Wael Hananeh

Pathological, immunological and molecular diagnosis of rabies in clinically suspected animals of different species using four detection techniques in Jordan.

A total of 29 brain tissue samples (BTS) were examined for rabies infection by different diagnostic techniques. None of the examined brain tissues were presented as a whole intact brain. Twenty-seven brain tissue samples from various animal species - dog (13 cases), cat (one case), fox (one case), pig (one case), cow (three cases), sheep (two cases), goat (one case), camel (one case), horse (one case) and donkey (three cases) - were provided by the Vaccine and Sera Department/Al-Bashir Central Hospital in Amman/Jordan from July 2009 up to May 2010. All these samples were frozen at -20°C, for a period of time and then fixed in 10% formalin after being tested for rabies virus by fluorescence antibody test (FAT). The results showed that 21 (77.77%) of 27 BTS were positive for rabies by FAT. Seventeen samples (58.62%) of 29 were positive by histopathology, 2 (6.90%) were positive by histopathology, immunohistochemistry (IHC) and of those which were fixed for 24h only, and 21 (72.42%) were positive using RT-PCR assay. Five of 29 BTS had no pathological lesions, 17 had Negri bodies and the remaining had non-suppurative encephalitis and necrosis. Thirteen BTS that were diagnosed positive by FAT were also positive by RT-PCR and histopathology, but negative by IHC. Four BTS that were positive by FAT were negative by histopathology, IHC and RT-PCR. Also, 3 BTS (cases 19, 22, and 25) that were negative by FAT were positive by RT-PCR and negative by IHC. One of these was negative, while two were positive by histopathology. Therefore, definitive diagnosis of rabies under these conditions in Jordan needs one or more other diagnostic tests in addition to FAT. Also, freezing and prolonged formalin fixation of BTS is not suitable for the detection of rabies virus antigen using IHC.

Investigation of the role of tumour necrosis factor-α, interleukin-1β, interleukin-10, nitric oxide and rheumatoid factor-immunoglobulin M in a rat model of arthritis

Collagen-induced arthritis (CIA) in rats is a widely used preclinical animal model of rheumatoid arthritis (RA). However, CIA development in Sprague-Dawley (SD) rats is less severe in terms of inflammatory response compared with other strains. Therefore, a modified CIA model called MCIA, using N-acetylmuramyl dipeptide (MDP), has been developed in the less sensitive SD rat strains. This work was conducted to better understand the immunopathological role and contributions of the pro-inflammatory T-helper type 1 (Th-1) cytokines and inflammatory mediators, interleukin-1 (IL-1β), tumour necrosis factor-alpha (TNF-α) and nitric oxide (NO); the anti-inflammatory T-helper type 2 (Th-2) cytokine, IL-10 and autoantibodies such as rheumatoid factor (RF)-immunoglobulin M (IgM) in this newly developed RA model. TNF-α, NO and RF-IgM levels were significantly increased, while IL-1β levels were not affected in this MCIA rat model. The levels of IL-10 were lower than the baseline when compared with controls. In conclusion: (1) the immunological features represented in the MCIA rat model favour the Th-1 cytokine profile over Th-2 and (2) RF-IgM can be used as a diagnostic test in preclinical RA models.

Small intestine mucosal immune system response to high-fat-high-cholesterol dietary supplementation in male rats

The immune effects of high-fat-high-cholesterol (HFHC) diet supplementation were investigated in Sprague–Dawley male rats. The rats were administered HFHC for 8 weeks via oral gavage. Two control groups received either a normal rat diet (NRD) or skim milk along with NRD. The levels of interleukin (IL)-1β, IL-10 and tumour necrosis factor (TNF)-α were assessed, using enzyme-linked immunosorbent assay, in the ileum, the mesenteric lymph node (MLN), and the splenic tissue homogenates, and in blood. Histopathological examination of the intestinal tissues was performed and showed no abnormalities among groups. An HFHC supplementation resulted in a significant increment in the levels of IL-1β and IL-10 in the intestinal tissues. TNF-α concentration was only changed in MLN tissue homogenates where it was downregulated after HFHC treatment. No other changes were reported in the spleen tissue homogenate and serum. The cytokine concentrations proposed using HFHC diet as a safe immune manipulator for gastrointestinal mucosal surfaces.

High-dose vitamin C: Does it exacerbate the effect of psychosocial stress on liver? Biochemical and histological study

AIM Chronic stress has been implicated as a contributing factor in liver injury. However, other factors that can contribute to the severity of stress effect in liver injury have not been well characterized. In this study, the combined effect of chronic psychosocial stress and variable dosing levels of vitamin C on liver injury, have been studied. METHODS Stress was chronically induced using intruder method. Vitamin C was administered by oral gavage. Both biochemical and histopathological measures were undertaken. RESULTS The results showed that low (50mg/kg/day) and moderate (150 mg/kg/day) doses of vitamin C alone or in combination with chronic stress had no effect on liver. However, combination of high dose of vitamin C (500 mg/kg/day) and chronic stress induced various histopathological liver lesions in most of animals in the group that was stressed and supplemented with high dose vitamin C. CONCLUSION Results of this study show a dose-dependent effect for vitamin C in exacerbating stress contribution to liver injury.

Investigation on the occurrence and pathology of paratuberculosis (Johne’s disease) in apparently healthy cattle in Jordan

Paratuberculosis (Johne’s disease) is an infectious, incurable, chronically progressive granulomatous enteritis which affects domestic and exotic ruminants. The causative agent is Mycobacterium avium paratuberculosis (Mycobacterium johnei), a slow growing mycobactin-dependent acid-fast bacillus. We investigated the occurrence of Johne’s disease in apparently healthy cattle, using 263 ileum and corresponding mesenteric lymph nodes, by histopathological examination, and 170 ileum and 120 mesenteric lymph nodes by immunohistochemical examination. The occurrence of the disease was 65% and 66% using immunohistochemistry and histopathology techniques, respectively. When Ziehl-Neelsen (ZN) and ELISA techniques were implemented, the occurrence was 1% (4/120) and 3% (8/278), respectively. Grading from I–IV of histopathological lesions based on type of cellular infiltrate and severity of lesions revealed that most of the positive cases were in grade I and II. Furthermore, staging I–III of immunohistochemistry results, has presented a high number of positive cases in stage I. ZN stain showed a very low occurrence; however, it is still used as a confirmatory test for clinical cases. On the other hand, ELISA technique showed a low occurrence of the disease (3%) in this study reflecting the low sensitivity of the technique in diagnosis of subclinical Johne’s disease (JD). These results showed that histopathology is a very good diagnostic method for subclinical paratuberculosis in cattle. From the present study, we conclude that the occurrence of JD in cattle is high in Jordan. It is interesting to note that this is the first study of JD in cattle in Jordan, and the results strongly suggest alarming fears of severity of the disease at the national level. The urgent need for national control strategies are well founded due to the economical importance of the disease.

Presumptive Secondary Ethylene Glycol (Antifreeze) Toxicity in a Turkey Vulture (A Case Report)

A presumptive diagnosis of secondary ethylene glycol toxicity in a female Turkey vulture (Cathartes aura) was made depending on the histopathological changes found in the renal tubules and presence of Isospora spp. of non-avian type within the colon. To the best of our knowledge, this is the first reported case of possible secondary ethylene glycol toxicity in a Turkey vulture in Louisiana State/USA.

Effects of Sidr honey on second-intention healing of contaminated full-thickness skin wounds in healthy dogs

Abstract The therapeutic effects of Sidr honey on second-intention healing of contaminated full-thickness skin wounds in dogs were investigated. Povidone-iodine was used as a standard treatment and served as a control. Healthy adult (two-to-four-year-old) mongrels, comprising six dogs and two bitches, were divided into four equal groups in order to obtain multi-aged wounds at the end of the study. Four 2cm × 2cm full-thickness skin wounds were created on both sides of the back area of each dog under general anaesthesia adhering to aseptic technique. Contaminated wounds were then divided into two treatment groups: Group 1, Sidr honey treated (right side wounds) and group 2, povidone-iodine treated (left side wounds). All wounds were evaluated grossly daily at the time of treatment application and digitally photographed once every week. Images were analysed using ImageJ software. The parameters of unhealed wound area and length of advancing epithelium were obtained. The epithelialisation areas, percentage of wound area, and wound contraction rate were then calculated. No significant differences were found between the two treatment groups in any of the parameters studied. Overall, both honey treated and iodine treated wounds healed well within the time period of the study (28 d). However, the study showed a beneficial effect of Sidr honey on second-intention healing of full thickness contaminated wounds in healthy dogs and the effect was comparable to that of Povidone iodine.

Review of animal models used to study effects of bee products on wound healing: findings and applications

Abstract Non-healing wounds are associated with high morbidity and might greatly impact a patient’s well-being and economic status. For many years, scientific research has focused on developing and testing several natural and synthetic materials that enhance the rate of wound healing or eliminate healing complications. Honey has been used for thousands of years as a traditional remedy for many ailments. Recently, honey has reemerged as a promising wound care product especially for infected wounds and for wounds in diabetic patients. In addition to its proposed potent broad-spectrum antibacterial properties, honey has been claimed to promote wound healing by reducing wound hyperaemia, oedema, and exudate, and by stimulating angiogenesis, granulation tissue formation and epithelialisation. Several animal models, including large animals, dogs and cats, and different species of laboratory animals have been used to investigate the efficacy and safety of various natural and synthetic agents for wound healing enhancement. Interpreting the results obtained by these studies is, however, rather difficult and usually hampered by many limiting factors including great variation in types and origins of honey, the type of animal species used as models, the type of wounds, the number of animals, the number and type of controls, and variation in treatment protocols. In this article, we provide a comprehensive review of the most recent findings and applications of published experimental and clinical trials using honey as an agent for wound healing enhancement in different animal models.

Molecular and Histopathological Characterization of Sheep-Associated Malignant Catarrhal Fever (SA-MCF) Outbreak in Beef Cattle

An outbreak of suspected malignant catarrhal fever (MCF) was investigated by molecular and histopathological assays. Of the 70 Holstein beef calf herds, 14 were affected by multiple clinical signs suggestive of MCF infection. These beef calves were housed next to sheep flocks. In the complete blood count, the 14 affected calves had severe anaemia with leucopaenia, lymphopaenia and neutropaenia. Upon PCR amplification using a hemi-nested PCR assay for the detection of the Ovine herpesvirus 2 (OvHV-2), bovine tissue samples from the mesenteric lymph nodes and spleen and ovine blood samples were shown to be positive with the expected PCR bands amplified. Direct sequencing of the hemi-nested PCR product confirmed the identity of the causative virus as OvHV-2. The histopathological findings confirmed the clinical and laboratory diagnosis of MCF. Collective clinical, PCR and histopathological data confirmed the identity of this outbreak to be a sheep-associated malignant catarrhal fever (SA-MCF).

What is your diagnosis? Lymphoadenopathy in a cow

A 4-year-old Holstein Friesian cow was presented to the Veterinary Health Center, Faculty of Veterinary Medicine at Jordan University of Science and Technology with the primary complaint of lethargy, inappetence, and reduced milk production for 2-week duration. On physical examination, the cow was febrile and lethargic, the mucous membranes were pale and the respiratory rate was increased. Many of the subcutaneous lymph nodes were enlarged. Smears were prepared from a fine-needle aspirate of the prescapular lymph nodes and submitted for cytologic examination (Figure 1, 2). Peripheral blood was collected from the jugular vein and submitted for a CBC including differential counts. Erythrogram changes showed mild macrocytic normochromic anemia. Leukogram changes showed mild leukopenia and neutropenia. A thrombocytopenia was also present. Smears of peripheral blood were also examined under the microscope for evaluation of cell morphology. Figure 2. Fine-needle aspiration smear from enlarged prescapular lymph node of a cow. Diff-3 stain, Bar = 10 lm. Figure 1. Fine-needle aspiration smear from enlarged prescapular lymph node of a cow. Diff-3 stain, Bar = 10 lm.