Positive Amplicons Research Articles

Molecular Detection and Identification of Piroplasms in Semi-Intensively Managed Cattle from Abeokuta, Nigeria

Abstract Piroplasmosis is a tick-borne haemolytic disease caused by different species of the Babesia and Theileria genera. Data on the prevalence of bovine piroplasms and their genetic diversity are scanty in Nigeria. Hence, this study reported the detection of some piroplasms in the blood of cattle in Abeokuta, Nigeria by the polymerase chain reaction (PCR). Blood samples were collected from 252 cattle and subjected to DNA extraction followed by PCR amplification of the partial region of 18S rRNA of the haemoprotozoans. Selected positive amplicons were unidirectionally sequenced and compared to the reference sequences from the Genbank. A total of 220 (87.3 %) cattle were positive for Theileria velifera and/or Babesia bigemina. The T. velifera was detected only in 163 (64.7 %) cattle, while 7 (2.8 %) cattle had a single infection with B. bigemina. Fifty cattle (19.8 %) had mixed infections with both parasites. There were no significant differences in piroplasm infections between the ages of cattle for both parasites. There were no significant differences in infection rates between the sexes for T. velifera, while the males had a significantly higher (P < 0.05) rate of infection for B. bigemina than the female cattle. The molecular detection of Babesia and Theileria species of cattle are reported for the first time in cattle in Abeokuta, Nigeria. This study, which confirmed the endemic nature of the parasites in cattle in the study area, stresses their importance in livestock health and production in Nigeria.

Serotyping versus genotyping in infected sheep and goats with small ruminant lentiviruses

Despite SRLV infection being endemic in Mexico, there is little information regarding which genotypes are present. We compared serotyping and PCR-sequencing results from sheep and goats infected with SRLV. We separated plasma and peripheral blood leukocytes (PBL) from 1940 blood samples from sheep and goats from 12 states across Mexico. To detect SRLV infection, we tested plasma samples using two commercial ELISA kits (VMRD and Eradikit SRLV Screening). Then, we serotyped the infecting virus (A/ B) using Eradikit SRLV Genotyping. PBL DNA was used to detect the proviral genome via PCR. Positive amplicons were sequenced to identify viral genotypes using a phylogenetic analysis. Also, we analysed for residues differences in the sequences of a capsid epitope between genotypes. The serological results indicated a higher detection of seropositive animals using the VMRD ELISA compared to Eradikit, with 21 % and 15.3 % more in sheep and goats respectively. Only 25.7 % of the ELISA serotyping results matched those from PCR-sequencing. PCR-sequencing was able to identify genotype A, B and coinfections in animals classified as indeterminate by the ELISA test. This lack of sensitivity may be related to the lack of epitopes from the matrix and transmembrane peptides used by ELISA screening. Sequences analysis revealed that SRLVs found in sheep cluster with genetic subtypes A2 and B1, while those in goats cluster with subtypes A1 and B1. Serotyping did not prove to be an adequate method for predicting the viral genotype (A and / or B) in infections caused by SRLV.

Isolation and Genetic Characterization of Cryptosporidium from Captive Wildlife of India

Background: Cryptosporidiosis is an emerging zoonotic protozoan disease caused by Cryptosporidium spp. The infection was reported worldwide from domestic animals and humans, including wild animals. From India, no such reports were published on Cryptosporidium infection in captive wildlife. Hence, a pilot study was conducted to report the occurrence of Cryptosporidium infection in captive wildlife of India. Methods: Faecal samples (n=788) were collected from 127 captive wildlife species of three zoological parks viz., Sri Venkateswara Zoological Park (SVZP), Tirupati (n=242); Indira Gandhi Zoological Park (IGZP), Visakhapatnam (n=218); Nehru Zoological Park (NZP), Hyderabad (n=328) and screened for Cryptosporidium infection. Preliminary screening of faecal samples was done for the detection of Cryptosporidium oocysts by modified Ziehl-Neelsen (mZN) staining method and the test positives were confirmed by nested PCR targeting 18S rRNA gene. Nested PCR amplicons were sequenced for determining the Cryptosporidium species. The resultant data were statistically analyzed by Fisher/Chi square, Fisher Exact test using SPSS software v 17.0.Result: In mZN staining method, 7.23 percent of isolates were found to be positive for Cryptosporidium and the highest rate of infection was detected in wildlife at NZP, Hyderabad (8.23%), followed by SVZP, Tirupati (7.44%) and IGZP, Visakhapatnam (5.50%). Cryptosporidium positive faecal samples by mZN staining were further confirmed by nested PCR and positive amplicons were sequenced for determination of Cryptosporidium species. Genetic characterization revealed five species viz., Cryptosporidium parvum; C. ryanae, C. suis, C. muris and Cryptosporidium avian genotype III. The study conclude that, Cryptosporidium infection was prevalent in the captive wildlife from the zoological parks of India and species variation was marked among the wildlife. Based on the available literature, the current study is the first of its kind on the prevalence of Cryptosporidium in captive wildlife from India.

Molecular detection and identification of eight potato viruses in Gansu province of China

Potato (Solanum tuberosum), a major food crop in the world is commonly infected with viral diseases, causing a loss in both quality and quantity of this crop. To determine the prevalence of eight important potato viruses in Gansu province of China, tuber samples were collected from Weiheyuan potato seed industry development Co., Ltd., and nineteen different commercial potato production fields at different locations in October 2018. Viruses were identified by RT-PCR using coat protein (CP)-specific primers, and validated by cloning three PCR positive amplicons of each virus followed by sequencing of three positive clones from each plasmid. Sequences were deposited in the GenBank. RT-PCR results demonstrated that mixed infections were very common in the collected tuber samples. Mixed infection of Potato virus X (PVX) and Potato virus S (PVS) caused 12.5 %; PVX and Potato aucuba mosaic virus (PAMV) caused 20.83 %; PVX, PVS and PAMV caused 12.5 %; PVX, Potato virus H (PVH) and PAMV caused 8.33 %; Potato leafroll virus (PLRV), PVX, PVS and PAMV caused 8.33 %; PVX, PVS, PVH and PAMV caused 16.67 %; PLRV, PVX, PVS, PVH and PAMV caused 8.33 %; and PVX, PVS, PVH, PAMV and Potato virus M (PVM) caused 4.17 % tuber infection, respectively; whereas, Potato virus Y (PVY) and Potato virus A (PVA) were not detected. PVY and PVA requires further testing in the future, and the result for these two viruses in the current study cannot be validated due to the lack of positive controls. PVS and PVH were also confirmed by western blot analysis. Phylogenetic analysis further revealed that the PLRV from Gansu was closely related to a Guangdong isolate (GenBank accession no. MF062487), PVX was closely related to a Shandong isolate (GenBank accession no. AF628555), PVS was closely related to a Guangxi isolate (GenBank accession no. KF011279) and an Australi isolate (GenBank accession no. GU319949), PVH was closely related to a Yunnan isolate (GenBank accession no. JQ904630), PAMV was closely related to a Bangladeshi isolate (GenBank accession no. MF133526), and PVM was closely related to a Tanzania isolate (GenBank accession no. KC866622). These findings will provide a basis for control of potato viruses in this region in the future.

Molecular characterisation of csgA gene among ESBL strains of A. baumannii and targeting with essential oil compounds from Azadirachta indica

ObjectivesA. baumannii is considered as a “red alert” nosocomial human pathogen and exhibits an extensive antibiotic resistance spectrum. The biofilm formation mediated by the csgA is a potent virulence factor in A. baumannii and targeting the same would be of a novel strategy to control A. baumannii infections. The aim of the present study is thus to evaluate the anti-biofilm activity of essential bio-compounds from Azadirachta indica against the ESBL producing strains of A. baumannii by in-vitro and in-silico studies. MethodsBiofilm formation by Semi-quantitative adherence assay was performed for the 73 strains of ESBL producing A. baumannii. Genomic DNA was extracted and molecular characterization of csgA gene was done by PCR amplification with further sequencing. In-vitro anti biofilm assay from crude extract of A.indica was performed which was then followed by the in-silico docking involving retrieval of csgA protein and ligand optimisation, molinspiration assessment on drug likeliness, docking simulations and visualisations. ResultsBiofilm assay showed 58.9%, 31.5% and 0.09% as high grade, low grade and non-biofilm formers respectively. 20.54% (15/73) of the screened genomes showed positive amplicons for the csgA gene associated with biofilm formation among the ESBL producing strains of A. baumannii. All the ceftazidime, cefipime and cefotaxime resistant strains showed the presence of csgA gene (100%; 15/15), followed by 46.6% (7/15) resistant isolates for ceftriaxone. In-vitro crystal violet viability assay showed MBEC50 and MBEC90 at a concentration of 20 µl and 40 µl respectively. In-silico assessments on the essential oil compounds from neem showed imidazole to exhibit the highest interaction with least docking energy and high number of hydrogen bonds. ConclusionThe current study emphasises thatimidazole from A.indica to be a promising candidate for targeting the csgA mediated biofilm formation in ESBL strains of A. baumannii. However, further in-vivo studies have to be implemented for the experimental validation of the same.

Diagnosis, genetic variations, virulence, and toxicity of AHPND-positive Vibrio parahaemolyticus in Penaeus monodon

Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp (Penaeus monodon) disease caused by Vibrio parahaemolyticus (VP) since 2013 in Bangladesh. The aim of this work was to evaluate a PCR and RT-PCR techniques as rapid methods for detecting V. parahaemolyticus AHPND-positive P. monodon using genetic markers. Healthy and diseased shrimp (P. monodon) samples were collected from three monitoring stations. The samples were enriched in TCBS plates and DNA extraction from the cultured bacteria. DNA quantifications, PCR amplification, RT-PCR, and gene sequencing were done for the detection of V. parahaemolyticus AHPND-positive P. monodon. The sequence of PCR amplicons showed 100% identity and significant alignment with V. parahaemolyticus. The primers used provided high specificity for V. parahaemolyticus in PCR detection compared with another Vibrio species. In the PCR, amplification resulted positive amplicons, whereas, non-AHPND isolates showed no amplicons. Neighbor-joining methods indicated that all genes evolved from a common ancestor and clades have different traits with very low genetic distance and low variability. The pairwise alignment scores of atpA, tox, blaCARB, 16S rRNA, and pirA genes were 100.0, 98.90, 98.89, 95.53, and 41.42, respectively. The RT-qPCR exposed variable expression levels for all genes in the AHPND-positive strain. Homology analysis and distance matrix exhibited all genes to have the lowest similarity and most divergence, offering the highest specificity. In this study, the expression and variability of target genes confirmed the presence of V. parahaemolyticus in all sampling sites. The results suggested that PCR amplification, RT-qPCR, and gene sequencing can be used for the rapid detection of V. parahaemolyticus in AHPND-positive P. monodon that may lead to subsequent prevention and treatment research in the future for managing this disease.

Frequency of Adhesive Virulence Factor fimH among the Clinical Isolates of Acinetobacter baumannii in India

The objective of the study was to detect the presence of fimH gene among the drug resistanst strains of Acinetobacter baumannii. fimH gene was found to be associated with a catch bond mechanism which led to better evolution of biofilm formation. Since there are not many studies done with this gene it would be a timely investigation and this study mainly aims in molecular characterization of fimH gene among clinical isolates of A. baumannii. Semi quantitative bio adherent assay was done by the multidrug resistant strains of A. baumannii to find the formation of biofilm. The DNA was extracted with the help of kit and PCR was performed for amplification. Pearson correlation analysis was done to find the existing correlation between the fimH gene and MDR strains of A. baumannii with significant p-value of (<0.05). From the screened 73 genomes of MDR A. baumannii 6.8% showed positive amplicons for the fimH gene which were related to biofilm and porin formation (Fig. 1). Correlation of its existence was high in beta lactamase (100%), cephems (100%), folate (100%) resistant strains, followed by aminoglycosides (80%), carbapenems (60%) and fluoroquinolones (60%) and efflux pumps (20%). In Spite of various measures undertaken to prevent the disease, the prevalence of the pathogen is multiplying. The current study recorded the presence of fimHgene (6.8%) among the clinical isolates of A.baumannii. This gene can be used as a target to develop new drugs and vaccines to combat the menace of A.baumannii infection.

Detection of tick-borne pathogens in Rhipicephalus sanguineus sensu lato and dogs from different districts of Portugal

Dogs are highly exposed to pathogens transmitted by ectoparasites. The Mediterranean climate of Southern Europe, together with the presence of stray and/or neglected pets in close proximity with humans, contribute for tick expansion and stand for increased risk to infections in humans due to the zoonotic potential of many of these agents.The aim of this study was to perform a molecular survey in dogs (suspected of tick-borne disease and/or infested with ticks), as well as in ticks collected from those animals, from 12 districts of Portugal to investigate the occurrence of Rickettsia spp. and other tick-borne pathogens (Babesia, Ehrlichia, Anaplasma and Hepatozoon). Additionally, a serological survey of spotted fever group Rickettsia in Portuguese dogs was performed using an in-house immunofluorescence assay (IFA).A total of 200 whole-blood samples and 221 Rhipicephalus sanguineus s. l. ticks were collected from dogs.A total of 14 (7 %) blood samples and 10 (4.5 %) ticks yielded presumptively positive 420-bp amplicons using the Rickettsia spp. partial ompB nested PCR. Screening of the ompB-positive samples using the gltA gene showed 8 positive ticks. All Rickettsia ompB and gltA sequences had the highest identity with R. massiliae. The Rickettsia-positive dogs were further tested for other tick-borne pathogens and were found to be infected with Babesia spp. (n = 5), but not with Ehrlichia, Anaplasma or Hepatozoon.Of the 149 dog serum specimens tested in the serological assay, 103 (69 %) were positive for IgG antibodies against spotted fever group Rickettsia. Antibodies were found in dogs from all the studied districts, in 55 (53 %) of the stray and in 48 (47 %) of the owned dogs.Our study detected and characterized for the first time R. massiliae in dogs from Portugal, broadening the geographical range of this canine pathogen and adding knowledge to the impact of this disease in dogs.

First identification of genotypes of Enterocytozoon bieneusi (Microsporidia) among symptomatic and asymptomatic children in Mozambique.

Enterocytozoon bieneusi is a human pathogen with a broad range of animal hosts. Initially, E. bieneusi was considered an emerging opportunistic pathogen in immunocompromised, mainly HIV-infected patients, but it has been increasingly reported in apparently healthy individuals globally. As in other African countries, the molecular epidemiology of E. bieneusi in Mozambique remains completely unknown. Therefore, we undertook a study to investigate the occurrence and genetic diversity of E. bieneusi infections in children with gastrointestinal symptoms as well as in asymptomatic children in Mozambique. Individual stool specimens were collected from 1,247 children aged between 0 and 14 years-old living in urban and rural settings in Zambézia (n = 1,097) and Maputo (n = 150) provinces between 2016 and 2019. Samples were analysed for E. bieneusi by nested-PCR targeting the internal transcribed spacer (ITS) region of the rRNA gene. All positive amplicons were confirmed and genotyped. Penalised logistic regression (Firth) was used to evaluate risk associations. The overall prevalence of E. bieneusi in this children population was 0.7% (9/1,247). A 10-fold higher prevalence was found in Maputo (4.0%; 6/150) than in Zambézia (0.3%; 3/1,097). All E. bieneusi-positive samples were from children older than 1-year of age, and most (8/9) from asymptomatic children. Nucleotide sequence analysis of the ITS region revealed the presence of four genotypes, three previously reported (Peru11, n = 1; Type IV, n = 2, and S2, n = 2) and a novel genotype (named HhMzEb1, n = 4). Novel genotype HhMzEb1 was identified in both asymptomatic (75%, 3/4) and symptomatic (25%, 1/4) children from a rural area in Maputo province in southern Mozambique. Genotypes HhMzEb1, Peru11, S2, and Type IV belonged to the Group 1 that includes genotypes with low host specificity and the potential for zoonotic and cross-species transmission. Being infected by enteric protozoan parasites and no handwashing were identified as risk associations for E. bieneusi infection. This study reports the first investigation of E. bieneusi genotypes in Mozambique with the identification of three previously reported genotypes in humans as well as a novel genotype (HhMzEb1). Findings highlight the need to conduct additional research to elucidate the epidemiology of E. bieneusi in the country, especially in rural areas where poor hygiene conditions still prevail. Special attention should be paid to the identification of suitable animal and environmental reservoirs of this parasite and to the characterization of transmission pathways.

Molecular detection, prevalence and risk factors of Theileria orientalis infection among cattle in Peninsular Malaysia

There is need to confirm the presence of Theileria orientalis among the cattle population in Peninsular Malaysia and to evaluate the risk factors associated with the infection. To this effect, blood samples were collected from 1045 cattle from 43 farms throughout the entire States of Peninsular Malaysia. The collected blood samples were subjected to DNA extraction and subsequent PCR amplification of the major piroplasm surface protein (MPSP) gene of the haemoprotozoan. Representative positive amplicons were purified, sequenced and compared with other sequences of the MPSP gene of T. orientalis curated from the GenBank. A well-structured questionnaire was used to get information about each cattle, it’s demography, the bio-security, environmental and management factors. Univariate and multivariate analysis were used for the statistical evaluation, with significance set at p < 0.05. A total prevalence of 49.76% (520/1045; 95% CI: 46.73 – 52.79) was obtained. Types of breeds, age, production type, herd size, level of farm biosecurity, farm size, presence of other animal species in the farm, management systems and prophylaxis were significantly (p < 0.05) associated with the prevalence of T. orientalis. This study confirmed the presence of T. orientalis and establish that the haemoprotozoan is endemic among cattle in Peninsular Malaysia.

Identification of Pathogenic Leptospira Species in the Urogenital Tract of Water Buffaloes (Bubalus bubalis) From the Amazon River Delta Region, Brazil.

In the current context of deforestation and fire in the Amazon, buffaloes could be a cost-effective and sustainable alternative for cattle production in the region, as they can convert low-quality foods and be raised in floodplain areas. However, little is known about the reproductive diseases that affect these animals; thus, the purpose of this study was to perform the molecular characterization of Leptospira spp. in the urogenital tract of water buffaloes (Bubalus bubalis) raised in the Amazon River Delta region in Brazil. Samples were collected from 114 kidneys, 204 ovaries, and 160 uterine swabs of slaughtered buffaloes in the Macapá microregion of Amapá State (Brazil) and were subjected to PCR to detect bacterial DNA. Positive amplicons were sequenced to identify Leptospira species. Among the total samples, 11/473 were PCR positive (2.3%), including 10 kidney samples and one uterine swab sample. DNA sequencing identified two pathogenic species from the kidney samples: L. interrogans, accounting for 60.0% (6/10) of these samples, and L. borgpetersenii, accounting for 20.0% (2/10), while 20.0% (2/10) were identified only at the genus level. The bacterium in the uterine swab sample was identified as L. interrogans with genetic proximity to strains belonging to the serovar Hardjo. This is the first report of leptospires species identified in buffaloes from the Amazon River Delta region and revealed that these animals may be carriers of different pathogenic Leptospira species, similar to bovines, including showing genital colonization.

Comparative Nucleotide Sequence Analysis of Glycoprotein B, C, and G of Infectious Laryngotracheitis Virus Isolated in Egypt During 2016-2018

Infectious Laryngotracheitis (ILT) is an economically important disease of poultry, causing a high mortality rate and /or reduced egg production. Tracheas from sever morbid and recently died unvaccinated household chicken were subjected to PCR assay to detect the presence of the virus through amplification of the glycoprotein B gene. All tested samples gave positive ct ranging from 13.32 to 25.53. Virus isolation was performed by inoculation—the processed tracheal swaps onto the chorioallantoic membrane. Positive pock lesion has been developed within 7 days post-inoculation. The pock lesions were subjected to PCR assay for amplification of both the full-length glycoprotein B, C, and G for sequencing analysis. Positive amplicons migrating about 2600, 1250, and 890 bp were amplified corresponding to the orf of gpB, gpC and gpG genes. Sequence alignment and phylogenetic tree revealed that all sequenced isolates gave a high degree with wild type isolate of ILT and a high degree of genetic stability was clearly evident among strains isolated in different periods (2016-2018), indicating that these glycoproteins could be used as a vaccine candidate.

Molecular Detection of Rickettsia Spp. and Coxiella Burnetii in Cattle, Water Buffalo, and Rhipicephalus (Boophilus) Microplus Ticks in Luzon Island of the Philippines.

Rickettsia and Coxiella burnetii are zoonotic, tick-borne pathogens that can cause febrile illnesses with or without other symptoms in humans, but may cause subclinical infections in animals. There are only a few reports on the occurrence of these pathogens in cattle and water buffalo in Southeast Asia, including the Philippines. In this study, molecular detection of Rickettsia and C. burnetii in the blood and in the Rhipicephalus (Boophilus) microplus ticks of cattle and water buffalo from five provinces in Luzon Island of the Philippines was done. A total of 620 blood samples of cattle and water buffalo and 206 tick samples were collected and subjected to DNA extraction. After successful amplification of control genes, nested PCR was performed to detect gltA of Rickettsia and com1 of C. burnetii. No samples were positive for Rickettsia, while 10 (cattle = 7, water buffaloes = 3), or 1.6% of blood, and five, or 1.8% of tick samples, were C. burnetii-positive. Sequence analysis of the positive amplicons showed 99–100% similarity to reported C. burnetii isolates. This molecular evidence on the occurrence of C. burnetii in Philippine ruminants and cattle ticks and its zoonotic nature should prompt further investigation and surveillance to facilitate its effective control.

First evidence of bovine immunodeficiency virus infection in Mexican cattle.

This study set out to identify the presence of bovine immunodeficiency virus (BIV) in animals geographically located in Mexico. BIV was first discovered in the United States in a dairy cow with persistent lymphocytosis, lymphoid hyperplasia and lymphocytic encephalitis. Many studies indicate that BIV infection is globally distributed, but its presence in Mexico remains unknown. We collected 1,168 heparinized blood samples from cattle in ten states across the Mexican Republic, then separated plasma using centrifugation and tested for antibodies against BIV. We used an indirect ELISA based on the use of a synthetic peptide derived from transmembrane glycoprotein (gp45/TM). In order to identify the viral genome, we designed a synthetic gene as a PCR control, as well as a pair of oligonucleotides for amplifying a 519bp product of the env gene which encodes the surface protein. Positive amplicons were purified and subjected to nucleotide sequencing. A total of 189 (28.94%) tested plasma samples suggest the presence of specific anti-BIV antibodies in all states studied except for Chiapas. Additionally, PCR results identified six positive cows in the states of Puebla and Coahuila. BIV in these cows was confirmed via nucleotide sequencing and in silico analysis of these samples. This is the first report of the presence of BIV in Mexican cattle.

Molecular characterization of ‘tubifex worms’ based on 16S rRNA and cytochrome c oxidase subunit I

‘Tubifex worms’ encompass extensive cryptic diversity that requires molecular characterization to confirm the species commonly found in Bangladesh including their lineages. Therefore, this study focused on the molecular characterization and phylogeny analysis at lineages level by sequencing the fragment of 16S rRNA and cytochrome c oxidase subunit I (COI) genes through standard cloning and sequencing techniques followed by phylogenetic characterization. All six isolates, belong to the genus Limnodrilus that is confirmed into two different species (L. hoffmeisteri and L. claparedianus-cervix) of the tubifex. The phylogenetic analysis estimated that the resident worms are under the same clade although transmissions from other countries might likely occur through different aquatic routes and birds. The drawbacks were the use of a complex colony rather than a single worm and taking representative isolates instead of all the positive amplicons for sequencing. However, this study is the first report of genetical confirmation of the genus Limnodrilus at lineage level from Bangladesh.

Tick-borne pathogens in Ixodidae ticks collected from privately-owned dogs in Italy: a country-wide molecular survey

BackgroundTicks and tick-borne diseases are increasingly recognized as a cause of disease in dogs worldwide. The epidemiology of ticks and tick-transmitted protozoa and bacteria has changed due to the spread of ticks to urban and peri-urban areas and the movement of infected animals, posing new risks for animals and humans. This countrywide study reports information on distribution and prevalence of pathogens in ticks collected from privately-owned dogs in Italy.We analyzed 2681 Ixodidae ticks, collected from 1454 pet dogs from Italy. Specific PCR protocols were used to detect i) Piroplasms of the genera Babesia and Theileria, ii) Gram-negative cocci of the family Anaplasmataceae and iii) Borrelia burgdorferi sensu lato. Sequencing of positive amplicons allowed for species identification.ResultsBabesia/Theileria spp. DNA was detected in 435 homogeneous tick-pools (Minimum Infection Rate (MIR) = 27.6%; 95% confidence interval (CI) = 25.4–29.8%) with higher prevalence in Ixodes ricinus and Rhipicephalus sanguneus group. The zoonotic B. venatorum was the most prevalent species (MIR = 7.5%; 95% CI = 6.3–9.0%). Anaplasma and Ehrlichia species were detected in 165 tick-pools (MIR = 10.5%; 95% CI = 9.3–11.8%) and specifically, A. phagocytophilum was identified with MIR = 5.1% (95% CI = 4.1–6.3%). Borrelia burgdorferi s.l. and B. afzelii were detected with MIR = 0.4% (95% CI = 0.2–0.8%) and MIR = 0.3% (95% CI 0.1–0.7%) respectively.ConclusionsZoonotic pathogens B. venatorum and A. phagocytophilum were the most frequently detected in ticks collected from privately-owned dogs which might be used as markers of pathogens presence and distribution.

Molecular identification of Jujube witches'-broom phytoplasma (16SrV) associated with witches'-broom disease of Ziziphus oenoplia in India

Severe witches’-broom disease of Ziziphus oenoplia was observed with significant disease incidence in Bhopal, India, during 2019. Phytoplasma was detected from symptomatic leaf samples by polymerase chain reaction (PCR) using phytoplasma 16S rRNA gene specific primers which revealed positive amplification of expected size ~1.2 kb DNA band. The positive amplicons of the phytoplasma 16S rRNA (1.2 kb) were sequence and sequenced data was submitted in GenBank database (Accession no. MK975463 and MK975462). On the basis of highest 99% sequence identities, closest phylogenetic relationships and In silico of the under study both the phytoplasma isolates associated with witches'-broom disease of Ziziphus oenoplia identified as a species of Jujube witches'-broom phytoplasma as a member of Elm yellows group (16SrV ). To the best of our knowledge, this is the first report on the association of Jujube witches'-broom phytoplasma species of Elm yellows group (16SrV) with witches'-broom disease of Z. oenoplia in India.

Toxoplasma gondii infection in selected species of free-living animals in Poland.

Free-living animals can play an important role as a reservoir of Toxoplasma gondi;, however, data concerning this issue in Poland are still limited.The aim of study was to assess the occurrence of T.gondii infection by using molecular methods in free-living animals in selected regions of Poland. Tissues samples of 396 animals (foxes, muskrats, birds, martens, badgers, polecats, raccoons, minks, raccoon dogs, otters, small rodents and insectivores, and grass snakes were collected from various regions of Poland. After samples digestion, DNA was isolated using QIAmp DNA Mini Kit (Qiagen). DNA extraction from small rodents and insectivores samples was performed without digestion. Next, nested PCR (B1 gene) and, for a part of nested PCR positive amplicons, RFLP PCR, were performed according to the method by Grigg and Boothroyd (2001). The other part of nested PCR positive DNA isolates were genotyped using 5 genetic markers: SAG1, SAG2 (5'- and 3'), SAG3, BTUB and GRA6, based on the method by Dubey et al. (2006). These PCR products were sequenced and compared with the NCBI database using Blast. In total, in 50 of the 396 examined animals DNA of T. gondii was detected (12.6%). The highest percentages of positive results in PCR was obtained in martens (40.9%) and badgers (38.5%), lower in birds (27.3%) and the lowest in foxes (7.4%). The RFLP and multilocus PCR analysis showed the dominance of T. gondii clonal type II (or II/III). The results of this study indicate the frequent T. gondii infection among free-living animals in Poland, especially martens and badgers, which may indirectly indicate that these animals contribute to the spread of the parasite in the sylvatic environment in Poland. The genotyping analysis showed the dominance of T. gondii clonal type II (or II/III).

Comparative evaluation of Cryptosporidium infection in malnourished and well-nourished children: Parasitic infections are affected by the interaction of nutritional status and socio-demographic characteristics

Cryptosporidium, as a small protozoan parasite, is a leading cause of persistent diarrhea in children in developing countries and has both a short and long-term impact on the growth of children. In the present study, Cryptosporidium infection was compared in malnourished and well-nourished children by modified acid-fast staining, nested-polymerase chain reaction (nested-PCR) and loop-mediated isothermal amplification (LAMP) methods. As a case-control study, Cryptosporidium infection in 94 malnourished children was evaluated and compared with those of 188 age and gender-matched well-nourished children. Oocysts of Cryptosporidium were detected by modified acid-fast staining method. The extracted DNA was amplified by nested-PCR and LAMP techniques. In addition, positive amplicons were directly sequenced for phylogenetic analysis. Cryptosporidium oocysts were found in the stools of two (2.12 %) children who were hospitalized and had diarrhea by nested-PCR while three isolates (3.2 %) were found by LAMP. Cryptosporidium-positive children were more malnourished compared to those who were negative for Cryptosporidium infection but this important finding was not statistically significant. C. parvum was the main species of Cryptosporidium detected in malnourished children in northwest Iran. LAMP can be considered as a sensitive field monitoring assay in patients with low parasite burden. Nutritional status and socio-demographic factors may have interactive effects on the incidence and severity of parasitic diseases.

Molecular Genetics and the Incidence of Transmitted Drug Resistance Among Pre-Treatment HIV-1 Infected Patients in the Eastern Cape, South Africa.

Transmitted drug resistance (TDR) remains a significant threat to Human immunodeficiency virus (HIV) infected patients that are not exposed to antiretroviral treatment. Although, combined antiretroviral therapy (cART) has reduced deaths among infected individuals, emergence of drug resistance is gradually on rise. To determine the drug resistance mutations and subtypes of HIV-1 among pre-treatment patients in the Eastern Cape of South Africa. Viral RNA was extracted from blood samples of 70 pre-treatment HIV-1 patients while partial pol gene fragment amplification was achieved with specific primers by RT-PCR followed by nested PCR and positive amplicons were sequenced utilizing ABI Prism 316 genetic sequencer. Drug resistance mutations (DRMs) analysis was performed by submitting the generated sequences to Stanford HIV drug resistance database. Viral DNA was successful for 66 (94.3%) samples of which 52 edited sequences were obtained from the protease and 44 reverse transcriptase sequences were also fully edited. Four major protease inhibitor (PI) related mutations (I54V, V82A/L, L76V and L90M) were observed in seven patients while several other minor and accessory PIs were also identified. A total of 11(25.0%) patients had NRTIs related mutations while NNRTIs were observed among 14(31.8%) patients. K103N/S, V106M and M184V were the most common mutations identified among the viral sequences. Phylogenetic analysis of the partial pol gene indicated all sequences clustered with subtype C. This study indicates that HIV-1 subtype C still predominates and responsible for driving the epidemic in the Eastern Cape of South Africa with slow rise in the occurrence of transmitted drug resistance.