Farmer's knowledge and suggested approaches for controlling aflatoxin contamination of raw milk in Pakistan.

Monitoring of aflatoxin levels in milk is often complicated in developing countries due to the dominance of informal markets channeling milk in raw form. Farmer's awareness and voluntary participation in aflatoxin mitigation can be critical in such scenarios. Therefore, the present study was conducted to understand the perceptions of dairy farmers about aflatoxins and link it with aflatoxin mitigation programs on milk in Pakistan. Information was collected from 450 peri-urban dairy farmers in seven cities using questionnaires. Majority (77.9%) of the farmers were aware of the negative impact of moldy feed on animal health. However, only 40.6% of the farmers were aware of the transferability of the toxins from moldy feed to milk. The farmers had almost no awareness of aflatoxins as 95% never heard of the term. After receiving an onsite briefing on effects of the toxin on animal and human health, and its transferability to milk, 98.3% farmers showed willingness to buy aflatoxin-safe feedstuffs, while 88.5% showed willingness to control aflatoxin in milk. Around half of the farmers considered aflatoxin control programs as affordable. On average, farmers agreed to pay 10.1% higher price for aflatoxin certified oilseed cakes. Availability of feedstuffs certified of low aflatoxin content was suggested by 22% of the participants as the critical step in reducing aflatoxins in milk. Other important suggestions included; subsidy on quality feeds (18%), raising awareness (18%), and legislation and monitoring (16%). The present results suggest that the current practice of milk monitoring in the country can yield desirable results only if it is coupled with feed certification programs ensuing availability of aflatoxin-safe feeds. Further, awareness can positively impact participation of producers in aflatoxin control programs. In this regard, awareness about effects of aflatoxins on animal health was found to be a more powerful trigger of voluntary control compared with the awareness of the toxin's transferability to milk.

Genetic characterization of peste des petits ruminants virus (Pakistani isolates) and comparative appraisal of diagnostic assays.

This study was designed to characterize N gene sequences of peste des petits ruminants virus (PPRV) isolates circulating in Pakistan and to evaluate the efficacy of available diagnostic assays on local isolates. During the study period, a total of sixty PPR outbreaks were investigated. A total of 20 selected samples from these outbreaks were sequenced for N gene. The result analysis and the phylogenetic trees indicated two different viral groups in N gene: one was closer to China and Tajikistan, while other group was similar to isolates from Iran and Saudi Arabia. Efficacy of three commercially available tests for the antigen detection of PPR, that is, peste test, enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) was compared. Keeping PCR as gold standard, sensitivity was calculated as 85% and 57% and specificity was calculated as 83% and 79% for ELISA and peste test, respectively. Value of K for ELISA was 0.67 which indicates good agreement between ELISA and RT-PCR. Value of K for peste test was 0.33 which indicates fair agreement between peste test and RT-PCR. In conclusion, study provides premier information about the use of different diagnostic tests and molecular situation of PPRV in Pakistan.

Aflatoxin Contamination of Milk Produced in Peri-urban Farms of Pakistan: Prevalence and Contributory Factors.

Aflatoxin M1 contamination of milk in Pakistan, like many developing countries, is poorly understood. The present study was therefore conducted to determine AFM1 contamination of milk and its contributory factors in Pakistan. We sampled milk and feedstuffs from 450 peri-urban dairy farms in seven major cities following a cross-sectional study design. Analysis of milk using ELISA revealed high contamination with an overall average of 3164.5 ng of AFM1/L, and significant differences (p < 0.001) between cities. The milk sampled from Gilgit, in northern hilly areas, had an average AFM1 level of 92.5 ng/L. Milk from other cities had 3529.7 ng/L average contamination, with only 5.7% samples qualifying the maximum tolerable limit of 500 ng of AFM1/L. Heavy mean aflatoxin contamination was found in bakery waste (724.6 µg/kg), and cottonseed cake (600.8 µg/kg). Rest of the other feedstuffs had moderate to low mean aflatoxin contamination, ranging from 66.0 µg/kg in maize stover to 3.4 µg/kg in wheat bran. The mean aflatoxin level in commercial dairy concentrates was 32.7 µg/kg. About 80% of the total aflatoxin intake of dairy animals was contributed by cottonseed cake alone due to its high aflatoxin contamination and proportion in dairy rations. On-farm storage time of oilseed cakes varied (p < 0.01) in different cities but was not associated with aflatoxin contamination. The exceptionally high AFM1 contamination suggests that milk from peri-urban dairy farms is a serious public health threat in Pakistan. This situation can be mitigated by reducing aflatoxin contamination in cottonseed cake and promoting the use of commercial concentrates and other feedstuffs with low contamination.

Aflatoxin Contamination of Milk Marketed in Pakistan: A Longitudinal Study.

A longitudinal one-year study was conducted to determine aflatoxin M1 levels in different types of milk marketed in Pakistan. Processed and raw liquid milk from 21 sources, two milk powder and six tea whitener brands were sampled on monthly basis from Islamabad. The aflatoxin M1 levels in liquid milk were lower (p < 0.05) in summer (April to July) compared with the levels in winter (January, November and December). The mean aflatoxin M1 levels were 254.9, 939.5, and 1535.0 ng/L in UHT, pasteurized, and raw milk, respectively (differing at p < 0.001). The mean toxin level in powdered milk after reconstitution was 522.1 ng/L. Overall, 12.9, 41.0, 91.9 and 50.0% of the UHT, pasteurized, raw and powdered milk samples, respectively, exceeded the Codex maximum tolerable limit of 500 ng of aflatoxin M1/L. It was estimated that consumers of raw and processed milk were exposed to 11.9 and 4.5 ng aflatoxin M1, respectively, per kg of body weight daily. The study indicates potential aflatoxin M1 exposure risks for the consumers of raw milk in the country. The levels of the toxin though comparatively lower in milk powder, requires attention as this type of milk is consumed by infants.

Comparison of Some ELISA Kits for Aflatoxin M1 Quantification.

Background: ELISA is a widely used method for aflatoxin M1 (AFM1) quantification in dairy products. This study was conducted to compare ELISA kits from different manufacturers for AFM1 quantification in milk. Methods: High sensitivity ELISA kits (for up to 250 ng AFM1/L), including AgraQuant (Romer Labs), Bioshield M1ES (Prognosis Biotech), Helica 96 (Helica Biosystems), Veratox (Neogen, Inc.), and the medium sensitivity kit Immunolab AM1E01 (Immunolab GmbH; for 10-1000 ng AFM1/L) were tested against a certified reference AFM1 whole milk having 44 ng AFM1/L and its 10-fold dilution, and a 50 ng AFM1/L standard. In another experiment, Prognosis Bioshield M1UF (70-1000 ng AFM1/L), Romer's AgraquantPlus (10-2000 ng AFM1/L), and Immunulab's AM1E01 kits were compared to test a 500 ng AFM1/L solution. Results: In both of the experiments, the quantification of the tested AFM1 levels did not differ (P ≥ 0.310) between the kits from various manufacturers. In the case of 4.4 ng AFM1/L, recovery of the toxin was close to the assigned value under the kit by Neogen, Inc. In case the of 44 ng level, better recoveries were seen under the kits by Immunolab and Prognosis Biotech. In both the cases, low relative standard deviation (RSD) values were obtained only for the kit by Prognosis Biotech. In the case of the 50 and 500 ng AFM1/L solutions, the kits by Prognosis Biotech and Romer Labs provided recoveries close to the assigned values as well as low RSD. Conclusions: These data indicate that all the studied ELISA kit brands had comparable efficacy for AFM1 quantification. Recovery of the toxin and RSD may, however, differ between kits from various manufacturers.

Serum cation profile of broilers at various stages of exposure to deoxynivalenol.

The present experiment was carried out to investigate if levels of serum cations in broilers are modulated differently at various stages of exposure to deoxynivalenol (DON). Male broiler chicks at 7 days of age were fed a basal diet (0.27 mg of DON; 0.01 mg of zearalenone/kg), or either a low DON diet (1.68 mg of DON; 0.15 mg of zearalenone/kg) or a high DON diet (12.21 mg of DON; 1.09 mg of zearalenone/kg) produced using extracts from Fusarium graminearum cultures. Blood samples from the birds were collected during weeks 2, 4, and 5 of exposure. The high DON diet resulted in lower serum calcium levels compared to the basal diet at all the 3 sampling stages, while the low DON diet resulted in lower serum calcium levels only during weeks 2 and 5. Serum potassium levels were reduced under both the DON diets during weeks 2 and 5, while no diet-associated changes were found for serum levels of magnesium, sodium, and zinc. Under the present experimental conditions, the serum levels of calcium were consistently modulated in the broilers exposed to the DON-contaminated diets. The modulation of serum levels of potassium was, however, dependent upon the stage of exposure to DON.

Electrophysiological response of chicken's jejunal epithelium to increasing levels of T-2 toxin.

The present investigations were conducted to test the effects of T-2 toxin on electrophysiological variables of jejunal epithelium of chicken. Jejunal segments of broilers were monitored in Ussing chambers in the presence of T-2 toxin at the levels of 0 (negative control), 0 (methanol/vehicle control), 0.1, 1, 5, and 10 µg/ml of buffer. T-2 toxin did not affect basal values of short circuit current (I(sc)), transmural potential difference, or tissue conductivity in the jejunal epithelium. T-2 toxin also did not statistically affect glucose-induced electrophysiological variables during the first 3 min of glucose induction. Compared to the vehicle control, the ouabain-sensitive I(sc) was negatively affected (P = 0.008) only under 5 µg of T-2 toxin/ml. Increasing levels of T-2 toxin negatively affected the ouabain-sensitive I(sc) in a cubic (P = 0.007) fashion. These data indicate that acute exposure to moderate levels of T-2 toxin may progressively impair the cation gradient across the jejunal epithelium.

Dose-dependent increase and decrease in active glucose uptake in jejunal epithelium of broilers after acute exposure to ethanol.

Little is known about the effects of ethanol on gastrointestinal tract of chicken. In this study, we investigated the effects of low levels of ethanol on electrophysiological variables of jejunal epithelium of commercial broilers. Jejunal tissues from 35- to 39-day-old broilers were exposed to either 0 or 0.1% ethanol in Ussing chambers, and electrophysiological variables were monitored for 40 min. After 40 and 60 min of incubation, glucose (20 mM) and carbamoylcholine (200 µM), respectively, were introduced into the chambers. The absolute and percent increase in short-circuit current (Isc) and potential difference (Vt) induced by glucose were increased significantly with 0.1% ethanol. There was no significant effect of 0.1% ethanol on carbamoylcholine-induced electrophysiological variables. To investigate if higher levels of ethanol have similar effects, we tested the effects of 0, 0.33, and 0.66% ethanol under similar experimental conditions until the glucose-addition step. Contrary to 0.1% ethanol, both the 0.33 and 0.66% ethanol levels significantly decreased the basal and glucose-induced Isc and Vt. Tissue conductivity remained unaffected in all cases. These results indicate that intestinal epithelia of chicken may be more sensitive to the effects of ethanol as compared with other species. This is the first report indicating dose-dependent increase and decrease in active glucose absorption in intestinal epithelia in the presence of ethanol.

Blood plasma levels of deoxynivalenol and its de-epoxy metabolite in broilers after a single oral dose of the toxin.

To evaluate the transfer of deoxynivalenol (DON) and its de-epoxy metabolite (de-epoxy-DON) in the plasma of chicken, mashed oats naturally contaminated with 9.5 mg DON/kg were fed to four broilers (35 days age) at a dose of 20 g/bird. Blood samples were then collected from two birds at 1 h, 3 h, and 5 h post-feeding, while from the other two birds at 2 h, 4 h, and 6 h post-feeding. Analysis of DON and de-epoxy-DON was carried out by using liquid chromatography-tandem mass spectrometry after clean-up with immunoaffinity columns. At 1 h, 3 h, and 5 h post-feeding, the average values of plasma DON were 0.35 ng/ml, 0.20 ng/ml, and 0.15 ng/ml, respectively. The corresponding average values of de-epoxy-DON at these time points were 0.70 ng/ml, 0.80 ng/ml, and 0.25 ng/ml, respectively. The sum of DON and de-epoxy-DON appearing in the plasma at 1 h post-feeding in these birds was estimated to be 0.044% of the total DON fed. At 2 h, 4 h, and 6 h post-feeding, the average values of plasma DON were 0.85 ng/ml, 0.45 ng/ml, and 0.30 ng/ml. De-epoxy-DON could not be detected in the birds sampled at 2 h, 4 h, and 6 h post-feeding. The total amount of DON appearing in the plasma at 2 h post-feeding in these birds was estimated to be 0.036% of the DON fed. These data show that the absorption rate of DON is very low in broilers and that there is also a rapid transformation, and clearance from plasma. Furthermore, there appeared to be individual variability in the capacity of birds to de-epoxidise DON.