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.     

Methicillin resistance genes and in vitro biofilm formation among Staphylococcus aureus isolates from bovine mastitis in India

IntroductionBiofilms, an assemblage of microbial cells irreversibly associated with a surface and enclosed in a matrix of polysaccharide material pose serious health challenges, resulting in high economic losses. The emergence of methicillin-resistant S. aureus (MRSA) infections and ability to form biofilms in dairy animals is of emerging concern for livestock and public health owing to their association with serious infections. The present study was undertaken to examine the presence of methicillin resistance genes among the biofilm forming Staphylococcus aureus strains isolated from cases of acute and subacute bovine mastitis. A total of 150 mastitic milk samples referred to Veterinary Clinical Complex, Shuhama (Aulesteng) SKUAST-K were screened in present study. The methicillin resistant Staphylococcus aureus isolates were also screened for in vitro biofilm forming ability.ResultsA total of 80 (53.33%) S. aureus isolates were recovered from cases of bovine mastitis of which 20 (25%) were methicillin (mecA) gene positive. Of the 20 mecA positive isolates, 20% were positive for SCCmec I, 35% for SCCmec IV and 45% for SCCmec V subtypes. In vitro antibiotic sensitivity testing of MRSA revealed complete resistance towards methicillin and other pencillin group of antibiotics.ConclusionA significant correlation was observed between in vitro biofilm formation and presence of methicillin resistance gene in S aureus isolates recovered from acute and subacute mastitis. The Staphylococcus aureus isolates positive for methicillin resistance gene (mecA) were either strong or moderate biofilm formers.     

Biofilm formation in Haemophilus parasuis: relationship with antibiotic resistance,serotype and genetic typing

Biofilms are surface-associated microbial communities, which are encased in self-synthesized extracellular environment. Biofilm formation may trigger drug resistance and inflammation, resulting in persistent infections. Haemophilus parasuis is the etiological agent of a systemic disease, Glässer's disease, characterized by fibrinous polyserositis, arthritis and meningitis in pigs. The purpose of this study was to examine the correlation between biofilm and antibiotic resistance among the clinical isolates of H. parasuis. In the present study, we tested biofilm-forming ability of 110 H. parasuis isolates from various farms using polystyrene microtiter plate assays. Seventy-three isolates of H. parasuis (66.4%) showed biofilm formation and most of them performed weak biofilm-forming ability (38/73). All isolates were tested for antimicrobial susceptibility to 18 antimicrobial agents by the broth microdilution method. H. parasuis isolates showed very high resistance (>90%) to sulfanilamide, nalidixic acid, and trimethoprim. Resistance to eight antibiotics such as penicillin (41.1% vs 8.1%), ampicillin (31.5% vs 8.1%), amoxicillin (28.8% vs 5.4%), gentamicin (46.6% vs 24.3%), cefazolin (19.2% vs 2.7%), doxycycline (19.2% vs 8.1%), cefotaxime (11% vs 2.7%), and cefaclor (13.7% vs 5.4%) was comparatively higher among biofilm producers than non-biofilm producers. Pulsed-field gel electrophoresis (PFGE) analyses could distinguish various isolates. Our data indicated that H. parasuis field isolates were able to form biofilms in vitro. In addition, biofilm positive strains had positive correlation with resistance to β-lactams antibiotics. Thus, biofilm formation may play important roles during H. parasuis infections.     

Investigation of biofilm production and icaA and icaD genes in Staphylococcus aureus isolated from heifers and cows with mastitis

Biofilm formation and antimicrobial resistance of Staphylococcus aureus are important virulence factors in cases of mastitis in dairy cows. However, few studies have investigated mastitis strains isolated from heifers. Within this context, the objective of the present study was to investigate biofilm formation on Congo red agar, the presence of the icaA and icaD genes by polymerase chain reaction (PCR), and the percentage of in vitro antimicrobial resistance of 110 S. aureus isolates from mammary gland secretions of heifers and cows with mastitis. PCR detected the icaA and icaD genes in 98% and 100% of isolates, respectively. However, only 55.5% of all isolates produced a biofilm on Congo red agar. Antimicrobial susceptibility testing revealed that 47.0% of isolates from heifers and 70.4% of isolates from cows were resistant to at least one of the antimicrobial agents tested. Resistance to penicillin and/or ampicillin was the most frequent (44.5%). These results indicate the need to implement prophylactic and control measures of mastitis for heifers. Heifers and cows can carry resistant strains with the capacity of biofilm production, a fact representing a threat to public health and animal well‐being and generating losses to dairy farmers.     

Prevalence and molecular characterization of Sarcocystis species in water buffaloes (Bubalus bubalus) in Egypt

The present study was planned to investigate the prevalence of Sarcocystis spp. among slaughtered water buffaloes (Bubalus bubalis) at Alexandria province, Egypt. Three hundred blood samples were collected from slaughtered buffaloes (5–7 years old). Two techniques were used to evaluate the seroprevalence of Sarcocystis spp., enzyme-linked immunosorbent assay (ELISA) and indirect haemagglutination assay (IHA). It was revealed that 203 (67.6 %) and 191 (63.6 %) of the tested serum samples were seropositive to Sarcocystis spp. by ELISA and IHA, respectively. The results of sensitivity and specificity of IHA relative to ELISA were 94 and 100 %, respectively. For molecular characterization of inter- and intra-species genetic polymorphism within Egyptian isolates of Sarcocystis spp. of water buffaloes, polymerase chain reaction (PCR) and polymerase chain reaction-restriction length polymorphisms (PCR-RFLPs) were performed on four macroscopic isolates. The isolates represented two different geographical regions of Egypt, Alexandria and Assuit provinces. Alexandria isolates (large and small-sized cyst of the same host) and Assuit isolates (large and small-sized cyst of the same host) were used. The 18S rDNA of the macroscopic cysts were characterized, in tandem, by four restriction endonucleases, RsaI, MboI, SspI and DraI. RsaI and MboI enzymes did not show any restriction sites for all isolates, leaving the amplified fragments without cutting. SspI showed two fragments in Alexandria and Assuit small-sized isolates cut by the enzyme at 600–700-bp fragments, while Alexandria and Assuit large-sized cysts amplicons were not digested by this enzyme. The fourth enzyme, DraI, cut the PCR product of Alexandria large-sized cysts into two fragments (420–780 bp), while Assuit large-sized amplicon was not cut. It could be concluded that there was a far distance between the two local isolates (small and large sized), but there were no differences between the large-sized isolates.     

上海市宠物猫杯状病毒、疱疹病毒及流感病毒的分离与鉴定

为了解上海市猫上呼吸道疾病病例中猫杯状病毒(FCV)、猫疱疹病毒1型(FHV-1)和猫流感病毒(FIV)的感染比例及其遗传变化特点,对上海市冬季53份表现上呼吸道症状宠物猫的眼结膜、口咽和鼻黏膜拭子,进行FCV、FHV-1和FIV分离与鉴定,并对分离的病毒进行遗传进化分析.结果显示:53份样品中,FCV分离率为58.4...     

Molecular identification of chlamydial cause of abortion in small ruminants in Jordan

Chlamydophila abortus (Ch. abortus) is the etiological agent of ovine enzootic abortion (OEA) and one of the most common infectious agents of abortion in small ruminants worldwide. RFLP-PCR analysis of the outer membrane protein gene (OMP2 gene) was used for diagnosis and characterization of chlamydial causes of abortion in small ruminants in Jordan. Sixty-six placental tissues and 15 vaginal swabs were collected from aborted ewes and does to identify cause of abortion in Jordan. Thirty-eight placental samples (58 %) and 13 vaginal swabs (87 %) were positive for chlamydial DNA. Shedding of bacteria in vaginal swabs was detected within 7 days after abortion. The results of this study showed that chlamydiosis is one of the important causes of abortion in small ruminants in Jordan. In addition, vaginal swab is an excellent sample for molecular diagnosis of chlamydiosis. DNA sequencing and RFLP analysis of the OMP2 reveal that all chlamydial cause of abortion in small ruminants in Jordan are due to Ch. abortus. While, Ch. pecorum was not detected in any sample. OMP2 gene of the isolated Jordanian strain was identical (100 %) to Ch. abortus FAS strain. In conclusion, Ch. abortus is an important cause of abortion in Jordan; vaginal swab within 7 days of abortion can be used for molecular diagnosis of chlamydiosis in small ruminants.     

Seroprevalence and molecular detection of Mycoplasma ovipneumoniae in goats in tropical China

In the present study, the seroprevalence and genetic identification of Mycoplasma ovipneumoniae infection in goats were investigated in Hainan Province, tropical China between October 2012 and October 2013. A total of 1,210 serum samples collected from 16 herds in various administrative regions in tropical China were evaluated using indirect hemagglutination assay (IHA). Antibodies to M. ovipneumoniae were tested in (31.7 %, 95 % confidence interval (CI) 29–34.3) 383 of 1,210 serum samples (IHA titer ≥1:16). The M. ovipneumoniae seroprevalence ranged from 26.8 % (95 % CI 20.8–32.9) to 39 % (95 % CI 30.8–47.2) among different regions in tropical China, and the difference was statistically significant (P?<?0.01). The seroprevalence of M. ovipneumoniae infection in goats was higher in winter (46.1 %, 95 % CI 39.6–52.5) and spring (33.8 %, 95 % CI 28.3–39.3) than in autumn (27.5 %, 95 % CI 22.6–32.3) and summer (24.7 %, 95 % CI 20.3–29.1), and the difference was statistically significant (P?<?0.01). In addition, DNA was extracted from nasal swab; lung samples and the 16S rRNA gene sequences were amplified by polymerase chain reaction (PCR) and then sequenced. Twenty-four of 329 (7.3 %) nasal swab samples and 73 of 280 (26.1 %) pneumonic lung tissues were found to contain M. ovipneumoniae, respectively. The results of the present survey indicate that M. ovipneumoniae infection is highly prevalent in goats in tropical China. This is the first report of the comprehensive survey of M. ovipneumoniae prevalence in goats in China.     

Investigation of chlamydophilosis from naturally infected cats

Background Chlamydophila felis, formerly known as Chlamydia psittaci var. felis, is frequently associated with ocular, respiratory, and occasionally reproduction tract infections. Even though the infection is sometimes asymptomatic, it potentially results in a latent immunosuppressive infection.ObjectiveThis study aimed to identify occurrences of feline chlamydophilosis, rarely reported in cats in Indonesia.MethodsThe observation was conducted in three cats with clinical signs of Cp. felis infection, particularly relapsing conjunctivitis. The cats'' histories were recorded based on owners'' information. Conjunctival swabs were sampled for cytology examination and molecular assay detection. A phylogenetic tree was generated using MEGA-X software to reveal group clustering. A post-mortem examination was performed on the cat that died during an examination.ResultsCp. felis was detected in both cytological examination and polymerase chain reaction assay. The phylogenetic tree demonstrated that the Cp. felis isolated in this study clustered with several other isolates from the other countries. Cp. felis can be isolated from cats with different clinical manifestations and levels of severity. The chronic fatal infection demonstrated interstitial broncho-pneumonia under histopathological examination.ConclusionsMolecular assay of Cp. felis is always recommended to obtain a definitive diagnosis of feline chlamydophilosis since the disease can have various clinical manifestations. Even though it may be subclinical and is often not fatal, an infected cat may be a carrier that could spread the pathogen in the surrounding environment. Serious disease management is suggested to avoid high costs associated with regularly relapsing disease.     

Diagnostic and prognostic utility of surface electrocardiography in cats with left ventricular hypertrophy

Objectives

To assess the ability of electrocardiography (ECG) to predict left ventricular hypertrophy (LVH) in the cat and to investigate the prognostic value of selected ECG variables in cats with LVH.

Prevalence,species distribution and antimicrobial resistance patterns of methicillin-resistant staphylococci in Lithuanian pet animals

Background

The bacterial genus Staphylococcus consists of many species that causes infections in pet animals. Antimicrobial resistant staphylococci cause infections that are difficult to treat and they are important from the point of one health perspective. The aim of this study was to determine the prevalence of methicillin-resistant Staphylococcus (MRS) species, including methicillin-resistant S. aureus (MRSA) in diseased pet animals (Group A) and kennel dogs (Group B) in Lithuania and to characterize the isolates according to their antimicrobial resistance.

Results

Twenty-one MRS isolates were obtained from 395 clinical samples (5.3 %; CI 95 % 3.5-8.0) of Group A animals. Sixteen, four and one isolates were from dogs, cats and a pet rabbit, respectively. The mecA gene was present in 20 isolates, whereas one isolate was positive for the mecC gene. Twenty-one MRS isolates (20.0 %; CI 95 % 13.5-28.6) were obtained from the vagina of female dogs (n = 105) (Group B). All isolates carried the mecA gene. Twelve MRS species were isolated of which S. pseudintermedius was the most common (18/42) followed by S. haemolyticus (8/42) and S. lentus (4/42). MRSA was not found. All MRS strains were susceptible to vancomycin, linezolid, daptomycin and quinupristin/dalfopristin. Resistance to tetracycline (16/21), clindamycin (15/21) and erythromycin (14/21) was the most common types of resistance in Group A animals. Three isolates also demonstrated resistance to rifampin. Resistance toward gentamicin (16/21), ciprofloxacin (15/21), macrolides (15/21) and tetracycline (12/21) was the most common in kennel dogs (Group B). The most common genes encoding resistance to antimicrobials (excluding beta-lactams) in isolates from Group A pets were tetK (21/42), aph(3′)-IIIa (11/42) and aac(6'')-Ie-aph(2'''')-Ia (9/42).

Conclusions

A wide range of MRS species were found in pet animals in Lithuania. MRSA was not found.     

Lack of association between p53 SNP and FISS in a cat population from Germany

One recent study indicates a significant association between certain single nucleotide polymorphisms (SNPs) in the genomic sequence of feline p53 and feline injection‐site sarcoma (FISS). The aim of this study was to investigate the correlation between a specific nucleotide insertion in p53 gene and FISS in a German cat population. Blood samples from 150 German cats were allocated to a control group consisting of 100 healthy cats and a FISS‐group consisting of 50 cats with FISS. All blood samples were examined for the presence of the SNP in the p53 gene. Results found the T‐insertion at SNP 3 in 20.0% of the cats in the FISS‐group and 19.2% of cats in the control‐group. No statistically significant difference was observed in allelic distribution between the two groups. Further investigations are necessary to determine the association of SNPs in the feline p53 gene and the occurrence of FISS.     

Real-time PCR carried out on DNA extracted from serum or blood sample is not a good method for surveillance of bovine brucellosis

Bovine brucellosis is endemic in many parts of the world including India. The disease diagnosis and surveillance are usually carried out by serological tests, which however have drawbacks. This study was undertaken to evaluate the potential of real-time PCR (RT-PCR) targeting bcsp31 gene for surveillance of bovine brucellosis. A total of 461 samples, which included 408 stored serum and 53 prospective blood samples, were used. It was found that 33 (7.15 %) samples were positive by RT-PCR, whereas 149 (32.32 %) and 132 (28.63 %) were positive by Rose Bengal plate test (RBPT) or standard agglutination test (STAT), respectively. The results of this study suggest that RT-PCR targeting bcsp31 gene carried out on DNA extracted from serum or blood may not be a suitable method for surveillance of brucellosis in bovines.     

Prevalence and risk factors for carriage of multi-drug resistant Staphylococci in healthy cats and dogs

We investigated the distribution of commensal staphylococcal species and determined the prevalence of multi-drug resistance in healthy cats and dogs. Risk factors associated with the carriage of multi-drug resistant strains were explored. Isolates from 256 dogs and 277 cats were identified at the species level using matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The diversity of coagulase-negative Staphylococci (CNS) was high, with 22 species in dogs and 24 in cats. Multi-drug resistance was frequent (17%) and not always associated with the presence of the mecA gene. A stay in a veterinary clinic in the last year was associated with an increased risk of colonisation by multi-drug resistant Staphylococci (OR = 2.4, 95% CI: 1.1~5.2, p value LRT = 0.04). When identifying efficient control strategies against antibiotic resistance, the presence of mechanisms other than methicillin resistance and the possible role of CNS in the spread of resistance determinants should be considered.     

Identification and genotyping of feline infectious peritonitis-associated single nucleotide polymorphisms in the feline interferon-γ gene

Feline infectious peritonitis (FIP) is an immune-mediated, highly lethal disease caused by feline coronavirus (FCoV) infection. Currently, no protective vaccine or effective treatment for the disease is available. Studies have found that some cats survive the challenge of virulent FCoV isolates. Since cellular immunity is thought to be critical in preventing FIP and because diseased cats often show a significant decrease in interferon-γ (IFN-γ) production, we investigated whether single nucleotide polymorphisms (SNP) in the feline IFN-γ gene (fIFNG) are associated with the outcome of infection. A total of 82 asymptomatic and 63 FIP cats were analyzed, and 16 SNP were identified in intron 1 of fIFNG. Among these SNP, the fFING + 428 T allele was shown to be a FIP-resistant allele (p = 0.03), and the heterozygous genotypes 01C/T and +408C/T were found to be FIP-susceptible factors (p = 0.004). Furthermore, an fIFNG + 428 resistant allele also showed a clear correlation with the plasma level of IFN-γ in FIP cats. For the identification of these three FIP-related SNP, genotyping methods were established using amplification refractory mutation system PCR (ARMS-PCR) and restriction fragment length polymorphisms (RFLP), and the different genotypes could easily be identified without sequencing. The identification of additional FIP-related SNP will allow the selection of resistant cats and decrease the morbidity of the cat population to FIP.     

Occurrence of antimicrobial resistant bacteria in healthy dogs and cats presented to private veterinary hospitals in southern Ontario: A preliminary study

The prevalence and patterns of antimicrobial susceptibility of fecal Escherichia coli, Salmonella spp., extended β-lactamase producing E. coli (ESBL-E. coli), methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-resistant Staphylococcus pseudintermedius (MRSP) were determined for healthy dogs (n = 188) and cats (n = 39) from veterinary hospitals in southern Ontario that had not had recent exposure to antimicrobials. The prevalence of antimicrobial resistance in E. coli was as follows: streptomycin (dogs — 17%, cats — 2%), ampicillin (dogs — 13%, cats — 4%), cephalothin (dogs — 13%, cats — < 1%), and tetracycline (dogs — 11%, cats — 2%). Eleven percent of dogs and 15% of cats had isolates that were resistant to at least 2 antimicrobials. Cephamycinase (CMY)-2 producing E. coli was cultured from 2 dogs. No Salmonella spp., ESBL-E. coli, MRSA, or MRSP isolates were recovered. The observed prevalence of resistance in commensal E. coli from this population was lower than that previously reported in companion animals, but a small percentage of dogs may be a reservoir for CMY-2 E. coli.     

Isolation and genetic characterization of Toxoplasma gondii from alpaca (Vicugna pacos) and sheep (Ovis aries)

Alpacas are important to the economy of several countries. Little is known of Toxoplasma gondii infection in alpacas worldwide. In the present study, T. gondii was isolated and genetically characterized from alpacas for the first time. Alpacas (n?=?16) and rams (n?=?12) pastured on a farm in Virginia, USA, were examined at necropsy. Antibodies to T. gondii were determined by the modified agglutination test (MAT, 1:25) and found in 6 of 16 alpacas with titers of 1:100 (2 alpaca), 1:400 (2 alpacas), 1:800 (1 alpaca), and 1:1,600 (1 alpaca), and 5 of 12 rams in titers of 1:50 in one, 1:400 in one, 1:800 in one, 1:1,600 in one, and 1:3,200 in one. Tissues of all 16 alpacas were bioassayed in mice or in cats. Muscles (heart, skeletal muscle) of nine alpacas with MAT titers of 1:25 were fed to T. gondii-free cats; the cats did not shed oocysts. Viable T. gondii was isolated from tissues of two of six seropositive alpacas by bioassay in mice. Viable T. gondii was isolated from three of three seropositive sheep by bioassay in mice. Genotyping using cell-cultured tachyzoites revealed four genotypes, including one for ToxoDB PCR-RFLP genotype #2 (type III), one for genotype #3 (type II variant), one for genotype #170, and two for a new genotype designated as ToxoDB PCR-RFLP genotype #230. Thus, four of the five T. gondii isolates in the present study belonged to different genotypes. These results indicate a higher genetic diversity among T. gondii isolates circulating in the USA than previously realized.     

Total sialic acid: An acute phase reactant in cats with a possible role in feline coronavirus infection

The aims of this study were to validate a colorimetric method to measure total sialic acid (TSA) in feline serum and to investigate the serum concentration of TSA in clinically healthy cats seronegative (n = 9) and seropositive (n = 48) for feline coronavirus (FCoV), and in cats affected by feline infectious peritonitis (FIP, n = 28), tumors (n = 20), or inflammation (n = 16). The correlation between TSA and α₁-acid glycoprotein (AGP) was also investigated. The method employed in this study is precise and accurate at TSA levels (in mg/L) commonly encountered in feline serum. No significant differences between seropositive (385.6 ± 192.2 mg/L) and seronegative (433.5 ± 179.0 mg/L) cats were detectable, suggesting that the simple infection by FCoVs does not influence TSA levels. Compared with seropositive controls, the concentration of TSA was higher in cats with FIP (556.7 ± 268.3 mg/L, P = 0.003), tumors (522.5 ± 294.4 mg/L, P = 0.028), and inflammation (546.8 ± 208.3 mg/L, P = 0.018). The discriminating power of TSA for FIP is moderate (area under the ROC curve = 0.65) and the likelihood ratio is higher than 3.0 only at high TSA levels. Consequently, TSA could support a diagnosis of FIP only at extremely high serum concentration (> 800 mg/L) or when the pre-test probability of FIP is high. No correlations were found between the TSA and AGP concentrations in cats with FIP, suggesting that sialylated proteins other than AGP are present. Both the antibody titre and the degree of AGP sialylation were negatively correlated with TSA levels, suggesting that increased TSA may contribute to reduce the burden of FCoVs.