Molecular Epidemiological Investigation Research Articles

P-885. Clinical Characteristics and Molecular Epidemiology of Invasive Nontypeable Haemophilus influenzae Disease at a Tertiary Medical Center in Washington, DC

Abstract Background The incidence of invasive nontypeable H. influenzae (NTHi) is on the rise, and NTHi now causes the majority of invasive H. influenzae infections across all age groups. While these presentations are usually among people at the extremes of age, we observed a recent increase in invasive NTHi cases in adults at our institution compared to previous years. Here, we report their clinical presentation and the subsequent molecular epidemiology investigation. Characteristics of Patients With Invasive Nontypeable Haemophilus influenzae (NTHi) Infection, GW Hospital, December 2022-August 2023 Abbreviations: HIV, human immunodeficiency virus; MSM, men who have sex with men; MSK, musculoskeletal; LOS, length of hospital stay; N/A, information not available; HBV, hepatitis B virus; C1, clonal cluster 1; C2 clonal cluster 2; NC, non-cluster * in cells/mm3 Methods We describe the demographics, clinical characteristics, and outcomes of individuals hospitalized at our institution with invasive NTHi disease between December 2022 and August 2023. Invasive NTHi disease was defined as infections beyond the respiratory tract or skin. Data was obtained from retrospective chart abstraction. Available bacterial isolates were sent to the Georgia Emerging Infections Program laboratory for polymerase chain reaction (PCR) and whole genome sequencing (WGS), and were categorized as belonging to clusters 1 or 2 (C1, C2) or as non-cluster, as described previously. Results Nine cases [median age 55 (32-76) years, 100% male, 89% Black] were identified. Five individuals were living with HIV (PWH) on antiretroviral therapy, with median CD4 count 348 (range 32-984) cells/mm3, and all except one were virologically suppressed. Six patients identified as men who have sex with men (MSM). Five patients presented with musculoskeletal manifestations. Seven isolates were available for PCR/WGS: one isolate belonged to C1, three to C2, and three as non-cluster. The mean hospital stay was 11 (range 3-28) days. Three patients required admission to the intensive care unit, all of which died. Conclusion This case series identified nine patients within a nine-month span presenting with invasive NTHi disease, an abrupt rise from our local historical data. We noted an overrepresentation of PWH and MSM, with over half of isolates belonging to two previously identified clonal clusters associated with similar demographics. Future research is needed to better understand novel transmission routes, clinical characteristics, genotypic relatedness, and geographic spread of invasive NTHi disease. Disclosures Rebecca Yee, Ph.D, Biomerieux: Grant/Research Support

High Prevalence of Transferable Integron-Associated Drug Resistance in Escherichia coli Strains Isolated from Blood Cultures in a University Hospital in xxxxxxxx, xxxxxxxx

Objective: In this study, we aimed to determine the the presence of transferable integron-associated drug resistance in Escherichia coli strains isolated from the blood cultures of patients with various infections at the Research and Application Center (………Hospital) of …………….., …………….., ……………... Methods: The presence of integrons was determined by integron-specific polymerase chain reactions, and the amplicons were analyzed by DNA sequencing. Plasmid transfer assays were carried out by the broth mating method. The clonal relationship of integron-containing strains was evaluated by pulsed-field gel electrophoresis (PFGE). Results: Resistance rates to the antibiotic groups used were between 0.9% to 63%. Thirty-eight (34,2%) strains contained class 1 integrons carrying dfrA7, dfr17-aadA5, dfrV, dfrA1-aadA1, and dfrA12-aadA2 gene cassette arrays. Seven (6,3%) strains contained class 2 integrons having dfrA1-sat2-aadA1 and dfrA1-sat2-aadA30 gene cassette arrays. Twenty-two conjugative resistance plasmids were harbored in integron-carrying isolates, and three of them were found to be in the IncN group. Four strains carrying class 1 integrons which were isolated from different clinics showed clonally similar patterns in PFGE analysis. Conclusion: We conclude that about 50% E. coli isolates in our hospital carry integron-associated transferable drug resistance, and may play a crucial role in horizontal dissemination. Further molecular epidemiological investigations are required in regard to explaining the relationship between the carriage of integrons in E. coli strains of blood origin in this hospital as well as countrywide.

Molecular Characterization and Pathogenicity Analysis of Porcine Rotavirus A.

Porcine rotavirus A (RVA) is one of the major etiological agents of diarrhea in piglets and constitutes a significant threat to the swine industry. A molecular epidemiological investigation was conducted on 2422 diarrhea samples from Chinese pig farms to enhance our understanding of the molecular epidemiology and evolutionary diversity of RVA. The findings revealed an average RVA positivity rate of 42% (943/2422), and the study included data from 26 provinces, primarily in the eastern, southern and southwestern regions. Genetic evolutionary analysis revealed that G9 was the predominant genotype among the G-type genotypes, accounting for 25.32% of the total. The VP4 genotypes were P[7] (36.49%) and P[23] (36.49%). The predominant genotypic combinations of RVA were G9P[23] and G9P[7]. Eleven RVA strains were obtained via MA104 cell isolation. A rat model was established to assess the pathogenicity of these strains, with three strains exhibiting high pathogenicity in the model. Specifically, the RVA Porcine CHN HUBEI 2022 (Q-1), RVA Porcine CHN SHANXI 2022 (3.14-E), and RVA Porcine CHN HUBEI 2022 (5.11-U) strains were shown to cause diarrhea in the rats and damage the intestinal villi during the proliferation phase of the infection, leading to characteristic lesions in the small intestine. These data indicate that continuous monitoring of RVA can provide essential data for the prevention and control of this virus.

Revealing caprine schistosomiasis and its One Health importance in Malawi: A molecular epidemiological investigation augmented with a praziquantel treatment and GPS animal tracking pilot sub-study

To shed first light on caprine schistosomiasis and its zoonotic potential in Malawi, we conducted a molecular epidemiological investigation, sampling goats (n = 230) across three districts, using faecal miracidia hatching test. Molecular genotyping of miracidia later revealed the prevalence of Schistosoma mattheei to be 0.0 % in Nsanje District (n = 30), 16.7 % in Chikwawa District (n = 30) and 25.3 % in Mangochi District (n = 170). Notably, a miracidium of Schistosoma haematobium was observed from a single goat in Chikwawa. Inspection of carcasses (n = 51) at two local abattoirs in Mangochi District did not find any evidence of caprine schistosomiasis where only a single herd, at Mangochi 3, was infected. Here, despite sampling several other herds nearby, the prevalence was 87.7 % (n = 49), with an animal found excreting 1000 miracidia per 5 g of faeces. At this location, our praziquantel treatment (n = 14) and GPS animal tracking (n = 2) pilot sub-study compared two local goat herds over a three-month period. The daily foraging ranges across a 10 km2 area were recorded, alongside targeted schistosome surveillance within local freshwater intermediate snail hosts. Analysis of GPS data revealed only one herd (infected) to have regular daily water contact with Lake Malawi whereas the other herd (not infected) totally avoided the lake. One week after praziquantel treatment administered at 40 mg/kg, anthelminthic cure rate was 92.3 % while at three months approximately a third of treated animals were shedding schistosome miracidia. Cercariae from several field-caught snails locally were genotyped, inclusive of finding a Schistosoma haematobium-mattheei hybrid. Our findings reveal the focalized nature of caprine schistosomiasis, signposting a novel alert for S. haematobium transmission, and highlight where zoonotic transmission can be intense. To better address zoonotic spill-over from S. mattheei (and/or S. haematobium), the national control programme for schistosomiasis should formally develop targeted surveillance of caprine schistosomiasis and where appropriate, attempt an integrated One Health intervention in future.

Genomic and phenotypic landscapes of X-linked hereditary hearing loss in the Chinese population

BackgroundHearing loss (HL) is the most common sensory birth deficit worldwide, with causative variants in more than 150 genes. However, the etiological contribution and clinical manifestations of X-linked inheritance in HL remain unclear within the Chinese HL population. In this study, we focused on X-linked hereditary HL and aimed to assess its contribution to hereditary HL and identify the genotype–phenotype relationship.MethodsWe performed a molecular epidemiological investigation of X-linked hereditary HL based on next-generation sequencing and third-generation sequencing in 3646 unrelated patients with HL. We also discussed the clinical features associated with X-linked non-syndromic HL-related genes based on a review of the literature.ResultsWe obtained a diagnostic rate of 52.72% (1922/3646) among our patients; the aggregate contribution of HL caused by genes on the X chromosome in this cohort was ~ 1.14% (22/1922), and POU3F4 variants caused ~ 59% (13/22) of these cases. We found that X-linked HL was congenital or began during childhood in all cases, with representative audiological profiles or typical cochlear malformations in certain genes. Genotypic and phenotypic analyses showed that causative variants in PRPS1 and AIFM1 were mainly of the missense type, suggesting that phenotypic variability was correlated with the different effects that the replaced residues exert on structure and function. Variations in SMPX causing truncation of the protein product were associated with DFNX4, which resulted in typical audiological profiles before and after the age of 10 years, whereas nontruncated proteins typically led to distal myopathy. No phenotypic differences were identified in patients carrying POU3F4 or COL4A6 variants.ConclusionsOur work constitutes a preliminary evaluation of the molecular contribution of X-linked genes in heritable HL (~ 1.14%). The 15 novel variants reported here expand the mutational spectrum of these genes. Analysis of the genotype–phenotype relationship is valuable for X-linked HL precise diagnostics and genetic counseling. Elucidation of the pathogenic mechanisms and audiological profiles of HL can also guide choices regarding treatment modalities.

The effect of the nucleolar protein ZNF385A on the ribosomal DNA copy number variation in response to Cr(VI)-induced DNA damage

Hexavalent chromium [Cr(VI)] is a widely distributed carcinogen in industrial contexts and general environmental contexts. Emerging research highlights the central role of ribosomal DNA (rDNA) in DNA Damage Responses (DDRs). However, there remains a lack of investigation into the potential dose-dependent relationship between exposure to Cr(VI) and alterations in rDNA copy number (CN), as well as the related mechanisms underlying these effects. A molecular epidemiological investigation involving 67 workers exposed to Cr(VI) and 75 unexposed controls was conducted. There was a notable increase in ZNF385A expression, variations in rDNA CN, and elevated γH2AX levels in the peripheral blood of Cr(VI)-exposed workers. Restricted cubic spline (RCS) models showed that blood Cr levels in the exposed population exhibited non-linear dose-dependent relationships with γH2AX, rDNA CN, and ZNF385A. Of considerable interest, there were robust and positive associations between ZNF385A and both γH2AX and rDNA CN. Further in vitro experiments provided concrete evidence that Cr(VI) simultaneously caused an increase in ZNF385A expression and variations in rDNA CN. ZNF385A-depleted cells showed increased sensitivity to Cr(VI)-mediated DDRs and alterations in rDNA CN. This study indicated that ZNF385A played a highly significant role in the rDNA CN variation in response to Cr(VI)-induced DNA damage.

Molecular epidemiological investigation of Salmonella isolated from the environment, animals, foods and patients in China

Salmonella as a foodborne pathogen, has a very important impact on human health. This study characterized the serotype, plasmid replicon, MLST typing, resistance phenotype and resistance gene of 282 strains of Salmonella isolated from the environment, animals, foods, and patients in China. These strains are classified into four sources: poultry farms (39/282), animals (30/282), foods (164/282), and hospitals (49/282) their resistance to ciprofloxacin, levofloxacin and ceftazidime is gradually increasing. Among them, ST11, ST19 and ST314 are dominant ST types; Salmonella enteritidis, Salmonella typhimurium and Kentucky Salmonella enteritidis serotypes are the dominant serotypes. This study conducted a comprehensive molecular epidemiological investigation of Salmonella from the unique perspective of Onehealth. It was revealed that the mcr-1 carried by Salmonella mainly relies on the horizontal transfer of Inc I2 and Inc Q plasmids. And a novel integron variable region cassette arrangement pattern aadA2-aadA23-catB3-tet(B)-dfrA12-eptB has emerged in Salmonella strains. More noteworthy is the outbreak of blaTEM-148 subtype in Salmonella from food sources, in which the Inc HI plasmid played an important role.

DNA Sequencing Accurately Diagnosed 146 Cases of Superficial Mycosis

Objective: Traditional fungal detection methods, such as fungal microscopy and cultivation, often have drawbacks such as high false negative rates and time-consuming cultivation. Using molecular biology methods for diagnosis can not only be used for identifying fungal strains in cultured colonies, but also for diagnosing diseased tissues, which can shorten the diagnosis time. There is a lack of systematic research on the clinical characteristics, susceptibility factors, and the composition and distribution of pathogenic fungi of superficial mycosis in Shiyan area. In order to understand the relevant situation of superficial mycosis and pathogenic fungi in this area, this study conducted a molecular epidemiological investigation on 146 patients with superficial mycosis who visited our outpatient department. Methods: From January 2022 to December 2022, the typical clinical manifestations of outpatient visits in our department were collected. 146 cases of superficial fungal patients with positive fungal microscopy were cultured and DNA was extracted. PCR technology was applied to compare the products in GeneBank after the amplification of ITS region. Results: A total of 23 pathogenic strains were obtained, including 112 strains of <i>Trichophyton rubrum</i> (76.71%), 5 strains of other dermatophytes (3.42%), 6 strains of <i>Candida</i> species (4.11%), 4 strains of <i>Aspergillus</i> species (2.74%), 8 strains of <i>Cladosporium</i> species (5.48%), and 11 strains of other fungi (7.53%). Conclusions: DNA sequencing combined with traditional fungal microscopic culture is helpful for more accurate diagnosis of superficial mycosis.

Molecular epidemiological analysis of Salmonella Schwarzengrund isolated in Japan by newly developed multi-locus variable-number tandem repeat analysis method

To establish molecular epidemiological analysis method for Salmonella Schwarzengrund and investigate its origin and genetic relationships among isolates in Japan, S. Schwarzengrund strains were examined by the pulsed-field gel electrophoresis (PFGE), the multi-locus sequencing typing (MLST), and the multi-locus variable-number tandem repeat analysis (MLVA) developed in this study. By the PFGE and MLST methods, the strains were typed into two clades (A and B) and two sequence types (STs) (96 and 241), respectively. Minimum spanning tree showed that the strains belonging ST241 genetically close to ST96, suggesting that ST241 originates from ST96. For MLVA, new primers for variable-number tandem repeat (VNTR) loci were designed, and five VNTR loci finally selected. In the MLVA method, the strains were typed into 11 sequence types. The MLVA method was able to differentiate strains belonging to the same clade of PFGE and MLST methods. These results indicate that the MLVA developed in this study shows a higher discriminatory power for S. Schwarzengrund compared to the PFGE and MLST methods and that it appears to be a useful method for the molecular epidemiological investigation of S. Schwarzengrund despite the low genetic diversity of this serotype in Japan.

Molecular characterization and epidemiological investigation of colistin resistance in carbapenem-resistant Klebsiella pneumoniae in a tertiary care hospital in Tehran, Iran

BackgroundCarbapenemase-producing Klebsiella pneumoniae (CRKP) presents a significant challenge to antimicrobial therapy, especially when compounded by resistance to colistin. The objective of this study was to explore molecular epidemiological insights into strains of clinical K. pneumoniae that produce carbapenemases and exhibit resistance to colistin. Eighty clinical isolates of CRKP were obtained from Milad Hospital in Tehran, Iran. Antimicrobial susceptibility and colistin broth disk elution were determined. PCR assays were conducted to examine the prevalence of resistance-associated genes, including blaKPC, blaIMP, blaVIM, blaOXA−48, blaNDM and mcr-1 to -10. Molecular typing (PFGE) was used to assess their spread.ResultsColistin resistance was observed in 27 isolates (33.7%) using the Broth Disk Elution method. Among positive isolates for carbapenemase genes, the most frequent gene was blaOXA−48, identified in 36 strains (45%). The mcr-1 gene was detected in 3.7% of the obtained isolates, with none of the other of the other mcr genes detected in the studied isolates.ConclusionTo stop the spread of resistant K. pneumoniae and prevent the evolution of mcr genes, it is imperative to enhance surveillance, adhere rigorously to infection prevention protocols, and implement antibiotic stewardship practices.

EPS6.10 Molecular epidemiologic investigation of nontuberculous mycobacteria: identifying healthcare-associated transmission and acquisition

EPS6.10 Molecular epidemiologic investigation of nontuberculous mycobacteria: identifying healthcare-associated transmission and acquisition

A Duplex PCR Assay for Rapid Detection of Klebsiella pneumoniae and Chryseobacterium in Large Yellow Croaker Fish.

Both Klebsiella pneumoniae and Chryseobacterium cause an increasing number of diseases in fish, resulting in great economic losses in aquaculture. In addition, the disease infected with Klebsiella pneumoniae or Chryseobacterium exhibited the similar clinical symptoms in aquatic animals. However, there is no effective means for the simultaneous detection of co-infection and discrimination them for these two pathogens. Here, we developed a duplex polymerase chain reaction (PCR) method based on the outer membrane protein A (ompA) gene of Klebsiella pneumoniae and Chryseobacterium. The specificity and validity of the designed primers were confirmed experimentally using simplex PCR. The expected amplicons for Klebsiella pneumoniae and Chryseobacterium had a size of 663 and 1404 bp, respectively. The optimal condition for duplex PCR were determined to encompass a primer concentration of 0.5 μM and annealing temperature of 57°C. This method was analytical specific with no amplification being observed from the genomic DNA of Escherichia coli, Vibrio harveyi, Pseudomonas plecoglossicida, Aeromonas hydrophila and Acinetobacter johnsonii. The limit of detection was estimated to be 20 fg of genomic DNA for Chryseobacterium and 200 fg for Klebsiella pneumoniae, or 100 colony-forming units (CFU) of bacterial cells in both cases. The duplex PCR was capable of simultaneously amplifying target fragments from genomic DNA extracted from the bacteria and fish liver. For practical validation of the method, 20 diseased fish were collected from farms, among which 4 samples were PCR-positive for Klebsiella pneumoniae and Chryseobacterium. The duplex PCR method developed here is time-saving, specific, convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of Klebsiella pneumoniae and Chryseobacterium in the field of aquaculture.

Molecular epidemiological investigation and recombination analysis of Cachavirus prevalent in China.

Chaphamaparvovirus carnivoran1 (canine Chaphamaparvovirus, also known as Cachavirus [CachaV]) is a novel parvovirus first reported in dog feces collected from the United States in 2017 and China in 2019. To continuously track its infection and evolution status, 276 canine anal swabs were obtained from pet hospitals in central, northern, and eastern China between 2021 and 2023 and screened via polymerase chain reaction; subsequently, a systematic study was conducted. Of these samples, nine (3.3%) were positive for CachaV. Using polymerase chain reaction, whole genome sequences of the nine CachaV-positive strains were amplified. The NS1 amino acid sequence identity between CachaV strains from China and other countries was 96.23-99.85%, whereas the VP1 protein sequence identity was 95.83-100%. CHN230521 demonstrated the highest identity for NS1 amino acids (99.85%) and VP1 amino acids (100%) with NWT-W88 and CP-T015. According to the model prediction of CHN220916-VP1 protein, Met64Thr, Thr107Ala, and Phe131Ser mutations may cause tertiary structural changes in VP1 protein. Interestingly, each of the nine CachaV strains harbored the same site mutations in NS1 (Ser252Cys, Gly253Leu, and Gly254Thr). Although no explicit recombination events were predicted, the clustering and branching of the phylogenetic tree were complicated. Based on the evolution trees for VP1 and NS1, the nine CachaV strains identified from 2021 to 2023 were closely related to those identified in gray wolves and cats. This study may be beneficial for evaluating the prevalence of CachaVs in China, thereby understanding the evolution trend of CachaVs.

Development and application of a TaqMan-probe-based multiplex real-time PCR assay for simultaneous detection of porcine circovirus 2, 3, and 4 in Guangdong province of China.

Porcine circoviruses disease (PCVD), caused by porcine circovirus (PCVs), is an important swine disease characterized by porcine dermatitis, nephrotic syndrome and reproductive disorders in sows. However, diseases caused by PCV2, PCV3, or PCV4 are difficult to distinguish, so a simple, rapid, accurate and high-throughput diagnostic and identification method is urgently needed to differentiate these three types. In this study, specific primers and probes were designed based on the conserved region sequences of the Rep gene of PCV2, and the Cap gene of PCV3 and PCV4. A multiplex qPCR assay was developed and optimized that the limit of detection concentration could reach as low as 3.8 copies/μL, with all correlation coefficients (R2) exceeding 0.999. Furthermore, the method showed no cross-reaction with other crucial porcine viral pathogens, and both intra-repeatability and inter-reproducibility coefficients of variation were below 2%. The assay was applied to the detection of 738 pig samples collected from 2020 to 2021 in Guangdong Province, China. This revealed positive infection rates of 65.18% for PCV2, 29.27% for PCV3, and 0% for PCV4, with a PCV2/PCV3 co-infection rate of 23.17%. Subsequently, complete genome sequences of 17 PCV2 and 4 PCV3 strains were obtained from the above positive samples and pre-preserved positive circovirus samples. Nucleotide sequence analysis revealed that the 17 PCV2 strains shared 96.7-100% complete nucleotide identity, with 6 strains being PCV2b and 11 strains being PCV2d; the 4 PCV3 strains shared 98.9-99.4% complete nucleotide identity, with 2 strains being PCV3a-1 and 2 strains being PCV3b. This research provides a reliable tool for rapid PCVs identification and detection. Molecular epidemiological investigation of PCVs in pigs in Guangdong Province will help us to understand PCV2 and PCV3 epidemiological characteristics and evolutionary trends.

A molecular epidemiological investigation of contagious caprine pleuropneumonia in goats and captive Arabian sand gazelle (Gazella marica) in Oman

BackgroundContagious caprine pleuropneumonia (CCPP) is a fatal WOAH-listed, respiratory disease in small ruminants with goats as primary hosts that is caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp). Twelve CCPP outbreaks were investigated in 11 goat herds and a herd of captive Arabian sand gazelle (Gazella marica) in four Omani governorates by clinical pathological and molecular analysis to compare disease manifestation and Mccp genetic profiles in goats and wild ungulates.ResultsThe CCPP forms in diseased and necropsied goats varied from peracute (5.8%), acute (79.2%) and chronic (4.5%) while all of the five necropsied gazelles showed the acute form based on the clinical picture, gross and histopathological evaluation. Colonies of Mccp were recovered from cultured pleural fluid, but not from lung tissue samples of one gazelle and nine goats and all the isolates were confirmed by Mccp-specific real time PCR. Whole genome-single nucleotide polymorphism (SNP) analysis was performed on the ten isolates sequenced in this study and twenty sequences retrieved from the Genbank database. The Mccp strains from Oman clustered all in phylogroup A together with strains from East Africa and one strain from Qatar. A low variability of around 125 SNPs was seen in the investigated Omani isolates from both goats and gazelles indicating mutual transmission of the pathogen between wildlife and goats.ConclusionRecent outbreaks of CCPP in Northern Oman are caused by Mccp strains of the East African Phylogroup A which can infect goats and captive gazelles likewise. Therefore, wild and captive ungulates should be considered as reservoirs and included in CCPP surveillance measures.

Rapid molecular epidemiology investigations into two recent measles outbreaks in Israel detected from October 2023 to January 2024.

Between late 2023 and early 2024, two measles outbreaks occurred in Israel, each caused by importation of measles virus strains of respective B3 and D8 genotypes. In this study, we validate transmission pathways uncovered by epidemiological investigations using a rapid molecular approach, based on complete measles virus genomes. The presented findings support this rapid molecular approach in complementing conventional contact tracing and highlight its potential for informing public health interventions.

Complete Mitogenome sequencing of the fish louse Argulus japonicus (Crustacea: Branchiura): Comparative analyses and phylogenetic implications.

The fish louse Argulus japonicus, a branchiuran crustacean of the Argulidae family, is attracting increasing attention because of its parasitic tendencies and significant health threats to global fish farming. The mitogenomes can yield a foundation for studying epidemiology, genetic diversity, and molecular ecology and therefore may be used to assist in the surveillance and control of A. japonicus. In this study, we sequenced and assembled the complete mitogenome of A. japonicus to shed light on its genetic and evolutionary blueprint. Our investigation indicated that the 15,045-bp circular genome of A. japonicus encodes 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), and 2 ribosomal RNAs (rRNAs) with significant AT and GC skews. Comparative genomics provided an evolutionary scenario for the genetic diversity of 13 PCGs: all were under purifying selection, with cox1 and nad6 having the lowest and highest evolutionary rates, respectively. Genome-wide phylogenetic trees established a close relationship between species of the families Argulidae (Arguloida) and Armilliferidae (Porocephalida) within Crustacea, and further, A. japonicus and Argulus americanus were determined to be more closely related to each other than to others within the family Argulidae. Single PCG-based phylogenies supported nad1 and nad6 as the best genetic markers for evolutionary and phylogenetic studies for branchiuran crustaceans due to their similar phylogenetic topologies with those of genome-based phylogenetic analyses. To sum up, these comprehensive mitogenomic data of A. japonicus and related species refine valuable marker resources and should contribute to molecular diagnostic methods, epidemiological investigations, and ecological studies of the fish ectoparasites in Crustacea.

Epidemiological investigation of coccidiosis and associated risk factors in broiler chickens immunized with live anticoccidial vaccines in China.

Coccidiosis is a costly intestinal disease of chickens caused by Eimeria species. This infection is associated with high mortality, reduced feed efficiency, and slowed body weight gain. The diagnosis and control of coccidiosis becomes challenging due to the fact that chickens can be infected by seven different Eimeria species and often occur mixed-species co-infections. Grasping the epidemiology of Eimeria species is crucial to estimate the efficiency of poultry management. This study aimed to explore the distribution of Eimeria species in broiler chickens in China after administering live anticoccidial vaccines. A total of 634 samples were obtained, and the survey results showed that the prevalence of Eimeria was 86.12% (546/634), and the most common species were E. acervulina (65.62%), E. necatrix (50.95%), E. mitis (50.79%), E. tenella (48.42%), and E. praecox (41.80%). Most samples indicated mixed-species infections (an average of 3.29 species per positive sample). Notably, 63.98% of samples contain 3 to 5 Eimeria species within a single fecal sample. The most prevalent combinations were E. acervulina-E. tenella (38.96%) and E. acervulina-E. necatrix (37.22%). Statistical analysis showed that flocks vaccinated with trivalent vaccines were significantly positive for E. necatrix in grower chickens (OR = 3.30, p < 0.05) compared with starter chickens, and tetravalent vaccinated flocks showed that starter chickens demonstrated a higher susceptibility to E. tenella-E. brunetti (OR = 2.03, p < 0.05) and E. acervulina-E. maxima (OR = 2.05, p < 0.05) compared with adult chickens. Geographically, in the case of tetravalent vaccine-immunized flocks, a substantial positive association was observed between E. necatrix infection rates and flocks from eastern (OR = 3.88, p < 0.001), central (OR = 2.65, p = 0.001), and southern China (OR = 3.17, p < 0.001) compared with southwestern China. This study also found a positive association between E. necatrix (OR = 1.64, p < 0.05), E. acervulina (OR = 1.59, p < 0.05), and E. praecox (OR = 1.81, p < 0.05) infection and coccidiosis occurrence compared with non-infected flocks in tetravalent vaccinated flocks. This molecular epidemiological investigation showed a high prevalence of Eimeria species in the field. The emergent species, E. brunetti and E. praecox, might be incorporated into the widely-used live vaccines in the future. These insights could be useful in refining coccidiosis control strategies in the poultry industry.

Molecular epidemiological investigation of Cryptococcus spp. carried by captive koalas (Phascolarctos cinereus) in Japan.

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a systemic mycosis that infects a wide range of species. Recent molecular biological investigations have allowed for the genotyping of these species, providing more detailed information on their pathogenicity and infection routes. Koalas (Phascolarctos cinereus) are frequently colonized by Cryptococcus spp., but molecular epidemiological studies have yet to be conducted in Japan. Here, we conducted multi-locus sequence typing (MLST) analysis on Cryptococcus spp. colonization isolates obtained from all koalas kept in seven parks across Japan. Out of 46 koalas examined, 10 (22%) were positive for C. gattii and 3 (6.5%) were positive for C. neoformans. All C. gattii isolates belonged to molecular type VGI and were either sequence type (ST) 51 or a novel ST, and all C. neoformans isolates belonged to molecular type VNI and ST23. Despite the frequent movement of koalas between parks, the STs were relatively park-specific, suggesting that the floor of the rearing barns is a source of infection and may act as a reservoir. MLST analysis confirmed that C. gattii was transported, established, and spread by koalas in areas where C. gattii was not originally present. MLST analysis is considered useful in assessing the pathogenicity and tracing the transmission routes of Cryptococcus spp. carried by koalas.IMPORTANCEThis is the first study to conduct a multi-locus sequence typing analysis on Cryptococcus spp. carried by captive koalas in Japan. Cryptococcosis remains a globally high-fatality fungal infection in humans, and captive koalas are known to carry a high percentage of Cryptococcus spp. Through this research, the molecular types and transmission routes of Cryptococcus spp. carried by koalas have been elucidated, revealing the potential role of enclosure flooring as a reservoir. It has been confirmed that Cryptococcus gattii, which is not endemic in Japan, has become established through koalas and is spreading to new individuals in Japan. This study is believed to provide valuable insights into koala conservation and contribute to the One Health approach for Cryptococcosis, a zoonotic infection.

Serological detection of Newcastle disease virus in backyard poultry production system in Woliso District South West Shewa, Central Ethiopia.

Newcastle disease (ND)is one of the most important respiratory viral diseases. The disease is endemic in many parts of Ethiopia. However, there is no clear record about the introduction of the virus to the country (Ethiopia). Hence, detail about the ND is very important in its (ND) control and prevention. Despite these facts, there is no available research work done on ND in the current research area that would help either as references for researchers or that could help in the control and prevention of the disease. Therefore, the objective of this study was to detect the ND virus (NDV), using serological methods in from December 2018 to November 2019. A cross-sectional type of study was conducted to detect the NDV. The convenience sampling method was used for sample data collection. Before data collection, chicken with previous history of vaccination against the NDV was excluded from the sampling animals. Then, a total of 348 blood samples of 2 mLwere collected from the brachial vein in 3 mLdisposable syringes. The serum was collected in labeled 2 mL cryovial tubes. Indirect enzyme-linked immunosorbent assay(ELISA) tests were performed to detect antibodies against NDV and to determine its antibody titer. The test was performed using (ID.vetinnovative version 2) procedure. In the indirect ELISA test, 37.64% (131/348) were positive and antibody titer mean value of (1761.9088) was scored. The standard deviation of 2592.42160 and a percentage coefficient of variation of147% was scored. From the finding, we conclude that indirect ELISA test detected the presence of the NDV in the study area and the heterogeneousity of antibody titer in the study area. Therefore, further molecular characterization and epidemiological investigation should be carried and vaccination of animals is critical in the study area.