Microscopic and molecular investigation of vector borne haemoprotozoan diseases in dromedary camel of North Gujarat, India.

BACKGROUND OBJECTIVES: Vector-borne haemoprotozoan diseases comprise diverse group of single celled organism transmitted by haematophagus invertebrates. The current study was aimed at the identification of major haemoprotozoan (Babesia, Theileria and Trypanosoma) in dromedary camel of North Gujarat region in India using microscopy and Polymerase Chain Reaction (PCR). METHODS: A total of 234 blood samples were screened by the microscopic and molecular detection assays. Molecular prevalence studies of Theileria, Trypanosoma spp and Babesia was undertaken using 18s ribosomal DNA, RoTat 1.2 and SS rRNA gene respectively. The data relating to microscopic and molecular prevalence along with associated risk factors were analysed by statistical methods. RESULTS: The overall prevalence of hamoprotozoan disease based on microscopic and molecular investigation was 23.50%. The sensitivity and specificity (95% Confidence Interval) of PCR assay was 100% in comparison to microscopy (45.45 % sensitive and 100 % specific). The kappa coefficient between PCR and microscopy indicated good level of agreement with a value of 0.704 and SE of 0.159. INTERPRETATION CONCLUSION: Despite holding much significance to the animal sector, little work has been undertaken in regional parts of India regarding camel parasites. The present study offers first preliminary research data investigating haemoprotozoan disease using parasitological and molecular methods in camels in the region.

Molecular detection of piroplasms in domestic donkeys in Xinjiang, China.

BACKGROUND: Piroplasmosis is a common and prevalent tick-borne disease that affects equids. OBJECTIVES: To determine the infection and molecular characteristics of the piroplasms in donkeys from Xinjiang, northwestern China, we undertook a cross sectional study by collecting representative samples across several counties within the region. METHODS: A total of 344 blood samples were collected from adult domestic donkeys from 13 counties in Xinjiang. PCR was conducted to test for T. equi and B. caballi in the blood samples based on the equine merozoite antigen-1 (Ema-1) gene and the 48 kDa rhoptry protein (BC48) gene, respectively. RESULTS: Sixteen blood samples tested positive for piroplasms and the overall infection rate was 4.7% (16/344). Seven of the 13 counties were positive for piroplasms. Among the 16 piroplasm-positive samples, 15 were singly infected with T. equi with an infection rate of 4.4% (15/344), and coinfection with T. equi and B. caballi was detected in one sample (0.3%, 1/344) from Wushi. Four T. equi sequence genotypes were identified and grouped into different branches of the evolutionary trees. CONCLUSION: These findings suggest that the infection rate of piroplasms is low in domestic donkeys in southern Xinjiang and that T. equi genotypes have a regional distribution.

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

BACKGROUND: Tick-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. RESULTS: We 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. CONCLUSION: This 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.

First molecular survey of tick-borne protozoan and bacterial pathogens in the questing tick population in Bangladesh.

Questing ticks carry various tick-borne pathogens (TBPs) that are responsible for causing tick-borne diseases (TBDs) in humans and animals around the globe, especially in the tropics and sub-tropics. Information on the distribution of ticks and TBPs in a specific geography is crucial for the formulation of mitigation measures against TBDs. Therefore, this study aimed to survey the TBPs in the questing tick population in Bangladesh. A total of 2748 questing hard ticks were collected from the pastures in Sylhet, Bandarban, Sirajganj, Dhaka, and Mymensingh districts through the flagging method. After morphological identification, the ticks were grouped into 142 pools based on their species, sexes, life stages, and collection sites. The genomic DNA extracted from tick specimens was screened for 14 pathogens, namely Babesia bigemina (AMA-1), Babesia bovis (RAP-1), Babesia naoakii (AMA-1), Babesia ovis (18S rRNA), Theileria luwenshuni (18S rRNA), Theileria annulata (Tams-1), Theileria orientalis (MPSP), Anaplasma marginale (groEL), Anaplasma phagocytophilum (16S rRNA), Anaplasma bovis (16S rRNA), Anaplasma platys (16S rRNA), Ehrlichia spp. (16S rRNA), Rickettsia spp. (gltA), and Borrelia (Bo.) spp. (flagellin B) using genus and species-specific polymerase chain reaction (PCR) assays. The prevalence of the detected pathogens was calculated using the maximum likelihood method (MLE) with 95 % confidence interval (CI). Among 2748 ixodid ticks, 2332 (84.86 %) and 416 (15.14 %) were identified as Haemaphysalis bispinosa and Rhipicephalus microplus, respectively. Haemaphysalis bispinosa was found to carry all the seven detected pathogens, while larvae of R. microplus were found to carry only Bo. theileri. Among the TBPs, the highest detection rate was observed in A. bovis (20/142 pools, 0.81 %, CI: 0.51-1.20), followed by T. orientalis (19/142 pools, 0.72 %, CI: 0.44-1.09), T. luwenshuni (9/142 pools, 0.34 %, CI: 0.16-0.62), B. ovis (4/142 pools, 0.15 %, CI: 0.05 - 0.34) and Bo. theileri (4/142 pools, 0.15 %, CI: 0.05-0.34), Ehrlichia ewingii (3/142 pools, 0.11 %, CI: 0.03-0.29), and Babesia bigemina (1/142, 0.04 %, CI: 0.00 - 0.16). This study reports the existence of T. luwenshuni, E. ewingii, and Bo. theileri in Bangladesh for the first time. The novel findings of this study are the foremost documentation of transovarian transmission of B. bigemina and E. ewingii in H. bispinosa and also provide primary molecular evidence on the presence of E. ewingii and Bo. theileri in H. bispinosa. Therefore, this study may shed light on the circulating TBPs in ticks in the natural environment and thereby advocate awareness among physicians and veterinarians to control and prevent TBDs in Bangladesh.

Molecular detection of tick-borne piroplasmids in camel blood samples collected from Cairo and Giza governorates, Egypt.

Piroplasmosis, a tick-borne disease affecting livestock, including camels, is caused by intracellular apicomplexan parasites belonging to the order Piroplasmida. Despite its importance, there's limited research on piroplasmosis among Egyptian camels. This study aimed to fill this gap by investigating tick-borne piroplasmids in camels from Cairo and Giza Governorates. Out of 181 blood samples collected between October 2021 and March 2022 from apparently healthy one-humped camels (Camelus dromedarius), PCR assays revealed a 41.4 % infection rate with various piroplasmids. Detected species included B. bovis (17.7 %), B. bigemina (12.2 %), B. caballi (8.3 %), B. naoakii (11.6 %), B. microti (1.7 %), T. equi (4.4 %), and Theileria spp. (28.7 %). Phylogenetic analysis revealed the first detection of T. equi genotype E in Egypt and identified a novel B. caballi genotype. Additionally, B. microti isolates were identified as the US-type. These findings shed lights on piroplasmosis among Egyptian camels, and provide valuable information for devising effective control strategies, especially B. microti, a pathogen with potential human health risks.

Tissue-specific localization of tick-borne pathogens in ticks collected from camels in Kenya: insights into vector competence.

Background: Tick-borne pathogen (TBP) surveillance studies often use whole-tick homogenates when inferring tick-pathogen associations. However, localized TBP infections within tick tissues (saliva, hemolymph, salivary glands, and midgut) can inform pathogen transmission mechanisms and are key to disentangling pathogen detection from vector competence. Methods: We screened 278 camel blood samples and 504 tick tissue samples derived from 126 camel ticks sampled in two Kenyan counties (Laikipia and Marsabit) for Anaplasma, Ehrlichia, Coxiella, Rickettsia, Theileria, and Babesia by PCR-HRM analysis. Results: Candidatus Anaplasma camelii infections were common in camels (91%), but absent in all samples from Rhipicephalus pulchellus, Amblyomma gemma, Hyalomma dromedarii, and Hyalomma rufipes ticks. We detected Ehrlichia ruminantium in all tissues of the four tick species, but Rickettsia aeschlimannii was only found in Hy. rufipes (all tissues). Rickettsia africae was highest in Am. gemma (62.5%), mainly in the hemolymph (45%) and less frequently in the midgut (27.5%) and lowest in Rh. pulchellus (29.4%), where midgut and hemolymph detection rates were 17.6% and 11.8%, respectively. Similarly, in Hy. dromedarii, R. africae was mainly detected in the midgut (41.7%) but was absent in the hemolymph. Rickettsia africae was not detected in Hy. rufipes. No Coxiella, Theileria, or Babesia spp. were detected in this study. Conclusions: The tissue-specific localization of R. africae, found mainly in the hemolymph of Am. gemma, is congruent with the role of this tick species as its transmission vector. Thus, occurrence of TBPs in the hemolymph could serve as a predictor of vector competence of TBP transmission, especially in comparison to detection rates in the midgut, from which they must cross tissue barriers to effectively replicate and disseminate across tick tissues. Further studies should focus on exploring the distribution of TBPs within tick tissues to enhance knowledge of TBP epidemiology and to distinguish competent vectors from dead-end hosts.

Detection of tick-borne pathogens in Rhipicephalus bursa ticks collected from the autochthonous Garrano breed of horses in Portugal.

The Garrano is a semi-feral horse breed native to several mountains in the northern Iberian Peninsula. Despite being endangered, this unique breed of pony has managed to survive in the wild and continues to be selectively bred, highlighting their remarkable resilience and adaptability to harsh environments. Wildlife plays a critical role in the survival of tick vectors in their natural habitats and the transfer of tick-borne pathogens, as they can serve as reservoir hosts for many agents and amplifiers for these vectors. The semi-feral lifestyle of the Garrano horses makes them particularly vulnerable to exposure to numerous tick species throughout the year. Therefore, the aim of this study was to investigate the occurrence of Anaplasma, Ehrlichia, Babesia, Theileria, and spotted fever rickettsiae in the Garrano horse ticks to obtain a knowledge of circulating agents in this host population. The collected ticks (n = 455) were identified as Rhipicephalus bursa. DNA specimens were organized in pools of 5 ticks, for molecular screening. Pools PCR results confirmed the presence of Candidatus Rickettsia barbariae (n = 12 for the ompB gene, n = 11 for the ompA gene and n = 6 for the gltA gene), Babesia bigemina (n = 1), Babesia caballi (n = 3), Theileria equi (n = 15) and Theileria haneyi (n = 1).These results confirm the circulation of an emerging rickettsial spotted fever group member, Candidatus R. barbariae, in R. bursa ticks. Our findings demonstrated that Candidatus R. barbariae co-circulates with B. bigemina and T. equi, which are vectored by R. bursa. We are reporting for the first time, the detection of T. haneyi among R. bursa ticks feeding in the Garrano horses in Portugal. Surveillance studies for tick-borne infections are essential to provide information that can facilitate the implementation of preventive and control strategies.

Metabarcoding of protozoa and helminth in black-necked cranes: a high prevalence of parasites and free-living amoebae.

Parasites and free-living amoebae (FLA) are common pathogens that pose threats to wildlife and humans. The black-necked crane (Grus nigricollis) is a near-threatened species and there is a shortage of research on its parasite diversity. Our study aimed to use noninvasive methods to detect intestinal parasites and pathogenic FLA in G. nigricollis using high-throughput sequencing (HTS) based on the 18S rDNA V9 region. A total of 38 fresh fecal samples were collected in Dashanbao, China, during the overwintering period (early-, middle I-, middle II-, and late-winter). Based on the 18S data, eight genera of parasites were identified, including three protozoan parasites: Eimeria sp. (92.1%) was the dominant parasite, followed by Tetratrichomonas sp. (36.8%) and Theileria sp. (2.6%). Five genera of helminths were found: Echinostoma sp. (100%), Posthodiplostomum sp. (50.0%), Euryhelmis sp. (26.3%), Eucoleus sp. (50.0%), and Halomonhystera sp. (2.6%). Additionally, eight genera of FLA were detected, including the known pathogens Acanthamoeba spp. (n = 13) and Allovahlkampfia spp. (n = 3). Specific PCRs were used to further identify the species of some parasites and FLA. Furthermore, the 18S data indicated significant changes in the relative abundance and genus diversity of the protozoan parasites and FLA among the four periods. These results underscore the importance of long-term monitoring of pathogens in black-necked cranes to protect this near-endangered species.

Title: Métabarcoding des protozoaires et des helminthes chez les grues à cou noir : forte prévalence de parasites et d'amibes libres. Abstract: Les parasites et les amibes libres sont des agents pathogènes courants qui constituent une menace pour la faune et les humains. La grue à cou noir (Grus nigricollis) est une espèce quasi menacée et les recherches sur sa diversité parasitaire sont insuffisantes. Notre étude visait à utiliser des méthodes non invasives pour détecter les parasites intestinaux et les amibes libres pathogènes chez G. nigricollis en utilisant le séquençage à haut débit basé sur la région V9 de l'ADNr 18S. Au total, 38 échantillons de matières fécales fraîches ont été collectés à Dashanbao, en Chine, au cours de la période d'hivernage (début, milieu I, milieu II et fin de l'hiver). Sur la base des données 18S, huit genres de parasites ont été identifiés, dont trois parasites protozoaires : Eimeria sp. (92,1 %) était le parasite dominant, suivi de Tetratrichomonas sp. (36,8 %) et Theileria sp. (2,6 %). Cinq genres d'helminthes ont été trouvés : Echinostoma sp. (100 %), Posthodiplostomum sp. (50,0 %), Euryhelmis sp. (26,3 %), Eucoleus sp. (50,0 %) et Halomonhystera sp. (2,6 %). De plus, huit genres d'amibes libres ont été détectés, y compris les agents pathogènes connus Acanthamoeba spp. (n = 13) et Allovahlkampfia spp. (n = 3). Des PCR spécifiques ont été utilisées pour identifier davantage les espèces de certains parasites et amibes libres. En outre, les données 18S ont indiqué des changements significatifs dans l'abondance relative et la diversité des genres des parasites protozoaires et des amibes au cours des quatre périodes. Ces résultats soulignent l'importance de la surveillance à long terme des agents pathogènes chez les grues à cou noir pour protéger cette espèce quasi menacée.

Hemopathogens in naturally infected bovine fetuses in Brazil.

The transplacental transmission of parasites and hemoparasites is crucial for understanding the epidemiology of diseases. This study aimed to assess the prevalence of hemopathogens in bovine fetuses at various gestational periods. Samples were obtained from a slaughterhouse in the state of Minas Gerais, Brazil, and a total of 236 fetuses were collected. DNA extracted from blood samples (145) and organ samples (a pool of brain and spleen) (236) underwent a nested PCR (nPCR) assay to detect Babesia spp., Theileria spp., Trypanosoma vivax, Anaplasma marginale, Anaplasma bovis, Anaplasma phagocytophilum, Ehrlichia minasensis, and hemotropic Mycoplasma spp. Additionally, serological analysis of 145 plasma samples was conducted using the indirect fluorescent antibody test-IFAT to detect IgG against Babesia bovis, Babesia bigemina, A. marginale, and Trypanosoma vivax. The observed prevalence of transplacental transmission was 19.3 %, 6.2 %, 42.7 % and 2.7 %, for A. marginale, B. bigemina, 'Candidatus M. haemobos', and Mycoplasma wenyonii, respectively. The prevalence of A. marginale by gestational trimester was 16 % (13/81) in the second trimester and 23 % (14/60) in the third trimester, with no positive samples in the first trimester. Regarding the species B. bovis and B. bigemina, all evaluated animals tested negative by nPCR, and no serological evidence for B. bovis was found by the IFAT. Babesia bigemina demonstrated an overall seroprevalence of 6.2 % (9/145), with 4.8 % (7/145) in the last trimester and 1.3 % (2/145) in the second trimester of pregnancy. In total, 42.7 % (62/145) of blood samples were positive for 'Candidatus M. haemobos', with 42 % (34/81) in the middle trimester, and 43 % (26/60) in the final trimester of pregnancy. Mycoplasma wenyonni was detected in 2.7 % (4/145) blood samples, all in coinfection with 'C. M. haemobos'. The prevalence by pregnancy trimester was 25 % (1/4) in the first trimester; 1.2 % (1/81) in the second trimester and 3.3 % (2/60) in the third trimester of pregnancy. Hemopathogen DNA was detected in fetus blood samples but not the brain or spleen samples. All the samples were negative for T. vivax, Theileria spp., Anaplasma spp. and Ehrlichia spp. Overall, in this study, approximately 70 % of fetuses were positive for one or more of the studied parasites. No significant associations were observed between pairs of pathogens, except 'C. M. haemobos' and A. marginale.

Molecular detection and characterization of prevailing Theileria equi genotype in equine from northern India.

Equine piroplasmosis caused by Theileria equi is a febrile, tick-borne disease of equids. However, there is limited literature about the genotyping of T. equi in India. Blood samples were collected from 202 horses and subjected to microscopy and PCR to detect T. equi. Initially, a universal screening primer pair targeting 18S ribosomal RNA genes common for Babesia caballi and T. equi was employed to amplify the DNA of both parasites. Thereafter additional primers were employed for species-specific detection resulting in amplification of approximately 435 bp specific for T. equi. T.equi was detected in 9.9% and 20.79% of horses screened by microscopy and PCR, respectively. The representative samples confirmed positive by PCR were sequenced, submitted to NCBI (OR651254, OR687254, OR685656, OR650830, OR650834), and used for genotype characterization and phylogenetic analysis. Employing Genetool and MEGA X software, the T. equi Indian isolates and across the globe were compared, and the results demonstrated 99.05-100% and 95.86-100% homologies, respectively. All the T. equi Indian isolates belonged to genotype A. Phylogeny based on the EMA-1 gene of five isolates (OR731831, OR731833, OR731829, OR731830, OR731832) were also characterized by sequencing and support the previous findings. Genotypes C and D, as well as genotypes B and E, exhibited lower levels of evolutionary divergence compared to other genotypes. The EMA-1 gene exhibited limited diversity and might not be the most suitable target for assessing variability within T. equi populations. The findings also reveal a significant association (p < 0.01) between T. equi infection and the presence of ticks.

Transboundary Tick and Tick-Borne Pathogen Threats to Cattle.

Transboundary incursions of ticks and tick-borne pathogens are ever present concerns for US cattle industries. Global trade in livestock and wildlife, historic and emerging transboundary issues with endemic tick populations and pathogens, and migratory bird flyways are pathways of concern. Transboundary challenges are presented for the Asian long-horned tick and Theileria orientalis Ikeda, for 2 cattle fever tick species [Rhipicephalus (Boophilus) annulatus and R (B) microplus] and Babesia bigemina and B bovis, and for the tropical bont tick and Ehrlichia ruminantium.

Equine Piroplasmosis in Asymptomatic Horses of Western Iran: Comparison of Microscopic Examination and Multiplex PCR.

PURPOSE: Piroplasmosis is responsible for anemia, fever, loss of physical activity and even death in equines. In epidemiological studies, accurate diagnostic tests are essential for detecting asymptomatic carriers. This study aimed to investigate the prevalence of infection in asymptomatic horses from Lorestan province, western Iran by developing a multiplex PCR. METHODS AND RESULTS: Blood samples were examined by microscopy and multiplex PCR targeting the SSU rRNA gene of Theileria equi and Babesia caballi. Out of the total of 165 horses, 19 (11.51%) and 31 (18.78%) cases were positive for piroplasms by microscopy and PCR, respectively. The detection rates of both genera were significantly higher in multiplex PCR compared to microscopy (p < 0.0001). Compared with multiplex PCR, the sensitivities of microscopy for the detection of Babesia were only 28.5%. The prevalence of T. equi infection was significantly higher in summer (p = 0.035). The prevalence of B. caballi was significantly higher in males (p = 0.038). CONCLUSION: Findings indicate that the multiplex PCR described here is a sensitive technique for the detection of piroplasm DNA in carriers. Furthermore, asymptomatic carriers must be considered as an important source of infection for equids living in this region.

Systematic Review and Meta-Analysis on Piroplasma spp. Infection and Co-infection with Anaplasma marginale in Domestic Ruminants from Algeria.

BACKGROUND: Piroplasmosis and anaplasmosis stand out as the primary diseases affecting livestock during periods of tick activity. These vector-borne diseases continue to emerge worldwide, exerting a detrimental impact on both animal health and national economies. The purpose of this study is to assess the prevalence of Piroplasma spp. and its co-occurrence with Anaplasma marginale in domestic ruminants in Algeria. METHODS: Three databases were systematically reviewed to identify eligible studies for the final meta-analysis, following the PRISMA statement. The 'meta' package in the R software was employed for the meta-analysis with the random effects model chosen for data pooling. RESULTS: The meta-analysis encompasses 14 research papers spanning a 19-year period (2004-2023). Theileria spp. was identified in all studies, covering 1675 cattle, 190 sheep, and 128 goats, yielding an overall Theileria infection rate of 45% (95% CI 26-65%). Specifically, cattle had a 59% infection rate, while sheep and goats had rates of 18% and 20%, respectively. Babesia spp. was found in nine studies, involving 1183 cattle and 190 sheep, resulting in an overall Babesia infection rate of 7% (95% CI 4-15%), with cattle and sheep having rates of 10% and 3%, respectively. Notably, eight Piroplasma species T. annulata, T. orientalis, T. buffeli, T. equi, Theileria sp., B. bovis, B. bigemina, and B. occultans were detected in cattle, with T. annulata being the most prevalent at 54%. Regional disparities and host factors also impacted infection rates, with higher rates in Northeastern Algeria and among suspected disease cattle. Additionally, gender, age, and breed influenced cattle susceptibility to Theileria infection. Furthermore, six distinct co-infections between Piroplasma spp. and A. marginale were observed, with T. annulata/A. marginale identified in six studies, demonstrating an 8.3% co-infection rate. CONCLUSION: This analysis offers crucial insights into the current status of Piroplasmosis and its co-infection with A. marginale in Algerian domestic ruminants, providing valuable data for surveillance and prevention strategies.

Development of a Test Card Based on Colloidal Gold Immunochromatographic Strips for Rapid Detection of Antibodies against Theileria equi and Babesia caballi.

Equine piroplasmosis (EP) is a serious problem in the horse industry, and controlling EP is critical for international horse trading. EP is caused by two apicomplexan protozoan parasites, Theileria equi and Babesia caballi. Rapid and accurate methods that are suitable for detecting these parasites in the field are crucial to control the infection and spread of EP. In this study, we developed a card to detect antibodies against T. equi and B. caballi based on two colloidal gold immunochromatographic strips according to the principle of the double-antigen sandwich. The proteins equi merozoite antigen 1 (EMA1) and rhoptry protein BC48 are commonly used as diagnostic antigens against T. equi and B. caballi, respectively. On the strip, the purified EMA1 or BC48 protein labeled with colloidal gold was used as the detector, and nitrocellulose membranes were coated with EMA1 or BC48 and the corresponding MAb as the test and control lines, respectively. The protocol takes 10 to 15 min and requires no specialized equipment or chemical reagents, and one test can detect two EP pathogens in one card. Specificity tests confirmed there was no cross-reactivity with sera positive for common equine pathogens. Using a commercial competitive enzyme-linked immunosorbent assay (cELISA) kit for comparison, 476 clinical samples were tested with the card. The coincidence rates were 96.43% and 97.90% for T. equi and B. caballi, respectively. The field trial feedback was uniformly positive, suggesting that this diagnostic tool may be useful for controlling the spread of T. equi and B. caballi. IMPORTANCE Equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi, is an important tick-borne disease of equines that is prevalent in most parts of the world. EP is considered a reportable disease by the World Organization for Animal Health (OIE). The accurate diagnosis and differentiation of T. equi and B. caballi are very important for the prevention, control, and treatment of EP. Therefore, we developed a double-antigen sandwich colloidal gold immunochromatography assay (GICG) to detect T. equi and B. caballi. Two GICG strips were assembled side by side on one card for the detection of T. equi and B. caballi, and the two EP pathogens could be detected in one test. This method was simple, rapid, and specific for the detection of EP; therefore, compared to the previous methods, this method is more suitable for pathogen diagnosis in the field.

Proportion and seasonality of blood parasites in animals in Mosul using the Veterinary Teaching Hospital Lab data.

Several local studies have examined evidence of blood parasites in different animals in Mosul; however, information about the most prevalent parasite and the seasonality of the infection remains limited. The objective of the study conducted here was to investigate the proportion and seasonality of blood parasites in animals in Mosul using the Veterinary Teaching Hospital Lab data. Laboratory records for a period of 25 months were used for data retrieval. In all included animals, Giemsa-stained blood smears were examined by an attending clinical pathologist for the presence of parasites. Seasons were assigned on a basis of examination date, and the seasonality was quantified by estimating season-to-season ratio. The results indicated that 61.77% of examined animals were tested positive for blood parasites. The most evident parasites were Trypanosoma spp., Theileria spp., Babesia spp., and then Anaplasma spp., with evidence of mixed infection. The odds of the infection did not significantly vary in different age groups. There was a marked linear pattern in the seasonality of the infection with Trypanosoma spp. and Anaplasma spp. An increase of the infection during spring and autumn with Theileria spp. and Babesia spp. was also evident. In conclusion, infection with blood parasites in different animals in Mosul is common with substantial burden, the effect of age-related infection is negligible, and the seasonality of the infection is evident.

Natural infection and molecular detection of Cytauxzoon felis in a free-ranging Puma concolor in the state of Goiás, Brazil / Infecção natural e detecção molecular de Cytauxzoon felis em Puma concolor de vida livre no estado de Goiás, Brasil

The puma (Puma concolor Linnaeus, 1771), the most widely distributed felid species in the Americas, can be found in all Brazilian biomes. Nevertheless, few studies have focused on hemoparasites in this species. Cytauxzoon felis, a hemoparasite that can infect domestic cats, has also been described in wild felids in Brazil. To the best of our knowledge, this study is the first to diagnose the natural infection and molecular detection of C. felis in a P. concolor in the state of Goiás. This animal presented non-regenerative anemia and inclusion suggestive of piroplasmids within red blood cells. The amplified 551 bp fragment of partial Piroplasmida 18S rRNA gene sequence was 100% identical to corresponding sequences of C. felis available in GenBank. No specific treatment for cytauxzoonosis was administered, and after rehabilitation, the animal was reintroduced into the wild. This finding provides some evidence that P. concolor may act as a natural host of the parasite. The epidemiology, vector and pathogenicity of this hemoparasite in wild and domestic cats in Brazil deserves further investigation.

O puma (Puma concolor Linnaeus, 1771) tem a maior distribuição entre os felídeos das Américas e é encontrado em todos os biomas do Brasil. No entanto, poucos estudos têm se concentrado nos hemoparasitos nesta espécie. Cytauxzoon felis, um hemoparasito que pode infectar gatos domésticos, também foi descrito em felídeos selvagens no Brasil. A saber, este estudo é o primeiro diagnóstico de infecção natural e detecção molecular de C. felis em um P. concolor do estado de Goiás. Este animal apresentou anemia arregenerativa e inclusão de piroplasmídeos nos glóbulos vermelhos. A amplificação do fragmento de 551 pb da sequência parcial do gene Piroplasmorida 18S rRNA foi 100% idêntica a sequências correspondentes de C. felis disponíveis no GenBank. Nenhum tratamento específico para citauxzoonose foi administrado e, após a reabilitação, o animal foi reintroduzido na natureza. Essa descoberta fornece algumas evidências de que P. concolor pode atuar como um hospedeiro natural do parasito. A epidemiologia, vetor e patogenicidade deste hemoparasito em gatos selvagens e domésticos no Brasil merecem uma investigação mais aprofundada.

Molecular detection of Anaplasma spp., Babesia spp. and Theileria spp. in yaks (Bos grunniens) and Tibetan sheep (Ovis aries) on the Qinghai-Tibetan Plateau, China.

BACKGROUND: Anaplasma, Babesia and Theileria are tick-borne pathogens (TBPs) that affect livestock worldwide. However, information on these pathogens in yaks (Bos grunniens) and Tibetan sheep (Ovis aries) on the Qinghai-Tibet Plateau (QTP), China, is limited. In this study, Anaplasma spp., Babesia spp. and Theileria spp. infections were assessed in yaks and Tibetan sheep from Qinghai Province. METHODS: A total of 734 blood samples were collected from 425 yaks and 309 Tibetan sheep at nine sampling sites. Standard or nested polymerase chain reaction was employed to screen all the blood samples using species- or genus-specific primers. RESULTS: The results showed that 14.1% (60/425) of yaks and 79.9% (247/309) of Tibetan sheep were infected with at least one pathogen. Anaplasma ovis, Anaplasma bovis, Anaplasma capra, Anaplasma phagocytophilum, Babesia bovis and Theileria spp. were detected in this study, with total infection rates for all the assessed animals of 22.1% (162/734), 16.3% (120/734), 23.6% (173/734), 8.2% (60/734), 2.7% (20/734) and 19.3% (142/734), respectively. For yaks, the infection rate of A. bovis was 6.4% (27/425), that of B. bovis was 4.7% (20/425) and that of Theileria spp. was 3.3% (14/425). Moreover, 52.4% (162/309) of the Tibetan sheep samples were infected with A. ovis, 30.1% (93/309) with A. bovis, 56.0% (173/309) with A. capra, 19.4% (60/309) with A. phagocytophilum and 41.4% (128/309) with Theileria spp. CONCLUSIONS: This study revealed the prevalence of Anaplasma spp., Babesia spp. and Theileria spp. in yaks and Tibetan sheep in Qinghai Province, China, and provides new data for a better understanding of the epidemiology of TBPs in these animals in this area of the QTP, China.

Molecular prevalence and risk factors associated with tick-borne pathogens in cattle in western Kenya.

BACKGROUND: Tick-borne pathogens (TBPs) are of global importance, especially in sub-Saharan Africa where they represent a major constraint to livestock production. Their association with human disease is also increasingly recognized, signalling their zoonotic importance. It is therefore crucial to investigate TBPs prevalence in livestock populations and the factors associated with their presence. We set out to identify TBPs present in cattle and to determine associated risk factors in western Kenya, where smallholder livestock production is important for subsistence and market-driven income. RESULTS: Tick-borne pathogen infections in blood samples collected from cattle at livestock markets and slaughterhouses between May 2017 and January 2019 were identified by high-resolution melting analysis and sequencing of PCR products of genus-specific primers. Of the 422 cattle sampled, 30.1% (127/422) were infected with at least one TBP, while 8.8% (37/422) had dual infections. Anaplasma spp. (19.7%) were the most prevalent, followed by Theileria (12.3%), Ehrlichia (6.6%), and Babesia (0.2%) spp. Sequence analysis of the TBPs revealed them to be Anaplasma platys-like organisms (13.5%), Theileria velifera (7.4%), Anaplasma marginale (4.9%), Theileria mutans (3.1%), Theileria parva (1.6%), and Babesia bigemina (0.2%). Ehrlichia ruminantium, Rickettsia spp., and arboviruses were not detected. Exotic breeds of cattle were more likely to be infected with A. marginale compared to local breeds (OR: 7.99, 95% CI: 3.04-22.02, p <  0.001). Presence of ticks was a significant predictor for Anaplasma spp. (OR: 2.18, 95% CI: 1.32-3.69, p = 0.003) and Ehrlichia spp. (OR: 2.79, 95% CI: 1.22-7.23, p = 0.022) infection. Cattle sampled at slaughterhouses were more likely to be positive for Anaplasma spp. (OR: 1.64, 95% CI: 1.01-2.70, p = 0.048) and A. marginale (OR: 3.84, 95% CI: 1.43-12.21, p = 0.012), compared to those sampled at livestock markets. CONCLUSION: This study reports TBP prevalence and associated risk factors in western Kenya, factors which are key to informing surveillance and control measures.

Tick distribution and detection of Babesia and Theileria species in Eastern and Southern Kazakhstan.

Piroplasmosis is an economically important tick-borne disease worldwide. However, little is known about the presence of Babesia spp. and Theileria spp. in ticks in Eastern and Southern Kazakhstan (ESK). During 2016 - 2019, adult ticks (at 26 sampling sites in 16 districts of 5 oblasts in ESK) were collected. Tick species were identified according to morphological and molecular characteristics. Two fragments (487 bp and 438 bp) of 18S ribosomal RNA (18S rRNA) were used to determine piroplasm species in representative 698 ticks. The genotype characteristics of Babesia caballi and Theileria equi were further analyzed by longer 18S rRNA gene fragments. A total of 6107 adult ticks (4558 parasitizing ticks and 1549 off-host ticks), including 4665 hard ticks and 1442 soft ticks, were collected from their natural hosts (cattle, horses, sheep, camels, shepherd dogs and hedgehogs) and the surrounding environment, respectively. Among the hard tick species, Dermacentor marginatus (62.59%, 2920/4665) was the most abundant, followed by Hyalomma asiaticum (19.36%, 903/4665) and Hyalomma detritum (9.95%, 464/4665). All soft ticks were identified as Argas persicus. 16S ribosomal DNA (16S rDNA) phylogenic analysis showed that several tick species in Kazakhstan, as exemplified by Haemaphysalis erinacei and D. marginatus, clustered together with conspecific ticks reported from China. Five species of piroplasms, i.e. Babesia occultans, Babesia caballi, Theileria ovis, Theileria annulata and Theileria equi, were detected in 698 representative ticks. Genotype E of T. equi in Almaty, and genotype A of B. caballi in Almaty and South Kazakhstan were identified.

Molecular detection and phylogenetic characterization of Theileria equi in horses (Equus caballus) from a peri-urban area of Argentina.

To investigate the presence of Theileria equi in an endemic area of equine piroplasmosis 42 horses (Equus caballus) from Corrientes City, Argentina were sampled. Eighty-one percent (34 blood samples) of the analyzed horses were tested positive to the presence of piroplasmid 18S rDNA. All these samples could be identified as T. equi by amplifying the specific EMA-1 (merozoite antigen 1) gene of this species. Phylogenetic analysis of an obtained 18S rDNA complete sequence from one strain resulted in the identification of this sample as T. equi sensu stricto (genotype A). This study presents the first molecular detection and characterization of T. equi by the complete 18S rDNA sequence in Argentina. Based on these results further studies should be carried out to investigate the distribution and heterogeneity of presented genotypes of T. equi in Argentina, which is essential for the diagnosis, prevention and treatment of equine piroplasmosis.