Mohie Haridy

An outbreak of a disease with external body lesions in Picasso triggerfish (Rhinecanthus assasi) and black damselfish (Neoglyphieodon melas) has been observed in the indoor aquarium of NIOF, Hurghada, Egypt. Thirty Picasso triggerfish and thirty-five black damselfish were clinically affected with gross skin lesions. The affected fishes were euthanized for gross, histological and bacteriological examinations. The disease onset started after exposing the fish to catching and indoor rearing stress. Anorexia, erratic swimming and hemorrhagic skin and mouth ulcers and fin rot were the main symptoms and lesions among Picasso triggerfish and black damselfish. Single species of bacteria was isolated from skin ulcer, liver and spleen and was diagnosed as Tenacibaculum maritimus. The gross lesions were irregular shallow erosions and deep ulcers on the dorsal and lateral skin, sometimes on the head, mouth and fins, and occasionally, on the cornea. Histological findings, the hepatic tissue revealed fatty degeneration and focal liquefactive necrosis. Congestion, hemorrhage, hemosidrosis and lymphoid depletion was observed in spleen. The deep skin ulcers characterized by epidermal and muscular necrosis. Picasso triggerfish and black damselfish had lesions similar to those of natural infection and mortality rates of 50 and 60%, respectively in experimental reproduction of the disease. The natural T. maritimus infection in Picasso triggerfish and black damselfish in Red Sea is not only a superficial skin lesion but also a systemic infection.

Supplementary Figure 1: Clinical signs of bacterial keratitis after treatment with extracts of C. procera latex and F. sycomorus leaves. Group 1 (a, g). received sterilized saline solution served as control (normal eyes view, with no clinical signs symptoms); groups from 2 to 6 were injected with 100 μl saline solution containing approximately 1500 cfu of MRSA (S. aureus). At the 5 th DPI, severe clinical signs were observed in groups 2 (b) (untreated) and 5 (e) (treated with aqueous extract of F. sycomorus leaves) and moderate signs in groups 3 (c) (treated with chloramephnicol), 4 (d) (treated with aqueous extract of C. procera latex) and 6 (f) (treated with ethanol extract of F. sycomorus leaves). At the 12 th DPI, severe signs of keratitis were still observed in group 2 (h) while mild signs were observed in groups 3 (i) and 5 (k). No clinical signs were observed in groups 4 (j) and 6 (l). In all treatments, the dose was 2 drops 3 times daily.

MRSA-induced keratitis in rabbit was used to evaluate the therapeutic effect of F. sycomorus leaves and C. procera latex extracts. Within the 6 rabbit groups tested, group 1 received sterilized saline, while other groups (2 to 6) received 100 μl of intrastromal injections of 1.5×10 3 colony forming unit (cfu) ml-1 of methicillin-resistant Staphylococcus aureus (MRSA). After 12 hours, groups 3 to 6 also received chloramphenicol, aqueous extract of C. procera latex, aqueous and alcoholic extracts of F. sycomorus leaves, respectively 3 times daily for 12 successive days. The tested extracts inhibited MRSA growth in vitro (i.e. on culture medium). Colony counts in cornea discs from groups 3 to 6 were significantly reduced (P ≤ 0.001) compared to group 2 (untreated). Clinical signs of keratitis were observed on group 2 until the end of experiment. In groups 3 to 6, gradual recovery was observed and signs disappeared by the 12 th DPI (days post inoculation). Only mild symptoms persisted in group 5 (aqueous extract of leaves). In group 3 and 5, cornea, iris, ciliary body and conjunctiva showed mild leukocytic infiltration and depigmentation of melanin cells while recovery of cornea and iris was observed in groups 4 and 6. In conclusion, the used extracts have potential therapeutic effects on MRSA-induced keratitis in rabbit.

In vitro antibacterial potential of chloroform, absolute ethanol, methanol, ethanol (70%) and aqueous extracts of Calotropis procera and Ficus sycomorus leaves and latex were evaluated against five Gram-negative bacteria (Neisseria lactamica ATCC 23970, Salmonella typhi ATCC 19430, Shigella flexenri ATCC 12022, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922) and two Gram-positive bacteria (methicillin-resistant Staphylococcus aureus MRSA ATCC 43300, S. aureus CONS ATCC 29213). The antibacterial activities were expressed as zone of inhibition; minimum inhibitory concentrations (MIC) and also the survival curve was determined as kinetic studies. Interestingly, among all the tested extracts, aqueous and ethanol (either absolute or 70%) of C. procera and F. sycomorus leaves and latex were the best solvents for elute polar antibacterial substances and showed bacteriocidal effect against most Gram-positive and negative bacteria. Also, latex extracts were more pronounced than leaf extracts on human pathogenic bacteria. The most resistant bacterium was E. feacalis against both plant extracts. On the other hand, S. areus MRSA was the most sensitive bacteria especially with ethanol 70% extract of leaves and latex for both plants. The results of MIC for these extracts show more or less values higher than the chloramephnicol. Our conclusion confirms that, susceptibility of Gram-positive bacteria to the aqueous or ethanolic extracts of leaves for both plants was more than those of Gram-negative bacteria. The activities of 70% ethanol extracts recorded highest activity against Gram-negative bacteria than those of other extracts. The results therefore established a good support for the use of C. procera and/or F. sycomorus in traditional medicine against Gram-positive and negative pathogenic bacteria.

The development of green synthesis of nanoparticles has received increasing attention due to ease of preparation, less chemical handling, and eco-friendly. In the present study, crystalllization of silver ions to nanosized particles by latex and leaf aqueous extract of Ficus sycomorus through bioreduction process was assessed. Strong plasmon resonance of silver nanoparticles was observed around 435 nm. UV–visible spectroscopy, transmission electron microscope (TEM) and energy dispersive X-ray fluorescence (EDXRF) were performed to examine the formation of silver nanoparticles (SNPs). The antibacterial activity of SNPs was tested against nine human pathogenic Gram −ve bacteria and one Gram +ve bacteria. Silver nanoparticles of extracts showed improved antibacterial activity on all the tested strains than for the extracts alone. This was confirmed either by optical density or zone of inhibition measurements. Silver nanoparticles was more effective in liquid than in solid medium probably because of better contact, for the higher silver content, with bacterial cells. It is concluded that fig (F. sycomorus) leaves and latex extracts can be used for the synthesis of SNPs that is environmentally friendly and cost effective. These preparations can be used for various biotechnology and medical applications for controlling pathogenic bacteria with better dispersion and, consequently, better efficiency in aqueous environment.

A single morphologic type of Sarcocystis cysts found in two out of 43 examined common coots, Fulica atra, is considered to represent a new species for which the name Sarcocystis atraii n. sp. is proposed and its description is provided. Coots were hunted from the vicinity of Brolos Lake located at KafrElsheikh province, Egypt. The structural morphology of the revealed sarcocysts was described using light and transmission electron microscopy. Sarcocysts were found in the leg and thigh muscles. The cysts were microscopic and measured 165−850 m in length × 50−85 m in width. Histologically; the sarcocyst wall was wavy and had minute undulations. Ultrastructurally, it measured 1−3 m in thickness and possessed many mushroom-like villar protrusions sometimes originating from other mushroom-like villar protrusions that measured approximately 0.5−2 m in length and up to 2 m in width, with the presence of electron dense ground substance of 300 nm to 1 m thick. The brady-zoites were elongated, banana-shaped and measured 7.5−14 × 1.5−2.5 m, with centrally or terminally located nuclei. The ul-trastructural features of the cyst wall belonged to type 24. On the basis of sequencing and phylogenic analyses for 18S rRNA , 28S rRNA genes and ITS-1 region; S. atraii n. sp. is considered a genetically distinct species, being most closely related to avian Sarcocystis spp. whose definitive hosts are predatory mammals.

Four Japanese forest green tree frogs (Rhacophorus arboreus) were presented with emaciation, abdominal distention, and ulcerative and nodular cutaneous lesions affecting the brisket, limbs, digits and ventral abdomen.  Another three frogs had been found dead in the same tank 1 year previously.  Necropsy examination of these seven frogs revealed splenomegaly and hepatomegaly, with multiple tan–yellow nodular foci present in the liver, spleen, heart, lungs, ovaries and kidneys.  Microscopically, five frogs had necrosis and surrounding granulomatous inflammation in the liver, spleen, kidneys, lungs, intestine and ovaries with numerous acid-fast bacilli in the areas of necrosis.  Two frogs had granulomatous lesions in the lungs, liver, spleen, heart, coelomic membrane, stomach and intestinal wall.  These lesions had no or minimal necrosis and few acid-fast bacilli.  Mycobacterium spp. was cultured from three frogs and identified as M. marinum by colony growth rate and photochromogenicity and DNA sequencing. This is the first report of M. marinum infection in Japanese forest green tree frogs.

Although MSB1 is the most-commonly used lymphoblastoid cell line for isolation of chicken anemia virus (CAV), some researchers have reported a few biological drawbacks. None of them were supported by the results of the present study. Another four avian (HP1, HP2, BK3 and CU10) and two mammalian (BTL-26 and KO-1) cell lines were investigated for susceptibility to the TK-5803 and AH-9409 strains. Both strains caused CPE on BK3 like MSB1. The mean number of positive cells for each strain in MSB1 and BK3 were not significantly different. The majority of the HP2, CU10 and HP1 cells showed no CPE. The virus titers of both strains were higher in MSB1 and BK3 (10(6.5-7.5) TCID(50)/0.1 ml) than in HP2, CU10 and HP1 (10(3.5-4.5) TCID(50)/0.1 ml). BTL-26 and KO-1 were resistant to CAV. BK3 could be used for isolation of CAV.

A 7-week-old SPF chicken inoculated at 4 weeks of age with chicken anemia virus was puffed up depressed and had ruffled feathers and a good body condition. Intestinal volvulus involving the jejunum and part of the duodenum forming two loops with one knob was observed. Microscopically, venous infarction of the obstructed loops, periportal and sublobular multifocal coagulative hepatic necrosis and granulomatous inflammation of the cecal tonsils were observed. Gram staining revealed no bacteria in hepatic tissue; however, gram-positive bacilli were detected in the necrotic debris in the intestinal lumen. Immunosuppression might have predisposed the chicken to intestinal and cecal tonsil infection that then progressed to volvulus. Loss of the mucosal barrier in infarction might allow bacterial toxins and vasoactive factors to escape into the systemic circulation (toxemia) and be responsible for the hepatic necrosis.