Birth weight, puppies' growth and health with limited-ingredient novel protein diet vs standard diet in late pregnancy.

Great attention has been given in the last years to the protein source of dog food, and commercial limited-ingredient diets with a single protein are available also for late pregnancy. This work compared the effect of a fish-based limited ingredient diet (LID), and of a standard mixed-protein diet (Mixed), fed to the bitches from the last three weeks of pregnancy and to the puppies at weaning, on birth weight, growth and health of the puppies. From a breeder's records, the weight of 22 Lagotto Romagnolo (LR) and 10 Appenzeller Cattle Dog (ACD) bitches on the day of mating, and of their 199 puppies, were extracted. The effect of diet on puppies' weight on day 0, 6, 30 and 60 was analyzed, considering litter size and sex. The analyses were repeated on puppies' weights normalized on the relative dam's non-pregnant bodyweight. Birth weight was available for 146 puppies, 82 LR and 64 ACD. Median birth weight of LR puppies was 287.5 g (170-400 g); sex ratio was 1.11 (males/females, N = 80). Median birth weight of ACD puppies was 390 g (240-525 g); sex ratio 1.15 (males/females, N = 58). Diet did not significantly affect birth weight in both breeds; however, it showed a significant effect on normalized birth weights (LR, P = 0.016; ACD, P = 0.034), with higher values for LID. At day 30, ACD puppies showed significantly higher weights with the Mixed diet (P = 0.002), and, at day 60, diet significantly affected the normalized weight in both breeds (LR, P = 0.019; ACD, P = 0.001), with higher values for the Mixed type. LID may help the dam to invest the energy in the growth of her litter, however, the same diet resulted in lower puppies' weights around weaning, compared to the Mixed diet. Although our results should be confirmed with larger numbers of animals and more breeds, they set some points worth to be further investigated. The choice of a limited-ingredient single-protein diet can affect litter weight and weight at weaning. Whether, administered to dams and puppies, it can prevent later pathologies, like chronic gastrointestinal diseases or food allergies, is a field of research deserving full attention.

Challenging the Hypothesis of in Utero Microbiota Acquisition in Healthy Canine and Feline Pregnancies at Term: Preliminary Data.

At present, there are no data on the presence of bacteria in healthy canine and feline pregnancies at term. Here, we investigated the uterine microbiome in bitches (n = 5) and queens (n = 3) undergoing elective cesarean section in two facilities. Samples included swabs from the endometrium, amniotic fluid, and meconium, and environmental swabs of the surgical tray as controls. Culture and 16S rRNA gene sequencing were used to investigate the presence of bacteria. Culture was positive for 34.3% of samples (uterus n = 3, amniotic fluid n = 2, meconium n = 4, controls n = 0), mostly with low growth of common contaminant bacteria. With sequencing techniques, the bacterial abundance was significantly lower than in environmental controls (p < 0.05). Sequencing results showed a species-specific pattern, and significant differences between canine and feline bacterial populations were found at order, family, and genus level. No differences were found in alpha and beta diversities between feto-maternal tissues and controls (p > 0.05). Dominant phyla were Bacteroidetes, Firmicutes, and Proteobacteria in different proportions based on tissue and species. Culture and sequencing results suggest that the bacterial biomass is very low in healthy canine and feline pregnancies at term, that bacteria likely originate from contamination from the dam's skin, and that the presence of viable bacteria could not be confirmed most of the time.

The evolution of dam-litter microbial flora from birth to 60 days of age.

BACKGROUND: Early bacterial colonization in puppies is still a poorly understood phenomenon. Although the topic is of considerable interest, a big gap in knowledge still exists on the understanding of timing and features of neonatal gut colonization. Thence, the purpose of this study was to evaluate the relationship between dam and litter microbial flora, in vaginally delivered puppies, from birth to two months of age. Bacteria were identified using MALDI-TOF, an accurate and sensitive method, and cluster analysis of data provided a new insight on the investigated topic. METHODS: Six dam-litter units of two medium size breeds were enrolled in the study. Vaginal and colostrum/milk samples were collected from dams after delivery and 48h post-partum, while rectal samples were taken from dams and puppies after delivery and at day 2, 30 and 60 (T2, T30 and T60, respectively) post-partum. Bacterial isolation and identification were performed following standard techniques, then the data were analyzed using a new approach based on bacterial genus population composition obtained using a wide MALDI-TOF screening and cluster analysis. RESULTS: Forty-eight bacteriological samples were collected from the dams and 145 from their 42 puppies. Colostrum/milk samples (n = 12) showed a bacterial growth mainly limited to few colonies. Staphylococci, Enterococci, E. coli, Proteus spp. were most frequently isolated. All vaginal swabs (n = 12) resulted in bacteria isolation (medium to high growth). Streptococci, Enterococci, E. coli were the most frequently detected. E. coli, Proteus mirabilis, Enterococcus spp., Streptococcus spp. were often obtained from dams' and puppies' rectal swabs. Clostridia, not isolated in any other sampling site, were rarely found (n = 3) in meconium while they were more frequently isolated at later times (T2: n = 30; T30: n = 17; T60: n = 27). Analysis of the bacterial genus pattern over time showed a statistically significant reduction (P < 0.01) in the heterogeneity of microbial composition in all time points if compared to birth for each dam-litter unit. These results were confirmed with cluster analysis and two-dimensional scaling. CONCLUSION: This novel data analysis suggests a fundamental role of the individual dam in seeding and shaping the microbiome of the litter. Thus, modulating the dam's microbiota may positively impact the puppy microbiota and benefit their health.

Does Bacteria Colonization of Canine Newborns Start in the Uterus?

The assumption that requires the uterus to be a sterile environment to sustain a successful pregnancy has been recently challenged in humans, and is still under debate. The aim of this study was to assess whether bacteria can be isolated from the pregnant uterus and from amniotic fluid and meconium of healthy canine fetuses at term, delivered through cesarean section. Fifteen dams of different breed, age and parity, undergoing either elective (n = 10) or emergency (n = 5) cesarean section after a healthy pregnancy, were included in the study. Swabs for bacterial culture were collected from the uterus, and from amniotic fluid and meconium. Bacteria were isolated from all the sampled sites and materials, irrespective of cesarean type. In most cases, different bacteria were isolated from the different sites. Acinetobacter spp., coagulase-negative Staphylococci and Bacillus spp. were frequently found while Pseudomonas aeruginosa, Micrococcus spp., Moraxella spp., Macrococcus spp., Glutamicibacter spp., Stenotrophomonas spp. and Psychrobacter spp. were only occasionally identified. Our data show that uterus and fetuses may not be sterile in healthy term canine pregnancies.

Effect of sterilization on the canine vaginal microbiota: a pilot study.

BACKGROUND: Surgical sterilization is the most effective method of contraception for dogs. It also prevents pyometra and reduces the risk of mammary tumour development. However, this procedure also has negative effects, such as urinary incontinence. Steroid hormone deprivation following gonadectomy could also affect canine vaginal mucosa conditions and the microbial community colonizing the vaginal tract. This hypothesis was tested by comparing the vaginal cytology and microbial community of two groups of bitches, including 11 in anoestrus and 10 sterilized bitches (post-pubertal sterilization in the last 4 years). Bacteria were identified through metataxonomic analysis, amplifying the V3-V4 regions of 16S rRNA gene, and culturing methods. RESULTS: Vaginal mucosa cytology was suggestive of dystrophic conditions in sterilized bitches, whereas a typical anoestrus pattern with parabasal and intermediate cells was appreciable in anoestrous animals. Metataxonomic analysis revealed large inter-individual variability. Salmonella, Mycoplasma and Staphylococcus were present in moderate quantities in almost all the samples in both groups. Mollicutes (class level) and Tenericutes (phylum level) were commonly present in moderate quantities in anoestrus samples, whereas these microbes were present at high levels in a single sample from the sterilized group. Based on culturing, a higher number of different species were isolated from the anoestrous bitches, and Mycoplasma canis was exclusively identified in an anoestrous bitch. Staphylococcus spp. was the most frequently isolated genus in both groups, followed by Streptococcus spp., and, among gram-negative bacteria, Escherichia spp. and Haemophilus spp. A comparison of the numbers of the most frequently isolated genera of bacteria from vaginal cultures of bitches revealed that Pasteurella and Proteus were the most frequently identified in sterilized animals based on metataxonomic analysis (p-value = 0.0497 and 0.0382, respectively), whereas Streptococcus was significantly and most frequently isolated from anoestrous bitches using culture methods (p value = 0.0436). CONCLUSIONS: In this preliminary investigation, no global patterns of the vaginal bacteria community were noted that characterized the condition of the bitches; however, cytology suggested local modifications. Sterilization after puberty caused minimal alterations in the vaginal microbial community of bitches within 4 years after surgery.

Serological survey of canine parvovirus 2 antibody titres in breeding kennels in northern Italy.

BACKGROUND: Current guidelines recommend parvovirus revaccination of adult dogs no more frequently than every 3 years. The aim of this study was to determine the prevalence of dogs showing protective serum antibody titres against canine parvovirus 2 in breeding kennels in Northern Italy and to assess the effect of time from vaccination and the sex of the dog on antibody titres. The study was carried out on 370 animals of different breeds kept in 33 breeding kennels. Antibodies to canine parvovirus 2 in serum samples were measured with an indirect immunoenzymatic assay validated by the manufacturer in relation to the 'gold standard' haemagglutination inhibition test. The number of months that had elapsed since the last vaccination was calculated for each animal and categorized into the following classes: < 12 months; 13-24 months; 25-36 months; 37-48 months; and > 49 months. RESULTS: The prevalence of 'unprotected' dogs was 4.6%. A satisfactory solid herd immunity was present in the majority of breeding kennels, although some vaccination failures were detected. A significant negative correlation was found between antibody titre and months since last vaccination. Comparable antibody titres were found in the first 3 years after vaccination. Although the antibody titre over time was not affected by the sex of the dog, 'unprotected' females had been vaccinated more recently than males with analogous low titres. CONCLUSIONS: Parvovirus revaccination of adult dogs every 3 years, as currently recommended, is also the appropriate recommendation for breeding kennels. Serological tests could be a useful tool to assess the effectiveness of vaccination.