Epidemiologic profile of hard ticks and molecular characterization of Rhipicephalus microplus infesting cattle in central part of Khyber Pakhtunkhwa, Pakistan.

Tick infestation is a major public and animal health concern causing significant financial losses, especially in tropical and subtropical regions of the world. This study aimed at investigating the epidemiologic profile of ticks infesting cattle and molecular identification of R. microplus in the centrally ignored part of Khyber Pakhtunkhwa, Pakistan. A total of 600 cattle from 20 farms were examined for the tick infestation, among them 358 (59.7%) cattle were infested with ticks. A total of 2118 nymph, larvae and adult tick stages were collected and morphologically identified followed by molecular confirmation of Rhipicephalus microplus. Host-based demographic and ecological parameter analysis revealed significantly higher tick infestation in adult, female, exotic, freely grazing, and with irregular/no acaricides treated cattle. The univariate logistic analysis showed that host age, gender, breed, acaricides use, and feeding method were significantly (P < 0.05) associated, whereas multivariate analysis revealed only host breed and feeding method were potential risk factors (P < 0.05) for tick infestation. Microscopy-based examination identified four different species of ticks including R. microplus (44.5%), Hyalomma anatolicum (38.5%), and Hyalomma marginatum (10.5%) and Hyalomma excavatum (6.5%). Tick infestation pattern showed that 55.9% of cattle was found co-infested with R. microplus and H. anatolicum followed by R. microplus and H. anatolicum and H. marginatum (29.3%) then R. microplus, H. anatolicum, H. marginatum, and H. excavatum (11.2%). Sequencing of the second internal transcribed spacer (ITS2-) and 16S rRNA gene fragments also confirmed the molecular identification of Rhipicephalus microplus. Phylogenetic analysis of ITS-2 revealed all sequences clustered in single clade of the R. microplus while the 16S rRNA nucleotide sequences showed that R. microplus in this study was clustered together in clade A along with other isolates from Pakistan, China, and India. The high tick infestation suggests the need for designing strategic and integrated control measures for ticks in order to ensure good health of domestic animals in this region of Pakistan.

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.

Genetic diversity, piroplasms and trypanosomes in Rhipicephalus microplus and Hyalomma anatolicum collected from cattle in northern Pakistan.

The two most important tick species in Pakistan are Rhipicephalus microplus and Hyalomma anatolicum. When associated with cattle, these have one or three host life cycles, respectively, with potential implications for their population genetics and for their vector role in the transmission of pathogens. To compare the two tick species in this context with molecular-phylogenetic methods, during the present study 123 ticks were collected from cattle in northern Pakistan. Two mitochondrial markers of 36 ticks were molecularly analyzed. All 11 R. microplus specimens had identical cox1 haplotypes, whereas the 25 H. anatolicum specimens had nine cox1 haplotypes. The latter belonged to two distinct phylogenetic lineages with high support. However, in the 16S rRNA gene these differences were less evident. Among the 113 ticks molecularly analyzed for tick-borne protozoa, the sequence of Babesia occultans was successfully amplified from two specimens of H. anatolicum. Theileria annulata was present in both R. microplus (10.4%) and H. anatolicum (27.3%), with significantly higher prevalence rate in the latter species. Only one tick, a H. anatolicum female, was positive in the PCR detecting Trypanosoma spp. Sequencing revealed the presence of a new genotype, with the closest phylogenetic relationship to stercorarian trypanosomes (in particular, to a tick-associated Trypanosoma sp. from Japan). In conclusion, the above differences between R. microplus and H. anatolicum may be partly related to their life cycles involving one host or three hosts, respectively. Among the others, host switching (reducing chances of inbreeding) and shorter periods spent on-host (reducing gene flow between cattle herds) are supposed to be important drivers of cox1 gene diversification in case of H. anatolicum as a three host tick species. These results highlight the importance of studying differences in intraspecific genetic diversity and piroplasm burdens between one host and three host ticks in the local scale. In addition, a Trypanosoma sp. molecularly identified in H. anatolicum is reported here for the first time from South Asia, deserving further evaluation concerning its host and vector species.

Molecular screening and genetic diversity of tick-borne pathogens associated with dogs and livestock ticks in Egypt.

BACKGROUND: The Middle East and North Africa (MENA) offer optimal climatic conditions for tick reproduction and dispersal. Research on tick-borne pathogens in this region is scarce. Despite recent advances in the characterization and taxonomic explanation of various tick-borne illnesses affecting animals in Egypt, no comprehensive examination of TBP (tick-borne pathogen) statuses has been performed. Therefore, the present study aims to detect the prevalence of pathogens harbored by ticks in Egypt. METHODOLOGY/PRINCIPAL FINDINGS: A four-year PCR-based study was conducted to detect a wide range of tick-borne pathogens (TBPs) harbored by three economically important tick species in Egypt. Approximately 86.7% (902/1,040) of the investigated Hyalomma dromedarii ticks from camels were found positive with Candidatus Anaplasma camelii (18.8%), Ehrlichia ruminantium (16.5%), Rickettsia africae (12.6%), Theileria annulata (11.9%), Mycoplasma arginini (9.9%), Borrelia burgdorferi (7.7%), Spiroplasma-like endosymbiont (4.0%), Hepatozoon canis (2.4%), Coxiella burnetii (1.6%) and Leishmania infantum (1.3%). Double co-infections were recorded in 3.0% (27/902) of Hy. dromedarii ticks, triple co-infections (simultaneous infection of the tick by three pathogen species) were found in 9.6% (87/902) of Hy. dromedarii ticks, whereas multiple co-infections (simultaneous infection of the tick by ≥ four pathogen species) comprised 12% (108/902). Out of 1,435 investigated Rhipicephalus rutilus ticks collected from dogs and sheep, 816 (56.9%) ticks harbored Babesia canis vogeli (17.1%), Rickettsia conorii (16.2%), Ehrlichia canis (15.4%), H. canis (13.6%), Bo. burgdorferi (9.7%), L. infantum (8.4%), C. burnetii (7.3%) and Trypanosoma evansi (6.6%) in dogs, and 242 (16.9%) ticks harbored Theileria lestoquardi (21.6%), Theileria ovis (20.0%) and Eh. ruminantium (0.3%) in sheep. Double, triple, and multiple co-infections represented 11% (90/816), 7.6% (62/816), and 10.3% (84/816), respectively in Rh. rutilus from dogs, whereas double and triple co-infections represented 30.2% (73/242) and 2.1% (5/242), respectively in Rh. rutilus from sheep. Approximately 92.5% (1,355/1,465) of Rhipicephalus annulatus ticks of cattle carried a burden of Anaplasma marginale (21.3%), Babesia bigemina (18.2%), Babesia bovis (14.0%), Borrelia theleri (12.8%), R. africae (12.4%), Th. annulata (8.7%), Bo. burgdorferi (2.7%), and Eh. ruminantium (2.5%). Double, triple, and multiple co-infections represented 1.8% (25/1,355), 11.5% (156/1,355), and 12.9% (175/1,355), respectively. The detected pathogens' sequences had 98.76-100% similarity to the available database with genetic divergence ranged between 0.0001 to 0.0009% to closest sequences from other African, Asian, and European countries. Phylogenetic analysis revealed close similarities between the detected pathogens and other isolates mostly from African and Asian countries. CONCLUSIONS/SIGNIFICANCE: Continuous PCR-detection of pathogens transmitted by ticks is necessary to overcome the consequences of these infection to the hosts. More restrictions should be applied from the Egyptian authorities on animal importations to limit the emergence and re-emergence of tick-borne pathogens in the country. This is the first in-depth investigation of TBPs in Egypt.

Fatigue: It Is Not Always in the Head.

Fatigue is a common presenting complaint in patients seen in clinics and same-day emergency care. Although it has a simple presentation, it can be challenging to diagnose and manage, particularly when an underlying medical condition presents atypically as fatigue. Here we present an interesting case of giant cell arteritis (GCA) with only fatigue as the presenting complaint. GCA is the inflammation of medium and large vessels in the body, including the aortic arch and its branches. It typically manifests above the age of 50 with headaches, jaw claudication, temporal tenderness, arthralgia, night sweats, and unintentional weight loss. Early diagnosis and treatment are of paramount importance to prevent complications, particularly permanent blindness.

Recent advancement, immune responses, and mechanism of action of various vaccines against intracellular bacterial infections.

Emerging and re-emerging bacterial infections are a serious threat to human and animal health. Extracellular bacteria are free-living, while facultative intracellular bacteria replicate inside eukaryotic host cells. Many serious human illnesses are now known to be caused by intracellular bacteria such as Salmonella enterica, Escherichia coli, Staphylococcus aureus, Rickettsia massiliae, Chlamydia species, Brucella abortus, Mycobacterium tuberculosis and Listeria monocytogenes, which result in substantial morbidity and mortality. Pathogens like Mycobacterium, Brucella, MRSA, Shigella, Listeria, and Salmonella can infiltrate and persist in mammalian host cells, particularly macrophages, where they proliferate and establish a repository, resulting in chronic and recurrent infections. The current treatment for these bacteria involves the application of narrow-spectrum antibiotics. FDA-approved vaccines against obligate intracellular bacterial infections are lacking. The development of vaccines against intracellular pathogenic bacteria are more difficult because host defense against these bacteria requires the activation of the cell-mediated pathway of the immune system, such as CD8+ T and CD4+ T. However, different types of vaccines, including live, attenuated, subunit, killed whole cell, nano-based and DNA vaccines are currently in clinical trials. Substantial development has been made in various vaccine strategies against intracellular pathogenic bacteria. This review focuses on the mechanism of intracellular bacterial infection, host immune response, and recent advancements in vaccine development strategies against various obligate intracellular bacterial infections.

Genetic diversity of vector-borne pathogens in ixodid ticks infesting dogs from Pakistan with notes on Ehrlichia canis, Rickettsia raoultii and Dirofilaria immitis detection.

BACKGROUND: Vector-/tick-borne pathogens (V/TBPs) pose a potential threat to human and animal health globally. Information regarding canine V/TBPs is scarce and no specific study has been conducted so far to explore the microbial diversity within ticks infesting dogs from Pakistan. Herein, this knowledge gap is addressed by assessing the genetic diversity and prevalence pattern of V/TBPs in ixodid ticks with special implications for public and canine health. METHODS: A total of 1150 hard ticks were collected from 300 dogs across central Khyber Pakhtunkhwa (KP), Pakistan. After morpho-molecular identification, 120 tick samples were screened for the presence of V/TBPs by amplifying 16S rRNA/gltA (Rickettsia/Ehrlichia and Wolbachia sp.), 18S rRNA (Theileria sp.) and cox1 (Dirofilaria sp.) genes through PCR followed by sequencing and phylogenetic study. RESULTS: In toto, 50 ixodid ticks (50/120, 41.7%) were found positive for V/TBPs DNA. The detected V/TBPs were categorized into five genera and eight species, viz. Ehrlichia (E. canis and Ehrlichia sp.), Rickettsia (R. massiliae, R. raoultii and Rickettsia sp.), Theileria (T. annulata), Dirofilaria (D. immitis) and Wolbachia (Wolbachia sp.). The pathogen prevalence patterns showed that R. massiliae was the most prevalent zoonotic V/TBP (19.5%), followed by E. canis (10.8%), Rickettsia sp. (7.5%), R. raoultii (6.7%), T. annulata (5.8%), D. immitis (5.8%), Wolbachia sp. (4.2%) and Ehrlichia sp. (3.3%), respectively. Among the screened tick species, most Rhipicephalus sanguineus sensu lato samples were found positive for V/TBP DNA (20/20,100%) followed by Rh. turanicus sensu stricto (13/20, 65%), Hyalomma dromedarii (8/20, 40%), Rh. haemaphysaloides (6/20, 30%), Hy. excavatum (2/20, 10%) and Rh. microplus (1/20, 5%). Co-occurrence of V/TBP was also detected in tick specimens (single V/TBP infection: 32 ticks; double and triple: 13 and 5 tick samples). The detected pathogens shared a phylogenetic relationship with similar isolates published in NCBI GenBank from Old and New World countries. CONCLUSION: Ixodid ticks infesting dogs harbor a diverse array of V/TBPs including zoonotic agents from Pakistan. Furthermore, the presence of D. immitis in ticks that infest dogs raises the possibility that this parasite has either attained its dead-end host (i.e. the tick) while feeding on dogs or has expanded its range of intermediate/paratenic hosts. Further research work is needed to investigate the epidemiology and confirm the vector competence of screened tick species for these pathogens from Pakistan.

First molecular confirmation of multiple zoonotic vector-borne diseases in pet dogs and cats of Hong Kong SAR.

In recent years, the incidence of vector-borne diseases (VBDs) has increased throughout the globe. In particular, tick-borne diseases (e.g., caused by Ehrlichia canis, E. ewingii, Anaplasma phagocytophilum, A. platys, Borrelia burgdorferi sensu stricto (s.s.) and Babesia gibsoni) and mosquito-borne diseases (e.g., caused by Dirofilaria immitis) diseases pose a burden on animal health. Nevertheless, there have been no studies undertaken on the occurrence of VBDs in pet dogs and cats in Hong Kong SAR. This study fills this gap, and is the first to determine the seroprevalence of major VBDs, such as those caused by D. immitis, E. canis, E. ewingii, A. phagocytophilum, A. platys and B. burgdorferi s.s, in dogs and cats through commercially available SNAP 4Dx plus testing. Infection by all these pathogens and Babesia sp. was further assessed through PCR and DNA sequencing. A total of 224 blood samples were collected from domestic dogs (n = 159) and cats (n = 65) in Hong Kong SAR during summer 2022. Hematocrit and platelet counts were determined in each blood sample and other hematological parameters were assessed using an automatic hematology analyzer and vortex the specimen for one to two minutes at or near the highest setting to minimize the clumping. All cat sera samples were negative for tested pathogens, but antibodies against some of the pathogens were detected in dog sera samples. Here, the highest figures were recorded for seroprevalence of E. canis/E. ewingii (10.7%), followed by D. immitis (5.7%), and A. phagocytophilum/A. platys (2.5%). No B. burgdorferi s.s. antibodies were detected in any of the dogs tested. Through molecular diagnostics, we detected the presence of B. gibsoni (3.7%), E. canis (3.1%), D. immitis (5.7%), and A. phagocytophilum (1.3%). Neighbor-Joining phylogenetic trees for vector-borne pathogens (i.e., genus Anaplasma sp.) showed 100% clustering to Japan, the USA and Germany, whereas genus Ehrlichia sp. showed 100% clustering to China, Turkey, Cuba, and Greece. Similarly, genus Babesia sp. clustered 100% to India, Sri Lanka and Austria, while D. immitis clustered in Iraq, South Korea, Portugal, France, the USA and Italy. This study provides the first evidence on the occurrence of tick-borne pathogens in pet dogs in Hong Kong SAR. Based on these findings, it is recommended that appropriate screening should be undertaken in domestic dogs to evaluate the prevalence of these pathogens and promote the timely control of VBDs.

Ticks Infesting Dogs in Khyber Pakhtunkhwa, Pakistan: Detailed Epidemiological and Molecular Report.

Ticks and tick-borne diseases are considered a major challenge for human and animal health in tropical, sub-tropical, and temperate regions of the world. However, only scarce information is available on the characterization of tick species infesting dogs in Pakistan. In this study, we present a comprehensive report on the epidemiological and phylogenetic aspects of ticks infesting dogs in Pakistan using the mitochondrial markers i.e. Cytochrome c oxidase subunit 1 (cox1) and 16S ribosomal RNA (16S rRNA) nucleotide sequences. A total of 300 dogs were examined and 1150 ixodid ticks were collected across central Khyber Pakhtunkhwa, Pakistan. The morpho-molecular characterization of hard ticks revealed the presence of two ixodid tick genera on dogs, i.e., Hyalomma and Rhipicephalus, including six tick species viz. Hyalomma dromedarii (15.9%), Hyalomma excavatum (3%), Rhipicephalus sanguineus s.l. (41.3%), Rhipicephalus turanicus s.s. (28.7%), Rhipicephalus haemaphysaloides (10.2%), and Rhipicephalus microplus (2%). The total prevalence of tick infestation in dogs was 61%. The district with the highest tick prevalence rate in dogs was Mardan (14.7%), followed by Peshawar (13%), Swabi (12%), Charsadda (11%), and Malakand (10.3%), respectively. Risk factors analysis indicated that some demographic and host management-associated factors such as host age, breed, exposure to acaricides treatment, and previous tick infestation history were associated with a higher risk of tick infestation on dogs. This is the first molecular report confirming the infestation of Hyalomma and Rhipicephalus tick species in the dog population from the study area. The present study also reported a new tick−host association between Hy. excavatum, Hy. dromedarii, and dogs. Phylogenetic analysis revealed that cox1 partial nucleotide sequences of Hy. excavatum in our dataset were 100% identical to similar tick specimens identified in Turkey, and those of Hy. dromedarii were identical to tick specimens from Iran. Whereas, Rh. haemaphysaloides and Rh. microplus' cox1 partial nucleotide sequences were identical to sequences previously published from Pakistan. Rhipicephalus turanicus s.s. 's cox1 isolates from the present study were 99.8−100% identical to Pakistani-reported isolates, and those of Rh. sanguineus s.l. were 100% identical to Chinese specimens. Results on the genetic characterization of ticks were further confirmed by 16S rRNA partial nucleotide sequences analysis, which revealed 100% identity between the tick isolates of this study and those of Hy. excavatum reported from Turkey; Hy. dromedarii specimens reported from Senegal; Rh. haemaphysaloides, Rh. microplus, and Rh. turanicus s.s., previously published from Pakistan, and Rh. sanguineus s.l., published from China. Furthermore, phylogenetic analysis showed that the Rh. sanguineus s.l. isolates of this study clustered with specimens of the tropical lineage with 7.7−10% nucleotide divergence from the specimens of the temperate lineage. Further molecular works need to be performed throughout Pakistan to present a more detailed map of tick distribution with information about dog host associations, biological characteristics, and pathogen competence.

Morphological identification and molecular characterization of economically important ticks (Acari: Ixodidae) from North and North-Western Egypt.

Ticks represent a major source of growing economic and public health concern, especially in the tropics and sub-tropics. Towards evidencing ticks' invasion in the North and North-Western parts of Egypt, the present study aimed to investigate the morpho-molecular aspects of those ectoparasites using stereomicroscopy, scanning electron microscopy (SEM) and sequencing of the mitochondrial 16S ribosomal DNA gene (rDNA) and nuclear second internal transcribed spacer (ITS2). Our analysis confirmed the presence and well-distribution of Rhipicephalus sanguineus s.l. infesting dogs and sheep (Alexandria governorate), Rhipicephalus annulatus infesting cattle (Beheira governorate), and Hyalomma dromedarii infesting camels (Marsa Matruh governorate) from North/North-Western Egypt. 16S rDNA and ITS2 sequences of the ticks were amplified using universal and gene-specific sets of primers, sequenced and analyzed. Lengths of amplified 16S rDNA sequences in all examined tick species were found to be similar in size (approximately 460 bp); however, they differed in base pair constitutions, whereas ITS2 lengths were 1,500 bp, 1,550 bp, and 1,800 bp for Rh. annulatus, Rh. sanguineus s.l., and Hy. dromedarii, respectively. Phylogenetically, based on the 16S rDNA results, Rh. sanguineus s.l. ticks clustered with the southeastern Europe lineage from Romania and Greece, Rh. annulatus ticks were similar to Turkish populations, and Hy. dromedarii were close to the isolates from Tunisia. Similarly, based on ITS2 sequences, Rh. sanguineus s.l. from dogs were showing 99% similarity to Nigerian populations; however, those collected from sheep were closer to Iranian populations with 4.1% nucleotide divergence between the two populations of different hosts. Rh. annulatus ticks were identical to a population from Romania, whereas Hy. dromedarii was close by 99.7% similarity to a population from Kenya. This is the first study reporting nucleotide sequences of 16S rDNA and ITS2 in integration with morphological identification of ticks from this part of Egypt.

Poultry Mites: Ubiquitous, Spreading, and Still a Growing Threat.

Poultry mites continue to be a major threat to poultry meat and egg production all over the world, with some species being blood-feeding arthropods that spend most of their time off-host and others burrowing under the bird's skin. Regardless of feeding strategy, these mites create welfare issues and production losses in poultry production systems in terms of bird growth, egg quality, and egg quantity. Furthermore, some species are able to transmit pathogens, introducing secondary infections that affect the birds' development and survival. Because of national restrictions on acaricide use and the development of mite resistance to available control products, the eradication of poultry mites is far from being achieved. However, new drugs and a better understanding of mite genetic and transcriptomic factors should aid the development of new control and treatment strategies. This review focuses on the main poultry mite species, their significance, and their current and future control.

Estudio recapitulativo- Los ácaros en avicultura: Ubicuos, con continua diseminación y persisten como una amenaza creciente. Los ácaros en avicultura continúan siendo una amenaza importante para la producción de carne de pollo y huevo en todo el mundo y algunas especies son artrópodos hematófagos y pasan la mayor parte de su tiempo fuera del hospedador y otras se esconden debajo de la piel de las aves. Independientemente de la estrategia de alimentación, estos ácaros crean problemas de bienestar y pérdidas de producción en los sistemas de producción avícola en términos de crecimiento de las aves, calidad y cantidad de huevos. Además, algunas especies pueden transmitir patógenos, introduciendo infecciones secundarias que afectan el desarrollo y la supervivencia de las aves. Debido a las restricciones nacionales sobre el uso de acaricidas y el desarrollo de resistencia de los ácaros a los productos de control disponibles, la erradicación de los ácaros de las aves está lejos de lograrse. Sin embargo, los nuevos fármacos y una mejor comprensión de los factores transcriptómicos y genéticos de los ácaros deberían ayudar al desarrollo de nuevas estrategias de control y tratamiento. Esta revisión se centra en las principales especies de ácaros de las aves, su importancia y su control actual y futuro.

Ehrlichiosis in Dogs: A Comprehensive Review about the Pathogen and Its Vectors with Emphasis on South and East Asian Countries.

Ehrlichiosis in dogs is an emerging vector borne rickettsial zoonotic disease of worldwide distribution. In general, three Ehrlichial species (Ehrlichia canis, E. ewingii, and E. chaffeensis) are involved in infecting dogs. Among them, E. canis is the well-known etiological pathogen affecting platelets, monocytes, and granulocytes. Dogs act as a reservoir, while the main vector responsible for disease transmission is Rhipicephalus sanguineus. However, in east Asian countries, Haemaphysalis longicornis is considered the principal vector for disease transmission. This disease affects multiple organs and systems and has three clinical manifestations, including acute, subclinical, and chronic. Definitive diagnosis involves visualization of morulae on cytology, detection of antibodies through an indirect immunofluorescence test (IFAT), and DNA amplification by polymerase chain reaction (PCR). In canine ehrlichiosis, no predilection of age or sex is observed; however, Siberian Huskies and German Shepherds are more likely to develop severe clinical manifestations. Doxycycline, rifampicin, and minocycline are proven to be effective drugs against canine ehrlichiosis. This review is intended to describe a brief overview of Ehrlichia infection in dogs, its reported prevalence in east and south Asian countries, and the latest knowledge regarding chemotherapy and associated vectors responsible for the disease transmission. This manuscript also identifies the prevailing knowledge gaps which merit further attention by the scientific community.

The Symbiotic Continuum Within Ticks: Opportunities for Disease Control.

Among blood-sucking arthropods, ticks are recognized as being of prime global importance because of their role as vectors of pathogens affecting human and animal health. Ticks carry a variety of pathogenic, commensal, and symbiotic microorganisms. For the latter, studies are available concerning the detection of endosymbionts, but their role in the physiology and ecology of ticks remains largely unexplored. This review paper focuses on tick endosymbionts of the genera Coxiella, Rickettsia, Francisella, Midichloria, and Wolbachia, and their impact on ticks and tick-pathogen interactions that drive disease risk. Tick endosymbionts can affect tick physiology by influencing nutritional adaptation, fitness, and immunity. Further, symbionts may influence disease ecology, as they interact with tick-borne pathogens and can facilitate or compete with pathogen development within the vector tissues. Rickettsial symbionts are frequently found in ticks of the genera of Ixodes, Amblyomma, and Dermacentor with relatively lower occurrence in Rhipicephalus, Haemaphysalis, and Hyalomma ticks, while Coxiella-like endosymbionts (CLEs) were reported infecting almost all tick species tested. Francisella-like endosymbionts (FLEs) have been identified in tick genera such as Dermacentor, Amblyomma, Ornithodoros, Ixodes, and Hyalomma, whereas Wolbachia sp. has been detected in Ixodes, Amblyomma, Hyalomma, and Rhipicephalus tick genera. Notably, CLEs and FLEs are obligate endosymbionts essential for tick survival and development through the life cycle. American dog ticks showed greater motility when infected with Rickettsia, indirectly influencing infection risk, providing evidence of a relationship between tick endosymbionts and tick-vectored pathogens. The widespread occurrence of endosymbionts across the tick phylogeny and evidence of their functional roles in ticks and interference with tick-borne pathogens suggests a significant contribution to tick evolution and/or vector competence. We currently understand relatively little on how these endosymbionts influence tick parasitism, vector capacity, pathogen transmission and colonization, and ultimately on how they influence tick-borne disease dynamics. Filling this knowledge gap represents a major challenge for future research.

A Review on the Marek's Disease Outbreak and Its Virulence-Related meq Genovariation in Asia between 2011 and 2021.

Marek's disease is an infectious disease in poultry that usually appears in neural and visceral tumors. This disease is caused by Gallid alphaherpesvirus 2 infection in lymphocytes, and its meq gene is commonly used in virulent studies for coding the key protein functional in oncogenic transformation of the lymphocytes. Although vaccines have been introduced in many countries to control its spread and are proven to be efficient, recent records show a decline of such efficiency due to viral evolution. In this study, we reviewed the outbreak of Marek's disease in Asia for the last 10 years, together with associated meq sequences, finding a total of 36 studies recording outbreaks with 132 viral strains in 12 countries. The visceral type is the most common (13 in 16 studies) form of Marek's disease, but additional unobserved neural changes may exist. MD induces liver lymphoma most frequently (11 in 14 studies), and tumors were also found in spleen, kidney, heart, gizzard, skin, intestine, lung, and sciatic nerve. Twelve viral strains distributed in China have been reported to escape the CVI988 vaccine, reaching a mortality rate of more than 30%. Phylogenetic analyses show the internal connection between the Middle East (Turkey, Iraq, Iran, Saudi Arabia), South Asia (India, Indonesia), and East Asia (China and Japan), while external viral communications might occasionally occur. In 18 strains with both sequential and mortality data, amino acid alignment showed several point substitutions that may be related to its virulence. We suggest more behavioral monitoring in Marek's disease-endemic regions and further studies on strain virulence, together with its Meq protein structural changes.

Microsatellite Analysis Revealed Potential DNA Markers for Gestation Length and Sub-Population Diversity in Kari Sheep.

Kari sheep inhabiting the Chitral district of Pakistan show variation in gestation length. In this study, we have analyzed the genetic differences between the three subtypes of Kari sheep (based on variation in gestation length) using microsatellite markers. Kari sheep samples were collected from their breeding tract and were characterized for gestation length and genetic diversity using microsatellite markers. A total of 78 Kari ewes were grouped into three categories based on gestation length (GL), i.e., Kari-S (with a shorter GL), Kari-M (with a medium GL), and Kari-L (with a longer GL). DNA from these samples was used to amplify 31 ovine-specific microsatellite loci through PCR. Of the total 78 Kari specimens, 24 were grouped in Kari-S (GL = 100.7 ± 1.8), 26 were from the Kari-M subtype (GL = 123.1 ± 1.0), and 28 were Kari-L (GL = 143.8 ± 1.5). Microsatellite analysis revealed an association of genotypes at two marker sites (MAF214 and ILSTS5) with variation in GL. A total of 158 alleles were detected across the 22 polymorphic loci with an average of 7.18 alleles per locus. Unique alleles were found in all three subtypes. The highest number of unique alleles was observed in Kari-L (15), followed by Kari-S (10) and Kari-M (8). The results indicated that Kari-S is a genetically distinct subtype (with higher genetic differentiation and distance) from Kari-M and Kari-L. The genetic uniqueness of Kari-S is important for further exploration of the genetic basis for shorter gestation length, and exploitation of their unique values.

First serological evidence of Q fever in large ruminants and its associated risk factors in Punjab, Pakistan.

Coxiellosis, also known as Q fever, is a zoonotic disease caused by Coxiella burnetii, a gram-negative bacterium that exerts a significant deleterious impact on the productive and reproductive capabilities of livestock, severely effecting the economics of this sector. In this study, 448 sera samples from cattle (n = 224) and buffalo (n = 224) were collected from 112 farms in Pakistan and examined for antibodies against C. burnetii using an indirect ELISA. Ticks were also collected from these animals. Serological analysis revealed a 23.66% and 27.23% seroprevalence of Q fever in cattle and buffalo, respectively. Odds ratio (OR) analysis of the factors associated with C. burnetii seropositivity was performed, and a multivariable logistic model identified five main variables associated with the seropositivity for coxiellosis. These were: (i) the absence of acaricide use (OR 5.61; 95% CI 2.97-10.94); (ii) the presence of ticks (OR 3.23; 95% CI 1.87-5.69); (iii) the abortion history during the preceding year on the farm (OR 14.96; 95% CI 8.09-29.34); (iv) the presence of sheep and goats (OR 2.47; 95% CI 1.20-5.35); and (v) the absence of a separate parturition area (OR 3.17; 95% CI 1.76-5.86). This study provides new insights into the seroprevalence of Q fever in large ruminants across seven studied districts of Punjab, Pakistan, also providing baseline data to inform improved herd management and on-farm practices for the prevention and control of Q fever in large ruminants in the region. Results of this work suggest that further molecular investigation of coxiellosis is warranted to provide a more thorough evaluation of C. burnetii epidemiology in Pakistan.

An Epidemiological Survey Regarding Ticks and Tick-Borne Diseases among Livestock Owners in Punjab, Pakistan: A One Health Context.

Recent global changes have led to an increase in the spread of ticks and tick-borne diseases (TBDs) affecting domestic ruminants and humans, with an annual loss of US $13.9-$18.7 billion. The current study determined the perception and practices of livestock farmers regarding tick infestation. A total of 112 livestock farms were surveyed in Punjab, Pakistan, among which animals from 42 (37.5%) farms were infested with ticks. Only 28.6% (n = 32) of the dairy farmers were consulting veterinarians for ticks control, while 86.7% (n = 97) of the respondents did not consider biosecurity measures in the control of tick transmission. Most of the respondents, 71.4% (n = 80), did not consider manual tick removal from their animals (i.e., by hand, followed by physically crushing) as a risky practice for spreading zoonotic diseases. Improper disposal of bottles of acaricides in the farm drainage was also observed, putting the environment and aquatic life at risk. These wrong practices may contribute to high disease burdens and economic losses, increasing the possibility of transmission of zoonotic TBDs and pollution of the environment. Therefore, an integrated One Health approach is required for the control of TBDs through environmentally friendly approaches.

CLABSI reduction using evidence based interventions and nurse empowerment: a quality improvement initiative from a tertiary care NICU in Pakistan.

OBJECTIVE: Central line associated bloodstream infection (CLABSI) is an important cause of morbidity and mortality in the neonatal intensive care unit (NICU). We designed a CLABSI Prevention Package (CPP) to decrease NICU CLABSI rates, using evidence-proven interventions. DESIGN: This was a quality improvement (QI) project. Data collection was divided into three phases (pre-implementation, implementation and post implementation). SQUIRE2.0 guidelines were used to design, implement and report this QI initiative. SETTING: A tertiary care level 3 NICU at the Aga Khan University Hospital (AKUH), Karachi, Pakistan. PATIENTS: All patients admitted to the AKUH NICU from 1 January 2016 to 31 March 2018 who had a central line in place during their NICU admission. INTERVENTIONS: CPP used evidence-based interventions focusing on hand hygiene, aseptic central line insertion techniques and central line care, prevention of fungal infections and nurse empowerment. MAIN OUTCOME MEASURES: CLABSI rates pre and post intervention were recorded. Secondary outcomes were risk factors for CLABSI, device (central line) utilisation ratio, CLABSI related mortality and micro-organism profile. RESULTS: CLABSI rates decreased from 17.1/1000 device days to 5.0/1000 device days (relative risk (RR)=0.36, CI=0.17-0.74). Device (central line) utilisation ratio declined from 0.30 to 0.25. Out of 613 patients enrolled in our study, 139 (22.7%) died. Mortality was higher in CLABSI group (n=20, 44%) as compared with non CLABSI group (n=119, 21.1%) (p<0.001). Gestational age of <27 weeks was an independent risk factor for CLABSI (RR=4.45, CI=1.10-18.25, p=0.03). A total of 158 pathogens were isolated among which 68 were associated with CLABSI. Gram-negative bacteria 31 (47.7%) were the most common cause of CLABSI. Ninety-seven (61%) micro-organisms were multi-drug resistant. CONCLUSIONS: CPP was effective in decreasing NICU CLABSI rates and can be used as a model to decrease NICU CLABSI rates in low or middle-income countries.

The Role of Ticks in the Emergence of Borrelia burgdorferi as a Zoonotic Pathogen and Its Vector Control: A Global Systemic Review.

Ticks are widely distributed across the globe, serving as hosts for numerous pathogens that make them major contributors to zoonotic parasitosis. Borrelia burgdorferi is a bacterial species that causes an emerging zoonotic tick-borne disease known as Lyme borreliosis. The role of ticks in the transmission of this pathogen was explored in this study. According to this systematic review, undertaken according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, 19 tick species are known to carry Borrelia burgdorferi, with more than half of the recorded cases in the last two decades related to Ixodes ricinus and Ixodes scapularis ticks. Forty-six studies from four continents, Europe, North America, Asia, and Africa, reported this pathogen in ticks collected from vegetation, animals, and humans. This study highlights an increasing distribution of tick-associated Borrelia burgdorferi, likely driven by accelerated tick population increases in response to climate change coupled with tick dispersal via migratory birds. This updated catalogue helps in compiling all tick species responsible for the transmission of B. burgdorferi across the globe. Gaps in research exist on Borrelia burgdorferi in continents such as Asia and Africa, and in considering environmentally friendly vector control strategies in Europe and North America.

A Review of Zoonotic Babesiosis as an Emerging Public Health Threat in Asia.

Zoonotic babesiosis poses a serious health risk in many parts of the world. Its emergence in Asia is thus a cause for significant concern, demanding that appropriate control measures are implemented to suppress its spread in this region. This study focuses on zoonotic Babesia species reported in Asia, offering an extensive review of those species reported in animals and humans. We reported 11 studies finding zoonotic Babesia species in animals and 16 in humans. In China, the most prevalent species was found to be Babesia microti, reported in both humans (n = 10) and wild and domesticated animals (n = 4). In Korea, only two studies reported human babesiosis, with a further two studies reporting Babesia microti in wild animals. Babesia microti was also reported in wild animal populations in Thailand and Japan, with evidence of human case reports also found in Singapore, Mongolia and India. This is the first review to report zoonotic babesiosis in humans and animals in Asia, highlighting concerns for future public health in this region. Further investigations of zoonotic species of Babesia in animal populations are required to confirm the actual zoonotic threat of babesiosis in Asia, as well as its possible transmission routes.