Search

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.

Anaplasma centrale (A. centrale) is an obligate red blood cell residing tick transmitted rickettsiae that has not been studied extensively for its prevalence in cattle along with its epidemiology. Aim of this investigation was to report the seasonal prevalence, phylogeny and epidemiological parameters associated with the prevalence of A. centrale in cattle breeds enrolled from District Layyah in Punjab, Pakistan. A total of 844 blood samples [Cross breed = 300, Holstein Friesian = 244, Sahiwal breed = 300)] were collected from apparently healthy cattle along with epidemiological data during 2017–18. PCR amplified 426 base pair fragment from 16S rRNA gene of A. centrale in 14.4% (122/844) of cattle. Amplified 16S rRNA partial gene sequence of A. centrale were confirmed by DNA sequencing and deposited to GenBank. Highest A. centrale prevalence was observed in spring (24%) followed by autumn (12.4%) summer (10%) and winter (7.1%) seasons. Sahiwal breed (18.3%) was most susceptible to A. centrale infection followed by cross (12.3%) and Holstein Friesian breed (12.3%). 69/844 (8.2%) of Giemsa stained cattle blood smears were also found positive for Anaplasma spp. Farms where animal use to drink pool water and farms where dogs and other dairy animals were living with cattle had higher A. centrale prevalence. Female cattle and dogs having tick burden were found associated with A. centrale infection. Hematological profile was severely disturbed in A. centrale positive cattle. It is recommended that A. centrale should be screened in cattle, in addition to A. marginale, for the effective control of tick born diseases in Pakistan.

The ikeda and chitose genotypes of

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.

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.

In this study, the presence, prevalence, and genotypes of Anaplasma phagocytophilum, A. ovis, and A. capra in sheep were investigated based on 16 S SSU rRNA, groEL, and gtlA gene-specific polymerase chain reaction (PCR), respectively. The sequences of the genes were used for detection of the phylogenetic position of the species. Additionally, a restriction fragment length polymorphism (RFLP) were carried out for discrimination of A. phagocytophilum and related variants (A. phagocytophilum-like 1 and 2). The prevalence of Anaplasma spp. was found as 25.8% (101/391), while it was found that A. ovis, A. phagocytophilum-like 1, and A. capra are circulating in the sheep herds in Kyrgyzstan, according to the PCRs, RFLP and the partial DNA sequencing results. The positivity rates of A. phagocytophilum-like 1, A. ovis, and A. capra genotype-1 were 6.9, 22.5, and 5.3%, respectively. A total of 32 (8.2%) sheep were found to be mix infected. Moreover, phylogenetic analyses and sequence comparison with those available in the GenBank showed that A. capra formed two distinct genetic groups (A. capra genotype-1 and A. capra genotype-2). Considering the zoonotic potential of these species, it may be necessary to make changes in the interpretation of anaplasmosis cases in animals and there is a need for further studies to determine the pathogenicity of the species/genotypes circulating in animals.

Simple Summary Pakistan is a tropical country where climate is favourable for tick growth and hence its incidence of tick-borne diseases is high, affecting the output of the livestock sector. In the present study the infection rate of Theileria annulata, the causative agent of bovine theileriosis, was compared in apparently healthy cattle enrolled from two different regions in Pakistan. Parasite prevalence was found to be higher in Dera Ghazi Khan District than in Lodhran. The infection rate was higher in cattle that were infested with ticks and in those animals that were housed indoors at dairy farms with other animals. This prophylactic detection of parasite will help to design strategies to control tick and tick-borne diseases in study areas. Abstract The present study was designed to report the molecular prevalence of T. annulata in cattle blood samples collected from Punjab in Pakistan. A total of 428 cattle blood samples were collected from Districts Lodhran (n = 218) and Dera Ghazi Khan (n = 210). The prevalence of T. annulata was determined by the amplification of a fragment from its cytochrome b gene and parasite prevalence was significantly higher (p = 0.03) in the blood samples of cattle collected from Dera Ghazi Khan (70/210; 33%) as compared to Lodhran (52/218; 24%). Presence of T. annulata was also confirmed by the amplification of a fragment from their 30 kDa gene. The amplified PCR products of both genes were confirmed by DNA sequencing and these partial DNA sequences were submitted to GenBank. Phylogenetic analysis revealed that amplified partial gene sequences resembled previously reported T. annulata sequences in cattle from India, China, Iran, Tunisia, Turkey and Egypt. The incidence of T. annulata infection was higher in Sahiwal cattle (p = 0.04) than the other enrolled cattle breed from Dera Ghazi Khan. Female cattle from Lodhran (p = 0.02), while males (p = 0.02), animals housed in close compounds (p = 0.04), animals with a tick burden (p = 0.005) and farms with only cattle (p = 0.01) in Dear Ghazi Khan were found to be more susceptible to T. annulata infection. We recommend that large-scale tick and tick-borne disease control strategies be implemented in both districts under investigation, especially in Dera Ghazi Khan.

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).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.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.

Anaplasmosis, caused by intracellular gram-negative bacteria

The Mediterranean tick, Hyalomma marginatum, is the most important vector of Crimean-Congo haemorrhagic fever virus and several pathogens that cause animal and human diseases and economic losses to livestock production. Given the medical and veterinary importance of this tick species, we sequenced and characterized its mitochondrial genome (mitogenome) for the first time. We designed two new primer sets and combined long-range PCR with next generation sequencing to generate complete mitogenomes with deep coverage from 10 H. marginatum adults. The mitogenomes contained 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal subunits, two control regions, and three tick-box motifs. The nucleotide composition of the H. marginatum mitogenomes were A+T biased (79.76%) and exhibited negative AT- and GC- skews across most PCGs. All PCGs were initiated by ATK codons and two truncated termination codons were seen in the COX2 and COX3 genes. All tRNAs exhibited typical cloverleaf structures, except for tRNACys and tRNASer1. A total of 62 polymorphic sites defined ten unique haplotypes. Phylogenetic analyses based on the 13 PCGs of 56 tick species revealed that four Hyalomma species (H. marginatum, H. asiaticum, H. rufipes, and H. truncatum) formed a monophyletic clade with strong support. The results of this study provide a comprehensive resource for further studies on the systematics, population genetics, molecular epidemiology, and evolution of ticks.

The present study was designed to check the molecular detection of Anaplasma marginale and Theileria annulata in blood samples of horses and donkeys collected from Dera Ghazi Khan District in Punjab and to document their phylogenetic origin and their association with studied epidemiological factors (sex and age) and complete blood count parameters, if any. A total of 195 blood samples were collected from apparently healthy horses (N = 141) and donkeys (N = 54). A. marginale DNA was detected by PCR in 4.9% (7/141) horse and in 9.2% (5/54) of donkey blood samples. Prevalence of T. annulata was 5.6% (8/141) and 11.1% (6/54) in horse and donkey samples, respectively. While 1.4% (N = 2) horses and 3.7% (N = 2) donkeys were found co-infected with both parasites. Representative amplicon for both parasites was confirmed by DNA sequenced and partial DNA sequence of the major surface protein–1b encoding gene of A. marginale and cytochrome b gene from T. annulata were submitted to the GenBank database under the accession number MK792344-MK792348. Epidemiological data analysis revealed that female horses were more prone to A. marginale (P = 0.02) while female donkeys were more susceptible to A. marginale (P

WOS:000472627700005 Tick-borne pathogens cause serious health problems and loss of productivity in domesticated and wild animals. A. molecular study was performed to detect the frequency of infection with Anaplasma/Ehrtichia (A/E) in cattle from Karaman province of Turkey. Venous blood samples were taken from 150 apparently healthy cattle in 2016. After amplification the hypervariable VI region of the 16S rRNA gene of A/E species, a reverse line blot (RLB) assay was performed using species-specific probes. Since some samples gave signal only to A/E catch-all probe, the samples analyzed in terms of major surface proteins (MSPs) of Anaplasma marginale. Genetic diversity and tandem repeat analysis were made for msp1 alpha gene sequences of A. marginale.Anaplasma-like bodies were detected in four (2.66%) animals via microscopic examination. Anaplasma centrale was detected in eight (5.33%) animals via RLB. When the samples were examined in terms of A. marginale msp 1 a gene with semi-nested PCR, a total of nine (6.00%) animals [six of them (4.00%) were positive for A. centrale with RLB] were found to be infected with A. marginale. In addition, the sequences of MSP 1a amplicons revealed one new tandem repeat (Tr70). According to these results, it was determined that A. marginale and A. centrale were found in cattle in Karaman province and this study provided the first evidence of genetic diversity of A. marginale with one new tandem repeat in cattle in the region. Kene kaynaklı patojenler evcil ve yabani hayvanlarda ciddi sağlık problemlerine ve verim kaybına neden olur. Karaman ilindeki sığırlarda Anaplasma / Ehrlichia (A/E) ile enfeksiyon sıklığını saptamak için moleküler bir çalışma yapıldı. Venöz kan numuneleri görünüşte sağlıklı olan 150 sığırdan 2016 yılında alınmıştır. A/E türlerinin 16S rRNA geninin değişken V1 bölgesi amplifiye edildikten sonra tür spesifik problar kullanılarak reverse line blot (RLB) deneyi gerçekleştirilmiştir. Bazı örnekler sadece A/E probuna sinyal verdiğinden, örnekler Anaplasma marginale'nin major surface proteinleri (MSPs) açısından analiz edilmiştir. A. marginale'nin msp1α gen dizileri için genetik çeşitlilik ve tandem tekrar analizi yapıldı. Mikroskobik inceleme ile dört (%2.66) hayvanda Anaplasma benzeri cisimler tespit edildi. Anaplasma centrale RLB ile sekiz (%5.33) hayvanda tespit edildi. Örnekler seminested PZR ile A. marginale msp1a geni açısından incelediğinde, toplam dokuz (%6.00) hayvanın [6'sı (%4.00) RLB ile A. centrale açısından pozitif olan] A. marginale ile enfekte olduğu bulunmuştur. Ek olarak, MSP1a amplikonlarının dizileri bir tane yeni tandem tekrarı (Tr70) ortaya çıkardı. Bu sonuçlara göre Karaman ilindeki sığırlarda A. marginale ve A. centrale'nin bulunduğu tespit edilmiş ve bu çalışma bölgedeki sığırlarda bir yeni tandem tekrarı ile A. marginale’nin genetik çeşitliliğinin ilk kanıtını sağlamıştır. Commission of Scientific Research Projects, Karamanoglu Mehmetbey UniversityKaramanoglu Mehmetbey University [41-M-16] This work was supported financially by a grant (41-M-16) from the Commission of Scientific Research Projects, Karamanoglu Mehmetbey University.

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.

Anaplasma marginale, Theileria ovis and Theileria lestoquardi are intracellular pathogens that infect a wide variety of animals and cause enormous economic losses worldwide. The present investigation aims to report the occurrence and the phylogeny of these pathogens infecting sheep (N = 330) from Rajanpur District in Punjab (Pakistan) by using msp1b gene for A. marginale and 18 S rRNA for T. ovis and T. lestoquardi. Results revealed that prevalence rates of A. marginale, T. ovis and T. lestoquardi were 7.3%, 6.1% and 1.2%, respectively. Four (1.2%) and one (0.3%) sheep were found to be co-infected with two and three pathogens, respectively. Risk factor analysis revealed that rams were more infected with A. marginale compared to ewes (P = 0.015). In addition, it was observed that animals located in herds having dogs and those in small herds were found more infected, respectively, with T. ovis (P = 0.001) and T. lestoquardi (P = 0.009). Phylogenetic analysis revealed that Pakistani isolates of each detected pathogens clustered together and were closely related to other Pakistani isolates and those from worldwide countries such as Iran, Iraq, Turkey, Sudan, Tanzania, South Africa, and USA. This is the first molecular study reporting the natural infection of Pakistani sheep with these three pathogens. These data need to be taken into account in order to improve the productivity of the livestock sector which is one of main sources of income in the country.

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.

Anaplasma centrale (A. centrale) is an obligate red blood cell residing tick transmitted rickettsiae that has not been studied extensively for its prevalence in cattle along with its epidemiology. Aim of this investigation was to report the seasonal prevalence, phylogeny and epidemiological parameters associated with the prevalence of A. centrale in cattle breeds enrolled from District Layyah in Punjab, Pakistan. A total of 844 blood samples [Cross breed = 300, Holstein Friesian = 244, Sahiwal breed = 300)] were collected from apparently healthy cattle along with epidemiological data during 2017–18. PCR amplified 426 base pair fragment from 16S rRNA gene of A. centrale in 14.4% (122/844) of cattle. Amplified 16S rRNA partial gene sequence of A. centrale were confirmed by DNA sequencing and deposited to GenBank. Highest A. centrale prevalence was observed in spring (24%) followed by autumn (12.4%) summer (10%) and winter (7.1%) seasons. Sahiwal breed (18.3%) was most susceptible to A. centrale infection followed by cross (12.3%) and Holstein Friesian breed (12.3%). 69/844 (8.2%) of Giemsa stained cattle blood smears were also found positive for Anaplasma spp. Farms where animal use to drink pool water and farms where dogs and other dairy animals were living with cattle had higher A. centrale prevalence. Female cattle and dogs having tick burden were found associated with A. centrale infection. Hematological profile was severely disturbed in A. centrale positive cattle. It is recommended that A. centrale should be screened in cattle, in addition to A. marginale, for the effective control of tick born diseases in Pakistan.

Back ground: Anaplasmosis is known as yellow bag or yellow fever and it is a tick borne disease caused by obligate intercellular gram negative bacteria, Anaplasma marginale. Aim: Present study is reporting seasonal prevalence, epidemiology and phylogeny of Anaplasma marginale in three cattle breeds from District Layyah, Southern Punjab, Pakistan. Methods and results: 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. Results: Polymerase chain reaction generated 265 base pair amplicon specific for Major surface protein–1b encoding gene of Anaplasma marginale in 8.6% (73/844) of enrolled cattle. 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). Its phylogenetic analysis had shown similarities with sequences submitted by other countries. 37/844 (4.3%) Giemsa stained blood smears were found positive for Anaplasma spp. 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 (%) heamatocrit, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were significantly disturbed in A. marginale positive than negative cattle. Conclusion: We recommend that this PCR protocol should be used for the detection of A. marginale in livestock for their proper diagnosis and treatment.

Anaplasma species are obligate intracellular rickettsial pathogens that affect the health of humans and animals. In this study, we investigated the presence and frequency of Anaplasma species by 16S rRNA PCR-RLB, RFLP, and DNA sequencing in 200 apparently healthy cattle. Anaplasma spp. overall infection rate was 38.5 % (77/200) by RLB. The frequency of single and mixed infections was 31.5 % (63/200) and 7% (14/200), respectively. The most common species was A. marginale (32.5 %), followed by A. centrale (5.5 %), Anaplasma/Ehrlichia catc-all (5.5 %) and Anaplasma sp. Omatjenne (2.5 %). No A. phagocytophilum and A. bovis were detected in the tested animals. Eleven of 77 PCR-positive amplicons gave positive reactions to the catch-all probes but did not show any signals to the species-specific probes, but PCR-RFLP results showed that these amplicons were A. phagocytophilum-like 1 and A. phagocytophilum-like 2 strains. Sequencing and phylogenetic analyses based on 16S rRNA gene validated RFLP findings and provided evidence for the circulation of A. phagocytophilum-like-1 and 2 strains in Turkish cattle. This is the first report of the presence of A. phagocytophilum-like strains in the country. These findings indicate that A. phagocytophilum-like 1 and 2 strains should be taken into account in the differential diagnosis with bovine anaplasmosis.

Babesia ovis is a tick‐transmitted protozoan haemoparasite causing ovine babesiosis in sheep and goats leading to considerable economic loss in Turkey and neighbouring countries. There are no vaccines available, therapeutic drugs leave toxic residues in meat and milk, and tick vector control entails environmental risks. A panel of eight mini‐ and micro‐satellite marker loci was developed and applied to study genetic diversity and substructuring of B. ovis from western, central and eastern Turkey. A high genetic diversity (He = 0.799) was found for the sample of overall B. ovis population (n = 107) analyzed. Principle component analysis (PCoA) revealed the existence of three parasite subpopulations: (a) a small subpopulation of isolates from Aydin, western Turkey; (b) a second cluster predominantly generated by isolates from western Turkey; and (c) a third cluster predominantly formed by isolates from central and eastern Turkey. Two B. ovis isolates from Israel included in the analysis clustered with isolates from central and eastern Turkey. This finding strongly suggests substructuring of a major Turkish population into western versus central–eastern subpopulations, while the additional smaller B. ovis population found in Aydin could have been introduced, more recently, to Turkey. STRUCTURE analysis suggests a limited exchange of parasite strains between the western and the central–eastern regions and vice versa, possibly due to limited trading of sheep. Importantly, evidence for recombinant genotypes was obtained in regionally interchanged parasite isolates. Important climatic differences between the western and the central/eastern region, with average yearly temperatures of 21°C versus 15°C, correspond with the identified geographical substructuring. We hypothesize that the different climatic conditions may result in variation in the activity of subpopulations of Rhipicephalus spp. tick vectors, which, in turn, could selectively maintain and transmit different parasite populations. These findings may have important implications for vaccine development and the spread of drug resistance. Instituto de Patobiología Fil: Mira, Anabela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Unlu, Ahmet Hakan. Van Yuzuncu Yil University. Vocational School of Gevas; Turquía Fil: Bilgic, Huseyin Bilgin. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; Turquía Fil: Bakirci, Serkan. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; Turquía Fil: Hacilarlioglu, Selin. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; Turquía Fil: Karagenc, Tulin. Aydin Adnan Menderes University. Faculty of Veterinary Medicine. Department of Parasitology; Turquía Fil: Carletti, Tamara. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina Fil: Weir, William. Universityof Glasgow. College of Medical, Veterinary and Life Sciences; Reino Unido Fil: Shiels, Brian. Universityof Glasgow. College of Medical, Veterinary and Life Sciences; Reino Unido Fil: Shkap, Varda. Kimron Veterinary Institute. Division of Parasitology; Israel Fil: Aktas, Munir. Firat University. Faculty of Veterinary Medicine. Department of Parasitology; Turquía Fil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

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.

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.

This study investigated possible transstadial transmission of Ehrlichia canis by Rhipicephalus sanguineus sensu lato collected from shelter dogs and the shelter grounds in Diyarbakır Province of south-eastern Turkey. Totally 225 engorged nymphs were collected from eight infected dogs with E. canis and incubated at 28°C for moulting. Unfed ticks from the shelter grounds comprising 1,800 larvae, 3,100 nymphs, and 85 adults were sorted according to sampling origin, life stage, and sex into 116 pools and screened by 16S rRNA PCR. Nine out of 26 pools of unfed adult ticks were positive for E. canis, with overall infection rate maximum likelihood estimation (MLE) of 4.83 (CI 2.39-8.87). E. canis was detected in three of 12 male pools (MLE 3.22, CI 0.86-8.83) and six of 14 female pools (MLE 6.16, CI 2.59-12.90). No adult pools collected from the shelter grounds were positive. Among 62 unfed nymph pools collected from the shelter, six were infected with E. canis (MLE 0.20, CI 0.08-0.42). No E. canis DNA was detected in any of the larva pools. Our results revealed molecular evidence for transstadial transmission of E. canis by R. sanguineus s.l. both from larva to nymph and from nymph to adult. We found no evidence of transovarial transmission.

Canine tick-borne pathogens are the source of emerging diseases and have important zoonotic relevance. Dogs play a major role in the transmission of several zoonotic tick-borne pathogens, as reservoirs and/or sentinels. To simultaneously detect Anaplasma and Ehrlichia species, a reverse line blot assay was conducted on 219 blood samples collected from autochthonous asymptomatic shelter dogs. One hundred and three (47.0%, CI 40.3-53.9) dogs were positive for one or both rickettsial pathogens. Seventy-one (32.4%, CI 26.3-39.0) dogs were infected with Anaplasma platys and 23 (10.5%, CI 6.8-15.3) with Ehrlichia canis. Concurrent infection with A. platys and E. canis was detected in nine (4.1%, CI 1.9-7.6) dogs. Partial sequences of the 16S ribosomal RNA gene shared 100% identity with the corresponding published sequences for A. platys and E. canis. Infection with Anaplasma phagocytophilum was not detected in the examined dogs. In total, 1018 (range 1-70, mean intensity 13.1, mean abundance 4.6) Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) sensu lato ticks (45.7% nymphs, 54.3% adults) were collected from the dogs. There was no significant association between Anaplasma/Ehrlichia infection and dog sex or age, but a significant correlation was found between rickettsia infection and presence of R. sanguineus. Improved tick control strategies to reduce the risk of these pathogens spreading among dogs and humans are needed in the region.

During early lactation, dairy cows may present a transient immunosuppressive state and develop anaplasmosis caused by Anaplasma marginale . In this study, clinical anaplasmosis in dairy cattle in the Thrace region of Turkey was investigated with respect to within-herd prevalence, vertical transmission, and genetic diversity. In March and September 2015, thirty lactating cows showed primary clinical signs of anaplasmosis, including fever, anaemia, decreased milk yield, anorexia, and laboured breathing. Symptoms disappeared in most cows after administration of long-acting oxytetracycline, but nine of them (30%) died. Following diagnosis based on clinical signs, microscopy and molecular findings, blood samples were collected from apparently healthy lactating cows (n = 184), pregnant heifers (n = 39) and newborn calves (n = 24). DNA was extracted from each sample and analyzed for the presence of major surface proteins (MSPs) of A. marginale , followed by sequencing to assess diversity of isolates. Microscopic examination of erythrocytes revealed A. marginale inclusion bodies in symptomatic cows. Examination of thin blood smears showed 3.8% of the lactating, clinically asymptomatic, cows to be infected with A. marginale , while nPCR detected 31.0% positive. A. marginale infection was not detected in pregnant heifers by either method. Congenital infection was found in one calf by nPCR. This is the first report of transplacental transmission of A. marginale in Turkey. The MSP4 sequence analyses showed high genetic diversity among the isolates, presenting 97.6-99.6% homology at the amino acid level. The sequences of MSP1a amplicons revealed genetic diversity providing three new tandem repeats.

This study aimed to assess the occurrence of canine haemoplasma infection in domestic dogs and its possible trans-stadial transmission by Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) in shelter dogs in Diyarbakir Province in southeast Turkey. Blood samples (n = 282) collected from domestic dogs were analysed by polymerase chain reaction (PCR) for the presence of canine haemoplasma. Fully engorged nymphs (n = 204) were removed from dogs that were positive for canine haemoplasma by PCR and maintained in an incubator at 28 °C for moulting. Unfed ticks (n = 2185) comprising 2100 nymphs and 85 adults collected from the grounds of the same shelter were also screened. Of 282 dogs, 108 [38.3%, 95% confidence interval (CI) 32.6-44.2] were PCR-positive for canine haemoplasmas. Mycoplasma haemocanis (Mhc) infection (26.2%, 95% CI 21.2-31.8) was observed in a significantly higher number of dogs than was Candidatus Mycoplasma haematoparvum (CMhp) infection (6.7%, 95% CI 4.1-10.3). Co-infections were seen in 15 (5.3%, 95% CI 3.0-8.6) dogs. None of the tick specimens examined were found to be positive for haemoplasma. Partial sequences of the 16S ribosomal RNA (rRNA) gene shared 99-100% identity with the corresponding published sequences for Mhc and CMhp. The present results revealed no trans-stadial transmission of canine haemoplasma species by R. sanguineus s.l. in field conditions.