Molecular prevalence, phylogeny and hematological impact of Toxoplasma gondii and Plasmodium spp. in common quails from Punjab, Pakistan.

This study investigates the molecular prevalence and phylogenetic characteristics of two prominent blood-borne pathogens, Toxoplasma gondii (T. gondii) and Plasmodium spp., in common quails (Coturnix coturnix) sampled from both wild (N = 236) and farmed (N = 197) populations across four districts (Layyah, Dera Ghazi Khan, Lahore, and Multan) in Punjab, Pakistan, during the hunting seasons from 2021 to 2023. Additionally, the impact of these pathogens on the complete blood count (CBC) of the hosts is examined. Out of 433 quails tested, 25 (5.8%) exhibited amplification of the internal transcribed spacer (ITS-1) gene for T. gondii, while 15 (3.5%) showed amplification of the Cytochrome b gene for Plasmodium spp. A risk factor analysis indicated that the prevalence of both pathogens was not confined to specific sampling sites or bird sexes (P > 0.05). District-wise analysis highlighted that hens were more susceptible to both T. gondii and Plasmodium spp. infections than cocks. Wild quails exhibited a higher susceptibility to T. gondii compared to farmed birds. Significant CBC variations were recorded in infected birds as compared to uninfected ones. BLAST analysis of generated sequences has confirmed the identity of recovered PCR amplicons as T. gondii and Plasmodium relictum. Phylogenetic analysis revealed that Pakistani isolates clustered with those reported from various countries globally. This study provides the first documentation of T. gondii and Plasmodium sp. infections in Pakistani quails, underscoring the need for detailed investigations across different regions to enhance our understanding of infection rates and the zoonotic potential of these parasites.

Molecular identification, risk factor assessment, and phylogenetic analysis of tick-borne pathogens in symptomatic and asymptomatic cattle from South-Eastern Iran.

Tick-borne pathogens (TBPs) represent a substantial threat to cattle globally, exerting adverse impacts on production, health, and economic viability. This study delves into the prevalence and implications of TTBPs in cattle sourced from resource-limited smallholder livestock farms situated in southeastern Iran, proximate to Afghanistan and Pakistan. Blood and tick specimens were systematically collected from a cohort of 230 cattle, comprising 150 asymptomatic and 80 symptomatic individuals. Genomic DNA isolated from blood samples underwent rigorous examination for the presence of key TBPs, including Anaplasma marginale, A. phagocytophilum, A. bovis, A. centrale, Babesia bigemina, and Theileria annulata, utilizing multiple genetic markers. Nucleotide sequence analysis facilitated the reconstruction of phylogenetic relationships. The study also evaluated various potential risk factors, such as clinical status, gender, age, breed, tick infestation, and management practices, to elucidate their associations with TTBPs. Among the cattle cohort, a staggering 87.8% (202/230) tested positive for at least one pathogen. Prevalence statistics encompassed A. marginale (72.2%), T. annulata (68.3%), A. phagocytophilum/A. platys-like complex (66.1%), A. centrale (16.7%), B. bigemina (10.0%), and A. bovis (6.1%). Remarkably, mixed infections involving two, three, and four pathogens were detected in 23%, 52.1%, and 2.2% of animals, respectively. Notably, all asymptomatic cattle were positive for at least one TBP. Tick infestation was observed in 62.2% (143/230) of cattle, predominantly caused by Hyalomma anatolicum (82.5%), Rhipicephalus (Boophilus) annulatus (13.1%), and R. sanguineus sensu lato (4.4%). Risk factors linked to TBPs encompassed tick infestation, older age, and crossbred animals. Clinical presentations among symptomatic cattle encompassed fever, anemia, weight loss, anorexia, jaundice, and enlarged superficial lymph nodes. This study underscores the pivotal role of asymptomatic carriers in the propagation of TTBPs within endemic regions. Furthermore, it emphasizes the potential for the implementation of molecular diagnostics to unmask subclinical infections, thereby affording the opportunity for targeted interventions aimed at ameliorating the burden of TTBPs in resource-constrained smallholder dairy farms.

Molecular prevalence of vector borne bacterial pathogens in the blood samples of wild rodent species trapped from Saudi Arabia.

Order Rodentia is the most speciose among mammals and the members of this order are known to host more than 60 zoonotic diseases and rodents are a potential health threat to humans. This study was designed to report the molecular prevalence and phylogenetic evaluation of various blood borne bacterial pathogens (Anaplasma ovis, Anaplasma phagocytophilum, Anaplasma marginale and Bartonella spp.) in the blood samples of four wild rodent species [Meriones rex (N = 27), Acomys dimidiatus (N = 18), Myomys yemeni (N = 6) and Rattus rattus (N = 3)] that were trapped during August till October 2020 from Al Makhwah governorate in Saudi Arabia. Results revealed by 9/54 (16.6%) rodents amplified Msp4 gene and 2/54 (3.7%) rodents amplified rpoB gene of Anaplasma ovis and Bartonella spp. respectively. Anaplasma phagocytophilum and Anaplasma marginale were not detected among enrolled rodent species. Meriones rex was the most highly infected rodent species. DNA sequencing and BLAST analysis confirmed the presence of Anaplasma ovis and the Bartonella koehlerae in rodent blood samples. Phylogenetic analysis of both pathogens showed that Saudi isolates were clustered together and were closely related to isolates that were reported from worldwide countries. Risk factor analysis revealed that prevalence of both bacterial pathogens was not restricted to a particular rodent species or a rodent sex (P > 0.05). In conclusion, we are reporting for the very first time that Saudi rodents are infected with Anaplasma ovis and rodents can be infected with Bartonella koehlerae. Similar studies at large scale are recommended in all those areas of Saudi Arabia that are unexplored for the incidence and prevalence of bacterial pathogens among the rodents that are living near human dwellings in order to prevent bacterial infections in local people as well as in livestock.

Understanding temporal and spatial distribution of intestinal nematodes of horses using faecal egg counts and DNA metabarcoding.

This study reports the spatial and temporal distribution of ascarid and strongylid nematodes in Thoroughbred horses by age category across different climatic zones in Australia over an 18-month period. Faecal samples (n = 2046) from individual horses were analysed using the modified McMaster technique for faecal egg counts (FECs). Strongylids were identified using PCR-directed next-generation sequencing of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA. Yearlings had the highest prevalence (82%) of strongyle eggs followed by weanlings (79%), foals (58%), wet mares (49%) and dry mares (46%). For Parascaris spp., foals had the highest prevalence (35%) followed by weanlings (21%) and yearlings (10%). The highest mean FECs for Parascaris spp. were observed in foals (525 eggs per gram [EPG] of faeces) while those for strongyles were in yearlings (962 EPG). Among horses that were classified as adults at the time of sampling, 77% (860 of 1119) of mares were low (i.e., <250 EPG) strongyle egg-shedders. Mean strongyle FEC counts were highest in the Mediterranean (818 EPG) followed by summer (599 EPG), winter (442 EPG), and non-seasonal (413 EPG) rainfall zones. Twenty-six nematode species were detected, with Cylicostephanus longibursatus (26.5%), Cylicocyclus nassatus (23.7%) and Coronocyclus coronatus (20.5%) being the most frequently detected species. Their richness and relative abundance varied with horse age, season and climatic zone. In addition, Strongylus equinus and Triodontophorus spp. (T. brevicauda and T. serratus) were also detected. This comprehensive study elucidates spatial (climatic zone) and temporal (i.e., seasonal) trends in prevalence and burdens of intestinal nematodes in Australian horses using non-invasive conventional and molecular methods. The information presented in this study is crucial for developing integrated management strategies to control horse parasites in farmed horses.

A national survey of anthelmintic resistance in ascarid and strongylid nematodes in Australian Thoroughbred horses.

This study quantified the extent of anthelmintic resistance (AR) in ascarid and strongylid nematodes against commonly used anthelmintics in Australian Thoroughbred horses. Faecal egg count reduction tests (FECRTs, n = 86) and egg reappearance period (ERP) tests were conducted on 22 farms across Australia. Faecal egg counts (FECs) were determined using the modified McMaster technique, and percent faecal egg count reduction (%FECR) was calculated using the Bayesian hierarchical model and hybrid Frequentist/Bayesian analysis method. The results were interpreted using old (published in 1992) and new (2023) research guidelines of the World Association for the Advancement of Veterinary Parasitology (WAAVP). The species composition of strongylid nematodes was detected utilising a DNA-metabarcoding method using pre- and post-treatment samples. Resistance was observed in strongylid nematodes to commonly used single-active and combination anthelmintics, including ivermectin (IVM %FECR range: 82%-92%; 95% lower credible interval (LCI) range: 80%-90%), abamectin (ABM: 73%-92%; 65%-88%), moxidectin (MOX: 89%-91%; 84%-89%), oxfendazole (OFZ: 0%-56%; 0%-31%) and its combination with pyrantel (OFZ + PYR: 0%-82%; 0%-78%). Resistance in Parascaris spp. was observed to IVM (10%-43%; 0%-36%), ABM (0%; 0%) and MOX (0%; 0%). When the new thresholds recommended by the WAAVP were used, AR was detected in six additional FECRTs for strongylids and three more tests for Parascaris spp., introducing resistance to OFZ and OFZ + PYR in the latter. Shortened ERPs (4-6 weeks) of strongylids were observed in 31 FECRTs in which AR was not detected at 2 weeks post-treatment for all the anthelmintics tested. Among cyathostomins, Cylicocyclus nassatus, Cylicostephanus longibursatus and Coronocyclus coronatus were the most prevalent species at 2 weeks post-treatment, whereas the main species appearing at five weeks following treatments with macrocyclic lactones were Cylicocyclus nassatus, Cylicostephanus longibursatus and Cylicocyclus ashworthi. After treatment with OFZ + PYR, the latter three, plus Coronocyclus coronatus and Cyathostomum catinatum, were detected at 5 weeks post-treatment. Overall, the study highlights the prevalence of AR in both ascarids and strongylid nematodes against commonly used anthelmintic products to control worms in Australian horses. The results indicate that ML combination products provided acceptable efficacy at 2 weeks. However, ERP calculations suggest that products work less effectively than previously measured. It is suggested to regularly monitor the efficacy of the anthelmintics and consider changing the worm control practices to better manage worms and AR in Australian horses.

Ticking off the Tick Vectors: Rhipicephalus microplus Fails to Transmit Theileria orientalis.

Theileria (T [...].

Prevalence and diversity of ascarid and strongylid nematodes in Australian Thoroughbred horses using next-generation sequencing and bioinformatic tools.

The study presents the results of a cross-sectional survey to describe the epidemiology of ascarid and strongylid nematodes in horses, the impact of diverse climatic conditions on parasite diversity and the levels of faecal egg shedding in different age groups of managed Thoroughbred horses. Individual faecal samples (n = 1377) collected from 62 Thoroughbred farms across four climatic zones in Australia were analysed using the modified McMaster technique for faecal egg counts (FECs) and strongylid nematodes were identified utilising PCR-directed next-generation sequencing (NGS) of the second internal transcribed spacer of the nuclear ribosomal DNA (ITS-2). Across all age groups, the prevalence of ascarid and strongylid nematodes was 12% (95% confidence interval 10-14%) and 72% (70-74%), respectively. Based on strongylid FECs, yearlings had the highest prevalence (89%) followed by weanlings (83%), foals (79%), wet mares (61%), dry mares (59%) and stallions (54%). However, for Parascaris spp., foals had the highest prevalence (46%) followed by weanlings (32%) and yearlings (13%). The highest mean FECs for Parascaris spp. were observed in foals (418 eggs per gram [EPG] of faeces) while those for strongylids were in yearlings (1002 EPG). Of the adult horses (mares and stallions), 67% (489 of 729) and 11% (77 of 729) were low (i.e., ≤250 EPG) and moderate (i.e., 251-500 EPG) strongylid egg-shedders, respectively. Strongylid egg shedding varied across climatic zones, with the highest mean FECs in the summer rainfall (723 EPG) followed by non-seasonal rainfall (629 EPG), winter rainfall (613 EPG), and Mediterranean (606 EPG) rainfall zones. Twenty-three nematode species were detected using NGS, with Cylicostephanus longibursatus (28%), Cylicocyclus nassatus (23%) and Coronocyclus coronatus (23%), being the most abundant species. Three species of Strongylus (i.e., S. vulgaris, S. equinus and S. edentatus) were also detected. The nemabiome composition, species richness and relative abundance varied within horse age and between climatic zones. These empirical findings provide a comprehensive understanding of the prevalence of parasites within horse populations and the multifaceted factors that influence their occurrence, thereby allowing for the formulation of tailored strategies aimed at parasite control in domestic horses.

Unravelling the Diversity of Microorganisms in Ticks from Australian Wildlife.

Ticks and tick-borne pathogens pose a significant threat to the health and welfare of humans and animals. Our knowledge about pathogens carried by ticks of Australian wildlife is limited. This study aimed to characterise ticks and tick-borne microorganisms from a range of wildlife species across six sites in Victoria, Australia. Following morphological and molecular characterisation (targeting 16S rRNA and cytochrome c oxidase I), tick DNA extracts (n = 140) were subjected to microfluidic real-time PCR-based screening for the detection of microorganisms and Rickettsia-specific real-time qPCRs. Five species of ixodid ticks were identified, including Aponomma auruginans, Ixodes (I.) antechini, I. kohlsi, I. tasmani and I. trichosuri. Phylogenetic analyses of 16S rRNA sequences of I. tasmani revealed two subclades, indicating a potential cryptic species. The microfluidic real-time PCR detected seven different microorganisms as a single (in 13/45 ticks) or multiple infections (27/45). The most common microorganisms detected were Apicomplexa (84.4%, 38/45) followed by Rickettsia sp. (55.6%, 25/45), Theileria sp. (22.2% 10/45), Bartonella sp. (17.8%, 8/45), Coxiella-like sp. (6.7%, 3/45), Hepatozoon sp. (2.2%, 1/45), and Ehrlichia sp. (2.2%, 1/45). Phylogenetic analyses of four Rickettsia loci showed that the Rickettsia isolates detected herein potentially belonged to a novel species of Rickettsia. This study demonstrated that ticks of Australian wildlife carry a diverse array of microorganisms. Given the direct and indirect human-wildlife-livestock interactions, there is a need to adopt a One Health approach for continuous surveillance of tick-associated pathogens/microorganisms to minimise the associated threats to animal and human health.

Molecular diagnostics for gastrointestinal helminths in equids: Past, present and future.

This review is aimed to (i) appraise the literature on the use of molecular techniques for the detection, quantification and differentiation of gastrointestinal helminths (GIH) of equids, (ii) identify the knowledge gaps and, (iii) discuss diagnostic prospects in equine parasitology. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews, we retrieved 54 studies (horses: 50/54; donkeys and zebras: 4/54) from four databases. Polymerase chain reaction (PCR) was employed in all of the studies whereas PCR amplicons were sequenced in only 18 of them. Other techniques used (including modifications of PCR) were reverse line blot, quantitative (q)PCR, restriction fragment length polymorphism, nested-PCR, PCR-directed next-generation sequencing, Southern blotting, single strand conformation polymorphism, PCR-enzyme linked immunosorbent assay, matrix-assisted laser desorption/ionisation-time of flight and random amplification of polymorphic DNA. Most of the studies (53/54) used nuclear ribosomal RNA (including the internal transcribed spacers, intergenic spacer, 5.8 S, 18 S, 28 S and 12 S) as target loci while cytochrome c oxidase subunit 1 and random genomic regions were targeted in only three and one studies, respectively. Overall, to date, the majority of molecular studies have focused on the diagnosis and identification of GIHs of equids (i.e. species of Anoplocephala, Craterostomum, cyathostomins, Oesophagodontus, Parascaris, Strongylus, Strongyloides and Triodontophorus), with a recent shift towards investigations on anthelmintic resistance and the use of high-throughput nemabiome metabarcoding. With the increasing reports of anthelmintic resistance in equid GIHs, it is crucial to develop and apply techniques such as advanced metabarcoding for surveillance of parasite populations in order to gain detailed insights into their diversity and sustainable control. To the best of our knowledge, this is the first systematic review that evaluates molecular investigations published on the diagnosis and quantification of equid GIHs and provides useful insights into important knowledge gaps and future research directions in equid molecular parasitology.

Egg reappearance periods of anthelmintics against equine cyathostomins: The state of play revisited.

Cyathostomins are the most common and highly prevalent parasites of horses worldwide. Historically, the control of cyathostomins has mainly relied on the routine use of anthelmintic products. Increasing reports on anthelmintic resistance (AR) in cyathostomins are concerning. A potential method proposed for detecting emerging AR in cyathostomins has been estimating the egg reappearance period (ERP). This paper reviews the data available for the ERP of cyathostomins against the three major classes of anthelmintics, macrocyclic lactones, tetrahydropyrimidines, and benzimidazoles. Published peer-reviewed original research articles were obtained from three databases (PubMed, CAB Direct and Web of Science) and were evaluated for their inclusion in a systematic review. Subsets of articles were then subjected to a review of ERP data. A total of 54 (of 134) studies published between 1972 and 2022 met the criteria for inclusion in the systematic review. Until the beginning of 2022, there was no agreed definition of the ERP; eight definitions of ERP were identified in the literature, complicating the comparison between studies. Additionally, potential risk factors for the shortening of the ERP, including previous anthelmintic use and climate, were frequently not described. Reports of shortened ERP for moxidectin and ivermectin are frequent: 20 studies that used comparable ERP definitions reported shortened moxidectin and ivermectin ERPs of 35 and 28 days, respectively. It is unclear whether the ERPs of these anthelmintics reduced to such levels are due to the development of AR or some biological factors related to horses, cyathostomin species, and/or the environment. The ERPs for other anthelmintics, such as fenbendazole and pyrantel, were frequently not reported due to established resistance against these drugs. Future research in horses is required to understand the mechanism(s) behind the shortening of ERP for cyathostomins. Based on this systematic review, we propose recommendations for future ERP studies.

Analysis and Simulation of Fractional Order Smoking Epidemic Model.

In recent years, there are many new definitions that were proposed related to fractional derivatives, and with the help of these definitions, mathematical models were established to overcome the various real-life problems. The true purpose of the current work is to develop and analyze Atangana-Baleanu (AB) with Mittag-Leffler kernel and Atangana-Toufik method (ATM) of fractional derivative model for the Smoking epidemic. Qualitative analysis has been made to `verify the steady state. Stability analysis has been made using self-mapping and Banach space as well as fractional system is analyzed locally and globally by using first derivative of Lyapunov. Also derive a unique solution for fractional-order model which is a new approach for such type of biological models. A few numerical simulations are done by using the given method of fractional order to explain and support the theoretical results.

Genetic characterisation of Echinocephalus spp. (Nematoda: Gnathostomatidae) from marine hosts in Australia.

We genetically characterised larval and adult specimens of species of Echinocephalus Molin, 1858 (Gnathostomatidae) collected from various hosts found within Australian waters. Adult specimens of Echinocephalus were collected from a dasyatid stingray [Pastinachus ater (Macleay); n = 2] from Moreton Bay, Queensland and larvae from a hydrophiine sea snake [Hydrophis peronii (Duméril); n = 3] from Cape York Peninsula, Queensland, from an octopus (Octopus djinda Amor & Hart; n = 3) from Fremantle, Western Australia and from a lucinid bivalve [Codakia paytenorum (Iredale); n = 5] from Heron Island, Queensland Australia. All nematode samples were identified morphologically and genetically characterised using the small subunit nuclear ribosomal DNA (SSU). Some morphological differences were identified between previous studies of Echinocephalus spp. and those observed herein but the significance of these differences remains unresolved. Molecular phylogenetic analyses revealed that larval Echinocephalus sp. from H. peronii and C. paytenorum in Australia were very similar (with strong nodal support) to larval Echinocephalus sp. infecting two fish species from Egypt, Saurida undosquamis (Richardson) (Synodontidae) and Pagrus pagrus (Linnaeus) (Sparidae). The SSU sequences of larval Echinocephalus sp. from O. djinda and adults from P. ater formed a well-supported clade with that of adult E. overstreeti Deardorff and Ko, 1983 from the Port Jackson shark, Heterodontus portusjacksoni (Meyer), as well as that of the larval Echinocephalus sp., from the common carp (Cyprinus carpio Linnaeus) from Egypt. This study extends the intermediate host range of Echinocephalus larvae by including a sea snake for the first time. Findings of this study highlight the importance of genetic characterisation of larval and adult specimens of Echinocephalus spp. to resolve the current difficulties in the taxonomy of this genus.

Fatal cases of bovine anaplasmosis in a herd infected with different Anaplasma marginale genotypes in southern Spain.

The genus Anaplasma contains various species capable of causing disease in animals and humans. Anaplasma marginale is one of the main tick-borne pathogens of bovines in tropical and subtropical regions; however, these bacteria are now being detected more frequently in other regions of the world including Europe. In July 2017, abortions, mortality and morbidity in Retinta breed of cattle were investigated in southwestern Spain. Based on clinical signs, the provisional clinical diagnosis of bovine anaplasmosis was made. A molecular-phylogenetic approach was used to characterize A. marginale using multiple markers, including 16S rRNA, msp1a, msp4 and msp5 genes. The msp1α sequence was different from the previously described sequences from Spain as well as other countries. The isolates of A. marginale were classified as Genotype C, with two of the five tandem repeats in the amino acid sequences MSP1α being novel. The highest variability was observed in the four sequences of msp5 which was depicted in their clustering into multiple clades on a phylogenetic tree. Comparison of msp5 nucleotide sequences and the corresponding amino acid sequences revealed the co-existence of different strains in the same region. This study highlights the occurrence of clinical bovine anaplasmosis in an endemic region of Spain.

Cyathostomin resistance to moxidectin and combinations of anthelmintics in Australian horses.

BACKGROUND: Cyathostomins are the most important and common parasitic nematodes of horses, with > 50 species known to occur worldwide. The frequent and indiscriminate use of anthelmintics has resulted in the development of anthelmintic resistance (AR) in horse nematodes. In this study we assessed the efficacy of commonly used anthelmintics against cyathostomins in Australian thoroughbred horses. METHODS: Two drug efficacy trials per farm were conducted on two thoroughbred horse farms in the state of Victoria, Australia. In the first trial, the horses on Farm A were treated with single and combinations of anthelmintics, including oxfendazole (OFZ), abamectin (ABM), abamectin and morantel (ABM + MOR), moxidectin (MOX) and oxfendazole and pyrantel (OFZ + PYR), at the recommended doses, whereas the horses on Farm B only received MOX, at the recommended dose. The faecal egg count reduction test (FECRT) was used to determine the efficacy and egg reappearance period (ERP) of anthelmintics. Based on the results of the first trial, the efficacies of MOX and a combination of ABM + MOR were reassessed to confirm their activities against cyathostomins. RESULTS: Of the five anthelmintic products tested on Farm A, resistance against OFZ, ABM and OFZ + PYR was found, with efficacies of - 41% (- 195% lower confidence limit [LCL]), 73% (60% LCL) and 82% (66% LCL) at 2 weeks post-treatment, respectively. The FECRT showed high efficacies of MOX and ABM + MOR (100%) at 2 week post-treatment and shortened ERPs for these anthelmintics (ABM + MOR: 4 weeks; MOX: 5 weeks). Resistance to MOX was found on Farm B, with a reduced efficacy of 90% (70% LCL) and 89% (82% LCL) at 2 weeks post-treatment in trials one and two, respectively. CONCLUSIONS: This study provides the first evidence of MOX- and multidrug-resistant (ABM and combinations of anthelmintics) cyathostomins in Australia and indicates the need for continuous surveillance of the efficacy of currently effective anthelmintics and large-scale investigations to assess the ERP for various anthelmintics.

Ticks and Tick-Borne Diseases in Central America and the Caribbean: A One Health Perspective.

Ticks have complex life cycles which involve blood-feeding stages found on wild and domestic animals, with humans as accidental hosts. At each blood-feeding stage, ticks can transmit and/or acquire pathogens from their hosts. Therefore, the circulation of tick-borne pathogens (TBPs), especially the zoonotic ones, should be studied in a multi-layered manner, including all components of the chain of infections, following the 'One Health' tenets. The implementation of such an approach requires coordination among major stakeholders (such as veterinarians, physicians, acarologists, and researchers) for the identification of exposure and infection risks and application of effective prevention measures. In this review, we summarize our current knowledge on the epidemiology of tick-borne diseases in Central America and the Caribbean and the challenges associated with the implementation of 'One Health' surveillance and control programs in the region.

Ticks and tick-borne diseases of bovines in a smallholder livestock context: The Pakistani example.

Ticks and tick-borne diseases (TTBDs) substantially affect the health and production of ruminants, particularly in resource-poor, small-scale farming systems worldwide. However, to date, there has been no critical appraisal of the current state of knowledge of TTBDs in such farming systems. In this article, we systematically reviewed the situation in Pakistan-as an example of a country that is highly reliant on agriculture to sustain its economy, particularly smallholder livestock farms, which are continually faced with challenges associated with TTBDs. The main aims of this review were to gain improved insights into the current status of TTBDs in small-scale farming systems, and to identify knowledge gaps, through the systematic evaluation of published literature on this topic from Pakistan, and to recommend future research directions. We searched publicly available literature from three databases (i.e. Web of Science, Google Scholar, and PubMed) on bovine TTBDs in Pakistan. Of 11,224 published studies identified, 185 were eligible for inclusion; these studies were published between August 1947 and June 2021. A critical analysis of these 185 studies revealed that the diagnosis of ticks and tick-borne pathogens (TBPs) in Pakistan has been based largely on the use of traditional methods (i.e. 'morpho-taxonomy'). At least 54 species of tick have been recorded, most of which belong to the genera Haemaphysalis, Hyalomma and Rhipicephalus. The prevalence of ticks was higher, particularly in young, exotic and crossbred female cattle, during the summer season. Major TBPs include species of Anaplasma, Babesia and Theileria, with prevalences being higher in cattle than buffaloes. Additionally, pathogens of zoonotic potential, including species of Anaplasma, Borrelia, the Crimean-Congo haemorrhagic fever virus, Coxiella, Ehrlichia and Rickettsia, have been recorded in both tick and bovine populations. Information on risk factors, spatial-temporal distribution, genetic diversity, and control of ticks and TBPs is limited, the vector potential of ticks and the distribution patterns of ticks and TBPs in relation to climate remains largely unexplored. Future research should focus on addressing these knowledge gaps and the key challenges of poverty, food security and disease outbreaks in a small-scale livestock farming context in order to provide sustainable, environment-friendly control measures for TTBDs.

Molecular detection of Strongyloides sp. in Australian Thoroughbred foals.

BACKGROUND: Strongyloides westeri is found in the small intestine of young horses, mainly in foals up to about 16 weeks of age. The main source of infection for foals is through transmammary transmission, and foals can develop acute diarrhoea, weakness, dermatitis and respiratory signs. The epidemiology of S. westeri in Australia is largely unknown. Further, molecular techniques have never been employed for detection of S. westeri in horses. This pilot study aimed to assess the utility of a molecular phylogenetic method for the detection of S. westeri in the faeces of foals. METHODS: Faecal samples were collected from a foal of less than 2 months of age, and eggs of Strongyloides sp. were detected using the modified McMaster technique. DNA was extracted from purified eggs, and a partial fragment of the small subunit of the nuclear ribosomal DNA (18S) was characterised using polymerase chain reaction, DNA sequencing and phylogenetic methods. RESULTS: Microscopic examination of faeces revealed small ellipsoidal eggs typical of Strongyloides sp. The 18S sequence generated by PCR in this study revealed 98.4% identity with that of a reference sequence of S. westeri available from GenBank. Phylogenetic analyses revealed a polyphyletic clustering of S. westeri sequences. CONCLUSION: This is the first study reporting the detection of DNA of Strongyloides sp. in faeces of a foal using a molecular phylogenetic approach targeting the variable region of 18S rDNA. It is anticipated that this study will allow future molecular epidemiological studies on S. westeri in horses.

Shared Odds of Borrelia and Rabies Virus Exposure in Serbia.

Lyme borreliosis (LB) is the most common tick-borne disease in Serbia and other European countries. Rabies is a fatal zoonosis distributed worldwide and is caused by the rabies virus. Professionals at risk of rabies-including veterinarians, hunters, communal service workers, and forestry workers-overlap with some professions at a higher risk of exposure to tick bites and tick-borne pathogen infections. We hypothesized that individuals identified by the public health system as at risk of rabies virus infection, and consequently vaccinated against rabies virus, also share a higher likelihood of Borrelia exposure. To test our hypothesis, a case-control study was carried out during 2019 in Serbia to determine the seroprevalence of anti-Borrelia antibodies in two case groups (individuals at risk and vaccinated against rabies virus) and a control group (individuals without risk of rabies). Individuals vaccinated against rabies following either "pre-exposure protocol" (PrEP, n = 58) or "post-exposure protocol" (PEP, n = 42) were considered as rabies risk groups and healthy blood donors (n = 30) as the control group. The results showed higher Borrelia seroprevalence in PrEP (17.2%; 10/58) and PEP (19.0%; 8/42) groups compared with the control group (6.67%; 2/30). Furthermore, odds ratio (OR) analysis showed that risk of rabies (in either the PrEP (OR = 2.91) or PEP (OR = 3.29) groups) is associated with increased odds of being seropositive to Borrelia. However, the difference in Borrelia seroprevalence between groups was not statistically significant (Chi-square (χ²) test p > 0.05). The shared odds of LB and rabies exposure found in this study suggest that, in countries where both diseases occur, the common citizen can be at risk of both diseases when in a risky habitat. These findings are important to guide physicians in targeting high-risk groups, and diagnose LB, and to guide decision-makers in targeting control and prevention measures for both infections in risk areas.

Comparative studies on faecal egg counting techniques used for the detection of gastrointestinal parasites of equines: A systematic review.

Faecal egg counting techniques (FECT) form the cornerstone for the detection of gastrointestinal parasites in equines. For this purpose, several flotation, centrifugation, image- and artificial intelligence-based techniques are used, with varying levels of performance. This review aimed to critically appraise the literature on the assessment and comparison of various coprological techniques and/or modifications of these techniques used for equines and to identify the knowledge gaps and future research directions. We searched three databases for published scientific studies on the assessment and comparison of FECT in equines and included 27 studies in the final synthesis. Overall, the performance parameters of McMaster (81.5%), Mini-FLOTAC® (33.3%) and simple flotation (25.5%) techniques were assessed in most of the studies, with 77.8% of them comparing the performance of at least two or three methods. The detection of strongyle, Parascaris spp. and cestode eggs was assessed for various FECT in 70.4%, 18.5% and 18.5% studies, respectively. A sugar-based flotation solution with a specific gravity of ≥1.2 was found to be the optimal flotation solution for parasitic eggs in the majority of FECT. No uniform or standardised protocol was followed for the comparison of various FECT, and the tested sample size (i.e. equine population and faecal samples) also varied substantially across all studies. To the best of our knowledge, this is the first systematic review to evaluate studies on the comparison of FECT in equines and it highlights important knowledge gaps in the evaluation and comparison of such techniques.

Targeted Next-Generation Sequencing and Informatics as an Effective Tool to Establish the Composition of Bovine Piroplasm Populations in Endemic Regions.

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.