Feline bartonellosis associated with some clinicopathological conditions in a veterinary hospital in Selangor, Malaysia.

A cross-sectional study was conducted to determine the prevalence of feline bartonellosis and the associated clinicopathological findings in cats presented to the University Veterinary Hospital, Selangor, Malaysia from 2013-2014. Out of 284 cats examined, Bartonella DNA was detected in 48 (16.9%) cats using a specific polymerase chain reaction (PCR) assay targeting the internal transcribed spacer of Bartonella species. Bartonella henselae strain Houston was identified through BLAST analyses of randomly selected amplicons. Univariable analysis showed significant association of feline bartonellosis with cats < 2 years of age (OR 1.37, 95% CI 0.982-1.927, p = 0.036) and those presenting with ocular discharge (OR 3.211, 95% CI 1.422-7.248, p = 0.003). Significant associations of neutrophilia (OR 2.244, 95% CI 1.131-4.452, p = 0.019) and monocytosis (OR 2.476, 95% CI 1.154-5.312, p = 0.017) with bartonella infection in cats were observed. This study reports for the first time the prevalence (approximately 17%) of feline bartonellosis in Malaysia and highlights several clinicopathological factors associated with the disease.

Feline bartonellosis associated with some clinico- pathological conditions in a veterinary hospital in Selangor, Malaysia

A cross-sectional study was conducted to determine the prevalence of feline bartonellosis and the associated clinicopathological findings in cats presented to the University Veterinary Hospital, Selangor, Malaysia from 2013–2014. Out of 284 cats examined, Bartonella DNA was detected in 48 (16.9%) cats using a specific polymerase chain reaction (PCR) assay targeting the internal transcribed spacer of Bartonella species. Bartonella henselae strain Houston was identified through BLAST analyses of randomly selected amplicons. Univariable analysis showed significant association of feline bartonellosis with cats < 2 years of age (OR 1.37, 95% CI 0.982–1.927, p = 0.036) and those presenting with ocular discharge (OR 3.211, 95% CI 1.422–7.248, p = 0.003). Significant associations of neutrophilia (OR 2.244, 95% CI 1.131–4.452, p = 0.019) and monocytosis (OR 2.476, 95% CI 1.154–5.312, p = 0.017) with bartonella infection in cats were observed. This study reports for the first time the prevalence (approximately 17%) of feline bartonellosis in Malaysia and highlights several clinicopathological factors associated with the disease.

Efficacy of four density gradient separation media to remove erythrocytes and nonviable sperm from canine semen.

The objective was to compare four commercially available density gradient centrifugation (DGC) media (ISolate [Irvine Scientific; Santa Ana, CA, USA], Percoll [Pharmacia; Uppsala, Sweden], PureCeption [SAGE In-Vitro Fertilization, Inc.; Trumbull, CT, USA], PureSperm 100 [Nidacon International AB; Molndal, Sweden]) for their ability to separate viable, motile sperm from contaminant nonviable (immotile and/or dead) sperm and red blood cells (RBC). Pooled sperm-rich fractions from four healthy dogs were assessed using Spermvison SAR (Minitube of America). For this, 1 mL of the blood/sperm admixture was pipetted over 4 mL of DGC media: 50%/90% ISolate (Irvine Scientific), 45%/90% Percoll (Pharmacia), 40%/80% PureCeption (SAGE In-Vitro Fertilization, Inc.), and 40%/80% PureSperm 100 (Nidacon International AB). After centrifugation, five 1-mL fractions (A, B, C, D, and E) and the sperm pellet (bottom fraction F) were separated. Sperm morphology and red blood cell/sperm ratio (RBC/S) per fraction were determined on stained slides. All DGC media separated RBC from sperm; the highest red blood cell/sperm ratio was present in ISolate (Irvine Scientific) and Percoll (Pharmacia) fraction A (29.4±29.7 and 28.2±20.8, respectively), and in fractions A and B of both PureCeption (SAGE In-Vitro Fertilization, Inc.) (37.0±22.8 and 39.6±24.3, respectively) and PureSperm 100 (Nidacon International AB) (25.2±5.9 and 23.0±3.9, respectively). The fractions with the highest total sperm recovery, motile sperm recovery, as well as overall motility were ISolate (Irvine Scientific) and Percoll (Pharmacia) fraction D (33.9±29.4%; 40.99±27.9%; 71.2±21.8% and 36.4±14.5%; 39.3±15.8%; 88.6±2.3%, respectively), and for PureCeption (SAGE In-Vitro Fertilization, Inc.) and PureSperm 100 (Nidacon International AB), the sperm pellet, fraction F (78.8±28.3%; 88.0±17.4%; 70.2±11.1% and 73.1±21.0%; 75.4±24.6%; 80.6±17.1%, respectively). In the pellet for PureCeption (SAGE In-Vitro Fertilization, Inc.), more sperm and motile sperm were recovered than in ISolate (Irvine Scientific) and Percoll (Pharmacia) fractions D (P<0.0163). Therefore, DGC media should be considered for canine semen purification when contaminated with blood or when separation of motile versus immotile sperm is needed.

The effects of endometrial scarification on uterine steroid receptors, bacterial flora and histological structure in the bitch.

Following laparotomy, the endometrium of six nulliparous Beagle bitches was scarified at the base of one uterine horn during early metoestrus, when the peripheral plasma P(4) concentration was >10 ng/ml; intrauterine swabs were taken at the same time for bacteriological culture. Twenty-one days later, a bilateral ovariohysterectomy was performed and segments of the scarified and non-scarified parts of the tubular genital tract removed; at the same time, swabs were taken from the uterine lumen. Tissue samples were collected and examined for histopathological structure, and the presence of nuclear oestrogen (ER) and progesterone (PR) receptors using an immunocytochemical method. The immunoreactivity was scored semiquantitatively, incorporating both the intensity and distribution of specific staining of the receptors using a simplified histoscore (H-score). All uterine swabs were sterile, and in three of the six bitches there were noticeable changes with distension of the uterine lumen with secretions and debris and distension of the endometrial gland ducts of the scarified uterine segment. There were no statistically significant differences in the H-scores of ER or PR between scarified and non-scarified segments, except for PR H-scores in the glandular epithelium where the values for the scarified were significantly higher than for the non-scarified endometrium (mean+/-S.E.M. is 129.9+/-22.8 versus 59.5+/-12.6; P<0.05). Thus, trauma can modify the structure of the endometrium and the characteristics of the PR. Whether changes in PR expression are involved in the pathogenesis of CEH/pyometra in the bitch could not be ascertained from this study.

Cystic endometrial hyperplasia/pyometra in dogs: a review of the causes and pathogenesis.

In this review the roles of endogenous and exogenous steroid hormones, steroid receptors, spontaneously occurring and experimentally induced genital tract infection, and endometrial trauma in the cause and pathogenesis of cystic endometrial hyperplasia/pyometra in bitches are considered. It is postulated that intrauterine bacteria, which ascend from the vagina during pro-oestrus and oestrus, induce the disease during metoestrus by acting on the progesterone-primed endometrium directly via toxin production, or indirectly by the release of inflammatory mediators. The lesion can be produced by endometrial trauma in a sterile uterus without the systemic effects associated with the disease. There is no association with premature or increased progesterone concentrations at the time that the disease is normally diagnosed in mid- to late metoestrus; however, such aberrant endocrine changes cannot be excluded from involvement in the pathogenesis of this disease, as there are no data from bitches preceding the onset of clinical signs. Expression of oestrogen and progesterone receptors are modified by endogenous steroid hormones but there is no clear evidence that changes in these receptors are involved in the pathogenesis of the disease; receptor expression can be modified by exogenous hormones.

Mitochondrial DNA deletion mutation levels are elevated in ALS brains.

This study was performed to explore the potential role of mitochondrial DNA mutations in the neurodegenerative process in amyotrophic lateral sclerosis (ALS). Using a semi-quantitative assay, a common mitochondrial DNA deletion mutation (mt DNA4977) was assayed in brain tissue obtained from six sporadic ALS patients and compared to four controls. In each brain, levels of this mutation were measured in a brain region affected by neurodegeneration, the motor cortex (Brodmann area 4), and compared to the temporal cortex (Brodmann area 17). In the ALS but not control brains, levels of mt DNA4977 were an average of more than 30-fold (range 15-250) higher in Brodmann area 4. These results support and extend those of previous studies implying that mitochondria may participate in the neurodegenerative process in ALS.

Oestrogen and progesterone receptors in the uterine wall of bitches with cystic endometrial hyperplasia/pyometra.

Fresh samples of uterine wall and peripheral plasma were obtained from 13 bitches of different breeds when they were treated for cystic endometrial hyperplasia/pyometra by ovariohysterectomy. The plasma samples were assayed for progesterone and 17beta-oestradiol, and the tissue samples were examined histopathologically and for the presence of oestrogen and progesterone receptors by an immunocytochemical method. The immunoreactivity was scored semiquantitatively, taking into account both the intensity and distribution of the specific staining of the receptors, by using a simplified scoring system. The scores for both oestrogen and progesterone receptors in the glandular epithelium were much lower, and in the endometrial stroma a mean (sd) score for oestrogen receptors was 46.0 (44.7) compared with 0 in comparable endometrial tissues from normal bitches at the same stage of the oestrous cycle.

The influence of exogenous steroid hormones on steroid receptors, uterine histological structure and the bacterial flora of the normal bitch.

Oestrogen (ER) and progesterone (PR) receptors have been shown to vary in both concentration and distribution during the oestrous cycle of the bitch, influenced by the normal changes in endogenous reproductive hormones. The influence of exogenous steroid hormones on steroid receptors and the histological structure of the uterus was studied in two groups of parous Beagle bitches. Group A (n = 6) were treated with progesterone (P4) in oil i.m. (3 mg/kg) in late metoestrus on the day that peripheral plasma P4 concentrations were first identified as <10 ng/ml, and subsequently once weekly on three other occasions. Group B (n = 6) were treated with a single i.m. injection of MPA (50 mg, 4.2-5.6 mg/kg) following the same protocol. Full-thickness uterine wall biopsies were obtained from the mid part of one horn 2-7 days after the last (fourth) injection of P4 or MPA. During the subsequent oestrus, when peripheral plasma P4 concentrations were between 8 and 10 ng/ml, each bitch in both groups (n = 12) received a single injection of oestradiol benzoate (ODB) in oil i.m. (7.5 mg, 0.63-0.84 mg/kg). All bitches had an ovariohysterectomy 7 days later. Full-thickness uterine wall samples were obtained from the mid part of the intact horn and other parts of the uterus. Swabs were taken from the uterine lumen for bacteriological examination; all were sterile. Tissue samples were sectioned and examined for evidence of lesions, and stained for ER and PR receptors using an immunocytochemical method. The immunoreactivity was scored semiquantitatively, incorporating both the intensity and distribution of specific staining of the receptors using a simplified histoscore (H-score). At the time of ovariohysterectomy, fluid had accumulated in the isolated section of the uterine horn distal to the point of biopsy; the volume was greater in the MPA-treated bitches. There was also evidence in some sections of histological changes in the endometrium. Variations in the expression of both ER and PR were seen between bitches, which may have been due to some not being in mid-metoestrus at the time of treatment. In general, ER scores were low after P4 and MPA treatment, but following ODB there was a significant (P<0.05) increase in ER expression in all parts of the endometrium. PR scores were zero in the glandular epithelium of all 12 bitches after P4, MPA and ODB treatment, whereas in the other parts of the endometrium they were generally moderate to high. Following treatment with ODB, PR generally increased in the three regions of the endometrium where PR were present. The study shows that ER and PR distribution and expression in the endometrium of bitches can be modified by P4, MPA and ODB, with evidence of individual variation.

Uterine bacterial flora and uterine lesions in bitches with cystic endometrial hyperplasia (pyometra).

Thirty-eight complete genital tracts were obtained fresh from local veterinary practices after bitches had been ovariohysterectomised for the treatment of cystic endometrial hyperplasia (pyometra). The aerobic and anaerobic culture of swabs obtained aseptically from the uterine lumen demonstrated the presence of Escherichia coli in 28 of the 29 cases from which bacteria were isolated and, from 25, a pure culture was obtained. Sixteen different serotypes were identified from 24 genital tracts; 032K+ CNF+ was isolated from four of 16 cases and 04K-; from three of 16 cases. Cytotoxin necrotising factors (CNFs) were identified in seven of the 16 serotypes; they included 02, 04, 06, 022, 025, 045 and 075, which except for 06 and 045 were haemolytic. Strains 04K- and 07K+ completely destroyed the integrity of the endometrial epithelium whereas 088K+ and 088K- had the least effect. The scores for the reduction in the integrity of the endometrial epithelium and the degree of infiltration of inflammatory cells were greater in the presence of CNF 1.

Immunocytochemical localization of oestrogen and progesterone receptors in the uterus of the normal bitch during oestrus and metoestrus.

Frozen uterine biopsy samples obtained from eight bitches during oestrus and metoestrus were used to develop immunocytochemical methods for identifying steroid receptors. The use of anti-steroid receptor monoclonal antibodies DAKO ID5 and NCL-PGR resulted in reliable detection of oestrogen and progesterone receptors, respectively. The distribution and heterogeneity of both oestrogen receptor and progesterone receptor expression varied during the oestrous cycle. During oestrus, steroid receptor expression was greatest as indicated by a distinct and homogeneous distribution of nuclear staining within cells of the endometrial glandular and luminal epithelium and myometrium. In contrast, during metoestrus there was an overall reduction in both the number and intensity of staining of nuclei. Distribution and intensity of nuclear staining was heterogeneous. This reduction was obvious within the luminal epithelial cells where nuclear staining was negligible compared with that of the glandular epithelial cells. This method could be used for further receptor studies, particularly in those diseases mediated by steroid hormones such as cystic endometrial hyperplasia and mammary neoplasia.

Observations on semen quality of dogs in the tropics.

Semen was obtained from eight mongrels and 17 purebred dogs kept in a tropical climate. The mean spermatozoal output per ejaculate was 303.6 +/- 138.2 and 602.9 +/- 466.2 million, respectively. Values for individual progressive motility, live and abnormal spermatozoa were comparable with those obtained in temperate regions. The type and frequency of spermatozoal abnormalities are illustrated.