Table 1 shows AFI frequencies distributed by ages, with most patients positive to at least one of the etio- logic agents being between 20 to 44 years old. However, the exhaustive analysis of frequency distributions by age and etiological agents (Fig. 2) shows that all distributions by ages correspond to Gaussian models. Patients infected with Bartonella bacilliformis showed a wide symmetric distribution centered in the range of 20-44 years, similar to the total population with AFI. However, the patients infected with Leptospiraspp. and Rickettsia spp. showed significantly different characteristics in their distributions (| 2 , p<0.05) in comparison to the total population with AFI. The distribution was wide and cen- tered between the ranges of 20-44 and 45-59 years old, indicating that adults were the most affected when infected with Leptospiraspp. Meanwhile, when Rickett- sia spp. was responsible for AFI, the distribution was narrower and centered in the group of young people, be- tween 5-19 and 20-44 years old (Fig. 2). Finally, the distribution of AFI bacterial etiology by sex did not show any significant differences. Only a slight tendency of a greater distribution of Leptospira in males can be ob- served with a frequency of 62.5% (CI95%: 38.6-81.5) (Table 1).
This PCR assay provides the potential to detect pathogenic Leptospiraspp. in a range of clinical specimens providing for earlier diagnosis and unequivocal evidence of active infection. As reported for the detection of meningococcal cells , the PCR assay developed in this work will pro- vide an enhanced capability for confirmation of cases where early antibiotic treatment has precluded detection by culture. Opportunity also exists for better postmortem testing where samples are generally of poor quality and do not lend themselves to culturing or serology.
In Southeast Asia, the number of reported cases and outbreaks of leptospirosis has increased dramatically in recent years, due in part to improvements in diagnosis and surveil- lance, but also as a result of the rapid environmental changes occurring in this region [19–21]. At least six zoonotic species have been detected in Southeast Asian rodents to date: L. borgpe- tersenii, L. interrogans, L. kirschneri, L. weilli, L. noguchii and L. wolfii [6,22]. In Malaysia, the annual number of reported cases increased more than 14-fold between 2004 and 2012, which led to the classification of leptospirosis as a mandatory notifiable disease at the end of 2010 . Although many recent Malaysian outbreaks have been associated with outdoor recrea- tional activities, human infections have also been documented in urban environments . Some studies have begun to assess the prevalence of Leptospiraspp. in urban reservoir species in Southeast Asia [22,25]), but none have yet compared how distribution and transmission varies with the degree of anthropogenic influence across an urban landscape. In this study, we screened native and invasive rodents found in urban, developing and rural locations around the city of Kuching, Sarawak for Leptospiraspp., to begin to explore how urbanization effects the presence and prevalence of Leptospira in Malaysian Borneo.
Leptospirosis is an endemic disease in Malaysia and in recent years there has been a dramatic increase in the number of reported cases. According to Ministry of Health, Malaysia a marked increase in the disease occurred from 12.5 per 100,000 population in 2012 to 15.0 per 100,000 population in 2013, with 71 of 4,457 cases resulting in mortality (Benacer et al., 2016a). Within the farming community, lepto- spirosis can potentially lead to economic losses, causing abortion, stillbirth, infertility, loss of milk production and death (Tilahun et al., 2013; Maleki et al., 2013). In Malaysia, Leptospiraspp. have been isolated from a wide variety of animals (Bahaman & Ibrahim, 1988) including rodents, cattle, pigs, and dogs, which serve as potential carriers (Thayaparan et al., 2013).
Strain and culture conditions. The pathogens Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130, a clinical isolate obtained from a leptospi- rosis outbreak in Salvador, Brazil (10), and L. borgpetersenii serogroup Mini strain 200801773, isolated from a patient with acute leptospirosis in Mayotte, France (3), were used in this study. Leptospires were cultivated in liquid Elling- hausen-McCullough-Johnson-Harris (EMJH) medium (6, 8). Additional refer- ence strains (n ⫽ 58) were obtained from the collection maintained by the National Reference Laboratory for Leptospira, which is also a WHO Collabo- rating Center, at the Institut Pasteur (Paris, France). We also included clinical strains (n ⫽ 91) isolated from human and animals of different geographical origins (mainland France and French overseas Territories) since 2007. Other bacterial genomic DNA (Escherichia coli, Borrelia burgdorferi, Borrelia hermsii, * Corresponding author. Mailing address: Institut Pasteur, Unite ´ de
2. Benacer D, Mohd Zain SN, Amran F, Galloway RL, Thong KL. Isolation and molecular characterization of Leptospira interrogans and Leptospira borgpetersenii isolates from the urban rat populations of Kuala Lumpur, Malaysia. Am J Trop Med Hyg 2013;88:704-9. 3. Bruce MG, Sanders EJ, Leake JA, Zaidel O, Bragg SL, Aye T, et al.
Hence, a second subtyping method, RAPD-PCR was used to improve the discrimination. In this study a greater genetic variation was observed among the members of the same serovar as the 39 isolates were represented by 14 dif- ferent patterns. Some of isolates represented the same ser- ovar isolated from different locations gave different RAPD patterns. Our study was in agreement with several previ- ous studies [46, 47]. RAPD-PCR is shown to be a useful technique in epidemiological investigation of leptospirosis. In several studies, RAPD-PCR had the ability to discrimin- ate between the strains at species and even at serovars level [20, 48]. Hence, based on discriminatory power, RAPD is more discriminative compared to PFGE which failed to distinguish strains within the same serovar. How- ever, the reproducibility of the PCR fingerprinting is mod- erate, which makes pattern analysis more difficult and tedious as compared to PFGE. PFGE examines the genetic variation throughout the genome and is highly reprodu- cible, which makes it the “gold standard” molecular typing of Leptospiraspp. . RAPD fingerprint could be an al- ternative tool in subtyping of Leptospira isolates, as it is easier and could generate results more rapidly than PFGE.
Leptospiraspp. belong to the bacterial phylum “Spiro- chaetes.” Members of the genus Leptospira are generally di- vided into a pathogenic species, Leptospira interrogans sensu lato, and a nonpathogenic species, Leptospira biflexa sensu lato (3, 12). Pathogenic members are the causal agents of leptospi- rosis, a widespread zoonosis that is a major public health di- lemma. In the natural reservoirs of the bacteria, such as ro- dents, infection produces chronic and persistent asymptomatic shedding in the renal tubules, and bacteria are then excreted in urine.
Due to unhygienic nature of the abattoir, faecal contaminations are likely to occur with the meat. Gram negative and gram positive bacteria such as E.coli, Staphylococcus spp, Klebsiella spp, Streptococcus spp, Salmonella spp, Clostridium spp, Bacillus spp, and Pseudomonas spp were found in abattoir site and some of these are food borne pathogens such as botulism, dysentery, gastroenteritis and typhoid (Fraze and Westhoff, 2004).
Mycoplasma sp. have been detected in a single dog liv- ing in Romania by Hamel and colleagues , who screened 29 local pet dogs. Additionally, the same study detected Mycoplasma sp. in 16 out of 109 dogs living in Germany but originating from Romania, as well as in one out of 78 dogs originating from Hungary . Al- though the geographical origin of infection remains un- clear, this study concluded that almost ten percent of sampled dogs carried the parasite. The findings by Hamel and colleagues were designated as Mycoplasma haemocanis based on the size of the amplified fragment, but the sequences of these fragments were however not determined. In the present study, we report two Myco- plasma species confirmed by sequencing: M. haemocanis and “ Ca. Mycoplasma haemoparvum ” . Both occurred in relatively high numbers, infecting almost one fifth of the dogs in the study, making Mycoplasma spp. the second most common pathogen. Travel history of the dogs in the present study was not reported, making it impossible to establish the actual origin of infection. However, the relatively high prevalence of both Mycoplasma species
Our study had some limitations. Only cats that were presented to veterinarians were used as the source of ticks and so this cannot be considered a random sample of domestic cat ticks, although the cats that were exam- ined were not brought into the veterinary surgery specif- ically for tick infestation. It could be argued that the cats sampled might also have been likely to receive preventa- tive tick treatment, since they were under veterinary care. No IAC PCR for the presence of tick DNA was available, but an artificial IAC was included to confirm the presence of amplifiable DNA . We were not able to use PCRs for the detection of feline Anaplasma or Ehrlichia spp. infection at a genus level on the tick DNA samples, due to the known presence of endogenous tick Ehrlichia/Anaplasma spp., as previously described , which confounds any positive results. Ideally, we would
The same protocol (above mentioned) was followed for isolation of fungi, instead of media the Potato dextrose Agar for Trichoderma spp and Sabouraud Dextrose Agar for Aspergillus spp was used with 10 day incubation period at 37°C. The samples were identified up to basic level grounded on their colony and spore morphology and microscopic examination as charted in the Bergey’s Manual .
In this study, bacteria isolate from ready-to-eat food and water samples were identified as Escherichia coli, Pseudomonas Spp, Enterobacter spp, Klebsiella spp, Salmonella spp, Bacillus spp, Staphylococcus aureus, Proteus spp, and Shigella spp. This observation was not surprising as it corroborates with that of Oluyege et al 2009; Oladipo and Adejemobi, 2010; and Majolagbe et al, 2011, who all identified Staphylococcus aureus, Bacillus, Marcescens, Streptococcus faccalis, Pseudomonas putida, Aeromonas hydrophila, Enterobacter aerogenes, Klebsiella spp, and Proteus spp from ready-to-eat food samples in Ado-ekiti and Ogbomoso, Nigeria respectively.
The present study was conducted for the isolation, identification and antibiotic sensitivity of the bacteria isolated from different street food. Presence of coliforms in the sample might be due to poor quality of water, unhygienic vendor places and poor personal hygiene of vendors. Most Street vendors were illiterate and they did not have a clear hygienic knowledge about the preparation, storage and serving of the food. All isolates found resistant to Cefixime, Cefalexin, Erythromycin, Fusaric acid, Cefuroxime and Aztreonam. The results of this study suggested that although RTE foods are cheap and economical but they are not healthy due to lack of hygienic measures, dirty utensils, and vendor‘s hygiene. These factors contributing many species of bacteria but major pathogen is E. coli, Salmonella spp. Shigella spp., Klebsiella spp. and Staphylococcus spp. Basic and main source of bacterial infection is poor hygienic measures and this problem may be solved by improving supervision in food handling procedure, extended consumer education on transmission of enteric food borne diseases and food safety risks. So that street vended RTE foods should be manufactured under Good Hygienic Practices and conservation practices should be developed in order to minimize the microbial contamination of food.
Our study had some limitations. First, although the sequences used as the targets in this multiplex PCR are from highly con- served regions of the genes, a weakness of this or any multiplex assay is that new variants of virulence genes could fail to amplify with the primers described. Second, this assay also identifies en- teroinvasive E. coli (EIEC), since both Shigella spp. and EIEC have the ipaH gene. Third, it was not possible to compare the perfor- mance of the assay between cultures and stool samples because the latter had been stored for a long time and the DNA might have been degraded.
fungi which is in the same family with Cladosporium spp. But in spring, the amount of Aspergillius was abundant. One reason for the development of this type of fungi is increase in environmental temperature which is the favorable condition for Aspergillius. There was also a significant difference between the amount and diversity of fungi in spring in comparison with winter perhaps due to the favorable weather and that the plants start to grow. Since the water source for city distribution system is surface water (river), the temperature of this source is influenced by the
ferent sites (the cheese-maker-stirrer and the milk filler tube), and at different time points (five months apart), were found to be closely related as demonstrated by PFGE (94% genetic similar- ity). Both isolates were identically ERY-resistant (ermC gene; iMLS B -resistance) and OXA-resistant (mecA gene) but differed in tetracycline resistance (PEN, TET, COT, CLI versus PEN, COT, CLI). In one biofilm-negative S. epidermidis isolate from the inside surfaces, multi-resistance to PEN, COT, and ERY (iMLS B ; ermC gene) was also detected. The genetic relatedness was also recorded in two S. aureus isolates found on the inside surfaces of the pasteurisation station and the milk filler tube, respectively. These isolates were of the same PFGE type and showed 96% similarity with the PFGE profile of one isolate from pasteurised milk filled with the device. This demonstrates the persistence of particular subpopulations of Staphylococcus spp. and their potential to spread in the dairy plant environment as well as dairy farms.