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 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.

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.

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.

Identification of CCp5 and FNPA as Novel Non-canonical Members of the CCp Protein Family in Babesia bovis.

Bovine babesiosis, caused by Babesia bovis, is an economically significant tick-borne disease that imposes restrictions to livestock production worldwide. Current methods to control bovine babesiosis have severe limitations and novel approaches, including transmission-blocking vaccines, are needed. Members of the widely conserved CCp family are multidomain adhesion proteins containing LCCL motifs, which are differentially expressed on gametocytes of apicomplexans, including Babesia spp. and Plasmodium spp. While Plasmodium parasites contain 6 distinct CCp genes, only three members (CCp 1-3) were previously identified in B. bovis. In this study, we describe the identification and characterization of two novel non-canonical members of the CCp gene family in B. bovis, named CCp5 and FNPA. The genes were identified in silico by TBLASTN using P. falciparum CCp family domains as queries. Unlike CCp1-3, the B. bovis CCp5 and FNPA proteins lack the LCCL canonical domain but contain other typical multidomain adhesion motifs which are present in classical CCp proteins. In addition, the B. bovis CCp5 and FNPA are in synteny with known CCp genes in related apicomplexans. Sequence analysis of these two proteins demonstrated high sequence conservation among B. bovis different isolates. Transcription, immunoblot, and immunofluorescence analyses demonstrated expression of CCp5 and FNPA in blood and in vitro induced sexual stages of B. bovis. The FNPA, in contrast to CCp5, has a predicted transmembrane domain, suggesting that it might be expressed in the surface of sexual stage parasites. Altogether, finding of this study support FNPA as a possible target of a transmission-blocking vaccine against B. bovis.

Seasonal survey, risk factor's analysis and genotyping of Theileria annulata infecting cattle in Punjab province, Pakistan.

Theileriosis is one of the most frequently reported tick borne diseases in tropical and subtropical regions and leads to annual economic losses, such as the reduced dairy products and increased casualties. Tropical theileriosis is caused by Theileria annulata and the present study was designed to improve our knowledge of Theileria annulata infection in Pakistani cattle. In order to assess the prevalence of Theileria annulata on cattle from Multan district in the Punjab province (Pakistan) according to seasons and other risk factors, a total of 1020 blood samples (340 samples each from cross, Holstein Frisian and Sahiwal breed) were collected between 2020 and 2022. Based on Tams-1 partial sequence amplification, the overall T. annulata prevalence was estimated at 11.3% (115/1020). The highest prevalence was observed in autumn season (14.1%), followed by winter (12.9%), summer (11.4%) and spring (6.7%) season. Sahiwal cattle were most susceptible to T. annulata infection (13.2%) followed by Crossbred (11.8%) and Holstein Frisian (8.8%). Epidemiological factor analysis revealed that female cattle, cattle rose with other dairy animals at farm, tick infested cattle, and cattle raised with dogs infested with ticks were associated with the prevalence of T. annulata. White blood cells, lymphocyte (%), Monocyte (%) hemoglobin, mean cell hemoglobin, mean corpuscular hemoglobin concentration, and platelet count were significantly affected blood parameters in T. annulata positive cattle of all three breeds. Representative partial Tams-1 sequences of four Pakistani T. annulata isolates revealed a single genotype genetically close to those infecting cattle from neighboring countries like Iran, Turkey and Egypt. Longitudinal survey and phylogenetic positioning of T. annulata is recommended for epidemiological correlation, diagnosis and treatment of theileriosis in such an agricultural region of Pakistan.

First report on molecular epidemiology, seasonality and phylogeny of Toxoplasma gondii infecting goats from Khanewal district in Punjab, Pakistan.

Raising small ruminants is the main source of income for farmers in Pakistan. Economic losses caused by Toxoplasma gondii to small ruminants have been reported worldwide, however reports on molecular detection of T. gondii are lacking in Pakistan despite a large goat population. The current study was carried out from March 2019 till February 2020 to report the seasonal and molecular prevalence of T. gondii in different breeds of goats located in Khanewal district of Punjab, Pakistan. A total of 898 blood goat samples were collected during the four seasons and screened for T. gondii DNA by using PCR based on the amplification of ITS-1 partial sequence. Out of 898 goats, 48 (5.3%) were found positive to T. gondii. The prevalence of T. gondii varied according to season (Chi square test,P = 0.016) and the highest prevalence was observed in goats tested during the summer (8.8%) followed by the spring (5.7%), the winter (4.4%) and the autumn season (2.2%). PCR products positive to T. gondii were confirmed by DNA sequencing and BLAST analysis. Phylogenetic study based on ITS 1 gene provided evidence that the amplified isolates of T. gondii were highly conserved in Pakistani goats. Buck (Fischer exact test, P = 0.002) and farms containing other dairy animals next to goats (Fischer exact test, P = 0.001) and farms with a water supply from pools (Fischer exact test, P = 0.001) were more infected with T. gondii. Infected goats had a reduction on red blood cell count (Two-sample t test, P = 0.01) and hemoglobin concentration (Two-sample t test, P = 0.03) and an increase in the number of monocytes (%) (Two-sample t test, P = 0.05), mean cell hemoglobin (Two-sample t test, P = 0.01) and serum creatinine (Two-sample t test, P = 0.01) as compared to T. gondii uninfected goats. In conclusion, we report a relatively low PCR based prevalence of T. gondii in goats from Khanewal district as previously the serum ELISA test based prevalence of T. gondii in Pakistani goats varied between 19-52%. Control measures should be taken to eradicate T. gondii infection in goats of the study area.

Identification of novel immune correlates of protection against acute bovine babesiosis by superinfecting cattle with in vitro culture attenuated and virulent Babesia bovis strains.

The apicomplexan tickborne parasites Babesia bovis and B. bigemina are the major causative agents of bovine babesiosis, a disease that negatively affects the cattle industry and food safety around the world. The absence of correlates of protection represents one major impediment for the development of effective and sustainable vaccines against bovine babesiosis. Herein we superinfected cattle with attenuated and virulent strains of B. bovis to investigate immune correlates of protection against acute bovine babesiosis. Three 6-month-old Holstein calves were infected intravenously (IV) with the in vitro culture attenuated Att-S74-T3Bo B. bovis strain (106 infected bovine red blood cells (iRBC)/calf) while three age-matched Holstein calves were inoculated IV with normal RBC as controls (106 RBC/calf). All Att-S74-T3Bo-infected calves showed a significant increase in temperature early after inoculation but recovered without treatment. Att-S74-T3Bo-infected calves also developed: (a) monocytosis, neutropenia, and CD4+ lymphopenia in peripheral blood on days 3 to 7 post-inoculation; (b) significant levels of TNFα, CXCL10, IFNγ, IL-4, and IL-10 in sera at day 6 after infection; and (c) IgM and IgG against B. bovis antigens, starting at days 10 and 30 post-inoculation, respectively. At 46 days post-Att-S74-T3Bo inoculation, all experimental calves were infected IV with the homologous virulent B. bovis strain Vir-S74-T3Bo (107 iRBC/calf). All Att-S74-T3Bo-infected calves survived superinfection with Vir-S74-T3Bo without displaying signs of acute babesiosis. In contrast, control animals showed signs of acute disease, starting at day 10 post-Vir-S74-T3Bo infection, and two of them were humanely euthanized at days 13 and 14 after inoculation due to the severity of their symptoms. Also, control calves showed higher (P<0.05) parasite load in peripheral blood compared to animals previously exposed to Att-S74-T3Bo. No significant alterations in the profile of leukocytes and cytokines were observed in Att-S74-T3Bo-inoculated after Vir-S74-T3Bo infection. In conclusion, data demonstrate novel changes in the profile of blood immune cells and cytokine expression in peripheral blood that are associated with protection against acute bovine babesiosis. These identified immune correlates of protection may be useful for designing effective and sustainable vaccines against babesiosis in cattle.

Prevalence of Anaplasma marginale in cattle blood samples collected from two important livestock regions in Punjab (Pakistan) with a note on epidemiology and phylogeny of parasite.

Anaplasmosis, caused by intracellular gram-negative bacteria Anaplasma marginale is one of the most frequently reported tick-borne disease (TBDs) in tropical and sub-tropical countries, including Pakistan. In the present study, a total of 428 cattle blood samples were collected to examine the prevalence and phylogenetic origin of A. marginale in two important livestock regions of Punjab Province in Pakistan, i.e. Lodhran and Dera Ghazi Khan Districts. In addition, association between occurrence of A. marginale in cattle blood and selected epidemiological factors has been also investigated. The presence of A. marginale genetic material was confirmed in 9% of the tested blood samples taken from cattle in Lodhran and in 17% from Dera Ghazi Khan. Prevalence of A. marginale was significantly higher in cattle from Dera Ghazi Khan. All the cattle breeds from both districts were equally susceptible to A. marginale infection. We reported higher prevalence of A. marginale in cattle living indoors or with other dairy animals in Dera Ghazi Khan district. However, no such relationship was observed in the Lodhran district. Sequencing of the msp1b gene shows 96-99% similarity of A. marginale in the study area to those reported from other parts of Pakistan, South Africa, and Israel. We recommend that large scale tick and tick-borne disease control strategies must be implemented in both districts.

Genetic diversity of major surface protein 1a of Anaplasma marginale in dairy cattle.

Major surface protein 1 alpha (Msp1α) is a stable genetic marker for identifying Anaplasma marginale. Our aims were to investigate the genetic diversity of A. marginale based on microsatellites and tandem repeats of the msp1α gene and to determine the phylogenetic relationships among six isolates obtained from 63 dairy cows positive for the pathogen from Turkey and from strains worldwide. Msp1α microsatellite analysis revealed infection with three genotypes (B, C, G). Genotype B was found in cows held in an intensive management system, while genotypes C and G were found in grazing cows. Microsatellite sequences produced SD-ATG distances of 19 and 23 nucleotides, with 23 nucleotides predominating, showing high infection potential. Fourteen different tandem repeats of A. marginale were found in the samples, and four msp1α gene repeats designated Tr4, Tr5, Tr6 and Tr7 were newly described. The majority of A. marginale isolates exhibited more than three Msp1a tandem repeats, with a maximum of seven. The Msp1a tandem repeats α-73-73-73-73-73-73 were commonly observed in intensively managed cows. The phylogenetic analysis revealed A. marginale strains from Turkey clustering with those of the Americas, Africa, Asia and Europe, suggesting a history of cattle trade and transport.

Molecular Detection and Phylogeny of Anaplasma phagocytophilum and Related Variants in Small Ruminants from Turkey.

Anaplasma phagocytophilum causes tick-borne fever in small ruminants. Recently, novel Anaplasma variants related to A. phagocytophilum have been reported in ruminants from Tunisia, Italy, South Korea, Japan, and China. Based on 16S rRNA and groEL genes and sequencing, we screened the frequency of A. phagocytophilum and related variants in 433 apparently healthy small ruminants in Turkey. Anaplasma spp. overall infection rates were 27.9% (121/433 analyzed samples). The frequency of A. phagocytophilum and A. phagocytophilum-like 1 infections was 1.4% and 26.5%, respectively. No A. phagocytophilum-like 2 was detected in the tested animals. The prevalence of Anaplasma spp. was comparable in species, and no significant difference was detected between sheep and goats, whereas the prevalence significantly increased with tick infestation. Sequencing confirmed PCR-RFLP data and showed the presence of A. phagocytophilum and A. phagocytophilum-like-1 variant in the sampled animals. Phylogeny-based on 16S rRNA gene revealed the A. phagocytophilum-like 1 in a separate clade together with the previous isolates detected in small ruminants and ticks. In this work, A. phagocytophilum-like 1 has been detected for the first time in sheep and goats from Turkey. This finding revealed that the variant should be considered in the diagnosis of caprine and ovine anaplasmosis.