Peripheral Blood Mononuclear Cells Regulated Cytokines and Transcription Factors Reduce Haemonchus contortus Larval Establishment in Black Bengal Goat

Peripheral Blood Mononuclear Cells Regulated Cytokines and Transcription Factors Reduce Haemonchus contortus Larval Establishment in Black Bengal Goat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Peripheral Blood Mononuclear Cells Regulated Cytokines and Transcription Factors Reduce Haemonchus contortus Larval Establishment in Black Bengal Goat Nurnabi Ahmed, Babul Chandra Roy, Amitav Biswas, Md. Rabbi, Md. Sajib, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4620150/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Black Bengal goat (BBG) is a native breed of Bangladesh and its ability in reducing the establishment of H. contortus infection through cellular and immune responses was unexplored. In vitro larval motility in response to PBMC, in vivo larval infectivity assay, differential blood cell counts, histopathology, cytokine and transcription expression have been investigated in BBG kids following H. contortus L 3 . In vitro motility experiment revealed that L 3 exposed to PBMC from primed kids had significantly (P < 0.05) reduced speed and straight-line distance compared to L 3 exposed to PBMC from naive kids. Kids with L 3 exposed to PBMC in primed and naive kids, reduced H. contortus egg shed. Upon 10,000 L 3 challenge, WBC count was higher in primed kids compared to naive and 35% of TC were neutrophils. Lymph nodes were increased in weight (P < 0.05) in primed kids except 1st day (D0). IL-4, IL-5, IL-13, IL-33, MCP1, CXCL1, TLR2 and GAL14 were expressed both naive and primed kids. Significant expression of IL-4, IL-5, IL-13, IL-33, MCP1 and CXCL1 at D3, D5 and D7, suggesting early T H 2 differentiation in primed kids. These results suggest that BBG can much resist H. contortus infectivity. Role of transcriptome differences in other resistant breeds should be investigated. Biological sciences/Cell biology Biological sciences/Genetics Biological sciences/Immunology Biological sciences/Molecular biology Health sciences/Diseases Health sciences/Gastroenterology Black Bengal goat PBMC H. contortus Resistance Cytokines TH2 Bangladesh Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Haemonchus contortus , is a voracious blood sucker trichostrongylid also known by its trivial name as barber's pool worm and considered the most pathogenic nematode due to its impact on small ruminant production worldwide 1,2 . This parasite causes severe anaemia, hypoprotenemia, oedema, and diarrhoea, which leads to significant morbidity and mortality sheep and goat farms located in tropical and subtropical climates 3,4 . Due to high prevalence (~ 80%), farmers are currently facing a severe challenge with this highly fecund parasite control measures 5 . Treatments rely on just a handful uses of anthelmintic drugs from the same class of drugs, such as benzimidazoles like albendazole and levamisole 3,6,7 . Over- and misuse of these anthelmintics causes strong selection pressures to the parasite 8 , which leads to the evolution and development of anthelmintic resistance (AR). Thus, the need for alternative and sustainable control strategies, such as exploration and selection of genetically resistant animal and the development of immunotherapeutic or imunoprophylactic tools, has increased dramatically 9 . In light of the aforementioned information, numerous independent studies have already been carried out to select resistant sheep breed in different countries, and it is shown that the immunity to this gastrointestinal nematode is primarily controlled by polarised helper T cell 1 (T H 1) and helper T cell (T H 2) responses that are directed towards the parasites' larval stages 10–13 . Resistance status against H. contortus were characterized by the participation of globular leucocytes, eosinophils, mast cells, and antibody production at the site of infection, as well as the type-2 hallmark cytokines produced by PBMC upon parasite stimuli such as IL-4, IL-5, IL-13 and IL-33 either in the peripheral blood or in the abomasum tissues or draining lymph nodes 14–20 . On the other hand, the T H 1 response is related to susceptibility and the induction of pro-inflammatory immune genes such as tumor necrosis factor (TNF), interferon gamma (IFN-γ) and IL-6, these changes contribute to nematode persistence 21 . In Mexico, it has been reported that Pelibuey sheep show resistance to GINs with low H. contortus EPG count and increased IL-4, IL-5, IL-10 and IL-13 and Fc fragment of IgE receptor Ia (FCER1A) gene expression 15 . It has been revealed that increased luminal flow coupled with intestinal muscle driven by IL-4 and IL-13 cytokines reduced parasite establishment 22 . H. contortus resistant sheep breed PBMC upregulated several germline-encoded receptors and T H 2-promoting transcription factors such as GATA-3, repressing the transcription of the pro-inflammatory cytokine IL-12 23 . Resistance traits observed in St. Croix sheep were associated with IL-4 up regulation and decreased H. contortus infection from the third day of post-infection 24 . The development of the lymphnode (LN) is one of the best physiological distinctions between susceptible and resistant animals against H. contortus 25 . Within five days of an initial H. contortus infection in resistant sheep, eosinophils, CD4 + T-cells, and B-cells in the abomasal mucosa rose, as did the size of the local lymph nodes 26 . In resistant sheep breeds, a new role for monocytes and nuetrophil in H. contortus infections were reported 22,27 . When Nippostrongylus brasiliensis and Heligmosomoides polygyrus infections were present in mice, neutrophils underwent phenotypic polarisation as a result of IL-4R signalling 18 . They also produced IL-13, which encouraged the differentiation of alternatively-activated macrophages, and they demonstrated a greater involvement in the primary response, helping to supply early T H 2 cytokines to support the T H 2 differentiation of adaptive immune cells. Neutrophils have been demonstrated to bind, form extracellular traps, and induce morbidity to larvae (Hc L 3 ) in vitro 10,22,28,29 . The early response of resistant sheep monocytes accelerates T H 2 immune activation to helminth larval stages reduce adult establishment and fecundity leading to reduced infection rates and pathogenicity 22,27 . In the first seven days of H. contortus infection, researchers found a significant increase in cellular infiltrate, notably eosinophil and neutrophil, when comparing abomasal tissue from resistant sheep to susceptible sheep 30 . The Black Bengal goat (BBG) is a native breed in Bangladesh that is well-known throughout the nation and around the world for its high-quality meat, milk, and skin 31 . The goat has a significant economic impact in the rural community of the country as it can produce four to eight offspring annually which helps to lower the unemployment and poverty 32 . This Black Bengal goat has been recently described as H. contortus resistant breed by exploring single nucleotide polymorphism in genes such as CIITA , ATP2A3 , HSPA8 , SERPING1 , STAT5B and ESYT1 which were responsible for lowering fecal egg count and expulsion of this parasite from the gastrointestinal track 33,34 . In sheep and goat, it has been also demonstrated recently that the early response to infection is a critical part of the protective host-immune response within seven days 12,13,35 . However, the distinction between naïve and primed BBG goat kids in response to GIN infection, as well as the underlying mechanisms, have not given enough attention. In order to determine the level of resistance, in vitro and in vivo experiment's goals were to investigate the mechanisms of how PBMC regulate immune response against infective stage larvae of H. contortus in BBG. Therefore, in vitro larval motility in response to PBMC, in vivo larval infectivity assay, differential blood cell counts, histopathology of abomasa and lymphnodes (LN), specific cytokine and transcription factors expression associated with resistance have been investigated in BBG kids following challenge H. contortus L 3 infection. Results Larval count Following oral administration of H. contortus 10,000 L 3 , number of L 4 retrieved from primed kids' abomasum was significantly (p<0.01) lower compared to naive kids at D7 (Figure 2 A). The average number of L 4 found in the primed and naive kids was 3,137 and 7,612. In neither group, any other stage of larval development was seen. Haematology There was a significant (p<0.05) variation in the mean PCV value between the primed and naïve group kids across all study time points. Compared to naïve kids, primed kids had greater PCV (Figure 2 B). There were statistically significant (p<0.05) differences in the mean and standard deviation of WBC, monocyte, eosinophil, and neutrophil between naïve and primed kids at D3, D5 and D7; however, there were no significant differences in the counts of lymphocytes and basophils at any time point in this study (Table 1). Lymphnode development Every slaughtered kid had all of its palpable abomasal lymph nodes (LN) counted and weighed. Figure 3A shows that there were no variations in LN counts between primed and naïve kids. Nevertheless, there were a notable variation in the LN numbers at D0, D3, D5, and D7. On D5 and D7, there was a significant difference (p<0.05) between naive and primed kids, although LN weight rose exponentially in both groups (Figure 3B). When comparing the weight variations of primed and naive kids following infection, the former experienced a three-fold rise in response of 4.8 g and the latter only showed one-and-a-half-fold increase of weight 1.4 g. Immune cell infiltration in abomasal mucosa Primed kids showed a higher abundance of neutrophils and eosinophils in the mucosal epithelium and submucosa compared to naïve kids (Figure 4). Globule leukocytes were present from the first day upon infection and counts rose in both kinds by D3 (p< 0.05) and continued to rise through the D7 (Figure 4.A). But there was no difference in globule leukocyte counts between primed kids and naive kids. Figure 4.B shows an exponential and significant increase in eosinophil counts to D7 (p<0.01). A deeper look revealed that neutrophils comprised a significant percentage of the cellular infiltration into the abomasal mucosa (Figure 4.C). Expression of cytokine and transcription factors in abomasal lymphnode Gene expression study in the abomasal lymph nodes revealed differences in cytokine and transcription factors expression bewteen naive and primed kids where day had a significant effect on these genes (Figure 5). IL-4 expression increased by D3 (p<0.05) in both naive and primed kids and was maintained until D7. The maximum expression was observed at D5 (p<0.01) in both groups. Primed kids exhibited increased IL-4 expression (5.7-fold vs 3.9-fold, p<0.05), indicating the beginning of T H 2 polarisation (Figure 5. A). IL-13 expression was confirmed from D0 to D7 in both group kids, with the highest expression observed at D5 (p<0.01) but no difference between naïve and primed kids (Figure 5.B). The lack of differential IL-12p40 expression contributes to T H 2 polarisation and indicates that the T H 1 response is down-regulated in both naïve and primed kids. The expression of transcription factor also contributes to T H 2 polarisation. The presence of GATA-3 from D0 to D7, with the greatest expression at D5, validated T H 2 polarisation. Primed kids showed higher expression of GATA-3 at D3 and D5 compared to naïve kids, indicating early T H 2 polarisation in primed kids (Figure 5.C). However, there was no significant increase in of GATA-3 was observed. Expression of T-bet gene decreased significantly from D5 to D7 (p< 0.05), indicating suppression of T H 1 at D7 (Figure 5.D). Gene expression in the abomasal mucosa IL-4, IL-5, IL-13, IL-33, GAL14, MCP1, CXCL1, and TLR-2 were expressed in the abomasal mucosa of the naïve and primed kids across all study time points (Figure 6). In both primed and naïve kids, significant differences were noted in the IL-4 gene expression at D5 (p<0.05) and D7 (p<0.01) in comparison to D0 (Figure 6A). After seven days of infection, the expression of IL-4 gene rose by over 16-fold in naïve kids and nearly 30-fold in primed kids. At D7, IL-5 gene expression was increased approximately 4-fold in naïve kids and 7-fold in primed kids (Figure 6 B). IL-13 and IL-33 were expressed from D0 to D7 and highest expression were recorded at D3 (Figure 6 C & D). From D3, expression of both cytokines was gradually decreased. Significant differences within groups were seen at D3 and D5 in naïve and primed kids compared to D0. The levels of significance for IL-13 and IL-33 in primed and naïve kids at D3 were different from those at D0 (p< 0.05 and p<0.01, respectively), although they both substantially declined from D3 to D5 and D7 (p<0.05). Between groups, significant difference was only seen at D3 (p<0.05) indicating more T H 2 polarisation in primed kid. In both primed and naïve kids, the expression and upregulation of GAL14, MCP1, CXCL1, and TLR2 genes were seen from D0 to D7. Primed kids showed approximately 1.5 times greater GAL14 expression at D5 and D7 compared to naïve kids and greatest expression were seen at D7 for naive kids while D7 for primed kids (Figure 6 E). MCP1 expression was also increased in naive and primed kids as day of infection progressed but greatest expression of MCP1 was observed at D7 in naive kids and interestingly similar fold changes were observed at D5 in primed kids indicating the activation of alternatively macrophage, which are the initial source of IL-4 in parasite infections. But highest expression of MCP1 in primed kids was recorded at D7. Between groups, significant amount of MCP1 expression were observed at D7 (6 vs. 12, p<0.01) in prime kids compared to naive kids (Figure 6 F). Similar findings were observed for CXCL1 (Figure 6 G) and TLR-2 (Figure 6 H) expression in the abomasal mucosa of naive and primed kids. There was a huge influx of CXCL1 and TLR-2 were observed from D5 both in naive and primed kids but the greatest expression for this gene were recorded at D7 though the level of expresseion of CXCL1 and TLR-2 of D7 in naive kids was similar to D5 in primed kids. Between naive and primed kids, significant differences in fold changes were only observed at D7 (TLR-2, 3 vs. 5.5, p<0.05; CXCL1, 10 vs. 13.3, p<0.05). In vitro larval motility The tracking data showed that, among study groups, levamisole treated larvae had lowest velocity and travelled the least straight-line distance than any other groups larvae. Furthermore, larvae exposed to primed kids PBMC had significantly (p<0.05) decreased velocity and straight-line distance compared to larvae exposed to naive and control goat kids PBMC (Figure 7 A & B). The average speed and straight-line distance covered by larvae exposed to primed kid's PBMC was 45.5 ± 6.9 μm and 56 ± 12 μm while larvae exposed to naïve PBMC was 16 ±2.5 μm and 13±1.3 μm. Larval infectivity following exposure to PBMC from naïve and primed kids To investigate larval infectivity in Black Bengal kid's, 5,000 L 3 treated with primed, naive and control groups PBMC were administered orally to kids of control group A, B and C. By week 5 following infection, differences were observed in FEC where group A kids shed an average of 2200 egg per gram (EPG) whereas the group B and C shed 3320 EPG and 7,100 EPG respectively (Figure 7 C). By week, the experiment was terminated. Group A kids shed then an average of 950 egg per gram (EPG) whereas the group B and C shed 2,560 EPG and 4,060 EPG respectively. Discussions In the present study, we investigated the cellular and immune response of BBG kids regarding H. contortus larval (L 3 ) infection for first seven days. We compared specific cytokines and transcription factors expression associated with H. contortus infection between naïve and primed BBG kids also. The present data demonstrated that following oral administration of 10,000 H. contortus L 3 to naive and primed kids, the number of larvae established into the abomasum of primed kids were markedly lower at 7 days (D7) compared to naïve kids. The finding indicates the development of mechanism specific to early-stage L 3 abolition after repeated infections. A similar study was conducted recently to detect early transcriptome differences between naïve and pre-infected goat kids and they showed that double timed challenged pre-infected H. contortus resistant (rHc) Creole goat kids had less larval count compared to single timed challenged naive kids after seven days of infection 10 . In this study, primed kids had higher mean PCV compared to naïve kids (41.5% vs 37.01%) indicating establishment of more protection during second exposure compared to first time exposure to H. contortus . The finding is in line with previous study where similar findings were observed between challenged and controlled rHc St. Croix hair sheep where mean PCV was 40 and 35 during 10 days infection 30 . In another study, Bambou et al. reported that in rHc Creole kids had higher mean PCV during second exposure (challenge 2) compared to primary infection (challenge 1) 50 . Again, current study finding is in accordance with previous study performed in the same animal model showed that, rHc goats produced better degree of protection in repeated exposure compared to single timed exposure 51 . Circulating total white blood cell count was found relatively higher in primed kids compared to naive kids across all study time points in this study. The present findings are in line with previous study demonstrated the differences in WBC count between resistant St. Croix and susceptible wool sheep at D0, D3, D5 and D7 and St. Croix sheep showed similar increasing trend of WBCs against H. contortus L 3 as Black Bengal kids showed 30 . Differential cell counts also revealed that activation of eosinophil in naive and primed kids started from same day but count was relatively higher in primed kids compared to naive kids. The present findings are in accordance with previous findings where increased eosinophils counts were observed in gastrointestinal track parasite (GTP) resistant breed by 3 days after infection compared to susceptible goat breeds 25 and mice model revealed that early activation of eosinophil happened in repeated exoposure and it was indicative by greater IL-5 expression 52 . The substantial contribution of neutrophils to total WBC in the present study, representing approximately 35% of total WBC, procasting a very special role for the development of protective immunity. This finding is an accordance with the findings of the previous study where similar count of neutrophils were observed in H. contortus resistant St. Croix sheep 30 . Recent data in murine models suggest a role of neutrophil phenotype priming of long-lived alternatively activated macrophages that facilitates parasites expulsion 53 . Another striking feature was observed during differential peripheral cell count and that was huge influx of monocyte counts were found at D3, D5 and D7 in naive and primed kids. This finding also may be a stone marker for the breed claimed itself much resistant against H. contortus and indicating larval trapping feature which was reported in rHc St. Croix sheep 27 . Considering the fact, hypothesizing that like St. Croix sheep, neutrophil and monocytes may indicate a joint role of macrophages and neutrophils in preventing larval establishment in Black Bengal goat 20,30 . The most notable physiological difference between resistant and susceptible animal against H. contortus is the extent of LN development 25 . In this study, LN number and weight were increased both in naive and primed kids across all time points. In a previous study, failure of lymphnode development in H. contortus infected susceptible wool shop compared with uninfected animals were recoreded 26 . Thus, the finding indicate that the Black Bengal goat have enough resistance against Haemonchus contortus 25,26,30 . Our study demonstrates that LN weight increased more than three times in primed kids whereas one and half times in naive kids at D7 compared to D0. These findings are in accordance with previous results in the sheep model where substantial increase were recorded in infected Hc resistant St Croix sheep 30 . The result also indicates that initiation of an appropriate immune response to the local lymph nodes was high in case of repeated exposure due to increased immune cells in LN 25 and the development of an acquired immune response by the clonal expansion of antigen specific B-cells into plasma cells capable of producing antibodies specific to H. contortus during repeated exposure 25 . Patterns of eosinophil and globule leukocyte infiltration of the abomasal mucosa observed in this experiment were consistent with other reports of eosinophil and globule leukocyte migration into abomasal mucosa during challenge with this parasite 18,26,30 and did not differ between naive and primed kids. Tissue damage caused by feeding may stimulate eosinophil migration to the abomasal mucosa 26 . Here, appearance of eosinophil in the abomasal epithelium was increased two times at D7 compared to D5. Similar trends were observed in H. contortus infected St. Croix sheep 30 though the infiltration of eosinophils and globule leukocytes seems to be associated with a normal secondary cellular immune response to GTP in sheep and not unique to resistant breeds. Cytokines and transcription factors such as IL-4, IL-13, GATA-3 and T-bet were expressed in the LN of naive and primed kids but IL-12p40 were not expressed. In both primed and naive kids group IL-4 and IL-13 were up regulated from D0. Primed kids had significantly greater expression of IL-4 and IL-13 from the day D3 to D5 and then down regulated compared to naive kids indicating that repeated exposure increases the intensity of these cytokines which impacts on immune cell recruitment in the local infection site such as neutrophil 28 . Again, these findings also validate the increasing number of neutrophils counts recorded in peripheral blood count in naive and primed kids. These findings are also in accordance with the previous reports in resistant St. Croix sheep 20,30 . Murine models showed that resistant breed responed against pathogenic nematode parasite such as Heligmosomoides polygyrus , Nippostrongylus brasiliensis and Trichuris muris through strong T H 2 responses and it was indicated by high levels of the cytokine's interleukin 4 (IL-4) and IL-13 while T H 1 response was high in suspectable breeds and high levels of interferon γ (IFNγ) and IL-12p40 were the markers with susceptibility 54-56 . Upregulated expression of GATA-3 and T-bet up to D5 and absence of IL-12p40 expression was indicative and responsible for the downregulation of T H 1 response and contributes to T H 2 polarisation and subsequently H. contortus expulsion 16,24 . IL-4, IL-5 and IL-13 are the hallmark cytokines of type-2 immunity, both at the innate and the adaptive level and have been associated to nematode resistance 57 . Our data revealed that both groups of Black Bengal goat kids upregulated IL-4, IL-5 and IL-13 expression in abomasal mucosa on D3, D5 and D7. These data were consistent with previous findings where increased IL-4 expression in parasite resistant St. Croix sheep at D3, D5 and D7 of infection were observed and no expression of IL-4, IL-5 were recorded for Haemonchus contortus susceptible wool sheep breed 24,58 . Primed kids had significant fold difference compared to naive kids suggested that due to repeated exposure to H. contortus, formed memory by plasma cells that initiated greater amount of IL-4 expression 57 . Primed kids had also greater IL-5 expression than naive kids that indicates early activation of eosinophil due to repeated exoposure 52 . Upregulation of GAL14, MCPI, CXCL1 and TLR2 genes were recorded in abomasal mucosa of naive and Black Bengal kids also. These cytokines were the essential cytokines for T H 2 activity. These findings are in line with the previous reports 20,24,52 . An increase in the expression of MCP1 in Black Bengal goats abomasal mucosa suggests differentiation of alternatively activated macrophages which are the initial source of IL-4. Murine studies have shown that alternatively activated macrophages are an early source of IL-4 in parasite infections 57 and therefore assist in T H 2 differentiation. Greater expression in CXCL1 in Black Bengal goats was consistent with published findings of increased neutrophil recruitment in St. Croix sheep during H. contortus infection 24,58 . Additionally, Black Bengal goats had greater expression of GAL14, recognized to be a parasite-specific immune mediator in ruminants 59 . In primed goat kids compared to naive kids, different immune pathways and upstream regulators were activated at the beginning of infection. Thereafter, here the findings of these cytokines associated with T H 2 responses increased expression in line with previous findings. A similar pattern was recorded for upstream regulator genes which were related to immunity like IL-4, IL-5, TLR-2 in second time infected goat abomasa 10 . These findings points that the breed Black Bengal initiate local T H 2 immune response during the first 7 days of H. contortus infection and facilitates parasite expulsion 14 . In this study, PBMC from primed and naive Black Bengal kids effectively reduced the larval speed as well as straight line distance which indicates partial larval morbidity. These findings are in line with previous findings where monocyte from rHc St. Croix sheep reduced larval speed and straight-line distance compared to susceptible wool sheep where monocyte induced IL-13 played a key role in larval paralysis 27,60 . Murine models of helminth infection have demonstrated that eosinophils trapped larvae and reduced larval movement in vitro 53,61 . This finding indicates that PBMC of BBG involved in larval trapping and killing at early stage of infection and PBMC from hosts with old infection showed more promising response but unable to specify the role and mechanism of particular PBMC in these events. Experimental data demonstrated significant reduction in FEC in kids after given an oral inoculation of primed PBMC-exposed H. contortus L 3 compared to kids where naive and controlled PBMC-exposed H. contortus L 3 were given orally. This finding is in line with previous finding 29 . In a similar study, Hc resistant Blackbelly sheep had reduced FEC and fewer established adult worms when sheep received an intraabomasal transfer of L 3 cultured with eosinophils 19 , further substantiating the role of innate cells on H. contortus L 3 viability, both in vitro and in vivo. Furthermore, recent exploration of single nucleotide polymorphism in immune genes such as CIITA , ATP2A 3, HSPA8 , SERPING1 , STAT5B and ESYT1 has been found responsible to reduce FEC in Black Bengal goat after H. contortus which supports the findings of current study 33 . Conclusions The present study revealed that Black Bengal goat mounts an immunological defense against H. contortus larvae during the first seven days of infection. Black Bengal goat exhibits an increased cellular and immune response by infiltrating immune cells into the abomasal mucosa, the growth of abomasal lymph nodes, along with increased expression of cytokines transcription factors that lead to the development of a highly polarised T H 2 response in the abomasum, which in turn creates an environment that is detrimental to the establishment of H. contortus . It is the first in depth study to explore the cellular and immune mechanisms lied behind the establishment and rejection of H. contortus in Black Bengal goat in Bangladesh. The study findings may not immediately benefit the farmers but definitely set the baseline to explore initiatives aimed at mitigating the impacts of H. contortus that may help the farmers to choose the Black Bengal goat in cross-breeding program. Materials and methodology Ethical Approval All tests and observations made on the animals were authorized by the Animal Welfare and Experimentation Ethics Committee of Bangladesh Agricultural University [AWEEC/BAU/2021(1)] and were conducted in compliance with the most recent legislation regarding animal experimentation and ethics. We confirmed that the study was conducted in accordance with ARRIVE guidelines. Animal Housing and Experimental design A total of 22 newborn Black Bengal goat kids were born and raised in the barn of the goat research farm of Parasitology Department, Bangladesh Agricultural University (BAU), Mymensingh. All 22 kids were divided into three groups, naïve group (n=8) primed group (n=8) and control group (n=6). Naïve and primed group kids were allowed to feed outdoor in green pasture in front of that barn for the weaning period while control goat kids were raised in a controlled, H. contortus free environment, with cemented stalls. Additionally, all group kids were limit fed 16% CP corn-soybean concentrate with ad libitum grass hay and water free from H. contortus . Furthermore, primed group kids were experimentally infected with a single dose of 2,000 H. contortus L 3 with a 10 ml syringe containing 2 ml of a suspension of L 3 at 1,000 L 3 /ml in tap water every week for 4 weeks. Fecal egg count (FEC) was performed on weekly basis after 21 days of post-infection in all three groups for ensuring the infection. After that kids from all group were treated orally with Levamisole (Sigma Aldrich, Germany) (8mg/kg) 27 and FEC were performed until it comes to zero (0) by using a modified McMaster’s technique 36,37 . All three groups were allowed to rest for three weeks (21 days). Naïve and primed goat kids were received a single dose of 10,000 H. contortus L 3 with a 10 ml syringe containing 10 ml of a suspension of L 3 at 1,000 L 3 /ml in tap water. Whole blood was collected from jugular vein from each kid of naïve and primed groups at day 0 post infection (D0), D3, D5 and D7 prior slaughtering with a view of differential blood cell count, isolating PBMC for in vitro larval motility assay. After slaughtering, abomasal tissue and abomasal lymphnode were isolated for RNA extraction. Naive and primed goat kids were randomly assigned to 4 blocks having two kids on each block and blocking was necessary because all kids could not be euthanized on a single day. On the other hand, control group kids were divided into three blocks (2/block) for in vivo larval infectivity assay. Fresh H. contortus larvae were cultured with PBMC collected from control, naïve and primed group kids for 18 hours. After that 5,000 PBMC exposed larvae from each group were administered orally to each block of control group kids (Figure 1). Haematology Right before sacrifice, blood samples were drawn from naïve, and primed groups via jugular venipuncture at D0, D3, D5, and D7 and were subsequently taken in vacutainer tubes treated with EDTA. The Packed Cell Volume (PCV), White blood cell (WBC), lymphocyte, monocyte, eosinophil, basophil and neutrophil count were measured using an automated hematology analyzer. The unit of cell count was cells x 10 3 /µl of whole blood. Separation of peripheral blood mononuclear cells from BBG kid’s blood Five ml of whole blood were collected via jugular venipuncture from each goat kid of control, naïve and primed group and pooled into 50 ml falcon eppendorf tube treated with ethylenediaminetetraacetic acid (EDTA) (Tyco, Mansfield, MA). Peripheral blood mononuclear cells (PBMC) were isolated by using density gradient medium Histopaque®-1077 (Sigma Aldrich, Germany) according to manufacturer instructions. After separation, viability of PBMC was checked by trypan blue through phase contrast microscopy 38 and cell count and dosing by hemacytometer and doses of PBMC were calculated for the larval culture 39 . Isolated PBMC were resuspended in heat-inactivated foetal bovine serum (FBS, Capricorn) containing 10% dimethylsulfoxide (DMSO), and cryopreserved in liquid N 2 until used 40 . Haemonchus contortus third stage larva (L 3 ) culture Abomasum of Black Bengal goats were collected from different slaughtered houses local markets of Mymensingh district, Bangladesh and adult female H. contortus were recovered from the abomasum (where available) at parasitology laboratory, BAU. After washing, the adult worms were incubated at 5% CO 2 and 37 °C for 30 min before use in supplemented RPMI culture medium 41 . Third-stage larvae (L 3 ) were obtained from H. contortus eggs by incubating humidified feces from infected sheep at 27 °C for 1 week 42 . Once collected, L 3 were kept in 100 cm 3 corning cell culture flask and maintained at 5-9 °C with approximately 1000 L 3 per ml phosphate buffer saline (PBS) 41,43 . In vitro cell culture assay After thawing, the PBMC were washed twice with PBS (pH 7.4) and reevaluating the viability, adjusted to the desired density (1 × 10 6 cell/ml) in the RPMI-1640 medium containing 10% fetal bovine serum, 100 U/ml streptomycin, and 100 mg/ml penicillin (UK Gibco, Paisley, UK) 27 . On the other hand, the isolated fresh larvae were washed three times in PBS followed by subsequent three washes in sterile PBS supplemented with 200 U/ml penicillin, and 200 μg/ml streptomycin. Cell suspensions of 5 x 10 5 cells (500 µL) from control (untreated), naïve and primed kids were added to a 24-well plate (Greiner CellStar, Frickenhausen, Germany) with an additional 400 µL of complete media and 100 L 3 Hc larvae (100 µL), for a total volume of 1 mL per well. Again, to compare the cell effects in terms of reducing straight line distance of L 3 larvae, 1µg/µL levamisole were added to the experiment plate and served as anthelmintic treated group 41 and placed in an incubator for 9 hours at 37°C with 5% CO 2 44 . Motility assessment Videos of larvae in cell culture were captured using Olympus Air wireless lens (Shinjuku, Tokyo, Japan) fixed to Motic AE2000 inverted microscope (Richmond, BC, Canada) at 40x magnification at department of Surgery and Obstetrics, BAU. Larval motility was assessed in terms of path length (straight line distance) using WormLabTM tracking software (MBF Bioscience, Williston, VT), measuring larval movement over 50 frames of video recording 27 . Abomasal and lymph node tissue histology All palpable lymph nodes were extracted from the lesser curvature of the abomasum by removing superficial fat. Lymph nodes were counted, weighed and the largest lymph node was cut longitudinally with a 4mm slice placed in 10% buffered formalin. The abomasum was cut along the greater curvature and contents were removed and washed gently in PBS (pH7.4). A section of the fundic region of the abomasum was removed, including a fold and all associated connective tissue, and placed in 10% buffered formalin. Small, longitudinal slices of both the lymph node and abomasal mucosa were embedded in paraffin, cut and stained with hematoxylin and eosin (H&E) in histopathology lab, department of Pathology, BAU. Eosinophils and neutrophils in the lower two-thirds and globule leukocytes in the upper one-third of the abomasal mucosa epithelium were visualized at 400X. Forty non-sequential views of each sample were counted using a grid reticule, and data are reported as the average cells/mm 2. RNA Extraction and Complementary DNA (cDNA) Synthesis from abomasal mucosa and lymphnode RNA was extracted from abomasal tissue and abomasal lymph nodeS by using QIAzol® Lysis Reagent (Qiagen) and Tissue Ruptor (Qiagen) at D0, D3, D5 and D7. In addition, RNA purification was then performed in silica columns using the RNeasy Mini Kit (Qiagen). Using the spectrophotometer NanodropTM 2000 (Thermo Scientific, Cleveland, USA), absorbance values at 260 nm (A260) and A260/A280 ratios, respectively, were used to quantify the concentration and purity of these RNA samples. Using 1,500 ng of total RNA, the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, USA), and oligo (dT) primers (IDT) in a T100TM thermal cycler (Bio-Rad, Redmond, USA), complementary DNA (cDNA) synthesis was carried out. Detection of cytokine and related pathway molecule expression by rt-PCR A total of 11 cytokines and transcription factors responsible for inducing T H 1 activity and T H 2 activity (e.g., IL4, IL-5, IL13, IL-33, GATA-3, T-bet, IL-12p40, GAL-14, MCP1, CXCL1, and TLR-2) either in abomasal mucosa and abomasal lymphnode were evaluated 27,45,46 . Primer pair sequences used in this study were obtained from those of other experiments (Ingham et al., 2008) or designed based on ovine or bovine mRNA sequences (Supplementary Table S1). GAPDH was used as the reference gene. RT 2 SYBR® Green qPCR Master Mix 2X was used for each reaction. All experiments were run in triplicate on a Roto-Gene Corbet 6000 using PCR-Arrays following the manufacturer’s instructions. The reaction was carried out under the following conditions: 95 o C for 20 sec, 40 cycles of 95 o C for 3 sec and 60 o C for 30 sec, and 60 o C for 20 sec. A melt curve was run for each gene to ensure amplification of a single product 20,27,40,47,48 . Relative fold changes (FC) were measured by using ΔΔCt relative to housekeeping gene GAPDH 49 . Data analysis Statistical analysis was performed using the GraphPad Premier 9.0 software package (GraphPad Prism; GraphPad Software Inc., San Diego, CA, USA). The results were presented as the mean ± standard deviation (SD) for fecal egg count and blood parameter analysis. The differences of cytokines and transcription factors between groups determined by two-way analysis of variance (ANOVA). Differences with P ≤ 0.05 were considered statically significant. Declarations Data availability Data will be available on request to Mr. Nurnabi Ahmed ( [email protected] ) and Dr. Md. Hasanuzzaman Talukder ( [email protected] ). In addition, the data files may be accessed in the following link: https://drive.google.com/drive/folders/10PsvqZAjiQlowoYVm64s5Jm_xIlmEyle?usp=sharing Acknowledgements The authors gratefully acknowledge the support of the National Science and Technology fellowship (NST) to Nurnabi Ahmed and Bangladesh Agricultural University Research System (BAURES). The authors also acknowledge the technical support of Departments of Pathology, Surgery and Obstetrics and Veterinary Teaching Hospital, Faculty of Veterinary Science, Bangladesh Agricultural University at different phases of the study. The abstract has been presented in person at 15th International Congress of Parasitology (ICOPA), during 21–26 August 2022 in Copenhagen, Denmark. Author’s contribution NA: Review of literature, Resources, Methodology, Formal analysis, Visualization, Original Draft Preparation, Validation, Software, Writing-Review and Editing BCR: Co-supervision, Resources, Methodology, Formal analysis, Writing-Review and Editing AB, MMRZ, MRRR, MMRS, MMH, MKR, HB: Review of literature, Methodology, Investigation, Writing-Review & Editing. MHT: Supervision, Conceptualization, Methodology, Project Administration, Validation, Visualization, Investigation, Resources, Writing-Review and Editing Financial support This study was funded by a project (/2021/77/BAU) from Bangladesh Agricultural University Research System (BAURES), Bangladesh Agricultural University, Mymensingh Bangladesh. Competing interests The authors declare that they have no conflict of interests related to this work. They are solely accountable for the content and writing of the report. References Adduci, I. et al. Haemonchosis in Sheep and Goats, Control Strategies and Development of Vaccines against Haemonchus contortus . Animals 12 , 2339 (2022). Ahmed, N. et al. Molecular and phylogenetic characterization of zoonotic Trichostrongylus species from goats for the first time in Bangladesh. Trans R Soc Trop Med Hyg 117 , 705-713 (2023). https://doi.org:10.1093/trstmh/trad034 Parvin, S. et al. Haemonchus contortus , an obligatory haematophagus worm infection in small ruminants: Population genetics and genetic diversity. Saudi Journal of Biological Sciences 31 , 104030 (2024). https://doi.org:https://doi.org/10.1016/j.sjbs.2024.104030 Saccareau, M. et al. Meta-analysis of the parasitic phase traits of Haemonchus contortus infection in sheep. Parasit Vectors 10 , 201 (2017). https://doi.org:10.1186/s13071-017-2131-7 Cruz-Tamayo, A. A. et al. Haemonchus contortus infection induces a variable immune response in resistant and susceptible Pelibuey sheep. Vet Immunol Immunopathol 234 , 110218 (2021). https://doi.org:10.1016/j.vetimm.2021.110218 Kaplan, R. M. Biology, Epidemiology, Diagnosis, and Management of Anthelmintic Resistance in Gastrointestinal Nematodes of Livestock. Vet Clin North Am Food Anim Pract 36 , 17-30 (2020). https://doi.org:10.1016/j.cvfa.2019.12.001 Parvin, S. et al. Frequency of benzimidazole resistance in Haemonchus contortus populations isolated from sheep and goats in Bangladesh. Ann Parasitol 68 , 563-568 (2022). https://doi.org:10.17420/ap6803.463 Kotze, A. C., Gilleard, J. S., Doyle, S. R. & Prichard, R. K. Challenges and opportunities for the adoption of molecular diagnostics for anthelmintic resistance. Int J Parasitol Drugs Drug Resist 14 , 264-273 (2020). https://doi.org:10.1016/j.ijpddr.2020.11.005 Rose Vineer, H. et al. Increasing importance of anthelmintic resistance in European livestock: creation and meta-analysis of an open database. Parasite 27 , 69 (2020). https://doi.org:10.1051/parasite/2020062 Aboshady, H. M., Félicité, Y., Hira, J., Barbier, C. & Bambou, J. C. Early Transcriptome Differences Between Pre-Infected and Naïve Kid Goats Infected With Haemonchus contortus . Front Vet Sci 9 , 873467 (2022). https://doi.org:10.3389/fvets.2022.873467 Bowdridge, S., MacKinnon, K., McCann, J. C., Zajac, A. M. & Notter, D. R. Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection. Veterinary Parasitology 196 , 172-178 (2013). https://doi.org:https://doi.org/10.1016/j.vetpar.2013.01.008 McRae, K. M. et al. Transcriptional profiling of the ovine abomasal lymph node reveals a role for timing of the immune response in gastrointestinal nematode resistance. Vet. parasitology 224 , 96-108 (2016). https://doi.org:10.1016/j.vetpar.2016.05.014 McRae, K. M., McEwan, J. C., Dodds, K. G. & Gemmell, N. J. Signatures of selection in sheep bred for resistance or susceptibility to gastrointestinal nematodes. BMC Genomics 15 , 637 (2014). https://doi.org:10.1186/1471-2164-15-637 Alba-Hurtado, F. & Muñoz-Guzmán, M. A. Immune responses associated with resistance to haemonchosis in sheep. Biomed Res Int 2013 , 162158 (2013). https://doi.org:10.1155/2013/162158 Estrada-Reyes, Z. et al. Cytokine and antioxidant gene profiles from peripheral blood mononuclear cells of Pelibuey lambs after Haemonchus contortus infection. Parasite Immunol 39 (2017). https://doi.org:10.1111/pim.12427 Gossner, A., Wilkie, H., Joshi, A. & Hopkins, J. Exploring the abomasal lymph node transcriptome for genes associated with resistance to the sheep nematode Teladorsagia circumcincta . Vet Res 44 , 68 (2013). https://doi.org:10.1186/1297-9716-44-68 Grencis, R. K. Immunity to helminths: resistance, regulation, and susceptibility to gastrointestinal nematodes. Annu Rev Immunol 33 , 201-225 (2015). https://doi.org:10.1146/annurev-immunol-032713-120218 Lacroux, C. et al. Haemonchus contortus (Nematoda: Trichostrongylidae) infection in lambs elicits an unequivocal T H 2 immune response. Vet Res 37 , 607-622 (2006). https://doi.org:10.1051/vetres:2006022 Terefe, G. et al. Immune response to Haemonchus contortus infection in susceptible (INRA 401) and resistant (Barbados Black Belly) breeds of lambs. Parasite Immunol 29 , 415-424 (2007). https://doi.org:10.1111/j.1365-3024.2007.00958.x Toscano, J. H. B. et al. Innate Immune Responses Associated with Resistance against Haemonchus contortus in Morada Nova Sheep. J Immunol Res 2019 , 3562672 (2019). https://doi.org:10.1155/2019/3562672 Jin, P. et al. Interferon-γ and Tumor Necrosis Factor-α Polarize Bone Marrow Stromal Cells Uniformly to a Th1 Phenotype. Sci Rep 6 , 26345 (2016). https://doi.org:10.1038/srep26345 Middleton, D., Garza, J. J., Greiner, S. P. & Bowdridge, S. A. Neutrophils rapidly produce Th2 cytokines in response to larval but not adult helminth antigen. Parasite Immunol 42 , e12679 (2020). https://doi.org:10.1111/pim.12679 Middleton, D. D. The effects of genetic differences in an innate immune receptor on immune responses to Haemonchus contortus in sheep, PhD thesis, West Virginia University (2021). Jacobs, J. R., Sommers, K. N., Zajac, A. M., Notter, D. R. & Bowdridge, S. A. Early IL-4 gene expression in abomasum is associated with resistance to Haemonchus contortus in hair and wool sheep breeds. Parasite Immunol 38 , 333-339 (2016). https://doi.org:10.1111/pim.12321 MacKinnon, K. M., Zajac, A. M., Kooyman, F. N. & Notter, D. R. Differences in immune parameters are associated with resistance to Haemonchus contortus in Caribbean hair sheep. Parasite Immunol 32 , 484-493 (2010). https://doi.org:10.1111/j.1365-3024.2010.01211.x Balic, A., Bowles, V. M. & Meeusen, E. N. Mechanisms of immunity to Haemonchus contortus infection in sheep. Parasite Immunol 24 , 39-46 (2002). https://doi.org:10.1046/j.0141-9838.2001.00432.x Shepherd, E., Greiner, S. P. & Bowdridge, S. A. Characterization of ovine monocyte activity when cultured with Haemonchus contortus larvae in vitro . Parasite Immunol 42 , e12773 (2020). https://doi.org:10.1111/pim.12773 Chan, L. et al. The Roles of Neutrophils in Cytokine Storms. Viruses 13 (2021). https://doi.org:10.3390/v13112318 Holt, R. M., Shepherd, E. A., Ammer, A. G. & Bowdridge, S. A. Effects of peripheral blood mononuclear cells on Haemonchus contortus larval motility in vitro . Parasite Immunol 37 , 553-556 (2015). https://doi.org:10.1111/pim.12219 Bowdridge, S. A., Zajac, A. M. & Notter, D. R. St. Croix sheep produce a rapid and greater cellular immune response contributing to reduced establishment of Haemonchus contortus . Vet. parasitology 208 , 204-210 (2015). https://doi.org:10.1016/j.vetpar.2015.01.019 Hossain, M. Performance of Black Bengal goat: a 50-year review. Trop Anim Health Prod 53 (2021). https://doi.org:10.1007/s11250-020-02477-2 Rakib, M. R. H. et al. Morphometric features and performances of Black Bengal goat in Bangladesh. Trop Anim Health Prod 54 , 341 (2022). https://doi.org:10.1007/s11250-022-03334-0 Alam, M. B. B. et al. Single nucleotide polymorphisms in candidate genes are significantly associated with resistance to Haemonchus contortus infection in goats. J Anim Science Biotechnology 10 , 30 (2019). https://doi.org:10.1186/s40104-019-0327-8 Kumar, R. et al. Variability of resistance in Black Bengal goats naturally infected with Haemonchus contortus. J Parasit Dis 39 , 76-79 (2015). https://doi.org:10.1007/s12639-013-0282-9 El-Ashram, S. et al. Early and late gene expression profiles of the ovine mucosa in response to Haemonchus contortus infection employing Illumina RNA-seq technology. Parasitol Int 66 , 681-692 (2017). https://doi.org:10.1016/j.parint.2017.05.007 Henriksen, S. & Aagaard, K. A simple flotation and McMaster method. (1977). Peña-Espinoza, M. et al. Efficacy of ivermectin against gastrointestinal nematodes of cattle in Denmark evaluated by different methods for analysis of faecal egg count reduction. Int J Parasit: Drugs and Drug Resistance 6 , 241-250 (2016). https://doi.org:https://doi.org/10.1016/j.ijpddr.2016.10.004 Pierce, L., Sarkar, S., Chan, L. L.-Y., Lin, B. & Qiu, J. Outcomes from a cell viability workshop: fit-for-purpose considerations for cell viability measurements for cellular therapeutic products. Cell & Gene Therapy Insights 7 , 551-569 (2021). Weinberg, A. et al. Viability and functional activity of cryopreserved mononuclear cells. Clin Diagn Lab Immunol 7 , 714-716 (2000). https://doi.org:10.1128/cdli.7.4.714-716.2000 Escribano, C. et al. Resistance to Haemonchus contortus in Corriedale sheep is associated to high parasite-specific IgA titer and a systemic Th2 immune response. Sci Rep 9 , 19579 (2019). https://doi.org:10.1038/s41598-019-55447-6 Munguía, B. et al. Sensitivity of Haemonchus contortus to anthelmintics using different in vitro screening assays: a comparative study. Parasit Vectors 15 , 129 (2022). https://doi.org:10.1186/s13071-022-05253-3 Johnston, C. et al. Cultivation of Heligmosomoides Polygyrus : An Immunomodulatory Nematode Parasite and its Secreted Products. J Vis Exp 2015 (2015). https://doi.org:10.3791/52412 García-Coiradas, L. et al. Immunization against Lamb Haemonchosis with a Recombinant Somatic Antigen of Haemonchus contortus (rHcp26/23). Vet Med Int 2010 , 852146 (2010). https://doi.org:10.4061/2010/852146 Wen, Z. et al. In vitro characterization of Haemonchus contortus trehalose-6-phosphate phosphatase and its immunomodulatory effects on peripheral blood mononuclear cells (PBMCs). Parasit Vectors 14 , 611 (2021). https://doi.org:10.1186/s13071-021-05115-4 Ingham, A., Reverter, A., Windon, R., Hunt, P. & Menzies, M. Gastrointestinal nematode challenge induces some conserved gene expression changes in the gut mucosa of genetically resistant sheep. Int J Parasitol 38 , 431-442 (2008). https://doi.org:10.1016/j.ijpara.2007.07.012 Walker, J. A. & McKenzie, A. N. J. T(H)2 cell development and function. Nat Rev Immunol 18 , 121-133 (2018). https://doi.org:10.1038/nri.2017.118 Ehsan, M. et al. Recombinant elongation factor 1 alpha of Haemonchus contortus affects the functions of goat PBMCs. Parasite Immunol 42 , e12703 (2020). https://doi.org:10.1111/pim.12703 He, L. et al. A TGF-β type II receptor that associates with developmental transition in Haemonchus contortus in vitro . PLoS Negl Trop Dis 13 , e0007913 (2019). https://doi.org:10.1371/journal.pntd.0007913 Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 , 402-408 (2001). https://doi.org:10.1006/meth.2001.1262 Bambou, J. C., Larcher, T., Ceï, W., Dumoulin, P. J. & Mandonnet, N. Effect of Experimental Infection with Haemonchus contortus on Parasitological and Local Cellular Responses in Resistant and Susceptible Young Creole Goats. BioMed Research International 2013 , 902759 (2013). https://doi.org:10.1155/2013/902759 Bambou, J.-C. et al. Peripheral immune response in resistant and susceptible Creole kids experimentally infected with Haemonchus contortus . Small Ruminant Research 82 , 34-39 (2009). https://doi.org:https://doi.org/10.1016/j.smallrumres.2009.01.008 Clutterbuck, E. et al. Recombinant human interleukin 5 is an eosinophil differentiation factor but has no activity in standard human B cell growth factor assays. Eur J Immunol 17 , 1743-1750 (1987). https://doi.org:10.1002/eji.1830171210 Chen, F. et al. Neutrophils prime a long-lived effector macrophage phenotype that mediates accelerated helminth expulsion. Nat Immunol 15 , 938-946 (2014). https://doi.org:10.1038/ni.2984 Anthony, R. M., Rutitzky, L. I., Urban, J. F., Jr., Stadecker, M. J. & Gause, W. C. Protective immune mechanisms in helminth infection. Nat Rev Immunol 7 , 975-987 (2007). https://doi.org:10.1038/nri2199 Cliffe, L. J. & Grencis, R. K. The Trichuris muris system: a paradigm of resistance and susceptibility to intestinal nematode infection. Adv Parasitol 57 , 255-307 (2004). https://doi.org:10.1016/s0065-308x(04)57004-5 Maizels, R. M. & Yazdanbakhsh, M. Immune regulation by helminth parasites: cellular and molecular mechanisms. Nat Rev Immunol 3 , 733-744 (2003). https://doi.org:10.1038/nri1183 La Flamme, A. C. et al. Type II-activated murine macrophages produce IL-4. PLoS One 7 , e46989 (2012). https://doi.org:10.1371/journal.pone.0046989 Jacobs, J. R., Greiner, S. P. & Bowdridge, S. A. Serum interleukin-4 (IL-4) production is associated with lower fecal egg count in parasite-resistant sheep. Vet parasitology 211 , 102-105 (2015). https://doi.org:10.1016/j.vetpar.2015.04.024 Benjamin, L. E., Hemo, I. & Keshet, E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. Development 125 , 1591-1598 (1998). https://doi.org:10.1242/dev.125.9.1591 Shepherd, E., Greiner, S. P., Russ, B. & Bowdridge, S. A. Interleukin-13 induces paralysis of Haemonchus contortus larvae in vitro. Parasite Immunol 42 , e12758 (2020). https://doi.org:10.1111/pim.12758 Martinez, F. O. & Gordon, S. The M1 and M2 paradigm of macrophage activation: time for reassessment. F1000Prime Rep 6 , 13 (2014). https://doi.org:10.12703/p6-13 Additional Declarations No competing interests reported. Supplementary Files Table1.docx SupplementaryTableS1.docx SupplementaryTableS1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4620150","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":327824937,"identity":"836e242a-2091-46a6-93a8-864d12012017","order_by":0,"name":"Nurnabi Ahmed","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Nurnabi","middleName":"","lastName":"Ahmed","suffix":""},{"id":327824938,"identity":"9c2f9351-f46b-4c94-94ef-902f3c8b3458","order_by":1,"name":"Babul Chandra Roy","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Babul","middleName":"Chandra","lastName":"Roy","suffix":""},{"id":327824939,"identity":"8a4a51f3-44cc-469d-a114-ae7c14b878df","order_by":2,"name":"Amitav Biswas","email":"","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Amitav","middleName":"","lastName":"Biswas","suffix":""},{"id":327824940,"identity":"273105f2-aaa6-4957-9b7e-b8a985f0f13f","order_by":3,"name":"Md. 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Hasanuzzaman Talukder","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYFAC5gbGBjYgLQHm2SSAKR4GhgSGA9g18DAwomhJI13LYcJa7NkPtknOKLPJ153d/PjDzx3n8/hnJDA+eNvGkMeHyxaexDbJDefSLLfdOWYm2XvmdrHEjQRmw7ltDMWSOB0G1PKw7bCB2Y0EMwbettuJDTcS2KR52xgSN+DSwv8QpOU/UEv6549/284lzr+RwP4brxYJoC0b2w4AteQYAA0/kLgBaAszXi03HjZbzjiXDNJSJi3bllxseOZhs+SccxI4/cLen3zwZk+ZHchhmz++bbPLkzuefPDDmzIbnCGGDTA2MMCiaRSMglEwCkYBeQAA7b9mkSA1feUAAAAASUVORK5CYII=","orcid":"","institution":"Bangladesh Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Md.","middleName":"Hasanuzzaman","lastName":"Talukder","suffix":""}],"badges":[],"createdAt":"2024-06-22 05:38:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4620150/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4620150/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60552936,"identity":"d5ee195f-9d46-4ab2-b2ad-80c7ca343820","added_by":"auto","created_at":"2024-07-18 05:45:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1623286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e and \u003cem\u003ein vitro \u003c/em\u003eexperimental design corresponding to the study of the immune response of Black Bengal Kids infected with \u003cem\u003eHaemonchus contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4620150/v1/ffc347d4fc3cbe51b8e01730.png"},{"id":60552418,"identity":"5d603d08-416a-41f1-adce-97fb65c3f77d","added_by":"auto","created_at":"2024-07-18 05:37:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":362496,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of \u003cem\u003eH. contortus\u003c/em\u003e larvae L\u003csub\u003e4 \u003c/sub\u003ecounts recovered from naïve and primed kids at 7 days post–infection (A), Measurements of anemia by PCV analysis is shown in (B). Significance levels were *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4620150/v1/d5aa7db3af9bcd6cc86f77b8.png"},{"id":60552417,"identity":"7efa1496-fabc-4f71-b8e2-b4bfdaf82310","added_by":"auto","created_at":"2024-07-18 05:37:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":349313,"visible":true,"origin":"","legend":"\u003cp\u003eLymph node development after \u003cem\u003eH. contortus\u003c/em\u003e challenge infection. Lymph nodes collected from the lesser curvature of the abomasum were counted (A) and weight of all palpable nodes was measured (B). Significance levels were *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4620150/v1/63e3c2412961f9bc40a547ae.png"},{"id":60553997,"identity":"4f6542a3-9b88-4b66-9dba-111abb06f0e5","added_by":"auto","created_at":"2024-07-18 06:01:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":468890,"visible":true,"origin":"","legend":"\u003cp\u003eComposition of cellular infiltrate into abomasal mucosa of naïve and primed kids at D0, D3, D5 and D7; globule leukocyte count (A), eosinophil count (B), neutrophil count (C)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4620150/v1/b6d3af77cae3b844f4d46d4f.png"},{"id":60552422,"identity":"dd818528-91d8-42fb-a81e-a4db08ff711b","added_by":"auto","created_at":"2024-07-18 05:37:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":567679,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of cytokine and transcription factor in abomasal lymphnode; fold change of IL-4 (A), fold change of IL-13 (B), fold change of GATA-3 (C), fold change of T-bet (D) Significance levels were *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4620150/v1/789df789981f1fb6d0820c52.png"},{"id":60552427,"identity":"6cc23b81-bf83-42e6-8191-25ccf7aa6477","added_by":"auto","created_at":"2024-07-18 05:37:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":801721,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential T\u003csub\u003eH\u003c/sub\u003e2 cytokine gene expression (IL-14, IL-5, IL-13, IL-33, GAL-14, MCP1, CXL1 and TLR-2) in the abomasal mucosa. Significance levels were *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4620150/v1/7744a7701d37b99f9f99c700.png"},{"id":60552937,"identity":"f5ff71b4-c7c5-42b4-b50c-8eab04406578","added_by":"auto","created_at":"2024-07-18 05:45:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1190629,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003elarval motility measurement after PBMC treatment (A \u0026amp; B) and \u003cem\u003ein vivo \u003c/em\u003einfectivity analysis\u003cem\u003e \u003c/em\u003ein kids orally administered with larva treated with different groups PBMC\u003cem\u003e.\u003c/em\u003e Error bars represent SEM. Asterisk indicates significant differences * P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4620150/v1/7d513d29ffb6dd2f983a8475.png"},{"id":82287791,"identity":"59c5cc56-cd71-4904-bbff-0a2fe979a244","added_by":"auto","created_at":"2025-05-08 16:46:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5837189,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4620150/v1/69a3e101-e01b-44b1-85a1-d835355b0bce.pdf"},{"id":60552420,"identity":"39f75e7b-058b-4577-b992-6fe378bddc6a","added_by":"auto","created_at":"2024-07-18 05:37:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16221,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4620150/v1/972262b815536474c88e29a8.docx"},{"id":60552419,"identity":"a04670bc-1d36-4ae3-ade6-4708588cc576","added_by":"auto","created_at":"2024-07-18 05:37:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14649,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4620150/v1/4b43104e017e2d7278a2a1ec.docx"},{"id":60553563,"identity":"f3153150-b743-4460-83a3-47794361e514","added_by":"auto","created_at":"2024-07-18 05:53:20","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":14649,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4620150/v1/f76261e838bd6e345e9db3d3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Peripheral Blood Mononuclear Cells Regulated Cytokines and Transcription Factors Reduce Haemonchus contortus Larval Establishment in Black Bengal Goat","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eHaemonchus contortus\u003c/em\u003e, is a voracious blood sucker trichostrongylid also known by its trivial name as barber's pool worm and considered the most pathogenic nematode due to its impact on small ruminant production worldwide \u003csup\u003e1,2\u003c/sup\u003e. This parasite causes severe anaemia, hypoprotenemia, oedema, and diarrhoea, which leads to significant morbidity and mortality sheep and goat farms located in tropical and subtropical climates \u003csup\u003e3,4\u003c/sup\u003e. Due to high prevalence (~\u0026thinsp;80%), farmers are currently facing a severe challenge with this highly fecund parasite control measures \u003csup\u003e5\u003c/sup\u003e. Treatments rely on just a handful uses of anthelmintic drugs from the same class of drugs, such as benzimidazoles like albendazole and levamisole \u003csup\u003e3,6,7\u003c/sup\u003e. Over- and misuse of these anthelmintics causes strong selection pressures to the parasite \u003csup\u003e8\u003c/sup\u003e, which leads to the evolution and development of anthelmintic resistance (AR). Thus, the need for alternative and sustainable control strategies, such as exploration and selection of genetically resistant animal and the development of immunotherapeutic or imunoprophylactic tools, has increased dramatically \u003csup\u003e9\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn light of the aforementioned information, numerous independent studies have already been carried out to select resistant sheep breed in different countries, and it is shown that the immunity to this gastrointestinal nematode is primarily controlled by polarised helper T cell 1 (T\u003csub\u003eH\u003c/sub\u003e1) and helper T cell (T\u003csub\u003eH\u003c/sub\u003e2) responses that are directed towards the parasites' larval stages \u003csup\u003e10\u0026ndash;13\u003c/sup\u003e. Resistance status against \u003cem\u003eH. contortus\u003c/em\u003e were characterized by the participation of globular leucocytes, eosinophils, mast cells, and antibody production at the site of infection, as well as the type-2 hallmark cytokines produced by PBMC upon parasite stimuli such as IL-4, IL-5, IL-13 and IL-33 either in the peripheral blood or in the abomasum tissues or draining lymph nodes \u003csup\u003e14\u0026ndash;20\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOn the other hand, the T\u003csub\u003eH\u003c/sub\u003e1 response is related to susceptibility and the induction of pro-inflammatory immune genes such as tumor necrosis factor (TNF), interferon gamma (IFN-γ) and IL-6, these changes contribute to nematode persistence \u003csup\u003e21\u003c/sup\u003e. In Mexico, it has been reported that Pelibuey sheep show resistance to GINs with low \u003cem\u003eH. contortus\u003c/em\u003e EPG count and increased IL-4, IL-5, IL-10 and IL-13 and Fc fragment of IgE receptor Ia (FCER1A) gene expression \u003csup\u003e15\u003c/sup\u003e. It has been revealed that increased luminal flow coupled with intestinal muscle driven by IL-4 and IL-13 cytokines reduced parasite establishment \u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eH. contortus\u003c/em\u003e resistant sheep breed PBMC upregulated several germline-encoded receptors and T\u003csub\u003eH\u003c/sub\u003e2-promoting transcription factors such as GATA-3, repressing the transcription of the pro-inflammatory cytokine IL-12 \u003csup\u003e23\u003c/sup\u003e. Resistance traits observed in St. Croix sheep were associated with IL-4 up regulation and decreased \u003cem\u003eH. contortus\u003c/em\u003e infection from the third day of post-infection \u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe development of the lymphnode (LN) is one of the best physiological distinctions between susceptible and resistant animals against \u003cem\u003eH. contortus\u003c/em\u003e \u003csup\u003e25\u003c/sup\u003e. Within five days of an initial \u003cem\u003eH. contortus\u003c/em\u003e infection in resistant sheep, eosinophils, CD4\u0026thinsp;+\u0026thinsp;T-cells, and B-cells in the abomasal mucosa rose, as did the size of the local lymph nodes \u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn resistant sheep breeds, a new role for monocytes and nuetrophil in \u003cem\u003eH. contortus\u003c/em\u003e infections were reported \u003csup\u003e22,27\u003c/sup\u003e. When \u003cem\u003eNippostrongylus brasiliensis\u003c/em\u003e and \u003cem\u003eHeligmosomoides polygyrus\u003c/em\u003e infections were present in mice, neutrophils underwent phenotypic polarisation as a result of IL-4R signalling \u003csup\u003e18\u003c/sup\u003e. They also produced IL-13, which encouraged the differentiation of alternatively-activated macrophages, and they demonstrated a greater involvement in the primary response, helping to supply early T\u003csub\u003eH\u003c/sub\u003e2 cytokines to support the T\u003csub\u003eH\u003c/sub\u003e2 differentiation of adaptive immune cells.\u003c/p\u003e \u003cp\u003eNeutrophils have been demonstrated to bind, form extracellular traps, and induce morbidity to larvae (Hc L\u003csub\u003e3\u003c/sub\u003e) \u003cem\u003ein vitro\u003c/em\u003e \u003csup\u003e10,22,28,29\u003c/sup\u003e. The early response of resistant sheep monocytes accelerates T\u003csub\u003eH\u003c/sub\u003e2 immune activation to helminth larval stages reduce adult establishment and fecundity leading to reduced infection rates and pathogenicity \u003csup\u003e22,27\u003c/sup\u003e. In the first seven days of \u003cem\u003eH. contortus\u003c/em\u003e infection, researchers found a significant increase in cellular infiltrate, notably eosinophil and neutrophil, when comparing abomasal tissue from resistant sheep to susceptible sheep \u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe Black Bengal goat (BBG) is a native breed in Bangladesh that is well-known throughout the nation and around the world for its high-quality meat, milk, and skin \u003csup\u003e31\u003c/sup\u003e. The goat has a significant economic impact in the rural community of the country as it can produce four to eight offspring annually which helps to lower the unemployment and poverty \u003csup\u003e32\u003c/sup\u003e. This Black Bengal goat has been recently described as \u003cem\u003eH. contortus\u003c/em\u003e resistant breed by exploring single nucleotide polymorphism in genes such as \u003cem\u003eCIITA\u003c/em\u003e, \u003cem\u003eATP2A3\u003c/em\u003e, \u003cem\u003eHSPA8\u003c/em\u003e, \u003cem\u003eSERPING1\u003c/em\u003e, \u003cem\u003eSTAT5B\u003c/em\u003e and \u003cem\u003eESYT1\u003c/em\u003e which were responsible for lowering fecal egg count and expulsion of this parasite from the gastrointestinal track \u003csup\u003e33,34\u003c/sup\u003e. In sheep and goat, it has been also demonstrated recently that the early response to infection is a critical part of the protective host-immune response within seven days \u003csup\u003e12,13,35\u003c/sup\u003e. However, the distinction between na\u0026iuml;ve and primed BBG goat kids in response to GIN infection, as well as the underlying mechanisms, have not given enough attention. In order to determine the level of resistance, \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiment's goals were to investigate the mechanisms of how PBMC regulate immune response against infective stage larvae of \u003cem\u003eH. contortus\u003c/em\u003e in BBG. Therefore, \u003cem\u003ein vitro\u003c/em\u003e larval motility in response to PBMC, \u003cem\u003ein vivo\u003c/em\u003e larval infectivity assay, differential blood cell counts, histopathology of abomasa and lymphnodes (LN), specific cytokine and transcription factors expression associated with resistance have been investigated in BBG kids following challenge \u003cem\u003eH. contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e infection.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLarval count \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing oral administration of \u003cem\u003eH. contortus\u003c/em\u003e 10,000 L\u003csub\u003e3\u003c/sub\u003e, number of L\u003csub\u003e4\u003c/sub\u003e retrieved from primed kids\u0026apos; abomasum was significantly (p\u0026lt;0.01) lower compared to naive kids at D7 (Figure 2 A). The average number of L\u003csub\u003e4\u003c/sub\u003e found in the primed and naive kids was 3,137 and 7,612. In neither group, any other stage of larval development was seen.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHaematology\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was a significant (p\u0026lt;0.05) variation in the mean PCV value between the primed and na\u0026iuml;ve group kids across all study time points. Compared to na\u0026iuml;ve kids, primed kids had greater PCV (Figure 2 B). There were statistically significant (p\u0026lt;0.05) differences in the mean and standard deviation of WBC, monocyte, eosinophil, and neutrophil between na\u0026iuml;ve and primed kids at D3, D5 and D7; however, there were no significant differences in the counts of lymphocytes and basophils at any time point in this study (Table 1).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLymphnode development\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEvery slaughtered\u0026nbsp;kid had all of its palpable abomasal lymph nodes (LN) counted and weighed. Figure 3A shows that there were no variations in LN counts between primed and na\u0026iuml;ve kids. Nevertheless, there were a notable variation in the LN numbers at D0, D3, D5, and D7. On D5 and D7, there was a significant difference (p\u0026lt;0.05) between naive and primed kids, although\u0026nbsp;LN weight rose exponentially in both groups (Figure 3B). When comparing the weight variations of primed and naive kids following infection, the former experienced a three-fold rise in response of 4.8 g and the latter only showed one-and-a-half-fold\u0026nbsp;increase of weight 1.4 g.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmune cell infiltration in abomasal mucosa\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimed\u0026nbsp;kids showed a higher abundance of neutrophils and eosinophils in the mucosal epithelium and submucosa compared to na\u0026iuml;ve kids (Figure 4). Globule leukocytes were present from the first day upon infection and counts rose in both kinds by\u0026nbsp;D3 (p\u0026lt; 0.05) and continued to rise through the D7\u0026nbsp;(Figure 4.A). But there was no difference in globule leukocyte counts between primed kids\u0026nbsp;and naive kids. Figure 4.B shows an exponential and significant increase in eosinophil counts to D7 (p\u0026lt;0.01). A deeper look revealed that neutrophils comprised a significant percentage of the cellular infiltration\u0026nbsp;into the abomasal mucosa (Figure 4.C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of cytokine and transcription factors in abomasal lymphnode\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene expression study in the abomasal lymph nodes revealed differences in cytokine and transcription factors expression bewteen naive and primed kids where day had a significant effect on these genes (Figure 5). IL-4 expression increased by D3 (p\u0026lt;0.05) in both naive and primed kids and was maintained until D7. The maximum expression was observed at D5 (p\u0026lt;0.01) in both groups. Primed kids exhibited increased IL-4 expression (5.7-fold vs 3.9-fold, p\u0026lt;0.05), indicating the\u0026nbsp;beginning of T\u003csub\u003eH\u003c/sub\u003e2 polarisation (Figure 5. A). IL-13 expression was confirmed from D0 to D7 in both group kids, with the highest expression observed at D5 (p\u0026lt;0.01) but no difference between na\u0026iuml;ve and primed kids (Figure 5.B). The lack of differential IL-12p40 expression contributes to T\u003csub\u003eH\u003c/sub\u003e2 polarisation and indicates that the T\u003csub\u003eH\u003c/sub\u003e1 response is down-regulated in both na\u0026iuml;ve and primed kids. The expression of transcription factor also contributes to T\u003csub\u003eH\u003c/sub\u003e2 polarisation. The presence of GATA-3 from D0 to D7, with the greatest expression at D5, validated T\u003csub\u003eH\u003c/sub\u003e2 polarisation. Primed kids showed higher expression of GATA-3 at D3 and D5 compared to na\u0026iuml;ve kids, indicating early T\u003csub\u003eH\u003c/sub\u003e2 polarisation in primed kids (Figure 5.C). However, there was no significant increase in of GATA-3 was observed. Expression of T-bet gene decreased significantly from D5 to D7 (p\u0026lt; 0.05), indicating suppression of T\u003csub\u003eH\u003c/sub\u003e1 at D7 (Figure 5.D).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression in the abomasal mucosa\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIL-4, IL-5, IL-13, IL-33, GAL14, MCP1, CXCL1, and TLR-2 were expressed\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ein the abomasal mucosa of the na\u0026iuml;ve and primed kids across all study time points (Figure 6). In both primed and na\u0026iuml;ve kids, significant differences were noted in the IL-4 gene expression at D5 (p\u0026lt;0.05) and D7 (p\u0026lt;0.01) in comparison to D0 (Figure 6A). After seven days of infection, the expression of IL-4 gene rose by over 16-fold in na\u0026iuml;ve kids and nearly 30-fold in primed kids.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt D7, IL-5 gene expression was increased approximately 4-fold in na\u0026iuml;ve kids and 7-fold in primed kids (Figure 6 B). IL-13 and IL-33 were expressed from D0 to D7 and highest expression were recorded at D3 (Figure 6 C \u0026amp; D). From D3, expression of both cytokines was gradually decreased. Significant differences within groups were seen at D3 and D5 in na\u0026iuml;ve and primed kids compared to D0. The levels of significance for IL-13 and IL-33 in primed and na\u0026iuml;ve kids at D3 were different from those at D0 (p\u0026lt; 0.05 and p\u0026lt;0.01, respectively), although they both substantially declined from D3 to D5 and D7 (p\u0026lt;0.05). Between groups, significant difference was only seen at D3 (p\u0026lt;0.05) indicating more T\u003csub\u003eH\u003c/sub\u003e2 polarisation in primed kid.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn both primed and na\u0026iuml;ve kids, the expression and upregulation of GAL14, MCP1, CXCL1, and TLR2 genes were seen from D0 to D7. Primed kids showed approximately 1.5 times greater GAL14 expression at D5 and D7 compared to na\u0026iuml;ve kids and greatest expression were seen at D7 for naive kids while D7 for primed kids (Figure 6 E). MCP1 expression was also increased in naive and primed kids as day of infection progressed but greatest expression of MCP1 was observed at D7 in naive kids and interestingly similar fold changes were observed at D5 in primed kids indicating the activation of alternatively macrophage, which are the initial source of IL-4 in parasite infections. But highest expression of MCP1 in primed kids was recorded at D7. Between groups, significant amount of MCP1 expression were observed at D7 (6 vs. 12, p\u0026lt;0.01) in prime kids compared to naive kids (Figure 6 F).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSimilar findings were observed for CXCL1 (Figure 6 G) and TLR-2 (Figure 6 H) expression in the abomasal mucosa of naive and primed kids. There was a huge influx of CXCL1 and TLR-2 were observed from D5 both in naive and primed kids but the greatest expression for this gene were recorded at D7 though the level of expresseion of CXCL1 and TLR-2 of D7 in naive kids was similar to D5 in primed kids. Between naive and primed kids, significant differences in fold changes were only observed at D7 (TLR-2, 3 vs. 5.5, p\u0026lt;0.05; CXCL1, 10 vs. 13.3, p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;larval motility\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tracking data showed that, among study groups, levamisole treated larvae had lowest velocity and travelled the least straight-line distance than any other groups larvae. Furthermore, larvae exposed to primed kids PBMC had significantly (p\u0026lt;0.05) decreased velocity and straight-line distance compared to larvae exposed to naive and control goat kids PBMC (Figure 7 A \u0026amp; B). The average speed and straight-line distance covered by larvae exposed to primed kid\u0026apos;s PBMC was 45.5 \u0026plusmn; 6.9 \u0026mu;m and 56 \u0026plusmn; 12 \u0026mu;m while larvae exposed to na\u0026iuml;ve PBMC was 16 \u0026plusmn;2.5 \u0026mu;m and 13\u0026plusmn;1.3 \u0026mu;m.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLarval infectivity following exposure to PBMC from na\u0026iuml;ve and primed kids\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate larval infectivity in Black Bengal kid\u0026apos;s, 5,000 L\u003csub\u003e3\u003c/sub\u003e treated with primed, naive and control groups PBMC were administered orally to kids of control group A, B and C. By week 5 following infection, differences were observed in FEC where group A kids shed an average of 2200 egg per gram (EPG) whereas the group B and C shed 3320 EPG and 7,100 EPG respectively (Figure 7 C). By week, the experiment was terminated. Group A kids shed then an average of 950 egg per gram (EPG) whereas the group B and C shed 2,560 EPG and 4,060 EPG respectively.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussions","content":"\u003cp\u003eIn the present study, we investigated the cellular and immune response of BBG kids regarding \u003cem\u003eH. contortus\u003c/em\u003e larval (L\u003csub\u003e3\u003c/sub\u003e) infection for first seven days. We compared specific cytokines and transcription factors expression associated with \u003cem\u003eH. contortus\u0026nbsp;\u003c/em\u003einfection between na\u0026iuml;ve and primed BBG kids also.\u003c/p\u003e\n\u003cp\u003eThe present data demonstrated that following oral administration of 10,000 \u003cem\u003eH. contortus\u0026nbsp;\u003c/em\u003eL\u003csub\u003e3\u0026nbsp;\u003c/sub\u003eto naive and primed kids, the number of larvae established into the abomasum of primed kids were markedly lower at 7 days (D7) compared to na\u0026iuml;ve kids. The finding indicates the development of mechanism specific to early-stage L\u003csub\u003e3\u003c/sub\u003e abolition after repeated infections. A similar study was conducted recently to detect early transcriptome differences between na\u0026iuml;ve and pre-infected goat kids and they showed that double timed challenged pre-infected \u003cem\u003eH. contortus\u003c/em\u003e resistant (rHc) \u003cem\u003eCreole\u003c/em\u003e goat kids had less larval count compared to single timed challenged naive kids after seven days of infection \u003csup\u003e10\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, primed kids had higher mean PCV compared to na\u0026iuml;ve kids (41.5% vs 37.01%) indicating establishment of more protection during second exposure compared to first time exposure to \u003cem\u003eH. contortus\u003c/em\u003e. The finding is in line with previous study where similar findings were observed between challenged and controlled rHc St. Croix hair sheep where mean PCV was 40 and 35 during 10 days infection \u003csup\u003e30\u003c/sup\u003e. In another study, Bambou et al. reported that in rHc\u003cem\u003e\u0026nbsp;Creole\u003c/em\u003e kids had higher mean PCV during second exposure (challenge 2) compared to primary infection (challenge 1) \u003csup\u003e50\u003c/sup\u003e. Again, current study finding is in accordance with previous study performed in the same animal model showed that, rHc goats produced better degree of protection in repeated exposure compared to single timed exposure \u003csup\u003e51\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCirculating total white blood cell count was found relatively higher in primed kids compared to naive kids across all study time points in this study. The present findings are in line with previous study demonstrated the differences in WBC count between resistant St. Croix and susceptible wool sheep at D0, D3, D5 and D7 and St. Croix sheep showed similar increasing trend of WBCs against \u003cem\u003eH. contortus\u0026nbsp;\u003c/em\u003eL\u003csub\u003e3\u003c/sub\u003e as Black Bengal kids showed \u003csup\u003e30\u003c/sup\u003e. Differential cell counts also revealed that activation of eosinophil in naive and primed kids started from same day but count was relatively higher in primed kids compared to naive kids. The present findings are in accordance with previous findings where increased eosinophils counts were observed in gastrointestinal track parasite (GTP) resistant breed by 3 days after infection compared to susceptible goat breeds \u003csup\u003e25\u003c/sup\u003e and mice model revealed that early activation of eosinophil happened in repeated exoposure and it was indicative by greater IL-5 expression \u003csup\u003e52\u003c/sup\u003e. The substantial contribution of neutrophils to total WBC in the present study, representing approximately 35% of total WBC, procasting a very special role for the development of protective immunity. This finding is an accordance with the findings of the previous study where similar count of neutrophils were observed in \u003cem\u003eH. contortus\u003c/em\u003e resistant St. Croix sheep \u003csup\u003e30\u003c/sup\u003e. Recent data in murine models suggest a role of neutrophil phenotype priming of long-lived alternatively activated macrophages that facilitates parasites expulsion \u003csup\u003e53\u003c/sup\u003e. Another striking feature was observed during differential peripheral cell count and that was huge influx of monocyte counts were found at D3, D5 and D7 in naive and primed kids. This finding also may be a stone marker for the breed claimed itself much resistant against \u003cem\u003eH. contortus\u003c/em\u003e and indicating larval trapping feature which was reported in rHc St. Croix sheep \u003csup\u003e27\u003c/sup\u003e. Considering the fact, hypothesizing that like St. Croix sheep, neutrophil and monocytes may indicate a joint role of macrophages and neutrophils in preventing larval establishment in Black Bengal goat \u003csup\u003e20,30\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe most notable physiological difference between resistant and susceptible animal against \u003cem\u003eH. contortus\u003c/em\u003e is the extent of LN development \u003csup\u003e25\u003c/sup\u003e. In this study, LN number and weight were increased both in naive and primed kids across all time points. \u0026nbsp;In a previous study, failure of lymphnode development in \u003cem\u003eH. contortus\u003c/em\u003e infected susceptible wool shop compared with uninfected animals were recoreded\u003csup\u003e26\u003c/sup\u003e. Thus, the finding indicate that the Black Bengal goat have enough resistance against\u003cem\u003e\u0026nbsp;Haemonchus contortus\u0026nbsp;\u003c/em\u003e\u003csup\u003e25,26,30\u003c/sup\u003e. Our study demonstrates that LN weight increased more than three times in primed kids whereas one and half times in naive kids at D7 compared to D0. These findings are in accordance with previous results in the sheep model where substantial increase were recorded in infected Hc resistant St Croix sheep\u003csup\u003e30\u003c/sup\u003e. The result also indicates that initiation of an appropriate immune response to the local lymph nodes was high in case of repeated exposure due to increased immune cells in LN \u003csup\u003e25\u003c/sup\u003e and the development of an acquired immune response by the clonal expansion of antigen specific B-cells into plasma cells capable of producing antibodies specific to \u003cem\u003eH. contortus\u003c/em\u003e during repeated exposure \u003csup\u003e25\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePatterns of eosinophil and globule leukocyte infiltration of the abomasal mucosa observed in this experiment were consistent with other reports of eosinophil and globule leukocyte migration into abomasal mucosa during challenge with this parasite \u003csup\u003e18,26,30\u003c/sup\u003e and did not differ between naive and primed kids. Tissue damage caused by feeding may stimulate eosinophil migration to the abomasal mucosa \u003csup\u003e26\u003c/sup\u003e. Here, appearance of eosinophil in the abomasal epithelium was increased two times at D7 compared to D5. Similar trends were observed in \u003cem\u003eH.\u003c/em\u003e \u003cem\u003econtortus\u0026nbsp;\u003c/em\u003einfected St. Croix sheep \u003csup\u003e30\u003c/sup\u003e though the infiltration of eosinophils and globule leukocytes seems to be associated with a normal secondary cellular immune response to GTP in sheep and not unique to resistant breeds. Cytokines and transcription factors such as IL-4, IL-13, GATA-3 and T-bet were expressed in the LN of naive and primed kids but IL-12p40 were not expressed. In both primed and naive kids group IL-4 and IL-13 were up regulated from D0. Primed kids had significantly greater expression of IL-4 and IL-13 from the day D3 to D5 and then down regulated compared to naive kids indicating that repeated exposure increases the intensity of these cytokines which impacts on immune cell recruitment in the local infection site such as neutrophil \u003csup\u003e28\u003c/sup\u003e. Again, these findings also validate the increasing number of neutrophils counts recorded in peripheral blood count in naive and primed kids. These findings are also in accordance with the previous reports in resistant St. Croix sheep \u003csup\u003e20,30\u003c/sup\u003e. Murine models showed that resistant breed responed against pathogenic nematode parasite such as \u003cem\u003eHeligmosomoides polygyrus\u003c/em\u003e, \u003cem\u003eNippostrongylus brasiliensis\u003c/em\u003e and \u003cem\u003eTrichuris muris\u003c/em\u003e\u0026nbsp; through strong T\u003csub\u003eH\u003c/sub\u003e2 responses and it was indicated by high levels of the cytokine\u0026apos;s interleukin 4 (IL-4) and IL-13 while T\u003csub\u003eH\u003c/sub\u003e1 response was high \u0026nbsp; in suspectable breeds and high levels of interferon \u0026gamma; (IFN\u0026gamma;) and IL-12p40 were the markers with susceptibility \u003csup\u003e54-56\u003c/sup\u003e. Upregulated expression of GATA-3 and T-bet up to D5 and absence of IL-12p40 expression was indicative and responsible for the downregulation of T\u003csub\u003eH\u003c/sub\u003e1 response and contributes to T\u003csub\u003eH\u003c/sub\u003e2 polarisation and subsequently \u003cem\u003eH. contortus\u003c/em\u003e expulsion \u003csup\u003e16,24\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIL-4, IL-5 and IL-13 are the hallmark cytokines of type-2 immunity, both at the innate and the adaptive level and have been associated to nematode resistance \u003csup\u003e57\u003c/sup\u003e. Our data revealed that both groups of Black Bengal goat kids upregulated IL-4, IL-5 and IL-13 expression in abomasal mucosa on D3, D5 and D7. These data were consistent with previous findings where increased IL-4 expression in parasite resistant St. Croix sheep at D3, D5 and D7 of infection were observed and no expression of IL-4, IL-5 were recorded for \u003cem\u003eHaemonchus contortus\u003c/em\u003e susceptible wool sheep breed \u003csup\u003e24,58\u003c/sup\u003e. Primed kids had significant fold difference compared to naive kids suggested that due to repeated exposure to \u003cem\u003eH. contortus,\u003c/em\u003e formed memory by plasma cells that initiated greater amount of IL-4 expression\u003csup\u003e57\u003c/sup\u003e. Primed kids had also greater IL-5 expression than naive kids that indicates early activation of eosinophil due to repeated exoposure\u003csup\u003e52\u003c/sup\u003e. \u0026nbsp;Upregulation of GAL14, MCPI, CXCL1 and TLR2 genes were recorded in abomasal mucosa of naive and Black Bengal kids also. These cytokines were the essential cytokines for T\u003csub\u003eH\u003c/sub\u003e2 activity. These findings are in line with the previous reports \u003csup\u003e20,24,52\u003c/sup\u003e. An increase in the expression of MCP1 in Black Bengal goats abomasal mucosa suggests differentiation of alternatively activated macrophages which are the initial source of IL-4. Murine studies have shown that alternatively activated macrophages are an early source of IL-4 in parasite infections\u003csup\u003e57\u003c/sup\u003e and therefore assist in T\u003csub\u003eH\u003c/sub\u003e2 differentiation. Greater expression in CXCL1 in Black Bengal goats was consistent with published findings of increased neutrophil recruitment in St. Croix sheep during \u003cem\u003eH. contortus\u003c/em\u003e infection \u003csup\u003e24,58\u003c/sup\u003e. Additionally, Black Bengal goats had greater expression of GAL14, recognized to be a parasite-specific immune mediator in ruminants \u003csup\u003e59\u003c/sup\u003e. In primed goat kids compared to naive kids, different immune pathways and upstream regulators were activated at the beginning of infection. Thereafter, here the findings of these cytokines associated with T\u003csub\u003eH\u003c/sub\u003e2 responses increased expression in line with previous findings. \u0026nbsp; A similar pattern was recorded for upstream regulator genes which were related to immunity like IL-4, IL-5, TLR-2 in second time infected goat abomasa\u003csup\u003e10\u003c/sup\u003e. These findings points that the breed Black Bengal initiate local T\u003csub\u003eH\u003c/sub\u003e2 immune response during the first 7 days of \u003cem\u003eH. contortus\u003c/em\u003e infection and facilitates parasite expulsion \u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study, PBMC from primed and naive Black Bengal kids effectively reduced the larval speed as well as straight line distance which indicates partial larval morbidity. These findings are in line with previous findings where monocyte from rHc St. Croix sheep reduced larval speed and straight-line distance compared to susceptible wool sheep where monocyte induced IL-13 played a key role in larval paralysis \u003csup\u003e27,60\u003c/sup\u003e. Murine models of helminth infection have demonstrated that eosinophils trapped larvae and reduced larval movement \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003e\u003csup\u003e53,61\u003c/sup\u003e. This finding indicates that PBMC of BBG involved in larval trapping and killing at early stage of infection and PBMC from hosts with old infection showed more promising response but unable to specify the role and mechanism of particular PBMC in these events.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExperimental data demonstrated significant reduction in FEC in kids after given an oral inoculation of primed PBMC-exposed \u003cem\u003eH. contortus\u003c/em\u003e L\u003csub\u003e3\u0026nbsp;\u003c/sub\u003ecompared to kids where naive and controlled PBMC-exposed \u003cem\u003eH. contortus\u003c/em\u003e L\u003csub\u003e3\u0026nbsp;\u003c/sub\u003ewere\u003csub\u003e\u0026nbsp;\u003c/sub\u003egiven orally. This finding is in line with previous finding \u003csup\u003e29\u003c/sup\u003e. In a similar study, Hc resistant Blackbelly sheep had reduced FEC and fewer established adult worms when sheep received an intraabomasal transfer of L\u003csub\u003e3\u003c/sub\u003e cultured with eosinophils \u003csup\u003e19\u003c/sup\u003e, further substantiating the role of innate cells \u003cem\u003eon H. contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e viability, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo.\u0026nbsp;\u003c/em\u003eFurthermore, recent exploration of single nucleotide polymorphism in immune genes such as \u003cem\u003eCIITA\u003c/em\u003e, \u003cem\u003eATP2A\u003c/em\u003e3, \u003cem\u003eHSPA8\u003c/em\u003e, \u003cem\u003eSERPING1\u003c/em\u003e, \u003cem\u003eSTAT5B\u003c/em\u003e and \u003cem\u003eESYT1\u003c/em\u003e has been found responsible to reduce FEC in Black Bengal goat after \u003cem\u003eH. contortus\u0026nbsp;\u003c/em\u003ewhich\u003cem\u003e\u0026nbsp;\u003c/em\u003esupports the findings of current study \u003csup\u003e33\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe present study revealed that Black Bengal goat mounts an immunological defense against \u003cem\u003eH. contortus\u003c/em\u003e larvae during the first seven days of infection. Black Bengal goat exhibits an increased cellular and immune response by infiltrating immune cells into the abomasal mucosa, the growth of abomasal lymph nodes, along with increased expression of cytokines transcription factors that lead to the development of a highly polarised T\u003csub\u003eH\u003c/sub\u003e2 response in the abomasum, which in turn creates an environment that is detrimental to the establishment of \u003cem\u003eH. contortus\u003c/em\u003e. It is the first in depth study to explore the cellular and immune mechanisms lied behind the establishment and rejection of \u003cem\u003eH. contortus\u003c/em\u003e in Black Bengal goat in Bangladesh. The study findings may not immediately benefit the farmers but definitely set the baseline to explore initiatives aimed at mitigating the impacts of \u003cem\u003eH. contortus\u003c/em\u003e that may help the farmers to choose the Black Bengal goat in cross-breeding program.\u003c/p\u003e "},{"header":"Materials and methodology ","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll tests and observations made on the animals were authorized by the Animal Welfare and Experimentation Ethics Committee of Bangladesh Agricultural University [AWEEC/BAU/2021(1)] and were conducted in compliance with the most recent legislation regarding animal experimentation and ethics. We confirmed that the study was conducted\u0026nbsp;in accordance with ARRIVE guidelines. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Housing and Experimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 22 newborn Black Bengal goat kids were born and raised in the barn of the goat research farm of Parasitology Department, Bangladesh Agricultural University (BAU), Mymensingh. All 22 kids were divided into three groups, na\u0026iuml;ve group (n=8) primed group (n=8) and control group (n=6). Na\u0026iuml;ve and primed group kids were allowed to feed outdoor in green pasture in front of that barn for the weaning period while control goat kids were raised in a controlled, \u003cem\u003eH. contortus\u003c/em\u003e free environment, with cemented stalls. Additionally, all group kids were limit fed 16% CP corn-soybean concentrate with ad libitum grass hay and water free from \u003cem\u003eH. contortus\u003c/em\u003e. Furthermore, primed group kids were experimentally infected with a single dose of 2,000 \u003cem\u003eH. contortus\u003c/em\u003e L\u003csub\u003e3\u0026nbsp;\u003c/sub\u003ewith a 10 ml syringe containing 2 ml of a suspension of L\u003csub\u003e3\u003c/sub\u003e at 1,000 L\u003csub\u003e3\u003c/sub\u003e/ml in tap water every week for 4 weeks. Fecal egg count (FEC) was performed on weekly basis after 21 days of post-infection in all three groups for ensuring the infection. After that kids from all group were treated orally with Levamisole (Sigma Aldrich, Germany) (8mg/kg) \u003csup\u003e27\u003c/sup\u003e and FEC were performed until it comes to zero (0) by using a modified McMaster\u0026rsquo;s technique \u003csup\u003e36,37\u003c/sup\u003e. All three groups were allowed to rest for three weeks (21 days). Na\u0026iuml;ve and primed goat kids were received a single dose of 10,000 \u003cem\u003eH. contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e with a 10 ml syringe containing 10 ml of a suspension of L\u003csub\u003e3\u003c/sub\u003e at 1,000 L\u003csub\u003e3\u003c/sub\u003e/ml in tap water. Whole blood was collected from jugular vein from each kid of na\u0026iuml;ve and primed groups at day 0 post infection (D0), D3, D5 and D7 prior slaughtering with a view of differential blood cell count, isolating PBMC for \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003elarval motility assay. After slaughtering, abomasal tissue and abomasal lymphnode were isolated for RNA extraction. Naive and primed goat kids were randomly assigned to 4 blocks having two kids on each block and blocking was necessary because all kids could not be euthanized on a single day. On the other hand, control group kids were divided into three blocks (2/block) for \u003cem\u003ein vivo\u003c/em\u003e larval infectivity assay. Fresh \u003cem\u003eH. contortus\u0026nbsp;\u003c/em\u003elarvae were cultured with PBMC collected from control, na\u0026iuml;ve and primed group kids for 18 hours. After that 5,000 PBMC exposed larvae from each group were administered orally to each block of control group kids (Figure 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHaematology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRight before sacrifice, blood samples were drawn from na\u0026iuml;ve, and primed groups via jugular venipuncture at D0, D3, D5, and D7 and were subsequently taken in vacutainer tubes treated with EDTA. The Packed Cell Volume (PCV), White blood cell (WBC), lymphocyte, monocyte, eosinophil, basophil and neutrophil count were measured using an automated hematology analyzer. The unit of cell count was cells\u0026nbsp;x 10\u003csup\u003e3\u003c/sup\u003e/\u0026micro;l\u0026nbsp;of whole blood.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSeparation of peripheral blood mononuclear cells from BBG kid\u0026rsquo;s blood\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFive ml of whole blood were collected via jugular venipuncture from each goat kid of control, na\u0026iuml;ve and primed group and pooled into 50 ml falcon eppendorf tube treated with ethylenediaminetetraacetic acid (EDTA) (Tyco, Mansfield, MA). Peripheral blood mononuclear cells (PBMC) were isolated by using density gradient medium Histopaque\u0026reg;-1077 (Sigma Aldrich, Germany) according to manufacturer instructions. After separation, viability of PBMC was checked by trypan blue through phase contrast microscopy \u003csup\u003e38\u003c/sup\u003e and cell count and dosing by hemacytometer and doses of PBMC were calculated for the larval culture \u003csup\u003e39\u003c/sup\u003e. Isolated PBMC were resuspended in heat-inactivated foetal bovine serum (FBS, Capricorn) containing 10% dimethylsulfoxide (DMSO), and cryopreserved in liquid N\u003csub\u003e2\u0026nbsp;\u003c/sub\u003euntil used \u003csup\u003e40\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHaemonchus contortus\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;third stage larva (L\u003csub\u003e3\u003c/sub\u003e) culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAbomasum of Black Bengal goats were collected from different slaughtered houses local markets of Mymensingh district, Bangladesh and adult female \u003cem\u003eH. contortus\u003c/em\u003e were recovered from the abomasum (where available) at parasitology laboratory, BAU. After washing, the adult worms were incubated at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37 \u0026deg;C for 30 min before use in supplemented RPMI culture medium\u003csup\u003e41\u003c/sup\u003e. Third-stage larvae (L\u003csub\u003e3\u003c/sub\u003e) were obtained from \u003cem\u003eH. contortus\u0026nbsp;\u003c/em\u003eeggs by incubating humidified feces from infected sheep at 27 \u0026deg;C for 1 week \u003csup\u003e42\u003c/sup\u003e. Once collected, L\u003csub\u003e3\u003c/sub\u003e were kept in 100 cm\u003csup\u003e3\u003c/sup\u003e corning cell culture flask and maintained at 5-9 \u0026deg;C with approximately 1000 L\u003csub\u003e3\u003c/sub\u003e per ml phosphate buffer saline (PBS) \u003csup\u003e41,43\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;cell culture assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter thawing, the PBMC were washed twice with PBS (pH 7.4) and reevaluating the viability, adjusted to the desired density (1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e cell/ml) in the RPMI-1640 medium containing 10% fetal bovine serum, 100 U/ml streptomycin, and 100 mg/ml penicillin (UK Gibco, Paisley, UK) \u003csup\u003e27\u003c/sup\u003e. On the other hand, the isolated fresh larvae were washed three times in PBS followed by subsequent three washes in sterile PBS supplemented with 200 U/ml penicillin, and 200 \u0026mu;g/ml streptomycin. Cell suspensions of 5 x 10\u003csup\u003e5\u003c/sup\u003e cells (500 \u0026micro;L) from control (untreated), na\u0026iuml;ve and primed kids were added to a 24-well plate (Greiner CellStar, Frickenhausen, Germany) with an additional 400 \u0026micro;L of complete media and 100 L\u003csub\u003e3\u003c/sub\u003e Hc larvae (100 \u0026micro;L), for a total volume of 1 mL per well. Again, to compare the cell effects in terms of reducing straight line distance of L\u003csub\u003e3\u003c/sub\u003e larvae, 1\u0026micro;g/\u0026micro;L levamisole were added to the experiment plate and served as anthelmintic treated group \u003csup\u003e41\u003c/sup\u003e and placed in an incubator for 9 hours at 37\u0026deg;C with 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e\u003csup\u003e44\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMotility assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVideos of larvae in cell culture were captured using Olympus Air wireless lens (Shinjuku, Tokyo, Japan) fixed to Motic AE2000 inverted microscope (Richmond, BC, Canada) at 40x magnification at department of Surgery and Obstetrics, BAU. Larval motility was assessed in terms of path length (straight line distance) using WormLabTM tracking software (MBF Bioscience, Williston, VT), measuring larval movement over 50 frames of video recording \u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbomasal and lymph node tissue histology\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll palpable lymph nodes were extracted from the lesser curvature of the abomasum by removing superficial fat. Lymph nodes were counted, weighed and the largest lymph node was cut longitudinally with a 4mm slice placed in 10% buffered formalin. The abomasum was cut along the greater curvature and contents were removed and washed gently in PBS (pH7.4). A section of the fundic region of the abomasum was removed, including a fold and all associated connective tissue, and placed in 10% buffered formalin. Small, longitudinal slices of both the lymph node and abomasal mucosa were embedded in paraffin, cut and stained with hematoxylin and eosin (H\u0026amp;E) in histopathology lab, department of Pathology, BAU. Eosinophils and neutrophils in the lower two-thirds and globule leukocytes in the upper one-third of the abomasal mucosa epithelium were visualized at 400X. Forty non-sequential views of each sample were counted using a grid reticule, and data are reported as the average cells/mm\u003csup\u003e2.\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Extraction and Complementary DNA (cDNA) Synthesis from abomasal mucosa and lymphnode\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was extracted from abomasal tissue and abomasal lymph nodeS by using QIAzol\u0026reg; Lysis Reagent (Qiagen) and Tissue Ruptor (Qiagen) at D0, D3, D5 and D7. In addition, RNA purification was then performed in silica columns using the RNeasy Mini Kit (Qiagen). Using the spectrophotometer NanodropTM 2000 (Thermo Scientific, Cleveland, USA), absorbance values at 260 nm (A260) and A260/A280 ratios, respectively, were used to quantify the concentration and purity of these RNA samples. Using 1,500 ng of total RNA, the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, USA), and oligo (dT) primers (IDT) in a T100TM thermal cycler (Bio-Rad, Redmond, USA), complementary DNA (cDNA) synthesis was carried out.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of cytokine and related pathway molecule expression by rt-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 11 cytokines and transcription factors responsible for inducing T\u003csub\u003eH\u003c/sub\u003e1 activity and T\u003csub\u003eH\u003c/sub\u003e2 activity (e.g., IL4, IL-5, IL13, IL-33, GATA-3, T-bet, IL-12p40, GAL-14, MCP1, CXCL1, and TLR-2) either in abomasal mucosa and abomasal lymphnode were evaluated \u003csup\u003e27,45,46\u003c/sup\u003e. Primer pair sequences used in this study were obtained from those of other experiments (Ingham et al., 2008) or designed based on ovine or bovine mRNA sequences (Supplementary Table S1). GAPDH was used as the reference gene. RT\u003csup\u003e2\u003c/sup\u003e SYBR\u0026reg; Green qPCR Master Mix 2X was used for each reaction. All experiments were run in triplicate on a Roto-Gene Corbet 6000 using PCR-Arrays following the manufacturer\u0026rsquo;s instructions. The reaction was carried out under the following conditions: 95\u003csup\u003eo\u003c/sup\u003eC for 20 sec, 40 cycles of 95\u003csup\u003eo\u003c/sup\u003eC for 3 sec and 60\u003csup\u003eo\u003c/sup\u003eC for 30 sec, and 60\u003csup\u003eo\u003c/sup\u003eC for 20 sec. A melt curve was run for each gene to ensure amplification of a single product \u003csup\u003e20,27,40,47,48\u003c/sup\u003e. Relative fold changes (FC) were measured by using \u0026Delta;\u0026Delta;Ct relative to housekeeping gene GAPDH \u003csup\u003e49\u003c/sup\u003e. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using the GraphPad Premier 9.0 software package (GraphPad Prism; GraphPad Software Inc., San Diego, CA, USA). The results were presented as the mean \u0026plusmn; standard deviation (SD) for fecal egg count and blood parameter analysis. The differences of cytokines and transcription factors between groups determined by two-way analysis of variance (ANOVA). Differences with P \u0026le; 0.05 were considered statically significant. \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be available on request to Mr. Nurnabi Ahmed ([email protected]) and Dr. Md. Hasanuzzaman Talukder ([email protected]). In addition, the data files may be accessed in the \u0026nbsp; following link: https://drive.google.com/drive/folders/10PsvqZAjiQlowoYVm64s5Jm_xIlmEyle?usp=sharing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the support of the National Science and Technology fellowship (NST) to Nurnabi Ahmed and Bangladesh Agricultural University Research System (BAURES). The authors also acknowledge the technical support of Departments of Pathology, Surgery and Obstetrics and Veterinary Teaching Hospital, Faculty of Veterinary Science, Bangladesh Agricultural University at different phases of the study. \u0026nbsp;The abstract has been presented in person at 15th International Congress of Parasitology (ICOPA), during 21\u0026ndash;26 August 2022 in Copenhagen, Denmark.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNA: Review of literature, Resources, Methodology, Formal analysis, Visualization, Original Draft Preparation, Validation, Software, Writing-Review and Editing\u003c/p\u003e\n\u003cp\u003eBCR: Co-supervision, Resources, Methodology, Formal analysis, Writing-Review and Editing\u003c/p\u003e\n\u003cp\u003eAB, MMRZ, MRRR, MMRS, MMH, MKR, HB: Review of literature, Methodology, Investigation, Writing-Review \u0026amp; Editing.\u003c/p\u003e\n\u003cp\u003eMHT: Supervision, Conceptualization, Methodology, Project Administration, Validation, Visualization, Investigation, Resources, Writing-Review and Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by a project (/2021/77/BAU) from Bangladesh Agricultural University Research System (BAURES), Bangladesh Agricultural University, Mymensingh Bangladesh. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interests related to this work. They are solely accountable for the content and writing of the report.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdduci, I.\u003cem\u003e et al.\u003c/em\u003e Haemonchosis in Sheep and Goats, Control Strategies and Development of Vaccines against \u003cem\u003eHaemonchus contortus\u003c/em\u003e. \u003cem\u003eAnimals\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 2339 (2022). \u003c/li\u003e\n\u003cli\u003eAhmed, N.\u003cem\u003e et al.\u003c/em\u003e Molecular and phylogenetic characterization of zoonotic \u003cem\u003eTrichostrongylus\u003c/em\u003e species from goats for the first time in Bangladesh. \u003cem\u003eTrans R Soc Trop Med Hyg\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 705-713 (2023). https://doi.org:10.1093/trstmh/trad034\u003c/li\u003e\n\u003cli\u003eParvin, S.\u003cem\u003e et al.\u003c/em\u003e \u003cem\u003eHaemonchus contortus\u003c/em\u003e, an obligatory haematophagus worm infection in small ruminants: Population genetics and genetic diversity. \u003cem\u003eSaudi Journal of Biological Sciences\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 104030 (2024). https://doi.org:https://doi.org/10.1016/j.sjbs.2024.104030\u003c/li\u003e\n\u003cli\u003eSaccareau, M.\u003cem\u003e et al.\u003c/em\u003e Meta-analysis of the parasitic phase traits of \u003cem\u003eHaemonchus contortus\u003c/em\u003e infection in sheep. \u003cem\u003eParasit Vectors\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 201 (2017). https://doi.org:10.1186/s13071-017-2131-7\u003c/li\u003e\n\u003cli\u003eCruz-Tamayo, A. A.\u003cem\u003e et al.\u003c/em\u003e \u003cem\u003eHaemonchus contortus\u003c/em\u003e infection induces a variable immune response in resistant and susceptible Pelibuey sheep. \u003cem\u003eVet Immunol Immunopathol\u003c/em\u003e \u003cstrong\u003e234\u003c/strong\u003e, 110218 (2021). https://doi.org:10.1016/j.vetimm.2021.110218\u003c/li\u003e\n\u003cli\u003eKaplan, R. M. Biology, Epidemiology, Diagnosis, and Management of Anthelmintic Resistance in Gastrointestinal Nematodes of Livestock. \u003cem\u003eVet Clin North Am Food Anim Pract\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 17-30 (2020). https://doi.org:10.1016/j.cvfa.2019.12.001\u003c/li\u003e\n\u003cli\u003eParvin, S.\u003cem\u003e et al.\u003c/em\u003e Frequency of benzimidazole resistance in \u003cem\u003eHaemonchus contortus\u003c/em\u003e populations isolated from sheep and goats in Bangladesh. \u003cem\u003eAnn Parasitol\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 563-568 (2022). https://doi.org:10.17420/ap6803.463\u003c/li\u003e\n\u003cli\u003eKotze, A. C., Gilleard, J. S., Doyle, S. R. \u0026amp; Prichard, R. K. Challenges and opportunities for the adoption of molecular diagnostics for anthelmintic resistance. \u003cem\u003eInt J Parasitol Drugs Drug Resist\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 264-273 (2020). https://doi.org:10.1016/j.ijpddr.2020.11.005\u003c/li\u003e\n\u003cli\u003eRose Vineer, H.\u003cem\u003e et al.\u003c/em\u003e Increasing importance of anthelmintic resistance in European livestock: creation and meta-analysis of an open database. \u003cem\u003eParasite\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 69 (2020). https://doi.org:10.1051/parasite/2020062\u003c/li\u003e\n\u003cli\u003eAboshady, H. M., F\u0026eacute;licit\u0026eacute;, Y., Hira, J., Barbier, C. \u0026amp; Bambou, J. C. Early Transcriptome Differences Between Pre-Infected and Na\u0026iuml;ve Kid Goats Infected With \u003cem\u003eHaemonchus contortus\u003c/em\u003e. \u003cem\u003eFront Vet Sci\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 873467 (2022). https://doi.org:10.3389/fvets.2022.873467\u003c/li\u003e\n\u003cli\u003eBowdridge, S., MacKinnon, K., McCann, J. C., Zajac, A. M. \u0026amp; Notter, D. R. Hair-type sheep generate an accelerated and longer-lived humoral immune response to \u003cem\u003eHaemonchus contortus\u003c/em\u003e infection. \u003cem\u003eVeterinary Parasitology\u003c/em\u003e \u003cstrong\u003e196\u003c/strong\u003e, 172-178 (2013). https://doi.org:https://doi.org/10.1016/j.vetpar.2013.01.008\u003c/li\u003e\n\u003cli\u003eMcRae, K. M.\u003cem\u003e et al.\u003c/em\u003e Transcriptional profiling of the ovine abomasal lymph node reveals a role for timing of the immune response in gastrointestinal nematode resistance. \u003cem\u003eVet. parasitology\u003c/em\u003e \u003cstrong\u003e224\u003c/strong\u003e, 96-108 (2016). https://doi.org:10.1016/j.vetpar.2016.05.014\u003c/li\u003e\n\u003cli\u003eMcRae, K. M., McEwan, J. C., Dodds, K. G. \u0026amp; Gemmell, N. J. Signatures of selection in sheep bred for resistance or susceptibility to gastrointestinal nematodes. \u003cem\u003eBMC Genomics\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 637 (2014). https://doi.org:10.1186/1471-2164-15-637\u003c/li\u003e\n\u003cli\u003eAlba-Hurtado, F. \u0026amp; Mu\u0026ntilde;oz-Guzm\u0026aacute;n, M. A. Immune responses associated with resistance to haemonchosis in sheep. \u003cem\u003eBiomed Res Int\u003c/em\u003e \u003cstrong\u003e2013\u003c/strong\u003e, 162158 (2013). https://doi.org:10.1155/2013/162158\u003c/li\u003e\n\u003cli\u003eEstrada-Reyes, Z.\u003cem\u003e et al.\u003c/em\u003e Cytokine and antioxidant gene profiles from peripheral blood mononuclear cells of Pelibuey lambs after \u003cem\u003eHaemonchus contortus\u003c/em\u003e infection. \u003cem\u003eParasite Immunol\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e (2017). https://doi.org:10.1111/pim.12427\u003c/li\u003e\n\u003cli\u003eGossner, A., Wilkie, H., Joshi, A. \u0026amp; Hopkins, J. Exploring the abomasal lymph node transcriptome for genes associated with resistance to the sheep nematode \u003cem\u003eTeladorsagia circumcincta\u003c/em\u003e. \u003cem\u003eVet Res\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 68 (2013). https://doi.org:10.1186/1297-9716-44-68\u003c/li\u003e\n\u003cli\u003eGrencis, R. K. Immunity to helminths: resistance, regulation, and susceptibility to gastrointestinal nematodes. \u003cem\u003eAnnu Rev Immunol\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 201-225 (2015). https://doi.org:10.1146/annurev-immunol-032713-120218\u003c/li\u003e\n\u003cli\u003eLacroux, C.\u003cem\u003e et al.\u003c/em\u003e \u003cem\u003eHaemonchus contortus\u003c/em\u003e (Nematoda: Trichostrongylidae) infection in lambs elicits an unequivocal T\u003csub\u003eH\u003c/sub\u003e2 immune response. \u003cem\u003eVet Res\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 607-622 (2006). https://doi.org:10.1051/vetres:2006022\u003c/li\u003e\n\u003cli\u003eTerefe, G.\u003cem\u003e et al.\u003c/em\u003e Immune response to \u003cem\u003eHaemonchus contortus\u003c/em\u003e infection in susceptible (INRA 401) and resistant (Barbados Black Belly) breeds of lambs. \u003cem\u003eParasite Immunol\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 415-424 (2007). https://doi.org:10.1111/j.1365-3024.2007.00958.x\u003c/li\u003e\n\u003cli\u003eToscano, J. H. B.\u003cem\u003e et al.\u003c/em\u003e Innate Immune Responses Associated with Resistance against \u003cem\u003eHaemonchus contortus\u003c/em\u003e in Morada Nova Sheep. \u003cem\u003eJ Immunol Res\u003c/em\u003e \u003cstrong\u003e2019\u003c/strong\u003e, 3562672 (2019). https://doi.org:10.1155/2019/3562672\u003c/li\u003e\n\u003cli\u003eJin, P.\u003cem\u003e et al.\u003c/em\u003e Interferon-\u0026gamma; and Tumor Necrosis Factor-\u0026alpha; Polarize Bone Marrow Stromal Cells Uniformly to a Th1 Phenotype. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 26345 (2016). https://doi.org:10.1038/srep26345\u003c/li\u003e\n\u003cli\u003eMiddleton, D., Garza, J. J., Greiner, S. P. \u0026amp; Bowdridge, S. A. Neutrophils rapidly produce Th2 cytokines in response to larval but not adult helminth antigen. \u003cem\u003eParasite Immunol\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, e12679 (2020). https://doi.org:10.1111/pim.12679\u003c/li\u003e\n\u003cli\u003eMiddleton, D. D. The effects of genetic differences in an innate immune receptor on immune responses to\u003cem\u003e Haemonchus contortus \u003c/em\u003ein sheep, PhD thesis, West Virginia University (2021).\u003c/li\u003e\n\u003cli\u003eJacobs, J. R., Sommers, K. N., Zajac, A. M., Notter, D. R. \u0026amp; Bowdridge, S. A. Early IL-4 gene expression in abomasum is associated with resistance to \u003cem\u003eHaemonchus contortus\u003c/em\u003e in hair and wool sheep breeds. \u003cem\u003eParasite Immunol\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 333-339 (2016). https://doi.org:10.1111/pim.12321\u003c/li\u003e\n\u003cli\u003eMacKinnon, K. M., Zajac, A. M., Kooyman, F. N. \u0026amp; Notter, D. R. Differences in immune parameters are associated with resistance to \u003cem\u003eHaemonchus contortus\u003c/em\u003e in Caribbean hair sheep. \u003cem\u003eParasite Immunol\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 484-493 (2010). https://doi.org:10.1111/j.1365-3024.2010.01211.x\u003c/li\u003e\n\u003cli\u003eBalic, A., Bowles, V. M. \u0026amp; Meeusen, E. N. Mechanisms of immunity to \u003cem\u003eHaemonchus contortus \u003c/em\u003einfection in sheep. \u003cem\u003eParasite Immunol\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 39-46 (2002). https://doi.org:10.1046/j.0141-9838.2001.00432.x\u003c/li\u003e\n\u003cli\u003eShepherd, E., Greiner, S. P. \u0026amp; Bowdridge, S. A. Characterization of ovine monocyte activity when cultured with \u003cem\u003eHaemonchus contortus\u003c/em\u003e larvae \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003eParasite Immunol\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, e12773 (2020). https://doi.org:10.1111/pim.12773\u003c/li\u003e\n\u003cli\u003eChan, L.\u003cem\u003e et al.\u003c/em\u003e The Roles of Neutrophils in Cytokine Storms. \u003cem\u003eViruses\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e (2021). https://doi.org:10.3390/v13112318\u003c/li\u003e\n\u003cli\u003eHolt, R. M., Shepherd, E. A., Ammer, A. G. \u0026amp; Bowdridge, S. A. Effects of peripheral blood mononuclear cells on \u003cem\u003eHaemonchus contortus\u003c/em\u003e larval motility \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003eParasite Immunol\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 553-556 (2015). https://doi.org:10.1111/pim.12219\u003c/li\u003e\n\u003cli\u003eBowdridge, S. A., Zajac, A. M. \u0026amp; Notter, D. R. St. Croix sheep produce a rapid and greater cellular immune response contributing to reduced establishment of \u003cem\u003eHaemonchus contortus\u003c/em\u003e. \u003cem\u003eVet. parasitology\u003c/em\u003e \u003cstrong\u003e208\u003c/strong\u003e, 204-210 (2015). https://doi.org:10.1016/j.vetpar.2015.01.019\u003c/li\u003e\n\u003cli\u003eHossain, M. Performance of Black Bengal goat: a 50-year review. \u003cem\u003eTrop Anim Health Prod\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e (2021). https://doi.org:10.1007/s11250-020-02477-2\u003c/li\u003e\n\u003cli\u003eRakib, M. R. H.\u003cem\u003e et al.\u003c/em\u003e Morphometric features and performances of Black Bengal goat in Bangladesh. \u003cem\u003eTrop Anim Health Prod\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 341 (2022). https://doi.org:10.1007/s11250-022-03334-0\u003c/li\u003e\n\u003cli\u003eAlam, M. B. B.\u003cem\u003e et al.\u003c/em\u003e Single nucleotide polymorphisms in candidate genes are significantly associated with resistance to \u003cem\u003eHaemonchus contortus\u003c/em\u003e infection in goats. \u003cem\u003eJ Anim Science Biotechnology\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 30 (2019). https://doi.org:10.1186/s40104-019-0327-8\u003c/li\u003e\n\u003cli\u003eKumar, R.\u003cem\u003e et al.\u003c/em\u003e Variability of resistance in Black Bengal goats naturally infected with Haemonchus contortus. \u003cem\u003eJ Parasit Dis\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 76-79 (2015). https://doi.org:10.1007/s12639-013-0282-9\u003c/li\u003e\n\u003cli\u003eEl-Ashram, S.\u003cem\u003e et al.\u003c/em\u003e Early and late gene expression profiles of the ovine mucosa in response to \u003cem\u003eHaemonchus contortus\u003c/em\u003e infection employing Illumina RNA-seq technology. \u003cem\u003eParasitol Int\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 681-692 (2017). https://doi.org:10.1016/j.parint.2017.05.007\u003c/li\u003e\n\u003cli\u003eHenriksen, S. \u0026amp; Aagaard, K. A simple flotation and McMaster method. (1977). \u003c/li\u003e\n\u003cli\u003ePe\u0026ntilde;a-Espinoza, M.\u003cem\u003e et al.\u003c/em\u003e Efficacy of ivermectin against gastrointestinal nematodes of cattle in Denmark evaluated by different methods for analysis of faecal egg count reduction. \u003cem\u003eInt J Parasit: Drugs and Drug Resistance\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 241-250 (2016). https://doi.org:https://doi.org/10.1016/j.ijpddr.2016.10.004\u003c/li\u003e\n\u003cli\u003ePierce, L., Sarkar, S., Chan, L. L.-Y., Lin, B. \u0026amp; Qiu, J. Outcomes from a cell viability workshop: fit-for-purpose considerations for cell viability measurements for cellular therapeutic products. \u003cem\u003eCell \u0026amp; Gene Therapy Insights\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 551-569 (2021). \u003c/li\u003e\n\u003cli\u003eWeinberg, A.\u003cem\u003e et al.\u003c/em\u003e Viability and functional activity of cryopreserved mononuclear cells. \u003cem\u003eClin Diagn Lab Immunol\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 714-716 (2000). https://doi.org:10.1128/cdli.7.4.714-716.2000\u003c/li\u003e\n\u003cli\u003eEscribano, C.\u003cem\u003e et al.\u003c/em\u003e Resistance to \u003cem\u003eHaemonchus contortus\u003c/em\u003e in Corriedale sheep is associated to high parasite-specific IgA titer and a systemic Th2 immune response. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 19579 (2019). https://doi.org:10.1038/s41598-019-55447-6\u003c/li\u003e\n\u003cli\u003eMungu\u0026iacute;a, B.\u003cem\u003e et al.\u003c/em\u003e Sensitivity of \u003cem\u003eHaemonchus contortus\u003c/em\u003e to anthelmintics using different in vitro screening assays: a comparative study. \u003cem\u003eParasit Vectors\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 129 (2022). https://doi.org:10.1186/s13071-022-05253-3\u003c/li\u003e\n\u003cli\u003eJohnston, C.\u003cem\u003e et al.\u003c/em\u003e Cultivation of \u003cem\u003eHeligmosomoides Polygyrus\u003c/em\u003e: An Immunomodulatory Nematode Parasite and its Secreted Products. \u003cem\u003eJ Vis Exp\u003c/em\u003e \u003cstrong\u003e2015\u003c/strong\u003e (2015). https://doi.org:10.3791/52412\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Coiradas, L.\u003cem\u003e et al.\u003c/em\u003e Immunization against Lamb Haemonchosis with a Recombinant Somatic Antigen of \u003cem\u003eHaemonchus contortus\u003c/em\u003e (rHcp26/23). \u003cem\u003eVet Med Int\u003c/em\u003e \u003cstrong\u003e2010\u003c/strong\u003e, 852146 (2010). https://doi.org:10.4061/2010/852146\u003c/li\u003e\n\u003cli\u003eWen, Z.\u003cem\u003e et al.\u003c/em\u003e In vitro characterization of \u003cem\u003eHaemonchus contortus\u003c/em\u003e trehalose-6-phosphate phosphatase and its immunomodulatory effects on peripheral blood mononuclear cells (PBMCs). \u003cem\u003eParasit Vectors\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 611 (2021). https://doi.org:10.1186/s13071-021-05115-4\u003c/li\u003e\n\u003cli\u003eIngham, A., Reverter, A., Windon, R., Hunt, P. \u0026amp; Menzies, M. Gastrointestinal nematode challenge induces some conserved gene expression changes in the gut mucosa of genetically resistant sheep. \u003cem\u003eInt J Parasitol\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 431-442 (2008). https://doi.org:10.1016/j.ijpara.2007.07.012\u003c/li\u003e\n\u003cli\u003eWalker, J. A. \u0026amp; McKenzie, A. N. J. T(H)2 cell development and function. \u003cem\u003eNat Rev Immunol\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 121-133 (2018). https://doi.org:10.1038/nri.2017.118\u003c/li\u003e\n\u003cli\u003eEhsan, M.\u003cem\u003e et al.\u003c/em\u003e Recombinant elongation factor 1 alpha of \u003cem\u003eHaemonchus contortus\u003c/em\u003e affects the functions of goat PBMCs. \u003cem\u003eParasite Immunol\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, e12703 (2020). https://doi.org:10.1111/pim.12703\u003c/li\u003e\n\u003cli\u003eHe, L.\u003cem\u003e et al.\u003c/em\u003e A TGF-\u0026beta; type II receptor that associates with developmental transition in \u003cem\u003eHaemonchus contortus\u003c/em\u003e \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003ePLoS Negl Trop Dis\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e0007913 (2019). https://doi.org:10.1371/journal.pntd.0007913\u003c/li\u003e\n\u003cli\u003eLivak, K. J. \u0026amp; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. \u003cem\u003eMethods\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 402-408 (2001). https://doi.org:10.1006/meth.2001.1262\u003c/li\u003e\n\u003cli\u003eBambou, J. C., Larcher, T., Ce\u0026iuml;, W., Dumoulin, P. J. \u0026amp; Mandonnet, N. Effect of Experimental Infection with \u003cem\u003eHaemonchus contortus\u003c/em\u003e on Parasitological and Local Cellular Responses in Resistant and Susceptible Young Creole Goats. \u003cem\u003eBioMed Research International\u003c/em\u003e \u003cstrong\u003e2013\u003c/strong\u003e, 902759 (2013). https://doi.org:10.1155/2013/902759\u003c/li\u003e\n\u003cli\u003eBambou, J.-C.\u003cem\u003e et al.\u003c/em\u003e Peripheral immune response in resistant and susceptible Creole kids experimentally infected with \u003cem\u003eHaemonchus contortus\u003c/em\u003e. \u003cem\u003eSmall Ruminant Research\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e, 34-39 (2009). https://doi.org:https://doi.org/10.1016/j.smallrumres.2009.01.008\u003c/li\u003e\n\u003cli\u003eClutterbuck, E.\u003cem\u003e et al.\u003c/em\u003e Recombinant human interleukin 5 is an eosinophil differentiation factor but has no activity in standard human B cell growth factor assays. \u003cem\u003eEur J Immunol\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1743-1750 (1987). https://doi.org:10.1002/eji.1830171210\u003c/li\u003e\n\u003cli\u003eChen, F.\u003cem\u003e et al.\u003c/em\u003e Neutrophils prime a long-lived effector macrophage phenotype that mediates accelerated helminth expulsion. \u003cem\u003eNat Immunol\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 938-946 (2014). https://doi.org:10.1038/ni.2984\u003c/li\u003e\n\u003cli\u003eAnthony, R. M., Rutitzky, L. I., Urban, J. F., Jr., Stadecker, M. J. \u0026amp; Gause, W. C. Protective immune mechanisms in helminth infection. \u003cem\u003eNat Rev Immunol\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 975-987 (2007). https://doi.org:10.1038/nri2199\u003c/li\u003e\n\u003cli\u003eCliffe, L. J. \u0026amp; Grencis, R. K. The \u003cem\u003eTrichuris muris\u003c/em\u003e system: a paradigm of resistance and susceptibility to intestinal nematode infection. \u003cem\u003eAdv Parasitol\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 255-307 (2004). https://doi.org:10.1016/s0065-308x(04)57004-5\u003c/li\u003e\n\u003cli\u003eMaizels, R. M. \u0026amp; Yazdanbakhsh, M. Immune regulation by helminth parasites: cellular and molecular mechanisms. \u003cem\u003eNat Rev Immunol\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 733-744 (2003). https://doi.org:10.1038/nri1183\u003c/li\u003e\n\u003cli\u003eLa Flamme, A. C.\u003cem\u003e et al.\u003c/em\u003e Type II-activated murine macrophages produce IL-4. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e46989 (2012). https://doi.org:10.1371/journal.pone.0046989\u003c/li\u003e\n\u003cli\u003eJacobs, J. R., Greiner, S. P. \u0026amp; Bowdridge, S. A. Serum interleukin-4 (IL-4) production is associated with lower fecal egg count in parasite-resistant sheep. \u003cem\u003eVet parasitology\u003c/em\u003e \u003cstrong\u003e211\u003c/strong\u003e, 102-105 (2015). https://doi.org:10.1016/j.vetpar.2015.04.024\u003c/li\u003e\n\u003cli\u003eBenjamin, L. E., Hemo, I. \u0026amp; Keshet, E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF. \u003cem\u003eDevelopment\u003c/em\u003e \u003cstrong\u003e125\u003c/strong\u003e, 1591-1598 (1998). https://doi.org:10.1242/dev.125.9.1591\u003c/li\u003e\n\u003cli\u003eShepherd, E., Greiner, S. P., Russ, B. \u0026amp; Bowdridge, S. A. Interleukin-13 induces paralysis of \u003cem\u003eHaemonchus contortus\u003c/em\u003e larvae in vitro. \u003cem\u003eParasite Immunol\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, e12758 (2020). https://doi.org:10.1111/pim.12758\u003c/li\u003e\n\u003cli\u003eMartinez, F. O. \u0026amp; Gordon, S. The M1 and M2 paradigm of macrophage activation: time for reassessment. \u003cem\u003eF1000Prime Rep\u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 13 (2014). https://doi.org:10.12703/p6-13\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Black Bengal goat, PBMC, H. contortus, Resistance, Cytokines, TH2, Bangladesh","lastPublishedDoi":"10.21203/rs.3.rs-4620150/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4620150/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBlack Bengal goat (BBG) is a native breed of Bangladesh and its ability in reducing the establishment of H. \u003cem\u003econtortus\u003c/em\u003e infection through cellular and immune responses was unexplored. \u003cem\u003eIn vitro\u003c/em\u003e larval motility in response to PBMC, \u003cem\u003ein vivo\u003c/em\u003e larval infectivity assay, differential blood cell counts, histopathology, cytokine and transcription expression have been investigated in BBG kids following \u003cem\u003eH. contortus\u003c/em\u003e L\u003csub\u003e3\u003c/sub\u003e. \u003cem\u003eIn vitro\u003c/em\u003e motility experiment revealed that L\u003csub\u003e3\u003c/sub\u003e exposed to PBMC from primed kids had significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduced speed and straight-line distance compared to L\u003csub\u003e3\u003c/sub\u003e exposed to PBMC from naive kids. Kids with L\u003csub\u003e3\u003c/sub\u003e exposed to PBMC in primed and naive kids, reduced \u003cem\u003eH. contortus\u003c/em\u003e egg shed. Upon 10,000 L\u003csub\u003e3\u003c/sub\u003e challenge, WBC count was higher in primed kids compared to naive and 35% of TC were neutrophils. Lymph nodes were increased in weight (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in primed kids except 1st day (D0). IL-4, IL-5, IL-13, IL-33, MCP1, CXCL1, TLR2 and GAL14 were expressed both naive and primed kids. Significant expression of IL-4, IL-5, IL-13, IL-33, MCP1 and CXCL1 at D3, D5 and D7, suggesting early T\u003csub\u003eH\u003c/sub\u003e2 differentiation in primed kids. These results suggest that BBG can much resist \u003cem\u003eH. contortus\u003c/em\u003e infectivity. Role of transcriptome differences in other resistant breeds should be investigated.\u003c/p\u003e","manuscriptTitle":"Peripheral Blood Mononuclear Cells Regulated Cytokines and Transcription Factors Reduce Haemonchus contortus Larval Establishment in Black Bengal Goat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 05:37:14","doi":"10.21203/rs.3.rs-4620150/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ea01cff5-83be-4f9b-9297-726e5295fa89","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34698140,"name":"Biological sciences/Cell biology"},{"id":34698141,"name":"Biological sciences/Genetics"},{"id":34698142,"name":"Biological sciences/Immunology"},{"id":34698143,"name":"Biological sciences/Molecular biology"},{"id":34698144,"name":"Health sciences/Diseases"},{"id":34698145,"name":"Health sciences/Gastroenterology"}],"tags":[],"updatedAt":"2025-05-08T16:38:33+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-18 05:37:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4620150","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4620150","identity":"rs-4620150","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}