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Open Access
Phenotypic and genotypic characterization of Staphylococcus aureus of animal origin
(CSK HPKV, Palampur, 2023-01-24) Barsain, Shivani; Verma, Subhash
Staphylococcus aureus is an opportunistic pathogen of humans and animals causing both acute and chronic infections. Several virulence-related factors have been described in S. aureus with multiple roles in attachment, multiplication, invasion, and evasion of host immune responses. The disease outcome therefore, does not merely depend on the host’s immune status but also on S. aureus phenotypic and genotypic virulence-associated factors. Several strains of S. aureus are being recovered from livestock suffering from various diseases; however, their true virulence potential and antibiotic resistance status never become known. This poses a huge challenge to veterinarians, owners and, the livestock industry because of losses associated with such isolates, their transmission and subsequent animal and human infections. Under this study, a total of 50 isolates of livestock origin were genotypically and phenotypically characterized. All isolates were nuc and catalase positive. Most of the isolates exhibited coagulase, biofilms, and hemolysins production. The mecA was detected in 38% of isolates; lukpv in 76%, tsst in 2%, sdrD in 80%, sdrE in 58%, clfA in 78%, cna in 34%, CC398 in 56% isolates. No isolate was positive for sea, scn, sak, vanA and icaA. The S. aureus were tested for their susceptibility to cloxacillin, amoxicillin, cephalexin, neomycin, tobramycin, doxycycline, erythromycin, vancomycin, enrofloxacin and levofloxacin. Only 12% isolates were susceptible to all the tested antibiotics; whereas 78% were resistant to one or more antibiotics. The isolates revealed 100% susceptibility to levofloxacin and enrofloxacin followed by vancomycin (98%), cephalexin (96%), erythromycin (94%), doxycycline (94%), neomycin (94%), tobramycin (84%), cloxacillin (70%), amoxicillin (46%). Amoxicillin was the least effective drug. About 16% isolates also exhibited MDR involving amoxicillin, erythromycin, doxycycline, tobramycin and cephalexin. A MAR index > 0.2 was recorded in 26% of isolates. Out of 50 isolates, 30% belonged to the highly virulent class, 54 % to the medium and 16% to low virulent class, respectively. This study confirmed that these isolates were livestock adapted and majority of them belonged to a single lineage comprising of CC398 cluster. In vivo pathogenic potential of isolates will allow S. aureus better classification enabling epidemiological tools in the hands of researchers and clinicians for better outcomes.
Open Access
Immunogenetic and biochemical profiling of Gaddi dogs
(CSK HPKV, Palampur, 2023-01-24) Yadav, Priyanka; Sharma, Mandeep
Gaddi dog is a generic term deriving its name from the nomadic shepherds referred to as Gaddis tribe (parts of Himachal Pradesh, Uttarakhand, J&K); who rear these dogs primarily to protect and guard their sheep and goats. It is a matter of great concern that this important germplasm is unknown to the masses and not recognized by any Kennel Club of India. This unique canine germplasm, therefore, needs to be documented and registered. The present study was designed to study the blood and haematological profiles; and parameters related to the innate immunity of Gaddi dogs that may help in the characterisation and registration of these dogs to a defined breed. The average physiological values for rectal temperature (101.4 ± 0.2 °F), respiratory rate (25.6 ± 0.60 breaths per min.), and heart rate (127.3 ± 1.20 beats per minute) were recorded. The values recorded for haematology and biochemical parameters from seven Gaddi dogs were within the normal range as has been reported for dogs of some other breeds. The mean percent values of major lymphocyte subsets were 44.50, 25.03, and 10.30 for T-helper cells (CD4+), cytotoxic T cells (CD8+) and B lymphocytes (CD21+), respectively. The respiratory burst activity of PMNCs (neutrophils) as measured by Nitroblue tetrazolium test and the DCFH-DA probe-based assay revealed around two-fold increase. Ferric reducing the ability of plasma assay showed higher FRAP values for Gaddi dogs (0.183-0.234 Fe2+ µmoles per ml) when compared with diseased dogs (0.172-0.201 Fe2+ µmoles per ml) of other breeds. The complement haemolytic activity (CH50) of Gaddi dogs was found to be in the range of (19-50.9 CH50 units) and the mean was 27.2 CH50 units. These values for dogs of some other breeds were in the range of 22.7-37.8 CH50 units. The expressions and sequences variation of two major antimicrobial peptides, CBD-103 and K9CATH were also studied. The expression of both CBD-103 and K9CATH were noticed in blood, oral and nasal mucosa. A 3-base pair deletion resulting in the loss of Glycine at position 23 of CBD-103 has been associated with black coat colour in certain breeds of dogs. The result of this study found that the coat colour in studied Gaddi dogs was independent of G23 deletion in CBD-103 and it was not associated with black coat colour in these dogs. This study also reported multiple alleles of K9CATH in Gaddi dogs.
Open Access
Microbiological studies on reproductive disorders of canines
(Palampur, 2022-12-03) Lali, Kasturi; Dhar, Prasenjit
For centuries, dogs have been considered to be man’s best friend. Diseases related to reproductive tract are quite common in canine practice. The present study was carried out with the objective to find out the association of different microbes in reproductive disorders of canines and determination of the antimicrobial susceptibility profile of the isolated microbes. Additionally, the bacterial enumeration of the obtained samples and minimum inhibitory concentration of the antimicrobials were also determined. Molecular detection of Brucella spp. was done employing PCR. A total number of 102 samples were collected from different places of Himachal Pradesh. These were accrued from dogs [both males (11) and females (91)] suffering from any reproductive disorders (purulent vaginitis, pyometra, dystocia, abortion, orchitis, etc.) as well as from apparently healthy dogs. The samples included vaginal swabs, uterine discharge, aborted foetus, preputial swabs and washings. The most frequently recorded reproductive disorder was of purulent vaginitis (15/102; 14.71 per cent) followed by pyometra (11/102;10.78 per cent). The age group of 2-5 years were mostly affected (46.07 per cent). A total of 124 isolates were recovered from 102 samples of which 112 (90.32 per cent) were bacterial isolates and 12 (9.68 per cent) were fungal isolates. The most prevalent bacteria obtained was E.coli (21.77 per cent) followed by Staphylococcus spp. (15.32 per cent) and Pseudomonas spp. (7.3%) while the most commonly isolated yeast was Candida spp. (4.84 per cent). The antimicrobial susceptibility profile of the recovered bacterial isolates revealed enrofloxacin (94.49 per cent) to be the most effective drug while fluconazole (100 per cent) was recorded as the most effective antimycotic agent. Bacterial enumeration revealed significant difference in the number of bacteria (cfu/ml) in diseased dogs in comparison to apparently healthy dogs which makes the quantitative analysis reliable. The obtained MIC value of different antimicrobials showed correlation with their resistance pattern in the AMST. 13 out of 102 samples (12.7 per cent) were positive for the genus Brucella (bcsp31) through PCR, however no isolates were obtained through standard procedures of isolation.. In conclusion, the study recorded plethora of microbial species associated with various reproductive disorders in canines along with their antimicrobial patterns.
Open Access
Molecular characterization and plasmid profiling of Pasteurella multocida of animal origin
(palampur, 2022-06-04) Parkhe, Prapti; Verma, Subhash
Pasteurella multocida, is a Gram negative opportunistic bacterial pathogen capable of causing several economically important diseases in animals including zoonosis. There are multiple capsular, LPS and virulence genotypes of this organism which are differently associated with various diseases in different hosts. In this study, a total of 75 isolates from different animal host species were studied for their capsule and lipopolysaccharide (LPS) genotypes, virulence-associated genes, antimicrobial susceptibility pattern and plasmid profile. A capsular type B was predominant genotype and its prevalence was recorded 85.3% in bovine, 40% in ovine, 66.7% in porcine and 20.8% in poultry isolates. The percent prevalence of capsular type A was 79.2 in avian followed by 40 in ovine, 33.3 in porcine and 12.2 in bovine isolates. Both rabbit and deer isolates were detected as capsular type A and only one isolate each from bovine and ovine belonged to capsular type D. The isolates were assigned to seven groups based on both capsule and LPS genotype, namely A:L1 (13/29), A:L3 (2/29), A:L6 (6/29), A:non-typeable (8/29), B:L2 (42/44), B:non-typeable (2/41) and D:L3 (2/2). When combining both the typing systems, L1 (44.8%), L2 (95.4%) and L3 (100%) were the most prevalent LPS genotypes found in capsular type A, B and D, respectively. P. multocida displayed significant association of a given capsule type with a particular LPS genotype. Many P. multocida remained untypeable for their LPS genotypes which suggested presence of genomic diversity in primer binding locations of LPS biosynthesis loci. Of the total 12 virulence genes, three (ompH, sodC, and ptfA) showed high prevalence in all capsular types whereas toxA was not detected in any of the genotypes. A large proportion of virulent genes (75 to 100%) were present in non-typeable strains. A given P. multocida genotype with a specific set of virulence associated genes clustered together suggesting a genetic relatedness despite their place of origin. Majority of isolates were susceptible to most antibiotics, however, resistance to tetracyclines, streptomycin and sulfonamides were displayed by 62%, 26% and 24% of isolates. Most MDR isolates belonged to capsular type B (15/17, 88.2%) and the MAR indices ranged from 0.04 to 0.39. Only a single isolate harboured a plasmid of molecular size around 11.6 kb, suggesting rarity of plasmids in studied isolates. Additional studies would be needed to understand completely the role of P. multocida genotypic combinations in disease outcome in different hosts and for their exploration as vaccine candidates. The detailed sequence analysis would provide more insight about various characteristics of the plasmid.
Open Access
Molecular characterization and plasmid profiling of Pasteurella multocida of animal origin
(palampur, 2022-03-31) Parkhe, Prapti; Verma, Subhash
Pasteurella multocida, is a Gram negative opportunistic bacterial pathogen capable of causing several economically important diseases in animals including zoonosis. There are multiple capsular, LPS and virulence genotypes of this organism which are differently associated with various diseases in different hosts. In this study, a total of 75 isolates from different animal host species were studied for their capsule and lipopolysaccharide (LPS) genotypes, virulence-associated genes, antimicrobial susceptibility pattern and plasmid profile. A capsular type B was predominant genotype and its prevalence was recorded 85.3% in bovine, 40% in ovine, 66.7% in porcine and 20.8% in poultry isolates. The percent prevalence of capsular type A was 79.2 in avian followed by 40 in ovine, 33.3 in porcine and 12.2 in bovine isolates. Both rabbit and deer isolates were detected as capsular type A and only one isolate each from bovine and ovine belonged to capsular type D. The isolates were assigned to seven groups based on both capsule and LPS genotype, namely A:L1 (13/29), A:L3 (2/29), A:L6 (6/29), A:non-typeable (8/29), B:L2 (42/44), B:non-typeable (2/41) and D:L3 (2/2). When combining both the typing systems, L1 (44.8%), L2 (95.4%) and L3 (100%) were the most prevalent LPS genotypes found in capsular type A, B and D, respectively. P. multocida displayed significant association of a given capsule type with a particular LPS genotype. Many P. multocida remained untypeable for their LPS genotypes which suggested presence of genomic diversity in primer binding locations of LPS biosynthesis loci. Of the total 12 virulence genes, three (ompH, sodC, and ptfA) showed high prevalence in all capsular types whereas toxA was not detected in any of the genotypes. A large proportion of virulent genes (75 to 100%) were present in non-typeable strains. A given P. multocida genotype with a specific set of virulence associated genes clustered together suggesting a genetic relatedness despite their place of origin. Majority of isolates were susceptible to most antibiotics, however, resistance to tetracyclines, streptomycin and sulfonamides were displayed by 62%, 26% and 24% of isolates. Most MDR isolates belonged to capsular type B (15/17, 88.2%) and the MAR indices ranged from 0.04 to 0.39. Only a single isolate harboured a plasmid of molecular size around 11.6 kb, suggesting rarity of plasmids in studied isolates. Additional studies would be needed to understand completely the role of P. multocida genotypic combinations in disease outcome in different hosts and for their exploration as vaccine candidates. The detailed sequence analysis would provide more insight about various characteristics of the plasmid
Open Access
DETECTION AND IDENTIFICATION OF EHRLICHIA SPECIES AMONG DOGS, INCONTACT HUMANS AND TICK VECTORS IN HIMACHAL PRADESH
(CSHHPKV Palampur, 2019-09-30) ANGARIA, SHIVANI; Chahota, Rajesh
Vector-borne diseases (VBDs) in animals and humans have gained worldwide importance in the climate change scenario. Ehrlichiosis is one of such rickettsial disease. Ehrlichia is an obligate, intra-cytoplasmic, gram-negative bacteria that cause ehrlichiosis in dogs and other vertebrates, transmitted by tick-vectors and occur mainly in the humid and warm regions. These bacteria multiply within the cytoplasm of White Blood Cells (WBCs) of vertebrate hosts and affect various organs. Canine ehrlichiosis is an important tick-borne disease of dogs worldwide and may lead to zoonosis. Till date, not much work has been done on ehrlichiosis, molecular identification and genetic characterization of native strains/species of Ehrlichia prevalent in Himachal Pradesh, therefore, this study was planned to detect the prevalence of Ehrlichia in dogs, tick vectors and humans in different locations of the state. A total of 215 samples were collected including blood samples from dogs (n = 177), blood samples from in-contact humans (n = 24) and ticks collected from the body of the dogs (n = 24). Samples were collected from 105 locations covering 38 tehsils of 9 districts of Himachal Pradesh. All the samples were tested using Ehrlichia genus and three Ehrlichia species (E. canis, E. ewingii and E. chaffeensis) specific PCR tests. 11 (6.2%) of blood samples from dogs were found positive for ehrlichiosis by examination of buffy-coat smears. By PCR tests, out of 177 blood samples of dogs, 107 (60.4%) were found positive for one or more Ehrlichia species. 61 (34.4%) samples were found harboring multiple Ehrlichia species. All the ticks were identified as Rhiphicephalus sanguineus. From ticks, 4.2 per cent samples were found positive, while no human blood sample was found positive. Phylogenetic positions of all the detected Ehrlichia species/strains were determined which were genetically characterized after nucleotide sequencing of partial 16S rRNA gene. It was found that all E. canis, E. platys species/strains were making clusters with earlier reported species/strains from different parts of India and abroad.
Open Access
DETECTION OF ENTERIC DNA VIRUSES FROM DIARRHEIC DOGS OF HIMACHAL PRADESH
(CSKHPKV. Palampur, 2019-07-20) THAKUR, DEEPIKA; Dhar, Prasenjit
Diarrhea is among the most common clinical conditions seen in dogs leading to high morbidity and mortality. It can have various causes, including infectious agents, like viruses, bacteria and parasites. The aim of the study was to isolate enteric DNA viruses from diarrheic dogs of Himachal Pradesh and characterize them at molecular level. A total of 238 fecal/other samples were collected from diarrheic / apparently healthy dogs along with brief clinical history of the animal. The faecal samples were subjected to DNA extraction and PCR for the detection of enteric DNA viruses viz. CPV, CAV-1 and Dog CV with primers targeting the VP2, pⅦ and replicase gene of CPV, CAV-1 and Dog CV respectively of each virus were used in the study. 109 (45.79 %) samples were found positive for CPV-2b and 4 (1.68%) samples were found positive for CAV-1. No samples were found positive for Dog CV. HA along with HI and DotELISA were standardized for virus detection from clinical samples and infected cell culture fluids using hyper immune sera raised in rabbits against CPV and CAV-1. Dot-ELISA was found to be almost similar to in sensitivity HA-HI for detection of viruses. Growth studies of CPV and CAV- 1 were done on MDCK, A-72 and Vero cells. CPV could be adapted to MDCK and A-72 cells but not to Vero cells while adaptation of CAV-1 was not possible on any cell culture. Physicochemical characterization of CPV was done by checking the effect of temperature, pH, chloroform and formalin on the virus. The VP2 (CPV-2) and pⅦ (CAV-1) gene fragment was sequenced directly which revealed close genetic similarity to Indian and Italian isolates of CPV and CAV, respectively. Microbiological studies on fecal samples revealed 37 bacterial isolates that were resistanct to penicillin and streptomycin in antibiogram and were found concurrently associated with clinical cases of viral diarrhea. In conclusion, diarrhea in dogs in H.P are mostly caused by CPV type 2b strain and occasionally by CAV-1 and is frequently associated with other bacterial species.
Open Access
DETECTION OF ENTERIC DNA VIRUSES FROM DIARRHEIC DOGS OF HIMACHAL PRADESH.
(CSKHPKV, Palampur, 2019-07) Thakur, Deepika; Dhar, Prasenjit
Diarrhea is among the most common clinical conditions seen in dogs leading to high morbidity and mortality. It can have various causes, including infectious agents, like viruses, bacteria and parasites. The aim of the study was to isolate enteric DNA viruses from diarrheic dogs of Himachal Pradesh and characterize them at molecular level. A total of 238 fecal/other samples were collected from diarrheic / apparently healthy dogs along with brief clinical history of the animal. The faecal samples were subjected to DNA extraction and PCR for the detection of enteric DNA viruses viz. CPV, CAV-1 and Dog CV with primers targeting the VP2, pⅦ and replicase gene of CPV, CAV-1 and Dog CV respectively of each virus were used in the study. 109 (45.79 %) samples were found positive for CPV-2b and 4 (1.68%) samples were found positive for CAV-1. No samples were found positive for Dog CV. HA along with HI and Dot- ELISA were standardized for virus detection from clinical samples and infected cell culture fluids using hyper immune sera raised in rabbits against CPV and CAV-1. Dot-ELISA was found to be almost similar to in sensitivity HA-HI for detection of viruses. Growth studies of CPV and CAV- 1 were done on MDCK, A-72 and Vero cells. CPV could be adapted to MDCK and A-72 cells but not to Vero cells while adaptation of CAV-1 was not possible on any cell culture. Physicochemical characterization of CPV was done by checking the effect of temperature, pH, chloroform and formalin on the virus. The VP2 (CPV-2) and pⅦ (CAV-1) gene fragment was sequenced directly which revealed close genetic similarity to Indian and Italian isolates of CPV and CAV, respectively. Microbiological studies on fecal samples revealed 37 bacterial isolates that were resistanct to penicillin and streptomycin in antibiogram and were found concurrently associated with clinical cases of viral diarrhea. In conclusion, diarrhea in dogs in H.P are mostly caused by CPV type 2b strain and occasionally by CAV-1 and is frequently associated with other bacterial species.
Open Access
MICROBIOLOGICAL INVESTIGATIONS ON RESPIRATORY INFECTIONS OF SHEEP AND GOATS WITH PARTICULAR EMPHASIS ON MYCOPLASMA
(CSKHPKV, Palampur, 2019-07) Sunaina; Verma, Subhash
In this study, a total of 163 of 198 morbid samples (nasal and tracheal swabs, lung tissues and pleural fluid) from sheep and goats showing respiratory disease signs were positive for bacteria belonging to seven genera and ten species. Staphylococcus aureus (34.94%) was major bacteria followed by E. coli (21.50%). Other bacteria included Bacillus spp., Pseudomonas aeruginosa, S. epidermidis etc. Nine (6.2%) samples also yielded growth resembling mollicutes which were confirmed by PCR. Out of 37, 83.8% S. aureus carried at least one virulence gene. The mecA was detected in 64.51% S. aureus followed by coa and lukPV in 14 (45.16%) and arcA in 12 (38.50%) isolates. Most S. aureus were sensitive to chloramphenicol, ceftriaxone and tetracycline. Penicillin was at least effective antibiotic. Out of 40, 55% isolates of E. coli carried at least one virulence gene. The eaeA, stx2 and UAL were detected in six, eleven and four E. coli isolates. Most E. coli were susceptible to chloramphenicol and ciprofloxacin. MAR index > 0.2 was recorded for S. aureus (75.6%) and E. coli (10%). Trueperella pyogenes was detected in 10 and Histophilus somni in one nasal swab out 50 nasal swabs of goats. In total, 64 of 198 samples were positive for Mycoplasma spp. using generic-specific primers targeting 16S rRNA gene. Also, 12.5% samples detected positive both for M. mycoides cluster and M. capricolum subsp. capripneumoniae, all of which emanated from an outbreak CCPP having case fatality of 45.07%. The 16S rRNA sequencing identified S. maltophilia, S. entericus and Enterococcus spp. These findings suggest that S. aureus is a major opportunistic pathogen of respiratory tract of sheep and goats harbouring many virulent and AMR genes with high MAR indices and MDR phenotype. This calls for rational usage of antibiotics in veterinary practice. Species-specific PCR should be first choice for diagnosis of CCPP, since MCCP is difficult to culture. Finally, the role of novel bacterial sequences recovered from respiratory tract of goats should be investigated in health and disease.

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