Investigating the Benefits of Target Selective Treatment by Average Daily Weight Gain Against Gastrointestinal Nematodes in Morada Nova Lambs

doi:10.21203/rs.3.rs-1499122/v1

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Investigating the Benefits of Target Selective Treatment by Average Daily Weight Gain Against Gastrointestinal Nematodes in Morada Nova Lambs

https://doi.org/10.21203/rs.3.rs-1499122/v1

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Gastrointestinal nematodes are the major obstacle to sheep farming, and their rational control depends on reliable parameters that guarantee both productivity and animal health. Thus, the objective of this study was to evaluate a method of targeted selective treatment (TST) of Morada Nova lambs, using the average daily weight gain (DWG). For this, 114 lambs in the rainy and 102 in the dry season were randomized into three treatments: Control (CT), Routine (RT - treated every 42 days), and Targeted Selective (TST - treated according to DWG). Packed cell volume (PCV) and eggs per gram of feces (EPG) tests were performed. Intending to investigate the resistance of parasites to anthelmintic, the EPG count reduction test (FECRT), RESISTA-Test© and molecular test (detection of a 63 bp indel in the acr-8 gene in Haemonchus contortus) were performed. For CT, RT and TST groups, the following results were obtained: mean EPG were 4665.1, 3063.5 and 3462.1 in the rainy season and 4475.1, 1341.7 and 2863.4 in the dry season, respectively; mean PCV were 32.1, 33.4 and 32.3% in the rainy season and 33.9, 36.0 and 35.1% in the dry season; mean DWG were 0.087, 0.101 and 0.094 kg in the rainy season and 0.102, 0.113 and 0.112 kg in the dry season; efficacies of levamisole in FECRT were 66.4, 24.1 and 76.4% in the rainy and 90.7, 12.4 and 64.8% in the dry season, respectively; in the RESISTA-Test© LC50 were 0.482, 1.926 and 0.117 µg.mL-1 in the rainy and 0.437, 0.851 and 0.045 µg.mL-1 in the dry season, respectively; in the molecular test, the frequencies of the homozygous resistant genotype were 57.1, 71.4 and 40.0% in the rainy season and 47.8, 55.9 and 41.9% in the dry season. In conclusion, TST allowed less resistance development through refugia maintenance, without productive losses, contributing to the sustainability of the sheep chain.

Haemonchus contortus

Happy FactorTM

RESISTA-Test©

acr-8 gene.

Infections by gastrointestinal nematodes (GIN) leads significant health problems and decrease in the productivity of small ruminants (Busin et al. 2013). Linked to the damage caused by these parasites, anthelmintic resistance to the main chemicals is also a huge constrain (Kaplan and Vidyashankar 2012). Adult animals of the Morada Nova breed are resistant to GIN (Issakowicz et al. 2016; Lôbo et al. 2011), but the lambs, in the growth phase, are considered resilient, needing attention concerning GIN control, because the reduction in weight gain in this stage has a direct impact on final weight (Haehling et al. 2020; Toscano et al. 2019a, 2019b).

Anthelmintic resistance has led to the search for more rational methods of parasite control, including selective treatment. Leaving part of the parasitic population without exposure to antiparasitic treatment (in refugia) is considered of critical importance to delay the evolution of resistant parasites (Van Wyk and Van Schalkwyk 1990; Van Wyk 2001). Parasites in refugia are those that have not been exposed to anthelmintics and include the stages of free living in the environment, parasites from untreated individuals, and those at any stage of the host life cycle that are not affected by anthelmintic treatments (Greer et al. 2020). Barnes et al. (1995) suggest a selective treatment method leaving out of the anti-helminthic treatment 20% of the animals with greater weight gain in the flock. With this new approach, the refugia’s conservation is extremely important to slow the establishment of resistance, as a portion of parasitic genetic material is not exposed to anthelmintics, which leads to the dilution of resistant genotypes present in helminth population (Greer et al. 2009).

In order to preserve refugia on farms it is necessary to design a selective treatment for sheep. This treatment, more personal, should be easy to implement and present a favorable cost-benefit to producers (Kenyon et al. 2013). To this end, a mathematical model must be developed with a production indicator that represents less loss to the flock and refugia maintenance (Berk et al. 2016). The Happy Factor™ is a targeted selective treatment (TST), which considers the average weight gain of developing lambs, the quality and quantity of the pasture and climatic factors (Greer et al. 2009). This model was considered efficient, because the reduction in weight gain occurs right at the beginning of infection, before the onset of clinical signs, when there is a reduction in voluntary food intake and in food conversion (Busin et al. 2013). Thus, weight gain is a sensitive indicator, affecting animal performance and the profitability of production systems. For most GIN, more complex interactions, including climate and management, make the use of these predictive tools more challenging, with the need to study models that support TST (Charlier et al. 2014). The consequences of anthelmintic treatment also depend on biology and ecology of parasites. Therefore, the validation of friendly and sustainable indicators at regional levels is essential (Edith et al. 2018).

As far as we are concerned, there are no studies aimed at developing TST in the Morada Nova sheep breed or that have evaluated it at tropical conditions. Thus, the objective of this study was to assess whether the average daily weight gain (DWG) can be a parameter for TST in Morada Nova lambs, favoring refugia and slowing down the establishment of anthelmintic resistance.

2.1. Experimental animals and management

The study was conducted at Embrapa Pecuária Sudeste - CPPSE, São Carlos, São Paulo, Brazil (22°01’S and 47°53’W). The region’s climate is classified as Cwa (Koëppen), with two well-defined seasons: rainy from October to March and dry from April to September. The average annual temperature is 21.2°C and the average annual air humidity is 75.6%.

The Morada Nova ewes were kept in 2.5 ha pasture, predominantly of Panicum maximum cv. Aruana, and managed in a rotational grazing system, with a period of occupation of 7 days and rest of 21 days. During the rainy season the ewes’ feeding was composed by pasture and supplementation with 300 g concentrate/animal. In the dry season, corn silage was also provided. Water and mineral salt were kept ad libitum.

To study the growth of lambs in the rainy season, the ewes were mated in March and April, so the births occurred in August and September. 100 ewes with 114 lambs in natural suckling, from single and twin births, were selected. Weaning occurred in the rainy season, in January. In the dry season, the mate was held in October and November, so that the ewes delivered between March and April. In this season, 76 females with 102 lambs met the same selection criteria. The animals were weaned in August, mid-dry season.

Immediately after birth, lambs were identified by conventional ear tags and by subcutaneous electronic chips and were weighed. Natural weaning was performed at 150 days of age in average. The ewes were removed from the Aruãna pasture, and the lambs remained in the same pasture until 210 days of age (D210) in average, under natural infections. The concentrate was supplied in a ratio of 1% of live weight (LW) per day and, in the dry season, corn silage was also provided gradually, as the supply of pasture decreased. Water and mineral salt were kept ad libitum.

2.2 Experimental design

At 63 days of age (D63), all lambs received anthelmintic treatment with Ripercol® L - 150F (levamisole 18.8% injectable, 9.4 mg.kg^-1) in order to initiate the experiment with all animals at the same parasitological conditions. This dose was higher than the recommended (6.2 mg.kg^-1) in order to reach a better efficacy in the sheep nematode control in Brazil (Costa et al. 2017) and make this trial possible. Levamisole presented the highest efficacy (81%) (albendazole - 54%, closantel - 79%, ivermectin - 44%, moxidectin - 35% and monepantel - 62%) in the FECRT performed with the Morada Nova lambs. These results reflect the resistance situation of the parasites from the Brazilian flocks (Veríssimo et al. 2012), as well as from the tropical countries (Sepúlveda-Vázquez et al. 2021), demonstrating the difficulty of carrying out experiments that require the use of highly effective anthelmintics.

The lambs were separated into three experimental groups in order to obtain similar means for: birth weight, type of birth (twin or single), sex, EPG and PVC, considering the data at D63 for the last traits. Anthelmintic treatments throughout the experimental period were performed with levamisole (9.4 mg.kg^-1), according to the criteria adopted for each treatment: 1) Control (CT): animals without anthelmintic treatment. 2) Routine (RT): animals treated every 42 days from D105 to D210, as usual at São Paulo state3) Targeted Selective (TST): animals treated according to the formula: lamb’s DWG ≤ DWG of the TSTgroup - standard deviation*0.5 (adapted from Cintra et al. 2018), every 21 days, from D105 to D210.

From D63 to D210, every 21 days, blood samples were collected for packed cell volume (PCV) determination, and feces samples for individual EPG counts (Ueno and Gonçalves 1998) and for group fecal cultures (Roberts and O'Sullivan 1950). In addition, the lambs were weighed every 21 days from birth to D210. To prevent deaths, precautionary anthelmintic treatment with the same drug was administered to lambs wherever they presented PCV ≤ 21% (adapted from Starling et al. (2019); CEUA CPPSE/ Protocol No. 01/2020), which occurred only in the CT. Their post-treatment data were removed from the statistical analyses.

2.3 Assessment of dry matter in forage and climatic factors

Forage collections occurred every 15 days in the rainy season and every 30 days in the dry season. All the material contained in a 1 m square was cut to 20 cm of the ground level and weighed for the determination of green matter production. After this procedure, sub-samples were placed in a forced-air oven at 65°C for 72 h, in order to determine the production of dry matter (DM), expressed in kg.ha^-1 (Nogueira and Souza 2005).

Climate data were assessed by the climate station at Embrapa Pecuária Sudeste. Temperature (ºC), relative humidity (%) and rainfall (mm) were measured.

2.4 Anthelmintic resistance analyses performed after 210 days of age

2.4.1 Parasitological necropsy

Two lambs from the rainy season and three from the dry season were selected from each treatment with the highest EPG counts. These animals were euthanized after stunning for parasitological necropsy according to technical criteria of the Ministry of Livestock Agriculture and Supply (MAPA), following humanitarian slaughter rules. The abomasum was recovered and cut into the larger curvature, and the small and large intestines were lined up and opened. The contents were then washed with water and recovered in a 0.297 mm sieve. Using a magnifying glass, the parasites were separated by genus and sex and stored in 70% alcohol for morphological identification (González-Garduño et al., 2013).

2.4.2 Diagnosis of levamisole resistance by the Fecal Egg Count Reduction Test (FECRT)

After D210, the lambs that were not selected for parasitological necropsy and had EPG higher than 200 were randomized for FECRT. The objective was to verify levamisole efficacy in each group, after its differential use.

Randomization for FECRT was based on EPG, PVC and animal weight data obtained on D210. Within each of the three treatments, the animals were separated into the control group (without treatment) and the group treated with levamisole. On post-treatment D7, feces were collected for individual EPG counts, fecal cultures and drug resistance evaluation according to the formula: % Efficacy = (Mean of EPG in the control group - Mean of EPG in the treated group/ Mean of EPG in the control group) x 100.

2.4.3 Diagnosis of levamisole resistance by the Larval Development Test (LDT) - RESISTA-Test^©

This test was performed on D210 with eggs from a pool of the feces collected from each experimental group and recovered by sequential sieves. About 70 GIN eggs were added to each well of a 96-well culture plate, which also received nutritive medium (Escherichia coli [EC11303] and amphotericin B [A9528] - Sigma-Aldrich). The plates were identified, sealed with PVC film and kept in an incubator for 24 h (27°C, RH 80%) for larval development (L₁). After this period, wells received 12 dilutions of levamisole hydrochloride (RESISTA-Test^©; Gainza et al. 2020; 2021). The plates were incubated for another six days. The negative control was composed of distilled water and nutritive medium. After the incubation period, first, second and third stage larvae (L₁, L₂, L₃) of each well were quantified using an inverted microscope to calculate the efficacy and lethal concentrations (LCs) of levamisole. All anthelmintic concentrations and negative control were evaluated in duplicate (Gainza et al. 2020; 2021).

2.4.4 Diagnosis of levamisole resistance by molecular testing

Fecal samples collected on D63 and D210 were submitted to fecal culture. After obtaining L₃, DNA extraction and genotyping of the 63 bp indel in the acr-8 gene was performed by polymerase chain reaction (PCR) specific for Haemonchus contortus, to evaluate the resistance to levamisole (Chagas et al. 2016).

In the rainy season, genotypes were assessed in 20 L₃ of all treatments (group-pooled fecal culture) on D63 and in 35, 21 and 20 L₃on D210 for CT, ST and TST, respectively. In the dry season, 39, 27 and 30 L₃ were used on D63, and 69, 35 and 43 L₃ on D210, respectively. The L₃ were submitted to DNA extraction according to Silvestre and Humbert (2000) and Coles et al. (2006) with modifications by Niciura et al. (2012). For DNA extraction, sodium hypochlorite solution was used for exsheating, and larvae were individually collected and incubated in digestion buffer (10 mM Tris-HCl pH 7.6, 10 mM EDTA pH 8.0, 50 mM NaCl, 2% SDS, 40 mM DTT and 0.4 mg/mL proteinase K) at 56ºC overnight. DNA was extracted with Phenol solution: Chloroform: Isoamyl alcohol (25:24:1), precipitated with 100% isopropanol and washed with 70% ethanol. DNA was then suspended in water, incubated at 37 ºC for 30 minutes and stored at -20 ºC (adapted from Sambrook et al. 2000).

PCR to detect the 63 bp deletion in the acr-8 gene associated with levamisole resistance was performed according to Barrère et al. (2014) with LEVF (5'-ACCTTACCTATACACCCGTC-3') and LEVR (5'-CTTGCCGTTATTACACCCTCG-3') primers. It consisted of 1x buffer, 0.25 µM each primer, 0.2 mM each dNTP, 1.5 mM MgCl₂, 1U Taq DNA polymerase and 2 µL DNA, in a final volume of 20 µl. Thermocycling conditions were initial denaturation at 94ºC for 2 minutes and 40 amplification cycles at 94ºC for 30 seconds, 55ºC for 30 seconds and 72ºC for 30 seconds, followed by final extension at 72ºC for 10 minutes.

Genotypes were attributed by banding pattern (256 bp for deletion and 319 bp no deletion) after 2% agarose gel electrophoresis in TBE buffer, ethidium bromide staining and observation in a UV transiluminator. The positive control was previously standardized (Chagas et al. 2016) and the negative control consisted of distilled water without DNA. The resistance genotype was assigned to animals homozygous for the 63 bp deletion.

2.5 Statistical analyses

Weight, DWG, PVC and EPG data were submitted to variance analysis by the SAS Proc Mixed procedure (SAS 2016) considering in the model the effects of treatments (CT, ST and TST), Day (from 0 to 210) and the respective interactions following a Split-Plot with repeated measurement in time. In selecting the best covariance matrix structure, the AIC (Smaller is Better - Akaike’s Information Criterion) criterion was used. Mean comparisons were performed by Tukey’s test at a significance level of 5%. The variable EPG was submitted to LOG (X+25) transformation.

Anthelmintic efficacy in RESISTA- Test® was determined based on the arithmetic mean of larval development according to the following equation (Coles et al. 1992): , whererefers to the number of larvae that has not reached the L₃ phase andcorresponds to the number of L₁ + L₂+ L₃. The results were analyzed by the Probit logistic regression model to determine the values of LC₅₀, LC₉₀, LC₉₅ and LC₉₉, which were defined as the anthelmintic concentrations in which 50%, 90%, 95% and 99% of larval development were inhibited. The analyses were performed with XLSTAT-Premium 2019.2.2 (Addinsoft 2019 - XLSTAT, Boston, USA) and a significance level of p ≤ 0.05 was considered. In the molecular test, Chi-square test was used to compare differences in frequency of the resistant genotype between D63 and D210 in each treatment group.

3.1 Infection analyses

Figure 1 presents the means of EPG obtained in CT, RT and TST groups. For 114 lambs (n = 38 animals/treatment) in the rainy season and 102 lambs (n = 34 animals/treatment) in the dry season, eight samples were taken. The EPG means were higher in the CT group, while the RT means were lower in both seasons. CT and TST did not differ on any experimental date of the rainy season but were different in the dry season (p ≤ 0.05). There was a statistical difference in the overall EPG mean between CT and RT in the rainy season and among the three treatment groups in the dry season (Table 1).

In addition to Trichostrongyloidea, eggs of Strongyloides sp. (only in the rainy season), Moniezia sp. and oocysts of Eimeria spp were also identified in EPG. Nematode genera frequencies in feces cultures were extremely homogeneous throughout the experimental period, in both seasons. Thus, the results are presented as means. The genera Haemonchus, Trichostrongylus, Cooperia and Oesophagostomum were identified, with averages of 81.6%, 10.3%, 5.0% and 3.1% in the rainy season and 83.3%, 12.6%, 3.5% and 0.6% in the dry season, respectively.

3.2 Hematological analyses

Blood collection for PCV also occurred on eight experimental dates, and the averages are shown in Figure 1. In lambs raised in the rainy season, PCV values did not undergo many changes, even with high EPG counts. The three treatments showed similar PCV until D105 in both seasons. From then on, CT maintained the lowest PCV and RT the highest, especially in the dry season. In the general mean, RT was statistically similar to TST only in the rainy season (p > 0.05) (Table 2).

3.3. Weight measurement and DWG

The weight measurement of lambs occurred every 21 days, from birth to D210, totaling 10 samples for each animal (Fig. 1). In the rainy season, statistical differences in mean weights were observed only in the two final dates. On D210, the mean RT weight exceeded those of the other treatments (p ≤ 0.05). In the dry season, CT averages were the lowest in relation to the other treatments on three dates (p ≤ 0.05). In D210 of the dry season and in the general average of both seasons, however, there was no difference between treatments (p > 0.05) (Table 3).

DWG in each treatment is shown in Figure 1. RT presented mean DWG statistically higher (p ≤ 0.05) than the other treatments in the rainy season, but it was not different from TST in the dry season (Table 4).

Table 5 shows the number of anthelmintic treatments performed in the experimental groups and its percentages. In a comparison with RT (100% of anthelmintic treatment), the TST presented an average percentage of 27.2% of lambs dewormed in the rainy season and 32.8% in the dry season, while the CT presented 10.5% and 4.4%, respectively, when PCV ≤ 21%.

At the end of the experiment, it was evaluated whether the criterion used in the TST (DWG ≤ DWG of the TST group - standard deviation*0.5) was adequate to identify lambs with higher reduction in body weight gain due to infection. The comparative criterion was that animals with EPG ≥ 4000 need anthelmintic treatment. For the TST group in the rainy season, 71% of the lambs would not have been treated because they had DWG > that formula; 69% of them agreed with the EPG, as they had EPG < 4000. Moreover, from the 29% who would have received anthelmintic treatment by DWG, 40% had EPG ≥ 4000. Therefore, there was 61% of agreement between DWG and EPG. In relation to the total number of evaluations performed in the TST (n = 217), 22% of the animals were at risk, that is, despite EPG ≥ 4000, they would not have received anthelmintic treatment because they presented high DWG, but at the same time they did not present symptoms, such as anemia or weight loss. In the dry season, 67% of the lambs would not have be treated for having DWG > that formula; 71% of them agreed because presented EPG < 4000. From the 33% who would have received treatment by DWG, 24% had EPG ≥ 4000. Therefore, there was a 55% of agreement between DWG and EPG, and of total performed evaluations, 20% of the animals were at risk in this season.

3.4. Forage and meteorological analyses

The average dry matter production (DM) of the pasture at the beginning of the rainy season was 1612 kg/ha. With the increase in rainfall rates from D84, DM production increased gradually, reaching an average of 4735 kg/ha on D168 to D210 (Fig. 1). In the dry season, an initial mean of 3262 kg/DM/ha was obtained, and as the rainfall declined throughout the experiment, on D210 the average was 1132 kg/DM/ha. In this period, the animals received corn silage supplementation with 6.5% crude protein and 63% total digestible nutrients.

The measured meteorological parameters indicated that the rainy season had higher rainfall rate, temperature and relative humidity. For the dry season, very low rainfall rates were observed (0 mm on three consecutive dates), associated with lower temperature and relative humidity (Fig. 1)

3.5 Diagnosis of resistance and infection profile on D210

Once the D210 data collection was completed, the separation into subgroups was established for FECRT. In the rainy season, levamisole presented an efficacy of 66.4% in CT, 24.1% in RT and 76.4% in TST, while in the dry season its efficacy was 90.7%, 12.4% and 64.8%, respectively. LCs obtained in both seasons in the RESISTA-Test^© showed a very high degree of resistance for RT. TST, on the other hand, was the one that presented the smallest values, demonstrating greater susceptibility to the anthelmintic treatment. There was no significant difference (p > 0.05) in the frequency of the resistant genotype between D63 and D210 in all treatment groups and in both seasons (Table 6). The Hardy-Weinberg Equilibrium test - HWE (data not shown) indicated that the polymorphism was under selection pressure (p ≤ 0.05) on D210 in the three treatments and in the two seasons.

In the parasitological necropsy, H. contortus, Trichostrongylus colubriformis, Oesophagostomum columbianum and Cooperia curticei were observed. Four lambs (66.6%) in the rainy season and two (22.2%) in the dry season presented C. curticei. One lamb (16.6%) presented O. columbianum (Table 7). In the rainy season there was a predominance of H. contortus (68.9%), while in the dry season T. colubriformis was the most prevalent (66.4%). The two species were responsible for practically all parasitic burden recovered from necropsied animals (98.1% - rainy season and 99.6% - dry season). Fecal cultures (D210) indicated predominance of H. contortus in the three experimental treatments, in the rainy and dry season. On D210, the percentages for Haemonchus, Trichostrongylus, Cooperia and Oesophagostomum, whose averages were 68%, 21%, 8% and 3% in the rainy season and 76%, 23%, 1% and 0% in the dry season, respectively, were extremely homogeneous in CT, RT and TST.

In the present study, TST approach was used to optimize weight gain for low-performance lambs and ensure longer life of anthelmintic drugs, by reducing the number of antiparasitic treatments. It was observed that the number of treatments in TST was almost half of the number in RT and, on average, 30% of the TST lambs received anthelmintics. Such findings are in agreement with those in a study in Scotland, in which DWG-treated lambs (or TST) received 50% less anthelmintic treatments than lambs submitted to the treatment recommended on the farm (every 42 days), without significant losses in DWG or time to reach the slaughter weight (Busin et al., 2014). In Great Britain, by means of a questionnaire answered by 383 sheep farmers, those who adopted the TST used significantly fewer drugs and reported less resistance than producers who did not adopt TST (Williams et al. 2021). For TST adoption, it’s important to highlight that the DWG is as a parameter easier obtained than EPG or PCV. Other studies have also shown that weight can be a reliable parameter for the decision-making of anthelmintic treatment (Greer et al., 2009; McBean et al., 2016).

The parasites identified in the fecal cultures in the present study were extremely aligned and corroborate with the previous studies conducted in the region, indicating higher percentage of H. contortus (Amarante et al. 2004; Chagas et al. 2008). However, the results from L₃ identification and necropsy also showed an important parasitism by Trichostrongylus, because despite the percentage being lower than Haemonchus, it’s important to consider that the Trichostrongylus female egg laying is around 200 eggs per parasite while for Haemonchus female is around 5000, e.i., 25 times more than that of Trichostrongylus (Ueno and Gonçalves 1998). Thus, if fecal cultures demonstrate 50% of each species for instance, it may mean the presence of one Haemonchus female and 25 Trichostrongylus females. The meteorological parameters showed that the two periods in which this study occurred were very typical of the rainy and dry seasons of tropical regions. The rainy season presented favorable conditions for the L₃ survival in the environment and, thus, greater sheep infection. Many L₄were also collected in the rainy season from the necropsied animals. These immature forms are probably due to the phenomenon of hypobiosis, in which they remain in a state of dormancy until the host presents better conditions. Immature forms may promptly replace adult parasites for instance and the phenomenon also avoids food disputes in cases of high infection ensuring thehelminth survival (Costa et al. 2011). On the other hand, dry season presented more unfavorable conditions for the L₃ survival in the environment. As a consequence, there was less infection of the lambs, visualized in the average EPG and in the number of parasites recovered from necropsy.

The results obtained for EPG indicated that anthelmintic treatments every 42 days (D105, D147 and D189) in RT generated a decrease in EPG counts on the subsequent dates (D126, D168 and D210), leading to statistical differences among groups. A similar pattern was observed for PCV, as those treatments promoted the maintenance of higher PCV in the RT throughout the experiment. The main clinical sign of the predominant infection by H. contortus is anemia, detected by PCV. Although Morada Nova breed is considered resistant to GIN (Issakowicz et al. 2016), such results prove that the lambs are resilient, that is, they remain healthy and productive despite high levels of infection. Even with high EPG counts, the mean PCV were similar to the one of healthy animals in the three groups, as already verified by Okino et al. (2021) and Toscano et al. (2019b) in Morada Nova breed.

In the weight gain evaluation, all groups presented similar averages in both seasons. The RT-treated animals showed better performance on D210 of the rainy season, but similar to the others in the dry season. Moreover, the animals reached higher mean live weights, demonstrating that the lowest infection, confirmed by mean EPG and post-necropsy counts, had a lower impact on production in this season. For DWG, there was a similarity between TST and RT in the dry season, with lower parasitic challenge. The amount of forage available in this season showed the need for animals’ supplementation, as expected. The concentrate was given throughout the experimental period of the two seasons and the silage was modulated according to the amount of dry matter measured in the pastures. Sheep should receive a supply of 2.8 kg DM/animal in Brazilian climatic conditions and also with adequate management and rotation of pastures (Pinto Filho et al. 2019). Such information corroborates with the management adopted in the present study, in which the average offer of 2.5 kg DM/animal/day was maintained during the dry season. Therefore, with corn silage supplementation offered in the dry season, it was verified that there was no deficit in the food supply that could reduce weight gain or DWG in the animals, reinforcing the hypothesis of a direct impact of GIN infection.

There are two studies evaluating the use of weight in the TST treatment in the South region of Brazil, in Ile de France and Texel animals. Both adopted the treatment of lambs raised in pasture with DWG lower than the mean DWG of the CT - standard deviation. The results indicated difficulty in achieving an ideal formula to decide to perform or not the anthelmintic treatment. Still, they concluded that DWG is a good parameter for decision-making, promoting significantly higher weight gain (Cintra et al. 2018; 2019). However, the adoption of the CT’s DWG as a reference in those studies, instead of the TST’s DWG, does not consider the specific conditions of the parasitic management of this group over time. The good results obtained in those studies are probably directly related to the evaluated breeds, which are larger and more susceptible to GIN than Morada Nova.

On D210 in the rainy season, TST presented lower DWG than RT and higher than CT. It also showed lower resistance rates, which ensures longer use of anthelmintic drugs. Although there was not a highlighted agreement between DWG and EPG, the weight measurement seems to be a more reliable parameter of the impact of infection than EPG counts. According to Love et al. (2017), EPG counts, performed by different methods, failed in the correct decision-making for the control of parasites, and it also interferes negatively in the use of the anthelmintics through FECRT. Therefore, a TST not based on EPG count can be a differential, especially in flocks with a predominance of resilient animals, and also, may be an important tool in the selection of more susceptible animals to be discarded. Mcbean et al. (2021) conducted studies altering the Happy Factor^TM coefficient in order to optimize the efficiency of this method to increase animal productivity in farms with different conditions and sheep breeds. The authors suggest that, if necessary, further refinements can be made from locally generated data. Thus, new studies would evaluate different situations, such as the benefits of avoiding anthelmintic treatment in a percentage of the animals with higher DWG.

FECRT, RESISTA-Test^© and molecular test indicated development of higher anthelmintic resistance in RT, mainly in the rainy season. The results were in agreement among RESISTA-Test^©, FECRT and molecular in the rainy season (RT > CT > TST). In the dry season, however, while RESISTA-Test^© and molecular test were concordant (RT > CT > TST), the FECRT was not so aligned (RT > TST > CT). In a study conducted in Brazilian flocks with a historical use of levamisole, the resistance detected was aligned in both FECRT and RESISTA-Test^© (Gainza et al. 2021). It is worth noting that the RESISTA-Test© and molecular test have been validated so far for H. contortus and, in the dry season, the necropsy indicated a higher percentage of T. colubriformis, which may explain this divergence.

Regarding the molecular evaluation, the observed differences in frequency of the homozygous resistant genotype was not significant between D63 and D210 for all treatment groups and in both seasons. It may suggest that the short interval (147 days) between evaluations was not enough to detect differences, even considering that the polymorphism was under selective pressure tested by HWE. Santos et al. (2019) detected 76.7% of the resistance allele frequencies after continuous use of the drug, which was similar to the values presented in the rainy season for the ST group. The allele for resistance was identified in properties in Bangladesh with a percentage ranging from 25% to 47% (Dey et al. 2020). However, as the characterization of levamisole resistance was not possible in some H. contortus parasitic populations studies suggest that the role of this polymorphism should be better investigated (Santos et al. 2019).

The present study exposes the need for local improvement of TST strategies in order to prolong efficacy of anthelmintics. Prioritize weight gain and ignore resistance development is the main factor affecting the sustainability of sheep production chain in several tropical and subtropical countries. The adoption of TST would imply in the acquisition of a scale that, in turn, reduces the need for animal management involving EPG and VG measurements, also resulting in less impact on the environment and less drug residues in animal products. Thus, it is possible to infer that, although not perfect for all situations, TST is useful. The fine-tuning to be carried out must consider, in addition to climate factors, different breeds and production systems, and the balance between resistance mitigation and DWG, that is, to obtain the highest animal performance with the lowest selection pressure on GIN.

The adoption of DWG in the TST allowed good weight performance, reduction in the use of anthelmintics and deceleration of resistance development in H. contortus in relation to the RT approach. The method provided the maintenance of zootechnical control of the flock, especially in the dry season, in which the parasite load was lower. The results suggest the need for studies to evaluate TST in various breeds and in environmental conditions with different parasitic challenges.

Animal welfare statement

The experimental protocols that were developed in this study fully complied with the ethical principles of animal experimentation prepared by Ethical Use of Animals Committee of Embrapa Pecuária Sudeste (CEUA CPPSE/ Protocol No. 01/2020).

Credit authorship contribution statement

Isabella Barbosa dos Santos: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft, Writing - review & editing. Luís Adriano Anholeto: Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing - original draft, Writing - review & editing. Gustavo Avelar de Sousa: Investigation, Methodology. Alessandra da Silva Nucci:Investigation, Methodology. Yousmel Alemán Gainza: Investigation, Methodology. Amanda Figueiredo:Investigation, Methodology. Leonardo Aparecido Lima dos Santos:Investigation, Methodology. Alessandro Pelegrine Minho: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft, Writing - review & editing. Waldomiro Barioni-Junior: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft, Writing - review & editing. Sérgio Novita Esteves: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft, Writing - review & editing.Simone Cristina Méo Niciura:Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft, Writing - review & editing. Ana Carolina de Souza Chagas: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing.

Competing Interest

The authors declare they have no conflict of interest.

Acknowledgements

We would like to thank the São Paulo Research Foundation (FAPESP) for the financial support and scholarships (grant no. 2019/02929-3; grant no. 2018/16070-1), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the scholarship (grant no. 131537/2018-0).

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Table 1.Means and standard error (SE) of eggs per gram of feces (EPG) counts in control (CT), routine (RT) and targeted selective (TST) treatments, in the different experimental days, in Morada Nova lambs, during the rainy (R) and dry (D) seasons (S).

S	Treat	D63	D84	D105	D126*	D147*	D168*	D189*	D210*	Mean*	SE
R	CT	620.0	730.9	6109.2	6301.3a	12448.0	6138.4a	3040.3	1932.6a	4665.1 a	490.7
	RT	663.2	587.3	3884.2	1091.2b	10096.0	1736.9b	4130.4	676.2b	3063.5b	493.6
	TST	686.8	554.0	3615.8	6237.4a	7317.1	5495.5a	2036.8	1753.1a	3462.0ab	492.1
D	CT	1746.6	823.3	6298.0	5916.2a	7573.0a	5044.6a	4947.9a	3450.9a	4475.1a	286.6
	RT	1816.3	886.1	3957.9	793.7c	1227.7c	647.9c	843.0c	561.4c	1341.7c	287.3
	TST	1782.4	919.1	4748.1	4682.5b	4016.2b	2968.1b	2232.1b	1558.7b	2863.4b	285.8

* Different letters in the same column indicate statistical differences between treatments by Tukey’s test (p ≤ 0.05), within each season.

Table 2. Means and standard error (SE) of packed cell volume (PCV) in the control (CT), routine (RT) and targeted selective (TST) treatments, in different experimental days, in Morada Nova lambs, during the rainy (R) and dry (D) seasons (S).

S	Treat	63	84	105	126*	147*	168*	189*	210*	Mean*	SE
R	CT	35.0	35.0	33.3	31.7b	28.2	30.5b	31.6	31.2b	32.1b	0.2
	RT	34.3	35.6	32.4	35.4a	29.2	34.0a	32.1	34.3a	33.4a	0.2
	TST	34.9	35.5	33.3	31.3b	29.8	30.8b	31.3	31.7b	32.3b	0.2
D	CT	36.4	36.2	34.5	33.0b	32.7b	31.9c	32.7b	33.7b	33.9b	0.5
	RT	36.7	35.7	33.9	36.0a	36.3a	36.4a	36.3a	36.4a	36.0a	0.5
	TST	37.1	36.3	33.9	34.5b	35.1a	34.4b	34.7a	34.8b	35.1a	0.5

* Different letters in the same column indicate statistical difference between treatments by Tukey’s test (p ≤ 0.05), within each season.

Table 3. Means and standard error (SE) of average live weight in control (CT), routine (RT) and targeted selective (TST) treatments, in different experimental days, in Morada Nova lambs, during the rainy (R) and dry (D) seasons (S).

S	Treat	0	21	42	63	84	105	126*	147*	168*	189*	210*	Mean*	SE
R	CT	2.43	5.32	8.17	11.23	13.77	16.18	17.52	18.22	17.96	19.75b	20.94b	13.77	0.41
	RT	2.47	5.51	8.36	11.41	13.72	16.03	18.48	19.12	19.63	21.71a	23.80a	14.57	0.41
	TST	2.40	5.69	8.47	11.63	14.08	16.37	17.98	19.03	18.69	20.51ab	22.12b	14.27	0.41
D	CT	2.48	5.51	8.36	10.54	13.08	15.56	17.52b	19.63c	21.29b	22.71	24.86	14.69	0.52
	RT	2.47	5.26	7.86	10.14	12.71	15.19	17.83a	20.46a	23.17a	24.80	26.59	15.13	0.52
	TST	2.45	5.70	8.33	10.50	12.99	15.37	17.77a	19.70b	22.33a	24.02	25.90	15.01	0.52

* Different letters in the same column indicate statistical difference between treatments by Tukey’s test (p ≤ 0.05), within each season.

Table 4. Means and standard error (SE) of average daily weight gain (DWG) in control (CT), routine (RT) and targeted selective (TST) treatments, in different experimental days, in Morada Nova lambs, during the rainy (R) and dry (D) seasons (S).

S	Treat	21	42	63	84	105	126*	147*	168*	189	210*	Mean*	SE
R	CT	0.13	0.14	0.15	0.12	0.12	0.06b	0.03	-0.01b	0.09	0.06c	0.087b	0.002
	RT	0.14	0.14	0.15	0.11	0.11	0.12a	0.03	0.02a	0.10	0.10a	0.101a	0.002
	TST	0.15	0.13	0.15	0.12	0.11	0.08b	0.05	-0.02b	0.09	0.08b	0.094b	0.002
D	CT	0.13	0.15	0.10	0.12	0.12	0.09b	0.05c	0.08b	0.07	0.11	0.102b	0.004
	RT	0.13	0.12	0.10	0.12	0.12	0.12a	0.12a	0.12a	0.08	0.09	0.113a	0.004
	TST	0.15	0.13	0.10	0.12	0.11	0.11a	0.09b	0.13a	0.08	0.09	0.112a	0.004

* Different letters in the same column indicate statistical difference between treatments by Tukey’s test (p ≤ 0.05), within each season.

Table 5. Number (percentage) of anthelmintic treatments used in control (CT), routine (RT) and targeted selective (TST) treatments, in different experimental days, in Morada Nova lambs, during the rainy (R) and dry (D) season (S).

S	Treat	D84	D105	D126	D147	D168	D189	D210	Total	Mean
R	CT	0	0	2(5.3%)	7(18.4%)	3(7.9%)	0	0	12	10.5%
	RT	0	38(100%)	0	38(100%)	0	38(100%)	0	114	100%
	TST	0	8(21.1%)	10(26.3%)	11(28.9%)	12(31.6%)	12(31.6%)	9(23.7%)	62	27.2%
D	CT	0	0	1(2.9%)	1(2.9%)	2(5.9%)	2(5.9%)	0	6	4.4%
	RT	0	34(100%)	0	34(100%)	0	34(100%)	0	102	100%
	TST	0	12(35.3%)	13(38.2%)	13(38.2%)	9(26.5%)	10(29.4%)	10(29.4%)	67	32.8%

Table 6. Lethal concentrations (LC, µg.mL^-1) obtained in RESISTA-Test^©, percentages of anthelmintic efficacy in FECRT and percentages of |Haemonchuscontortuswith resistant genotypes by PCR (D63 and D210) in control (CT), routine (RT) and targeted selective (TST) treatments in the rainy (R) and dry (D) seasons (S).

	Rainy							Dry
	RESISTA-Test^©				FECRT	PCR		RESISTA-Test^©				FECRT	PCR
Treat	LC₅₀	LC₉₀	LC₉₅	LC₉₉	D210	D63*	D210	LC₅₀	LC₉₀	LC₉₅	LC₉₉	D210	D63	D210
CT	0.482	4.471	8.424	27.712	66.4	50.0	57.1	0.437	5.914	12.377	49.463	90.7	59.0	47.8
RT	1.926	17.277	32.34	105.593	24.1		71.4	0.851	11.793	24.849	100.57	12.4	51.9	55.9
TST	0.117	0.797	1.374	3.821	76.4		40.0	0.045	0.441	0.839	2.806	64.8	42.4	41.9

*Fecal culture performed in pool and not by group.

Table 7. Nematode classification, sex and mean after necropsy of the lambs in control (CT), routine (RT) and targeted selective (TST) treatments in the rainy (R) and dry (D) seasons (S).

S	Treat	H. contortus				T. colubriformis			O. columbianum				C. curticei
S	Treat	F	M	L₄	T	F	M	T	F	M	T	F		M	T
	CT	1445	985	1155	3585	735	510	1245	0	0	0	110		140	250
R	RT	1200	1360	2500	5060	1685	1185	2870	0	0	0	5		5	10
	TST	1900	1400	1635	4935	945	690	1635	15	10	25	40		50	90
Total		4545	3745	5290	13580 (68.9%)	3365	2385	5750 (29.2%)	15	10	25 (0.1%)	155		195	350 (1.8%)
	CT	477	397	77	951	583	493	1076	3	3	6	3		3	6
D	RT	30	37	0	67	367	270	637	0	0	0	0		0	0
	TST	120	110	10	240	453	350	803	0	0	0	3		3	6
Total		627	544	87	1258 (33.2%)	1403	1113	2516 (66.4%)	3	3	6 (0.1%)	6		6	12 (0.3%)

F: female; M: male; T: total.

No competing interests reported.

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You are reading this latest preprint version

Investigating the Benefits of Target Selective Treatment by Average Daily Weight Gain Against Gastrointestinal Nematodes in Morada Nova Lambs

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Version 1

Abstract

Figures

1. Introduction

2. Material And Methods

2.1. Experimental animals and management

2.2 Experimental design

2.3 Assessment of dry matter in forage and climatic factors

2.4 Anthelmintic resistance analyses performed after 210 days of age

2.4.1 Parasitological necropsy

2.4.2 Diagnosis of levamisole resistance by the Fecal Egg Count Reduction Test (FECRT)

2.4.3 Diagnosis of levamisole resistance by the Larval Development Test (LDT) - RESISTA-Test^©

2.4.4 Diagnosis of levamisole resistance by molecular testing

2.5 Statistical analyses

3. Results

3.1 Infection analyses

3.2 Hematological analyses

3.3. Weight measurement and DWG

3.4. Forage and meteorological analyses

3.5 Diagnosis of resistance and infection profile on D210

4. Discussion

5. Conclusion

Declarations

References

Tables

Additional Declarations

Status:

Version 1

Investigating the Benefits of Target Selective Treatment by Average Daily Weight Gain Against Gastrointestinal Nematodes in Morada Nova Lambs

Status:

Version 1

Abstract

Figures

1. Introduction

2. Material And Methods

2.1. Experimental animals and management

2.2 Experimental design

2.3 Assessment of dry matter in forage and climatic factors

2.4 Anthelmintic resistance analyses performed after 210 days of age

2.4.1 Parasitological necropsy

2.4.2 Diagnosis of levamisole resistance by the Fecal Egg Count Reduction Test (FECRT)

2.4.3 Diagnosis of levamisole resistance by the Larval Development Test (LDT) - RESISTA-Test©

2.4.4 Diagnosis of levamisole resistance by molecular testing

2.5 Statistical analyses

3. Results

3.1 Infection analyses

3.2 Hematological analyses

3.3. Weight measurement and DWG

3.4. Forage and meteorological analyses

3.5 Diagnosis of resistance and infection profile on D210

4. Discussion

5. Conclusion

Declarations

References

Tables

Additional Declarations

Status:

Version 1

2.4.3 Diagnosis of levamisole resistance by the Larval Development Test (LDT) - RESISTA-Test^©