Goat Isolate Research Articles

Antibacterial Efficacy of Plant Aqueous Extracts Against Escherichia coli O157:H7 Bacteria in Grenada, West Indies

Antimicrobial resistance has developed as a major health problem worldwide, endangering the efficacy of antibiotics and imposing a severe burden of resistant infections. The progressive increase in the prevalence of multiple resistant bacterial strains paves the way for the selection of natural plants with antibacterial properties as an alternative substitution for the treatment of bacterial infections. Hence, this study was conducted to determine the antibacterial efficacy of neem (Azadirachta indica), moringa (Moringa oleifera), and curry leaf (Murraya koenigii) against Escherichia coli (E coli) O157:H7 isolated from a fecal sample of a goat from Grenada. This is the first medicinal plant-based research study in Grenada on this important zoonotic pathogen. Aqueous extracts of medicinal plants were evaluated for antibacterial efficacy in vitro, using the agar well diffusion method, and subsequently compared with the antibacterial drug gentamicin. The resulting data demonstrated that aqueous plant extracts of neem leaves, moringa leaves, and curry leaves exhibited in vitro antibacterial efficacy comparable to that of gentamicin against E coli O157:H7 goat isolate. Hence, these plants could be used as an effective substitute for antibacterial agents in prophylactic or therapeutic control of bacterial infections in Grenada.

Sparse Evidence for Giardia intestinalis, Cryptosporidium spp. and Microsporidia Infections in Humans, Domesticated Animals and Wild Nonhuman Primates Sharing a Farm-Forest Mosaic Landscape in Western Uganda.

Zoonotic pathogen transmission is considered a leading threat to the survival of non-human primates and public health in shared landscapes. Giardia spp., Cryptosporidium spp. and Microsporidia are unicellular parasites spread by the fecal-oral route by environmentally resistant stages and can infect humans, livestock, and wildlife including non-human primates. Using immunoassay diagnostic kits and amplification/sequencing of the region of the triosephosphate isomerase, small ribosomal subunit rRNA and the internal transcribed spacer genes, we investigated Giardia, Cryptosporidium, and microsporidia infections, respectively, among humans, domesticated animals (livestock, poultry, and dogs), and wild nonhuman primates (eastern chimpanzees and black and white colobus monkeys) in Bulindi, Uganda, an area of remarkably high human–animal contact and spatial overlap. We analyzed 137 fecal samples and revealed the presence of G. intestinalis assemblage B in two human isolates, G. intestinalis assemblage E in one cow isolate, and Encephalitozoon cuniculi genotype II in two humans and one goat isolate. None of the chimpanzee and colobus monkey samples were positive for any of the screened parasites. Regular distribution of antiparasitic treatment in both humans and domestic animals in Bulindi could have reduced the occurrence of the screened parasites and decreased potential circulation of these pathogens among host species.

Genetic characterization and ex-vivo neutralization behavior of bluetongue virus serotype-16 recovered from apparently healthy goat

Bluetongue virus (BTV) infects almost all the domestic and wild ruminants though the clinical disease is most commonly reported in sheep and some species of deer. Goat and cattle are the most common asymptomatic reservoir of the virus. Full genome sequencing and serological characterization of the virus isolates are emphasized for understanding the phylogenetic relationship and molecular epidemiology of bluetongue (BT). In this study, we report phylogenetic and phenotypic antigenic relationship of a BTV serotype-16 (PDP2/13/Ind) recovered from an apparently healthy goat from the state of Uttarakhand, a hilly terrain of sub-Himalayan India with four other BTV-16 isolates. The full genome sequence data was analyzed and the phylogenetic relationship of the goat isolate with other BTV-16 was established. Phylogenetic analysis revealed cluster of PDP2/13/Ind along with other Indian BTV-16 isolates indicating their close ancestral relationship. A cohesive ancestral relationship, irrespective of the genome segments analyzed, was also observed between Indian and Mediterranean BTV-16. The mean substitution rate of different segments of BTV-16 isolates varied from 3.231 × 10−5 (seg-2) to 1.129 × 10-3 (seg-6) substitutions per site per year. Timescale analysis indicated that all the segments had an older ancestor. No statistically significant geographic structuring of BTV-16 isolates was observed indicating frequent gene flow. The goat isolate shares highest identity (99.5%–99.8%) with G53/ABT/HSR, a BTV-16 recovered from the western part of the country whereas high level of divergence (11.9%–33.3%) at genomic segment level was observed with a Nigerian BTV-16 (NIG1982/10). Phenotypic antigenic relationship (r) of PDP2/13/Ind with other isolate-specific hyperimmune serum (HIS) determined from serum neutralization titer was 0.672 ± 0.058 to 0.948 ± 0.09. On other hand, the calculated ‘r’ score was 0.636 ± 0.063 to 0.814 ± 0.201 when HIS against PDP2/13/Ind was used to neutralize the other BTV-16 isolates. The percentage antigenic similarity (R) of the PDP2/13/Ind with other BTV-16 isolates was 65.39 ± 5.38–87.67 ± 14.86. Data suggests presence of subtype antigenic variation amongst the BTV-16 isolates recovered from the goats of a geographically restricted area of the state of Uttarakhand, India.

Molecular typing of Brucella species by PCR-RFLP and SSCP

PCR-RFLP and PCR-SSCP help to understand variabilities in Brucella spp. genome which in turn assist in planning epidemiological strategies for control of brucellosis in animal population and thereby human transmission. Restriction fragment length polymorphism (RFLP) analysis was carried out for BCSP31 (223 bp) gene of 15 isolates using restriction enzyme HaeII and BsaBI. All isolates yielded a similar restriction pattern as that of reference strains i.e. 189 + 34 bp and 164 + 60 bp fragments with HaeII and BsaBI restriction enzyme, respectively. PCR- SSCP analysis of BCSP 31 gene of 33 Brucella spp. isolates was carried out. Seven different SSCP band patterns designated from “A” to “F” were observed. Out of 33 isolates of Brucella spp., 19 isolates showed SSCP profile similar to ATCC reference strains of Brucella spp. designated as band pattern “A” indicating no polymorphism. Whereas, 14 isolates showed polymorphism in SSCP band pattern designated from “B” to “G”. Band pattern “A” was the most common in 19 (57.58 %) isolates followed by “B” in 6 (18.18 %) isolates and “G” in 4 (12.12 %) isolates, whereas “C”,”D”,”E”, and “F” band patterns were found in each of 1 (3.03 %) isolate. SSCP band pattern “A” was observed most commonly in buffalo (10) followed by human (6) and cattle (3). The SSCP band pattern “B” was found in equal proportion in cattle and buffalo (3 each). The “C” and “E” SSCP band patterns were observed in cattle, whereas “D” and “F” SSCP band pattern was noticed in buffalo and human isolates, respectively. The “G” SSCP band pattern was observed in 3 cattle and 1 goat isolate. In cattle-ABCEG, buffalo-ABD, goat-G and in human-AF band patterns were seen. Comparative results of RFLP and SSCP in 15 isolates showed that, PCR-SSCP is more sensitive than RFLP for detection of polymorphism in BCSP31 gene.

Comparative Response of the West African Dwarf Goats to Experimental Infections with Red Sokoto and West African Dwarf Goat Isolates of Haemonchus contortus.

Response of the West African Dwarf (WAD) goats to two different isolates of Haemonchus contortus, the Red Sokoto (RS) goat isolate (RSHc) and the WAD goat isolate (WADHc) (isolated from WAD goats), was studied by experimental infections of 4–6-month-old male WAD goat kids. Group 1 and Group 2 goats were each infected with 4500 infective larvae (L3) of RSHc and WADHc, respectively. Group 3 animals served as uninfected control. Prepatent period (PPP), faecal egg counts (FEC), worm burden (WB), body weight (BW), packed cell volume (PCV), and body condition score (BCS) were determined. WAD goats infected with RSHc isolate and the ones infected with WADHc isolate had mean PPP of 19.63 ± 0.26 and 19.50 ± 0.19, respectively. Goats infected with WADHc isolate had significantly higher FEC (P = 0.004) and WB (P = 0.001). BW were significantly higher (P = 0.004) both in the controls and in Group 2 goats infected with WADHc isolate than in Group 1 goats infected with the RSHc isolate. BCS of animals in both infected groups dropped significantly (P = 0.001). There was a significant drop in PCV (P = 0.004) of both infected groups in comparison. Both isolates of H. contortus were pathogenic to the host.

Comparative analyses of the complete mitochondrial genomes of the two ruminant hookworms Bunostomum trigonocephalum and Bunostomum phlebotomum

Bunostomum trigonocephalum and Bunostomum phlebotomum are blood-feeding hookworms of sheep and cattle, causing considerable economic losses to the live stock industries. Studying genetic variability within and among hookworm populations is critical to addressing epidemiological and ecological questions. Mitochondrial (mt) DNA is known to provide useful markers for investigations of population genetics of hookworms, but mt genome sequence data are scant. In the present study, the complete mitochondrial DNA (mtDNA) sequences of the sheep and goat hookworm B. trigonocephalum were determined for the first time, and the mt genome of B. phlebotomum from yak in China was also sequenced for comparative analyses of their gene contents and genome organizations. The lengths of mt DNA sequences of B. trigonocephalum sheep isolate, B. trigonocephalum goat isolate and B. phlebotomum China yak isolate were 13,764bp, 13,771bp and 13,803bp in size, respectively. The identity of the mt genomes was 99.7% between B. trigonocephalum sheep isolate and B. trigonocephalum goat isolate. The identity of B. phlebotomum China yak isolate mt genomes was 85.3% with B. trigonocephalum sheep isolate, and 85.2% with B. trigonocephalum goat isolate. All the mt genes of the two hookworms were transcribed in the same direction and gene arrangements were consistent with those of the GA3 type, including 12 protein-coding genes, 2 rRNA genes and 22 tRNA genes, but lacking ATP synthetase subunit 8 gene. The mt genomes of B. trigonocephalum and B. phlebotomum were similar to prefer bases A and T, the contents of A+T are 76.5% (sheep isolate), 76.4% (goat isolate) and 76.9% (China yak isolate), respectively. Phylogenetic relationships reconstructed using concatenated amino acid sequences of 12 protein-coding genes with three methods (maximum likelihood, Bayesian inference and neighbor joining) revealed that the B. trigonocephalum and B. phlebotomum represent distinct but closely-related species. These data provide novel and useful genetic markers for studying the systematics, and population genetics of the two ruminant hookworms.

In Vitro and In Vivo Infectious Potential of Coxiella burnetii: A Study on Belgian Livestock Isolates

Q-fever is a zoonosis caused by the gram-negative obligate intracellular pathogen Coxiella burnetii. Since its discovery, and particularly following the recent outbreaks in the Netherlands, C. burnetii appeared as a clear public health concern. In the present study, the infectious potential displayed by goat and cattle isolates of C. burnetii was compared to a reference strain (Nine Mile) using both in vitro (human HeLa and bovine macrophage cells) and in vivo (BALB/c mice) models. The isolates had distant genomic profiles with one - the goat isolate - being identical to the predominant strain circulating in the Netherlands during the 2007–2010 outbreaks. Infective doses were established with ethidium monoazide-PCR for the first time here applied to C. burnetii. This method allowed for the preparation of reproducible and characterized inocula thanks to its capacity to discriminate between live and dead cells. Globally, the proliferative capacity of the Nine Mile strain in cell lines and mice was higher compared to the newly isolated field strains. In vitro, the bovine C. burnetii isolate multiplied faster in a bovine macrophage cell line, an observation tentatively explained by the preferential specificity of this strain for allogeneic host cells. In the BALB/c mouse model, however, the goat and bovine isolates multiplied at about the same rate indicating no peculiar hypervirulent behavior in this animal model.

Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species

The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468 bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P 52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).

Genetic and antigenic characterization of bovine viral diarrhea virus type 2 isolated from Indian goats ( Capra hircus)

Recent studies have shown that bovine viral diarrhea virus (BVDV) type 1 is widely prevalent in Indian cattle. In a surveillance of randomly collected 562 blood samples from seven states during 2004–2006, BVDV type 2 was detected in two native Indian goats by nested reverse transcription polymerase chain reaction (nRT-PCR). The virus isolated from them was classified antigenically as BVDV 2 on the basis of virus neutralization test and reactivity with monoclonal antibodies. Phylogenetic analysis of three different genomic regions, 5′ un-translated region (5′ UTR), E rns structural coding region and NS5B nonstructural coding region typed Indian goat isolate as BVDV 2a having close similarity with strains from North America and Europe suggesting its probable introduction through trade. It was placed in a separate clade within the 2a branch having unique mutations in E rns and NS5B region. This is the first report of BVDV 2 in India and only second time recorded in goat species. The isolation of BVDV 2 from goat warrants intensive surveillance in cattle and sheep.

Efficacy of spheroplastic and cell-wall competent vaccines for Mycobacterium avium subsp. paratuberculosis in experimentally-challenged baby goats

A Mycobacterium avium subspecies paratuberculosis (MAP) vaccine that reduced the incidence of clinical disease or reduced fecal shedding of MAP would aid control of Johne's disease (JD). The objective of the present study was to evaluate the efficacy of four MAP vaccine combinations, including cell-wall competent (CWC) alum adjuvant, CWC-QS21 adjuvant, cell-wall deficient (CWD) alum adjuvant and CWD-QS21 adjuvant vaccines. Eighty baby goats were vaccinated at 1 and 4 weeks of age with one of these vaccines or a sham control vaccine consisting of alum adjuvant. Kids were challenged orally with approximately 6.0 × 10 9 organisms in four divided doses of 1.5 × 10 9 organisms using a goat isolate of MAP. Vaccinated challenged and challenged control groups had 10 and 6 kids per group, respectively. Half of the kids within each group were necropsied at either 6 or 9 months post-challenge. Gross and microscopic lesions and relative number of acid-fast bacilli were evaluated and scored at necropsy. Results indicated all challenged kids had some lesions compatible with JD suggesting none of the vaccines prevented infection. Three vaccines (CWC-alum, CWC-QS21 and CWD-QS21) reduced lesion scores by 46–51% at 9 months. CWD-alum vaccine resulted in a more severe (+33.5%) lesion score than sham-vaccinated challenged control. Lesion scores were greater at 9 months than at 6 months post-challenge in the sham-vaccinated challenged group and CWD-alum vaccinated group, while lesion scores were generally stable with remaining vaccines. Mean fecal CFU/g were significantly different across time from challenge to 9-month necropsy ( p = 0.043) and the CWC-QS21 vaccine group had a marked reduction in fecal CFU/g at all time points post-challenge. A reduction in MAP CFU/g was also detected in necropsy tissues from kids given the CWC-alum, CWC-QS21 and CWD-QS21 vaccines, and increased CFU/g were detected in tissues from kids given the CWD-alum vaccine. Immunological tests evaluated included, humoral response evaluation by AGID, ELISA and Western blot, and cell mediated response by comparative PPD skin testing ( M. avium, Old Johnin, M. bovis and Lot 2 Johnin PPD's), and production of MAP induced γ-interferon. Vaccination also resulted in false-positive PPD skin test reactions for M. avium PPD, Old Johnin PPD and γ-interferon tests. When a 2-mm cutoff above normal skin thickness was used to define positive skin test reactions, false-positive reactions for M. bovis were detected in only 2 of 32 kids given a vaccine with QS21 adjuvant.

OS-1. Are conventional clinical markers outperformed by microarray gene expression profilers in breast cancer prognosis?

HindIII, PstI, AvaI, and SalI sites were mapped on the genomes of six isolates of capripoxvirus from sheep, goats, and cattle. Genome pairs were aligned by the alignment of cross-hybridizing HinIII fragments and the introduction of padding fragments (pads) at specific locations. The majority of these pads represent the approximate positions of relative deletions or insertions. Three possible phylogenetic networks were generated for seven capripoxvirus isolates by Wagner parsimony analysis of the nonconserved HindIII sites on their genomes, and confidence limits were calculated for the network nodes. Nucleotide sequence divergence values, calculated from the numbers of nonconserved HindIII, PstI, AvaI, and SalI sites on the genomes of typical sheep, goat, and cattle isolates, indicated that the typical sheep and cattle isolates are more closely related to one another than to the typical goat isolate. Nonconserved HindIII, PstI, AvaI, and SalI sites were shown to be distributed throughout the genomes. Evidence that isolates YG-1 and OS-1 are descended from an isolate whose genome arose by recombination is discussed. Terminally repeated regions were identified on each of the capripoxvirus genomes mapped here. By mapping BamHI, ClaI, EcoRI, HindII, HindIII, and SalI sites present within the terminal 10 kb of the genome of isolate InS-1, the terminal repeats of this genome ware shown to be between 2.25 and 3.40 kb in length and inverted with respect to one another.

Intraspecies polymorphism of Cryptosporidium parvum revealed by PCR-restriction fragment length polymorphism (RFLP) and RFLP-single-strand conformational polymorphism analyses.

A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C. parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C. parvum.

Characteristic differences in reverse transcription-polymerase chain reaction products of ovine, bovine, and human respiratory syncytial viruses.

In reverse transcription-polymerase chain reactions (RT-PCR) and DNA hybridizations using primers and an oligonucleotide probe to the fusion (F) protein mRNA of bovine respiratory syncytial virus (BRSV), all the BRSV isolates and a goat isolate could be distinguished from prototype isolates of human respiratory syncytial viruses (HRSV) and ovine (sheep and bighorn sheep) respiratory syncytial viruses (RSV). However, RT-PCR amplifications with primers to sequences of the HRSV F protein mRNA resulted in amplified products of approximately 243 bp if mRNA templates of subgroup A HRSV strains were present and slightly larger amplified products with subgroup B HRSV strains. No amplified products were observed in HRSV-primed RT-PCR with BRSV or goat or ovine RSV mRNA templates. Although the ovine RSV isolates were antigenically cross-reactive with the goat RSV, HRSV and BRSV isolates, they were no amplified with either HRSV- or BRSV-specific primers in RT-PCR. These results confirm previous immunological comparisons suggesting that some ovine RSV isolates should be considered as distinct respiratory syncytial viruses.

Examination of Tissue Cyst Formation by Toxoplasma gondii in Cell Cultures Using Bradyzoites, Tachyzoites, and Sporozoites

Tissue cyst formation by a goat isolate (GT-1) of Toxoplasma gondii was examined in bovine monocyte, human fetal lung, and Madin-Darby bovine kidney cell cultures. Transmission electron microscopy (TEM) and cat feeding studies indicated that tissue cysts were present in all 3 cell lines examined. Tissue cysts were first seen 3 days postinoculation (PI) using TEM. Standard cell culture procedures were used and no additional condition was needed to induce tissue cyst formation. Cats fed cell cultures excreted T. gondii oocysts in their feces 5-7 days PI. These oocysts caused lethal infections in mice. Tissue cysts were produced in cell cultures regardless if the initiating inoculum consisted of bradyzoites, sporozoites, or a mixture of bradyzoites and tachyzoites. Tissue cyst formation has been followed through 40 subpassages of infected cells. By TEM tissue cysts still were present after 40 passages, but when 40th-passaged cultures were fed to cats, oocytsts were not excreted. This indicates that the parasite had become oocystless after repeated passage in vitro.

A comparison of the genomes of capripoxvirus isolates of sheep, goats, and cattle

HindIII, PstI, AvaI, and SalI sites were mapped on the genomes of six isolates of capripoxvirus from sheep, goats, and cattle. Genome pairs were aligned by the alignment of cross-hybridizing HinIII fragments and the introduction of padding fragments (pads) at specific locations. The majority of these pads represent the approximate positions of relative deletions or insertions. Three possible phylogenetic networks were generated for seven capripoxvirus isolates by Wagner parsimony analysis of the nonconserved HindIII sites on their genomes, and confidence limits were calculated for the network nodes. Nucleotide sequence divergence values, calculated from the numbers of nonconserved HindIII, PstI, AvaI, and SalI sites on the genomes of typical sheep, goat, and cattle isolates, indicated that the typical sheep and cattle isolates are more closely related to one another than to the typical goat isolate. Nonconserved HindIII, PstI, AvaI, and SalI sites were shown to be distributed throughout the genomes. Evidence that isolates YG-1 and OS-1 are descended from an isolate whose genome arose by recombination is discussed. Terminally repeated regions were identified on each of the capripoxvirus genomes mapped here. By mapping BamHI, ClaI, EcoRI, HindII, HindIII, and SalI sites present within the terminal 10 kb of the genome of isolate InS-1, the terminal repeats of this genome ware shown to be between 2.25 and 3.40 kb in length and inverted with respect to one another.

Experimental infection of castrated lambs with Mycoplasma agalactiae.

The course of experimental infection in groups of 6-month-old castrated lambs with field isolates of Mycoplasma agalactiae from France was followed culturally and serologically for 7 months. Infection with an ovine field isolate following inoculation by different routes and contact exposure was compared with that caused similarly by a caprine field isolate. The prolonged infections produced were symptomless apart from limited arthritis in one animal inoculated with the isolate from sheep and increased lachrymation in another associated with the goat isolate. The ovine isolate was more virulent in that ante- and post-mortem recoveries of the organism were more consistent and the serological responses more pronounced. Serological responses varied between animals and between strain infections, and the results of the film inhibition test were more consistent than those of the complement fixation test. The limitations of both these tests for detecting carrier infections are discussed.