Ribotyping of Indian Isolates of <Emphasis Type="Italic">Pasteurella multocida</Emphasis> Based on 16S and 23S rRNA Genes

The applicability of ribotyping based on 16S and 23S rRNA was evaluated for molecular epidemiological studies. Forty-eight isolates of Pasteurella multocida isolated from different hosts and geographical locations and one reference isolate were ribotyped. Only four ribotypes were found. All the isolates including reference isolate from wild carnivores had the same ribotype, though they had different serotypes. The isolate from a tiger had one band in addition to the bands present in the major ribotype. The isolates from lions represented two ribotypes; of these ribotypes, one (r2) had an additional band of 3.6 kbp, which was absent in all other ribotypes. The second ribotype (r4) from a lion had one band missing (6 kbp) that was present in the other ribotypes. These isolates were further typed using ERIC-PCR and REP-PCR. With ERIC-PCR and REP-PCR, higher D values of 0.83 and 0.89 were obtained. The current study revealed that ribotyping is not a very efficient typing tool for use in molecular epidemiology for differentiation of isolates.     

Prevalent Serotypes of Pasteurella multocida Isolated from Different Animal and Avian Species in India

Identification and estimation of the prevalence of Pasteurella multocida organisms in different animal and avian species in India during November 2000 to July 2003 was carried out. Out of 418 samples collected from different outbreaks suspected to be caused by P. multocida, a total of 206 bacterial cultures were identified as P. multocida on the basis of cultural, morphological and biochemical characteristics. All the 206 cultures were isolated from different domestic animal species (cattle, buffalo, sheep, goat, pig and rabbit), avian species (chicken, duck, quail, turkey, goose) and wild animals such as leopard and deer. Serotyping of P. multocida cultures revealed the presence of various serotypes (A:1, A:3, A:1,3, A:4, B:2, D:1 and -:1) among the livestock population. P. multocida polymerase chain reaction (PCR) assay applied on different forms of bacterial cultures (bacterial culture lysate, direct bacterial colony and mixed bacterial culture lysate) yielded an amplified product of approximately 460 bp specific for P. multocida. The results of PCR assay correlated well with conventional methods of identification. The present investigation revealed the presence of varied serotypes among livestock and PCR assay was found to be useful for rapid, sensitive and specific diagnosis of pasteurellosis in animals and avian species.     

Specific identification of Pasteurella multocida serogroup-A isolates by PCR assay

A polymerase chain reaction (PCR) assay targeting the hyaC-hyaD gene was developed and used to identify strains of Pasteurella multocida belonging to serogroup-A. A set of serogroup-specific-PCR primers amplified a 564 bp product from genomic DNA prepared from bacterial cells or directly from bacterial colonies. This method detected as low as 10 ng of bacterial DNA and had a specificity of 100% for P. multocida serogroup-A. A nested PCR method yielded a single 374 bp product. All fifty isolates were also shown to be identical by restriction fragment length polymorphism (RFLP) analysis of the PCR products after digestion with BglII.     

The effects of a single injection of dexamethasone-21-isonicotinate on the lymphocyte functions of dairy cows at two weeks post partum

Dexamethasone is a potent therapeutic for treatment of the fatty liver syndrome or primary ketosis in post partum dairy cows. Reservations exist, however, among practitioners with respect to the risk of immunosuppression induced by corticosteroids. The aim of this study was to investigate the effect of a single injection of dexamethasone-21-isonicotinate on distinct immune functions of postpartum dairy cows because only scarce information is available on the effects of corticosteroid preparations when administered at a dosage and frequency for treatment of the fatty liver syndrome or primary ketosis. Sixteen Swedish red-pied dairy cows, between days 9 and 15 post partum, were allotted to either a control group (n = 8) or a treatment group (n = 8). The cows in the treatment group received a single intramuscular injection of a dexamethasone-21-isonicotinate suspension at a dosage of 0.02 mg/kg i.m. at the start of the experiment. White blood cell counts and selected lymphocyte functions (lymphocyte proliferation, expression of lymphocyte markers and the b2 and a4 chain of adhesion molecules belonging to the integrin family) and some parameters of the energy metabolism (glucose, insulin) were determined before the administration of corticosteroids (day 0) and subsequently at days 2, 4, 7 and 9 of the experiment. Changes in glucose and insulin were within the target range for treatment of the fatty liver syndrome or primary ketosis. Significant (P < 0.05) increases in the number of circulating white blood cells were observed in treated cows on the second day following treatment which was exclusively caused by an increase in the number of circulating neutrophils. Lymphocyte blastogenesis in response to ConA and the percentages of lymphocytes positive for CD2, CD4, CD8, CD49d and CD18 as well as the intensity of CD49d expression did not differ between the treatment and control groups. There was, however, a significant reduction (P < 0.01) in the intensity of CD18 expression on lymphocytes in the treated animals on the fourth day after treatment. In conclusion, a single administration of dexamethasone-21-isonicotinate in a dosage of 0.02 mg/kg i.m. at two weeks post partum in healthy cows had a significant but highly transient effect on CD18 expression on lymphocytes and the number of peripheral blood neutrophils, but did not affect lymphocyte blastogenesis or lymphocyte subpopulation patterns in peripheral blood.     

MOSFET-Embedded microcantilevers for measuring deflection in biomolecular sensors

A promising approach for detecting biomolecules follows their binding to immobilized probe molecules on microfabricated cantilevers; binding causes surface stresses that bend the cantilever. We measured this deflection, which is on the order of tens of nanometers, by embedding a metal-oxide semiconductor field-effect transistor (MOSFET) into the base of the cantilever and recording decreases in drain current with deflections as small as 5 nanometers. The gate region of the MOSFET responds to surface stresses and thus is embedded in silicon nitride so as to avoid direct contact with the sample solution. This approach, which offers low noise, high sensitivity, and direct readout, was used to detect specific binding events with biotin and antibodies.     

Effects of probiotics and maternal vaccination on Salmonella enteritidis infection in broiler chicks

The effects of probiotics and maternal vaccination with an inactivated Salmonella Enteritidis (SE) vaccine on day-old chicks challenged with SE were evaluated. A 2 X 3 factorial arrangement was used (with or without probiotics; breeders nonvaccinated, vaccinated intramuscularly, or vaccinated intraperitoneally). Three trials were conducted in isolation cabinets and SE challenge was different between trials. The number of SE organisms per chick and the time interval between housing and introduction of seeder birds (hereafter called challenge) were 1.6 X 10(8) and 1 hr (Trial I), 1.8 X 10(6) and 12 hr (Trial II), and 1.2 X 10(4) and 24 hr (Trial III). SE recovery was assessed in ceca and liver at 3, 5, and 7 days postchallenge, and the number of colony-forming units (CFU) in ceca was evaluated at 5 and 7 days postchallenge. The number of SE (log CFU) in the ceca reduced 0.56 log (from 7.59 to 7.03) and 1.45 log (7.62 to 6.17) because of the treatment with probiotics in Trials II and III, respectively. The greater reduction in Trial III indicates the importance of the early use of probiotics on the prevention of SE infection. Treatment with probiotics resulted in a smaller number of SE-positive livers after 5 days postchallenge on Trial III. Although there was no significant effect of maternal vaccination on the number of SE CFU in the ceca, a significant effect of maternal vaccination on the SE CFU was observed in the liver, but not in the ceca at 5 days after challenge.     

Identification of avian strains of Pasteurella multocida in India by conventional and PCR assays

The prevalence of capsular and somatic serotypes were studied among 123 Pasteurella multocida strains isolated from chickens (n = 94), ducks (22), quails (4), turkeys (2) and geese (1) from different geographical regions of India. All strains exhibited similar cultural and morphological characteristics. Ninety-two of the isolates belonged to serotype A:1, the most prevalent serotype, with serotypes A:3, A:1,3, D:3 and F:3 having two isolates each. Only one isolate was positive for serotypes A:4 and D:1. Twenty isolates were untyped. A multiplex capsular PCR assay generated amplicons of sizes 460, 1044, 657 and 854 bp in 106 isolates identified as capsular serotype-A, 15 in serotype D and two in serotype F. Capsular types B and E were not detected in any of the avian isolates studied. The present findings suggest that a multiplex capsular PCR assay may be suitable for the rapid initial identification serotypes P. multocida during epidemiological studies of fowl cholera.     

Photocatalytic production and processing of conjugated linoleic acid-rich soy oil

Daily intake of conjugated linoleic acid (CLA), an anticarcinogenic, antiatherosclerotic, antimutagenic agent, and antioxidant, from dairy and meat products is substantially less than estimated required values. The objective of this study was to obtain CLA-rich soybean oil by a customized photochemical reaction system with an iodine catalyst and evaluate the effect of processing on iodine and iodo compounds after adsorption. After 144 h of irradiation, a total CLA yield of 24% (w/w) total oil was obtained with 0.15% (w/w) iodine. Trans,trans isomers (17.5%) formed the majority of the total yield and are also associated with health benefits. The isomers cis-9,trans-11 and trans-10,cis-12 CLA, associated with maximum health benefits, formed approximately 3.5% of the total oil. This amount is quite significant considering that total CLA obtained from dairy sources is only 0.6%. ATR-FTIR, 1H NMR, and GC-MS analyses indicated the absence of peroxide and aldehyde protons, providing evidence that secondary lipid oxidation products were not formed during the photochemical reaction. Adsorption processing vastly reduced the iodine and iodocompounds without CLA loss. Photocatalysis significantly increased the levels of CLA in soybean oil.     

Downgrading of heavy broiler chicken carcasses due to myodegeneration of the anterior latissimus dorsi: pathologic and epidemiologic studies

Recently, in some Brazilian poultry companies, a dorsal cranial muscular lesion has been increasingly detected in broilers, causing heavy economic losses due to carcass downgrading. The observed gross lesions located in the anterior latissimus dorsi (ALD) muscle are characterized by yellowish discoloration of the skin and swelling on the dorsal cranial region of that muscle. When the ALD muscle is cut, subcutaneous edema, muscular superficial hemorrhage, pallor, adherence, and increased thickness and density are observed. Microscopically, findings indicate degenerative and polyphasic features, variation in fiber size and splitting, presence of hyaline, necrotic and regenerative myofibers, extensive fibrosis, and adipose tissue with lymphohistiocytic infiltration in all ALD muscles affected. The etiology of the lesion is unknown, and no detailed report was found in literature. The highest frequency of carcass downgrading due to this lesion was found in the heaviest and the oldest males of high-yield broiler strains (P < 0.01). This study is the first to describe the pathologic and some epidemiologic aspects of this new myopathy.     

High Mycobacterium bovis genetic diversity in a low prevalence setting

The genetic diversity among South African Mycobacterium bovis isolates from cattle was determined by genetic fingerprinting. The restriction fragment length polymorphism (RFLP) markers IS6110 and polymorphic GC-rich sequence (PGRS) as well as spoligotyping and determination of variable number of tandem repeats (VNTR) were used to characterize sub samples of 91 M. bovis field isolates. PGRS RFLP was the single most discriminatory method and combinations of typing methods, which included IS6110 and/or PGRS had the highest discriminatory power, able to reveal 29 distinct genotypes among 35 farms with no epidemiological link. Three of the farms were co-infected with two genetically unrelated strains. In contrast to reports from European and also other colonised countries on the African continent our findings are suggestive of a high genetic diversity of M. bovis in South Africa's cattle population, implying a variety of unrelated ancestor strains. Despite effective intervention through test-and-slaughter campaigns no indication of a 'founder effect' was apparent in the panel of isolates derived from all infected provinces.