Hypobiosis and Development of Haemonchus contortus and Trichostrongylus colubriformis Infection in Lambs under Different Levels of Nutrition

Abstract

The influence of four levels of dietary supplementation on the development of the immune response and on the biology of Haemonchus contortus and Trichostrongylus colubriformis was evaluated in Dorper lambs under serial artificial infections with 1000 infective larvae (L3) of each species; the supplement was given every 3 days for 12 weeks. For each of the four diets formulated, one infected group (n = 7) and one control group (n = 4) were set up. Diets 1, 2, 3, and 4 contained 0%, 25%, 50%, and 75% of concentrate, respectively. There was an evident immune response to infection in all groups, manifested through eosinophilia and the production of anti-L3 immunoglobulins. There was a significant inverse relationship between the levels of nutrients in the diets and the numbers of late fourth-stage larvae, immature adult stages, and adults of H. contortus. Following 12 weeks of serial infections, most of the H. contortus population was found at the early fourth stage, indicating the occurrence of hypobiosis. The early fourth stage represented 59.5%, 75.8%, 83.3%, and 86.3% of the total Haemonchus worm burden, respectively, in Diets 1, 2, 3, and 4. In the case of T. colubriformis, hypobiosis was not observed, and diet had no influence on the establishment of parasites.

1. Introduction

Haemonchus contortus and Trichostrongylus colubriformis are considered to be the most important gastrointestinal nematodes of sheep in Brazil [1,2]. T. colubriformis causes disorders in the mucosa of the small intestine; these disorders affect the digestion and absorption of nutrients and lead to the impairment of feed utilization [3,4,5]. Blood-sucking H. contortus parasitism leads to anemia, weakness, and, frequently, deaths, particularly among young sheep and periparturient ewes [6,7]. Populations of both H. contortus and T. colubriformis with multidrug resistance are widespread [8,9,10], which makes it challenging to provide proper prophylaxis for parasitic infections and, in some circumstances, impractical to have sheep production under the existing grazing conditions.

Significant reductions in performance occur among young sheep that are mix-infected with H. contortus and T. colubriformis. Dorper lambs under conditions of serial artificial infection showed a 13.7% reduction in food consumption, 21.3% reduction in daily body weight gain, and 16.6% reduction in cold carcass weight [11]. A study on grazing lambs showed that supplemented animals had lower fecal egg counts (FECs) compared with the unsupplemented groups [12].

Under a climate characterized by warm, rainy summers and dry winters, there is no seasonality in the transmission of H. contortus and T. colubriformis to sheep. Large worm burdens may develop even during periods of drought due to the presence of infective larvae (L3) that are able to survive despite the low environmental humidity [1]. L3 are considerably resilient and can survive on pasture and inside sheep fecal pellets for a few months, provided that temperatures are not extreme and moisture is sufficient [13].

Once L3 have been ingested by the host, their success in becoming established in the digestive tract depends on the factors related to the host’s immune response. The optimal situation occurs when larvae are ingested by a naïve host. Conversely, in sheep that have developed immunity as a result of ongoing infections, larval establishment may be drastically reduced [14,15]. In this case, the infective larvae are often unable to settle in the mucosa and are eliminated. The development of the immune response is related to the release of epithelial-derived cytokines that activate a variety of innate immune cells that promote adaptive Th2 responses. Type 2 cytokines, either from innate or adaptive origin, activate effector mechanisms in the gut tissue. IL-5 and IL-9 recruit eosinophils and mast cells, and IL-13 enhances the turnover of the epithelium. Goblet cell hyperplasia, augmented mucus production, along with changes in its composition, the production of resistin-like molecule b (RELM-b), and increased muscle contractility are common effector mechanisms against helminths driven by both IL-4 and IL-13. All these local changes favor parasite dislodging and clearance [16].

As a way to evade the host’s immune response, the development of established larvae becomes inhibited, usually when the larvae are at the early fourth stage (early L4). This process of inhibition is called hypobiosis, dormancy, or arrested development. In the case of H. contortus, about 90% of hypobiotic larvae are recovered from the abomasal contents and washings, thereby suggesting that early L4 may be only loosely attached to the abomasal mucosa, from which they may be dislodged during the processing of the abomasa for worm examination [17].

Some evidence suggesting a connection between the presence of adult worms and the inhibition of development exists. Hypobiosis might involve a threshold of infection that is regulated by the adult worm population already established in the gastrointestinal tract. It was found that greater numbers of inhibited H. contortus larvae were present when a challenge was superimposed on extant infections, thus indicating that resident worms or a factor activated by their presence induced developmental arrest [14,18,19].

Hypobiosis may also be triggered when the environmental conditions are adverse for the development and survival of the free-living stages. Because the infective stage of H. contortus is highly susceptible to winter conditions, hypobiosis is usually the major factor in overwinter survival in cold temperate zones, with the arrested development of the majority of L3 ingested occurring in autumn. Rapid development during short periods of high summer temperatures can lead to haemonchosis [7].

There are also indications that hypobiosis can occur due to a lack of moisture in markedly seasonal climates with long and hot dry seasons in semi-arid regions, which are also adverse for the development and survival of the free-living stages. Under such conditions, hypobiosis largely explains the common, though sporadic, outbreaks of haemonchosis. There have been instances when acute haemonchosis was reported during the dry season, even before the onset of the rains in semi-arid areas of Kenya. The source of this disease was attributed to the resumption of the development of hypobiotic larvae [17].

Although less frequent, there are also reports of the inhibited development of Trichostrongylus spp. While other trichostrongylids are inhibited as early L4 larvae, Trichostrongylus spp. is found to be inhibited as L3 [20,21]. The main cause of the inhibition of Trichostrongylus spp. in the Netherlands is, presumably, the immunity of adult sheep [20].

Because of the importance of hypobiosis in the epidemiology of parasitic gastroenteritis, the present experiment was carried out under controlled conditions with the aim of determining the influence of dietary supplementation on the development of both the immune response and the biology of H. contortus and T. colubriformis in Dorper lambs.

2. Materials and Methods

All the procedures involving animals in this study were conducted in accordance with the international ethical standards and were approved by the local ethics committee on animal use (protocol number 78/2014-CEUA; FMVZ/UNESP).

2.1. Description of the Experiment

Forty-four lambs raised in pens from lambing and with low exposure to helminth infections were used. All animals were treated orally with a combination of monepantel (2.5 mg/kg; Zolvix^®, Novartis, Dundee, UK), albendazole (10 mg/kg; Valbazen^®, Pfizer, Campinas, Brazil), and levamisole (10 mg/kg; Ripercol^®, Fort Dodge, Campinas, Brazil). A qualitative fecal examination [22] upon arrival at the university facilities demonstrated that 11 animals were infected with strongylids and 42 were infected with Strongyloides papillosus. No nematode eggs were found in fecal examinations performed 14 days after the anthelmintic treatment.

The lambs were fed with one of the following four diets: Diet 1—hay alone (7.4% metabolizable protein (MP) and 1861 kcal/kg of metabolizable energy (ME)); Diet 2—proportions of 75% hay and 25% concentrate (8.8% MP and 2133 kcal/kg ME); Diet 3—proportions of 50% hay and 50% concentrate (10.2% MP and 2418 kcal/kg ME); and Diet 4—proportions of 25% hay and 75% concentrate (11.5% MP and 2726 kcal/kg ME). The experiment used a completely randomized 2 × 4 factorial design (infected or control × four diets) with 44 Dorper lambs randomized to the eight treatments as follows:

• Diet 1—infected (n = 7) and control (n = 4);
• Diet 2—infected (n = 7) and control (n = 4);
• Diet 3—infected (n = 7) and control (n = 4);
• Diet 4—infected (n = 6) and control (n = 4).

One of the initial seven animals in the infected group that received Diet 4 was excluded from the experiment because it did not adapt to the diet and showed clinical signs of ruminal acidosis.

The diets for this experiment consisted of ground coast cross hay (Cynodon dactylon) and concentrate formulated with ground corn, soybean meal, calcitic limestone, urea, and ammonium chloride [11]. Before the beginning of the serial infections, the animals were subjected to a period of adaptation to the diets over three weeks. The lambs were kept in individual pens (3 m²), where they had free access (ad libitum) to the diets and water. At the time of the initial infection, the lambs were four months of age and had a mean body weight of 31.5 ± 3.24 kg.

2.2. Nematode Isolates, Production of L3, and Serial Infections

The T. colubriformis isolate was obtained in 2003 from a sheep that was naturally infected with Haemonchus spp. and Trichostrongylus spp. In order to eliminate Haemonchus and thus isolate the T. colubriformis L3, two treatments with organophosphate were carried out (12 June 2003 and 4 July 2003) at a dose of 100 mg/kg body weight (Neguvon-Bayer), which caused the elimination of the Haemonchus infection [23].

The H. contortus isolate, previously characterized as multiple-resistant, was obtained from two male lambs acquired in Pratânia, state of São Paulo, in May 2006 [24]. Upon arrival at the UNESP facilities, the animals were treated with moxidectin (200 μg/kg, Cydectin NF*, 1% injectable solution, Fort Dodge^®). On that day, the two lambs presented 1100 and 3200 eggs per gram (EPG). Only Haemonchus larvae were present in the coprocultures. Seven days after treatment, the animals showed increases in fecal egg counts (FECs), respectively, to 5600 and 7400 EPG, and the coprocultures confirmed that they were only infected with Haemonchus spp. Since the time of isolation, the L3 of both isolates were kept frozen in liquid nitrogen.

Immediately after the preserved L3 were thawed, donor lambs for each nematode species were infected to replicate the mono-infections and obtain the necessary number of L3 for the serial infections later on. The monospecific infections of donor lambs were confirmed based on L3 morphology [22].

Each lamb assigned to the four diet groups received 1000 L3 of H. contortus and 1000 L3 of T. colubriformis every three days, thus totaling 28,000 L3 of each species over the 12 weeks of the infection period (Figure 1).

2.3. Blood Samples

Once a week, blood samples (5 mL) were collected by means of jugular vein puncture into Vacutainer^® tubes containing anticoagulant (EDTA). Blood eosinophils were quantified, and the excess blood was centrifuged to collect plasma, which was stored at −20 °C for further immunological tests.

2.4. Worm Examination

All the four groups started the trial at the same time (i.e., Day 0), and all 43 lambs were slaughtered on the same day (Day 84), four days after the last infection, for the recovery and enumeration of parasites (Figure 1). After they had been euthanized, the abomasa and small intestines were frozen at −20 °C. Two weeks later, the organs started to be thawed (samples of six lambs were processed per day); each organ was washed separately in saline solution and their contents were fixed in 5% formaldehyde. The abomasal and small intestine contents were collected, and the parasites present in a 10% subsample were counted, sexed, and classified according to their stage of development and species [22].

2.5. Enzyme-Linked Immunosorbent Assay (ELISA)

The plasma levels of IgG antibodies against the total antigens of H. contortus and T. colubriformis L3 were estimated using an ELISA. The antigen production method used has been previously described [25]. When measuring the parasite-specific plasma IgG levels, we followed a protocol previously described [26] with the following modifications: plates were coated with 2.5 µg/mL of antigen and then incubated with peroxidase-conjugated rabbit-anti sheep IgG diluted at 1:40,000 (A130-101P, Bethyl Laboratories, Inc., Montgomery, TX, USA). The results were expressed as the percentage of the optical density value (OD) of the positive standard serum by means of the following formula [27]:

% OD = [(OD mean of the tested serum − OD mean of blank)/(OD mean of the positive standard serum − OD mean of blank)] × 100.

2.6. Statistical Analysis

Data were subjected to a normality test (Shapiro–Wilk test) and, when necessary, transformed to log₁₀ (x + 1), this being the case of the worm counting and eosinophils data. Data were assessed using an analysis of variance for the variables with just one measurement (worm counting). For the weekly measurements (eosinophils and IgG), repeated-measurement analysis using the general linear models (GLMs) procedure of the Statistical Analysis System, version 9.4 [28], was used. Diet and infection status were the classes evaluated. Averages were compared by means of Tukey’s test at a 5% significance level, and only significant interactions at this level were reported in the results.

The association between the average number of worms and the chemical composition of the diets (metabolizable protein and metabolizable energy) was evaluated by means of linear regression.

3. Results

3.1. Haemonchus contortus

The average total worm counts (with minimum–maximum values) of H. contortus were: 10,269 (6380–13,240) for Diet 1, 6916 (530–9470) for Diet 2, 8153 (350–13,090) for Diet 3, and 7227 (830–12,730) for Diet 4, which corresponded to mean (upper and lower confidence interval limits (95%)) rates of establishment, respectively, of 36.7% (29–44), 24.7% (15–34), 29.1% (15–43), and 25.9% (11–41) of the 28,000 L3 administered to the lambs during the trial. Within the H. contortus worm population that became established, most specimens were found at the early L4 stage (Figure 2), which represented 59.5% (39.6–79.4), 75.8% (62.7–89.0), 83.3% (71.8–94.7), and 86.3% (69.3–103.2) of the total Haemonchus worm burden in the Diet 1, 2, 3, and 4 groups, respectively. The averages of late-L4 males, early L5 males, and adult females were significantly lower in Diet 4 in comparison with the Diet 1 group (p < 0.05).

Figure 3 presents the results regarding the association between H. contortus worm counts and the levels of metabolizable protein (MP). There was no association between the level of MP in the diet and the early L4 counts (r = −0.25; p > 0.05). Conversely, significant inverse associations were detected between MP and the other stages for the development of Haemonchus with correlation coefficients ranging from −0.92 to −0.99. For example, the numbers of adults corresponded to 10.6%, 5.1%, 4.8%, and 3.3% of the numbers of L3 given, respectively, in Diets 1, 2, 3, and 4. The slopes of the regression analysis were negative and statistically significant (p < 0.05) for late-L4 males (−0.355), late-L4 females (−0.355), early L5 males (−0.301), adult males (−0.289), and adult females (−0.344). The correlation coefficients between metabolizable energy (ME) and the total number of H. contortus worms matched those obtained using MP. To avoid repetition, they are not presented here.

3.2. Trichostrongylus colubriformis

No group differences were detected regarding T. colubriformis worm counting (p > 0.05), and there were no significant correlations between worm counts and levels of MP or ME in the diets. The T. colubriformis worm burdens were as follows: 8191 (6800–10,490) in Diet 1; 9493 (6780–11,830) in Diet 2; 8654 (4240–12,230) in Diet 3; and 7897 (3320–9690) in Diet 4. These corresponded to the establishment of 29.3% (25–33), 33.9% (28–40), 30.9% (22–40), and 28.2% (19–37), respectively, of the 28,000 L3 specimens that were given to the lambs during the trial. Differently from Haemonchus, most of the T. colubriformis worms were found in the adult stage (Figure 2) with percentages of 95.4%, 94.6%, 96.2%, and 98.0% of the total worm burden in the groups with Diets 1, 2, 3, and 4, respectively. Therefore, there was no evidence of arrested development in T. colubriformis.

The non-infected control lambs did not present worms at the end of the trial.

3.3. Immune Response

Infection had a significant effect on eosinophil counts (p < 0.001). The averages in the non-infected control groups remained below 350 eosinophils/µL of blood during the study, while the infected lambs showed increases in blood eosinophil counts (Figure 4), with significant differences (p < 0.05) being detected between infected and non-infected animals from day 14 until the end of the trial (Figure 4). No significant diet * infection interaction and no diet effect were recorded in relation to blood eosinophils (p > 0.05).

Significant time * infection interactions (p < 0.0001) occurred in relation to IgG values. Sharp increases in the averages for IgG anti-H. contortus and anti-T. colubriformis L3 antigens were observed, with significant infection effects (p < 0.05), starting at days 28 and 42 post-infection, respectively, for T. colubriformis and H. contortus, and continuing until the end of the study (Figure 5). The IgG levels remained high and stable in the last three samplings from the infected animals. No diet effect was recorded (p > 0.05).

4. Discussion

An inverse relationship between the levels of nutrients in the diets (MP and ME) and the numbers of late-L4, early L5, and adults of H. contortus was observed based on the high negative correlation coefficient recorded between the average numbers of specimens of each stage of development and the level of metabolizable protein in the four diets. These results clearly showed that the quality of the diet had a significant impact on H. contortus parasitism. This finding was in agreement with those of other studies, which also showed that resistance to nematodes was influenced by diet [29], and particularly by the supply of metabolizable protein [30]. There was an evident immune response to infection in the lambs of all diet groups, manifested by eosinophilia and the production of anti-L3 immunoglobulins in comparison with the non-infected controls. These results are in agreement with studies conducted with infected and non-infected sheep, demonstrating an important role of innate and adaptative response against helminth infection [16]. However, even the animals fed only with hay were able to produce antibodies and inflammatory cells in numbers similar to those observed in the other supplemented groups. Therefore, it was not possible to detect any influence from the diets on these immunological variables.

Multiple pathways related to the immune response and tissue repair play critical roles in the resistance process. The type 2 immune response (Th2) has evolved to direct wound-healing machinery not only to repairing and remodeling tissue but also to mediating the containment, destruction, or expulsion of helminths [31]. The Th2 response induces mast cell hyperplasia, eosinophilia, and the production of anti-parasite immunoglobulins, as demonstrated in the infected lambs of the present trial. Resistance has been correlated also with hemostasis, which is important for stopping bleeding, thereby deterring parasite feeding. This, in association with increased mucus production, could accelerate parasite expulsion [32]. Therefore, it is possible that with an increase in diet quality, parasites may find a less friendly environment in well-nourished lambs, which would lead to a reduction in the numbers of larvae under development and the numbers of adult worms. In this context, animals kept on a nutrient-rich diet would be able to respond more efficiently, with a greater capacity for clotting and for repairing tissue damage, thus reducing the harm caused by H. contortus.

There was a marked difference between the species regarding the developmental stages at which the parasites were found. While we did not find evidence of hypobiosis involving T. colubriformis, most of the H. contortus parasite population was in the early fourth stage, thus indicating the inhibition of development. However, several larvae administered at the end of the study (especially at days 78 and 81) could be developing and not hypobiotic at day 84, when animals were euthanized. In the case of H. contortus, most L3 moult to L4 by day 3 and L4 to L5 by day 9–11 [33]. Therefore, it is not possible to assume that all early L4 recovered at necropsy in this study were indeed arrested. Nevertheless, in the present trial, in which infection was administered over a 12-week period, the early L4 stage represented 56.4%, 67.2%, 78.4%, and 77.4% of the total Haemonchus worm burden, respectively, in the groups fed with Diets 1, 2, 3, and 4. In other studies conducted at the authors’ same laboratory, the proportion of H. contortus early L4 larvae was relatively high (around 20–30%) when lambs acquired natural infections while grazing on contaminated pasture [1,34]. Interestingly, in the animals of the same trials that were also infected with T. colubriformis, virtually the entire population of T. colubriformis recovered was made up of adults [1,34]. Similar results were reported in the United States, from St. Croix × Dorset lambs that were kept on contaminated pasture for eight weeks [35]. Those lambs had parasite counts of 1535 adults and 2964 early L4 larvae of H. contortus. The same animals also had a high T. colubriformis worm burden (17,471 adult worms), but without evidence of inhibited development in this species.

Our results, as well as those of Barger et al. [19], indicated that in the case of H. contortus, the existence of an adult population established in the abomasum is important for the induction of hypobiosis. Similarly, some evidence suggesting a connection between the presence of adult worms and the inhibition of the development of Ostertagia ostertagi in cattle exists [36]. The elimination of adult worms may be a factor that triggers the resumption of development. The removal by anthelmintic treatment of adult worms from calves that had been infected daily for 204 days with larvae of O. ostertagi stimulated the resumed development of inhibited early fourth-stage larvae [36]. Possibly, H. contortus larvae in hypobiosis resume development to replace the adults lost due to anthelmintic treatments or senescence. There is evidence that adult H. contortus worms start to be eliminated gradually after a peak in egg output around 7 weeks after a single infection with 4000 L3, and only a few adults remain by 16 weeks post-infection in lambs [15].

In the present experiment, as well as in other studies on T. colubriformis, adult worms predominated, regardless of the type of infection (natural or artificial) [3,35]. Therefore, hypobiosis of T. colubriformis does not seem to occur in lambs, or it is far less important in comparison with its occurrence in H. contortus. However, Dobson et al. [21] found L3 of T. colubriformis in a state of hypobiosis and drew attention to the difficulty of quantifying the parasites at this stage, which could lead to underestimating the real number of worms in hypobiosis.

Brazilian studies on the importance of hypobiosis in the epidemiology of parasitic gastroenteritis are scarce. It is likely that the disinhibition of hypobiotic larvae plays an important role in outbreaks during the periparturient relaxation of immunity in ewes. Outbreaks of haemonchosis have also been reported in small ruminants during periods of prolonged drought, when it is unlikely that there is any source of environmental infection. In this case, the resumption of the development of hypobiotic larvae is possibly involved in these outbreaks. Therefore, the present results show that more attention should be given to hypobiosis in epidemiological studies and also to designing approaches for the sustainable control of parasitic gastroenteritis. The efficacy of anthelmintic treatments against hypobiotic larvae is possibly reduced. A similar problem can occur with the vaccine against haemonchosis (Barbervax^®) because the worms are affected when they ingest blood with antibodies that attach to the lining of the worm intestine, blocking digestion and starving the parasite. Because the hypobiotic larvae do not feed, they might survive. These issues need to be addressed in further studies.

5. Conclusions

Following 12 weeks of serial infections, most of the H. contortus population was found at the early L4 stage, indicating the occurrence of hypobiosis. As the amount of metabolizable energy and metabolizable protein in the diet of Dorper lambs increased, there were decreases in the recovery of H. contortus at the late-L4, early L5, and adult stages. However, there was no effect from the diet on the numbers of early L4, which became the predominant stage in the H. contortus abomasal population. In the case of T. colubriformis, the phenomenon of hypobiosis was not observed, and the diet had no influence on the establishment of the parasite.

There was an evident immune response to infection in the lambs of all diet groups, manifested by eosinophilia and the production of anti-L3 immunoglobulins in comparison with the non-infected controls, but it was not possible to detect any influence from the diets on these immunological variables.