nPCR Assay Research Articles

Efficacy of amniotic fluid, blood and urine samples for the diagnosis of toxoplasmosis in pregnant women candidates for amniocentesis using serological and molecular techniques

BackgroundsToxoplasmosis, a prevalent parasitic infection, is primarily caused by Toxoplasma gondii (T. gondii). This infection poses a significant threat to neonates during pregnancy and individuals with compromised immune systems. Consequently, it is imperative to develop a novel diagnostic approach that combines high sensitivity with low-risk sampling to effectively manage patients. The aim of this study is to utilize serological and molecular techniques for the diagnosis of T. gondii infection in 100 pregnant women who were under the care of a gynecologist and were candidates for amniocentesis.MethodsDuring the 15-19th weeks of pregnancy, a total of 100 samples each of amniotic fluid, buffy coat, plasma, and urine simultaneously were collected from pregnant women candidates for amniocentesis in Mazandaran province, northern Iran. This study involved various assessments: (1) detecting anti-T. gondii IgM and IgG in plasma through chemiluminescence assay (2) determining IgG avidity in plasma using the Enzyme-linked immunosorbent assay technique (3) identifying of T. gondii DNA in amniotic fluid, buffy coat and urine by nested PCR (nPCR) and quantitative real-time PCR (qPCR) methods targeting the REP-529 gene, as well as genotyping using GRA6 target genes, and (4) assessing the sensitivity and specificity of the nPCR and qPCR tests.ResultsOut of 100 pregnant women screened, 70 were between the ages of 31 to 40 years old. Among them, 23 and 44 had one and two previous pregnancies. Additionally, 13 and 8 women had one and two history of abortions, respectively. Following serologic testing, 52% of the individuals were positive for T. gondii antibodies. Of these, 52 samples were positive for IgG antibodies, and one sample was positive for both IgG and IgM antibodies. Notably, all 52 cases with IgG positivity exhibited a high level of IgG avidity. Regarding the molecular testing of amniotic fluid samples, two pregnant women tested positive in the nPCR assay, while three tested positive in the qPCR assay. Furthermore, genotyping revealed that all positive samples belonged to type I of the T. gondii genotype. Moreover, none of the 100 buffy coat and urine samples tested positive for T. gondii using the nPCR and qPCR techniques.ConclusionThe findings of the current study suggest that serological methods alone may not be reliable in diagnosing congenital toxoplasmosis and cannot rule out the diagnosis of toxoplasmosis and must be approved by molecular tests.

Genetic diversity of Theileria spp. in deer (Artiodactyla: Cervidae) from Brazil.

Babesia spp. and Theileria spp. are tick-borne apicomplexan protozoa that can cause disease in animals and humans. Deer are considered reservoirs for a wide variety of Piroplasmida species, including some potentially zoonotic. This study aimed to investigate the occurrence and genetic diversity of piroplasmids in wild deer sampled in four Brazilian states (São Paulo, Mato Grosso do Sul, Paraná and Goiás). For this purpose, extracted DNA samples from 181 deer buffy coat samples (138 Blastocerus dichotomus, 26 Subulo gouazoubira, 4 Mazama jucunda, 3 Mazama rufa and 10 Ozotocerus bezoarticus) were subjected to a nested PCR (nPCR) assay based on the 18S rRNA gene in order to perform a screening for piroplasmids and characterized based on the near-complete 18S rRNA, hsp70 and cox-3 genes. As a result, 75.14% (136/181) samples were positive for piroplasmids. Of these, 108 (79.41%), 101 (74.26%) and 67 (49.26%) were positive to near complete 18S rRNA, hsp70 and cox-3 genes, respectively. Phylogenetic analyses based on three molecular markers showed similar topology to each other. All sequences obtained in the present study were positioned into the Theileria sensu stricto clade, forming a distinct clade, albeit close to T. cervi. Most sequences grouped together into a large clade divided into subclades, which were often related to deer genus/species, showing that Theileria lineages seemed to show specificity according to deer genus/species. Two 18S rRNA sequences (one obtained from S. gouazoubira and another from M. jucunda) were positioned into a different clade, apart from other sequences detected in this study, indicating that different species of Theileria occur in deer from Brazil. Two subclusters were observed in the phylogenetic analysis based on the hsp70 gene: the first containing only sequences detected in marsh deer and the second grouping sequences detected in brocket deer (Mazama spp. and S. gouazoubira). The latter was also divided into smaller clades that grouped Theileria genotypes according to deer species (M. jucunda, M. rufa and S. gouazoubira). This study provides the first molecular evidence of Theileria infection in M. jucunda, as well as co-infection by distinct Theileria (sub)species/genotypes in the same deer was evidenced. Finally, this study expanded the knowledge on the diversity of Theileria spp. infecting deer from South America.

First report of infectious spleen and kidney necrosis virus (ISKNV) in two native cichlids cultured in Brazil

Peacock bass (syn.: tucunaré, Cichla ocellaris) and the pearl cichlids (syn.: acará, Geophagus brasiliensis) are South American cichlids that are highly valued in both the ornamental and sport fish industries. Since 2017, a number of outbreaks of infectious spleen and kidney necrosis virus (ISKNV) have been reported on Brazilian food and ornamental fish farms. In this study, we detected ISKNV in farmed peacock bass and pearl cichlid by PCR and sequence analysis of the partial major capsid protein (MCP) gene. Moribund peacock bass (n=10) and pearl cichlids (2) from a farm experiencing elevated mortality among juveniles and adults of these species, were submitted for bacteriological and molecular diagnostics. Spleen, liver, brain, and kidney tissues were cultured on 5% sheep blood agar and cystine heart agar with 1% glucose and bovine haemoglobin. No bacteria were isolated from the 12 fish. Additionally, DNA extracts from the liver and spleen of all animals were tested for ISKNV using two conventional polymerase chain reaction (cPCR) assays and two nested PCR (nPCR) assays. ISKNV DNA was amplified in all 12 fish DNA extracts tested, in two or more of the PCR assays. Selected ISKNV amplicons were confirmed by Sanger sequencing. The nucleotide sequences derived from these animals were identical to ISKNV strains previously detected in food (e.g., tilapia and carp) and ornamental species, including strains previously detected in fish from Brazil. To the authors’ knowledge, this is the first report of ISKNV in these native Brazilian cichlids.

Incidence and Genetic Investigation of Avian Coronaviruses in Migratory Ducks From South Korea

Coronaviruses (CoVs) belonging to the Gamma-CoV and Delta-CoV genera are widespread in poultry and wildfowl. Migratory birds, particularly duck species, serve as hosts for CoVs and play a pivotal role in transmitting the viruses to other species, including mammals. Despite the potential risks to animals and humans, there remains a narrow knowledge of the genetic and epidemiological properties of CoVs in wild birds. The current research aimed to detect and characterize CoVs present in migratory duck species (Anas acuta, Anas platyrhynchos, and Anas poecilorhyncha) from South Korea. Employing two rounds of pan‐CoV real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) and nested PCR (nPCR) assays amplifying the conserved RNA‐dependent RNA polymerase (RdRp) portion common to all known CoVs, we screened 2120 duck fecal samples collected during 2022–2023. The results indicated the presence of CoVs in 4.2% (91/2120) of samples from migratory ducks. Nucleotide sequencing of the RdRp gene revealed that all identified CoVs were clustered within the Gamma-CoV genus. Further phylogenetic analysis suggested that South Korean gamma‐CoVs belong to the Igacovirus subgenus and share similarities with those found worldwide, highlighting the critical role of migratory ducks in introducing and exporting avian CoVs. We discovered two clade VII igacovirus strains in wild ducks closely related to those in pigeons, implying potential cross infection between these avian species. Overall, our study underscores the importance of active surveillance and monitoring of avian CoVs in wild birds as a preemptive response against the forthcoming emergence of new CoV species that can threaten both animal and human health.

Development of a Real-Time Quantitative PCR Based on a TaqMan-MGB Probe for the Rapid Detection of Theileria haneyi.

Equine piroplasmosis (EP) is a parasitic disease caused by Theileria equi (T. equi), Babesia caballi (B. caballi) and Theileria haneyi (T. haneyi). This disease is considered to be reportable by the World Organization for Animal Health (WOAH). Real-time quantitative PCR (qPCR) is regarded as a straightforward, rapid and sensitive diagnostic method to detect pathogens. However, qPCR has not been employed in the various epidemiological investigations of T. haneyi. In this study, we developed a new qPCR method to detect T. haneyi based on the chr1sco (chromosome 1 single-copy open reading frame (ORF)) gene, which has no detectable orthologs in T. equi or B. caballi. A TaqMan MGB probe was used in the development of the qPCR assay. A plasmid containing the chr1sco gene was constructed and used to establish the standard curves. The novel qPCR technique demonstrated great specificity for detecting additional frequent equine infectious pathogens and sensitivity for detecting diluted standard plasmids. This qPCR was further validated by comparison with an optimized nested PCR (nPCR) assay in the analysis of 96 clinical samples. The agreement between the nPCR assay and the established qPCR assay was 85.42%. The newly established method could contribute to the accurate diagnosis of T. haneyi infections in horses.

Molecular Detection and Characterization of Ehrlichia canis Isolates from Three Geographic Regions in Mexico: A Retrospective Study.

Canine monocytic ehrlichiosis (CME) is the most common tick-borne disease affecting domestic dogs and other wild canids. It has a worldwide distribution and is associated with the presence of the brown dog tick. Few studies have been conducted in Mexico to identify and characterize Ehrlichia canis genetic variability. In the present study, 111 dogs of different sex, breed, and age from three geographic regions in Mexico were included. All of them had a previous history of tick infestation and/or the presence of one or more clinical signs compatible with CME. All dogs were tested by a commercial ELISA and nested PCR assay for the detection of E. canis. In addition, we analyzed the E. canis genetic diversity from the 16S rRNA gene sequences obtained in this study, along with 15 additional sequences described for E. canis in Mexico and obtained from GeneBank. Serological detection by commercial ELISA results showed overall infection rates of 85.58% (95/111), including 73.1% (30/41) in samples from Guerrero state; 75% (15/20) in Morelos; and 100% (50/50) in Chihuahua. On the other hand, molecular detection (nPCR assay) showed 31.5% (35/111) overall infection rate, with 41.4% (17/41) in Guerrero state; 55% (11/20) in Morelos; and 14% (7/50) in Chihuahua. We observed a high 16S rRNA gene sequence conservancy in most of the E. canis isolates in the three geographical areas from Mexico, including those analyzed in this research, suggesting a common geographic origin among isolates.

Development of loop-mediated isothermal amplification for rapid detection of sporotrichosis caused by Sporothrix schenckii.

Sporothrix schenckii is the causative agent of sporotrichosis, which most commonly causes lymphocutaneous infections in immunocompromised hosts. This pathogen infects dogs, cats, cattle, and buffaloes and can potentially infect humans. Diagnosis by fungal culture is lengthy, and although there are several clinical diagnoses and molecular methods, these are complicated and time-consuming for veterinarians. This study aimed to develop a visual diagnostic assay that is less time-consuming and can be used by veterinarians to screen for sporotrichosis. To develop a loop-mediated isothermal amplification (LAMP) assay for sporotrichosis, primers specific for fragments of the 18S rRNA gene of S. schenckii were designed. Then, the time and temperature were optimized to successfully achieve LAMP. Ten-fold serial dilutions of DNA were used to determine the detection limit using both LAMP and nested polymerase chain reaction (nPCR) assays. The optimal LAMP conditions were incubation at 73°C for 30 min. Agarose gel electrophoresis revealed a ladder-like pattern of the LAMP product, and a sky-blue color indicated a positive result. A comparison of the LAMP assay with nPCR revealed that it was 10 times more sensitive than nPCR, with a detection limit of 10 pg. The use of a heat box compared with a thermocycler gave the same results. Loop-mediated isothermal amplification gives good results and may represent a future alternative diagnostic tool for screening fungal pathogens before the results of conventional fungal cultures are received. However, this method should be further studied to clarify its use with clinical samples.

Development and field evaluation of an ELISA to differentiate Anaplasma marginale-infected from A. centrale-vaccinated cattle.

Immunization of calves with Anaplasma centrale is used to prevent acute anaplasmosis caused by A. marginale. Natural and vaccine-acquired immunity is detected through serologic tests based primarily on A. marginale recombinant major surface protein 5 (MSP5m) because it has 91% identity with MSP5 from A. centrale (MSP5c). We developed a displacement, double-antigen, sandwich ELISA (ddasELISA) to detect antibodies against A. marginale or A. centrale. For ddasELISA validation, we analyzed serum samples positive for antibodies against Anaplasma spp. from cattle naturally infected with A. marginale (n = 300) or vaccinated with A. centrale (n = 255). Species-specific nested PCR (nPCR) assays were used to confirm infection. The optical density (OD) values obtained from antibodies directed at unique epitopes of A. marginale (ODAm) or A. centrale (ODAc) were used in the formula ODAm/ODAc. If the derived ratio was >0.38, the serum sample was considered positive for antibodies against A. marginale, with 98.9% sensitivity and 98.0% specificity. In a field evaluation, we analyzed 702 Anaplasma spp. antibody-positive serum samples from 34 herds by ddasELISA and nPCR; 571 were classified by ddasELISA as A. marginale-infected or A. centrale-vaccinated, with 84% agreement (κ = 0.70) between ddasELISA and nPCR. Our results indicate that ddasELISA could be used as a cost-effective alternative to molecular techniques to confirm infection with A. marginale in countries in which prevention is based on vaccination with A. centrale.

Molecular-Genotyping Detection of Entamoeba histolytica in Diarrheic Patients.

Amebiasis is caused by Entamoeba histolytica, a protozoan that is found worldwide. The degree of pathogenesis of clinical isolates varies greatly. This study was aimed to molecular identification of E. histolytica in children using the nested polymerase chain reaction (nPCR), and then, a genotyping of positive E. histolytica isolates using the quantitative PCR (qPCR) assay through targeting serine-rich E. histolytica protein (SREHP) gene. For this purpose, a total of 50 bloody diarrheic stool samples were collected from the children attended to the Al-Zahraa' Teaching Hospital and Alkut Hospital for Gynecology, Obstetric and Pediatrics (Alkut, Wasit, Iraq) were subjected to the present study from September to December 2021. Firstly, the extracted DNAs that amplified using specific primers through targeting 18S rRNA gene and tested using nPCR assay were revealed an overall 48% (24/50) positive samples for E. histolytica. For genotyping, our results were detected an existence of four different genotypes (I, II, III and IV) with a significant prevalence of Genotype-II (54.17%) when compared to Genotype-I (20.83%), Genotype-III (12.5%) and Genotype- IV (12.5%). In addition, results of melting temperature of targeted genotypes were 84ºC, 83 - 83.5ºC, 82.5ºC and 81ºC for Genotype-I, II, III and IV, respectively. In conclusion, molecular amplification of 18S rRNA gene was revealed the large prevalence of E. histolytica among bloody diarrheic children of study areas; while, amplification of SREHP gene was reflected the widespread phenotypic variation of the Genotype-II suggesting the high ability of this genotype to spread infection in children. In different endemic areas as Iraq, the utilization of high-resolution genotyping methods showed the extremely polymorphic genetic structure of this parasite.

Detection of the Mycobacterium avium complex in dogs with lymphadenitis.

Mycobacterium avium complex (MAC) is connected to human immunosuppressive diseases, including HIV-AIDS, and may pose a zoonotic threat. MAC causes lymphadenopathy in children, respiratory infection in adults, and generalized infection in immunocompromised individuals. Infection with nontuberculous Mycobacteria (NTM) in humans is now primarily brought on by MAC. Recently, MAC members have emerged as pathogenic organisms for animals and humans. While dogs are generally resistant to mycobacterial infections, there have been some cases of infection that result in systemic or disseminated diseases. The organisms can be transmitted to dogs through oral contact, and their faeces can be a possible source of infection for dog owners. It is important to note that this ailment is zoonotic, especially if infected pet dogs are in prolonged contact with their humans. The study was planned to demonstrate the occurrence of MAC organisms and other Mycobacteria in dogs associated with lymphadenopathy cases with special emphasis on lymphadenitis. A total of 123 samples (100 lymph node aspirates, 15 lymph node tissues, and 8 blood samples) from 83 dogs suspected of lymphadenitis accompanied by gastroenteritis, chronic skin infections, immunosuppression, chronic pulmonary diseases, and other chronic undiagnosed diseases were studied. The samples were processed for cytological and microscopic examination by Ziehl-Neelsen staining. Following the decontamination procedure, the aspiration and lymph node tissue samples were inoculated into Middlebrook 7H11 media for up to 8 weeks. The aspirated material was also directly used for molecular detection by triplex-nested polymerase chain reaction (nPCR) assay. A cytological study revealed pyogranulomatous inflammation of the lymph node tissue. Impression smears from lymph node tissues displayed the presence of acid-fast organisms. Out of 83 cases of dogs, 8 were found to be positive for Mycobacterium spp. Among those 8 positive cases, 3 were confirmed to belong to MAC, and 5 belonged to the Mycobacterium tuberculosis complex (MTB complex). MAC and MTB are the underestimated bacteria that could be the causative agents of lymphadenitis in animals.

Comparison of molecular and serological assays on cerebrospinal fluid for the diagnosis of neurosyphilis.

Many assays are available on cerebrospinal fluid (CSF) for the diagnosis of neurosyphilis (NS) but there is no 'gold standard'. The aim of this study was to evaluate different molecular and serological assays used in NS. We evaluated two PCR assays and three serological techniques in parallel on CSF samples collected between 2019 and 2020 from patients suspected of NS. The study included 143 patients comprising 30 early NS, 7 late NS and 106 patients without a diagnosis of NS. All patients with NS were symptomatic and had either neurological (67.6%) or ophthalmological signs (54.1%). The qPCR and nPCR assays had overall sensitivities (Se) of 41% and 27%, respectively; with each an overall specificity (Sp) of 100%. VDRL had a Se of 51% and a Sp of 92%. Immunoblot had a Se of 62% and a Sp of 85%. Finally, treponemal tests (TT) had a Se of 96% and a Sp of 69%. Our study confirms the excellent specificity of molecular techniques allowing to avoid overdiagnosis of NS, and thus, unjustified intensive antibiotic therapy protocols. CSF TT, although not very specific, has an excellent Se confirming that there is almost never NS with negative CSF TT. VDRL and immunoblot tests have better overall diagnostic performance. However, none of these techniques has sufficient diagnostic performance to represent a 'gold standard'. Thus, the diagnosis of NS relies on a combination of clinical and biological parameters with the association of PCR with serology, associating VDRL and immunoblot, in CSF.

High-throughput Plasmodium falciparum hrp2 and hrp3 gene deletion typing by digital PCR to monitor malaria rapid diagnostic test efficacy.

Most rapid diagnostic tests for Plasmodium falciparum malaria target the Histidine-Rich Proteins 2 and 3 (HRP2 and HRP3). Deletions of the hrp2 and hrp3 genes result in false-negative tests and are a threat for malaria control. A novel assay for molecular surveillance of hrp2/hrp3 deletions was developed based on droplet digital PCR (ddPCR). The assay quantifies hrp2, hrp3, and a control gene with very high accuracy. The theoretical limit of detection was 0.33 parasites/µl. The deletion was reliably detected in mixed infections with wild-type and hrp2-deleted parasites at a density of >100 parasites/reaction. For a side-by-side comparison with the conventional nested PCR (nPCR) assay, 248 samples were screened in triplicate by ddPCR and nPCR. No deletions were observed by ddPCR, while by nPCR hrp2 deletion was observed in 8% of samples. The ddPCR assay was applied to screen 830 samples from Kenya, Zanzibar/Tanzania, Ghana, Ethiopia, Brazil, and Ecuador. Pronounced differences in the prevalence of deletions were observed among sites, with more hrp3 than hrp2 deletions. In conclusion, the novel ddPCR assay minimizes the risk of false-negative results (i.e., hrp2 deletion observed when the sample is wild type), increases sensitivity, and greatly reduces the number of reactions that need to be run.

Longitudinal dynamics and health impact of Hepatozoon procyonis (Apicomplexa: Hepatozoidae) on naturally infected ring-tailed coatis Nasua nasua (Carnivora: Procyonidae) from Midwestern Brazil

This study aimed to morphologically and molecularly detect Hepatozoon procyonis in ring-tailed coatis’ (Nasua nasua) blood and associated ticks from central-western Brazil, Campo Grande, Mato Grosso do Sul state and also evaluate the impact of the protozoa in blood parameters and coati´s health. Samplings were performed in a conservation area Parque Estadual do Prosa (PEP) and in a Brazilian Air Force Private Area namely Vila da Base Aérea (VBA), between March 2018 and April 2019. We collected 165 blood samples, 61 from recaptured coatis. Peripheral blood smears were stained with Romanovsky-type stain for H. procyonis parasitemia assessment. DNA extracted from blood samples and ticks (Amblyomma spp.) were submitted to a nested PCR (nPCR) assay based on the 18S rRNA gene for Hepatozoon spp. Out of 104 individuals sampled, 80 (77%) were positive for H. procyonis in at least one capture. Overall, 67/165 (40.6%) blood smears showed H. procyonis gametocytes (PEP: 41/63 - 65%; VBA: 26/102 - 25.5%). Parasitemia based on 500 assessed leucocytes ranged from 1 (0.2%) to 50 (10%) and 1 (0.2%) to 25 (5%), from animals sampled in PEP and VBA, respectively. Fluctuation on the parasitemia was observed during recaptures. nPCR results showed higher positivity when compared to blood smears, i.e. 112/165 (68%) positive blood samples [PEP: 41/63 (65%), VBA: 26/102 (25.5%)]. In total, 63/248 (25.4%) tick DNA samples were positive at nPCR for Hepatozoon sp., including 32/87 (37%) pools (1 to 10 larvae) of Amblyomma larvae, 21/105 (20%) pools (1 to 5 nymphs) of Amblyomma sculptum nymphs, 9/43 (21%) pools (1 to 5 nymphs) of Amblyomma dubitatumnymphs, and 1/12 (8%) A. sculptum adult female. The partial 18S rRNA sequence from one coati's blood sample and one representative of each positive tick species randomly selected from each area for sequencing (1,000 bp) showed 100% identity with sequences of H. procyonis from GenBank previously detected in coatis. Regarding H. procyonis infection, no statistical differences were obtained when comparing males vs. females (p-value 0.67), immature animals vs. adults (p-value 0.31), rainy vs. dry season (p-value 0.51) and sampling location (p-value 0.42). No noticeable alteration in blood parameters or heath status was observed in parasite animals. H. procyonis circulates in a high prevalence in coatis from central-western Brazil. Parasitemia fluctuates among different coatis’ recaptures and apparently the infection has no influence in coatis’ hematological and clinical parameters.

Detection of bovine gammaherpesvirus 6 in tissues of aborted fetuses from dairy cows concomitantly infected by Histophilus somni

Bovine gammaherpesvirus 6 (BoGHV6), formerly known as bovine lymphotropic virus, is a member of the Macavirus genus, subfamily Gammaherpesvirinae, family Herpesviridae, that was initially associated with proliferative diseases in cattle. While the Macavirus genus contains agents, including alcelaphine gammaherpesvirus 1 (AlGHV1), ovine gammaherpesvirus 2 (OvGHV2), and caprine gammaherpesvirus-2 (CpGHV2), known to cause malignant catarrhal fever (MCF), and are collectively referred to as MCF virus (MCFV) group of organisms, diseases and/or clinical syndromes have not been associated with BoGHV6 and porcine lymphotropic herpesvirus (PLHV). This report investigated the occurrence of BoGHV6 in tissues of aborted dairy fetuses known to be infected by Histophilus somni to identify possible disease patterns associated with infection by this Macavirus. A nested-PCR (nPCR) assay was used to amplify the BoGHV6 polymerase gene from multiple tissues of 13 fetuses and the cow of one of these which were derived from seven dairy herds located in three geographical regions of Brazil. Direct sequencing confirmed the results of the nPCR assays. Additionally, all fetal tissues were previously investigated for the presence of H. somni, Listeria monocytogenes, Neospora caninum, Brucella abortus, Leptospira spp., bovine alphaherpesvirus 1, and bovine viral diarrhea virus (BVDV) by PCR and/or RT-PCR assays. The nPCR assay amplified BoGHV6 DNA from fetuses of most dairy herds (85.7%; 6/7) investigated, resulting in the amplification of BoGHV6 from 76.9% (10/13) of all fetuses evaluated from two geographical and important cattle-producing regions of Brazil. Furthermore, only BoGHV6 was identified in the spleen (n = 3), myocardium, and kidney (n = 2) of five fetuses, and BoGHV6 was the only agent associated with myocarditis in one of these. Nevertheless, dual, triple, and quadruple infections (including BVDV, B. abortus, and N. caninum) were identified in fetuses that were concomitantly infected by H. somni. Phylogenetic analysis revealed that the strain herein identified has 100% nucleotide (nt) sequence identity with wild type strains of BoGHV6 circulating in ruminants from Brazil and 99.8% nt identity with the reference strain of BoGHV6 but was 72.2–73.3% and 67.4–68.2% different from members of the MCFV group and PLHV, respectively. These results demonstrated that 76.9% of the fetuses evaluated were infected by BoGHV6, most likely via vertical infection resulting in transplacental transmission. Considering that most fetuses were concomitantly infected by BoGHV6 and H. somni the real impact of this viral infection cannot be efficiently determined. However, since BoGHV6 was the only pathogen identified in the myocardium of one fetus with myocarditis by histopathology, the possible participation of this Macavirus in the etiopathogenesis of the myocardial disease observed in this fetus cannot be ignored or discarded. However, the mere amplification of BoGHV6 DNA from the myocardium is not enough to establish a definite association between cause and effect, since in situ evaluations and experimental studies would be needed to confirm this agent in the etiopathogenesis of fetal diseases and/or abortions in cattle. Consequently, additional studies are needed to determine the exact role, if any, of BoGHV6 in the development of fetal disease, and possibly fetal mortality.

Molecular identification of Theileria species in naturally infected sheep using nested PCR-RFLP.

Ovine theileriosis is an important tick-borne haemoprotozoan disease of sheep in tropical and subtropical regions, causing severe productivity and economic loss. There is a paucity of information related to molecular studies of ovine theileriosis from India. The present study identified different Theileria spp. in naturally infected sheep using nested PCR-restriction fragment length polymorphism (nPCR-RFLP). Blood samples and ticks were collected from 204 sheep in different agro-climatic zones of Haryana state, India, during the tick active season. Microscopic examination of thin blood smears revealed 33.3% (68/204) infections with Theileria spp., while 44.6% (91/204) of blood samples were positive by nPCR assay. Different Theileria spp. were identified based upon RFLP patterns using four restriction enzymes: Hpa II, Bsh 1285I, Hae II and Rsa I. Out of 91 positive samples, 50.5% (46/91), 23.08% (21/91), 11% (10/91) and 2.2% (2/91) were positive for T. ovis, T. lestoquardi, T. luwenshuni (Theileria sp. China 1/Theileria sp. China) and T. annulata, respectively. Mixed infection was detected in 13.2% (12/91) of cases. Based upon HpaII enzymatic digestion pattern, two samples with T. lestoquardi and T. annulata, nine samples with T. lestoquardi and T. ovis and one sample with T. ovis and T. annulata were detected. The presence of these Theileria spp. was further confirmed by sequence analysis. The majority of ticks collected from sheep were identified as Rhipicephalus spp. followed by Hyalomma anatolicum and Hemaphysalis spp. The present investigation depicts the first comprehensive molecular report of naturally infected sheep with T. ovis, T. lestoquardi, T. annulata and T. luwenshuni from northern India.

Development of a duplex real-time PCR assay for simultaneous detection and differentiation of Theileria equi and Babesia caballi.

Equine Piroplasmosis (EP) is a tick-borne disease caused by three apicomplexan protozoan parasites, Theileria equi (T. equi), Babesia caballi (B. caballi) and T. haneyi, which can cause similar clinical symptoms. There are five known 18S rRNA genotypes of T. equi group (including T. haneyi) and three of B. caballi. Real-time PCR methods for detecting EP based on 18S rRNA analysis have been developed, but these methods cannot detect all genotypes of EP in China, especially genotype A of T. equi. In this study, a duplex real-time PCR detection method was developed for the simultaneous detection and differentiation of T. equi and B. caballi. The primers and probes for this duplex real-time PCR assay were designed based on the conserved 18S rRNA gene sequences of all genotypes of T. equi and B. caballi including Chinese strain. Double-quenched probes were used in this method, which provide less background and more signal to decrease the number of false positives relative to single-quenched probes. The newly developed real-time PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. The real-time PCR assays were further validated by comparison with a nested PCR assay and a previous developed real-time PCR for EP and sequencing results in the analysis of 506 clinical samples collected from 2019 to 2020 in eleven provinces and regions of China. Based on clinical performance, the agreements between the duplex real-time PCR assay and the nPCR assay or the previous developed real-time PCR assay were 92.5% (T. equi) and 99.4% (B. caballi) or 87.4% (T. equi) and 97.2% (B. caballi). The detection results showed that the positivity rate of T. equi was 43.87% (222/506) (10 genotype A, 1 genotype B, 4 genotype C, 207 genotype E), while that of B. caballi was 5.10% (26/506) (26 genotype A), and the rate of T. equi and B. caballi co-infection was 2.40% (12/506). The established method could contribute to the accurate diagnosis, pathogenic surveillance and epidemiological investigation of T. equi and B. caballi infections in horses.

Wastewater-Based Epidemiology and Long-Read Sequencing to Identify Enterovirus Circulation in Three Municipalities in Maricopa County, Arizona, Southwest United States between June and October 2020.

We used wastewater-based epidemiology and amplicon-based long-read high-throughput sequencing for surveillance of enteroviruses (EVs) in Maricopa County, Arizona, Southwest United States. We collected 48 samples from 13 sites in three municipalities between 18 June and 1 October 2020, and filtered (175 mL each; 0.45 µm pore size) and extracted RNA from the filter-trapped solids. The RNA was converted to cDNA and processed through two workflows (Sanger sequencing (SSW) and long-read Illumina sequencing (LRISW)) each including a nested polymerase chain reaction (nPCR) assay. We subjected the ~350 bp amplicon from SSW to Sanger sequencing and the ~1900–2400 bp amplicon from LRISW to Illumina sequencing. We identified EV contigs from 11 of the 13 sites and 41.67% (20/48) of screened samples. Using the LRISW, we detected nine EV genotypes from three species (Enterovirus A (CVA4, EV-A76, EV-A90), Enterovirus B (E14) and Enterovirus C (CVA1, CVA11, CVA13, CVA19 and CVA24)) with Enterovirus C representing approximately 90% of the variants. However, the SSW only detected the five Enterovirus C types. Similarity and phylogenetic analysis showed that multiple Enterovirus C lineages were circulating, co-infecting and recombining in the population during the season despite the SARS-CoV-2 pandemic and the non-pharmaceutical public health measures taken to curb transmission.

Detection rate and genotyping of Cryptosporidium spp. and its relation to copro TNF-α in elderly Egyptians attending outpatient clinics of Cairo University Hospitals

Background: Elderly individuals are considered an at-risk population, susceptible to enteric infections;and Cryptosporidium spp. is an apicomplexan protozoan considered to be one of the most commonprotozoa causing diarrhea. Cryptosporidiosis causes elevation of many pro-inflammatory cytokines liketumor necrosis factor-alpha (TNF-α) which may play a role in pathogenesis of the disease.Objectives: This study was designed for detection and genotyping of Cryptosporidium spp. in elderlypatients and the relationship of infection with copro TNF-α. Diagnosis was by evaluation of permanentacid-fast cold Kinyoun's (AF) staining, immunochromatographic detection (ICT), and ELISA in comparisonto molecular diagnosis as gold standard diagnostic method.Subjects and Methods: Stool samples were collected from 270 elderly patients aged above 60 yearsold attending outpatient clinics of Internal Medicine Hospital, Cairo University. Samples were examinedmicroscopically by direct wet mount, and AF staining, and then subjected to ICT, ELISA, and nested PCR(nPCR) assays. Positive samples by nPCR were then subjected to Restriction fragment length polymorphism(RFLP) to detect Cryptosporidium genotypes. Copro-levels of TNF-α were measured to assess theirrelationship with cryptosporidiosis.Results: Cryptosporidiosis detection rates of 3.7%, 6.3%, 6.7%, 3.7% were determined by microscopicexamination after AF staining, ICT, ELISA and nPCR, respectively. When RFLP was performed on nPCRpositive samples, eight and two samples were assigned as genotype 1 and 2, respectively. Moreover, TNF-αwas significantly correlated with cryptosporidiosis.Conclusion: The elderly are highly vulnerable to cryptosporidiosis. Immunodiagnosis and moleculartechniques are fundamental for the diagnosis of cryptosporidiosis. Cryptosporidiosis significantly affectscopro TNF-α.

Detection of Rickettsia lusitaniae Among Ornithodoros sawaii Soft Ticks Collected From Japanese Murrelet Seabird Nest Material From Gugul Island, Republic of Korea.

In a follow-up to the investigations of soft ticks identified from seabird nest soil and litter collected from coastal islands of the Republic of Korea (ROK), Ornithodoros sawaii and Ornithodoros capensis were assessed for the presence and identification of rickettsiae. Ticks collected from samples of 50-100 g of nest litter and soil from seabird nests were identified individually by morphological techniques, and species confirmed by sequencing of the mt-rrs gene. Subsequently, tick DNA preparations were screened for the presence of rickettsiae using a genus-specific nested PCR (nPCR) assay targeting the 17 kDa antigen gene. The amplicons from the 17 kDa assay and two additional nPCR assays targeting the gltA and ompB gene fragments were sequenced and used to identify the rickettsiae. A total of 134 soft ticks belonging to two species, O. sawaii Kitaoka & Suzuki 1973 (n = 125) and O. capensis Neumann 1901 (n = 9), were collected. Rickettsia lusitaniae DNA was detected and identified among O. sawaii ticks (n = 11, 8.8%) collected from nest litter and soil of the Japanese murrelet (Synthliboramphus wumizusume Temminck 1836) at Gugul Island along the western coastal area of the ROK. This study confirmed for the first time the presence of R. lusitaniae associated with O. sawaii collected from migratory seabird nests in the ROK.

Molecular Detection and Identification of Babesia spp., Theileria spp., and Anaplasma spp. in Sheep From Border Regions, Northwestern China.

Babesia, Theileria, and Anaplasma are important causative agents of tick-borne diseases that severely affect sheep. However, there is paucity in the occurrence genetic diversity of the infections of tick-borne diseases in sheep in border regions, northwestern China. In this study, nested polymerase chain reaction (nPCR) assays and gene sequencing were used to identify tick-borne Babesia spp., Theileria spp., and Anaplasma spp. infections in border regions, northwestern China. Out of 323 samples tested in this study, 225 (69.7%) sheep were infected with Babesia spp., Theileria spp., and Anaplasma spp. Two hundred six (63.8%), 60 (18.6%), 54 (16.7%), 51 (15.8%), 32 (9.9%), 19 (5.9%), and 16 (5.0%) were positive for A. ovis, B. motasi-like, A. bovis, T. uilenbergi, A. phagocytophilum, T. luwenshuni, and B. motasi-like Xinjiang, respectively. The most common dual infection was with A. ovis and B. motasi-like while the most frequent triple coinfection was A. ovis, B. motasi-like, and T. uilenbergi with coinfection rates of 17.0% (55/323) and 5.0% (16/323), respectively. Sequencing analysis indicated that A. ovis MSP4, A. phagocytophilum epank1, A. bovis 16S rRNA, B. motasi-like rap1-b, B. motasi-like Xinjiang rap1-a, T. luwenshuni 18S rRNA, and T. uilenbergi 18S rRNA from border regions, northwestern China, showed 99–100% identity with documented isolates from other countries. To the best of the authors' knowledge, this is the first report of T. uilenbergi and T. luwenshuni infections of sheep in border regions, northwestern China. Furthermore, these findings provide important data for understanding the distribution of Babesia, Theileria, and Anaplasma in sheep between border countries and China.