Nemaline myopathy in a six-month-old Pomeranian dog.

ABSTRACT: Nemaline myopathy - a clinically and genetically complex heterogenous group of disorders - is described uncommonly in humans and rarely in animals, and is characterised by progressive muscle weakness. The diagnosis is confirmed by histological and/or ultrastructural identification of subsarcolemmal, thread-like, rod-shaped structures called nemaline rod bodies within more than 40% of skeletal muscle fibres. These rods contain the Z-line protein, α-actinin, that can be effectively stained in skeletal muscles using Gomori or Masson trichrome and negatively stained with periodic acid-Schiff. Similar rod-like bodies have been found in smaller numbers in dogs with endocrine disorders and occasionally in other conditions in humans. This report is of a six-monthold Pomeranian dog which had progressive exercise intolerance over a two-month period associated with severe disuse muscle atrophy of the thoracic limbs, as well as gradual pelvic limb weakness and regurgitation of food. Baseline diagnostics ruled out endocrinopathies and after histological and ultrastructural evaluation of thoracic limb muscles and nerve biopsies confirmed nemaline myopathy. The clinical course, diagnostic test results, ultrastructure of skeletal muscle and peripheral nerve, gross necropsy findings and histopathology using various stains are described and illustrated.

Prevalence of subclinical bactibilia in apparently healthy shelter dogs.

OBJECTIVES: To determine the prevalence of subclinical bactibilia in apparently healthy shelter dogs and characterise serum liver enzymes activities, hepatobiliary histopathology and bile cytology in apparently healthy dogs with and without bactibilia. MATERIALS AND METHODS: Healthy, abandoned dogs euthanased for non-medical reasons were prospectively recruited for this cross-sectional study. Whole blood, collected immediately before euthanasia, was submitted for serum liver enzyme activity (alkaline phosphatase, alanine aminotransferase and gamma-glutamyl transferase) analyses. Bile, gall bladder and liver samples were collected aseptically from dogs within 25 minutes of euthanasia. Bile was submitted for bacterial culture and cytology in all dogs. Gall bladder and liver samples were submitted for histopathological examination in bactibilic dogs and nine randomly selected non-bactibilic dogs. RESULTS: Sixty-five healthy dogs were included in this study. Bactibilia was diagnosed in 10.8% (7/65) of dogs, with 9.2% (6/65) of dogs diagnosed on cytological examination and 4.6% (3/65) on culture. Elevated alanine aminotransferase activities were present in one bactibilic and five non-bactibilic dogs; and elevated gamma-glutamyl transferase activities in one bactibilic and two non-bactibilic dogs. No dogs had elevated alkaline phosphatase activities. Histopathological changes in bactibilic and non-bactibilic dogs included lymphoplasmocellular cholecystitis (7/7 and 9/9), gall bladder oedema (7/7 and 9/9), hepatic vacuolar degeneration (6/7 and 8/9), cholangitis (5/7 and 7/9), hepatic nodular hyperplasia (3/7 and 5/9) and hepatic cholestasis (2/7 and 4/9). CLINICAL SIGNIFICANCE: This study confirms that subclinical bactibilia occurs in a small number of apparently healthy shelter dogs and that subclinical hepatobiliary histopathological abnormalities can occur in apparently healthy bactibilic and non-bactibilic dogs.

Fusarium graminearum Species Complex Associated with Maize Crowns and Roots in the KwaZulu-Natal Province of South Africa.

Thirty-three isolates of the Fusarium graminearum species complex obtained from diseased maize (Zea mays) crowns and roots in the Winterton district, KwaZulu-Natal province of South Africa were identified to species level. Their pathogenicity and virulence to maize 'PHI 32D96B' seedlings were determined under glasshouse conditions, with seedling survival and growth and crown and root rot as criteria. Phylogenetic analyses using the 3-O-acetyltransferase (Tri101) gene region sequences revealed the presence of F. boothii (2 isolates), F. graminearum sensu stricto (26 isolates), and F. meridionale (5 isolates) in the F. graminearum species complex associated with diseased maize crowns and roots. Pathogenicity results showed that F. boothii was the most and F. meridionale the least virulent of the three species. F. boothii and F. graminearum sensu stricto significantly reduced survival of seedlings and all three species caused significant reduction in growth and significantly more crown and root rot than the control (uninoculated). This is the first report of F. boothii, F. graminearum sensu stricto, and F. meridionale associated with diseased maize crowns and roots and their pathogenicity and virulence as soilborne pathogens on maize seedlings in South Africa.

Phytophthora taxa associated with cultivated Agathosma, with emphasis on the P. citricola complex and P. capensis sp. nov.

Agathosma species, which are indigenous to South Africa, are also cultivated for commercial use. Recently growers experienced severe plant loss, and symptoms shown by affected plants suggested that a soilborne disease could be the cause of death. A number of Phytophthora taxa were isolated from diseased plants, and this paper reports their identity, mating type, and pathogenicity to young Agathosma plants. Using morphological and sequence data seven Phytophthora taxa were identified: the A1 mating type of P. cinnamomi var. cinnamomi, P. cinnamomi var. parvispora and P. cryptogea, the A2 mating type of P. drechsleri and P. nicotianae, and two homothallic taxa from the P. citricola complex. The identity of isolates in the P. citricola complex was resolved using reference isolates of P. citricola CIT groups 1 to 5 sensu Oudemans et al. (1994) along with multi-locus phylogenies (three nuclear and two mitochondrial regions), isozyme analyses, morphological characteristics and temperature-growth studies. These analyses revealed the isolates from Agathosma to include P. multivora and a putative novel species, P. taxon emzansi. Furthermore, among the P. citricola reference isolates the presence of a new species was revealed, described here as P. capensis. Findings of our study, along with some recent other studies, have contributed to resolving some of the species complexity within the P. citricola complex, resulting in the identification of a number of phylogenetically distinct taxa. The pathogenicity of representative isolates of the taxa from Agathosma was tested on A. betulina seedlings. The putative novel species, P. taxon emzansi, and P. cinnamomi var. parvispora were non-pathogenic, whereas the other species were pathogenic to this host.

Isolation and Identification of the Causal Agent of Brown Stalk Rot, A New Disease of Maize in South Africa.

During 2004 to 2005, an unreported disease of maize (Zea mays) was observed on commercial fields in the Northwest and Mpumalanga Provinces of South Africa. Infected plants were stunted, with a vertical crack at the first internode. Inside the stem, a dark-brown, narrow lesion was present along the crack. Internal browning inside the stem extended upward, reaching the top internode in some plants. Seed cobs were underdeveloped. Diseased plants were scattered in the fields and 10 to 70% of the crop was affected. Gram-negative, facultatively anaerobic bacteria were consistently isolated from diseased tissues. Pathogenicity tests established that representative strains induced disease symptoms similar to those observed on maize plants in the field. Physiological and biochemical characterization using the API 20E and API 50CHE systems and 16S rRNA gene sequence analyses showed that the strains belonged to the genus Pantoea. The results of these tests also separated the strains into two groups. The first group, giving a positive reaction in the indole test, was similar to Pantoea ananatis. The second group of strains was indole negative and resembled P. agglomerans. The fluorescent amplified fragment length polymorphism (F-AFLP) genomic fingerprints generated by the indole-positive strains and P. ananatis reference strains were similar and clustered together in the dendrogram, confirming that the indole-positive bacteria causing brown stalk rot on maize were P. ananatis. The F-AFLP fingerprints produced by the indole-negative strains were distinctly different from those generated by P. ananatis, P. agglomerans, P. dispersa, P. citrea, P. stewartii subsp. stewartii, and P. stewartii subsp. indologenes. The results indicated that indole-negative bacteria causing brown stalk rot on maize might belong to a previously undescribed species of the genus Pantoea. This is the first report of a new disease on maize, brown stalk rot, caused by two bacterial species, P. ananatis and an undescribed Pantoea sp.

First Report of Stem and Root Rot of Tomato Caused by Phytophthora capsici in South Africa.

Tomato plants, grown in open hydroponic systems under shadecloth and plastic near Barberton and Pretoria in South Africa and Srelebi Phikwe in Botswana, developed symptoms of wilting with brown-to-black cankers on the lower stems, blackening of the vascular tissues, and root rot. Pathogens isolated from affected tissues were identified as Phytophthora capsici Leonian (1) and Pythium aphanidermatum (Edson) Fitzp. (2).They occurred separately or together. Pythium aphanidermatum has previously been recorded on tomato in South Africa. P. capsici isolates were papillate, caducous, grew at >36°C, had tapered sporangial bases, and a maximum sporangial length of >70 µm. Koch's postulates were confirmed by inoculating 4-week-old tomato seedlings (cv. Floradade) grown at 22 to 30°C in a steam-pasteurized mixture of sawdust compost, pine bark, and vermiculite (3:2:1). Plugs from V8 juice agar cultures of P. capsici were placed on wounds made on the stems of 10 seedlings. Ten wounded uninoculated plants served as controls. Water-soaked lesions were visible on the stems of all inoculated plants after 2 days. Control plants remained healthy. After 4 days, lesions turned dark brown with affected plants wilted or dead. Reisolation yielded P. capsici. The experiment was repeated with similar results. To our knowledge, this is the first report of P. capsici on tomatoes in South Africa. References: (1) A. H. Thompson et al. S. Afr. J. Bot. 60:257, 1994.(2) W. Dick. Keys to Pythium. University of Reading Press, Reading, U.K., 1990.

Root Rot and Stunting of Hydroponically Grown Endive, Fennel, and Sorrel Caused by Pythium F-group in South Africa.

Specialized vegetable crops such as endive (Cichorium endiva), fennel (Foeniculum vulgare), and sorrel (Rumex spp.) are being cultivated hydroponically in South Africa to be marketed as admixtures in salads. Stunted growth accompanied by browning and rotting of the root tips has been observed at a commercial recirculating gravel bed hydroponic system near Pretoria during the warm summer months. Root segments excised from symptomatic plants were rinsed in sterile water and plated on Pythium selective medium (1). Pythium F-group, characterized by the production of noninflated filamentous sporangia and no oospores (2), was isolated from 40% of endive, 60% of fennel, and 7% of sorrel root segments. Koch's postulates were confirmed by inoculating 4-week-old seedlings of each crop in the greenhouse with a Pythium F-group isolate from the particular crop. Inoculations were performed by adding 3 ml of suspension (105 zoospores per ml) to each liter of aerated nutrient solution. Control plants received no inoculum, and the experiment was repeated once. After 4 weeks, inoculated plants showed stunting of the foliage and slight to moderate root rot. Pythium F-group could readily be reisolated from roots of inoculated plants. To our knowledge, this is the first report of Pythium F-group on these crops in South Africa. References: (1) W. J. Botha and R. L. J. Coetzer. S. Afr. J. Bot. 62:196, 1996. (2) M. W. Dick. Keys to Pythium. University of Reading Press, Reading, UK, 1990.

Colony variation in Sinorhizobium meliloti inoculant strain U 45.

A culture of Sinorhizobium meliloti strain U 45, maintained on yeast extract-mannitol (YM) agar, produced a mixture of Congo red-absorbing (R1) and non-absorbing (W1) colonies when grown on YM medium containing Congo red. The original freeze-dried (FD) culture formed gummy (G), white (W2) and small red (R2) colony types on the above medium. All colonies were stable except G, which segregated into G and W2-like types. Immune diffusion patterns of all colony types were identical. The W1 colony type dominated R1 when a 1:1 combination was sub-cultured on YM agar. The parent cultures and their variants exhibited a range of N2-fixing effectiveness and competitiveness when inoculated onto two cultivars of Medicago sativa. Variant R2 from the FD culture was ineffective on both cultivars. Genomic DNA fingerprinting with insertion elements ISRm3 and ISRm2011-2 suggested that transposition of these elements was not a cause of variation, but a DNA band was absent in the profiles of two out of three W2-like colonies. Protein profile comparisons showed high similarity (r = 0.98) between the colony types when grown in YM broth. When grown on Tryptone-Yeast extract medium, variants from the FD and agar-maintained cultures formed separate clusters with r = 0.79. Polymerase chain reaction fingerprinting using repetitive, site-directed and arbitrary primers failed to differentiate the variants. The results emphasize the need to monitor culture variability to maintain the quality of legume inoculants.

Pythium spp. Infecting Hydroponically Grown Lettuce in South Africa.

Root rot and wilting of lettuce (Lactuca sativa L. var. capitata L.) commonly occur in hydroponic systems in South Africa. Plants showing stunted growth, wilt, and root rot were collected at two commercial hydroponic systems in Gauteng Province, and root segments were plated on a Pythium selective medium (1). P. irregulare and three species groups without oogonia (F, HS, and T) were identified (2). Pythium groups F, HS, and T, and P. irregulare were represented by 63, 6, 5, and 26% of the isolates, repectively. Koch's postulates were confirmed in two greenhouse experiments by inoculating 4-week-old lettuce cv. Lutetia seedlings grown in aerated nutrient solution at 21°C. Two V8 juice agar cultures of an isolate of each species or species group were blended in 500 ml of sterile water, and 166 ml of suspension was added to the nutrient solution in each of three 5-liter receptacles containing four lettuce seedlings. Control plants received sterile V8 juice agar suspension. After 4 weeks, all Pythium isolates caused root rot and reduced fresh plant weight significantly, although no wilting occurred. Symptoms were more severe with P. irregulare and Pythium group HS than with groups F and T. Compared to the control, P. irregulare reduced fresh root and shoot weight of plants by 51 and 38%, Pythium group HS by 41 and 33%, Pythium group T by 29 and 26%, and Pythium group F by 30 and 24%, respectively. References: (1) W. J. Botha and R. L. J. Coetzer. S. Afr. J. Bot. 62:196, 1996. (2) M. W. Dick. Keys to Pythium. University of Reading Press, Reading UK, 1990.

Report of Root Rot Caused by Pythium F-Group on Hydroponically Grown Celery in South Africa.

Celery (Apium graveolens) is commonly grown in hydroponic systems in South Africa. During the warm summer months, plants often become stunted or die, with mortality as high as 70% in some systems. Affected plants show severe root rot and yellowing of the foliage. Root segments excised from symptomatic celery plants collected from various hydroponic systems in Gauteng Province, South Africa, were rinsed in sterile distilled water and plated on PARP medium (2). Pythium was isolated consistently from both rotted and symptomless roots. Isolates produced only noninflated filamentous sporangia and no oospores, conforming to the description of Pythium F-group (1). Koch's postulates were confirmed by inoculating 4-week-old celery seedlings cultivated in aerated nutrient solution in a hydroculture system in the greenhouse. A zoospore suspension (105 zoospores per ml) of Pythium F-group cultured on V8 juice agar was added to the nutrient solution at the rate of 3 ml/1iter. Control plants received no inoculum, and the experiment was repeated once. Plants were assessed after 4 weeks. All inoculated plants showed symptoms of root rot, stunting, and yellowing, whereas control plants remained healthy. Pythium F-group was recovered on PARP medium only from the roots of inoculated plants. Pythium F-group has been described as a pathogen of celery elsewhere, but to our knowledge, this is the first report of root rot caused by the fungus on celery in South Africa. References: (1) M. W. Dick. Keys to Pythium. University of Reading Press, Reading, UK, 1990. (2) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986.

The effect of passive immunization on active immunity against Clostridium perfringens type D in lambs.

Lambs in different stages of development of active immunity against Clostridium perfringens type D were treated with partially purified immunoglobulin in an attempt to superimpose a passive immunity on an existing or developing active immunity. Three different studies were undertaken to determine the impact of partial purified immunoglobulins on these vaccinated animals. In 2 of the 3 studies, active immunity was induced by administering the normal routine enterotoxaemia vaccinations and allowing the basic immunity to become established, for a period ranging from 2 weeks for the animals in study 1 and 4 months for those in study 2, before passive immunization with the partially purified immunoglobulins took place. An increase in the epsilon antibody titre occurred in each of the 2 studies after the animals were passively immunized with immunoglobulin, though this increase was not statistically significant (P greater than 0.05). In the 3rd study, when the animals were given the initial vaccination of the Onderstepoort enterotoxaemia oil adjuvant vaccine together with the immunoglobulin, an immediate increase in the epsilon antitoxin titre occurred that was statistically significant (P less than 0.05) 2-14 days after administration. No negative effects were noted on the development of an initial active immunity or an existing active immunity against Clostridium perfringens type D when they were passively immunized with partially purified immunoglobulin.

Horizontal transmission in sheep and delayed clearance in guinea pigs and mice of a Brucella melitensis Rev. I mutant.

Virulence assays in guinea-pigs, mice and sheep with a Rev. I mutant strain (FSA) demonstrated that the latter had increased virulence in comparison with a reference strain. The mutant showed slower clearance in guinea-pigs and mice when compared with standard Rev. I strains and horizontal transmission was achieved in 2 sheep.

The passive protection of lambs against Clostridium perfringens type D with semi-purified hyperimmune serum.

Weaned lambs, having a detectable level of maternal antibodies (1-2 units/ml) against C. perfringens type D, showed protective antitoxin levels lasting for 29 days after receiving a single parenteral dose of 200 units/kg hyperimmune serum. Lambs, having no maternal antibodies (less than 0,07 units/ml) to C. perfringens type D but receiving the same dose of hyperimmune serum, maintained protective antibody levels for only 21 days. Three weeks after the titres fell below the minimum protective level of 0,15 units/ml, both these groups were treated again in the same manner. The passive immunity conferred in both groups now lasted for 42 days. When the hyperimmune serum was administered to lambs already immunized by vaccination, a slight increase was noted in the antibody titre.

Antibody response in cattle to oil emulsion rabies and ephemeral fever vaccines.

A stable oil emulsion rabies vaccine with a low viscosity was composed by a formula previously employed for Newcastle disease vaccine. Cattle developed high and sustained antibody levels, and guinea pigs were found to be solidly immune after a single injection of this vaccine. Antibody responses in cattle to 2 oil emulsion ephemeral fever vaccines were not satisfactory after a single injection, and severe local reactions were encountered when booster injections were applied.

Immunization of guinea-pigs and cattle with a reduced dose Clostridium chauvoei vaccine produced in a semi-synthetic medium.

A semi-synthetic culture medium and method are described for the production of a reduced dose Clostridium chauvoei vaccine. The vaccine gave excellent results in guinea-pigs, and 2 injections of 2.0 ml protected cattle against challenge with 2 M.L.D. of a virulent culture for at least 12 months. The suitability of C. chauvoei Strain OP64 as a vaccine strain was confirmed.

Comparison of oil adjuvant and aluminium phosphate-adsorbed toxoid for the passive immunization of lambs against tetanus.

Immunization of ewes with oil emulsion toxoid followed by an aluminium phosphate-adsorbed toxoid both containing 10 Lf per dose, resulted in a very high antitoxin level in their lambs. Two injections of aluminium phosphate-adsorbed toxoid also imparted a passive immunity to lambs which is considered to be adequate to protect them against tetanus for 4 weeks after birth.