Iranian Journal of Veterinary Research
Volume:23 Issue: 3, Summer 2022

Identification of genotypic characteristics and pathogenicity of Staphylococcus aureus isolates is very important in the epidemiological study of its related diseases.

The present study was done to compare the S. aureus isolates from different sources on the basis of virulence gene properties, biofilm production ability, and phylogenetic variations.

Seventy S. aureus isolates (including 25 human, 25 raw milk, and 20 pet animal isolates) were subjected to slime production ability testing, polymerase chain reaction (PCR) detection of 14 different virulence genes, and DNA fingerprinting using restriction fragment length polymorphism (RFLP) of coa gene PCR products.

Among 70 S. aureus, 64 (91.4%) isolates were slime producers on Congo red agar (CRA) medium. The spa and icaD virulence genes were present in all isolates and the seD and etaA genes were not detected in any of the isolates. In total, 22 different virulence gene patterns and nine distinct clusters of coa-PCR-RFLP were identified among isolates.

According to the results, S. aureus strains of human origin showed a significant association with specific virulence gene profiles and genotypes. seB and seC were the most responsible genes for S. aureus enterotoxin among human and animal isolates, respectively. Coa-RFLP showed partially appropriate results in the classification and source detection of S. aureus isolates.

Information on the prevalence of infectious agents in dairy farms forms the basis for formulating a suitable control strategy; especially in endemic situations.

A cross-sectional study was undertaken to determine the prevalence of six economically important bovine diseases, causing reproductive disorders including bovine abortion in organized dairy herds in India.

A total of 1,075 animals (cattle and buffaloes) from 09 dairy farms were screened by ELISA tests.

Bovine viral diarrhoea (BVD) was the most prevalent (56.5%) disease followed by infectious bovine rhinotracheitis (IBR) (45.4%). Prevalence of Q-fever (5.4%) and neosporosis (6.1%) were less on the farms. Although 16.3% of the samples turned positive for brucellosis, the contribution of calf-hood vaccination (B. abortus S19 vaccine) to the prevalence of antibodies cannot be ruled out. The overall prevalence of bovine anaplasmosis, known to cause sporadic abortions in dairy herds, was 34.1% in the 9 farms with a prevalence of less than 20% in 5 farms. Infection of multiple abortifacient (seroprevalence to more than two pathogens) was recorded in 56.8% of animals. A very strong association was observed between BVD and brucellosis (Odds ratio 14.2; P<0.001). Further, a positive association was also seen between seroprevalence of IBR and anaplasmosis, and neosporosis and Q fever (P<0.05).

Viral diseases were found to be more common in the dairy herds than bacterial and protozoan diseases. Increased susceptibility of IBR seropositive cows to other bacterial and viral infections was observed.

The addition of antioxidants to semen extenders is fundamental to limit oxidative changes resulting from cryopreservation.

This experiment was designed to clarify the effects of cysteine and L-carnitine (LC) on post-thaw sperm criteria.

Semen samples were collected once weekly from five mature Zaraibi bucks for 10 successive weeks. Fresh semen samples were evaluated for basic semen characteristics, and the accepted samples were pooled and divided into seven aliquots: control (Tris egg yolk extender), cysteine (2.5, 5 and 10 mM) and LC (2.5, 5 and 7.5 mM). Aliquots were diluted and subjected to cryopreservation procedures. After thawing, sperm criteria were evaluated regarding motility, viability, plasma membrane (hypo-osmotic swelling test, HOST) and acrosome integrity, total antioxidant capacity (TAC), level of malondialdehyde (MDA), and DNA comet assay.

Addition of both 10 mM cysteine and 7.5 mM LC significantly increased post-thaw sperm motility, viability, HOST, and acrosome integrity. Comet assay revealed significant decreases in DNA damage with best results at 2.5 mM cysteine and 7.5 mM LC. Besides, 10 mM cysteine and 7.5 mM LC exhibited significant diminish in MDA levels. Cysteine was positively correlated with sperm viability, acrosome integrity, and comet, but negatively correlated with MDA and tail length. Positive correlations were declared between LC and progressive motility, viability, HOST and TAC, while negative correlations were found with MDA, comet, tail DNA, tail moment, and olive tail moment.

Supplementing cysteine and LC to frozen buck semen improved sperm criteria and decreased DNA damage, possibly due to their effectiveness in the suppression of MDA formation.

Bovine tuberculosis (bTB) is a major bacterial disease that causes significant economic disruption across the globe.

Our study was based on using a fluorescence polarization assay (FPA) that used fluorescein-labeled ESAT-6 protein to detect Mycobacterium bovis antibodies in bovine serum.

The ESAT-6 protein was used in a FPA. Positive TB reactors were determined by the comparative intradermal test (CID) and interferon gamma test (IFN-γ). Antibodies against M. bovis were detected using a fluorescein isothiocyanate (FITC) labeled tracer and a whole culture FITC labeled tracer in the positive cattle.

Of the 192 animals tested for bTB, 37 were found to be positive by either the CID or IFN-γ assays. Using the mP values from five culture-positive serum samples, a cutoff value of more than >127 mp provided the best discrimination between positive reactors and negative bTB animals. The ESAT-6 results of FPA in comparison with CID results revealed sensitivity of 92.9% and specificity of 64.6%, and in comparison with results IFN-γ, showed sensitivity of 95.7% and specificity of 49%. FPA using FITC labelled ESAT-6 as a tracer has better sensitivity (95.7%) and specificity (49.1%) than IFN-γ test in humoral immune response in animals.

This work revealed that the ESAT-6 protein as an antigen can be used in diagnosing bTB using a practical and sensitive humoral test.

Mycobacterium bovis is a zoonotic member of the Mycobacterium tuberculosis complex with a wide range of hosts, mainly cattle. Molecular epidemiological studies should be conducted to determine the transmission route, zoonotic risk factors, and phylogenetic relationships of M. bovis strains.

This study aimed to characterize bovine and human M. bovis isolates by molecular methods.

Molecular characterization and clonal relationship of strains isolated from tissue and organ samples of 76 cattle with positive tuberculin tests were collected from a slaughterhouse, and four M. bovis strains isolated from clinical materials of patients with suspected pulmonary TB isolates were analyzed using 24-locus MIRU-VNTR and spoligotyping methods. QuantiFERON-TB Gold Plus (QFT-Plus; Qiagen) was used to determine the prevalence of latent TB infection among 21 slaughterhouse personnel including 7 veterinarians, 12 butchers, 1 caretaker, and 1 veterinary technician.

SB0288/SIT685 type was detected in both cattle and humans by the spoligotyping method. When evaluating MIRU-VNTR, the presence of a 100% compatible pattern between human and bovine isolates was not detected, but some human samples were found to be 91.6% similar to a bovine sample. In addition, 21 slaughterhouse workers were screened with the interferon gamma-released assay (IGRA) and a 23.8% positivity was detected.

Clonal similarity was determined between the bovine and human isolates using the MIRU-VNTR and spoligotyping methods and IGRA positivity in the occupational group suggested that M. bovis might be associated with pulmonary tuberculosis in humans.

Nile tilapia is a highly valuable fish in the aquaculture sector. A culture farm has reported heavy mortalities of tilapia.

The present study aimed to identify the etiological agent responsible for the heavy mortality in cage cultured tilapia.

The moribund and freshly dead fishes were analyzed for clinical signs. Biochemical and molecular characterizations were performed to identify the etiological agents of the disease. Also, polymerase chain reaction (PCR) assay was used to detect the presence of the virulence genes. The susceptibility of the isolates to various antibiotics was tested by the disk diffusion method.

The results of the biochemical tests and PCR assay confirmed that co-infection with Aeromonas hydrophila, and Streptococcus iniae was responsible for the disease severity. Phylogenetic analysis of the 16S rRNA gene showed that A. hydrophila and S. iniae isolates shared 99% and 98% sequence homology with A. hydrophila and S. iniae previously deposited in the Genbank database. The multiple antibiotic resistance (MAR) index of A. hydrophila was 0.16 and that of S. iniae was 0.71. The PCR test revealed that both pathogens harbored numerous virulence factors. The experimental infection study confirmed that the synergistic action of A. hydrophila and S. iniae led to increased mortality in tilapia. Histopathological changes were observed in the liver and spleen tissues of the co-infected fishes.

These findings indicate that the disease outbreak in the tilapia culture farm occurred as a result of co-infection by A. hydrophila and S. iniae.

Clostridium perfringens commonly resides in the gastrointestinal tract and can survive in different environmental conditions. This pathogen produces several protein toxins including the potent ε-toxin which is classified as a category B toxin by the Centers for Disease Control and Prevention (CDC). In several studies, the induction of C. perfringens type C or D to produce toxins much more rapidly by close contact of bacteria with Caco-2 cells has been reported.

The effect of close contact of enterocyte-like Caco-2 cells with C. perfringens type D (vaccine strain) on the production time of ε- and α-toxins was studied.

During C. perfringens type D contact with Caco-2 cells for 5 h, ε- and α-toxins expressions (at 0, 2, and 5 h) were evaluated by a quantitative real-time PCR assay. Non-contacted bacteria with cells were included as the negative control in this research.

Bacterial contact with the Caco-2 cells induces a significant effect on the mean expression of the ε-toxin gene (etx) (P<0.05). Two h after contact, the highest level of gene expression was detected in the experimental group. Bacterial harvesting time, cell treatment, and their interactions did not affect significantly the mean expression of the α-toxin gene (cpa) (P>0.05).

According to the findings of the present study, 2 h of bacterial contact with Caco-2 cells could stimulate etx gene expression in the C. perfringens type D vaccine strain.

Dogs are the favorite companion animals among humans. The close interaction between dogs and people increases the risk of antibiotic resistance spreading. Surveillance for antimicrobial resistance and the identification of ESBL-producing Escherichia coli as an indicator bacterium is an important tool for managing antimicrobial drug therapy.

The present study targeted to identify and characterize ESBL-producing E. coli among dogs suffering from diarrhea in and around Kolkata.

Isolation and identification of E. coli from dogs suffering from diarrhea (n=70) along with screening for the production of both ESBL and AmpC. The isolates were further characterized through antimicrobial resistance profiling, resistance genes (blaCTX-M, blaTEM, and blaSHV) screening, and phylogenetic group study.

Among the 70 isolates, 21 (30%) were confirmed ESBL producers. An antibiogram typing of ESBL-producing E. coli revealed that the majority of them were resistant to norfloxacin (85.7%) followed by tetracycline (61.90%), doxycycline (57.14%), piperacillin/tazobactam (52.38%), cotrimoxazole (47.62%), gentamicin (42.62%), amikacin (23.81%), and chloramphenicol (19.05%). Major resistance genes included blaCTX-M (100%), blaTEM (28.57%), and blaSHV (9.50%). The predominant phylogenetic groups were phylogroup A (76%) followed by phylogroup D (24%).

The current investigation reported a high prevalence of both ESBL and AmpC β-lactamase (AmpC) producing E. coli, co-resistance to a distinct group of antibiotics, and co-existence of different ESBL genes in dogs. Our findings highlight the importance of diagnostic antimicrobial susceptibility testing for proper antimicrobial therapy and to prevent antimicrobial resistance from spreading to humans from dogs in Kolkata and the surrounding area.

Cryptosporidium, an opportunistic, zoonotic, apicomplexan parasite, is one of the most common causes of diarrhea in neonatal bovine calves around the globe. Bovine calves act as a major source of infection by excreting huge numbers of highly resistant oocysts in faeces, which can survive for a long time in extreme environmental conditions. As low as ten oocysts can cause disease and mortality, leading to the requirement of an early and accurate diagnosis for proper and favorable prognosis, management, and control.

The current study was conducted with the objective to evaluate various diagnostic techniques (acid fast staining, negative staining, fluorescent, ELISA, PCR, nested PCR, and qPCR) for the detection of Cryptosporidium in the faecal samples of diarrheic bovine calves.

Two hundred diarrheic faecal samples from bovine calves were collected and subjected to these techniques for Cryptosporidium diagnosis. Results of these were evaluated for diagnostic comparison.

Out of 200 faecal samples evaluated, 24% (48/200) were detected positive for Cryptosporidium using a combination of two techniques as gold standard criteria. Cohen’s kappa value indicated moderate to almost perfect agreement (0.616 to 0.986) among all the techniques used in the present study. Leishman staining showed the lowest sensitivity (54.17%), while nested PCR and qPCR showed the highest sensitivity (97.92%). Diagnostic specificity of all these tests ranged from 98.68 to 100%.

Auramine stain was used for the first time in the bovine calves in India for the detection and diagnostic comparison of Cryptosporidium. It showed strong agreement with the molecular as well as classical diagnostic techniques, and can be used for primary screening for better diagnosis.

Vaccines have been widely exploited to prevent tick-borne infections in cattle. Most vaccines have faced failure in the field because of inconsistency in an immune response. It is presumed that the cement-cone proteins of ticks that participate in the acquisition of blood meal for ticks possess strong immune-stimulating properties and, hence, could be a useful candidate in vaccine development.

We evaluated cement-cone proteins of tick Hyalomma anatolicum as a vaccine candidate against infestations of H. anatolicum and H. aegyptium in cattle.

The cement-cone proteins were extracted from H. anatolicum to develop stage-reactive and immunogenic cross-reactive vaccine against the infestation of two species of ticks H. anatolicum and H. aegyptium. The immune response of the vaccine was tested against cement-cone proteins starved, partially fed, and richly fed ticks.

The findings of the present study demonstrated the cross-reactivity among the two species of ticks that belonged to the same genus (Hyalomma). The antigenic similarity between the two ticks species suggests that a common antigen may possibly be suitable for a vaccine against the two different species of ticks. The results have also indicated that the 23 kDa cement-cone protein of H. anatolicum and H. aegyptium may be responsible for the induction, or elicitation of immunogenic, common stage reactive, and cross-reactive host immune responses with consistent intensity throughout the life stages of ticks.

The vaccine based upon cement-cone proteins of ticks may be a useful deterrent against tick-borne infections in cattle in countries like Pakistan.

Establishment of experimental cystic echinococcosis (CE) in laboratory mice is required for the study of CE. Experimental CE may be established in primary or secondary forms; however, the primary form is more reliable for the study of CE.

The aim of the study was to assess the possibility of establishment of primary CE in Balb/c mice via the oral administration of Echinococcus granulosus s.l. eggs.

E. granulosus s.s. eggs were obtained from the gravid segments of the adult worms collected from an experimentally infected dog. Thirty-four female Balb/c mice were allocated into 4 groups and were orally infected with E. granulosus s.s. eggs as follows: 1) the mice with normal immunity, a single dose of 1000 eggs, 2) the mice with normal immunity, 3 doses of 500 eggs, 3) immunosuppressed mice, a single dose of 1000 eggs, and 4) immunosuppressed mice, 3 doses of 500 eggs. After 6.5 months, all the mice were opened and their internal organs were examined for the presence of CE cysts. The livers of infected mice were also examined for the presence of E. granulosus s.l. compartments by the PCR method.

There was no developed or developing CE cyst in the abdominal cavities or on the internal organs of all the mice in four groups. In addition, in the molecular study, all the examined liver samples were negative for the parasite material.

The results of the present study revealed that Balb/c mice are not a suitable host for establishment of primary CE following the oral administration of E. granulosus s.s. eggs.

Despite advances in food management techniques, foodborne illness remains a major concern. Contamination of Salmonella and Escherichia coli pathogens, especially in the poultry sector, is responsible for salmonellosis and other gastrointestinal illness, leading to millions of deaths worldwide. Overuse of antibiotics and other chemical treatments have further increased the emergence of antibiotic resistant bacteria.

This study aimed to study the efficacy of phages cocktail to reduce the load of E. coli and Samlonella spiked on poultry meat.

In this study, a broad spectrum cocktail of phages was used to lyse E. coli and Salmonella spiked on chicken meat.

Based on the result of the CFU drop assay, phages like E. coli 153T 3ii and Salmonella 191(3) were selected. Phage concentration of 0.01 MOI showed a reduction in E. coli and Salmonella count to 6 h and 2 h, respectively. Further, phages were tested on the surface of chicken meat. E. coli showed a 90% reduction up to 4 h, whereas Salmonella showed a 90% reduction up to 6 h. When phages were treated in combination, a significant reduction of up to 12 h was found with Salmonella phage, showing better antimicrobial activity.

The suitable concentration of a specific phage or phage cocktail can significantly reduce the bacterial count on chicken meat. Phage mediated biocontrol can be used as an alternative approach to eliminate enteric pathogens in the poultry industry.

Mastitis is one of the most expensive diseases in the dairy industry. It causes heavy monetary losses by decreasing milk production and treatment cost. Streptococcus agalactiae, the cause of contagious bovine mastitis, possesses various virulence factors that contribute to pathogenicity.

The main aim of the study was to evaluate the distribution of virulence genes of S. agalactiae.

In the current work, 98 Streptococcus species were isolated from 320 milk samples, collected from Veterinary Clinical Complex, Junagadh. Out of the isolates, 42 S. agalactiae isolates were used for virulence genes detection.

All Streptococcus spp. were confirmed at genus level by targeting tuf gene, and S. agalactiae was identified at species level by targeting 16S rRNA gene. For virulence gene detection, scpB, cfb, and cylE genes were targeted. Of 42 S. agalactiae isolates, 15, 16, and 10 isolates possessed scpB, cfb, and cylE genes, respectively.

This study aids us to know virulence characteristics and mechanisms responsible for the development of new strains in mastitis epidemiology in response to prevention and control strategies.

Klebsiella pneumoniae can cause high mortality in birds. In recent years, small farming of canaries has been developed in Iran and infectious diseases are the main obstacle in the progress of these occupations which increases the importance of identification of pathogenic bacterial agents.

Case description:

 A flock with 250 one-year-old canaries presented a history of anorexia, lethargy, mild diarrhea, and approximately 30% mortality. Physical examination revealed that the birds were severely debilitated, cachectic, dehydrated with ruffled feathers, and wet discolored stool around the cloaca. Necropsy findings revealed enlarged liver with multiple pale white, irregular foci on the surface of parenchyma, serosal petechial hemorrhages, and enlargement of lungs, liver, and kidneys. The entire intestine was intensely reddened with fibrinonecrotic exudate content. Histopathological findings of the liver elicited multifocal hepatocellular necrosis, hemorrhage, and also basophilic bacterial colonies. The results of biochemical and molecular tests confirmed K. pneumonia as the causative agent.

Findings/treatment and outcome:

 Based on antimicrobial susceptibility test, K. pneumonia isolates were susceptible to gentamycin and ciprofloxacin which were administrated for the considered treatment protocol.

K. pneumoniae is one of the most important causes of mortality in canaries with multiple antibiotic resistance, therefore assessments of health conditions can supply suitable information to help decision-making about the sanitary processes, control, prevention, and treatment.