Moisture contents in poultry ration samples collected from areas under study
However, other samples collected from Sana’a did not differ significantly in either for their moisture contents or for their mean of colonization. In Taiz, moisture contents of samples differed significantly and ranged 9.1–18.3%. For ration samples collected from farmer’s poultry farms, range of moisture contents for the same samples was 7.7–18.3%. However, the highest colonization was coincided with the highest level of moisture content percentage. The least samples colonization significance was given by all commercial samples. This result was in agreement with the reports by Gloria et al., Shetty (1997), and Bhat (1997).
Isolation and identification of fungi
Data on fungi occurring in poultry ration obtained in Yemen are coincided with those reported in different countries by Abdel-mallek et al. (1993), Nijs and De-Nijs (1997), and Bottalico (1997). Results of the present work showed a predominance of the genera obtained (F. moniliforme, A. flavus) with those reported by Dasilva et al. (2000) who reported the predominance of the genera Phoma (57.1%), Aspergillus (42.7%), Fusarium (25.0%), and Rhizopus (21.4%).The most frequently found species were Aspergillus flavus and Fusarium moniliforme. Survey of mold count and fungal species on feed samples proved that Aspergillus, Penicillium, and Fusarium were the most frequent genera. These results are also in agreement with the report by Castella et al. (1999a) and Dasilva et al. (2000). Therefore, only Fusarium moniliforme and Aspergillus flavus were subjected for further studies.
Occurrence frequency of isolated fungi in samples collected from farmer’s poultry farms in four governorates
Occurrence and frequencies of fungi isolated from samples collected from poultry ration supply companies in Sana’a and Taiz showed that Fusarium moniliforme ranked the most contaminant frequently occurring in samples collected from poultry ration supply companies in Sana’a followed by Aspergillus flavus. For samples collected from Taiz poultry ration supply companies, data proved that Aspergillus flavus exhibited the most frequently occurring contaminant followed by Fusarium moniliforme. Other contaminants are either negligible or absent. Contamination with fungi increased in samples collected from poultry farms mostly in all governorates. On the other hand, Fusarium moniliforme was also dominant in samples collected from farmer’s poultry farms in all governorates followed by Aspergillus flavus. Results strongly indicated that poultry rations distributed to poultry farmers were already contaminated at the source of origin. Three genera of fungi—Aspergillus, Penicillium, and Fusarium (Gibberella)—are the ones involved most frequently in cases of mycotoxin contamination in corn, small grains, and soybeans which are the major constituent elements of poultry rations, and the Fusarium was detected in 83% of 69 cereal samples from batches intended for food or feed production Nijs and De-nijs (1997) reported that several Fusarium species occurring on pre-harvest cereals in Europe cause widespread stalk and ear rot of maize and head blight of small cereals. They also lead to the accumulation of mycotoxins in infected ears. There is increasing evidence for the occurrence of toxigenic strains of Fusarium moniliforme [Gibberella fujikuroi] and FB1 mycotoxin in infected maize ears all over Europe. Such reported data are strong evidence explaining how the poultry ration constituents imported from abroad into Yemen are born with primary source of infection and molds. Such fact inconceivably also harden the beliefs in that contamination of poultry ration formulated from infected maize, soybeans, and cereals used in poultry ration formulation starts at the growing duration in the field. In case of maize contamination with Aspergillus flavus, three Aspergillus species were isolated from different agroecological zones, with Aspergillus flavus being the most prevalent. The country-wide mean percentage of kernel infection was c. 20% in two successive years (Setamou et al. 1997). Data obtained in the current study in Yemen also are in agreement with these reports by Magnoli et al. (1999) and Orsi et al. (2000) who reported that microbiological analysis revealed a predominance of Fusarium species, which presented the greatest total number of CFUs per gram in three maiz hybrids, followed by Penicillium spp., Aspergillus spp., and 10 other fungal genera. Fusarium moniliforme [Gibberella fujikuroi] was the most prevalent species (59.2% of Fusarium isolates in Br 201, 55.4% in C 125 and 69.2% in Cx 322 (Chau et al. 1997). On the other hand, soybeans which is a major element of poultry ration constituent is also subjected to infection by several contaminant and has been evaluated by Park et al. (1999) who isolated a total of 52 isolates of Fusarium species obtained from soybean seeds from various parts of the Korea Republic and identified as F. oxysporum, F. moniliforme [Gibberella fujikuroi], F. semitectum [F. pallidoroseum], F. solani, F. graminearum [Gibberella zeae], or F. lateritium [G. baccata]. Moreover, data are in agreement with findings reported by Orsi et al. (2000), Proctor et al. (1999), Jindal et al. (1999), and Ono et al. (1999).
The performance of fungi colonization and the level of moisture content in the collected poultry ration samples collected from four governorates under study were investigated. All rates of colonization differed significantly by samples. However, the highest rate of colonization (83 colonies) was reported in sample delivered by a farmer owing a poultry farm suffering from chicken dwarfism or growth loss in Sana’a District. Data also indicated that the high degree of colonization was for samples collected from farmer’s poultry farms in general. Although contamination was less at the origin of distribution by poultry ration supply companies, samples were still significantly high. It can be concluded that under such circumstances, farmers are used to buy already contaminated poultry rations and moreover, source of contamination is the poultry ration raw materials imported from abroad. Such assumption was supported by the uniformity of contaminant genera isolated from approximately all samples collected from four governorates under study. Such findings in fact support such assumption data reported by several investigators who confirmed that infection of the imported raw materials used in formulating locally poultry rations started usually at the source of origin during crop growing in fields, (Bottalico, 1997; Park et al. 1999; Orsi et al. 2000; Dasilva et al. 2000; Chau et al. 1997; Jindal et al. 1999; Ono et al. 1999; Gonzalez et al. 1999).
Determination of aflatoxins and fumonisins in poultry ration samples collected from poultry ration supply companies
It is concluded that contamination of samples with mycotoxins at Sana’a poultry ration supply companies was less than that detected at Taiz supply companies. Data are in agreement with reports raised by Dasilva et al. (2000) who showed that 12.8% of samples tested were contaminated with aflatoxin B1 and 74.2% were contaminated with fumonisin B1. Aflatoxin production is conditioned by abiotic and biotic (genetic) parameters. Among the isolates, Aspergillus flavus and Aspergillus parasiticus produced AFB, Aspergillus flavus produced AFG 1, Aspergillus ochraceus produced ochratoxin-A, Aspergillus japonicus produced sterigmatocystin, and Penicillium citrinum produced citrinin. AFB in the range 333–10,416 μg/kg was produced by Aspergillus spp. in rice, pulses, and oilseeds (Martins et al. 1999). Data also are in agreement with findings reported by Zhang et al. (1997) who studied 246 samples and found that of 164 maize samples collected from areas in which the risk of human esophageal cancer (HEC) is high, fumonisins were detected in 106 samples at 0.5–16.0 ppm, but of 82 samples collected from HEC low risk areas, fumonisins at 0.5–1.5 ppm were found in 23 samples. The highest level of aflatoxins detected in Sana’a area was 22 ppb. A sample delivered by a farmer owing a poultry farm suffering from chicken dwarfism or loss growth. However, mean of aflatoxins in ration samples collected from Sana’a poultry farms was 12 ppb.
Determination of aflatoxin and fumonisin levels in poultry ration samples collected from Farmer’s poultry farms in four governorates
In general, aflatoxin concentrations in ration samples differed significantly, but no significant different was detected in the case of fumonisins.
Similar results were reported by Magnoli et al. (1999) who investigated the production of fumonisins by toxigenic Fusarium species strains and in 158 samples of poultry feeds collected from a factory located in the department of Rio Cuarto, Cordoba province, Argentina. Also, they showed that toxin produced in highest levels by the majority of the strains was FB1. Moreover, they confirmed that the low level of fumonisins detected (0.3–5.0 ppm.) in all samples collected indicates that a genetically component of F. moniliforme biotype is responsible for the pattern of Fusarium moniliforme toxigenic effect in poultry ration samples tested. Also Bottalico et al. (1997) in Accra reported that 15 maize samples from 4 markets and processing sites were analyzed for fumonisins B1, B2, and B3. All samples contained fumonisins ranged from 70 to 4222 mg/kg. Additional studies on maize samples from 15 processing sites in Accra revealed a co-occurrence of both fumonisins and aflatoxins in 8/15 samples (Kpodo et al. 2000). Data are also in agreement with these reported by Ali et al. (1998) and Proctor et al. (1999).
Effect of three moisture content levels on concentrations of aflatoxins and fumonisins produced in poultry rations
Data showed that effect of three levels of moisture contents on means of aflatoxins and fumonisins production was different by the level of sample moisture content. No significant difference for aflatoxins produced at 15–20% moisture content was determined. Aflatoxins, zearalenone, and ochratoxin A, and moisture levels and average contamination by aflatoxins B1 + G1 were 14.9, 13.9, and 4.3 μg/kg, respectively when 27.7% of the samples had moisture levels above 14.5% (Gloria et al. 1997). Also Magan et al. (1984) concluded that Aspergillus flavus grows best on corn at 18.0–18.5% moisture. Moisture content below 13% prevents invasion by Aspergillus flavus. Fungal growth may begin on corn at moisture content lower than 18.0%. As the fungus grows, the respiration occurs, releasing heat and moisture into the surrounding environment in the grain mass. An increase in the moisture content and temperature of the surrounding corn cause a hot spot. If moisture content and temperature continue to rise, the environment for Aspergillus flavus becomes more favorable. Fungal growth is best at 18% moisture. At 20% moisture content and above, other fungi grow better and crowd out Aspergillus flavus.
Data reported here also indicated that at 25% moisture content, a significant difference between aflatoxins produced at higher moisture contents and at the lower ones tested was determined. Mean of fumonisin levels produced at the three levels of moisture contents used. No significant difference for fumonisin levels detected was given at all moisture contents tested. Data obtained are supported by Shetty and Bhat (1997) who reported that fumonisins B1 contamination in the poultry feed samples ranged from 0.02 to 0.26 mg/kg. Such findings emphasized the importance of small differences in MC in determining safe storage conditions. The rapid moisture equilibration between wet and dry corn indicated that corn could be blended with minimal risk of mold damage or aflatoxins contamination if the average MC of the blend is low enough to prevent mold growth (Sauer and Burroughs, 1980).
Effect of three levels of temperature on concentrations of aflatoxins and fumonisins produced in poultry rations
Thus, finding presented here proved that aflatoxins secretion by (A.) flavus decreased as temperature increased over 25 °C up to 35 °C. Fusarium tricinctum and some strains of F. graminearum, F. equiseti, F. sporotrichioides, F. poae, and F. lateritium produced T-2 and other toxic trichothecenes. These fungi commonly attack grains and can grow at temperatures from slightly above freezing to about 86 °F. T-2 and HT-2 toxins are produced over a temperature range of 46 °F to 77 °F, with the maximum production at temperatures below 59 °F. There was a trend toward higher rate of aflatoxin accumulation per percentage of Aspergillus flavus infection (MEdi et al. 1997). The growth rate of G. fujikuroi var. subglutinans was highest at 20 °C and 25 °C in maize cultures and at 15 C° in rice cultures than in maize, with maximal yield at 25 °C of 979 μg/g after 2 weeks and 143 μg/g after 3 weeks in maize and rice cultures, respectively (Setamou et al. 1997), Castella et al. (1999b).
Conditions that favor aflatoxin production and the invasion of corn by Aspergillus flavus in the field include drought stress or damage to the corn ear by ear worms or other insects, birds, hail, or early frost. High temperatures, high relative humidity around the kernels, and kernel moisture below 30% (wet basis) are ideal conditions for fungal invasion of the kernel. The optimum temperature for aflatoxin production in storage is between 25 °C and 32 °C (77 °F and 90 °F). Kernels with a moisture content below 15% are at less risk of mold growth and aflatoxin production, while an optimum kernel moisture is around 18% and an optimum relative humidity in the bin is 85% or higher. But the field conditions that favor Fusarium to invade these grain crops include warm, moist weather. Blight symptoms can develop within 3 days after infection when temperatures range between 25 °C and 30 °C and moisture is continuous. Plants appear most susceptible when they are infected at the flowering stage of development. Ochratoxins occur most readily in storage of high-moisture (greater than 22%) grains. Therefore, the only recommended control is to keep grains cool and dry in storage (Proctor et al. 1999). The amount of aflatoxin produced in storage is determined by storage conditions. The most important factors are grain moisture content and temperature. Optimum temperatures for Aspergillus flavus to grow are 80–90 °F; optimum grain moisture content is 18%. Damaged corn also favors the growth of (A.) flavus. Importantly, aflatoxin concentration never decreases in storage; it only increases or remains the same (Sauer and Burroughs, 1980; Magan et al. 1984).