The relationship between the standard passive mouse protection test or serum antibody titres measured by indirect haemagglutination or enzyme-linked immunosorbent assays and active protection in buffaloes immunized with different types of haemorrhagic septicaemia bacterins was investigated. Groups of 2-3 buffaloes were immunized with the bacterins currently in use in Asia, viz., broth bacterin (BB), alum precipitated vaccine (APV) and oil adjuvant vaccine (OAV) either subcutaneously (BB, APV) or intramuscularly (OAV) and challenged subcutaneously with virulent organisms at different periods post-immunization. Although the passive mouse protection and indirect haemagglutination tests carried out with the pre-challenge sera from vaccinated buffaloes revealed no relationship with active protection in buffaloes, a relationship was observed between the ELISA antibody titres and protection. In contrast, a dose-response relationship was observed between the homologous active and passive mouse protection test.
An investigation into the epidemiology of Trypansoma evansi infection in crossbred dairy cattle was conducted for a period of 12 months on a dairy cattle farm in Penninsular Malaysia. The prevalence of parasitaemia was highest in lactating animals (13.4%), followed by those in the dry herd (8.8%), late pregnant animals (8.1%), early pregnant animals (4.7%), calves (0.3%) and heifers (0.2%). The prevalence of antigenaemia was highest in the lactating animals (54.7%), followed by that in dry animals (53.7%), heifers (51.1%), late pregnant animals (47.7%), early pregnant animals (46.5%) and calves (24.2%).
A study to determine the immunoglobulin and cellular responses in the respiratory tract of goats following intranasal exposures to formalin-killed Pasteurella haemolytica A2 was carried out. Forty-two goats were divided into two groups. Goats in Group 1 were subjected to double intranasal exposures to formalin-killed P. haemolytica A2 while goats in Group 2 were the unexposed control. Prior to and at weekly intervals post-exposure, three goats from each group were killed, serum samples were collected while the lungs were flushed with 50 ml normal saline before the right apical lobes were fixed in 10% buffered formalin. Both serum and lung lavage fluid were subjected to enzyme-linked immunosorbent assay (ELISA) to determine the levels of IgA, IgM and IgG while the formalin-fixed tissues were examined histologically. IgA levels in the lung lavage fluid increased rapidly to reach a significantly (p < 0.05) high level as early as Week 2 post-exposure and remained significantly (p < 0.05) high throughout the study period. The IgM levels increased at an intermediate rate to reach a significantly (p < 0.05) high level at Week 3 post-exposure before they decreased to an insignificant (p > 0.05) level the following week and the weeks thereafter. IgG levels increased gradually and only reached a significantly (p < 0.01) high level at Weeks 5 and 6 of the study. The size of the bronchus-associated lymphoid tissue (BALT) and the number of lymphocytes in BALT increased significantly from Week 2 and remained high thereafter. However, differences in the numbers of BALT were insignificant (p > 0.05) initially before becoming significantly (p < 0.05) high at Weeks 5 and 6. The BALT responses were parallel to those of imunoglobulins in the lung lavage fluid.
The outer membrane proteins (OMP) were extracted from the P. haemolytica A2, A7 and A9 to determine their potential as immunogens and their capability for cross-protection. Sixty lambs of approximately 9 months old were divided into four main groups. Animals in Group 1 were vaccinated with 2ml vaccine containing 100microg/ml of the outer membrane proteins of P. haemolytica A2. Animals in Group 2 were similarly vaccinated with the OMPs of P. haemolytica A7 while Group 3 with OMPs of P. haemolytica A9. Animals in Group 4 were unvaccinated control. During the course of the study, serum was collected to evaluate the antibody levels toward each OMP. There appeared to be good immune responses. However, high antibody levels did not necessarily result in good protection of the animals, particularly against cross-infection with P. haemolytica A9 in animals vaccinated with the OMPs of P. haemolytica A2. It seemed that the antibody responses were more specific toward the homologous challenge but generally did not cross-protect against heterologous serotype challenge. However, the OMPs of P. haemolytica A7 produced good in vivo cross-protection and excellent correlations when good antibody responses against all serotypes led to successful reductions of the extent of lung lesions following homologous and heterologous challenge exposures. Thus, the OMPs of P. haemolytica A7 was effective in protecting animals against homologous and heterologous infection by live P. haemolytica A2, A7 and A9.
1. Hubbard x Hubbard (HH) and Shaver x Shaver (SS) chicks given a dietary supplement of either 50 mg/kg oxytetracycline (OTC) or 1 g/kg Lactobacillus culture (LC) were exposed to 36 +/- 1 degrees C for 3 h daily from day (d) 21 to 42. 2. Prior to heat treatment, body weight (d 21) and weight gain (d 1 to d 21) of OTC and LC birds were greater than those fed the control diet. Chicks given LC had the best food efficiency followed by OTC and control birds during d 1 to d 21. Body weight (d 1 and d 21) and weight gain (d 1 to d 21) were greater for HH tlhan SS chicks. 3. After 3 weeks of heat exposure, birds receiving the LC diet had greater body weight and weight gain, higher food intake and lower food efficiency than OTC and control chicks. 4. Antibody production against Newcastle discase vaccine on d 21 was not affected by strain or diet. On d 42, while diet had negligible effect on this variable among the SS broilers, HH birds fed LC had higher antibody production than those on the control diet. 5. Neither strain nor diet had a significant effect on mortality.
The vp28 gene encoding an envelope protein (28 kDa) of white spot syndrome virus (WSSV) was amplified from WSSV-infected tiger shrimp that originated from Malaysia. Recombinant VP28 protein (r-28) was expressed in Escherichia coli and used as an antigen for preparation of monoclonal antibodies (MAbs). Three murine MAbs (6F6, 6H4 and 9C10) that were screened by r-28 antigen-based enzyme-linked immunosorbent assay (ELISA) were also able to recognize viral VP28 protein as well as r-28 on Western blot. Three non-overlapping epitopes of VP28 protein were determined using the MAbs in competitive ELISA; thus, an antigen-capture ELISA (Ac-ELISA) was developed by virtue of these MAbs. Ac-ELISA can differentiate WSSV-infected shrimp from uninfected shrimp and was further confirmed by a polymerase chain reaction (PCR) and Western blot. Approximately 400 pg of purified WSSV sample and 20 pg of r-28 could be detected by Ac-ELISA, which is comparable in sensitivity to PCR assay but more sensitive than Western blot in the detection of purified virus. Hemolymph and tissue homogenate samples collected from a shrimp farm in Malaysia during December 2000 and July 2001 were also detected by Ac-ELISA and PCR with corroborating results.
The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specificpathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.
House crows (Corvus splendens) in Selangor, Malaysia were examined for the presence of Campylobacter species, Salmonella species, Mycoplasma gallisepticum and Mycoplasma synoviae by serology, culture and pcr. For the detection of Campylobacter and Salmonella species swabs were taken either from the intestine or cloaca. For the detection of mycoplasmas, swabs were taken either from the choanal cleft or trachea for culture and pcr and serum samples were tested by the rapid serum agglutination (rsa) and monoclonal antibody-blocking elisa (mbelisa) for antibodies to M gallisepticum and M synoviae. For campylobacter, 25.3 per cent of the crows were positive by culture, and the species identified were Campylobacter jejuni and Campylobacter coli. No Salmonella species were isolated. Four of 24 swabs were positive for M gallisepticum dna but none gave positive results for M synoviae dna. No M gallisepticum or M synoviae antibodies were detected by rsa but 60 per cent of the sera gave positive reactions for M gallisepticum and 13 per cent gave positive reactions for M synoviae by mbelisa.
This report describes the proliferation and transmission patterns of Pasteurella multocida B:2 among stressful goats, created through dexamethasone injections. Thirty seven clinically healthy adult goats were divided into three groups consisted of 15 goats in group A, 11 goats in group B and the remaining 11 in group C. At the start of the study, all goats of group A were exposed intranasally to 1.97 x 10(10) CFU/ml of live P multocida B:2. Dexamethasone was immediately administered intramuscularly for 3 consecutive days at a dosage rate of 1 mg/kg. The exposed goats were observed for signs of HS for a period of 1 month. At the end of the 1-month period, 11 goats from group B were introduced into and commingled with the surviving goats of group A before all goats from both groups were immediately injected intramuscularly with dexamethasone for 3 consecutive days. The treatment with dexamethasone was then carried out at monthly interval throughout the 3-month study period. Goats of group C were kept separately as negative control. Three surviving goats from each group were killed at 2-week interval for a complete post-mortem examination. Two (13%) goats of group A were killed within 24 hours after intranasal exposure to P multocida B:2 while another two (13%) goats from the same group were killed on day 40, approximately 10 days after the second dexamethasone injection. All four goats showed signs and lesions typical of haemorrhagic septicaemia. Bacteraemia was detected in 3 goats of group A that were having rectal temperature higher than 41degrees C. The P. multocida B:2 isolation pattern was closely associated with dexamethasone injections when significantly (p < 0.05) higher rate of isolations from both groups were observed after each dexamethasone injection. Transmission of P multocida B:2 from goats of group A to group B was successful when P multocida B:2 was isolated from goats of group B for a period of 28 days. There was a strong correlation between dexamethasone injections, rate of bacterial isolation and serum cortisol level. The IgG level showed an increasing trend 2 weeks after exposure to P multocida B:2 and remained high throughout the study period.
Following a series of H5N1 cases in chickens and birds in a few states in Malaysia, there was much interest in the influenza A viruses subtypes that circulate among the local pig populations. Pigs may act as a mixing vessel for avian and mammal influenza viruses, resulting in new reassorted viruses. This study investigated the presence of antibodies against influenza H1N1 and H3N2 viruses in pigs from Peninsular Malaysia using Herdcheck Swine Influenza H1N1 and H3N2 Antibody Test Kits. At the same time, the presence of influenza virus was examined from the nasal swabs of seropositive pigs by virus isolation and real time RT-PCR. The list of pig farms was obtained from the headquarters of the Department of Veterinary Services, Malaysia, and pig herds were selected randomly from six of 11 states in Peninsular Malaysia. A total of 727 serum and nasal swab samples were collected from 4- to 6-month-old pigs between May and August 2005. By ELISA, the seroprevalences of swine influenza H1N1 and H3N2 among pigs were 12.2% and 12.1% respectively. Seropositivity for either of the virus subtypes was detected in less than half of the 41 sampled farms (41.4%). Combination of both subtypes was detected in 4% of all pigs and in 22% of sampled farms. However, no virus or viral nucleic acid was detected from nasal samples. This study identified that the seropositivity of pigs to H1N1 and H3N2 based on ELISA was significantly associated with factors such as size of farm, importation or purchase of pigs, proximity of farm to other pig farms and the presence of mammalian pets within the farm.
We had examined the immunogenicity of a series of plasmid DNAs which include neuraminidase (NA) and nucleoprotein (NP) genes from avian influenza virus (AIV). The interleukin-15 (IL-15) and interleukin-18 (IL-18) as genetic adjuvants were used for immunization in combination with the N1 and NP AIV genes. In the first trial, 8 groups of chickens were established with 10 specific-pathogen-free (SPF) chickens per group while, in the second trial 7 SPF chickens per group were used. The overall N1 enzyme-linked immunosorbent assay (ELISA) titer in chickens immunized with the pDis/N1+pDis/IL-15 was higher compared to the chickens immunized with the pDis/N1 and this suggesting that chicken IL-15 could play a role in enhancing the humoral immune response. Besides that, the chickens that were immunized at 14-day-old (Trial 2) showed a higher N1 antibody titer compared to the chickens that were immunized at 1-day-old (Trial 1). Despite the delayed in NP antibody responses, the chickens co-administrated with IL-15 were able to induce earlier and higher antibody response compared to the pDis/NP and pDis/NP+pDis/IL-18 inoculated groups. The pDis/N1+pDis/IL-15 inoculated chickens also induced higher CD8+ T cells increase than the pDis/N1 group in both trials (P<0.05). The flow cytometry results from both trials demonstrated that the pDis/N1+pDis/IL-18 groups were able to induce CD4+ T cells higher than the pDis/N1 group (P<0.05). Meanwhile, pDis/N1+pDis/IL-18 group was able to induce CD8+ T cells higher than the pDis/N1 group (P<0.05) in Trial 2 only. In the present study, pDis/NP was not significant (P>0.05) in inducing CD4+ and CD8+ T cells when co-administered with the pDis/IL-18 in both trials in comparison to the pDis/NP. Our data suggest that the pDis/N1+pDis/IL-15 combination has the potential to be used as a DNA vaccine against AIV in chickens.
Hendra virus (HeV) and Nipah virus (NiV) are the causative agents of emerging transboundary animal disease in pigs and horses. They also cause fatal disease in humans. NiV has a case fatality rate of 40 - 100%. In the initial NiV outbreak in Malaysia in 1999, about 1.1 million pigs had to be culled. The economic impact was estimated to be approximately US$450 million. Worldwide, HeV has caused more than 60 deaths in horses with 7 human cases and 4 deaths. Since the initial outbreak, HeV spillovers from Pteropus bats to horses and humans continue. This article presents a brief review on the currently available diagnostic methods for henipavirus infections, including advances achieved since the initial outbreak, and a gap analysis of areas needing improvement.
This study was carried out to determine the antibody responses and protective capacity of an inactivated recombinant vaccine expressing the cell wall surface anchor family protein of Streptococcus agalactiae following oral vaccination against streptococcosis in tilapia. Tilapia were vaccinated orally with 10(6) CFU/mL of the recombinant vaccine incorporated in feed (feed-based recombinant vaccine) (vaccinated group or Group 1), 10(6) CFU/mL of pET-32 Ek/LIC vector without cell wall surface anchor family protein (control group or Group 2), 10(6) CFU/mL of formalin-killed cells of S. agalactiae vaccine incorporated in feed was also prepared (feed-based vaccine) (vaccinated group or Group 3), and unvaccinated control group or Group 4 (fed with commercial pellets). During the course of study, serum, mucus and gut lavage fluid were collected to evaluate the antibody levels via enzyme-linked immunosorbent assay (ELISA). The results showed that tilapia immunized with the feed-based recombinant vaccine developed a strong and significantly (P
Recent reports indicate Neospora caninum has a possible role in causing abortions in sheep in New Zealand. Knowledge about the epidemiology of neosporosis in sheep is limited. This study aimed to adapt and validate a commercially available ELISA assay as an IgG avidity assay to discriminate between acute (primary and re-inoculated) and chronic N. caninum infections in sheep. In addition, it was used to compare the antibody avidity values between lambs from ewes inoculated with N. caninum either during the pregnancy or in the previous year. The avidity assay was undertaken by using 6M urea for the first wash after incubation with the primary antibody in the commercial ELISA (Chekit* Neospora antibody test kit, IDEXX Laboratories, Australia). Sequential serum samples were obtained from naïve ewes (n=16) experimentally inoculated with live N. caninum tachyzoites. All ewes were seropositive by two weeks post-inoculation and remained seropositive for 20 weeks post-inoculation. There was a linear relationship between time after inoculation and avidity values (p<0.05) over the first 24 weeks. In Week 4, all animals had avidity values <35% and by Week 8, 8/16 animals had avidity values of >35%. These results suggest that an avidity value of <35% indicates a recent primary infection while a value of >35% is indicative of a chronic infection. The assay was then validated using samples from other groups of experimentally inoculated sheep as well as samples from naturally infected ewes. When comparing sample to positive ratio (S/P) and avidity values from lambs born from recently inoculated ewes with those from ewes inoculated the previous year and re-inoculated in the current year, it was possible to differentiate the lambs at 2 weeks of age. Lambs from recently inoculated ewes had low S/P and avidity values at 2 weeks of age which increased by 12 weeks of age. In comparison, lambs from re-inoculated ewes had high S/P and avidity values at 2 weeks of age, due to maternal antibody influence but values were similar to those from lambs that were born from recently inoculated ewes at 12 weeks of age. Avidity values for four naturally infected ewes were all >60% indicating chronic infection. These results suggest that the assay is able to discriminate between recent and chronic infection in sheep as well as able to differentiate lambs with maternal immunity compared to their own de novo immunity. As such it can be utilized to understand the kinetics of N. caninum infection in sheep.
To determine if toltrazuril was effective in eliminating Neospora caninum infection from congenitally infected lambs. Twenty-eight ewes were allocated to 3 groups where animals in Groups A and B were inoculated with 1 × 10(7)N. caninum tachyzoites on Day 120 of gestation and Group C was maintained as a negative control group. Lambs born from ewes in Group A were treated with toltrazuril (20mg/kg) on Days 0, 7, 14 and 21 after birth. Lambs in Groups B and C were untreated. All lambs in Groups A and B were seropositive at 12 weeks of age. At 12 weeks of age, no differences between lambs in Group A and Group B were observed in serological results (ELISA and western blot), presence of N. caninum-related brain histopathological lesions or the number of organisms detected by qPCR. Group C remained negative for serology, detection of N. caninum DNA as well as histopathology throughout the study. Results indicate that N. caninum congenitally-infected lambs had a continuing infection with N. caninum despite being treated with toltrazuril.
Porcine reproductive and respiratory syndrome (PRRS) is a disease that is both highly contagious and of great economic importance in Malaysia. Therefore, reliable and improved diagnostic methods are needed to facilitate disease surveillance. This study compared PRRSV antibody responses in oral fluid versus serum samples following PRRS modified live (MLV) vaccination using commercial antibody ELISA kits (IDEXX Laboratories, Inc.). The study involved two pig farms located in Perak and Selangor, Malaysia. Both farms were vaccinated with PRRS MLV 1 month prior to sample collection. Thirty-five animals were used as subjects in each farm. These 35 animals were divided into 7 different categories: gilts, young sows, old sows, and four weaner groups. Oral fluid and serum samples were collected from these animals individually. In addition, pen oral fluid samples were collected from weaner groups. The oral fluid and serum samples were tested with IDEXX PRRS Oral Fluid Antibody Test Kit and IDEXX PRRS X3 Antibody Test Kit, respectively. The results were based on sample to positive ratio (S/P ratio of the samples). Results revealed a significant and positive correlation between serum and oral fluid samples for both farm A (p = 0.0001, r = 0.681) and farm B (p = 0.0001, r = 0.601). In general, oral fluids provided higher S/P results than serum, but the patterns of response were highly similar, especially for the sow groups. Thus, the use of oral fluids in endemic farms is effective and economical, particularly for large herds. In conclusion, the authors strongly recommend the use of oral fluids for PRRS monitoring in endemic farms.
Brucella melitensis is the most important pathogenic species of Brucella spp. which affects goats and sheep and causes caprine and ovine brucellosis, respectively. Serological tests for diagnosis of brucellosis such as Rose Bengal plate test (RBPT) and enzyme-linked immunosorbent assay (ELISA) usually utilize smooth lipopolysaccharides (S-LPS) as a diagnostic antigen which could give false positive serological reactions. Outer membrane proteins (OMP) of B. melitensis have been used as alternative diagnostic antigens rather than S-LPS for differential serological diagnosis of brucellosis, mainly in ELISA with single recombinant OMP (rOMP) as a diagnostic antigen. Nevertheless, the use of single format mainly showed lack of sensitivity against the desired rOMP. Therefore, this study aimed to determine whether a newly developed rOMPs indirect ELISA (rOMPs I-ELISA), based on combination of rOMP25, rOMP28 and rOMP31of B. melitensis, has a potential benefit for use in the serodiagnosis of brucellosis.
Rotavirus B (RVB) infection in cattle is poorly understood. The objective of this study was to describe the epidemiological features of repeated outbreaks of epidemic diarrhea due to RVB infection in adult cattle on a large dairy farm complex in Japan. In October 2002, approximately 550 adult cows and approximately 450 in February 2005 had acute watery diarrhea at several farms on the complex. Four months before the first outbreak, RVB antibody-positive rates at subsequently affected farms were significantly lower than at non-affected farms (30% to 32% versus 61% to 67%). During the acute phase of both outbreaks, RVB antibody-positive rates in diarrheal cows tested were as low as 15% to 26%. Most of the farms affected in the second outbreak were also involved in the first outbreak. Some adult cows with RVB diarrhea in the first outbreak showed not only RVB seroresponse, but also RVB shedding in the second outbreak, although none of these cows developed diarrhea. Nucleotide sequences of the VP7 and VP4 genes revealed a close relationship between RVB strains in both outbreaks. Taken together, these results indicate that outbreaks of epidemic RVB diarrhea in adult cows might be influenced by herd immunity and could occur repeatedly at the same farms over several years. To our knowledge, this is the first report on repeated RVB infections in the same cattle.
Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero-surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans-infected fish, particularly asymptomatic fish. In this study, we proposed a serum-based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens.
Diagnosing tuberculosis (TB) in farmed red deer (Cervus elaphus) is challenging and might require combining cellular and humoral diagnostic tests. Repeated skin-testing with mycobacterial purified protein derivatives (PPDs) might sensitize or desensitize the subjects to both kinds of diagnostic tools. We evaluated the effect of repeated (every 6 months) comparative tuberculin skin testing on skin test and ELISA responsiveness in farmed red deer hinds from a TB-free herd. Eighteen 8-month old hinds were inoculated with bovine and avian PPDs and the mitogen phytohaemagglutinin (PHA), as positive control and concurrently tested by ELISA for antibodies against avian (avian PPD, aPPD and protoplasmatic antigen 3, PPA3) and bovine antigens (bPPD and MPB70). Blood serum was also sampled three weeks after each skin testing round and tested for antibodies against aPPD and bPPD, in order to detect eventual antibody level boosts. Testing took place every six months from winter 2012 until winter 2015.