Viral and bacterial infections associated with camel (Camelus dromedarius) calf diarrhea in North Province,Saudi Arabia

Diarrhea and deaths in new-born camel calves were noticed by veterinary investigators and pastoralist in Saudi Arabia to be very high. Hence, it is thought to be necessary to investigate this problem from the virological and bacteriological point of view. The role of pathogenic bacteria and viruses in six different towns of North Province (Al-Assafia, Arar, Domat Aljandal, Hail, Skaka and Khoa) in Saudi Arabia was studied. Survey was conducted in diarrheic camel calves aged 12 months or younger. In our study calf diarrhea was reported in 184 out of 2308 camels examined clinically during one year, the prevalence of diarrhea was found to be 8.0% in calves ranging from one month to one year. In the present study group A rotavirus and Brucella abortus were detected in 14.7% and 8.98%, respectively, using ELISA technique. Escherichia coli was isolated from diarrheic calf camel (58.2%) 99/170 samples during dry and wet season. Salmonella spp. and Enterococcus spp. were detected in 12% and 8.8% of the specimens, respectively. In this study enterotoxogenic E. coli (ET E. coli) was isolated from 7% of diarrheic camel, which indicates the strong correlation between the camel calf diarrhea and the detection of enterotoxogenic E. coli. This study represented the first report for the detection of group A rotavirus and B. abortus antigen and antibodies in calf camels in Saudi Arabia. It is recommended that the disease should be controlled by vaccination in calf camels.     

Amino acids content and electrophoretic profile of camel milk casein from different camel breeds in Saudi Arabia

This study aimed to evaluate amino acids content and the electrophoretic profile of camel milk casein from different camel breeds. Milk from three different camel breeds (Majaheim, Wadah and Safrah) as well as cow milk were used in this study.Results showed that ash and moisture contents were significantly higher in camel milk casein of all breeds compared to that of cow milk. On the other hand, casein protein of cow milk was significantly higher compared to that of all camel milk breeds. Molecular weights of casein patterns of camel milk breeds were higher compared to that of cow milk.Essential (Phe, Lys and His) and non-essential amino acids content was significantly higher in cow milk casein compared to the casein of all camel milk breeds. However, there was no significant difference for the other essential amino acids between cow casein and the casein of Safrah breed and their quantities in cow and Safrah casein were significantly higher compared to the other two breeds. Non-essential amino acids except Arg and the essential amino acids (Met, Ile, Lue and Phe) were also significantly higher in cow milk α-casein compared to α-casein from all camel breeds. Moreover, essential amino acids (Val, Phe and His) and the non-essential amino acids (Gly and Ser) content was significantly higher in cow milk β-casein compared to the β-casein of all camel milk breeds and the opposite was true for Lys, Thr, Met and Ile. However, Met, Ile, Phe and His were significantly higher for β-casein of Majaheim compared to the other two milk breeds. The non-essential amino acids (Gly, Tyr, Ala and Asp) and the essential amino acids (Thr, Val and Ile) were significantly higher in cow milk κ-casein compared to that for all camel milk breeds. There was no significant difference among all camel milk breeds in their κ-casein content of most essential amino acids.Relative migration of casein bands of camel milk casein was not identical. The relative migration of α_s-, β- and κ-casein of camel casein was slower than those of cow casein. The molecular weights of α_s-, β- and κ-casein of camel caseins were 27.6, 23.8 and 22.4 KDa, respectively. More studies are needed to elucidate the structure of camel milk.     

Purification and partial characterisation of camel milk xanthine oxidoreductase

Xanthine oxidoreductase (XOR) was purified in the presence of dithiothrietol from camel milk with yields of up to 22.2mg/l that were comparable to those obtained from bovine and human milk sources. On SDS-PAGE, the freshly purified camel milk XOR had a protein flavin (A280/A450) ratio of 5.3 +/- 0.4 and appeared homogenous with a single major band of approximately Mr 145.3 KDa. Surprisingly, in all the batches (n = 8) purified camel milk XOR showed no detectable activity towards xanthine or NADH. The molybdenum content of camel XOR was comparable to human and goat milk enzymes. After resulphuration, camel milk XOR gave a specific activity of 1.1 nmol/min/mg and 13.0 nmol/min/mg enzyme towards pterin (fluorimetric assay) and xanthine (spectrophotometric assay) respectively. This activity was markedly lower than that of human, bovine and goat enzymes obtained under the same conditions. These findings suggest that the molybdo-form of camel enzyme is totally under desulpho inactive form. It is possible that camel neonates are equipped with an enzymic system that reactivates XOR in their gut and consequently generates antibacterial reactive oxygen species.     

Identification of a new Indian camel germplasm by microsatellite markers based genetic diversity and population structure of three camel populations

Camel invokes fascinating chapter of Indian desert history and is integral component of its ecosystem. Camel population has reached a crisis point after three decades of decline (75%) causing major concern to the policy makers. >28% of Indian camel is not yet characterized. It is imperative to describe country’s camel germplasm and its existing diversity for designing conservation plan. One such population is Sindhi, distributed along border with Pakistan. Twenty five microsatellite markers being valuable tool for estimating genetic diversity were selected to elucidate genetic variability and relationship of Sindhi with two registered camel breeds of India- Marwari and Kharai. The standard metrics of genomic diversity detected moderate variability in all the three populations. A total of 303 alleles with a mean of 8.116 ± 0.587 alleles per locus were found in total of 143 animals. Sindhi population had intermediate allelic diversity with 8.522 ± 1.063 alleles per locus. Corresponding values in Marwari and Kharai were 8.783 ± 0.962 and 7.043 ± 1.030, respectively. Genetic variability within the breeds was moderate as evidenced by the mean observed heterozygosity of 0.556 ± 0.025. Sindhi camel population harbors higher genetic variability (Ho = 0.594) as compared to the two registered camel breeds (Marwari, 0.543 and Kharai, 0.531). Mean expected heterozygosity under Hardy-Weinberg equilibrium was higher than the observed values across the three camel groups, indicating deviations from assumptions of this model. In fact, average positive F value of 0.084 to 0.206 reflected heterozygote deficiency in these populations. These Indian camel populations have not experienced serious demographic bottlenecks in the recent past. Differences among populations were medium and accounted for 7.3% of total genetic variability. Distinctness of three camel populations was supported by all the approaches utilized to study genetic relationships such as genetic distances, phylogenetic relationship, correspondence analysis, clustering method based on Bayesian approach and individual assignment. Sindhi camel population was clearly separated from two registered breeds of Indian camel. Results conclude Sindhi to be a separate genepool. Moderate genetic diversity provides an optimistic viewpoint for the survival of severely declining indigenous camel populations with appropriate planning strategies for conserving the existing genetic variation and to avoid any escalation of inbreeding.     

Mapping Potential Amplification and Transmission Hotspots for MERS-CoV,Kenya

Dromedary camels have been implicated consistently as the source of Middle East respiratory syndrome coronavirus (MERS-CoV) human infections and attention to prevent and control it has focused on camels. To understanding the epidemiological role of camels in the transmission of MERS-CoV, we utilized an iterative empirical process in Geographic Information System (GIS) to identify and qualify potential hotspots for maintenance and circulation of MERS-CoV, and produced risk-based surveillance sites in Kenya. Data on camel population and distribution were used to develop camel density map, while camel farming system was defined using multi-factorial criteria including the agro-ecological zones (AEZs), production and marketing practices. Primary and secondary MERS-CoV seroprevalence data from specific sites were analyzed, and location-based prevalence matching with camel densities was conducted. High-risk convergence points (migration zones, trade routes, camel markets, slaughter slabs) were profiled and frequent cross-border camel movement mapped. Results showed that high camel-dense areas and interaction (markets and migration zones) were potential hotspot for transmission and spread. Cross-border contacts occurred with in-migrated herds at hotspot locations. AEZ differential did not influence risk distribution and plausible risk factors for spatial MERS-CoV hotspots were camel densities, previous cases of MERS-CoV, high seroprevalence and points of camel convergences. Although Kenyan camels are predisposed to MERS-CoV, no shedding is documented to date. These potential hotspots, determined using anthropogenic, system and trade characterizations should guide selection of sampling/surveillance sites, high-risk locations, critical areas for interventions and policy development in Kenya, as well as instigate further virological examination of camels.     

Molecular Characterization of the <Emphasis Type="Italic">Camelus dromedarius</Emphasis> Putative Cytochrome P450s Genes

The expression levels of cytochrome P450s were examined in different camel tissues by western blotting and semi-quantitative polymerase chain reaction. Camelus dromedarius liver microsomes were found to express different P450s isoenzymes constitutively. The maximum expression of P450 protein was seen in the camel liver in the order of P450 2E1, 1A1, 3A and 2B1/2. Camel extrahepatic tissues, kidney, spleen and the lung showed detectable levels of P450s 1A1 but lower than that noticed in liver. Detectable level of P450 2B1/2 was also observed in camel lung (29.5 vs. 58% liver microsomes). P450scc and 21-hydroxylase were found to be differentially expressed only in camel testis. Partial sequences of these P450s genes showed high similarities with the human P450s. These results demonstrate that the multiple forms of P450s are differentially expressed in camel tissues and that the relative levels of expression are comparable with other mammals. These observations might be important in understanding the differential susceptibility of camel tissues to the toxic effects of xenobiotics and environmental pollution.     

Testicular development and serum sex steroid profiles during the annual sexual cycle of the male sturgeon hybrid the bester

Testicular development in the adult male F₁ sturgeon hybrid, the bester ( Huso huso L. female x Acipenser ruthenus L. male), was examined monthly in relation to serum sex steroid levels. Spermatogenesis lasted for 1 year, with meiosis generally starting in September and spermiogenesis in November, although there was considerable variation in testicular developmental stages between fish sampled monthly. Testicular development continued, slowly, during the winter months until April. Fish did not exhibit spontaneous spermiation, and phagocytotic activity of Sertoli cells became prominent from May onwards. Androgen levels increased during Spermatogenesis and remained high throughout the pre-spermiation period. In the degeneration stage, 11-ketotestosterone concentrations declined to low levels, while testoster- one levels remained high. The serum concentration of 17,20β-dihydroxy-4-pregnen-3-one was low throughout the reproductive cycle. Based on these results, it is suggested that the time appropriate for induction of final maturation would be from November–December to April when the testes are in the late stage of development.     

Diversity and distribution of CYP gene family in Bactrian camel

Cytochrome P450 (CYP) enzymes belong to a superfamily of monooxygenases which are phase I enzymes responsible for the first pass metabolism of about 90% of drugs in animals. However, these enzymes are often polymorphic and metabolism of the same drug in different species or different individuals is influenced by genetic and non-genetic factors. Bactrian camels are capable of survival in harsh living environments, being able to consume diets that are often toxic to other mammals and can tolerate extreme water and food deprivation. The aim of this study was to investigate whether the Bactrian camel’s special metabolic pathways and unique detoxification capabilities are attributable to particularities of the CYP gene family. The Bactrian camel’s whole genome sequencing data were systemically analyzed and annotated, and then, CYP gene family was searched from the whole protein database and compared with CYP gene families of cattle, horse, chicken, and human. The total of 63 CYP gene copies were found in Bactrian camel’s whole genome and were classified into 17 families and 38 subfamilies. Among them, 9 multi-gene families were found, and CYP2, CYP3, and CPY4 have 27, 6, and 7 subfamilies, accounting for 43, 10, and 11% in camel CYP gene, respectively. In comparison with cattle, chicken, horse, and human, the distribution of CYP gene subfamilies in camel is different, with more CYP2J and CYP3A copies in the Bactrian camel, which may contribute to the Bactrian camel’s specific biological characteristics and metabolic pathways. Comparing to the cow, horse, chicken, and human CYP genes, the distribution of CYP gene subfamilies is distinct in the Bactrian camel. The higher copy number of CYP2J gene and CYP3A gene in Bactrian camel may be the important factors contributing to the distinct biological characteristics and metabolic pathways of Bactrian camels for adaptation to the harsh environments.     

Tissue Specific Expression of Glutathione S-transferases, Glutathione Content and Lipid Peroxidation in Camel Tissues

Differential expression of glutathione S-transferase (GST) enzyme activity in various tissues of the camel was observed with a maximum activity in the liver. Compared with the rat and human livers, GST activity in camel liver was 50% lower than that of rat liver and similar to that of human liver. Extrahepatic tissues in camel have a comparable GST activity with those of similar tissues in the rat. Assay of GST activity using ethacrynic acid as substrate demonstrated maximum activity in the camel brain followed by intestine, liver and kidney. Microsomal GST activity in camel tissues was expressed in the order of liver > testis > intestine ≈ kidney ≈ brain. Phenotyping of GST was performed in camel hepatic and extrahepatic tissues using human specific antibodies to class α, μ, and π cytosolic GST isoenzymes and rat specific antibody to the microsomal GST. Western immunoblot and immunohistochemical analyses showed an abundant expression of GST α and μ in the camel liver, while π was very poorly expressed. Camel extrahepatic tissues however, had a significant expression of GST π. The camel GST isoenzymes were found to be predominantly expressed in the hepatocytes around the central vein with a gradual decrease in expression in the hepatocytes located toward the periphery. Kidney cortex exhibited a greater expression of the enzyme protein in the proximal tubules as compared to the glomeruli. Glutathione (GSH) concentration in rat tissues, except in the brain, was about 2-fold higher than that of camel tissues. Rate of NADPH-dependent microsomal lipid peroxidation was comparable both in the rat and camel tissues with the highest activity in the brain and lowest activity in the intestine. The differential expression of GST isoenzymes in different organs of the camel, GSH concentration and the rate of lipid peroxidation in different tissues may be important factors in determining the differential susceptibility of camel tissues to the toxic effects of xenobiotics.     

Ultrastructure of the gametogenesis of the common Mediterranean starfish,Echinaster (Echinaster) sepositus

Echinaster (Echinaster) sepositus is one of the most abundant sea stars in western Mediterranean rocky bottoms, yet its reproductive biology remains virtually unknown. Here we report the ultrastructure of its gametogenesis over 2 consecutive years. It is a gonochoric species with an annual reproductive cycle spawning gametes in late summer and early autumn. Each arm of every individual contained two gonads (dark red in females and yellow in males). In both sexes, the gonad was a single, large sac composed of several smaller sacs. The gonad wall consisted of two multilayered sacs, outer and inner, separated by the genital haemal sinus. The histology of the gonad wall was consistent with that found in other asteroids. Oogenesis was continuous during the year, but eggs were spawned only in late summer. Oocytes were in close relationship with follicular cells that are suggested to transfer nutrients to the oocytes. Spermatogenesis was restricted to 5–6 months in spring-summer. It occurred in columns with an axial interstitial cell supporting each column, and producing processes towards the lumen that remained connected to spermatogenic cells by intercellular junctions. Developing sperm cells were found along the length of the column, while spermatozoa were found free in the testis lumen. Spermatogenesis followed the pattern described for echinoderms, to give rise to an acrosome-bearing, round-shaped spermatozoon. The histology and cytology of the reproductive process in E. sepositus followed the general pattern found for asteroids.     

Studies on purine turnover in the camel (Camelus dromedarius) and zebu (Bos indicus)

Significantly higher hypoxanthine over uric acid ratios were found in camel plasma and urine, with respect to those of zebu. Enzyme levels of purine catabolism were markedly lower in camel than in zebu liver. Oxidation of hypoxanthine appears to be the limiting step of purine metabolism in camel liver. Any hepatic hypoxanthine appears to be actively converted into IMP in camel liver, rather than oxidized to uric acid.     

Variability of Some Milk-Associated Genes and Proteins in Several Breeds of Saudi Arabian Camels

To gain knowledge on the molecular basis of diversity of several clans of Saudi camel (Camelus dromedarius) characterization of these animals was conducted at both genetic and protein levels. To this end, blood and milk samples were collected from several camel breeds at different Saudi Arabia locations (northern Jeddah, Riyadh, and Alwagh governorates). Genomic DNA was extracted from blood of four Saudi camel breeds (Majahem, Safra, Wadha, and Hamara), and DNA fragments of the casein and α-lactalbumin genes were amplified. The retrieved DNA sequences were analyzed for genetic variability. The inter-simple sequence repeat technique was used for confirming the relationships among the analyzed camel breeds, and the PCR–RFLP with two restriction enzymes was utilized for exploring their molecular variations. The number of haplotypes, gene diversity, nucleotide diversity, average number of nucleotide differences, and sequence conservation were calculated for all the analyzed DNA sequences. These analyses revealed the presence of several single nucleotide polymorphisms in the analyzed DNA sequences. A group of neighbor joining trees was built for inferring the evolutionary variations among the studied animals. Protein profiling of milk from different camel clans was also conducted, and differences between and within the Saudi camel clans were easily found based on the isoelectric focusing (IEF) profiles using ampholytes with different IEF range. This study revealed that analyzed camel breeds show low levels of genetic differences. This may be a reflection of the evolutionary history of C. dromedarius that was domesticated based on a highly homogeneous ancestor ecotype.     

Molecular analysis of some camel cytochrome P450 enzymes reveals lower evolution and drug-binding properties

Camels bear unique genotypes and phenotypes for adaptation of their harsh environment. They have unique visual systems, sniffing, water metabolism, and heat-control mechanisms that are different from other creatures. The recent announcement for the complete sequence of camel genome will allow for the discovery of many secrets of camel life. In this context, the genetic bases of camel drug-metabolizing enzymes are still unknown. Furthermore, the genomic content of camel that rendered it highly susceptible to some drugs (as monensin and salinomycin) and became easily intoxicated needs to be investigated. The objectives of this work are the annotation of camel genome and retrieval of camel for cytochrome P450 (CYP) 1A1, 2C, and 3A enzymes. This is followed by comprehensive phylogenetic, evolution, molecular modeling, and docking studies. In comparison with the human enzymes, camel CYPs showed lower evolution rate, especially CYP1A1. Furthermore, the binding of monensin, salinomycin, alfa-naphthoflavone, felodepine, and ritonavir was weaker in camel enzymes. Interestingly, rerank score indicated instable binding of monensin and salinomycin with camel CYP1A1 as well as salinomycin with camel CYP2C. The results of this work suggest that camels are more susceptible to toxicity with compounds undergoing metabolic oxidation. This conclusion was based on lower evolution rate and lower binding potency of camels compared with the human enzymes.     

Comparison of virokine from camel pseudocowpoxvirus (PCPV) with Interleukin 10 of the Dromedary camel (Camelus dromedarius)

Cellular interleukin-10 (IL-10) gene from the peripheral blood mononuclear cells of the healthy Dromedary camel (Camelus dromedarius) and viral IL-10 (vIL-10) from the skin scabs of the Dromedary camels infected with contagious ecthyma (a parapoxviral infection in the camels) were amplified by polymerase chain reaction, cloned and characterized. Sequence analysis revealed that the open reading frame (ORF) of dromedarian camel IL-10 is 537 bp in length, encoding 178 amino acid polypeptide while open reading frame of vIL-10 from camel is 561 bp, encoding 187 amino acid polypeptide. The Dromedary camel IL-10 exhibited 62.6% and 68.5% sequence identity at the nucleotide and amino acid level, respectively, with vIL-10 from camel. Sequence analysis also revealed that the Dromedary camel IL-10 shared 99.4% and 98.3% identity at the nucleotide and amino acid level, respectively, with the Bactrian camel (Camelus bactrianus). But vIL-10 from camel shared 84.7% and 83.4% sequence identity at the nucleotide and amino acid level, respectively, with vIL-10 from reindeer (Rangifer tarandus), which is a ruminant species belonging to the order Artiodactyla. The present study was conducted to evaluate the evolutionary origin of the camel parapoxvirus with parapoxviruses of cattle and sheep and the resultant sequence analysis revealed that camel parapoxvirus is closely related to cattle parapoxvirus than sheep parapoxvirus (Orf virus).     

Metabolism of benzo(a)pyrene,dimethylbenzanthracene and aflatoxin B1 by camel liver microsomes

The ability of camel liver microsomes to metabolise a range of common environmental carcinogens including benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 has been investigated. The camel liver has shown the ability to metabolise benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 to a number of metabolites. The major metabolites of benzo(a)pyrene produced by camel liver enzymes were identified as its mono-hydroxy derivatives and suggest that the metabolic detoxification pathways of carcinogen metabolism are predominant in this species. Benzo(a)pyrene metabolising activity in camel liver required NADPH and was inhibited by CO and alpha-naphthoflavone suggesting the involvement of cytochrome P450 in the metabolism of this carcinogen by camel liver. The cytochrome P450-dependent metabolism of carcinogen and other specific substrates such as ethoxyresorufin and ethoxycoumarin, by camel liver enzymes, was about 50% higher than that of rat liver enzymes. The cytochrome P450-dependent metabolism of a variety of carcinogenic and other substrates by camel liver demonstrated that there are multiple forms of cytochrome P450 enzymes involved in the metabolism of a wide array of xenobiotics and pollutants.     

The distribution of phosphofructokinase isoenzymes in the liver of camel, rat and rabbit

1. The distribution of phosphofructokinase isoenzymes have been compared among camel, rat and rabbit livers. 2. Only a single phosphofructokinase isoenzyme is present in the camel liver which has shown different physical and regulatory properties from the isoenzymes of rat and rabbit liver. 3. The ammonium sulphate precipitation curves of the camel and rabbit enzymes were monophasic, whereas the rat enzyme was biphasic. 4. Rabbit liver phosphofructokinase was slightly more anodic than the rat enzyme, whereas the camel enzyme was the least anodic as shown by the techniques of DEAE-cellulose chromatography and cellulose acetate electrophoresis. 5. Partially purified camel liver phosphofructokinase showed different regulatory properties from the rabbit and rat isoenzymes as the apparent Km values were 0.58, 0.45 and 0.82 mM respectively.     

Purification and functional characterization of pancreatic insulin from camel (Camelus dromedarius)

Large-scale production of insulin still represents the key step in helping diabetic patients throughout the world. Many species and approaches have been used for the production of insulin. In this study, we purified and characterized for the first time pancreatic insulin from the Arabian camel (Camelus dromedarius) using a modified acid-alcohol extraction method. After extraction insulin was purified using a one-step gel filtration on a Sephadex G-50 column leading to a purification yield of 80 mg/kg (20%) of camel pancreas. The purity of camel insulin was assessed by SDS–PAGE and HPLC using insulin from human, bovine and porcine as standards. Molecular weight was determined for purified camel insulin as 5800 Daltons and its amino acid composition is similar to that known for other species. The functional characterization of purified crude camel insulin was demonstrated in vitro by positive competition by radioimmunoassay and in vivo showing camel insulin inducing acute hypoglycaemia in mice. Together, our study reports for the first time the successful purification of functional insulin from camel pancreas with similar properties compared to other insulin species. This is of great interest given that the camel represents considerable economic worth in many countries.     

Studies on camel hemoglobin. 1. Physico-chemical properties and some structural aspects of camel hemoglobin (Camelus dromedarius).

Hemoglobin from an adult camel (Camelus dromedarius) was prepared from the red cell lysate by CM- and DEAE-cellulose chromatography. The purified hemoglobin showed a lesser mobility on starch gel electrophoresis at pH 8.5 than that of human hemoglobin C. Native camel hemoglobin contains 95-99% alkali-resistant hemoglobin and in soluble in 2.94 M K2HPO4/KH2PO4 buffer. Different forms of camel hemoglobin show similar ammonium sulfate precipitation curves. Indirect evidence for the stability of camel hemoglobin solutions was obtained from several sources. Spontaneous met-hemoglobin formation is extremely slow and minimal quantities of degradation products appear on starch gel electrophoresis and on chromatographic separation. The alpha and beta chains of camel hemoglobin A were separated on a CM-23 column by the use of a pyridine formate gradient. Large peptide fragments were obtained by tryptic digestion of maleylated alpha and beta chains. The N-terminal structure of the alpha and beta chains and of tryptic maleylated peptides derived from alpha and beta chains are presented. Between adult camel hemoglobin and adult human hemoglobin six amino acid differences in the N-terminal 20 amino acid residues of the alpha chain, at residues: 4, 5, 12, 14, 17, and 19; eight amino acid substitutions were found in the beta chain at positions: 4, 5, 6, 9, 12, 13, 16, and 19. Substitutions at alpha5 Ala leads to Lys, and beta19 Asn leads to Lys, increase the net positive charge of camel hemoglobin by two, while other substitutions result in no charge differences. The molecular basis of the stability of camel adult hemoglobin is discussed.