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Isolation, Identification, and Molecular Characterization of Haemonchus contortus in Slaughtered Small Ruminants in the Chattogram Metropolitan Area, Bangladesh

VSRR_9_2_121-131

Isolation, Identification, and Molecular Characterization of Haemonchus contortus in Slaughtered Small Ruminants in the Chattogram Metropolitan Area, Bangladesh

Muhammad Abdul Mannan1,2, Sharmin Chowdhury2, Md Abul Hashem3,4 and Md. Hazzaz Bin Kabir1,5*

1Department of Microbiology and Parasitology, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh; 2Department of Pathology and Parasitology, Faculty of Veterinary Medicine, Chattogram Veterinary and Animal Sciences University, Khulshi-4225, Chittagong, Bangladesh; 3Department of Health, Chattogram City Corporation, Bangladesh; 4Feinberg School of Medicine, Northwestern University, Chicago, USA; 5Laboratory of Sustainable Animal Environment, Graduate School of Agricultural Science, Tohoku University, 232-3 Yomogida, Naruko-onsen, Osaki, Miyagi, 989-6711, Japan.

Abstract | Haemonchus contortus is a highly destructive parasitic nematode in small ruminants that causes significant economic losses to sheep and goat breeding operations worldwide. To combat this problem and minimize economic damage, effective preventative measures should be aimed at high-risk populations and a thorough understanding of the epidemiology is crucial. This study aimed to improve the understanding of the molecular epidemiology of Haemonchus contortus in sheep and goat carcasses in the Chattogram Metropolitan area in Bangladesh. A sample of 400 abomasa (150 from sheep and 250 from goats) was collected from five different slaughterhouses in the Chattogram Metropolitan area and analyzed using morphological and microscopic methods. The Polymerase Chain Reaction (PCR) was then employed to amplify the sec internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA and the mitochondrial nicotinamide dehydrogenase subunit 4 gene (nad4) to identify H. contortus, followed by direct sequencing. Out of the 400 samples tested, 186 (46.5%) were positive for H. contortus. The parasite was found in 51.3% of sheep and 43.6% of goats. Through the sequencing analysis, 45 nad4 genotypes and 10 ITS-2 genotypes were discovered. The prevalence of Haemonchus contortus isolated was not influenced by demographic factors such as age, gender, breed, and seasons in the study. Our results highlight the state of our knowledge on the spread of H. contortus parasite among small ruminants in Chattogram slaughterhouses.


Editor | Muhammad Abubakar, National Veterinary Laboratories, Park Road, Islamabad, Pakistan.

Received | August 19, 2023; Accepted | October 02, 2023; Published | October 14, 2023

*Correspondence | Md. Hazzaz Bin Kabir, Laboratory of Sustainable Animal Environment, Graduate School of Agricultural Science, Tohoku University 232-3 Yomogida, Naruko-onsen, Osaki, Miyagi 989-6711, Japan; Email: md.hazzaz.bin.kabir.c1@tohoku.ac.jp

Citation | Mannan, M.A., S. Chowdhury, M.A. Hashem and M.H.B. Kabir. 2023. Isolation, identification, and molecular characterization of Haemonchus contortus in slaughtered small ruminants in the Chattogram metropolitan area, Bangladesh. Veterinary Sciences: Research and Reviews, 9(2): 121-131.

DOI | https://dx.doi.org/10.17582/journal.vsrr/2023/9.2.121.131

Keywords | Chittagong, Haemonchus contortus, ITS-2, nad4 gene, Slaughterhouse, Small ruminant

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).



Introduction

Haemonchus contortus is a prevalent blood-sucking parasite among other gastrointestinal nematodes in small ruminants (O’Connor et al., 2006). This parasite inhabits the abomasum of the host and causes haemonchosis, leading to significant economic losses from decreased meat, milk, wool, and leather production, as well as from clinical symptoms of blood loss (Kumsa et al., 2008). Haemonchus contortus can cause anemia, edema, and in severe cases, death in affected animals, particularly during hot and humid summer months (Easwaran et al., 2009). In some regions, other species such as H. placei and H. longistipes of Haemonchus can coexist in shared pastures used by all ruminants (Brasil et al., 2012; Jacquiet et al., 1998). Each worm is estimated to suck approximately 0.05 mL of blood daily and waste 0.1 mL of blood due to leakage (Soulsby, 1982; Urquhart et al., 2000). The parasite has a remarkable ability to survive due to its ecological adaptability (Troell et al., 2006).

Bangladesh is the fourth largest producer of goats (26.9 million) and sheep (3.8 million) in Asia, and the livestock sector contributes 1.85% to the country’s gross domestic product (DLS, 2023). Livestock is crucial for reducing poverty and generating income in Bangladesh. The majority of these animals are owned by nomads who are able to roam freely, while small farms are mostly found in big cities. This results in increased contact between different animal species during pasture and irrigation (Hussain et al., 2014; Shen et al., 2017). Despite this, parasitic infections remain a major challenge in livestock management. Effective control of H. contortus infections requires the development of appropriate management strategies. However, overreliance on anthelmintics has led to widespread drug resistance in H. contortus populations, making the development of effective control strategies more challenging (Kaplan, 2004). Molecular identification of H. contortus species is crucial for implementing effective control strategies. In general, H. contortus has a complex genetic structure, and there are different strains and species of this nematode. Different species or strains of H. contortus may have varying susceptibility to environmental factors, such as temperature or humidity (Kaplan, 2004). Targeted control measures can be implemented based on this information to prevent or manage H. contortus infections. Morphological identification of Haemonchus contortus using microscopy has long been a reliable method for detecting and distinguishing the parasite. However, Molecular techniques can differentiate between closely related species or strains of parasites that may have varying levels of pathogenicity. This information is crucial for designing targeted control strategies that are specific to the particular species or strain present in a given region.

Molecular tools provide valuable information including determining the source of infection, tracking transmission routes, and identifying potential risk factors. This knowledge helps in developing effective control measures that target specific stages or sources of transmission (Yin et al., 2013). It provides insights into the prevalence, distribution, and changes in parasite populations over time, enabling the assessment of control program effectiveness and the identification of areas requiring intensified interventions. In Bangladesh, there is a lack of information about parasitism in slaughterhouses. A recent study found the presence of Haemonchus contortus in goats at local slaughterhouses in Sylhet (Nath et al., 2021).

Molecular identification of H. contortus can provide valuable information on the transmission dynamics and epidemiology of the parasite, which can inform the design of targeted control strategies (Sargison, 2011). Moreover, identifying the species and strains of H. contortus present in a particular area can help establish baseline data for monitoring changes in the prevalence and distribution of the parasite over time, allowing for early detection of new outbreaks (Besier et al., 2016). Conducting molecular identification on Haemonchus contortus is crucial for evaluating effective control strategies and understanding the epidemiology of the parasite. The current study focuses on identifying H. contortus in small ruminants in slaughterhouses and examining the factors related to infection such as age, sex, breed, host, body condition, seasons and regions.

Materials and Methods

Study area and specimen collection

This study was conducted on goats and sheep that were raised in different parts of Bangladesh and slaughtered in the abattoirs of the Chattogram Metropolitan area. Samples of the abomasa were collected from five different abattoirs located in Jhautala, Firingebazar, Pahartali, Colonethat, and Halishahar on a weekly basis from January to December 2015. Data on the demographic factors (age, sex, breed, host, body condition, seasons, and regions) was recorded for each abattoir through questionnaires during the time of sampling. A total of 250 goats and 150 sheep were sampled, with adult worms being extracted from the abomasa following the protocol described by Iqbal (Iqbal et al., 1993) and (MAFF, 1986) with slight modifications. In brief, adult worms were recovered from the abomasa of sheep and goats obtained from the slaughterhouse. After slaughtering, the abomasum was removed from the other stomach parts and ligated at both ends. After that, the abomasum was transported directly to the Chittagong Veterinary and Animal Sciences University’s Department of Parasitology laboratory for more research. It was dissected along its greater curvature, and the contents were poured into a glass beaker. The mixture was then put through a series of washings, sedimentations, and decants until the supernatant was sufficiently clear to make worm collecting simple. Both the abomasum and its contents were carefully examined, and individual adult male worms were collected and identified by microscopic examination of spicules, according to MAFF (1986). They were then preserved in 70% ethanol and stored at -20°C until DNA extraction was performed. To reduce the possibility of inaccurate DNA amplification from female worm eggs, only mature male worms were employed.

Specimen isolation and microscopic examination

The collected parasite samples were washed with normal saline and put into sterile petridishes for examination. The worms were visually inspected before being studied under a microscope for species identification, following the process outlined by Soulsby (1982). In brief, the worms were placed in a glycerine alcohol solution (90 mL 70% ethanol and 10 mL glacial glycerine) for 24 hr until they become transparent, then mounted with glycerin jelly (10 g gelatin, 500 mL glycerine, 10 g phenol, and 60 mL distilled water). Observations and measurements were conducted under a light microscope (Olympus BX-53, Tokyo, Japan) with an ocular micrometer. Out of the 400 collected samples, only 186 (109 goats and 77 sheep) that showed positive results under the microscope were subjected to a conventional polymerase chain reaction (PCR) for further confirmation.

DNA extraction and PCR amplification of specimens

Total genomic DNA was extracted from adult worms using G-spinTM Total DNA Extraction Kit (REF-17045, iNtRON Biotechnology, Korea) according to the manufacturer’s instruction, which was then stored at -20°C. To confirm the species of Haemonchus, a conventional PCR was conducted to amplify 321 bp of the ITS-2 of the nuclear rDNA using the forward primer NC1-F (5’-ACGTCTGGTTCAGGGTTGTT-3’) and the reverse primer NC1-R (5’-TTAGTTTCT TTTCCTCCGCT-3’), as described previously by Stevenson et al. (1995) and Akkari et al. (2013), and the 800 bp of nad4 gene was also amplified using Primer1-F (5’-GGATTTGGTCAGCAAATTGAA-3’) and Primer2-R (5’-GCCTGCAAATGAATTAACA-3’), (Yin et al., 2013).

A 50 μL reaction was used in each PCR amplification and it consisted of 25 μL of master mix (which included DNA polymerase, buffer, and four types of nucleotides in certain quantities, as well as MgCl2), 5 μL of DNA sample (with a concentration of over 30 ng/μL), 2.5 μL of forward primer (10 pmol/μL), 2.5 μL of reverse primer (10 pmol/μL), and 15 μL of distilled water. The PCR reaction for the amplification of 321 bp of ITS-2 involved an initial heating at 95°C for 2 min, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 7 min. The nad4 gene amplification started with an initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 secs, annealing at 55°C for 30 secs, extension at 72°C for 1 min, and a final extension at 72°C for 5 min. The amplified fragments were separated through 1.5% gel electrophoresis, stained with ethidium bromide, and the bands were visualized on a UV transilluminator. The positive PCR products were then purified using a Favor PrepTM PCR Clean-Up Mini Kit and 40 μL of elution buffer was used to retrieve the pure DNA.

Sequencing and phylogenetic analysis

The purified DNA amplicons of ITS-2 (10) and nad4 (45) gene samples from the PCR were directly sequenced in both directions with the primers used for the PCR by the ABI 3100 Genetic Analyzer and the BigDyeTM Terminator v.3.1 Sequencing Kit on an automated sequencer (ABI3730XL, Applied Biosystems, USA). DNA sequences were assembled with Codoncode Aligner version 7.1.1 software (CodonCode Corporation). Nucleotide sequences were aligned using Clustal W (Larkin et al., 2007). The obtained sequences of the ITS-2 and nad4 genes were compared with the published reference sequences of H. contortus deposited in GenBank using NCBI BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Evolutionary analysis and phylogeny were conducted using the ITS-2 and nad4 gene in MEGA 7.0, with the reliability of the tree confirmed through bootstrap analysis with 1000 replicates (Kumar et al., 2016). The phylogenetic tree was constructed using the Tamura 3-Parameter model (Tamura, 1992), taking into account the host and country, with an appropriate outgroup included in the analysis.

Statistical analysis

The data collected was analyzed statistically to determine the correlation between the prevalence and sampling of animals. The data was recorded in a Microsoft Excel 2010 spreadsheet and analyzed using STATA version 11.0 software (Stata Corporation, 2009; website URL: http://www.stata.com). The Chi-square test was used to determine the significance of factors that are related to the infection. A multivariable logistic regression analysis was carried out to evaluate the impact of various variables on the results. The significance level was set at a 95% confidence interval and a P value of less than 0.05 (Schwartz, 1993).

Results and Discussion

Microscopic detection of H. contortus

We noticed that the female worm had a “barber’s pole” appearance to the naked eye. The average length and width of male worms were 10.65 mm and 0.22 mm respectively, while the average length and width of female worms were 21.33 mm and 0.48 mm respectively (as shown in Figure 1a). The body of the worm appeared slender and tapered at the anterior end in males and both ends in females under the microscope (Figure 1b). Overall, 46.5% of the total population were positive. Among them, H. contortus was found in 51.3% (77/150) of Sheep and 43.6% (109/250) of goats of the populations we studied (Table 1). The positive cases of H. contortus observed under the microscope were 51.3%, 43.6%, 50.8%, 42.5%, 47.3%, 44.3%, 41.9, and 75.0% in sheep, goat, young, adult, male, female, healthy, and anemic groups of animals respectively (as seen in Table 1).

H. contortus is a type of ruminant gastrointestinal nematode that typically resides in the real stomach. Out of all the Haemonchus parasites, H. contortus is considered the most common and pathogenic species (Troell et al., 2006). In order to establish successful control measures, it is crucial to have a complete understanding of the molecular epidemiology of the parasite. During the investigation, H. contortus was found in 46.5% of the small ruminants. Compared to previous reports from other countries, such as 55.5% in Benin (Attindehou et al., 2012), 78% in China’s Heilongjiang region (Wang et al., 2006), and 80.2% in Ethiopia (Fentahun and Luke, 2012), the infection rate in Bangladesh is relatively low. In the study area, sheep were more infected (51.3%) than goats (43.6%). This supports the findings of Fentahun and Luke (2012) who found that sheep had a higher prevalence rate (81.2%) than goats (73.5%). Factors such as close grazing, which increases exposure risk, poor hygiene, and contaminated wool in the perineal region, can all contribute to a higher prevalence of H. contortus in sheep.

 

Table 1: Detection of Haemonchus contortus by microscopic and PCR.

Variable

Categories

(Total No.)

Microscopic observation

PCR technique

No. infected

Percentage

No. infected

Percentage

Host

Sheep (150)

77

51.3

77

51.3

Goat (250)

109

43.6

109

43.6

Age

Young (191)

97

50.8

97

50.8

Adult (209)

89

42.5

89

42.5

Sex

Male (285)

135

47.3

135

47.3

Female (115)

51

44.3

51

44.3

Body condition

Healthy (344)

144

41.9

144

41.9

Anaemic (56)

42

75.0

42

75.0

Total

186

46.5

186

46.5

 

Identification of H. contortus by PCR

Out of 400 sheep and goats, 186 (46.5%) were found to be infected with H. contortus using PCR (Table 1). The amplicon length was determined by comparing it to known lengths of 321 bp for ITS-2 and 800 bp for nad4 gene on a 1.5% gel electrophoresis under UV light (Figure 2). The infection rate varied depending on the sample location, but in all slaughterhouses except SH-1, sheep had a higher infection rate than goats (Table 2). Sheep proved to be more susceptible to H. contortus with 51.3% (77/150) positive results, while goats had 43.8% (109/250) positive results (Table 1). The infection rate slightly differed when comparing age, sex, and health status, but there was no significant difference in the prevalence of H. contortus between young and adult animals, sex, or sheep and goat breeds (Table 3). However, the body condition of the animals (healthy or anaemic) greatly impacted the prevalence of H. contortus, with higher rates in anaemic sheep (70.8%) and goats (78.1%) (Table 3). No significant seasonal influence was observed (P = 0.543).

The prevalence in the coastal area was slightly higher at 58.6% compared to the hilly area (45.5%) and the plain area (35.3%). The rainy season posed a higher risk compared to other seasons, with a recorded prevalence of 69.9% during August, September, and October. In contrast, the prevalence was 45.5% during the summer months of April, May, and June. The lowest prevalence of 24% was observed in winter, specifically December, January, and February (Table 3). However, no significant associations (p > 0.05) were found between the prevalence of haemonchosis and both the environmental seasons and regions.

 

Table 2: Prevalence of Haemonchus contortus in different slaughterhouse (SH) at Chattogram.

Location

Slaughterhouse (SH)

Host

No. of

samples

No. of H. contortus positive

(%)

Jhautala

SH-1

Sheep

30

11

36.6

Goat

50

23

46.0

Firingebazar

SH-2

Sheep

30

16

53.3

Goat

50

21

42.0

Pahartali

SH-3

Sheep

30

15

50.0

Goat

50

24

48.0

Colonethat

SH-4

Sheep

30

12

40.0

Goat

50

19

38.0

Halishahar

SH-5

Sheep

30

23

76.6

Goat

50

22

44.0

Total

400

186

46.5

 

Table 3: Prevalence of Haemonchur contortus based on demographic characteristics (age, sex, breed, body condition, and seasonal influence).

Variable

Host

Group

No. examined

No. infected

Percentage (%)

P value

Age

Sheep

Young (< 1year)

38

26

68.4

0.100

Adult (> 1year)

112

51

45.5

Goat

Young (< 1year)

153

71

46.4

0.079

Adult (> 1year)

97

38

39.1

Sex

Sheep

Male

103

54

52.4

0.584

Female

47

23

48.9

Goat

Male

182

81

44.5

0.682

Female

68

28

41.1

Breed

Sheep

Local

150

77

51.3

0.158

Goat

Black Bengal

174

82

47.1

Jamunapari

49

18

36.7

Saanen

27

9

33.3

Body condition*

Sheep

Healthy

126

60

47.6

0.03

Anaemic

24

17

70.8

Goat

Healthy

218

84

38.5

0.03

Anaemic

32

25

78.1

Season

Winter

133

32

24.0

Rainy

133

93

69.9

0.543

Summer

134

61

45.5

Region

Coastal

133

78

58.6

Plain

133

47

35.3

0.664

Hilly

134

61

45.5

 

*Significant risk factor.

 

 

According to the results of the study, the prevalence of H. contortus was higher in young sheep (68.4%) and goats (46.4%) compared to adult sheep (45.5%) and goats (39.1%). Qamar et al. (2009) also found a similar pattern, with a slightly higher prevalence in young (39.9%) compared to adult (33.2%) small ruminants. This pattern has been reported in other studies as well (Gamble and Zajec, 1992; Colditz et al., 1996; Magona and Musisi, 2002). The higher prevalence in young animals is attributed to their lower level of immunity compared to adults who have built immunity through gradual exposure to these parasites. In this study, male sheep had a higher estimated prevalence of H. contortus compared to female sheep (52.4%), although this relationship was not statistically significant, unlike in some prior studies where female hosts had higher prevalence (Maqsood et al., 1996; Gauly et al., 2006). A study conducted in Benin (Attindehou et al., 2012) found almost identical occurrence of haemonchosis in both sexes (55.4% and 55.6%).

Furthermore, the local sheep breed (51.3%) and the Black Bengal goat breed (47.1%) were more susceptible to H. contortus than the Jamunapari (36.7%) and Saanen (33.3%) sheep breeds. In Bangladesh, the prevalence of haemonchosis was investigated using coproscopic examination and found to be higher in Black Bengal goats (57.1%) and other crossbred goats (55.8%), as reported by Nuruzzaman et al. (2012). However, in this study, none of the host-specific characteristics such as age, sex, or breed had a significant (p > 0.05) impact on the prevalence of H. contortus, which may occur in small ruminants due to the shared pastures and similar risk of infection for all (Attindehou et al., 2012). The body condition of the host was significantly associated with the parasitic status in this study. The prevalence of H. contortus was significantly higher in anemic sheep (70.8%) and goats (78.1%) compared to healthy sheep (47.6%) and goats (38.5%). In South Africa’s scarce areas, goats were examined for clinical anemia caused by Haemonchus parasites and found to be a health-threatening worm (Vatta et al., 2002). These findings were supported by Attindehou et al. (2012), who found that haemonchosis was present in 90.7% of anemic small ruminants and 36% of non-anemic small ruminants.

 

The prevalence of haemonchosis varied among different regions, with the highest recorded in the coastal region (58.6%), followed by the hilly region (45.5%) and the plain region (35.3%). The environmental conditions, such as relative humidity and ambient temperature, in the coastal areas may provide suitable conditions for the hatching of gravid eggs, resulting in the presence of infective larvae. Animals become infected when they ingest grasses containing these larvae during grazing. In this study, the burden of haemonchosis exhibited significant seasonal variation. The infection intensity was higher during the rainy season (69.9%), followed by the summer season (45.5%) and the winter season (24%). These findings align with a study by Attindehou et al. (2012), which reported a higher prevalence during the rainy season (79.4%) compared to the dry season (36%). Adequate rainfall and moisture during the rainy season may enhance larval development and survival on the grazing lands, resulting in an increased occurrence of haemonchosis. Conversely, the winter season, characterized by suboptimal temperature and moisture, may induce hypobiosis, leading to a decreased number of hatched gravid eggs. These factors likely contribute to the lower prevalence of infection during the dry season.

Sequence and phylogenetic analyses

A total of 45 nad4 (25 goats and 20 sheep) and 10 ITS-2 samples with positive results were selected for sequencing analysis. Samples with high-quality DNA were preferred for sequencing to minimize errors and improve accuracy. We successfully obtained 800-bp nad4 and 321-bp ITS-2 nuclear rDNA sequences through sequencing and alignment analysis. The genetic analysis revealed 45 different nad4 genotypes among the H. contortus sequences and found that these sequences showed 98.5-100% identity with reference sequences in the GenBank database. Then constructed the phylogenetic tree by using the Maximum Likelihood (ML) method with identified 45 nad4 gene sequences (Figure 3). The tree was constructed using three sequences from each nation (China, Pakistan, Yemen, Italy, and the United States) and five randomly selected sequences from the study. The ML method, combined with bootstrap analysis using 1000 replicates, revealed major clades with over 50% nodal support, using Haemonchus placei (AF070820) as the outgroup. Most isolates from the same continent clustered together, but one Yemeni isolate merged with US isolates, and the Bangladeshi isolates formed a group with a nearly identical Chinese isolate. The Pakistani and Chinese isolates also formed the same cluster with strong nodal support.

A 321-bp product of the H. contortus ITS-2 gene was obtained and ten genotypes were identified with a similarity range of 99.8-100% among the sequences analyzed. A phylogenetic tree was constructed using 25 randomly selected ITS-2 gene sequences and the Maximum Likelihood (ML) method (Figure 4). Five sequences were randomly selected from each nation (Bangladesh, China, Pakistan, India, and Thailand). The dendrogram showed that the H. contortus haplotypes were randomly spread among haplotypes from different countries, with significant nodal support of 66%. No unique relationships were found in the analysis of country phylogenetics. The representative sequences from this study were submitted to the DNA Data Bank of Japan and the European Nucleotide Archive (ENA), with the accession numbers: H. contortus nad4, KY031955-KY031964, KY041808-KY041842; H. contortus ITS-2, KU558755-KU558759.

 

The evolutionary relationships between sheep and goats were observed using a phylogenetic tree (Figure 3) based on 5 nad4 haplotype sequences. There was no clear grouping between the two populations of H. contortus, supporting the lack of host specificity reported by Gharamah et al. (2012). Further analysis (Figure 4) based on 5 ITS-2 sequences showed no distinct grouping of H. contortus among the host populations. Dey et al. (2019) reported the absence of phylogeographic structuring among H. contortus populations from sheep and goats in seven geographical areas in Bangladesh, suggesting that multiple hosts share common grazing pastures. Significant gene flow between populations may also result from animal movements (Shen et al., 2017). Thus, further research is needed to better understand the molecular properties of H. contortus. The tree revealed low genetic variation in the H. contortus population in Bangladesh, echoing the findings in Malaysia (Gharamah et al., 2012), where H. contortus isolated from Malaysia and Yemen formed distinct clades with strong gene flow and limited genetic variation. In conclusion, the study highlights the presence of H. contortus infection in slaughterhouse animals in Bangladesh.

The limitation of this study was the limited availability of high-quality samples for selection in sequencing. Furthermore, we preferred samples with high-quality DNA that were free of contaminants and had a high yield for sequencing. The cost of sequencing also influenced sample selection, as sequencing, all available samples may have been too expensive.

Regarding the economic impact of Haemonchosis, the disease can lead to reduced productivity in livestock, including decreased growth rates, decreased milk production, and even death in severe cases. This can have significant economic implications for the agricultural sector in Bangladesh where livestock farming is an important source of income and food. In Bangladesh, where agriculture and livestock farming are vital to the economy and the livelihoods of many people, the economic impact of haemonchosis could be substantial. Infected animals may require medical treatment, which can incur costs for farmers in terms of veterinary services and medications. Additionally, reduced productivity and potential losses due to mortality can affect the income and food security of farmers who rely on their livestock.

Conclusions and Recommendations

It may be concluded that the total prevalence of H. contortus in small ruminants in the research area was alarmingly high, but the causes for this high incidence could not be determined in this investigation due to the lack of significant relationship among the majority of parameters studied. During the study of the nad4 gene, only minor genetic variants were found. This study recovered DNA sequences of H. contortus from a slaughterhouse in Chattogram, Bangladesh for the first time and could serve as a starting point for further investigations aimed at developing control strategies for H. contortus in the country. A farm-based extended study may be necessary in the future to uncover other suspected causes.

Acknowledgements

The authors express cordial thanks Dr Shama Rangan Barua and Dr Omer Faruk from department of livestock service (DLS) for their help during the research work. Thanks Dr Saiful Bari and Dr Abdul Mannan from Chittagong Veterinary and Animal Sciences University (CVASU), Bangladesh for statistical knowledge of data analysis. The funding from University Grants Commission (UGC-HEQEP, subproject CP 3220) of Bangladesh was cordially acknowledged for financial support during this research work.

Novelty Statement

This study presents a unique exploration into the prevalence and genetic diversity of Haemonchus contortus, a parasitic nematode that affects small ruminants in the Chattogram Metropolitan area. This research contributes to the understanding of parasitic infections in this region, shedding light on the specific strains of Haemonchus contortus present and their potential impact on livestock health. Furthermore, it lays the foundation for the development of targeted control and prevention strategies, with potential implications for the livestock industry and overall agricultural sustainability in Bangladesh.

Author’s Contribution

Muhammad Abdul Mannan: Conceptualization, methodology, data curation, software, formal analysis, writing-original draft.

Sharmin Chowdhury: Data curation, investigation, funding acquisition.

Md Abul Hashem: Formal analysis, writing, review and editing.

Md Hazzaz Bin Kabir: Formal analysis, software, writing, review and editing, Supervision.

Ethics statement

This study was approved by the ethical committee of Chattogram Veterinary and Animal Sciences University authority (ethical approval number was CVASU- PAPA: 0007.2014-15/02) and the Chattogram City Corporation, Bangladesh.

Conflict of interest

The authors have declared no conflict of interest.

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