Analysis of the Nasal Microbiota in Healthy and Diseased Pigs.

Massive sequencing of a fragment of 16S rRNA gene allows the characterization of bacterial communities in different body sites: the microbiota. Nasal microbiota can be analyzed by DNA extraction from nasal swabs, amplification of the specific fragment of interest, and posterior sequencing. The raw sequences obtained need to go through a computational process to check their quality and then assign the taxonomy. Here, we will describe the complete process from sampling to get the microbial diversity of nasal microbiota in health and disease.

Gut-associated microbes are present and active in the pig nasal cavity.

The nasal microbiota is a key contributor to animal health, and characterizing the nasal microbiota composition is an important step towards elucidating the role of its different members. Efforts to characterize the nasal microbiota composition of domestic pigs and other farm animals frequently report the presence of bacteria that are typically found in the gut, including many anaerobes from the Bacteroidales and Clostridiales orders. However, the in vivo role of these gut-microbiota associated taxa is currently unclear. Here, we tackled this issue by examining the prevalence, origin, and activity of these taxa in the nasal microbiota of piglets. First, analysis of the nasal microbiota of farm piglets sampled in this study, as well as various publicly available data sets, revealed that gut-microbiota associated taxa indeed constitute a substantial fraction of the pig nasal microbiota that is highly variable across individual animals. Second, comparison of herd-matched nasal and rectal samples at amplicon sequencing variant (ASV) level showed that these taxa are largely shared in the nasal and rectal microbiota, suggesting a common origin driven presumably by the transfer of fecal matter. Third, surgical sampling of the inner nasal tract showed that gut-microbiota associated taxa are found throughout the nasal cavity, indicating that these taxa do not stem from contaminations introduced during sampling with conventional nasal swabs. Finally, analysis of cDNA from the 16S rRNA gene in these nasal samples indicated that gut-microbiota associated taxa are indeed active in the pig nasal cavity. This study shows that gut-microbiota associated taxa are not only present, but also active, in the nasal cavity of domestic pigs, and paves the way for future efforts to elucidate the function of these taxa within the nasal microbiota.

Intensive antibiotic treatment of sows with parenteral crystalline ceftiofur and tulathromycin alters the composition of the nasal microbiota of their offspring.

The nasal microbiota plays an important role in animal health and the use of antibiotics is a major factor that influences its composition. Here, we studied the consequences of an intensive antibiotic treatment, applied to sows and/or their offspring, on the piglets' nasal microbiota. Four pregnant sows were treated with crystalline ceftiofur and tulathromycin (CTsows) while two other sows received only crystalline ceftiofur (Csows). Sow treatments were performed at D-4 (four days pre-farrowing), D3, D10 and D17 for ceftiofur and D-3, D4 and D11 for tulathromycin. Half of the piglets born to CTsows were treated at D1 with ceftiofur. Nasal swabs were taken from piglets at 22-24 days of age and bacterial load and nasal microbiota composition were defined by 16 s rRNA gene qPCR and amplicon sequencing. Antibiotic treatment of sows reduced their nasal bacterial load, as well as in their offspring, indicating a reduced bacterial transmission from the dams. In addition, nasal microbiota composition of the piglets exhibited signs of dysbiosis, showing unusual taxa. The addition of tulathromycin to the ceftiofur treatment seemed to enhance the deleterious effect on the microbiota diversity by diminishing some bacteria commonly found in the piglets' nasal cavity, such as Glaesserella, Streptococcus, Prevotella, Staphylococcus and several members of the Ruminococcaceae and Lachnospiraceae families. On the other hand, the additional treatment of piglets with ceftiofur resulted in no further effect beyond the treatment of the sows. Altogether, these results suggest that intensive antibiotic treatments of sows, especially the double antibiotic treatment, disrupt the nasal microbiota of their offspring and highlight the importance of sow-to-piglet microbiota transmission.

Ceftiofur treatment of sows results in long-term alterations in the nasal microbiota of the offspring that can be ameliorated by inoculation of nasal colonizers.

BACKGROUND: The nasal microbiota of the piglet is a reservoir for opportunistic pathogens that can cause polyserositis, such as Glaesserella parasuis, Mycoplasma hyorhinis or Streptococcus suis. Antibiotic treatment is a strategy to control these diseases, but it has a detrimental effect on the microbiota. We followed the piglets of 60 sows from birth to 8 weeks of age, to study the effect of ceftiofur on the nasal microbiota and the colonization by pathogens when the treatment was administered to sows or their litters. We also aimed to revert the effect of the antibiotic on the nasal microbiota by the inoculation at birth of nasal colonizers selected from healthy piglets. Nasal swabs were collected at birth, and at 7, 15, 21 and 49 days of age, and were used for pathogen detection by PCR and bacterial culture, 16S rRNA amplicon sequencing and whole shotgun metagenomics. Weights, clinical signs and production parameters were also recorded during the study. RESULTS: The composition of the nasal microbiota of piglets changed over time, with a clear increment of Clostridiales at the end of nursery. The administration of ceftiofur induced an unexpected temporary increase in alpha diversity at day 7 mainly due to colonization by environmental taxa. Ceftiofur had a longer impact on the nasal microbiota of piglets when administered to their sows before farrowing than directly to them. This effect was partially reverted by the inoculation of nasal colonizers to newborn piglets and was accompanied by a reduction in the number of animals showing clinical signs (mainly lameness). Both interventions altered the colonization pattern of different strains of the above pathogens. In addition, the prevalence of resistance genes increased over time in all the groups but was significantly higher at weaning when the antibiotic was administered to the sows. Also, ceftiofur treatment induced the selection of more beta-lactams resistance genes when it was administered directly to the piglets. CONCLUSIONS: This study shed light on the effect of the ceftiofur treatment on the piglet nasal microbiota over time and demonstrated for the first time the possibility of modifying the piglets' nasal microbiota by inoculating natural colonizers of the upper respiratory tract.

Longitudinal Study of Fecal Microbiota in Calves with or without Diarrhea Episodes before Weaning.

The microbiota plays an important role in the development of diarrhea in pre-weaned calves. The characterization of the fecal microbiota in health and disease can be critical to unravel the bacterial dynamics associated with diarrhea and help with its prevention and control. In this study, we aimed to detect changes in the fecal microbiota of calves that experienced early-life diarrhea episodes. Fecal samples were taken from calves remaining healthy and calves with an episode of diarrhea during the study. We sampled at arrival (12 days of age) and after one and two months of life; also, at the time of the diarrhea episode for the diarrheic calves (day 17). Samples were processed to extract total DNA, submitted to 16S rRNA gene sequencing, and bioinformatically analyzed to infer the bacterial populations. Microbiota changes through time were reported for both groups. However, we detected an earlier stabilization in the healthy group. Moreover, we detected changes within low abundant taxa that may play a role in the subsequent health status of the animals. The fecal microbiota of healthy and diarrheic calves showed different dynamics in the diversity through time that may be the reflections of the variations within low-abundant taxa.

Sow Contact Is a Major Driver in the Development of the Nasal Microbiota of Piglets.

The nasal microbiota composition is associated with the health status of piglets. Sow-contact in early life is one of the factors influencing the microbial composition in piglets; however, its impact has never been assessed in the nasal microbiota of piglets reared in controlled environmental conditions. Nasal microbiota of weaning piglets in high-biosecurity facilities with different time of contact with their sows (no contact after farrowing, contact limited to few hours or normal contact until weaning at three weeks) was unveiled by 16S rRNA gene sequencing. Contact with sows demonstrated to be a major factor affecting the nasal microbial composition of the piglets. The nasal microbiota of piglets that had contact with sows until weaning, but were reared in high biosecurity facilities, was richer and more similar to the previously described healthy nasal microbiota from conventional farm piglets. On the other hand, the nasal communities inhabiting piglets with no or limited contact with sows was different and dominated by bacteria not commonly abundant in this body site. Furthermore, the length of sow-piglet contact was also an important variable. In addition, the piglets raised in BSL3 conditions showed an increased richness of low-abundant species in the nasal microbiota. Artificially rearing in high biosecurity facilities without the contact of sows as a source of nasal colonizers had dramatic impacts on the nasal microbiota of weaning piglets and may introduce significant bias into animal research under these conditions.