Carrier Sows Research Articles

A natural knockout of the MYO7A gene leads to pre-weaning mortality in pigs.

SummaryThe pig breeding system provides a unique framework to study recessive defects and the consequence on the phenotype. We examined a commercial synthetic Duroc population for recessive defects and identified a haplotype on chromosome 9 significantly affecting pre‐weaning mortality. To identify the causal variant underlying the mortality, we examined sequence data of four carrier animals and 21 non‐carrier animals from the same population. The results yield a strong candidate causal stop‐gained variant (NM_001099928.1:c.541C>T) affecting the MYO7A gene in complete linkage disequilibrium with the lethal haplotype. The variant leads to an impaired (p.Gln181*) MYO7A protein that truncates 2032 amino acids from the protein. We examined a litter from a carrier sow inseminated by a carrier boar. From the resulting piglets, two confirmed homozygous piglets suffered from severe balance difficulties and the inability to walk properly. The variant segregates at a carrier frequency of 8.2% in the evaluated population and will be gradually purged from the population, improving animal welfare. Finally, this 'natural knockout' will increase our understanding of the functioning of the MYO7A gene and provides a potential model for Usher syndrome in humans.

DNA-type results of Landrace sows for RYR1-gene and its association with productivity

Recently the assessment of QTL genes has been a relevant focus of research. Among other genes, the RYR1 is one of the most important. Research on this gene for Landrace sows of Ukrainian selection has been insufficient. This fact confirms the need for our work. A total of 63 Landrace sows from different families were evaluated by reproductive qualities for two generations. A comprehensive assessment of the sows’ reproductive ability was performed using the SIRQS-index. Determination of polymorphism in the RYR1 gene in pigs was performed by using DNA-typing of animals. Genetic potential was calculated between two generations “mother-daughter”. By assessment of polymorphism of the RYR1 gene, it was found that 6.3% of the Landrace sows were the carriers for the mutant allele of the RYR1 gene. No animals with the RYR1nn genotype were detected. Accordingly, animals with the RYR1NN genotype accounted for 93.6%. The frequency of the N allele of the RYR1 gene was 0.97, the n frequency of the RYR1 gene allele was 0.03. Sows with RYR1NN genotype had a higher level of reproductive ability compared to the RYR1Nn genotype. RYR1NN genotype also had a higher level of genetic potential. The greatest progress was established between generations of Landrace sows which were carriers for the mutant allele. The highest values of this progress were by the NBA, the lowest – by the NW. On the contrary, there was regression between populations for part of the population (Landrace sows of Ukrainian selection of RYR1Nn genotype) by the all estimated indicators of reproductive ability. Sows that were free of the mutant allele of the RYR-1 gene had high SIRQS index. The phenotypic consolidation coefficients by the NBA were lower for sows free of the mutant allele than for its carrier. The advantage of sows free of the mutant allele of the RYR-1 gene over its carrier sows was established in almost all assessed indicators of reproductive ability. No significant differences in the level of consolidation of reproductive ability between sows with different allelic variants of the RYR-1 gene were established. The higher productive level of sows with RYR1NN genotype is reflected in the indicators of economic efficiency of production. It allows higher levels of profitability to be obtained and net profit to be increased by 1093 UAH compared to sows with RYR1Nn genotype. The further monitoring of the studied gene and the gradual elimination of carriers of the mutant allele is a promising direction in breeding work.

Detection of a Frameshift Deletion in the SPTBN4 Gene Leads to Prevention of Severe Myopathy and Postnatal Mortality in Pigs.

Piglet mortality is a complex phenotype that depends on the environment, selection on piglet health, but also on the interaction between the piglet and sow. However, also monogenic recessive defects contribute to piglet mortality. Selective breeding has decreased overall piglet mortality by improving both mothering abilities and piglet viability. However, variants underlying recessive monogenic defects are usually not well captured within the breeding values, potentially drifting to higher frequency as a result of intense selection or genetic drift. This study describes the identification by whole-genome sequencing of a recessive 16-bp deletion in the SPTBN4 gene causing postnatal mortality in a pig breeding line. The deletion induces a frameshift and a premature stop codon, producing an impaired and truncated spectrin beta non-erythrocytic 4 protein (SPTBN4). Applying medium density single nucleotide polymorphism (SNP) data available for all breeding animals, a pregnant carrier sow sired by a carrier boar was identified. Of the resulting piglets, two confirmed homozygous piglets suffered from severe myopathy, hind-limb paralysis, and tremors. Histopathological examination showed dispersed degeneration and decrease of cross-striations in the dorsal and hind-limb muscle fibers of the affected piglets. Hence, the affected piglets are unable to walk or drink, usually resulting in death within a few hours after birth. This study demonstrates how growing genomic resources in pig breeding can be applied to identify rare syndromes in breeding populations, that are usually poorly documented and often are not even known to have a genetic basis. The study allows to prevent carrier-by-carrier matings, thereby gradually decreasing the frequency of the detrimental allele and avoiding the birth of affected piglets, improving animal welfare. Finally, these “natural knockouts” increase our understanding of gene function within the mammalian clade, and provide a potential model for human disease.

Persistent Infection and Transmission of Senecavirus A from Carrier Sows to Contact Piglets.

Senecavirus A (SVA) is a picornavirus that causes acute vesicular disease (VD), that is clinically indistinguishable from foot-and-mouth disease (FMD), in pigs. Notably, SVA RNA has been detected in lymphoid tissues of infected animals several weeks following resolution of the clinical disease, suggesting that the virus may persist in select host tissues. Here, we investigated the occurrence of persistent SVA infection and the contribution of stressors (transportation, immunosuppression, or parturition) to acute disease and recrudescence from persistent SVA infection. Our results show that transportation stress leads to a slight increase in disease severity following infection. During persistence, transportation, immunosuppression, and parturition stressors did not lead to overt/recrudescent clinical disease, but intermittent viremia and virus shedding were detected up to day 60 postinfection (p.i.) in all treatment groups following stress stimulation. Notably, real-time PCR and in situ hybridization (ISH) assays confirmed that the tonsil harbors SVA RNA during the persistent phase of infection. Immunofluorescence assays (IFA) specific for double-stranded RNA (dsRNA) demonstrated the presence of double-stranded viral RNA in tonsillar cells. Most importantly, infectious SVA was isolated from the tonsil of two animals on day 60 p.i., confirming the occurrence of carrier animals following SVA infection. These findings were supported by the fact that contact piglets (11/44) born to persistently infected sows were infected by SVA, demonstrating successful transmission of the virus from carrier sows to contact piglets. Results here confirm the establishment of persistent infection by SVA and demonstrate successful transmission of the virus from persistently infected animals.IMPORTANCE Persistent viral infections have significant implications for disease control strategies. Previous studies demonstrated the persistence of SVA RNA in the tonsil of experimentally or naturally infected animals long after resolution of the clinical disease. Here, we showed that SVA establishes persistent infection in SVA-infected animals, with the tonsil serving as one of the sites of virus persistence. Importantly, persistently infected carrier animals shedding SVA in oral and nasal secretions or feces can serve as sources of infection to other susceptible animals, as evidenced by successful transmission of SVA from persistently infected sows to contact piglets. These findings unveil an important aspect of SVA infection biology, suggesting that persistently infected pigs may function as reservoirs for SVA.

Creating effective biocontainment facilities and maintenance protocols for raising specific pathogen-free, severe combined immunodeficient (SCID) pigs.

Severe combined immunodeficiency (SCID) is defined by the lack of an adaptive immune system. Mutations causing SCID are found naturally in humans, mice, horses, dogs, and recently in pigs, with the serendipitous discovery of the Iowa State University SCID pigs. As research models, SCID animals are naturally tolerant of xenotransplantation and offer valuable insight into research areas such as regenerative medicine, cancer therapy, as well as immune cell signaling mechanisms. Large-animal biomedical models, particularly pigs, are increasingly essential to advance the efficacy and safety of novel regenerative therapies on human disease. Thus, there is a need to create practical approaches to maintain hygienic severe immunocompromised porcine models for exploratory medical research. Such research often requires stable genetic lines for replication and survival of healthy SCID animals for months post-treatment. A further hurdle in the development of the ISU SCID pig as a biomedical model involved the establishment of facilities and protocols necessary to obtain clean SPF piglets from the conventional pig farm on which they were discovered. A colony of homozygous SCID boars and SPF carrier sows has been created and maintained through selective breeding, bone marrow transplants, innovative husbandry techniques, and the development of biocontainment facilities.

Antimicrobial Resistance Profile and Genotypic Characteristics of Streptococcus suis Capsular Type 2 Isolated from Clinical Carrier Sows and Diseased Pigs in China.

Streptococcus suis serotype 2 is an important zoonotic pathogen. Antimicrobial resistance phenotypes and genotypic characterizations of S. suis 2 from carrier sows and diseased pigs remain largely unknown. In this study, 96 swine S. suis type 2, 62 from healthy sows and 34 from diseased pigs, were analyzed. High frequency of tetracycline resistance was observed, followed by sulfonamides. The lowest resistance of S. suis 2 for β-lactams supports their use as the primary antibiotics to treat the infection of serotype 2. In contrast, 35 of 37 S. suis 2 with MLSB phenotypes were isolated from healthy sows, mostly encoded by the ermB and/or the mefA genes. Significantly lower frequency of mrp+/epf+/sly+ was observed among serotype 2 from healthy sows compared to those from diseased pigs. Furthermore, isolates from diseased pigs showed more homogeneously genetic patterns, with most of them clustered in pulsotypes A and E. The data indicate the genetic complexity of S. suis 2 between herds and a close linkage among isolates from healthy sows and diseased pigs. Moreover, many factors, such as extensive use of tetracycline or diffusion of Tn916 with tetM, might have favored for the pathogenicity and widespread dissemination of S. suis serotype 2.

A recent L1 insertion withinSPEF2gene is associated with changes inPRLRexpression in sow reproductive organs

The immotile short tail sperm (ISTS) defect was recognized in the Finnish Yorkshire population at the end of the 1990s when several affected boars were identified. The causal mutation for this defect is a recent L1 insertion within the SPEF2 gene. In 2001, the insertion frequency was already 0.23. Even though all homozygous boars are eliminated from the population due to infertility, the amount of affected boars increased rapidly until marker-assisted selection against the defect was established. Previously we identified an association between the L1 insertion and litter size in the first parity. In this study, we analyzed the expression of the genomic region adjacent to the L1 insertion on porcine chromosome 16. Based on the RNA-seq data analysis, prolactin receptor (PRLR) was identified as down-regulated in the oviduct of ISTS homozygous sows. Quantitative PCR (qPCR) analysis confirmed the significant down-regulation of PRLR in the ovary, oviduct, and uterus of ISTS homozygous and carrier sows compared with controls. In addition, three unannotated loci between PRLR and SPEF2 showed some transcription activity in the analyzed samples. We further investigated the possible mechanisms of the L1 influence on the decrease in the identified gene expression. The methylation pattern of the PRLR gene region appeared unaffected. However, reads mapping to the L1 sequence indicated an increase in L1 antisense promoter expression in the ISTS homozygous animals. The current data suggest that the presence of the L1 affects by some mechanism the expression patterns upstream of the insertion site.

Prevalence of Streptococcus suis Isolated from Clinically Healthy Sows in China

Streptococcus suis is an important pathogen in pigs. Transmission of this pathogen is generally believed to occur between healthy carrier sows and their offspring, so the carrier status of S. suis in healthy sows is important for the control of S. suis infections in pigs, especially in suckling and growing pigs. In this study, the prevalence of S. suis isolated from clinically healthy sows in China was studied for the first time. A total of 1 043 tonsil samples were collected from clinically healthy sows from 10 regions in China from 2005 to 2007. Among the 421 S. suis isolates, 31 strains were identified as capsular type 2. The results showed that S. suis was widespread in swine herds in China with the carrier rates in different herds ranging from 19.5 to 93.9%. Overall, 40.4 and 3.0% of clinically healthy sows harbored S. suis and capsular type 2 in their palatine tonsils, respectively. Statistically significant differences of carrier rates of S. suis and capsular type 2 between the different farms were observed, which was independent of herd sizes and geographic distributions of different herds.

In vitro antimicrobial susceptibility of Streptococcus suis strains isolated from clinically healthy sows in China

Streptococcus suis is an important pathogen in the swine industry. Because transmission is generally thought to occur between healthy carrier sows and their offspring, it is important to understand which antimicrobial agents are likely to be effective against the strains isolated. This study is the first to report on the antimicrobial susceptibility of S. suis isolated from clinical healthy sows. From 2005 to 2007 a total of 421 S. suis isolates were recovered from sows in China and subjected to antimicrobial susceptibility testing in accordance with the Clinical and Laboratory Standards Institute (CLSI) standards. High-level resistance were found with tetracycline (91.7%) and sulfisoxazole (86.7%), followed by clindamycin (68.4%), erythromycin (67.2%), tilmicosin (66.7%) and trimethoprim/sulfamethoxazole (59.1%). These six antimicrobial agents presented the highest MIC 50 values and the antibiogram (19.2%) most frequently observed. Lower resistance rates among the β-Lactams support their use as the primary drugs to treat the infection of S. suis. However, appropriate dosing or combination antibiotic therapeutic regimens should be adhered to in view of the resistant and intermediate strains to penicillin (9.5% and 42.3%), ampicillin (4.0% and 29.9%) and ceftiofur (22.1% and 37.3%), respectively.

Elimination of porcine reproductive and respiratory syndrome virus from a seedstock breeding farm and a supplying boar stud by a modified test and removal method

To eliminate porcine reproductive and respiratory syndrome virus (PRRSV) from a supplying boar stud, samples of serum and semen from 118 boars were assessed three times a month by an indirect fluorescent antibody (IFA) test to detect antibodies, and by a nested reverse transcriptase-PCR (nRT-PCR) to detect the genome of PRRSV. The boars detected as persistently infected carriers were culled. A PRRSV-negative population of boars was established after three months and no semen positive for the virus was detected for six months. Subsequently, a three-step plan was introduced to eliminate PRRSV from the seedstock breeding farm during three parity cycles on the farm over 15 months, each step taking five months. In step 1, umbilical cords taken from piglets at birth and serum samples taken from their dams at the start of weaning were subjected to ifa and nRT-pcr analysis. The sows with PRRSV detected in serum by nRT-pcr were regarded as carrier sows and culled. The rates of detection of PRRSV were reduced from 5 percent to 2.5 percent in the sera of the sows, and from 14.8 percent to 1.8 percent in the umbilical cords of the piglets. In step 2, the sows that had farrowed the piglets with PRRSV detected by nRT-PCR in their cords were considered to have transmitted the infection and removed. During step 2, the virus detection rates in umbilical cords by nRT-pcr were reduced, but not completely eliminated. In step 3, 10-week-old nursery pigs with antibodies to PRRSV in their serum by ifa and elisa were culled. The three steps established the PRRSV-negative state of the multisite farm containing the breeding and nursery farm, and the PRRSV-negative state of both the multisite farm and the supplying boar stud was evaluated by monthly monitoring over at least one parity cycle of the farm for five months.

Elimination of strains of Streptococcus suis serotype 2 from the tonsils of carrier sows by combined medication and vaccination

The effect of vaccination with a killed whole-cell vaccine of extracellular factor-positive Streptococcus suis serotype 2 (S suis 2 ef+) combined with medication with amoxicillin on the presence of virulent...

Significance of determining intrauterine infections with classical swine plague virus within programme of curbing and eradicating this disease

Intrauterine infection of the fetus occurs if a pregnant non-vaccinated sow is infected with the virus of classical swine plague (CSF). The infection of the fetus results in the occurrence of viremia and the distribution of the virus in fetal tissue is similar to the distribution which is established in post-natal infected swine. The objective of these investigations was to determine intrauterine (transplacental) infection in the event of the appearance of CSF in different periods of pregnancy in non-vaccinated and vaccinated sows. The examined material were organs and tissue of fetuses within two examined cases of CSF in non-immune pregnant sows. In the third examined case of CSF, the material comprised the blood of piglets before suckling the colostrum, animals originating from vaccinated sows, at a farm in which CSF had been diagnosed. Samples of tissue and blood of the piglets were examined for the presence of antigens and specific antibodies against the CSF virus using the immunoenzyme technique (ELISA). Even though the investigations were performed on a small number of samples, the obtained results raise the question of the possibility of the occurrence of intrauterine infection with a CSF field virus in sows vaccinated with the C-strain of CSF. The syndrome of a carrier sow and persistent infections are the chief problem factors that need to be considered within the programme of curbing and eradicating classical swine plague.

Status of oocytes and embryos of X‐autosome translocation carrier sows

The impact of an X‐autosome translocation t(Xp+;14q‐), on ovulation, fertilization and embryo survival in carrier sows, was examined and compared with these parameters of normal sows. Corpora lutea counts during week‐2 and week‐4 of gestation were similar in normal and carrier sows (14.4 ± 1.36 and 15.5 ±2.18) although embryo recovery (11.0 ± 1.87 and 6.0 ± 1.47) was lower than that from normal sows (12.8± 1.46 and 11.5 ±0.87), at these stages. Among the embryos karyotyped from the week‐2 embryos of carrier sows, 42 % were normal, 26.4% were carriers and 31.6% were of unbalanced chromosome make‐up, and of the week‐4 embryos of carriers, 33.3% were normal, 57.1% were carriers and 9.1% were chromosomally unbalanced females. The preponderance of females among the unbalanced embryos recovered at week‐2 of gestation (11_ and 1_) and the total absence of males among those recovered at week‐4, suggest that oocytes with unbalanced chromosome constitution are eliminated before week‐2 of gestation if they are fertilized by Y bearing sperm, and that the unbalanced oocytes fertilized by X bearing sperm survive up to the peri‐attachment stage even though all chromosomally unbalanced embryos are eliminated before term regardless of their sex.

An on-farm study of the epidemiology of Actinobacillus pleuropneumoniae infection in pigs as part of a vaccine efficacy trial

Thirty cohort pigs were followed from birth to slaughter to study epidemiological patterns of porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae. The study was conducted within a larger 380-animal study of vaccination against Mycoplasma hyopneumoniae and A. pleuropneumoniae in a 340-sow farrow-to-finish piggery with 4-month weaning, operating a continuous system of intensive production in the North Island of New Zealand. The cohort pigs were randomly allocated into two equal groups: vaccinated and control. Pigs in the first group were vaccinated at 2 and 4 weeks of age with both M. hyopneumoniae vaccine and A. pleuropneumoniae vaccine at separate vaccination sites. A series of nasal swabs was taken at 4, 8, 10, 11, 12, 14, 16 and 18 weeks of age. Each swab was streaked onto the surface of a selective medium on the farm and the plates were immediately transported to a laboratory and incubated at 37°C for 5 days. After the trial, pigs were slaughtered at an average of 132 days of age, lungs were examined and samples taken for bacteriological culture and isolation. Thirty-five out of 256 samples produced haemolytic colonies which were Gram-negative, V-factor-dependent and positive to the CAMP test. A. pleuropneumoniae was first isolated at 4 weeks of age from one vaccinated pig. This finding suggests that piglets became infected in the farrowing pen and the source of infection might be a carrier sow. The interval-specific cumulative incidence of A. pleuropneumoniae infection reached a maximum of 54% and 40% at 11 weeks of age in the vaccinated and control groups, respectively. Infection status of the litter is considered to be a factor influencing morbidity in infected herds during weaner and grower periods. Our results suggest that simultaneous vaccination with M. hyopneumoniae and A. pleuropneumoniae vaccines at 2 and 4 weeks of age might lessen the prevalence but cannot absolutely prevent A. pleuropneumoniae infection during the weaner or grower-finisher periods.

Early embryonic loss in x-autosome translocation carrier sows

Early embryonic loss in x-autosome translocation carrier sows

<I>Actinobacillus pleuropneumoniae</I>1 型の新生豚感染

Suckling piglets delivered from sows that had complement-fixing antibody titers of 1: 32 againstA. pleuropneumoniaeserotype 1 were infected with this organism at as early as 10-day-old. Maternal antibody gradually declined and was not demonstrated hereafter 40-day-old, and pneumonic lung lesions were first detected in a piglet sacrificed at 46-day-old. From the results obtained here it was suggested that piglets were infected with A. pleuropneumoniae from their carrier sows at the early stage of suckling but the onset of illness might be delayed until maternal antibody disappeared.

An inherited congenital goitre in pigs.

A diagnosis of congenital goitre was confirmed histologically in piglets which were born hairless and swollen, and with significantly enlarged thyroid glands. The iodine content of the thyroid glands and the serum total thyroxine concentrations were very low. No evidence was found of iodine deficiency or significant goitrogenic activity in the diet fed to the sows. An investigation of the parentage of six affected litters revealed that they all had one or other of two boars as the sire, grandsire or great grandsire. The suspicion of an inherited disorder was confirmed when a test mating of a suspect carrier boar and sow resulted in the birth of two affected piglets. The ratio of the numbers of affected and unaffected piglets was statistically consistent with an autosomal recessive mode of inheritance.

Characterization of a translocation and its impact on fertility in the pig.

A chromosome anomaly originally detected in two intersex pigs and previously reported as a Robertsonian translocation was characterized, and its transmission pattern was assessed by analyzing the progeny and immediate relatives of a sow. Cytogenetic investigations on four phenotypically normal relatives of the intersexes, including a sow, showed that the rearrangement was a reciprocal translocation involving the X chromosome and an autosome. Giemsa-banding and reverse-banding techniques indicated that the break in the X chromosome may have occurred in the terminal segment of the short arm (Xp) and that in the autosome in the proximal segment of the acrocentric chromosome 14 (14q). Reverse-banding techniques also revealed that the normal X is the late-replicating X in female translocation carriers. Centromere banding revealed an intercalary band on the long arm of the submetacentric chromosome representing the altered X in translocation carriers. The translocation was designated as rcp (X;14) (p+;q-). Chromosome analysis on 45 of 72 live offspring of the carrier sow showed a 20:25 distribution of carriers to normal piglets, with a carrier to normal ratio of 5:13 among females and 15:12 among male piglets, indicating an overall reduction of females (18) compared with males (27). Male carriers from one litter at sexual maturity showed hypoplastic testes and no spermatozoa in their ejaculates or fluid aspirates from the cauda epididymis. Their seminiferous tubules were narrow, spermatogenesis was impaired, and pyknotic and giant nuclei were abundant in the germinal epithelium. Meiotic preparations showed no stage beyond pachytene, suggesting that the absence of spermatozoa in the ejaculates may be due to the arrest of cells at the pachytene stage.

A review on classical swine fever infections in pigs: Epizootiology, clinical disease and pathology

A review is given on classical swine fever (CSF) including epizootiology, clinical disease and pathology. Under the item of epizootiology the history of CSF is briefly summarized. Ways of transmission are described with special reference to CSF in wild boars. The chapter about clinical disease includes the description of different courses of CSF such as peracute, acute, subacute form and chronic disease with reference to the course of transplacental infection and fate of the progeny associated with the “carrier sow syndrome”. The most typical lesions in CSF are summarized in the chapter of pathology.

Epizootiology of swine fever

Summary In countries with an intensive pig production system, swine fever (SF), where present, may be regarded as one of the economically most important pig diseases. Although reservoirs of virus and pathways of transmission are well understood, field experience has shown that the disease is extremely difficult to eradicate, especially in countries with intensive pig breeding. The virulence of the strains circulating in the field and the measures applied to control the disease determine to a large extent the course of an epizootic. Outbreaks of highly virulent strains are readily recognised. Epizootics caused by low virulent strains are characterised by indistinct signs of disease, slow spread of virus through the herd and the comparatively important role of the 'carrier sow syndrome'. The latter phenomenon may result in the birth of healthy looking but persistently infected and immune-tolerant piglets. This and the occurrence of chronic infections are largely responsible for the perpetuation of the virus in the pig population. The 1982-1985 epizootic of SF in the Netherlands showed an almost equal prevalence of outbreaks in breeding and fattening herds. In proportion to the number of herds in the country, there was in both categories an approximately linear relationship between the risk of infection and the size of the herds. The proportion of affected breeding herds increased as mass vaccination of areas at risk was started earlier in the epizootic. In close-knit communities with large breeding units, man should be regarded as the single most important factor in the herd-to-herd transmission of the virus, whereas in fattening units and in areas with small pig farms, transport and introduction of infected pigs into herds accounts for the majority of outbreaks and for the spread of the disease. Farms visited by persons handling pigs subsequent to contact with an infected herd should, therefore, be included in the epizootiological investigations. Virological and serological methods are indispensable in order to confirm or to rule out the possibility of infection of suspected contacts, especially in outbreaks caused by strains of low virulence. Continuous education of farmers regarding the importance of maintaining precautionary measures against the introduction of contagious diseases and, in the case of an advancing epizootic, special instructions to all people entering piggeries, would contribute greatly to reducing the untraceable pathways of SF spread.