Msp2 Gene Research Articles

Cladistics and population structure analysis of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in Haryana, India based on genes encoding the major surface proteins

Anaplasma marginale, a tick-borne obligate intracellular rickettsia is incriminated to cause heavy economic losses throughout the tropical and subtropical regions, including India. However, studies highlighting the phylogeography and demographic dynamics of A. marginale are very scant from India. Thus, the present study assessed the cladistics and population structure of Anaplasma marginale based on the genes encoding the major surface proteins (MSP) 4 and 5. The cladistics based on phylogenetic tree including the sequences generated herein with the GenBank archived sequences of A. marginale was performed. The relationship between A. marginale haplotypes based on both the genetic markers was estimated by performing median joining (MJ) haplotype network analysis. Demographic dynamics involving population diversity indices and neutrality indices was also performed. The cladistics and MJ haplotype network analysis corresponding to both the markers demonstrated the presence of three distinct clades (1-3) of A. marginale. Population structure analysis revealed low nucleotide (0.00236 ± 0.00064 and 0.00955 ± 0.00101) and haplotype (0.321 ± 0.073 and 0.493 ± 0.083) diversities for the MSP4 and MSP5 genes, respectively. High genetic structuring and low gene flow [Nm values ranging between 0.044481 and 0.208337 for the MSP4 gene and 0.032735 (clades 1 and 2) for the MSP5 gene] was also recorded among the different clades of A. marginale, based on both genetic markers. The present study highlighted the presence of different clades of A. marginale distributed worldwide. The isolates circulating in north India belonged to the dominant clade prevalent throughout the globe.

Plasmodium falciparum genetic diversity; implications for malaria control in Ethiopia: Systematic review and meta-analysis.

In malaria endemic regions, Plasmodium falciparum infection is characterized by variable genetic diversity at different settings. The parasite's various forms are found at varied frequency in different geographic areas. Understanding malaria parasite diversity and transmission is vital to evaluate control interventions. The aim of this study was under taken to determine the status of P. falciparum genetic diversity and MOI in different regions of Ethiopia. Relevant publications were identified from electronic databases such as; PubMed, EMBASE, Google scholar and Google. Besides, an online search was done using the above databases for all articles published in English on genetic diversity of P. falciparum in Ethiopia. STATA software was used for data analysis. The pooled estimates were calculated using random effect model. The summary estimates were presented using forest plots and tables. A total of 11 studies were included in the systematic review. However, only 8, 10 and 2 studies were included for Pfmsp-1, Pfmsp-2 and glurp gene meta-analysis, respectively. However, the meta-analysis result showed that the pooled prevalence of Pfmsp-1, msp-2 and glurp gene were 84% for both msp-1/2% and 51%, respectively. The pooled prevalence of msp-1 gene was higher in Amhara followed by Oromia region and lower in SNNPR while, for msp-2 gene the pooled prevalence was higher in Benshangul gumez region. Among the allelic family of msp-1 and msp-2 genes, MAD20 (34%) and FC27 (44%) were the most predominant respectively. Based on the review, there is evidence of the presence of high genetic diversity of P. falciparum parasites in Ethiopia, suggesting that malaria transmission remain high and that strengthened control efforts are needed. The approaches and methods used for investigation of diversified parasites have similarity between studies and should use advanced molecular techniques, like microsatellite, to assess the genetic diversity of P. falciparum for better results.

Therapeutic efficacy of artemether–lumefantrine in the treatment of uncomplicated Plasmodium falciparum malaria in Arba Minch Zuria District, Gamo Zone, Southwest Ethiopia

BackgroundArtemether–lumefantrine (AL) has been the primary anti-malarial drug used to treat uncomplicated Plasmodium falciparum malaria in Ethiopia since 2004. However, there have been recent reports of AL resistance mutations in different African countries, including Ethiopia. This is concerning and requires periodic monitoring of anti-malarial drug resistance. Therefore, the current study aimed to evaluate the therapeutic efficacy of AL in treating uncomplicated P. falciparum malaria in the Arba Minch Zuria District, Gamo Zone, Southwest Ethiopia.MethodsA single-arm prospective study with a 28-day follow-up period was conducted from July to October 2022. Capillary blood samples were collected for RDT and microscopic examination. The study enrolled monoinfected P. falciparum patients aged ≥ 18 years at Ganta Sira Health Post. Sociodemographic and clinical data were recorded, and a dried blood spot (DBS) was prepared for each participant. Nested polymerase chain reaction (nPCR) genotyping of the msp-1 and msp-2 genes was only performed for recurrent cases to distinguish between recurrence and reinfection. Data entry and analysis were performed using the WHO Excel spreadsheet and SPSS version 26.ResultsA total of 89 patients were enrolled, and 67 adequately completed the 28-day follow-up period. AL showed a 100% clearance rate for fever on day 2 and asexual parasites on day 3. Gametocytes were detected in 13.5% (12/89) of the participants. The gametocyte clearance rate was 58.3% (7/12) until day 7 and 100% (12/12) until day 14. Five participants developed recurrent malaria, three of whom experienced relapse and two of whom experienced reinfection. Based on the Kaplan–Meier survival analysis, the PCR-uncorrected and PCR-corrected cumulative incidence of success were 93.7% (95% CI 85.5–97.3) and 96.2% (95% CI 85.5–98.7), respectively.ConclusionAL was efficacious in treating uncomplicated P. falciparum malaria in the study area. However, the detection of recurrent patients highlights the need for continuous efficacy studies in this area.

Rhipicephalus (Boophilus) microplus Infestation and its Implications in Anaplasma marginale Infection among Cattle in South Gujarat, India

Background: This study aims to identify risk factors for Rhipicephalus (Boophilus) microplus ticks’ infestation in South Gujarat’s cattle and explore its implications on Anaplasma marginale. Methods: An approach was employed to retrieve the crucial insights from the owners and examinations of 2305 cattle (crossbred=1495, indigenous=810) of South Gujarat, India. Species specific PCR was used to detect 576 bp of msp5 gene of A. marginale in the oviposited ticks. Result: Crossbred cattle (73.91%) had higher tick prevalence than indigenous ones (55.80%), with R. (B.) microplus being the dominant species. Highest tick infestation was noted in cattle greater than 3 years (74.43%), followed by less than 1 year (70.23%) and 1-3 years old (55.60%). Ticks collected from crossbred exhibited higher prevalence of A. marginale compared to indigenous cattle. Significantly highest (p greater than 0.05) ticks infestation rate was noted in cattle with pale conjunctiva, poor body condition and black coat colour. Infection rate of both the parasites was highest during summer, followed by monsoon and winter. Kachcha house and earthen floor favoured the survival of the pathogen. Owners in surveyed regions combat tick infestations using mechanical (7.69%), chemical (83.51%) and herbal/no treatments (10.07%). These findings emphasize the intricate link between tick infestations and A. marginale infection, offering crucial insights for cattle health management.

Epitope prediction of malarial MSP-1 protein of plasmodium sp. using in silico method

Due to its pivotal function in parasite adherence to the RBC membrane during erythrocyte invasion, Msp1 represents a potential vaccine candidate. Three or four fragments of MSP1 are produced during the proteolytic maturation process, and these fragments stay together as a complex on the cell surface. Msp1 fragments have been investigated as potential vaccine candidate, and malaria antibody immunoassays have made use of the fragmented polypeptide, which exhibits a considerable degree of intra-species conservation. Because of its polymorphism, Msp1 is also useful in strain differentiation and carries a crucial genetic marker. To comprehend genetic variation among species and find epitopes for the development of a disease vaccine, Msp1 needs to be investigated. The preliminary investigation on the evolutionary history using the Maximum Likelihood method and the General Time Reversible model (GTR) models were used. The phylogenetic relationship of the MSP1 gene was inferred and it represents the level of similarity between the MSP1 gene of Plasmodium species from various locations in South-East Asia. The top 30 MHC class II epitopic regions were predicted using Bepipred server with epitope mean score above 0.05.

High genetic diversity of Anaplasma ovis in sheep from Bosnia and Herzegovina.

Ovine anaplasmosis (sensu stricto) is a rickettsial blood disease caused by the tick-borne species Anaplasma ovis. The disease is characterized by mild anemia, fever, and icterus. A more severe clinical presentation is possible in non-endemic areas. There is no existing data on the presence of Anaplasma ovis in Bosnia and Herzegovina. However, given the country's location within the Mediterranean Basin and the recent molecular detection of Babesia ovis, it is plausible that sheep in the region could naturally be infected with this tick-borne pathogen. Blood samples from 81 sheep in the Podrinje and Herzegovina areas were examined by PCR. PCR positivity was found in 38 (46.9%) cases indicating a high number of infected sheep. Mixed infections with Babesia ovis and A.ovis were observed in 63.3% of cases. A higher number of positive sheep was recorded in the area of Herzegovina. Phylogenetic analysis of the gltA, groEL, and msp4 genes of A. ovis revealed numerous genotypes and significant genetic variability. This diversity was not related to geographic origin, tick-borne infection status, or sheep breeding practices in Podrinje and Herzegovina. The data obtained in this study suggest that the emergence of new genotypes and the high genetic variability of A. ovis are driven by specific local and micro-environmental factors.

Quality Control of Microscopic Diagnosis of Malaria in Healthcare Facilities and Submicroscopic Infections in Mossendjo, the Department of Niari, the Republic of the Congo.

The control and management of malaria are linked to the quality of diagnosis. We sought to estimate the performance of routine microscopy for malaria diagnosis and assess the prevalence of submicroscopic Plasmodium (P.) falciparum infection among febrile patients in two healthcare facilities in Mossendjo, the Republic of the Congo. A cross-sectional study was conducted between January and December 2022. A total of 650 and 234 patients with signs of uncomplicated malaria were enrolled at the Centre de Sante Intégré (CSIMSJ) and Hôpital de Base (HBMSJ), respectively. Two thick blood smears were performed for each patient, one analyzed by routine microscopists and the other by an expert. The msp-1 and msp-2 genes were genotyped to detect submicroscopic P. falciparum infection. At the CSIMSJ, the sensitivity was 49.5% and the specificity was 88.6%. The positive and negative predictive values were 77.7% and 68.7%, respectively. At the HBMSJ, the sensitivity was 32.9% and the specificity was 79.4%. The positive and negative predictive values were 44.8% and 69.5%, respectively. P. falciparum was the only species detected by routine microscopists, while experts identified some cases with P. malariae and P. ovale. The proportion of submicroscopic infections was 35.75%. Children under 5 years old had higher rates of parasitemia. However, submicroscopic infections were more pronounced in the adult group. The performance of routine malaria microscopists at Mossendjo was inaccurate at both sites. With the large proportion of submicroscopic infection, malaria management at Mossendjo requires the improvement of microscopists' skills and the concomitant use of RDTs.

Molecular detection and characterization of Anaplasma marginale and Babesia canis vogeli infecting dogs in Luxor, Egypt

Tick-borne diseases in animals are increasing rapidly worldwide, but there is insufficient information about tick-borne diseases infecting dogs in southern Egypt. Thus, in the current study, we detected the presence of Anaplasma marginale (A. marginale) and Babesia canis vogeli (B. canis vogeli) in the blood of dogs. The results revealed that 4/100 (4%) were positive, and a higher infection rate was found in males (75%), than females (25%). The phylogenetic analysis for the major surface protein 4 (msp4) gene in this study was compared with amplicons separate from other reported isolates with alignment by identity 100% with cattle and camels from Egypt, and the phylogenetic analysis for the B. canis vogeli small subunit ribosomal RNA (SSU rRNA) gene in this study identified identity by 99.89% with dogs from Egypt. This report is considered the first report in southern Egypt about A. marginale in dogs based on the sequence analysis of the msp4 gene, providing new data for the classification and identification of A. marginale in dogs compared to A. marginale isolated from other animals in southern Egypt.

Plasmodium falciparum transmission based on merozoite surface protein 1 (msp1) and 2 (msp2) gene diversity and antibody responses in Ibadan, Nigeria

BackgroundNigeria is a major contributor to the global malaria burden. The genetic diversity of malaria parasite populations as well as antibody responses of individuals in affected areas against antigens of the parasite can reveal the transmission intensity, a key information required to control the disease. This work was carried out to determine the allelic frequency of highly polymorphic Plasmodium falciparum genes and antibody responses against schizont crude antigens in an area of Ibadan, Nigeria. Materials and methodsBlood was collected from 147 individuals with symptoms suspected to be malaria. Malaria infection was determined using a rapid diagnostic test (RDT), and msp1 and msp2 were genotyped by a nested PCR method. In addition, levels of IgG directed against P. falciparum FCR3S1.2 schizont extract was measured in ELISA. ResultsApproximately 25% (36/147) were positive for a P. falciparum infection in RDT, but only 32 of the positive samples were successfully genotyped. MAD20 was the most prevalent and K1 the least prevalent of the msp1 alleles. For msp2, FC27 was more prevalent than 3D7. The mean multiplicities of infection (MOI) were 1.9 and 1.7 for msp1 and msp2, respectively. IgG levels correlated positively with age, however there was no difference in median antibody levels between RDT-positive and RDT-negative individuals. ConclusionLow MOI has before been correlated with low/intermediate transmission intensity, however, in this study, similar levels of P. falciparum-specific antibodies between infected and non-infected individuals point more towards a high level of exposure and a need for further measures to control the spread of malaria in this area.

Molecular detection and characterization of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in sheep and goats in Luxor, Egypt

BackgroundTick-borne diseases cause economically significant losses to animal production globally, and anaplasmosis and theileriosis are associated with the greatest losses. However, the spread of the relevant pathogens in flocks of domesticated animals in southern Egypt is little understood. Accordingly, in this study, we aimed to determine the prevalences of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in southern Egyptian sheep and goats through blood tests, and to make a molecular characterization of the A. ovis detected in sheep targeting a specific gene.ResultsWe collected blood samples collected from 300 sheep and goats (n=150 /species) in Luxor Province in southern Egypt, and analyzed them for the presence of A. ovis, T. ovis and T. lestoquardi with screening by conventional and nested PCR targeting the msp4 and msp5, 18S rRNA, and merozoite surface protein genes. For A. ovis 140/300 samples (46.66%) were positive overall, with 90/150 (60%) and 50/150 (33.33%) positive samples in sheep and goats, respectively. Two major surface protein genes of A. ovis, msp4 and msp5, were sequenced using DNA extracted from sheep and goat blood samples, for phylogenetic analysis and genotyping. The msp4 gene sequence revealed no significant genetic diversity, to contrast to data on A. ovis strains from other countries. For T. lestoquardi, 8/150 (5.33%) samples were positive in sheep, but no samples were positive in goats (0%). For T. ovis, 32/150 (21.33%) samples were positive in sheep, but no samples were positive in goats (0%). Sequencing targeting the merozoite surface protein gene for T. lestoquardi and the small subunit ribosomal RNA gene for T. ovis revealed no significant genetic diversity in the study, another contrast to data on A. ovis strains from other countries.ConclusionThis study provides valuable data on phylogenetic and molecular classifications of A. ovis, T. ovis and T. lestoquardi found in southern Egyptian sheep and goats. It also represents the first report on detection and molecular characterization of T. lestoquardi in southern Egyptian sheep based on the specific merozoite surface protein gene, thus providing valuable data for molecular characterization of this pathogen in southern Egypt.

Expression Profiling of MSP1 Gene Involved in Mega-gametophyte Development in Ploidy Series of Guinea Grass (Panicum maximum Jacq.)

Hybrid seeds are playing important role in increasing agricultural production. However, process of hybrid seed development involves high cost for most of the major crops. Under such situation apomixis can play crucial role in creation of one-time hybrid and maintain it for multiple generations. Apomixis and polyploidy are interrelated with each other and expression of apomixis completely relies on polyploidy. Guinea grass (Panicum maximum Jacq.) provides ideal system for research on apomixis and polyploidy because of its extensive diversity in morphological, cytological, biochemical, and molecular characteristics. The experiment was carried out in the eight-ploidy series i.e. 3x, 4x, 5x, 6x, 7x, 8x, 9x, 11x and one obligate sexual Guinea grass genotype SRP75. Considering role of MSP1 gene in apomeiosis, its expression was studied in guinea grass at three developmental stages of spikelet viz., pre-meiotic, meiotic and post-meiotic. Findings of this study highlight the complex regulation of MSP-1 expression during different developmental stages and across various ploidy levels in guinea grass. At pre-meiotic stage, except 4x, downregulation pattern of MSP1 was observed in all the ploidy levels. But opposite was observed in the post-meiotic stage.

Therapeutic efficacy and tolerability of artemether–lumefantrine for uncomplicated Plasmodium falciparum malaria in Niger, 2020

BackgroundMonitoring therapeutic efficacy is important to ensure the efficacy of artemisinin-based combination therapy (ACT) for malaria. The current first-line treatment for uncomplicated malaria recommended by the National Malaria Control Program in Niger is artemether–lumefantrine (AL). In 2020, an in vivo study was carried out to evaluate clinical and parasitological responses to AL as well as the molecular resistance to the drug in three sentinel sites: Agadez, Tessaoua and Gaya, in Niger.MethodsA multi-center, single-arm trial was conducted according to the 28-day World Health Organization (WHO) 2009 therapeutic efficacy study protocol. Children between 6 months and 15 years with confirmed uncomplicated Plasmodium falciparum infection and 1000–200,000 asexual parasites/μL of blood were enrolled and followed up for 28 days. Uncorrected and PCR-corrected efficacy results at day 28 were calculated, and molecular correction was performed by genotyping the msp1, msp2, and glurp genes. The pfk13, pfdhfr, pfdhps, pfcrt and pfmdr genes were analyzed by PCR and Sanger sequencing. The Kaplan–Meier curve assessed parasite clearance.ResultsA total of 255 patients were enrolled in the study. The adequate clinical and parasitological response after PCR correction was 98.9% (95% CI 96.4–101.0%), 92.2% (85.0–98.5%) and 97.1% (93.1–101.0%) in Gaya, Tessaoua and Agadez, respectively. No adverse events were observed. Ten mutations (SNP) were found, including 7 synonyms (K248K, G690G, E691E, E612E, C469C, G496G, P718P) and 3 non-synonyms (N594K, R255K, V714S). Two mutations emerged: N594K and V714S. The R255K mutation detected in Southeast Asia was also detected. The pfdhpsK540E and pfdhfrI164L mutations associated with high levels of resistance are absent. There is a reversal of chloroquine resistance.ConclusionThe study findings indicate that AL is effective and well tolerated for the treatment of uncomplicated malaria in three sites in Niger. The emergence of a pfk13 mutation requires additional testing such as the Ring Stage Assay and CRISPR/Cas9 to confirm the role of these emerging mutations.Trial registration NCT05070520, October 7, 2021.

Plasmodium falciparum AMA1 and CSP antigen diversity in parasite isolates from southern Ghana.

Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic antigen are usually not fully effective against other variants due to altered epitopes. This study aimed to evaluate diversity in the Plasmodium falciparum antigens apical membrane antigen 1 (PfAMA1) and circumsporozoite protein (PfCSP) from circulating parasites in a malaria-endemic community in southern Ghana and to determine the effects of polymorphisms on antibody response specificity. The study involved 300 subjects, whose P. falciparum infection status was determined by microscopy and PCR. Diversity within the two antigens was evaluated by msp2 gene typing and molecular gene sequencing, while the host plasma levels of antibodies against PfAMA1, PfCSP, and two synthetic 24mer peptides from the conserved central repeat region of PfCSP, were measured by ELISA. Of the 300 subjects, 171 (57%) had P. falciparum infection, with 165 of the 171 (96.5%) being positive for either or both of the msp2 allelic families. Gene sequencing of DNA from 55 clonally infected samples identified a total of 56 non-synonymous single nucleotide polymorphisms (SNPs) for the Pfama1 gene and these resulted in 44 polymorphic positions, including two novel positions (363 and 365). Sequencing of the Pfcsp gene from 69 clonal DNA samples identified 50 non-synonymous SNPs that resulted in 42 polymorphic positions, with half (21) of these polymorphic positions being novel. Of the measured antibodies, only anti-PfCSP antibodies varied considerably between PCR parasite-positive and parasite-negative persons. These data confirm the presence of a considerable amount of unique, previously unreported amino acid changes, especially within PfCSP. Drivers for this diversity in the Pfcsp gene do not immediately seem apparent, as immune pressure will be expected to drive a similar level of diversity in the Pfama1 gene.

Identification of Anaplasma marginale adhesins for entry into Dermacentor andersoni tick cells using phage display.

Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host-pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A. marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b, mlp2, and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.

Anaplasma phagocytophilum in urban and peri-urban passerine birds in Ile-de-France

Wild animals in general, birds in particular, play a key role in transporting ticks and propagating tick-borne pathogens. Several studies have confirmed the infection of birds with Anaplasma phagocytophilum, with overall prevalence varying widely from country to country and/or study to study. This zoonotic bacterium, transmitted mainly by ticks of the genus Ixodes, is responsible for granulocytic anaplasmosis in humans (HGA) and domestic animals (cats, dogs, horses). The disease is also called tick-borne fever (TBF) in ruminants. Extremely rare in the USA, TBF is very common in Europe, where it causes economic losses in livestock. Conversely, HGA is well established in the USA whereas only a few less severe cases have been observed in Europe. Current typing techniques support the existence of multiple variants with differences in virulence/pathogenicity and tropism for certain tick and host species. However, epidemiological cycles remain difficult to characterize in Europe. Several studies describe a cycle apparently involving only birds in Europe, but no such study has been conducted in mainland France. Our objectives were to search for A. phagocytophilum in passerine birds in the Ile-de-France region and to explore their diversity using groEL and ankA gene typing and multilocus sequence typing (MLST). Various tissues (spleen, liver, and skin) were collected from cadavers of 680 passerines between March and December 2021. The presence of A. phagocytophilum was detected by qPCR Taqman targeting the msp2 gene. Three blackbirds (Turdus merula) were found positive, representing detection rates of 0.4 % in all birds tested and 3.3 % in blackbirds. The higher frequency of detection in blackbirds could be at least partially explained by their lifestyle, as they feed on the ground. Analysis of the results of groEL and ankA typing and MLST from positive blackbirds support the hypothesis that the avian A. phagocytophilum strains in Ile-de-France are distinct from those found in mammals, and that they form their own cluster in Europe.

Anaplasmosis outbreaks in taurine and zebu cattle in Brazil: causes, treatments, and Anaplasma marginale genetic diversity.

This study aimed to investigate seven outbreaks of A. marginale infection in two regions of Brazil, affecting taurine, zebu, and crossbred cattle. We assessed the possible causes, treatment measures, and genetic diversity of A. marginale. These outbreaks occurred in two states (Goiás: outbreaks 1-7; Mato Grosso do Sul: outbreak 3), breeds (Holstein, Nellore, and crossbreed), age groups (beef cattle: 18-25 days old and 7-8 months; dairy cattle: 18-25 days old, 13-14 months, and cow after the first birth) and rearing systems (feedlot, pasture, pen in a wood shaving bedding system and compost bedded-pack barns). Metaphylactic or prophylactic treatments varied according to outbreak (imidocarb dipropionate: outbreaks 1-4 and 6; enrofloxacin: outbreaks 5 and 7; diminazene diaceturate: outbreak 5). In outbreaks 6 and 7, the packed cell volume was monitored. In all outbreaks, the practice of needle/syringe sharing was discontinued. For outbreaks 1-3, clinical signs and mortality (range, 4.8-13.3%) occurred 36-45 days after entry into the feedlot. In outbreak 4, A. marginale was diagnosed in 66.2% of the calves (bacteremia, 0-4.5%), with a mortality of 8.6%. Among nursing calves aged 60 days during outbreak 5, 53.8% were infected with A. marginale, with average bacteremia of 2.7% (range, 0-21.3%), and a mortality of 13.8%. In dairy heifers aged 14 months, raised in paddocks lacking vegetation cover and infested with R. microplus, then transitioned to a rotational grazing system also infested with R. microplus, the A. marginale bacteremia ranged from 3.2 to 6.7%, with a mortality of 20%. Before monitoring during outbreak 7, the mortality was 17.9%, but no further deaths were observed after monitoring initiation. In conclusion, possible causes triggering the outbreaks included primary tick infestation, needle/syringe sharing, and stress factors which may have affected the immunological statues of animals in the feedlots. Control measures performed in all outbreaks were effective. The partial msp4 gene sequences of A. marginale generated herein belonged to two haplotypes, but further research would be needed to investigate if this finding has any clinical significance.

RPA-CRISPR/Cas12a assay for the diagnosis of bovine Anaplasma marginale infection

Anaplasma marginale infection is one of the most common tick-borne diseases, causing a substantial loss in the beef and dairy production industries. Once infected, the pathogen remains in the cattle for life, allowing the parasites to spread to healthy animals. Since clinical manifestations of anaplasmosis occur late in the disease, a sensitive, accurate, and affordable pathogen identification is crucial in preventing and controlling the infection. To this end, we developed an RPA-CRISPR/Cas12a assay specific to A. marginale infection in bovines targeting the msp4 gene. Our assay is performed at one moderately high temperature, producing fluorescent signals or positive readout of a lateral flow dipstick, which is as sensitive as conventional PCR-based DNA amplification. This RPA-CRISPR/Cas12a assay can detect as few as 4 copies/μl of Anaplasma using msp4 marker without cross-reactivity to other common bovine pathogens. Lyophilized components of the assay can be stored at room temperature for an extended period, indicating its potential for field diagnosis and low-resource settings of anaplasmosis in bovines.

Molecular Study of Anaplasma spp. in Horses, Sheep, and Goats with Phylogenetic Analysis in Northwest Iran.

Anaplasmosis, a tick-borne disease with worldwide distribution, impacts ruminants, equines, carnivores, and humans. This study aimed to investigate Anaplasma phagocytophilum in horses from Ardabil province and Anaplasma ovis in small ruminants from East Azerbaijan province using the Nested Polymerase Chain Reaction (PCR) method. Blood samples were taken from the jugular vein of 100 healthy horses in the Ardabil province and 156 healthy sheep and goats (116 sheep and 40 goats) in the East Azerbaijan province during the spring and summer seasons of 2016 in northwest Iran. The collected blood samples were stored at -20°C until the molecular experiments were conducted. Nested PCR was employed to detect A. phagocytophilum in horses and A. ovis in small ruminants using extracted DNA and amplifying 16S rRNA and msp4 genes. The Chi-square test of independence was used to determine the relationship between Anaplasma spp., infection, and independent variables, including age, gender, animal species, and sampling location. None of the 100 samples collected from horses in the Ardabil province were positive for A. phagocytophilum. In the East Azerbaijan province, 11 out of the 156 (7.05%) blood samples collected from sheep and goats tested positive for A. ovis. In addition, A. ovisinfection was not significantly related to the independent variables. Phylogenetic analysis showed that the sequence obtained in this study (MH790273) had 100% homology with the sequence obtained from sheep infected with Anaplasma in Ahvaz province (JQ621903.1). The findings of this study can contribute to the prevention and control of anaplasmosis in farm animals in northwestern Iran.

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.

Beninese Plant Extracts with Antiplasmodial Activity Select New Allele Variants Msp1 and Msp2 in Plasmodium falciparum.

Natural medicinal products are commonly used as a remedy against malaria infections in African populations and have become a major source of information for the screening of new and more effective antiplasmodial molecules. Therefore, in vitro studies are needed to validate the efficacy of these medicinal products and to explore the potential effects of such drugs on the genetic diversity of Plasmodium falciparum. The current study has investigated the impact of some Beninese plant extracts with antiplasmodial activity on the genetic diversity of P. falciparum. Five (5) ethanolic plant extracts (Dissotis rotundifolia, Ehretia cymosa Thonn, Hibiscus surattensis L., Cola millenii K. Shum, and Costus afer Ker Gawl) and a compound extracted from Ehretia cymosa Thonn (encoded CpE2) were tested against asexual stage parasites of a culture-adapted strain of P. falciparum. Subsequently, the P. falciparum Msp1 and Msp2 markers were genotyped, and the number of allelic variants and the multiplicity of infection (MOI) were compared between drug-exposed and unexposed parasites. All plant extracts have shown inhibitory activity against asexual P. falciparum and selected new allelic variants of the Msp1 and Msp2 genes compared to unexposed parasites. The newly selected allelic variants were K1_100bp and RO33_300bp of the Msp1 gene and FC27_150bp, FC27_300bp, FC27_400bp, and FC27_600bp of the Msp2 gene. However, there was no significant difference in MOI between drug-exposed and unexposed parasites. Our study highlights a source for the selection of new Msp1 and Msp2 alleles after exposure to antimalarial drugs. These findings pave the way for further studies investigating the true roles of these newly selected alleles in P. falciparum.