Multi-Drug Resistant Pathogenic Escherichia coli Isolated from Wild Birds, Chicken, and the Environment in Malaysia.

Transmission of pathogenic microorganisms in the last decades has been considered a significant health hazard and pathogenic E. coli, particularly antibiotic-resistant strains, have long been identified as a zoonotic problem. This study aimed to investigate multidrug resistant pathogenic E. coli isolates from wild birds, chickens, and environment in selected Orang Asli and Malay villages in Peninsular Malaysia. The bacteriological culture-based technique, disc diffusion method, and multiplex Polymerase Chain Reaction (mPCR) assay was used to determine the occurrence of pathogenic E. coli strains in the several samples in the study. E. coli isolates showed a variety of multi-drug resistant (MDR) antibiotypes and Enteropathogenic E. coli (EPEC) and Enteroinvasive E. coli (EIEC) were the most predominantly identified pathogenic E. coli strains. The findings of this study demonstrated the significance of animal reservoirs and the environment as sources of pathogenic E. coli, resistant bacteria, and resistance genes. Hence, there is a need for adoption of a practical surveillance approach on MDR pathogens to control foodborne contamination.

Molecular Detection, Risk Factors and Public Awareness of Avian Bornavirus among Captive and Non-captive Birds in Peninsular Malaysia.

Introduction: Proventricular dilatation disease (PDD) is caused by avian bornavirus (ABV) has been identified in psittacine, non-psittacine birds and waterfowl. Birds may show signs of gastrointestinal tract deficit or neurological dysfunction or even both. The objectives of this study were to determine the molecular prevalence, risk factors and public awareness of ABV and PDD among captive and non-captive birds in Peninsular Malaysia. Material and Methods: A total of 344 cloacal swabs or faeces were collected and subjected to detection using the RT-PCR assay. Meanwhile, KAP questionnaires were distributed by using the Google forms platform. Results: Molecular prevalence studies revealed that 4.5% (9/201) of the pet birds were ABV-positive, whereas 0% (0/143) in waterfowl. Nine positive pet birds were identified to be PaBV-2, which is closest to ABV isolates EU781967 (USA). Among the risk factors analysed, category, age and, location, were found to show an association with the ABV positivity. The KAP survey result showed: the respondents have low knowledge (32.9%), however, they showed positive attitude (60.8%) and good practice (94.9%). The association between knowledge, attitude and practice showed that there was a significant association between knowledge-attitude and also attitude-practice (P<0.05). Conclusion: This study proved that avian bornavirus (ABV) causes proventricular dilatation disease (PDD) among a group of pet birds of Psittaciformes, but it is present in Peninsular Malaysia with a low prevalence rate. Furthermore, in addition to the useful databases obtained from this study, the level of public awareness on the importance of avian bornavirus that causes fatal disorders among a wide range of bird species is satisfactorily raised.

Isolation of Mycobacterium avium and other nontuberculous mycobacteria in chickens and captive birds in peninsular Malaysia.

BACKGROUND: Mycobacterium avium complex (MAC) causes a chronic infectious in the birds known as avian mycobacteriosis. Almost all species of the birds are susceptible to MAC which consists of two closely related species of mycobacteria, that is, M. avium and M. intracellulare. This study aimed to determine the occurrence of Mycobacterium avium subsp. avium (MAA) in chickens and captive birds in selected states of Peninsular Malaysia. RESULTS: A 300 fecal samples were collected from village chickens (n = 100), layer chickens (n = 100) and captive birds (n = 100). Fecal samples were split into two aliquots for microbiological and molecular detection of MAA. Microbiology detection consisted of microscopy (Ziehl-Neelsen staining) and culture of samples decontaminated with 1% Cetylperidinium chloride and vancomycin, nalidixic acid and amphotericin B (VNA) antibiotic cocktail [vancomycin (VAN) 100 µg/ml, nalidixic acid (NAL) 100 µg/ml and amphotericin B (AMB) 50 µg/ml] onto Löwenstein-Jensen (L-J). Molecular detection (PCR-IS901) was performed to detect MAA DNA from the feces and PCR-16S rRNA and IS901 for identification of genus Mycobacterium and Mycobacterium avium sub species avium isolated onto L-J. All samples (296) were AFB negative smear. M. avium was isolated in 0.3% (1/296) samples by culture and detected in 2.5% (6/242) samples by PCR (IS901). Other mycobacteria were found in 1.7% (5/296) chickens. Of five isolates, two were identified as Mycobacterium terrae and M. engbaekii and remaining isolates were not sequenced. Birds positive for M. avium included White Pelican (n = 1) Black Hornbill (n = 1), Macaw (n = 2), Cockatoo (n = 2) and village chicken (n = 1). CONCLUSION: It is concluded that chickens and birds were infected with M. avium in selected areas of Peninsular Malaysia. Although, PCR is rapid, reliable and cost effective method for detection of M. avium in a subclinical stage, the culture of the avian feces should still be used as a reference test for the diagnosis of avian tuberculosis.

Loop-Mediated Isothermal Amplification (LAMP) as a Promising Point-of-Care Diagnostic Strategy in Avian Virus Research.

Over the years, development of molecular diagnostics has evolved significantly in the detection of pathogens within humans and their surroundings. Researchers have discovered new species and strains of viruses, while mitigating the viral infections that occur, owing to the accessibility of nucleic acid screening methods such as polymerase chain reaction (PCR), quantitative (real-time) polymerase chain reaction (qPCR) and reverse-transcription qPCR (RT-qPCR). While such molecular detection methods are widely utilized as the benchmark, the invention of isothermal amplifications has also emerged as a reliable tool to improvise on-field diagnosis without dependence on thermocyclers. Among the established isothermal amplification technologies are loop-mediated isothermal amplification (LAMP), recombinant polymerase amplification (RPA), strand displacement activity (SDA), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA) and rolling circle amplification (RCA). This review highlights the past research on and future prospects of LAMP, its principles and applications as a promising point-of-care diagnostic method against avian viruses.

Exposure to Zoonotic West Nile Virus in Long-Tailed Macaques and Bats in Peninsular Malaysia.

The role of wildlife such as wild birds, macaques, and bats in the spreading and maintenance of deadly zoonotic pathogens in nature have been well documented in many parts of the world. One such pathogen is the mosquitoes borne virus, namely the West Nile Virus (WNV). Previous research has shown that 1:7 and 1:6 Malaysian wild birds are WNV antibody and RNA positive, respectively, and bats in North America may not be susceptible to the WNV infection. This study was conducted to determine the status of WNV in Malaysian macaques and bats found in mangrove forests and caves, respectively. Archive sera and oropharyngeal swabs from long-tailed macaques were subjected to the antibody detection using WNV competitive enzyme-linked immunosorbent assay (c-ELISA) and WNV RNA using RT-PCR, respectively, while the archive oropharyngeal and rectal swabs from bats were subjected to RT-PCR without serological analysis due to the unavailability of serum samples. The analysis revealed a WNV seropositivity of 29.63% (24/81) and none of the macaques were positive for WNV RNA. Meanwhile, 12.2% (5/41) of the bats from Pteropodidae, Emballonuridae, and Rhinolophidae families tested positive for WNV RNA. Here, we show a high WNV antibody prevalence in macaques and a moderate WNV RNA in various Malaysian bat species, suggesting that WNV circulates through Malaysian wild animals and Malaysian bat species may be susceptible to the WNV infection.

Antibiogram Profiles and Risk Factors for Multidrug Resistance of Salmonella enterica Recovered from Village Chickens (Gallus gallus domesticus Linnaeus) and Other Environmental Sources in the Central and Southern Peninsular Malaysia.

The emergence of multidrug resistance (MDR), including colistin resistance, among Enterobacteriaceae recovered from food animals poses a serious public health threat because of the potential transmission of these resistant variants to humans along the food chain. Village chickens or Ayam Kampung are free-range birds and are preferred by a growing number of consumers who consider these chickens to be organic and more wholesome. The current study investigates the antibiogram profiles of Salmonella isolates recovered from village chicken flocks in South-central Peninsular Malaysia. A total of 34 isolates belonging to eight serotypes isolated from village chickens were screened for resistance towards antimicrobials including colistin according to the WHO and OIE recommendations of critical antibiotics. S. Weltevreden accounted for 20.6% of total isolates, followed by serovars Typhimurium and Agona (17.6%). The majority of isolates (73.5%) demonstrated resistance to one or more antimicrobials. Eight isolates (23.5%) were resistant to ≥3 antimicrobial classes. Colistin resistance (minimum inhibitory concentrations: 4-16 mg/L) was detected among five isolates (14.7%), including S. Weltevreden, S. Albany, S. Typhimurium, and Salmonella spp. Univariable analysis of risk factors likely to influence the occurrence of MDR Salmonella revealed that the flock size, poultry production system, and use of antibiotics in the farm were not significantly (p > 0.05) associated with MDR Salmonella. The current study highlights that MDR Salmonella occur at a lower level in village chickens compared to that found in live commercial chickens. However, MDR remains a problem even among free-range chickens with minimal exposure to antibiotics.

Evidence of West Nile virus infection in migratory and resident wild birds in west coast of peninsular Malaysia.

West Nile virus (WNV) is a zoonotic mosquito-borne flavivirus that is harbored and amplified by wild birds via the enzootic transmission cycle. Wide range of hosts are found to be susceptible to WNV infection including mammals, amphibians and reptiles across the world. Several studies have demonstrated that WNV was present in the Malaysian Orang Asli and captive birds. However, no data are available on the WNV prevalence in wild birds found in Malaysia. Therefore this study was conducted to determine the serological and molecular prevalence of WNV in wild birds in selected areas in the West Coast of Peninsular Malaysia. Two types of wild birds were screened, namely migratory and resident birds in order to explore any possibility of WNV transmission from the migratory birds to the resident birds. Thus, a cross-sectional study was conducted at the migratory birds sanctuary located in Kuala Gula, Perak and Kapar, Selangor by catching 163 migratory birds, and 97 resident birds from Kuala Gula and Parit Buntar, Perak at different time between 2016 and 2017 (Total, n = 260). Blood and oropharyngeal swabs were collected for serological and molecular analysis, respectively. Serum were screened for WNV antibodies using a commercial competitive ELISA (c-ELISA) (ID Screen® West Nile Competition Multi-species ELISA, ID VET, Montpellier, France) and cross-reactivity towards Japanese Encephalitis virus (JEV) was also carried out using the JEV-double antigen sandwich (DAS) ELISA. Oropharyngeal swabs were subjected to one-step RT-PCR to detect WNV RNA, in which positive reactions were subsequently sequenced. WNV seropositive rate of 18.71% (29/155) at 95% CI (0.131 to 0.260) and molecular prevalence of 15.2% (16/105) at 95% CI (0.092 to 0.239) were demonstrated in migratory and resident wild birds found in West Coast Malaysia. Phylogenetic analyses of the 16 WNV isolates found in this study revealed that the local strains have 99% similarity to the strains from South Africa and were clustered under lineage 2. Evidence of WNV infection in resident and migratory birds were demonstrated in this study. As a summary, intervention between migratory birds, resident birds and mosquitoes might cause the introduction and maintenance of WNV in Malaysia, however the assumption could be further proven by studying the infection dynamics in the mosquitoes present in the studied areas.

Occurrence of Campylobacter species from broiler chickens and chicken meat in Malaysia.

Campylobacter is reported as a major cause of foodborne illness worldwide. Consumption of contaminated chicken meat is considered a significant risk factor of Campylobacter infection in humans. This study investigated the occurrence of non-Campylobacter jejuni-Campylobacter coli, in broiler chickens (n = 210) and chicken meat (n = 109). The samples were collected from seven broiler chicken farms (n = 210 cloacal swabs), 11 markets (n = 84 chicken meat), and 5 supermarkets (n = 25 chicken meat) located in different districts of Selangor State. Campylobacter were isolated from cloacal swabs using the Cape Town Protocol and from meat samples using the method of Duffy et al. (2007) with some modifications for Campylobacter isolations which were reported effective in the isolation of non-C. jejuni-C. coli Campylobacter species. The isolates were identified by Gram staining for cellular morphology, wet mount for motility and biochemical tests. Confirmation of presumed Campylobacter isolates was carried out using multiplex PCR (mPCR). One hundred seven (107/210) or 50.9% and twenty-nine (29/109) or 26.6% of chickens and chicken meat samples respectively were positive for Campylobacter species. Among the Campylobacter isolates from chickens, C. jejuni was the most predominantly isolated species (69.5%), followed by C. coli (16.2%). Campylobacter fetus and C. upsaliensis were the non-C. jejuni-C. coli Campylobacter species isolated in this study, at 9.3% and 2.5% respectively. Overall, the findings indicated broiler chickens were colonized not only by the common Campylobacter species but also by other Campylobacter species. We found the Cape Town Protocol useful to detect the occurrence of non-C. jejuni-C. coli isolates in chickens.

Salmonella in native "village" chickens (Gallus domesticus): prevalence and risk factors from farms in South-Central Peninsular Malaysia.

Village chicken or Ayam Kampung, common to Southeast Asian countries, has always been regarded as superior in comparison to commercial broiler chicken in terms of wholesomeness and health benefits. The current study investigates the prevalence and risk factors of Salmonella among village chicken flocks from the central and southern states of Peninsular Malaysia. A total of 35 village flocks were sampled from Selangor (n = 19), Melaka (n = 10), Johor (n = 4), and Negeri Sembilan (n = 2). In total, 1,042 samples were collected; these included cloacal swabs (n = 675), eggs (n = 62), pooled drinking water (n = 175), pooled feeds (n = 70), and pooled flies (n = 60). Isolation of Salmonella from cloacal swabs, poultry drinking water, and feeds was carried out according to the protocols and recommendations of the World Organization for Animal Health (OIE) terrestrial manual. The prevalence of Salmonella at an individual bird-level was 2.5% (17/675, 95% CI: 1.6 to 4.0). All eggs screened were negative; in the case of environmental samples, however, Salmonella was detected in 5.14% (9/175), 7.14% (5/70), and 5.0% (3/60) for water, feed, and flies, respectively. A total of 34 isolates and 8 Salmonella serotypes were identified. Weltevreden (20.6%) was the most common, followed by Typhimurium and Agona (17.6%), Albany and Enteritidis (8.8%), Molade (5.9%), Corvallis and Schleissheim (2.9%), and others grouped as Salmonella spp. (11.8%). Multivariable logistic regression models revealed that Salmonella positivity among flocks could be strongly predicted by storage of feeds (uncovered feeds; OR = 10.38; 95% CI: 1.25 to 86.39; p = 0.030) and uncovered water tanks (uncovered tank; OR = 6.43; 95% CI: 1.02 to 40.60; p = 0.048). The presence of Salmonella in village chickens in the study area was lower than that of commercial chickens in Malaysia.

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.

Evaluation of six decontamination procedures for isolation of Mycobacterium avium complex from avian feces.

Culture is considered the gold standard for definitive diagnosis of mycobacterial infections. However, consensus about the most suitable culture procedure for isolation of nontuberculous mycobacteria is lacking. The study compared the recoveries of mycobacteria after decontamination of spiked and fresh avian feces with 4% sodium hydroxide (NaOH), 12% sulfuric acid (H2SO4), or 1% cetylperidinium chloride (CPC), with and without mixture of three antibiotics, namely vancomycin (VAN, 100 µg/ml), nalidixic acid (NAL, 100 µg/ml), and amphotericin B (AMB, 100 µg/ml). The antibiotic mixture was referred to as VNA. Decontamination procedures were evaluated using two (n = 2) avian fecal samples spiked with 106, 104, and 102 CFU/ml of Mycobacterium avium subsp. avium (ATCC 15769) and fresh avian feces (n = 42). M. avium subsp. avium was detected on the culture media from spiked samples (106 and 104 CFU/ml) decontaminated with NaOH, NaOH-VNA, H2SO4, and H2SO4 -VNA for 2-6 weeks. These bacteria were detected in 2-4 weeks when using CPC and CPC-VNA. M. avium subsp. avium cannot be isolated on culture media from spiked samples (102 CFU/ml) decontaminated with any decontaminating agent. Two mycobacterial isolates, namely, Mycobacterium terrae and M. engbaekii, were isolated from field samples decontaminated with NaOH and CPC-VNA. With regard to the contamination rate, the use of CPC-VNA showed lower contamination rates (5.5% and 19.0%) from spiked and field samples than those of the other methods (NaOH: 22.2% and 59.5%, NaOH-VNA: 16.7% and 21.4%, H2SO4: 11.1% and 40.5%, H2SO4-VNA: 5.5% and 21.4%, and CPC: 66.7% and 50%). In conclusion, the decontamination of fecal samples following a two-step procedure with 1% CPC and VNA can ensure high recovery rate of many mycobacteria with the lowest contamination in cultures.

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.

Optimization of a closed rat tibial fracture model.

BACKGROUND: The use of a closed fracture model has become the preferred model to study the fracture healing process, given that the periosteum and the soft tissue surrounding the fracture site play an important role in the fracture healing process. Some techniques like osteotomy, drilling the long bones and the use of the guillotine-like apparatus to induce fracture are characterized by some undesirable effects and complications. The aim of this study is to optimize and evaluate an in vivo fracture model using three-point bending pliers that can be used to study secondary bone fracture healing in rats. METHODS: Modified three-point bending pliers were used as a device to create the closed rat tibial bone fracture that was prefixed with an intramedullary pin (23 G × 11/2″) in rats. The exact location of the induced closed fracture was along the long bone. The presence of bone comminution, and the fracture bone alignment were immediately examined after the induction of the fracture until the 6th week. RESULTS: All fractures induced were transverse, located in the middle to proximal one third of the tibia, and they all healed without complications. Bone union as shown radiographically occurred within 2-3 weeks postoperative. The average angle of the fracture line with the axis of the tibia was 89.41 ± 2.11°. The lateral and anterio-posterior pin angulation views were 167.33 ± 3.67° and 161.60 ± 4.87° respectively. The average length of proximal end of the fractured bone in comparison with the whole length of intact bone was 41.02 ± 3.27%. There was a significant difference in percentage of the gross callus area and gross callus index, while there was no significant difference in X-ray callus index. There was no significant difference of the gross callus area between slight comminution (n = 4) and non comminution (n = 21). CONCLUSION: The optimized rat tibial fracture model resulted in mainly transverse tibial mid-shaft fractures with minimal bone comminution and absence of surrounding soft tissue damage. The size area of consequent soft callus formation and the extent to which the closed fracture model was reproducible are very good outcomes making it feasible for in vivo laboratory research use.

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.

Potential Reservoir and Associated Factors for West Nile Virus in Three Distinct Climatological Zones.

West Nile virus (WNV) is a zoonotic single-strand RNA arbovirus (family Flaviviridae: Flavivirus), transmitted among avian hosts in enzootic cycles by a mosquito vector. The virus has a significant disease effect on humans and equines when it bridges into a cycle with various sequelae with epidemic potential. This study was carried out to identify the potential spectrum of WNV hosts in three geographic areas with climatologically distinct features: Malaysia, Qatar, and the United States of America (U.S.). Serum samples were collected from avian and mammal species suspected to be reservoirs for the virus at these areas in a cross-sectional epidemiologic study. The samples were tested for the presence of antibodies against the virus using an enzyme-linked immunosorbent assay. Data on putative risk factors were also collected and analyzed for significance of association with seropositivity using the logistic regression analysis. Among the tested avian and mammalian species, raccoons had the highest seroconversion rate (54%) followed by crows (30%), horses (27%), camels (10%), other avian species (7%), and canine species (3%). It was almost twice as likely to detect seroconversion among these mammalian and avian species in the fall in comparison to other seasons of the year. Only mammalian and avian species and seasons of the year were significantly associated with the likelihood of seroconversion to WNV when we controlled for other factors in the multivariate analysis. Our data from the U.S. showed that raccoons and camels are susceptible to infection by the virus and may play a role in the perpetuation of endemic foci for the disease.

Evaluation of Osteogenic Potentials of Avian Demineralized Bone Matrix in the Healing of Osseous Defects in Pigeons.

OBJECTIVES: To evaluate avian allogeneic demineralized bone matrix (DBM) in the healing of long bone defects as a function of geometry and time in a pigeon model. STUDY DESIGN: Experimental. ANIMALS: Adult rock pigeons (n = 60). METHODS: Midshaft ulnar osseous defects were grafted with 2 geometric forms of DBM (tubular vs. chipped) and stabilized with a hybrid fixator. Autologous chips of sternal keel were used in a third group as control. Outcomes were evaluated by radiography and histology/histomorphometry at 4, 8, 12, and 24 weeks postoperatively. RESULTS: Despite an early rapid healing response, autografts plateaued (histologic score and new bone area) by 8 weeks with no significant improvement afterwards. Conversely, allogeneic DBM implants demonstrated continuous temporal improvement in bone healing, and tubular DBM finally outpaced autograft implants after week 12 with values for metrics achieving statistical significance by week 24. Chip DBM was inferior to tubular DBM and autograft. CONCLUSIONS: Avian DBM is osteogenic, biocompatible, and safe in orthotopic sites with potential usefulness in avian bone grafting. Implant geometry (shape and size) affects such osteogenic potentials.

Evaluation of a lytic bacteriophage, Φ st1, for biocontrol of Salmonella enterica serovar Typhimurium in chickens.

In this study, a Salmonella Typhimurium lytic bacteriophage, Φ st1, which was isolated from chicken faecal material, was evaluated as a candidate for biocontrol of Salmonella in chickens. The morphology of Φ st1 showed strong resemblance to members of the Siphoviridae family. Φ st1 was observed to be a DNA phage with an estimated genome size of 121 kbp. It was found to be able to infect S. Typhimurium and S. Hadar, with a stronger lytic activity against the former. Subsequent characterisation of Φ st1 against S. Typhimurium showed that Φ st1 has a latent period of 40 min with an average burst size of 22 particles per infective centre. Approximately 86.1% of the phage adsorbed to the host cells within the initial 5 min of infection. At the optimum multiplicity of infection (MOI) (0.1), the highest reduction rate of S. Typhimurium (6.6 log10 CFU/ml) and increment in phage titre (3.8 log10 PFU/ml) was observed. Φ st1 produced adsorption rates of 88.4-92.2% at pH7-9 and demonstrated the highest bacteria reduction (6.6 log10 CFU/ml) at pH9. Φ st1 also showed an insignificant different (P>0.05) reduction rate of host cells at 37 °C (6.4 log10 CFU/ml) and 42 °C (6.0 log10 CFU/ml). The in vivo study using Φ st1 showed that intracloacal inoculation of ~10¹² PFU/ml of the phage in the chickens challenged with ~10¹° CFU/ml of S. Typhimurium was able to reduce (P<0.05) the S. Typhimurium more rapidly than the untreated group. The Salmonella count reduced to 2.9 log10 CFU/ml within 6h of post-challenge and S. Typhimurium was not detected at and after 24h of post-challenge. Reduction of Salmonella count in visceral organs was also observed at 6h post-challenge. Approximately 1.6 log10 FU/ml Φ st1 was found to persist in the caecal wall of the chicks at 72 h of post-challenge. The present study indicated that Φ st1 may serve as a potential biocontrol agent to reduce the Salmonella count in caecal content of chickens.

Qualitative and quantitative evaluation of avian demineralized bone matrix in heterotopic beds.

OBJECTIVE: To evaluate the osteogenic potential of avian demineralized bone matrix (DBM) in the context of implant geometry. STUDY DESIGN: Experimental. ANIMALS: Rock pigeons (n = 24). METHODS: Tubular and chipped forms of DBM were prepared by acid demineralization of long bones from healthy allogeneic donors and implanted bilaterally into the pectoral region of 24 pigeons. After euthanasia at 1, 4, 6, 8, 10, and 12 weeks, explants were evaluated histologically and compared by means of quantitative (bone area) and semi quantitative measures (scores). RESULTS: All explants had new bone at retrieval with the exception of tubular implants at the end of week 1. The most reactive part in both implants was the interior region between the periosteal and endosteal surfaces followed by the area at the implant-muscle interface. Quantitative measurements demonstrated a significantly (P = .012) greater percentage of new bone formation induced by tubular implants (80.28 ± 8.94) compared with chip implants (57.64 ± 3.12). There was minimal inflammation. CONCLUSIONS: Avian DBM initiates heterotopic bone formation in allogeneic recipients with low grades of immunogenicity. Implant geometry affects this phenomenon as osteoconduction appeared to augment the magnitude of the effects in larger tubular implants.

Heterotopic implantation of autologous bone marrow in rock pigeons (Columba livia): possible applications in avian bone grafting.

Autologous bone marrow, alone or as a composite marrow graft, has received much attention in various species. To assess the potential osteogenicity of autologous, extramedullary bone marrow implants in an avian model, 24 adult pigeons (Columba livia) were given intramuscular implantations of autologous marrow aspirated from the medial tibiotarsus. Birds were euthanatized at 1, 4, 6, 8, 10, and 12 weeks after surgery to evaluate whether ectopic bone had formed at the implant sites. Primary evaluations by in situ radiography and postmortem histologic examinations showed no evidence of bone formation. Further evaluation with histologic scores and histomorphometry revealed a significantly increased rate of angiogenesis at the implant sites by the sixth and tenth week postimplantation (P < .05). No significant differences between the treatment and control sites were present at any other endpoints. Results of this study show that, although autologous bone marrow lacks heterotopic osteogenic potentials in this avian model, it could still function as a useful adjunct to routine bone grafting techniques because of its unique capabilities to promote early angiogenesis.

Management of a cutaneous squamous cell carcinoma in an American flamingo (Phoenicopterus ruber).

A 32-year-old female American flamingo (Phoenicopterus ruber) was presented with a squamous cell carcinoma of the middle digit of the right foot. No clinical, hematologic, or radiologic evidence of metastasis was present. Salvage amputation of the digit resulted in complete cure, whereas previous electrosurgery and radiation therapy were unsuccessful. Three years later, another squamous cell carcinoma was diagnosed in the middle digit of the left foot. The digit was also amputated. Seven months after the second amputation, the bird did not have any recurrence or signs of metastasis.