Morphometry and Molecular Identification of Haemonchus Cobb, 1898 (Trichostrongylidae: Nematoda) Isolates from Small Ruminants in Tanzania Based on Mitochondrial cox 1 and rRNA-ITS genes

The genus Haemonchus is the major abomasal parasite of ruminants responsible for substantial economic losses in tropical and temperate regions. This study was conducted to clarify the morphometry and molecular characterisation of Haemonchus species isolated from sheep in Babati district, Tanzania. A total of 486 trichostrongylid nematodes were recovered from five sheep. Of the total worms, 106 nematodes were distinguished by 37 males and 69 females. The asymmetrical length of dorsal ray and the distance of bulb at the apex of spicules were used for identification of males. In females, the linguiform vulvar flap was the most predominant with 33 out of 69 (48%) compared with knobbed morph type which was 25/69 (36%) and smooth morph type with 11/69 (16%). Partial cox1 sequence fragments of Haemonchus contortus isolates showed 98.8%, 99.3%, 99.7%, 99.5%, 99.3%, and 98.4% in male, smooth, knobbed, linguiform A, linguiform B, and linguiform C, respectively; with the average nucleotide divergence ranged from 1.03 to 2.35%. The amplified fragments of ITS-2 genes in knobbed, linguiform A, and smooth morphotypes revealed 99.4%, 98.5%, and 98.3%, respectively. Phylogenetic analysis was evaluated by employing Bayesian inference and maximum-likelihood, and the tree was distinctly separated into three clusters focusing on H. contortus in cluster I within the family Haemonchidae. Genetic drifting, mutation, and modification of the morphological features of the Haemonchus species described to have an impact on the development of drug resistance. Species identification is necessary to understand which species infect animal host. We recommend more studies on the parasites intensity and the strategies for controlling Haemonchus species in Tanzania.


Introduction
The genus Haemonchus (Trichostrongylidae) has bloodsucking nematodes of domestic and wild ruminants. The adult worm is cylindrical in shape and yellowish in colour with alternating red and white barber-pole; Male spicules have bulb shaped blunt pointed anterior end [1]. The Haemonchus species are potential harmful and significant threat in tropical, subtropical, and warm temperate regions [2]. The genus is orally transmitted to all classes of ruminants by consuming contaminated pastures with L3 infective larvae that develop into adult worm whilst burrow into the internal layer of the abomasum [3]. The adult worm feeds on the host's blood in the abomasum causing anaemia and oedema, which reduce animal production and ultimate deaths [4].
Synlophe patterns, spicules morphometry, vulvar flap, and cervical papillae are important morphological features in the identification of Haemonchus species [12]. In addition, the host feeding habit [13], host immunity [14], host strain [15], host gender [16], and coexisting parasitic infection described to manipulate their morphological features [17]. The clarification of Haemonchus species by morphological features is still debatable, which demand the use of molecular techniques to unveil specific and intraspecific variations associated with their geographical distribution [18]. The investigation on nematodes through nucleus and mitochondrial deoxyribonucleic acid (DNA) provides genetic information on the infection of subpopulation of a parasitic species to the host species [19].
H. contortus, H. placei, and H. similis have been reported in domesticated ruminants [20], with limited clarification on their morphological features in Tanzania. Regardless of developing low-cost molecular tools for investigating Haemonchus species with other trichostrongylid nematodes [21], there is no clear description on the morphology and phylogenetic information of Haemonchus species isolated from sheep and other small ruminants in Tanzania.
Therefore, this study was taken to clarify the identification of Haemonchus species based on morphology and genetics by using mitochondrial cytochrome oxidase subunit 1 gene (cox 1) and the ribosomal RNA-internal transcribed spacer (rRNA-ITS).

Study Area and Animal Selection.
This study was conducted in three villages namely Kiru, Ayasanda, and Magugu villages in Babati district located in Manyara region. Babati district is confined between 3°and 4°South and 35°and 36°East [22]. Five sheep were selected randomly from the selected villages from June 2020 to January 2021. The selected animals were 8-10 months old considering the choice of young animals and those that have not been recently dewormed [23].

Climate
Condition of the Area. Warm-summer Mediterranean and tropical climate is mostly dominated in the area.
The warm season lasts for 5.5 months, from October to March, with an average daily temperature above 27°C [22]. The cool season lasts from June to August, with an average daily temperature below 23°C. The rainy period of the year lasts for 7.3 months, from October to May, whereby the high rainfall period begins from March and ends in May. The dry period lasts for 4.7 months, from May to October [24].
2.3. Animal Processing. The isolation of worms was conducted based on Hansen and Perry [23] and Maff [25] with some modification. The gastrointestinal tracts of selected sheep were double ligated at the abattoir into three sections to prevent mixing of the abomasal, small intestinal, and large intestinal contents. Each tract was placed in a bucket, labelled before being processed. The nematode worms were isolated, counted, and placed in collecting tubes containing 70% ethanol for molecular studies and 10% formalin for morphological studies [23].

Morphological Identification of Haemonchus Species.
Prior to identification, the nematode worms were washed in normal saline to free them from mucus and then fixed in hot 70% ethanol. Dorsal ray and spicules were considered as important key for identification for the male worms. The posterior parts of the male worms were cut before the bursa and mounted in lactophenol for clear examination of the spicules under a microscope [26]. For the female worms, the cuticular process and vulvar shapes were examined based on Rose [27] and Le Jambre and Whitlock [28] procedures.

Molecular Identification of Haemonchus Species
2.5.1. PCR and DNA Sequencing. Collected nematodes were sorted by grouping parasites per host species. Individual nematode was selected from each host group and washed in Phosphate buffered saline (PBS). Prior to the extraction, worms were ground with a pestle in adaptive transport layer buffer and Proteinase K in 1.5 ml microcentrifuge tube and allowed to melt into suspension overnight at 56°C. The incubated nematode sample was extracted using QIAamp DNA mini-Kit following the manufacturer's procedures (Qiagen, Valencia, CA, USA). Genomic DNA was dissolved in 50 μl of Tris-EDTA (TE) buffer (10 mM Tris/1 mM Ethylenediaminetetraacetic Acid (EDTA)). The mitochondrial cox 1 fragment was amplified by primers JB3 (5 ′ -TTT TTT GGG CAT CCT GAG GTT TAT-3 ′ ) and JB4.5 (5 ′ -TAA AGA AAG AAC ATA ATG AAA ATG-3 ′ ) [29]. The ITS gene primers NC2 (TTA GTT TCT TTT CCT CCG CT) and NC5 (GTA GGT GAA CCT GCG GAA GGA TCA TT) were used for amplification of rRNA-ITS genes [30]. The polymerase chain reaction (PCR) amplification was performed using 50 ng of genomic DNA template in 25 μl reaction mixtures consisting of 1 μl of each primer (10 pmol), 1 μl of generic DNA, 2.5 μl of 10× buffer [200 mM Tris-HCl (pH 8.4) and 500 mM KCl], 12.25 μl of 2× buffer (MgCl 2 , dNTP) and 1.25 units Taq polymerase (Takara Bio Inc. ® , Kusatsu, Shiga, Japan), and 6 μl of distilled water.
The genomic DNA was initially denaturized at 95°C for 2 minutes followed by 30 cycles of denaturation at 95°C for 60 seconds, annealing at 55°C for 60 seconds, and extension at 72°C for 60 seconds with a final extension at 72°C for 7 minutes. PCR condition for ITS-2 region was 3 minutes at 95°C, over 35 cycles of 1 minute at 95°C, 1 minute at 58°C, 60 seconds at 72°C, and a final extension step of 10 minutes at 72°C. Phylogenetic analysis was evaluated by employing Bayesian inference (BI) and maximum-likelihood (ML) using the partial sequences of cox 1/ITS 1 in the Molecular Evolution Genetics Analysis (MEGA) software version 7.0 [31]. The HKY+G substitution model was used for sampling of cox1 and ITS sequences. BI analyses were used in the Bayesian Evolutionary Analysis Sampling Trees program version 1.10.4 [32]. The HKY substitution model sampling was chosen according to the MEGA. The nodes were assessed by bootstrapping with 1,000 pseudoreplicates.

Results
A total of 486 trichostrongylid nematodes were recovered from five sheep in Babati district abattoir. Of the total worms recovered, 106 nematodes were distinguished by 37 males and 69 females. The high number of parasites recovered in abomasa were similar size except those from female sheep observed to be larger than in the male sheep. The faint pale-yellowish and slender nematodes tapering towards both ends with alternating red and white "barber-pole" in female 3 Journal of Parasitology Research were observed, whereas the filiform in the anterior parts of the males and bursal lobes in the posterior part were observed. The buccal cavity was relatively small without buccal capsules; rather there is pronounced dorsal lancet protracted from dorsal wall (Figure 1). Both male and female worms had cervical papillae with spine structure-like protruding outside more or less parallel to longitudinal cuticular ridges, with highly visible transverse striations on the body (Figure 1).
The morphmetic features of female Haemonchus contortus associated with the vulvar position with or without prominent features along its structure. The linguiform vulvar flap was the most predominant count for 33 out of 69 (48%) compared with knobbed females with 25/69 (36%) and smoothed females that count for 11/69 (16%; Figure 2). In addition, the linguiform vulvar flap also could be categorized into three subtypes the linguiform A, B, and C ( Figure 2). For the purpose of this study, the linguiform A is the vulvar morph type with one cuticular inflation; linguiform B is the vulvar flap with no cuticular inflation; and linguiform C is the vulvar flap with cuticular inflation raised from the linguiform process ( Figure 2).  Journal of Parasitology Research vestibular. The vulvar flap is well demarcated with or without linguiform processes ( Figure 2; Table 2). The tail is long without projectory spine with simple dorsal and muscular ventral rims developed along the anal pore at the posterior end of the tail (Figure 1).

3.2.
Male. The filiform males were small compared with females, with an average length of 14,039 μm and width of 298 μm. The cervical papillae are 210 μm from the anterior end along with the oesophagus with a length of 1,232 μm; in the posterior end, the male bursa revealed elongated lobes with long and muscular slender rays of variations in their lobes ( Figure 3). The dorsal ray is asymmetrically branched to its length. Each branch divides again approximately at the end of its length giving rise to a short external branch node parallel to the long branches projecting to the dorsal lobe margin. The two spicules measure 434 μm with small bulb and pore near its end supported by gubernaculum, which is spindle shaped in dorsal view narrowing to the posterior extremity measuring 212 μm (Figure 3; Table 1).

Molecular Descriptions.
Phylogenetic analysis was determined by the ML and BI. The phylogenetic tree was distinctly separated into three clusters (Figure 4) (Table 3). The nucleotide identities between the Haemonchus species were revealed by percentage composition of 98.6-99.7%.  Figure 5). The sequences were deposited in Genbank with accession numbers OK181226, OK181227, and OK181228 for knobbed, linguiform A, and smooth morph type, respectively.

Discussion
Morphological study has been widely used for understanding the biology, population, and ecological adaptation of Haemonchus species infestation in the small ruminants   Journal of Parasitology Research [28]. The main focus is to reduce the burden of worms in the gastrointestinal tract of small ruminants by rescinding morphological features, such as length and width of the worm, cervical papillae, vulvar flap, spicule, and gubernaculum length [36]. The asymmetrical length of dorsal ray with branch nodes towards the dorsal lobe margin and the distance of bulb at the apex of spicules were important structures for the identification of males. The comparative study of the female morph types, such as knobbed, smooth, and linguiform, is related to geographical locations and population density of the Haemonchus species [37,38]. In addition, the presence of morph types with additional inflations in females is associated with the multiple phenotypic expression caused by adaptive characteristics of the Haemonchus species based on environmental factors [39]. We provide the phylogenetic relations among the linguiform and other morphometric types observed in this study. Linguiform was highly observed compared with other morph types possibly caused by the dominance modifiers that differ from locality [40]. It is still uncertain to conclude the significance of phenotypes as genetic markers due to limited research illustrating the dominance hierarchy of Haemonchus species in Tanzania. The availability and use of genetic information are vital tool for the management of parasitic diseases for easy identification of specific parasite infecting the host [41]. The molecular information works as promising outstanding markers for species description and determination of genetic population. For instance, the use of ITS genes as one of the most variable nuclear loci for evolutionary studies becomes more important in discriminating nematode species [42,43]. The use of cox 1 and ITS-2 with some portion of 5.8S sequences in this study provides a significant step in discriminating H. contortus especially in Tanzania.   The variability of H. contortus in the present study is associated with the area of study that the sheep were selected randomly in the population, which were not recently dewormed and shared the same pastures with other ruminants, and the timing of sampling, which is associated with the transmission risks (June to January) similar to what has been proposed in previous studies [19,42]. In addition, the use of cox 1 and ITS-2 with some portion of 5.8s, which are stable conserved regions among the trichostrongyloid genomes, revealed the clear relationship among the monophyletic in Haemonchidae. The present study showed a high percentage of similarity with Haemonchus species that was confirmed from previous study to contribute to the development of haemonchosis. The present study is signifying that the comparative sequence analysis of Haemonchus species is a powerful tool for inferring the function cox1 and ITS-2 with portion of 5.8s as the novel functional genes for identifying Haemonchus species. During sampling, dewormed sheep were selected randomly in the same pastures with other ruminants and the timing of sampling was associated with the transmission risks (June to January) similar to what has been proposed in previous studies [44][45][46] on the comparison of Haemonchidae and Molineidae families to see the genetic relationship on the site of infection that H. contortus infect abomasum are closer to intestine parasites. The present study is emphasising on the use of cox 1 sequences due to its high affinity in the accumulation of substitutions more than ITS genes. However, the use of ITS genes has significant importance on the quick distinguishing between known species (lower level of intraspecific polymorphism) [19].

Conclusion
The continue persistence of Haemonchus is related to the genetic drifting, mutation, and modification of their morphological features described to have impact on the development of drug resistance. Species identification is necessary to understand which species infect animal host [46]. The use of molecular marker in identification could provide more information on the genetic variation for the species that could provide more information on the treatment procedures include drug discovery. Therefore, we recommend more studies on the parasites intensity and the strategies for controlling Haemonchus species in Tanzania.

Data Availability
Nematodes samples and Genomic DNA were stored in Tanzania Parasite Resource Bank and the International Parasite Resource Bank (iPRB) in Korea and obtainable on special request. All sequences from the Haemonchus contortus isolates from sheep were deposited in GenBank with accession numbers OK236357, OK178541, OK178542, OK178543, OK236356, and OK236358 for the male, knobbed morph type, linguiform A, linguiform B, smooth morph type, and linguiform C, respectively. In addition, the accession numbers OK181226, OK181227, and OK181228 for knobbed, linguiform A, and smooth morph type, respectively, can be retrieved.

Disclosure
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflicts of Interest
The author(s) declare(s) that they have no conflicts of interest.