Beyond parasitism: Exploring the microbial profile of Haemonchus contortus and its predilection site (abomasum) in Kashmir Merino sheep.

Gastrointestinal helminth infection, particularly by Haemonchus contortus, poses significant challenges to sheep farming worldwide. While anthelmintic drugs have been traditional control measures, the emergence of resistance calls for alternative strategies. Understanding the interaction between parasites, host, and their microbiome is crucial for management of helminth infection. This study intricately explores the interactions between microbial communities in Kashmir Merino sheep infected with H. contortus, to understand the complex interplay between host, parasite, and their microbiome. Sheep abomasal contents and H. contortus were collected from infected and control groups, processed for DNA extraction, and subjected to metagenomic sequencing of the 16 S rRNA gene. Downstream analysis unveils distinct microbial patterns, where Proteobacteria were dominant in H. contortus, while Bacteroidota and Firmicutes prevailed in the sheep abomasum. The revelation of unique genera and shifts in diversity indices underscored helminth-induced disruptions in the host. Beta diversity analysis further showed significant variations in bacterial profiles, providing insights into the intricate host, parasite, and microbiome dynamics. Additionally, this study elucidated the presence of pathogenic bacteria within H. contortus, accentuating their potential role in exacerbating sheep health issues. This finding underscores the complexity of the host-parasite-microbiome interaction showing helminth-induced microbiome alterations of the host.

Mannheimia haemolytica-associated fibrinonecrotizing abomasitis in lambs.

Mannheimia haemolytica-associated abomasitis has been clinically described as a cause of sudden death in lambs, but it is poorly characterized. We describe the pathological features of a severe fibrinonecrotizing abomasitis in 3 lambs that died suddenly. All 3 abomasums had a thickened submucosa due to edema and necrotic areas delimited by bands of degenerate neutrophils with slender nuclei (oat cells) and angiocentric distributions. The overlying mucosa was congested. Myriads of gram-negative coccobacilli were observed within the oat cell bands. M. haemolytica was isolated from the abomasum in all 3 animals and was serotyped as A2 in one of them. Pericarditis and pleuritis were observed in 2 of the lambs. Clostridium spp. were isolated in 1 lamb and detected by immunohistochemistry in the 3 animals, suggesting clostridial co-infection. M. haemolytica should be considered among the differential diagnoses of necrotizing abomasitis in lambs.

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.

The Effects of Artificially Dosed Adult Rumen Contents on Abomasum Transcriptome and Associated Microbial Community Structure in Calves.

This study aimed to investigate the changes in abomasum transcriptome and the associated microbial community structure in young calves with artificially dosed, adult rumen contents. Eight young bull calves were randomly dosed with freshly extracted rumen contents from an adult cow (high efficiency (HE), n = 4), or sterilized rumen content (Con, n = 4). The dosing was administered within 3 days of birth, then at 2, 4, and 6 weeks following the initial dosing. Abomasum tissues were collected immediately after sacrifice at 8 weeks of age. Five genera (Tannerella, Desulfovibrio, Deinococcus, Leptotrichia, and Eubacterium; p < 0.05) showed significant difference in abundance between the treatments. A total of 975 differentially expressed genes were identified (p < 0.05, fold-change > 1.5, mean read-counts > 5). Pathway analysis indicated that up-regulated genes were involved in immune system process and defense response to virus, while the down-regulated genes involved in ion transport, ATP biosynthetic process, and mitochondrial electron transport. Positive correlation (r > 0.7, p < 0.05) was observed between TRPM4 gene and Desulfovibrio, which was significantly higher in the HE group. TRPM4 had a reported role in the immune system process. In conclusion, the dosing of adult rumen contents to calves can alter not only the composition of active microorganisms in the abomasum but also the molecular mechanisms in the abomasum tissue, including reduced protease secretion and decreased hydrochloric acid secretion.

Dynamic change of the gastrointestinal bacterial ecology in cows from birth to adulthood.

The gut microbiota plays multiple critical roles in maintaining the health of the host, especially in ruminants. However, our understanding of the establishment of gut microbiota from birth to adulthood is still limited. To address this, the bacterial ecology of the rumen, abomasum, duodenum, and rectum in Holstein cows ranging in age from 1 week to 5 years old was investigated using 16S rRNA gene sequencing in this study. A major change in the composition, diversity, and abundance of bacteria was observed with increased age (p < 0.05). Microbiota gradually matured in each gut segment and followed the Gompertz model when the Chao1, Shannon, and maturity indexes (p < 0.05, r > 0.94) were applied. Importantly, the Gompertz model parameter differed between the gut segments, with the highest microbiota growth rate found in the rectum, followed by the rumen, abomasum, and duodenum. Compared to older animals, greater microbiota similarities were found in the adjacent gut segments of younger animals (p < 0.05). Our findings indicate that gut microbiotas are established quickly when cows are young and then slow with age and that early in life, hindgut microbiota may be more easily affected by the foregut microbiota.

Can the foregut nematode Haemonchus contortus and medicinal plants influence the fecal microbial community of the experimentally infected lambs?

The abomasal parasitic nematode Haemonchus contortus can influence the abomasal microbiome of the host. On the other hand, no information occurs on the influence of the parasite on the hindgut microbiome of the host. We evaluated the impact of Haemonchus contortus on the fecal microbial community of the experimentally infected lambs treated with a mixture of medicinal herbs to ameliorate the haemonchosis. Twenty-four female lambs were divided into four groups: infected animals (Inf), infected animals supplemented with a blend of medicinal herbs (Inf+Herb), uninfected control animals (Control), and uninfected animals supplemented with medicinal herbs (C+Herb). Inf and Inf+Herb lambs were infected orally with approximately 5000 L3 larvae of a strain of H. contortus susceptible to anthelmintics (MHco1). Herb blend (Herbmix) consisted of dry medicinal plants of Althaea officinalis, Petasites hybridus, Inula helenium, Malva sylvestris, Chamomilla recutita, Plantago lanceolata, Rosmarinus officinalis, Solidago virgaurea, Fumaria officinalis, Hyssopus officinalis, Melisa officinalis, Foeniculum vulgare, and Artemisia absinthium. Each animal was fed meadow hay and a commercial concentrate (600 + 350 g DM/d). Inf+Herb and C+Herb lambs were fed Herbmix (100 g DM/d and animal). Treatment lasted for 50 days. The fecal microbial fermentation parameters (short-chain fatty acids, ammonia, and pH) were evaluated at intervals of 0, 20, 32, and 50 days. The fecal eubacterial populations were evaluated by denaturing gradient gel electrophoresis (DGGE) at day 32 when H. contortus infection was the highest. No substantial effects of the H. contortus infection and the herbal treatment on fecal microbial fermentation parameters and fecal eubacterial populations were observed. Evaluation of DGGE patterns by Principal component analysis pointed to the tendency to branch the C+Herb group from the other experimental groups on Day 32. The results indicate that hindgut microbial activity was not disturbed by H. contortus infection and herbal treatment.

Microbiome analysis as a platform R&D tool for parasitic nematode disease management.

The relationship between bacterial communities and their host is being extensively investigated for the potential to improve the host's health. Little is known about the interplay between the microbiota of parasites and the health of the infected host. Using nematode co-infection of lambs as a proof-of-concept model, the aim of this study was to characterise the microbiomes of nematodes and that of their host, enabling identification of candidate nematode-specific microbiota member(s) that could be exploited as drug development tools or for targeted therapy. Deep sequencing techniques were used to elucidate the microbiomes of different life stages of two parasitic nematodes of ruminants, Haemonchus contortus and Teladorsagia circumcincta, as well as that of the co-infected ovine hosts, pre- and post infection. Bioinformatic analyses demonstrated significant differences between the composition of the nematode and ovine microbiomes. The two nematode species also differed significantly. The data indicated a shift in the constitution of the larval nematode microbiome after exposure to the ovine microbiome, and in the ovine intestinal microbial community over time as a result of helminth co-infection. Several bacterial species were identified in nematodes that were absent from their surrounding abomasal environment, the most significant of which included Escherichia coli/Shigella. The ability to purposefully infect nematode species with engineered E. coli was demonstrated in vitro, validating the concept of using this bacterium as a nematode-specific drug development tool and/or drug delivery vehicle. To our knowledge, this is the first description of the concept of exploiting a parasite's microbiome for drug development and treatment purposes.

Isolation and molecular identification of Lactobacillus with probiotic potential from abomasums driven rennet.

Lactobacillus species are beneficial for the functional food industry and preventive medicine. The complex microflora of traditional cheese depends on the cheese types (e.g., homemade rennets). Here, the abomasum driven rennet was assessed for the existence of lactobacilli. For differentiating lactobacilli, the bacterial suspension was screened for the acid and bile resistance. The isolated bacteria were evaluated for antibiotic susceptibility and antagonistic impacts on other pathogenic bacteria. The 16S rDNA gene was evaluated by the amplified ribosomal DNA restriction analysis (ARDRA) recruiting the restriction enzyme Taq I and compared to the virtually digested patterns of previous reports on lactobacilli. The isolates were examined by random amplified polymorphic DNA (RAPD) and distinctive lactobacilli were sequenced. ARDRA and RAPD data showed three distinct lactobacilli strains, including L. acidophilus, L. planetarum, and L. fermentum. The homemade rennet is proposed as the novel source of probiotic strains as an alternative to the traditional cheeses.

Microbial diversity in the rumen, reticulum, omasum, and abomasum of yak on a rapid fattening regime in an agro-pastoral transition zone.

The ruminant digestive system harbors a complex gut microbiome, which is poorly understood in the case of the four stomach compartments of yak. High-throughput sequencing and quantitative PCR were used to analyse microbial communities in the rumen, reticulum, omasum, and abomasum of six domesticated yak. The diversity of prokaryotes was higher in reticulum and omasum than in rumen and abomasum. Bacteroidetes predominated in the four stomach compartments, with abundance gradually decreasing in the trend rumen > reticulum > omasum > abomasum. Microorganism composition was different among the four compartments, all of which contained high levels of bacteria, methanogens, protozoa and anaerobic fungi. Some prokaryotic genera were associated with volatile fatty acids and pH. This study provides the first insights into the microorganism composition of four stomach compartments in yak, and may provide a foundation for future studies in this area.

Clostridial Abomasitis and Enteritis in Ruminants.

Clostridial abomasitis and enteritis are important alimentary diseases observed in all domestic ruminant species. These diseases most commonly result from overgrowth of Clostridium perfringens types A, B, C, D, and E with the associated release of bacterial exotoxins that result in necrosis of the abomasal or intestinal mucosa. Clostridium difficile may also be associated with enteritis in calves but is much less common than disease caused by C perfringens. This article reviews the causes, pathophysiology, clinical signs, diagnosis, treatment, and prevention of clostridial gastrointestinal diseases in ruminants. Particular emphasis is given to describing the various forms of disease and treatment of individual cases.

Microbial community and ovine host response varies with early and late stages of Haemonchus contortus infection.

The interactions between gastric microbiota, ovine host, and Haemonchus contortus portray the ovine gastric environment as a complex ecosystem, where all factors play a pertinent role in fine-tuning each other and in haemeostasis. We delineated the impact of early and late Haemonchus infection on abomasal and ruminal microbial community, as well as the ovine host. Twelve, parasite-naive lambs were divided into four groups, 7 days post-infection (dpi) and time-matched uninfected-control groups; 50 dpi and time-matched uninfected control groups were used for the experiment. Six sheep were inoculated with 5000 H. contortus infective larvae and followed for 7 or 50 days with their corresponding uninfected-control ones. Ovine abomasal tissues were collected for histological analysis and gastric fluids were collected for PH value measurements, microbial community isolation and Illumina MiSeq platform and bioinformatic analysis. Our results showed that Haemonchus infection increased the abomasal gastric pH (P = 0.05) and resulted in necrotizing and inflammatory changes that were more severe during acute infection. Furthermore, infection increased the abomasal bacterial load and decreased the ruminal microbiome. A 7-day infection of sheep with H. contortus significantly altered approximately 98% and 94% of genera in the abomasal and ruminal bacterial profile, respectively (P = 0.04-0.05). However, the approximate altered genera 50 days after infection in the ovine abomasal and ruminal microbiome were about 62% and 69%, correspondingly (P = 0.04-0.05) with increase in some bacteria and decrease in others. Overall, these results indicate that Haemonchus infection plays a crucial role in shaping stomach microbial community composition, and diversity.

Amplicon sequencing of bacterial microbiota in abortion material from cattle.

Abortions in cattle have a significant economic impact on animal husbandry and require prompt diagnosis for surveillance of epizootic infectious agents. Since most abortions are not epizootic but sporadic with often undetected etiologies, this study examined the bacterial community present in the placenta (PL, n = 32) and fetal abomasal content (AC, n = 49) in 64 cases of bovine abortion by next generation sequencing (NGS) of the 16S rRNA gene. The PL and AC from three fetuses of dams that died from non-infectious reasons were included as controls. All samples were analyzed by bacterial culture, and 17 were examined by histopathology. We observed 922 OTUs overall and 267 taxa at the genus level. No detectable bacterial DNA was present in the control samples. The microbial profiles of the PL and AC differed significantly, both in their composition (PERMANOVA), species richness and Chao-1 (Mann-Whitney test). In both organs, Pseudomonas was the most abundant genus. The combination of NGS and culture identified opportunistic pathogens of interest in placentas with lesions, such as Vibrio metschnikovii, Streptococcus uberis, Lactococcus lactis and Escherichia coli. In placentas with lesions where culturing was unsuccessful, Pseudomonas and unidentified Aeromonadaceae were identified by NGS displaying high number of reads. Three cases with multiple possible etiologies and placentas presenting lesions were detected by NGS. Amplicon sequencing has the potential to uncover unknown etiological agents. These new insights on cattle abortion extend our focus to previously understudied opportunistic abortive bacteria.

Use of Brucella abortus species specific polymerase chain reaction assay for the diagnosis of bovine brucellosis.

Serology is primarily used in the diagnosis of bovine brucellosis. Bacterial culture and isolation is the gold standard in diagnosing brucellosis but, like serology, it does not offer complete (100%) diagnostic sensitivity and specificity. Polymerase chain reaction (PCR) has been suggested to offer better specificity and sensitivity. In this study, we evaluated the performance of Brucella abortus species specific (BaSS) PCR directly from different samples in the diagnosis of bovine brucellosis in naturally infected cattle in KwaZulu-Natal province of South Africa with known infectious status from culture. The BaSS PCR had a low diagnostic sensitivity (DSe) of 70%, but was able to identify vaccine strains using abomasal fluid from aborted foetuses and detect Brucella DNA from decomposing samples. The best sample for the BaSS PCR was abomasal fluid.

Microbial community composition along the digestive tract in forage- and grain-fed bison.

BACKGROUND: Diversity and composition of microbial communities was compared across the 13 major sections of the digestive tract (esophagus, reticulum, rumen, omasum, abomasum, duodenum, jejunum, ileum, cecum, ascending colon, transverse colon, descending colon, and rectum) in two captive populations of American bison (Bison bison), one of which was finished on forage, the other on grain. RESULTS: Microbial diversity fell to its lowest levels in the small intestine, with Bacteroidetes reaching their lowest relative abundance in that region, while Firmicutes and Euryarchaeota attained their highest relative abundances there. Gammaproteobacteria were most abundant in the esophagus, small intestine, and colon. The forage-finished bison population exhibited higher overall levels of diversity, as well as a higher relative abundance of Bacteroidetes in most gut sections. The grain-finished bison population exhibited elevated levels of Firmicutes and Gammaproteobacteria. Within each population, different sections of the digestive tract exhibited divergent microbial community composition, although it was essentially the same among sections within a given region of the digestive tract. Shannon diversity was lowest in the midgut. For each section of the digestive tract, the two bison populations differed significantly in microbial community composition. CONCLUSIONS: Similarities among sections indicate that the esophagus, reticulum, rumen, omasum, and abomasum may all be considered to house the foregut microbiota; the duodenum, jejunum, and ileum may all be considered to house the small intestine or midgut microbiota; and the cecum, ascending colon, transverse colon, descending colon, and rectum may all be considered to house the hindgut microbiota. Acid from the stomach, bile from the gall bladder, digestive enzymes from the pancreas, and the relatively low retention time of the small intestine may have caused the midgut's low microbial diversity. Differences in microbial community composition between populations may have been most strongly influenced by differences in diet (forage or grain). The clinical condition of the animals used in the present study was not evaluated, so further research is needed to establish whether the microbial profiles of some bison in this study are indeed indicative of dysbiosis, a predisposing factor to ruminal acidosis and its sequelae.

The effect of helminth infection on the microbial composition and structure of the caprine abomasal microbiome.

Haemonchus contortus is arguably the most injurious helminth parasite for small ruminants. We characterized the impact of H. contortus infection on the caprine abomasal microbiome. Fourteen parasite naive goats were inoculated with 5,000 H. contortus infective larvae and followed for 50 days. Six age-matched naïve goats served as uninfected controls. Reduced bodyweight gain and a significant increase in the abosamal pH was observed in infected goats compared to uninfected controls. Infection also increased the bacterial load while reducing the abundance of the Archaea in the abomasum but did not appear to affect microbial diversity. Nevertheless, the infection altered the abundance of approximately 19% of the 432 species-level operational taxonomic units (OTU) detected per sample. A total of 30 taxa displayed a significantly different abundance between control and infected goats. Furthermore, the infection resulted in a distinct difference in the microbiome structure. As many as 8 KEGG pathways were predicted to be significantly affected by infection. In addition, H. contortus-induced changes in butyrate producing bacteria could regulate mucosal inflammation and tissue repair. Our results provided insight into physiological consequences of helminth infection in small ruminants and could facilitate the development of novel control strategies to improve animal and human health.

[Nasal, pulmonary, and abomasal aspergillosis (Aspergillus fumigatus) in a calf]. / Nasale, pulmonale und abomasale Aspergillose (Aspergillus fumigatus) bei einem Kalb.

This study presents a case of nasal aspergillosis in a 17-days old calf (German Fleckvieh): it had been admitted moribund to the Clinic for Ruminants of the University of Munich, and died after a short time. Pathologically, the calf was diagnosed with purulent-necrotizing rhinitis, necrotizing pneumonia, and diphtheroid-necrotizing abomasitis. Histologically, fungal elements were found in all the localizations mentioned before, and mycologically, Aspergillus fumigatus was cultured from nasal cavity. Pathogenesis is discussed.

Characteristics of fluid composition of left displaced abomasum in beef cattle fed high-starch diets.

To clarify the pathophysiology of left displaced abomasum (LDA), beef cattle fed high-starch diets were examined. The abomasal pH in beef cattle with LDA was lower than that in non-LDA reference animals (data from beef cattle at an abattoir), suggesting that it facilitated acidity. Bacteriological examinations of the abomasal fluid in cattle with LDA revealed the presence of Pseudomonas spp., Clostridium spp. and Candida spp., presumably reflecting the accelerated influx of ruminal fluid into the abomasum. Biochemical analyses of serum revealed that LDA cattle had higher lactic acid and lower vitamin A and E levels than non-LDA reference animals. These results indicate that beef cattle with LDA may suffer from vitamin A and E deficiencies due to maldigestion of starch and the high acidity of abomasal fluid.

Clinical, microbiological and histological findings in lambs affected by 'salivary abomasum disease'.

'Salivary abomasum disease' is a common syndrome in Greece affecting lambs and kids from three to 17 days of age. In this case series, we present clinical and laboratory findings from 37 affected lambs presented alive and subsequently euthanased for welfare reasons and necropsied, and also from 24 other lambs submitted dead that were also necropsied. The clinical signs in the 37 lambs presented alive included lethargy (100 per cent), absence of sucking (83.8 per cent), weakness (37.8 per cent), abdominal distension (40.5 per cent) and increased frequency of urination (24.3 per cent). Diarrhoea was not observed in any affected lambs. At necropsy of these 37 lambs, the abomasum was distended with gas (70.3 per cent), saliva (43.2 per cent) along with mixed milk clots and gastric secretions; while multiple small mucosal and serosal haemorrhages with blood clots ('coffee grains') were recorded (91.9 per cent). Eight of 37 lambs that were examined alive, had elevated blood urea nitrogen concentrations (21.6 per cent). The pH of the abomasal contents ranged from 1.0 to 2.8; Escherichia coli was cultured from six of 37 (16.2 per cent) abomasal fluid samples. A mild to moderate inflammatory cell infiltrate was present in the mucosal lamina propria of 13 of 15 abomasal samples (86.6 per cent). Kidneys were paler than normal in 13 of the total 61 lambs necropsied (21.3 per cent); while acute tubular necrosis was evident on histopathological examination of 11 of 12 examined pale kidneys (91.6 per cent). The low abomasal pH and reported successful treatment with oral sodium bicarbonate suggest that metabolic acidosis may develop during the disease; however, further studies, including blood gas analysis, and determination of D- and L-lactic acid concentrations, are necessary to confirm this hypothesis.

Effects of abomasal infusion of flaxseed (Linum usitatissimum) oil on microbial ß-glucuronidase activity and concentration of the mammalian lignan enterolactone in ruminal fluid, plasma, urine and milk of dairy cows.

Ruminal microbiota plays an important role in the conversion of plant lignans into mammalian lignans. The main mammalian lignan present in the milk of dairy cows fed flax products is enterolactone (EL). The objectives of the present study were to investigate the effects of abomasal infusion of flax oil on the metabolism of flax lignans and concentrations of EL in biological fluids of dairy cows. A total of six rumen-cannulated dairy cows were assigned within a 2 × 3 factorial arrangement of six treatments utilising flax hulls (0 and 15·9 % of DM) and abomasal infusion of flax oil (0, 250 and 500 g/d). There were six periods of 21 d each. Samples were collected during the last 7 d of each period and subjected to chemical analysis. Flax hull supplementation increased concentrations of EL in ruminal fluid, plasma, urine and milk, while flax oil infusion had no effect. Post-feeding, ß-glucuronidase activity in the ruminal fluid of cows infused with 250 g flax oil was significantly lower for cows fed hulls than for those fed the control diet. The present study demonstrated that the presence of a rich source of n-3 fatty acids such as flax oil in the small intestine does not interfere with the absorption of the mammalian lignan EL and that lower ruminal ß-glucuronidase activity had no effect on the conversion of flax lignans into EL in the rumen of dairy cows.

Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses.

Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle.