Ca. M. haematomacacae” in Assamese macaques in Thailand. These results hold significance as they enhance our understanding of hemoplasma infection distribution among macaque populations in Thailand.

Investigation of SARS-CoV-2 infection in domesticated animals from COVID-19 affected households in Bangladesh

The emergence and rapid global spread of the novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has sparked significant interest in understanding its zoonotic origins. This study aims to investigate animal and environmental samples in Bangladesh to identify the zoonotic source and reverse zoonosis of SARS-CoV-2. This research adopted a comprehensive approach to gather and analyze samples from various animal species, including cattle, sheep, goats, cats, and dogs that are peridomestic in nature or remain in close contact, ensuring biosafety and avoiding cross-contamination. SARS-CoV-2 or related coronaviruses were identified using immunological detection and real-time reverse transcription-polymerase chain reaction (RT-qPCR). A total of 1028 samples from different regions of Bangladesh were collected from cattle (552), sheep (135), goats (112), cats (118), and dogs (111), of which 465 were rectal, 363 nasal swabs, and 200 blood samples. The samples were collected from those areas where there were human records of COVID-19 during July 2021- December 2022. The blood samples were collected and immediately used for antibody screening. No positive samples were found in both antibody test screens and RT-qPCR in fecal and nasal swab samples. The study indicated no COVID-19 infection was found in domestic animals and pets despite staying in close contact with COVID-19 patients or households, suggesting a limited risk of transmission from these animals to humans within the studied context. It's possible that domestic animals are not significantly involved in the COVID-19 epidemic in natural environment exposure conditions.

Detection of small ruminant Lentivirus proviral DNA in red deer from Poland

Small ruminant lentiviruses (SRLVs) are widespread and infect goats and sheep. Several reports also suggest that SRLVs can infect wild ruminants. The presence of specific antibodies against SRLVs has been identified in wild ruminants from Poland, but no studies have been conducted to detect proviral DNA of SRLVs in these animals. Therefore, the purpose of this study was to examine samples from Polish wild ruminants to determine whether these animals can serve as reservoirs of SRLVs under natural conditions. A total of 314 samples were tested from red deer (n = 255), roe deer (n = 52) and fallow deer (n = 7) using nested real-time PCR. DNA from positive real-time PCR samples was subsequently used to amplify a CA fragment (625 bp) of the gag gene, a 1.2 kb fragment of the pol gene and an LTR-gag fragment. Three samples (0.95%) were positive according to nested real-time PCR using primers and probe specific for CAEV (SRLV group B). All the samples were negative for the primers and probe specific for MVV (SRLV A group). Only SRLV LTR-gag sequences were obtained from two red deer. Phylogenetic analysis revealed that these sequences were more closely related to CAEV than to MVV. Our results revealed that deer can carry SRLV proviral sequences and therefore may play a role in the epidemiology of SRLVs. To our knowledge, this is the first study describing SRLV sequences from red deer.

Reliability of the rapid antigen test for diagnosis of SARS-CoV-2 in Tunisia

Background: Early and accurate diagnosis is crucial for preventing the spread of SARS-CoV-2 infection. The rapid antigen test was developed for testing infection, and it was necessary to assess its performance before widespread use in Tunisia. Aim: To evaluate the effectiveness of a rapid antigen test for the detection of SARS-CoV-2 in nasopharyngeal swabs in Tunisia. Methods: Nasopharyngeal samples were taken from COVID-19 suspected cases between October and December 2020 and tested using the Standard Q COVID-19 Ag test (SD-Biosensor, Republic of Korea) and real-time reverse transcription polymerase chain reaction (RT-PCR). Results: Overall, 4539 patients were tested. Of the total study population (N = 4539), 82.5% of positive samples remained positive with the rapid antigen test, while 20.2% (470/2321) of samples that were negative with rapid antigen test were confirmed positive with RT-PCR, giving a negative predictive value of 79.8% for the rapid antigen test. The sensitivity and negative predictive value of the rapid antigen test were 70.2% and 65.8%, respectively. These results improved to 96.4% and 92.8%, respectively, when considering the cycle threshold value by RT-PCR below 25. Conclusion: Although the rapid antigen test was less sensitive than RT-PCR, its ability to rapidly detect individuals with high viral loads makes it suitable for use during an epidemic.

Development of a Bead-Based Multiplex Fluorescent Immunoassay to Detect Antibodies against Maedi-Visna Virus in Sheep

The Maedi-visna virus (MVV) causes a persistent infection in small ruminants, and its high genetic heterogeneity affects the performance of diagnostic tests when used in different populations. Therefore, the aim of this study was to develop a bead-based multiplex immunoassay tailored to detect antibodies against a Norwegian MVV strain. We used tissue samples from 14 PCR-positive sheep from a recent MVV outbreak in Norway to sequence the viral strain and produced recombinant antigens based on sequences from one animal. The assay included commercial TM-A and recombinant Norwegian p25, p16–25 and SU5 antigens. Cut-off values for each antigen were determined using receiver operating characteristic curves on 40 ELISA-negative and 67 ELISA-positive samples from the outbreak. The intraplate and interplate repeatability were investigated by testing a quadruplicate of five samples over three days, while the analytical sensitivity (aSe) and specificity (aSp) were measured in comparison to a commercial ELISA. The repeatability showed a coefficient of variation below 15% for most positive samples. The aSe was equal or higher for the multiplex assay than the ELISA, and the aSp of each antigen was 91.7, 93.3, 95.0 and 93.3% for p25, p16–25, SU5 and TM-A, respectively. The assay shows promising results; however, further evaluations of diagnostic characteristics are necessary before implementation in the Norwegian surveillance programme.

Improving Targeted Mass Spectrometry Data Analysis with Nested Active Machine Learning

Targeted mass spectrometry (MS) holds promise for precise protein and protein‐representative peptide identification and quantification, enhancing disease diagnosis. However, its clinical application is hindered by complex data analysis and expert review requirements. It is hypothesized that machine learning (ML) models can automate data analysis to accelerate the clinical application of MS. The approach involves an ML‐driven pipeline that extracts statistical and morphological features from an MS target region and feeds these features into ML algorithms to generate and assess predictive models. The findings demonstrate ML prediction models exhibit superior performance when trained on extracted features versus raw spectra intensity data and that random forest models exhibit robust classification performance in both internal and external validation datasets. These models remain effective across varying training dataset sizes and positive sample rates and are enhanced by a nested active learning approach. This approach can thus revolutionize clinical MS applications.

Occurrence and distribution of Meloidogyne spp. in fields rotated with sweetpotato and host range of a North Carolina population of Meloidogyne enterolobii.

Root-knot nematodes (RKN, Meloidogyne spp.) are some of the most economically important and common plant parasitic nematodes in North Carolina (NC) cropping systems. Soil samples collected from fields planted with crops rotated with sweetpotato [Ipomoea batatas (L.) Lam.] in 39 NC counties in 2015-2018 were processed at the NC Nematode Assay Laboratory. The occurrence of second-stage juvenile (J2) RKN populations was examined based on collection year, month, county, and previous planted crop. The highest number of RKN positive samples originated from Cumberland (53%), Sampson (48%), and Johnston (48%) counties. The highest average RKN population density was detected in Sampson (147 J2/500 cm3 soil) and Nash (135 J2/500 cm3 soil) counties, while Wayne (7 J2/500 cm3 soil) and Greene (11 J2/500 cm3 soil) counties had the lowest average RKN population density. Meloidogyne enterolobii is a new invasive species that is impacting sweetpotato growers of NC. The host status of a NC population of M. enterolobii, the guava-root knot nematode, was determined by examining eggs per gram of fresh root (ER) and the final nematode egg population divided by the initial population egg count (reproductive factor, RF) in greenhouse experiments. This included eighteen vegetable, field, cover crops and weed species. The tomato 'Rutgers' was used as a susceptible control. Cabbage 'Stonehead', pepper 'Red bull', and watermelon 'Charleston gray' and 'Fascination' were hosts and had similar mean ER values to the positive control, ranging from 64 to 18,717. Among field crops, cotton, soybean 'P5018RX', and tobacco were hosts with ER values that ranged from 185 to 706. Members of the Poaceae family such as sweet corn (Zea mays) and sudangrass (Sorghum x drummondii) were non-hosts to M. enterolobii and the mean ER values ranged from 1.85 to 7. The peanut 'Tifguard' and winter wheat (Triticum aestivum) also had lower ER values than the vegetable hosts. Growers should consider planting less susceptible or non-hosts such as peanut, sudangrass, sweet corn, and winter wheat in 2-3 year crop rotations to lower populations of this invasive nematode.

Molecular diagnosis and characterization of Anaplasma marginale and Ehrlichia ruminantium infecting beef cattle of Maputo Province, Mozambique

BackgroundMembers of the Anaplasmataceae family, such as the Anaplasma and Ehrlichia species, cause economic losses and public health risks. However, the exact economic impact has not been comprehensively assessed in Mozambique due to limited data available on its basic epidemiology. Therefore, we investigated the molecular occurrence and identity of Anaplasma and Ehrlichia spp. infecting beef cattle in Maputo province, Mozambique.MethodsA total of 200 whole blood samples were collected from apparently healthy beef cattle. Whole blood DNA was extracted and tested for presence of Anaplasma spp. and Ehrlichia ruminantium DNA through amplification of the 16S rRNA and map1 genes. Positive samples to Anaplasma spp. were subject to PCR assay targeting the A. marginale-msp5 gene. Amplicons obtained were purified, sequenced and subject to phylogenetic analyses.ResultsAnaplasma spp., A. marginale and E. ruminantium were detected in 153 (76.5%), 142 (71%) and 19 (9.5%) of all the samples analyzed, respectively. On this same sample group, 19 (9.5%) were co-infected with A. marginale and E. ruminantium. The 16S rRNA sequences of Anaplasma spp. obtained were phylogenetically related to A. marginale, A. centrale and A. platys. Phylogenetic analysis revealed that A. marginale-msp5 nucleotide sequences were grouped with sequences from Asia, Africa and Latin America, whereas E. ruminantium-map1 DNA nucleotide sequences were positioned in multiple clusters.ConclusionCattle in Maputo Province are reservoirs for multiple Anaplasma species. A high positivity rate of infection by A. marginale was observed, as well as high genetic diversity of E. ruminantium. Furthermore, five new genotypes of E. ruminantium-map1 were identified.

The prevalence of human papillomavirus in ocular surface squamous neoplasia in HIV positive and negative patients in a South African population.

To assess the prevalence and subtypes of Human Papillomavirus (HPV) in Ocular Surface Squamous Neoplasia (OSSN) in Human Immunodeficiency Virus (HIV) positive and negative patients in South Africa. This study was a single center retrospective cross-sectional study, conducted at Tygerberg Hospital, Western Cape, South Africa. We assessed 63 histopathologically confirmed OSSN formalin-fixed paraffin-embedded (FFPE) tissue blocks from 2015-2023. The presence of HPV was determined using the Hybrispot Direct Flow Chip Kit. Corresponding clinical data was retrieved from the National Health Laboratory Service (NHLS) central data warehouse. Of the confirmed OSSN samples, 66.7% tested positive for HPV (95% confidence interval [CI] 54-77.3%). Of the 42 HPV positive samples, 38 (90.5%) had one or more known genotypes detected and 4 had unknown genotypes. The most prevalent subtypes were HPV 11, 16 and 18 (found in 61.9%, 52.4% and 33.3% of HPV positive samples respectively). 88.9% of the lesions biopsied were from HIV positive patients, of whom 56.4% had a CD4 + count of < 200 cells/µL. A lower median CD4 + count was detected among HIV positive patients with invasive squamous cell carcinoma compared to those with moderate dysplasia (p < 0.0198). There is a high prevalence of HPV in OSSN in South Africa. Certain subtypes namely, 11, 16, 18, 31, 33 and 35 may be more carcinogenic. HIV with HPV co-infection may be linked as a causative factor in the development of OSSN.

Clinicopathological and Molecular Analysis of Aqueous Humor for the Diagnosis of Feline Infectious Peritonitis

Background: This study was designed to assess the diagnostic utility for FIP of cytology, protein measurement and RT-PCR for feline coronaviruses (FCoV) on aqueous humor (AH), since little information is currently available. Methods: AH samples (n = 85) were collected post-mortem from 13 cats with effusive FIP (E-FIP), 15 with non-effusive FIP (NE-FIP) and 16 without FIP, to perform cytology (n = 83) and RT-PCR (n = 66) and to calculate their sensitivity, specificity and positive and negative likelihood ratios (LR+ and LR−). The protein concentration was measured on 80 fluids. Results: The proportion of RT-PCR positive samples did not differ among groups, while positive cytology was more frequent in samples with FIP (p = 0.042) or positive RT-PCR (p = 0.007). Compared with other groups, the protein concentration was higher in samples with NE-FIP (p = 0.017), positive RT-PCR (p = 0.005) or positive cytology (p &lt; 0.001). The specificity of cytology together with RT-PCR, cytology alone, RT-PCR alone and cytological proteinaceous background were 90.0%, 84.6%, 70.0%, 61.5%, and the LRs 3.48, 2.65, 1.83, 1.64, respectively. However, their sensitivities were low (34.8–63.0%) and their LR− high (0.60–0.72). Conclusions: Based on the LR+, cytology and/or RT-PCR may support the diagnosis when the pre-test probability of FIP is high. The concentration of intraocular protein is a promising marker, especially in NE-FIP.

Molecular markers of artemisinin resistance during falciparum malaria elimination in Eastern Myanmar

BackgroundArtemisinin resistance in Plasmodium falciparum threatens global malaria elimination efforts. To contain and then eliminate artemisinin resistance in Eastern Myanmar a network of community-based malaria posts was instituted and targeted mass drug administration (MDA) with dihydroartemisinin-piperaquine (three rounds at monthly intervals) was conducted. The prevalence of artemisinin resistance during the elimination campaign (2013–2019) was characterized.MethodsThroughout the six-year campaign Plasmodium falciparum positive blood samples from symptomatic patients and from cross-sectional surveys were genotyped for mutations in kelch-13—a molecular marker of artemisinin resistance.ResultThe program resulted in near elimination of falciparum malaria. Of 5162 P. falciparum positive blood samples genotyped, 3281 (63.6%) had K13 mutations. The prevalence of K13 mutations was 73.9% in 2013 and 64.4% in 2019. Overall, there was a small but significant decline in the proportion of K13 mutants (p < 0.001). In the MDA villages there was no significant change in the K13 proportions before and after MDA. The distribution of different K13 mutations changed substantially; F446I and P441L mutations increased in both MDA and non-MDA villages, while most other K13 mutations decreased. The proportion of C580Y mutations fell from 9.2% (43/467) before MDA to 2.3% (19/813) after MDA (p < 0.001). Similar changes occurred in the 487 villages where MDA was not conducted.ConclusionThe malaria elimination program in Kayin state, eastern Myanmar, led to a substantial reduction in falciparum malaria. Despite the intense use of artemisinin-based combination therapies, both in treatment and MDA, this did not select for artemisinin resistance.

Pigs in southern Italy are exposed to three ruminant pathogens: an analysis of seroprevalence and risk factors analysis study

BackgroundPigs are susceptible to several ruminant pathogens, including Coxiella burnetti, Schmallenberg virus (SBV) and bovine viral diarrhea virus (BVDV). These pathogens have already been described in the pig population, although the dynamics of the infection and the impact on pig farms are currently unclear. The aim of this work was to evaluate the presence of these infections in the pig population of the Campania region, southern Italy, and to evaluate the risk factors associated with a greater risk of exposure.ResultsA total of 414 serum samples belonging to 32 herds were tested for the presence of antibodies against SBV, Coxiella, and BVD using commercial multispecies ELISA kits. SBV (5.3%) was the most prevalent pathogen, followed by Coxiella (4.1%) and BVD (3%). The risk factors included in the study (age, sex, province, farming system, ruminant density and major ruminant species) had no influence on the probability of being exposed to BVD and Coxiella, except for the location, in fact more pigs seropositive to Coxiella were found in the province of Caserta. However, the univariate analysis highlighted the influence of age, location, and sex on exposure to SBV. The subsequent multivariate analysis statistically confirmed the importance of these factors. The presence of neutralizing antibodies for SBV and BVDV, or antibodies directed towards a specific phase of infection for Coxiella was further confirmed with virus-neutralization assays and phase-specific ELISAs in a large proportion of positive samples. The presence of high neutralizing antibody titers (especially for SBV) could indicate recent exposures. Twelve of the 17 positive samples tested positive for antibodies against Coxiella phase I or II antigens, indicating the presence of both acute and chronic infections (one animal tested positive for both phases antibodies).ConclusionsOur study indicates a non-negligible exposure of pigs from southern Italy to the above pathogens. Further studies are necessary to fully understand the dynamics of these infections in pigs, the impact on productivity, and the public health consequences in the case of Coxiella.

Development and evaluation of time-resolved rapid immunofluorescence test for detection of TSOL18 specific antibody in porcine cysticercosis infections

BackgroundPorcine cysticercosis, a serious zoonotic parasitic disease, is caused by the larvae of Taenia solium and has been acknowledged by the World Organization for Animal Health. The current detection methods of Cysticercus cellulosae cannot meet the needs of large-scale and rapid detection in the field. We hypothesized that the immunofluorescence chromatography test strip (ICS) for detecting Cysticercus cellulosae, according to optimization of a series of reaction systems was conducted, and sensitivity, specificity, and stability testing, and was finally compared with ELISA. This method utilizes Eu3+-labeled time-resolved fluorescent microspheres (TRFM) coupled with TSOL18 antigen to detect TSOL18 antibodies in infected pig sera.ResultsICS and autopsy have highly consistent diagnostic results (n = 133), as determined by Cohen’s κ analysis (κ = 0.925). And the results showed that the proposed ICS are high sensitivity (0.9459) with specificity (0.9792). The ICS was unable to detect positive samples of other parasites. It can be stored for at least six months at 4℃.ConclusionsIn summary, we established a TRFM-ICS method with higher sensitivity and specificity than indirect ELISA. Results obtained from serum samples can be read within 10 min, indicating a rapid, user-friendly test suitable for large-scale field detection.

Epidemic Features and Megagenomic Analysis of Childhood Mycoplasma Pneumoniae post COVID-19 Pandemic: A 6-year Study in Southern China

ABSTRACT With the atypical rise of Mycoplasma pneumoniae infection (MPI) in 2023, prompt studies are needed to determine the current epidemic features and risk factors with emerging trends of MPI to furnish a framework for subsequent investigations. This multicentre, retrospective study was designed to analyse the epidemic patterns of MPI before and after the COVID-19 pandemic, as well as genotypes and the macrolide resistance-associated mutations in MP sampled from pediatric patients in Southern China. Clinical data was collected from 133674 patients admitted into investigational hospitals from June 1, 2017, to November 30, 2023. Metagenomic next-generation sequencing (mNGS) data were retrieved based on MP sequence positive samples from 299 pediatric patients for macrolide resistance-associated mutations analysis. Pearson’s chi-squared test was used to compare categorical variables between different time frames. The monthly average cases of pediatric common respiratory infection diseases were increased without enhanced public health measures after the pandemic, especially for influenza, respiratory syncytial virus infection, and MPI. The contribution of MPI to pneumoniae was similar to that in the outbreak in 2019. Compared mNGS data between 2019-2022 and 2023, the severity of MP did not grow stronger despite higher rates of macrolide-resistance hypervariable sites, including loci 2063 and 2064, were detected in childhood MP samples of 2023. Our findings indicated ongoing surveillance is necessary to understand the impact of post pandemic on MP transmission disruption on epidemic season and severity of clinical outcomes in different scenarios.

CogCol: Code Graph-Based Contrastive Learning Model for Code Summarization

Summarizing source code by natural language aims to help developers better understand existing code, making software development more efficient. Since source code is highly structured, recent research uses code structure information like Abstract Semantic Tree (AST) to enhance the structure understanding rather than a normal translation task. However, AST can only represent the syntactic relationship of code snippets, which can not reflect high-level relationships like control and data dependency in the program dependency graph. Moreover, researchers treat the AST as the unique structure information of one code snippet corresponding to one summarization. It will be easily affected by simple perturbations as it lacks the understanding of code with similar structure. To handle the above problems, we build CogCol, a Code graph-based Contrastive learning model. CogCol is a Transformer-based model that converts code graphs into unique sequences to enhance the model’s structure learning. In detail, CogCol uses supervised contrastive learning by building several kinds of code graphs as positive samples to enhance the structural representation of code snippets and generalizability. Moreover, experiments on the widely used open-source dataset show that CogCol can significantly improve the state-of-the-art code summarization models under Meteor, BLEU, and ROUGE.

Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1

BackgroundInfectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain’s gC, TK, gG, gD, and gE genes.ResultsThe study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/μL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28.ConclusionsThe established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

First molecular detection of Borrelia sp. in tapirs (Tapirus terrestris).

Borrelia theileri is a tick-borne spirochete causative agent of fever, apathy and reduced food consumption in cattle. Molecular diagnosis has expanded the understanding of Borrelia theileri with new hosts and geographical locations being described. The present study aimed to describe the first molecular detection of B. theileri in wild tapirs (Tapirus terrestris) from South America. Blood DNA samples obtained from 99 tapirs sampled in Pantanal (n = 61) and Cerrado (n = 38) biomes were screened using a qPCR assay based on the 16S rRNA gene of Borrelia sp. Positive samples in the qPCR assay were subjected to PCR assays to allow characterization of fragments from 16S rRNA and flaB genes. Two (2/99; 2.0%) animals from Pantanal biome were positive in the qPCR and one sample presented bands of expected size for the flaB protocol. Amplicons from this sample were successfully cloned and sequenced. In the phylogenetic analysis, Borrelia sp. from T. terrestris grouped together with B. theileri sequences previously detected in Rhipicephalus microplus ticks and cattle from Minas Gerais State in Brazil, Rhipicephalus geigyi from Mali, and R. microplus and Haemaphysalis sulcata from Pakistan. This finding contributes to our knowledge regarding susceptible hosts species for B. theileri. More studies are necessary to understand the potential effects of B. theileri on tapir's health.

One-year monitoring of potentially pathogenic microorganisms in the waters and sediments of the Lesina and Varano lagoons (South-Est Italy)

In this study, two Mediterranean coastal lagoons (Lesina and Varano) of Southern Italy, located in the north of the Apulia region, were investigated for the presence of Shiga toxin Escherichia coli (STEC) and potentially enteropathogenic Vibrio species in parallel with norovirus (NoV), hepatitis A virus (HAV), hepatitis E virus (HEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to evaluate the presence of potentially pathogenic bacteria and viruses in the water and sediments of these ecosystems. From March 2022 to February 2023, a total of 98 samples were collected: 49 water samples and 49 sediment samples. STEC strains were isolated in three samples (3.1%), of which one (2%) was water (stx1 and stx2 positive) and two (4.1%) were sediment (both stx2 positive) samples. Vibrio spp. were detected in twenty samples (20.4%), of which nine were water (18.4%) and eleven were sediment (22.4%) samples. The species detected included V. parahaemolyticus, V. cholerae, and V. vulnificus. NoV was detected in 25 (25.5%) samples, while none of the water or sediment samples were positive for HAV, HEV, and SARS-CoV-2. The results of this study provide an overview of the presence of potentially pathogenic microorganisms in areas influenced by anthropogenic pressure. Monitoring the circulation of these pathogens could be useful to evaluate the water flowing into the lagoons, in particular discharge waters (i.e., urban, agricultural, and livestock runoff), considering the presence of fish and shellfish farms in these sites.

Incidence of Cryptosporidium: A Neglected Parasite in Immunocompetent Adults in Swabi

Objective: To highlight the increasing incidence of Cryptosporidium in immunocompetent adults and the resultant altered epidemiology. Methodology: This study was conducted over a period of one year, from 1st January 2023 to 1st of January, 2024 at Microbiology Laboratory MTI- GKMC/BKMC, Swabi. Modified Ziehl Neelsen staining was performed on the received stool samples from patients presenting with wide range of symptoms associated with Irritable Bowel Syndrome (IBS). Abdominal pain, recurrent episodes of diarrhea and weight loss were the main symptoms. Modified ZN stain being one of the most reliable, quick and cost effective methods for the detection of cysts of Cryptosporidium using 40X and oil immersion lens was used for diagnosis. Results: A total of 62 samples were received from immunocompetent patients in the said period out of which 34% were positive for the oocysts of Cryptosporidium species. Out of a total of 21 positive samples 62% were males and 38% were females. The age range of the patients spanned between 20 to 60 years. Conclusion: Knowing this high incidence 34% of Cryptosporidium spp. in the healthy adults, necessitates to prioritize steps for the prevention of this infection by implementation of public health measures.