Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

Research (Published online: 20-01-2016)

11. Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification - B. Radhika, N. Vinod Kumar and D. Sreenivasulu

Veterinary World, 9(1): 60-64

   doi: 10.14202/vetworld.2016.60-64

Abstract

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Aim: The loop mediated isothermal amplification (LAMP) was standardized for rapid detection of Clostridium perfringens.

Materials and Methods: A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening ofC. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnIfor alpha toxin gene.

Results: Out of 120 samples screened 112 (93.3%) samples were positive by both LAMP and polymerase chain reaction (PCR) for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis.

Conclusion: Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases.

Keywords: Clostridium perfringens, enterotoxemia, lambs, loop mediated isothermal amplification.

Genetic structure of Mugil cephalus L. populations from the northern coast of Egypt

Research (Published online: 15-01-2016)

10. Genetic structure of Mugil cephalus L. populations from the northern coast of Egypt - Mahmoud Magdy, Mariam Gergis Eshak and Mohamed Abdel-Salam Rashed

Veterinary World, 9(1): 53-59

   doi: 10.14202/vetworld.2016.53-59

Abstract

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Aim: The gray mullet, Mugil cephalus, has been farmed in semi-intensive ponds with tilapia and carps in Egypt for years. The current study used the fluorescent amplified fragment length polymorphism (F-AFLP) technique to search for genetic differences between the populations of M. cephalus in the northern region of Egypt and to detect the gene flow between sampled locations and the homogeneity within M. cephalus genetic pool in Egypt.

Materials and Methods: To fulfill the study objectives 60 (15/location) samples were collected from four northern coast governorates of Egypt (Alexandria “sea,” Kafr El-Sheikh “farm,” Damietta “farm” and Port Said “sea”). Three replicates of bulked DNA (5 samples/replicate) for each location were successfully amplified using the standard AFLP protocol using fluorescent primers. DNA polymorphism, genetic diversity, and population structure were assessed while positive outlier loci were successfully detected among the sampled locations. Based on the geographical distribution of sampling sites, the gene flow, the genetic differentiation, and correlations to sampling locations were estimated.

Results: A total of 1890 polymorphic bands were scored for all locations, where 765, 1054, 673, and 751 polymorphic bands were scored between samples from Alexandria, Kafr El-Sheikh, Damietta and Port Said, respectively. The effective number of alleles (ne) for all bulked samples combined together was 1.42. The expected heterozygosity under Hardy–Weinberg assumption (He) for all bulked samples combined together was 0.28. Bulked samples from Damietta yielded the lowest ne (1.35) and the lowest He (0.23) when inbreeding coefficient (FIS) = 1. Bulked samples from Kafr El-Sheikh scored the highest ne (1.55) and the highest He (0.37). Bulked samples from Alexandria scored 1.40 for ne and 0.26 for He, while bulked samples from Port Said scored 1.39 for ne and 0.26 for He. The observed bulked samples formed three sub-population groups, where none is limited to a certain sampling location. A high differentiation among locations was detected, however, is not fully isolating the locations. Gene flow was 0.58. Positive outliers loci (117) were detected among the four sampled locations while weak significant correlation (r=0.15, p=0.03) was found for the distance between them.

Conclusion: Even though this species is cultivated in Egypt, the wild population is still present and by the current study a flow of its genes is still exchanged through the northern coast of Egypt. Which contribute to the cultivated populations leading to heterogeneity in its genetic pool and consequently affects the production consistency of M. cephalus in Egypt.

Keywords: fluorescent amplified fragment length polymorphism, isolation by distance, Mantel test, marine fish, Mugil cephalus, natural selection pressure, population structure.

Epidemiological study of bovine brucellosis in three senatorial zones of Bauchi State, Nigeria

Research (Published online: 15-01-2016)

9. Epidemiological study of bovine brucellosis in three senatorial zones of Bauchi State, Nigeria - S. G. Adamu, N. N. Atsanda, A. O. Tijjani, A. M. Usur, A. G Sule and I. A. Gulani

Veterinary World, 9(1): 48-52

   doi: 10.14202/vetworld.2016.48-52

Abstract

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Aim: To determine the seroepidemiological patterns of bovine brucellosis in three senatorial zones of Bauchi State, Nigeria.

Materials and Methods: Blood samples were aseptically collected from the anterior jugular vein of 336 slaughtered cattle, between September 2013 and March 2014 in three senatorial zones of Bauchi State, Nigeria. The sera obtained were screened for brucellosis using rose Bengal plate test (RBPT) and serum agglutination test (SAT) in parallel. The data generated was subjected to Chi-square and Fishers exact test analysis to establish whether there is a relationship between the breeds, sex, and location of the animals sampled.

Results: Of the 336 cattle screened, 18 (5.4%) and 13 (3.9%) were positive by RBPT and SAT, respectively. There was no statistically significant association (p>0.05) between the sex, age, and location of cattle with seropositivity of brucellosis in the state. It was concluded that brucellosis is prevalent in Bauchi State. Further study is recommended in other abattoirs and herds of cattle in Bauchi State for confirmation of the status of the disease among cattle slaughtered in the state.

Conclusion: A high seroprevalence of brucellosis among the cattle in Bauchi state indicates that the disease is endemic and cattle are one of the animals that perpetuate and sustain the disease.

Keywords: cattle, Bauchi state, brucellosis, Nigeria, seroprevalence.

Retrospective analyses of dogs found serologically positive for Ehrlichia canis in Cebu, Philippines from 2003 to 2014

Research (Published online: 13-01-2016)

8. Retrospective analyses of dogs found serologically positive for Ehrlichia canis in Cebu, Philippines from 2003 to 2014 - Adrian P. Ybañez, Rochelle Haidee D. Ybañez, Rex R. Villavelez, Honey Pearl F. Malingin, Dana Natasha M. Barrameda, Sharmaine V. Naquila and Shiella Mae B. Olimpos

Veterinary World, 9(1): 43-47

   doi: 10.14202/vetworld.2016.43-47

Abstract

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Aim: The study aimed to document the clinical and hematological observations of dogs found serologically positive for Ehrlichia canis and to identify parameters or factors that are associated with the disease with focus on the anemic and thrombocytopenic state of the infected dogs.

Materials and Methods: From 7 participating veterinary establishments, a total of 913 cases from 2003 to 2014 were initially assessed using inclusion criteria, including E. canis diagnosis by the attending veterinarian and the presence of ticks or history of infestation, thrombocytopenia, and/or anemia. From these, 438 cases that were found serologically positive for E. canis using commercial test kits were selected. Profile, clinical observations and hematological test results were obtained from the selected cases. Computations for statistical associations between the anemic and thrombocytopenic state of the infected dogs and their profile, observed clinical signs and other hematological values were performed.

Results: Most of the dogs were purebred (60.0%) and female (51.1%) and were within the age range of 1-5 years (38.4%). The mean packed cell volume (PCV), red blood cell (RBC) count, and platelet count were lower than the normal values while the absolute count of basophils were higher than normal values. Creatinine and blood urea nitrogen (BUN) appear to be elevated. The most common clinical signs observed were inappetence (41.3%), lethargy/depression (35.6%), vomiting (32.4%), fever (18.5%), paleness (8.2%), and epistaxis (6.6%). Analyses showed that there were no significant differences on the hematological values and clinical signs between thrombocytopenic and non-thrombocytopenic seropositive dogs. Moreover, very weak correlations between platelet count and RBC count, absolute lymphocyte count, and neutrophil count were found. On the other hand, only paleness (p=0.008) and epistaxis (p=0.004) were found to be significantly different between anemic and non-anemic patients. This coincided with the linear regression results where PCV (p=0.000, R=0.787, R2=0.619) was moderately correlated with the RBC count. In addition, eosinophil count was found weakly correlated.

Conclusion: E. canis infection in dogs may produce varied clinical signs that may be influenced by the thrombocytopenic and anemic states of affected animals. Complete blood counts remain important in the diagnosis of the disease, especially the platelet and RBC counts. Creatinine, BUN and alanine aminotransferase can be of value in the diagnosis of the infection. Several cases were lost to follow-up and appeared to be a challenge for handling veterinarians to monitor compliance of owners and progress of infected patients.

Keywords: Cebu, clinical signs, dog, Ehrlichia canis, hematology, Philippines.

Effect of age, sex and physiological stages on hematological indices of Banni buffalo (Bubalus bubalis)

Research (Published online: 13-01-2016)

7. Effect of age, sex and physiological stages on hematological indices of Banni buffalo (Bubalus bubalis) - Mehul D. Patel, Abdul Lateef, Hemen Das, Ajay S. Patel, Ajay G. Patel and Axay B. Joshi

Veterinary World, 9(1): 38-42

   doi: 10.14202/vetworld.2016.38-42

Abstract

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Aim: To determine the physiological baseline values for hematological indices of Banni buffalo Bubalus bubalis) as well as to assess their alteration due to age, sex and physiological stages.

Materials and Methods: A total of 42 clinically healthy Banni buffaloes were categorized into seven groups (n=6): Group I (male calves ≤1 year), Group II (bulls >1 year), Group III (female calves ≤1 year), Group IV (pregnant lactating buffaloes), Group V (non-pregnant lactating buffaloes), Group VI (pregnant dry buffaloes), and Group VII (non-pregnant dry buffaloes). Blood samples collected aseptically from all the experimental groups were analyzed employing automated hematology analyzer. The data obtained were statistically analyzed; the mean and standard deviations were calculated and set as the reference values.

Results: The erythrocytic indices viz. total erythrocytes count (TEC), hemoglobin, and packed cell volume (PCV) were significantly higher in bulls as compared to that of male calves unlike mean corpuscular volume, mean corpuscular hemoglobin (MCH), and MCH concentration. The female calves had higher TEC and PCV than the adult buffaloes irrespective of sex. The total leukocyte count (TLC) and neutrophil counts in male calves were significantly lower than the bulls unlike the eosinophil, while monocyte and basophil remained unchanged with age. The TLC, differential leukocyte count and platelet count varied non-significantly among the adult female groups at different physiological stages. However, neutrophils were found to be apparently higher in lactating buffaloes.

Conclusion: The present study would be helpful for physiological characterization of this unique buffalo breed of Gujarat. Further, data generated may be a tool for monitoring the health and prognosis as well as diagnosis of diseases.

Keywords: age, banni, erythrocytic, lactation, leukocytic indices, pregnancy, sex.

Effect of different culture systems on the production of foot and mouth disease trivalent vaccine

Research (Published online: 12-01-2016)

6. Effect of different culture systems on the production of foot and mouth disease trivalent vaccine - Amr Ismail Hassan

Veterinary World, 9(1): 32-37

   doi: 10.14202/vetworld.2016.32-37

Abstract

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Aim: This study aims to determine the effect of the stationary rawx, roller, and the suspension cell culture systems on the total virus yield infectivity and antigenicity.

Materials and Methods: Three serotypes of foot and mouth disease virus (FMDV) (serotype A, O and SAT-2) were inoculated separately into baby hamster kidney-21 cell line in rawx, roller, and suspension cultivation systems using multiplicity of infection (1:100). Samples were taken from the total virus yield from each system at 15, 18, 21, and 24 h post-inoculation. Testing the total virus yield infectivity through virus titration and antigenicity through estimation of complement fixing titer and 146S content and evaluation of the potency of the vaccine prepared from the different cultivation systems were done.

Results: The results showed that the FMDV titer of serotype A, O, and SAT-2 obtained from the roller cultivation system showed the highest level followed by suspension cultivation system then the rawx cultivation system. The FMDV titer showed its highest level at 21 h post-inoculation in all the cultivation systems and then decline at 24 h post-inoculation. The antigenicity reached its highest value content at 18 h post-inoculation either by complement fixation test or by quantifying the 146S intact virion. Montanide ISA 206 oil inactivated trivalent vaccines were prepared from the tested serotypes (A Iran O5. O Panasia and SAT-2/EGY/2012) harvested at 18 h post-inoculation from the 3 culture systems. The results of tracing the antibody response showed that the mean antibody response from the roller cultivation system start its protective antibody titer earlier at 2 weeks post-vaccination (WPV) than the vaccine prepared from the other two cultivation system and the immune protection period lasts longer for 36 WPV for the roller cultivation system vaccine than the other two cultivation systems.

Conclusion: The best cultivation system used for the production of FMD vaccine regarding its highest infectivity and antigenicity is the roller system.

Keywords: baby hamster kidney-21 cell culture, foot and mouth disease virus, monolayer tissue culture cells, suspension tissue culture cells.

Analysis of viral protein-2 encoding gene of avian encephalomyelitis virus from field specimens in Central Java region, Indonesia

Research (Published online: 12-01-2016)

5. Analysis of viral protein-2 encoding gene of avian encephalomyelitis virus from field specimens in Central Java region, Indonesia - Aris Haryanto, Ratna Ermawati, Vera Wati, Sri Handayani Irianingsih and Nastiti Wijayanti

Veterinary World, 9(1): 25-31

   doi: 10.14202/vetworld.2016.25-31

Abstract

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Aim: Avian encephalomyelitis (AE) is a viral disease which can infect various types of poultry, especially chicken. In Indonesia, the incidence of AE infection in chicken has been reported since 2009, the AE incidence tends to increase from year to year. The objective of this study was to analyze viral protein 2 (VP-2) encoding gene of AE virus (AEV) from various species of birds in field specimen by reverse transcription polymerase chain reaction (RT-PCR) amplification using specific nucleotides primer for confirmation of AE diagnosis.

Materials and Methods: A total of 13 AEV samples are isolated from various species of poultry which are serologically diagnosed infected by AEV from some areas in central Java, Indonesia. Research stage consists of virus samples collection from field specimens, extraction of AEV RNA, amplification of VP-2 protein encoding gene by RT-PCR, separation of RT-PCR product by agarose gel electrophoresis, DNA sequencing and data analysis.

Results: Amplification products of the VP-2 encoding gene of AEV by RT-PCR methods of various types of poultry from field specimens showed a positive results on sample code 499/4/12 which generated DNA fragment in the size of 619 bp. Sensitivity test of RT-PCR amplification showed that the minimum concentration of RNA template is 127.75 ng/μl. The multiple alignments of DNA sequencing product indicated that positive sample with code 499/4/12 has 92% nucleotide homology compared with AEV with accession number AV1775/07 and 85% nucleotide homology with accession number ZCHP2/0912695 from Genbank database. Analysis of VP-2 gene sequence showed that it found 46 nucleotides difference between isolate 499/4/12 compared with accession number AV1775/07 and 93 nucleotides different with accession number ZCHP2/0912695.

Conclusions: Analyses of the VP-2 encoding gene of AEV with RT-PCR method from 13 samples from field specimen generated the DNA fragment in the size of 619 bp from one sample with sample code 499/4/12. The sensitivity rate of RT-PCR is to amplify the VP-2 gene of AEV until 127.75 ng/μl of RNA template. Compared to Genbank databases, isolate 499/4/12 has 85% and 92% nucleotide homology.

Keywords: avian encephalomyelitis, reverse transcription polymerase chain reaction, viral protein 2 gene.