Anthelmintic activity of three selected ethnobotanical plant extracts against Strongyloides venezuelensis.

The agropastoral farmers have employed Turraea vogelii(TVL),Senna podocarpa(SPL), and Jaundea pinnata (JPL) leaves for treating various diseases, including intestinal parasites in livestock and the human population in Nigeria. Gastrointestinal nematodes are highly significant to livestock production and people's health, and natural products are interesting as sources of new drugs. In this study, we evaluated the effectiveness of extracts derived from these plants in treating parasitic infections using third-stage infective larvae (L3) of Strongyloides venezuelensis. We obtained crude extracts using n-gexane (Hex), ethyl acetate (Ea), and methanol (Met). The extracts were analyzed for their phytochemical composition, and their ability to prevent hemolysis were tested. The mean concentrations of total phenols in SPL Hex, SPL Ea, and SPL Met were 92.3 ± 0.3, 103.0 ± 0.4, and 128.2 ± 0.5 mg/100 g, respectively. Total tannin concentrations for JPL Ea, SPL Ea, SPL Hex, and TVL Hex were 60.3 ± 0.1, 89.2 ± 0.2, 80.0 ± 0.1, and 66.6 ± 0.3 mg/100 g, respectively. The mean lethal concentration (LC50) at 72 h for JPL Ea 39 (26-61) µg/mL. SPL Ea was 39 (34-45) µg/mL, and TVL Hex 31 (26-36) µg/mL. The antiparasitic activities of the extracts against L3 were dose- and time-dependent. All the extracts were slightly hemolytic to the erythrocytes. In this study, the plant extract tested demonstrated significant anti-S. venezuelensis activity. These phytobotanical extracts could be used to create formulations for the potential treatment of helminthiasis in animals and humans.

Nemabiome metabarcoding shows a high prevalence of Haemonchus contortus and predominance of Camelostrongylus mentulatus in alpaca herds in the northern UK.

Gastrointestinal nematodes (GINs) are a common threat faced by pastoral livestock. Since their major introduction to the UK in the early 1990s, South American camelids have been cograzed with sheep, horses, and other livestock, allowing exposure to a range of GIN species. However, there have been no molecular-based studies to investigate the GIN populations present in these camelids. In the current study, we sampled nine alpaca herds from northern England and southern Scotland and used high-throughput metabarcoded sequencing to describe their GIN species composition. A total of 71 amplicon sequence variants (ASVs) were identified representing eight known GIN species. Haemonchus contortus was the most prevalent species found in almost all herds in significant proportions. The identification of H. contortus in other livestock species is unusual in the northern UK, implying that alpacas may be suitable hosts and potential reservoirs for infection in other hosts. In addition, the camelid-adapted GIN species Camelostrongylus mentulatus was identified predominantly in herds with higher faecal egg counts. These findings highlight the value of applying advanced molecular methods, such as nemabiome metabarcoding to describe the dynamics of gastrointestinal nematode infections in novel situations. The results provide a strong base for further studies involving cograzing animals to confirm the potential role of alpacas in transmitting GIN species between hosts.

Molecular identification and phylogenetic analysis of gastrointestinal nematodes in different populations of Kazakh sheep.

Gastrointestinal nematode (GIN) infection in sheep has been recognized globally as a major problem challenging animal health and production. The objective of this study is to use a molecular diagnosis of the prevalence for gastrointestinal nematode (GIN) dominant species of Kazakh sheep and its hybrid (Kazakh × Texel). The internal transcribed spacer 2 (ITS-2) sequences of ribosomal DNA (rDNA) were used as the target sequence. In the study, three dominant species of nematodes, namely Haemonchus contortus, Trichostrongylus spp., and Teladorsagia (Ostertagia) circumcincta from the Kazakh sheep and the F1 and F2 generations of Texel × Kazakh sheep hybrids were subjected to molecular identification and phylogenetic analysis. The fecal and single larva genomic DNA were extracted and amplified by PCR using specific primers to determine the infection rate of the three nematode species. In addition, the PCR products were sequenced and analyzed using bioinformatics methods to construct a phylogenetic tree. The results showed that all the three species had their ITS-2 specific amplified. According to the sequence homology analysis of PCR products, the results showed a high homology (above 98.5% homology) with H. contortus, Trichostrongylus spp., T. circumcincta ITS-2 sequences in GenBank. Phylogenetic analysis showed that the ITS-2 sequences of the three species were on the same branch as the ITS-2 sequences of the same species in NCBI. And on different branches from those of the ITS-2 sequences of different families, genera and species. Sequences carried out on three species from different samples showed a close relationship and little genetic difference in phylogenetic tree. The infection rates based on fecal DNA were 35.59, 25.55, and 11.24% for H. contortus, Trichostrongylus spp., and T. circumcincta, respectively. While the infection rates based on larva DNA, were 24.07, 18.89, and 13.26% for H. contortus, Trichostrongylus spp., and T. circumcincta, respectively. The seasonal prevalence of the three dominant species in spring was significantly higher than that in autumn and winter. And there was no significant difference between Kazakh, F1 and F2 sheep considering the infection rate of the studied three species of nematodes. This study provides valuable molecular approaches for epidemiological surveillance and for assisting in the control of Nematodirus infection in sheep.

An in vitro rumen incubation method to study exsheathment kinetics of Haemonchus contortus third-stage infective larvae.

This study developed and evaluated an in vitro rumen incubation (IVRI) method to describe the exsheathment kinetics of Haemonchus contortus third-stage infective larvae (L3) in ruminal liquor (RL). The specific objectives were (i) to standardize the IVRI method to facilitate the contact between L3 and RL as well as the larval recovery, and (ii) to apply the IVRI method to describe the exsheathment kinetics of H. contortus and to select the best fitting nonlinear model. Incubation devices containing H. contortus larvae were incubated according to the IVRI technique in cattle RL or PBS. The incubation conditions included RL mixed with a nitrogen-rich media, maintained at 39 °C, with pH = 7.0, vented with CO2 and manual agitation. The larvae were recovered after 0, 1, 3, 6, 9, 12, and 24 h. The exsheathed and ensheathed larvae were counted to estimate the exsheathment (%) in RL or PBS. Exsheathment in RL was analyzed with nonlinear regression models: Exponential, Gompertz, Logistic, Log-Logistic, and Weibull. The models' fit was compared to select the one that best described the exsheathment kinetics. The exsheathment in RL reached 6.52%, 20.65%, 58.22%, 69.24%, 73.08%, and 77.20% in 1, 3, 6, 9, 12, and 24 h, respectively. Although the Gompertz, Weibull, and Logistic models were adequate to describe the observed exsheathment, the Log-Logistic model had the best fit. The IVRI method using bovine RL represents a suitable tool for the study of the in vitro exsheathment kinetics of H. contortus L3.

Ovine PAPPA2 gene coding variants are linked to decreased fecal egg shedding in native Turkish sheep naturally infected with gastrointestinal nematodes.

In this study, the association between PAPPA2 coding variants and gastrointestinal (GI) nematode fecal egg count (FEC) score in adult Turkish sheep was investigated. For this purpose, the FEC score was determined in adult sheep from six breeds: Karacabey Merino (n = 137), Kivircik (n = 116), Cine capari (n = 109), Karakacan (n = 102), Imroz (n = 73), and Chios (n = 50). Sheep were classified as shedders or non-shedders within breeds and flocks. The first group was the fecal egg shedders (> 50 per gram of feces), and the second group was the no fecal egg shedders (≤ 50 per gram of feces). The exon 1, exon 2, exon 5, exon 7, and a part of 5'UTR of the ovine PAPPA2 gene were genotyped by Sanger sequencing of these two groups. Fourteen synonymous and three non-synonymous single-nucleotide polymorphisms (SNPs) were found. The non-synonymous SNPs, D109N, D391H, and L409R variants, are reported for the first time. Two haplotype blocks were constructed on exon 2 and exon 7. The specific haplotype, C391G424G449T473C515A542 on the exon 2 that carries the 391H variant, was tested against four other common haplotypes. Our results indicate that C391G424G449T473C515A542 haplotype was significantly associated with fecal egg shedding status in adult Turkish sheep (p-value, 0.044).

Dietary protein supplementation results in molecular and cellular changes related to T helper type 2 immunity in the lung and small intestine in lactating rats re-infected with Nippostrongylus brasiliensis.

Acquired immunity to gastrointestinal nematodes reduces during late pregnancy and lactation which is known as periparturient relaxation of immunity (PPRI). Protein supplementation reduces the degree of PPRI in a rat model re-infected with Nippostrongylus brasiliensis, but the underlying molecular mechanisms have yet to be elucidated. Here, we hypothesized that protein supplementation will enhance T helper type 2 immunity (Th2) in the lung and small intestine. Nulliparous Sprague-Dawley rats were given a primary infection of N. brasiliensis prior to mating and restrictedly fed diets with either low protein (LP) or high protein (HP) during pregnancy and lactation. Dams were secondary infected with N. brasiliensis on day 2 post-parturition, and histology and gene expression were analysed for tissue samples collected at days 5, 8 and 11. Genes related to Th2 immunity in the lung, Retnla, Il13 and Mmp12, and in the intestine, Retnlb, were upregulated in HP dams compared to LP dams, which indicates the effect of dietary protein on Th2 immunity. HP dams also had increased splenic CD68+ macrophage populations compared to LP dams following secondary infection, suggesting enhanced immunity at a cellular level. Our data assist to define strategic utilization of nutrient supply in mammals undergoing reproductive and lactational efforts.

Naturally acquired ovine-adapted nematode infections in young cattle and their treatment with eprinomectin 5% w/v extended-release injection.

The objective of this controlled study was to determine the anthelmintic efficacy of eprinomectin 5% w/v extended-release injection (Eprinomectin ERI; LONGRANGE®, Boehringer Ingelheim Animal Health) against primarily ovine-adapted gastrointestinal strongylid nematode (GIN) parasites in naturally infected young cattle. Eighteen calves which grazed on a sheep-dominated mixed sheep-cattle farm were randomly allocated into two equal groups (saline [control] or Eprinomectin ERI, administered each at 1 mL/50 kg body weight once subcutaneously), treated and euthanized 14 days thereafter for a differential GIN count. Ten species of strongylid nematodes were recovered from the control calves (ovine-adapted Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Cooperia curticei, Nematodirus battus, Chabertia ovina; bovine-adapted Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus; 'generalist' Trichostrongylus axei). Adult GIN counts ranged from 1,540 to 5,244 for the control calves and from zero to 110 for the Eprinomectin ERI-treated calves. Accepting the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products anthelmintic guidelines criteria on adequacy of infections to be demonstrated in the control animals, this study showed that Ch. ovina, C. curticei, H. contortus, N. battus, T. axei, and T. colubriformis were reduced significantly (p < 0.0001) by ≥ 98.7% in the animals treated with Eprinomectin ERI. In conclusion, Eprinomectin ERI treatment was efficacious against a range of ovine-adapted nematode parasites in naturally infected young cattle.

Variability in the Response against Teladorsagia circumcincta in Lambs of Two Canarian Sheep Breeds.

The increasing resistance to anthelmintics has necessitated the exploration of alternative control strategies of gastrointestinal nematode (GIN) infections. A sustainable option is genetic selection based on differences in susceptibility to GIN infection between and within breeds of sheep. Here, three-month-old Canaria Hair breed (GIN-resistant) and Canaria Sheep breed (GIN-susceptible) showed no significant between-breed differences after trickle infection with Teladorsagia circumcincta, whereas considerable individual variability was found in both breeds. Next, data from lambs of both breeds were used to explore the relationships between parasitological variables and T. circumcincta-specific IgA levels, local immune cell populations, and abomasal lymph node gene expression to understand the possible mechanisms underlying resistance. Mucosal IgA levels as well as numbers of globular leukocytes and MHC-II+ cells were associated with protection. Analysis of lymph node gene expression revealed the associations between lower parasite numbers and cumulative fecal egg counts and several immune pathways, such as leukocyte cell adhesion, activation and differentiation of T cells, in particular CD4+ and IL-4 production. The data obtained here may inform on the relationship between phenotypic resistance variability and protective responses at the humoral, cellular, and transcriptomic levels, thus contributing to identifying immune responses in young lambs that could be used as markers for selection.

Gastrointestinal nematodes and mineral deficiencies in yearling cattle in Santiago del Estero, northern Argentina.

The aims were to study the epidemiology and the effect of gastrointestinal nematodes (GINs) on yearling cattle in a semiarid region in Argentina and to evaluate the mineral serum levels. Ten- to twelve-month-old calves were monitored between November and April 2019. Animals were divided into three groups: untreated control (CONG; n = 20); group treated with moxidectin (MXD, 200 µg/kg) every 30-40 days (MXDG; n = 20); group equally MXD treated and mineral supplementation administered in November and January (MMG; n = 20). Individual GIN egg counts (epg) and fecal cultures were made every 30-40 days. Animal live weight gain was determined. On 7-11-18, 25-1-19, and 3-4-19, serum levels of Se, Cu, Zn, Mg, and Ca were determined by atom absorption spectrophotometry. At the start of the trial, epg values were low (x̄ = 5.5), which increased only in CONG towards the end of the assay (x̄ = 229). In both GMXD and MMG, epg remained very low (x̄ = 4.1). Cooperia spp. (81%) prevailed over Haemonchus spp. (9%) and Oesophagostomum spp. (9%). Deficiency of Se ranged between marginal and important, except for MMG, although mean values were always below normal. Cu was marginally deficient in CONG and MXDG on the first two sampling dates, whereas MMG had normal values after treatment. Mg had low values in the three groups (x̄ = 1.5 mg/dl), whereas Ca and Zn exhibited normal values. Live weight gain (LWG) was higher in MMG than in MXDG, with significant differences (p < 0.05) by mid-January and then, when epg increased, dewormed groups showed higher LWG (p < 0.034) than CONG. A negative effect of GINs on LWG was observed, as well as low to marginal levels of Se, Cu, and Mg.

Epidemiology, prevention and control of gastrointestinal helminths of small ruminants in the Caribbean region-a scoping review.

This review gathered information from peer-reviewed publications on the epidemiology, prevention and control of gastrointestinal helminths (GIHs) parasites of small ruminants in the Caribbean region from 1990 to 2021. Literature search was performed on four electronic databases using a combination of search terms and Boolean operators. Results showed that gastrointestinal nematodes (GINs) were the most common parasites, with seven genera documented across six reviewed countries. Haemonchus contortus was the most common species occurring in all six countries, and predominant in occasions where mixed infections were observed. Moniezia species were the only Platyhelminth species documented in Trinidad and Grenada. The overall prevalence of GIH infections was observed to be high in goats than in sheep. A high level of anthelminthic resistance (AR) with H. contortus was reported, whilst other nematodes showed to be effectively controlled by one or more anthelmintic drugs. FAMACHA© method was applied in the identification of anaemic animals for selective treatment, hence contributing to the decreased use of anthelmintic drugs. There is a need for national surveys of GIHs and AR in small ruminants in the Caribbean countries. Surveys, coupled with the use of molecular techniques to detect and identify species of GIHs present in the Caribbean region, as well as their epidemiology which will inform development of integrated control strategies is recommended. There is also a need to create awareness to small ruminant farmers in the region on the prevailing challenge of AR and limit wanton use of anthelminthics to reduce or prevent AR in small ruminants.

Recombinant Paraprobiotics as a New Paradigm for Treating Gastrointestinal Nematode Parasites of Humans.

Gastrointestinal nematodes (GINs) of humans, e.g., hookworms, negatively impact childhood growth, cognition, nutrition, educational attainment, income, productivity, and pregnancy. Hundreds of millions of people are targeted with mass drug administration (MDA) of donated benzimidazole anthelmintics. However, benzimidazole efficacy against GINs is suboptimal, and reduced/low efficacy has been seen. Developing an anthelmintic for human MDA is daunting: it must be safe, effective, inexpensive, stable without a cold chain, and massively scalable. Bacillus thuringiensis crystal protein 5B (Cry5B) has anthelmintic properties that could fill this void. Here, we developed an active pharmaceutical ingredient (API) containing B. thuringiensis Cry5B compatible with MDA. We expressed Cry5B in asporogenous B. thuringiensis during vegetative phase, forming cytosolic crystals. These bacteria with cytosolic crystals (BaCC) were rendered inviable (inactivated BaCC [IBaCC]) with food-grade essential oils. IBaCC potency was validated in vitro against nematodes. IBaCC was also potent in vivo against human hookworm infections in hamsters. IBaCC production was successfully scaled to 350 liters at a contract manufacturing facility. A simple fit-for-purpose formulation to protect against stomach digestion and powdered IBaCC were successfully made and used against GINs in hamsters and mice. A pilot histopathology study and blood chemistry workup showed that five daily consecutive doses of 200 mg/kg body weight Cry5B IBaCC (the curative single dose is 40 mg/kg) was nontoxic to hamsters and completely safe. IBaCC is a safe, inexpensive, highly effective, easy-to-manufacture, and scalable anthelmintic that is practical for MDA and represents a new paradigm for treating human GINs.

Prevalence of anthelmintic resistance of gastrointestinal nematodes in Polish goat herds assessed by the larval development test.

BACKGROUND: Helminthic infections, in particular those caused by gastrointestinal nematodes (GIN), are found worldwide and are among the most economically important diseases of goats. Anthelmintic resistance (AR) in GIN of goats is currently present worldwide, and single- or multidrug resistant species are widespread. The aim of this study was to determine the prevalence of AR to benzimidazoles (BZ), macrocyclic lactones (ML) and imidazothiazoles represented by levamisole (LEV) in the Polish goat herds by using an in vitro larval development test, which is useful especially in large-scale epidemiological surveys. RESULTS: This cross-sectional study was conducted from September 2018 to June 2019 and enrolled 42 dairy goat herds scattered over the entire country. The most commonly used anthelmintic class in goat herds in Poland were BZ (92%), followed by ML (85%) and LEV (13%). BZ-resistant GIN populations were found in 37 herds (88%, CI 95%: 75 to 95%), ML-resistant GIN populations in 40 herds (95%, CI 95, 84 to 99%), and LEV-resistant GIN populations in 5 herds (12%, CI 95%: 5 to 25%). Multidrug resistance involving all three anthelmintic classes was found in 5 herds (12%, CI 95, 5 to 25%). Based on the morphological features of stage 3 larvae the main resistant GIN turned out to be Haemonchus contortus and Trichostrongylus spp. The use of BZ and frequency of anthelmintic treatments were significantly related to the presence of AR to BZ in Polish goat herds. CONCLUSIONS: This cross-sectional study demonstrates the existence of AR to BZ, ML and LEV on Polish goat farms. Resistance to BZ and ML is widespread, while AR to LEV is currently at a low level. A considerable proportion of herds harbours multidrug resistant GIN, which requires further consideration. An effective anthelmintic treatment strategy, reasonable preventive measures and better understanding of the resistance-related management practices by farmers and veterinarians may delay further development of AR.

Resistance of strongylid nematodes to anthelmintic drugs and driving factors at Czech goat farms.

BACKGROUND: Strongylid nematode infections may negatively affect both animal health and welfare, with deleterious consequences for livestock productivity. Many farmers in recent decades have relied on anthelmintics as the sole strategy of control, but the intensive use of these chemotherapeutics has led to the development of anthelmintic resistance (AR). Knowledge of both the efficacy of anthelmintics and factors promoting AR are essential to effectively control nematode infections, but no information on these topics for goats in the Czech Republic (CR) is available. This survey aimed to determine the occurrence of AR at Czech goat farms and to identify risk factors for the development of AR. A total of 24 herds of dairy goats across the CR were evaluated using in vitro tests for detecting AR, and a questionnaire survey was carried out to evaluate factors associated with AR. RESULTS: Resistance against benzimidazoles was confirmed at 18 (75%) farms, and the level of resistance was high in four (22%) of the affected herds based on the egg hatch test. Ivermectin-resistant nematodes were detected in 13 (54%) herds using the larval development test; Teladorsagia/Trichostrongylus and Haemonchus were the predominant types of resistant larvae. Eight (62%) of the affected herds were evaluated as highly resistant to ivermectin. Eleven (46%) of the herds were resistant to both benzimidazoles and ivermectin. This report is the first on dual AR in the CR. A univariate logistic regression analysis indicated that a high stocking rate and farmer inexperience were significantly associated with ivermectin and benzimidazole resistance, respectively. CONCLUSIONS: The results of our survey suggest that AR is widespread amongst herds of dairy goats in the CR, likely due to inappropriate practices of pasture and health management. AR may be an issue for expanding dairy-goat production in the CR in the near future unless both veterinary practitioners and farmers widely adopt strategies to prevent the development of AR.

Anthelmintic resistance: is a solution possible?

More than 50 years after anthelmintic resistance was first identified, its prevalence and impact on the animal production industry continues to increase across the world. The term "anthelmintic resistance" (AR) can be briefly defined as the reduction in efficacy of a certain dose of anthelmintic drugs (AH) in eliminating the presence of a parasite population that was previously susceptible. The main aim of this study is to examine anthelmintic resistance in domestic herbivores. There are numerous factors playing a role in the development of AR, but the most important is livestock management. The price of AH and the need to treat a high number of animals mean that farmers face significant costs in this regard, yet, since 1981, little progress has been made in the discovery of new molecules and the time and cost required to bring a new AH to market has increased dramatically in recent decades. Furthermore, resistance has also emerged for new AH, such as monepantel or derquantel. Consequently, ruminant parasitism cannot be controlled solely by using synthetic chemicals. A change in approach is needed, using a range of preventive measures in order to achieve a sustainable control programme. The use of nematophagous fungi or of plant extracts rich in compounds with anthelmintic properties, such as terpenes, condensed tannins, or flavonoids, represent potential alternatives. Nevertheless, although new approaches are showing promising results, there is still much to do. More research focused on the control of AR is needed.

In vitro efficacy of different concentrations of Duddingtonia flagrans on varying egg densities of gastrointestinal nematodes of cattle.

The nematophagous fungus Duddingtonia flagrans, used for the biological control of gastrointestinal nematodes in livestock, is fed to infected animals so its chlamydospores and the parasite eggs are voided together with faeces where the fungus preys on nematode larvae, thus reducing pasture infectivity. The number of chlamydospores needed for the fungus to be efficient in the presence of a wide range in numbers of parasitic eggs is largely unknown and a matter of discussion. The aim of this study was to determine the fungal efficacy of four different chlamydospore concentrations against three different levels of cattle faecal egg counts. Fungal concentrations of 11000, 6250, 3000 and 1000 chlamydospores/gram of faeces (cpg) were added to cultures containing 840, 480 or 100 eggs/gram of faeces (epg). After 14 days of incubation, the efficacy of D. flagrans, in decreasing order of chlamydospore concentrations, ranged from 100% (P < 0.0001) to 77% (P > 0.0999) in the 100 epg groups; 100% (P < 0.0001) to 92% (P = 0.4625) in the 480 epg groups and 100% (P < 0.0001) to 96% (P = 0.7081) in the 840 epg groups. The results indicate that the numbers of eggs in cattle faeces were not a determining factor on the fungal efficacy against gastrointestinal nematodes.

Survival of the nematophagous fungus Duddingtonia flagrans to in vitro segments of sheep gastrointestinal tract.

The nematophagous fungus Duddingtonia flagrans is used in integrated management of gastrointestinal nematodes in ruminants. The chlamydospores of the fungus, orally administered, pass through the segments of the ruminant digestive tract and, in the feces, capture the nematodes preventing their migration to grasslands. The drastic conditions of the gastrointestinal segments can negatively affect the fungus' biocontrol activity. The aim of this study was to assess the effect of in vitro conditions of the sheep's main gastrointestinal segments on the concentration, viability and nematode predatory ability of D. flagrans chlamydospores. The segments evaluated separately in vitro were the oral cavity, rumen, abomasum, and small intestine. The results showed that chlamydospores concentration was not affected by exposure to the different segments. The viability of the chlamydospores after exposure to the oral cavity (2.53 × 106 CFU/mL) and small intestine (1.24 × 105 CFU/mL) was significantly lower than its control treatment, with values of 6.67 × 106 CFU/mL and 2.31 × 105 CFU/mL respectively. Nematode predatory ability after rumen exposure was reduced by 7% compared to the control treatment, by 25% after abomasum exposure and by 17% after small intestine. This study revealed the individual in vitro effect of each segment of ovine gastrointestinal tract on the integrity of this strain of the fungus D. flagrans affecting its viability and nematode predatory ability under the evaluated conditions. Delivery systems could be designed to protect chlamydospores considering the impact of each gastrointestinal segment.

A simple molecular method to identify and quantify genera of gastrointestinal nematodes of cattle.

Classic approaches for antemortem identification of gastrointestinal nematodes (GIN) require coproculture of eggs and morphological examination. While adequate for diagnosis, many PCR techniques cannot easily quantify mixed infections without controls and/or standard curves. Herein, we developed a simple and rapid test for differentiating and quantifying mixed infections of GIN using PCR products separated by capillary electrophoresis. Among the cattle GIN, the ITS2 region is sufficiently distinct in length to delineate among the most common infecting genera, Ostertagia ostertagi = 373 bases (b), Haemonchus contortus (placei) = 366b, Cooperia punctata (oncophora) = 376b, Trichostrongylus axei = 372b, and Oesophagostomum radiatum = 357b. Conserved primers were synthesized that span the ITS2 where one primer was fluorescently labeled with 6-FAM. DNAs from infective L3 were PCR amplified then loaded onto an ABI 3130 sequencer adapted for size fragment analysis. Resulting peak amplitudes were both diagnostic and quantitative on a relative basis. As proof of principle, quantification was performed on PCR fragments from mixed species pairs of Ostertagia ostertagi, Cooperia punctata, and Haemonchus contortus and analyzed using Gene Marker V1.85 software. In all cases, linear responses were observed where R2 > 0.97 and line slopes ranged between 0.90 and 1.1. When tested on eggs from naturally infected animals, the assay showed superior results on two farms when compared to coproculture and morphological identification. Using wildlife-derived samples, results coincided well with deep amplicon sequencing. The assay is adaptable to large-scale studies, does not require comparative PCR controls, and should be compliant with GIN from small ruminant livestock.

Dietary protein degradability effect on performance of lambs experimentally infected with Haemonchus contortus and Trichostrongylus colubriformis.

The study investigated the effects of dietary protein degradation rate on growth performance and immune response of crossbred Dorper × short-tail Han ram lambs experimentally infected with Haemonchus contortus and Trichostrongylus colubriformis. Eighteen lambs were randomly assigned to three dietary treatments, rapidly degradable protein (RDP), moderately degradable protein (MDP), and slowly degradable protein (SDP) diets. Feed intake and body weight of the lambs were recorded weekly until 42 days post-infection. The fecal egg count (FEC), FAMACHA scores, and immunoglobulins (IgG, IgM and IgA) were also monitored during the experimental period. A metabolic trial was conducted to assess apparent digestibility and volatile fatty acids were also determined. The lambs in SDP and MDP groups had higher feed, nutrient intake, weight gain, and feed efficiency than those in the RDP group. Feed conversion ratio (FCR) of the lambs in RDP group was higher than those in the SDP and MDP groups. A significant (P < 0.001) decrease in FEC was observed in the SDP and MDP groups. Dietary treatment had no significant effect on FAMACHA scores and concentration of serum antibodies. Concentration of acetic acid was higher (P < 0.013) in the lambs fed RDP than those fed the SDP and MDP diets. The lambs fed SDP diet had higher apparent digestibility than those fed the RDP diet. The poor performances in RDP group could be overcome by including SDP that ensures adequate post-ruminal protein supply reaching the small intestine.

Increased susceptibility to Haemonchus contortus infection by interleukin-5 modulation of eosinophil responses in sheep.

Eosinophils are prominent effector cells in immune responses against gastrointestinal nematode infections in ruminants, but their in vivo role has been hard to establish in large animals. Interleukin-5 is a key cytokine in the induction and stimulation of anti-parasitic eosinophil responses. This study attempted to modulate the eosinophil response in sheep through vaccination with recombinant interleukin-5 (rIL-5) and determine the effect on subsequent Haemonchus contortus infection. Nematode-resistant Canaria Hair Breed (CHB) sheep vaccinated with rIL-5 in Quil-A adjuvant, had lower blood eosinophil counts and higher mean worm burdens than control sheep vaccinated with Quil-A adjuvant alone. In addition, adult worms in IL-5-vaccinated sheep were significantly longer with higher eggs in utero in female worms, supporting an active role of eosinophils against adult parasites in CHB sheep. These results confirm that eosinophils can play a direct role in effective control of H contortus infection in sheep and offer a new approach to study immune responses in ruminants.

Experimental parasite community perturbation reveals associations between Sin Nombre virus and gastrointestinal nematodes in a rodent reservoir host.

Individuals are often co-infected with several parasite species, yet measuring within-host interactions remains difficult in the wild. Consequently, the impacts of such interactions on host fitness and epidemiology are often unknown. We used anthelmintic drugs to experimentally reduce nematode infection and measured the effects on both nematodes and the important zoonosis Sin Nombre virus (SNV) in its primary reservoir (Peromyscus spp.). Treatment significantly reduced nematode infection, but increased SNV seroprevalence. Furthermore, mice that were co-infected with both nematodes and SNV were in better condition and survived up to four times longer than uninfected or singly infected mice. These results highlight the importance of investigating multiple parasites for understanding interindividual variation and epidemiological dynamics in reservoir populations with zoonotic transmission potential.