Species-specific Polymerase Chain Reaction Research Articles

First molecular survey of tick-borne protozoan and bacterial pathogens in the questing tick population in Bangladesh.

Questing ticks carry various tick-borne pathogens (TBPs) that are responsible for causing tick-borne diseases (TBDs) in humans and animals around the globe, especially in the tropics and sub-tropics. Information on the distribution of ticks and TBPs in a specific geography is crucial for the formulation of mitigation measures against TBDs. Therefore, this study aimed to survey the TBPs in the questing tick population in Bangladesh. A total of 2748 questing hard ticks were collected from the pastures in Sylhet, Bandarban, Sirajganj, Dhaka, and Mymensingh districts through the flagging method. After morphological identification, the ticks were grouped into 142 pools based on their species, sexes, life stages, and collection sites. The genomic DNA extracted from tick specimens was screened for 14 pathogens, namely Babesia bigemina (AMA-1), Babesia bovis (RAP-1), Babesia naoakii (AMA-1), Babesia ovis (18S rRNA), Theileria luwenshuni (18S rRNA), Theileria annulata (Tams-1), Theileria orientalis (MPSP), Anaplasma marginale (groEL), Anaplasma phagocytophilum (16S rRNA), Anaplasma bovis (16S rRNA), Anaplasma platys (16S rRNA), Ehrlichia spp. (16S rRNA), Rickettsia spp. (gltA), and Borrelia (Bo.) spp. (flagellin B) using genus and species-specific polymerase chain reaction (PCR) assays. The prevalence of the detected pathogens was calculated using the maximum likelihood method (MLE) with 95 % confidence interval (CI). Among 2748 ixodid ticks, 2332 (84.86 %) and 416 (15.14 %) were identified as Haemaphysalis bispinosa and Rhipicephalus microplus, respectively. Haemaphysalis bispinosa was found to carry all the seven detected pathogens, while larvae of R. microplus were found to carry only Bo. theileri. Among the TBPs, the highest detection rate was observed in A. bovis (20/142 pools, 0.81 %, CI: 0.51-1.20), followed by T. orientalis (19/142 pools, 0.72 %, CI: 0.44-1.09), T. luwenshuni (9/142 pools, 0.34 %, CI: 0.16-0.62), B. ovis (4/142 pools, 0.15 %, CI: 0.05 - 0.34) and Bo. theileri (4/142 pools, 0.15 %, CI: 0.05-0.34), Ehrlichia ewingii (3/142 pools, 0.11 %, CI: 0.03-0.29), and Babesia bigemina (1/142, 0.04 %, CI: 0.00 - 0.16). This study reports the existence of T. luwenshuni, E. ewingii, and Bo. theileri in Bangladesh for the first time. The novel findings of this study are the foremost documentation of transovarian transmission of B. bigemina and E. ewingii in H. bispinosa and also provide primary molecular evidence on the presence of E. ewingii and Bo. theileri in H. bispinosa. Therefore, this study may shed light on the circulating TBPs in ticks in the natural environment and thereby advocate awareness among physicians and veterinarians to control and prevent TBDs in Bangladesh.

Rapid molecular identification of Rana dybowskii by species-specific primers

Oviductus Ranae is the dried oviduct from Rana dybowskii, a forest frog species with medicinal, tonic, and cosmetic properties. Due to the high price and resource shortage, counterfeit varieties of Oviductus Ranae often appear in the market. However, traditional identification methods cannot accurately differentiate between Oviductus Ranae and its adulterants. In this study, a rapid molecular identification method has been established. The method involves extracting genomic DNA in just 30 s using filter paper purification, species-specific rapid polymerase chain reaction (PCR) amplification, and finally, fluorescence detection of the products. It can accurately identify Oviductus Ranae and its three common adulterants in about 30 min, making the process simple, fast, and highly specific.

Comparison of qPCR and metabarcoding for environmental DNA surveillance of a freshwater parasite.

Analysis of environmental DNA (eDNA) has been successfully used across freshwater ecological parasitology to inform management of ecologically and economically important species. However, most studies have used species-specific quantitative polymerase chain reaction (qPCR) assays to detect target taxa. While generally effective, this approach limits the amount of community and management-supporting data that can be obtained from eDNA samples. If eDNA metabarcoding could be conducted with the same accuracy of a single species approach, researchers could simultaneously detect a target species while obtaining vast community data from eDNA samples. We sampled 38 freshwater sites on Fort McCoy, Wisconsin and compared qPCR to metabarcoding for eDNA detection of the ectoparasitic gill louse Salmincola edwardsii, an obligate parasite of Salvelinus fishes (chars). We found no evidence to suggest S. edwardsii occupancy or detection probabilities differed between qPCR and metabarcoding. Further, we found that the number of S. edwardsii reads from metabarcoding were negatively predictive of C T values from qPCR (C T value indicates cycle a significant amount of target eDNA is detected, with lower C Ts indicative of more DNA), demonstrating that our metabarcoding reads positively predicted qPCR DNA quantities. However, the number of reads was not predictive of overall qPCR score (number of positive qPCR replicates). In addition to S. edwardsii, metabarcoding led to the detection of a vast community of over 2600 invertebrate taxa. We underscore the necessity for conducting similar analyses across environments and target species, as the ecology of eDNA will vary on a per-study basis. Our results suggest that eDNA metabarcoding provides a highly sensitive and accurate method for detecting parasitic gill lice while also illuminating the broader biological community and co-occurrence of species in the environment.

Transposon insertion in Rothia dentocariosa

ObjectivesRothia spp. are emerging as significant bacteria associated with oral health, with Rothia dentocariosa being one of the most prevalent species. However, there is a lack of studies examining these properties at the genetic level. This study aimed to establish a genetic modification platform for R. dentocariosa. MethodsRothia spp. were isolated from saliva samples collected from healthy volunteers. Subsequently, R. dentocariosa strains were identified through colony morphology, species-specific polymerase chain reaction (PCR), and 16S ribosomal RNA gene sequencing. The identified strains were then transformed with plasmid pJRD215, and the most efficient strain was selected. Transposon insertion mutagenesis was performed to investigate the possibility of genetic modifications. ResultsA strain demonstrating high transforming ability, designated as R. dentocariosa LX16, was identified. This strain underwent transposon insertion mutagenesis and was screened for 5-fluoroorotic acid-resistant transposants. The insertion sites were confirmed using arbitrary primed PCR, gene-specific PCR, and Sanger sequencing. ConclusionThis study marks the first successful genetic modification of R. dentocariosa. Investigating R. dentocariosa at the genetic level can provide insights into its role within the oral microbiome.

Exploring the relationship between flumethrin resistance and Anaplasma marginale infection in Rhipicephalus (Boophilus) microplus ticks of cattle.

The study explores the relationship between flumethrin resistance and Anaplasma marginale infection in Rhipicephalus (Boophilus) microplus of cattle in South Gujarat, India. Adult Immersion Test (AIT) was used to assess flumethrin resistance and polymerase chain reaction (PCR) to confirm A. marginale infection. Species-specific PCR resulted in the amplification of 576bp of msp5 gene of A. marginale in 17.69% (49/277) groups of ticks, and subsequent digestion with EcoRI cleaved it into two distinct segments. Navsari district, noted level Ι resistance [resistance factors (RF) = 1.78-3.34], and A. marginale prevalence was 16.67, 15.38, 23.08, 15.38, and 11.76% in Navsari, Jalalpore, Gandevi, Chikhli, and Vansda sub-districts, respectively. Similarly, Vyara and Dolvan sub-districts of Tapi observed level Ι resistance (RF = 1-3.63), with A. marginale positivity of 21.43 and 22.22%, while Valod and Songhad demonstrated susceptibility, with 14.29 and 12.50% of A. marginale, respectively. Moving to Surat, the Mahuva, Bardoli, Mandvi, Palsana, and Kamrej sub-districts observed the level Ι resistance (RF = 1.94-2.89), coupled with 14.29, 17.65, 20, 20, and 21.43% of A. marginale, respectively. Lastly, in Valsad district, Dharampur, Kaparada, Valsad, and Umbergaon noted level Ι resistance (RF = 1.67-1.81), and corresponding A. marginale positivity rates of 18.18, 19.23, 15.00, and 20.00%. The scatter plot unveiled a significant moderate positive correlation between RF and A. marginale positivity% (p = 0.0362), characterized by a Pearson correlation coefficient (r) of 0.4963. The covariance (1.1814) highlighted fluctuations, while the coefficient of determination (r2) (0.2463) clarified that 24.63% of the variability in A. marginale positivity% could be attributed to the RF.

Evaluating Malaria Rapid Diagnostic Tests and Microscopy for Detecting Plasmodium Infection and Status of Plasmodium falciparum Histidine-Rich Protein 2/3 Gene Deletions in Southeastern Nigeria.

Delays in malaria diagnosis increase treatment failures and deaths. In endemic regions, standard diagnostic methods are microscopy and malaria rapid diagnostic tests (mRDTs) detecting Plasmodium falciparum histidine-rich protein 2/3 (PFHRP2/PFHRP3), but gene deletions can allow certain parasites to remain undetected. We enlisted a cohort comprising 207 symptomatic individuals, encompassing both children and adults, at a hospital in Nnewi, Nigeria. The prevalence of parasites was determined using a highly sensitive, species-specific quantitative polymerase chain reaction (SS-qPCR). Within a subset of 132 participants, we assessed the sensitivity and specificity of microscopy and HRP2-mRDTs in comparison to SS-qPCR for the detection of P. falciparum. We also investigated the prevalence of pfhrp2/pfhrp3 gene deletions. Greater sensitivity was achieved with mRDTs (95%) compared with microscopy (77%). Also, mRDTs exhibited greater specificity (68%) than microscopy (44%). The positive predictive value of mRDTs (89%) surpassed that of microscopy (80%), suggesting a greater probability of accurately indicating the presence of infection. The negative predictive value of mRDTs (82%) was far greater than microscopy (39%). Of the 165 P. falciparum-positive samples screened for pfhrp2/pfhrp3 gene deletions, one gene deletion was detected in one sample. Regarding infection prevalence, 84% were positive for Plasmodium spp. (by reverse transcription [RT]-qPCR), with P. falciparum responsible for the majority (97%) of positive cases. Thus, exclusive reliance on microscopy in endemic areas may impede control efforts resulting from false negatives, underscoring the necessity for enhanced training and advocating for high-throughput molecular testing such as RT-qPCR or qPCR at referral centers to address limitations.

Surveillance of tick-borne bacteria infection in ticks and forestry populations in Inner Mongolia, China.

Ticks are one of the most important vectors that can transmit pathogens to animals and human beings. This study investigated the dominant tick-borne bacteria carried by ticks and tick-borne infections in forestry populations in Arxan, Inner Mongolia, China. Ticks were collected by flagging from May 2020 to May 2021, and blood samples were collected from individuals at high risk of acquiring tick-borne diseases from March 2022 to August 2023. The pooled DNA samples of ticks were analyzed to reveal the presence of tick-borne bacteria using high-throughput sequencing of the 16S rDNA V3-V4 region, and species-specific polymerase chain reaction (PCR) related to sequencing was performed to confirm the presence of pathogenic bacteria in individual ticks and human blood samples. All sera samples were examined for anti-SFGR using ELISA and anti-B. burgdorferi using IFA and WB. A total of 295 ticks (282 Ixodes persulcatus and 13 Dermacentor silvarum) and 245 human blood samples were collected. Rickettsia, Anaplasma, Borrelia miyamotoi, and Coxiella endosymbiont were identified in I. persulcatus by high-throughput sequencing, while Candidatus R. tarasevichiae (89.00%, 89/100), B. garinii (17.00%, 17/100), B. afzelii (7.00%, 7/100), and B. miyamotoi (7.00%, 7/100) were detected in I. persulcatus, as well the dual co-infection with Candidatus R. tarasevichiae and B. garinii were detected in 13.00% (13/100) of I. persulcatus. Of the 245 individuals, B. garinii (4.90%, 12/245), R. slovaca (0.82%, 2/245), and C. burnetii (0.41%, 1/245) were detected by PCR, and the sequences of the target genes of B. garinii detected in humans were identical to those detected in I. persulcatus. The seroprevalence of anti-SFGR and anti-B. burgdorferi was 5.71% and 13.47%, respectively. This study demonstrated that Candidatus R. tarasevichiae and B. garinii were the dominant tick-borne bacteria in I. persulcatus from Arxan, and that dual co-infection with Candidatus R. tarasevichiae and B. garinii was frequent. This is the first time that B. miyamotoi has been identified in ticks from Arxan and R. solvaca has been detected in humans from Inner Mongolia. More importantly, this study demonstrated the transmission of B. garinii from ticks to humans in Arxan, suggesting that long-term monitoring of tick-borne pathogens in ticks and humans is important for the prevention and control of tick-borne diseases.

An emerging pathogen found in farm-cultured fourfinger threadfin (Eleutheronema tetradactylum), Edwardsiella piscicida, its genetic profile, virulent genes, and pathogenicity

The diseased fourfinger threadfin (Eleutheronema tetradactylum) exhibited clinical symptoms, such as hemorrhaging in the buccal area and skin. Gross pathological examination revealed hepatic hemorrhage, spleen enlargement, and brain congestion. Histopathological examination revealed multifocal necrosis and the presence of bacteria in various organs, including the heart, liver, spleen, and head kidney. Severe blood vessel congestion was also observed. All bacterial isolates obtained from the liver, spleen, head kidney, and brain were identified as gram-negative, motile, rod-shaped bacilli that displayed α-hemolysis. Utilizing a combination of the API 20 E system and species-specific primer-based polymerase chain reaction assays (EDtA, EDtD, and EDi primer sets), as well as sequencing of the 16S ribosomal DNA (16S rDNA) and iron-cofactor superoxide dismutase (sodB), the findings unequivocally established the identity of all isolates as E. piscicida. Examination of the E. piscicida isolates using transmission electron microscopy revealed the presence of flagella. Furthermore, genetic fingerprinting using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) demonstrated that the E. piscicida strains isolated from the fourfinger threadfin constituted a distinct genotype separate from the previously identified strains associated with Edwardsiellosis in different fish hosts. The pathogenicity of the representative E. piscicida isolates (E8–E12) was evaluated by subjecting the fourfinger threadfin to intraperitoneal injections. The gross lesions and histopathological alterations observed in experimentally infected fourfinger threadfins closely mirrored those observed in naturally infected fish. This study confirmed that E. piscicida-induced Edwardsiellosis could lead to disease outbreaks in farm-cultured fourfinger threadfin.

Belgian Cross-Sectional Epidemiological Study on Zoonotic Avian Chlamydia spp. in Chickens.

Chlamydia psittaci, Chlamydia gallinacea, and Chlamydia abortus are the most common Chlamydia spp. in chickens and have a confirmed or suggested zoonotic potential. No recent data are available on their prevalence and impact in the Belgian chicken industry or in the recreational chicken branch. Therefore, a cross-sectional epidemiological study was executed where samples were collected from both factory-farmed and backyard chickens. More specifically, pharyngeal chicken swabs were obtained from 20 chicken farms, 5 chicken abattoirs, and 38 different backyard locations and were analyzed using species-specific Polymerase Chain Reactions (PCRs) for the presence of the three avian Chlamydia spp. To investigate their zoonotic potential, samples were simultaneously collected from 54 backyard chicken caretakes and 37 professional chicken caretakers or abattoir employees and analyzed using species-specific PCRs as well. This study confirmed the presence of DNA of all three Chlamydia species in both the chicken industry and backyard settings. Chlamydia psittaci was the most prevalent in the industry chickens (11.0%), whereas Chlamydia gallinacea was the dominant species in the backyard chickens (14.5%). Chlamydia abortus infections were more common in the commercial chickens (9.0%) compared to the backyard chickens (2.6%). The DNA of all three species was also detected in humans (3.9% Chlamydia psittaci, 2.9% Chlamydia gallinacea, and 1.0% Chlamydia abortus).

Simultaneous Identification of Water Buffalo, Cattle, and Yak Meat and Horns by Multiplex Polymerase Chain Reaction (PCR)

Beef is an important source of protein for human consumption globally; in addition to beef, water buffalo and yak meat are widely consumed in Asia. Additionally, water buffalo horns are an integral part of traditional Chinese medicine. Adulteration of water buffalo horn with cattle and yak horn for economic gain has become a prevalent and pressing concern. This study used multiplex polymerase chain reaction (PCR) for the simultaneous identification of meat and horns from water buffalo, cattle, and yaks within a single PCR test. Species-specific primers were designed for multiplex PCR based on a comprehensive comparison of mitochondrial gene sequences with those of commonly edible and horned animals. Multiplex PCR conditions were optimized by considering the concentration of components and the annealing temperatures for each set of species-specific primer pairs. A total of 220 bp amplicons were obtained for water buffalo, 269 bp for cattle, and 346 bp for yak. Validation measures were used to eliminate interference from other animal sources. Simultaneous identification of the three species in a single assay presents substantial advantages over conventional species-specific PCR methods.

First report of dog ticks and tick-borne pathogens they are carrying in Malawi.

Ticks are vectors for transmitting tick-borne pathogens (TBPs) in animals and humans. Therefore, tick identification is necessary to understand the distribution of tick species and the pathogens they carry. Unfortunately, data on dog ticks and the TBPs they harbor in Malawi are incomplete. This study aimed to identify dog ticks and the TBPs they transmit in Malawi. One hundred thirty-two ticks were collected from 87 apparently healthy but infested domestic dogs in four districts of Malawi, which were pooled into 128 tick samples. The ticks were morphologically identified under a stereomicroscope using identification keys, and species identification was authenticated by polymerase chain reaction (PCR) through the amplification and sequencing of 12S rRNA and cytochrome c oxidase subunit I (CO1) genes. The tick species identified were Rhipicephalus sanguineus sensu lato (58.3%), Haemaphysalis elliptica (32.6%), and Hyalomma truncatum (9.1%). Screening for TBPs using species-specific PCR assays revealed that 48.4% of the ticks were infected with at least one TBP. The TBP detection rates were 13.3% for Anaplasma platys, 10.2% for Babesia rossi, 8.6% for B. vogeli, 6.3% for Ehrlichia canis, 3.9% for A. phagocytophilum, 3.1% for B. gibsoni, 2.3% for B. canis and 0.8% for Hepatozoon canis. Co-infections of up to three pathogens were observed in 48.4% of the positive samples. This is the first study to identify dog ticks and the TBPs they harbor in Malawi. These findings provide the basis for understanding dog tick distribution and pathogens they carry in Malawi. This study necessitates the examination of ticks from more study locations to have a better picture of tick challenge, and the development of ticks and tick-borne disease control methods in Malawi.

Acquisition and transmission of Grapevine fanleaf virus (GFLV) by Xiphinema index and Xiphinema italiae (Longidoridae).

Grapevine fanleaf virus (GFLV) is one of the most severe virus diseases of grapevines, causing fanleaf degeneration that is transmitted by Xiphinema index. This paper aims to isolate Xiphinema species from Tunisian vineyard soil samples and assess their ability to acquire and transmit GFLV under natural and controlled conditions. Based on morphological and morphometric analyses, Tunisian dagger nematodes were identified as X. index and Xiphinema italiae. These results were confirmed with molecular identification tools using species-specific polymerase chain reaction primers. The total RNA of GFLV was extracted from specimens of Xiphinema and amplified based on real-time polymerase chain reaction using virus-specific primers. Our results showed that X. index could acquire and transmit the viral particles of GFLV. This nepovirus was not detected in X. italiae, under natural conditions; however, under controlled conditions, this nematode was able to successfully acquire and transmit the viral particles of GFLV.

Characterization of genotypes and antimicrobial resistance profiles of clinical isolates of Shigella from patients in the southern region of Iran

BackgroundShigella spp., which are facultative anaerobic bacilli within the Enterobacteriaceae family, present a significant public health burden due to their role as prominent contributors to diarrheal diseases worldwide. A molecular analysis can facilitate the identification and assessment of outbreaks involving this bacterium. So, we aimed to investigate the antibiotic susceptibility pattern and clonal relatedness of clinical Shigella spp. isolates obtained from patients with diarrhea in Hormozgan province, South of Iran.MethodsFrom 2019 to 2021, a cross-sectional investigation was conducted on 448 stool samples obtained from patients who were experiencing diarrhea, in the southern region of Iran. Shigella spp. isolates were identified based on biochemical and serological tests. All Shigella species were verified using species-specific polymerase chain reaction (PCR), followed by susceptibility testing to antimicrobial agents. Subsequently, genotyping of all Shigella species was conducted using ERIC-PCR.ResultsOut of a total of 448 stool samples, the presence of Shigella was detected in 62 cases, accounting for a prevalence rate of 13.84%. Among the identified isolates, the majority were attributed to S. flexneri, representing 53.23% of the cases. This was followed by S. sonnei at 24.19% and S. boydii at 22.58%. Notably, no instances of S. dysenteriae were found. The highest prevalence of Shigella isolates was observed in infants and children under the age of five. A significant proportion of the identified isolates demonstrated resistance to various antibiotics. Specifically, high resistance rates were noted for ampicillin (90.78%), piperacillin–tazobactam (87.1%), cefixime (83.87%), trimethoprim–sulfamethoxazole (83.87%), cefotaxime (82.26%), and ceftriaxone (80.65%). In addition, a substantial number (87.1%) of the isolates exhibited a multidrug-resistant (MDR) phenotype. Using the ERIC-PCR method, a total of 11 clusters and 6 distinct single types were identified among all the Shigella isolates.ConclusionA notable occurrence of antibiotic-resistant Shigella species has been noted, with multi-drug resistant (MDR) strains presenting an increasing challenge for treating shigellosis worldwide, and this includes Iran. Techniques such as ERIC-PCR are useful for assessing the genetic variation and connections between Shigella strains, which indirectly contributes to understanding antimicrobial resistance patterns. Further research is needed to explore the specific correlation between resistance genes and ERIC genotyping patterns in Shigella strains.

Optimization of culture conditions for and assessment of kimchi-originated lactic acid bacterial isolates toward their extracellular GABA-producing ability

The objectives of this study were to optimize the medium and culture conditions using a strong γ-aminobutyric acid (GABA) producer as a reference lactic acid bacterial strain, to screen and identify GABA-producing lactic acid bacterial isolates from kimchi, and to determine their extracellular GABA-producing abilities. Thin-layer chromatography was used to screen GABA-producing bacterial isolates and high-performance liquid chromatography was used to evaluate the bacterial GABA production abilities. Species-specific polymerase chain reaction analyses were used to identify GABA-producing bacterial isolates. The optimal medium and culture conditions were found to be the modified Man–Rogosa–Sharpe (MRS) broth (with an initial pH of 6.5) containing 4% sucrose, 5% glutamate, and 1% yeast extract at 37°C for 5 days. After incubation under the optimized culture conditions, 217 kimchi bacterial isolates were screened to evaluate their respective GABA-producing abilities. Screening the 217 kimchi bacterial isolates identified 24 GABA-producing lactic acid bacterial isolates (11%): Lactobacillus plantarum (17), Lactobacillus brevis (six), and Leuconostoc mesenteroides (one), indicating that only a small proportion of the strains produce GABA in the culture broth. The extracellular GABA-producing abilities of the bacterial strains identified in this study varied even within the same species, ranging from 5.8 to 101.7 mM among the 17 GABA-producing L. plantarum isolates and from 8.5 to 88.6 mM among the six GABA-producing L. brevis isolates. In summary, three species of the 24 kimchi GABA-producing bacterial isolates were identified, including one rare species (L. mesenteroides) and the two most dominant species (L. brevis and L. plantarum).
 Keywords: Lactobacillus brevis; Lactobacillus plantarum; Leuconostoc mesenteroides; Optimization; γ-aminobutyric acid (GABA)

The outbreak of migratory goat's brucellosis in the Swat ecosystem of Khyber Pakhtunkhwa.

Brucellosis is a major threat to public health especially in developing countries including Pakistan. This study reveals the characterisation ofBrucellaspecies affecting humans and goats in the Swat region of Khyber Pakhtunkhwa, Pakistan. Blood samples were collected from shepherds and goats and analysed by Rose Bengal precipitation test (RBPT), standard plate agglutination test (SPAT), polymerase chain reaction (PCR) and Sanger sequencing of 16S rRNA gene. The findings of the study indicated 24% (36/150) and 11.3% (17/150) positivity forBrucella abortusandBrucella melitensis,respectively, in human samples. In samples of goats, 26.66% (40/150) were positive forB. abortusand 16.66% (25/150) samples were positiveB. melitensisby SPAT. The species-specific PCR confirmedB. abortusin 24% (36/150) of human samples and 26.66% (17/150) of goat samples by targeting the IS711 locus. The remaining seropositive samples were confirmed asB. melitensisusing IS711 M species-specific primer. The sequences of the amplified fragments of the 16S rRNA gene were blasted, and phylogenetic analysis revealed thatBrucellaspecies circulating in the Swat district were closely related toB. melitensisandB. abortusreported from India, China, Philippines, and the United States (US) showing the existence of the possible epidemiological linkage among theBrucellaspecies. This study concluded that there was a higher prevalence ofB. abortus(26.6%) in humans and goats compared toB. melitensis(16.6%). These results revealed that theBrucellaspecies were circulating in both humans and goats in the study areas. The findings of the study concluded thatB. abortusandB. melitensiswere circulating in goats and shepherds with a higher prevalence ofB. abortusthanB. melitensis. Furthermore, theBrucellaspecies identified in Swat were phylogenetically related to theBrucellaspecies reported from India, China, Philippines and the US.Contribution:The proposed study covers the scope of the journal. The species of the genusBrucellaaffect both animals and shepherds. This study investigates the seroprevalence of brucellosis in shepherds and goats in different geographical areas in the Swat district. The phylogenetic analysis of theBrucellaspp. identified in Swat showed close relationships to theBrucellaspecies reported in India, China, Philippines and the US, which shows the possible epidemiological linkages between theBrucellaspp.

Development and validation of a two-step assay for differentiation of Penis et testis cervi from cervi nippon Temmink and cervi elaphus Linnaeus based on specific-species polymerase chain reaction-restriction fragment length polymorphism patterns

Penis et testis cerviis one of the precious traditional Chinese medicines (TCMs) in China, which, whenauthentic, is sourcedfrom species ofCervi nippon Temmink(C. Nippon)andCervi elaphus Linnaeus(C.elaphus L).Authentication of species ofPenis et testis cerviremainstechnically challenging. This study attempted to modify the species-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) method using restriction enzymeHpy188III for the identification ofPenis et testis cervispecies.First, thecytochrome b (Cytb)and cytochrome c oxidase subunit I (COI) genes in mitochondrial DNAfrom two authentic species and their counterfeitsor adulterantswere downloaded and analyzed with bioinformatics tools to establish the phylogenetic tree, then a series of primers were designed, and the restriction enzymesites were confirmed. Genomic DNA was extracted fromall samplesby the improved SDS method. Species-specific PCR reactions were optimizedto differentiate authentic Sika deer and ReddeerPenis et testis cervifrom their counterfeits or adulterants. PCR-RFLP using single restriction enzyme Hpy188III was performed inCytb regionsto identify the species of Sika deer and Reddeer. Simultaneously, recombination plasmid carrying thecytb fragmentcould be cloned, sequenced, and blasted.The assay specificity and sensitivity were evaluated, and30 batches of commercial samples werevalidated. Thephylogenetic treeshowed that the physico-chemical properties of Sika deer and Reddeer variants were similar.Sufficient DNA extracts were extracted from all samples used inthe improved SDS method.Establishment and optimization of a species-specific PCR reactionsand PCR-RFLPsystem were undertaken to identify deer species inPenis et testis cervisamples. Aspecific PCR-RFLP pattern with three distinct products of sizes 155, 113, and 204 bp was exhibited in Sika deerspecies,and two distinct products of 204 and 268 bp in size were evidentin Reddeerspecies,which were significantly different from the pattern of thecounterfeitsor adulterants.The distinctive PCR-RFLP fingerprint pattern using the restriction enzyme Hpy188III is a rapid and reliable assay for identification of thePenis et testis cervispecies.

Sibling Species Composition and Susceptibility Status of Anopheles gambiae s.l. to Insecticides Used for Indoor Residual Spraying in Eastern Uganda.

Malaria remains one of the most critical disease causing morbidity and mortality in Uganda. Indoor residual spraying (IRS) and the use of insecticide-treated bed nets are currently the predominant malaria vector control interventions. However, the emergence and spread of insecticide resistance among malaria vectors threaten the continued effectiveness of these interventions to control the disease, particularly in high transmission areas. To inform decisions on vector control, the current study evaluated the Anopheles malaria vector species and their susceptibility levels to 0.1% bendiocarb and 0.25% pirimiphos-methyl insecticides used in IRS intervention program in Namutumba district, Eastern Uganda. Anopheles larvae were collected between March and May 2017 from different breeding sites in the parishes of Nsinze and Nawaikona in Nsinze sub-county and reared to adults to assess the susceptibility status of populations in the study area. Mosquitoes were identified using morphological keys and species-specific polymerase chain reaction (PCR) assays. Susceptibility tests were conducted on 2- to 5-day-old non-blood-fed adult female Anopheles that emerged using insecticide-impregnated papers with 0.1% bendiocarb and 0.25% pirimiphos-methyl following standard World Health Organization (WHO) insecticide susceptibility bioassays. A Log-probit regression model was used to derive the knock-down rates for 50% and 95% of exposed mosquitoes. A total of 700 mosquito larvae were collected from different breeding sites. Morphological identification showed that 500 individuals that emerged belonged to Anopheles gambiae sensu lato (s.l.), the main malaria vector. The PCR results showed that the dominant sibling species under the A. gambiae complex was Anopheles arabiensis 99.5% (395/397). WHO bioassay tests revealed that the population of mosquitoes exhibited high levels of susceptibility (24-hour post-exposure mortality 98-100%) to both insecticides tested. The median knock-down time, KDT50, ranged from 6.6 to 81.4 minutes, while the KDT95 ranged from 21.6 to 118.9 minutes for 0.25% pirimiphos-methyl. The KDT50 for 0.1% bendiocarb ranged from 2.8 to 62.9 minutes, whereas the KDT95 ranged from 36.0 to 88.5 minutes. These findings indicate that bendiocarb and pirimiphos-methyl are still effective against the major malaria vector, A. arabiensis in Nsinze sub-county, Namutumba district, Uganda and can be effectively used for IRS. The study has provided baseline information on the insecticide susceptibility status on malaria vectors in the study area. However, routine continuous monitoring program of insecticide susceptibility and malaria vector composition is required so as to guide future decisions on insecticide use for IRS intervention toward malaria elimination and to track future changes in vector population.

A novel qPCR-based technique for identifying avian sex: An illustration within embryonic craniofacial bone.

Sex is a biological variable important to consider in all biomedical experiments. However, doing so in avian embryos can be challenging as sex can be morphologically indistinguishable. Unlike humans, female birds are the heterogametic sex with Z and W sex chromosomes. The female-specific W chromosome has previously been identified in chick using a species-specific polymerase chain reaction (PCR) technique. We developed a novel reverse transcription quantitative PCR (RT-qPCR) technique that amplifies the W chromosome gene histidine triad nucleotide-binding protein W (HINTW) in chick, quail, and duck. Accuracy of the HINTW RT-qPCR primer set was confirmed in all three species using species-specific PCR, including a novel quail-specific HINTW PCR primer set. Bone development-related gene expression was then analyzed by sex in embryonic lower jaws of duck and quail, as adult duck beak size is known to be sexually dimorphic while quail beak size is not. Trends toward sex differences were found in duck gene expression but not in quail, as expected. With these novel RT-qPCR and PCR embryo sexing methods, sex of chick, quail, and duck embryos can now be assessed by either/both RNA and DNA, which facilitates analysis of sex as a biological variable in studies using these model organisms.

Meat Species Identification in Chicken Blood Meal using Polymerase Chain Reaction Technique

A study was conducted to identify the species of chicken in high heat processed chicken secondary by-product comprising of poultry blood meal. A total of 30 samples of poultry blood meal were collected from different poultry processing plants in Tamil Nadu. Species specific polymerase chain reaction was employed to validate the meat species identification in heat processed secondary poultry by-products. Custom designed primer sequences of mitochondrial cyt b gene were utilised for the present study. The results revealed successful amplification of chicken species at 227 base pairs in samples of all the blood meal samples. Thus, it was concluded that species specific polymerase chain reaction technique can be effectively used in the screening of chicken meat species in different heat processed secondary by-products for quick and reliable results.

Molecular investigation of malaria-infected patients in Djibouti city (2018–2021)

BackgroundThe Republic of Djibouti is a malaria endemic country that was in pre-elimination phase in 2006–2012. From 2013, however, malaria has re-emerged in the country, and its prevalence has been increasing every year. Given the co-circulation of several infectious agents in the country, the assessment of malaria infection based on microscopy or histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDT) has shown its limitations. This study, therefore, aimed to assess the prevalence of malaria among febrile patients in Djibouti city using more robust molecular tools.MethodsAll suspected malaria cases reported to be microscopy-positive were randomly sampled (n = 1113) and included in four health structures in Djibouti city over a 4-year period (2018–2021), mainly during the malaria transmission season (January–May). Socio-demographic information was collected, and RDT was performed in most of the included patients. The diagnosis was confirmed by species-specific nested polymerase chain reaction (PCR). Data were analysed using Fisher’s exact test and kappa statistics.ResultsIn total, 1113 patients with suspected malaria and available blood samples were included. PCR confirmed that 788/1113 (70.8%) were positive for malaria. Among PCR-positive samples, 656 (83.2%) were due to Plasmodium falciparum, 88 (11.2%) Plasmodium vivax, and 44 (5.6%) P. falciparum/P. vivax mixed infections. In 2020, P. falciparum infections were confirmed by PCR in 50% (144/288) of negative RDTs. After the change of RDT in 2021, this percentage decreased to 17%. False negative RDT results were found more frequently (P < 0.05) in four districts of Djibouti city (Balbala, Quartier 7, Quartier 6, and Arhiba). Malaria occurred less frequently in regular bed net users than in non-users (odds ratio [OR]: 0.62, 95% confidence interval [CI]: 0.42–0.92).ConclusionsThe present study confirmed the high prevalence of falciparum malaria and, to a lesser extent, vivax malaria. Nevertheless, 29% of suspected malaria cases were misdiagnosed by microscopy and/or RDT. There is a need to strengthen the capacity for diagnosis by microscopy and to evaluate the possible role of P. falciparum hrp2 gene deletion, which leads to false negative cases of P. falciparum.