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Helicobacter pylori (Hp) is an organism commonly present worldwide in the human population, sometimes causing serious illnesses such as duodenal and gastric ulcers, adenocarcinoma of the stomach, and low-grade B-cell mucosa-associated lymphoid tissue lymphoma of the stomach. This article describes a multiplex-touchdown PCR method for the identification and genotyping (vacA—s1/m1, s1/m2, and s2/m2—and cagA genes) of Hp directly from sheep milk artificially contaminated with Hp strains from human gastric biopsies and with lip ATCC 43504. The strains from humans carried s1/m2 cagA+ and s2/m2 cagA+ allelic combinations, while the ATCC strains carried an s1/m1 cagA+ allelic combination. The technique showed a sensitivity of 15 CFU/ml for species identification and of 1,500 CFU/ml for the detection of genes encoding for VacA and CagA. It has proven to be specific and rapid, and the authors suggest that it be used as a rapid screening method to ensure that sheep milk is uncontaminated with this organism. [ABSTRACT FROM AUTHOR]

• Despite extensive vaccination, canine parvovirus (CPV) still represents one of the major causes of pups' mortality. • CPV immunisation failures occur frequently and recognize different reasons. • Interference by maternally-derived antibodies is the main cause of CPV immunisation failures. • Eradication of CPV infection is a challenge for the future, but it will not be achieved in a short time Despite extensive vaccination, canine parvovirus (CPV) remains a leading infectious cause of canine mortality, especially among juveniles. This review provides an update on CPV vaccine types and vaccination protocols. The design of CPV prevention strategies and vaccination programs with a goal of herd immunity has been hampered by deficiencies of studies that model companion animal viral infections and inform an understanding of the basic reproduction number. However, the most important issue in eradication of CPV disease is represented by immunisation failures including: i) the presence of interfering titres of maternally-derived antibodies; ii) the presence of non-responders; and iii) possible reversion to virulence. In contrast, the role of the CPV variants in immunisation failures is widely debated. Taking into account the reduced circulation of canine distemper virus and canine adenovirus type 1 in countries where extensive vaccination is carried out, more effort should be made to aim for CPV eradication, including antibody testing to determine the optimal time for vaccinations of pups and adults and homogeneous vaccine coverage of dog population. [ABSTRACT FROM AUTHOR]

Diarrhoea in lambs and kids is often a complex, multi-factorial syndrome. Common infectious causes of diarrhoea in lambs and kids during the first month of life are of bacterial or parasite nature. However, despite appreciable improvements in management practices and prevention and treatment strategies over the last decades, diarrhoea is still a common and costly syndrome affecting newborn small ruminants. Recent advances in the diagnostics and metagenomic investigations of the enteric environment have allowed discovering a number of novel viruses, although their pathobiological properties remain largely unknown. Assessing more in depth the impact of these viruses on the health and productions of these livestock animals is necessary and requires the development of accurate diagnostic tools and updating of the diagnostic algorithms of enteric pathological conditions. [ABSTRACT FROM AUTHOR]

Abstract: Caliciviruses are an important cause of gastroenteritis in humans and animals. Molecular analysis of the polymerase and capsid genes of porcine caliciviruses, sapoviruses (SaVs) and noroviruses (NoVs), has demonstrated a broad range of genetic diversity but information on their epidemiology and pathogenic role in pigs is limited. In this study, 292 faecal samples were obtained from 4–5 to 8–9 week old asymptomatic pigs from four porcine herds in Ireland during 2005–2007 and were screened by RT-PCR using calicivirus-specific primers. Only seven samples from two porcine herds tested positive for porcine calicivirus. By sequence analysis of the partial RNA-dependent RNA polymerase (RdRp) fragment, six samples from one such herd were closely related to each other (>98% nucleotide identity) and were characterised as genogroup (GG) III (Cowden-like) porcine SaVs. These viruses demonstrated an amino acid (aa) identity of 81.3–98.6% to GGIII SaVs. Conversely, one calicivirus strain, 9/07/Ire (identified from a different herd in 2007), was distantly related to GIII SaVs and displayed 94.6–98.6% aa identity to rare K7-like porcine caliciviruses, representatives of a potential novel SaV genogroup (GGVII), described previously in Japan and the USA. Circulation of SaVs in asymptomatic animals might be a mechanism of virus persistence in porcine populations and should be considered with respect to understanding the epidemiology of these viruses in porcine herds. [Copyright &y& Elsevier]

• Interfering MDA levels can hamper the immune response against CPV-2 vaccination. • Fourteen pups with different MDA titres were orally administered an MLV CPV-2b vaccine. • All pups but one seroconverted after a single vaccine administration. • The remaining pup raised an immune response after the second vaccination. • Oral administration of CPV-2 vaccines can help overcome MDA interference. Different strategies have been proposed to overcome maternally derived antibody (MDA) interference with canine parvovirus type 2 (CPV-2) immunisation, including intranasal vaccination, which presents some practical limitations. In the present study, the results of the oral administration of a commercial CPV-2b modified live virus (MLV) vaccine in pups with MDA are reported. The CPV-2b vaccine was orally administered to 14 6-week-old pups with a bait. Blood samples and rectal swabs were collected at different days post-vaccination (dpv) to determine CPV-2 antibody titres and DNA loads. Thirteen pups were positive to serological and virological tests after the first vaccination and one pup became positive after the second vaccine administration. The findings of this study suggest that systemic immunity against CPV-2 may be achieved by the use of an MLV CPV-2b vaccine administered orally even in the presence of MDA titres that usually interfere with vaccination. [ABSTRACT FROM AUTHOR]

Highlights • Bubaline herpesvirus 1 (BuHV-1) was detected in trigeminal ganglia of experimentally infected goats. • Latent infection was reactivated with desamethasone in a goat 10 months after the experimental infection. • The reactivated virus was shed through nasal and ocular secretions. • Reactivation of BuHV-1 in goat resulted in a 4-fold increase in the antibody titer. Abstract Latent infection is a common mechanism used by several alphaherpesviruses to persist in their host but it is not clear whether this mechanism is also triggered in heterologous infections. Cross-species infections have been documented repeatedly for alphaherpesviruses of ruminants, a group of closely related viruses. Herewith we report latent infection with bubaline alphaherpesvirus 1 (BuHV-1) in experimentally infected goats and subsequent virus reactivation after treatment with dexamethasone (DMS) at 10 months after infection. After DMS treatment, the virus was isolated in one such animal in the nasal swabs from day 3 to 9 post treatment and in the ocular swabs at day 6. The goat was euthanized 48 days after DMS treatment and viral DNA was detected by PCR in the trigeminal ganglia and in two cervical ganglia. Additionally, BuHV-1 DNA was detected by PCR in the trigeminal ganglia of the other 3 goats. [ABSTRACT FROM AUTHOR]

Herpesvirus infections are generally subjected to strong host species restriction, although virological and serological investigations have revealed the possibility of cross-species infections in closely related animal species. In this study we evaluated susceptibility of goats to infection by Bubaline alphaherpesvirus 1 (BuHV-1). Four goats were inoculated intra-nasally with BuHV-1 and monitored clinically, virologically and serologically for 42 days. None of the goats displayed clinical signs although all the animals variably shed the virus by the nasal route during the first 12 days after infection. BuHV-1 was also detected in the white blood cells of two animals in the first week post infection. The results suggest that goats are susceptible to BuHV-1 infection and that they could play an epidemiological role in the circulation/transmission of the virus among domestic and wild ruminants and impact to some extent on the control plans for herpesviruses in cattle. [ABSTRACT FROM AUTHOR]

Bocaparvovirus is a newly established genus within the family Parvoviridae and has been identified as a possible cause of enteric, respiratory, reproductive/neonatal and neurological disease in humans and several animal species. In this study, metagenomic analysis was used to identify and characterise a novel bocaparvovirus in the faeces of rabbits with enteric disease. To assess the prevalence of the novel virus, rectal swabs and faecal samples obtained from rabbits with and without diarrhoea were screened with a specific PCR assay. The complete genome sequence of the novel parvovirus was reconstructed. The virus was distantly related to other bocaparvoviruses; the three ORFs shared 53%, 53% and 50% nucleotide identity, respectively, to homologous genes of porcine bocaparvoviruses. The virus was detected in 8/29 (28%) and 16/95 (17%) samples of rabbits with and without diarrhoea, respectively. Sequencing of the capsid protein fragment targeted by the diagnostic PCR identified two distinct bocaparvovirus populations/sub-types, with 91.7–94.5% nucleotide identity to each other. Including these novel parvoviruses in diagnostic algorithms of rabbit diseases might help inform their potential pathogenic role and impact on rabbit production and the virological profiles of laboratory rabbits. [ABSTRACT FROM AUTHOR]

Caprine herpesvirus 1 (CpHV-1) infection in goats induces genital vesicular-ulcerative lesions that strictly resemble the lesions induced by herpesvirus 2 in the human host. The immunosuppressive drug Mizoribine (MIZ) was found to increase the antiviral activity of Acyclovir (ACV) against herpesvirus infections, raising interesting perspectives on new combined therapeutic strategies. In this study the anti-CpHV-1 activity in vitro of ACV alone or in combination with MIZ was characterized. When applied alone at non-toxic concentrations, ACV had a slight effect on CpHV-1 replication while in combination with MIZ a dose-dependent inhibition of the virus yield was observed with an IC50 of ACV of 28.5 μM. These findings suggest that combined therapy of ACV and MIZ is potentially exploitable in the treatment of genital infection by herpesviruses. Caprine herpesvirus 1 (CpHV-1) infection in goats induces genital vesicular-ulcerative lesions that strictly resemble the lesions induced by herpesvirus 2 in the human host. The immunosuppressive drug Mizoribine (MIZ) was found to increase the antiviral activity of Acyclovir (ACV) against herpesvirus infections, raising interesting perspectives on new combined therapeutic strategies. In this study the anti-CpHV-1 activity in vitro of ACV alone or in combination with MIZ was characterized. When applied alone at non-toxic concentrations, ACV had a slight effect on CpHV-1 replication while in combination with MIZ a dose-dependent inhibition of the virus yield was observed with an IC50 of ACV of 28.5 μM. These findings suggest that combined therapy of ACV and MIZ is potentially exploitable in the treatment of genital infection by herpesviruses. [ABSTRACT FROM AUTHOR]

The results of a study designed to evaluate the performance of an in-clinic test for the detection of canine parvovirus (CPV) are reported. A total of 150 faecal samples collected from dogs with acute diarrhoea were tested using the in-clinic test, a haemagglutination assay (HA) and a real-time PCR assay for CPV detection, quantification and characterisation. CPV was detected in 66, 73, and 101 faecal samples by in-clinic, HA and PCR testing, respectively. The relative sensitivity and specificity of the in-clinic test were 86.3% and 96.1%, respectively, when the test was compared to HA, and 65.3% and 100%, respectively, when compared to real-time PCR. The sample distribution according to the virus type was CPV-2a, n = 44; CPV-2b, n = 11; CPV-2c, n = 44, CPV-2, n = 2, as determined by minor groove binder probe assays and/or sequence analysis. The percentage of positive in-clinic tests was 70.5% for CPV-2a, 72.7% for CPV-2b and 75.0% for CPV-2c (P> 0.05). Using real-time PCR as the reference standard for CPV detection, the in-clinic test was more specific than HA and had comparable sensitivity to HA, demonstrating detection rates similar to those previously observed for other rapid in-clinic tests. The in-clinic test was also able to detect all CPV types at equivalent rates. [ABSTRACT FROM AUTHOR]

Abstract: Canine coronavirus (CCoV) is an enveloped RNA virus, responsible for gastrointestinal infection in dogs. To date, two different CCoV genotypes have been recognized, CCoV type I and CCoV type II. Recently, CCoV type II strains of potential recombinant origin with transmissible gastroenteritis virus (TGEV) were detected and characterized as a new subtype (CCoV-IIb) of canine coronavirus, in order to be differentiated from the “classical” CCoV type II strains (CCoV-IIa). In the present study, two CCoV-IIb strains were detected in the faeces and internal organs of two puppies, which died after presenting gastrointestinal symptoms. Mixed infection of both subtypes (CCoV-IIa/IIb) was detected in the faeces, while only CCoV-IIb was detected in the organs. Puppies were also infected by canine parvovirus type 2 (CPV-2). Both CCoV-IIb strains were isolated on cell cultures and subjected to sequence analysis and phylogeny. By means of RT-PCR and real time RT-PCR assays, tissue distribution and quantitation of viral loads took place. These cases represent the first description of tissue distribution and quantitation of CCoV-IIb strains, detected in the organs. The detection of CCoV-IIa strains, which is restricted to the faeces, suggests that CCoV-IIb strains may have an advantage in disseminating throughout a dog with CPV-2 coinfection, in contrast to common enteric CCoV-IIa strains. [Copyright &y& Elsevier]

Abstract: Both egg- and cell-adapted canine distemper virus (CDV) vaccines are suspected to retain residual virulence, especially if administered to immuno-suppressed animals, very young pups or to highly susceptible animal species. In the early 1980s, post-vaccine encephalitis was reported in dogs from various parts of Britain after administration of a particular batch of combined CDV Rockborn strain/canine adenovirus type-1 vaccine, although incrimination of the Rockborn strain was subsequently retracted. Notwithstanding, this, and other reports, led to the view that the Rockborn strain is less attenuated and less safe than other CDV vaccines, and the Rockborn strain was officially withdrawn from the markets in the mid 1990s. By sequencing the H gene of the strain Rockborn from the 46th laboratory passage, and a commercial vaccine (Candur® SH+P, Hoechst Rousell Vet GmbH), the virus was found to differ from the commonly used vaccine strain, Onderstepoort (93.0% nt and 91.7% aa), and to resemble more closely (99.6% nt and 99.3% aa) a CDV strain detected in China from a Lesser Panda (Ailurus fulgens). An additional four CDV strains matching (>99% nt identity) the Rockborn virus were identified in the sequence databases. Also, Rockborn-like strains were identified in two vaccines currently in the market. These findings indicate that Rockborn-like viruses may be recovered from dogs or other carnivores with distemper, suggesting cases of residual virulence of vaccines, or circulation of vaccine-derived Rockborn-like viruses in the field. [ABSTRACT FROM AUTHOR]

Abstract: Water buffalo (Bubalus bubalis) are affected by high rates of embryonic mortality and abortion related to infectious diseases and non-infectious factors. A number of viral and bacterial infections have been associated with reproductive failure, but there is limited information on the role of chlamydial infections. In order to investigate the presence and the role of Chlamydiaceae in water buffalo a retrospective study was performed in a herd with a history of reproductive failure. During an 11-month period, the pregnant heifers suffered an abortion rate of 36.8% between the 3rd and 7th month of pregnancy. Antibodies to Chlamydiaceae were detected in 57% of the aborted cows, and in 0% of the overtly healthy cows used as control. By a nested-PCR assay, three of 14 vaginal swabs from aborted animals tested positive for Chlamydophila agents and, additionally, three out of seven aborted fetuses tested positive for Chlamydophila spp., with two being co-infections by Cp. abortus and Cp. pecorum and one being characterised as Cp. abortus. Sequence analysis of the amplicons confirmed the results of the nested-PCR. The presence of anti-Chlamydiaceae antibodies in more than half of the aborting animals (P <0.002) and the detection of Chlamydophila agents in several fetal organs and in the vaginal swabs are consistent with the history of abortions observed in the herd and suggest an abortifacient role by Chlamydophila spp. in water buffalo (B. Bubalis) herds. [Copyright &y& Elsevier]

Abstract: Caprine herpesvirus 1 provides a unique virus–animal model to investigate potential tools applicable for the therapy and prophylaxis of genital herpesvirus infections of humans. In order to evaluate the efficacy of mucosal immunization in the goat model, an inactivated CpHV-1 vaccine was adjuvated with the enzymatically inactive mutant of the heat-labile enterotoxin of Escherichia coli, LTK63, and used to immunize goats by the vaginal route, by administering two doses at a 3-week interval. The mucosal vaccine was safe, as neither local nor systemic reactions were associated with the vaccine administration. The vaccinated animals displayed high levels of secretory IgA and were significantly protected after challenge with the virulent CpHV-1 strain, with marked decrease in virus shedding, while the unvaccinated goats were not. These findings suggest that mucosal immunization is potentially exploitable in the control of genital infection by herpesviruses. [Copyright &y& Elsevier]

Abstract: Four outbreaks of infectious canine hepatitis (ICH) occurring in Italy between 2001 and 2006 are reported. Three outbreaks were observed in animal shelters of southern Italy, whereas a fourth outbreak involved two purebred pups imported from Hungary few days before the onset of clinical symptoms. In all outbreaks canine adenovirus type 1 (CAV-1) was identified by virus isolation and PCR. In three outbreaks, other canine viral pathogens were detected, including canine distemper virus, canine parvovirus or canine coronavirus. The present study shows that CAV-1 is currently circulating in the Italian dog population and that vaccination is still required. [Copyright &y& Elsevier]

Abstract: A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (101 copies of standard DNA and 3×101 infected erythrocytes ml−1 of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n =51; sheep, n =2; goats, n =1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60×103 to 5.70×108 copiesml−1 of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs. [Copyright &y& Elsevier]

Abstract: Canine coronavirus (CCoV) is widespread in dogs in several countries and causes mild enteric illness evolving to severe enteritis in young pups. In in vitro cultures canine coronaviruses generally induce extensive cell death, however nature of the events leading to cell death remains largely unknown. We analysed the induction of cytopathic effect by CCoV in a canine fibrosarcoma cell line (A-72) in order to characterize the apoptotic effect in homologous cell system. Following CCoV infection A-72 cell line, which is permissive to CCoV, showed reduced growth rate, as detected by MTT assay, a standard colorimetric assay for measuring cellular proliferation, and underwent to apoptotic death. Starting from 24h after CCoV infection, cells morphology appeared dramatically changed, with cells rounding and detachment from culture surface. Morphologic and biochemical features of apoptosis, such as blebbing of the plasma membrane, translocation of phosphatidilserine to cell surface and annexin V positive staining, nuclear fragmentation, apoptotic bodies formation and DNA laddering, were detected in CCoV-infected cells. Propidium iodide staining of infected culture indicated the appearance of hypodiploid DNA peak corresponding to apoptotic cell population. Commonly to other animal coronavirus infection caspase-3 is likely to contribute to the execution phase of apoptosis induced by CCoV in A-72 cells since we found activation of enzymatic activity as well as procaspase-3 activating cleavage. Apoptotic death of infected cells is detrimental as it causes cell and tissue destruction as well as inflammatory responses. Therefore in the case of CCoV associated gastroenteritis, apoptosis of epithelial mucosa cells may be responsible for pathology induced by CCoV infection. [Copyright &y& Elsevier]

Abstract: Canine distemper virus (CDV) is a highly contagious viral pathogen causing lethal disease in dogs and other mammalians. A high degree of genetic variation is found between recent CDV strains and the old CDV isolates used in the vaccines and such genetic variation is regarded as a possible cause of the increasing number of CDV-related diseases in dogs. The H gene shows the greatest extent of genetic variation that allows for distinction of various lineages, according to a geographical pattern of distribution and irrespective of the species of identification. In the present study, hemagglutinin (H) genes obtained from field strains detected from clinical specimens of Italian dogs were analyzed genetically. Phylogenetic analysis revealed that a homogeneous group of CDV strains is widespread in Italian dogs, all which are included into the European lineage. Unexpectedly, strains 179/04 and 48/05 clustered along with CDVs of the Arctic lineage, the highest identity being to strain GR88 (98.0 and 98.4%aa, respectively). The full-length sequence of a red fox CDV strain, 207/00 was also determined and analyzed. The H protein of the fox CDV strain was unrelated to strains within the major European lineage. These results suggest that at least three different CDV lineages are present in Italy. [Copyright &y& Elsevier]

Abstract: An epidemiological survey was carried out to investigate the distribution of the VP7 and VP4 specificities of lapine rotaviruses (LRVs) in rabbitries from different geographical regions of Italy. Almost all the strains were characterized as P[22],G3, confirming the presence of the newly-recognized rotavirus P[22] VP4 allele in Italian rabbits. Only one P[14],G3 LRV strain was identified and two samples contained a mixed (P[14]+[22],G3) rotavirus infection. All the LRV strains analyzed exhibited a genogroup I VP6 specificity and a long dsRNA electropherotype. However, one of the P[14],G3 strains possessed a super-short pattern. Altogether, these data highlight the epidemiological relevance of the P[22] LRVs in Italian rabbitries. [Copyright &y& Elsevier]

The safety and the efficacy of a modified-live (ML) canine coronavirus (CCoV) vaccine strain 257/98-3c was evaluated in 14 dogs seronegative and virus negative for CCoV. For the safety test, four dogs were inoculated, two by intramuscular and two by oronasal route, with 10 times the vaccinal dose. During the observation period (28 days) all dogs did not display any local or systemic reaction. For the efficacy test, eight dogs were vaccinated by intramuscular (four dogs—group A) or by oronasal route (four dogs—group B). Two dogs were maintained as non-vaccinated controls. In the dogs of group A, vaccinal virus was not detected in faecal samples by virus isolation (VI) and by PCR assay, while in the dogs of group B, the virus was revealed for six median days only by PCR. Twenty-eight days later, the vaccinated and control dogs were challenged with a field CCoV strain. After the challenge, the vaccinated dogs did not display clinical signs and the dogs of group A shed virus for 5.5 median days, evaluated by VI, and for 10 median days evaluated by PCR. Virus shedding was not observed, both by VI and PCR assay, in the dogs of group B. The two control dogs displayed moderate clinical signs and the virus was detected by VI for 14.5 median days starting from day 3 post-challenge (dpc 3) and by PCR assay for 23 median days starting from dpc 1. [Copyright &y& Elsevier]

Three goats from a group of five caprine herpesvirus 1 (CpHV.1) seronegative pregnant goats were inoculated intranasally with a virulent BA.1 strain of CpHV.1. Goat n.1 was infected on day 45 of pregnancy, goat n.2 on day 92 and goat n.3 on day 127. Each of the three goats produced a single foetus 10–60 days after infection. Foetus n.1 was never found and so it could not be examined for virological findings. Goat n.2 delivered at term of gestation and CpHV.1 was detected by PCR and isolated from most of the foetal organs. Foetus n.3 was partially autolysed and the virus was only detected by PCR but not isolated from foetal organs. The results confirm the damaging effect of CpHV.1 infection on pregnancy, the difficulty in diagnosing the CpHV.1 induced abortion, and the importance developing appropriate prophylactic programmes. [Copyright &y& Elsevier]

• Evaluation of passive immunity facilitates effective vaccination in puppies. • A haemagglutination inhibition assay was modified to improve interpretation. • Pre-treatment of sera with red blood cells prevented nonspecific hemagglutination. • Lowering the concentration of red blood cells prevented artefactual precipitation. Canine parvovirus type 2 (CPV-2) infection is associated with severe gastroenteritis in puppies. Quantification of CPV-2 specific antibodies before vaccination can reveal the presence of interfering maternal-derived immunity and facilitate timing of effective immunisation. Inhibition of haemagglutination (HI) is commonly used to measure CPV-2-specific antibody levels in serum. However, the presence of nonspecific agglutinins in canine serum and artefactual precipitation of red blood cells (RBC) are both limitations of the assay. In this study, we compared the standard HI protocol with a refined HI protocol, in which canine serum was pre-incubated with porcine RBC for 12 h to remove nonspecific agglutinins and a lower concentration (0.1% vs. 0.8%) of porcine RBC suspensions was used to limit artefactual precipitation of RBC. A panel of canine sera, collected from 80 dogs of different ages and with different neutralising antibody titres, was analysed. Nonspecific agglutinins were identified in most (97%) serum samples from puppies <4 months of age and in only 7% dogs 6 months old. Pre-treatment of serum samples was effective in removing nonspecific agglutinins from all samples and artefactual precipitation of RBCs was not noted when 0.1% RBC suspensions were used. Refinement of the HI protocol has increased the accuracy of interpretation and reduced the interference of nonspecific agglutinins, primarily seen in puppies. This reduces the likelihood of incorrect assessment of passive or active immunity in puppies when deciding whether to administer or defer vaccination, which could potentially leave them susceptible to CPV-2 infection. [ABSTRACT FROM AUTHOR]