Karina Mallardo

Comparison of the cytobrush, cottonswab, and low-volume uterine flush techniques to evaluate endometrial cytology for diagnosing endometritis in chronically infertile mares.

Endometritis is the most important cause of infertility in barren mares. The quick method of endometrial cytology (EC) has a relatively high reliability in diagnosing endometrial inflammation in the mare. For reliable cytological results, a collection technique that yields many well-preserved cells representative of a large uterine surface area without causing harm to the reproductive tract is required. The aim of the study was to compare three usually employed techniques for collection of endometrial and inflammatory cells (guarded cotton swab, uterine lavage, and cytobrush) in chronically infertile mares. Twenty Standardbred mares were used. In each mare, samples for EC were collected, first by a cotton swab (DGS), then by a cytobrush (CB), and finally by low volume flush (LVF). The slides were stained using the Diff Quick stain. The following parameters were assessed for each tested technique: background content of the slides; quality of the cells harvested; total cellularity; neutrophils; ratio PMN/uterine epithelial cells; inflammatory cells; vaginal epithelium cells. Categorical variables were compared using contingency tables and Pearson Chi-square tests, whereas continuous variables were compared using one-way analysis of variance (ANOVA); P<0.05 was considered significant. Samplings by DGS and CB resulted easy and quick to perform via a single operator in all cases. LVF was performed easily, but required the presence of 2-3 players and took more time. The background content of the slides prepared by DGS appeared proteinaceous, slides prepared by LVF appeared contaminated by red blood cells or debris, whereas slides prepared by CB appeared clear. All smears showed a good total cellularity. The CB yielded significantly more cells (P<0.0001) than DGS and LVF. The DGS produced significant more cells than LVF (P<0.0001). The DGS produced significantly more (P=0.003) intact cells than CB and LVF. Distorted cells were significantly (P=0.001) more frequent in smears by LVF. The CB harvested significantly (P=0.009) more fragmented cells. CB and LVF produced significantly (P<0.0001; P=0.02) more PMNs/HPF than DGS. In smears collected by LVF the proportion of PMNs/uterine epithelial cells was significantly (P=0.0062; P=0.0023) higher than in smears by CB and DGS. CB collected a significantly higher (P=0.0011) proportion of PMNs than DGS. Acute endometritis was diagnosed in 50% (10/20) of the mares by DGS cytological samples, 25% (5/20) by CB, and 75% (15/20) by LVF. Inflammatory cells other than PMN (lymphocytes, macrophages, eosinophils) were collected exclusively by CB method. Epithelial cells from the vagina were only detected in LVF slides. The agreement of the diagnosis of endometritis between the three techniques of collection and between the different criteria adopted to evaluate smears obtained with the same technique was poor (k≤0.3). In conclusion, results show that cytobrush and flush specimens were superior in all parameters to cotton swab smears. Even though the cytobrush technique requires specialized equipment, sample collection by this method was easier, more consistent, and quicker than the lavage method, indicating that the brush would be the preferred collection method for use on field in the mare. More studies are needed to establish criteria for interpretation of inflammation in the mare on cytobrush samples.

Methicillin-Resistant Staphylococci Isolated from Healthy Horses and Horse Personnel in Italy

Methicillin-resistant staphylococci (MRS) were isolated from nasal swabs of 56 of 159 (35.2%; 95% confidence interval [CI]: 27.9–43.2%) healthy horses. Two nasal swabs were collected from each horse; 43 of 159 (27%; 95% CI: 20.5–34.8%) of the cohort were colonized by MRS strains in 1 nostril, while in the remaining 13 of 159 (8.2%; 95% CI: 4.6–13.9%), different or identical MRS strains were isolated in both nostrils. Of the 29 humans in close contact with the horses tested, 4 (13.8%; 95% CI: 4.5–32.6%) were found to be carriers of MRS. All isolates were coagulase negative with the exception of 2 coagulase-positive MRS strains, Staphylococcus aureus and Staphylococcus pseudintermedius, both isolated from horses. To assay the methicillin resistance, a susceptibility test to oxacillin with standardized disk diffusion method, a PBP-2a latex agglutination test, and a methicillin resistance gene (mecA) polymerase chain reaction assay were performed. Pulsed-field gel electrophoresis patterns of isolates from horses and humans in close contact with the horses revealed similarity. The results suggest evidence of transmission between animals, from animals to humans, and vice versa.

Effect of dietary mannan‐oligosaccharides on in vivo performance, nutrient digestibility and caecal content characteristics of growing rabbits

To evaluate the effect of mannan-oligosaccharides (MOS) on in vivo performance, nutrient digestibility, fermentation characteristics and caecal microbial populations of rabbits, 144 thirty-five days old hybrid Hyla were equally divided into three groups, one of which was fed the same diet without additives (control group), one with antibiotics (colistin sulphate, 144 mg/kg; tylosin, 100 mg/kg; oxytetracyclin, 1000 mg/kg) and one with MOS (1 g/kg of diet). Mortality rate, live weight, feed intake and feed conversion ratio were recorded up to 62 days of age. At 60 days nutrient digestibility was measured by acid insoluble ash method. The caecal content of 10 rabbits per group was collected at 62 days and analysed for volatile fatty acids production, ammonia content and microbial count. Rabbits from the control group had a significantly (p < 0.01) lower body weight at 62 days (1638.9 g vs. 1779.4 g and 1862.5 g, respectively for the control, MOS and antibiotic groups) while the antibiotic group showed a higher (p < 0.05) feed intake than the control group (127.9 g/day vs. 109.3 g/day). Rabbits from the MOS group had a higher apparent digestibility of cellulose (34.27% vs. 29.61% and 27.49%, respectively for the MOS, control and antibiotic groups) and, as a consequence a higher level of acetate in the caecal content (39.93 mmol/l vs. 34.21 mmol/l and 23.09 mmol/l, respectively for the MOS, control and antibiotic groups). Caecal microflora of the MOS group rabbits also had a higher fermentative activity in respect of protein source, as demonstrated by the higher productions of branched chain fatty acids. MOS and antibiotics significantly reduced the colonies of Coliformis (2.32 vs. 3.20 vs. 2.40 logCFU/g, respectively for the MOS, control and antibiotic groups, p < 0.01). Mannan-oligosaccharides at 1 g/kg of diet can be used as an alternative to antibiotics during the rabbits growth period.

Evaluation of a fluorescence polarization assay for the detection of serum antibodies to Brucella abortus in water buffalo (Bubalus bubalis)

The fluorescence polarization assay (FPA) was evaluated for the serological diagnosis of brucellosis in water buffalo (Bubalus bubalis) in southern Italy. This assay uses O-polysaccharide prepared from Brucella abortus lipopolysaccharide conjugated with fluorescein isothiocyanate as a tracer. It has many methodological advantages over older, more established tests and can be performed in a fraction of the time. Sera from 890 buffalos from the Campania Region - 526 positive sera and 364 negative sera according to the complement fixation test (CFT) - were evaluated in this study. All samples were tested with the Rose Bengal test (RBT), CFT, and FPA in parallel and in blind fashion. Sensitivities (Sn) were 84.5% and 92.6%, and specificities (Sp) were 93.1% and 91.2% for RBT and FPA, respectively, relative to CFT. Finally, receiver operating characteristic (ROC) analysis suggested a cut-off value of 117 millipolarization (mP) units. On the whole, these results suggested that FPA might replace RBT in the diagnosis of buffalo brucellosis for its better performance relative to CFT, its adjustable cut-off useful in different epidemiological situations, its reliability, ease of performance, and for its potential application in field and high-throughput laboratories.

Shedding of Brucella abortus rough mutant strain RB51 in milk of water buffalo (Bubalus bubalis).

The objective of this study was to determine if Brucella abortus rough mutant strain RB51 (SRB51) is eliminated in buffalo milk. Thirty Brucella-free female buffaloes were used in this study: ten 4-5 years old were inoculated with the triple of the recommended calfhood dose of SRB51 by subcutaneous route, ten 2-3 years old at the first lactation were previously vaccinated twice as calves with triple the recommended calf dose of RB51, while five 4-5 years old and five 2-3 years old not vaccinated Brucella-free female buffaloes served as controls. Milk samples were taken aseptically on a daily basis for the first 30 days and weekly for the second and third months. The samples were inoculated on selective media for isolation of SRB51 and incubated for 11 days. Moreover, PCR analysis was also performed directly on milk samples. SRB51 was isolated from milk samples only during the first week post-vaccination while RB51 DNA was detected during the first week till the fourth week post-vaccination only in water buffaloes vaccinated as adults. The identification of Brucella RB51 in milk samples, strongly suggests that this Brucella vaccine could be excreted in milk of buffalo cows vaccinated as adults, while our data demonstrate that the vaccine is safe for use in buffaloes vaccinated as calves in which it was not excreted in milk.

A comparative evaluation of methicillin-resistant staphylococci isolated from harness racing-horses, breeding mares and riding-horses in Italy

OBJECTIVE To investigate the prevalence of methicillin-resistant staphylococci (MRS) which is a potencial risk factor of transmission between animals and humans in different types of horses (harness racing-horses, breeding mares and riding-horses) and to compare the antimicrobial resistance of the isolates. METHODS A total of 191 healthy horses, housed at different locations of the Campania Region (Italy), were included in the study. Nasal swab samples were collected from each nostril of the horses. The mecA gene was detected by a nested PCR technique. Antibiotic susceptibility was tested for each isolate. RESULTS MRS was isolated from nasal samples of 68/191 (35.6%; 95% CI: 28.9%-42.9%) healthy horses. All isolates were coagulase-negative with the exception of two coagulase-positive MRS strains, identified as Staphylococcus aureus and Staphylococcus pseudintermedius, 2/83 (2.4%; 95% CI: 0.4%-9.2%). Interestingly, both coagulase-positive MRS isolates were from harness racing-horses. These horses also presented a significantly higher positivity for MRS (53.3%; 95% CI: 40.1%-66.1%) than the breeding mares and riding-horses groups. Antibiotic susceptibility testing showed difference between isolates due to different origins except for an almost common high resistance to aminopenicillins, such as ampicillin and amoxicillin. CONCLUSIONS It can be concluded that harness racing-horses may act as a significant reservoir of MRS as compared to breeding mares and riding-horses.

Effect of spray application of Lactobacillus plantarum on in vivo performance, caecal fermentations and haematological traits of suckling rabbits

Two days before kindling, 228 New Zealand White rabbit does were homogeneously divided into two groups (114 does per group) and fed the same diet. After delivery, the litters were equalized to 8 pups. From 1 to 35 days of age (weaning), the control group (CONT) did not receive any treatment while in the experimental group (LAC) the nests were sprayed with a commercial product containing lyophilized Lactobacillus plantarum dissolved in water (12 g/L). L. plantarum was sprayed on the litters (5 mL per rabbit) once a day during seven consecutive days after delivery. After one week of rest, the treatment was repeated for another week according to the same experimental protocol. Mortality rate, recorded on all the litters (912 rabbits per group) was significantly lower in the LAC group (9.9 vs 17.2%; P<0.05). There were no significant differences in in vivo performance of the 24 litters per group, and rabbits of both groups reached a similar weight at weaning (938 vs 932 g for LAC and CONT groups, respectively). Rabbits from the LAC group showed fermentative activity of caecal microflora (total volatile fatty acids 24.8 vs 14.5 mmol/L; P<0.01) and higher percentage of lymphocytes (73.7 vs 63.9% of total white blood cells; P<0.05). Among the microflora population of rabbit caecal content from the LAC group, it was possible to identify L. plantarum (1.25×106 CFU/g). It might be supposed that the changes in caecal microflora can affect our results and improve the sanitary status of Lactobacillus-sprayed rabbits in the period 1–35 days of age.

Antibiotic susceptibility of haemolytic E. coli strains isolated from diarrhoeic faeces of buffalo calves

We investigated the antibiotic resistance of a collection of 94 strains (55.6%) of haemolytic Escherichia coli (E. coli) isolated in 169 diarrhoeic faecal samples from buffalo (Bubalus bubalis) calves. Bacterial colonies on McConkey and EHLY agar that showed the morphology of E. coli were biochemically tested and then, furtherly classified as haemolytic, using PCR-based assays for enterohemorrhagic E. coli hly (hlyEHEC) virulence gene. When the pathogenic isolates were tested for their susceptibility to 13 different antibiotics, each tested isolate was found to be highly resistant to more than three antibiotics. In fact, absolute resistance (100% of resistance) to penicillin G, lincomycin, neomycin, was detected. Amoxicillin/clavulonic acid and ampicillin were found to be moderately effective against the majority of isolates (46.8% of resistance). Thirty-two (34%) of the haemolytic E. coli strains were phenotypically resistant to tetracycline. None of the isolated strains of E. coli was resistant to colistin sulfate. We conclude that the high prevalence of antimicrobial resistance detected in our study is a source of concern, and cautious use of antibiotics in food producing animals is highly recommended.

Use of mannan oligosaccharides during "post-weaning enteric syndrome" in rabbits: effect on in vivo performance from 35 to 60 days

Abstract Four groups, each consisting of 684 weaned (35 days) hybrid Hyla rabbits were fed ad libitum the same commercial concentrate supplemented, respectively, with antibiotics (AGP group: colistin sulphate 144 mg/kg; tylosin 100m g/kg and oxytetracyclin 1000 mg/kg) or with mannan oligosaccharides (MOS) at 0.5 (group MOS_0.5), 1.0 (group MOS_1.0) and 1.5 g/kg (group MOS_1.5). Up to 60 days, mortality rate was recorded daily. For each group, 64 rabbits were controlled weekly for live weight to calculate daily weight gain (DWG). Feed intake (and, by consequence, feed conversion ratio) was measured, weekly, per group. No differences were observed for live weight during the trial, while DWG showed an alternate trend, in general, significantly lower for AGP group, exclusive of the third week (49-56 days). Exclusive of the first week of the trial feed intake was higher for AGP than the other groups and the feed conversion ratio was more favourable for MOS groups. Mortality rate was significantly higher (34.2%) in AGP groups. The lowest mortality was recorded in MOS_1.0 group (7.75%).

Safety of B. abortus rough mutant strain RB51 administration in Buffalo cows

Abstract The objective of this study was to determine if B. abortus rough mutant strain RB51 is eliminated in Buffalo milk. Five milk buffaloes were inoculated with the triple of the recommended calfhood dose (3.0 – 10.2 x 1010 cfu/ml) of B. abortus RB51 strain by subcutaneous route in the right axillary region. Milk samples were taken aseptically on a daily basis for the first 30 days and weekly for the second and third months. The samples were inoculated on Brucella Medium Base (BMB) (Oxoid) and Rifampin Brucellae Medium (RBM) and incubated under 10% CO2 at 37°C for 10 days. The suspicious colonies were recultured in BMB and RBM. PCR analysis was also performed on milk samples. There were no isolations of bacteria with characteristics of Brucella from any of the milk samples collected during 90 days of the study. However Brucella RB51 DNA was detected on day 2 and 3 post vaccination in one buffalo cow and on day 21 post vaccination in another buffalo cow. It was concluded that the strain used at this dose wasn’t eliminated by milk in Buffaloes inoculated during lactation, however PCR positive results underline the necessity of milk pasteurization in order to minimize food-chain exposure.