Coordination of prophage and global regulator leads to high enterotoxin production in staphylococcal food poisoning-associated lineage.

Staphylococcus species in food produce Staphylococcal enterotoxins (SEs) that cause Staphylococcal food poisoning (SFP). More than 20 SE types have been reported, among which Staphylococcal enterotoxin A (SEA) has been recognized as one of the most important SEs associated with SFP. However, the regulatory mechanisms underlying its production remain unclear. Previously, we identified a major SFP clone in Japan, CC81 subtype-1, which exhibits high SEA production. In this study, we attempted to identify the factors contributing to this phenomenon. Thus, we demonstrated that the attenuation of the activity of endogenous regulator, Staphylococcal accessory regulator S (SarS), and the lysogenization of a high SEA-producing phage contributed to this phenomenon in CC81 subtype-1. Furthermore, our results indicated that SarS could directly bind to the promoter upstream of the sea gene and suppress SEA expression; this low SarS repression activity was identified as one of the reasons for the high SEA production observed. Therefore, we revealed that both exogenous and endogenous factors may probably contribute to the high SEA production. Our results confirmed that SE production is a fundamental and critical factor in SFP and clarified the associated production mechanism while enhancing our understanding as to why a specific clone frequently causes SFP. IMPORTANCE: The importance of this study lies in its unveiling of a molecular regulatory mechanism associated with the most important food poisoning toxin and the evolution of Staphylococcal food poisoning (SFP)-associated clone. SFP is primarily caused by Staphylococcus aureus, with Staphylococcal enterotoxin A (SEA) being commonly involved in many cases. Thus, SEA has been recognized as a major toxin type. However, despite almost a century since its discovery, the complete mechanism of SEA production is as yet unknown. In this study, we analyzed an SEA-producing SFP clone isolated in East Asia and discovered that this strain, besides acquiring the high SEA-producing phage, exhibits remarkably high SEA production due to the low activity of SarS, an intrinsic regulatory factor. This is the first report documenting the evolution of the SFP clone through the coordinated action of exogenous mobile genetic factors and endogenous regulators on this notorious toxin.

Investigation of Staphylococcus aureus positive for Staphylococcal enterotoxin S and T genes.

Staphylococcus aureus produces staphylococcal enterotoxins (SEs) and causes food poisoning. It is known that almost all SE-encoding genes are present on various types of mobile genetic elements and can mobilize among S. aureus populations. Further, plasmids comprise one of SE gene carriers. Previously, we reported novel SEs, SES and SET, harbored by the plasmid pF5 from Fukuoka5. In the present study, we analyzed the distribution of these SEs in various S. aureus isolates in Japan. We used 526 S. aureus strains and found 311 strains positive for at least one SE/SE-like toxin gene, but only two strains (Fukuoka5 and Hiroshima3) were positive for ses and set among the specimens. We analyzed two plasmids (pF5 and pH3) from these strains and found that they were different. Whereas these plasmids partially shared similar sequences involved in the ser/selj/set/ses gene cluster, other sequences were different. A comparison of these plasmids with those deposited in the NCBI database revealed that only one plasmid had the ser/selj/set/ses cluster with a stop mutation in set similar to that in pH3. In addition, the chromosomes of Fukuoka5 and Hiroshima3, positive for ses and set, were classified into different genotypes. Despite the low rate of gene positivity for these SEs, it is suggested that there is diversity in plasmids and strains carrying these two SEs. Consequently, regarding the entire feature of SE prevalence, we improved the multiplex PCR detection method for the SE superfamily to obtain further insight.

Development of an Immunoassay for Detection of Staphylococcal Enterotoxin-Like J, A Non-Characterized Toxin.

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases.

Complete Sequence of a Staphylococcus aureus Clonal Complex 81 Strain, the Dominant Lineage in Food Poisoning Outbreaks in Japan.

Staphylococcus aureus No. 10 is an isolate from a staphylococcal food poisoning outbreak in Japan, classified as clonal complex 81 subtype 1. It preferentially produces larger quantities of staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin H (SEH) in foods and media. Here, we report the complete annotated genome sequence of the chromosome and a plasmid.

The emetic activity of staphylococcal enterotoxins, SEK, SEL, SEM, SEN and SEO in a small emetic animal model, the house musk shrew.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are the most recognizable causative agents of emetic food poisoning in humans. New types of SEs and SE-like (SEl) toxins have been reported. Several epidemiological investigations have shown that the SEs and SEl genes, particularly, SEK, SEL, SEM, SEN and SEO genes, are frequently detected in strains isolated from patients with food poisoning. The purpose of the present study was to evaluate the emetic activity of recently identified SEs using a small emetic animal model, the house musk shrew. The emetic activity of these SEs in house musk shrews was evaluated by intraperitoneal administration and emetic responses, including the number of shrews that vomited, emetic frequency and latency of vomiting were documented. It was found that SEs induce emetic responses in these animals. This is the first time to demonstrate that SEK, SEL, SEM, SEN and SEO possess emetic activity in the house musk shrew.

Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning.

We previously demonstrated the clonal complex 81 (CC81) subtype 1 lineage is the major staphylococcal food poisoning (SFP)-associated lineage in Japan (Y. Sato'o et al., J Clin Microbiol 52:2637-2640, 2014, http://dx.doi.org/10.1128/JCM.00661-14). Strains of this lineage produce staphylococcal enterotoxin H (SEH) in addition to SEA. However, an evaluation of the risk for the recently reported SEH has not been sufficiently conducted. We first searched for staphylococcal enterotoxin (SE) genes and SE proteins in milk samples that caused a large SFP outbreak in Japan. Only SEA and SEH were detected, while there were several SE genes detected in the samples. We next designed an experimental model using a meat product to assess the productivity of SEs and found that only SEA and SEH were detectably produced in situ. Therefore, we investigated the regulation of SEH production using a CC81 subtype 1 isolate. Through mutant analysis of global regulators, we found the repressor of toxin (Rot) functioned oppositely as a stimulator of SEH production. SEA production was not affected by Rot. seh mRNA expression correlated with rot both in media and on the meat product, and the Rot protein was shown to directly bind to the seh promoter. The seh promoter sequence was predicted to form a loop structure and to hide the RNA polymerase binding sequences. We propose Rot binds to the promoter sequence of seh and unfolds the secondary structure that may lead the RNA polymerase to bind the promoter, and then seh mRNA transcription begins. This alternative Rot regulation for SEH may contribute to sufficient toxin production by the CC81 subtype 1 lineage in foods to induce SFP.

Identification and Characterization of a Novel Staphylococcal Emetic Toxin.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have superantigenic and emetic activities, which cause toxic shock syndrome and staphylococcal food poisoning, respectively. Our previous study demonstrated that the sequence of SET has a low level of similarity to the sequences of other SEs and exhibits atypical bioactivities. Hence, we further explored whether there is an additional SET-related gene in S. aureus strains. One SET-like gene was found in the genome of S. aureus isolates that originated from a case of food poisoning, a human nasal swab, and a case of bovine mastitis. The deduced amino acid sequence of the SET-like gene showed 32% identity with the amino acid sequence of SET. The SET-like gene product was designated SElY. In the food poisoning and nasal swab isolates, mRNA encoding SElY was highly expressed in the early log phase of cultivation, whereas a high level of expression of this mRNA was found in the bovine mastitis isolate at the early stationary phase. To estimate whether SElY has both superantigenic and emetic activities, recombinant SElY was prepared. Cell proliferation and cytokine production were examined to assess the superantigenic activity of SElY. SElY exhibited superantigenic activity in human peripheral blood mononuclear cells but not in mouse splenocytes. In addition, SElY exhibited emetic activity in house musk shrews after intraperitoneal and oral administration. However, the stability of SElY against heating and pepsin and trypsin digestion was different from that of SET and SEA. From these results, we identified SElY to be a novel staphylococcal emetic toxin.

Molecular typing of Salmonella enterica serovar 4,[5],12:i:- isolates from humans, animals and river water in Japan by multilocus variable-number tandem repeat analysis and pulsed-field gel electrophoresis.

Fifty-one Salmonella enterica serovar 4,[5],12:i:- (S. 4, [5],12:i:-) isolates (14 human strains, 34 animal strains and 3 river water strains) which are assumed to be monophasic variants of S. Typhimurium were analyzed using pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) in order to investigate their genetic diversities and relationships. PFGE, MLVA and combination of them identified 28, 27 and 34 profiles (Simpson's diversity indices [DI]=0.94, 0.96 and 0.97), respectively. No correlations were detected between MLVA clustering and PFGE clustering or phage typing. These results suggested that S. 4,[5],12:i:- originated from multiple S. Typhimurium ancestors. Two cattle and one pig isolates showing identical phage types as well as PFGE and MLVA profiles to human isolates S. 4,[5],12:i:- suggested the existence of the links between human infections and animal reservoirs.

Molecular epidemiological characterization of Staphylococcus aureus isolates originating from food poisoning outbreaks that occurred in Tokyo, Japan.

Staphylococcal food poisoning (SFP), one of the commonest food-borne diseases, results from the ingestion of one or more staphylococcal enterotoxins (SEs) produced in foods by Staphylococcus aureus. In the present study, 203 S. aureus strains originating from 83 outbreaks that had occurred in Tokyo were examined for their coagulase type and genotype of SEs to analyze their molecular epidemiological characteristics. The representative subsets of the 83 S. aureus isolates were analyzed by multilocus sequence typing (MLST) and S. aureus pathogenicity island (SaPI) scanning. The isolates were integrated into eight specific clonal complexes (CC) s; CC81, CC8, CC6, CC5, CC508, CC59, CC20 and CC30. The profiles of the coagulase type, SE/SEl genotype and the suspected type of enterotoxin-encoding mobile genetic element (MGE) indicated a correlation with each CC. SaPI scanning showed fixed regularity between the distributions of genomic islands, including SaPIs, and the phylogenetic lineage based on MLST. These results indicate that the S. aureus isolates, which classified into eight CCs, have distinguishable properties concerning specific coagulase type, enterotoxin genotype and MGE type. Strains of S. aureus harboring these particular elements possess the potential to cause SFP.

Molecular epidemiology and identification of a Staphylococcus aureus clone causing food poisoning outbreaks in Japan.

Molecular characterization of isolates from staphylococcal food poisoning (SFP) outbreaks in Japan showed that the dominant lineage causing SFP outbreaks is clonal complex 81 (CC81), a single-locus variant of sequence type 1, coagulase type VII, positive for sea and/or seb, and positive for seh. Among various CC lineages producing staphylococcal enterotoxin A, CC81 showed the highest toxin productivity.

Emetic potentials of newly identified staphylococcal enterotoxin-like toxins.

Staphylococcal enterotoxins (SEs) are a common causative agent of food poisoning. Recently, many new SE-like (SEl) toxins have been reported, although the role of SEls in food poisoning remains unclear. In this study, the emetic potentials of SElK, SElL, SElM, SElN, SElO, SElP, and SElQ were assessed using a monkey-feeding assay. All the SEls that were tested induced emetic reactions in monkeys at a dose of 100 µg/kg, although the numbers of affected monkeys were significantly smaller than the numbers that were affected after consuming SEA or SEB. This result suggests that these new SEs may play some role in staphylococcal food poisoning.

A novel comprehensive analysis method for Staphylococcus aureus pathogenicity islands.

Staphylococcus aureus pathogenicity islands (SaPIs) form a growing family of mobile genetic elements (MGEs) in Staphylococci. Horizontal genetic transfer by MGEs plays an important role in the evolution of S. aureus. Several SaPIs carry staphylococcal enterotoxin and SE-like toxin genes. To comprehensively investigate the diversity of SaPIs, a series of primers corresponding to sequences flanking six SaPI insertion sites in S. aureus genome were designed and a long and accurate (LA)-PCR analysis method established. LA-PCR products of 13-17 kbp were observed in strains with seb, selk or selq genes. Restriction fragment length polymorphism (RFLP) analysis showed that the products have different RFLP characteristics than do previously described SaPIs; they were therefore predicted to include new SaPIs. Nucleotide sequencing analysis revealed seven novel SaPIs: seb-harboring SaPIivm10, SaPishikawa11, SaPIivm60, SaPIno10 and SaPIhirosaki4, selk and selq-harboring SaPIj11 and non-superantigen-harboring SaPIhhms2. These SaPIs have mosaic structures containing components of known SaPIs and other unknown genes. Strains carrying different SaPIs were found to have significantly different production of superantigen toxins. The present results show that the LA-PCR approach can comprehensively identify SaPI diversity and is useful for investigating the evolution of S. aureus pathogenicity.

Staphylococcal enterotoxin A has potent superantigenic and emetic activities but not diarrheagenic activity.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are pyrogenic superantigenic toxins that are involved in human diseases including food poisoning and toxic shock syndrome. Although the superantigenic activity of SEs has been well characterized, its role and mechanism in clinical symptoms of food poisoning remain poorly understood. In this study, house musk shrews (Suncus murinus), a small emetic animal model, were used to study the role of SEs in clinical manifestations of food poisoning. Administration of SEA induced a potent emetic response in vivo and showed significant superantigenic activity in vitro in house musk shrews. However, SEA revealed no diarrheagenic activity. SEA directly injected into the intestinal loops of house musk shrews failed to induce fluid exudation and consequent dilation of the intestinal segments. Rabbit intestinal loop experiments were further carried out to confirm the results and also showed that SEA induced no fluid exudation and consequent dilation. Furthermore, the SEA-producing S. aureus also failed to induce fluid exudation in the administered loops of these animal models. These results indicate that SEA has potent superantigenic and emetic activities, but does not have a diarrheagenic activity.

Submucosal mast cells in the gastrointestinal tract are a target of staphylococcal enterotoxin type A.

Staphylococcal enterotoxin A (SEA) is a leading causative toxin of staphylococcal food poisoning. However, it remains unclear how this toxin induces emesis in humans, primates, and certain experimental animals. To understand the mechanism of SEA-induced emesis, we investigated the behavior of SEA in the gastrointestinal (GI) tract in vivo using the house musk shrew (Suncus murinus). Immunofluorescence of GI sections showed that perorally administered SEA translocated from the lumen to the interior tissues of the GI tract and rapidly accumulated in certain submucosa cells. These SEA-binding cells in the submucosa were both tryptase- and FcεRIα-positive, suggesting these SEA-binding cells were mast cells. These SEA-binding mast cells were 5-hydroxytryptamine (5-HT)-positive, but the intensity of the 5-HT signal decreased over time compared to that of mast cells in the negative control. Furthermore, toluidine blue staining showed the number of metachromatic mast cells was decreased in the duodenal submucosa, suggesting that SEA binding induced degranulation and release of 5-HT from submucosal mast cells. These observations suggest that the target cells of SEA are submucosal mast cells in the GI tract and that 5-HT released from submucosal mast cells plays an important role in SEA-induced emesis.

Social Environment and Control Status of Companion Animal-Borne Zoonoses in Japan.

Changing social and environmental factors have been the cause of an increase in the number and variety of animals are being imported into Japan. Moreover, the number of Japanese households are keeping companion animals has also risen. These factors, along with the high density of the Japanese population and the low percentage of registered dogs, have increased the risk of animal-to-human transmission of zoonoses. To control zoonosis outbreaks, the Japanese government has implemented a three-stage approach for the border control of zoonoses and has stipulated the monitoring and reporting of eight companion animal-borne zoonoses under the Rabies Prevention Law and the Infectious Diseases Control Law. The fact that no case of human and animal rabies has been reported over the past 50 years indicates that these measures are highly effective in preventing rabies transmission. Although it is known that the total number of possible companion animal-borne zoonosis outbreaks decreased between 2005 and 2009 when compared with numbers between 2001 and 2004, the number of zoonosis cases that can be attributed to transmission by companion animals remains unclear. Active surveillance should be conducted on a national level to collect the data necessary to determine this number and identify trends in companion-animal transmitted diseases. Using the data collected, regulation systems should be evaluated to determine whether they have met reasonable goals and policy planning conducted for the control of emerging diseases.

Superantigenic toxin genes coexist with specific staphylococcal cassette chromosome mec genes in methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is a leading cause of human disease in the hospital setting and the community. Superantigenic toxin-producing methicillin-resistant Staphylococcus aureus (MRSA) is currently important for nosocomial infections and food-borne diseases worldwide because of its global spreading and difficulty in therapy. Superantigenic toxins can bypass normal antigen presentation and have strong T cell mitogenic activity, leading to massive release of proinflammatory cytokines and contributing to the severity of S. aureus sepsis. In this study, a total of 131 MRSA isolates from patients in the University Hospital were searched for staphylococcal cassette chromosome mec (SCCmec) genes and the staphylococcal superantigenic toxin genes by multiplex polymerase chain reactions. The MRSA isolates were classified into SCCmec type II (74.8%), type I (13.0%), type IV (3.8%), type V (2.3%), and type I and type II (3.8%). MRSA isolates (102/131) also carried a number of superantigenic toxin genes including staphylococcal enterotoxin (se) and toxic shock syndrome toxin-1 (tst-1) genes. The most frequent superantigen gene profile (55/131, 42.0%) of the MRSA isolates includes staphylococcal enterotoxin C (sec), seg, sei, staphylococcal enterotoxin-like L (sell), selm, seln, selo, and tst-1. Furthermore, SCCmec type I or type II MRSA isolates more frequently harbor sec, seg, sei, sell, selm, seln, selo, and tst-1 genes, compared to other types of MRSA. These results indicate that the selected superantigenic toxin genes are linked to SCCmec type I and type II. The coexistence of these toxins and the SCCmec genes in S. aureus may contribute to the biological fitness and pathogenicity of MRSA.

High affinity of interaction between superantigen and T cell receptor Vbeta molecules induces a high level and prolonged expansion of superantigen-reactive CD4+ T cells.

In mice implanted with an osmotic pump filled with the superantigen (SAG) staphylococcal enterotoxin A (SEA), the Vß3(+)CD4(+) T cells exhibited a high level of expansion whereas the Vß11(+)CD4(+) T cells exhibited a mild level of expansion. In contrast, in mice implanted with an osmotic pump filled with SE-like type P (SElP, 78.1% homologous with SEA), the Vß11(+)CD4(+) T cells exhibited a high level of expansion while the Vß3(+)CD4(+) T cells exhibited a low level of expansion, suggesting that the level of the SAG-induced response is determined by the affinities between the TCR Vß molecules and SAG. Analyses using several hybrids of SEA and SElP showed that residue 206 of SEA determines the response levels of Vß3(+)CD4(+) and Vß11(+)CD4(+) T cells both in vitro and in vivo. Analyses using the above-mentioned hybrids showed that the binding affinities between SEA and the Vß3/Vß11 ß chains and between SEA-MHC class II-molecule complex and Vß3(+)/Vß11(+) CD4(+) T cells determines the response levels of the SAG-reactive T cells both in vitro and in vivo.

New journal selection for quantitative survey of infectious disease research: application for Asian trend analysis.

BACKGROUND: Quantitative survey of research articles, as an application of bibliometrics, is an effective tool for grasping overall trends in various medical research fields. This type of survey has been also applied to infectious disease research; however, previous studies were insufficient as they underestimated articles published in non-English or regional journals. METHODS: Using a combination of Scopus and PubMed, the databases of scientific literature, and English and non-English keywords directly linked to infectious disease control, we identified international and regional infectious disease journals. In order to ascertain whether the newly selected journals were appropriate to survey a wide range of research articles, we compared the number of original articles and reviews registered in the selected journals to those in the 'Infectious Disease Category' of the Science Citation Index Expanded (SCI Infectious Disease Category) during 1998-2006. Subsequently, we applied the newly selected journals to survey the number of original articles and reviews originating from 11 Asian countries during the same period. RESULTS: One hundred journals, written in English or 7 non-English languages, were newly selected as infectious disease journals. The journals published 14,156 original articles and reviews of Asian origin and 118,158 throughout the world, more than those registered in the SCI Infectious Disease Category (4,621 of Asian origin and 66,518 of the world in the category). In Asian trend analysis of the 100 journals, Japan had the highest percentage of original articles and reviews in the area, and no noticeable increase in articles was revealed during the study period. China, India and Taiwan had relatively large numbers and a high increase rate of original articles among Asian countries. When adjusting the publication of original articles according to the country population and the gross domestic product (GDP), Singapore and Taiwan were the most productive. CONCLUSION: A survey of 100 selected journals is more sensitive than the SCI Infectious Disease Category from the viewpoint of avoiding underestimating the number of infectious disease research articles of Asian origin. The survey method is applicable to grasp global trends in disease research, although the method may require further development.

Interaction between superantigen and T-cell receptor Vbeta element determines levels of superantigen-dependent cell-mediated cytotoxicity of CD8(+) T cells in induction and effector phases.

Specific superantigens activate different T-cell fractions with distinct TCR V beta elements in association with MHC class II molecules and also induce SDCC against MHC class II(+) target cells. In the present study, to determine whether the responsiveness of each CD8(+) T-cell fraction expressing a different TCR V beta element is primarily determined by the TCR V beta, we compared the levels of proliferation and SDCC in V beta3(+) and V beta11(+) T cells upon stimulation with SEA. Upon stimulation with SEA(wt), the levels of proliferation were higher in V beta3(+) T cells than in V beta11(+) T cells. The levels of SDCC were also higher for the combination of V beta3(+) T cells and SEA(wt) than for the combination of V beta11(+) T cells and SEA(wt) during both the induction phase and the effector phase. In addition, upon stimulation with SEA(m), the levels of proliferation were higher in V beta11(+) T cells than in V beta3(+) T cells. And then, the levels of SDCC were also higher for the combination of V beta11(+) T cells and SEA(m) than for the combination of Vbeta3(+) T cells and SEA(m) during both the induction phase and the effector phase. These results suggest that the SAG-responsiveness of each CD8(+) T-cell fraction expressing a different TCR V beta element is primarily determined by the interaction between the TCR V beta element and the SAG.

Canine neutrophil dysfunction caused by downregulation of beta2-integrin expression without mutation.

Canine leukocyte adhesion deficiency (CLAD) in Irish setters is caused by genetic defects of leukocyte integrin CD18 leading to recurrent bacterial infections. We report clinical features and analysis of neutrophil function from two mixed-breed canine littermates (one female and one male dog) similar to CLAD. The symptoms of pyogenic infection were first recognized at 3 months of age and since then the patients suffered from recurrent bacterial infections. These clinical findings were strongly suggestive of genetic phagocyte dysfunction. Neutrophil function tests revealed a marked reduction of serum-opsonized zymosan-mediated superoxide production in the two littermates. Neutrophils of the male dog revealed impaired integrin-mediated adherence and phagocytic activity, whereas ability of serum opsonization was normal. There was also a profound decrease of surface expression of CD11b/CD18 and beta2-integrin transcript level, detected by real-time RT-PCR without missense mutations unlike CLAD. Immunoblot analysis indicated that protein expression of cytochrome b(558) component gp91(phox), the cytosolic components p47(phox) and p67(phox) of NADPH oxidase components increased profoundly in the male. Our study suggests that decreased transcriptional levels of beta2-integrin without mutations, lead to downregulation of surface expression, resulting in multiple defects in adhesion-related neutrophil functions and consequently, recurrent bacterial infections from puppyhood.