Hypoallergenic infant formula lacks transforming growth factor beta activity and has a lower anti-inflammatory activity than regular infant formula.

Hypoallergenic formulas are recommended for infants who are not breastfed and cannot tolerate cow milk formulas due to allergy. These formulas are hydrolyzed to break down larger protein chains into shorter, easy-to-digest, and potentially less allergenic proteins. Hydrolysis, however, possibly occurs at the expense of the transforming growth factor beta (TGF-ß) and anti-inflammatory activity that is inherent in regular formula. Our objective was to determine the TGF-ß and the anti-inflammatory activity of commercially available hypoallergenic and regular formulas. Human gingival fibroblasts were incubated with reconstituted formulas followed by detection of TGF-ß target genes and activation of Smad2/3 signaling. Gingival fibroblasts and the oral squamous cell carcinoma cell line HSC-2 were also exposed to formulas before adding interleukin (IL)1ß and tumor necrosis factor (TNF)α to provoke expression of pro-inflammatory cytokines. For murine bone marrow-derived macrophages, pro-inflammatory cytokine expression was stimulated with saliva. Changes in p65 nuclear translocation and phosphorylation of smad3 and p38 were analyzed by immunostaining. Our study demonstrated that regular formula, but not hypoallergenic formula, enhanced the expression of TGF-ß target genes IL11, PRG4, and NOX4 in gingival fibroblasts. Hypoallergenic formulas also failed to initiate nuclear translocation of Smad2/3 and phosphorylation of Smad3. Moreover, regular formulas were more potent than hypoallergenic formulas in reducing the expression of pro-inflammatory cytokines in gingival fibroblasts, HSC-2 epithelial cells, and murine bone marrow macrophages. Hypoallergenic and regular formulas had a similar capacity to reduce p65 nuclear translocation and phosphorylation of p38 in fibroblasts. These findings suggest that hypoallergenic formulas lack in vitro TGF-ß activity and have a lower anti-inflammatory activity compared with regular formulas.

Differential phenotype of immune cells in blood and milk following pegylated granulocyte colony-stimulating factor therapy during a chronic Staphylococcus aureus infection in lactating Holsteins.

Neutrophils are principal host innate immune cell responders to mastitis infections. Thus, therapies have been developed that target neutrophil expansion. This includes the neutrophil-stimulating cytokine granulocyte colony-stimulating factor (gCSF). Pegylated gCSF (PEG-gCSF; Imrestor, Elanco Animal Health, Greenfield, IN) has been shown to reduce the natural incidence of mastitis in periparturient cows in commercial settings and reduce severity of disease against experimental mastitis challenge. Pegylated gCSF stimulates neutrophil expansion but also induces changes in monocyte and lymphocyte circulating numbers, surface protein expression changes, or both. We hypothesized that PEG-gCSF modulates surface expression of monocytes and neutrophils and facilitates their migration to the mammary gland. We challenged 8 mid-lactation Holsteins with approximately 150 cfu of Staphylococcus aureus (Newbould 305) in a single quarter via intramammary infusion. All animals developed chronic infections as assessed by bacteria counts and somatic cell counts (SCC). Ten to 16 wk postchallenge, 4 of the animals were treated with 2 subcutaneous injections of PEG-gCSF 7 d apart. Complete blood counts, SCC, bacterial counts, milk yield, feed intake, neutrophils extracellular trap analysis, and flow cytometric analyses of milk and blood samples were performed at indicated time points for 14 d after the first PEG-gCSF injection. The PEG-gCSF-treated cows had significantly increased numbers of blood neutrophils and lymphocytes compared with control cows. Flow cytometric analyses revealed increased surface expression of myeloperoxidase (MPO) on neutrophils and macrophages in milk but not in blood of treated cows. Neutrophils isolated from blood of PEG-gCSF-treated cows had decreased surface expression of CD62L (L-selectin) in blood, consistent with cell activation. Surprisingly, CD62L cell surface expression was increased on neutrophils and macrophages sourced from milk from treated animals compared with cells isolated from controls. The PEG-gCSF-treated cows did not clear the S. aureus infection, nor did they significantly differ in SCC from controls. These findings provide evidence that PEG-gCSF therapy modifies cell surface expression of neutrophils and monocytes. However, although surface MPO+ cells accumulate in the mammary gland, the lack of bacterial control from these milk-derived cells suggests an incomplete role for PEG-gCSF treatment against chronic S. aureus infection and possibly chronic mammary infections in general.

Yeast colonization of voice prostheses: pilot study investigating effect of a bovine milk product containing anti-Candida albicans immunoglobulin A antibodies on yeast colonization and valve leakage.

OBJECTIVES: Our goals were to determine whether a bovine milk product containing anti-Candida albicans immunoglobulin A antibodies ("immune milk") could reduce the adherence of C albicans to voice prosthesis silicone in vitro, and whether administration of the milk could reduce C albicans colonization and voice prosthesis damage in vivo. METHODS: An in vitro assay of C albicans attachment to silicone was developed with radiolabeled C albicans. A pilot crossover in vivo trial, over 3 periods of 3 months, was also undertaken for 4 patients with voice prostheses, comparing daily administrations of immune milk and a control milk product. The prosthesis valves were replaced at each changeover and were assessed for wet weight of removable biofilm, yeast numbers in removable biofilm, valve leakage, and valve damage. RESULTS: Immune milk inhibited C albicans adherence to silicone in vitro. However, in a small clinical pilot study, this effect was not replicated. CONCLUSIONS: There is scope to further investigate the topical use of immune milk for management of voice prosthesis biofilms.

Light chain-deficient mice produce novel multimeric heavy-chain-only IgA by faulty class switching.

Recently, we identified that diverse heavy chain (H-chain)-only IgG is spontaneously produced in light chain (L-chain)-deficient mice (L(-/-) with silenced kappa and lambda loci) despite a block in B cell development. In murine H-chain IgG, the first Cgamma exon, C(H)1, is removed after DNA rearrangement and secreted polypeptides are comparable with camelid-type H-chain IgG. Here we show that L(-/-) mice generate a novel class of H-chain Ig with covalently linked alpha chains, not identified in any other healthy mammal. Surprisingly, diverse H-chain-only IgA can be released from B cells at levels similar to conventional IgA and is found in serum and sometimes in milk and saliva. Surface IgA without L-chain is expressed in B220(+) spleen cells, which exhibited a novel B cell receptor, suggesting that associated conventional differentiation events occur. To facilitate the cellular transport and release of H-chain-only IgA, chaperoning via BiP association seems to be prevented as only alpha chains lacking C(H)1 are released from the cell. This appears to be accomplished by imprecise class-switch recombination (CSR) from Smu into the alpha constant region, which removes all or part of the Calpha1 exon at the genomic level.

Ingestion of Streptococcus mutans induces secretory immunoglobulin A and caries immunity.

Ingestion of killed cells of a highly cariogenic strain of Streptococcus mutans induced specific antibodies in both saliva and milk but not in serum of gnotobiotic rats. These antibodies were associated with the immunoglobulin A class. When infected with Streptococcus mutans, orally immunized animals developed significantly fewer carious lesions than nonimmunized infected controls.

Breastfeeding stimulates total and cow's milk-specific salivary IgA in infants.

Breastfeeding may increase the rate of mucosal maturation and IgA production. We sought to determine the effect of breastfeeding vs. formula-feeding on the maturation of oral mucosa by measuring the salivary total antibodies and cow's milk protein-specific IgA. Fifty-eight saliva samples were collected from 39 healthy, full term infants. At the age of 3 months (n = 25) eight infants received only breast milk and seventeen formula (cow's milk based n = 10, hydrolysed n = 7) and breast milk; and at the age of 6 months (n = 33) eleven received breast milk, seventeen formula and breast milk and five were not breastfed any more (cow's milk based n = 14, hydrolysed n = 8). Total IgA, IgG, IgM and protein, and beta-lactoglobulin specific IgA were measured from saliva with enzyme-linked immunoassay (ELISA). The antibody results were proportioned to total protein. No differences in antibody levels between the feeding groups were found at 3 months of age. At 6 months, total IgA, total IgM and beta-lactoglobulin-specific IgA were higher among the breastfed infants compared to those receiving formula as supplement to breast milk or not breastfed any more (breast milk vs. any formula p = 0.029, p = 0.015, p = 0.058; breast milk vs. cow's milk formula p = 0.025, p = 0.044, p = 0.038). To conclude, breastfeeding stimulated the mucosal immune system to produce IgA to saliva, which is a marker for immunological maturation and likely provides protection against environmental antigens.

High levels of IgG4 antibodies to foods during infancy are associated with tolerance to corresponding foods later in life.

Children with eczema and sensitization to foods are recommended skin care and, if food allergy is proven by challenge, an elimination diet. For most children the diet period is transient, but the process behind tolerance development and the influence of decreased allergen exposure is not fully known. The aim of the study was to investigate the effect of elimination diet on serum and salivary antibodies and to identify immunological parameters related to the ability to tolerate foods. Eighty-nine children, below 2 yr of age, with eczema and suspected food allergy were included. Recommended treatment was skin care to all children, and 60 children had a period of elimination diet. At 4(1/2) yr of age, the children were divided into two groups, based on if they had been able to introduce the eliminated foods, or not. Serum and salivary antibodies were analyzed with enzyme-linked immunosorbent assay and UniCAP before and after a 6-wk treatment period and at 4(1/2) yr of age. Children sensitized to egg and/or milk that could eat and drink the offending foods at 4(1/2) yr of age, had higher levels of Immunoglobulin G(4) antibodies to ovalbumin and beta-lactoglobulin and also higher IgG(4)/Immunoglobulin E ratios on inclusion in the study, than those who had to eliminate egg and/or milk from their diet, beyond 4(1/2) yr of age. The highest IgG(4)/IgE ratios were found in children with circulating IgE antibodies to egg and/or milk but negative skin prick test on inclusion. The 6-wk treatment period did not significantly affect the levels of serum and salivary antibodies. In conclusion, eczematous, food sensitized infants with high levels of IgG(4) and high ratios of IgG(4)/IgE antibodies to food allergens are more likely to consume these foods at 4(1/2) yr than infants with low levels and ratios.

Site specific PEGylation of ß-lactoglobulin at glutamine residues and its influence on conformation and antigenicity.

ß-lactoglobulin (ß-LG) is one of the main allergens in milk. Polyethylene glycol (PEG) modification (PEGylation) was found to have the ability to reduce the antigenicity of proteins. To determine the effect of site specific PEGylation on ß-LG antigenicity and conformation, we applied 5 kDa methoxy polyethylene glycol-amine (mPEG-NH2) to modify ß-LG at glutamine (Gln) residues under the catalysis of transglutaminase. The antigenicity of ß-LG was measured using rabbit IgG antibodies by indirect competitive ELISA. The result indicated that the antigenicity of ß-LG was decreased from 72.2 µg/mL to 22.7 µg/mL after PEGylation. SDS-PAGE and MALDI-TOF-MS showed that the molecular mass of native ß-LG was about 18.3 kDa while the PEGylated ß-LG had a molecular mass of 23.4 kDa, which meant that mono-PEGylated ß-LG was obtained after PEGylation and purification by cation exchange chromatography. Additionally, the circular dichroism spectrum of the PEGylated ß-LG was approximately superimposed on that of ß-LG and the secondary structure content of ß-LG also had no significant changes after PEGylation, which indicated that the secondary structure of ß-LG was preserved. After PEGylation, the intrinsic fluorescence intensity of ß-LG decreased from 6361 to 5159 while the surface hydrophobicity increased, which indicated that the tertiary structure of ß-LG was slightly changed. PEGylation site analysis result showed that Gln 155 or Gln 159 might be the most possible binding site. In conclusion, the decrease of the antigenicity of ß-LG induced by the PEGylation is mainly due to the steric shielding effect of PEG chain rather than conformational changes of ß-LG.

Detection of serum and salivary IgE and IgG1 immunoglobulins specific for diagnosis of food allergy.

Given the growing incidence and prevalence of life-threatening food allergies, health concerns have raised new perspectives for in vivo and in vitro diagnostic methodologies, pointing to saliva as a promising material, already used to diagnose other pathologies. Based on the above considerations, this study aimed to verify the possible use of saliva for the detection of IgE and IgG1 in the diagnosis of food allergy. This was a randomized, cross-sectional clinical study with a quantitative approach, developed at a hospital referral center in allergy in the state of Ceará, from January to July 2015. The sample consisted of 36 children of both sexes, aged between 1 and 60 months, with a diagnosis of cow's milk protein allergy (CMPA) by the RAST test. Children hospitalized or under immunosuppressive drugs were excluded from the study. Serum and saliva samples of the participants were collected and subsequently subjected to the indirect immunoenzymatic assay (ELISA) for the detection of specific serum and salivary immunoglobulins for food: corn, papaya, cow's milk, egg white, wheat, soybeans, peanuts, nuts, kiwi, cacao, fish, shrimp, bananas and tomatoes. For comparison of serum and saliva results, the T-test of independent samples and Mann-Whitney were adopted, for samples with normal and non-normal distribution respectively. A confidence interval of 95% was adopted for significant results. It was observed that 100% (n = 36) of the participants presented cow's milk allergy through the indirect ELISA, detecting IgE or IgG1 in serum and saliva. When serum IgE and IgG1 concentrations were compared, there was no statistical difference (p > 0.05) in 12 of the 14 foods evaluated. The same amount (n = 12) of non-significant differences (p > 0.05) was observed in the comparison of the 14 foods under IgE and IgG1 contractions in saliva. In the verification of the average values of IgE present in the serum and saliva of the foods, only cow's milk, fish and papaya showed statistically significant differences (p < 0.05). Of the total food evaluated, only the average levels of IgG1 present in serum and saliva showed a significant value (p < 0.05) in banana and tomato. These findings indicate that the detection of IgE and IgG1 in saliva proves to be as efficient as in the serum. The use of the salivary technique for use in the diagnosis of food allergy is suggested.

Does Neospora caninum cause reproductive problems in pigs?

Neospora caninum is known to cause reproductive disturbances in several animal species, such as cattle, sheep, and goats. However, research on the effects of N. caninum on reproduction in pigs is limited. The objective of this study was to verify the transplacental transmission of N. caninum in pigs during several gestational stages. Twelve healthy Toxoplasma gondii and N. caninum seronegative female pigs were selected and separated into four groups of three animals each. Group A was maintained as a control group. Groups B, C, and D were inoculated intravenously with 2.9 × 107 tachyzoites of the N. caninum strain Nc1, 30 days before conception and at 45 and 90 days of gestation, respectively. Blood samples were collected from females periodically through IFAT for IgG and IgM screening to confirm the infection. At birth, after blood samples were collected from the piglets, they were then euthanized for the collection of the brain, heart, lung, liver, and diaphragm, which were then subjected to PCR. All inoculated gilts seroconverted (IgG) from the seventh day after inoculation. Nine of the 12 females expelled 24 mummified fetuses at the time of delivery, two in group A (eight), two in group B (four), three in group C (nine), and two in group D (three). Of the 24 mummified fetuses, nine were positive for N. caninum (one (25%) fetus of group B, seven (77.8%) of group C, and one (33.3%) of group D). A total of 126 live piglets were born. When the organs of the piglets from the inoculated females were analyzed by PCR for N. caninum, 88 (93.61%) were positive. All gilts inoculated produced at least one positive piglet. This demonstrates that there is transplacental transmission of N. caninum in all phases of gestation, regardless of the time of infection.

Food allergy in childhood (infancy to school age).

Food allergy is a potentially life-threatening condition affecting almost 10% of children, with an increasing incidence in the last few decades. It is defined as an immune reaction to food, and its pathogenesis may be IgE mediated, mixed IgE and non-IgE mediated, or non-IgE mediated. Potentially all foods can cause food allergy, but a minority of foods are responsible for the vast majority of reactions reported. A good clinical history is crucial for an accurate diagnosis. Allergy tests, including the skin prick test and measurement of specific IgE antibodies, are useful tools in the case of IgE-mediated or mixed allergy but have not been shown to be of any help in delayed allergic reactions to foods.

Changed sensitivity to antigen in a gut epithelium treated with bile salts.

Colonic epithelia from guinea-pigs, sensitized by feeding with cow milk, responded to antigen (beta-lactoglobulin) challenge when applied to the serosal, but not the mucosal, side of the tissue. The response, under short circuit conditions, was an inwardly directed current due to chloride secretion. Two detergents, deoxycholate and Triton X-100, caused the basal short circuit current to decrease and transepithelial conductance to increase when applied to the mucosal surface. After removing detergents from the bathing solution tissues now responded to antigen challenge from the mucosal side, without impairment of the overall response. There was a correlation between the conductance change induced by detergents and the fraction of the total response which could be elicited form the mucosal side of the tissue. It was concluded that models of local hypersensitivity reactions to ingested foodstuffs require both development of immunological sensitivity plus increased permeability to antigen. The role of bile salts in inducing the latter is discussed.

Food antibodies in oral disease: a study of serum antibodies to food proteins in aphthous ulceration and other oral diseases.

We have investigated the incidence of antibodies to food antigens in patients with recurrent minor aphthous ulceration and in patients with other oral ulcerative diseases. The incidence of these antibodies was the same in both groups of patients and was significantly greater than the incidence in a control group of normal people. There was no evidence to support the hypothesis that aphthous ulceration is primarily due to hypersensitivity to food antigens. The factors which might contribute to the absorption of antigenic molecules from the mouth and to the increased immune response in patients with oral disease have been considered.

Validation of the specific isotype assay to detect antibodies against foot-and-mouth disease virus in bovine milk.

The specific isotype assay (SIA) detects IgG1 against foot-and-mouth disease (FMD) virus in bovine milk. A strong correlation was demonstrated between milk antibody titres, and those in serum as measured by the liquid phase blocking ELISA. Thus the SIA would be useful on a herd basis to monitor the milk of vaccinated cattle to determine when re-immunisation is advisable. The SIA titration ELISA was then simplified to a single dilution test and optimised to differentiate the reactions in the milk of FMD-naive cows from those in animals which had been infected with FMD or vaccinated against the disease. For milk from immunised cattle, the pH of the sample was important and borderline positive specimens with a pH of 6.0 or below gave negative results. For milk from naive animals, the optical density (OD) registered in the SIA varied according to the time of year that samples were collected which, in turn, influenced the OD above which milks might be considered positive. Studies showed that the pH of milk could be maintained within the range suitable for the SIA by either storing for up to 1 week at 4 degrees C or by freezing at -20 degrees C for an indefinite period.

Predicting herd protection against foot-and-mouth disease by testing individual and bulk tank milk samples.

Four groups of cattle were tested for antibodies against foot-and-mouth disease (FMD) virus type O(1) over three 70 day vaccination cycles using the liquid-phase-blocking-ELISA (LPBE). First lactation cows showed the lowest titres and group protection levels (GPLs) against FMD virus strains with 'r' values < or =0.5 while second lactation animals gave the highest results. When mean serum titres for each group and sampling date were plotted against GPL a strong correlation was found. Revaccination was indicated at a mean titre of approximately log10 2.88 (1:760; R=0.93; n=86) if the herd was threatened by field strains with an 'r' value of 0.25, or log10 2.62 (1:420; R=0.83; n=48) if this ratio was 0.5. Significant overall correlation (R=0.53; n=624) was obtained between serum titres and milk IgG(1) results derived from the modified specific isotype assay (SIA). Milk titres equivalent to 1:100, 1:200, 1:400 and 1:800 were 1:3.8, 1:6.3, 1:10.4 and 1:17.1, respectively, in first lactation cows. Bulk tank milk samples demonstrated a repeating pattern of results corresponding to the vaccination cycle with no titre lower than log10 1.05 (1:11). Colostrum from first lactation animals showed mean SIA results of log10 4.06 (1:11,480) and early milk titres only levelled off approximately 11 days post partum (dpp).

Modulation of a specific humoral immune response and changes in intestinal flora mediated through fermented milk intake.

This study was undertaken to elucidate whether eating a fermented milk containing Lactobacillus acidophilus La1 and bifidobacteria could induce changes in intestinal flora and modulate the immune response in man. Volunteers consumed a fermented milk containing L. acidophilus La1 and bifidobacteria over a period of three weeks during which an attenuated Salmonella typhi Ty21a was administered to mimic an enteropathogenic infection. A control group ate no fermented foods but received the S. typhi Ty21a. Faecal flora analyses showed an increase in L. acidophilus and bifidobacterial counts during fermented milk intake. The specific serum IgA titre rise to S. typhi Ty21a in the test group was > 4-fold and significantly higher (P = 0.04) than in the control group. An increase in total serum IgA was also observed. These results indicate that lactic acid bacteria which can persist in the gastrointestinal tract can act as adjuvants to the humoral immune response.

Effective immunity to dental caries: studies of active and passive immunity to Streptococcus mutans in malnourished rats.

The present studies suggest that rat dams passively transfer IgG to their offspring via milk. Furthermore, rat dams hyperimmunized to S mutans after intravenous administration of this bacterium have serum-precipitating antibody to S mutans group-specific antigen. This serum precipitin was also observed in serums of their offspring during the suckling period and was detectable for a week after weaning. When these offspring were infected with S mutans on the day of weaning, significantly fewer smooth surface lesions developed in them than in infected rats reared on nonimmunized mothers. These results suggest that anti-S mutans antibody, perhaps of the IgG2a class, is passively transferred from mother to offspring via the milk. Furthermore, this antibody is probably important in protection against S mutans infection. In this regard, recent studies by Lehner, Challacombe, and Caldwell have suggested that crevicular fluid transudating serum antibodies are important in the prevention of dental caries in rhesus monkeys. From our studies and others, it is becoming clear that at least two sources and classes of antibody are important in caries immunity. Secretory IgA, produced and secreted into saliva after local injection can be correlated with protection. At the same time, serum antibody (presumably IgG) either passively or actively derived also gives immune protection. Further studies must clarify the precise role of these Ig's and their possible synergistic activity with other specific immune factors in saliva in order to determine the mechanism(s) involved in effective caries immunity.

Observations of absorption of immunolactoglobulins in calves.

The aim of this study was to ascertain whether administration of one liter of stilulated saliva from a healthy cow as first food to newborn calves blocks absorption of immunolactoglobulins administered later in colostrum or milk. The study material consisted of 12 calves, divided into two groups, one of which received colostrum, and the other milk. Blood samples were drawn immediately after birth and 3 and 6 hours after administration of saliva and 3 and 6 hours after administration of colostrum (group I) or milk (group II). Only some of the calves of each group absorbed immunolactoglobulins from colostrum or milk. In these calves, immunoglobulins could be demonstrated in their serum as early as 3 hours after birth. Absorption of immunolactoglobulins was independent of their concentration in food as their levels were similar in calves fed colostrum or milk. The experiment failed, however, to give an unequivocal answer to the question whether feeding calves before the first administration of colostrum restricts or inhibits absorption of immunolactoglobulins from colostrum.

The metabolism and transport of bovine serum SIgA.

Experiments were undertaken in six Holstein-Friesian cattle to determine whether secretory IgA (SIgA) could be transported from serum into exocrine body fluids. Preliminary data indicated that when IgG1, IgG2, IgM and SIgA were administered i.v., only SIgA and IgG1 appeared in bile 90 min later at concentrations equal to or exceeding those in serum at the same time. Two hours post-injection, 70% of the SIgA recovered in bile was intact however only 30% co-precipitable with anti-secretory component (SC) while greater than 90% of the administered IgA was precipitated by this method. All recovered IgG1 was of low molecular weight. More detailed studies indicated that the IgA recovered in bile 7 h post-injection or in milk 3 h post-injection, was predominantly lower molecular weight than intact SIgA. Most of this low molecular weight radioactivity was TCA precipitable and ca 50% was dialyzable; these data indicate that TCA-precipitability is an inadequate criterion for determining whether intact SIgA is transported. The radioactivity recovered in parotid saliva was almost entirely non-TCA precipitable and dialyzable. Almost all SIgA recovered in bovine serum remained intact and had a t1/2 of 15.7 h. When transport into milk and bile was calculated from total, recovered radioactivity (i.e. 29% and 2.7, respectively), data compared favorably with those conducted in sheep in which dimeric IgA (without SC) was administered i.v. When we calculated transport on the basis of recovered intact IgA, only 1.47 and 0.54% of the injected dose had been transported into milk and bile, respectively, 24 h later. Most IgA in ruminant bile may be of serum origin although the same appears to be unlikely for the IgA in milk.

Effect of immune bovine milk on Streptococcus mutans in human dental plaque.

The use of a mouthrinse of bovine milk containing antibodies to Streptococcus mutans resulted in an initial reduction in the numbers of recoverable Strep. mutans in a group of 9 individuals. Ten volunteers who used control bovine milk that contained no antibody activity to Strep. mutans had variable levels of plaque Strep. mutans. In addition, after culture on Mitis Salivarius and Gold's agar, the plaque Strep. mutans from subjects who used the immune bovine milk rinse formed smaller colonies than those from pre-treatment plaque and from all plaque samples of subjects who used the control rinse.