RHDV2 epidemic in UK pet rabbits. Part 2: PCR results and correlation with vaccination status.

OBJECTIVE: To report PCR results and vaccination status of rabbits with rabbit haemorrhagic disease following an investigation into sudden or unexpected death. MATERIALS AND METHODS: PCR testing for RHDV2 and RHDV1 was performed on rabbit liver samples at two laboratories. Laboratory A reported results as positive or negative; Laboratory B reported results quantitatively as RNA copies per mg liver, categorised as negative, inconclusive or positive. The vaccination status of rabbits with both histopathological features of rabbit haemorrhagic disease and positive PCR test results were collated. RESULTS: PCR results matched histopathological findings in 188 of 195 (96%) cases. Seven individuals showed equivocal results, all of which had histopathological features of RHD but three tested PCR-negative and four results conflicted between laboratories. RHDV2 was the serotype detected in all PCR-positive cases. Histological features of rabbit haemorrhagic disease and PCR test results were positive in 125 rabbits; 51 unvaccinated, 56 in-date with Nobivac Myxo-RHD and 13 vaccinated against RHDV2 - although nine of these were vaccinated within 10 days of death. CLINICAL SIGNIFICANCE: PCR testing complements histopathology in cases of sudden death in rabbits by confirming the diagnosis and identifying virus serotype, but there can be false negatives. Although RHDV2 is currently prevalent in UK pet rabbits, vaccination against both RHDV1 and RHDV2 is recommended. Failures of RHDV2 vaccine are infrequent.

RHDV2 epidemic in UK pet rabbits. Part 1: clinical features, gross post mortem and histopathological findings.

OBJECTIVES: To report clinical features, gross post mortem and histopathological findings from an investigation into sudden or unexpected death in rabbits that was undertaken during an outbreak of rabbit haemorrhagic disease. MATERIALS AND METHODS: Using a standard protocol, veterinarians were invited to submit case histories and results of their post mortem examination of pet rabbits that died unexpectedly. Histopathological examination of heart, lungs, liver, spleen and kidney samples was collated with macroscopic appearance and clinical details. RESULTS: Hepatocellular necrosis, characteristic of rabbit haemorrhagic disease, was observed in 185 of 300 (62%) submissions, often accompanied by glomerular thrombosis and changes in other organs. Evidence of rabbit haemorrhagic disease was not apparent on histopathology in 113 of 300 (38%) rabbits. Gross post mortem examination by veterinary practitioners did not always reflect reported histopathological changes. No macroscopic abnormalities were seen in 78/185 (42%) of rabbit haemorrhagic disease cases. Rapid death and death of other rabbits in the household were common features of rabbit haemorrhagic disease. Ante mortem clinical signs included anorexia, collapse, lethargy, seizures, icterus, bleeding from the mouth, dyspnoea, hypothermia, pyrexia, bradycardia or poor blood clotting. CLINICAL IMPORTANCE: Rabbit haemorrhagic disease can be suspected from a history of sudden death, especially if multiple rabbits are affected. There is not always macroscopic evidence of the disease but histopathology is useful to support or refute a diagnosis of rabbit haemorrhagic disease and provide information about other causes of death.

Health survey of 167 pet rabbits (Oryctolagus cuniculus) in Finland.

Only a limited amount of information is available about health status of pet rabbits. The aim of this study was to obtain data about the health status of pet rabbits considered healthy by the owners in Finland. Physical examination and lateral abdominal and lateral skull radiography were performed on 167 pet rabbits of which 118 (70.7 per cent) had abnormal findings in at least one examination. The most common findings were acquired dental disease (n=67, 40.1 per cent), vertebral column deformities and degenerative lesions (n=52, 31.1 per cent), skin disorders (n=28, 16.8 per cent) and eye disorders (n=12, 7.2 per cent). Vertebral column angulating deformities were significantly more common in dwarf lop rabbits (P≤0.001). The prevalence of health disorders was significantly higher in rabbits over three years of age of which 51 (82.3 per cent) had findings in at least one examination (P<0.05). Rabbits as prey animals hide their illness, which cause difficulties to owners to recognise health problems. Because of the high prevalence of clinical and radiological findings in apparently healthy pet rabbits, regular physical examinations are advised, especially for animals over three years old.

Association of pre-pregnancy BMI and postpartum weight retention with postpartum HbA1c among women with Type 1 diabetes.

AIM: To examine the association of pre-pregnancy BMI and postpartum weight retention with postpartum HbA(1c) levels in women with Type 1 diabetes. METHODS: We longitudinally evaluated 136 women with Type 1 diabetes who received prenatal, pregnancy, and postpartum care through Joslin Diabetes Center's Diabetes and Pregnancy Program between 2004 and 2009. Weight, BMI and HbA(1c) concentrations were assessed before the index pregnancy and repeatedly monitored after delivery until 12 months postpartum. We used linear mixed models to assess the association of postpartum HbA(1c) with pre-pregnancy BMI and postpartum weight retention. RESULTS: The mean HbA(1c) concentration increased from 49 mmol/mol (6.6%) at 6 weeks postpartum to 58 mmol/mol (7.5%) by 10 months postpartum, a level similar to the mean pre-pregnancy HbA(1c) concentration. Postpartum weight retention showed a linearly decreasing trend of 0.06 kg/week (P < 0.0001), with -0.1 kg average postpartum weight retention by 1 year postpartum. Compared with women with a pre-pregnancy BMI ≥ 25 kg/m², women with a lower pre-pregnancy BMI maintained a 3.4 mmol/mol (0.31%) lower HbA(1c) concentration, after adjusting for several sociodemographic, reproductive and diabetes-related factors (P = 0.03). There was a suggestion of a time-varying positive association between HbA1c and postpartum weight retention, with the most significant difference of 3.7 mmol/mol (0.34%; P = 0.05) at 30 weeks postpartum among women with postpartum weight retention ≥ 5 kg vs those with postpartum weight retention < 5 kg. CONCLUSIONS: Pre-pregnancy BMI and postpartum weight retention were positively associated with HbA(1c) during the first postpartum year in women with Type 1 diabetes. Interventions to modify the behaviours associated with these body weight factors before pregnancy and after delivery may help women with Type 1 diabetes maintain good glycaemic control after pregnancy.

Clinical value of blood glucose measurement in pet rabbits.

Blood glucose was measured with a portable glucose meter in 907 rabbits, including 238 clinically healthy ones. Blood glucose concentrations ranged from 1.2 to 30.1 mmol/l. Diabetes mellitus was not encountered. No significant effect of sex or sedation was found. Hypoglycaemia was seen in 16 rabbits including one with an insulinoma. There was a significant relationship between blood glucose, food intake, signs of stress and severity of clinical disease. Rabbits showing signs of stress had higher blood glucose than rabbits with no signs and rabbits that were totally anorexic had higher blood glucose values than those that were eating normally or those with reduced food intake. Severe hyperglycaemia (>20 mmol/l) was associated with conditions with a poor prognosis. Rabbits with confirmed intestinal obstruction had a mean blood glucose of 24.7 mmol/l (n=18). This was significantly higher than the rabbits with confirmed gut stasis, which had a mean value of 8.5 mmol/l (n=51). The conclusion of the study was that blood glucose is a measurable parameter that can be used to assess the severity of a rabbit's condition and help to differentiate between gut stasis and intestinal obstruction in rabbits that are anorexic.

Gastric dilation and intestinal obstruction in 76 rabbits.

Eighty-four incidents of gastric dilation (bloat) were investigated in 76 pet rabbits, and an intestinal obstruction was confirmed in 64 of them. In 49 the obstruction was due to pellets of compressed hair, in four to locust bean seeds, in five to neoplasia, in two to postspay adhesions, and in one case each to carpet fibre, tapeworm cysts, a strangulated hernia and diverticulosis. In all but four cases, the obstruction was in the small intestine. The condition affected a variety of breeds fed on a variety of diets. Radiography was a useful diagnostic tool because gas and/or fluid in the digestive tract outlined the dilated stomach and intestines. Twenty-nine of the rabbits died or were euthanased without treatment, and 40 underwent exploratory surgery; of these, 10 died during surgery, three were euthanased because of intestinal neoplasia, eight died postoperatively and 19 recovered. Fifteen rabbits in which radiography indicated that a foreign body had passed out of the small intestine did not undergo surgery; of these, 13 recovered and two died.

Biotransformation of beta-ketosulfides to produce chiral beta-hydroxysulfoxides.

The biotransformations of a series of substituted phenylthio-2-propanone and benzylthio-2-propanone were carried out using Helminthosporium sp. NRRL 4671, Mortierella isabellina ATCC 42613, or Rhodococcus erythropolis IGTS8. Several products gave microbial oxidation of sulfide to sulfoxide and reduction of carbonyl to secondary alcohol, producing beta-hydroxysulfoxides in medium to high enantiomeric and diastereomeric purities. Fungal biotransformations using Helminthosporium sp. and M. isabellina resulted in the opposite sulfoxide configurations of various beta-hydroxysulfoxide products.

Encephalitozoon cuniculi in pet rabbits.

The results of a serological test for Encephalitozoon cuniculi in 125 pet rabbits are reviewed, together with follow-up studies of clinical cases. Blood samples were taken from 38 asymptomatic rabbits and 87 rabbits showing neurological, renal or ocular signs suggestive of encephalitozoonosis. In the asymptomatic group, six of 26 (23 per cent) apparently healthy rabbits, sampled as part of a health screen, were seropositive; of the remaining 12 asymptomatic rabbits, sampled because they lived with seropositive companions, eight (66 per cent) were seropositive. Fifty-eight of the rabbits with clinical disease showed neurological signs, including head tilt, seizures, ataxia and swaying; three of them also showed renal signs and two showed ocular signs, and these five rabbits were all seropositive. Head tilt was the most common neurological sign in 21 of 23 (91 per cent) of the seropositive cases. All nine rabbits with ocular lesions were seropositive. In follow-up studies of clinical cases, the rabbits showed variable responses to treatment with albendazole, fenbendazole, antibiotics or corticosteroids, and some cases recovered without treatment.

Gestational diabetes mellitus.

In summary, much controversy exists surrounding the diagnosis, treatment, and even existence of GDM. At present, there is not enough evidence to advocate the Carpenter and Coustan criteria over the NDDG criteria. In univariate analysis, the Toronto Tri-Hospital Study demonstrated an increased incidence of cesarean section, macrosomia, and preeclampsia with increasing carbohydrate intolerance in those who did not meet NDDG criteria for GDM. Multivariate analysis, however, showed that this contribution is small relative to other nonmodifiable risk factors. A shift to the Carpenter and Coustan criteria would identify a larger population of patients with GDM and increase treatment costs. In addition, although treatment of these borderline GDM patients might reduce macrosomia, there is no evidence to indicate that it reduces the cesarean section rate. The precise threshold at which glucose intolerance adversely affects pregnancy outcomes and increases the risk for the development of type 2 diabetes in the mother is unknown. The perinatal risks associated with hyperglycemia seem to increase continuously with increasing maternal hyperglycemia. More randomized intervention trials are needed to define the effects of graded increases in glucose intolerance on maternal and fetal morbidity.

Parathyroid hormone, haematological and biochemical parameters in relation to dental disease and husbandry in rabbits.

During a two-year period between 1995 and 1997, over 80 blood samples were collected from pet rabbits in order to investigate an apparent osteodystrophy affecting the skulls of rabbits with acquired dental disease. A series of haematological and biochemical analyses relating to calcium metabolism were performed and samples were taken for parathyroid hormone (PTH) assay. The rabbits were categorised according to the condition of their teeth and the manner in which the pets were kept. PTH concentrations were higher and calcium concentrations lower in hutch-kept rabbits with advanced dental disease in comparison with those kept in free-range conditions. No dental problems were detected in the free-range rabbits on radiological or clinical examination. During the course of the study, differences in haematological pictures and albumin values emerged among rabbits kept under the different husbandry regimes. Complete blood counts from free-range rabbits were comparable with laboratory reference ranges, whereas there were significantly lower red cell and lymphocyte counts in rabbits exhibiting advanced dental disease. Serum albumin values were significantly higher in rabbits kept in free-range conditions than in those with advanced dental disease or those unaffected by dental disease but kept in hutches. Rabbits kept in hutches showed trends towards anaemia and lymphopenia. Results indicated that acquired dental disease of pet rabbits is related to husbandry and is associated with alterations in calcium metabolism.

Extensively keratinized squamous intraepithelial lesions of the cervix are difficult to grade.

We tested the hypothesis that extensively keratinized squamous intraepithelial lesions (SILs) are difficult to grade precisely by identifying 100 Papanicolaou smears with a keratinizing SIL that had been originally judged difficult to grade. Of these, 65 were confirmed as low-grade SIL (LSIL) or high-grade SIL (HSIL) on subsequent biopsy. The 65 smears were reviewed independently by 3 cytopathologists who graded each case as LSIL or HSIL (by Bethesda System criteria). The accuracy of the grade was determined by the subsequent biopsy results; accuracy was compared with that of a historic control group of SILs with biopsy follow-up. In the study group, biopsies showed LSIL in 41 cases and HSIL in 24. The mean accuracy for a smear diagnosis of LSIL was 60% for the study group and 92% for the control group. For a smear diagnosis of HSIL, the accuracy was 60% for the study group and 95% for the control group. The overall kappa value for the study group confirmed poor interobserver agreement. Some keratinizing SILs are difficult if not impossible to grade precisely using standard criteria. For such lesions, the diagnosis "SIL, grade cannot be determined due to extensive keratinization" is justified.

Urine cytology. It is still the gold standard for screening?

Urine cytology remains the gold standard for bladder cancer screening. It is the test against which all others are compared when evaluating potential bladder tumor markers. The answer to whether urine cytology possess the optimal combination of sensitivity and specificity to retain consideration as the best screening device depends on the goals of the clinical practice. Urine cytology has excellent specificity with few false-positive cases. Its overall sensitivity is poor, but this drawback is explained for the most part by poor criteria for identifying well-differentiated, low-grade TCC. The natural history of such lesions is the occurrence of multiple superficial recurrences in 70% to 80% of patients, with only a minority (10% to 15%) progressing to muscle invasive or metastatic disease. Because patients with low-grade TCC are at low risk for progression, they are monitored primarily for the development of a subsequent tumor. One might argue that the detection of new low-grade lesions is of secondary importance to the early detection of disease progression. The performance characteristics of urine cytology in this regard are much improved. Urine cytology often results in the identification of high-grade malignant cells even before a cystoscopically distinguishable gross lesion is present. Routinely diagnosing grade I TCC may be clinically irrelevant. Ancillary techniques to improve the sensitivity of urine cytology have been insufficiently additive to have much clinical value. Several promising bladder tumor markers have been investigated as potential screening tools and are summarized in Table 3. BTA, nuclear matrix proteins, and fibrin/fibrinogen degradation products share lower specificities than urine cytology and may have high rates of false positivity. Telomerase is highly sensitive and highly specific but is not readily available as a point-of-service test. Hyaluronidase and hyaluronic acid are promising prognostic markers, but hyaluronidase does not detect grade I TCC. Early results from studies of this marker await verification. Combining some of these new markers may optimize their performance status, allowing the advantages of one test to correct the shortcomings of another. Likewise, their combination with urine cytology may prove beneficial. Although adding urine cytology has not increased the sensitivity of some point-of-service tests, few studies have addressed the effect on specificity. Until an obvious winner is declared in the race to find a bladder tumor marker, urine cytology will remain the gold standard screening method because of its comfortable familiarity.

Myxoid neoplasms of the adrenal cortex: a rare histologic variant.

The myxoid variant of adrenocortical carcinoma is a rare neoplasm described previously in only two case reports. Because of the rarity of these lesions, the presence of myxoid changes in adrenal cortical neoplasms usually raises the possibility of malignancy. We studied the histopathologic features of 14 cases of myxoid adrenocortical neoplasms, including six adenomas and eight carcinomas. All patients with adenomas with sufficient follow-up (n = 5) were alive with no recurrence of their tumors or evidence of metastatic disease. Four patients with carcinomas died of their disease, two were alive with metastatic disease, and one was alive with no evidence of recurrence or metastatic disease. Histologically, the 14 tumors varied in their myxoid composition, ranging from 10% to 95%. The myxoid foci stained positively with Alcian blue and were usually negative with periodic acid-Schiff and mucicarmine stains. As a group, the immunophenotype of the lesions was typical of other adrenal cortical neoplasms, with positive immunostaining for vimentin, synaptophysin, and alpha-inhibin. One tumor was focally positive for keratin. Myxoid adrenal cortical neoplasms should be included in the differential diagnosis of myxoid retroperitoneal neoplasms. Myxoid changes in adrenal cortical neoplasms may be present in both adenomas and carcinomas, and the usual clinical and histopathologic features for adrenocortical neoplasms should be used to diagnose these neoplasms.

Problems in grading soft tissue sarcomas.

Clinicians, particularly medical oncologists, place increasing importance on histologic grading as a means of informing their treatment decisions in patients with soft tissue sarcoma. Numerous different grading systems (with a variable number of grades) have been described. Although these have often used different parameters, it is nevertheless generally accepted that histologic grade, however derived, is probably the best prognostic indicator in these sarcoma patients. Unfortunately, however, there is no consensus as to the best grading system, and even those schemes that are most widely applied (those of the French National Cancer Centers and the National Cancer Institute in the United States) have important and acknowledged limitations. Furthermore, assessment of many of the most popular grading parameters is necessarily subjective, and, to date, potentially more objective measures of proliferative activity and ploidy (eg, using immunohistochemistry or DNA flow cytometry) have not improved on experienced morphologic grading. In addition, the confounding (and often misleading) effect on histologic grade of preoperative therapy, which is increasingly used, has not been addressed in any meaningful way. This overview highlights the limitations, problems, and concerns regarding histologic grading of soft tissue sarcomas.

LSIL biopsies after HSIL smears. Correlation with high-risk HPV and greater risk of HSIL on follow-up.

Can the risk associated with a high-grade cervical smear be disregarded when followed by a low-grade biopsy? We examined the distribution of human papillomavirus (HPV) types in such cases to see whether they segregated preferentially with low-risk or high-risk viruses and compared the distribution with that reported in the literature for women with high-grade squamous intraepithelial lesions (HSILs) and low-grade squamous intraepithelial lesions (LSILs). We identified 48 cases of HSIL smears with corresponding LSIL biopsy specimens. Biopsy specimens were tested and typed for HPV by polymerase chain reaction amplification with consensus primers followed by restriction fragment length polymorphism analysis, and HPVs were scored as low-risk or high-risk types. Thirty-seven cases scored positive for HPV DNA: 2 for low-risk HPV types, 17 for high-risk types, and 18 for types of unknown oncogenicity. The prevalence of high-risk HPV was significantly higher than that of low-risk HPV. There was a higher rate of high-risk HPV than that seen in historic unselected LSIL cases. Cases of HSIL cytology/LSIL histology represent a group distinct from unselected LSILs by virtue of their higher prevalence of high-risk HPV types and, therefore, warrant closer clinical follow-up.

Preliminary investigation of the vitamin D status of pet rabbits.

The plasma concentrations of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) (vitamin D3) were measured in blood samples taken from one wild rabbit and 13 pet rabbits at different times of the year. Some pet rabbits had low or undetectable plasma concentrations of 1,25-(OH)2D3 especially if they were kept in hutches. Rabbits with more access to sunlight had higher concentrations of 1,25-(OH)2D3.

The effect of hormonal replacement therapy on the vascular reactivity and endothelial function of healthy individuals and individuals with type 2 diabetes.

Estrogens protect healthy women from cardiovascular disease. However, epidemiological data suggest that women with diabetes are denied the cardioprotection associated with estrogens. Whether or not hormonal replacement therapy (HRT) confers cardiovascular benefits in postmenopausal women with diabetes is not known. The aim of this study was to examine the effects of HRT on the microvascular reactivity and endothelial function of individuals with and without diabetes. We studied the following groups of individuals: premenopausal healthy women [n = 28, age 41 +/- 8 yr (mean +/- SD)], premenopausal women with type 2 diabetes (n = 16, age 43 +/- 6 yr); postmenopausal healthy women (n = 12, age 57 +/- 4 yr), postmenopausal women with diabetes (n = 17, age 62 +/- 5 yr); postmenopausal healthy women on HRT (n = 13, age 51 +/- 5 yr), postmenopausal women with diabetes on HRT (n = 11, age 57 +/- 7 yr). We used laser Doppler flowmetry to measure forearm cutaneous vasodilatation in response to iontophoresis of 1% acetylcholine (endothelium dependent) and 1% sodium nitroprusside (endothelium independent). The endothelium-dependent vasodilation was significantly higher in premenopausal healthy women (180 +/- 67%; increase over baseline) compared to premenopausal diabetic women (87 +/- 41%; P < 0.001). endothelium-dependent vasodilation was also higher in postmenopausal healthy women on HRT (143 +/- 52) compared with postmenopausal diabetic women on HRT (86 +/- 61), postmenopausal healthy women without HRT (104 +/- 43), and postmenopausal diabetic women without HRT (74 +/- 28; P < 0.001). A similar pattern of responses was observed in the endothelium-independent vasodilation (premenopausal healthy women, 126 +/- 56; premenopausal diabetic women, 88 +/- 26; postmenopausal healthy women on HRT, 121 +/- 37; postmenopausal diabetic women on HRT, 88 +/- 41; postmenopausal healthy women without HRT, 84 +/- 36; and postmenopausal diabetic women without HRT, 73 +/- 36; P < 0.001). Soluble intercellular adhesion molecule (sICAM) was also measured among all the women with diabetes. Premenopausal women with diabetes (248.9 +/- 56 ng/ml) and postmenopausal women with diabetes on HRT (257.7 +/- 49 ng/ml) had lower sICAM levels compared with the postmenopausal diabetic women without HRT (346.4 +/- 149 ng/ml; P < 0.05). We conclude that menopausal status and type 2 diabetes are associated with impaired microvascular reactivity. HRT substantially improves microvascular reactivity in postmenopausal healthy women. In contrast, the effect of HRT on the microvascular reactivity of postmenopausal diabetic women is less apparent. However, the use of HRT among women with diabetes is associated with lower sICAM levels, suggesting an attenuation in endothelial activation.