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

doi:10.21203/rs.3.rs-4620150/v1

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

https://doi.org/10.21203/rs.3.rs-4620150/v1

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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 L₃. In vitro motility experiment revealed that L₃ exposed to PBMC from primed kids had significantly (P < 0.05) reduced speed and straight-line distance compared to L₃ exposed to PBMC from naive kids. Kids with L₃ exposed to PBMC in primed and naive kids, reduced H. contortus egg shed. Upon 10,000 L₃ 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_H2 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

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_H1) and helper T cell (T_H2) 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_H1 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_H2-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_H2 cytokines to support the T_H2 differentiation of adaptive immune cells.

Neutrophils have been demonstrated to bind, form extracellular traps, and induce morbidity to larvae (Hc L₃) in vitro ^10,22,28,29. The early response of resistant sheep monocytes accelerates T_H2 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 L₃ infection.

Larval count

Following oral administration of H. contortus 10,000 L₃, number of L₄ retrieved from primed kids' abomasum was significantly (p<0.01) lower compared to naive kids at D7 (Figure 2 A). The average number of L₄ 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_H2 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_H2 polarisation and indicates that the T_H1 response is down-regulated in both naïve and primed kids. The expression of transcription factor also contributes to T_H2 polarisation. The presence of GATA-3 from D0 to D7, with the greatest expression at D5, validated T_H2 polarisation. Primed kids showed higher expression of GATA-3 at D3 and D5 compared to naïve kids, indicating early T_H2 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_H1 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_H2 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 L₃ 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.

In the present study, we investigated the cellular and immune response of BBG kids regarding H. contortus larval (L₃) 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 L₃to 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 L₃ 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 L₃ 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 recoreded²⁶. 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 sheep³⁰. 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_H2 responses and it was indicated by high levels of the cytokine's interleukin 4 (IL-4) and IL-13 while T_H1 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_H1 response and contributes to T_H2 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 expression⁵⁷. Primed kids had also greater IL-5 expression than naive kids that indicates early activation of eosinophil due to repeated exoposure⁵². 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_H2 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 infections⁵⁷ and therefore assist in T_H2 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_H2 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 abomasa¹⁰. These findings points that the breed Black Bengal initiate local T_H2 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 L₃compared to kids where naive and controlled PBMC-exposed H. contortus L₃weregiven 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 L₃ cultured with eosinophils ¹⁹, further substantiating the role of innate cells on H. contortus L₃ viability, both in vitro and in vivo. Furthermore, recent exploration of single nucleotide polymorphism in immune genes such as CIITA, ATP2A3, 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.

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_H2 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.

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 L₃with a 10 ml syringe containing 2 ml of a suspension of L₃ at 1,000 L₃/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 L₃ with a 10 ml syringe containing 10 ml of a suspension of L₃ at 1,000 L₃/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 10³/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 N₂until used ⁴⁰.

Haemonchus contortus third stage larva (L₃) 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% CO₂ and 37 °C for 30 min before use in supplemented RPMI culture medium⁴¹. Third-stage larvae (L₃) were obtained from H. contortus eggs by incubating humidified feces from infected sheep at 27 °C for 1 week ⁴². Once collected, L₃ were kept in 100 cm³ corning cell culture flask and maintained at 5-9 °C with approximately 1000 L₃ 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 × 10⁶ 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 10⁵ 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 L₃ 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 L₃ 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% CO₂⁴⁴.

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_H1 activity and T_H2 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. RT² 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^oC for 20 sec, 40 cycles of 95^oC for 3 sec and 60^oC for 30 sec, and 60^oC 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.

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.

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

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