Molecular characterization and pathology of field isolates of foot-and-mouth virus in Swiss albino mice.

Foot-and-mouth disease is responsible for severe economic losses to the livestock industry of Pakistan. This study aimed to use Swiss albino mice as a cost-effective experimental animal model to study different immunological and histopathological aspects of FMDV instead of natural targeted species like cattle. After isolation of field isolates FMDV on BHK-21 cell line, biological titer of the virus and mice infectious dose50 was calculated. Virus was injected in 45 Swiss albino mice (group A) through intraperitoneal route. The gross, histopathological and immunopathological lesions in heart, trachea and lungs were recorded at different day's intervals. Histopathologically, the heart showed congestion, hemorrhages and necrosis of cardiac muscles. Trachea showed deciliated epithelium and lungs showed hemorrhages, bronchial edema and alveolar emphysema. Immunohistochemical studies revealed the presence of virus in cardiac muscles, tracheal and bronchial epithelium and alveolar lumen. The findings evoked a thought that laboratory animals could be an alternative to large animals to meet budget limitations for further research on foot-and-mouth-disease.

Risk factors for outbreaks caused by variant strain of Newcastle disease on environmentally controlled broiler chicken farms in Lahore, Pakistan.

Newcastle disease (ND) is a frequently reported disease in poultry among both vaccinated and non-vaccinated flocks in Pakistan. During 2011-2012 poultry industry in Punjab, mainly in Lahore region, faced fatal outbreaks of ND caused by a variant strain. An analytical study was conducted during outbreak period in Lahore region. A total of 114 environmentally controlled farms were selected with the help of convenient sampling method. A questionnaire was designed about the potential risk factors associated with the spread of ND outbreak. The bivariate relationships between ND status and independent variables were investigated by applying the Chi-square and Fisher's exact test. Multivariable logistic model was used to estimate the effect of each studied variable on the outcome by adjusting the other variables in the model. The variables which showed an association with ND outbreaks at commercial poultry farms were improper method for dead birds disposal (OR=4.96; 95% CI 1.63-15.12), use of same feed transporting vehicle at multiple poultry farms (OR=4.92; 95% CI 1.58-15.33), farm to farm distance of less than 1 km (OR=9.32; 95% CI(1.19-73.12), number of sheds at one farm (OR=2.31; 95% CI 0.93-5.69), labor type (OR=2.72; 95% CI 0.83-8.88) and biosecurity (OR= 4.47; 95% CI 0.56-35.66).

RETRACTED ARTICLE: Assessment of aflatoxins in peanuts grown in the Pothohar area of Pakistan.

A survey was carried out to assess the mycotoxin (aflatoxins) contamination in locally grown peanuts. A total of 72 samples of raw, roasted and salty peanuts were collected randomly from the Pothohar Plateau of Pakistan. The samples were dried, ground and extracted by adding acetonitrile:water (84:16; v/v%). The filtered extracts were cleaned with MycoSep-226 columns and analysed by high-performance liquid chromatography with a fluorescence detector. The limit of quantification for aflatoxin B1 was 1 µg/kg with 70% recovery observed in spiked samples with a concentration range of 1-10 µg/kg. The results indicated that aflatoxins were present in almost 82% of the samples tested, with levels ranging from 14.3 to 98.8 µg/kg. This reflects that optimal conditions for fungal growth and mycotoxin contamination are frequent in peanut crop fields as well as in storehouses. Human exposure to such toxins can be controlled through appropriate measures, creating awareness and implementing regulations.

Sequence analysis and biological characterization of virulent Avian avulavirus 1 isolated from asymptomatic migratory fowl.

Continuous monitoring and surveillance of avian avulaviruses (AAvVs) in water/migratory fowl is imperative to ascertain the evolutionary dynamics of these viruses. Here, we report genomic and amino acid characteristics of two AAvVs strains isolated from asymptomatic waterfowl (Anas carolinensis). Sequence characteristics including the presence of virulent motif (112RRQKR↓F117) and biological assessment confirmed the virulent nature of study isolates. Phylogenetic analysis of complete fusion (F) and hemagglutinin-neuraminidase (HN), and hyper-variable region of F gene revealed clustering of both strains within genotype VII and sub-genotype VIIi. The inferred residue analysis of complete F and HN genes revealed a number of substitutions in functionally and structurally important motif/s compared to reference strains of each genotype (I-XI). This study concludes an evolutionary nature of avian avulavaris 1 (AAvV-1), ascertaining continuous surveillance of migratory fowl to better elucidate their infection, epidemiology and subsequent impacts on commercial and backyard poultry. Keywords: virulent AAvV-1; migratory fowl; genetic characterization; evolutionary analysis; Pakistan.

Seroprevalence of Bluetongue Virus in small ruminants in Balochistan province, Pakistan.

Bluetongue (BT), caused by bluetongue virus (BTV), is a vector-borne disease of small ruminants that has the potential to spread across international borders. Despite large populations of susceptible animals and borders with BTV endemic countries, little is known of the disease burden and prevalent serotypes in the province of Balochistan in Pakistan. We conducted a cross-sectional study to determine seroconversion and prevalent serotypes in selected districts of the province using a competitive enzyme-linked immunosorbent assay (cELISA) and real-time polymerase chain reaction (RT-PCR). Sera (n = 876) were collected from clinically healthy sheep and goats originating from the districts of Quetta (n = 300), Mastung (n = 201), Killa Saifullah (n = 75) and Kech (n = 300). None of the study herds (n = 97) were seronegative for BTV, and at the individual level, the overall prevalence of BTV seroconversion was 47.26% (n = 414/876, 95% CI = 43.92%-50.63%). A higher percentage of goats (50.87%, 95% CI = 45.99%-55.73%) were seropositive for anti-VP7 immunoglobulins (IgG) than sheep (44.21%, 95% CI = 39.81%-48.70%). Odds ratios of seroconversion for goats were associated with breed type (χ2  = 16.84, p = .01), parity (χ2  = 23.66, p = .00) and presence of vector (χ2  = 2.63, p = .10), whereas for sheep, it was associated with breed type (χ2  = 13.80, p = .01) and parity (χ2  = 53.40, p = .00). Serotype 8 was the most prevalent (26.82%, 95% CI = 14.75%-43.21%) followed by an equal prevalence of serotypes 2 and 9 (7.31%, 95% CI = 1.91%-21.01%). To the best of our knowledge, this is the first study conducted in Balochistan province and the results indicate that there is a necessity to initiate intervention strategies to control BT disease burden not only in this region of Pakistan but also in adjacent areas of the neighbouring countries, Iran and Afghanistan.

Molecular evidence shows that the liver fluke Fasciola gigantica is the predominant Fasciola species in ruminants from Pakistan.

Fascioliasis is an important disease affecting livestock, with great costs to producers worldwide. It has also become a serious issue for human populations in some endemic areas as an emerging zoonotic infection. There are two Fasciola species of liver fluke responsible for this disease, which occur worldwide, Fasciola hepatica and Fasciola gigantica. Identifying these two species on the basis of adult or egg morphology requires specialist knowledge due to the similarity of characters, and may misidentify putative intermediate or hybrid forms. In this study we sequenced the internal transcribed spacer 2 (ITS-2) rDNA of liver flukes collected from multiple species of hosts from seven localities in the Punjab and Baluchistan provinces of Pakistan, to determine the distribution of these two species. All 46 flukes processed in this study, collected from seven sites, showed the rDNA ITS-2 genotype corresponding to F. gigantica, contradicting previous reports, based on adult and egg morphology, that both species are present in Pakistan, with F. hepatica being the more common.

Prevalence of Neospora caninum antibodies in sheep and goats in Pakistan.

The purpose of the present study was to obtain seroepidemiological information on the Neospora caninum infection status of sheep and goats in different areas of Punjab Province and Azad Kashmir (Pakistan). A cross-sectional study, with the use of a competitive ELISA, showed an overall 27.7% (35 of 128) (95% confidence interval [CI] ± 7.7%) and 8.6% (13 of 142) (95% CI ± 4.6%) seroprevalence of N. caninum antibodies in sheep and goats, respectively. The difference in seroprevalence between sheep and goat populations was statistically significant (P < 0.05). The highest prevalence (37.4% ± 13.2%) was recorded in the tailless breed of sheep.

Single or combined effects of mannan-oligosaccharides and probiotic supplements on the total oxidants, total antioxidants, enzymatic antioxidants, liver enzymes, and serum trace minerals in cyclic heat-stressed broilers.

This study was intended to explore the oxidative status of broilers under cyclic heat stress (HS) as modulated by supplementation of mannan-oligosaccharides (MOS) and a probiotic mixture (PM). Two hundred fifty 1-d-old chicks were randomly divided into 5 groups. From d 22, birds were either kept in a thermoneutral zone (TN; 26.7°C for 24 h/d) or subjected to HS (35 ± 1.1°C and 75 ± 5% RH for 8 h/d from 1000 to 1800 h) to the conclusion of the study on d 42. Birds were fed either a corn-based diet (TN and HS groups) or the same diet supplemented with 0.5% MOS (HS-MOS group), 0.1% PM (HS-PM group), or their combination as a symbiotic (SYN; HS-SYN group). On d 42, birds were killed by cervical dislocation to collect serum for the determination of total oxidants, total antioxidants, paraoxonase, arylesterase, ceruloplasmin, aspartate aminotransferase, alanine aminotransferase, and trace minerals. Heat stress increased (P < 0.05) total oxidants and total antioxidants and decreased (P < 0.05) paraoxonase and arylesterase, with no change in ceruloplasmin, aspartate aminotransferase, and alanine aminotransferase activities. Dietary supplementation decreased (P < 0.05) total oxidants and total antioxidants, with no effect on the activities of other enzymes. Heat stress did not influence serum copper, zinc, and manganese concentrations of birds when compared with those in the TN group. However, MOS increased (P < 0.05) concentrations of all the trace minerals, whereas SYN increased (P < 0.05) concentrations of only zinc and copper. It was concluded that MOS or PM supplementation, alone or as a SYN, may reduce some of the detrimental effects of HS, whereas MOS alone or as a SYN may improve the absorption of trace minerals.

Seroprevalence of Neospora caninum in dairy buffaloes in Lahore District, Pakistan.

Seroprevalence of Neospora caninum in dairy buffaloes ( Bubalus bubalis ) was assessed in the Lahore District of Punjab Province, Pakistan. The study revealed an overall prevalence of 54.7% for N. caninum antibodies determined through a competitive enzyme-linked immunosorbent assay performed on randomly collected serum samples. The highest prevalence was observed in buffaloes >3-5 yr of age (64.1%), followed by 57.9% for 5 to 6 yr olds, and 55.8% in 1-yr-old neonates, with high probability of infection under intensive dairy farming conditions. The pattern of prevalence was closely associated with the season as reflected by the highest prevalence (70.5%) in summer (May-August) and lowest (39.6%) in winter (November-January). Aborting buffaloes illustrated significantly higher (78.9%) exposure compared with non-aborting dams (59.8%). Prevalence in animals with canine contact was significantly higher (60.3%) than without contact (48.1%). This is the first reported prevalence of N. caninum in Pakistan.