SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice.

Since the beginning of the COVID-19 pandemic, there has been a significant need to develop antivirals and vaccines to combat the disease. In this work, we developed llama-derived nanobodies (Nbs) directed against the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Most of the Nbs with neutralizing properties were directed to RBD and were able to block S-2P/ACE2 interaction. Three neutralizing Nbs recognized the N-terminal domain (NTD) of the S-2P protein. Intranasal administration of Nbs induced protection ranging from 40% to 80% after challenge with the WA1/2020 strain in k18-hACE2 transgenic mice. Interestingly, protection was associated with a significant reduction in virus replication in nasal turbinates and a reduction in virus load in the brain. Employing pseudovirus neutralization assays, we identified Nbs with neutralizing capacity against the Alpha, Beta, Delta, and Omicron variants, including a Nb capable of neutralizing all variants tested. Furthermore, cocktails of different Nbs performed better than individual Nbs at neutralizing two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest the potential of SARS-CoV-2 specific Nbs for intranasal treatment of COVID-19 encephalitis.

Nanobodies against SARS-CoV-2 reduced virus load in the brain of challenged mice and neutralized Wuhan, Delta and Omicron Variants.

In this work, we developed llama-derived nanobodies (Nbs) directed to the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Nanobodies were selected after the biopanning of two VHH-libraries, one of which was generated after the immunization of a llama (lama glama) with the bovine coronavirus (BCoV) Mebus, and another with the full-length pre-fused locked S protein (S-2P) and the RBD from the SARS-CoV-2 Wuhan strain (WT). Most of the neutralizing Nbs selected with either RBD or S-2P from SARS-CoV-2 were directed to RBD and were able to block S-2P/ACE2 interaction. Three Nbs recognized the N-terminal domain (NTD) of the S-2P protein as measured by competition with biliverdin, while some non-neutralizing Nbs recognize epitopes in the S2 domain. One Nb from the BCoV immune library was directed to RBD but was non-neutralizing. Intranasal administration of Nbs induced protection ranging from 40% to 80% against COVID-19 death in k18-hACE2 mice challenged with the WT strain. Interestingly, protection was not only associated with a significant reduction of virus replication in nasal turbinates and lungs, but also with a reduction of virus load in the brain. Employing pseudovirus neutralization assays, we were able to identify Nbs with neutralizing capacity against the Alpha, Beta, Delta and Omicron variants. Furthermore, cocktails of different Nbs performed better than individual Nbs to neutralize two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest these Nbs can potentially be used as a cocktail for intranasal treatment to prevent or treat COVID-19 encephalitis, or modified for prophylactic administration to fight this disease.

Dynamics of the ruminal microbial ecosystem, and inhibition of methanogenesis and propiogenesis in response to nitrate feeding to Holstein calves.

It is known that nitrate inhibits ruminal methanogenesis, mainly through competition with hydrogenotrophic methanogens for available hydrogen (H2) and also through toxic effects on the methanogens. However, there is limited knowledge about its effects on the others members of ruminal microbiota and their metabolites. In this study, we investigated the effects of dietary nitrate inclusion on enteric methane (CH4) emission, temporal changes in ruminal microbiota, and fermentation in Holstein calves. Eighteen animals were maintained in individual pens for 45 d. Animals were randomly allocated to either a control (CTR) or nitrate (NIT, containing 15 g of calcium nitrate/kg dry matter) diets. Methane emissions were estimated using the sulfur hexafluoride (SF6) tracer method. Ruminal microbiota changes and ruminal fermentation were evaluated at 0, 4, and 8 h post-feeding. In this study, feed dry matter intake (DMI) did not differ between dietary treatments (P > 0.05). Diets containing NIT reduced CH4 emissions by 27% (g/d) and yield by 21% (g/kg DMI) compared to the CTR (P < 0.05). The pH values and total volatile fatty acids (VFA) concentration did not differ between dietary treatments (P > 0.05) but differed with time, and post-feeding (P < 0.05). Increases in the concentrations of ruminal ammonia nitrogen (NH3-N) and acetate were observed, whereas propionate decreased at 4 h post-feeding with the NIT diet (P < 0.05). Feeding the NIT diet reduced the populations of total bacteria, total methanogens, Ruminococcus albus and Ruminococcus flavefaciens, and the abundance of Succiniclasticum, Coprococcus, Treponema, Shuttlewortia, Succinivibrio, Sharpea, Pseudobutyrivibrio, and Selenomona (P < 0.05); whereas, the population of total fungi, protozoa, Fibrobacter succinogenes, Atopobium and Erysipelotrichaceae L7A_E11 increased (P < 0.05). In conclusion, feeding nitrate reduces enteric CH4 emissions and the methanogens population, whereas it decreases the propionate concentration and the abundance of bacteria involved in the succinate and acrylate pathways. Despite the altered fermentation profile and ruminal microbiota, DMI was not influenced by dietary nitrate. These findings suggest that nitrate has a predominantly direct effect on the reduction of methanogenesis and propionate synthesis.

Changes in hematological, biochemical, and blood gases parameters in response to progressive inclusion of nitrate in the diet of Holstein calves.

BACKGROUND AND AIM: Nitrate (NO3 -) reduces enteric methane emissions and could be a source of non-protein nitrogen in ruminant feeds. Nonetheless, it has a potential toxic effect that could compromise animal health and production. The purpose of this study was to determine the effects of progressive inclusion of NO3 - in the diet on the hematological, biochemical, and blood gases parameters, in turn, the effects on feed intake and live weight gain (LWG) in Holstein calves. MATERIALS AND METHODS: Eighteen Holstein heifers and steers (nine animals/treatment) were maintained in individual pens for 45 days. Animals were randomly allocated to either a control or nitrate diet (ND) (containing 15 g of NO3 -/kg of dry matter [DM]). The biochemical parameters and blood gases were analyzed only in the NO3 - group on days: -1, 1, 7, 13, 19, and 25 corresponding to 0, 20, 40, 60, 80, and 100% of the total inclusion of NO3 - in the diet, respectively. In addition, DM intake (DMI) and LWG were evaluated among dietary treatments. RESULTS: Feeding the ND did not influence DMI or LWG (p>0.05). Methemoglobin (MetHb) and deoxyhemoglobin increased according to the NO3 - concentrations in the diet (p<0.05), while an opposite effect was observed for oxyhemoglobin and carboxyhemoglobin (p<0.05). Hematocrit levels decreased (p<0.05), while albumin, alanine aminotransferase, and gamma-glutamyl transpeptidase concentrations were not modified (p>0.05). However, glucose, urea, aspartate aminotransferase (AST), and retinol concentrations increased (p<0.05) according to the NO3 - concentrations in the diet. CONCLUSION: This study confirmed that the progressive inclusion of 123 g of NO3 -/animal/day in the diet could be safe without affecting DMI and LWG of Holstein calves. In turn, a dose-response effect of the MetHb, glucose, urea, AST, and retinol was observed, but these values did not exceed reference values. These results highlighted the importance of using a scheme of progressive inclusion of NO3 - in the diet of calves to reduce the risks of NO3 - toxicity.

Physiological and behavioral indices of short-term stress in wild vicuñas (Vicugna vicugna) in Jujuy Province, Argentina.

The management of wild vicuñas can trigger a stress response that may compromise welfare. In Santa Catalina, Jujuy Province, Argentina, indices of short-term stress associated with capture, handling, and shearing were studied in 105 wild vicuñas (Vicugna vicugna). The study included 2 groups (n = 59 and n = 46) of wild vicuñas captured in 2 consecutive days. Independent variables analyzed included sex, restraint time, and groups. Cortisol, creatine kinase, glucose, white blood cells, temperature, heart rate, and respiratory frequency were higher than published values. Respiratory rate increased during handling and correlated with holding time and group size, while heart rate decreased. Packed cell volume was higher in females. Cortisol concentrations differed between restraint groups and sex and inversely correlated with agonistic behavior. The most common behavior was increased vigilance. Sternal recumbency increased over holding time. During handling procedures, frequency of sudden movements like kicking and attempts to stand increased as restraint time increased. Females vocalized more than males. In conclusion, the methods used triggered measurable changes suggestive of short-term stress that appeared to be physiologically tolerated by the vicuñas.

Ciliate protozoa of the forestomach of llamas (Lama glama) from locations at different altitude in Argentina.

This study describes the diversity and concentration of the protozoal population from the forestomach of llamas in Argentina at three altitudinal locations. Protozoal diversity was studied in samples from eight llamas from Hurlingham (Buenos Aires, 43 m altitude), four from Tilcara (Jujuy, 2465 m altitude) and six llamas from Cieneguillas (Jujuy, 3800 m altitude). The total concentrations of protozoa in the forestomach contents were 7.9, 9.1 and 4.1 cells x 104 ml-1 in Hurlingham, Tilcara and Cieneguillas, respectively (P>0.05). Entodinium spp. represented 97.9, 92.3 and 71.4% of the protozoal community in Hurlingham, Tilcara and Cieneguillas, respectively, and the remaining protozoa belonged to the Eudiplodinium genus. Entodinium spp. were identified as E. caudatum (mostly morphotype dubardi), E. longinucleatum, E. parvum, E. bovis, E. exiguum, E. dubardi, and a minor presence of E. bimastus (in three animals) and E. ovibos (in one animal). In regards to the rest of protozoal species, Eudiplodinium maggii is the first reported host record for the genus in llamas. This species was present in the forestomach of 14 out of 18 llamas tested, and in one case it was the unique protozoal species. The vestibuliferids, Dasytricha and Isotricha were absent from the forestomach of llamas. Similarly, other species such as those from the Caloscolex genus, Diplodinium cameli and Entodinium ovumrajae, commonly found in Old World Camelids, were also absent from llamas.

Molecular identification of Sarcocystis aucheniae as the macrocyst-forming parasite of llamas.

The domestic South American camelids (SACs), llama (Lama glama) and alpaca (Lama paco), are frequently found to be infected with Sarcocystis parasites. Infections give rise in skeletal muscle to macroscopic cysts (1-5mm long) that resemble rice seeds, each containing several million living bradyzoites. The finding of cysts prevents commercialization of SAC meat, an important source of income for rural families in the Andean flatlands. Thus, development of diagnostic methods to facilitate the control of these infections is highly desirable, and the first step to this end is the unequivocal species identification of the causative agent. Based on the cyst form and size, the infecting parasite has been described as Sarcocystis aucheniae; however, this traditional approach is not reliable as similar cysts may contain different species. To date, molecular identification has been done for a single isolate of S. aucheniae from an alpaca in Australia. In order to verify the identity of the species present in SACs of South America, the complete 18S rRNA gene was PCR-amplified and sequenced from macrocyst DNA obtained from three llamas of the Andean flatlands. A phylogenetic Bayesian analysis was carried out using the analyzed and available 18S rRNA sequences of Sarcocystis spp. In the constructed tree, all of the new 18S rRNA gene sequences segregated in a single clade together with the 18S rRNA gene sequence reported from an alpaca in Australia, demonstrating that the isolated parasite is S. aucheniae, and that this parasite indiscriminately infects both domestic SACs. This work represents the first molecular identification of the causative agent of SAC sarcocystiosis in South America, and can contribute to the development of control methods for this neglected parasitosis.

Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR.

The high fibrolytic activity and large biomass of strictly-anaerobic bacteria that inhabit the rumen makes them primarily responsible for the degradation of the forage consumed by ruminants. Llamas feed mainly on low quality fibrous roughages that are digested by an active and diverse microflora. The products of this fermentation are volatile fatty acids and microbial biomass, which will be used by the animals. The aim of this study was to detect the three major fiber-digesting anaerobic bacteria in the forestomach contents of llamas by PCR. In this study, we detected Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in the forestomach contents of eight native llamas from Argentina.

Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR / Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR.

The high fibrolytic activity and large biomass of strictly-anaerobic bacteria that inhabit the rumen makes them primarily responsible for the degradation of the forage consumed by ruminants. Llamas feed mainly on low quality fibrous roughages that are digested by an active and diverse microflora. The products of this fermentation are volatile fatty acids and microbial biomass, which will be used by the animals. The aim of this study was to detect the three major fiber-digesting anaerobic bacteria in the forestomach contents of llamas by PCR. In this study, we detected Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in the forestomach contents of eight native llamas from Argentina.

Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR. / Detection of fiber-digesting bacteria in the forestomach contents of llamas (Lama glama) by PCR.

The high fibrolytic activity and large biomass of strictly-anaerobic bacteria that inhabit the rumen makes them primarily responsible for the degradation of the forage consumed by ruminants. Llamas feed mainly on low quality fibrous roughages that are digested by an active and diverse microflora. The products of this fermentation are volatile fatty acids and microbial biomass, which will be used by the animals. The aim of this study was to detect the three major fiber-digesting anaerobic bacteria in the forestomach contents of llamas by PCR. In this study, we detected Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in the forestomach contents of eight native llamas from Argentina.

Antibodies to pathogenic livestock viruses in a wild vicuña (Vicugna vicugna) population in the Argentinean Andean altiplano.

Serum samples from 128 wild vicuñas (Vicugna vicugna) were tested for antibodies (Ab) to rotavirus (RV), bovine parainfluenza virus 3 (BPIV-3), bovine herpesvirus-1 (BHV-1), bovine viral diarrhea virus (BVDV-1), foot-and-mouth disease virus (FMDV), bluetongue virus (BTV), equine herpesvirus-1 (EHV-1), and influenza A virus equine (EIV). Samples were collected in Cieneguillas Province of Jujuy, in northern Argentina. Feces from 44 vicuñas were also collected to investigate RV shedding. Llamas (Lama glama) and domestic cattle (Bos taurus) from the area studied also were tested for antibodies to these viruses. Antibodies against RV (100%) and BPIV-3 (37%) were detected in the vicuñas sampled. No RV antigen was detected in any of the fecal samples tested. One vicuña was positive for Ab to BHV-1 (0.8%) and another for BVDV-1 (0.8%). The Ab prevalences detected in llamas were: 100% (16/16) for RV, 47% (8/17) for BPIV-3, 17.6% (3/17) for BHV-1, and 5.9% (1/17) for BVDV-1. However, domestic cattle had high antibody prevalences for RV and BPIV-3, 100% (13/13) and 73% (11/15), respectively, but were negative for Ab to BHV-1 and BVDV. No antibodies against FMDV, BTV, EHV-1, or EIV were detected in wild vicuñas or domestic species. Because no data of viral circulation on wild vicuñas are available, this report represents the first evidence of viral infection in wild vicuñas from the Argentinean Andean Puna.

Llama-derived single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice.

Group A rotavirus is one of the most common causes of severe diarrhea in human infants and newborn animals. Rotavirus virions are triple-layered particles. The outer capsid proteins VP4 and VP7 are highly variable and represent the major neutralizing antigens. The inner capsid protein VP6 is conserved among group A rotaviruses, is highly immunogenic, and is the target antigen of most immunodiagnosis tests. Llama-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity and can therefore be expected to have properties different from conventional antibodies. In this study a library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated. Binders directed to VP6, in its native conformation within the viral particle, were selected and characterized. Four selected VHH directed to conformational epitopes of VP6 recognized all human and animal rotavirus strains tested and could be engineered for their use in immunodiagnostic tests for group A rotavirus detection. Three of the four VHH neutralized rotavirus in vivo independently of the strain serotype. Furthermore, this result was confirmed by in vivo partial protection against rotavirus challenge in a neonatal mouse model. The present study demonstrates for the first time a broad neutralization activity of VP6 specific VHH in vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool for the prevention and treatment of rotavirus diarrhea.