Mg Collett

Effect of Sample Quality on the Sensitivity of Endoscopic Biopsy for Detecting Gastric and Duodenal Lesions in Dogs and Cats

BACKGROUND The quality of histopathology slides of endoscopic biopsies from different laboratories varies, but the effect of biopsy quality on outcome is unknown. HYPOTHESIS The ability to demonstrate a histologic lesion in the stomach or duodenum of a dog or cat is affected by the quality of endoscopic biopsy samples submitted. More endoscopic samples are needed to find a lesion in poor-quality tissue specimens. ANIMALS Tissues from 99 dogs and 51 cats were examined as clinical cases at 8 veterinary institutions or practices in 5 countries. METHODS Histopathology slides from sequential cases that underwent endoscopic biopsy were submitted by participating institutions. Quality of the histologic section of tissue (inadequate, marginal, adequate), type of lesion (lymphangiectasia, crypt lesion, villus blunting, cellular infiltrate), and severity of lesion (normal, mild, moderate, severe) were determined. Sensitivity of different quality tissue samples for finding different lesions was determined. RESULTS Fewer samples were required from dogs for diagnosis as the quality of the sample improved from inadequate to marginal to adequate. Duodenal lesions in cats displayed the same trend except for moderate duodenal infiltrates for which quality of tissue sample made no difference. Gastric lesions in dogs and mild gastric lesions in cats had the same trend, whereas the number of tissue samples needed to diagnose moderately severe gastric lesions in cats was not affected by the quality of tissue sample. CONCLUSIONS AND CLINICAL IMPORTANCE The quality of endoscopically obtained tissue samples has a profound effect on their sensitivity for identifying certain lesions, and there are differences between biopsies of canine and feline tissues.

Infection of sheep with adult and larval Ostertagia circumcincta: abomasal morphology.

The infection of parasite-naive sheep with approximately 15,000 adult Ostertagia circumcincta via abomasal cannulae resulted in marked changes in the structure and function of the abomasum. The functional changes, which have been characterised previously, included elevated abomasal pH and increased serum concentrations of pepsinogen and gastrin. Eight days after the transplant of adult worms, the abomasa of recipient animals were significantly heavier than those of controls (P < 0.001), the thickness of the fundic mucosa was greater (P < 0.01), there were fewer parietal cells (P < 0.01) and increases in the numbers of mitotic figures and mucus-producing cells. Mucous cell hyperplasia was also evident in the fundic mucosae of sheep receiving a trickle infection of infective, third-stage O. circumcincta larvae and was prominent within nodules associated with larval development. In non-nodular mucosa, there was hyperplasia of mucous cells and changes in the distribution of parietal cells. Decreases in the number of parietal cells at the gland base were offset by increases at a mid-gland level, probably due to chronic hypergastrinaemia, so that, overall, total parietal cell number was unaffected. Mucous cell hyperplasia and the diminution of parietal cell number are seen in a diverse range of disease states and may be mediated by host growth factors such as Transforming growth factor-alpha. Alternatively, the cellular and/or the secretory changes in response to the presence of adult worms are mediated by chemicals that are cytotoxic/inhibitory for parietal cells, and released by the parasites themselves.

Effect of Tissue Processing on Assessment of Endoscopic Intestinal Biopsies in Dogs and Cats

BACKGROUND Prior studies failed to detect significant association between hypoalbuminemia and small intestinal lesions. HYPOTHESIS Use of pictorial templates will enhance consistency of interpathologist interpretation and identification of intestinal lesions associated with hypoalbuminemia. ANIMALS Tissues from 62 dogs and 25 cats examined as clinical cases at 7 referral veterinary practices in 4 countries. METHODS Retrospective, observational study. Histopathology slides from sequential cases undergoing endoscopic biopsy were examined by 4 pathologists by pictorial templates. Changes for 9 microscopic features were recorded as normal, mild, moderate or severe, and 2- and 4-point scales were tested for consistency of interpretation. Logistic regression models determined odds ratios (OR) of histologic lesions being associated with hypoalbuminemia while kappa statistics determined agreement between pathologists on histologic lesions. RESULTS There was poor agreement (kappa = -0.013 to 0.3) between pathologists, and institution of origin of slides had effect (kappa = 1.0 for 3 of 4 lesions on slides from Institution 5) on agreement between pathologists on selected histologic features. Using 2 point as opposed to 4-point grading scale increased agreement between pathologists (maximum kappa = 0.69 using 4-point scale versus maximum kappa = 1.0 using 2-point scale). Significant association (P = .019- .04; 95% OR = 3.14-10.84) between lacteal dilation and hypoalbuminemia was found by 3 pathologists. CONCLUSIONS AND CLINICAL IMPORTANCE Substantial inconsistency between pathologists remains despite use of pictorial template because of differences in slide processing. Distinguishing between mild and moderate lesions might be important source of the disagreement among pathologists.

Mucopolysaccharidosis IIIA (Sanfilippo syndrome) in a New Zealand Huntaway dog with ataxia.

Abstract Aim: To investigate the nature of a progressive ataxia in a New Zealand Huntaway dog. Methods: The affected dog was examined clinically before being humanely killed and necropsied. Selected tissues were submitted to light and electron microscopy and to biochemical analyses. Results: The histological lesions were interpreted as indicative of one of the forms of mucopolysaccharidosis type-III (dMPS-III), a lysosomal storage disease. Biochemically there was a deficiency of heparan sulphamidase. All the heparan sulphate chains had non-reducing-end glucosamine-N-sulphate residues. Conclusion: The disease is dMPS-IIIA (Sanfilippo syndrome). An autosomal recessive mode of inheritance can be provisionally assumed from the nature of this disease in other species.

Campylobacter fetus fetus abortions in vaccinated ewes

Abstract Aims: To investigate the cause of an outbreak of ovine abortion in 1996 in a flock of 300 two-tooth (rising 2-year-old) ewes vaccinated against Campylobacter fetus fetus infection and to subsequently characterise the strain of C. fetus fetus isolated from aborted foetuses. Methods: Standard bacteriological methods were used to identify C. fetus fetus isolates which were then antigenically typed and subjected to pulsed-field gel electrophoresis (PFGE) and compared to the vaccine strain. Results: C. fetus fetus was identified as the causal agent of the abortions despite the ewes having been vaccinated before ram introduction and at the time of ram removal. Four isolates cultured from aborted material were indistinguishable when compared using antigenic typing and PFGE, but all differed from the vaccine strain. Conclusions: Within the limitations of the available typing systems, it is proposed that PFGE maybe a useful tool to establish the distribution and strain variation of C. fetus fetus. Clinical relevance: This field case indicates the need for further study of non-vaccine C. fetus fetus strains which cause abortion in vaccinated ewes, and of the importance of these strains to the New Zealand sheep industry.

Potential involvement of Neospora caninum in naturally occurring ovine abortions in New Zealand

Neospora caninum is an obligate intracellular parasite and is recognised as the leading cause of bovine abortion worldwide. Natural infection with N. caninum has been described in sheep but it has generally not been regarded as a significant cause of abortion. Recently, there have been several New Zealand cases of foetal abortions where N. caninum was detected which strongly suggested the involvement of Neospora in these abortions. However, there is minimal information about the prevalence of N. caninum infection naturally occurring in New Zealand sheep flocks and particularly its impact on reproduction success. Thus, this present study provides preliminary data on the role that Neospora is playing in ovine reproductive failure by establishing the prevalence of N. caninum antibodies and DNA in ewe blood and foetal material present in 21 New Zealand sheep farms with ongoing unexplained abortion problems and 10 farms with consistently high fertility levels. The results of this study demonstrated an overall seroprevalence of 1.4% which varied between Aborting/non-pregnant (1.8%), age-matched pregnant controls (0.6%) and high fertility (2.1%) ewes. However, despite the variation observed, there was no statistical difference between the three groups. In addition, Neospora DNA was detected by PCR in 13% of submitted foetal brains and in ewe blood from aborting/non-pregnant (6.9%), age-matched pregnant controls (3.6%) and high fertility pregnant (2.1%) ewes. When the PCR results were considered with the IFAT and IDEXX ELISA results, there was no correlation between serology positive and PCR positive blood samples. Taken together, these results reveal that reliance on ELISA-based serology or PCR alone may underestimate the involvement of Neospora. Furthermore, determining the involvement of Neospora appears to require a multi-facetted approach where diagnostic methods and serological cut-off values may need to be adjusted as further information about the effect of natural infections with N. caninum in the ovine host is elucidated.

Dose-titration challenge of young pregnant sheep with Neospora caninum tachyzoites.

Pregnant ewe lambs (7-8 months old at breeding) were inoculated intravenously at 90 days after joining with the ram with 50, 5 x 10(3), 10(6), or 10(8)Neospora caninum tachyzoites and outcomes were compared to a control group. Seroconversion was measured by indirect fluorescent antibody test (IFAT) at fortnightly intervals and by enzyme-linked immunosorbent assay (ELISA) at 28 days post-challenge. Seroconversion (by IFAT) occurred in all animals except for 2 sheep in the lowest dose group, including the 9 sheep in the control group. IFAT antibody titres rose and fell rapidly, peaking at 1:800 and in 8 animals was negative within 7 days of abortion. ELISA results more closely reflected infection dose and outcome of pregnancy than IFAT serology did. All 10 ewes in each of the 2 highest dose groups aborted, 5 of 10 ewes in the mid-dose group aborted and no ewes in the lowest dose or control groups aborted. Histological lesions consistent with N. caninum infection were seen in the brains of all 25 aborted lambs, as well as in 2 live-born premature lambs from the group receiving 5 x 10(3) tachyzoites and 2 clinically normal lambs (one from the control group). Polymerase chain reaction (PCR) detected N. caninum DNA in a variety of tissues from lambs, aborted foetuses and dams including 3 of 9 ewe-lamb pairs from the control group. The results from this study showed a strong relationship between challenge dose of N. caninum tachyzoites, the sample to positive (S/P) percentage in an ELISA test 28 days after challenge and pregnancy outcome. IFAT results did not correlate well with the level of challenge or the outcome of pregnancy and their relevance in studies of this kind should be questioned.

Photodynamic chlorophyll a metabolites, including phytoporphyrin (phylloerythrin), in the blood of photosensitive livestock: Overview and measurement

Abstract AIM: To validate a spectrofluorometric method for measuring chlorophyll a metabolites, specifically phytoporphyrin (= phylloerythrin), as well as the chlorins, pheophorbide a and pyropheophorbide a, in the blood of photosensitive cattle and sheep. METHODS: Standard methanolic solutions of pheophorbide a (25 µM), pyropheophorbide a (25 µM), and phytoporphyrin (<3.7 µM) were prepared. Serum and plasma samples were obtained from cattle (n=5), sheep (n=3), and one alpaca, with clinical facial eczema (i.e. photosensitive), as well as from clinically normal (n=2 of each) adult cows, recently weaned calves, and sheep (controls). Standard solutions of the three metabolites were characterised using high-performance liquid chromatography (HPLC), with mass spectrometry, in conjunction with absorption and emission spectral data, and were compared with sera from photosensitive animals. In the latter, phytoporphyrin was the only metabolite detected. Calibration curves were prepared by adding different ratios of methanol and standard solutions of phytoporphyrin in methanol to diluted serum from control animals. Peak areas of fluorescence spectra were determined in samples from photosensitive animals. RESULTS: Pheophorbide a and pyropheophorbide a produced typical chlorin spectra, and had excitation/emission maxima of 408/669 nm and 409/669 nm, respectively. Phytoporphyrin showed a typical porphyrin fluorescence spectrum, with excitation/emission maxima of 425/644 nm. Pyropheophorbide a and phytoporphyrin had very similar chromatographic retention times, the same chemical formula and same mass, but were distinguishable by differences in their absorption spectra. In sera from photosensitive animals, the fluorescence emission at 644 nm was shown to arise solely from phytoporphyrin and not from any other chlorophyll a metabolites. Calibration curves using sera and plasma from control animals gave reliable measurements of phytoporphyrin in the range 0.4–6 µM. The sera of facial eczema-affected cattle and sheep had concentrations of phytoporphyrin ranging from 0.4 to 1.8 and 0.9 to 2.8 µM, respectively. Haemolysed serum samples were not suitable for determination of phytoporphyrin with this method. CONCLUSIONS AND CLINICAL RELEVANCE: A spectrofluorometric method for the quantification of phytoporphyrin in the blood of photosensitive animals has been validated, and can be applied to the measurement of other chlorophyll a metabolites in blood. This will be a useful tool in the further investigation of the cause and pathogenesis of idiopathic photosensitivities of farm animals.

Toxicity of white snakeroot (Ageratina altissima) and chemical extracts of white snakeroot in goats.

White snakeroot (Ageratina altissima) is a sporadically toxic plant that causes trembles in livestock and milk sickness in humans that drink tainted milk. The putative toxin in white snakeroot is tremetone and possibly other benzofuran ketones, even though it has not been demonstrated in vivo. Toxic white snakeroot was dosed to goats, and they developed clinical signs of poisoning, exercise intolerance, significant increases in serum enzyme activities, and histological changes. Tremetone and the other benzofuran ketones were extracted with hexane; the extracts and residues were analyzed for tremetone and dosed to goats at tremetone and benzofuran ketone concentrations similar to the original plant material. However, none of the dosed goats developed the disease. The results demonstrate for the first time that white snakeroot is a potent myotoxin in goats and that other compound(s), which may be lost or modified during the extraction process, could be involved in causing trembles and milk sickness.

Suspected zinc-induced copper deficiency in growing kittens exposed to galvanised iron.

Abstract AIM: To investigate the possible causes of fading coat colour and an acquired hind-limb ataxia affecting sixteen 4- to 5-month-old kittens in a closed feline colony during 1993 and 1994. METHODS: Records of kittens and litters born in the colony between 1991 and 1997 were analysed. The kittens had been kept from birth until approximately 5 months of age in plastic cages with galvanised iron bar doors. Histopathological sections from 4 of the worst affected ataxic kittens necropsied in 1993 were re-examined. In addition, 6 of the original 16 affected kittens that survived were re-examined as 4- to 5-year-old adults, which were moderately ataxic; these cats were then humanely killed for necropsy. RESULTS: In the kittens, clinical signs included lordosis, dysmetria, ataxia of the hind-limbs and fading coat colour; histopathological lesions included Wallerian-type degeneration in the spinal cord, pons and medulla, and neuronal degeneration in the vestibular nuclei and ventral horns of the spinal cord. Analysis of colony data ruled out an inherited disease, and there was no evidence of dietary inadequacy orexcess. Similar, though milder, clinical and histopathological changes were noted in the affected adults. CONCLUSIONS: Circumstantial evidence is consistent with a diagnosis of zinc-induced copper deficiency caused by the ingestion of zinc oxide from the galvanised iron bar doors. CLINICAL RELEVANCE: Because of the possibility of zinc- induced copper deficiency, galvanised iron should be avoided when designing and constructing cages for cats in veterinary clinics, pet shops and boarding facilities.