Sheep Isolates Research Articles

Molecular detection of Cryptosporidium parvum in sheep and goats in rural areas of Al-Muthanna Province, Iraq

Background: Cryptosporidium species are widespread protozoa and are considered an important cause of diarrhea in humans and animals. Aim: The aim of this study was to identify the occurrence of Cryptosporidium parvum in diarrheic sheep and goats from the rural areas of the Al-Muthanna Province, southern Iraq, based on molecular screening using PCR. The outcomes would help in determining the distribution of C. parvum in sheep and goats in the area, as well as the risk factors for the infection. Methodology: Sixty fecal samples were collected from clinically ill sheep and goats. The samples were submitted to PCR and sequencing that targeted the 18S rRNA gene in the protozoan. Results: The PCR revealed a 60% (18 samples) and 76.67% (23 samples) positive identification of C. parvum in sheep and goats, respectively. The sequencing demonstrated nucleotide similarities with isolates deriving from Iraq (MG878399.1) and India (JN836324.1) for the sheep isolates, and with isolates deriving from South Korea (DQ054819.1) and the USA (AF159112.1) for the goat isolates. Conclusion: C. parvum is the dominant species in the sheep and goats of Al-Muthanna Province, in southern Iraq. Moreover, the phylogenetic analysis demonstrates that the current study’s isolates may share the same ancestor with local and global isolates.

A pilot study on the epidemiology, diagnosis and characterization of Echinococcus granulosus sensu lato in sheep, goats and dogs in Siirt province of Türkiye revealed remarkable adaptation of Echinococcus canadensis (G6/G7) in goats

Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato (s.l.), represents one of the most significant zoonotic diseases globally, affecting both humans and animals. The objective of this study was to ascertain the prevalence of E. granulosus sensu lato in sheep and goats in a pilot region with a one-year slaughterhouse follow-up period and to determine the genetic differences and haplotypes among sheep, goat, and dog isolates. To this end, the prevalence of CE cysts was determined by monitoring the slaughter of sheep and goats at least three days a week at a slaughterhouse in the Siirt province of Türkiye during 2023. Additionally, faecal samples were collected from stray dogs and analysed using both flotation and molecular techniques. The presence of CE cysts was identified in 569 (11.12 %) of the 5119 sheep and 66 (2.31 %) of the 2860 goats after slaughtering. The highest positivity was observed in November (20.39 %), while the lowest was recorded in July (5.62 %). Of the sheep that detected positive, 25 (4.39 %) were less than one year old, while 544 (95.61 %) were older than one year. Of the infected sheep, 26 (4.57 %) were male and 543 (95.43 %) were female. 204 (35.85 %) sheep exhibited fluid-filled CE cysts, 338 (59.40 %) displayed calcification, and 27 (4.75 %) demonstrated the presence of newly developed cysts. The highest positivity was observed in December (5.83 %), while the lowest was recorded in May (0.62 %) in goats. Of the positive goats, two (3 %) were less than one year old, while the remaining 64 (97 %) were older than one year. Of the goats infected with CE cysts, 10 (15.15 %) were male and 56 (84.85 %) were female. Of the cysts, 56.1 % were fluid-filled, 42.4 % were calcified and 1.5 % were newly developed. Following DNA sequence analysis of CE cyst isolates obtained from the slaughterhouse, all 61 sheep sequences were identified as E. granulosus s.s. (G1/G3). Of the 13 goat isolates, seven were identified as E. granulosus s.s. (G1/G3), while the remaining six were classified as E. canadensis (G6/G7). The centrifugal flotation method was employed to detect the presence of Isospora spp. oocysts in eight dogs, Toxocara canis and hookworm eggs in three dogs each, and Dipyllidium caninum eggs in one dog. A total of 54 dog faeces were examined. No Taeniid eggs were observed in any of the dogs. Following PCR analysis of the mt-CO1 gene region in the dog faecal samples, four samples were positive for a 875 bp band. Only one of these bands was suitable for sequence analysis, which confirmed it as E. granulosus s.s. (G1/G3).

First report on the occurrence of psoroptic mange in llamas (Lama glama) of the Andean region.

An outbreak of Psoroptes sp.-caused mange was detected in a llama herd of Larcas, Jujuy province, Argentina. Infested llamas showed alopecia, erythema, hyperpigmentation, hyperkeratosis, and inflammation of the ear pinnae, as well as crusts and serous, serosanguineous, or purulent drainage with unpleasant smell in the external ear canal. Microscopic evaluation of skin scrapings revealed 0.5- to 0.7-mm-long acari identified as Psoroptes sp. based on their morphology. Histology showed a typical allergic reaction with perivascular to periadnexal mixed inflammatory infiltrate. Phylogenetic tree analysis showed that the cytochrome c oxidase subunit I gene sequences analyzed from the sampled acari clustered into a single P. ovis clade including sequences isolated from rabbits and bighorn sheep, with P. natalensis as a sister taxon that infested bighorn sheep from the USA. Phylogenetic analysis of cytochrome b sequences showed three well-supported clades, one of which contained the sequences of the Larcas llamas and US bighorn sheep isolates. This is the first report on P. ovis infestation of llamas raised in their original location. Investigations on mange etiological agents acting on South American camelids and their distribution are necessary to implement control strategies to mitigate the negative impacts of these parasitic infections.

Prevalence and Antibiogram of Clostridium perfringens in Small Ruminants of District Kalat, Balochistan

Background: Among life-threatening infections of small ruminants (sheep/goat), enterotoxaemia has vital importance. This disease is caused by certain serotype of Clostridium perfringens and commonly known as pulpy kidney or overeating infection. The current study was carried out to determine the prevalence of C. perfringens in small ruminants in different zones (Kalat, Mangochar, Johan, and Gazg) of district Kalat, Balochistan. Methods: Fecal samples (n=100) were collected aseptically, from rectum of enterotoxaemia suspected animals. The samples were cultured in Reinforced Clostridial Media (RCM). Biochemically identified isolates were further confirmed through polymerase chain reaction (PCR). Result: Overall prevalence of C. perfringens was noticed 48%, with higher occurrence rate (p less than 0.01) in sheep (60%) than goat (36%). Among fifty samples of each specie, 30 and 18 isolates recovered from sheep and goat respectively. The area wise analysis showed that, in goats, the highest prevalence was found in Kalat (50%) followed by the Mangochar (38.46%), Johan (30.77%) and Gazg (25%). The statistical analysis revealed the significant differences (p less than 0.05; 95% confidence interval (CI) 18.80-68.91) in C. perfringens prevalence in various areas of Kalat district. The results of the gender-wise prevalence of C. perfringens exhibited a numerically higher (p greater than 0.05; 95% CI 9.23-49.02) prevalence in female goats (40%) than male goats (32%). Whereas, the age-wise analysis indicated that, in less than 1 year age group (44.44%), the prevalence of C. perfringens was higher (p less than 0.05; 95% CI 8.00-57.89) than greater than 1 but less than 2 years (29.41%) and ³ 2-year (33.33%) age groups. Similarly, in sheep, the highest prevalence (p less than 0.05) was found in Kalat (76.92%) followed by Mangochar (66.67%), Johan (58.33%) and Gazg (38.46%). The age wise and gender-wise differences (p less than 0.05) were also observed in various age and gender groups of sheep similar to goat. Among 30 sheep isolates of C. perfringens, a highest number (28 isolates) showed resistance against amikacin, followed by amoxicillin/penicillin (25 isolates) and colistin sulphate (22 isolates); while among 18 isolates of goat 17 were found resistant against colistin sulphate and 16 were resistant against amoxicillin and amikacin. In conclusion enterotoxaemia is prevailing in small ruminants of district Kalat, Balochistan thus mass scale vaccination campaign should be carried out to prevent losses.

Phylogenetic analysis of Klebsiella pneumoniae isolates of respiratory tract infections in humans and sheep.

Klebsiella pneumoniae is an important opportunistic pathogen, which is capable of colonizing the respiratory system in both humans and animals causing mild to severe infections. This study aims to isolate K. pneumoniae from the nasal discharges of human and sheep as well as identify the antibiotic resistance and molecular phylogeny of local isolates. A total of 100; 50 humans and 50 sheep, positive nasal swab isolates were selected, and confirmed biochemically and by the VITEK-2 system. Molecular testing using the polymerase chain reaction (PCR) and phylogeny was conducted. On MacConkey agar, Klebsiella isolates appeared as large, pinkish, and mucoid colonies; while microscopically, it appeared as Gram-negative rods. Traditional biochemical tests revealed that 62% and 78% of human and sheep isolates were positive Klebsiella isolates, whereas respectively, 54.84% and 71.8% of these isolates were positive by VITEK-2. Antibiotic susceptibility tests showed that the human isolates were sensitive to aztreonam, piperacillin-tazobactam, ciprofloxacin, and cefuroxime. Subsequently, sheep isolates were sensitive to cefuroxime, ciprofloxacin, piperacillin-tazobactam, ampicillin, cefoxitin, and tetracycline. Targeting 16S rRNA gene, a total of 17 human and 28 sheep isolates were molecularly positive K. pneumoniae. Phylogenetic analysis of study human and sheep isolates showed their identity to NCBI Indian (LC747146.1) and Iraqi (LC711141.1) isolates, respectively. Comparative analysis between the local human and sheep isolates revealed a significant identity that ranged from 99.82% to 99.88% with a percentage of mutation ranging from 0.008% to 0.002%. Klebsiella pneumoniae is a highly prevalent bacterium in both human and sheep with an observable resistance to antibiotics. Molecular phylogeny of study isolates demonstrated their close relation, suggesting the possible direct or indirect transmission of the bacterium from sheep to human or vice versa. Moreover, studies are greatly important to estimate the routes of bacterial transmission. Also, extensive hygiene practices could be lowered the spreading of K. pnuemoniae to farm workers.

The canonical Brucella species-host dependency is changing, however, the antibiotic susceptibility profiles remain unchanged

Brucellosis is a chronic disease caused by Brucella species with a wide range of hosts, from marine mammals to terrestrial species, but with strict host preferences. With the zoonotic character, the prevalence of human brucellosis cases is a reflection of animal infections. This study aimed to identify 192 Brucella isolates obtained from various sources by Bruce-ladder PCR and to determine their antibiotic susceptibilities by gradient diffusion method (E-test). As a result of the PCR, all human isolates (n = 57) were identified as B. melitensis. While 58 (82.9%) of the cattle isolates were identified as B. abortus, 59 (90.8%) of the sheep isolates were identified as B. melitensis. In addition, 12 (17.1%) of the cattle isolates and 6 (9.2%) of the sheep isolates were determined as B. melitensis and B. abortus, respectively. The primary host change behavior of B. melitensis was 1.9 times higher than that of B. abortus. While gentamicin and ciprofloxacin susceptibilities of Brucella isolates were 100%, tetracycline, doxycycline, streptomycin, trimethoprim/sulfamethoxazole and rifampicin susceptibilities were 99%, 99%, 97.4%, 91.7% and 83.9%, respectively. The lowest sensitivity of the isolates was determined against to cefoperazone as 26%. A triple-drug resistance was detected in 1 B. abortus isolate that included simultaneous resistance to cefoperazone, rifampicin, and trimethoprim/sulfamethoxazole. The high susceptibility profiles we found against to antibiotics such as tetracycline, doxycycline gentamicin and ciprofloxacin, used widely in treatment, are encouraging. However, the change in the canonical Brucella species-primary host preference suggests the need to reconsider eradication program, including updating vaccine formulations.

Molecular Characterization and Antimicrobial Susceptibilities of Corynebacterium pseudotuberculosis Isolated from Caseous Lymphadenitis of Smallholder Sheep and Goats.

Caseous lymphadenitis (CLA) is a bacterial infection caused by Corynebacterium pseudotuberculosis (C. pseudotuberculosis) that affects sheep and goats, leading to abscess formation in their lymph nodes. The present study aimed to isolate and identify C. pseudotuberculosis from CLA in smallholder sheep and goats, and determine the resistance patterns, virulence, and resistance genes of the isolates. Additionally, genotypic and phylogenetic analysis of the isolates was conducted using ERIC-PCR and DNA sequencing techniques. A cross-sectional study examined 220 animals (130 sheep and 90 goats) from 39 smallholder flocks for clinical signs of CLA. Fifty-four (24.54%) animals showed CLA-compatible lesions, confirmed by C. pseudotuberculosis isolation and PCR identification. Sheep had a lower infection rate of CLA (18.46%) compared with goats (33.3%). Antimicrobial susceptibility testing of 54 C. pseudotuberculosis isolates to 24 antimicrobial drugs revealed that they were 100% resistant to bacitracin and florfenicol, while none of the isolates were resistant to norfloxacin. A high resistance rate was observed for penicillin and erythromycin (92.6% each). Interestingly, 16.7% of C. pseudotuberculosis isolates recovered from sheep showed vancomycin resistance. Molecular characterization of C. pseudotuberculosis isolates revealed that PLD, PIP, and FagA virulence genes were present in all examined isolates. However, the FagB, FagC, and FagD genes were detected in 24 (100%), 20 (83%), and 18 (75%) of the sheep isolates, and 26 (87%), 26 (87%), and 18 (60%) of the goat isolates, respectively. The β-lactam resistance gene was present in all isolates. Furthermore, 83% of the sheep isolates carried the aminoglycoside (aph(3″)-lb), chloramphenicol (cat1), and bacitracin (bcrA) resistance genes. Among the isolates recovered from goats, 73% were found to contain macrolides (ermX), sulfonamide (sul1), and bacitracin (bcrA) resistance genes. It is worrisome that the glycopeptide (vanA) resistance gene was detected in 8% of the sheep isolates as a first report. ERIC-PCR genotyping of 10 multi-drug-resistant C. pseudotuberculosis isolates showed a high similarity index of 83.6% between isolates from sheep and goats. Nucleotide sequence analysis of partial 16S rRNA sequences of C. pseudotuberculosis revealed 98.83% similarity with biovar Ovis of globally available reference sequences on the Genbank database. Overall, our findings might indicate that C. pseudotuberculosis infection in smallholders in Egypt might be underestimated despite the significant financial impact on animal husbandry and potential health hazards it poses. Moreover, this study highlights the importance of implementing a sustainable control strategy and increasing knowledge and awareness among smallholder breeders to mitigate the economic impact of CLA.

Molecular characterization of cattle and sheep isolates of Echinococcus granulosus from Elazig province in Turkey and expression analysis of the non-coding RNAs, egr-miR-7, egr-miR-71 and egr-miR-96

Cystic Echinococcosis (CE) is a common zoonotic disease seen in human and animals worldwide, caused by the larval form of Echinococcus granulosus. In this study, E. granulosus s.l. species and haplotypes were determined in hydatid cysts isolated from cattle and sheep, and the expression levels of egr-miR-7, egr-miR-71 and egr-miR-96 miRNAs were compared in different cyst structures. A total of 82 (cattle, n = 41; sheep, n = 41) hydatid cyst isolates (germinal membranes and/or protoscoleces) were collected from a slaughterhouse in Elazig province of Turkey. After mt-CO1 gene sequences were made, 81 out of 82 hydatid cyst isolates were determined as E. granulosus s.s. (G1 and G3), while an isolate of cattle origin was determined as Echinococcus canadensis (G6/7). A total of 26 nucleotide polymorphisms and 29 haplotype groups were identified in the samples. miRNA expressions in germinal membranes of sterile cysts and germinal membrane and protoscoleces of fertile cysts were investigated by qRT-PCR and Real Time PCR analyses. It was determined that miRNAs were expressed at high levels in 79.31% of the 29 haplotype groups and at low levels in the remaining 10.34%. In 10 fertile samples of sheep origin, egr-miR-7, egr-miR-71 and egr-miR-96 miRNAs were found to be 44, 168, and 351-fold higher in expression, respectively, in the germinal membrane compared to the protoscoleces. Especially egr-miR-96 may have the potential to be used as biomarkers in the diagnosis of active CE.

High prevalence of multidrug-resistant Campylobacter jejuni in sheep and goats milk in Jordan

The prevalence and resistance of Campylobacterjejuni in small ruminants in Jordan to antimicrobials of human clinical importance and the management factors that associate with C. jejuni in the flocks were determined. Flock-level prevalence of C. jejuni was 28.5%; individual animals’ prevalence was 11.0%. Flocks that reported abortions within the immediate last season had 2.7 × higher likelihood of C. jejuni occurrence than flocks that did not report abortions in the immediate last season. More than 75% of C. jejuni isolates were resistant to ampicillin, nalidixic acid, streptomycin, and erythromycin; the lowest resistance was toward amoxicillin-clavulanic acid. Overall, 70.1% of the isolates were multidrug-resistant (resistant to ≥3 antimicrobial classes); 51.7% were resistant to ≥5 antimicrobial classes. Sheep isolates generally showed significantly higher resistance percentages than goat isolates. The results highlight the probability of small ruminants as a source of campylobacteriosis in human and the importance of the heat treatment of milk.

High multidrug resistance of Listeria monocytogenes and association with water sources in sheep and goat dairy flocks in Jordan

Listeria monocytogenes is a significant pathogen that causes listeriosis in humans and small ruminants. This study aimed to estimate the prevalence, antimicrobial resistance and risk factors of L. monocytogenes in small dairy ruminants in Jordan. A total of 948 milk samples were collected from 155 sheep and goat flocks across Jordan. L. monocytogenes was isolated from the samples, confirmed and tested for 13 clinically important antimicrobials. Data were also collected on the husbandry practices to identify risk factors for the presence of L. monocytogenes. The results showed that the flock-level prevalence of L. monocytogenes was 20.0% (95% CI; 14.46%−26.99%) and the prevalence in the individual milk samples was 6.43% (95% CI; 4.92%−8.36%). The univariable (UOR=2.65, p = 0.021) and multivariable (AOR=2.49, p = 0.028) analyses showed that using water from municipality pipelines as a water source in the flock reduced L. monocytogenes prevalence. All L. monocytogenes isolates were resistant to at least one antimicrobial. High percentages of the isolates were resistant to ampicillin (83.6%), streptomycin (79.3%), kanamycin (75.0%), quinupristin/dalfopristin (63.8%) and clindamycin (61.2%). About 83.6% of the isolates (94.2% and 75% of the sheep and goat isolates) exhibited multidrug resistance (resistance to ≥3 antimicrobial classes). In addition, the isolates exhibited fifty unique antimicrobial resistance profiles. Thus, it’s recommended to restrict the misuse of clinically important antimicrobials and to chlorinate and monitor the water sources in sheep and goat flocks.

Comparison of Various Diagnostic Techniques for the Detection of Blastocystis Spp. and its Molecular Characterisation in Farm Animals in the United Arab Emirates.

Blastocystis spp. is a common anaerobic intestinal parasite infecting humans and a diverse range of animals. The aim of the study was to compare different diagnostic methods for the detection of Blastocystis and survey the occurrence of its subtypes in farm animals, namely sheep, cows and camels, in Al-Ain, United Arab Emirates. Ninety-seven faecal samples comprised of 69 from sheep, 12 from cows and 16 from camels were submitted to DNA extraction, PCR and sequencing. Blastocystis was screened for microscopically in 65 samples using direct wet-mount, modified acid-fast staining, trichrome staining and in vitro culture techniques. Fifteen (15.5%) samples were positive by PCR, twelve of which were confirmed by sequencing. Using PCR as a comparison standard, the sensitivity and specificity of the direct wet-mount, modified acid-fast staining, trichrome staining and in vitro culture methods were 40.0% and 78.3%, 40.0% and 83.3%, 80.0% and 80.0%, and 80.0% and 76.7% respectively. Only culture and trichrome tests were significantly associated with PCR (odds ratio (OR) = 13.14; 95% confidence interval (CI): 1.35-127.4; P = 0.007 and OR = 16; 95% CI: 1.63-156.5; P = 0.003, respectively) with trichrome detecting more positive cases than in vitro culture. The subtype (ST)10 was the only one found in all 12 sequenced sheep isolates. The study corroborated previous data indicating that sheep are the natural hosts for ST10. No zoonotic subtypes nor mixed-subtype colonisation were found. The report also confirmed the superiority of trichrome staining in detecting Blastocystis spp.

A longitudinal study reveals persistence of antimicrobial resistance on livestock farms is not due to antimicrobial usage alone.

There are concerns that antimicrobial usage (AMU) is driving an increase in multi-drug resistant (MDR) bacteria so treatment of microbial infections is becoming harder in humans and animals. The aim of this study was to evaluate factors, including usage, that affect antimicrobial resistance (AMR) on farm over time. A population of 14 cattle, sheep and pig farms within a defined area of England were sampled three times over a year to collect data on AMR in faecal Enterobacterales flora; AMU; and husbandry or management practices. Ten pooled samples were collected at each visit, with each comprising of 10 pinches of fresh faeces. Up to 14 isolates per visit were whole genome sequenced to determine presence of AMR genes. Sheep farms had very low AMU in comparison to the other species and very few sheep isolates were genotypically resistant at any time point. AMR genes were detected persistently across pig farms at all visits, even on farms with low AMU, whereas AMR bacteria was consistently lower on cattle farms than pigs, even for those with comparably high AMU. MDR bacteria was also more commonly detected on pig farms than any other livestock species. The results may be explained by a complex combination of factors on pig farms including historic AMU; co-selection of AMR bacteria; variation in amounts of antimicrobials used between visits; potential persistence in environmental reservoirs of AMR bacteria; or importation of pigs with AMR microbiota from supplying farms. Pig farms may also be at increased risk of AMR due to the greater use of oral routes of group antimicrobial treatment, which were less targeted than cattle treatments; the latter mostly administered to individual animals. Also, farms which exhibited either increasing or decreasing trends of AMR across the study did not have corresponding trends in their AMU. Therefore, our results suggest that factors other than AMU on individual farms are important for persistence of AMR bacteria on farms, which may be operating at the farm and livestock species level.

Molecular Epidemiology and Phylogenetic Analysis of Peste des Petits Ruminants Virus Circulating in Sheep in Bangladesh

Peste des petits ruminants (PPR) is a viral disease of small ruminants that is highly contagious, severe, reportable, and economically important. The present study was conducted to detect the PPR virus (PPRV) circulating in sheep in Bangladesh to determine its association with epidemiological risk factors and the degree of relationship between the F and H genes of the PPRV of sheep with those of other sheep and goat isolates. A cross-sectional study was conducted in five selected districts of Bangladesh to collect data on locations, ecological zones, breeds, age, sex, sources, time period, and farming systems using a structured questionnaire accompanied by face-to-face interviews. During sampling, 250 nasal swab samples were collected from live sheep with the typical clinical signs of PPR. Thereafter, a reverse-transcriptase polymerase chain reaction (RT-PCR) assay was employed to detect PPRV using the F and H genes. Risk factors were determined using bivariable and multivariable logistic regression analyses. Phylogenetic analysis of the detected PPRV was performed using MEGA software after sequencing both F and H genes. Using RT-PCR, 35.6% (89/250, 95% CI: 29.6%–41.6%) of the samples were found to be positive for PPRV. Locations, breeds, sources, and feeding systems were identified as potential molecular epidemiological risk factors for PPRV infection in a multivariate logistic regression model. Nucleotide sequencing and phylogenetic analysis showed that the PPRV strain was genetically related to the lineage IV virus isolates. For the F gene, the sequence divergence of our gene and other selected genes ranged from 0.01% to 0.018% within lineage IV, and the similarity ranged from 98.2% to 99.0%. In the case of the H gene, similar results were also observed in divergence, ranging from 0.017% to 0.083% among lineage IV and others, and similarity varied from 91.7% to 98.3%. To the best of our knowledge, this is the first study in Bangladesh conducted to determine the RT-PCR-based molecular epidemiology of PPRV in sheep. This study highlights the importance of establishing successful interventions for managing PPRV infections in small ruminants in Bangladesh.

Whole Genome Sequencing of Escherichia coli and Enterococcus spp. in wildlife-livestock interface: a pilot study

ObjectivesThis pilot study provides a multidisciplinary investigation to monitor livestock-wildlife interface. Ecological data, microbiological investigations, and whole genome sequencing were used to characterize eight bacterial isolates obtained from sympatric domestic and wild ruminants in Maiella National Park (Italy) in terms of genetic patterns of antimicrobial resistance. MethodsUsing selective culturing of fresh fecal samples of monitored and georeferenced populations of Apennine chamois, goats, red deer, and sheep, Escherichia coli, Enterococcus faecium, and Enterococcus faecalis isolates were isolated and subjected to minimum inhibitory concentration determination and whole genome sequencing. ResultsThe analyzed isolates showed phenotypic and genotypic resistance to tetracycline and critically important antibiotics such as linezolid and carbapenems. Virulence genes related to biofilm regulation and Shiga toxins were also detected. Furthermore, serotypes related to nosocomial infections, harbouring plasmids recognized as important mobile resistance gene transmitters, were identified. ConclusionsThis multidisciplinary pilot study represents a promising initial step to identify the environmental drivers and the transmission routes of antimicrobial resistance and virulence factors, providing new data on bacteria from rare and endangered species such as Apennine chamois.

Molecular evaluation of sheep and goats isolates of Pasteurella multocida and their antibiotic resistance.

Pasteurella multocida exists as a commensal in the upper respiratory tracts of livestock, and poultry, and causes a wide variety of diseases in humans and animals. This study aimed to investigate the incidence of P. multocida by bacteriological and molecular characterization in sheep and goats and screening the existence of capsule-specific genes and their antibiotic resistance pattern. Totally, 1650 nasopharyngeal swabs were collected from apparently healthy sheep and goats and 460 lung tissues were collected from slaughtered animals in Fars province, Iran. All samples were cultured and suspected colonies were examined by biochemical tests, antimicrobial assay and polymerase chain reaction (PCR). Among 165 P. multocida (104 sheep and 61 goats) isolates, the capA, capD, and capB genes were amplified in 98, 48, and 12 isolates, respectively. The occurrence of four virulence-associated genes of P. multocida isolates were determined by PCR. Most isolates harbored the toxA (79.40%) and hgbB genes (70.90%) and 59.40% of isolates had the pfhA gene. Almost half of the isolates (46.10%) contained the tbpA gene. According to the current study, P. multocida capsular type A had the most frequency followed by type D. In addition, the high frequency of tbpA, pfhA, toxA, and hgbB genes revealed that these genes are possibly important in the pathogenesis of P. multocida. Oxytetracycline, enrofloxacin, florfenicol, and tilmicosin were the most effective drugs.

Vancomycin-variable enterococci in sheep and cattle isolates and whole-genome sequencing analysis of isolates harboring vanM and vanB genes.

Vancomycin resistance encoded by the vanA/B/M genes in enterococci is clinically important because of the transmission of these genes between bacteria. While vancomycin resistance is determined by detecting only vanA and vanB genes by routine analyses, failure to detect vanM resistance causes vancomycin resistance to be overlooked, and clinically appropriate treatment cannot be provided. The study aimed to examine the presence of vanM-positive enterococcal isolates in Ankara, Turkey, and to have detailed information about them with sequence analyses. Caecal samples were collected from sheep and cattle during slaughter at different slaughterhouses in Ankara, Turkey. Enterococci isolates were identified, confirmed, and analyzed for the presence of vanA/B/M genes. Antibiotic resistance profiles of isolates were determined by the broth microdilution method. A whole genome sequence analysis of the isolates harboring the vanM and vanB genes was performed. 13.7% of enterococcal isolates were determined as Enterococcus faecium and Enterococcus faecalis. 15% of these isolates contained vanB, and 40% were vanM-positive. S98b and C32 isolates were determined to contain 16 CRISPR-Cas elements. 80% of the enterococci isolates were resistant to nitrofurantoin and 15% to ciprofloxacin. The first vanM-positive vancomycin-variable enterococci (VVE) isolates from food-producing animals were identified, and the S98b strain has been assigned to Genbank with the accession number CP104083.1. Therefore, new studies are needed to facilitate the identification of vanM-resistant enterococci and VVE strains.

Genotypic evaluation of Pasteurella multocida isolated from cattle and sheep by pulsed-field gel electrophoresis.

Pasteurella multocida a Gram-negative bacterium exists as a commensal in the upper respiratory tracts of livestock, and poultry. It is causative agent of a range of diseases in mammals and birds including fowl cholera in poultry, atrophic rhinitis in pigs and bovine hemorrhagic septicemia in cattle and buffalo. This study aimed to isolate P. multocida from sheep and cattle lungs sampled and assessed by bacteriological procedures and pulse field gel electrophoresis (PFGE) characterization. In this study 52 isolated of P. multocida were obtained (2016-2017) from clinically healthy and diseased animals (sheep and cattle) evaluated by PFGE for determining the relationship between them. According to the results of this study 12 sheep isolates had similarities above 94.00% and two cattle isolates showed similarities above 94.00%. When compared between sheep and cattle, most isolates showed a similarity of less than 50.00% indicating the great differences between isolates. It is noteworthy that in the present study, performed by PFGE to determine the type of P. multocida isolates, a very high distinction was made to determine the type of isolates and the relationship between isolates based on fragments in their genome using enzymes.

Phenotype of Coxiella burnetii Strains of Different Sources and Genotypes in Bovine Mammary Gland Epithelial Cells.

Despite the high prevalence of C. burnetii in dairy herds and continuous shedding via milk by chronically infected cows, bovine milk is not recognized as a relevant source of human Q fever. We hypothesized that the bovine mammary gland epithelial cell line PS represents a suitable in vitro model for the identification of C. burnetii-strain-specific virulence properties that may account for this discrepancy. Fifteen C. burnetii strains were selected to represent different host species and multiple loci variable number of tandem repeat analysis (MLVA) genotypes (I, II, III and IV). The replication efficiencies of all strains were similar, even though strains of the MLVA-genotype II replicated significantly better than genotype I strains, and bovine and ovine isolates replicated better than caprine ones. Bovine milk isolates replicated with similar efficiencies to isolates from other bovine organs. One sheep isolate (Cb30/14, MLVA type I, isolated from fetal membranes) induced a remarkable up-regulation of IL-1β and TNF-α, whereas prototypic strains and bovine milk isolates tended to suppress pro-inflammatory responses. While infection with strain Nine Mile I rendered the cells partially refractory to re-stimulation with E. coli lipopolysaccharide, Cb30/14 exerted a selective suppressive effect which was restricted to IL-6 and TNF-α and spared IL-1β. PS cells support the replication of different strains of C. burnetii and respond in a strain-specific manner, but isolates from bovine milk did not display a common pattern, which distinguishes them from strains identified as a public health concern.

A Comprehensive in silico Analysis of mt-CO1 gene of Fasciola hepatica

This study was conducted for the aim of comprehensive evaluation of the genetic diversity and phylogenetic relations of F. hepatica among various hosts. In this study, published sequences of mt-CO1 gene fragments belonging to final hosts of F. hepatica were used to create the dataset. First of all, 478 sequences were obtained with PubMed search. Then, some shorter sequences were removed after alignment, and 319 sequences which included cattle (n=242), sheep (n=46), goat (n=5), donkey (n=8), bison (n=7), water buffalo (n=7), human (n=2) and camel (n=2) sequences were analysed in the MEGA X program. The existence of 72 haplotype groups was detected. The most polymorphic sites (n=42) containing 31% (13/42) parsimony informative sites were detected in Iranian isolates. The mt-CO1 network consisted of 35 haplotypes, 80% out of which were geographically unique. However, a main haplotype consisting of 39.2% of the total isolates was formed. The bison isolates of F. hepatica showed the highest haplotype diversity within the final host isolates, followed by the sheep, water buffalo, cattle, goat and donkey isolates. The genetic diversity of F. hepatica in different hosts and countries revealed the possibility of new strains appearing in the future.

The first Mycoplasma ovipneumoniae recovered from a sheep with respiratory disease in Brazil - draft genome and genomic analysis.

Mycoplasma ovipneumoniae is an important etiological agent of sheep respiratory disease worldwide. Here, we describe the first isolation and draft genome sequence of M. ovipneumoniae strain USP-BR2017 retrieved from tracheobronchial lavage of a sheep showing clinical signs of respiratory disease in the Rio de Janeiro State, Brazil. The culture of tracheobronchial lavage resulted in glucose-fermenting fried egg colonies, which were identified as M. ovipneumoniae by polymerase chain reaction. The genome was sequenced using the Illumina NextSeq 2000 and de novo assembled using SPAdes. The genome of the sequenced organism presented an approximate size of 1,122,253bp.The annotation revealed 773 coding DNA sequences (CDSs), 806 genes, three rRNAs, and 30 tRNAs. Data analysis revealed M. ovipneumoniae strain USP-BR2017 contains a few virulence genes, including the hemolysing C gene (hlyC). In addition, strain USP-BR2017 showed high identity over the 16S rRNA gene with other sheep isolates from China and United States. This first description of M. ovipneumoniae in diseased Brazilian sheep demonstrates the importance of continuous surveillance and diagnostics of pathogens causing respiratory disease in sheep in Brazil.