Re: Use of polymerase chain reaction for the differentiation of Group A bovine rotavirus G6, G8, and G10 genotypes in the North Island of New Zealand

Extract

In the paper by L Howe, H Sugiarto and RA Squires published in the New Zealand Veterinary Journal 56, 218–221, 2008, entitled, “Use of polymerase chain reaction for the differentiation of Group A bovine rotavirus G6, G8, and G10 genotypes in the North Island of New Zealand”, the authors concluded that “Currently, the rotavirus vaccine available in New Zealand contains only the G6 strain, and may not provide optimal protection for animals infected with G10 rotavirus. … This information may be of assistance in the development and optimisation of vaccines, and enhancement of effective control strategies.”     

Re: Re: Use of Polymerase chain reaction for the differentiation of Group A bovine rotavirus G6, G8, and G10 genotypes in the North Island of New Zealand

Abstract

Extract

The authors of the scientific article entitled “Use of polymerase chain reaction for the differentiation of Group A bovine rotavirus G6, G8, and G10 genotypes in the North Island of New Zealand” (Howe et al. 2008 Howe, L, Sugiarto, H and Squires, RA. 2008. Use of polymerase chain reaction for the differentiation of Group A bovine rotavirus G6, G8, and G10 genotypes in the North Island of New Zealand. New Zealand Veterinary Journal, 56: 218–221. [Taylor &; Francis Online], [Web of Science ®] , [Google Scholar]) would like to thank Dr Moffat for his letter of correspondence, and would like to take the opportunity to address the concerns raised by him in the New Zealand Veterinary Journal 57, 68, 2009, entitled, “Re: Use of polymerase chain reaction for the differentiation of Group A bovine rotavirus G6, G8, and G10 genotypes in the North Island of New Zealand”.     

Determination of bovine rotavirus G serotype by polymerase chain reaction

A total of 113 diarrheic samples comprising of 68 buffalo calves and 45 cow calves were screened by RNA-PAGE for the detection of presence of rotavirus. RNA-PAGE analysis of these samples revealed 11 (9.73%) was found positive for rotavirus. Out of 68 faecal samples of buffalo calves tested for viral gastroenteritis, 8 (11.76%) were found positive for rotavirus. Similarly, out of 45 faecal samples of cattle calves tested for viral gastroenteritis, 3 (6.66%) was found positive for rotavirus. Rotavirus-positive samples represented long electropherotype. All RNA-PAGE-positive faecal samples for rotavirus subjected to RT-PCR for VP7 gene, ten samples yielded a specific product of 1,013 bp of VP7 gene. All the PCR-positive samples of the present study were subjected to genotyping with primers for G6, G8 and G10 genotype. All positive samples showed G10 genotype. This indicates that G10 may be predominant genotype among bovine calves in Mumbai region in India.     

Detection and differentiation of bovine group A rotavirus serotypes using polymerase chain reaction-generated probes to the VP7 gene.

Dot and Northern blot hybridization assays were developed to detect and differentiate group A bovine rotavirus serotypes using radiolabeled serotype 6 (Nebraska calf diarrhea virus [NCDV] and United Kingdom [UK] strains) or serotype 10 (Crocker [Cr] strain) VP7 gene probes. Partial length VP7-specific cDNA encompassing areas of major sequence diversity were generated by the polymerase chain reaction (PCR) using either cloned VP7 genes (NCDV and UK strains) or reverse transcribed mRNA (Cr strain) as templates. Radiolabeled probes prepared from the PCR-generated cDNA were tested at various stringency conditions to optimize the hybridization assays. At high stringency conditions (52 C, 50% formamide, 5 x standard saline citrate), the NCDV, UK, and Cr probes serotypically differentiated bovine rotavirus isolates in RNA samples prepared from cell culture propagated viruses or in fecal specimens from infected gnotobiotic calves. The sensitivity and specificity of NCDV and Cr VP7 probes were characterized in dot blot hybridization assays, and the probes were estimated to detect at least 1 ng of viral RNA. The serotyping results obtained using VP7 probes were similar to those obtained using serologic assays. Further development of these assays may provide a useful means for the rapid detection and differentiation of bovine rotavirus serotypes in fecal samples from calves in the field.     

Use of restriction fragment length polymorphism, immunoblotting, and polymerase chain reaction in the differentiation of avian poxviruses.

Restriction deoxyribonucleic acid (DNA) fragment profile analysis coupled with immunogenic protein profile analysis has provided useful information in determining the differences between vaccine strains and field isolates of fowlpox virus (FPV). The DNA of strains examined in this study clearly fell into 3 minor groups of restriction patterns similar but distinct from one another: restriction patterns exhibited by the vaccine strains except 1 vaccine strain, Vac-82; restriction profiles indicated by Vac-82 and field isolates FI-38 and FI-42; and restriction patterns indicated by field isolates FI-43, FI-51, FI-54, and FI-56. Furthermore, when the strains were analyzed and compared by immunoblotting analysis, they showed group differences similar to the differences in restriction profiles. Both techniques provided high sensitivity in verifying differences between vaccine strains and field isolates of FPV. The disparity found in restriction fragments or immunogenic protein profile between vaccine strains and field isolates does not exclude the appreciable high degree of DNA sequence conservation and homology. However, the minor disparity observed in these strains suggests a molecular basis for why vaccinated commercial flocks could have continually been infected by variant strains of FPV. A rapid and sensitive polymerase chain reaction method, which amplified a product from the 4b core protein gene of the FPV genome, was developed for identification and differentiation of members of the genus Avipoxvirus. Whereas total DNA from either vaccine strains or field isolates was used as template for amplifying a predicted product of 578 or 1409 bp, only cleavage of the amplified product (1409 bp) represented an additional detection technique for species differentiation. An attempt to distinguish between strains on the basis of amplification product was partially successful.     

Prevalence and strain differentiation of Giardia intestinalis in calves in the Manawatu and Waikato regions of North Island, New Zealand

Giardia intestinalis has been reported in newborn calves world-wide; however, information on the extent of G. intestinalis in New Zealand calves has to date been very limited. The current study attempted to establish the prevalence rate of G. intestinalis in calves up to 8 weeks old in New Zealand. More than 700 calf fecal specimens were collected during the spring calving seasons of 1998 and 1999 from two regions in North Island, New Zealand (Manawatu and Waikato) and tested for the presence of G. intestinalis. In addition to determining the presence of G. intestinalis in newborn calves, sequence analysis was performed using specific amplification primers developed to target a section of the ribosomal DNA (rDNA). This locus is considered to be rapidly evolving, and therefore, suitable for use in the elucidation of phylogenetic relationships between G. intestinalis isolates. Sequencing was performed using G. intestinalis DNA extracted from cysts collected directly from the calf fecal matter. There was no culturing of the G. intestinalis isolates either in vivo or in vitro. Over 40% of all collected calf fecal specimens contained G. intestinalis cysts and rDNA sequence analysis revealed two different sequences among calf isolates. These sequence differences were not found to correspond to a particular season, geographical region or farming practice. Preliminary phylogenetic analysis suggests that these two rDNA sequence types are indicative of calf hosts.     

Use of a polymerase chain reaction assay to detect bovine leukosis virus in dairy cattle

OBJECTIVE: To evaluate the use of a polymerase chain reaction (PCR) assay in detecting bovine leukosis virus (BLV) in adult dairy cows. DESIGN: Prospective study. ANIMALS: 223 adult dairy cows. PROCEDURE: Cows were tested for BLV status by use of an ELISA and a PCR assay. Sensitivity, specificity, predictive values of positive and negative tests, and the percentage of cows correctly classified by PCR assay were calculated. Ninety-five percent confidence intervals were calculated for sensitivity and specificity. RESULTS: Sensitivity and specificity were 0.672 and 1.00, respectively. Prevalence of BLV in this herd was 0.807. Predictive value of a positive test was 1.00, and predictive value of a negative test was 0.421. The percentage of cows correctly classified by PCR assay was 73.5%. CONCLUSIONS AND CLINICAL RELEVANCE: A positive PCR assay result provided definitive evidence that a cow was infected with BLV. Sensitivity and negative predictive value for PCR assay were low. Consequently, PCR assay alone is unreliable for routine detection of BLV in herds with high prevalence of the disease.     

Use of a polymerase chain reaction to subtype Mycobacterium avium subspecies paratuberculosis, an increasingly important pathogen from farmed deer in New Zealand

AIMS: To review the number of microbiologically-confirmed cases of Johne's disease in farmed deer since 2000, and determine the prevalence of the bovine and ovine subtypes of Mycobacterium avium subsp paratuberculosis (M. paratuberculosis), using a highly specific polymerase chain reaction (PCR) test on samples from infected herds. METHODS: The number of cases of M. paratuberculosis in farmed deer identified by culture or IS900 PCR was documented. A highly specific PCR test was applied to subtype M. paratuberculosis from BACTEC 12B cultures selected on the basis of one culture per deer herd, to give a wide coverage of herds in New Zealand. RESULTS: From January 2001 to October 2005, M. paratuberculosis was isolated from 1,141 farmed deer, and has now been identified by microbiological testing in over 600 deer herds in New Zealand. The bovine subtype of M. paratuberculosis was shown by a highly specific PCR test to be present in 91/95 herds examined; the ovine subtype was found in the remaining four herds. CONCLUSIONS: Since 2000, there has been a substantial increase in both the number of microbiologically-confirmed cases of Johne's disease in farmed deer and the number of infected herds. Johne's disease is now widespread and common in deer herds throughout New Zealand. Whilst the bovine subtype of M. paratuberculosis predominates in deer herds in New Zealand in which Johne's disease has been confirmed, the occasional finding of the ovine subtype highlights the need to consider both sheep and cattle as potential sources of infection for farmed deer.     

A polymerase chain reaction assay for the detection of Leptospira spp. in bovine semen.

A rapid and specific method for the detection of pathogenic Leptospira spp. in bovine semen using the polymerase chain reaction (PCR) is described. The primers used were derived from an EcoR1/BamH1 fragment that hybridized strongly to chromosomal DNA from the hardjobovis serovar. Three different extraction methods were evaluated in this study: phenol-chloroform extraction method, proteinase K (PK) in 1% SDS, followed by phenol-chloroform, and phenol-chloroform followed by 1% cetyltrimethylammonium bromide (CTAB). A PCR product of approximately 500 base pairs (bp) in length was obtained when DNA from pure Leptospira culture was used as a template for PCR, regardless of the DNA extraction method used. The product was consistent with that predicted from the gene sequence. However, in semen seeded in vitro, as well as in semen from infected bulls, a PCR product was obtained only when the leptospiral DNA was extracted from the specimen using the CTAB method. In contrast, other methods used for DNA extraction did not generate suitable templates for the PCR procedure. This is the first PCR protocol developed to detect Leptospira in bovine semen. The PCR protocol provided a direct and unequivocal demonstration that Leptospira can be detected in semen of infected animals. The CTAB method was also used successfully in detecting Leptospira in the urine of infected animals. The PCR procedure was shown to be more sensitive than either the fluorescent antibody test (FAT) or culture for detecting the organism in urine.     

A serosurvey for antibodies to Leptospira in dogs in the lower North Island of New Zealand

AIMS: To investigate the prevalence of antibodies to endemic and exotic Leptospira serovars in samples from a serum bank, collected from dogs in the lower North Island of New Zealand. METHODS: Sera (n=466), which had been collected from apparently healthy dogs, were screened using the microscopic agglutination test (MAT) for antibodies to serovars L. borgpeterseni serovar hardjo, L. interrogans serovars pomona, copenhageni and canicola, and L. kirschneri serovar grippotyphosa. RESULTS: Antibody to Leptospiral antigen was found in 14.2% of dogs tested. The highest level of reactivity was with serovar copenhageni, to which 9.5% (41/433) of sera were positive. Antibodies to serovars grippotyphosa and canicola were not detected in this population of dogs. CONCLUSIONS: Leptospira infection is relatively common in dogs in the lower North Island . CLINICAL RELEVANCE: Vaccination of dogs against leptospirosis should be considered using vaccine containing antigen to serovars hardjo, pomona and copenhageni. The presence of moderate levels of copenhageni antibody in dogs in the lower North Island raises the possibility that this serovar has become established in rodent populations in this region.     

Frequency of group A rotavirus with mixed G and P genotypes in bovines: predominance of G3 genotype and its emergence in combination with G8/G10 types

The present study describes the genotypic distribution of rotaviruses (RVs) in an Indian bovine population with unexpectedly higher proportions of G3 alone or in combination of G8/G10. PCR-genotyping confirmed that 39.4% (13/33) of the prevalent RVs were the G3 type while 60.6% (20/33) were dual G3G10 or G3G8 types. P typing revealed that 93.9% (31/33) of the samples were P[11] while 6.1% (2/33) possessed a dual P[1]P[11] type. Sequence analysis of the VP7 gene from G3 strains viz. B-46, 0970, and BR-133 showed that these strains had sequence identities of 90.5% to 100% with other bovine G3 strains. The highest identity (98.9% to 100%) was observed with RUBV3 bovine G3 strains from eastern India. The G3 strains (B-46, 0970, and BR-133) showed 97.5% to 98.8% sequence homologies with the Indian equine RV strain Erv-80. Phylogenetic analysis demonstrated that G3 strains clustered with bovine RUBV3 and J-63, and equine Erv-80 G3. Overall, these results confirmed that the incidence of infection by RVs with the G3 genotype and mixed genotypes in the bovine population was higher than previously predicted. This finding reinforces the importance of constantly monitoring circulating viral strains with the G3 genotype in future surveillance studies.     

A cross-sectional survey of Thoroughbred stud farm management in the North Island of New Zealand

AIM: To obtain initial baseline data on the management of Thoroughbred stud farms in the North Island of New Zealand. METHODS: Data on the management of Thoroughbred stud farms were collected from a sample of 22 stud farms located in the south Auckland/Waikato region (n=15) and lower North Island (n=7) of New Zealand, using a face-to-face survey. The studmaster provided information on the size, scope and management of the farms during the 2004/2005 breeding season. Analysis was based on the location of the farm and size of the breeding operation (number of resident mares). RESULTS: Effective farm size ranged from 20 to 526 ha and averaged 167 (standard error (SE) 36) and 88 (SE 49) ha in the south Auckland/Waikato and lower North Island areas, respectively. Some farms in the Auckland/Waikato region stood shuttle stallions. The median number of stallions per farm was three (range 0-9), and the median mare-to-stallion ratio was 43 (range 10-250). The farms had a mean of 50 (range 7-180) wet mares and 21 (range 0-100) dry mares. The number of mares per breeding stallion increased with increasing size of breeding operation (p=0.04), being 28 (95% confidence interval (CI) = 10-56) vs 40 (95% CI=16-74) vs 74 (95% CI=44-113) for moderate (or=200 mares in total) operations, respectively. Seventy-one percent of farms aimed to breed dry mares early in the breeding season, and used a combination of lights, hormone therapy, and rising plane of nutrition to achieve this. Foaling took place in foaling paddocks monitored using a night foaling attendant (17/22) or with foaling alarms (5/22). At birth, 17/22 studmasters routinely administered antibiotics, 14/22 administered tetanus antitoxin, 9/22 administered an enema to foals, and 2/22 did not routinely administer prophylactic treatments. Weaning occurred at 5 (range 3.7-7) months of age, and foals were confined to a box for 1-2 weeks on 16/22 farms. Weaned foals were drenched with anthelmintics every 7 (range 4-9) weeks, and were fed 2.9 (range 1-6) kg of concentrate feed while at pasture until intensive management associated with preparation of the horses for auction began 13 (range 6-20) weeks before the yearling sales. Eight farms weighed the weanlings, at least monthly, to monitor growth. CONCLUSIONS AND CLINICAL RELEVANCE: The management of Thoroughbred horses was relatively consistent throughout the regions surveyed. Utilisation of breeding stallions tended to be more efficient on the larger stud farms in the south Auckland/Waikato region. Even though foals are grown at pasture they are often provided with large quantities of concentrate feed.     

A cross-sectional survey of Thoroughbred stud farm management in the North Island of New Zealand

AIM: To obtain initial baseline data on the management of Thoroughbred stud farms in the North Island of New Zealand.

METHODS: Data on the management of Thoroughbred stud farms were collected from a sample of 22 stud farms located in the south Auckland/Waikato region (n=15) and lower North Island (n=7) of New Zealand, using a face-to-face survey. The studmaster provided information on the size, scope and management of the farms during the 2004/2005 breeding season. Analysis was based on the location of the farm and size of the breeding operation (number of resident mares).

RESULTS: Effective farm size ranged from 20 to 526 ha and averaged 167 (standard error (SE) 36) and 88 (SE 49) ha in the south Auckland/Waikato and lower North Island areas, respectively. Some farms in the Auckland/Waikato region stood shuttle stallions. The median number of stallions per farm was three (range 0.9), and the median mare-to-stallion ratio was 43 (range 10.250). The farms had a mean of 50 (range 7.180) wet mares and 21 (range 0.100) dry mares. The number of mares per breeding stallion increased with increasing size of breeding operation (p=0.04), being 28 (95% confidence interval (CI) = 10.56) vs 40 (95% CI=16.74) vs 74 (95% CI=44.113) for moderate (≤70 mares), medium (90–199 mares) and large (≥200 mares in total) operations, respectively. Seventy-one percent of farms aimed to breed dry mares early in the breeding season, and used a combination of lights, hormone therapy, and rising plane of nutrition to achieve this.

Foaling took place in foaling paddocks monitored using a night foaling attendant (17/22) or with foaling alarms (5/22). At birth, 17/22 studmasters routinely administered antibiotics, 14/22 administered tetanus antitoxin, 9/22 administered an enema to foals, and 2/22 did not routinely administer prophylactic treatments. Weaning occurred at 5 (range 3.7–7) months of age, and foals were confined to a box for 1–2 weeks on 16/22 farms. Weaned foals were drenched with anthelmintics every 7 (range 4–9) weeks, and were fed 2.9 (range 1–6) kg of concentrate feed while at pasture until intensive management associated with preparation of the horses for auction began 13 (range 6–20) weeks before the yearling sales. Eight farms weighed the weanlings, at least monthly, to monitor growth.

CONCLUSIONS AND CLINICAL RELEVANCE: The management of Thoroughbred horses was relatively consistent throughout the regions surveyed. Utilisation of breeding stallions tended to be more efficient on the larger stud farms in the south Auckland/Waikato region. Even though foals are grown at pasture they are often provided with large quantities of concentrate feed.     

A survey of feeding , management and faecal pH of Thoroughbred racehorses in the North Island of New Zealand

AIM: To identify feeding and management variables associated with variation in faecal pH within a population of intensively managed Thoroughbred racehorses in New Zealand. METHODS: A cross-sectional survey was conducted of 16 racehorse trainers in the North Island of New Zealand. Interviews were conducted at the trainers' stables to obtain information on feeding and management of horses, and faecal samples were collected and faecal pH measured. RESULTS: Ninety-seven percent of the horses surveyed were confined in an area or=12 h/day. Trainer's age, number of years they had trained horses, age and gender of horses, weeks in race training, racing class, frequency of feeding, bedding type, and exercise workload had no effect on mean faecal pH. Acidic faecal pH (pH 12 horses. Acidic faecal pH was associated with trainers who offered 4 kg of grain as the only form of concentrate fed, or offered 12 horses. Irrespective of management system, it appears important to provide at least 2.25 kg of hay/day ad libitum, to buffer hindgut acidosis associated with diets high in soluble carbohydrate.     

Development of a shuttle polymerase chain reaction for the detection of bovine herpesvirus 2

Three different polymerase chain reaction (PCR) protocols were evaluated for their ability to detect bovine herpesvirus 2 (BoHV-2): single-step PCR with 3 reaction stages (denaturation, annealing and extension), 2 reaction stages (denaturation and annealing/extension; shuttle PCR), and semi-nested PCR with 3 reaction stages. All the PCR protocols showed the same sensitivity (detection limit of 0.4 TCID(50)). A non-specific band sometimes appeared in mock cell DNA at annealing temperatures below 64 degrees C. The shuttle PCR was found to be superior to the other protocols under consideration because of the speed of its application. Furthermore, no non-specific band was detected in DNAs of eight other DNA viruses. Thus, the shuttle PCR seems to be an excellent diagnostic tool for BoHV-2 infections.     

牛新孢子虫病PCR检测方法的建立和应用

犬新孢子虫感染是导致妊娠母牛流产的主要原因之一，准确快速地诊断是有目的治疗该病的前提。本研究根据已知的犬新孢子虫种属特异性基因片段Nc-5基因序列，设计了一对特异性引物，建立了检测犬新孢子虫的PCR技术。利用本方法可以从感染牛胎儿的脑脊液及实质脏器中扩增出1条大小为350bp的特异性核酸片段。在吉林省延边龙井地区的应用检测结果表明，流产牛胎儿中新孢子虫感染率为17％（15／90）。本方法在实际应用中快速、灵敏、特异性高，可以用于牛和其它动物新孢子虫病的快速诊断和流行病学调查。     

A survey of feeding,management and faecal pH of Thoroughbred racehorses in the North Island of New Zealand

AIM: To identify feeding and management variables associated with variation in faecal pH within a population of intensively managed Thoroughbred racehorses in New Zealand.

METHODS: A cross-sectional survey was conducted of 16 racehorse trainers in the North Island of New Zealand. Interviews were conducted at the trainers' stables to obtain information on feeding and management of horses, and faecal samples were collected and faecal pH measured.

RESULTS: Ninety-seven percent of the horses surveyed were confined in an area ≤5 × 5 m for ≥12 h/day. Trainer's age, number of years they had trained horses, age and gender of horses, weeks in race training, racing class, frequency of feeding, bedding type, and exercise workload had no effect on mean faecal pH. Acidic faecal pH (pH ≤6.32) was associated with stables with ≤12 horses, and trainers at stables with ≤12 horses offered more concentrate feed than those at stables with >12 horses. Acidic faecal pH was associated with trainers who offered 4 kg of grain as the only form of concentrate fed, or offered ≤2.25 kg hay/day. Horses that displayed stable vices had less acidic faecal pH than horses that did not display stable vices, viz pH 6.70 (standard error of the mean (SEM) 0.135) vs 6.43 (SEM 0.029) (p=0.04).

CONCLUSIONS AND CLINICAL RELEVANCE: Racehorse management in New Zealand is similar to that observed in other major racing countries. Trainers with ≤12 horses fed more concentrates and their horses had lower faecal pH than those of trainers with >12 horses. Irrespective of management system, it appears important to provide at least 2.25 kg of hay/day ad libitum, to buffer hindgut acidosis associated with diets high in soluble carbohydrate.