Shahzad Munir

Quantification of antibodies against poultry haemagglutinating viruses by haemagglutination inhibition test in Lahore

A study was conducted for quantification of antibodies to haemagglutinating viruses such as AIV (H9N2 and H7N3), NDV and IBV in vaccinated poultry flocks. A total of 198 serum samples from different commercial broiler and layer farms in Lahore, Pakistan, were checked for their serological statuses. Haemagglutination inhibition (HI) test was performed to determine the titer of serum antibodies against the above mentioned viruses. It was observed that 25 and 44% samples were positive for H7N3 and H9N2 in broilers, respectively. The seropositivity of H7N3 and H9N2 in serum samples of commercial layers were 66 and 94%, respectively. It was recorded that 96% of the samples in broilers and 100% of those in layers, were found seropositive for antibodies against NDV. Moreover, 69 and 86% were seropositive for IBV in broilers and layers, respectively. Calculated geometric mean titers for groups of 0-2, 2-4 and 4-6 weeks of age were found to be 36.8, 78.8, and 111.4 for H9N2; 48.5, 157.6, 222.9 for NDV; and 97, 84.4, 111.4 for IBV, respectively. It was concluded that the samples were not having protective antibody titers indicating unsuccessful vaccination practiced in these poultry farms which may lead to significant economic losses.

Epidemic outbreak of anthroponotic cutaneous leishmaniasis in Kohat District, Khyber Pakhtunkhwa, Pakistan

An epidemiological and molecular study was carried out for the first time in Kohat District of Khyber Pakhtunkhwa (KP) province, Pakistan from April 2015 to May 2016 to determine the prevalence of Cutaneous Leishmaniasis (CL) in local population and Internally Displaced People (IDPs). In 13 different villages, a total of 1359 (out of 26,250 individuals belonging to local population) and 140 (out of 3615 IDPs residing in these villages) cases were recorded and 300 samples were collected. The total prevalence of CL in local population was 5.17% with active lesions and scar prevalence of 3.91% and 1.26% respectively. Similarly a prevalence of 3.86% for CL was recorded in IDPs. Highest number of IDPs having CL active lesions and scars were recorded in villages Sherkot, Surgul, and Jarma and their presence was positively correlated with CL in local population. Age wise prevalence was highest in young children of age group 1-15 years. The microscopic examination showed 64.33% (193/300) positive samples while kinteoplastic PCR showed 84.66% (254/300) positive. For the first time in KP province, 2/784 sandflies trapped from the study villages was found positive for Leishmania by PCR. Restriction Fragment Length Polymorphism of patients and sandflies samples revealed L. tropica as the prevalent Leishmania species in this district. The results of sequencing and RFLP identified L. tropica in Phlebotomus sergenti. This is the first ever report of molecular identification of L. tropica from sandflies of genus P. sergenti in Khyber Pakhtunkhwa province. This data can be helpful for health authorities in finding out new CL foci and to plan effective strategies for the provision of health facilities to poor people of this area.

First report on molecular characterization of Leishmania species from cutaneous leishmaniasis patients in southern Khyber Pakhtunkhwa province of Pakistan

OBJECTIVE To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis (CL) is endemic and was thought to be caused by Leishmania tropica only. METHODS Biopsy samples from 432 CL suspected patients were collected from 3 southern districts of Khyber Pakhtunkhwa during years 2011-2016. Microscopy on Giemsa stained slides were done followed by amplification of the ribosomal internal transcribed spacer 1 gene. RESULTS Leishmania amastigotes were detected by microscopy in 308 of 432 samples (71.3%) while 374 out of 432 samples (86.6%) were positive by ribosomal internal transcribed spacer 1 PCR. Subsequent restriction fragment length polymorphism confirmed L. tropica in 351 and L. major in 6 biopsy samples. CONCLUSIONS This study is the first molecular characterization of Leishmania species in southern Khyber Pakhtunkhwa. It confirmed the previous assumptions that anthroponotic CL is the major CL form present in Khyber Pakhtunkhwa province. Furthermore, this is the first report of L. major from a classical anthroponotic CL endemic focus identified in rural areas of Kohat district in southern Khyber Pakhtunkhwa.

Epidemiology of Cutaneous Leishmaniasis Outbreak, Waziristan, Pakistan

During 2013–2015, prevalence of cutaneous leishmaniasis in war-affected Waziristan areas was 3.61% by PCR. Youths (1–15 years of age) were more susceptible. Internal transcribed spacer 1 PCR followed by restriction fragment length polymorphism analysis identified Leishmania tropica in 215 samples and Leishmania major in 6 samples.

Identification for the First Time of Cyclo(d-Pro-l-Leu) Produced by Bacillus amyloliquefaciens Y1 as a Nematocide for Control of Meloidogyne incognita

The aim of the current study was to describe the role and mechanism of Bacillus amyloliquefaciens Y1 against the root-knot nematode, Meloidogyne incognita, under in vitro and in vivo conditions. Initially, the exposure of the bacterial culture supernatant and crude extract of Y1 to M. incognita significantly inhibited the hatching of eggs and caused the mortality of second-stage juveniles (J2), with these inhibitory effects depending on the length of incubation time and concentration of the treatment. The dipeptide cyclo(d-Pro-l-Leu) was identified in B. amyloliquefaciens culture for the first time using chromatographic techniques and nuclear magnetic resonance (NMR 1H, 13C, H-H COSY, HSQC, and HMBC) and recognized to have nematocidal activity. Various concentrations of cyclo(d-Pro-l-Leu) were investigated for their effect on the hatching of eggs and J2 mortality. Moreover, the in vivo nematocidal activity of the Y1 strain was investigated by conducting pot experiments in which tomato plants were inoculated with M. incognita. Each and every pot was amended 50 mL of fertilizer media (F), or Y1 culture, or nematicide (N) (only once), or fertilizer media with N (FN) at 1, 2, 3, 4 and 5 weeks after transplantation. The results of the pot experiments demonstrated the antagonistic effect of B. amyloliquefaciens Y1 against M. incognita as it significantly decreases the count of eggs and galls per root of the tomato plant as well as the population of J2 in the soil. Besides, the investigation into the growth parameters, such as the length of shoot, shoot fresh and dry weights of the tomato plants, showed that they were significantly higher in the Y1 strain Y1-treated plants compared to F-, FN- and N-treated plants. Therefore, the biocontrol repertoire of this bacterium opens a new insight into the applications in crop pest control.

Characterization of dengue virus in Aedes aegypti and Aedes albopictus spp. of mosquitoes: A study in Khyber Pakhtunkhwa, Pakistan

Dengue is a vector-borne disease caused by dengue virus. According to the recent report of CDC that one-third population of the world are at high risk with Dengue fever. The prevalence of the dengue hemorrhagic fever was found more in tropical and sub-tropical regions of the world. Aedes mosquitoes was reported as the main cause of transmission of dengue virus. So the current study was planned to characterize the virus in Aedes mosquitoes collected from different area of Pakistan. In current investigation, Aedes mosquitoes and larvae were trapped under conducive conditions which are counted as 495 Aedes mosquitoes and 260 Aedes larvae. First of all, adult mosquitoes were identified morphologically under microscopy, counted as 73.3% Ae. aegypti and 26.7% Ae. albopictus. Finally, reverse transcriptase polymerase chain reaction analyses that only 4 adults of Aedes mosquitoes and 10 Aedes larvae as naturally infected with dengue virus with possible source Ae. aegypti. This study basically uncovered the presence of virus in different species of mosquitoes in southern regions of Pakistan. The present study will also give us an insight for vector control programs of dengue virus in the affected area.

Huanglongbing Control: Perhaps the End of the Beginning

Huanglongbing (HLB) is one of the most destructive citrus plant diseases worldwide. It is associated with the fastidious phloem-limited α-proteobacteria ‘Candidatus Liberibacter asiaticus’, ‘Ca. Liberibacter africanus’ and ‘Ca. Liberibacter americanus’. In recent years, HLB-associated Liberibacters have extended to North and South America. The causal agents of HLB have been putatively identified, and their transmission pathways and worldwide population structure have been extensively studied. However, very little is known about the epidemiologic relationships of Ca. L. asiaticus, which has limited the scope of HLB research and especially the development of control strategies. HLB-affected plants produce damaged fruits and die within several years. To control the disease, scientists have developed new compounds and screened existing compounds for their antibiotic and antimicrobial activities against the disease. These compounds, however, have very little or even no effect on the disease. The aim of the present review was to compile and compare different methods of HLB disease control with newly developed integrative strategies. In light of recent studies, we also describe how to control the vectors of this disease and the biological control of other citrus plant pathogens. This work could steer the attention of scientists towards integrative control strategies.

Acaricidal Activity of Cyclodipeptides from Bacillus amyloliquefaciens W1 against Tetranychus urticae

Bioassay-guided fractionation of the supernatant of the biocontrol strain Bacillus amyloliquefaciens W1 led to the isolation of eight acaricidal cyclodipeptides from the active fractions by column chromatography separation and HPLC purification. The chemical structures of these compounds were identified as cyclo-(Gly-l-Phe), 2, cyclo-(l-Phe- trans-4-OH-l-Pro), 3, cyclo-(Gly-l-Tyr), 4, cyclo-(l-Ala-l-Pro), 5, cyclo-(l-Pro- trans-4-OH-l-Pro), 6, cyclo-(Gly-l-Pro), 7, cyclo-(l-Pro-l-Pro), 8, and cyclo-(l-Tyr- trans-4-OH-l-Pro), 9. Those cyclodipeptides displayed significant acaricidal activities with LC50 values of 13.85-98.24 μM. Cyclo-(l-Tyr- trans-4-OH-l-Pro) (LC50 13.85 μM) was five times more effective than the positive control abamectin (LC50 72.06 μM). The results indicated that the hydroxyl group is an important component. This is the first report on the acaricidal capabilities of cyclodipeptides against Tetranychus urticae. The results revealed that the acaricidal activity of the biocontrol strain B. amyloliquefaciens W1 was dependent on its constituent cyclodipeptides, which have the potential to be safe and environmentally friendly acaricides.

Molecular detection of Leishmania species in human and animals from cutaneous leishmaniasis endemic areas of Waziristan, Khyber Pakhtunkhwa, Pakistan

Objectives: To detect Leishmania species in human patients, animal reservoirs and Phlebotomus sandflies in Waziristan, Pakistan. Methods: Tissue smears and aspirates from 448 cutaneous leishmaniasis (CL) suspected patients were analyzed. To sort out role of the reservoir hosts, skin scrapings, spleen and liver samples from 104 rodents were collected. Furthermore, buffy coat samples were obtained from 60 domestic animals. Sandflies were also trapped. All human, animals and sandfly samples were tested by microscopy, kinetoplastic PCR and internal transcribed spacer 1 (ITS1) PCR followed by restriction fragment length polymorphism for detection of Leishmania species. Results: An overall prevalence of 3.83% and 5.21% through microscopy and ITS1 PCR respectively was found. However, the statistically non-significant correlation was found between area, gender, and number of lesions. The presence of rodents, sandflies, domestic animals and internally displaced people increased the risk of CL. Using ITS1-PCR-RFLP, Leishmania tropica (L. tropica) was confirmed in 106 samples while 25 of the isolates were diagnosed as Leishmania major (L. major). Similarly, 3/104 rodents were positive for L. major and 14 pools of DNA samples containing Phlebotomus sergenti sandflies were positive for L. tropica. None of samples from domestic animals were positive for leishmaniasis. Conclusions: In the present study, L. tropica and L. major are found to be the main causative agents of CL in study area. Movement of internally displaced people from CL endemic areas presents a risk for nearby CL free areas. To the best of our knowledge, we report for the first time L. major infection in rodents (Rattus rattus) and L. tropica in Phlebotomus sergenti sandflies trapped in Waziristan, Pakistan.