Detection of ruminant meat and bone meals in animal feed by real-time polymerase chain reaction: result of an interlaboratory study

The commercialization of animal feeds infected by prions proved to be the main cause of transmission of bovine spongiform encephalopathy (BSE). Therefore, feed bans were enforced, initially for ruminant feeds, and later for all feeds for farmed animals. The development and validation of analytical methods for the species-specific detection of animal proteins in animal feed has been indicated in the TSE (Transmissible Spongiform Encephalopathies) Roadmap (European Commission. The TSE (Transmissible Spongiform Encephalopathy) roadmap. URL: http://europa.eu.int/comm/food/food/biosafety/bse/roadmap_en.pdf, 2005) as the main condition for lifting the extended feed ban. Methods based on polymerase chain reaction (PCR) seem to be a promising solution for this aim. The main objective of this study was to determine the applicability of four different real-time PCR methods, developed by three National expert laboratories from the European Union (EU), for the detection and identification of cattle or ruminant species in typical compound feeds, fortified with meat and bone meals (MBM) from different animal species at different concentration levels. The MBM samples utilized in this study have been treated using the sterilization condition mandatory within the European Union (steam pressure sterilization at 133 degrees C, 3 bar, and 20 min), which is an additional challenge to the PCR methods evaluated in this study. The results indicate that the three labs applying their PCR methods were able to detect 0.1% of cattle MBM, either alone or in mixtures with different materials such as fishmeal, which demonstrates the improvement made by this technique, especially when compared with results from former interlaboratory studies.     

Supplementation of various meals to fish meal diet for chum salmon fry

In a 6-week feeding experiment, chum salmon, Oncorhynchus keta, swim-up fry, were fed fish meal diets supplemented with silkworm pupae powder (5%), dried beef liver (5%), krill meal (5%) or earthworm powder (5%) at the expense of fish meal, or substituting glucose (13%) for dextrin. Fish fed the diet with earthworm powder showed the best growth performance (675% weight gain in 6 weeks) and feed efficiency (117%). The growth rate and feed efficiency of the krill meal group were also significantly better than those of the control group (without supplement or substitution). Substitution of glucose and supplementation of silkworm pupae or beef liver failed to improve the growth rate, but significantly improved feed efficiency. Food consumption of fish fed the diet supplemented with earthworms was lowest, and none of the dietary treatments appreciably increased food intake of the fish over that of the control group. The dietary group receiving earthworm powder also showed significantly higher body fat content than the remaining dietary groups. No significant differences were noted in body protein and ash contents among all dietary treatments.     

Pleiotropic effect of the dwarfing gene d18-k on cool tolerance at booting stage under the genetic background of an extremely cool-tolerant line Norin-PL8 in rice

Norin‐PL8 (‘PL8’) is an extremely cool‐tolerant line of rice in Japan that contains genes for cool tolerance originating from a javanica landrace. It was investigated to see whether the dwarfing gene d18‐k (kotaketamanishiki dwarf) exerts its pleiotropic effect on enhancing the cool tolerance at the booting stage in the genetic background of PL8. The d18‐k isogenic line of the recurrent parent PL8 (D8), PL8, and two commercial cultivars ‘Hayayuki’ and ‘Kirara 397’ were used. For each line/cultivar, the 12°C‐5‐day treatment was conducted at various times during the booting stage. In addition to spikelet fertility, the ratio (%) of the fertilized‐spikelet number of each treated panicle to the varietal mean of fertilized‐spikelet number per panicle in the control (FS‐T/C) was adopted to estimate cool temperature damage. For FS‐T/C, the lines‐cultivars ranked in the order of D8 > PL8 > ‘Hayayuki’ > ‘Kirara 397’, reflecting their cool tolerances. D8 exceeded PL8 in both pollen grain number per anther in the control and as an indicator of pollen fertility after the treatment, as a result of the effects of d18‐k. Consequently, d18‐k can be used to develop super‐highly cool‐tolerant cultivars for cool‐weather areas.     

Factors responsible for levels of male sterility in photoperiod-sensitive cytoplasmic male sterile (PCMS) wheat lines

A `two-line system' using photoperiod-sensitivecytoplasmic male sterility (PCMS) caused by Aegilops crassa cytoplasm has been proposed as a newmeans of producing hybrid wheat. The PCMS line ismaintained by self-pollination under short-dayconditions (14.5 h light period), and F<sub>1</sub> seedscan be produced by outcrossing of the PCMS line witha pollinator under long-day conditions (15 h lightperiod). As the levels of male sterility in PCMSlines under the long-day conditions is a crucialfactor in determining hybrid purity of the F<sub>1</sub>seeds, a study was conducted into the effect ofseeding rate on male sterility in PCMS lines. Threedifferent density levels were tested using analloplasmic line of Japanese wheat cultivar `Norin 26'which exhibits PCMS. Levels of male sterility of thePCMS line increased at sparse planting, because tiller(ear) number per plant increased at low seedingdensity and late-appearing ears tended to exhibithigher levels of male sterility than early-appearingears. On the other hand, male sterility levels of thePCMS lines depended on genotype, e.g., the PCMS`Fujimikomugi' was completely male sterile, whereasthe PCMS `Norin 26' showed partial male sterility. APCMS line showing complete male sterility, such as thePCMS `Fujimikomugi', should produce F<sub>1</sub> seeds withhigh purity. However, the PCMS `Fujimikomugi' showeda lower female fertility. For practical use, it isnecessary to produce PCMS lines having high malesterility with high female fertility under long-dayconditions.     

Identification on commercialized products of AFLP markers able to discriminate slow- from fast-growing chicken strains

The European chicken meat market is characterized by numerous quality marks: "Label de Qualité Wallon" in Belgium, "Label Rouge" in France, denominations of geographical origin, organic agriculture, etc. Most of those certified productions have specifications requiring the use of slow-growing chicken strains. The amplified fragment length polymorphism (AFLP) technique has been used to search molecular markers able to discriminate slow-growing chicken strains from fast-growing ones and to authenticate certified products. Two pairs of restriction enzymes (EcoRI/MseI and EcoRI/TaqI) and 121 selective primer combinations were tested on individual DNA samples from chicken products essentially in carcass form that were ascribed as belonging to either slow- or fast-growing strains. Within the resulting fingerprints, two fragments were identified as type-strains specific markers. One primer combination gives a band (333 bp) that is specific for slow-growing chickens, and another primer pair generates a band (372 bp) that was found to be characteristic of fast-growing chickens. The two markers were isolated, cloned, and sequenced. The effectiveness and the specificity of the two interesting determinants were assessed on individuals of two well-known strains (ISA 657 and Cobb 500) and on commercialized products coming from various origins.     

Changes in allergenicity and digestibility of squid tropomyosin during the Maillard reaction with ribose

The effect of the Maillard reaction on the allergenicity of squid tropomyosin (TM) was investigated. When TM was reacted with ribose (TM-ribose), its human-specific IgE-binding ability decreased markedly and alpha-chymotryptic digestibility of TM was also altered at the early stage of the Maillard reaction. On the other hand, the modification of the lysine residues in TM using 2,4,6-trinitrobenzenesulfonic acid had no effect on the allergenicity and alpha-chymotryptic digestibility of TM. Therefore, the structural change in TM induced by the Maillard reaction would cause the reduction of the allergenicity, rather than the block of lysine residues. Although peptic digestion diminished the specific IgE-binding ability of TM, the reduction of the allergenicity by the Maillard reaction remained after peptic digestion. These results suggest that hypersensitive reaction of TM-ribose in the human body might be lower than that of native TM.     

Mastitis caused by Mycobacterium kansasii infection in a dog

A 2‐year, 7‐month‐old female Chihuahua was admitted for a mammary mass measuring one cm in diameter. The dog had a history of demodicosis for 4 months and showed signs of pseudopregnancy at the time of the visit. Cytologic examination of an aspirate of the mass revealed a large number of macrophages containing nonstaining bacterial rods, which were acid‐fast in a Ziehl–Neelsen stain, suggesting mycobacterial infection. Histologic examination of the mass revealed a pyogranulomatous mastitis characterized by an infiltration with macrophages containing acid‐fast bacteria. Mycobacterium kansasii was subsequently cultured and identified by PCR. Surgical excision of the mass resulted in the growth of other dermal masses, but antimycobacterial treatment with rifampin and clarithromycin resolved these masses within 1 month. Three months after discontinuation of the treatment, similar organisms were found in aspirates of the enlarged bilateral inguinal lymph nodes by cytologic examination. Despite antimycobacterial treatment for another 4 months, there was no improvement and demodicosis also recurred. The dog eventually died of lymphoma 5 months after the relapse of mycobacterial infection. Although M kansasii is considered an important pathogen for pulmonary and cutaneous disease in people, there is only one report in a dog with an infection in a pleural effusion. As both adult‐onset demodicosis in dogs as well as mycobacterial infection in people have been associated with T‐lymphocyte deficiency, the M kansasii infection in this dog may have been associated with a condition of immune compromise.     

Retrospective diagnosis of feline GM2 gangliosidosis variant 0 (Sandhoff-like disease) in Japan: possible spread of the mutant allele in the Japanese domestic cat population

GM2 gangliosidosis variant 0 (human Sandhoff disease) is a lysosomal storage disease caused by simultaneous deficiencies of acid beta-hexosaminidase (Hex) A and Hex B due to an abnormality of beta-subunit, a common component in these enzyme molecules, which is coded by the HEXB gene. In the present study, a retrospective diagnosis was performed in 2 previous suspected cases of feline Sandhoff-like disease using a DNA test to detect the causative mutation identified previously in 4 cats in 2 other families of Japanese domestic cats. Enzymic analysis was also performed using stored leukocytes and plasma collected from the subject families in order to investigate the usefulness of enzymic diagnosis and genotyping of carriers. The DNA test suggested that the 2 cases were homozygous recessive for the mutation. Consequently, 6 cats homozygous for the same mutation have been found in 4 separate locations of Japan, suggesting that this mutant allele may be spread widely in the Japanese domestic cat populations. In enzymic analysis, Hex A and Hex B activities in leukocytes and plasma measured using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide as a substrate were negligible in affected cats, compared with those in normal and carrier cats. However, there was a wide overlap in enzyme activity between normal and carrier cats. Therefore, it was concluded that enzymic analysis is useful for diagnosis of affected cats, but is not acceptable for genotyping of carriers.     

Clinical course of pyruvate kinase deficiency in Abyssinian and Somali cats

The objective of this study was to examine clinical signs, laboratory parameters, and course of disease in Abyssinian and Somali cats with pyruvate kinase (PK) deficiency. The clinical course of 25 PK-deficient cats was followed over a time period of 0.8-11.3 years (median 4.3). Eleven cats (age 0.8-7.8 years, median 4.4) did not show signs according to the owners. In 14 cats (age 0.1-5 years, median 1.7) the owners noted lethargy (10), diarrhoea (seven), pale mucous membranes (six), inappetence (six), poor coat quality (six), weight loss (four), icterus (four), and pica (two). Sixteen cats had been used for breeding at least once before diagnosis. Laboratory abnormalities included anaemia (70%), increased aggregated reticulocyte counts (94%), hyperglobulinaemia (80%), hyperbilirubinaemia (53%), and increased liver enzymes (47%). Six of 25 affected cats died (four) or were euthanased (two) at ages ranging from 1.3 to 11.3 years (median 4.1) presumably because of PK-deficiency. These findings emphasise that PK deficiency shows variation in age of onset and severity of signs. As PK-deficient cats can be asymptomatic testing for PK deficiency before breeding is strongly recommended.