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.

Human and animal brucellosis in Jordan between 1996 and 1998: a study.

Between 1996 and 1998, a total of 2,494 samples of blood from humans and animals were collected and tested for brucellosis. This total included 1,594 samples of animal blood, collected from 1,050 sheep from 20 flocks, and 544 goats from eight herds. The serum samples were tested using the Rose Bengal test, the tube agglutination test, the complement fixation test and an enzyme-linked immunosorbent assay. Moreover, a complete history was compiled from each flock/herd. The rate of abortions in sheep due to brucellosis ranged from 0.5% to 56%, with a mean of 33.2%. The goats had a higher abortion rate. Thirty-four aborted sheep foetuses collected from these 20 flocks were bacteriologically and pathologically examined. A pure culture of Brucella melitensis biotype 3 was isolated from 21 of the aborted foetuses. The human blood samples were collected from two groups: first, from 800 apparently healthy people who were reporting to community hospitals for routine health checks and secondly, from 100 people from groups with a high-risk of contracting brucellosis, such as veterinarians, sheep-herders and laboratory technicians. The Brucella antibody titres for the 900 human serum samples were obtained using the microtitre agglutination test. The cumulative percentage of the serum samples showing a titre reading greater than 1:80 was higher in the at-risk group than among the normal population (7% compared to 4.1%). Although these results were not statistically significant, the higher percentage of positive reactors among the high-risk group may indicate an increased risk factor among professional agricultural and veterinary personnel in Jordan. It was concluded that brucellosis is common in sheep and goats in Jordan, subjecting the human population to high risks. Brucella melitensis Rev. 1 vaccination has been internationally recognised as the key to successfully controlling the disease. All animals in Jordan were repeatedly vaccinated between 1996 and 1998 on a trial basis, using a reduced dose of 1 x 10(5) colony-forming units (CFU). Cumulative data on the annual rate of human cases of brucellosis indicate that fewer people are affected each year. The same is true for the rate of abortions in animals. Such evidence strongly suggests that the vaccination programme has been successful. However, as wild strains of Brucella have also been isolated from vaccinated animals, the authors recommend increasing the amount of vaccine to a full dose of 1 to 2 x 10(9) CFU and vaccinating young female animals between the ages of three and eight months. To avoid brucellosis in humans, people should be educated about the dangers of contact with infected animals and the consumption of raw milk and milk products.

Equine babesiosis associated with strenuous exercise: clinical and pathological studies in Jordan.

Clinical, haematological and pathological studies were undertaken in Jordan in a stud of 103 racing horses clinically suffering from babesiosis and apparently healthy animals. Out of 47 horses which participated in strenuous exercise, three mares showed sudden onset of immobility and reluctance to move and two mares died. Clinical examination revealed that these five horses (group 1) had fever, anorexia, weakness and severe icterus and, in two mares, haemoglobinuria. Haematological examination revealed that all five horses were heavily parasitized with Babesia equi. This was also found in four horses (group 2) with no evidence of clinical babesiosis. In group 3 (94 horses), neither clinical signs nor B. equi were observed in the blood. The horses in group 1 and 2 recovered after treatment with imidocarb. When the mean values of white blood cell count, red blood cell count, haemoglobin and packed cell volume in group 1 were compared with those for groups 2 and 3, a significant difference was found (P < 0.05). A significant difference was also found when the mean values were compared before and after treatment. Examination of serum total protein, bilirubin and serum enzymes revealed a significant decrease in the mean value of total serum protein (P < 0.05), and a significant increase in the mean values of bilirubin (P < 0.05) in group 1 compared to groups 2 and 3. A significant elevation in the mean value of aspartate aminotransaminase, gamma-glutamyltransferase and creatine phosphokinase and a substantial elevation in the mean value of alkaline phosphatase was also observed in group 1 compared to groups 2 and 3. Postmortem examination of the dead horses showed that the animals had icterus, hepatomegaly and full urinary bladder with deep-red urine. Histopathological examination of the liver showed massive centrilobular degeneration and necrosis. The bile canaliculi and bile ducts were prominent and plugged with dark-brown to canary-coloured bile pigments. The lungs had congestion, oedema, and thrombosis of pulmonary veins. Our results suggest that the horses suffered from B. equal with clinical manifestation following exercise. The clinical, haematological and pathological findings indicate that the animals suffered from haemolytic anaemia which responded to imidocarb therapy.

A technique for identification of cellular proteins by western immunoblotting and immunoautoradiography of 2-D PAGE.

A major effort of our work has been devoted to the identification, using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), of lymphoid polypeptides that are involved in lymphoid proliferation or differentiation. We have encountered problems during this effort pertaining to the exact localization of a polypeptide(s) in the silver stained gels that is recognized by western immunoblotting or proteins detected by autoradiography. In this paper, we present a method, using India ink stained/immuno-staining replica of 2-D gel nitrocellulose membrane (NCM) or India ink stained coupled with autoradiography in the case of phosphoproteins, which allows us to exactly localize the polypeptide spots detected by these methods in the silver stained 2-D PAGE. This method is expected to popularize and widen the use of 2-D PAGE technology in the investigation of cellular polypeptides.