Case Report: Strawberry Disease in Farmed Chilean Rainbow Trout

Strawberry disease is a chronic, nonlethal skin condition that affects Rainbow Trout Oncorhynchus mykiss in the United States and several European countries, where it is also known as red-mark syndrome. We provide the first identification and characterization of three strawberry disease outbreaks occurring at two aquaculture farms in southern Chile. Clinically affected fish weighing an average of 400 g presented multiple bright-red, usually raised, skin lesions on the flank, ventral surface, and dorsal surface. A PCR using Rickettsia-like-organism (RLO)-specific primers was performed on nine affected fish, and all skin samples were positive for the RLO 16S ribosomal RNA sequence. All PCR results for Flavobacterium psychrophilum and other bacterial and viral pathogens were negative. Histopathological examination of the skin lesions revealed extensive dermatitis, with severe lymphocytic infiltration in advanced cases. This report is the first to describe strawberry disease in farmed Chilean Rainbow Trout. Additional studies are needed to evaluate the risk for Rainbow Trout culture; fish challenge experiments should be performed to fulfill Koch’s postulates and to demonstrate that RLO is the cause of this disease.

Received December 27, 2014; accepted October 23, 2015.     

Colorimetric Method of Loop-Mediated Isothermal Amplification with the Pre-Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms

Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP–calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10² F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP–calcein method had 97% homology with the DNA sequence of F. columnare.

Received May 21, 2014; accepted August 10, 2014     

Distribution of Infectious Pancreatic Necrosis Virus (IPNV) Based on Surveillance Programs in Freshwater Trout Farms of Mexico

Diagnostic testing was performed between 2000 and 2012 to determine the distribution of infectious pancreatic necrosis virus (IPNV) in the main states of the Mexican Republic with freshwater Rainbow Trout Oncorhynchus mykiss (Walbaum) farms. This virus was positively identified from Rainbow Trout farms in seven of the eight states assessed. Due to nonnormal data distribution, a logistic regression model was applied for statistical analysis, the results of which indicated that virus prevalence was variable between states, with moderate but significant differences. Regarding the time periods evaluated, IPNV prevalence was higher during the first years of the study. The susceptible, infected, removed model was used to examine this phenomenon, which indicated that the decreased prevalence during the latter years of the study could be associated with a real elimination of the infection. The information of the cases analyzed also suggests a relationship with the irregularity in the submission of samples to the laboratory and emphasizes other factors that have contributed to the transmission of IPNV throughout the country.

Received November 10, 2014; accepted December 5, 2015.     

Assessment of Detoxifying Markers for Florfenicol in Rainbow Trout Liver

Florfenicol (FF) is employed in fish farms to contest or prevent bacterial infections. However, this pharmaceutical may produce reactive oxygen species that may cause biochemical changes in antibiotic-treated fish. The aim of this study was to evaluate the effect of FF on Rainbow Trout Oncorhynchus mykiss treated for 10 d with 7.5 and 15 mg/kg FF followed by a withdrawal period of 5 d. Superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, glyoxalase I and glyoxalase II, total glutathione, lactic dehydrogenase, and alkaline phosphatase were investigated in the livers of treated and untreated fish. A general impairment of antioxidant enzymes and metabolic indicators was measured in FF-treated Rainbow Trout. Onset of oxidative damage may have occurred during the antibiotic treatment as a consequence of the effect of FF toxicity at mainly the highest dose. Nevertheless, the rise in levels of total glutathione and glutathione S-transferase even after the withdrawal period may shield the antibiotic-mediated oxidative processes.

Received December 22, 2015; accepted May 26, 2016 Published online October 28, 2016     

Respiratory phagocytes are implicated in enhanced colibacillosis in chickens co-infected with influenza virus H9N2 and Escherichia coli

1. The aim of this study was to determine the most likely time interval after infection with influenza virus H9N2 for co-infection with Escherichia coli to cause colibacillosis, the importance of lung load of E. coli and the involvement of respiratory phagocytes.

2. Specific pathogen free chickens were inoculated intranasally with 10⁶EID₅₀ of influenza virus or uninfected. After specified time intervals, 10⁷ CFU E. coli or phosphate-buffered saline was inoculated. The presence of lesions, the number of respiratory phagocytes in the respiratory lavage fluid and the E. coli load in the lung were determined after different time intervals.

3. Compared with the number of lesions in chickens receiving only E. coli inoculation, the number lesions in co-infected chickens were increased at 0- and 3-d time intervals, but reduced in the groups at 6- and 9-d intervals between co-infection.

4. At 1–3 d after E. coli inoculation, the number of lesions chickens was correlated with the number of respiratory phagocytes harvested and related to the E. coli load in the lungs at 5 d.

5. These results suggest that the lesions caused by E. coli in chickens were increased within a 0–3 d interval following H9N2 virus inoculation and that this effect is related to the number of respiratory phagocytes.     

The Impact of Egg Ozonation on Hatching Success,Larval Growth,and Survival of Atlantic Cod,Atlantic Salmon,and Rainbow Trout

The direct exposure of fish eggs to ozonated water has generated interest as a means of ensuring pathogen-free eggs without the use of harsh chemicals. However, there are numerous knowledge gaps, including safe contact times, exposure levels, and potential long-term effects on aquaculture species in both freshwater and seawater. The effect of different ozone (O₃) doses (0.5–1.0, 1.5–2.0, and 2.5–3.0 mg of O₃/L for 90 s) on recently fertilized eggs of Atlantic Cod Gadus morhua and eyed eggs of Atlantic Salmon Salmo salar and Rainbow Trout Oncorhynchus mykiss was evaluated in comparison with the effects of two commercial disinfectants: Perosan (0.004 mg/L) and Ovadine (100 mg/L). The impact of ozone application was evaluated based on hatching success, larval nucleic acid concentration, larval growth, and survival. Overall, results indicated that ozonation of Atlantic Cod eggs at a dose less than 3.0 mg/L for 90 s produced no negative effect on the larvae up to 30 d posthatch. Furthermore, ozonation of Atlantic Salmon and Rainbow Trout eggs generated no negative effect on the larvae, based on monitoring until 85% yolk sac re-absorption (16 d posthatch).

Received May 6, 2014; accepted October 24, 2014     

Dermatomycosis caused by Paranannizziopsis australasiensis in five tuatara (Sphenodon punctatus) and a coastal bearded dragon (Pogona barbata) in a zoological collection in New Zealand

CASE HISTORY: Health monitoring of tuatara (Sphenodon punctatus) at Auckland Zoo between 2001 and 2009 showed that 58/93 tuatara had been affected by dermatitis of unknown origin. From 2011 onwards, cases of suspected fungal dermatitis underwent extensive diagnostic investigations.

CLINCAL FINDINGS: Six cases of dermatomycosis were attributed to Paranannizziopsis australasiensis, five in tuatara and one in a coastal bearded dragon (Pogona barbata). Cases presented typically as raised, yellow to brown encrustations on the skin. Severe cases progressed to necrotising ulcerative dermatitis, and in the bearded dragon to fatal systemic mycosis. Following topical and systemic treatments, lesions resolved in all five tuatara.

LABORATORY FINDINGS: Histopathological examination of skin biopsy samples revealed dermatitis with intralesional septate branching hyphae. Fungal culture yielded isolates morphologically resembling Chrysosporium species, and isolates were submitted for molecular confirmation and sequencing of DNA.

DIAGNOSIS: All six cases were confirmed as dermatitis due to infection with P. australasiensis, on the basis of fungal culture and DNA sequencing of isolates.

CLINICAL RELEVANCE: These are the first reported cases of dermatomycosis associated with P. australasiensis infection in tuatara, and the first cases in which systemic therapeutic agents have been used in the treatment of such disease. Tuatara at the Auckland Zoo are now routinely examined every 3 months and tissue samples from any lesions sent for histopathology and fungal culture. Further work to elucidate the epidemiology and significance of P. australasiensis infections in reptiles in New Zealand is important for both welfare and conservation purposes.     

Hydrogenated oil decreases tissue concentrations of n‐6 polyunsaturated fatty acids and may contribute to dyschondroplasia in broilers

1. In a factorial design of dietary treatments, male Ross broilers were given diets containing soyabean oil, hydrogenated soya‐bean oil (as a source of trans‐fatty acids) or feed fat with either 0 or 300 μg of added D‐biotin/kg.

2. Growth to 28 d was not influenced by the dietary treatments.

3. Length of tibiotarsal bones was reduced (P<0.05) and severity of leg bone cartilage lesions, characteristic of dyschondroplasia, was highest (P<0.05) in broilers fed on diets containing hydrogenated soyabean oil.

4. Feeding hydrogenated soyabean oil lowered (P< 0.05) the concentrations of C₂₀:_4n6 and the ratios of C_20:4n6/C_18:2n6 in liver and growth plate cartilage.

5. Growth plate cartilage from birds affected with dyschondroplasia contained lower proportions of prostaglandin precursor fatty acids compared with normal growth plate.

6. It is speculated that an inhibition of prostaglandin biosynthesis brought about by the presence of trans‐fatty acids might contribute to the occurrence of lesions similar to dyschondroplasia.     

Assessment of health and welfare of Austrian laying hens at slaughter demonstrates influence of husbandry system and season

1. Selected health and welfare variables of laying hens from 79 flocks kept in alternative husbandry systems were assessed at the end of lay.

2. Investigations were carried out on 100 hens per flock at the slaughter line and recorded as part of a novel scoring system. In addition, post-mortem investigations as well as parasitological examinations were conducted on 10 birds from each flock.

3. Birds with access to free range had better plumage than birds kept in barn systems. Housing in aviaries was associated with a higher prevalence of foot pad lesions and keel bone deformations. In addition, poorer plumage, more and worse skin and foot pad lesions were documented in hens slaughtered during the winter months.

4. Oophoritis and/or salpingitis were the most prevalent pathomorphological changes observed. Intestinal parasites, especially Heterakis gallinarum and Ascaridia galli, were frequent. Hens kept indoors had fewer nematoda and cestoda than those from conventional and organic free range.

5. The slaughterhouse protocol allowed direct comparison of variables between flocks and specific problems to be identified in particular flocks. The results could be used to initiate detailed investigations into problematic issues on selected farms.     

Natural resistance of Sri Lankan village chicken to Salmonella gallinarum infection

1. An experiment was conducted to compare the natural resistance of an indigenous breed of local village chickens to Salmonella gallinarum with two commercial breeds: ISA Brown and ISA White layers under experimental conditions.

2. A total of 72 chickens from each of these breeds were randomly distributed to 4 pens to provide equal numbers of two replicate pens maintained as infected and control (uninfected). All chickens in infected groups were inoculated orally with 1 × 10⁸ CFU (1 ml dose) of a field isolate of S. gallinarum, at the age of 8 and 16 weeks given over 5 consecutive days. Growth performance, clinical signs, gross pathological lesions and antibody responses were measured.

3. A significantly higher mortality was observed in the brown layers compared with the white layers, and clinical signs and mortality were absent in village chickens. However, a large number of birds with gross lesions and high antibody titres were detected in village chickens, indicating that birds had the disease subclinically. Commercial breeds had a significantly higher body weight, feed intake and feed conversion efficiency.

4. There was a significantly lower proportion of positive reactors in village chickens in the whole-blood agglutination test (35%) compared to brown (100%) and white (90%) layers even after the second inoculation. Uninfected birds were negative in all groups. The indirect enzyme-linked immunosorbent assay confirmed these observations.

5. These results suggest that the indigenous breed had superior natural resistance to S. gallinarum than the commercial breeds.     

Detection of Infectious Pancreatic Necrosis Virus from the Leeches Hemiclepsis marginata and Hirudo medicinalis

Leeches have been reported to harbor several important fish pathogens, including spring viremia of carp virus, infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV), and also may contain blood protozoa. In the present study, leeches were collected from water bodies located in Kurdistan province, Iran. The specimens were tested for IHNV, VHSV, and infectious pancreatic necrosis virus (IPNV) using the PCR method. The results showed that two different species of leeches, Hemiclepsis marginata and Hirudo medicinalis, were infected by IPNV among the seven species studied. The infected leeches were found in areas that were polluted with untreated sewage coming from upstream fish farms culturing Rainbow Trout Oncorhynchus mykiss. In addition, the fish at fish farms in the vicinity had been infected with IPNV 9 months previously. Our results showed that the virus causing infectious pancreatic necrosis is present in the leeches H. marginata and H. medicinalis, suggesting that leeches are a potential source of IPNV in fish farms.

Received October 14, 2015; accepted June 1, 2016 Published online September 29, 2016     

Effects of short intermittent lighting on food consumption and performance of dwarf broiler breeders and progeny

1. Two hundred and sixty four dwarf broiler breeder hens were subjected to ad libitum or restricted feeding and to four lighting patterns: 15L (bright light):9D (dark), 15L:9d (dim light), 2L:10d: 1L:11d, (0.5L:3.5d) × 6.

2. Breeder hens fed ad libitum and subjected to either conventional or intermittent lighting ingested respectively, 25 g and 14 to 17 g more than hens restricted to 115 g/d.

3. Body weight was greater in hens fed ad libitum irrespective of the lighting pattern and of the amount of food intake.

4. Persistency of egg production was impaired by intermittent lighting.

5. Ad libitum feeding reduced egg fertility. The decrease was larger in breeder hens on intermittent lighting.

6. Hatchability was increased in hens submitted to the symmetrical lighting pattern (0.5L:3.5d).

7. Egg and chick weights were higher in hens fed ad libitum.

8. Shell index and shell breaking strength increased in restricted hens on the intermittent lighting pattern (0.5L:3.5d) × 6.

9. The best performance was obtained in restricted hens on the conventional lighting pattern.     

Toxic effects of high concentrations of zinc oxide in the diet of the chick and laying hen

1. The effects of high dietary concentrations of zinc oxide on chicks and hens were studied in four experiments.

2. Chicks given 2 000, 4 000 or 6 000 mg zinc/kg diet from 2 to 6 weeks of age grew poorly, many showing gizzard erosion and lesions of the exo‐crine pancreas. Dissecting aneurysms occurred in a few birds receiving 6 000 mg zinc/kg.

3. Pancreatic lesions were found in chicks maintained on 1 000, 2 000 or 4 000 mg zinc/kg from 0 to 4 weeks and the two higher concentrations reduced body weight and increased the incidence of gizzard erosion.

4. Histologically, gizzard lesions varied from excessive desquamation of epithelial cells, heterophils and erythrocytes into an abnormally spongi‐form koilin, to erosion of koilin, glands and pits. Pancreatic lesions could be detected only microscopically and consisted of dilation of the acinar lumina, cytoplasmic vacuolation, cytoplasmic globule formation and necrosis of the exocrine cells with interparenchymal fibrosis.

5. Similar lesions of the gizzard and pancreas were found in hens after they had received 10 000 or 20 000 mg zinc/kg diet for only 4 d.

6. Hens given 10 000 mg zinc/kg for 4 d followed by a normal zinc intake for 28 d had normal gizzards and pancreata.     

Gene expression profile of IGF1 and MSTN mRNA and their correlation with carcass traits in different breeds of geese at 70 d of age

1. The expression of insulin-like growth factor-1 (IGF1) and myostatin (MSTN) mRNA in breast and leg muscle was quantified in 70-d-old Taihu and Wanxi geese by using a Multiplex Competitive Fluorescent–PCR method and the correlations between mRNA levels and carcass traits were analysed.

2. IGF1 mRNA expression in breast muscle in Taihu geese was significantly higher than that in Wanxi geese and the MSTN mRNA level in leg muscle in Taihu geese was significantly higher than that in Wanxi geese.

3. There was no significant difference in breast muscle MSTN or leg muscle IGF1 mRNA expression between the two breeds.

4. Within the same breed, the IGF1 mRNA expression in leg muscle of male geese was significantly higher than that in female geese, and MSTN mRNA expression in leg muscle was significantly higher than that in breast muscle.

5. There was no difference in the IGF1 mRNA expression between tissues.

6. There was a positive correlation between IGF1 mRNA and MSTN mRNA and a negative correlation between IGF1 mRNA expression of breast muscle and leg muscle ratio.

7. In Wanxi geese, MSTN mRNA expression in leg muscle was negatively associated with body weight and leg muscle weight.     

Aspects of metabolism related to the occurrence of skin lesions in biotin‐deficient chicks

1. Chicks were fed on biotin‐deficient low‐ and high‐protein diets supplemented with increasing concentrations of biotin.

2. Biotin deficiency decreased hepatic activity of pyruvate carboxylase [EG 6.4.1.1] but activity of acetyl‐CoA carboxylase [EG 6.4.1.2] was comparatively unaffected.

3. Increasing dietary protein increased the severity of biotin deficiency as assessed by skin lesions and decreased plasma biotin concentrations.

4. The severity of the skin lesions over all the treatments was most closely related to plasma biotin concentration.     

Flavobacterium psychrophilum Infections in Salmonid Broodstock and Hatchery-Propagated Stocks of the Great Lakes Basin

Bacterial coldwater disease (BCWD), caused by Flavobacterium psychrophilum, threatens wild and propagated salmonids worldwide and leads to substantial economic losses. In addition to being horizontally transmitted, F. psychrophilum can be passed from infected parents to their progeny, furthering the negative impacts of this pathogen. In Michigan, both feral and captive salmonid broodstocks are the gamete sources used in fishery propagation efforts. A 5-year study was initiated to follow the prevalence of systemic F. psychrophilum infections in feral broodstocks of four species (steelhead Oncorhynchus mykiss [potadromous Rainbow Trout]; Coho Salmon O. kisutch; Chinook Salmon O. tshawytscha; and Atlantic Salmon Salmo salar) residing in three Great Lakes watersheds. Additionally, captive broodstocks of four species (Rainbow Trout, Brown Trout Salmo trutta, Lake Trout Salvelinus namaycush, and Brook Trout Salvelinus fontinalis) maintained at two facilities were assessed for the presence of F. psychrophilum. The resultant offspring from each broodstock population were sampled for F. psychrophilum infections multiple times throughout hatchery residency. Using selective flavobacterial culture and PCR confirmation, F. psychrophilum was detected in all broodstocks except the captive Lake Trout and Brook Trout. Logistic regression analysis demonstrated that among the infected feral broodstocks, Chinook Salmon from the Lake Michigan watershed had the highest prevalence of systemic F. psychrophilum infection (mean = 63.2%). Among the captive broodstocks, the Gilchrist Creek strain of Brown Trout had the highest infection prevalence (mean = 5%). Collectively, the captive broodstocks were found to have significantly lower infection prevalence than the feral broodstocks. Despite the high prevalence of systemic F. psychrophilum infections in many broodstock populations, the bacterium was rarely detected in their progeny during hatchery rearing. However, heavy losses associated with clinical BCWD outbreaks did occur. Collectively, our results reinforce that BCWD continues to threaten Great Lakes basin salmonids.

Received April 6, 2015; accepted August 25, 2015     

Effects of dietary supplementation with Gynura procumbens (Merr.) on egg yolk cholesterol,excreta microflora and laying hen performance

1. The present study investigated the effects of dietary supplementation with Gynura procumbens on egg yolk and serum cholesterol and triglycerides, excreta microflora, laying performance and egg quality.

2. A total of 160 Hy-Line Brown layers (45 weeks old) were randomly assigned into 4 treatments on the basis of laying performance. Each treatment had 4 replicates with 10 birds each.

3. Dietary treatments were basal diet supplemented with 0 (control), 2.5, 5.0 and 7.5 g/kg diet G. procumbens during 56-d feeding period.

4. Serum (d 21, 42 and 56) and egg yolk (d 28, 42 and 56) cholesterol and triglycerides concentrations were linearly reduced with increasing dietary concentrations of G. procumbens.

5. Increasing dietary concentrations of G. procumbens linearly reduced the excreta total anaerobic bacteria (d 28), Clostridium sp. and Escherichia coli (d 28 and 56) populations.

6. Overall egg production and egg mass were linearly increased, and overall feed efficiency was linearly improved with increase in dietary G. procumbens.

7. Dietary increasing concentrations of G. procumbens linearly improved egg yolk colour (d 28 and 56) and breaking strength of eggs (d 56).

8. The results obtained in the present experiment indicate that dietary supplementation with G. procumbens could reduce the egg yolk cholesterol, suppresses harmful excreta microflora and improves layers performance.     

The bacterial flora of unhatched eggs

1. The methods of bacteriological examination of hatchery waste eggs were compared.

2. Using one of these methods a survey was conducted on eggs which had failed to hatch from commercial hatcheries.

3. An assessment of the level of contamination in the eggs examined indicated an average level of contamination of 12.7%.

4. The contaminating flora was composed mainly of Micrococcus sp. and Enterobacteriaceae, with Streptococcus sp., Staphylococcus sp., Bacillus sp. and Pseudomonas sp. present at lower levels.

5. Various interrelationships between contamination and hatchability are discussed.     

The occurrence of giant fibres in different muscles of two chicken genotypes

1. The occurrence of Giant Fibres (GF) in three muscles (Pectoralis major (PM), Iliotibialis lateralis and Semimembranosus) with different types of energy metabolism was studied in slow- and fast-growing chicken strains.

2. A total of 20 one-day-old Leghorn chicks (slow-growing) and 20 broiler (Ross 508) chicks (fast-growing) were reared to 100 and 45 d, respectively.

3. A small percentage of GF was seen in pre rigor muscle samples even at 3 min post mortem in both genotypes and in all muscle types studied.

4. From 3 min to 24 h post mortem GF increased both in Leghorn and broiler chickens but to a different extent according to muscle type and genotype.

5. The highest GF 24 h post mortem value was found in the PM muscles belonging to the fast-growing broiler line.

6. It was concluded that every type of muscle can develop GF, but this phenomenon is more evident in the PM especially in animals selected for increased growth rate.     

Behaviour of egg shell under compression in relation to deformation measurements1

1. The egg does not completely obey Hook's law when compressed between flat surfaces.

2. Deformation was not completely reversible due to crumbling at the points of contact with the force.

3. There are three components of non‐destructive deformation: (a) local deformation at the points of contact, (b) local surface crumbling and (c) deformation of the over‐all shell structure.

4. The latter two are uncontrolled, contributing to errors in the prediction of fracture force and depending on the non‐destructive force applied.

5. These effects should be considered in test selection and interpretation.

6. The optimum non‐destructive force was found to be 1 kg.