Antimicrobials can be overused in dog breeding facilities, leading to increased resistance among commensal bacteria. The aim of this study was to investigate antimicrobial use intensity in a professional canine reproduction setting by assessing the resistance profiles of Escherichia coli and Staphylococcus pseudintermedius isolated from healthy breeding bitches, as well as through a questionnaire for breeders. Five bitches from each of 15 kennels in Northern Italy were sampled from the perivulvar skin and rectum, and the susceptibility of the isolated bacteria to a panel of different antimicrobials was determined (Minimum Inhibitory Concentration). Kennels were classified according to their reported intensity of antimicrobial use, and the association between antimicrobial use and resistance in S. pseudintermedius and E. coli was evaluated using Fisher’s exact test (p < 0.05). E. coli exhibited the highest resistance to ampicillin (around 50%), moderate resistance to amoxicillin-clavulanic acid (~24%) and cefalexin (~47%), while resistance to the remaining agents was low (generally ≤10–15%); the prevalence of Extended Spectrum Beta-Lactamase (ESBL)-producing E. coli was 7.76%. More than 82% of S. pseudintermedius strains were resistant to penicillin and ampicillin; mecA-positive methicillin-resistant S. pseudintermedius accounted for 17.65%. Multi-drug resistant (MDR, i.e., acquired non-susceptibility to at least one agent in three or more antimicrobial categories) E. coli and S. pseudintermedius were 22.53 and 41.18%, respectively. MDR E. coli were significantly more frequent in kennels reporting ‘high antimicrobial use’ (p = 0.0160). The generally high levels of acquired resistance suggest extensive antimicrobial use, especially beta-lactams. Breeder responses were partly inconsistent, indicating that complementary strategies should be adopted to monitor antimicrobial use in dog breeding facilities.

Escherichia coli (E. coli) is the bacterium most frequently isolated from canine pyometra. The Endometrial Pathogenic E. coli (EnPEC) show virulence traits that cause tissue damage through adhesion/invasion of cells, and toxin production. Phosphoinositide 3-kinases (PI3K) are intracellular signal transducers with a fundamental role in the innate immune defense. Bacteria that inhibit the PI3K pathway can escape the host defenses. The aim of this study was (a) to investigate the association among the characteristics of E. coli isolated from pyometra (phylogroup, hemolysis, CNF—cytotoxic necrotizing factor, CDT—cytolethal distending toxin), the severity of tissue lesions and the expression of PI3K; (b) to assess the resistance profile. E. coli was isolated from 17/21 uteri, always in pure culture. All E. coli belonged to the phylogroup B2 and 12/17 uteri contained hemolytic strains. CNF was evidenced only in the hemolytic strains. CDT was never detected. Four uteri showed perforation: all contained CNF-positive hemolytic E. coli and PI3K expression was negative. No significant association was found between the characteristics of E. coli and the degree of tissue damage or PI3K expression. The non-hemolytic E. coli tended to show higher resistance toward some antimicrobials. Our results confirm that the highly virulent phylogenetic group B2 is often involved in canine pyometra. PI3K was not expressed in the perforated uteri, suggesting a potential interference of the pathogen with the host cells defenses. Future research should be focused on the mechanism of host defense evasion through the inactivation of the PI3K signaling pathway.

The study investigated whether bacterial seeding occurs in utero in dogs and cats using a multi-technique approach, including 16S rRNA gene sequencing, culture, and fluorescence in situ hybridization (FISH). Healthy pregnant bitches and queens (n = 8) undergoing ovariohysterectomy between 30 and 45 days of gestation were included. Placenta and amniotic fluid samples from two foetuses per dam, along with multiple controls (uterine serosa, sampling table, and surgeon's gloves), were collected and analysed. Bacterial sequences were detected in all foetal samples, with no significant differences in intra- and inter-sample diversity (i.e., alpha and beta diversities, respectively) based on sample type or species. However, the surgeon's gloves showed species-specific differences in bacterial composition. After removing control sequences, significant differences based on foeto-maternal units emerged. Moraxella spp. was cultured from the canine placenta with the highest bacterial load, and Burkholderia cepacia was isolated from two feline placentae. FISH showed low bacterial presence in 50 % of placentae without histological signs of inflammation. No bacterial growth was observed in amniotic fluid or control samples. A multi-technique approach, including multiple controls, is essential in studies involving low-biomass samples, as the results of the present study indicated that contamination could mask real bacterial composition of foetal samples. After decontamination, differences were observed based on the foeto-maternal unit. This study confirmed the presence of bacterial DNA in placental and amniotic fluid samples during mid-term, healthy pregnancies in dogs and cats. Bacterial DNA is not equivalent to living bacteria, hence bacterial DNA fragments in the uterus probably originate from the dam but are unlikely to indicate the presence of a resident foetal microbiome.

The vaginal microbiota of the queen (i.e., female cat) has never been described using culture independent methods. The objectives of the present research were to describe the vaginal microbiota of healthy domestic shorthair queens using both 16S rRNA sequencing and culture, and to assess the effects of age, living environment, and reproductive season on its composition. Thirty queens undergoing elective ovariectomy were included in the study. The vaginal samples were collected just before surgery, from animals under general anaesthesia. Two consecutive mini-swabs were introduced in the queens’ vaginal tract. A preliminary study with 10 healthy queens aimed to negate sampling order's effect. Two consecutive samples for sequencing (5 queens, 10 swabs) and culture (5 queens, 10 swabs) were collected, confirming a match (100% in culture, Bray-Curtis P=0.96 in sequencing). The experiment included 20 queens that were prospectively grouped based on age (prepubertal N = 10, adult N = 10), living environment (indoor N = 10, outdoor N = 10), and time of the year, whether during the reproductive season (N = 10) or during seasonal anoestrous (N = 10). Bacteria were identified through metataxonomic analysis, amplifying the V1-V2 regions of 16S rRNA gene, and through standard culture followed by MALDI-TOF MS.The feline vaginal microbiota is dominated by Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteria. Escherichia-Shigella, Streptococcus, and Pasteurella were the most abundant genera. Although culture underestimated bacterial richness and diversity compared to sequencing, Escherichia and Streptococcus were the most isolated bacteria. No bacterial growth was observed in 15% of samples (N = 3/20), whereas growth of one or two bacterial species was observed in 64.7% (N=11/17) and 35.3% (N=6/17) of cases, respectively. No differences in terms of alpha (Kruskal-Wallis rank sum test P = 0.65) and beta diversity (Bray-Curtis, Unweighted and Weighted UniFrac analyses P>0.5) were observed. Although a difference in alpha diversity based on phylogenetic tree (P=0.02) was detected between indoor and outdoor queens. In conclusion, mixed and monoculture of Escherichia coli, Streptococcus canis, Staphylococcus felis, and Enterococcus spp. are normal findings within the cat vagina. Age and reproductive season do not influence the feline vaginal microbiota, whereas further research is needed to elucidate the role of the living environment.

Pyometra is a bacterial uterine infection that frequently affects intact older bitches. The treatment of choice is ovariohysterectomy, often associated with perioperative antimicrobial therapy. The aim of this retrospective investigation was to evaluate the antibiotic choice at different administration times (pre-surgery, at surgery/during hospitalization, and post-surgery) against complications and outcomes, considering the susceptibility profile of bacteria.The medical records of 51 bitches referred to the Veterinary Teaching Hospital of the University of Turin (January 2021-November 2023) and subjected to ovariohysterectomy and bacteriological examination (bacterial culture and susceptibility tests) of the uterine exudate, were analysed. All animals had a positive outcome without surgical site infections, with a longer hospitalization time in case of peritonitis or sepsis; antimicrobials were administered for an average of 7 days. Comparing the susceptibility of the isolated bacteria towards the antimicrobials administered before, during and after surgery, complete efficacy was observed in 42%, 46% and 50% of cases, respectively. In addition, 5/16 peritonitis cases were treated with an in vitro ineffective antimicrobial, and 30% of the bitches never received a fully effective antibiotic, according to susceptibility tests using the minimum inhibitory concentration method (MIC). Cephazolin resulted the best option for Escherichia coli, the most frequently isolated bacterium. Our study confirms that pyometra has a good prognosis following ovariohysterectomy and antibiotic administration. Monitoring the clinical evolution without changing the antibiotic according to bacterial susceptibility could represent the right choice in uncomplicated cases. Evaluation of outcomes without postoperative antibacterial treatment or with a shorter course would be another topic worth investigating, with the aim to judiciously reduce the use of antibiotics.

IntroductionShelters for stray dogs and cats deserve careful monitoring for zoonotic risk, as they represent a crucial point for prevention and control of infection spread. Data sorting to prioritize zoonotic agents in a geographic area need constant updating, but no regular official programs are ongoing, to allow an efficient risk survey for these animal species. This study aimed to conduct a comprehensive investigation of the prevalence of certain known, potential and emerging zoonoses within the framework of the routine monitoring of dog and cat shelters in North-East Italy.MethodsA total of 389 cats and 257 dogs housed in public veterinary services shelters and feline colonies were included in the present investigation. The animals originated from the provinces of Padua, Venice, Rovigo, Vicenza, Verona, Trento and Bolzano. Serological, molecular and microbiological diagnostics were implemented to investigate the prevalence of Leptospira sp., Brucella canis, Leishmania infantum, dermatophytes, gastrointestinal parasites, antimicrobial-resistant bacteria, Capnocytophaga sp., Bartonella sp., Norovirus, Rotavirus A, Cowpox virus, Mammalian Orthoreovirus, Hepatitis E virus, SARS-CoV-2 and Influenza A virus.ResultsData about some known zoonoses (e.g., serological positivity of Leishmania infantum 25% and Leptospira sp. 44.3% in dogs, and Bartonella henselae 70% in cats) resulted aligned with previous research and recent reports, whereas there was a notable occurrence of some potential, emerging and neglected pathogens (e.g., Mammalian Orthoreovirus 0.38% in dogs and 2.83% in cats). For some other agents (e.g., dermatophytes in dogs and in cats) the prevalence resulted lower than expected.DiscussionThe prevention of the zoonotic risk requires a re-examination of the complex interaction between humans, animals, and environment. This is of particular importance in settings like companion animal shelters, which serve as key sites for disease monitoring and zoonotic risk mitigation. The study highlights the need to monitor and prioritize the zoonotic pathogens, to implement and constantly update surveillance and specific training programs for the kennels’ operators, and management of epidemiological risks.

BackgroundMicrobial colonization during early life has a pivotal impact on the host health, shaping immune and metabolic functions, but little is known about timing and features of this process in dogs. The objectives of this study were to characterize the first step of intestinal microbiota development in naturally delivered canine puppies and to investigate its relationship with the maternal bacterial flora, using traditional culture and molecular analyses. Sixty puppies of two breeds, Appenzeller Cattle Dog (n = 3 dams) and Lagotto Romagnolo (n = 6), housed in the same breeding kennel, were included in the study. Swabs were collected in duplicate (for culture and for molecular analysis) from the dams' vagina and rectum at the end of parturition, from puppies' rectum, before maternal care, and from the environment (floor of the nursery and parturition box).Results93.3% meconium samples showed bacterial growth, limited to a few colonies in 57.0% of cases. High growth was detected for Enterococcus faecalis, which was the most frequently isolated bacterium. The genus Enterococcus was one of the most represented in the dams' rectum and vagina (88.9% and 55.6%, respectively). The genera Staphylococcus, Enterococcus, Escherichia and Proteus were also often isolated in meconium but were usually present in maternal samples as well, together with ubiquitous bacteria (Acinetobacter, Psychrobacter). In the environmental samples, just a few bacterial species were found, all with low microbial load. Additionally, bacteria of the phyla Proteobacteria, Firmicutes, and Actinobacteria were identified in meconium through molecular analysis, confirming the culture results and the early colonization of the newborn gut. Maternal, meconium and environmental samples had similar alpha diversity, while beta-diversity showed differences among families (i.e. a dam and her litter), and association indexes revealed a significant correlation between family members and between sample origin, suggesting a strong contribution of the maternal flora to the initial seeding of the canine neonatal gut and a strong individual dam imprint.ConclusionThis study showed that the meconium of vaginally delivered puppies has its own microbiota immediately after birth, and that it is shaped by the dam, which gives a specific imprint to her litter.

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.

An 8-month-old female Main Coon with a history of recurrent behavioral changes and anorexia was presented with sternal recumbency and depression. Within 5 days, the cat progressively worsened with symptoms of stupor and coma and was euthanized. At post-mortem examination, a solid, grayish infratentorial mass located in the midline rostrally to the cerebellum, was observed. Histologically, highly cellular clusters of small-to-medium undifferentiated cells were intermingled with paucicellular areas with fibrillary eosinophilic (neuropil-like) appearance. Numerous multilayered (ependymoblastic) true rosettes were present. The mitotic activity was frequent (up to 15 mitoses/HPF), involving both undifferentiated cells and rosettes. By immunohistochemistry (IHC), tumor cells were diffusely positive for vimentin, variably for synaptophysin, S-100, and NSE, and focally for NeuN; they were negative for GFAP and CK AE1/AE3. The histological and IHC aspects were consistent with an Embryonal Tumor with Abundant Neuropil and True Rosettes (ETANTR). Embryonal neoplasms of the central nervous system (CNS) are characterized by primitive undifferentiated cells, able to develop toward neuronal, glial, ependymal, and mesenchymal lines. Although extremely rare, juvenile embryonal tumors should be considered in the differentials of CNS disorders in young cats.

Canine parvovirus Type 2 (CPV-2) is a worldwide distributed virus considered the major cause of viral gastroenteritis in dogs. Studies on Italian CPV-2 are restricted to viruses circulating until 2017. Only one study provided more updated information on CPV-2 but was limited to the Sicily region. No information regarding the circulation and genetic characteristics of CPV-2 in Northeast Italy has been made available since 2015. The present study investigated the genetic characteristics of CPV-2 circulating in the dog population of Northeast Italy between 2013 and 2019. The VP2 gene of 67 CPV-2 was sequenced, and phylogenetic analysis was performed to identify patterns of distribution. Phylogenetic and molecular analysis highlighted unique characteristics of Northeast Italian CPV-2 and interestingly depicted typical genetic clustering of the Italian CPV-2 strains, showing the existence of distinct CPV-2 genetic groups. Such analysis provided insights into the origin of some Italian CPV-2 genetic clusters, revealing potential introductions from East European countries and the spread of CPV-2 from South/Central to North Italy. This is the first report that describes the genetic characteristics of recent Italian CPV-2. Tracking the genetic characteristics of CPV-2 nationally and globally may have impact on understanding the evolution and distribution of CPV-2, in particular in light of the current humanitarian emergency involving Ukraine, with the massive and uncontrolled movement of people and pet animals.

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.

Although usually harmless, Staphylococcus spp. can cause nosocomial and community-onset skin and soft tissue infections in both humans and animals; thus, it is considered a significant burden for healthcare systems worldwide. Companion animals have been identified as potential reservoirs of pathogenic Staphylococcus with specific reference to Methicillin Resistant Staphylococcus aureus (MRSA). In this study, we investigated the circulation and the genetic relationships of a collection of Staphylococcus spp. isolates in a family composed of four adults (a mother, father, grandmother, and grandfather), one child, and a dog, which were sampled over three years. The routes of transmission among humans and between humans and the dog werelyzed. The results displayed the circulation of many Staphylococcus lineages, belonging to different species and sequence types (ST) and being related to both human and pet origins. However, among the observed host-switch events, one of them clearly underpinnthroponotic route from a human to a dog. This suggests that companion animals can potentially have a role as a carrier of Staphylococcus, thus posing a serious concern about MRSA spreading within human and animal microbial communities.

A 15-day-old, female, captive Panthera tigris cub was hospitalized after developing severe hyperthermia, depression, and lack of appetite. The clinical condition rapidly worsened, and the tiger cub died in 72 h after the onset of neurological symptoms, septic shock, and multiple organ dysfunction syndrome. The postmortem main gross findings consisted of a severe and diffuse bilateral fibrino-suppurative meningoencephalitis and ventriculitis, mild fibrinous and sero-hemorrhagic polyserositis and cystitis, severe pulmonary edema, and hemorrhages. Microscopically, the meninges, ependyma, and choroid plexuses were diffusely expanded by abundant infiltration of neutrophils and macrophages, with multifocal fibrinous exudation. Histiocytic interstitial pneumonia, fibrinous and neutrophilic polyserositis, and pyelocystitis were also observed. Vascular thrombosis with multifocal vasculitis and vascular necrosis were frequently observed. Aerobic and anaerobic cultures performed on the brain, lungs, intestine, kidneys, and in pericardial effusion reported the presence of Salmonella enterica subsp. enterica serovar Enteritidis. Environmental and nutritional contamination were identified as putative sources of infections. To the best of the authors' knowledge, this is the first report of Salmonella Enteritidis septicemia with meningoencephalitis in a tiger cub, which highlights the need to further investigate the cause of acute perinatal death to reduce the risk of infectious disease outbreaks.

Information on weaning techniques in the tropics is scarce, particularly regarding the long-term effect of temporary early cow-calf separation or restricted suckling. Therefore, we studied the effects of these two handling practices on well-being and performance at 150 days postpartum in fifteen zebu cow-calf pairs randomly assigned to three treatments. Continuous suckling (CS) where calves remained with their dams from birth to weaning; restricted suckling (RS) calves were allowed to suckle 30 min/day from Day 34 until weaning at Day 150 and kept separated the rest of the time; temporary separation (TS) calves were separated for 72 h from their dams from Day 33 to 36 but remained with their dams the rest of the time. Blood samples and behavioral data were collected on Days 32-36 (1st period) and 149-153 (2nd period). In the 1st period, a greater percentage of RS and TS calves were observed dose to the fence line (<10 m) that separated them from their dams (P <0.0001) and vocalized more than CS calves (P < 0.0001), while in the 2nd period, RS calves had the highest cortisol concentration and vocalization rate (P <0.05). Similarly, during the 1st period, a greater percentage of RS and TS cows were observed close to the fence line than CS cows (P < 0.0001), with TS cows vocalizing the most (P - 0.001). In the 2nd period, RS cows had greater cortisol concentration than TS (P = 0.037) and CS cows (P = 0.003). More TS and CS cows than RS were observed close to the fence line (P = 0.03 and P = 0.05). On Day 150, TS calves and cows vocalized more than RS and CS animals (P < 0.0001). Before calf-cow separation, 27 out of 45 cows were cycling (CS - 10; RS = 6; TS = 11). After separation, 12 of the remaining 18 cows resumed ovarian activity (CS = 3; RS = 5; TS = 4), and all cows were cyding after estrous synchronization treatment. The pregnancy rate was similar between CS, RS, and TS (60, 53, and 60% respectively). In conclusion, temporary separation increased calf distress response to definitive weaning even four months later, while restricted suckling seemed to reduce it. (C) 2020 The Authors. Published by Elsevier Inc. on behalf of The Animal Consortium.

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.

A 5-year-old healthy pluriparous pregnant Flat-coated Retriever bitch was monitored by ultrasound on post-ovulation days 30 and 57: no deviation from normality picture were observed. On day 60, one of the three most caudal fetuses showed ultrasonographic signs of fetal anasarca: subcutaneous edema and anechoic fluid in thoracic and abdominal cavities. There was an increased volume of extra-fetal fluids. On day 64 a Cesarean section was performed and one of the seven pups that were delivered, a female, showed generalized subcutaneous edema and died soon after birth. She weighed 660 g, compared to a mean of 472 g for the other 6 normal fetuses. A total of 295, 40 and 27.5 mL of liquid were collected from subcutaneous tissue, abdominal and thoracic cavity, respectively. Liver showed sub-glissonian necrotic areas. Molecular analyses with PCR method for canine Herpesvirus, Parvovirus, Adenovirus, Leptospira interrogans, Chlamydia spp., Neospora caninum and Toxoplasma gondii from pools of organs (spleen, kidney and brain) and pleural effusion tested negative. This is the first reported case of fetal anasarca with a rapid onset diagnosed on day 60 post-ovulation just three days after observing a normal ultrasonographic pattern in Flat-coated Retriever. Ultrasonographic diagnosis of fetal anasarca is of primary importance when assisting parturition, due to its inherent risk of dystocia. Ultrasonographic monitoring in the immediate prepartum period may be useful in all breeds as it may help to detect ultrasonographic alterations occurring right before term such as anasarca.

BackgroundSurgical 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.ResultsVaginal 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).ConclusionsIn 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.

Two studies were performed to determine (1) the role of endogenous progesterone (P4) concentration during the follicular, luteal and mid gestation phases of zebu type cattle (B. indicus) in the response to the exit velocity (EV) and flight distance (FD) tests and (2) the response of non-cyclic female beef heifers (B. taurus) to the same tests during treatments with P4 and immediately after P4 withdrawal. In Study 1, 31 Brahman cows; 17 pregnant and 14 cycling were used. Previous to the study, all cycling animals were treated with 2 PGF2 alpha injections, 11 days apart. Exit velocity and FD were performed in cycling cows, at days 11 and 21 of their natural cycle after the induced oestrous (day 0), and at day 125.9 +/- 1.8 days (range: 117 to 139)] in the pregnant cows. The EV was slower and the FD was shorter in pregnant cows than in the cows in their luteal phase (EV: 0.83 +/- 0.07 m.s(-1) vs. 0.55 +/- 0.06 m.s(-1): P = 0.007; FD: 3.8 +/- 0.4 m vs. 2.6 +/- 0.5 m: P = 0.05 for cows in their luteal phase and gestating cows respectively). In Study 2, 24, 9-m old Hereford-Aberdeen Angus cross heifers were used. Twelve heifers were treated with P4 intravaginal releasing devices (CIDR, 1.38 g of P4, Pfizer, Auckland, NZ) for 5 days (P4G), and other 12 heifers remained as untreated controls (CG). The EV and FD were assessed in both groups before inserting the CIDR, 1 and 5 days after the treatment began, and 16 h after devices removal. Before placing the CIDRs there were no differences in the EV or FD. One day after CIDRs were inserted, FD was shorter in P4G than in CG heifers (5.0 +/- 0.8 m vs. 7.0 +/- 0.6 m, P = 0.04). Sixteen h after CIDRs removal the EV was greater in P4G than in CG heifers (2.2 +/- 0.5 m.s(-1) vs 1.2 +/- 0.1 m.s(-1), P = 0.05), and FD tended to be greater in P4G than in CG heifers (4.1 +/- 0.5 m vs. 3.2 +/- 0.4 m, P = 0.09). In conclusion, female zebu type cattle (B. indicus) reacted less during mid-gestation than during their mid-luteal phase when they were subjected to two tests to measure temperament traits. Additionally, female beef calves (B. taurus) also responded less to those tests when they were treated with P4, and they responded more immediately after P4 withdrawal.