Isolation of Lactobacillaceae bacteria from feces of ostrich (Struthio camelus).

The ostrich (Struthio camelus) is an herbivorous bird with a long and developed hindgut. In the hindgut, there is a dense and highly diverse population of anaerobic bacteria, and active fermentation produces high concentrations of short-chain fatty acids. Bacteria in the hindgut of the ostrich are considered vital for both their nutritional contribution and health benefits, such as benefits to the immune and defense system of the host. We attempted to isolate Lactobacillaceae, which might be involved in improving immune function and in inhibiting pathogens. The number of colonies from ostrich feces observed on LBS agar medium was 3.64×103 per gram of feces. Three strains of Lactobacillaceae were isolated from the feces. Nearly the entire length of the 16S ribosomal RNA gene of these isolates was sequenced, and a homology search showed high identity with L. brevis (identity=99.93%), L. coryniformis (98.39%), and L. paracasei (100.0%). These isolates may be deemed potential probiotics for the ostrich. 2022, Japan Poultry Science Association.

Introduction

Unlike other birds, the ostrich (Struthio camelus) is an herbivorous bird with a long and well-developed hindgut (Matsui ). In its hindgut, there is a dense and highly diverse population of anaerobic bacteria (Matsui , b), and active fermentation produces high concentrations of short-chain fatty acids (Skadhauge ; Swart ), components which provide 76% of the metabolizable energy to the host (Swart ). Bacteria in the hindgut of the ostrich are vital for both their nutritional contribution and health benefits, such as benefits to the immune and defense systems of the host.

Among the gastrointestinal microorganisms, lactic acid bacteria, identified as Lactobacillaceae, contribute to the host's defense system by improving immune function and by suppressing pathogens. The genus Lactobacillus is now classified into 25 genera (Zheng et al., 2020). If these genera can be isolated from the gastrointestinal tract of ostrich and characterized, the contribution of Lactobacillaceae can be clarified. Furthermore, isolates of Lactobacillaceae from ostrich may serve as probiotics for the animal.

The objective of this study was to isolate and identify members of the Lactobacillaceae from ostrich feces.

Materials and Methods

Animals and Feed

Three adult ostriches (two males and one female) reared at Hisai Norin High School (Mie Prefecture, Japan) were used in this study. The birds were fed with a self-mixed feed. The ingredients and composition of the feed are listed in Table 1. Water was provided ad libitum.

Table 1.

Ingredients and composition of the feed.

Ingredients	Composition (%)
Maize	27.23
Wheat bran	11.98
Soybean meal	2.18
Lucerne pellet	43.57
Lucerne meal	10.89
Calcium phosphate	1.09
Calcium carbonate	2.18
Sodium chloride	0.44
Vitamin premix	0.44

Sample Collection

Sample collection was performed in a non-invasive manner. Fecal samples that were released/excreted were collected at approximately 11:00 am. The fecal samples were immediately placed in a clean plastic bag and kept at 39°C. The collected samples were transferred to the laboratory within 30 minutes.

Isolation of Lactobacillaceae

Lactobacillaceae members were isolated as per protocols described by Tsuchida with a few modifications. One gram of feces from each ostrich was diluted in 9 mL of sterilized saline and mixed thoroughly. The slurry was further diluted to 10−2 with sterilized saline. A portion (100 µL) of the diluted samples was spread onto an LBS agar plate (Becton, Dickinson and Company, Sparks, MD, USA). The plates were then transferred into the BBL GasPak Anaerobic System (Becton, Dickinson and Company) and incubated anaerobically for 6 days at 39°C. Colonies on the agar plate were selected and inoculated into 2 mL of MRS broth (Becton, Dickinson and Company). The culture was then incubated for 4 d at 39°C. A full loop (10 µL) of the culture fluid was again streaked onto LBS agar and incubated anaerobically in a GasPak anaerobic system at 39°C. Finally, a single colony was selected, inoculated into 10 mL of MRS broth, and incubated for 4 d at 39°C.

Extraction of DNA and Sequencing

Ten milliliters of the culture fluid was centrifuged (8,000 ×g for 10 min, 4°C), and the pellet was used for genomic DNA extraction using the NucleoSpin Microbial DNA Kit (Macherey-Nagel GmbH & Co. Düren, Germany). The 16S ribosomal RNA gene fragment was amplified as per methods described in our previous study (Hattori and Matsui 2008). The reaction mixture was purified using ExoProStar (GE Healthcare, Buckinghamshire, UK). The purified DNA was sequenced with B35rev (5′-GGTATGGGATGAGCTTGC-3′; Guettler ), C01rev (5′-GGTTGCGCTCGTTGC-GGG-3′; Guettler ), and 926f (5′-AAACTCAAA-GGAATTGACGG-3′; Leser ). The sequencing chemistry and sequencer used have been described previously (Abrar ). Sequenced fragments were combined into one sequence using the DNADynamo software (Blue Tractor Software Ltd, UK). The combined DNA sequence obtained was explored for homology in BlastN (Altschul ).

Results and Discussion

To the best of our knowledge, this is the first report on the isolation of Lactobacillaceae members from the hindgut of ostrich.

The number of colonies enumerated based on observation on the LBS agar plate was 3.64×103±1.65×103 per gram of feces (mean±SE, n=3). Nikravesh-Masouleh reported that the number of Lactobacillus bacteria in the feces of ostrich chicks ranged between 106 and 108 colony forming units/g. This difference may be attributable to the age of the birds. Videvall reported that the composition of Lactobacillus decreased with the growth of ostrich chicks. The present results support this finding. In our previous study conducted on microbial diversity in the hindgut of adult ostrich, there was no Lactobacillaceae clone sequence observed in the 16S ribosomal RNA gene library (Matsui ). Therefore, Lactobacillaceae is a minor constituent of the adult ostrich hindgut.

A total of 87 colonies were selected for analysis. Within these colonies, the partial 16S ribosomal RNA gene was sequenced using the B35rev primer of 17 isolates. Three Lactobacillaceae isolates (HNost011, HNost031, and HNost082) were obtained from ostrich feces (Table 2). The almost full-length 16S ribosomal RNA genes of these isolates were sequenced and searched for homology against the public database. HNost011, HNost031, and HNost082 showed high identity with Levilactobacillus (formerly Lactobacillus) brevis (identity=99.93%), Loigolactobacillus (formerly Lactobacillus) coryniformis (98.39%), and Lacticaseibacillus (formerly Lactobacillus) paracasei (100.0%). Lactobacillaceae isolates from chicken cloaca and feces were identified as Ligilactobacillus (formerly Lactobacillus) agilis, Lactobacillus crispatus, Latilactobaillus (formerly Lactobacillus) curvatus, Limosilactobacillus (formerly Lactobacillus) fermentum, Lactobacillus gallinarum, Limosilactobacillus (formerly Lactobacillus) ingluviei, Lactobacillus johnsonii, Lactobacillus kitasatonis, Lactiplantibacillus (formerly Lactobacillus) plantarum, Ligilactobacillus (formerly Lactobacillus) salivarius, and Limosilactobacillus (formerly Lactobacillus) vaginalis (Kobierecka ). Experimentally, L. plantarum showed the ability to decrease colonization of the Campylobacter jejuni strain (Kobierecka ). None of the ostrich isolates overlapped with the isolates from chickens. One reason for this is the small number of isolated Lactobacillaceae members. Isolation of more Lactobacillaceae members should be performed in the future.

Table 2.

Sequence identity of the 16S ribosomal RNA gene of isolates from ostrich feces against nucleotide database

Isolate	Nearest known species	Identity (%)
HNost011	Levilactobacillus (formerly Lactobacillus) brevis	99.93
HNost031	Loigolactobacillus (formerly Lactobacillus) coryniformis	98.39
HNost055	Enterococcus faecium	100
HNost056	Enterococcus faecium	100
HNost057	Enterococcus faecium	100
HNost058	Enterococcus faecium	100
HNost059	Enterococcus faecium	100
HNost062	Enterococcus faecium	100
HNost065	Bacillus coagulans	100
HNost066	Enterococcus faecium	100
HNost067	Bacillus coagulans	99.79
HNost069	Enterococcus faecium	100
HNOst076	Bacillus coagulans	100
HNost078	Bacillus coagulans	100
HNost082	Lacticaseibacillus (formerly Lactobacillus) paracasei	100
HNost085	Enterococcus faecium	100
HNost086	Enterococcus faecium	100

The body weight (BW) and BW gain of ostrich chicks that received commercial probiotics (not for ostrich) were higher than those of chicks fed with a control diet (Karimi-Kivi ). Moreover, the abundance of Lactobacillaceae was significantly higher in chickens in the better feed efficiency group (Yan ). If the development of probiotics designated for ostrich can be achieved, ostrich productivity may be improved. The Lactobacillaceae isolates in the present study may be deemed potential candidates for this purpose.