Species Specificity of Adherent Lactobacilli Isolated from Psittacine Birds
Jeanne Smith, DVM and Sheikh A. Selim, DVM, MPVM, PhD.From Avian Health Products

Abstract: Adherent lactobacilli were isolated from crop epithelial cells of a cockatiel (Nymphicus hollandicus), a budgerigar (Melopsittacus undulates), and a green-cheeked conure (Pyrrhura molinae molinae). A minimum of 1.5 x 106 live organisms/ml drinking water was administered to the same species of origin and birds from species of varying levels of relatedness. Colonization was determined by recovering the test lactobacillus from feces after discontinuing dosing. The conure isolate colonized birds from the same genus as the bird of origin, but not in birds from the same family or subfamily. The cockatiel isolate colonized in a red-tailed black cockatoo (Calyptorhynchus banksii), considered to be in a different subfamily, but not in other birds in the family Cacatuidae. The budgerigar isolate colonized in the budgerigar only, not in birds from the same family or subfamily.

Key words: lactobacillus, species specificity

Introduction

Lactobacilli species have formed the cornerstone for probiotic and beneficial bacterial products based on research in mammals and birds. Research in poultry showed that only birds had lactobacilli strains that adhered to chicken (Gallus gallus) gastrointestinal endothelial cells, mammals did not. Mammalian lactobacilli do not colonize the gastrointestinal tract of the bird species studied. Adherent lactobacilli isolated from chickens , Carolina wood duck (Aix sponsa), pigeon (unreported species), quail (unreported species), and pheasant (unreported species) all adhered to chicken crop endothelial cells in vitro but colonization in vivo was reported to be species specific.1,2 The species in those studies were from different orders or related at the family level at best. In mammalian research, probiotic strains of lactobacilli are also described as species specific, but the species studied (rats, mice, pigs, dogs, and humans) are widely divergent.3

There has been very little research on adherence or colonization of lactobacilli in psittacine birds. One study administered a chicken origin lactobacillus-based probiotic to neonates of several psittacine species, but viable lactobacilli were not found in the inoculum and the test lactobacillus was not recovered from cloaca cultures.4 Although research in poultry has not revealed a minimum dose of live lactobacillus required to result in colonization or to provide beneficial effects, significant benefits have generally been seen when chicks are dosed with 106-109 live organisms per bird or in a similar range per liter of drinking water or gram of food. Another study administered two formulations of a chicken origin lactobacillus to cockatiel neonates. Live organisms were recovered from the inoculum, the dose of live organisms was calculated to be 6 x 106 for one formulation and 2.5 x 106 for the other based on manufacturer claims. Crop swabs yielded greater numbers of lactobacilli in the treated birds, but adherence or colonization of the gastrointestinal tract with the test lactobacillus was not evaluated.5 The authors administered an adherent lactobacillus isolated from cockatiels to neonate cockatiels at a dose rate of 5 x 105 live organisms per chick every 3 days. Crop epithelial cell adherence was demonstrated and there was a statistically significant reduction in gram negative bacteria found in crop contents.6

The purpose of this study was to determine if adherent lactobacilli isolated from psittacine birds can colonize other species of psittacine birds related at the family, subfamily, and genus level.

Materials and Methods

Bacteria: Parent-raised young adult or juvenile birds were anesthetized with isoflurane and given intracoelomic injections of euthanasia solution. A cockatiel, budgerigar, and green-cheeked conure were used due to availability of a bird meeting the above criteria being euthanized for diagnostic purposes. The crops were removed aseptically and washed with sterile saline three times. The supernatant from the third washing was retained. The crops were macerated and the macerate and the third wash supernatant were cultured on Rogosa agar under microaerophilic conditions. Adherent lactobacilli were identified by recovering significantly higher numbers from the macerate than the wash solution. The bacteria were speciated, grown in selective liquid media, and lyophilized. Numbers of live organisms were determined by titration of reconstituted bacteria within one month of the start of each trial. A Lactobacillus salivarius was isolated from the cockatiel and from the budgerigar. A Lactobacillus reuteri was isolated from the green-cheeked conure.

Birds: All birds except the Pyrrhura conures were individual pet birds. Their feces were cultured on blood agar plates under microaerophilic conditions prior to administration of lactobacilli. Any lactobacilli present were identified as a different species from the test lactobacillus prior to inclusion in the study. The Pyrrhura species were breeding birds from the same facility the L reuteri was obtained. These birds were treated with enrofloxacin at a rate of 50 mg/120 ml drinking water for 10 days. Feces were cultured and birds were included in the study if their feces yielded no bacterial growth. The cockatiel origin L salivarius was administered to a cockatiel, a red-tailed black cockatoo (Calyptorhynchus banksii), a black palm cockatoo (Probosciger aterrimus), a Goffin’s cockatoo (Cacatua goffini), a budgerigar, a Solomon Island eclectus (Roratus solomonenesis), an orange-winged Amazon parrot (Amazona amazonica), and a sun conure(Aratinga solstitialis). The budgerigar origin L salivarius was given to two budgerigars, a Solomon Island eclectus, an orange-winged Amazon parrot, a sun conure, and a cockatiel. The green-cheeked conure origin L reuteri was given to a green-cheeked conure, a fiery-shouldered conure (P egrefia egregia), a maroon-bellied conure (P frontalis frontalis), a pearly conure (P perlata lepida), a black-capped conure (P rupicola sandiae), a rose-crowned conure (P rhodocephala), a sun conure, an orange-winged Amazon parrot, a Solomon Island eclectus, a budgerigar, and a cockatiel.

Treatment and Sampling: Birds were housed individually and fed dry pellets or seed mix only. The inoculum was reconstituted the first day of treatment and administered at a rate of 1.5 x 106 to 4 x 107 live organisms per ml non-chlorinated drinking water. Treated water was made fresh daily for 10 days. Previous stability testing of the inoculum showed insignificant loss of titer over ten days of refrigeration. Feces were collected from each bird on the last day of treatment and weekly for up to four weeks thereafter. The feces were cultured on blood agar plates under microaerophilic conditions (candle jar) and any bacterial growth morphologically similar to the test lactobacillus was speciated. In some cases sampling was discontinued if a bird had negative samples (no test lactobacillus isolated) the previous two samplings.

Results

Table 1 shows the results of administration of the cockatiel origin lactobacillus. The bird species are listed in descending order of relatedness to the cockatiel. In the on-treatment sampling, the test lactobacillus was recovered from the feces of all the birds except the Goffin’s cockatoo and the orange-winged Amazon parrot. After ending treatment, the test lactobacillus was isolated from the feces of the cockatiel and the red-tailed black cockatoo weekly for four weeks.

Table 2 shows the results of administration of the budgerigar origin lactobacillus. The bird species are listed in descending order of relatedness to the budgerigar. During treatment, the test lactobacillus was recovered from the budgerigars, the Solomon Island eclectus, and the sun conure, but not the orange-winged Amazon parrot or the cockatiel. The test lactobacillus was recovered from the Solomon Island eclectus one week after ending treatment but not at later samplings. The budgerigars remained positive for the four weeks post-treatment.

Table 3 shows the results of administration of the green-cheeked conure origin lactobacillus. The bird species are listed in descending order of relatedness to the green-cheeked conure. The test lactobacillus was recovered from feces from all the birds tested while on the treatment. The green-cheeked conure and the fiery-shouldered conure remained positive for the four weeks post-treatment. The maroon-bellied, pearly, and rose-crowned conures tested positive at one week post-treatment and at four weeks post-treatment. The sun conure, orange-winged Amazon parrot, and the Solomon Island eclectus remained positive at the one week post-treatment sampling only. The cockatiel tested negative after treatment ended.

Discussion

Recovery of the test lactobacilli from feces while it was still being administered indicates the bacteria’s ability to survive the environment of the gastrointestinal tract. One of the requirements for an effective probiotic is this ability to survive passage.7 In this study, the lactobacilli were recovered in most of the birds. The cockatiel origin lactobacillus was not recovered from the Goffin’s cockatoo or the orange-winged Amazon parrot, and the budgerigar origin lactobacillus was not recovered from the orange-winged Amazon parrot or the cockatiel. This would not seem to be affected by relatedness as the bacteria survived in other members of less related species.

Recovery of the test lactobacilli from feces after treatment was discontinued is an indicator of the bacteria’s ability to colonize the gastrointestinal tract. Colonization can be achieved either by adherence to the gastrointestinal endothelial cells, adherence to material in close association with the endothelial cells, or by rapid reproduction that allows the bacteria to replace itself before it is swept out of the gastrointestinal tract.2,3 Because birds must be sacrificed to determine adherence, we decided that colonization would be an acceptable measure of the bacteria’s adaptation to a bird species. The cockatiel origin lactobacillus successfully colonized the red-tailed black cockatoo but not the black palm cockatoo or other members of the family Cacatuidae. The taxonomy of the cockatiel is controversial. It is the only species in its genus and has been classified as the only member of its subfamily; however, DNA sequencing suggests that the cockatiel and the Calyptorhinchus cockatoos should be reclassified within the same subfamily.7 If that is the case, the cockatiel origin lactobacillus was able to colonize birds in the same subfamily. The budgerigar is also the only species in its genus. It belongs to the same subfamily (Psittacinae) as the Solomon Island eclectus and the orange-winged Amazon parrot . The budgerigar origin lactobacillus only colonized the budgerigars, not members of the same subfamily. The pyrrhura conures belong to the same subfamily (Arinae) as the sun conure, but the green-cheeked conure origin lactobacillus colonized all of the Pyrrhura conures. The pearly, rose-crowned, maroon-bellied, and black-capped conures tested negative for the test lactobacillus at two weeks post treatment and three weeks post treatment. This failure to recover the test lactobacillus those two weeks may have been due to the habits of those four birds. They all dipped their pellets into their water bowls until their water was pellet sludge. Their water was changed twice daily, but this may have affected the fecal cultures. The pellets typically have Bacillus species. and the fecal cultures of these four birds consistently yielded high numbers of Bacillus species. The lactobacillus, being more fastidious, may not have grown on media in competition with the Bacillus. The test lactobacillus reappeared among the Bacillus at four weeks post-treatment.

The ability to adhere, or at least colonize, has been found to be necessary to obtain many of the benefits of lactobacillus treatment in poultry.2,3 Previously, this ability has been described as species specific. This study identified a cockatiel origin adherent lactobacillus capable of colonizing a red-tailed black cockatoo, a bird possibly from the same subfamily; and a green-cheeked conure origin adherent lactobacillus capable of colonizing birds within the same genus. Research in poultry has shown that not all adherent lactobacilli are created equal. They vary in their reproductive rates, ability to adhere (even in the same species of origin), production of metabolites, and conference of beneficial effects.2,3,8 This study was limited in terms of bird numbers and bird species. More research is needed to determine if other lactobacilli can be found with a broader ability to colonize our many companion bird species.

References

1.            Fuller R. Ecological studies on the lactobacillus flora associated with the crop epithelium of the fowl. J Applied Bact. 1973;36:131-139.

2.            Fuller R. Epithelial attachment and other factors controlling the colonization of the intestine of the gnotobiotic chicken by lactobacilli. J of Applied Bact. 1978;45:389-395.

3.            Gomez-Gil B, Roque A, Turnbull JF, Inglis V. A review on the use of microorganisms as probiotics. Rev Latinoamericana de Microbiologica. 1998;40:166-172.

4.            Joyner KL, Swanson JL. The use of a lactobacillus product in a psittacine hand-feeding diet: its effects on normal aerobic microflora, early weight gain, and health. Proc Annu Conf Assoc Avian Vet. 1988;127-137.

5.            Tully TN, Shane SM, Kearney MT. Evaluation of two Lactobacillus acidophilus formulations as dietary supplements in neonatal cockatiels (Nymphicus hollandicus). J Avian Med Surg. 1998;12:25-29.

6.            Smith JM, Selim SA. Performance of neonate cockatiels given cockatiel derived lactobacillus. Proc Annu Conf Assoc Avian Vet. 2003;157-159.

7.            Christidis L, Boles W. Systematics and Taxonomy of Australian Birds. Collingwood, Australia: Csiro Publishing; 2008:150.

8.            Garriga M, Pascual M, Monfort JM, Hugas M. Selection of lactobacilli for chicken probiotic adjuncts. J Appl Micro. 1998;84:125-132.

Tables

Table 1. Recovery of cockatiel origin adherent L. salivarius from feces.

Species             Relationa  CFU/cc         During tx           1 wk             2 wk              3 wk              4 wk

                                                       water                                       post-tx        post-tx           post-tx           post-tx

a S=same species, F=same family, O=same order.

Table 2. Recovery of budgerigar origin adherent L. salivarius from feces.

Species                 Relationa     CFU/cc         During tx          1 wk               2 wk               3 wk               4 wk

                                                  water                                 post-tx           post-tx           post-tx         post-tx

a S=same species, SF=same subfamily, F=same family, O=same order.

Table 3. Recovery of green-cheeked conure origin adherent L. reuteri from feces.

Species             Relationa   CFU/cc         During tx             1 wk                 2 wk               3 wk               4 wk

                                                 water                                    post-tx             post-tx           post-tx         post-tx

a S=same species, G=same genus, SF=same subfamily, F=same family, O=same order.

Conure-Bac®TM

Conure-bac is a strain of Lactobacillus reuteri isolated from the intestinal tract lining cells of healthy green-cheeked conures (Pyrrhura molinae). It has been found to colonize the intestinal tracts of not only green-cheeked conures, but other Pyrrhura species conures, such as fiery-shouldered conures (Pyrrhura egregia). Lactobacillus reuteri has been included in many probiotics for a variety of species of animals, including humans, and is renowned for its production of a metabolite called reuterin which shows strong antibacterial properties. It can be expected that the Conure-bac strain of Lactobacillus reuteri will offer the same benefits in green-cheeked conures as other strains offer in other animals.

Our research showed that Conure-bac was recovered from the droppings of green-cheeked and fiery-shouldered conures during and after drinking water treatment. It remained the predominant bacteria in the droppings five weeks after treatment ended when testing was discontinued. This indicates that the bacteria survives passage through the intestinal tract and it colonizes the intestinal tract, allowing the bacteria to provide benefits well after treatment has ended.

Normal, beneficial bacterial flora help maintain a bird’s health by preventing harmful bacteria and yeasts from binding to intestinal lining cells. They do this by four processes:

1.Competitive exclusion. The good bacteria takes up all the binding sites in the intestinal lining, leaving no place for the harmful bacteria and yeasts.

2.Altering the intestinal environment. Lactobacilli create metabolites that acidify the intestinal tract, making the environment hostile for certain bacteria and yeasts.

3.Production of antibacterial metabolites. Some probiotics are better at this than others. Lactobacullus reuteri is one of the best.

4.Improving immunity of the intestinal tract. Research in avian probiotics shows that colonization of the intestinal tract with beneficial bacteria can increase the local immune reactions found in the intestinal tract.

Conure-bac is recommended for use in handfed pyrrhura species conures who aren’t getting normal bacteria from their parents and parent birds feeding chicks in the nest. Because antibiotic treatment eliminates beneficial bacteria as well as harmful bacteria, treatment after antibiotic use is recommended. Research has shown that as birds age they gradually lose their normal bacterial flora and it’s replaced by potentially harmful bacteria. So we also recommend treating with Conure-bac on a yearly basis. Birds also lose their normal bacterial flora under conditions of stress. Treat with Conure-bac whenever your conure has gone through a significantly stressful period.

Conure-bac is available freeze dried in 15 cc vials. When reconstituted, it will supply ½ cup of treated drinking water for 10 days, enough to treat a cage or flight with up to 10 adult conures. Or it can be individually administered to up to 20-25 handfed chicks for 10 days.

Budgie-bac™ Probiotic for Budgies

Budgie-bac is a strain of Lactobacillus salivarius isolated from the intestinal tract lining cells of healthy budgerigars (Melopsittacus undulatus).   Our research shows that Budgie-bac can be recovered from the feces of budgies during treatment as well as for 6 weeks after treatment ended. These results indicate that Budgie-bac survives the environment of the intestinal tract and it colonizes the intestinal tract. In addition, Budgie-bac became the predominant bacteria in the birds’ intestinal tract. Lactobacillus salivarius is a common lactobacillus used in many probiotics. Our Tiel-bac is also a Lactobacillus salivarius. As we continue to research bird probiotics we find that these adherent lactobacilli are at least genus specific. So, for example, the Tiel-bac L. salivarius doesn’t colonize in budgies and the Budgie-bac L. salivarius doesn’t colonize in cockatiels. This finding is consistent with the vast research in poultry that has indicated poultry adherent lactobacilli are also species specific.

Budgies are often infected with a variety of organisms from their parents or their flock of origin. These infections can remain latent (undetected) for years. With stress or exposure to feces (even their own) these diseases can become clinically apparent. Organisms such as Candida, Avian Gastric Yeast, hexamita, giardia, and gram-negative bacteria can all be reduced by having a healthy gastrointestinal tract. Adherent lactobacilli provide protection in the following ways:

1. 1. Competitive exclusion. The good bacteria takes up all the binding sites in the intestinal lining, leaving no place for the harmful organisms to attach.
2. 2.Altering the intestinal environment. Lactobacilli create metabolites that acidify the intestinal contents, making the environment hostile to certain bacteria and yeasts. Most of these adherent lactobacilli also produce metabolites that are antibacterial to other bacteria.
3. 3.Improving immunity of the intestinal tract. Research in poultry shows that adherent lactobacilli can increase the local immune reactions found in the intestinal tract.

Budgie-bac is recommended for use in handfed budgies, parents feeding chicks in the nest or prior to breeding season, budgies that have been treated with antibiotics, pet budgies that have not had exposure to other budgies for a year or more. Birds also lose their normal bacterial flora under stressful conditions, so budgies should be treated with Budgie-bac after any significantly stressful period.

Budgie-bac is easy to administer. It comes freeze-dried in 15 cc vials. When reconstituted it will supply ½ cup of treated drinking water for 10 days, enough to treat a cage or flight of up to 20 adult budgies. It can also be individually dosed in chicks being handfed (up to 20-25 chicks) or in pet budgies. It has no objectionable flavor and is readily accepted in drinking water or by mouth.