Fowl adenovirus: history, emergence, biology and development of a vaccine against hydropericardium syndrome.

The poultry industry has emerged as one of the largest and fastest growing public sectors in the developed and developing countries. Unfortunately, this industry is under a major threat from diseases that are viral (Newcastle disease, infectious bursal disease, influenza, hydropericardium syndrome), bacterial (colibacillosis, pasteurellosis, salmonellosis, mycoplasmosis), parasitic (coccidiosis, histoplasmosis) or nutritional (dyschondroplasia, osteoporosis). Among these diseases, hydropericardium syndrome (HPS) is one of the important emerging diseases occurring in the specific areas of the world where broilers (chickens) are reared under intensive conditions. HPS was first observed in 1987 at Angara Goth, an area near Karachi, Pakistan, where broilers are raised. Since then, HPS has been reported in many countries of the world. From these reported cases, an adenovirus that was either isolated from or visualized electron microscopically in the liver of affected broilers has been implicated in the syndrome. The syndrome has been reproduced by inoculation of isolated fowl adenovirus (FAdV) strains, and hence, the syndrome is also called infectious hydropericardium syndrome. To our knowledge, HPS has not been observed in humans, so it is not considered a zoonotic disease, but it is of economic importance and causes huge losses to the poultry industry. Efforts have been made to develop conventional vaccines against this disease, which were formulated from infected liver homogenate. Formalin-inactivated liver organ vaccines have failed to protect the poultry industry. Hence, there is a dire need to develop a suitable vaccine to combat this disease. Currently, recombinant vaccine candidates are being developed by using molecular biology and biotechnological approaches for the prevention and control of infectious diseases, including HPS. Therefore, it is suggested that the immunogenicity of these recombinant proteins should be evaluated for their use as subunit vaccines.

Molecular cloning, expression and characterization of 100K gene of fowl adenovirus-4 for prevention and control of hydropericardium syndrome.

Fowl adenovirus-4 is an infectious agent causing Hydropericardium syndrome in chickens. Adenovirus are non-enveloped virions having linear, double stranded DNA. Viral genome codes for few structural and non structural proteins. 100K is an important non-structural viral protein. Open reading frame for coding sequence of 100K protein was cloned with oligo histidine tag and expressed in Escherichia coli as a fusion protein. Nucleotide sequence of the gene revealed that 100K gene of FAdV-4 has high homology (98%) with the respective gene of FAdV-10. Recombinant 100K protein was expressed in E. coli and purified by nickel affinity chromatography. Immunization of chickens with recombinant 100K protein elicited significant serum antibody titers. However challenge protection test revealed that 100K protein conferred little protection (40%) to the immunized chicken against pathogenic viral challenge. So it was concluded that 100K gene has 2397 bp length and recombinant 100K protein has molecular weight of 95 kDa. It was also found that the recombinant protein has little capacity to affect the immune response because in-spite of having an important role in intracellular transport & folding of viral capsid proteins during viral replication, it is not exposed on the surface of the virus at any stage.

Insecticide sprays, natural enemy assemblages and predation on Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae).

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama is considered a key citrus pest due to its role as vector of 'huanglongbing' (HLB) or citrus greening, probably the most economically damaging disease of citrus. Insecticidal control of the vector is still considered a cornerstone of HLB management to prevent infection and to reduce reinoculation of infected trees. The severity of HLB has driven implementation of intensive insecticide programs against ACP with unknown side effects on beneficial arthropod fauna in citrus agroecosystems. We evaluated effects of calendar sprays directed against this pest on natural enemy assemblages and used exclusion to estimate mortality they imposed on ACP populations in citrus groves. Predator exclusion techniques were used on nascent colonies of D. citri in replicated large untreated and sprayed plots of citrus during the four major flushing periods over 2 years. Population of spiders, arboreal ants and ladybeetles were independently assessed. Monthly sprays of recommended insecticides for control of ACP, adversely affected natural enemy populations resulting in reduced predation on ACP immature stages, especially during the critical late winter/early spring flush. Consequently, projected growth rates of the ACP population were greatest where natural enemies had been adversely affected by insecticides. Whereas, this result does not obviate the need for insecticidal control of ACP, it does indicate that even a selective regimen of sprays can impose as yet undetermined costs in terms of reduced biological control of this and probably other citrus pests.

Essential medicines management during emergencies in Pakistan.

This paper illustrates the experiences of essential medicine management in providing cure and care to victims of Pakistan's 2005 earthquake in a safe, rational and effective mode. The health interventions assured access to essential medicine, sustained supply, inventory control through a computerized logistic support system and rational use of medicines. World Health Organization Pakistan outlined modalities for acceptance of donated medicines, assisted in speedy procurement of medicines and designed customized kits. Proper storage of medicines at controlled temperature was ensured in warehousing facilities in 12 locations. A steady supply of medicines and their consumption without stock-outs in the 56 first-level care facilities of calamity-hit areas helped to ascertain the average consumption and cost of essential medicines and supplies for the catchment population. Tools for quantification and forecasting of medicines and supplies were developed and shared. Medicines and medical supplies were efficiently used resulting in minimum wastage.

Human organ and tissue transplantation in Pakistan: when a regulation makes a difference.

Organ transplantation must be viewed in relation to the prevailing cultural, religious and economic conditions of a nation. Over the past two decades, Pakistan has emerged as one of the largest centres for commercial renal transplantation. Government efforts, supported by professional associations, civil society organizations and the media, along with World Health Organization technical assistance, have led to the development of legislation regulating this practice and curbing organ trade in conformity with international guidelines. Although only two years have passed since the enactment of the law, there is evidence that conditions have significantly improved, raising hopes for ethical and safe organ transplantation in Pakistan. This study reviews the salient features of the legislation and lists the foreseeable evolving challenges and opportunities.

Comparison of laboratory colonies and field populations of Tamarixia radiata, an ectoparasitoid of the Asian citrus psyllid, using internal transcribed spacer and cytochrome oxidase subunit I DNA sequences.

The genetic diversity of Tamarixia radiata Waterston (Hymenoptera: Eulophidae) laboratory colonies derived from collections in China, northern Vietnam, Pakistan, and a mixed colony from Taiwan and southern Vietnam was evaluated using the internal transcribed spacer (ITS) region 1, ITS-2, and the 5' end of the cytochrome oxidase subunit I gene. The strains share the same ITS sequence, consistent with the morphological hypothesis that the collections represent a single species. The COI marker was variable and could distinguish the northern Vietnam and Pakistan colonies from each other and from the other colonies. Comparison of COI sequences from field-collected populations of Puerto Rico, Guadeloupe, and Texas indicates that Florida is not a likely source of the introduction into Puerto Rico but is a likely source of the introduction into Texas.

Prevention of beta-thalassemia in a large Pakistani family through cascade testing.

BACKGROUND: We report cascade testing of a large Pakistani family for beta-thalassemia alleles. The family was still practicing consanguineous marriages and was at risk of having more affected births. OBJECTIVE: The objective of this study was to show that identification of disease carriers in families with index cases in order to create awareness about disease and provide genetic counseling would result in reduction of the frequency of beta-thalassemia in Pakistan. METHODS: In this large family with an index case, 27 available living members were tested for beta-thalassemia. Carriers of the disease were detected by measuring hemoglobin indices, and amplification refractory mutation system polymerase chain reaction was used for mutation analysis. Genetic counseling was provided to members of this family. RESULTS: There were already 3 marriages between the carrier members and 1 between a carrier and noncarrier in this large family; 12 (44.4%) members were found to carry the mutant gene, representing a very high carrier rate compared to the 5.4% carrier frequency of beta-thalassemia in the general population of Pakistan. The family was counseled for prevention of affected births. The initially reluctant family gradually became cooperative and seriously attended the genetic counseling sessions. CONCLUSION: Cascade testing is more practical than general population screening in a country with limited health facilities where consanguineous marriages are practiced. This report emphasizes the need of extensive testing within families with index cases to identify the carriers of beta-thalassemia in order to reduce disease occurrence through awareness and genetic counseling.

Positive correlation of fitness with group size in two biotypes of Russian wheat aphid (Homoptera: Aphididae).

Changes in fitness parameters as a function of colony size (one versus 10 aphids) were measured in two biotypes (RWA1 and RWA2) of the Russian wheat aphid, Diuraphis noxia (Mordvilko) (Homoptera: Aphididae), feeding on three cultivars of wheat, Triticum aestivum L., at two temperatures. 'Trego' is a cultivar with no specific resistance to D. noxia, whereas, 'Stanton' and 'Halt' express Dny and Dn4 resistance sources, respectively. Feeding in a group accelerated the development of RWA1 on Trego and Stanton at 20 degrees C, but not at 24 degrees C, whereas grouped RWA2 developed faster than solitary RWA2 on all three cultivars at 24 degrees C, but not at 20 degrees C. Survival (first instar-adult) of RWA2 also was improved by grouping on Stanton and Halt at 24 degrees C, but solitary RWA2 survived better at 20 degrees C on all three cultivars. The reproductive rate of RWA1 was improved by grouping on Trego and Stanton at both temperatures, but only on Halt at 24 degrees C. Lifetime fecundity of RWA1 also was increased by grouping in all cases except for Trego at 20 degrees C. Grouped development increased the reproductive rate of RWA2 on all three cultivars at 24 degrees C, but had no effect at 20 degrees C. Grouped RWA2 developed and reproduced faster than grouped RWA1 on all three cultivars at 24 degrees C. Thus, the fitness of D. noxia was positively correlated with group size during colony establishment, but the effects were sensitive to temperature, being more pronounced at 20 degrees C for RWA1 and at 24 degrees C for RWA2.

Detailed deletion mapping of chromosome 9p and p16 gene alterations in human borderline and invasive epithelial ovarian tumors.

We used PCR amplification of tandem repeats to study the pattern of allelic loss in borderline and invasive ovarian epithelial tumors using 12 primer pairs to generate a detailed deletion map of chromosome 9p. In the invasive ovarian carcinomas, there were three regions displaying high frequency of loss of heterozygosity (LOH) ranging from 31-38%. In contrast, LOH was a rare event among the borderline ovarian tumors, with one region revealing a rate of 20% and the remaining regions only 0-8% LOH. Therefore, allelic loss does not seem to be important for the development of borderline ovarian tumors. We also examined p16 gene expression and mutations in ovarian cancer cell lines and invasive and borderline ovarian tumor tissues. Southern blot analysis revealed no losses of the p16 gene in either the invasive or borderline ovarian tumors. However, the ovarian carcinoma cell lines showed a 50% homozygous deletion rate. SSCP analysis detected a mobility shift in only one (borderline) tumor. Since the primary invasive ovarian tumors did not show any deletions or mutations, it appears that p16 does not play a role in the pathogenesis of these tumors.

Sampling Methods for Detection and Monitoring of the Asian Citrus Psyllid (Hemiptera: Psyllidae).

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama is a key pest of citrus due to its role as vector of citrus greening disease or "huanglongbing." ACP monitoring is considered an indispensable tool for management of vector and disease. In the present study, datasets collected between 2009 and 2013 from 245 citrus blocks were used to evaluate precision, sensitivity for detection, and efficiency of five sampling methods. The number of samples needed to reach a 0.25 standard error-mean ratio was estimated using Taylor's power law and used to compare precision among sampling methods. Comparison of detection sensitivity and time expenditure (cost) between stem-tap and other sampling methodologies conducted consecutively at the same location were also assessed. Stem-tap sampling was the most efficient sampling method when ACP densities were moderate to high and served as the basis for comparison with all other methods. Protocols that grouped trees near randomly selected locations across the block were more efficient than sampling trees at random across the block. Sweep net sampling was similar to stem-taps in number of captures per sampled unit, but less precise at any ACP density. Yellow sticky traps were 14 times more sensitive than stem-taps but much more time consuming and thus less efficient except at very low population densities. Visual sampling was efficient for detecting and monitoring ACP at low densities. Suction sampling was time consuming and taxing but the most sensitive of all methods for detection of sparse populations. This information can be used to optimize ACP monitoring efforts.

An epidemic of Dengue fever in Karachi--associated clinical manifestations.

Dengue is a mosquito borne disease with worldwide distribution. Depending on virulence factors and host immune status, infection can manifest as a severe but non fatal viral syndrome or a rapidly progressive and a frequently fatal haemorrhagic fever. During the course of an outbreak of viral fever in Karachi, Pakistan from June, 1994 to September, 1995, we studied 145 cases admitted at the Aga Khan University Hospital. 43% of the cases were between the age group of 20-30 years, majority of these being male (75%). Amongst the clinical signs and symptoms, the most frequent findings were fever, vomiting and abdominal pain. Spontaneous hemorrhagic manifestations occurred in 66 patients and of these petechiae and mucosal bleed were the commonest, that is, 42% and 38% respectively. At presentation thrombocytopenia (platelet count < or = 50,000) per cubic millimeter) was present in 78%, leucopenia (white cell count < 4,000 per cubic millimeter) in 34%. Apart from one patient who died from hemorrhagic shock on the 5th day of admission, the remaining patients recovered and their platelet counts normalized on an average in 9 days. This is the first reported epidemic in Karachi of dengue infection.

Early detection of typhoid by polymerase chain reaction.

BACKGROUND: Typhoid is a common problem in developing countries. Cultivation of bacteria and serology (especially Widal test) give unacceptable levels of false-negative and false-positive results, respectively. PATIENTS AND METHODS: In this study, a recently introduced polymerase chain reaction-based technique (which has 100% specificity for Salmonella typhi) was compared with blood culture and Widal test during the first week of illness of 82 suspected cases of typhoid. RESULTS: The respective figures of positivity for PCR, blood culture and Widal test were 71.95%, 34.1%, and 36.5%. A control group of 20 healthy persons gave figures of 0%, 0%, and 33.3%, respectively. CONCLUSION: We conclude that this PCR-based technique is not only absolutely specific, but also very sensitive and, therefore, much superior to blood culture and Widal test for the early diagnosis of typhoid.

A subunit vaccine against hydropericardium syndrome using adenovirus penton capsid protein.

Hydropericardium syndrome (HPS) is a disease of poultry that is caused by fowl adenovirus-4. Inactivated liver homogenate from diseased birds is still the choice of vaccine in some countries which disseminates numerous pathogens along with inactivated virus. Moreover incomplete attenuation or inactivation, reversion to virulence and the oncogenic potential/genetic instability of the adenoviruses have prevented their use in routine vaccines. To address this problem an effort is made to develop a subunit vaccine. For this purpose penton base protein of HPS virus was expressed in Escherichia coli and used as subunit vaccine in broilers. Immunogenicity of the recombinant penton base protein and challenge protection test against pathogenic virus demonstrated the ability of recombinant penton base protein to confer (90%) protection. Results suggest that the recombinant penton base protein is a candidate for subunit vaccine against HPS.

Identification of hepatitis B virus core mutants by PCR-RFLP in chronic hepatitis B patients from Punjab, Pakistan.

Chronic hepatitis B virus (HBV) infection remains a major health issue worldwide. Several factors including core gene variation are responsible for the development of chronicity of HBV infection. The present study was designed to identify the variations in the core region of the HBV genome in a local population of chronic hepatitis B patients (n = 57) using a PCR-based restriction fragment length polymorphism (PCR-RFLP) method. Fifty subjects were found to be positive for the presence of HBV DNA. For the core region genotyping, the Ava II and Msp I restriction enzymes were used. Mutations at nucleotide (nt) 2147 and nt 2362 in the HBV genome in the core region for Ava II (A4 type, 74%) and nt 2331 for Msp I (M1 type, 66%) were observed as the most common pattern. These results are different from those of previously reported studies on other populations and thus appear to be unique to the Pakistani population. This type of characterization of core mutants may be useful for the design of vaccines based on viral epitopes that are effective for the Pakistani population. Moreover, these unique genotypic patterns for the HBV core gene might be some of the main factors responsible for understanding the underlying mechanism by which HBV chronicity is developed in the Pakistani population.

Adventitious shoot induction and somatic embryogenesis with intact seedlings of several hybrid seed geranium (Pelargonium x hortorum Bailey) varieties.

Intact seedlings of hybrid seed geranium (Pelargonium x hortorum Bailey) were tested for their ability to produce adventitious shoots and somatic embryos by direct culture of mature seeds on Murashige and Skoog (1962) medium (MS) supplemented with growth regulators BAP, BAP + IAA, or thidiazuron (TDZ). Ten varieties were tested in the presence of different BAP concentrations, four with BAP + IAA, and two with TDZ. Varieties used in this study differed in their response to BAP in the medium. Multiple adventitious shoots were produced by seven of the ten varieties tested. Multiple adventitious shoots were induced at all levels of TDZ in the medium. TDZ also induced callusing from roots and direct embryogenesis from intact hypocotyls. Adventitious shoots were separated, rooted and transferred to soil where they grew as normal healthy plants and flowered.

Phenylacetic acid-induced somatic embryogenesis in cultured hypocotyl explants of geranium (Pelargonium x hortorum Bailey).

The effect of a non-indole compound, phenylacetic acid (PAA), on the induction of somatic embryogenesis in tissue cultures of geranium (Pelargonium x hortorum Bailey cv. Scarlet Orbit Improved) was investigated. Hypocotyl explants derived from young, dark-grown seedlings were cultured on Murashige and Skoog (1962) medium (MS) supplemented with PAA or IAA (0.01-120 µM) alone or in combination with BAP (8 µM). Somatic embryogenesis was induced by both PAA and IAA at 0.01-20 µM with 8 µM BAP, however, the optima differed considerably for the two compounds. Maximal activity of IAA for somatic embryogenesis was found at 0.1-2.5 µM, whereas PAA gave best results at 10 and 20 µM under identical culture conditions. Higher concentrations (30-120 µM) of IAA or PAA in the medium induced callusing in the explants, but the callus was neither embryogenic nor morphogenic.

Cryopreservation of immature spring wheat zygotic embryos using an abscisic acid pretreatment.

Spring wheat (Triticum aestivum L.) zygotic embryos were successfully cryopreserved, without the addition of exogenous cryoprotectants, using only an abscisic acid (ABA) pretreatment. Optimum survival was obtained when embryos were cultured in vitro for 10 days on semisolid Murashige and Skoog (MS) nutrient medium supplemented with 0.5 mg/L (±) ABA prior to cryopreservation. The embryos resumed growth within three days when returned to MS medium devoid of ABA but containing 2mg/L 2,4-dichlorophenoxyacetic acid. The embryogenic calli produced from these embryos exhibited normal plant regeneration on auxin-free media. Changes in dw/fw ratio, as well as the esterified fatty acid and sucrose concentrations correlated positively with the development of tolerance to cryopreservation.

Characterization ofAzospirillum and related diazotrophs associated with roots of plants growing in saline soils.

Diazotrophs, especially of genusAzospirillum were readily isolated from roots of many plants using semi-solid nitrogen free malate medium (NFM). These isolates formed fine, white sub-surface pellicle in nitrogen-free malate medium within 24h, which gradually moved to the surface, and exhibited high acetylene reduction rates. Using selected cultural and biochemical tests, most of the isolates were identified asAzospirillum brasilense. Four isolates from Kallar grass root surface and one isolate fromAtriplex root interior formed phenotypically a homogenous group. It shared many characteristics with the species of genusAzospirillum except shape. All the biochemical tests performed, categorized them withA. brasilense. However, the shape and the protein profile on SDS polyacrylamide gel electrophoretograms suggested that the group of these five isolates is clearly distinct and differs widely from all the type strains, belonging to various genera.

Morphoregulatory role of thidiazuron : substitution of auxin and cytokinin requirement for the induction of somatic embryogenesis in geranium hypocotyl cultures.

Somatic embryogenesis was induced in hypocotyl explants of geranium (Pelargonium x hortorum) cultured on media supplemented with various concentrations of N-phenyl-N'-1,2,3-thiadiazol-5-ylurea (thidiazuron). In less than 2 weeks, somatic embryos were observed in treatments containing levels of thidiazuron (TDZ) ranging from 0.2 to 1.0 micromolar. The use of N(6)-benzylaminopurine in combination with indole-3-acetic acid also evoked embryogenesis, but the efficiency of somatic embryo production was significantly lower than that obtained with TDZ. Hypocotyl culture for only 2 days on TDZ-supplemented medium before transfer to a basal medium was sufficient for inducing somatic embryogenesis. This distinction between the induction and expression of embryogenesis may provide an experimental system for studying the developmental biology of somatic embryogenesis. Substitution of the auxin-cytokinin requirement for the induction of somatic embryogenesis by TDZ suggests the possibility of a novel mode of its action by modulation of endogenous growth regulators.

Metal tolerance in tissue cultures of anthoxanthum odoratum.

Tissue cultures were initiated from one non-tolerant (S20) and two zinc and lead tolerant (T92 and T94) clones of Anthoxanthum odoratum. Growth of callus from the non-tolerant clone was reduced by the presence of zinc, lead, copper and nickel, whereas callus from the two tolerant clones showed no reduction of growth in the presence of zinc and lead but growth was reduced by copper and nickel. The specificity of metal tolerance shown by the parental material was maintained in the callus. Tolerant and non-tolerant callus accumulated similar amounts of zinc and lead.