Bio-typing of Mycobacterium avium subspecies paratuberculosis isolates recovered from the Himalayan sheep and goats.

Information on bio-type profile of Mycobacterium avium subspecies paratubeculosis (MAP) in sheep flocks and goat herds of Himalayan region is not reported earlier. The aim of our study was to determine the bio-type of MAP infecting livestock of this region. A total of 71 faecal samples (sheep-57, goats-14) were screened by Ziehl-Neelsen (ZN) staining and IS900 PCR, and then processed for culture on Herrold's egg yolk medium (HEYM) having mycobactin J (MJ). Out of 71 faecal samples, MAP colonies were seen only in four samples (sheep-3 and goat-1). Isolates were confirmed as MAP on the basis of slow growth, acid fastness, MJ dependency, IS900 and IS1311 PCR. All the IS900 and IS1311 PCR positive samples were bio-typed by IS1311 PCR-REA (restriction endonuclease analysis), which confirmed all four isolates as 'bison type.' In IS1311 based phylogeny of MAP isolates by ClustalW method of the MegAlign program of DNASTAR Lasergene software, the four sequences of MAP isolates (NCBI sequence nos. MH988763, MH988765, MH988766 and MH988764) did not show any distinct clustering/grouping pattern. However, these four isolates showed a bit of closeness to the MAP sequences (KC990353.1 and KC990352.1) of 'bison type' isolated from wood bison in Canada. In conclusion, this is the first report on isolation and bio-type profile of MAP infecting sheep and goats of Himalayan region. Study will help in devising prevention and control strategies against spread of MAP infection in livestock population of Himalayan region.

Development of diagnostic assays for differentiation of atypical Aeromonas salmonicida vapA type V and type VI in ballan wrasse (Labrus bergylta, Ascanius).

Aeromonas salmonicida (As) is a highly heterogeneous bacterial species, and strains' host specificity has been reported. Ballan wrasse (Labrus bergylta Ascanius, 1767) is susceptible to atypical As (aAs) vapA type V and type VI in Scotland and Norway. Identification of the bacterium is achieved by culture and molecular techniques; however, the available methods used to distinguish the As types are costly and time-consuming. This paper describes the development of a PCR and a restriction enzyme assay for the detection of aAs vapA type V and type VI in ballan wrasse, respectively. Type V-specific primers were designed on conserved regions of the vapA gene, and the restriction enzyme assay was performed on the PCR products of the hypervariable region of vapA gene for the detection of type VI isolates. Amplification product was produced for type V (254 bp) and restriction bands (368 and 254 bp) for type VI isolates only. In addition, the assays detected type V and type VI isolates in spiked water samples and type V in diagnostic tissue samples. The assays are fast, specific and cost-effective and can be used as specific diagnostic tools for cleaner fish, to detect infectious divergence strains, and to manage and mitigate aAs disease outbreaks through vaccine development.

First Report of Sex Chromosomes in Night Lizards (Scincoidea: Xantusiidae).

Squamate reptiles (lizards, snakes, and amphibians) are an outstanding group for studying sex chromosome evolution-they are old, speciose, geographically widespread, and exhibit myriad sex-determining modes. Yet, the vast majority of squamate species lack heteromorphic sex chromosomes. Cataloging the sex chromosome systems of species lacking easily identifiable, heteromorphic sex chromosomes, therefore, is essential before we are to fully understand the evolution of vertebrate sex chromosomes. Here, we use restriction site-associated DNA sequencing (RADseq) to classify the sex chromosome system of the granite night lizard, Xantusia henshawi. RADseq is an effective alternative to traditional cytogenetic methods for determining a species' sex chromosome system (i.e., XX/XY or ZZ/ZW), particularly in taxa with non-differentiated sex chromosomes. Although many xantusiid lineages have been karyotyped, none possess heteromorphic sex chromosomes. We identified a ZZ/ZW sex chromosome system in X. henshawi-the first such data for this family. Furthermore, we report that the X. henshawi sex chromosome contains fragments of genes found on Gallus gallus chromosomes 7, 12, and 18 (which are homologous to Anolis carolinensis chromosome 2), the first vertebrate sex chromosomes to utilize this linkage group.

A novel PCR protocol for detection and differentiation of neuropathogenic and non-neuropathogenic equid alphaherpesvirus 1.

Equid alphaherpesvirus 1 (EHV-1) infections can have a major impact on the horse industry and equine welfare by causing abortion or respiratory or neurologic disease. A single nucleotide polymorphism (A2254→G2254) in open reading frame (ORF) 30, encoding the catalytic subunit of the DNA polymerase, has been shown to be a strong predictive marker for neuropathogenicity. Given that a previously established real-time PCR (rtPCR) protocol yielded unsatisfactory results concerning determination of the EHV-1 genotype, we developed and evaluated a new conventional PCR protocol enabling identification of the genotype by sequencing and restriction enzyme analysis (REA). Thirty samples from horses with signs typical for EHV-1 infection were tested by rtPCR and our new conventional PCR. The results showed that compared to rtPCR, the conventional PCR protocol combined with sequencing and REA was more reliable concerning unambiguous determination of the EHV-1 genotype. Results of our new assay confirmed previous findings, according to which the non-neuropathogenic genotype A2254 is predominantly found in animals with fever, respiratory signs, and abortions or perinatal mortality, whereas the neuropathogenic genotype G2254 is primarily detected in animals suffering from neurologic disease. In some samples, results pointed towards coinfection with both genotypes. Further studies are required in order to elucidate the significance of infections with genotype A2254 and G2254 in neurologic and non-neurologic cases, respectively.

Development and Validation of Sex-Specific Markers in Pelodiscus Sinensis Using Restriction Site-Associated DNA Sequencing.

The sex of an animal influences its economic traits, especially in species displaying sexual dimorphism. The Chinese soft-shelled turtle, Pelodiscus sinensis, is an economically important aquatic species that shows significant male sexual dimorphism, with a large body size, faster growth, a thick and wide calipash, and lower body fat. In this study, ten male and ten female turtles were subjected to restriction site-associated DNA sequencing (RAD-seq) using the Hi-Seq 4000 sequencing platform to isolate female-specific DNA fragments. We identified 5967 bp and 6532 bp fragments using genome walking. Three female-specific markers designed from these two fragments were confirmed to separate the sexes of Pelodiscus sinensis perfectly. One of the female-specific markers showed dosage association in female and male individuals. Individuals from different populations (n = 296) were used to validate that the female-specific markers could identify the genetic sex of Pelodiscus sinensis with 100% accuracy. The results of the present study demonstrated that RAD-seq was useful to develop sex-related markers in animals, and verified that the sex determination system of Pelodiscus sinensis belonged to the ZZ/ZW heterogametic system. Importantly, the developed markers could lead to a method for sex-controlled breeding in the Chinese soft-shelled turtle.

Comparative restriction enzyme mapping of Campylobacter jejuni isolates from turkeys and broilers based on flaA flagellar gene using HpyF3I endonuclease.

Turkeys and broilers have been identified as important reservoirs for Campylobacter jejuni which is of public health significance. The evaluation of the genotypes among C. jejuni strains within different reservoirs is critical for our understanding of the epidemiology of this infectious agent. The present study aimed to compare the genetic diversity and differences of C. jejuni isolates from turkeys and broilers using flagellin PCR-RFLP typing (flaA typing) technique, in terms of the ease of use and discriminatory power. Sixty C. jejuni isolates were detected biochemically and confirmed by duplex-PCR from turkeys and broilers (30 strains from each bird species). Then, a flaA gene fragment (1725 bp) of C. jejuni isolates was amplified and amplicons were digested with HpyF3I enzyme. Restriction analysis by HpyF3I gave four different flaA patterns (H1, H2, H3, H4) among all tested C. jejuni isolates. In broiler isolates, all four patterns were observed but in turkey isolates, only H2 and H4 patterns were present. The results clearly demonstrated that distribution of the flaA typing patterns differed depending on the host species (broiler/turkey). H1 and H3 flaA types are more prevalent in broiler than turkey isolates, while H2 type is significantly more prevalent within isolates from turkey (p < 0.05). The flaA typing technique by digestion with HpyF3I enzyme can almost give us a clue to the source of infection in local outbreaks.

Comparison of whole genome sequencing to restriction endonuclease analysis and gel diffusion precipitin-based serotyping of Pasteurella multocida.

The gel diffusion precipitin test (GDPT) and restriction endonuclease analysis (REA) have commonly been used in the serotyping and genotyping of Pasteurella multocida. Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis has become the gold standard for other organisms, offering higher resolution than previously available methods. We compared WGS to REA and GDPT on 163 isolates of P. multocida to determine if WGS produced more precise results. The isolates used represented the 16 reference serovars, isolates with REA profiles matching an attenuated fowl cholera vaccine strain, and isolates from 10 different animal species. Isolates originated from across the United States and from Chile. Identical REA profiles clustered together in the phylogenetic tree. REA profiles that differed by only a few bands had fewer SNP differences than REA profiles with more differences, as expected. The GDPT results were diverse but it was common to see a single serovar show up repeatedly within clusters. Several errors were found when examining the REA profiles. WGS was able to confirm these errors and compensate for the subjectivity in analysis of REA. Also, results of WGS and SNP analysis correlated more closely with the epidemiologic data than GDPT. In silico results were also compared to a lipopolysaccharide rapid multiplex PCR test. From the data produced in our study, WGS and SNP analysis was superior to REA and GDPT and highlighted some of the issues with the older tests.

RAD SNP markers as a tool for conservation of dolphinfish Coryphaena hippurus in the Mediterranean Sea: Identification of subtle genetic structure and assessment of populations sex-ratios.

Dolphinfish is an important fish species for both commercial and sport fishing, but so far limited information is available on genetic variability and pattern of differentiation of dolphinfish populations in the Mediterranean basin. Recently developed techniques allow genome-wide identification of genetic markers for better understanding of population structure in species with limited genome information. Using restriction-site associated DNA analysis we successfully genotyped 140 individuals of dolphinfish from eight locations in the Mediterranean Sea at 3324 SNP loci. We identified 311 sex-related loci that were used to assess sex-ratio in dolphinfish populations. In addition, we identified a weak signature of genetic differentiation of the population closer to Gibraltar Strait in comparison to other Mediterranean populations, which might be related to introgression of individuals from Atlantic. No further genetic differentiation could be detected in the other populations sampled, as expected considering the known highly mobility of the species. The results obtained improve our knowledge of the species and can help managing dolphinfish stock in the future.

Development and characterization of microsatellite markers for Tibouchina hatschbachii (Melastomataceae), an endemic and habitatrestricted shrub from Brazil / Desenvolvimento e caracterização de marcadores microssatélites para Tibouchina hatschbachii (Melastomataceae), um arbusto endêmico e com restrição de habitat da região subtropical do Brasil

Tibouchina hatschbachii Wurdack (Melastomataceae) is an autogamous shrub restricted to granite (GO) and sandstone (SO) rock outcrops from subtropical Brazil. We designed primers for the amplification of microsatellite regions for T. hatschbachii, and characterized these primers to estimate genetic diversity parameters and contemporary genetic structure patterns. Eight loci were successfully amplified and were characterized using 70 individuals from three natural populations. Polymorphic information content ranged from 0.200 to 0.772 per locus. All loci were polymorphic, with allele numbers ranging from two to eight. The low degree of polymorphism may be explained by the fact that T. hatschbachii has disjunct populations and a recent genetic bottleneck, and also that it is self-pollinated. The observed and expected heterozygosities ranged from 0.115 to 1.000 and from 0.112 to 0.800, respectively. We observed private alleles in all loci. These are important features that enable us to identify population differentiation and help to us understand gene flow patterns for T. hatschbachii in subtropical Brazil. Eight microsatellite loci from other species of Tibouchina amplified positively in T. hatschbachii.

Tibouchina hatschbachii Wurdack (Melastomataceae) é um arbusto autógamo, com ocorrência restrita em afloramentos rochosos graníticos (GO) e areníticos (SO) na região subtropical do Brasil. Neste trabalho, foram desenvolvidos marcadores para a amplificação de regiões microssatélites para T. hatschbachii e caracterizados esses primers para estimar parâmetros de diversidade genética. Oito loci foram amplificados com sucesso e caracterizados, utilizando 70 indivíduos de três populações naturais. O conteúdo de informação polimórfica variou de 0,200 a 0,772 por locus. Todos os loci foram polimórficos, com números de alelos que variam de dois a oito. O baixo grau de polimorfismo pode ser explicado pelo fato de que T. hatschbachii possui populações disjuntas e uma história recente de gargalo genético populacional, e também pelo fato de apresentar um sistema reprodutivo de autopolinização, tendendo a favorecer a baixa variação. As heterozigosidades observadas e esperadas variaram entre 0,115-1,000 e 0,112-0,800, respectivamente. Também foi observada a presença de alelos privados em todos os loci. Estas são características importantes que nos permitirão identificar a diferenciação entre populações e poderão ajudar na compreensão dos padrões de fluxo gênico atual de T. hatschbachii na região subtropical do Brasil. Oito loci microssatélites de outras espécies de Tibouchina amplificaram

Rapid microsatellite development in Gekko japonicus using sequenced restriction-site associated DNA markers.

Twelve polymorphic microsatellite loci were isolated in the Japanese gecko, Gekko japonicus. We genotyped one population from Wenzhou, Zhejiang Province, China (N = 36). The mean number of observed alleles per locus was 7.3 (range 4 to 13). Observed and expected heterozygosity values ranged from 0.200 to 0.944 and from 0.395 to 0.797, respectively. One locus (GJ20) showed significant departure from Hardy-Weinberg equilibrium; no linkage disequilibrium was found between any two loci. These informative microsatellite markers will be useful for population genetic analyses of G. japonicus and other species in the genus Gekko.

Modified 16S-23S rRNA intergenic region restriction endonuclease analysis for species identification of Enterococcus strains isolated from pigs, compared with identification using classical methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Fast and reliable identification of bacteria to at least the species level is currently the basis for correct diagnosis and appropriate treatment of infections. This is particularly important in the case of bacteria of the genus Enterococcus, whose resistance profile is often correlated with their species (e.g. resistance to vancomycin). In this study, we evaluated restriction endonuclease analysis of the 16S-23S rRNA gene intergenic transcribed spacer (ITS) region for species identification of Enterococcus. The utility of the method was compared with that of phenotypic methods [biochemical profile evaluation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)]. Identification was based on 21 Enterococcus reference strains, of the species E. faecalis, E. faecium, E. hirae, E. durans, E. casseliflavus, E. gallinarum, E. avium, E. cecorum and E. columbae, and 47 Enterococcus field strains isolated from pigs. Restriction endonuclease analysis of the ITS-PCR product using HinfI, RsaI and MboI, in the order specified, enabled species differentiation of the Enterococcus reference and field strains, and in the case of the latter, the results of species identification were identical (47/47) to those obtained by MALDI-TOF MS. Moreover, as a result of digestion with MboI, a unique restriction profile was also obtained for the strains (3/3) identified by MALDI-TOF MS as E. thailandicus. In our opinion, restriction endonuclease analysis of the 16S-23S rRNA gene ITS region of Enterococcus may be a simple and relatively fast (less than 4 h) alternative method for identifying the species occurring most frequently in humans and animals.

Mycobacterium avium subsp. paratuberculosis sheep strains isolated from Cyprus sheep and goats.

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic incurable infection of intestinal tract of animals. Molecular characterization of Map isolates classifies them into two major groups, 'Cattle' or Type II and 'Sheep' or Type I/III with a different phenotype, epidemiology, virulence and pathogenesis. The aim of this study was to examine 192 Map ELISA-positive sheep and goats from Cyprus using faecal culture and genotype Map isolates using IS1311 PCR and restriction endonuclease analysis (IS1311 PCR-REA) with HinfI restriction enzyme. Map was isolated from only four (4.6%) faecal samples out of 88 sheep and 15 (14.4%) faecal samples out of 104 goats. Genotyping of the isolates using IS1311 PCR-REA revealed that sheep and goat populations on the island are infected primarily by 'Sheep' strains. Only three Map isolates from goats originated from one farm were characterized as 'Cattle' strains.

First report of genotyping of Toxoplasma gondii in free-living Microtus fortis in northeastern China.

Toxoplasma gondii is an intracellular protozoan parasite which imperils the health of almost all warm-blooded animals, including humans. The objective of this study was to determine genetic characterization of T. gondii in free-living Microtus fortis (reed vole) in Jilin province, northeastern China. A total of 104 DNA samples, 74 from Gongzhuling and 30 from Baicheng, were extracted from lung tissues of M. fortis , and 56 (53.8%) of them were positive for T. gondii by semi-nested polymerase chain reaction of the B1 gene. These positive DNA samples were typed at 10 genetic markers including SAG1, 5'- and 3'-SAG2, alternative SAG2, BUTB, GRA6, L358, PK1, c22-8, c29-2, and Apico. Four samples were successfully genotyped at all genetic loci and grouped to 2 distinct genotypes; 2 samples belonged to ToxoDB Genotype no. 10 (Type I) and the other 2 presented ToxoDB Genotype no. 9 ( http://toxodb.org/toxo/ ); 4 samples were genotyped at 8 genetic loci, in which 2 samples belonged to ToxoDB Genotype no. 10 and 2 presented ToxoDB Genotype no. 9. To our knowledge, this is the first report of genetic typing of T. gondii from free-living M. fortis in northeast China. The results suggest that the Type I and ToxoDB Genotype no. 9 could be a potential risk factor for transmission through the reed vole in this region.

A rapid profiling assay for avian leukosis virus subgroup E proviruses in chickens.

Endogenous retroviral elements (ERVs) are prolific components of the genomes of complex species, typically occupying more sequence space than do essential, protein-encoding genes. Much of what we know today about the structure and function, as well as the evolution and pathogenic potential, of ERVs was fleshed out over several decades during the last century using the avian leukosis virus subgroup E-related (ALVE) family of endogenous retroviruses of chickens as a model system. A critical enabling factor in the elucidation of ALVE structure and function is the ability to detect and unambiguously identify specific ALVE proviral elements and to develop accurate element profiles for individual chickens under study. Currently, the most common approach for ALVE locus detection involves element-specific PCR assays carried out using primers that target host DNA near the insertion site of the provirus (i.e., the upstream and downstream flanks of the unoccupied site). Here we describe a new approach for proviral detection that exploits restriction enzyme sites in flanking DNA to develop ALVE element profiles more rapidly than with assays currently in use. Moreover, unlike element-specific PCR tests, the "profiling" assay detects novel ALVEs for which insertion sites have not yet been identified as well as previously characterized elements.

Prevalence, molecular characterization and zoonotic potential of Cryptosporidium spp. in goats in Henan and Chongqing, China.

To estimate the prevalence and public health significance of cryptosporidiosis in goats in China, 1265 fecal samples from seven farms in Henan province and Chongqing city were examined for Cryptosporidium oocysts. The overall infection rate of Cryptosporidium spp. was 3.48% (44/1256). Significant difference was observed among age groups, with the post weaned kids having the highest infection rate (4.58%; ρ<0.01). Cryptosporidium spp. were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis and DNA sequence analysis of the small subunit (SSU) rRNA gene. The SSU rRNA-based PCR identified three Cryptosporidium species, including Cryptosporidium ubiquitum (24/44) in Henan and Chongqing, and Cryptosporidium andersoni (16/44) and Cryptosporidium xiaoi (4/44) in Henan. Among which, the C. ubiquitum and C. andersoni were first identified in goats thus far and were found in all age groups except no C. andersoni being found in the postparturition nannies, whereas the C. xiaoi was detected in pre-weaned kids and pregnant nannies. Subtyping C. ubiquitum by DNA sequence analysis of the 60 kDa glycoprotein (gp60) gene suggested the isolates identified all belonged to zoonotic XIIa subtype 2. Thus, the dominant C. ubiquitum found in this study and the XIIa subtype 2 has been found in humans indicated goats are a potential source for zoonotic infections with the C. ubiquitum. More studies are needed for better understanding of differences in the transmission and public health significance of cryptosporidiosis in goats.

Genital form of pasteurellosis in breeding turkeys infected during artificial insemination and isolation of an unusual strain of Pasteurella multocida.

A genital and potentially fatal form of Pasteurella multocida infection was reported on two turkey-breeding farms on which birds were vaccinated against Pasteurella multocida. Both outbreaks were linked to the use of semen from young vaccinated toms with a history of respiratory pasteurellosis followed by treatment during rearing. Typing by agar gel immunodiffusion and rapid slide agglutination of P. multocida isolated from cloacal swabs was completed by multilocus sequence typing. Restriction enzyme analysis showed that that the isolates were clonal. They belonged to sequence type (ST) 30, described in chickens, cats, and ducks. This strain differed in sequence type from the ones used in the vaccine (ST8, ST60, ST53, and ST235), which might have limited its effectiveness. No contamination of the semen (n = 30) was found, suggesting fecal contamination during semen collection.

Contrasting results of culture-dependent and molecular analyses of Mycobacterium avium subsp. paratuberculosis from wood bison.

Reduced to near extinction in the late 1800s, a number of wood bison populations (Bison bison athabascae) have been re-established through reintroduction initiatives. Although an invaluable tool for conservation, translocation of animals can spread infectious agents to new areas or expose animals to pathogens in their new environment. Mycobacterium avium subsp. paratuberculosis, a bacterium that causes chronic enteritis in ruminants, is among the pathogens of potential concern for wood bison management and conservation. In order to inform translocation decisions, our objectives were to determine the M. avium subsp. paratuberculosis infection status of wood bison herds in Canada and to culture and genetically characterize the infective strain(s). We tested fecal samples from bison (n = 267) in nine herds using direct PCR for three M. avium subsp. paratuberculosis-specific genetic targets with different copy numbers within the M. avium subsp. paratuberculosis genome. Restriction enzyme analysis (REA) and sequencing of IS1311 were performed on seven samples from five different herds. We also evaluated a panel of different culture conditions for their ability to support M. avium subsp. paratuberculosis growth from feces and tissues of direct-PCR-positive animals. Eighty-one fecal samples (30%) tested positive using direct IS900 PCR, with positive samples from all nine herds; of these, 75% and 21% were also positive using ISMAP02 and F57, respectively. None of the culture conditions supported the growth of M. avium subsp. paratuberculosis from PCR-positive samples. IS1311 REA and sequencing indicate that at least two different M. avium subsp. paratuberculosis strain types exist in Canadian wood bison. The presence of different M. avium subsp. paratuberculosis strains among wood bison herds should be considered in the planning of translocations.

Comparison of pre- and post-vaccination ovine Johne's disease prevalence using a Bayesian approach.

This study was conducted to evaluate the effectiveness of Gudair™ vaccine in decreasing the prevalence of shedding of Mycobacterium avium subsp. paratuberculosis (MAP) in flocks of varying initial prevalence. Thirty-seven self-replacing Merino flocks from New South Wales and Victoria (Australia) that had been vaccinating lambs with Gudair™ for at least five years were enrolled in the study. These flocks had been tested prior to or at commencement of vaccination using pooled faecal culture, agar gel immunodiffusion or both tests. These pre-vaccination test results were used to estimate pre-vaccination prevalence. Post-vaccination prevalence was estimated from culture of usually 7 pools of 50 sheep collected from the enrolled flocks in 2008-2009, approximately five or more years after commencement of vaccination. A Bayesian model was developed to estimate and compare the pre- and post-vaccination prevalences for the enrolled flocks. Apparent pre- and post-vaccination prevalences for flocks were modelled as functions of the true pre- and post-vaccination prevalences, respectively, and the sensitivities and specificities of the respective diagnostic tests. Logit-normal models were specified on pre- and post-vaccination true prevalences and were then used to make inferences about the median and 90th percentile of the prevalence distributions and their differences. Priors were mostly specified based on published literature or analysis of abattoir surveillance data for this population of flocks. The analysis found a significant decline in ovine Johne's disease prevalence from a pre-vaccination median prevalence of 2.72% [95% probability interval (PI): 1.40; 6.86%] to a post-vaccination median prevalence of 0.72% (0.39; 1.27%). However 30 of the 37 flocks still contained sheep that were shedding MAP in their faeces. The results suggest that vaccination with Gudair™ is usually effective in reducing the prevalence of faecal shedding but the response to vaccination is variable among flocks. The Bayesian approach reported here could be implemented in similar situations to compare prevalences where information from multiple diagnostic tests with varied sensitivities and specificities is available.

The effects of polymorphisms in 7 candidate genes on resistance to Salmonella Enteritidis in native chickens.

Salmonella enterica serovar Enteritidis infection is a common concern in poultry production for its negative effects on growth as well as food safety for humans. Identification of molecular markers that are linked to resistance to Salmonella Enteritidis may lead to appropriate solutions to control Salmonella infection in chickens. This study investigated the association of candidate genes with resistance to Salmonella Enteritidis in young chickens. Two native breeds of Malaysian chickens, namely, Village Chickens and Red Junglefowl, were evaluated for bacterial colonization after Salmonella Enteritidis inoculation. Seven candidate genes were selected on the basis of their physiological role in immune response, as determined by prior studies in other genetic lines: natural resistance-associated protein 1 (NRAMP1), transforming growth factor ß3 (TGFß3), transforming growth factor ß4 (TGFß4), inhibitor of apoptosis protein 1 (IAP1), caspase 1 (CASP1), lipopolysaccharide-induced tumor necrosis factor (TNF) α factor (LITAF), and TNF-related apoptosis-inducing ligand (TRAIL). Polymerase chain reaction-RFLP was used to identify polymorphisms in the candidate genes; all genes exhibited polymorphisms in at least one breed. The NRAMP1-SacI polymorphism correlated with the differences in Salmonella Enteritidis load in the cecum (P = 0.002) and spleen (P = 0.01) of Village Chickens. Polymorphisms in the restriction sites of TGFß3-BsrI, TGFß4-MboII, and TRAIL-StyI were associated with Salmonella Enteritidis burden in the cecum, spleen, and liver of Village Chickens and Red Junglefowl (P < 0.05). These results indicate that the NRAMP1, TGFß3, TGFß4, and TRAIL genes are potential candidates for use in selection programs for increasing genetic resistance against Salmonella Enteritidis in native Malaysian chickens.

Detection and differentiation of pigeon paramyxovirus serotype-1 (PPMV-1) isolates by RT-PCR and restriction enzyme analysis.

Detection and pathotyping of Newcastle disease virus (NDV) is extremely important because the appearance of virulent virus has significant economic consequences. During 1981 to 1985, infections of racing and show pigeons with an avian paramyxovirus serotype-1 (APMV-1) hit worldwide, and a panzootic occurred due to a variant form of classical NDV. On the basis of pathogenicity and monoclonal antibody binding studies, the virus was termed 'pigeon PMV-1' (PPMV-1). In the past, number of Newcastle disease outbreaks in poultry and other birds has been attributed to PPMV-1. PPMV-1 viruses are known to present difficulty when assessed by conventional in vivo pathogenicity tests. In this study, the technique of reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme (RE) analysis was used to detect and differentiate PPMV-1 isolates of Indian origin. Restriction enzyme digestion analysis of RT-PCR-amplified fusion protein (F) gene, encoding for the cleavage activation sites of fusion protein, was carried out with restriction enzymes BglI, HhaI, HaeIII, HinfI, MboI, MspI, PvuII and StyI. A set of only four enzymes HhaI, MspI or HaeIII, MboI and BglI alone were sufficient to differentially detect APMV-1 and PPMV-1 viruses and their pathotypes. In conclusion, RT-PCR followed by RE analysis proved to be useful for detection and differentiation of APMV-1 and PPMV-1 isolates at genomic level.