Effects of resveratrol topical ointment on wound healing of full-thickness cutaneous burns in albino rats.

OBJECTIVE: In this study, the effects of resveratrol topical ointment on wound contraction and histopathology of full-thickness cutaneous burn wounds were evaluated. METHOD: Adult albino rats were grouped into four equal-sized groups of 15 rats each, as follows: Group A-no wound, no treatment (control); Group B-1% silver sulphadiazine; Group C-5% resveratrol, and Group D-wound without treatment (control). A burn wound measuring 23.5mm was created on the skin at the dorsum of all rats in groups B-D after shaving. The percentage of wound contraction was measured using a digital Vernier Caliper on days 1, 3, 5, 7, 10, 14, 16, 18 and 21, post-wounding. From each group, five rats were then euthanised and tissue samples of the skin, liver and kidney were collected in 10% buffered formalin for histopathology. RESULTS: The percentage of wound contraction was significant (p<0.05) on 7, 14 and 18 days post treatment. Histopathologically, 5% resveratrol topical ointment application resulted in a thicker epidermis with neovascularisation and an increased collagen distribution. Resveratrol topical ointment ameliorated the extent of hepatocellular and nephrotubular injuries following burn-induced hepatocellular and acute kidney injuries. CONCLUSION: In this study, topical application of 5% resveratrol ointment appeared to enhance burn wound healing by increasing the rate of wound contraction through collagen fibre synthesis, granulation tissue formation and epithelial regeneration.

Changes in selected cytokines, acute-phase proteins, gonadal hormones and reproductive organs of non-pregnant does challenged with Mannheimia haemolytica serotype A2 and its LPS endotoxin.

Previous investigations have revealed that lipopolysaccharide (LPS) endotoxin from certain Gram-negative bacteria could adversely affect the reproductive system of female animals. However, it is unknown whether LPS endotoxin of Mannheimia haemolytica serotype A2, the principal causative bacteria that cause pneumonic mannheimiosis in small ruminants, may also induce similar insidious effects. Therefore, this study aimed to investigate the effects of M. haemolytica serotype A2 and its LPS endotoxin on the responses of female gonadal hormones (progesterone and oestrogen), pro-inflammatory cytokines (interleukin-1ß, interleukin-6), acute-phase proteins (haptoglobin and serum amyloid A) and cellular changes via histopathology study of female reproductive organs of the treatment does. Twelve clinically healthy, non-pregnant, crossbred does were randomly allocated into three equal groups. Group 1 was administered intranasally with 2 ml of sterile phosphate-buffered saline (PBS) and served as a negative control group. Group 2 was challenged intranasally with 2 ml of bacterial inoculum containing 109 colony-forming units (CFU)/ml of M. haemolytica serotype A2, while Group 3 was challenged intravenously with 2 ml of LPS endotoxin extracted from 109 CFU/ml of M. haemolytica serotype A2. Following that, blood samples were collected serially at pre-determined intervals for serological analyses. All does were euthanised 60 days post-challenges, and tissue samples from the ovaries, oviducts, uterine horns, uterine body, cervix and vagina were collected for histopathological study. The serological result revealed a significant increase (p < 0.05) in the mean concentrations of progesterone, oestrogen, interleukin-1ß, interleukin-6, haptoglobin and serum amyloid A for both challenged groups. Histopathologically, all reproductive organs (except the cervix and vagina) from both challenged groups displayed significant cellular alterations (p < 0.05) characterised by haemorrhage and congestion, necrosis and degeneration, inflammatory cell infiltration and oedema. This study provides new information that elucidates the potential role of pneumonic mannheimiosis in the pathogenesis of female infertility amongst small ruminants.

Further Insights Into Caprine Arthritis Encephalitis (CAE): The Current Status of Seroprevalence Among Small Ruminants in Two Selected States of Peninsular Malaysia.

Caprine arthritis-encephalitis virus (CAEV) is a member of the genus lentivirus causing caprine arthritis-encephalitis (CAE), a chronic inflammatory condition affecting the lungs, joints, udder and central nervous system of small ruminants such as sheep and goats. CAE is distributed worldwide and is recognised as a significant cause of morbidity and decreased milk production in dairy goats. Earlier studies highlighted the clinicopathological features and supplied preliminary serological evidence for the existence of CAE among selected goat herds in Malaysia. Therefore, this study aims to provide further insights into the seroprevalence and contributing factors of CAE among sheep and goat herds in two states of Peninsular Malaysia. The blood samples and biodata were randomly collected from a total of 262 individual sheep (40) and goat (222) in seven smallholder farms. Blood sera were tested for specific anti-CAEV antibodies using Qayee-Bio CAEV sandwich-ELISA test kits according to standard procedures. Our results of the study revealed 21.4% (95% CI: 15.8-28.6) apparent and 20.6% (95% CI: 14.5-27.8) true seroprevalence with significant differences (p < 0.05) in seroconversion rates between the states, farms, production systems and breeds of small ruminants. The prevalence of CAE in the Malaysian Peninsular is a potential threat to the small ruminant industry and developing agricultural economy. Further studies are required to determine the genetic characteristics, distribution and risk factors of CAEV for effective prevention and control in Malaysia.

Virus Kaprin Artritis Ensefalitis (CAEV) merupakan ahli kumpulan dalam genus virus lentivirus dimana akan menyebabkan penyakit kaprin artritis ensefalitis (CAE) di mana penyakit ini akan menyebabkan keradangan kronik pada paru-paru, sendi, kelenjar mamari dan sistem saraf pusat bagi haiwan ruminan kecil seperti bebiri dan kambing. CAE telah merebak ke seluruh dunia dan penyakit ini akan menyebabkan penularan wabak pada kadar morbiditi yang tinggi dan mengurangkan kuantiti penghasilan susu bagi kambing tenusu. Kajian terdahulu memberi penekanan kepada dapatan klinikal patologi dan data bukti serologi kewujudan penyakit CAE dalam kalangan gerompok kambing di Malaysia. Maka, kajian kini bertujuan memberikan pendedahan awal berkaitan kadar kelaziman serologi dan faktor yang menyumbang penyakit CAE dalam kalangan bebiri dan kambing bagi dua negeri di Semanjung Malaysia. Sampel darah dan data biologi telah dikumpulkan secara rawak dengan jumlah sampel 262 ekor (40 ekor bebiri dan 222 ekor kambing) daripada tujuh buah ladang peternak kecil. Serum yang telah dikumpul diuji dengan antibodi spesifik anti-CAEV dengan menggunakan prosedur piawai kit elisa daripada Qayee-Bio CAEV. Keputusan kajian ini menunjukkan kadar kelaziman serologi 21.4% (95% CI: 15.8­28.6) jelas dan 20.6% (95% CI: 14.5­27.8) kadar kelaziman benar dengan perbezaan ketara (p < 0.05) dalam kadar perubahan kelaziman serologi antara negeri, ladang, sistem produksi dan baka haiwan ruminan kecil. Dengan wujudnya kelaziman penyakit CAE di Semenanjung Malaysia ini akan menyumbang kepada kemungkinan ancaman negatif terhadap industri ruminan kecil dan sektor ekonomi dalam bidang penternakan. Lebih banyak kajian diperlukan untuk menentukan ciri genetik virus penyebab penyakit ini, taburan penyakit dan faktor penyumbang bagi CAEV supaya dapat mengadakan kawalan dan pencegahan efektif bagi penyakit ini di Malaysia.

Development and immunogenic potentials of chitosan-saponin encapsulated DNA vaccine against avian infectious bronchitis coronavirus.

Infectious Bronchitis (IB) is an economically important avian disease that considerably threatens the global poultry industry. This is partly, as a result of its negative consequences on egg production, weight gain as well as mortality rate.The disease is caused by a constantly evolving avian infectious bronchitis virus whose isolates are classified into several serotypes and genotypes that demonstrate little or no cross protection. In order to curb the menace of the disease therefore, broad based vaccines are urgently needed. The aim of this study was to develop a recombinant DNA vaccine candidate for improved protection of avian infectious bronchitis in poultry. Using bioinformatics and molecular cloning procedures, sets of monovalent and bivalent DNA vaccine constructs were developed based on the S1 glycoprotein from classical and variants IBV strains namely, M41 and CR88 respectively. The candidate vaccine was then encapsulated with a chitosan and saponin formulated nanoparticle for enhanced immunogenicity and protective capacity. RT-PCR assay and IFAT were used to confirm the transcriptional and translational expression of the encoded proteins respectively, while ELISA and Flow-cytometry were used to evaluate the immunogenicity of the candidate vaccine following immunization of various SPF chicken groups (A-F). Furthermore, histopathological changes and virus shedding were determined by quantitative realtime PCR assay and lesion scoring procedure respectively following challenge of various subgroups with respective wild-type IBV viruses. Results obtained from this study showed that, groups vaccinated with a bivalent DNA vaccine construct (pBudCR88-S1/M41-S1) had a significant increase in anti-IBV antibodies, CD3+ and CD8+ T-cells responses as compared to non-vaccinated groups. Likewise, the bivalent vaccine candidate significantly decreased the oropharyngeal and cloacal virus shedding (p < 0.05) compared to non-vaccinated control. Chickens immunized with the bivalent vaccine also exhibited milder clinical signs as well as low tracheal and kidney lesion scores following virus challenge when compared to control groups. Collectively, the present study demonstrated that bivalent DNA vaccine co-expressing dual S1 glycoprotein induced strong immune responses capable of protecting chickens against infection with both M41 and CR88 IBV strains. Moreso, it was evident that encapsulation of the vaccine with chitosan-saponin nanoparticle further enhanced immune responses and abrogates the need for multiple booster administration of vaccine. Therefore, the bivalent DNA vaccine could serve as efficient and effective alternative strategy for the control of IB in poultry.

Responses of female reproductive hormones and histopathology in the reproductive organs and associated lymph nodes of Boer does challenged with Corynebacterium pseudotuberculosis and its immunogenic corynomycolic acid extract.

BACKGROUND: Corynebacterium pseudotuberculosis biotype ovis is a bacterium that causes caseous lymphadenitis (CLA), a chronic disease of sheep and goats characterized by the formation of suppurative abscesses in superficial and visceral lymph nodes and internal organs of small ruminants. This study was designed to evaluate the reproductive hormonal changes (estrogen and progesterone) and histopathology in the reproductive organs and associated lymph nodes of does challenged with C. pseudotuberculosis biotype ovis and its immunogen; corynomycolic acid. A total of 12 healthy non-pregnant female goats were grouped into three: A, B and C consisting of four does each. Group A was intradermally inoculated with 2 mL of sterile phosphate buffered saline (PBS) pH 7 (negative control group); group B was intradermally inoculated with 2 mL of corynomycolic acid extract (CMAs), while group C was intradermally inoculated with 2 mL of 109 colony-forming unit (cfu) of live C. pseudotuberculosis. Blood samples were also collected at predetermined intervals for estrogen and progesterone hormonal assays. The does were euthanized 90 days post challenge and tissue samples of the uterus, ovaries, fallopian tubes, cervix and associated lymph nodes were collected and fixed in 10% neutral buffered formalin for histopathological processing. The result showed various degrees of histopathological changes (hemorrhage, congestion, degeneration, necrosis, edema, leucocytic infiltrations) in the reproductive organs and associated lymph nodes of both inoculation groups. Increases in estrogen hormone concentration were observed in both inoculation groups in comparison to the control group. However, progesterone concentration was only increased in group C. This study highlighted that corynomycolic acid extract from C. pseudotuberculosis biotype ovis resulted in significant histopathology in the reproductive organs and associated lymph nodes of does and increase estrogen concentration.

Responses of pro-inflammatory cytokines, acute phase proteins and cytological analysis in serum and cerebrospinal fluid during haemorrhagic septicaemia infection in buffaloes.

Sudden death is usually the main finding in field animals during haemorrhagic septicaemia outbreaks caused by Pasteurella multocida type B:2 that causes acute, fatal and septicaemic disease in cattle and buffaloes. This situation may be due to failure in early detection of the disease where early treatment of antibiotics may improve the prognosis of the animal and other surviving animals. Thus, there is a grey area on the knowledge on the potential usage of pro-inflammatory cytokines and acute phase proteins as early biomarkers in the diagnosis of haemorrhagic septicaemia. In addition, exploration of the cerebrospinal fluid during infection has never been studied before. Therefore, this study was designed to fill up the grey areas in haemorrhagic septicaemia research. Twenty-one buffalo calves were divided into seven treatment groups where group 1 was inoculated orally with 10 mL of sterile phosphate-buffered saline pH 7 which act as a negative control group. Groups 2 and 3 were inoculated orally and subcutaneously with 10 mL of 1012 colony-forming unit of P. multocida type B:2. Group 4 and 5 buffaloes were inoculated orally and intravenously with 10 mL of lipopolysaccharide broth. Groups 6 and 7 were administered orally and subcutaneously with 10 mL of outer membrane protein broth. During the post-infection period of 21 days, blood and cerebrospinal fluid were sampled for the analyses of pro-inflammatory cytokines, acute phase proteins and cytological examination. Buffalo calves infected with P. multocida and its immunogens via different routes of inoculation showed significant changes (p < 0.05) of pro-inflammatory cytokines, acute phase proteins and cytological changes in both the serum and cerebrospinal fluid. Buffalo calves from groups 3 and 7 showed the highest pro-inflammatory cytokines, whereas group 6 had the highest acute phase protein concentration and group 5 revealed the highest value for cytology changes. In summary, results obtained in this study could be used as a profiling study to add novel knowledge to the haemorrhagic septicaemia research as well as the development of biomarkers.

The distribution of important sero-complexes of flaviviruses in Malaysia.

Flaviviruses (FVs) are arthropod-borne viruses of medical and veterinary importance. Numerous species of FVs have been isolated from various host; mainly humans, animals, ticks, and mosquitoes. Certain FVs are extremely host-specific; at the same time, some FVs can infect an extensive range of species. Based on published literatures, 11 species of FVs have been detected from diverse host species in Malaysia. In humans, dengue virus and Japanese encephalitis virus have been reported since 1901 and 1942. In animals, the Batu Cave virus, Sitiawan virus, Carey Island, Tembusu virus, Duck Tembusu virus, and Japanese encephalitis viruses were isolated from various species. In mosquitoes, Japanese encephalitis virus and Kunjin virus were isolated from Culex spp., while Zika virus and Jugra virus were isolated from Aedes spp. In ticks, the Langat virus was isolated from Ixodes spp. One of the major challenges in the diagnosis of FVs is the presence of sero-complexes as a result of cross-reactivity with one or more FV species. Subsequently, the distribution of specific FVs among humans and animals in a specific population is problematic to assess and often require comprehensive and thorough analyses. Molecular assays such as quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and digital droplet RT-PCR (ddRT-PCR) have been used for the differentiation of flavivirus infections to increase the accuracy of epidemiological data for disease surveillance, monitoring, and control. In situations where sero-complexes are common in FVs, even sensitive assays such as qRT-pCR can produce false positive results. In this write up, an overview of the various FV sero-complexes reported in Malaysia to date and the challenges faced in diagnosis of FV infections are presented.

Establishment of lung auscultation scoring method and responses of acute phase proteins and heat shock proteins in vaccinated and non-vaccinated goats.

Pneumonic pasteurellosis is an economically important infectious disease in the small ruminant industry which causes sudden death and loss for farmers. Nonetheless, this disease is still a common sight in sheep and goats in Malaysia, probably due to the unpopular usage of pasteurellosis vaccine or inappropriate vaccination practices. The aim of this study was designed to classify the severity of pneumonia via the establishment of auscultation scoring method and to quantify the acute phase proteins and heat shock proteins responses from vaccinated and non-vaccinated goats. Goat farms, consist of vaccinated and non-vaccinated farms, were selected in this study: where 15 clinically normal healthy goats and 9 pneumonic goats were selected from vaccinated farms whereas 15 clinically normal healthy goats and 31 pneumonic goats from non-vaccinated farms were selected for this study. Crackle lung sounds were not detected in both vaccinated and non-vaccinated normal goats. However, vaccinated pneumonic goats showed mild crackle lung sound while non-vaccinated pneumonic goats exhibited moderate crackle lung sound. There were significant increases (p < 0.05) in acute phase proteins and heat shock proteins concentrations for the non-vaccinated pneumonic goats group. In this study, conclusion can be made that the vaccinated goats exhibited very mild clinical responses of pneumonia and non-significant biomarker responses compared to the non-vaccinated goats. Thus, vaccination is an effective preventive measure to control pneumonic pasteurellosis and acute phase proteins and heat shock proteins can be considered as future biomarkers in screening and rapid diagnostic method for this particular disease.

In vivo efficacy of tobramycin-loaded synthetic calcium phosphate beads in a rabbit model of staphylococcal osteomyelitis.

BACKGROUND: Osteomyelitis is an acute or chronic inflammatory process of the bone following infection with pyogenic organisms like Staphylococcus aureus. Tobramycin (TOB) is a promising aminoglycoside antibiotic used to treat various bacterial infections, including S. aureus. The aim of this study was to investigate the efficacy of tobramycin-loaded calcium phosphate beads (CPB) in a rabbit osteomyelitis model. METHODS: Tobramycin (30 mg/mL) was incorporated into CPB by dipping method and the efficacy of TOB-loaded CPB was studied in a rabbit osteomyelitis model. For juxtaposition, CPB with and without TOB were prepared. Twenty-five New Zealand white rabbits were grouped (n = 5) as sham (group 1), TOB-loaded CPB without S. aureus (group 2), S. aureus only (group 3), S. aureus + CPB (group 4), and S. aureus + TOB-loaded CPB (group 5). Groups infected with S. aureus followed by CPB implantation were immediately subjected to surgery at the mid-shaft of the tibia. After 28 days post-surgery, all rabbits were euthanized and the presence or absence of chronic osteomyelitis and the extent of architectural destruction of the bone were assessed by radiology, bacteriology and histological studies. RESULTS: Tobramycin-loaded CPB group potentially inhibited the growth of S. aureus causing 3.2 to 3.4 log10 reductions in CFU/g of bone tissue compared to the controls. Untreated groups infected with S. aureus showed signs of chronic osteomyelitis with abundant bacterial growth and alterations in bone architecture. The sham group and TOB-loaded CPB group showed no evidence of bacterial growth. CONCLUSIONS: TOB-incorporated into CPB for local bone administration was proven to be more successful in increasing the efficacy of TOB in this rabbit osteomyelitis model and hence could represent a good alternative to other formulations used in the treatment of osteomyelitis.

Dermatopathology of Orf Virus (Malaysian Isolates) in Mice Experimentally Inoculated at Different Sites with and without Dexamethasone Administration.

Orf is a clinical manifestation of parapoxvirus infection often fatal in goats and sheep especially when they are under stress or influenced by unfavorable environment. This study investigated the pathogenicity of two Orf virus isolates (ORFV UPM1/14 and UPM2/14) and host response in mouse model by using different inoculation sites with/without prior exposure to dexamethasone. Treatments with dexamethasone served as an immunosuppressant that may mimic stress situation in affected animals. Groups of five mice were given intradermal injection of 0.2 mL of tissue culture infective dose 50 (TCID50) of UPM1/14 (Group 1) and UPM2/14 (Group 2) at the dorsum (Group 1A; Group 2A), ear pinna (Group 1B; Group 2B), and labial commissure (Group 1C; Group 2C). An inoculum 0.2 mL of UPM1/14 was administered to animals treated with dexamethasone (n=5; 5 mg/kg/day intraperitoneally) and nondexamethasone (n=5) groups at the dorsum, ear pinna, and labial commissure. No significant difference (p>0.05) was observed in the mean lesion scores among the groups of different inoculation sites or between dexamethasone-treated and nontreated groups. However, there was a significant difference (p<0.05) in the mean stratum thickness of affected skin following inoculation with UPM2/14 isolate at the ear pinna and labial commissure. Histopathology examination revealed keratosis, acanthosis, and ballooning degeneration in the skin of affected mice. Orf virus DNA was detected in the skin samples by targeting F1L and B2L virus-specific genes in polymerase chain reaction (PCR) assay. Intradermal inoculation with UPM1/14 or UPM2/14 isolate produced a mild skin lesion in mice, and there was no significant difference in orf disease manifestation despite variation of inoculation sites. Similarly, short-term dexamethasone administration gave no adverse effects on pathogenicity of orf virus isolates.

Japanese encephalitis in Malaysia: An overview and timeline.

Japanese encephalitis (JE) is a vector-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). It causes encephalitis in human and horses, and may lead to reproductive failure in sows. The first human encephalitis case in Malaya (now Malaysia) was reported during World War II in a British prison in 1942. Later, encephalitis was observed among race horses in Singapore. In 1951, the first JEV was isolated from the brain of an encephalitis patient. The true storyline of JE exposure among humans and animals has not been documented in Malaysia. In some places such as Sarawak, JEV has been isolated from mosquitoes before an outbreak in 1992. JE is an epidemic in Malaysia except Sarawak. There are four major outbreaks reported in Pulau Langkawi (1974), Penang (1988), Perak and Negeri Sembilan (1998-1999), and Sarawak (1992). JE is considered endemic only in Sarawak. Initially, both adults and children were victims of JE in Malaysia, however, according to the current reports; JE infection is only lethal to children in Malaysia. This paper describes a timeline of JE cases (background of each case) from first detection to current status, vaccination programs against JE, diagnostic methods used in hospitals and factors which may contribute to the transmission of JE among humans and animals in Malaysia.

Seroprevalence of small ruminant caprine arthritis encephalitis lentivirus among goats from selected small ruminant farms in Selangor, Malaysia.

BACKGROUND AND AIM: Caprine arthritis encephalitis (CAE) is an important viral disease of small ruminants particularly in dairy goats with severe social and economic implication. Hence, this study was designed to determine the seroprevalence of CAE virus (CAEV) among goat population in selected small ruminant farms in Selangor and the risk factors associated with the occurrence of the disease. MATERIALS AND METHODS: Blood samples were collected from a total of 91 goats selected at random. Blood serum was harvested and used for competitive enzyme-linked immunosorbent assay test to detect antibodies against CAE virus. RESULTS: The result obtained showed that 8/91 (8.8%) of the goats were seropositive for CAEV. In addition, biosecurity management, source of origin and sex of the animal were observed to be important risk factors associated with the occurrence of CAE in goats. CONCLUSION: The findings of this study affirmed that the seroprevalence of CAEV infection among goat population in small ruminant farms in Selangor, Malaysia, is low. However, there is need to institute strict control measures such as testing and culling positive animals or separation of infected animals from those that tested negative to the disease for effective eradication of the disease.

Prevalence and risk factors of Japanese encephalitis virus (JEV) in livestock and companion animal in high-risk areas in Malaysia.

Japanese encephalitis (JE) is vector-borne zoonotic disease which causes encephalitis in humans and horses. Clinical signs for Japanese encephalitis virus (JEV) infection are not clearly evident in the majority of affected animals. In Malaysia, information on the prevalence of JEV infection has not been established. Thus, a cross-sectional study was conducted during two periods, December 2015 to January 2016 and March to August in 2016, to determine the prevalence and risk factors in JEV infections among animals and birds in Peninsular Malaysia. Serum samples were harvested from the 416 samples which were collected from the dogs, cats, water birds, village chicken, jungle fowls, long-tailed macaques, domestic pigs, and cattle in the states of Selangor, Perak, Perlis, Kelantan, and Pahang. The serum samples were screened for JEV antibodies by commercial IgG ELISA kits. A questionnaire was also distributed to obtain information on the animals, birds, and the environmental factors of sampling areas. The results showed that dogs had the highest seropositive rate of 80% (95% CI: ± 11.69) followed by pigs at 44.4% (95% CI: ± 1.715), cattle at 32.2% (95% CI: ± 1.058), birds at 28.9% (95% CI: ± 5.757), cats at 15.6% (95% CI: ± 7.38), and monkeys at 14.3% (95% CI: ± 1.882). The study also showed that JEV seropositivity was high in young animals and in areas where mosquito vectors and migrating birds were prevalent.

Evaluation of in vitro efficacy of docetaxel-loaded calcium carbonate aragonite nanoparticles (DTX-CaCO3NP) on 4T1 mouse breast cancer cell line.

Cockle shell-derived calcium carbonate nanoparticles have shown promising potentials as slow drug-releasing compounds in cancer chemotherapy. In this study, we evaluated the in vitro efficacy of docetaxel (DTX)-loaded CaCO3NP on 4T1 cell line. This was achieved by evaluating the following: cytotoxicity using MTT assay, fluorescence imaging, apoptosis with Annexin V assay, cell cycle analysis, scanning (SEM) and transmission electron microscopy (TEM), and scratch assay. Based on the results, DTX-CaCO3NP with a DTX concentration of 0.5 µg/mL and above had comparable cytotoxic effects with free DTX at 24 h, while all concentrations had similar cytotoxic effect on 4T1 cells at 48 and 72 h. Fluorescence and apoptosis assay showed a higher (p < 0.05) number of apoptotic cells in both free DTX and DTX-CaCO3NP groups. Cell cycle analysis showed cycle arrest at subG0 and G2/M phases in both treatment groups. SEM showed presence of cellular blebbing, while TEM showed nuclear fragmentation, apoptosis, and vacuolation in the treatment groups. Scratch assay showed lower (p < 0.05) closure in both free DTX and DTX-CaCO3NP groups. The results from this study showed that DTX-CaCO3NP has similar anticancer effects on 4T1 cells as free DTX, and since it has a slow release rate, it is a more preferred substitute for free DTX.

Histological analysis of anti-cancer drug loaded, targeted Mn:ZnS quantum dots in metastatic lesions of 4T1 challenged mice.

5-Fluororaucil (5-FU) as anti-cancer drug was reported to induce thymidine synthase (TS) overexpression and cancer cell resistance. To improve its therapeutic efficacy and selective targeting, here we developed a targeted delivery system mediated by the active ligand-folate receptor chemistry to deliver the 5-FU drug selectively into the tumor microenvironment. The preparation was achieved by exploring chitosan (CS)-biopolymer based system with folic acid (FA)-conjugation. The 5-FU@FACS-Mn:ZnS quantum dots (QDs) based on the histological assessment conducted in the 4T1 challenged mice showed an improved tumor remission in the liver, spleen and lungs. The 5-FU@FACS-Mn:ZnS composite induced anti-proliferative properties in these organs as compared to the free 5-FU drug. Unlike the 5-FU@FACS-Mn:ZnS treated groups which showed some specific morphological changes such as cell shrinkage without obvious presence of adipocytes, the excised section of the tumor in the untreated control group and the free 5-FU drug treated group showed necrotic and degenerated cells; these cells are multifocally distributed in the tumor mass with evidence of widely distributed adipocytes within the tumor mass. These findings suggest that the 5-FU@FACS-Mn:ZnS composite has a superior role during the induction of apoptosis in the 4T1 cells as compared to the free 5-FU drug treated groups. The results of the study therefore suggest that the impregnation of 5-FU anti-cancer drug within the FACS-Mn:ZnS system significantly improves its selective targeting efficacy, in addition to improving the anti-proliferative properties and attenuate possible tumor resistances to the 5-FU drug. The work discusses about the anti-metastatic effects of folic acid-bound 5-Fluororacil loaded Mn:ZnS quantum dots towards 4T1 cell line proliferation in mice based on the histological analysis.

Identification and comparison of RCMV ALL 03 open reading frame (ORF) among several different strains of cytomegalovirus worldwide.

BACKGROUND: Rat cytomegalovirus ALL-03 (Malaysian strain) which was isolated from a placenta and uterus of a house rat, Rattus rattus diardii has the ability to cross the placenta and infecting the fetus. To further elucidate the pathogenesis of the Malaysian strain of Rat Cytomegalovirus ALL-03 (RCMV ALL-03), detailed analysis on the viral genome sequence is crucial. METHODS: Genome sequencing of RCMV ALL-03 was carried out in order to identify the open reading frame (ORF), homology comparison of ORF with other strains of CMV, phylogenetic analysis, classifying ORF with its corresponding conserved genes, and determination of functional proteins and grouping of gene families in order to obtain fundamental knowledge of the genome. RESULTS: The present study revealed a total of 123 Coding DNA sequences (CDS) from RCMV ALL-03 with 37 conserved ORF domains as with all herpesvirus genomes. All the CDS possess similar function with RCMV-England followed by RCMV-Berlin, RCMV-Maastricht, and Human CMV. The phylogenetic analysis of RCMV ALL-03 based on conserving genes of herpes virus showed that the Malaysian RCMV isolate is closest to RCMV-English and RCMV-Berlin strains, with 99% and 97% homology, respectively. Similarly, it also demonstrated an evolutionary relationship between RCMV ALL-03 and other strains of herpesviruses from all the three subfamilies. Interestingly, betaherpesvirus subfamily, which has been shown to be more closely related with gammaherpesviruses as compared to alphaherpesviruses, shares some of the functional ORFs. In addition, the arrangement of gene blocks for RCMV ALL-03, which was conserved among herpesvirus family members was also observed in the RCMV ALL-03 genome. CONCLUSION: Genomic analysis of RCMV ALL-03 provided an overall picture of the whole genome organization and it served as a good platform for further understanding on the divergence in the family of Herpesviridae.

Safety assessments of subcutaneous doses of aragonite calcium carbonate nanocrystals in rats.

Calcium carbonate nanoparticles have shown promising potentials in the delivery of drugs and metabolites. There is however, a paucity of information on the safety of their intentional or accidental over exposures to biological systems and general health safety. To this end, this study aims at documenting information on the safety of subcutaneous doses of biogenic nanocrystals of aragonite polymorph of calcium carbonate derived from cockle shells (ANC) in Sprague-Dawley (SD) rats. ANC was synthesized using the top-down method, characterized using the transmission electron microscopy and field emission scanning electron microscope and its acute and repeated dose 28-day trial toxicities were evaluated in SD rats. The results showed that the homogenous 30 ± 5 nm-sized spherical pure aragonite nanocrystals were not associated with mortality in the rats. Severe clinical signs and gross and histopathological lesions, indicating organ toxicities, were recorded in the acute toxicity (29,500 mg/m2) group and the high dose (5900 mg/m2) group of the repeated dose 28-day trial. However, the medium- (590 mg/m2 body weight) and low (59 mg/m2)-dose groups showed moderate to mild lesions. The relatively mild lesions observed in the low toxicity dosage group marked the safety margin of ANC in SD rats. It was concluded from this study that the toxicity of CaCO3 was dependent on the particulate size (30 ± 5 nm) and concentration and the route of administration used.

Isolation and identification of bacterial populations of zoonotic importance from captive non-venomous snakes in Malaysia.

AIM: Captivity of non-venomous snakes such as python and boa are common in zoos, aquariums and as pets in households. Poor captivity conditions expose these reptiles to numerous pathogens which may result in disease conditions. The purpose of this study was to investigate the common bacteria isolated from necropsied captive snakes in Malaysia over a five year period. MATERIALS AND METHODS: A total of 27 snake carcasses presented for necropsy at the Universiti Putra Malaysia (UPM) were used in this survey. Samples were aseptically obtained at necropsy from different organs/tissues (lung, liver, heart, kindey, oesophagus, lymph node, stomach, spinal cord, spleen, intestine) and cultured onto 5% blood and McConkey agar, respectively. Gram staining, morphological evaluation and biochemical test such as oxidase, catalase and coagulase were used to tentatively identify the presumptive bacterial isolates. RESULTS: Pythons had the highest number of cases (81.3%) followed by anaconda (14.8%) and boa (3.7%). Mixed infection accounted for 81.5% in all snakes and was highest in pythons (63%). However, single infection was only observed in pythons (18.5%). A total of 82.7%, 95.4% and 100% of the bacterial isolates from python, anaconda and boa, respectively were gram negative. Aeromonas spp was the most frequently isolated bacteria in pythons and anaconda with incidences of 25 (18%) and 8 (36.6%) with no difference (p > 0.05) in incidence, respectively, while Salmonella spp was the most frequently isolated in boa and significantly higher (p < 0.05) than in python and anaconda. Bacteria species were most frequently isolated from the kidney of pythons 35 (25.2%), intestines of anacondas 11 (50%) and stomach of boa 3 (30%). CONCLUSION: This study showed that captive pythons harbored more bacterial species than anaconda or boa. Most of the bacterial species isolated from these snakes have public health importance and have been incriminated in human infections worldwide.

Formulation of a Sustained Release Docetaxel Loaded Cockle Shell-Derived Calcium Carbonate Nanoparticles against Breast Cancer.

PURPOSE: Here, we explored the formulation of a calcium carbonate nanoparticle delivery system aimed at enhancing docetaxel (DTX) release in breast cancer. METHODS: The designed nano- anticancer formulation was characterized thorough X-ray diffraction (XRD), Fourier transformed infrared (FTIR), transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM) and Brunauer-Emmett-Teller (BET) methods. The nano- anticancer formulation (DTX- CaCO3NP) was evaluated for drug delivery properties thorough in vitro release study in human body simulated solution at pH 7.4 and intracellular lysosomal pH 4.8. RESULTS: Characterization revealed the successful synthesis of DTX- CaCO3NP, which had a sustained release at pH 7.4. TEM showed uniformly distributed pleomorphic shaped pure aragonite particles. The highest entrapment efficiency (96%) and loading content (11.5%) were obtained at docetaxel to nanoparticles ratio of 1:4. The XRD patterns revealed strong crystallizations in all the nanoparticles formulation, while FTIR showed chemical interactions between the drug and nanoparticles with negligible positional shift in the peaks before and after DTX loading. BET analysis showed similar isotherms before and after DTX loading. The designed DTX- CaCO3NP had lower (p < 0.05) cytotoxity against MCF-7 cells than DTX at 24 h but comparable (p > 0.05) effects at 48 h and 72 h. However, the DTX- CaCO3NP released less than 80% of bond DTX at 48 and 72 h but showed comparable effects with free DTX. CONCLUSIONS: The results showed that the developed DTX- CaCO3NP released DTX slower at pH 7.4 and had comparable cytotoxicity with free DTX at 48 and 72 h in MCF-7 cells.

Reproductive hormonal variations and adenohypophyseal lesions in pre-pubertal buffalo heifers inoculated with Pasteurella multocida type B: 2 and its immunogens.

BACKGROUND: Hemorrhagic septicemia is a fatal disease of cattle and buffaloes caused by P. multocida. Although the pathogenesis of the bacteria has been well established in literature, there is a paucity of information on the possible role of the bacteria and its immunogens; lipopolysaccharide (LPS) and outer membrane proteins (OMPs) on the reproductive capacity of buffalo heifers. METHODS: In this study, twenty one healthy prepubertal female buffaloes aged 8 months were divided into seven groups of 3 buffaloes each (G1-G7). Group 1 (G1) served as the negative control group and were inoculated orally with 10 mL sterile Phosphate Buffer Saline (PBS), groups 2 (G2) and 3 (G3) were inoculated orally and subcutaneously with 10 mL of 1012 colony forming unit (cfu) of P.multocida type B: 2, while groups 4 (G4) and 5 (G5) received 10 mL of bacterial LPS orally and intravenously, respectively. Lastly, groups 6 (G6) and 7 (G7) were orally and subcutaneously inoculated with 10 mL of bacterial OMPs. Whole blood was collected in EDTA vials at stipulated time points (0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, 120, 168, 216, 264, 312, 360, 408, 456 and 504 h), while tissue sections of the pituitary glands were collected and transported to the histopathology laboratory in 10% buffered formalin for processing and Hematoxylin and eosin staining. Plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), progesterone (PG), estradiol (EST) and gonadotrophin releasing hormone (GnRH) were determined. RESULTS: The histopathological lesions observed in the pituitary gland included hemorrhage, congestion, inflammatory cell infiltration, hydropic degeneration, necrosis and edema. These changes were higher (p < 0.05) in distribution and severity in G3, G6 and G7. Hormonal concentrations of LH, FSH, PG, EST and GnRH declined in all inoculation groups as time elapsed and were lower (p < 0.05) than that of the control group. CONCLUSION: Based on these findings, P.multocida B: 2 and its immunogens can be said to negatively affect the hypothalamic-pituitary-gonadal axis, resulting in decreased levels of reproductive hormones which may predispose to infertility in buffalo heifers.