Chromosomal localization of the lysozyme gene cluster in river buffalo (Bubalus bubalis L.)

Lysozyme (LYZ) is an antibacterial enzyme which allows the digestion of bacteria present in tears and saliva. In the true stomach of ruminants LYZ breaks open the bacteria of the foregut, which are subsequently digested by typical mammalian digestive enzymes, allowing the incorporation of nutrients from the bacteria. Southern analysis with a single exon from a cow lysozyme gene revealed that there are about 10 genes in ruminants (Irwin & Wilson 1989), while pig and primates have a single lysozyme gene (Swansonet al. 1991) and camels have two (Irwinet al. 1992). The higher number ofLYZ genes in ruminants is believed to be the result of gene duplication associated with the evolution of foregut fermentation (Irwinet al. 1992). Recently, the genomic organization of the lysozyme gene family has been determined in domestic cattle, and, using a cocktail of genomic clones, the lysozyme gene cluster (LYZ@) was assigned to chromosome (Chr) 5, band 23 by fluorescencein situ hybridization (FISH) (Gallagheret al. 1993). In our continued effort to test the genetic homology of conserved chromosome banding regions between cattle and river buffalo, and to extend the river buffalo physical gene map, we have mapped theLYZ@ by FISH and R-banding.     

Cloning and biological characterization of buffalo (Bubalus bubalis) interferon-gamma

Interferon-gamma, a major immunomodulatory cytokine, of Indian water buffalo (Bubalus bubalis) was characterized at molecular level. Complementary DNA and essential promoter region were cloned and sequenced, and functional recombinant protein was expressed in bacterial system. The cDNA has 97.8% nucleotide identity with 11-nucleotide and four-amino acid variations, and the essential promoter region has 98.4% identity with five-nucleotide variations and a four-nucleotide deletion in comparison with the corresponding bovine sequences. All the major promoter elements such as NF IL-2 like motif, cyclosporin sensitive binding element and GATA motif are strictly conserved. Recombinant buffalo-IFN-gamma expressed in bacterial system reacted with an anti-bovine-IFN-gamma monoclonal antibody in Western blot and showed antiviral activity against buffalo pox virus in cultured Madin-Darby bovine kidney (MDBK) cells by inhibiting virus induced cytopathic effect. The study shows high level sequence similarity of IFN-gamma among ruminants. In view of the immunomodulatory and antiviral activities of IFN-gamma, this molecule will be useful in better understanding of the immune system of water buffaloes.     

Onset and sequence of RBA-band replication on the inactive X-chromosomes of cattle (Bos taurus L.), river buffalo (Bubalus bubalis L.) and goat (Capra hircus L.)

This study was undertaken to provide cytogenetic information about onset and sequence of RBA-band replication on the inactive X-chromosomes of cattle, river buffalo and goat. Blood cultures were synchronized overnight with thymidine after 48 hours of growth. The cell block was released with fresh medium and the cells allowed to grow in the presence of BrdU and H33258 for 1, 2, 4, 6, 8, 10, 12 and 14 hours, including 20 minutes colcemide. Results show that: (a) the onset of RBA-banding replication was 12 hours before mitosis in cattle and river buffalo, 14 hours in the goat; (b) the replication process was still on in cattle and river buffalo one hour before mitosis, whereas it was off in the goat; consequently the length of the G2 phase was less than one hour in cattle and river buffalo and one hour or slightly longer in the goat; (c) the first band undergoing replication was identified as band Xq31 in cattle, homologous to band Xq34-36 in river buffalo and Xq24 in the goat; (d) the second replicating band was the Xp22 in cattle, homologous to band Xq21 in river buffalo and Xq34 in the goat, respectively; (e) the sequence of RBA-band replication was quite similar between cattle and river buffalo, but reversed in the goat, due to the wide chromosomal rearrangements which differentiated the X-chromosome of Caprinae from that of Bovinae.     

Quantitative aspects of water buffalo (Bubalus bubalis) spermatogenesis.

In the buffalo, seminiferous tubules occupy about 82% of the testis. Spermatogenesis can be divided into 6 stages according to characteristic cellular associations in the seminiferous epithelium. A-spermatogonia have a volume of approximately 1,400 microns3 and the highest absolute mitochondrial volume of all spermatogenic cells. B-spermatogonia display cellular, nuclear and mitochondrial volumes of approximately half the values of A-spermatogonia. From preleptotene (approximately 470 microns3) to late diplotene (approximately 2,300 microns3), the volume* of primary spermatocytes increases nearly five-fold; their nuclear volumes increase by 3.5 times within the same period. During zygotene mitochondrial cristae start to dilate. Grouping of mitochondria by a dense intermitochondrial substance is most prominent during pachytene and diplotene. In pachytene the absolute size of the Golgi apparatus more than doubles, indicating a high secretory activity. Through zygotene only rER is encountered; in pachytene and diplotene a tubular sER makes its first appearance. Secondary spermatocytes are found only in stage 4 of the cycle. Due to partial cell necrosis and autolytic events, late maturation phase spermatids display no more than 25% of the size of cap phase spermatids. There is no morphological evidence for an active uptake and digestion of residual bodies by the Sertoli cells. Also, no lipid cycle is present in the buffalo seminiferous epithelium. Morphometric evaluations reveal that 63% of all theoretically possible germ cells disappear from the seminiferous epithelium during spermatogenesis. Heavy cell loss is observed in stage 4 of the cycle in the spermatogonial fraction as well as during the second meiotic division.     

Molecular characterization and prokaryotic expression of buffalo (Bubalus bubalis) Interleukin-6

The study describes the characterization of Interleukin-6 cDNA and essential promoter sequences of the Indian Water Buffalo (Bubalus bubalis) and expression of the recombinant IL-6 in Escherichia coli. Buffalo IL-6 shows very high nucleotide level identity of the cDNA (98.7%) and promoter (98%) sequences with the corresponding cattle sequences. All the major regulatory elements of IL-6 promoter like AP-1, Multiple Response Element, NF-IL6, ETS binding domain and NF-kappaB binding sites show absolute conservation. Basal level IL-6 mRNA is detected in organs like liver, lung and spleen. Concanavalin A stimulated splenocytes produced maximum IL-6 mRNA at 8h poststimulation. Recombinant IL-6 production in JM109 (DE3) and BL21 (DE3) pLysS bacterial system is substantially enhanced by supplementation of rare codon tRNAs through co-transformation with a second plasmid. BL21 (DE3) pLysS strain is a more efficient producer of the IL-6 as it expressed two-fold more protein than by JM109 (DE3) cells. The study shows high-level conservation of IL-6 regulatory and coding sequences between cattle and buffalo, and indicates the use of a common reagent for studying the effects of this cytokine in these species.     

Experimental transmission of foot-and-mouth disease among Indian buffalo (Bubalus bubalis) and from buffalo to cattle

Indian buffalo and cattle were infected experimentally with a serotype O strain of foot-and-mouth disease virus of buffalo origin. Whereas intradermolingual inoculation of buffalo produced largely sub-clinical infection, inoculation in the dental pad produced vesicles in the mouth and on the feet. A buffalo infected via the dental pad transmitted infection to cattle and buffalo by direct contact with them for 24h. The contact-exposed buffalo developed (1) delayed-onset clinical signs, and (2) shedding of virus from the nose, commencing before the appearance of vesicles and continuing until the experiment was terminated 10 weeks after exposure. The covert nature of the disease in Indian buffalo, coupled with the prolonged shedding of virus, suggests that this species represents a host of epidemiological importance.     

Biological and genetic analysis of a bovine-like coronavirus isolated from water buffalo (Bubalus bubalis) calves

We describe the isolation, biological and genetic characterization of a host-range variant of bovine coronavirus (BCoV) detected in water buffalo (Bubalus bubalis). By conventional and real-time RT-PCR assays, the virus was demonstrated in the intestinal contents of two 20-day-old buffalo calves dead of a severe form of enteritis and in the feces of additional 17 buffalo calves with diarrhea. Virus isolation, hemagglutination and receptor-destroying enzyme activity showed that the buffalo coronavirus (BuCoV) is closely related to BCoV but possesses some different biological properties. Sequence and phylogenetic analyses of the 3' end (9.6 kb) of the BuCoV RNA revealed a genomic organization typical of group 2 coronaviruses. Moreover, the genetic distance between BuCoV and BCoV was proven to be the same or even higher than the distance between other ruminant coronaviruses and BCoV. In conclusion, our data support the existence of a host-range variant of BCoV associated with enteritis in buffaloes.     

Concurrent Cryptosporidium and Candida infections in a buffalo calf (Bubalus bubalis)

Cryptosporidiosis and candidiasis are opportunistic infections of neonates. An investigation was contemplated to study the enteric lesions associated with concurrent infections by the pathogens in a male bubaline calf, aged 8 weeks, which died on March 23, 2010. The gross lesions were comprised of hyperplastic changes and erosion of the microvillus surface of the enteric epithelium. The histological sections revealed invasion of the mucosal cells by two types (Cryptosporidium spp. and Candida spp.) of pathogens. The deeper layers of the intestine did not reveal appreciable architectural changes, except for the presence of inflammatory cellular elements in the lamina propria. The sections stained with Ziehl–Neelsen revealed bright red, oval to spherical bodies (3–5 μm), and those stained with Gomori's Methanamine silver nitrate revealed black-colored fungal spores and pseudohyphae. Based on morphological and in vitro cultural characteristics, the pathogens were identified as Cryptosporidium spp. and Candida spp., respectively. The lesions seem to be consequential events to synergistic invasion of the enterocytes by the pathogens. Complex changes in the microenvironment of the gut, incidental to in situ release of hydrolytic enzymes, toxins, and products of host–parasite interaction liberated at the site of infection by the pathogens, have been reported and discussed.     

Histological studies on certain autonomic ganglia in the buffalo (Bubalus bubalis).

In the buffalo the sympathetic ganglionic neurons were multipolar and contained finely granular or vesicular Nissl material and eccentric nuclei with more than one nucleolus. They measured (mean values) 23.62 X 14.30 mum in the cranial cervical ganglion, 22.51 X 13.58 mum in the vertebral ganglion, 25.25 X 15.75 mum in the cervicothoracic ganglion and 22.84 X 14.27 mum in the fourth thoracic ganglion. Their density values were 256 neurons/mm2 in the cranial cervical ganglion, 250 neurons/mm2 in the vertebral ganglion, 248 neurons/mm2 in the cervicothoracic ganglion and 252 neurons/mm2 in the fourth thoracic ganglion.     

Genetic resistance to Brucella abortus in the water buffalo (Bubalus bubalis)

Brucellosis is a costly disease of water buffaloes (Bubalus bubalis). Latent infections and prolonged incubation of the pathogen limit the efficacy of programs based on the eradication of infected animals. We exploited genetic selection for disease resistance as an approach to the control of water buffalo brucellosis. We tested 231 water buffalo cows for the presence of anti-Brucella abortus antibodies (by the agglutination and complement fixation tests) and the Nramp1 genotype (by PCR-denaturing gradient gel electrophoresis). When the 231 animals (58 cases and 173 controls) were divided into infected (seropositive) and noninfected (seronegative) groups and the Nramp1 genotypes were compared, the seropositive subjects were 52 out of 167 (31%) in the Nramp1A+ (Nramp1AA or Nramp1AB) group and 6 out of 64 (9.4%) in the Nramp1A- (Nramp1BB) group (odds ratio, 4.37; 95% confidence limits, 1.87 to 10.19; chi2, 11.65 for 1 degree of freedom). Monocytes from Nramp1BB subjects displayed significantly (P < 0.01) higher levels of Nramp1 mRNA than Nramp1AA subjects and also a significantly (P < 0.01) higher ability in controlling the intracellular replication of several Brucella species in vitro. Thus, selection for the Nramp1BB genotype can become a valuable tool for the control of water buffalo brucellosis in the areas where the disease is endemic.     

Histochemical studies on the spinal cord of the Indian buffalo (Bubalus bubalis).

The distribution pattern of glycogen, alkaline phosphatase and acid phosphatase has been studied and recorded in normal and operated spinal cord of the Indian buffalo (Bubalus bubalis). Based on the observations, the following conclusions have been arrived at i) Glycogen is present in the cytoplasm and on the surface of the spinal neurons. Following injury, it is depleted from these sites. ii) Alkaline phosphatase is located in the nucleus and on the neuronal surface. It is not traceable at these sites following spinal cord injury. iii) Acid phosphatase is mostly confined to the neuronal cytoplasm and tends to increase in injured neurons.     

Anatomical studies on the optic chiasma in the Indian buffalo (Bubalus bubalis).

The optic chiasma of the buffalo was a transversely oval body, averaging 9.0 +/- 0.89 X 12.3 +/- 0.48 X 5.0 +/- 0.48 mm. Its shape was like a letter "X". This was located 2--3 mm cranial to the pituitary. Following unilateral enucleation, the degenerating myelinated fibres were noted throughout the optic chiasma, and in both the optic tracts. The degeneration in the contralateral optic tract was more dense. By enumerating the degenerating fibres in the remnant optic nerve, in the ipsilateral and contralateral optic tracts it was noted that 57.34% of the optic nerve fibres crossed over and only 12.29% of the fibres remained uncrossed.     

Isolation,characterization, and interaction of lignin-degrading bacteria from rumen of buffalo (Bubalus bubalis)

The purpose of this study was to isolate lignin-degrading bacteria from buffalo rumen and to explore their interactions further. Using lignin as the carbon source, three bacteria, B-04 (Ochrobactrum pseudintermedium), B-11 (Klebsiella pneumoniae), and B-45 (Bacillus sonorensis), which have shown lignin degradation potential, were successfully isolated and identified from the rumen fluid of buffalo by colony morphology, 16S ribosomal RNA gene sequencing, and biochemical and physiological analyses. The degradation rates of lignin were determined, and the maximum values were 4.86%, 11.1%, and 7.68% for B-04, B-11, and B-45, respectively. The maximum laccase activities were 0.65, 0.93, and 1.15 U/ml, while the maximum lignin peroxidase activities were 5.72, 8.29, and 18.69 U/ml, respectively. Pairwise interaction studies showed inhibitory interaction between B-04 and B-45, inhibitory interaction between B-04 and B-11, and symbiotic interaction between B-11 and B-45. This is the first report on the lignin degradation ability of bacteria isolated from the buffalo's rumen, which provides a new understanding for revealing the mechanism of roughage tolerance of buffalo.     

In vitro comparison of soybean lecithin-based extenders for cryopreservation of buffalo (Bubalus bubalis) semen

The objective of the present study was to compare the quality of frozen buffalo semen processed in tris-citric egg yolk extender with two different commercially available soybean lecithin-based extenders. Split pooled ejaculates, possessing more than 70 % visual sperm motility were divided in three aliquots and diluted in AndroMed®, Bioxcell®, or tris-citric egg yolk (TCE) extenders. Post-thaw motion characteristics, viability, plasma membrane integrity, acrosome morphology, and DNA integrity of buffalo sperm were studied after thawing and incubation for 4 h. Results demonstrate that total motility in frozen–thawed semen processed in TCE extender were similar when compared with AndroMed® or Bioxcell® extenders (P?>?0.05). Kinematic parameters, such as average path velocity and linearity index in the soybean lecithin-based extenders were comparatively superior to TCE extender. After 4 h of incubation, proportions of overall and progressive motility decreased in all extenders but were comparatively superior in soybean lecithin-based extenders than TCE extender (P?<?0.05). The proportion of post-thaw sperm viability, plasma membrane integrity and normal apical ridge remained similar in all extenders (P?>?0.05). The post-thaw sperm viability was comparatively superior in soybean lecithin-based extenders than TCE after incubation at 37 °C for 4 h (P?<?0.05). The type of extender had not been the significant effect on the percentage of spermatozoa with DNA damage after thawing and incubation for 4 h. In addition, our results suggest that soybean lecithin base extenders can be used as a substitute for tris-citric egg yolk extender in cryopreservation of buffalo semen.     

Anatomical studies on the optic tract (tractus opticus) of the Indian buffalo (Bubalus bubalis).

The optic tract of the buffalo was found to begin from the posterolateral angle of the chiasma, from where it extended laterally for about 8 mm. Each tract was round in its initial portion, but was flattened subsequently. In its course to the corpus geniculatum laterale (CGL), it swept round the homolateral cerebral peduncle. The tract fibres entered the CGL chiefly on its ventrolateral aspect. The optic fibres appeared to enter the CGL as individual fibres. The fibres in the optic tract were mostly myelinated. There were 253823 +/- 27534-254800 +/- 29232 myelinated fibres in it. There was no fascicular arrangement of these fibres. The calibre of the myelinated fibres in the optic tract varied from 1-12 microns. A fibre diameter analysis revealed an unimodal distribution of these fibres with a peak at 2 microns. Thinner fibres predominated in the peripheral portion of the optic tract. In the central portion, fibres of larger diameter were seen in greater numbers.