Antibiotic susceptibility of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from subclinical bovine mastitis cases and in vitro efficacy of bacteriophage

Staphylococcus aureus is an opportunistic pathogen that may cause severe infections in livestock, and represents the major cause of mastitis in dairy cows. Currently, instead of using antibiotics, new strategies are sought to reduce this clinical health problem. The aim of this study was to determine the efficacy of phage therapy to kill S. aureus strains obtained from farms located at the State of Guanajuato, México. Thirty-six S. aureus strains from cow milk with subclinical mastitis were isolated and identified, and the susceptibility to antibiotics and four phages also isolated in this work was tested. It was found that more of 90% of S. aureus isolates were not susceptible to six or more antibiotics, and 100% were resistant to penicillin, dicloxacillin, cefotaxime, ampicillin and cephalothin, and 81 and 77%, to tetracycline and cefuroxime, respectively. Fortunately, 100% of S. aureus isolates were susceptible to phages used in this work, which was detected as clear zones using specific phage. It was shown for the first time, that phages used in this study are active against pathogenic S. aureus and might be incorporated into the therapy as an important tool for the control of staphylococcal bovine mastitis, specially to antibiotic-resistant S. aureus strains isolated in farm located at the state of Guanajuato, México; and its use might be extended to other regions inside or outside the country.     

Methicillin and aminoglycoside resistance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from bovine mastitis and sequence analysis of their <Emphasis Type="Italic">mecA</Emphasis> genes

Although methicillin-resistant Staphylococcus aureus (MRSA) were generally isolated from human beings; these agents were recently isolated from various animal species. It has been shown that MRSA isolates are not only resistant to beta-lactam antibiotics, but can also be resistant to the other commonly used antibiotics. In this study, 18 phenotypic methicillin resistant S. aureus isolates from bovine mastitis cases were analyzed by PCR for the presence of mecA gene encoding methicillin resistance and aac(6′)/aph(2″), aph(3′)-IIIa and ant(4′)-Ia genes encoding aminoglycoside resistance. Out of 18 S. aureus isolates (oxacillin MICs, ≥4 μg/ml), 3 were positive for mecA gene. Only one from 3 mecA positive isolates was positive for genes encoding aminoglycoside-modifying enzymes and this isolate carried aac(6′)/aph(2″) in combination with aph(3′)-IIIa gene. The aph(3′)-IIIa gene was detected in 3 isolates. These three isolates carrying the aminoglycoside-modifying enzyme genes were resistant to gentamicin, kanamycin and neomycin. The mecA gene of 3 MRSA isolates was sequenced. All three mecA genes of these isolates were identical to that found in human MRSA strains, except a one-base substitution at nucleotide position 757. From the data presented in this study, it can be concluded that MRSA isolated from bovine mastitis may be originated from human beings, but further studies are needed to investigate the possibility of zoonotic transfer of MRSA.     

Genotypic and phenotypic detection of capsular polysaccharide and biofilm formation in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from bovine milk collected from Brazilian dairy farms

Staphylococcus aureus is a pathogen that frequently causes mastitis in bovine herds worldwide. This pathogen produces several virulence factors, including cell-associated adhesins, toxic and cytolytic exoproteins, and capsular polysaccharides. The aim of the present study was to test for the presence of genes involved in capsular polysaccharide production and biofilm formation in S. aureus isolated from bovine mastitis samples collected from 119 dairy herds located in three different Brazilian regions, as well as to assay the production of capsular polysaccharides and biofilm, in vitro. The detection of the cap, icaAD, and bap genes was performed using PCR. The detection and quantification of capsular polysaccharide production was performed using ELISA assays. The ability of the isolates to form a biofilm was examined using the polystyrene surface of microtiter plates. All 159 S. aureus isolates investigated harboured the cap gene: 80 % carried the cap5 gene and 20 % carried the cap8 gene. Sixty-nine percent of the isolates expressed capsular polysaccharide (CP) in vitro, 58 % expressed CP5 and 11 % expressed CP8. All of the isolates harboured the icaA and icaD genes, and 95.6 % of the isolates carried the bap gene. Of the 159 isolates analysed, 97.5 % were biofilm producers. A significant association between the capsular genotype and phenotype and the amount of biofilm formation was detected: cap5/CP5 isolates tended to form more biofilm and to produce a thinner CP layer than cap8/CP8 isolates. The results indicate a high potential for pathogenicity among S. aureus isolated from bovine milk collected from three different regions in Brazil.     

Milk yield and associated economic losses in quarters with subclinical mastitis due to <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Ethiopian crossbred dairy cows

The objective of the study was to estimate the prevalence and losses associated with subclinical mastitis (SCM) caused by Staphylococcus aureus in Ethiopian crossbred dairy cows. A split-udder trial was carried out to determine milk yield losses in udder quarter with S. aureus-caused SCM. Each quarter of the study cows was examined using the California Mastitis Test (CMT) and quarter milk production was measured over a period of 8 days. Milk yield losses for CMT positive quarters were estimated by comparing production of quarters with CMT score 0. Mean milk yield for uninfected healthy quarters was 1.66 kg per milking (95% CI, 1.66–1.55 kg per milking), and the rate of milk reduction for quarters with CMT scores of 1+, 2+, and 3+ was 25%, 33%, and 48%, respectively. Economic losses at different farm-size levels were calculated by multiplying the prevalence of CMT scores with milk yield losses associated with respective CMT scores. In Debre Ziet dairy herds, a quarter with SCM due to S. aureus lost an average of 34.5% of its potential milk production while the total milk yield loss per cow was estimated at 6.8%. Losses were highest in large-scale (13%) farms and lowest (3.7%) in small-scale. Based on the prevalence, the overall financial loss for each cow per lactation was 984.64 Eth Birr (US78.65) and losses in large farms (1,882.40 Eth Birr or US78.65) and losses in large farms (1,882.40 Eth Birr or US150.35) were over 3.5 times the loss in small-size farms. These figures possibly underestimate the potential benefits of mastitis control program as they do not include other direct and indirect costs.     

Antibiotic resistance and molecular characteristics of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from backyard-raised pigs and pig workers

Staphylococcus aureus is a commensal and pathogenic bacterium with impact on public health and livestock industry. The study investigated nasal carriage, antibiotic resistance, and molecular characterization of S. aureus in pigs and pig workers. Nasal swabs from 300 backyard-raised pigs and 101 pig workers were used for the study. Resulting isolates were confirmed using MALDI-TOF MS, tested for antibiotic resistance, and three different multiplex PCRs were used to detect enterotoxin, mecA, spaA, scn, and pvl genes. spa typing was used to annotate the isolates into MLST clonal complexes (CC). Structured questionnaire was used to access possible risk factors for S. aureus carriage. The prevalence of S. aureus in pigs and pig workers were 5.3 and 12.9%, respectively. The isolates were resistant to beta-lactams (97%), tetracycline (62%), sulfonamide (52%), aminoglycoside (20.6%), fluoroquinolone (24%), and mupirocin (3.4%). Twenty seven (93%) of the isolates carried scn, 7(24%) pvl, and 12 (41%) enterotoxin genes, respectively. Questionnaire survey showed medical-related occupation of household members was associated (p?<?0.5) with S. aureus carriage. This study suggests the presence of human multidrug resistant strains of S. aureus, high carriage of pvl, and enterotoxin genes, and CC5, CC15, and CC152 were the CC-groups shared among pigs and pig workers.     

<Emphasis Type="Italic">Theileria</Emphasis> (<Emphasis Type="Italic">Babesia</Emphasis>) <Emphasis Type="Italic">equi</Emphasis> and <Emphasis Type="Italic">Babesia caballi</Emphasis> Infections in Horses in Galicia,Spain

The control of equine piroplasmosis is becoming increasingly important to maintain the international market open to the horse industry. The purpose of this study was to demonstrate the occurrence of equine piroplasmosis (Theileria equi and Babesia caballi) in Galicia, north-west Spain, and to compare haematological and serum biochemistry parameters between non-parasitaemic horses and horses parasitaemic with T. equi and B. caballi. Sixty serum samples (control group) were taken from healthy horses pastured on two farms, and examined for evidence of equine T. equi and B. caballi infection by indirect fluorescent antibody test (IFAT). Of the 60 samples, 24 (40%) and 17 (28.3%) samples were positive for T. equi and B. caballi, respectively. Twelve (20%) samples were positive for both parasites. Haematology and serum biochemistry were compared between controls and a series of 36 horses clinically affected by T. equi (25) or B. caballi (11). Compared with the healthy group, there was a 43% and 37% decrease in the haematocrit for T. equi and B. caballi infection, respectively. Parasitaemic horses presented an intense anaemia and serum biochemistry signs of liver damage. The anaemia was more severe in T. equi-infected than in B. caballi-infected horses. Our results suggest that equine piroplasmosis is widespread in the region and is a cause for concern.     

SCC <Emphasis Type="Italic">mec</Emphasis> typing and antimicrobial resistance of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (MRSA) from pigs of Northeast India

Staphylococcus aureus is one of the most important pathogens of both humans and animal. Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that causes serious infections both in hospitals and communities due to its multidrug resistance tendency. This study was undertaken to characterize the MRSA isolates from pigs and to determine the antimicrobial resistance of these isolates. Forty nine MRSA strains (one strain per positive pig) isolated from pigs of Northeast India were characterized by SCCmec typing and antimicrobial resistance. The overall prevalence of MRSA was 7.02 % with the highest prevalence recorded in pigs aged 1–3 months (P = 0.001) and in nasal samples (P = 0.005). Two SCC mec types (type III and V) were found in Indian pigs with predominance of type V. All isolates were resistant to penicillin. Seventeen resistance groups were observed where 87.75 % isolates showed multidrug resistance (showed resistance to three or more classes of antimicrobials). The most predominant resistance pattern observed was Oxytetracycline + Penicillin + Sulfadiazine + Tetracycline accounting 12.24 % of the isolates. The present study contributes to the understanding of characteristics and antimicrobial resistance of porcine MRSA isolates which in turn will help in devising strategy for the control of this pathogen. Findings of the study also throw light on multidrug resistance MRSA and emphasize the need for judicious use of antimicrobials in animal practice.     

Alterations in coagulation parameters in dairy cows affected with acute mastitis caused by <Emphasis Type="Italic">E. coli</Emphasis> and <Emphasis Type="Italic">S. aureus</Emphasis> pathogens

This study was conducted to evaluate alterations in coagulation parameters in dairy cows affected with acute Escherichia coli (E. coli) mastitis and to compare those values to cows affected with Staphylococcus aureus (S. aureus ) mastitis. Twenty-four, adult Holstein-Friesian dairy cows affected with acute E. coli mastitis and 17 cows affected with S. aureus mastitis were studied. Cows affected with E. coli mastitis had significantly prolonged activated partial thromboplastin time (APTT) (P < 0.01), prothrombin time (PT) (P < 0.05) and decreased (P < 0.05) platelets numbers. Cows with S. aureus mastitis had only significantly prolonged APTT (P < 0.05) and decreased (P < 0.05) platelet counts. In the hematology evaluation, cows affected with E. coli and those affected with S. aureus mastitis had elevated hematocrit values but only significantly (P < 0.05) so in mastitic cows caused by E. coli. Both groups of mastitic cows had significantly (P < 0.05) lower leukocyte counts. Only cows with E. coli mastitis had significantly (P < 0.05) lower neutrophil count. In the plasma biochemical evaluation, creatinine concentrations were significantly (P < 0.05) elevated in both groups of cows. Blood urea nitrogen (BUN) concentration was only significantly elevated in cows affected with E. coli mastitis. Results of this study indicated that dairy cows affected with acute E. coli mastitis are more likely to develop clinical manifestations of disseminated intravascular coagulation than cows affected with S. aureus mastitis.     

Frequency of enterotoxins,toxic shock syndrome toxin-1, and biofilm formation genes in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from cows with mastitis in the Northeast of Brazil

Staphylococcus aureus is among the microorganisms more frequently associated with subclinical bovine mastitis. S. aureus may produce several virulence factors. This study aimed at determining the frequency of virulence factors such as enterotoxins, toxic shock syndrome toxin 1, and ica adhesion genes. In addition, we assessed antimicrobial drug resistance in S. aureus isolated from clinical and subclinical cases of mastitis. A total of 88 cows with clinical or subclinical mastitis were sampled, resulting in 38 S. aureus isolates, from which 25 (65.78%) carried toxin genes, including seb, sec, sed, tst, and icaD adhesion gene. These S. aureus isolates belong to 21 ribotypes and three S. aureus strains belonged to the same ribotype producing ica adhesion gene. Approximately 90% of S. aureus strains obtained in our study demonstrated multiple resistance to different antimicrobial agents. The most efficacious antimicrobial agents against the isolates were gentamicin, amoxicillin, and norfloxacin. Gentamicin was the most efficacious agent inhibiting 78.95% of the S. aureus isolates. The least efficacious were penicillin, streptomycin, and ampicillin. Our results can help in understanding the relationship between virulence factors and subclinical mastitis caused by S. aureus. Further research about diversity of S. aureus isolates and genes responsible for the pathogenicity of subclinical mastitis is essential.     

Evaluation of mammary gland immunity and therapeutic potential of <Emphasis Type="Italic">Tinospora cordifolia</Emphasis> against bovine subclinical mastitis

Enhancement of the diseased mammary gland immunity and therapeutic potential of hydro-methanolic extract of Tinospora cordifolia (T. cordifolia; stem) in bovine subclinical mastitis was investigated. Somatic cell count (SCC), total bacterial count (TBC), phagocytic activity, and leukocyte lysosomal enzymes like myeloperoxidase and acid phosphatase activity and Interleukin-8 (IL-8) level were evaluated after intramammary infusion of hydro-methanolic extract (stem) of T.cordifolia in diseased cows. The qualitative analysis of the extract revealed the presence of polysaccharide, phenol, alkaloid, and protein. Intramammary infusion of hydro-methanolic extract of T. cordifolia treatment initially enhanced the SCC; thereafter, significant reduction in cell count (P < 0.05) was observed on day 15 of the treatment period, however, reduction in TBC was observed from day 3 onwards. The phagocytic activity of milk polymorphonuclear cells enhanced in the diseased cows treated with the T. cordifolia extract. Similarly, the lysosomal enzyme content of the milk polymorphonuclear cells enhanced significantly (P < 0.05) in diseased cows treated with T. cordifolia. The IL-8 level in milk serum also increased significantly (P < 0.05) in diseased cows treated with the herb extract. The results suggest that the hydro-methanolic extract of T.cordifolia (stem) possesses antibacterial and immunomodulatory properties. In the present study, the biological activity of the Tinospora cordifolia extract at standardized dose against bovine subclinical mastitis is reported for the first time. Development of alternative therapy with medicinal plants is an option for livestock farmers who are not allowed to use the conventional allopathic drugs under certain farming system or cannot afford to use allopathic drugs.     

<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> intramammary infection affects milk yield and SCC of dairy cows

Staphylococcus aureus (S. aureus) is the most prevalent infectious microorganism affecting dairy cattle worldwide, and its pathogenic characteristics facilitate its spread in dairy herds. S. aureus intramammary infections (IMI) are mainly subclinical, and associated losses can exceed average herd losses where the pathogen is not isolated. However, the extent it affects milk composition at udder and quarter levels is still unknown, and cow composite milk losses may be underestimated due to the dilution effect. The aim of this study was to investigate the effects of S. aureus subclinical mastitis on mammary quarter milk yield and composition. In order to determine the effects of the pathogen on milk yield and composition at quarter level, a pairwise comparison of infected and non-infected mammary quarters (n?=?28) from two dairy herds was carried out. Quarters were individually milked, and milk production and composition were assessed. S. aureus has increased somatic cell counts at quarter level; however, no effect of S. aureus IMI on milk lactose, fat, and protein contents was observed. Fat yield from infected quarters decreased, but losses due to the infection caused by S. aureus were not associated with quarter positioning in cows.     

Isolation and characterization of two lytic bacteriophages against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> from India: newer therapeutic agents against Bovine mastitis

Bovine mastitis causes severe economic losses to dairy farmers. Staphylococcus aureus, is one of the most important pathogen implicated in etiology of clinical and subclinical mastitis in bovines. In view of increasing antimicrobial resistance alternatives to antibiotic therapy are much needed. The present decade has witnessed a renewed interest in phage based therapeutics and diagnostics. The present study, describes isolation and characterization of two lytic phages SAJK-IND and MSP against Staphylococcus aureus having a potential to be used in therapy against mastitis. SAJK-IND and MSP phages belonged to Myoviridae and Podoviridae families, respectively. TEM imaging of the two phages revealed an iscosahedral head. MSP phage has a short non contractile tail. SAJK-IND and MSP have a burst size of 44?±?3 and 25?±?5 PFU/ infected cell, respectively. SAJK-IND and MSP phages revealed ? 12 and ?16 proteins, respectively on SDS-PAGE analysis. The lytic activity of the phages was specific for Staphylococcus aureus. SAJK-IND revealed 100% lytic activity against several strains of Staphylococcus aureus isolated from mastitis milk samples whereas, MSP had only 40% lytic activity. SAJK-IND phage genome was sequenced, assembled and deposited in Genbank under accession no MG010123.     

The analysis of <Emphasis Type="Italic">groEL</Emphasis> gene in <Emphasis Type="Italic">Salmonella enterica</Emphasis> subspecies <Emphasis Type="Italic">enterica</Emphasis> serovar Typhimurium isolated from avians by PCR-Restriction Fragment Length Polymorphism method

Salmonella enterica subspecies enterica serovar Typhimurium causes food-borne outbreaks and systemic diseases in humans and animals. groEL gene (also known as mopA gene in S. Typhimurium), possessing conserved sequence, plays an important role in invasion of bacteria. The purpose of present study was to identify the polymorphism of groEL gene among different avians in different regions by PCR-RFLP method. Fifty two S. Typhimurium isolates (Broiler (n = 13), Layer (n = 12), Duck (n = 5), Goose (n = 5), Sparrow (n = 8), Canary (n = 3), Pigeon (n = 5) and Casco parrot (n = 1). were identified using serotyping as well as multiplex-PCR. Then, amplification of groEL gene performed and amplified products subjected to restriction digestion with BsuRI enzyme. Three RFLP profiles, A, B and C, generated DNA fragments between approximately 100–1,000 bp in size, were observed. The RFLP profile A was observed in 35 (67.3%), profile B in 14 (26.9%) and profile C in 3 (5.77%) of isolates. S. Typhimurium isolates recovered from 13 broilers (two of which profile A, 9 profile B and 2 profile C) and from 8 sparrows (two of which profile A, 5 profile B and 1 profile C) showed all three profiles, but 12 layers and other avians (including Canary (n = 3), Goose (n = 5), Duck (n = 5), Pigeon (n = 5) and Casco parrot (n = 1)) showed profile A. None of these profiles was allotted for a special region. The result of present study showed that S. Typhimurium undergoes genetic mutations in groEL gene under unpleasant milieu in different regions and in different avians. Thus, genetic diversity, despite conserved nature of groEL gene in S. Typhimurium, may exist but it depends on the condition where bacteria have settled. To our knowledge, three RFLP profiles of groEL gene generated by BsuRI restriction enzyme were not reported previously.     

Prevalence of thin-walled <Emphasis Type="Italic">Sarcocystis cruzi</Emphasis> and thick-walled <Emphasis Type="Italic">Sarcocystis hirsuta</Emphasis> or <Emphasis Type="Italic">Sarcocystis hominis</Emphasis> from cattle in Iran

Bovine sarcocystosis is caused by Sarcocystis cruzi and is known to cause considerable morbidity and mortality in cattle. This species is distributed worldwide in cattle and is the most prevalent of the Sarcocystis species infecting cattle. There is high infection rate of sarcocyst in cattle in Iran, but to our knowledge, there is no study about identification of Sarcocystis species. This work aimed to survey prevalence of S. cruzi cyst in slaughtered cattle of Isfahan, Iran. In this study, esophageal and diaphragmatic muscles of 100 cattle were collected from Fesaran abattoir of Isfahan and examined for the presence of Sarcocystis spp. cysts macroscopically and microscopically. No macroscopic sarcocysts were found in any of the samples. In light microscopy, 89 out of 100 cattle (89%) had thin-walled cysts of S. cruzi, while 21 out of them (21%) had thick-walled sarcocysts. In addition to light microscopy, ultrastructural features of the thin-walled cyst confirmed the presence of S. cruzi.     

Pathological,serological, and virological findings in sheep infected simultaneously with <Emphasis Type="Italic">Bluetongue</Emphasis>, <Emphasis Type="Italic">Peste-des-petits-ruminants</Emphasis>, and <Emphasis Type="Italic">Sheeppox viruses</Emphasis>

In this study, pathological, serological and virological examinations were performed on 15 sheep from a flock of 250 sheep and lambs that suffer from simultaneous naturally occurring BTV, PPRV and SPV outbreaks. SPV was diagnosed macroscopically and histopathologically, BTV was diagnosed by ELISA, and PPRV was diagnosed pathologically and by ELISA. Clinically fever, diarrhea, depression, polypnea, conjunctivitis, lacrimation, rhinitis, erosive stomatitis, edema of eyelids, photophobia, cutaneous eruption with erythematous areas especially noticeable in wool-free parts of the body and axilla lesions evolving into papules were observed. At necropsy, the most effected organs were lungs and gut. Subepicardial hemorrhages were also commonly seen. While typical pox lesions were observed in some lambs, usually fibrinous pleuropneumonia was more prominent lung lesion. SPV and PPRV lesions were seen at the histopathological examination of the lesioned tissues, BT lesions were mild than SPV and PPRV microscopically. Serum and leukocyte samples of 15 animals were examined for PPRV and BTV by ELISA; 5 samples were positive for PPRV and 6 BTV, 4 were positive for both PPRV and BTV simultaneously. One hundred animals died, most were lambs. Mortality rates were 100% in lambs and 80% in the herd.     

Detection of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> in tissue samples of cattle and buffaloes

Tissue samples were collected at random from cattle (Bos taurus) and buffalo (Bubalus bubalis) from an abattoir of the district of Lahore and were analyzed for the presence of Mycobacterium avium subsp. paratuberculosis and Mycobacterium bovis through acid-fast staining and polymerase chain reaction (PCR). Body condition of animals and diarrhea were recorded. Most of the animals were emaciated. Diarrhea was noticed in 15.6% of buffaloes and 19.2% of cattle. Intestinal pathology was observed in 29% of buffaloes and 32.8% of cattle. Number of mesenteric lymph node (MLN) showing gross lesions was a bit higher (35.6%) in cattle than buffalo (31.2%). Acid-fast staining of tissue scraping smears revealed the presence of acid-fast bacilli (AFB) in 17.4% intestinal and 16.4% MLN tissue samples in buffalo, while in cattle 19.2% intestinal and 17.8% MLN were found positive for AFB. In buffaloes, PCR confirmed 12.8% intestinal and 12.4% MLN positive samples for M. avium subsp. paratuberculosis. However, in cattle, PCR analysis demonstrated 14.2% positive results for M. avium subsp. paratuberculosis in both MLN and intestinal tissue samples. PCR also confirmed M. bovis in 5.8% of cattle and 5% of buffalo MLN and intestinal tissues. PCR positive tissue samples for M. avium subsp. paratuberculosis were from those animals which were emaciated, having diarrhea, and severe gross lesions. AFB were also detected in tissue scraping smears of these animals. It is concluded that infection by various mycobacterium species can be differentiated by PCR, which is not possible by acid-fast staining technique.