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.

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.

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.

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.

A review: Surveillance of lumpy skin disease (LSD) a growing problem in Asia.

Lumpy skin disease (LSD) is a viral disease caused by lumpy skin disease virus (LSDV), a member of Capripoxvirus, genus of Poxviridae family. It is a transboundary infection of monetary significance that primarily affects water buffaloes and cattle. LSD was known to be once endemic in Saharan regions of Africa but later on reported in central Asian and neighboring countries of Pakistan like, India, Iran and China. It is a vector borne disease and arthropods are believed to be the main perpetrators. It is discernible by its high morbidity and low mortality. Characteristic lumps on skin and high fever are considered as major signs while reduced milk production, infertility, early embryonic death and anorexia are some of the salient clinical manifestations of the disease. Additionally, nodules on mucosa of oro-pharynx, udder, genitalia and rectum are usually observed on examination. This article summarizes LSD outbreaks across Asia during last fifteen years. It is a general consensus amongst the veterinary community that disease is endemic in Pakistan as it shares borders with India, Iran and China where recent outbreaks are reported. Historically Pakistan is free of LSD, however it is at high risk of a LSDV outbreak as neighboring regions are becoming endemic. Vaccination, strict quarantine measures, limited movement of livestock along with vector control could be effective for preventing the spread of the disease. This review aims to summarize the latest developments in the epidemiology of LSD with the focus on transboundary spread, possible emergence and economic implications on Pakistan.

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.

Molecular evidence of Anaplasma infection in naturally affected domestic cats of Pakistan.

Feline anaplasmosis is considered as an emerging tick-borne disease of zoonotic potential. The aim of current study was to investigate the molecular prevalence of anaplasmosis, associated risk factors, and alterations in hematological parameters of domestic cats from Lahore, Pakistan. Blood samples of 100 domestic cats from district Lahore were examined microscopically and the extracted genomic DNA from each sample was processed for the amplification of 16 S rRNA gene of Anaplasma. PCR confirmed isolates were purified for sequencing. The data regarding the risk factors was collected in a predesigned questionnaire and statistically analyzed by logistic regression analysis. The study found a molecular prevalence of 13% (13/100) among analyzed blood samples. The nucleotide analysis of Anaplasmataceae species sequences amplified by PCR showed high resemblance (99%) with isolates from Korea, Japan, Malaysia, Philippines, and India. The potential risk factors found to be significantly associated (p < 0.05) with disease dynamics based on the Chi-Square test were tick infestation on studied animals, previous tick history, tick control status, house hygiene, and housing type. A significant (p < 0.05) decrease in the number of platelets, erythrocytes, hemoglobin level, and pack cell volume was observed in cats suffering from anaplasmosis compared to the healthy ones. The current study is the first report of Anaplasma infection in domestic cats of Pakistan. This study will be effectual in designing the control strategies for this disease.

Molecular Analysis and Risk Factors Associated With Theileria equi Infection in Domestic Donkeys and Mules of Punjab, Pakistan.

Theileriosis is an important disease of economic significance which badly affects the equine husbandry of developing countries. The present study was planned to investigate the molecular prevalence of theileriosis, associated risk factors, and alterations in hematological parameters of donkeys and mules from district Mianwali, Punjab, Pakistan. Blood samples from 150 equids (n = 75 donkeys; n = 75 mules) were examined microscopically, and the genomic DNA from each sample was processed for the amplification of the 18S rRNA gene of Theileria. The polymerase chain reaction confirmed isolates were purified followed by sequencing. The data regarding the analysis of risk factors were collected in a predesigned questionnaire and statistically analyzed by logistic regression analysis. An overall prevalence of 17.33% was noted in this study. Donkeys showed more prevalence followed by mules being 20.0% and 14.7%, respectively. The study isolates showed high resemblance (99%) with isolates from the United States of America, Spain, Brazil, Israel, Cuba, France, South Africa, Korea, Turkey, Tunisia, India, E. Caribbean, and Nigeria. The potential risk factors found to be significantly associated (P < .05) with disease dynamics were tick infestation on study animals, previous tick history, and house hygiene. A significant (P < .05) decrease in the number of platelets, erythrocytes, hemoglobin level, and packed cell volume was observed in donkeys and mules suffering from theileriosis compared with the healthy ones. The study is the first report regarding the molecular characterization of theileriosis in donkeys and mules in Pakistan. The findings will be effectual in designing effective control strategies for this disease in Punjab, Pakistan.

Balantidium coli in domestic animals: An emerging protozoan pathogen of zoonotic significance.

Balantidium coli (B. coli) is an emerging ciliated protozoan parasite of zoonotic importance which causes a disease balantidiasis in a variety of host species including pigs, camels, ruminants, equines and even human. This disease has a cosmopolitan distribution with high prevalence rates in tropical and sub-tropical areas of the world due to favorable geo-climatic conditions for the development and survival of the parasite in these regions. Main reservoir hosts for this pathogen are pigs and animals; acquire infection mainly by the ingestion of the food or water contaminated with the porcine feces. The infected animal manifests clinical signs of anorexia, dehydration, profuse watery diarrhea and retarded growth. Wet mount slide prepared from intestinal scrapings and fecal material is used for the identification of trophozoites and cysts stages of this parasite. PCR can also be used to confirm the parasite. Secnidazole, oxytetracycline and metronidazole have varying efficacy against B. coli infection in various domestic animal species. There is no comprehensive literature available on the occurrence and distribution of the infection at international level. Therefore, the published data between 1989 and 2019 regarding this disease is critically analyzed to provide a detailed overview on this pathogen with special emphasis on geographical distribution of B. coli in domestic animals and different therapeutic agents used to treat this infection. This review will pinpoint the endemic regions which may be a source of potential disease outbreaks and will also help in application of more effectual control strategies against balantidiasis.

A comprehensive review on past, present and future aspects of canine theileriosis.

Canine theileriosis is a notorious tick borne piroplasmid infection of wild and domestic canines. The causative agent has not yet been accurately classified. PCR studies revealed that causative agent resembles to Theileria genus and thus provisionally named as Theileria annae. The other Theileria species reported in canines is Theileria annulata, Theileria equi and unnamed Theileria specie. This emergent canine infection is considered to be endemic in most of the European countries. However in Asia this disease has not been reported till date. The vectors responsible for transmission of this disease have not been determined. It has been suggested that DNA of Theileria annae has been detected in hard tick Ixodes hexagonus in Northwestern Spain and several other tick species. Clinically canine theileriosis is characterized by severe weakness, fever, hemoglobinuria and anemia. Recently atovaquone or buparvaquone plus azithromycin therapy showed better clinical efficacy. This comprehensive review is intended to summarize the current knowledge on prevalence and epidemiology of canine theileriosis in different countries of the world and associated tick vectors.