The current study was approved from Kohat University of Science and Technology Kohat ethical approval committee for sample collection from animals. Written informed consent has been granted by the owners for their animal’s participation in the current study.

The present study was done at Kohat, Khyber Pakhtunkhwa. It lies 489 m above sea level between 32° 47′ and 33° 53′ north latitude and 70° 34′ and 72° 17′ east longitude (Figure 1). Approximately 5 g of feces was directly obtained from the rectum of 180 sheep and goats (90 each) under strict aseptic conditions in the study area. All information, including the animal type, sex, age, location, and collection month, was recorded on the mandated proforma for each sheep and goat. The Molecular Parasitology and Virology Laboratory, Department of Zoology, KUST, received appropriately labeled samples in cold boxes. Prior to further processing, the samples were preserved at −20°C.

Three grams of feces were added to container 1 with 50 mL of PBS dispensed into the same container, and a tongue blade was used to mix the feces with the PBS solution. The fecal suspension was transferred from the jar to container 2, and finally to the test tube using a double layer of cheesecloth. A convex meniscus formed on top of the test tube after the solution was¹ gently tapped off. A delicate coverslip was set atop the test tube and left undisturbed for 20 min. Twenty minutes later, the coverslip and drop of fluid attached to it were carefully removed from the test tube and placed on a clean slide. For microscopic positive samples, the tube was filled with 2 mL of PBS and spun in a microcentrifuge for 5 min at 3000 rpm to rinse the eggs. After the supernatant was removed, the rinsing procedure was repeated. Once the supernatant was removed, 300 𝜇l of the ova in the pellet was collected for genome extraction (46). Coverslips adhering to the slide were stained with iodine and examined for eggs and larvae using a light compound microscope under 10X and 40X objective lens, according to Ebrahim (47).

Haemonchus positive samples were extracted using the “QIAamp Fast DNA Stool Mini Kit” (Qiagen GmbH, Hilden, Germany). The manufacturer’s protocol was used to extract the complete genomic DNA. Amplification of the prepared DNA was carried out as described by Hussain et al. (9). The ITS2 gene was amplified using NC1F (5’-ACGTCTGGTTCAGGGTTGTT-3′) and NC1R (5’-TTAGTTTCTTTTCCTCCGCT-3′) with an amplicon size of 350 bp (23). To achieve the necessary gene amplification, a thermal cycler was utilized with a 25 𝜇l PCR reaction volume comprising of 13 𝜇l PCR Master mix (dNTPs, MgCl2, Taq DNA polymerase), 1 𝜇l forward and reverse primers, 5 𝜇l PCR water (dH2O), and 5 𝜇l extracted DNA. The initial temperature was 96°C for 7 min, followed by 40 cycles of 95°C for 45 s, 57.5°C for 45 s, and 72°C for 45 s, with a final extension temperature of 72°C for 7 min. A 1.5% agarose gel was used for DNA resolution. Bands (Fermentas, United States) were compared to a 2000 bp DNA ladder marker (23). A gel documentation device was used to resolve the bands under UV illumination.

Adherence to Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) was maintained throughout the execution of this review on articles related to H. contortus epidemiology in ruminants across Pakistan (10). Research articles published in English were retrieved from different databases, including ISI Web of Science, Mendeley, PubMed, ScienceDirect, EBSCHO, and Google Scholar. Databases were searched for articles using keywords such as prevalence, epidemiology, infection rate/infestation rate, Haemonchus contortus, Haemonchus, sheep, goat, ruminants, and Pakistan. Boolean operators “AND” and “OR” were also used to retrieve articles (“Prevalence” OR “epidemiology” OR “infection rate” OR “infected” AND “Haemonchus contortus” AND “Pakistan” AND “ruminants” OR “sheep” OR “goat”) on the H. contortus distribution rate across Pakistan from January 2000 till July 2023. The article references and PDFs were downloaded to a folder created using Mendeley reference manager software for the current study. Duplicate studies were removed from the folder after checking the title and references, followed by irrelevant article removal due to the non-availability of data in the abstract.

We checked the article titles and abstracts to determine whether they had any data on the prevalence of H. contortus in Pakistan. The inclusion criteria were as follows: i) articles written in English, ii) articles containing H. contortus distribution in ruminants, and iii) articles published between January 2000 and July 2023. However, we excluded reviews, duplicate articles, studies dealing with other species of Haemonchus prevalence data, articles involving two species without confirmation of a single species outcome, publications with vague data such as trichostrongylid/strongyles infection rate, or sources of samples without complete text.

The current systematic review included studies that were evaluated using the following criteria to ascertain quality scores: (a) random sampling/not, (b) sampling method clear/not, (c) sampling method detailed/not, (d) number of samples ≥80/not, and (e) ≥3 risk factors. By calculating the aforementioned answers, each study was given a score; a score of 0 for “no” and a score of 1 was given for “yes,” aggregating a total score of 5. Studies with a score of 0–1, 2–3, and 4–5 were classified as below average, average, and good quality, respectively.

To ensure that no papers were overlooked, two writers (NA and RA) separately searched the English databases for titles and abstracts of articles that met the inclusion criteria. The eligibility of the study for inclusion was determined by the same writers who independently reviewed the entire text. Potentially eligible studies were excluded if (i) the prevalence data results were not provided in terms of the number of samples and (ii) the dataset was ambiguously presented based on ruminants. Another author (AM) made the ultimate choice following a debate in which the two writers could not reach an agreement. Finally, the data were extracted by NA and RA, which included pertinent information about the initial author, publication year, host species, type of sample, method of study, number of examined samples, positive sample, prevalence rate, random sampling/not, clear sampling method/not, detailed sampling method/not, number of samples ≥80/not, and ≥ 3 risk factors studies/not to calculate the score and quality of included studies using a pre-designed Microsoft Excel spreadsheet (Figure 2).

After amplicon confirmation through gel electrophoresis, 4 randomly selected amplified DNA products (two each sheep and goat samples) were shipped to Macrogen Inc. (Seoul, South Korea) for purification and sequencing. The chromatograms of the sequenced samples were interpreted, and noise was removed at the start and end of each sequence using BioEdit software. The resulting sequences were subjected to BLAST in NCBI² for confirmation of H. contortus. After confirmation of H. contortus, the reference sequences were retrieved and transferred to Mega11 for further processing. Sequences were trimmed and aligned using Clustal W, followed by the inclusion of an outgroup in the software, and the maximum composite likelihood technique was used for phylogenetic analysis. The sequences from this study and those from the NCBI gene repository were used to construct a phylogenetic tree. Through the addition of a maximal composite probability parameter and 1,000 bootstrap replications, an evolutionary tree was constructed using the maximum composite likelihood approach. Nucleotide sequences reported in the present study are available in GenBank^™, an NCBI database, under the accession numbers OK447878, OK481181, OM276841, and OM276825.

To determine the differences between the observed and expected data, numerous factors, including type of ruminant, age, sex, month, and distribution by locality, were analysed in relation to the prevalence of haemonchosis using Chi-squared and Fisher exact tests in R software. A p-value of less than 0.05 was used to determine the statistical significance of the findings (11).

A total of 180 fecal samples were collected from both sheep and goats of different age groups, sex, months, and area from July to December, 2021. Forty-six (46) small ruminants were found positive for haemonchosis. The documented overall prevalence of H. contortus in small ruminants was 25.55%. However, sheep was non-significantly more susceptible (28.89%) to haemonchosis in comparison to goats (22.22%) p > 0.05 (Figure 3A). Both goats and sheep were divided into male and female categories based on sex. The sex-wise infection of H. contortus recorded in the current research work was higher in female sheep and goats (28.82%) as compared to male sheep and goats (20.28%) p > 0.05 (Figure 3B).

Small ruminants were divided into three age groups: <1 year, 1–3 year, and > 3 years named as juvenile, young and adult, respectively. The highest prevalence rate was recorded in juvenile (33.33%) and adult age groups (29.82%), while the lowest was in the young age group (19.54%) however, the differences were non-significant p > 0.05 (Figure 3C). The present study was divided into 5 different localities of district Kohat named Lachi, Ghamkol Camp, Arid Zone Small Ruminant Research Institute (SRRI), Paracha Town, and Gumbat. The highest occurrence of Haemonchus was observed in Ghamkol camp (29.41%), while the lowest was in the Arid Zone (SRRI) (17.5%) of district Kohat p < 0.02 (Figure 3D). The current study was comprised of 6 months of sample data from July to December. The highest incidence of 38.70% of H. contortus was documented in the month of July, whereas the lowest was 11.11% in December p < 0.01 (Figure 3E).

We identified and added a total of 118 articles PDF with references from our searched databases using Mendeley Web Importer. We removed 35 duplicate articles from the Mendeley reference manager software dedicated folder for the current study. After removing duplicate articles, 83 articles remained. Primary screening of the title and abstract resulted in the exclusion of 24 papers not concerning with Pakistan; the remaining 59 articles were then chosen for full-text reading. In addition, 38 studies were removed according to our inclusion and exclusion parameters. Finally, 21 eligible papers based on H. contortus epidemiology in Pakistani ruminants were eventually included in the current systematic review, as shown in Table 1.

Out of 21 articles, the highest number of studies 13 (61.90%) were reported from Punjab, followed by Khyber Pakhtunkhwa 5(23.80%) and 1(4.76%) each from Baluchistan, Sindh, and Islamabad capital territory. The current systematic review consists of Haemonchus contortus studies on the ruminant population, which includes sheep, goats, cattle, buffalo, camels, and markhor. The most common types of samples used for H. contortus identification were fecal 17, followed by worm 3, and a single study collected both blood and fecal samples. Multiple methods were used for H. contortus evaluation, including direct smear microscopy, key identification, floatation, sedimentation, McMaster, and the Eggs per gram method. The most common evaluation method was direct microscopy, while the least common was key identification, as shown in Table 1. Based on risk factors 12 studies fall in the average category, followed by 6 in the good category and 3 in the below-average category.

The 21 selected Pakistani studies on ruminants show a 38.06% overall prevalence rate of H. contortus across Pakistan. Studies performed in all 5 regions of Pakistan, namely Khyber Pakhtunkhwa, Punjab, Baluchistan, Sindh, and Islamabad capital territory area, accounted for 34.49, 40.53, 10.41, 14.64, and 88.5%, respectively, p < 0.001. Based on the type of animal, sheep, goat, cattle, buffalo, and markhor show 44.99, 27.09, 15.08, 14.29, and 40% H. contortus with significant difference p < 0.001 respectively, Figure 4.

Followed by DNA extraction and amplification of the ITS-2 gene, a PCR product of approximately 350 bp was found under UV light. The top hits and highly similar sequences were retrieved for downstream phylogenetic tree construction. A dendrogram was constructed for the ITS-2 gene of Haemonchus spp. and other related nematode genera, including Marshallagia and Trichostrongylus. The dendrogram is comprised of 4 sequences from Kohat and 24 published sequences of different geographical localities, including Pakistan, China, India, Malaysia, Iran, Bangladesh, Thailand, Mongolia, Yemen, Italy, Denmark, and Japan, among others.

The phylogenetic analysis clustered H. contortus into a major clade with different subclades (Figure 5). H. contortus is grouped separately from other related species, i.e., H. bedfordi, H. longistipes, and H. placei. The H. contortus of the current study was closely related to the H. contortus of the neighboring Asian countries, including Iran, India, China, Mongolia, and Malaysia. This subclade also comprised a sequence from district Jhang of Pakistan, United Kingdom, Italy, and Mongolia. Surprisingly, our study’s neighbor joining dendrogram (Figure 5) showed that the isolates under investigation were quite closely related to those from Italy and the United Kingdom.