Pakistani Strains Research Articles

Toxicity and resistance of field collected Musca domestica (Diptera: Muscidae) against insect growth regulator insecticides

The house fly, Musca domestica, is a serious pest of public health importance with the ability to develop insecticide resistance. The focus of the present study was to evaluate toxicity and resistance of the field collected house flies from Punjab, Pakistan, against insect growth regulator (IGR) insecticides. House flies collected from six different localities exhibited very low levels of resistance to cyromazine, triflumuron, and methoxyfenozide compared with the Lab-susceptible reference strain, with resistance ratios (RRs) ranging between 3.56- to 8.19-fold, 1.45- to 3.68-fold, and 2.20- to 8.60-fold, respectively. However, very low to low levels of resistance were observed for pyriproxyfen and very low to moderate levels for lufenuron with RRs ranged from 4.13- to 11.63-fold to 8.57- to 22.75-fold, respectively. There was a significant correlation between RRs of cyromazine and triflumuron (r = 0.976, p < 0.001), suggesting the possibility for cross-resistance. Susceptibility status of different IGRs in Pakistani strains of house flies is reported here for the first time. The trend observed in decreasing susceptibility towards different IGR insecticides tested will continue unless resistance management practices are followed.

Epidemiology and Genetic Diversity of Rotavirus Strains in Children with Acute Gastroenteritis in Lahore, Pakistan

Pakistan harbors high disease burden of gastro-enteric infections with majority of these caused by rotavirus. Unfortunately, lack of proper surveillance programs and laboratory facilities have resulted in scarcity of available data on rotavirus associated disease burden and epidemiological information in the country. We investigated 1306 stool samples collected over two years (2008–2009) from hospitalized children under 5 years of age for the presence of rotavirus strains and its genotypic diversity in Lahore. The prevalence rate during 2008 and 2009 was found to be 34% (n = 447 out of 1306). No significant difference was found between different age groups positive for rotavirus (p>0.05). A subset of EIA positive samples was further screened for rotavirus RNA through RT-PCR and 44 (49.43%) samples, out of total 89 EIA positive samples, were found positive. G and P type prevalence was found as follows: G1P [4] = 3(6.81%); G1P [6] = 9(20.45%); G1P [8] = 1(2.27%); G2P [4] = 21(47.72%); G2P [8] = 1(2.27%); G9P [4] = 1(2.27%); G9P [6] = 1(2.27%) and G9P [8] = 7(15.90%). Phylogenetic analysis revealed that the VP7 and VP4 sequences clustered closely with the previously detected strains in the country as well as Belgian rotaviruses. Antigenic characterization was performed by analyzing major epitopes in the immunodominant VP7 and VP4 gene segments. Although the neutralization conferring motifs were found variable between the Pakistani strains and the two recommended vaccines strains (Rotarix™ and RotaTeq™), we validate the use of rotavirus vaccine in Pakistan based on the proven and recognized vaccine efficacy across the globe. Our findings constitute the first report on rotavirus’ genotype diversity, their phylogenetic relatedness and epidemiology during the pre-vaccination era in Lahore, Pakistan and support the immediate introduction of rotavirus vaccine in the routine immunization program of the country.

Comparison of the virulence markers of helicobacter pylori and their associated diseases in patients from Pakistan and Afghanistan

Background/Aim:Helicobacter pylori is a Gram-negative bacteria, which is associated with development of gastroduodenal diseases. The prevalence of H. pylori and the virulence markers cytotoxin-associated gene A and E (cagA, cagE) and vacuolating-associated cytotoxin gene (vacA) alleles varies in different parts of the world. H. pylori virulence markers cagA, cagE, and vacA alleles in local and Afghan nationals with H. pylori-associated gastroduodenal diseases were studied.Patients and Methods:Two hundred and ten patients with upper gastrointestinal symptoms and positive for H. pylori by the urease test and histology were included. One hundred and nineteen were local nationals and 91 were Afghans. The cagA, cagE, and vacA allelic status was determined by polymerase chain reaction.Results:The nonulcer dyspepsia (NUD) was common in the Afghan patients (P = 0.025). In Afghan H. pylori strains, cagA was positive in 14 (82%) with gastric carcinoma (GC) compared with 29 (45%) with NUD (P = 0.006), whereas cagE was positive in 11 (65%) with GC and 4 (67%) with duodenal ulcer (DU) compared with 12 (18%) with NUD (P < 0.001 and 0.021, respectively). The vacA s1a/b1 was positive in 10 (59%) of GC compared with 20 (31%) in NUD (P = 0.033). In Pakistani strains, cagE was positive in 12 (60%) with GC, 7 (58%) with GU, 12 (60%) with DU compared with 11 (16%) with NUD (P < 0.001, 0.004, and < 0.001, respectively). In Pakistani strains, cagA/s1a/m1 was 39 (33%) compared with Afghans in 17 (19%) (P = 0.022). Moderate to severe mucosal inflammation was present in 51 (43%) Pakistani patients compared with 26 (28%) (P = 0.033) in Afghans. It was also associated with grade 1 lymphoid aggregate development in Pakistani patients 67 (56%) compared with 36 (40%) (P = 0.016) in Afghans.Conclusion:Distribution of H. pylori virulence marker cagE with DU was similar in Afghan and Pakistan H. pylori strains. Chronic active inflammation was significantly associated with Pakistani H. pylori strains.

Amino acid diversity of antigenic sites of Iranian type O foot-and-mouth disease virus

Foot-and-mouth disease (FMD) is a major cause of endemic outbreaks in livestock in Iran. In this study, clinical field samples of foot-and-mouth disease virus were collected from an outbreak in Khorasan Razavi Province during April and August of 2010, and subjected to indirect sandwich ELISA and RT-PCR. The viral serotype circulating during the period was confirmed to be type O. The virus was then genetically characterized for its complete P1 genomic sequences to compare with nine corresponding nucleotide sequences of representative foot-and-mouth disease viruses (FMDVs) registered in the GenBank. The P1-coding region was 2208 nucleotides in length with 736 encoded amino acid residues. Phylogenetic analysis revealed two major lineages of A (with three additional clusters) and B. Iranian field isolate was grouped within cluster I, most closely related to Pakistani strains PAK/39/2008 and PAK/29/2008 sharing 98.37 and 98.1% amino acid identity, respectively, demonstrating the close epidemiological links between countries in the region. In contrast, our isolate showed low amino acid identity with Italian isolate O-2-Brescia (93.48%) and Argentinean isolate O1 Caseros (93.75%). Based on multiple sequence alignments, comparison of sequences showed that the characteristic amino acid mutations were found in the VP1, VP2 and VP3 proteins of isolated virus. This article is the first to report on the complete P1 genomic characterization of type O FMDV circulating in Iran

PVII-13 Waterborne outbreaks of acute gastroenteritis in January-May 2009, Turkey

As a part of strategy to control diarrheal diseases, World Health Organization (WHO) recommends to include rotavirus vaccines in national immunization programs. Sentinel surveillance networks have been established to monitor rotavirus disease burden and genotype distribution in both pre and post vaccine era in many countries. Unfortunately, due to lack of proper surveillance programs, data on rotavirus disease burden and genotype distribution from Pakistan is scarce. We investigated 502 stool samples from children (< 5 years) hospitalized due to gastroenteritis in Rawalpindi, Pakistan during 2014 for the presence of group A rotavirus (RVA) and its genotypic diversity. Among 147 ELISA positive samples, 131 were successfully genotyped for RVA. Common G types detected were G1 (23.6%), followed by G3 (22.9%), G12 (19.8%), G2 (19.08%) and G9 (9.9%). The most common P-type was P[8] (41.2%), followed by P[6] (29%) and P[4] (28.24%). G3P[8] (17.55%) was the most prevalent genotype combination followed by G12P[6] (16.7%), G2P[4] (15.2%) and G1P[8] (14.5%). Mixed infection of rotavirus G-P types was also observed in 6% of samples. Phylogenetic analysis of VP7 and VP4 genes of Pakistani strains showed that G1, G2, G9 and P[4], P[6], P[8] were closely related to strains circulating worldwide as well as previously reported strains from Pakistan. Pakistani G12P[8] strains NIH-BBH-3981 and NIH-BBH-4003 belonged to lineage 3 cluster 3a along with strains from USA and Italy whereas G12P[6] strains NIH-BBH-3978, NIH-BBH-4052 and NIH-BBH-4444 were closely related to strains from Italy, Thailand, United Kingdom and with previously reported G12 strains from Pakistan within lineage 3 cluster 3b. This pre-vaccination data supports the need for RVA vaccine inclusion at our national level and will be helpful in assessing the effect of vaccination on RVA genotype diversity due to vaccine selection pressure once post-vaccination data becomes available.

Potent Bactericidal Constituents from Mallotus philippinensis against Clarithromycin and Metronidazole Resistant Strains of Japanese and Pakistani Helicobacter pylori

In the quest for potent anti-Helicobacter pylori agents, we found 70% EtOH extract of Mallotus philippinensis (LAM.) MUELL. (MPM) with strong bactericidal activity at the concentration of 15.6-31.2 mg/l against eight H. pylori strains. Further fractionation and purification of 70% EtOH extract of MPM led to the isolation of 5 compounds, namely 5,7-dihydroxy-8-methyl-6-prenylflavanone (1), 3'-prenylrubranine (2), red compound (3), isorottlerin (4), and rottlerin (5) which were elucidated on the basis of nuclear magnetic resonance and mass spectroscopy. Among the isolated compounds, rottlerin exhibited most potent bactericidal activity with minimum bactericidal concentration (MBC) value of 3.12-6.25 mg/l against several clinical H. pylori isolates including Japanese and Pakistani strains, nine clarithromycin resistant (CR), and seven metronidazole resistant (MR) strains. Minimum inhibitory concentration (MIC) values of CR (8->256 mg/l) and MR (>256 mg/l) strains were analyzed by E test. Moreover, the clarithromycin resistant strains were evaluated for A2143G and A2144G point mutations of 23s rRNA gene to correlate the MBC values with mutation type. Our results revealed the potent in vitro anti-H. pylori activity of MPM and rottlerin, specially against CR and MR strains, which could be gainfully utilized for the development of novel antimicrobials to prevent H. pylori related disorders.

Delayed cuticular penetration and enhanced metabolism of deltamethrin in pyrethroid‐resistant strains of Helicoverpa armigera from China and Pakistan

The penetration and metabolism of [14C]deltamethrin was studied in susceptible and resistant Chinese and Pakistani strains of Helicoverpa armigera (Hübner), which were resistant to deltamethrin by 330- and 670-fold, respectively. The penetration of deltamethrin into resistant individuals was significantly slower than into susceptible individuals over a 24-h period. The time taken for 50% penetration of the applied deltamethrin was 1 h for the susceptible strain and 6 h for both of the resistant strains. The internal radioactivity was reduced by the larvae of resistant strains much faster than by the susceptible larvae. After 48 h, 40% of the penetrated deltamethrin was still inside the larvae of the susceptible strain, in comparison with 1.5-5% in the Pakistani strain and 8-14% in the Chinese strain. Both of the resistant strains produced methanol-soluble and water-soluble metabolites, but the susceptible strain produced methanol-soluble metabolites only. By 12, 24 and 48 h after dosing, the amount of methanol-soluble metabolites excreted by the resistant strains was almost double that of the susceptible strain. Both of the resistant strains also excreted 5-7% of the penetrated dose as a water-soluble metabolite after 48 h. In comparison with the Chinese strain, the Pakistani strain exhibited slower penetration, lower internal content and faster excretion of deltamethrin, which correlated with the higher resistance of the Pakistani strain. These findings show that the resistant Pakistani and Chinese strains of H. armigera possess mechanisms of reduced cuticular penetration and enhanced metabolism of deltamethrin and perhaps other pyrethroids.

Characterization of the glycoproteins of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) virus is the cause of an important tick-borne disease of humans throughout regions of Africa, Europe, and Asia. Like other members of the genus Nairovirus, family Bunyaviridae, the CCHF virus M genome RNA segment encodes the virus glycoproteins. Sequence analysis of the CCHF virus (Matin strain) M RNA segment revealed one major open reading frame that potentially encodes a precursor polyprotein 1,689 amino acids (aa) in length. Comparison of the deduced amino acid sequences of the M-encoded polyproteins of Nigerian, Pakistani, and Chinese CCHF virus strains revealed two distinct protein regions. The carboxyl-terminal 1,441 aa are relatively highly conserved (up to 8.4% identity difference), whereas the amino-terminal 243 to 248 aa are highly variable (up to 56.4% identity difference) and have mucin-like features, including a high serine, threonine, and proline content (up to 47.3%) and a potential for extensive O-glycosylation. Analysis of released virus revealed two major structural glycoproteins, G2 (37 kDa) and G1 (75 kDa). Virus protein analysis by various techniques, including pulse-chase analysis and/or reactivity with CCHF virus-specific polyclonal and antipeptide antibodies, demonstrated that the 140-kDa (which contains the mucin-like region) and 85-kDa nonstructural proteins are the precursors of the mature G2 and G1 proteins, respectively. The amino termini of the CCHF virus (Matin strain) G2 and G1 proteins were established by microsequencing to be equivalent to aa 525 and 1046, respectively, of the encoded polyprotein precursor. The tetrapeptides RRLL and RKPL are immediately upstream of the cleavage site for mature G2 and G1, respectively. These are completely conserved among the predicted polyprotein sequences of all the CCHF virus strains and closely resemble the tetrapeptides that represent the major cleavage recognition sites present in the glycoprotein precursors of arenaviruses, such as Lassa fever virus (RRLL) and Pichinde virus (RKLL). These results strongly suggest that CCHF viruses (and other members of the genus Nairovirus) likely utilize the subtilase SKI-1/S1P-like cellular proteases for the major glycoprotein precursor cleavage events, as has recently been demonstrated for the arenaviruses.

Relative Activity of Various Esterases in Six Pakistani Strains of the Lesser Grain Borer, Rhyzopertha dominica (Fabricius)

Acetylcholinesterase (AChE), arylesterase (AE), carboxylesterase (CE), and cholinesterase (ChE) activities were determined in six adult populations of the lesser grain borer, Rhyzopertha dominica (Fabricius) (Coleoptera: Bostrichidae) collected from grain warehouses in various areas of Punjab, namely Lahore (L), Sialkot (S), Wazirabad (W), Multan (M), Chichawatni (C), and Karachi (K). The multiple forms of esterases were resolved by polyacrylamide gel electrophoresis (PAGE) and activities determined biochemically, using a spectrophotometer. All the strains had the same AChE activity except Sialkot strain, which had 46% more activity than the susceptible K strain. Similarly K, S, W, L, and C strains exhibited almost the same AE level, whereas the M strain showed 29% lower activity than K strain. The CE activity of L (352%), S (198%) and C (214%) strains and ChE activity of L (178%) and C (317%) strains were significantly higher than that of K strain. The total esterases of L, S and C strains showed respectively 152%, 63% and 73% higher activity than the susceptible K strain. The PAGE pattern of various esterases coincided with the biochemical analysis. The variable thickness of the bands in the gel indicated relative esterase induction that could be correlated with the development of resistance in R. dominica.

Characterization of Foxtail Millet, Setaria italica (L.) P. Beauv., in Pakistan Based on Intraspecific Hybrid Pollen Semi-sterility.

Sixteen strains of foxtail millet, Setaria italica (L.) P. Beauv., originally collected from Pakistan were crossed with three strains which had previously been used as testers for classifying six landrace groups based on hybrid pollen sterility. Wide variation was observed in pollen fertility of the F1 hybrids, ranging from 14.2% to 94.7%, while pollen fertility of all the selfed Pakistani strains and testers was normal. Pakistani strains were classified into type C (8 strains), type BC (2) and type X (6) on the basis pollen fertility of more than 75% is normal fertility. No A, B or AC type strains were detected. Type C was found in land-races collected from Punjab Province, the southern part of the North-West Frontier Province (N. W. F. P.) and Baltistan. Type X was distributed in the northern part of N. W. F. P. and an adjacent area of the western part of Gilgit Agency, in the northeastern part of Gilgit Agency and in Baltistan. Different landrace groups were suggested among the strains of type X by the variation in hybrid sterility observed with each tester. This observation was strongly supported by an almost sterile hybrid between type X strains from N. W. F. P. and the northeastern part of Gilgit Agency. Type BC was found in Gilgit Agency and Baluchistan. Landrace groups classified by hybrid pollen fertility did not show any clear relationship with morphological groups which had a different geographical distribution. The present results imply that S. italica, with various phylogenetic backgrounds, might have been introduced into this region several times, and subsequently evolved into three distinct morphological groups.