Uptake of intermittent preventive treatment of malaria in pregnancy and risk factors for maternal anaemia and low birthweight among HIV-negative mothers in Dschang, West region of Cameroon: a cross sectional study.

BACKGROUND: Approximately 32 million pregnant women are at risk of malaria with up to 10,000 maternal deaths and 200,000 neonates at risk annually. Intermittent Preventive Treatment (IPT) with sulfadoxine-pyrimethamine (SP) is recommended by the World Health Organization (WHO) to reduce disease in pregnancy and adverse maternal and newborn outcomes. At least three doses of SP should be taken by pregnant women during antenatal consultation (ANC) beginning from the thirteenth week of pregnancy till parturition. The aim of this study was to assess uptake of IPT during pregnancy and risk factors for maternal anaemia and infant birth weight in Dschang, West region of Cameroon. METHODS: A total of 380 consenting pregnant women at delivery were recruited in a cross- sectional prospective survey between January to December 2021. Data on ANC attendance, total dose of IPT and history of malaria were abstracted from hospital ANC records while socio-demographic characteristics, bed net use and obstetrics history of each participant were also recorded through an interview. Further, blood samples were collected from the intervillous space for assessment of maternal anaemia and microscopic parasitology. Nested PCR based on amplification of the Plasmodium 18S sRNA was carried out to detect submicroscopic infection. IPTp coverage was calculated per WHO recommendation and the prevalence of anaemia and low birth weight were estimated as proportions in the total sample of pregnant women and live births, respectively. Crude and adjusted odds ratios and their 95% confidence intervals were used to estimate associations between pregnancy outcomes considered and risk factors in specific and general models. A p < 0.05 was considered significant. The R software (V4.1.4) was used for all analyses. RESULTS: A majority of pregnant women was aged between 24 and 34 years old (59.2%) and had secondary education (58.8%). Uptake of ≥ 3 IPTp was 64.99% with 77.20% of all who received at least one IPTp doses taking a mix of SP and DP or DP alone in successive ANC contacts. Those with four or more ANC contacts (73.42%) were more likely to have received at least one IPTp. Furthermore, 13.9% of live births had low birthweights (BW < 2500 g) and one in four parturient women with moderate anaemia by WHO criteria. Microscopy (blood smear examination) and PCR-based diagnosis revealed between 0% and 1.57% of parasite-infected placental samples, respectively. Reported malaria in pregnancy predicted maternal anaemia at birth but not birth weight. Only gestational age (< 37 weeks) and bed net use (< 5 months) significantly predicted infant birth weight at delivery. CONCLUSION: The uptake of WHO recommended IPT doses during pregnancy was moderately high. Reported malaria in pregnancy, poor bed net coverage, gestational age less than 37 weeks adversely affect maternal haemoglobin levels at birth and infant birth weight. Asymptomatic and submicroscopic placental parasite infections was found at low prevalence. Together these results highlight the importance of maintaining aggressive measures to prevent malaria in pregnancy and protect the health of mother and baby.

Prevalence, epidemiology, seasonality, and phylogeny of Anaplasma marginale in blood samples of goats collected from Punjab, Pakistan.

PURPOSE: In Pakistan, a major constrain to goat farming is the tick and tick-borne diseases that results in financial losses to livestock farmers. This study was conducted to report the molecular prevalence of Anaplasma (A.) marginale in goat blood samples collected during four seasons from Khanewal district in Punjab (Pakistan). METHODS AND RESULTS: The mps1 gene of A. marginale was targeted in 900 blood samples that were collected on seasonal basis (n = 225 per season) and 6.6% (61/900) goats were found positive with A. marginale. Anaplasma marginale positive PCR products were sequenced and submitted to the GenBank. Prevalence of A. marginale varied with sampling season (P = 0.002) and it was highest in the summer (11.5%) followed by the autumn (7.6%), spring (5.3%), and winter seasons (2.7%) respectively. Anaplasma marginale prevalence varied significantly between goat breeds during the autumn (p = 0.01) and summer seasons (p = 0.02). Goats more than 2 years old and livestock farms where only goats were kept and dogs were associated with herds were risk factors for ovine anaplasmosis during different seasons. White and red blood cell counts and parameters associated with their counts were affected in A. marginale infected goats while studied serum parameters remained unaffected. CONCLUSION: PCR is a reliable tool for the detection of A. marginale in goat blood samples. A relatively low prevalence of A. marginale in goats of Khanewal district was observed and the parasite prevalence in goats was higher in the summer (May until September) and autumn (October and November) seasons. Control measures are required to prevent tick-borne diseases in ruminants from Pakistan.

A Report on Molecular Detection and Phylogenetic Evaluation of Anaplasma marginale in Ticks and Blood Samples Collected from Cattle in District Layyah in Punjab (Pakistan).

Anaplasmosis is a tick-borne disease caused by obligate intercellular gram-negative bacteria, Anaplasma (A.) marginale. The present study reports on seasonal prevalence, epidemiology, and phylogeny of A. marginale in three cattle breeds from District Layyah, Southern Punjab, Pakistan. A total of 844 blood samples (Cross = 300, Holstein Friesian = 244, Sahiwal breed = 300) from apparently healthy cattle on seasonal basis were collected along with epidemiological data during May 2018 till April 2019. Polymerase chain reaction generated 265 base-pair amplicon specific for major surface protein-1b encoding gene of A. marginale in 8.6% (73/844) of enrolled cattle. The highest prevalence was observed during autumn (18.3%) followed by summer (9.7%) and winter season (7.1%). Holstein Friesian breed was most susceptible to A. marginale infection (13.1%) followed by Sahiwal (7.6%) and cross breed (6%). Representative amplified partial gene sequences of A. marginale were submitted to GenBank (Accession numbers MK032842 and MK032843). 37/844 (4.3%) Giemsa-stained blood smears were found positive for Anaplasma spp. Small number of ticks including Hyalomma anatolicum, Hyalomma excavatum, Rhipicephalus microplus, Haemaphysalis punctata were identified from cattle but none of them was found PCR positive for the presence of A. marginale. Analysis of epidemiological factors revealed that female cattle and farm with water supply from pool, farms where other dairy animals and dogs were living with cattle and dogs having ticks load on them had significant association with A. marginale prevalence. It was observed that white blood cell, lymphocytes (%), monocytes (%) hematocrit, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were significantly disturbed in A. marginale-positive than negative cattle.

Molecular epidemiology of Theileria annulata infection of cattle in Layyah District, Pakistan.

Theileria annulata is the cause of tropical theileriosis in cattle in Pakistan, where it has a significant impact on the cattle industry. Here we report the molecular detection and seasonal prevalence and blood parameters of T. annulata infection in crossbred, Holstein Frisian and Sahiwal breed in Layyah District in the Punjab. In total, 844 blood samples (cross = 244, Holstein Frisian = 300, Sahiwal breed = 300) collected in 2017 and 2018 were tested. Blood smear screening revealed 125/844 (15%) of cattle positive for Theileria species. PCR amplification of cytochrome b gene indicated an overall T. annulata prevalence of 21% (174/844). The highest prevalence was observed in autumn season (53%), followed by winter (20%), summer (14%) and spring (3%). Crossbred cattle were the most susceptible to T. annulata (28%) followed by Sahiwal (19%) and Holstein Frisian. Representative partial cytochrome b gene sequences of T. annulata revealed phylogenetic similarities with sequences submitted from India, Iran, China, Turkey and Spain. Small numbers of ticks, including Hyalomma anatolicum, Hyalomma excavatum, Rhipicephalus microplus, and Haemaphysalis punctata, were identified from cattle but none of them was found PCR positive for the presence of T. annulata. Analysis of the hematology data indicated that red blood cell, hemoglobin, mean cell hemoglobin, mean corpuscular hemoglobin, lymphocyte (%), monocyte (%) and platelet count were significantly altered in T. annulata-positive cattle of all three breeds. Screening of cattle by PCR for the detection of T. annulata is recommended for diagnosis and treatment.

Molecular detection and prevalence of Theileria ovis and Anaplasma marginale in sheep blood samples collected from Layyah district in Punjab, Pakistan.

Theileria ovis and Anaplasma marginale are intracellular pathogens affecting a wide range of animals, causing huge economic losses worldwide. The present study reports the molecular evidence of Theileria ovis and Anaplasma marginale in sheep blood samples (N = 218) collected from Layyah district in Punjab (Pakistan), where economy heavily relies on livestock. A 520 base pair fragment specific for 18S ribosomal RNA gene of Theileria ovis was PCR amplified in 23/218 (10.6%) sheep blood samples, while for Anaplasma marginale, a 265 base pair fragment specific for msp1b gene was generated in 15/218 (6.9%) sheep blood samples. Two blood samples were found co-infected (0.9%) with both parasites. Amplified PCR products of both parasites were confirmed by DNA sequencing and submitted to GenBank. Prevalence of both Theileria ovis (p = 0.3) and Anaplasma marginale (p = 0.4) varied non-significantly among the investigated sheep breeds. Tick burden on dogs present with sheep herds was found associated with Theileria ovis infection in sheep (p = 0.05). It was observed that lambs (p = 0.009), sheep in small herds (p = 0.04), and tick burden on dogs present with sheep herds (p = 0.01) were associated with Anaplasma marginale infection in sheep during the present study. In conclusion, we are reporting a higher prevalence of Theileria ovis than Anaplasma marginale in blood samples of sheep collected from Layyah district. Tick-infested dogs were found to be risk factors for the transmission of both pathogens in sheep, and tick control strategies should be extended to dogs associated with sheep herds in this area.

Molecular detection of small ruminant piroplasmosis and first report of Theileria luwenshuni (Apicomplexa: Theileridae) in small ruminants of Pakistan.

Theileriosis is a widespread and economically important disease of small ruminants in Pakistan. Ruminants are the intermediate hosts in the lifecycle of Theileria spp., with ticks of the family Ixodidae being the definitive hosts. To better understand the distribution and prevalence of theileriosis in Pakistan, a molecular survey was performed in small ruminants from the Lower Dir district of the Khyber Pakhtunkhwa province. A total of 200 healthy sheep and goats were screened from Maidan, Samar Bagh and Munda districts of district Dir Lower, Pakistan during December (2017) to April (2018). DNA samples were screened through nested PCR using universal primers. The amplified 492-498 bp amplicon was subjected to RLB analysis which was based on the hypervariable of the 18S rRNA gene to test for the presence of genotypes of Theileria in blood samples. A phylogeny was constructed to determine the species of Theileria genotypes. Nested PCR results indicated 53.5% prevalence of one or more Theileria genotypes in the blood of the host animal. From RLB assay, 27 animals (13.5%) showed infection with only a single species of Theileria while 80 animals (40%) showed coinfection by multiple Theileria spp. Based on the 18S rRNA phylogeny, the unknown genotype is of the species Theileria luwenshuni and is closely related to Chinese isolates. The present finding is the first report on molecular diagnosis of Theileria luwenshuni in small ruminants in Pakistan.

First molecular survey of piroplasm species in cattle from Kyrgyzstan.

Bovine piroplasmosis is a tick-borne disease caused by apicomplexan hemoparasites of the genera Theileria and Babesia. This study was carried out to assess the presence and frequency of piroplasm parasites in apparently healthy cattle in Kyrgyzstan. A total of 454 blood samples were collected from animals of various ages in eight villages located in the Chu valley and around the Lake Issyk Kul. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers specific targeting members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic and species-specific oligonucleotide probes were covalently linked. The results revealed the presence of three piroplasm species (Theileria orientalis, Babesia major, Theileria annulata). Theileria orientalis was the most prevalent species (32.8%; CI 28.5-37.3). Babesia major was the only species of Babesia found in any of the samples (1.3%; CI 0.5-2.8). The co-existence of Theileria annulata and T. orientalis was detected in nine animals (1.9%; CI 0.9-3.7). BLAST search revealed that the Theileria sequences shared 100% identity with the recently reported sequences for T. buffeli and T. annulata. The sequence of B. major was also 100% identical to an existing B. major sequence. This molecular survey provides important epidemiological data for control of bovine piroplasmosis caused by T. orientalis, B. major, and T. annulata in Kyrgyzstan.

Molecular identification of Theileria and Babesia in ticks collected from sheep and goats in the Black Sea region of Turkey.

A molecular survey was undertaken in the Black Sea region of Turkey to determine the presence of Theileria and Babesia species of medical and veterinary importance. The ticks were removed from sheep and goats, pooled according to species and locations, and analyzed by PCR-based reverse line blot (RLB) and sequencing. A total of 2241 ixodid ticks belonging to 5 genus and 12 species were collected and divided into 310 pools. Infection rates were calculated as the maximum likelihood estimation (MLE) with 95% confidence intervals (CI). Of the 310 pools tested, 46 (14.83%) were found to be infected with Theileria or Babesia species, and the overall MLE of the infection rate was calculated as 2.27% (CI 1.67-2.99). The MLE of the infection rates were calculated as 0.691% (CI 0.171-1.78) in Haemaphysalis parva, 1.47% (CI 0.081-6.37) in Rhipicephalus sanguineus, 1.84% (CI 0.101-7.87) in Ixodes ricinus, 2.86% (CI 1.68-4.48) in Rhipicephalus turanicus, 5.57% (CI 0.941-16.3) in Hyalomma marginatum, and 6.2% (CI 4.02-9.02) in Rhipicephalus bursa. Pathogens identified in ticks included Theileria ovis, Babesia ovis, Babesia bigemina, and Babesia microti. Most tick pools were infected with a single pathogen. However, five pools displayed mixed infections with T. ovis and B. ovis. This study provides the first molecular evidence for the presence of B. microti in ticks in Turkey.

Survey of tick-borne pathogens in grazing horses in Kyrgyzstan: phylogenetic analysis, genetic diversity, and prevalence of Theileria equi.

Introduction: Tick-borne pathogens (TBP) are an important group of organisms that can affect animals and humans all over the world. Equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi, is considered one of the most important tick-borne diseases and can cause significant clinical symptoms and mortality in horses. Moreover, EP plays a restrictive role in international horse traditions and transportation. Although these species can cause similar symptoms, there are different 18S rRNA genotypes of T. equi (five genotypes) and B. caballi (three genotypes). Besides piroplasma species, Anaplasma and hemotropic mycoplasmas (HM) are known as other important tick-borne pathogens reported in horses. Methods: In this study, we investigated the presence, prevalence, genetic diversity, and phylogenetic analyses of TBPs using PCRs and DNA sequencing in grazing horses in Kyrgyzstan. For these purposes, a total of 311 blood samples were collected from Chuy, Issyk-Kul, Naryn, Osh, Talas, and Jalal-Abad. Results: DNA amplification of TBP revealed that 23 (7.40%) out of 311 samples were found to be positive for T. equi. However, B. caballi, HM, A. phagocytophilum, and A. capra were not detected in this study. The infection rate of T. equi was higher in males (8.11%) than in females (6.35%) (p=0.2880) and in those older than 5 years (9.02%) than in the 1-4 age group (6.35%) (p=0.1950). Phylogenetic analysis of 18S rRNA revealed that A and E genotypes of T. equi have circulated in grazing horses in Kyrgyzstan. Discussion: Information about the genetic diversity of T. equi is important for understanding the population dynamics of the species and developing effective control strategies against this pathogen. This is the first molecular investigation of A. capra in horses in Kyrgyzstan. Although this pathogen has been detected in different hosts in Kyrgyzstan, it was not detected in this study. However, considering the wide host spectrum of A. capra, it is thought that more large-scale studies are needed to understand the effect of horses on the epidemiology of this pathogen.

Pseudo-plaque reduction neutralization test (PPRNT) for the measurement of neutralizing antibodies to Crimean-Congo hemorrhagic fever virus.

BACKGROUND: Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus family Bunyaviridae, which are enveloped viruses containing tripartite, negative polarity, single-stranded RNA. CCHF is characterized by high case mortality, occurring in Asia, Africa, the Middle East and Europe. Currently, there are no specific treatments or licensed vaccines available for CCHFV. Recently, two research groups have found adult mice with defective interferon responses allowed to lethal CCHFV infection. These mouse models could provide invaluable information for further studies. Efforts to develop a vaccine against CCHFV are being made. To determine the efficacy of vaccine candidates it is important to conduct serological studies that can accurately measure levels of protective antibodies. In the present study, a pseudo-plaque reduction neutralization test (PPRNT) based on enzyme-catalyzed color development of infected cells probed with anti-CCHFV antibodies was used to measure neutralization antibody of CCHFV. METHODS: Sixty-nine human serum samples (20 acute and 49 convalescent) were tested. The presence of CCHFV antibodies was determined and confirmed by a commercial ELISA kit. CCHFV RNA was determined by RT-PCR. All the samples were analyzed by PPRNT and fluorescent focus reduction neutralization test (FFRNT) to measure of CCHFV-neutralizing antibodies. RESULTS: Pseudo-plaque reduction neutralization test showed a high sensitivity (98%), specificity (100%) and agreement (96,6%) in qualitative comparison with those of the FFRNT. There was a high correlation between the titers obtained in PPRNT and FFRNT (R2 = 0.92). The inter- and intra-assay variation of PPRNT revealed good reproducibility and positive cut-off of PPRNT was defined as 1:4 by the geometric mean titers for the individual samples distributed. CONCLUSION: The pseudo-plaque reduction neutralization test described in this study is a fast, reproducible and sensitive method for the measurement of CCHF neutralizing antibodies. This novel assay could serve as useful tools for CCHF research in epidemiology, vaccine development and other studies of immunity. It also provides an alternative to PRNT when viruses with no or poor CPE in cell culture.

Molecular investigations of Hepatozoon species in dogs and developmental stages of Rhipicephalus sanguineus.

The occurrence and distribution of Hepatozoon species in stray dogs, and the developmental stages of Rhipicephalus sanguineus detached from the same dogs in Diyarbakir Province, Turkey is reported. A total of 328 ticks, including 133 adults (55 males and 75 females consist of 63 partially engorged and 15 fully engorged) and 195 nymphs (91 partially engorged and 104 fully engorged) were detached from the dogs. Fully engorged nymphs and females were incubated at 27 °C and relative humidity of 85 % to molt to adult stage and recover eggs. The ticks were pooled according to sex and developmental stage. No Hepatozoon gamonts were found, whereas, by PCR, 15.87 % (10/63) of the dogs were infected with Hepatozoon canis. Of the 68 tick pools tested, 14 (20.58 %) pools were infected with Hepatozoon spp., an overall maximum likelihood estimation of prevalence of 4.9 % (95 % confidence intervals (CI) = 2.85-7.93 %) per 100 ticks. Maximum likelihood estimation of the infection rate varied by tick sex and developmental categories, ranging from 1.75 % (95 % CI = 0.11-8.11 %) in fed males to 6.81 % (95 % CI = 2.07-17.46 %) in unfed females. One amplicon from a fed adult female was 99 % identical to the sequence for Hepatozoon felis. The remaining sequences isolated from both dogs and ticks shared 99-100 % similarity with the corresponding H. canis isolates. This is the first detection of H. canis and H. felis in the tick R. sanguineus in Turkey.

Molecular identification of Theileria and Babesia in sheep and goats in the Black Sea Region in Turkey.

This study was carried out to investigate presence and distribution of Theileria and Babesia species via microscopic examination and reverse line blotting (RLB) techniques in sheep and goats in the Black Sea region of Turkey. For this purpose, 1,128 blood samples (869 sheep and 259 goats) were collected by active surveillance from sheep and goats in different provinces of various cities in the region in the years 2010 and 2011. Smears were prepared from the blood samples, stained with Giemsa, and examined under the light microscope for Theileria and Babesia piroplasms. The genomic DNAs were extracted from blood samples. The length of 360-430-bp fragment in the variable V4 region of 18S SSU rRNA gene of Theileria and Babesia species was amplified using the gDNAs. The polymerase chain reaction products were hybridized to the membrane-connected species-specific probes. A total of 38 animals (3.37%) including 34 sheep (3.91%) and 4 goats (1.54%) were found to be positive for Theileria spp. piroplasms in microscopic examination of smears while Babesia spp. piroplasm could not detected. Infection rates were 34.64% in sheep, 10.04% in goats, and totally 28.99% for Theileria ovis while 0.58% in sheep and totally 0.44% for Babesia ovis. However, Theileria sp. OT3 was detected in 2.65% of sheep and 2.04% of all animals; besides Theileria sp., MK had 0.58% prevalence in sheep and 0.77% in goats, with a total 0.62% with RLB. Although T. ovis and Theileria sp. MK were determined in both sheep and goats, B. ovis and Theileria sp. OT3 were observed only in the sheep. These results provide the first detailed molecular data for sheep and goat theileriosis and babesiosis in the region.

Discovery of a Novel Species Infecting Goats: Morphological and Molecular Characterization of Babesia aktasi n. sp.

A novel Babesia sp. infecting goats was discovered based on the molecular findings obtained in the current study, which was conducted in the Mediterranean region of Türkiye. The goal of this study was to isolate this species of Babesia (Babesia sp.) infecting goats in vivo and to assess the genetic and morphological characterization of the parasite. To identify the animal naturally infected with Babesia sp. and isolate the parasite from this animal, field studies were conducted first, and genomic DNA were extracted from blood samples taken from goats (n = 50). The Theileria, Babesia, and Anaplasma species were identified using a nested PCR-based reverse line blotting (RLB) method. The study included one goat that was determined to be infected with Babesia sp. (single infection) in RLB for in vivo isolation. A blood smear was prepared to examine the parasite's morphology, but it was found to be negative microscopically. Following that, a splenectomy operation (to suppress the immune system) was performed to make the parasites visible microscopically in this animal. Parasitemia began after splenectomy, and the maximum parasitemia was determined to be 1.9%. The goat displayed no significant symptoms other than fever, loss of appetite, and depression. During a period when parasitemia was high, blood from this goat was inoculated into another splenectomized goat (Theileria-Babesia-Anaplasma-Mycoplasma spp. free). On the third day of inoculation, 10% parasitemia with high fever was detected in the goat, and on the fourth day, the goat was humanely euthanized due to severe acute babesiosis symptoms. Except for mild subcutaneous jaundice, no lesions were discovered during the necropsy. According to the microscopic measurement results, ring, double pyriform, spectacle-frame-like, and line forms were observed, and it was observed to be between 1.0-2.5 µm (1.38 ± 0.17 to 0.7 ± 0.21-all forms). A phylogenetic analysis and sequence comparison using the 18S rRNA and cox1 genes revealed that this species is distinct from the small ruminant Babesia species (18S rRNA 92-94%, cox1 79-80%) and has the highest similarity to Babesia sp. deer, which has been reported in deer. Furthermore, it was determined to resemble B. venatorum, B. divergens, Babesia sp. FR1 and Babesia sp. MO1 species, all of which are zoonotic. Additional research is needed to clarify the clinical status of this parasite in goats and other hosts (mountain goat, sheep, calf).

Molecular Prevalence and Genetic Diversity Based on Msp1a Gene of Anaplasma ovis in Goats from Türkiye.

Anaplasma ovis is a tick-borne obligated intraerythrocytic bacterium that infects domestic sheep, goats, and wild ruminants. Recently, several studies have been carried out using 16S rRNA and msp4 genes to identify the genetic diversity of A. ovis. Instead of these genes, which are known to be highly stable among heterologous strains, Msp1a, which is accepted as a stable molecular marker to classify A. marginale strains, was used in A. ovis genetic diversity studies. The genetic diversity of A. ovis strains according to the Msp1a gene has not been extensively reported. Therefore, the purpose of this study was to examine the genetic diversity of A. ovis in goats specifically using analysis of the Msp1a gene. Blood samples were taken from the vena jugularis to the EDTA tubes from 293 randomly selected goats (apparently healthy) in the Antalya and Mersin provinces of Mediterranean region of Türkiye. The Msp1a gene of A. ovis was amplified in all DNA samples through the use of PCR, using a specific set of primers named AoMsp1aF and AoMsp1aR. Among the amplified products, well-defined bands with different band sizes were subjected to sequence analysis. The obtained sequence data were converted into amino acid sequences using an online bioinformatics program and the tandem regions were examined. The Msp1a gene of A. ovis was amplified in 46.1% (135 out of 293) of the goats. Through tandem analysis, five distinct tandems (Ao8, Ao18, Tr15-16-17) were identified, and it was found that three of these tandems (Tr15-16-17) were previously unknown and were therefore defined as new tandems. The study also involved examination of ticks from goats. It was observed that the goats in the area were infested with several tick species, including Rhipicephalus bursa (888/1091, 81.4%), R. turanicus (96/1091, 8.8%), Dermacentor raskemensis (92/1091, 8.4%), Hyalomma marginatum (9/1091, 0.8%), and R. sanguineus s.l. (6/1091, 0.5%). This study provides important data for understanding the genetic diversity and evolution of A. ovis based on tandem repeats in the Msp1a protein.

Small Ruminant Piroplasmosis: High Prevalence of Babesia aktasi n. sp. in Goats in Türkiye.

Small ruminant piroplasmosis is the hemoparasitic infection of sheep and goats caused by Babesia and Theileria species responsible for clinical infections with high mortality outcomes. The disease is transmitted by ixodid ticks and prevalent in the tropical and subtropical regions of the world, including Türkiye. A prevalence survey, using molecular methods, is conducted in this study to determine the frequency of newly defined Babesia aktasi n. sp. and other tick-borne piroplasm species in small ruminants in Turkiye. A total of 640 blood samples from sheep (n = 137) and goats (n = 503) were analyzed by nested PCR-based reverse line blot (RLB) hybridization. The results show that 32.3% (207/640) of apparently healthy, small ruminants are infected with three Theileria and two Babesia species. Babesia aktasi n. sp. was the most prevalent species in goats, with 22.5% of samples being positive, followed by B. ovis (4%), T. ovis (2.8%), T. annulata (2.6%), and Theileria sp. (0.6%). None of the sheep samples were positive for Babesia aktasi n. sp.; however, 51.8% were infected with T. ovis. In conclusion, the findings reveal that B. aktasi n. sp. is highly prevalent in goats, but absent in sheep. In future studies, experimental infections will determine whether B. aktasi n. sp. is infectious to sheep, as well as its pathogenicity in small ruminants.

Experimental infection of non-immunosuppressed and immunosuppressed goats reveals differential pathogenesis of Babesia aktasi n. sp.

Babesiosis is an acute and persistent tick-borne disease caused by protozoan parasites of the genus Babesia. These hemoparasites affect vertebrates globally, resulting in symptoms such as high fever, anemia, jaundice, and even death. Advancements in molecular parasitology revealed new Babesia species/genotypes affecting sheep and goats, including Babesia aktasi n. sp., which is highly prevalent in goats from Turkiye's Mediterranean region. The objective of this study was to investigate the pathogenesis of B. aktasi infection in immunosuppressed (n=7) and non-immunosuppressed (n=6) goats. These animals were experimentally infected with fresh B. aktasi infected blood, and their clinical signs, hematological and serum biochemical parameters were monitored throughout the infection. The presence of parasites in the blood of immunosuppressed goats was detected by microscopic examination between 4 and 6 days after infection, accompanied by fever and increasing parasitemia. Goats that succumbed acute disease exhibited severe clinical signs, such as anemia, hemoglobinuria, and loss of appetite. However, the goats that survived showed milder clinical signs. In the non-immunosuppressed group, piroplasm forms of B. aktasi were observed in the blood within 2-5 days after inoculation, but with low (0.01-0.2%) parasitemia. Although these goats showed loss of appetite, typical signs of babesiosis were absent except for increased body temperature. Hematological analysis revealed significant decreases in the levels of red blood cells, leukocytes and platelet values post-infection in immunosuppressed goats, while no significant hematological changes were observed in non-immunosuppressed goats. In addition, serum biochemical analysis showed elevated transaminase liver enzymes levels, decreased glucose, and lower total protein values in the immunosuppressed group post-infection. Babesia aktasi, caused mild disease with minor clinical symptoms in non-immunosuppressed goats. However, in immunosuppressed goats, it exhibited remarkable pathogenicity, leading to severe clinical infections and death. In conclusion, this study provides valuable insights into the pathogenicity of the parasite and will serve as a foundation for future research aimed at developing effective prevention and control strategies against babesiosis in small ruminants. Further research is required to investigate the pathogenicity of B. aktasi in various goat breeds, other potential hosts, the vector ticks involved, and its presence in natural reservoirs.

Babesia ovis secreted antigen-1 is a diagnostic marker during the active Babesia ovis infections in sheep.

Ovine babesiosis caused by Babesia ovis is an economically significant disease. Recently, a few B. ovis-specific proteins, including recombinant B. ovis secreted antigen-1 (rBoSA1), have been identified. Immunological analyses revealed that rBoSA1 resides within the cytoplasm of infected erythrocytes and exhibits robust antigenic properties for detecting anti-B. ovis antibodies. This protein is released into the bloodstream during the parasite's development. It would be possible to diagnose active infections by detecting this secretory protein. For this purpose, a rBoSA1-specific polyclonal antibody-based sandwich ELISA was optimized in this study. Blood samples taken from the naturally (n: 100) and experimentally (n: 15) infected sheep were analyzed for the presence of native BoSA1. The results showed that native BoSA1 was detectable in 98% of naturally infected animals. There was a positive correlation between parasitemia level in microscopy and protein density in sandwich ELISA. Experimentally infected animals showed positive reactions from the first or second day of inoculations. However, experimental infections carried out by Rhipicephalus bursa ticks revealed the native BoSA1 was detectable from the 7th day of tick attachment when the parasite began to be seen microscopically. Sandwich ELISA was sensitive enough to detect rBoSA1 protein at a 1.52 ng/ml concentration. Additionally, no serological cross-reactivity was observed between animals infected with various piroplasm species, including Babesia bovis, B. bigemina, B. caballi, B. canis, B. gibsoni, Theileria equi, and T. annulata. Taken collectively, the findings show that the rBoSA1-specific polyclonal antibody-based sandwich ELISA can be successfully used to diagnose clinical B. ovis infections in sheep at the early stage.

Seasonality, epidemiology and phylogeny of Theileria ovis with a note on hematological and biochemical changes in asymptomatic infected goats from Pakistan.

Caprine theileriosis, caused by Theileria ovis is a serious production issue, especially in the areas that depend on goats and sheep for milk, meat, and other economic benefits. Pakistan has a large goat population, but few reports have been documented from this country regarding PCR-based detection of T. ovis. The molecular prevalence of T. ovis, on a seasonal basis, in various goat breeds enrolled from Muzaffar Garh district of Punjab in Pakistan was determined from October 2018 to September 2019. In this study, 1084 goat blood samples were screened for the detection of T. ovis DNA through PCR-based amplification of 18S rRNA gene. Out of 1084 goats, 12 (1.11%) were infected with T. ovis. The parasite prevalence varied with the sampling seasons (Chi square test, P = 0.008), and the parasite prevalence was highest in goat blood samples collected in summer (2.39%) followed by winter (1.88%). DNA sequencing and BLAST analysis confirmed the presence of T. ovis, and the amplified isolates from the 18S rRNA gene of T. ovis were found to be highly conserved during phylogenetic analysis. Young goats (Fischer exact test, P = 0.022) were found more infected with T. ovis during the winter season. Infected goats had elevated white blood cell counts (Two-sample t-test, P = 0.04), blood urea nitrogen to Creatinine ratio (Two-sample t-test, P = 0.02) and decreased serum Creatinine (Two-sample t-test, P = 0.001) as compared to T. ovis negative goats. We report a relatively low molecular prevalence of T. ovis in goats from the Muzaffar Garh district. However, it is recommended that control measures to eradicate T. ovis infection in goats in this area should be taken.

A study on ovine tick-borne hemoprotozoan parasites (Theileria and Babesia) in the East Black Sea Region of Turkey.

In this study, the frequency of Theileria and Babesia species was assessed via reverse line blotting and blood smear-based diagnostic methods in small ruminants. A total of 201 apparently healthy animals from 26 randomly selected herds located in 4 locations (Artvin, Giresun, Gumushane, and Tokat) of East Black Sea Region of Turkey were investigated for the blood protozoans. In a polymerase chain reaction (PCR), the hypervariable V4 region of the 18S ribosomal RNA gene was amplified with a set of general primers specific for all Theileria and Babesia species. The PCR products were hybridized against catchall and species-specific (Theileria spp., Theileria lestoquardi, Theileria ovis, Theileria sp. OT1, Theileria sp., OT3, Theileria sp., MK, Theileria luwenshuni, Theileria uilenbergi, Babesia spp., Babesia ovis, Babesia motasi, and Babesia crassa) probes. Theileria piroplasms were identified in nine (4.47%) samples by microscopic examination. Reverse line blotting (RLB) detected the infection in 19.90% of the samples. The infection rate of sheep (28.90%) was higher than goats (4.10%). T. ovis, Theileria sp., MK, and Theileria sp. OT3 were detected by RLB. The most prevalent Theileria species was T. ovis (18.90%) followed by Theileria sp. MK (0.99%). Theileria sp. OT3 was detected in one sample (0.43%). A single animal was infected as mix with T. ovis and Theileria sp. MK. The other Theileria (T. lestoquardi, Theileria sp. OT1, T. luwenshuni, and T. uilenbergi) and Babesia (B. ovis, B. motasi, and B. crassa) species were not detected. This study is the first molecular survey on ovine tick-borne protozoans in East Black Sea Region of Turkey.

Detection of Theileria orientalis Genotypes from Cattle in Kyrgyzstan.

The ikeda and chitose genotypes of Theileria orientalis, which for many years were thought to be benign, cause a disease that results in significant economic losses in the cattle industry. This study was carried out in order to determine the genotypes of T. orientalis in cattle in Kyrgyzstan, and 149 archived DNA samples known to be T. orientalis were analyzed by the PCR amplification of the major piroplasm surface protein (MPSP) gene region. Single-Strand Conformation Polymorphism (SSCP) analysis was performed to uncover the nucleotide changes in the archived DNA samples, and 15 samples showing different band profiles were subjected to sequence analysis. As a result of the sequence analysis, it was seen that the samples belonged to the buffeli and chitose A genotypes. In order to identify mixed genotypes, PCR was performed using primers specific for these genotypes, and buffeli (type 3), chitose (type 1) and buffeli+chitose were found to be positive in 26.2%, 2% and 71.8% of samples, respectively. As a result of this study, we showed the presence of buffeli (type 3) and chitose (type 1) genotypes of T. orientalis in cattle in Kyrgyzstan. Comprehensive epidemiological studies are needed to understand the clinical infections caused by the pathogenic chitose A and to determine the geographical distribution and different genotypes of T. orientalis.