Specific Pathogen-free Status Research Articles

Monitoring swine virus transmission in embryos derived from commercial abattoir oocytes.

Pigs are pivotal in agriculture and biomedical research and hold promise for xenotransplantation. Specific-pathogen-free (SPF) herds are essential for commercial swine production and xenotransplantation research facilities. Commercial herds aim to safeguard animal health, welfare, and productivity, and research facilities require SPF status to protect immunocompromised patients. Somatic cell nuclear transfer (SCNT) embryos are the norm for producing cloned and genetically edited animals. Oocytes for embryo reconstruction are most conveniently sourced from commercial abattoirs with unclear disease statuses. However, research on viral clearance from donor oocytes during embryo reconstruction remains limited. SCNT has previously been shown to reduce the transmission of Porcine reproductive and respiratory syndrome virus, Bovine viral diarrhea virus, Porcine Circovirus type 2, and Porcine parvovirus. Still, it is lacking for other pathogens, including endogenous viruses. This project contains two preliminary studies investigating the polymerase chain reaction (PCR) assay detection of common swine viruses through the phases of producing parthenogenic and SCNT embryos. Exogenous pathogens detected in oocyte donor tissue or the oocyte maturation media were not detected in the produced embryos. Porcine endogenous retrovirus type C (PERVC) was not removed by parthenogenic embryo activation and was detected in 1 of the 2 tested SCNT embryos reconstructed using a PERVC-negative cell line. SCNT and parthenogenic embryo construction similarly reduced exogenous virus detection. SCNT embryo construction helped reduce endogenous virus detection. This project demonstrates the importance of screening embryos for endogenous viruses and shows the usefulness of parthenogenic embryos in future exogenous virus clearance studies.

The relationship of maternal rank, 5-HTTLPR genotype, and MAOA-LPR genotype to temperament in infant rhesus monkeys (Macaca mulatta).

Temperament is a construct whose manifestations are quantifiable from an early age, and whose origins have been proposed as "biological." Our goal was to determine whether maternal rank and infant genotype are associated with five measures of temperament in 3- to 4-month old rhesus monkeys (Macaca mulatta), all of whom were born and reared by their mothers in large, outdoor, half-acre cages. Maternal rank was defined as the proportion of animals outranked by each female, and the two genes of interest to us were monoamine oxidase and serotonin transporter, both of which are polymorphic in their promoter regions (MAOA-LPR and 5-HTTLPR, respectively), with one allele of each gene considered a "plasticity" allele, conferring increased sensitivity to environmental events. Our large sample size (n = 2014-3140) enabled us to examine the effects of individual genotypes rather than combining genotypes as is often done. Rank was positively associated with Confident temperament, but only for animals with the 5-repeat allele for MAOA-LPR. Rank had no other effect on temperament. In contrast, genotype had many different effects, with 5-HTTLPR associated with behavioral inhibition, and MAOA-LPR associated with ratings-based measures of temperament. We also examined the joint effect of the two genotypes and found some evidence for a dose-response: animals with the plasticity alleles for both genes were more likely to be behaviorally inhibited. Our results suggest phenotypic differences between animals possessing alleles for MAOA-LPR that show functional equivalence based on in vitro tests, and our data for 5-HTTLPR revealed differences between short/short homozygotes and long/short heterozygotes, strongly suggesting that combining genotypes for statistical analysis should be avoided if possible. Our analysis also provides evidence of sex differences in temperament, and, to our knowledge, the only evidence of differences in temperament based on specific pathogen-freestatus. We suggest several directions for future research.

Specific Pathogen-Free Animals for Civilian and Military Trauma: a Cautionary Note in the Translation of New Drug Therapies.

Specific-pathogen free (SPF) animals were introduced into biomedical research in the early 1960s to reduce the incidence of disease into experimental design. The goal was to provide animals with selected microbiota compatible with sustained health. Sixty years later, SPF status has become a variable itself in biomedical research. Alterations in the gut microbiome-host relationship can profoundly influence basic physiology, immune/inflammatory function, susceptibility to infection and disease, and behavior. In addition, it can influence the translational success of a drug or technology from animal models to humans. We discuss this aspect of SPF status in animal models used for military or civilian trauma and shock research. Currently, there is a broad spectrum of SPF exclusion and inclusion criteria which vary from one supplier or animal husbandry facility. If translation to humans is the end-game of trauma research, we recommend replicating a gut microbiome similar to the wild-type for optimal success. We further suggest that at the end of each publication a URL access be provided on Animal Microbial/Pathogen Exclusion Status that a study was based upon. This may help address the differences in results within a single laboratory or between laboratories around the world and improve translation success.

Facts, truths and myths about SPF shrimp in Aquaculture

AbstractShrimp domestication and genetic improvement programmes began in late 1980s, in the United States of America, under the United States Marine Shrimp Farming Program (USMSFP), using the Pacific whiteleg shrimp Penaeus vannamei. The USMSFP was based on proven concepts from the livestock and poultry industries and began with establishing a specific pathogen‐free (SPF) shrimp stock. The original shrimp stock was obtained using rigorous screening of captured wild shrimp for selection of individuals naturally free of major shrimp pathogens. Although the concept of SPF animals was well defined for terrestrial animals, it was relatively new for aquaculture, and it took some time to be adopted by the aquaculture community. In the early 1990s, parallel to USMSFP, several other programmes on genetic improvement of shrimp were also initiated in Latin America. Subsequently, several new terminologies and products, such as specific pathogen resistant (SPR) shrimp, specific pathogen tolerant (SPT) shrimp and even ‘all pathogen exposed’ (APE) shrimp, entered the shrimp industry vocabulary and became commercial. This led to confusion in the shrimp industry about the meaning, relationship and significance of these new terms with respect to SPF. This position paper attempts to clarify these concepts, provide science‐based definitions, reconfirms the importance of developing, maintaining and using domesticated, specific pathogen‐free (SPF) shrimp stocks (which may also achieve SPR and/or SPT status) to reduce the risk of disease outbreaks and increase production and profit. The same principles would apply to development of domesticated SPF stocks for other species used in aquaculture. The paper also discusses the difficulties of confirming and certifying SPF status due to the presence of endogenous viral elements (EVEs) and calls for internationally agreed science and evidence‐based technical guidelines for producing healthy shrimp.

Detection of IHHN Virus in a Specific Pathogen Free Bonafide <i>Penaeus vannamei</i> (Boone, 1931) Broodstock Consignment Imported to India

The World Organization for Animal Health (formerly Office International des Epizooties OIE) listed pathogenic shrimp virus, infectious hypodermal haematopoetic necrosis virus (IHHNV), has been detected in a specific pathogen free (SPF) declared consignment imported from an approved SPF vannamei broodstock supplier to India. The Aquatic Quarantine Facility (AQF), Chennai the only Government-approved quarantine premise for ensuring the SPF status of imported Penaeus vannamei broodstock detected this pathogen during its routine screening process. The AQF, which has so far screened about 1068 SPF batches of vannamei consignment, recorded that one particular batch imported to India was not SPF, though the exporting country declared the stock as SPF. The presence of the viral pathogen was confirmed by both nested PCR and quantitative PCR (qPCR). Sequencing data of the target viral genome revealed 100% similarity with the already published decapod penstyldensovirus-1 isolate IHHNV partial genome (GenBank accession no. KJ862253). The test results declared by AQF were further validated by the Central Institute of Brackishwater Aquaculture, Chennai, which is the referral laboratory of AQF. As the import of non-SPF stock into the country is a source of biosecurity threat, the entire batch was incinerated according to the standard operating procedure of AQF. Thus the stringent biosecurity measures and disease diagnostic strategies followed at AQF served to safeguard the industry from this virus, which would have otherwise gained entry into the country through the falsely claimed SPF stock. This communication highlights the importance of SPF stock and detection of IHHNV.

Effect of Zbtb33 gene knockout and bacterial lipopolysaccharide on home cage behavior in mice

The Zbtb33 gene encodes the bimodal transcriptional repressor Kaiso, which causes epigenetic repression of genes by binding to methylated mCpG islets in the promoters of the genes. Despite the fact that Kaiso is intensively expressed in the central nervous system, its participation in the regulation of behavior is still poorly understood. Only the participation of Kaiso in the regulation of the behavioral response to emotional stress in the open field and forced swimming tests has been shown. The aim of this study is to elucidate the role that Kaiso plays in regulating daily activity, as well as the behavioral response to stimulation of nonspecific immunity. Experiments were performed on adult male mice with Zbtb33 gene knockout (KO) and animals of the C57BL/6 line (wild type, WT). All animals were 11 weeks old, weighed 26 ± 1 g and had SPF (specific pathogen free) status throughout the experiment. The animals of each genotype were divided into three weighted groups of 8 animals each. Initially, the daily dynamics of motor activity, sleep, food and water intake of intact animals was measured using the PhenoMaster software-hardware complex. The animals of each group were then injected with saline (control), 0.1 or 1.0 mg/kg of bacterial lipopolysaccharide (LPS) dissolved in saline, and again measured for their daily activity, food and water intake. Intact KO and WT mice did not differ in the average daily motor activity and sleep duration. However, intact KO mice were less active in the dark time, and also consumed less food and water as compared to intact WT animals. LPS at both doses suppressed motor activity, prolonged sleep duration and caused anorexia in mice of both genotypes. However, the effect of low dose of LPS (0.1 mg/kg) on the food and water intake was more pronounced in KO mice than in WT animals. The results shed light on the biological significance of the Kaiso gene and serve as a justification for the necessity of the normal functioning of this gene in natural populations.

Effects of reproductive technologies and SPF status on some physiological and behavioral characteristics in rats with arterial hypertension (ISIAH Strain)

Modern standards of Laboratory Animal Science include working with laboratory animals of a high quality, in particular, with specific pathogen-free (SPF) mice and rats. On the other hand, assisted reproductive technologies (ART) are widely used in modern medicine for human infertility treatment and genome resource banking. In the present study, a comparison of body weight, blood pressure (BP), and behavior in the elevated plus maze (EPM) test was made between three groups of ISIAH (inherited stress induced arterial hypertension) rats: a group of animals that were born and raised in a conventional animal facility and two groups from an SPF animal facility (animals born naturally and animals resulting from ART). There were no changes in BP between the groups, but the behavior of ISIAH rats differed depending on rearing conditions. In particular, the grooming time, as well as the number of defecations and the number of urinations during the test decreased in both groups of ISIAH rats born in the SPF animal facility as compared to ISIAH rats born in the conventional animal facility. The behavior of the ISIAH rat offspring resulting from ART was different from that of the naturally born group: the EPM test revealed reduced anxiety in the former. The results of the present study indicate that the rearing conditions and the reproductive technologies affect some behavioral characteristics in adult ISIAH rats, although they posessed arterial hypertension in all the conditions used in this study.

Very Virulent Infectious Bursal Disease Virus Produces More-Severe Disease and Lesions in Specific-Pathogen-Free (SPF) Leghorns Than in SPF Broiler Chickens.

Infectious bursal disease virus (IBDV) is an important pathogen of chickens causing negative economic impacts in poultry industries worldwide. IBDV has a variable range of virulence, with very virulent (vvIBDV) strains being responsible for the greatest losses from mortality and decreased performance. Previous vvIBDV studies using conventional broilers reported resistance to lethal effects and decreased performance as compared to specific-pathogen-free (SPF) layers, but the potential contribution of the conventional vs. SPF status to resistance has not been examined. In this study we compared differences in the acute pathologic effects of infection by the California rA strain of vvIBDV for SPF white leghorn egg-laying chickens and SPF white Plymouth Rock broiler chickens over a 7-day experimental period. Based on the clinical signs and mortality observed, as well as on themore-severe pathologic changes in lymphoid tissues and kidneys, white leghorns were shown to be more susceptible to thedeleterious effects of vvIBDV infection than were white Plymouth Rocks. This study provides important information on theimpact of chicken breed on susceptibility to vvIBDV and the absence of impact from conventional vs. SPF status on the outcome.

Improving immunity in the elderly: current and future lessons from nonhuman primate models

The immune system must overcome daily challenges from pathogens to protect the body from infection. The success of the immune response to infection relies on the ability to sense and evaluate microbial threats and organize their elimination, while limiting damage to host tissues. This delicate balance is achieved through coordinated action of the innate and adaptive arms of the immune system. Aging results in several structural and functional changes in the immune system, often described under the umbrella term "immune senescence". Age-related changes affect both the innate and adaptive arms of the immune system and are believed to result in increased susceptibility and severity of infectious diseases, which is further exacerbated by reduced vaccine efficacy in the elderly. Therefore, multiple strategies to improve immune function in the aged are being investigated. Traditionally, studies on immune senescence are conducted using inbred specific pathogen free (SPF) rodents. This animal model has provided invaluable insight into the mechanisms of aging. However, the limited genetic heterogeneity and the SPF status of this model restrict the successful transfer of immunological discoveries between murine models and the clinical setting. More recently, nonhuman primates (NHPs) have emerged as a leading translational model to investigate immune senescence and to test interventions aimed at delaying/reversing age-related changes in immune function. In this article, we review and summarize advances in immuno-restorative approaches investigated in the NHP model system and discuss where the NHP model can support the development of novel therapeutics.

Role of Housing Modalities on Management and Surveillance Strategies for Adventitious Agents of Rodents

Specific pathogen-free (SPF) rodents for modern biomedical research need to be free of pathogens and other infectious agents that may not produce disease but nevertheless cause research interference. To meet this need, rodents have been rederived to eliminate adventitious agents and then housed in room- to cage-level barrier systems to exclude microbial contaminants. Because barriers can and do fail, routine health monitoring (HM) is necessary to verify the SPF status of colonies. Testing without strict adherence to biosecurity practices, however, can lead to the inadvertent transfer of unrecognized, inapparent agents among institutions and colonies. Microisolation caging systems have become popular for housing SPF rodents because they are versatile and provide a highly effective cage-level barrier to the entry and spread of adventitious agents. But when a microisolation-caged colony is contaminated, the cage-level barrier impedes the spread of infection and so the prevalence of infection is often low, which increases the chance of missing a contamination and complicates the corroboration of unexpected positive findings. The expanding production of genetically engineered mutant (GEM) rodent strains at research institutions, where biosecurity practices vary and the risk of microbial contamination can be high, underscores the importance of accurate HM results in mitigating the risk of the introduction and spread of microbial contaminants with the exchange of mutant rodent strains among investigators and institutions.

The use of endogenous and exogenous reference RNAs for qualitative and quantitative detection of PRRSV in porcine semen

Semen is known to be a route of porcine reproductive and respiratory syndrome virus (PRRSV) transmission. A method was developed for qualitative and quantitative detection of the seminal cell-associated PRRSV RNA in relation to endogenous and exogenous reference RNAs. As endogenous control for one-step real-time reverse transcription (RT)-PCR UBE2D2 mRNA was selected. Particularly for the analysis of persistent infections associated with low copy numbers of PRRSV RNA, UBE2D2 mRNA is an ideal control due to its low expression in seminal cells and its detection in all samples analysed ( n = 36). However, the amount of UBE2D2 mRNA in porcine semen varied (up to 106-fold), thus its use is limited to qualitative detection of PRRSV RNA. For quantitation, a synthetic, non-metazoan RNA was added to the RNA isolation reaction at an exact copy number. The photosynthesis gene ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit ( rbcL) from Arabidopsis thaliana was used as an exogenous spike. Unexpectedly, PRRSV RNA was detected in a herd of specific pathogen-free (SPF) boars which were tested ELISA-negative for anti-PRRSV antibodies. Therefore, RT-PCR for seminal cell-associated PRRSV is a powerful tool for managing the SPF status during quarantine programs and for routine outbreak investigations.

Age-related morphological changes in kidneys of SPF C57BL/6Cr mice maintained under controlled conditions.

Age-related morphological changes were examined in the kidneys of inbred C57BL/6Cr mice maintained in a controlled environment. The specific pathogen free status of animals used in the present study was confirmed by microbiological monitoring. Kidneys were histologically and histometrically investigated at 3, 5, 12, 15, 24 and 27-months-old. Kidney weights did not change with age. Renal corpuscles increased in number at 24- and 27-months-old, but diameter remained constant. The percentage of renal corpuscles with a cuboidal glomerular capsule decreased at 24- and 27-months-old. Score indicating glomerular damage increased from 5- to 27-months-old. Changes to the proximal convoluted tubules were severe. Vacuolar degeneration was observed from 12-months-old. Tubular atrophy was observed at 24- and 27-months-old, with number of nuclei per unit area increasing at the same ages. Amyloidosis and scar lesions were observed at 27-months-old. Focal cell infiltration around vessels was found at 24- and 27-months-old. Electron microscopy at 27-months-old revealed expansion of the mesangial matrices and fusion of foot processes in the glomeruli. Enlarged lysosomes with lipid content were observed in the proximal convoluted tubules.

Prevalence of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in Litopenaeus vannamei in the Pacific Ocean off the Coast of Panama

Abstract.— In March 2000, 104 wild caught Litopenaeus wannamei broodstock, captured off the Pacific coast of Panama, were screened for the following penaeid viruses: infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV). The purpose of this study was to determine the prevalence of IHHNV and WSSV in wild shrimp in this area of the Western Hemisphere and to acquire specific pathogen free (SPF) L. vannamei for inclusion into the Oceanic Institute's genetic breeding program. The prevalence of the viruses was determined using the dot blot hybridization format, which is a commercially available molecular method for detecting these viruses. Dot blot hybridization assays can be used as an initial screening method to detect moderately to highly infected shrimp. The results from the dot blot assays indicated the prevalence of IHHNV in 28% and WSSV in 2% of the 104 hemolyrnph samples tested. Results from this study were used to establish the initial candidate SPF status of the animals that were assessed and to determine the prevalence of two serious pathogens of penaeid shrimp captured from the wild of the Pacific Ocean in the Central American region off the coast of Panama.

Associations of Ascaris suum and Oesophagostomum spp. infections of sows with management factors in 83 Danish sow herds

Associations of the occurrence of Ascaris suum and Oesophagostomum spp. in sows with herd management factors were examined using logistic-binomial regression. The information used was from 11 to 78 sows sampled from each of 83 breeding herds (2961 sows in total) and examined for eggs per gram of faeces (EPG). A sow excreting at least 20 EPG was defined as ‘infected’. Management factors of the study herds were recorded using a questionnaire. A total of 263 sows from 50 herds (8.9% of sows) and 375 sows from 20 herds (12.7% of sows) were infected with A. suum and Oesophagostomum spp., respectively. For A. suum, sows from herds with more than 85 sows had significantly higher odds of being infected compared with those from 30 to 85 sows ( P < 0.05). When bedding was provided for sows, the odds of A. suum infection was 5.4 compared with sows from herds in which bedding was not provided ( P < 0.05). For Oesophagostomum spp., sows from herds with different specific pathogen free status had about one tenth the odds of being infected compared with those from conventional herds ( P < 0.05). Sows that had been treated with anthelmintics had very low odds of being infected with Oesophagostomum spp. compared with those that were not treated ( P < 0.05). The effects of these management factors in both final models did not differ when the definition of an ‘infected’ sow was changed. The present results suggest the importance of disposal of bedding material from pens in reducing the prevalence of A. suum in larger sow herds. Anthelmintic treatment is important in reducing the prevalence of Oesophagostomum spp. infection of sows.

The effect of vitamin A on leukotriene production in the ischemic rat brain

Human plasma leukotriene C4 (LTC4) levels of cerebral infarcted patients showed a significant increase and plasma vitamin A (VA) levels showed a significant decrease compared to the normal plasma obtained from age-matched control. Therefore, the effect of VA on leukotriene (LT) levels and the progress of cerebral edema were investigated in VA deficient Wistar rat brains. Incomplete global cerebral ischemia was induced by occluding the bilateral common carotid arteries (BLCO) with clips. Wistar rats were made VA deficient by feeding them a vitamin A deficient diet for 5 weeks on a specific pathogen free status. After 3 hours of BLCO the blood was reperfused by removing the clips. After each period of reperfusion, the rat brain was fixed by freezing in situ and used for assaying leukotrienes, vitamin A, and water content. Slow reacting substance of anaphylaxis (LTC4 + LTD4 + LTE4) levels showed an increase at the end of BLCO in the VA deficient group and the high levels persisted for 30 min and then decreased to the control level. Brain water contents were elevated significantly at the 30 min phase of reperfusion. With VA administration, the water contents tended to be lower than in the VA deficient group at any phase. Histologically, after ischemia and reperfusion, evans blue extravasation and marked spongioid formations around small vessels were observed in the VA deficient rats only. These facts indicate that VA functions to stabilize cell membranes and suppress ischemic cerebral damage.